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Merhod for the production of a sealing cover and sealing cover produced by said method |
The sealing cover (1) comprises a planar support (3), on which a number of sealing elements (4) are arranged, each of which is provided for the sealing of a sample container (2). The planar support (3) and the sealing elements (4) are produced from different materials in the same injection moulding tool (21) by means of two-component injection moulding. The planar support (3) is detachably connected to the sealing elements (4) by means of adhesion. The planar support (3) and the sealing elements (4) are preferably made from related plastics. |
1. Method of producing a sealing mat (1) having a carrier sheet (3) on which are arranged multiple sealing elements (4), each being provided for sealing a test tube (2), characterized in that the carrier sheet (3) and the sealing elements (4) are made of different materials in the same injection die mold (21) by two-component injection molding, and the carrier sheet (3) is thus detachably joined to the sealing elements (4) by adhesion. 2. Method as claimed in claim 1, characterized in that the carrier sheet (3) and the sealing elements (4) are made of related plastics. 3. Method as claimed in claim 1 or 2, characterized in that each sealing element has a flange (8) projecting outward on an upper edge, the carrier sheet (3) being integrally molded onto this flange. 4. Method as claimed in claim 3, characterized in that the flange (8) has an upper surface (14) onto which the carrier sheet (3) is integrally molded. 5. Method as claimed in any one of claims 1 through 4, characterized in that the carrier sheet (3) is made of a thermoplastic elastomer. 6. Method as claimed in any one of claims 1 through 4, characterized in that the carrier sheet (3) is made of silicone rubber. 7. Method as claimed in any one of claims 1 through 4, characterized in that the carrier sheet (3) is made of SEBS or SBS. 8. Method as claimed in any one of claims 1 through 4, characterized in that the sealing elements (4) are made of polypropylene or polyethylene. 9. Method as claimed in any one of claims 1 through 7, characterized in that the sealing elements (4) are made of EVA. 10. Method as claimed in any one of claims 1 through 9, characterized in that the bonding strength between the carrier sheet (3) and one of the sealing elements (4) is essentially greater than the holding force of a sealing element (4) in a test tube (2). 11. Method as claimed in any one of claims 1 through 10, characterized in that first the sealing elements (4) are injected and then the carrier sheet (3) is injected. 12. Sealing mat produced by the method as claimed in claim 1, characterized in that each sealing element (4) has a flat connection (28) on the upper edge, where the sealing (4) is detachably joined to the carrier sheet (3) by adhesion. 13. Sealing mat as claimed in claim 12, characterized in that the flat connection (28) is arranged on an upper edge of the sealing element (4). 14. Sealing mat as claimed in claim 13, characterized in that the sealing element (4) has a flange (8) protruding outward on said upper edge and the flat connection (28) is formed by an end face (14) of this flange (8). 15. Sealing mat as claimed in any one of claims 12 through 14, characterized in that the sealing elements (4) are made of polypropylene or polyethylene and the carrier sheet (3) is made of a thermoplastic elastomer. 16. Sealing mat as claimed in any one of claims 12 through 15, characterized in that the carrier sheet (3) and the sealing elements (4) are made of different but related plastics. 17. Sealing mat as claimed in any one of claims 12 through 16, characterized in that the carrier sheet (3) is made of SEBS or SBS and the sealing elements are made of EVA. 18. Sealing mat as claimed in any one of claims 12 through 17, characterized in that each sealing element (4) has a nose (31) on the top side of a bottom (11) on which the sealing element (4) can be gripped and pulled away from the test tube (2). |
Method of diagnosing or monitoring saccharometabolic error |
This invention provides a method for diagnosing geriatric diseases associated with insulin resistance, such as type II diabetes, at an early stage and a method for monitoring the therapeutic effects of agents for type II diabetes by quantitatively assaying GBP28 with the use of a monoclonal antibody that can assay adiponectin (GBP28) in a sample. |
1. A method for diagnosing or monitoring carbohydrate metabolism disorders by assaying adiponectin (GBP28) in a sample. 2. The method according to claim 1, wherein the carbohydrate metabolism disorder is type II diabetes. 3. The method according to claim 1, wherein the GBP28 is of a naturally-occurring type. 4. A method for monitoring the therapeutic effects of agents for type II diabetes by assaying GBP28 in a sample. 5. The method according to claim 4, wherein the 0BP28 is of a naturally-occurring type. 6. The method according to claim 4, wherein the agents for type II diabetes are thiazolidine derivatives. 7. The method according to claim 5, wherein the assay is conducted through antigen-antibody reactions. 8. A method for assaying GBP28, wherein naturally-occurring GBP28 in a sample is assayed. 9. The method according to claim 1, wherein GBP28 is assayed using a monoclonal antibody that specifically reacts with naturally-occurring GBP28. 10. The method according to claim 9, wherein the monoclonal antibody used is produced from hybridoma FERM BP-7660 or FERM BP-7661. 11. The method according to claim 1, wherein GBP28 is assayed by the solid phase method, competitive assay, agglutination assay, turbidimetric assay, or sandwich enzyme immunoassay. 12. The method according to claim 1, wherein the sample is serum, plasma, synovial fluid, pleural effusion, tissue extract, tissue, culture supernatant, or urine. 13. A monoclonal antibody that specifically reacts with naturally-occurring GBP28 having a trimer structure of GBP28 and/or a structure of aggregated GBP28 trimers. 14. The monoclonal antibody according to claim 13, which is produced from hybridoma PERM BP-7660 or FERM BP-7661. 15. A kit for assaying naturally-occurring GBP28 in a sample, which comprises a monoclonal antibody that specifically reacts with naturally-occurring GBP28. |
<SOH> BACKGROUND ART <EOH>The number of patients afflicted with diabetes is as many as 100,000,000 or more throughout the world, and 90% or more thereof are afflicted with type II diabetes (Schoonjans, K. and Auwerx, J., Lancet 355: 1008-1010, 2000). Type II diabetes is characterized by impaired insulin secretion and/or insulin resistance (DeFronzo, R, A. ct al., Diabetes Care 15: 318-368, 1992). Thiazolidinediones had been found both in animal experiments and in clinical studies to improve insulin resistance. When such agents were administered to patients with type II diabetes, insulin action was elevated. This adversely resulted in lowered levels of blood glucose, glycohemoglobin, and serum insulin (Schwartz, S. et al., N. Engl. J. Med. 338: 861-866, 1998). Adiponectin, which is a plasma protein derived from the adipocyte (Maeda, K. et al., Biochem. Biophys. Res. Commun. 221: 286-289, 1996; Arita, Y. et al., Biochem. Biophys. Res. Commun. 257: 79-83, 1999) (also known as a gelatin-binding protein of 28 kDa (GBP28) (Nakano, Y et al., J. Biochem. 120: 803-812, 1996; Saito, K. et al., Gene 229: 67-73, 1999; Saito, K. et al., Biol. Pharm. Bull. 22: 1158-1162, 1999)), adheres to a damaged vascular wall in vitro, blocks endodermal NF-κB signal transmission (Ouchi, N. et al., Circulation 102: 1296-1301, 2000), and inhibits cell proliferation of smooth muscles induced by HB-EGF and PDGF (Matsuzawa, Y. et al., Ann. N. Y. Acad. Sci. 892: 146-154, 1999). In the case of obesity patients, the levels of this protein in serum bad been reported to be low (Arita Y. et al., Biochem. Biophys. Res. Commun. 257: 79-83, 1999). In the case of patients with type II diabetes and those of coronary artery diseases, the aforementioned serum levels bad been reported to be lowered (Hotta, K. et al., Arterioscler Thromb. Vasc. Biol. 20: 1595-1599, 2000). In the past, blood glucose, HbA IC , serum insulin, HOMA-IR, and the like had been known as factors associated with type II diabetes. These factors were, however insufficient in terms of operability and/or sensitivity as indicators for diagnosing type II diabetes or for monitoring therapeutic effects. As mentioned above, association of GBP28 with type II diabetes had been also suggested. However, it was not sufficiently clarified whether or not GBP28 was actually applicable to the diagnosis of type II diabetes and the monitoring of therapeutic effects in the clinical field. The ELISA technique of Ohmoto et al. is known as a method for assaying GBP28 (Ohmoto et al., BIO Clinica 15(10) 758-761, 2000; Japanese Laid-open Patent Publication (Kokai) No. 200-304748). Naturally occurring GBP28 in blood is constituted by 3 monomers, and 4 to 6 trimers are aggregated (J. Biochem. 120, 803-812, 1996). However, the process of the aforementioned conventional technique was complicated due to the use of a monoclonal antibody to GBP28 having a monomeric structure. This required naturally occurring GBP28 in a sample to be denatured, a specimen to be mixed with an SDS solution at the time of assay, and thermal treatment to be conducted at 100° C. This conventional technique was to assay the naturally occurring GBP28 in a denatured state. Therefore, it could not directly assay the GBP28 in its natural state. |
<SOH> SUMMARY OF THE INVENTION <EOH>In order to overcome the above problems, the present inventors have assayed the blood concentrations of a variety of parameters associated with type II diabetes in the patients with this ailment. They have also conducted concentrated studies in order to search for factors that could be clinical indicators for the diagnosis of carbohydrate metabolism disorders, such as type II diabetes, and the monitoring of therapeutic effects of agents for type II diabetes. As a result, they have found that the assay of GBP28 was very effective for the diagnosis of carbohydrate metabolism disorders and for the monitoring of the therapeutic effects of the agents therefor as described in the Examples below. They have also found that GBP28 in a sample can be simply and rapidly assayed in its natural state by assaying the GBP28 in an aggregated form. This has led to the completion of the present invention. More specifically, the present invention provides the following (1) to (13). (1) A method for diagnosing or monitoring carbohydrate metabolism disorders by assaying adiponectin (GBP28) in a sample. (2) The method according to (1) above, wherein the carbohydrate metabolism disorder is type II diabetes. (3) The method according to (1) or (2) above, wherein the GBP28 is of a naturally occurring type. (4) A method for monitoring the therapeutic effects of agents for type II diabetes by assaying GBP28 in a sample. (5) The method according to (4) above, wherein the GBP28 is of a naturally occurring type. (6) The method according to (4) or (5) above, wherein the agents for type II diabetes are thiazolidine derivatives. (7) The method according to (5) or (6) above, wherein the assay is conducted through antigen-antibody reactions. (8) A method for assaying GBP28, wherein naturally-occurring GBP28 in a sample is assayed. (9) The method according to any of (1) to (8) above, wherein GBP28 is assayed using a monoclonal antibody that specifically reacts with naturally occurring GBP28. (10) The method according to (9) above, wherein the monoclonal antibody used is produced from hybridoma FERM BP-7660 or FERM BP-7661. (11) The method according to any of (1) to (10) above, wherein GBP28 is assayed by the solid phase method, competitive assay, agglutination assay, turbidimetric assay, or sandwich enzyme immunoassay. (12) The method according to any of (1) to (11) above, wherein the sample is serum, plasma, synovial fluid, pleural effusion, tissue extract, tissue, culture supernatant, or urine. (13) A monoclonal antibody that specifically reacts with naturally occurring GBP28 having a trimer structure of GBP28 and/or a structure of aggregated GBP28 trimers. (14) The monoclonal antibody according to (13) above, which is produced from hybridoma FERM P-7660 or FERM-7661. (15) A kit for assaying naturally occurring GBP28 in a sample, which comprises a monoclonal antibody that specifically reacts with naturally occurring GBP28. This description includes part or all of the contents as disclosed in the description and/or drawings of Japanese Patent Application No. 2001-248047, which is a priority document of the present application. |
Alkyne-aryl phosphodiesterase-4 inhibitors |
Compounds represented by Formula (I) or a pharmaceutically acceptable salt thereof, are phosphodiesterase 4 inhibitors useful in the treatment of asthma and inflammation. |
1-28. (canceled). 29. A method of treatment or prevention of asthma; chronic bronchitis; chronic obstructive pulmonary disease; adult respiratory distress syndrome; infant respiratory distress syndrome; cough; chronic obstructive pulmonary disease in animals; adult respiratory distress syndrome; ulcerative colitis; Crohn's disease; hypersecretion of gastric acid; bacterial, fungal or viral induced sepsis or septic shock; endotoxic shock; laminitis or colic in horses; spinal cord trauma; head injury; neurogenic inflammation; pain; reperfusion injury of the brain; psoriatic arthritis; rheumatoid arthritis; ankylosing spondylitis; osteoarthritis; inflammation; or cytokine-mediated chronic tissue degeneration; allergic rhinitis, allergic conjunctivitis, eosinophilic granuloma, osteoporosis, arterial restenosis, atherosclerosis, reperfiision injury of the myocardium chronic glomerulonephritis, vernal conjunctivitis, cachexia, transplant rejection, or graft versus host disease; or memory impairment, monopolar depression, Parkinson disease, Alzheimer's disease, acute and chronic multiple sclerosis, psoriasis, benign or malignant proliferative skin diseases, atopic dermatitis, urticaria, cancer, tumor growth or cancerous invasion of normal tissues: comprising the step of administering a therapeutically effective amount, or a prophylactically effective amount, of the compound for Formula (I) or a pharmaceutically acceptable salt thereof, wherein: R is H, —C1-6alkyl or —C3-6cycloalkyl; R1 is H, or a —C1-6alkyl, —C3-6cycloalkyl, —C1-6 alkoxy, —C2-6alkenyl, —C3-6alkynyl, —C(O)—C1-6alkyl, C(O)-aryl, C0-6alkyl)SOn—(C1-6alkyl), —(C0-6alkyl)SOn-(aryl), phenyl, wherein aryl is selected from phenyl or naphthyl and wherein any of the groups is optionally substituted with 1-3 independent —C1-6alkyl, —C1-6alkoxy, OH, —N(C0-6alkyl)(C0-6alkyl), —(C0-6alkyl)SOnC1-6alkyl), nitro, CN, ═N—O—C1-6alkyl, —O—N═C1-6alkyl, or halogen substituents; R2 is absent, H, halogen, —C1-6alkyl, —C3-6cycloalkyl, —C1-6alkyl(C3-6cycloalkyl)(C3-6cycloalkyl), —C1-6alkoxy, phenyl, nitro, CN, ═N—O—C1-6alkyl, —O—N═C1-6alkyl, —N(C0-6alkyl)(C0-6alkyl), —NHSOn—(C1-6alkyl), —NHC(O)C1-6alkyl, —C(O)C1-6alkyl, —C(O)—O—C1-6alkyl, —C1-6alkyl(═N—OH), —C(N═NOH)C1-6alkyl, —C0-6alkyl(oxy)C1-6alkyl-phenyl, —SOnNH(C0-6alkyl), or —(C0-6alkyl)SOn—(C1-6alkyl), wherein the phenyl, is optionally substituted with halogen, —C1-6alkyl, —C1-6alkoxy, hydroxy, —N(C0-6alkyl)(C0-6alkyl), or —C(O)—O—C1-6 alkyl, and any alkyl is optionally substituted with 1-6 independent halogen or —OH substituents; n is 0, 1, or 2; R3 is absent, H, OH, —N(C0-6alkyl)(C0-6alkyl), halogen or C1-6alkyl, wherein any alkyl is optionally substituted with 1-6 independent halogen, OH, or —N(C0-6alkyl)(C0-6alkyl) substituents; R4, R5, R6, and R7 each independently is H, halogen, —C1-6alkyl, —C1-6alkoxy, —SOn—(C1-6alkyl), nitro, CN, or —N(C0-6alkyl)(C0-6alkyl), and any alkyl is optionally substituted with 1-6 independent halogen or —OH substituents; and R8 is pyridyl or pyridonyl, or pyridyl N-oxide. 30. A method according to claim 29 wherein: R8 is pyridyl, or pyridyl N-oxide. 31. A method according to claim 29 wherein: R is Hydrogen. 32. A method according to claim 29 wherein R4, R5, R6 and R7 are each hydrogen. 33. A method according to claim 29 wherein R1 is —C3-6cycloalkyl. 34. A method according to claim 29 wherein R is Hydrogen; and R4, R5, R6 and R7 are each hydrogen. 35. A method according to claim 34 wherein R8 is pyridyl or pyridyl N-oxide. 36. A method according to claim 34 wherein R1 is 3 cycloalkyl. 37. A method according to claim 29 wherein the compound is selected from N-Isopropyl-1-[3-(2-pyridinylethynyl)phenyl]-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; N-Isopropyl-1-[3-(4-pyridinylethynyl)phenyl]-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; N-Isopropyl-1-[3-(1-oxido-4-pyridinylethynyl)phenyl]-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; N-Isopropyl-1-[3-(3-pyridinylethynyl)phenyl]-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; N-Isopropyl-1-[3-(1-oxido-3-pyridinylethynyl)phenyl]-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; N-Cyclopropyl-1-[3-(3-pyridinylethynyl)phenyl]-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; N-Cyclopropyl-1-[3-(1-oxido-3-pyridinylethynyl)phenyl]-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; N-Isopropyl-1-[3-(6-amino-3-pyridinylethynyl)phenyl]-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; N-Isopropyl-1-{3-[5-(1-hydroxy-1-methylethyl)-1-oxido-3-pyridinylethynyl]phenyl}-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; N-Isopropyl-1-{3-[6-(1-hydroxy-1-methylethyl)-3-pyridinylethynyl]phenyl}-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; N-Isopropyl-1-{3-[6-(1-hydroxy-1-methylethyl)-1-oxido-3-pyridinylethynyl]phenyl}-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; N-Isopropyl-1-{3-[4-(1-hydroxy-1-methylethyl)-2-pyridinylethynyl]phenyl}-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; N-Isopropyl-1-{3-[5-(1-hydroxy-1-methylethyl)-2-pyridinylethynyl]phenyl}-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; N-Isopropyl-1-{3-[6-(1-hydroxy-1-methylethyl)-2-pyridinylethynyl]phenyl}-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; N-Cyclopropyl-1-{3-[6-(1-hydroxy-1-methylethyl)-1-oxido-3-pyridinylethynyl]phenyl}-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; 1-[3-(1-Oxido-3-pyridinylethynyl)phenyl]-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; or 1-[3-(1-Oxido-3-pyridinylethynyl)phenyl]-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxylic acid; or a pharmaceutically acceptable salt thereof. 38. A method according to claim 29 wherein the compound is N-Cyclopropyl-1-[3-(3-pyridinylethynyl)phenyl]-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; or a pharmaceutically acceptable salt thereof. 39. A method according to claim 29 wherein the compound is N-Cyclopropyl-1-[3-(1-oxido-3-pyridinylethynyl)phenyl]-1,4-dihydro[1,8]naphthyridin-4-one-3-carboxamide; or a pharmaceutically acceptable salt thereof. 40. A method according to claim 29 selected from asthma; chronic bronchitis; chronic obstructive pulmonary disease; adult respiratory distress syndrome; infant respiratory distress syndrome; cough and adult respiratory distress syndrome; arthritis; rheumatoid arthritis; osteoarthritis and inflammation. 41. A method according to claim 29 selected from memory impairment, monopolar depression, Parkinson disease and Alzheimer's disease. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention is directed to compounds that are alkyne-aryl substituted 1,8-naphthyridin-4(1H)-ones. In particular, this invention is directed to phenyl substituted 1,8-naphthyridin-4(1H)-ones which are phosphodiesterase-4 inhibitors wherein the phenyl group is at the 1-position and contains a 2-alkyne substituent group further optionally substituted. 2. Related Background Hormones are compounds that variously affect cellular activity. In many respects, hormones act as messengers to trigger specific cellular responses and activities. Many effects produced by hormones, however, are not caused by the singular effect of just the hormone. Instead, the hormone first binds to a receptor, thereby triggering the release of a second compound that goes on to affect the cellular activity. In this scenario, the hormone is known as the first messenger while the second compound is called the second messenger. Cyclic adenosine monophosphate (adenosine 3′,5′-cyclic monophosphate, “cAMP” or “cyclic AMP”) is known as a second messenger for hormones including epinephrine, glucagon, calcitonin, corticotrophin, lipotropin, luteinizing hormone, norepinephrine, parathyroid hormone, thyroid-stimulating hormone, and vasopressin. Thus, cAMP mediates cellular responses to hormones. Cyclic AMP also mediates cellular responses to various neurotransmitters. Phosphodiesterases (“PDE”) are a family of enzymes that metabolize 3′, 5′ cyclic nucleotides to 5′ nucleoside monophosphates, thereby terminating cAMP second messenger activity. A particular phosphodiesterase, phosphodiesterase-4 (“PDE4”, also known as “PDE-IV”), which is a high affinity, cAMP specific, type IV PDE, has generated interest as potential targets for the development of novel anti-asthmatic and anti-inflammatory compounds. PDE4 is known to exist as at lease four isoenzymes, each of which is encoded by a distinct gene. Each of the four known PDE4 gene products is believed to play varying roles in allergic and/or inflammatory responses. Thus, it is believed that inhibition of PDE4, particularly the specific PDE4 isoforms that produce detrimental responses, can beneficially affect allergy and inflammation symptoms. It would be desirable to provide novel compounds and compositions that inhibit PDE4 activity. A major concern with the use of PDE4 inhibitors is the side effect of emesis which has been observed for several candidate compounds as described in C. Burnouf et al., (“Burnouf”), Ann. Rep. In Med. Chem., 33:91-109(1998). B. Hughes et al., Br. J. Pharmacol., 118:1183-1191(1996); M. J. Perry et al., Cell Bioclhem. Biophys., 29:113-132(1998); S. B. Christensen et al., J. Med. Chem., 41:821-835(1998); and Burnouf describe the wide variation of the severity of the undesirable side effects exhibited by various compounds. As described in M. D. Houslay et al., Adv. In Pharmacol., 44:225-342(1998) and D. Spina et al., Adv. In Pharmacol., 44:33-89(1998), there is great interest and research of therapeutic PDE4 inhibitors. International Patent Publication WO9422852 describes quinolines as PDE4 inhibitors. International Patent Publication WO9907704 describes 1-aryl-1,8-naphthylidin-4-one derivatives as PDE4 inhibitors. A. H. Cook, et al., J. Chem. Soc., 413-417(1943) describes gamma-pyridylquinolines. Other quinoline compounds are described in Kei Manabe et al., J. Org. Chem., 58(24):6692-6700(1993); Kei Manabe et al., J. Am. Chem. Soc., 115(12):5324-5325(1993); and Kei Manabe et al., J. Am. Chem. Soc., 114(17):6940-6941(1992). Compounds that include ringed systems are described by various investigators as effective for a variety of therapies and utilities. For example, International Patent Publication No. WO 98/25883 describes ketobenzamides as calpain inhibitors, European Patent Publication No. EP 811610 and U.S. Pat. Nos. 5,679,712, 5,693,672 and 5,747,541 describe substituted benzoylguanidine sodium channel blockers, U.S. Pat. No. 5,736,297 describes ring systems useful as a photosensitive composition. U.S. Pat. Nos. 5,491,147, 5,608,070, 5,622,977, 5,739,144, 5,776,958, 5,780,477, 5,786,354, 5,798,373, 5,849,770, 5,859,034, 5,866,593, 5,891,896, and International Patent Publication WO 95/35283 describe PDE4 inhibitors that are tri-substituted aryl or heteroaryl phenyl derivatives. U.S. Pat. No. 5,580,888 describes PDE4 inhibitors that are styryl derivatives. U.S. Pat. No. 5,550,137 describes PDE4 inhibitors that are phenylaminocarbonyl derivatives. U.S. Pat. No. 5,340,827 describes PDE4 inhibitors that are phenylcarboxamide compounds. U.S. Pat. No. 5,780,478 describes PDE4 inhibitors that are tetra-substituted phenyl derivatives. International Patent Publication WO 96/00215 describes substituted oxime derivatives useful as PDE4 inhibitors. U.S. Pat. No. 5,633,257 describes PDE4 inhibitors that are cyclo(alkyl and alkenyl)phenyl-alkenyl (aryl and heteroaryl) compounds. However, there remains a need for novel compounds and compositions that therapeutically inhibit PDE4 with minimal side effects. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is directed to alkyne-aryl substituted 1,8-naphthyridin-4(1H)-ones represented by Formula (I): or pharmaceutically acceptable salts thereof, which are phosphodiesterase-4 inhibitors. This invention also provides a pharmaceutical composition which includes an effective amount of the novel alkyne-aryl substituted 1,8-naphthyridin-4(1H)-ones and a pharmaceutically acceptable carrier. This invention further provides a method of treatment in mammals of, for example, i) Pulmonary disorders such as asthma, chronic bronchitis, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome, infant respiratory distress syndrome, cough, chronic obstructive pulmonary disease in animals, adult respiratory distress syndrome, and infant respiratory distress syndrome, ii) Gastrointestinal disorders such as ulcerative colitis, Crohn's disease, and hypersecretion of gastric acid, iii) Infectious diseases such as bacterial, fungal or viral induced sepsis or septic shock, endotoxic shock (and associated conditions such as laminitis and colic in horses), and septic shock, iv) Neurological disorders such as spinal cord trauma, head injury, neurogenic inflammation, pain, and reperfusion injury of the brain, v) Inflammatory disorders such as psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, inflammation and cytokine-mediated chronic tissue degeneration, vi) Allergic disorders such as allergic rhinitis, allergic conjunctivitis, and eosinophilic granuloma, vii) Psychiatric disorders such as depression, memory impairment, and monopolar depression, viii) Neurodegenerative disorders such as Parkinson disease, Alzheimer's disease, acute and chronic multiple sclerosis, ix) Dermatological disorders such as psoriasis and other benign or malignant proliferative skin diseases, atopic dermatitis, and urticaria, x) Oncological diseases such as cancer, tumor growth and cancerous invasion of normal tissues, xi) Metabolic disorders such as diabetes insipidus, xii) Bone disorders such as osteoporosis, xiii) Cardiovascular disorders such as arterial restenosis, atherosclerosis, reperfusion injury of the myocardium, and xiv) Other disorders such as chronic glomerulonephritis, vernal conjunctivitis, transplant rejection and graft versus host disease, and cachexia—maladies that are amenable to amelioration through inhibition of the PDE4 isoenzyme and the resulting elevated cAMP levels—by the administration of an effective amount of the novel alkyne-aryl substituted 1,8-naphthyridin-4(1H)-ones or a precursor compound which forms in vivo the novel alkyne-aryl substituted 1,8-naphthyridin-4(1H)-ones which are phosphodiesterase-4 inhibitors. detailed-description description="Detailed Description" end="lead"? |
Device for turning objects |
The invention relates to an object turner or a turning apparatus (4) for end-turning of e.g. ice-cream cones (22) after selective deflection of the objects (22) from an object conveyor (2) on which the objects are placed lying in horizontal trays (6). The novelty of the turning apparatus (4) according to the invention is that the turning apparatus (4) has selection means, which makes the turning process selective, and means ensuring that the process operates in parallel and synchronously with the object conveyor (2). The selected objects (22) are forwarded into a turning unit (8), where the object (22) is end-turned around a horizontal axis (38), and returned onto the object conveyor (2) in the same tray (6) previously occupied by the object such a turning apparatus (4) makes it possible for objects (22) to be end-turned in a predetermined pattern of e.g. all objects, every second or third object. The turning apparatus (4) shows a high level of reliability and at the same time gentle processing of the objects (22). The turning process is carried out quickly, and the apparatus (2, 4) typically has a capacity of 300 to 600 objects/minute |
1. Turning apparatus for end turning objects, preferably ice-cream cones, after selective deflection of the objects from an object conveyor on which the objects are placed lying in horizontal trays, wherein the turning apparatus (4) has selection means, which makes the turning process selective, and means ensuring that the process proceeds in parallel and synchronous with the object conveyor (2), on which objects (22) are forwarded in object trays (6), and where the selection means selects the objects (22) and forward these objects (22) separately across a turning unit (8), where the objects (22) and associated means (8, 16, 18) are turned in the orthogonal plane of the direction of movement about a horizontal axis, which is parallel with the object conveyor (2) and by the use of delivery means are delivered back on the object conveyor (2), preferably in the same object tray (6) as the object (22) previously were in; and wherein the selection means performs a selective and gentle “sweeping, pushing or deflection” of objects (22) into a turnable tube (8), where the deflection occurs by a transverse band on which adjustable means for different selection patterns are provided. 2. Turning apparatus according to claim 1, characterised in that the selection means of the turning apparatus are adapted to selective deflection according to a predetermined pattern, for instance all objects (22), every second or every third object (22) to a turning unit (8). 3. Turning apparatus according to the claims 1 or 2, characterised in that the turning apparatus (4) is divided into three sections, namely a receiving section (10), an end turning section (12) and a returning section (14), where the end turning is performed gently during coupling to the turning apparatus (8), where the complete installation (2, 4) has a capacity on 300-600 objects/minute. 4. Turning apparatus according to the claims 1-3, characterised in that the turning units (8) are controlled for end turning, wherein the rotation of each turn tube (20) is controlled by fixed guide rails (18), which end turn the tube (20) after it has left a receiving section (10) and before it enters into a delivery section (14), where the turning tube (20) immediately after the passage of a delivery section (14) is controlled by the guide rails (18) to an inclined position and is rotated back to the initial position before returning to the receiving section (10) where the turning tube (20) during the entire cycle is controlled by guide rails (18). 5. Tuning apparatus according to the claims 1-4, characterised in that the turning apparatus (4) comprises means for returning objects (22) to the object conveyor (2) where air under high pressure and an air current drives the object (22) out of the turning unit (8). 6. Turning apparatus according to the claims 1-5, characterised in that the turning apparatus (4) comprises a number of turning units (8) consisting of at least one turning tube (20) where the turn tube (20) is 360° rotatable about its rotation axis (38) and where the turn tube (20) is mounted (26) in a consol (24) at points (26) which are displaced relative to the centre line of the tube (20), so that the bottom of the turn tube lays under the level of the bottom of the object tray by the reception of an object (22). 7. Turning apparatus according to claim 6, characterised in that the turn tube (20) is provided with a funnel-shaped receiving end (40) and a tapered delivery end (42). 8. Turning apparatus according to the clams 6 and 7, characterised in that a turning unit (8) consists of at least one turn tube (20) and at least one console part (24) with a console foot (30) for fastening it to driving means of the pulling station and support arms (32) which extend from the console foot (30) for retention of at least one bottom plate (34) and where a tube holding arm (28) extend from the console (24) connecting the console (24) and the turn tube (20) via a bearing (26). 9. Turning apparatus according to the claims 6-8, characterised in that a turning unit (8) comprises at least one bottom plate (34) which is fastened to the console (24) and to support arms (32) protruding from the console (24) and formed in a shape, which corresponds to the circular arc which is described by the turn tube (20) when it is rotated about its rotation axis (38). |
Diagnostic testing process and apparatus |
A method and apparatus for use in a flow through assay process is disclosed. The method is characterised by a “pre-incubation step” in which the sample which is to be analysed, (typically for the presence of a particular protein), and a detection analyte (typically an antibody bound to colloidal gold or a fluorescent tag) which is known to bind to the particular protein may bind together for a desired period of time. This pre incubation step occurs before the mixture of sample and detection analyte come into contact with a capture analyte bound to a membrane. The provision of the pre-incubation step has the effect of both improving the sensitivity of the assay and reducing the volume of sample required for an assay. An apparatus for carrying out the method is disclosed defining a pre-incubation chamber for receiving the sample and detection analyte having a base defined by a membrane and a second membrane to which a capture analyte is bound. In one version the pre-incubation chamber is supported above the second membrane in one position but can be pushed into contact with the membrane carrying the capture analyte thus permitting fluid transfer from the incubation chamber through the capture membrane. In another version the membrane at the base of the incubation chamber is hydrophobic and its underside contacts the capture membrane and when a wetting agent is applied to the contents of the pre-incubation chamber fluid transfer occurs. |
1. a vertical flow through assay test apparatus comprising: a first member comprising a first, porous, membrane to which is bound a capture analyte for binding to a reagent to be detected, the member having an upper surface and a lower surface; a second member being a body of absorbent material disposed below and touching the lower surface of the first member; a vessel for containing a liquid sample spaced above the first member said vessel having side walls and a base, the base being defined by a second membrane, the vessel being capable of retaining a liquid sample for a predetermined incubation period; and means for supporting the vessel above the first member in two positions, a first position in which the membrane is spaced a sufficient distance from the first member so as to not permit fluid transfer from the vessel to the body of absorbent material, and a second position in which the second membrane is in contact with the first member, such contact permitting fluid transfer from the vessel through the first and second membranes to the body of absorbent material. 2. An apparatus as claimed in claim 1 wherein the second membrane defined at the base of the vessel is a hydrophilic membrane. 3. An apparatus as claimed in claim 1 wherein the second membrane defined at the base of the vessel is a hydrophobic membrane. 4. An apparatus as claimed in claim 2 wherein the first membrane is a nitrocellulose membrane. 5. An apparatus as claimed in claim 1 wherein the capture analyte is a ligand such as an antigen or antibody. 6. A vertical flow through assay test apparatus for use in an assay process comprising a housing including: a first member comprising a first, porous, membrane to which is bound a capture analyte for binding to a reagent to be detected, the member having an upper surface and a lower surface; a second member being a body of absorbent material such as tissue paper or the like disposed below and touching the lower surface of the first member; a vessel for containing a liquid sample located above the first member, said vessel having side walls, and a base defined by a second, hydrophobic, membrane, having an upper and a lower surface, the vessel being supported above the first member with the lower surface of the hydrophobic membrane in contact with the upper surface of the first member, the base being capable of retaining an aqueous sample in the vessel for a predetermined incubation period. 7. An apparatus as claimed in claim 6 wherein the vessel is a well. 8. An apparatus as claimed in claim 6 wherein the capture analyte is a ligand such as an antigen or antibody. 9. An apparatus as claimed in claim 6 wherein the body of absorbent material using a vertical flow through assay test apparatus comprises multiple layers of absorbent tissue. 10. A method for assaying for the presence of a pre-determined reagent in a liquid sample comprising the steps of: a) providing a first porous membrane to which capture analytes for binding to the reagent have been bound; b) placing a liquid sample to be assayed and a detection analyte in a vessel having a base defined by a second porous membrane, the vessel being capable of retaining the liquid sample for a predetermined incubation period; c) allowing a sufficient period of time to pass for the detection analyte to bind to the reagent, if present in the liquid sample; d) contacting the base of the vessel with the first porous membrane; and e) causing the liquid sample to flow through the membranes to allow the reagent to bind to the capture analyte carried on the first membrane. 11. A method as claimed in claim 10 further comprising the steps of: removing the vessel and washing the first membrane with a buffer prior to inspecting the membrane for the presence of the detection analyte. 12. A method as claimed in claim 10 further comprising the steps of: washing the first membrane with a buffer by addition to the vessel before removal and inspection of the first membrane for the presence of the detection analyte. 13. A method for assaying for the presence of a pre-determined reagent using a vertical flow through assay test apparatus comprising: a first member comprising a first, porous, membrane to which is bound a capture analyte for binding to a reagent to be detected, the member having an upper surface and a lower surface; a second member being a body of absorbent material disposed below and touching the lower surface of the first member; a vessel for containing a liquid sample spaced above the first member said vessel having side walls and a base, the base being defined by a second membrane, the vessel being capable of retaining a liquid sample for a predetermined incubation period; and means for supporting the vessel above the first member in two positions, a first position in which the membrane is spaced a sufficient distance from the first member so as to not permit fluid transfer from the vessel to the body of absorbent material, and a second position in which the second membrane is in contact with the first member, such contact permitting fluid transfer from the vessel through the first and second membranes to the body of absorbent material, comprising the steps of: a) placing a sample to be assayed and a detection analyte in the vessel, with the vessel disposed in the first position; b) allowing a sufficient period of time to pass for the detection analyte to bind to the reagent, if present; c) depressing the vessel to the second position to contact the base of the vessel with the first porous membrane; d) allowing the sample to flow through the first and second membranes to allow the reagent, if present to bind to the capture analyte carried on the first membrane. 14. A method as claimed in claim 13 wherein the second membrane defined at the base of the vessel is hydrophobic and the step of allowing the sample to flow through the first and second membranes includes the addition of a wetting agent on the upper surface of the second membrane prior to moving the vessel to the second position. 15. A method for assaying for the presence of a pre-determined reagent using a vertical flow through assay test apparatus comprising: a first member comprising a first, porous, membrane to which is bound a capture analyte for binding to a reagent to be detected, the member having an upper surface and a lower surface; a second member being a body of absorbent material disposed below and touching the lower surface of the first member; a vessel for containing a liquid sample spaced above the first member said vessel having side walls and a base, the base being defined by a second membrane which is a hydrophobic membrane, the vessel being capable of retaining a liquid sample for a predetermined incubation period; and means for supporting the vessel above the first member in two positions, a first position in which the membrane is spaced a sufficient distance from the first member so as to not permit fluid transfer from the vessel to the body of absorbent material, and a second position in which the second membrane is in contact with the first member, such contact permitting fluid transfer from the vessel through the first and second membranes to the body of absorbent material comprising the steps of: a) placing a sample to be assayed and a detection analyte in the vessel; b) allowing a sufficient period of time to pass for the detection analyte to bind to the reagent, if present; c) adding a wetting agent to the vessel; d) allowing the sample to flow through the first and second porous membranes to allow the reagent, if present to bind to the capture analyte carried on the first membrane. 16. A method as claimed in claim 10 wherein the sample is whole blood wherein whole red blood cells are removed in the vessel by the second membrane acting as a filter and plasma in the blood is allowed to flow-through to the first membrane. 17. A method as claimed in claim 10 wherein the sample contains particulate materials selected from the group including grain extracts, cell or microbial extracts wherein particulate materials are removed in the vessel by the second membrane acting as a filter. 18. A method as claimed in claim 10 wherein the sample comprises body fluids such as plasma, sera, urine, saliva and sputum and wherein the second membrane is a hydrophobic membrane. 19. A method as claimed in claim 10 including the step of removing or neutralising unwanted analytes in the sample to be tested that may interfere with the binding of the sample analyte with the capture analyte on the first membrane in the vessel. 20. A method as claimed in claim 19 wherein antibodies or other analytes which bind to the unwanted analytes are bound to the walls or the base of the vessel. 21. A method as claimed in claim 10 wherein the sample to be tested is carried on an absorbent surface such as a swab or the like and including the steps of swirling the swab in an extraction solution in the vessel. 22. Apparatus as claimed in claim 1 wherein the capture analytes comprise ligands selected from the group consisting of ligands for detecting tuberculosis, ligands for detecting HIV, ligands for detecting hepatitis, ligands for detecting syphilis and ligands for detecting malaria. 23. Apparatus as claimed in claim 22 wherein the first membrane carries capture analytes for detecting more than one disease. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Lateral flow and flow-through technology have been used for diagnostic assays for almost twenty years. Lateral flow technology is currently dominant because lateral flow devices are easy to produce and the assay can be performed in a simple 2-step process that can be adapted for whole blood separation. This results in a simple device that can be used in the field as a rapid point-of-care diagnostic (Cole et al 1996 Tuberc. Lung. Dis. 77:363-368). However, multiple disease diagnosis using lateral flow technology is very difficult because of differences in lateral diffusion between samples and variation in flow rates between batches of the partitioning membrane. This means that antigen or antibody signal strengths may vary both within tests and between batches of tests, resulting in inconsistent results. Existing flow-through diagnostic tests can be completed in less than two minutes compared with typical times of five to fifteen minutes for lateral flow tests. This advantage in speed however, is often at the expense of sensitivity. A further disadvantage is that higher volumes of sample are required to achieve the same sensitivity as lateral flow. This may be problematic in some situations. For example, the diagnosis of analytes (reagents) in whole blood requires the separation of plasma from whole blood cells. The higher volumes of whole blood required for this would quickly block the membranes in the flow-through format. The basic principal of flow-through assays is well established. The tests are designed to determine the existence of, and in some cases, the quantity of, a predetermined analyte/reagent in a sample. Often the reagent will be a protein but other reagents can be tested for. If the assay is to test for the existence of a particular disease in a patient, the patient's body fluids may be tested for an antibody or other protein produced by the patient in response to the infection, or for a protein which is expressed by the bacterium or viral agent or the like causing the disease. In a typical flow through assay a liquid sample which is believed to contain the reagent is sucked into an absorbent pad via a membrane to which is bound a capture analyte which is known to bind to the reagent. The membrane is then typically washed with a buffer and a liquid containing a detection analyte which also binds to the reagent and which includes a tracer or marker which is detectable, is applied to the membrane. The detection analyte binds to the immobilised reagent bound to the membrane and can be seen or otherwise detected to indicate the presence of the reagent. U.S. Pat. No. 4,246,339 discloses a test device for assaying liquid samples for the presence of a predetermined reagent. The device includes telescoping top and bottom members defining a liquid reservoir therebetween and resilient means for biasing the members in the open position. The top member defines a series of test wells each of which has a base defined by a microporous membrane with a capture analyte immobilised on the membrane surface. Absorbent means are located in the bottom member, spaced from the membrane in the open position but in contact therewith in the closed position. U.S. Pat. No. 4,246,339 discloses the adding the test serum diluted with a buffer to a test well, and incubating the device at room temperature for ten minutes prior to depressing the cassette to the closed position to pass the sample through the membranes into the absorbent material. When the membranes are dry, the membrane is washed and then covered with a solution containing a detection analyte which binds to the immobilised reagent followed by a subsequent step in which a stain is applied. It will be appreciated that the process described in U.S. Pat. No. 4,246,339, is a somewhat long drawn out, time consuming and tedious process and also lacks sensitivity. A more recent flow through device is described in U.S. Pat. No. 5,185,127 which discloses an assay device including a filter stack and an enclosure having a base portion and a lid. The filter stack has a hydrophilic membrane having a capture analyte thereon, referred to in U.S. Pat. No. 5,185,127 as a binder. A hydrophobic membrane is located under the hydrophilic membrane and a pad of absorbent material is located under the hydrophobic membrane. The lid includes an upwardly extending rib which defines a recess having an insert therein. In use, a sample containing the reagent (referred to in U.S. Pat. No. 5,185,127 as the analyte) is placed in the well of the assay device at which time the reagent/analyte binds to the capture analyte/binder. Flow of the assay solution however, does not take place because the aqueous solution does not wet the hydrophobic membrane placed under the hydrophilic membrane in the filter stack. Thus as much time is necessary to complete the binding of the detection analyte to the reagent is allowed. When binding is judged to be complete, flow may be initiated by adding a wetting agent which wets the hydrophobic membrane. After which time the aqueous liquid flows into pad of absorbent material. The membrane may then be washed and treated with a detection analyte/tracer which may be an antibody which specifically binds to the analyte, the antibody having a label covertly conjugated thereto. Again the sensitivity of U.S. Pat. No. 5,185,127 is lacking and is not equivalent to that obtainable in lateral flow or ELISA formats. It is one object of the present invention to provide an improved method and apparatus for use in an assay process such as an immunoassay, diagnostic assay or the like in which the process and apparatus are capable of screening a wide range of samples such as whole blood, plasma/serum, or samples with a high particulate load such as crushed grain (eg wheat heads) and which is simple and rapid to perform whilst still maintaining sensitivities at least equivalent to that obtainable in lateral flow or ELISA formats. A related object of the present invention is to provide a method and apparatus which can be successfully used for multiple disease diagnosis from a single whole blood or other sample in a single test. An extension would be successful screening of a sample in a single test for the presence of multiple analytes not necessarily related to disease (e.g drugs, agriculture, veterinary testing). Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application. Because the prior art is not consistent in its terminology, for the avoidance of doubt and for the purpose of clarity, the following terms used in the specification below, are defined as follows. The term “reagent” is used to refer to the compound protein or the like which is to be detected by the assay. The term “capture analyte” is used to refer to a compound which is bound to a membrane and to which the reagent will bind. The term “detection analyte” is used to refer to a compound which will also bind to the reagent and which carries a tracer or some other element whose presence may be detected, typically visually detected whether under visible light, or fluorescence. |
<SOH> SUMMARY OF THE INVENTION <EOH>In a first broad aspect, the present invention provides an apparatus and method for use in an assay process which is characterised by providing a “pre-incubation step” in which the sample and detection analyte (which may typically be an antibody bound to colloidal gold or a fluorescent tag) may bind together, which has the effect of both improving the sensitivity of the assay and reducing the volume of sample required for an assay prior to reaction of the sample/analyte complex with a reaction membrane to which one or more ligands are bound. Thus, in one aspect of the present invention there is provided an apparatus for use in an assay process comprising: a first member comprising a first, porous, membrane to which is bound a capture analyte for binding to a reagent to be detected, the member having an upper surface and a lower surface; a second member being a body of absorbent material disposed below and touching the lower surface of the first member; a vessel for containing a liquid sample spaced above the first member said vessel having side walls and a base, the base being defined by a second membrane, the vessel being capable of retaining a liquid sample for a predetermined incubation period; and means for supporting the vessel above the first member in two positions, a first position in which the membrane is spaced a sufficient distance from the first member so as to not permit fluid transfer from the vessel to the body of absorbent material, and a second position in which the second membrane is in contact with the first member, such contact permitting fluid transfer from the vessel through the first and second membranes to the body of absorbent material. In a related aspect the present invention provides a method for assaying for the presence of a pre-determined reagent in a liquid sample comprising the steps of: a) providing a first porous membrane to which capture analytes for binding to the reagent have been bound; b) placing a sample to be assayed and a detection analyte in a vessel having a base defined by a second porous membrane, the vessel being capable of retaining the liquid sample for a predetermined incubation period; c) allowing a sufficient period of time to pass for the detection analyte to bind to the reagent, if present in the liquid sample; d) contacting the base of the vessel with the first porous membrane; and e) causing the liquid sample to flow through the membranes to allow the reagent to bind to the capture analyte carried on the first membrane. Thus, the present invention provides a chamber which may serve as a pre-incubation chamber in which a pre-incubation step can occur where the sample and detection analyte combine, which improves the sensitivity of the test and reduces the volume of sample required for the assay. It has been found that the pre-incubation step increases the test sensitivity for a typical existing flow-through apparatus by approximately ten times to equivalent levels of sensitivity compared with lateral flow technology, while still allowing the assay to be completed in around two minutes compared to 10 minutes for lateral flow formats. For example a ground wheat head suspension may be solubilised, and mixed and pre-incubated in the chamber with a detection analyte in the form of an antibody against alpha-amylase linked to a colloidal gold particle. The contents of the chamber may then be allowed to flow through to the first membrane containing a capture analyte in the form of an immobilised anti-amylase antibody, and anti-amylase antibody/gold complexes will bind to the immobilised antibody forming a detectable signal. The signal can be detected by the removal of the pre-incubation unit and washing of the reaction membrane with buffer. This format can also be used for detecting reagents in whole blood since whole red blood cells can be removed in the pre-incubation chamber and the plasma allowed to flow-through to the reaction membrane containing a bound capture analyte. In this format, the base membrane defined at the base of the pre-incubation chamber will typically be a membrane which has the correct pore size to retain the red blood cells and allow the plasma to pass through on contact with the first membrane. Similarly particulate samples containing grain extracts, cell or microbial extracts can be analysed with this flow-through format since particulate matter can be removed in the pre-incubation chamber and therefore cannot block the reaction area on the upper surface of the reaction membrane. The apparatus can also be used for detecting analytes in body fluids other than blood, such as plasma, sera, urine, saliva and sputum. In this case, the sample can be retained in the pre-incubation chamber by use of a hydrophobic membrane. To obtain efficient flow through capillary action to the second member when the pre-incubation chamber is lowered, the reaction membrane is pre-wet with a wetting agent containing a detergent or the reaction membrane is blocked with a hygroscopic solution such as sucrose, trehalose, fructose, or alternatively, glycerol. This changes the characteristics of the reaction membrane from a non-hygroscopic to a hygroscopic membrane allowing the sample to flow through to the second member upon contact of the membrane at the base of the pre-incubation chamber with the reaction membrane. In a yet further embodiment, if a hydrophobic membrane is used as the base of the pre-incubation chamber, the apparatus may be used with the hydrophobic membrane and reaction membrane in contact, with the operator adding a wetting agent to the sample to cause flowthrough, when desired. Thus, in a related aspect, there is provided an apparatus for use in an assay process comprising a housing including: a first member comprising a first, porous, membrane to which is bound a capture analyte for binding to a reagent to be detected, the member having an upper surface and a lower surface; a second member being a body of absorbent material such as tissue paper or the like disposed below and touching the lower surface of the first member; a chamber located above the first member said chamber having side walls, and a base including a second, hydrophobic, membrane, having an upper and a lower surface, the pre-incubation chamber being supported above the first member with the lower surface of the hydrophobic membrane in contact with the upper surface of the first member. The pre-incubation chamber can also be used to remove analytes that may interfere with the assay, such as human anti-mouse antibodies (HAMAS), in solution or by binding anti-analyte antibodies to the surface of the chamber. The chamber can also be used to extract the analyte of interest from an absorbent surface such as a swab, which has been taken from the throat of a patient, by swirling the swab in an extraction solution in the chamber. The pre-incubation chamber may be part of a pre-filter unit which acts also to pre-filter the sample prior to contact with the upper surface of the first member. Examples of assays that can be preformed by this method where two reaction steps are involved (the incubation of the analyte with the labeled anti-analyte followed by the binding of this complex to a solid-phase anti-analyte), are: Direct Antigen Assay 1. Ag* (analyte)+Ab* 1 (anti-Ag)-label 2. Solid phase-Ab 2 (anti-Ag)+Ag/Ab 1 (anti-Ag)-label complex Direct Antibody Assay (i) 1. Ab 1 (analyte=anti-Ag)+Ab 2 (anti-Ab 1 )-label 2. Solid phase-Ag+Ab 1 (anti-Ag)/Ab 2 (anti-Ab 1 )-label complex Direct Antibody Assay (ii) 1. Ab 1 (analyte=anti-Ag)+Ab 2 (anti-Ab 1 )-label 2. Solid-phase-Ab 3 (anti-Ag)/Ag+Ab 1 (anti-Ag)/Ab 2 (anti-Ab 1 )-label complex Indirect Antigen Assay 1. Ag (analyte)+Ab 1 (anti-Ag)+Ab 2 (anti-Ab 1 )-label 2. Solid-phase-Ab 3 (anti-Ag)+Ag/Ab 1 (anti-Ag)/Ab 2 (anti-Ab 1 )-label complex Indirect antibody Assay (i) 1. Ab 1 (analyte=anti-Ag)+Ab 2 (anti-Ab 1 )+Ab 3 (anti-Ab 2 )-label 2. Solid phase Ag+Ab 1 (anti-Ag)/Ab 2 (anti-Ab 1 )/Ab 3 (anti-Ab 2 )-label complex Indirect Antibody Assay (ii) 1. Ab 1 (analyte=anti-Ag)+Ab 2 (anti-Ab 1 )+Ab 3 (anti-Ab 2 )-label 2. Solid phase Ab 4 (anti-Ag)/Ag+Ab 1 (anti-Ag)/Ab 2 (anti-Ab 1 )/Ab 3 (anti-Ab 2 )-label complex *Ag Indicates Antigen *Ab Indicates Antibody A piezoelectric driven printer may be used to dispense precise amounts of multiple disease ligands such as antigens or antibodies or an analyte as a micro array onto a reaction membrane for use in the apparatus of the first aspect of the present invention. The ligands or analytes may be dispensed in particular patterns, e.g. letters for ease of recognition of results. Typically, 100 pl of fluid reagent (1 drop), or multiples thereof, is dispensed, but this will vary depending on the application. The resultant size of the spot on the membrane is about 55 microns or more in diameter subject to fluid diffusion on the membrane, but again this will vary depending on the application. It is possible to dispense droplets with diameters of 5-10 microns, and hence lower volumes of fluid reagent (for example, 1-10 pl) can be applied. Using precise quantitative printing of micro arrays of antibodies, antigens, or other analytes means that tests using precise quantities of these reagents can be produced for multi disease diagnosis of a single sample. This array technology can be applied to tests for drugs or other markers across all diagnostic fields. Alternatively, an adult/neonatal syringe pump 1235 from ATOM Medical Corporation, Japan, typically used to administrator small quantities of intravenous liquids through a catheter to hospital patients can be adapted to apply single or multiple lines of a capture analyte to the first membrane eg nitrocellulose. In one preferred embodiment, ligands for detecting tuberculosis, HIV, hepatitis, syphilis and malaria antibodies may be deposited onto a reaction membrane. This would allow the simultaneous diagnosis of tuberculosis, HIV, hepatitis, syphilis and malaria from a single blood sample without the need for intermediate sample treatment steps. Utilising the present invention allows the assaying of small volumes of whole blood and thus the present invention provides a very rapid diagnostic assay device that is simple to use and can be used in both laboratory and point-of-care field diagnostic locations. For example, a finger prick of blood would be sufficient to perform an assay. Similarly large volumes of sample can be used in this device by increasing the amount of absorbent material (second member). For instance, 10 mls of dilute fluids like urine can be can be assayed to detect low abundance molecules. Analytes and/or ligands (e.g. antigens or antibodies) can be printed down in titrating amounts and/or concentrations. Thus, in an individual screen, this would provide a means of quantitating analyte-ligand levels within the sample solution. The pre-incubation step may also be carried out with a multi-analyte detector where any number of detection analytes can be attached to a gold particle or a similar detectable tag e.g. a fluorescent marker. |
Seat-based weight sensor |
An apparatus and method for measuring the weight of an occupant of a seat is described. The weight of the occupant can be determined by measuring the deflection of a portion of the seat. This may be accomplished through the use of a magnet and Hall-effect sensor, wherein deflection of a portion of the seat may cause a change in the distance between the magnet and the Hall-effect sensor. This change in distance may result in a change in intensity measured by the sensor, which can then be correlated to the weight of the occupant. Particular application may be made to measuring the weight of an occupant of an automobile, for purposes such as safety and ergonomics, and more readily to assist in the development of improved airbag deployment strategies. |
1. An apparatus for measuring an occupant's weight, comprising: an occupant support, having a first portion and a second portion, wherein the first portion is deflected due to the occupant's weight and the second portion is not deflected due to the occupant's weight; a magnet mounted to the first portion; and a linear Hall-effect sensor attached to the second portion, wherein the magnet opposes the linear Hall-effect sensor. 2. The apparatus as in claim 1, wherein the first portion comprises a spring, and the second portion comprises a seat pan. 3. The apparatus as in claim 1, wherein the first portion comprises seat posts, and the second portion comprises a seat track. 4. An apparatus for measuring an occupant's weight, comprising: an occupant support, having a first portion and a second portion, wherein the first portion is deflected due to the occupant's weight and the second portion is not deflected due to the occupant's weight; a linear Hall-effect sensor mounted to the first portion; a magnet attached to the second portion, wherein the magnet opposes the linear Hall-effect sensor. 5. The apparatus as in claim 4, wherein the first portion comprises a spring, and the second portion comprises a seat pan. 6. The apparatus as in claim 4, wherein the first portion comprises a seat post, and the second portion comprises a seat track. 7. A method for measuring the weight of an occupant of a seat, comprising: providing an occupant support, having a first portion and a second portion, wherein the first portion is deflected due to the occupant's weight and the second portion is not deflected due to the occupant's weight; mounting a magnet to the first portion; and attaching a linear Hall-effect sensor to the second portion, wherein the magnet opposes the linear Hall-effect sensor. 8. The method of claim 7, wherein the first portion comprises a spring, and the second portion comprises a seat pan. 9. The method of claim 7, wherein the first portion comprises seat posts, and the second portion comprises a seat track. 10. A method for measuring the weight of an occupant of a seat, comprising: providing an occupant support, having a first portion and a second portion, wherein the first portion is deflected due to the occupant's weight and the second portion is not deflected due to the occupant's weight; mounting a linear Hall-effect sensor to the first portion; attaching a magnet to the second portion, wherein the magnet opposes the linear Hall-effect sensor. 11. The method of claim 10, wherein the first portion comprises a spring, and the second portion comprises a seat pan. 12. The method of claim 10, wherein the first portion comprises a seat post, and the second portion comprises a seat track. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates to an apparatus and method for measuring the weight of an occupant of a seat. More specifically, it relates to measuring the weight of an occupant of a seat through the use of a magnet and a linear Hall-effect sensor. 2. Description of the Related Art There are a number of uses for knowing the weight of an occupant of a seat, and further, the weight of an occupant of a car seat. These may fall into several categories: safety, ergonomics, among others. Knowledge of the occupant's weight can also contribute to both improved functionality of safety systems and more user-friendly positioning of the occupants among other uses. An active area of engineering research and development involves developing low-risk airbag deployment strategies. It is acknowledged that many airbag deployments are inappropriate to the situation. For example, in many cases the airbag system deploys the passenger seat airbag when the passenger seat is unoccupied, leading to unnecessary airbag reinstallation costs for the user and/or the insurance company. Perhaps more importantly, injury can occur to an occupant due to an amount of extra energy in the airbag deployment. One area of ongoing research involves developing a control module which is capable of tailoring various aspects of the airbag deployment to the specific occupant of the seat. An important input to such a control module is the weight of the occupant which is a variable discussed in the present invention. Conventional systems may rely on pressure sensors and strain gages to measure loads. While pressure sensors and strain gages vary in design, each having its own advantages, disadvantages and specific utilities, they can suffer from a number of general drawbacks. For example, some desirable strain gage materials are also sensitive to temperature variations. Therefore, they may require a temperature-compensation technique to be added to the system. Also, strain gages tend to change resistance as they age, requiring adjustment and/or replacement. For the case of semiconductor strain gages, the resistance-to-strain relationship is likely nonlinear, requiring software compensation to overcome nonlinearity. Therefore, what is needed is an apparatus and method for measuring the weight of an occupant of a car seat, having a linear response, and which is robust with respect to variations in ambient conditions and aging of the materials, thus addressing and solving problems associated with conventional systems. |
<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the invention disclosed herein to provide a method and apparatus for measuring the weight of an occupant of a car seat, which uses a magnet and linear Hall-effect sensor. The invention can determine the weight of the occupant by measuring the deflection of certain portions, e.g, in one embodiment, the bottom surface, of the seat. In one embodiment, the deflection of the bottom surface of a car seat is measured as follows. A magnet is mounted to the springs on the bottom of the seat. Opposing the magnet is a linear Hall-effect sensor. As the seat bottom deflects, the magnet moves closer to the Hall-effect sensor. This increases the intensity of the magnetic field at the Hall-effect sensor, causing the sensor to change its response. This change in response is measured and correlated with the weight of the occupant of the car seat. Clearly, the design may be modified so that upon deflection of the seat, the magnet moves farther from the Hall-effect sensor, and the consequent change in response (a decrease in this case) is correlated with the weight of the occupant. An advantage of this embodiment is its use of established technology for a novel purpose. There is no electronics development needed, and the technique benefits from the many advantages of linear Hall-effect sensors. For instance, linear Hall-effect sensors may enjoy relative insensitivity to certain ambient conditions, e.g. they can be stable with respect to changes in temperature, humidity, vibration and dust. They also have many properties which are constant over time, whereas many other sensor types degrade much more rapidly with age. Furthermore, since they lack mechanical contacts, Hall effect sensors are more robust than other sensors whose contacts wear and can become an interference source due to arcing. Moreover, since Hall-effect sensors are based on semiconductors, carrier mobility can be controlled by adding impurities, thus making it possible to obtain a repeatable Hall coefficient. |
Production and use of a polar lipid-rich fraction containing omega-3 and/or omega-6 highly unsaturated fatty acids from microbes, genetically modified plant seeds and marine organisms |
The production and use, and in particular, the extraction, separation, synthesis and recovery of polar lipid-rich fractions containing eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA(n-3) or DPA(n-6)), arachidonic acid (ARA), and eicosatetraneonoic acid (C20:4n-3) from microorganisms, genetically modified seeds and marine organisms (including fish and squid) and their use in human food applications, animal feed, pharmaceutical applications and cosmetic applications. |
1. A method for providing a human, animal or aquaculture organism diet supplement enriched with at least one of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (n-3)(DPAn-3), eicosatetraenoic acid (n-3), docosapentaenoic acid (n-6)(DPAn-6) or arachidonic acid (ARA) comprising the steps: a) producing a polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA from genetically modified seeds or marine animals; and b) providing said polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA in a form consumable or usable by humans or animals. 2. The method of claim 1, wherein the animal is a companion animal. 3. A method for treating a deficiency in at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA comprising the steps: a) producing a polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA from microbes, genetically modified seeds or marine animals; and b) providing said polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA to treat said deficiency. 4. The method of claim 3, wherein said deficiency results in an inflammatory condition, an immune system imbalance, a cardiovascular disease, a developmental deficit related to nervous system development, a woman's health condition or an infant's health condition. 5. A method for treating a chronic inflammatory disease state of the lung comprising the steps: a) producing a purified phospholipid fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA from microbes, genetically modified seeds or marine animals; b) blending said phospholipid fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA with at least one of EPA-, GLA- or SDA-rich oils; and c) producing an aerosol comprising the blend of step (b) for the treatment of said disease states. 6. The method of claim 5, wherein the chronic inflammatory disease state of the lung is COPD, asthma or cystic fibrosis. 7. A method for the treatment of skin lesions, induced burn, UV-irradiation or other skin disorders comprising the steps: a) producing a purified phospholipid fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA from microbes, genetically modified seeds or marine animals; b) blending said phospholipid fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA with at least one EPA-, GLA- or SDA-rich oil; and c) producing a lotion or cream for the treatment of said skin disorders. 8. A method for treating cachexia or fat malabsorption comprising the steps: a) producing a purified phospholipid enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA; b) blending said purified phospholipid enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA with at least one other purified phospholipid; c) blending said purified phospholipid enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA with at least one DHA, EPA, GLA- or SDA-rich oil; and d) producing a liquid or dried dietetic product for the treatment of said disease states. 9. The method of claim 8, wherein the cachexia or fat malabsorption is a result of cancer or Crohn's disease. 10. The method of claim 8, wherein the at least one other purified phospholipid is obtained from the group consisting of soy, rape seed, evening primrose, safflower, sunflower, canola, peanut, egg or mixtures thereof. 11. A method for the treatment of H. pylori-infection of gastrointestinal tract comprising the steps: a) producing a purified phospholipid fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA from microbes, genetically modified seeds or marine animals; b) blending said phospholipid fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA with at least one EPA-, GLA- or SDA-rich oil; and c) producing a fat emulsion or a dietetic product for the treatment of said disease. 12. A method for providing a fat blend enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA comprising the steps: a) extracting a polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA from microbes, genetically modified seeds or marine animals; and b) mixing said polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA with another oil. 13. The method of claim 12, wherein said another oil is selected from the group consisting of fish oil, microbial oil, vegetable oil, GLA-containing oil, SDA-containing oil or and mixtures thereof. 14. A method for providing a blend of polar lipids enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA comprising the steps: a) extracting a polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA from microbes, genetically modified seeds or marine animals; and b) mixing said polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA with another polar lipid. 15. The method of claim 14, wherein said another polar lipid is selected from the group consisting of soy polar lipids, rape seed polar lipids, sunflower polar lipids, safflower polar lipids, canola polar lipids, peanut polar lipids or egg yolk polar lipids and mixtures thereof. 16. A fat blend enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA comprising: a) a polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA from microbes, genetically modified seeds or marine animals; and b) another oil. 17. The fat blend of claim 16, wherein said another oil is selected from the group consisting of fish oil, microbial oil, vegetable oil, GLA-containing oil, SDA-containing oil and mixtures thereof. 18. A blend of polar lipids enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA comprising the steps: a) extracting a EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA-enriched polar lipid-rich fraction from microbes, seeds or marine animals; and b) mixing said EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA-enriched polar lipid-rich fraction with another polar lipid. 19. The blend of polar lipids of claim 18, wherein said another polar lipid is selected from the group consisting of soy polar lipids, rape seed polar lipids, sunflower polar lipids, safflower polar lipids, canola polar lipids, peanut polar lipids or egg yolk polar lipids and mixtures thereof. 20. Purified phospholipids enriched with at least one EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA derived from polar lipid-rich fraction extracted from genetically modified seeds or marine animals. 21. The purified phospholipids of claim 20, wherein said EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA-enriched phospholipid-fraction is in a form consumable or usable by humans or animals. 22. A method for providing a human, animal or aquaculture organism diet supplement enriched with at least one of eicosapentaenoic acid (EPA), docosapentaenoic acid (n-3)(DPAn-3), eicosatetraenoic acid (n-3) or docosapentaenoic acid (n-6)(DPAn-6) comprising the steps: a) producing a polar lipid-rich fraction enriched with at least one of EPA, DPA(n-3), DPA(n-6) or eicosatetraenoic acid from microbes, genetically modified seeds or marine animals; and b) providing the polar lipid-rich fraction enriched with at least one of EPA, DPA(n-3), DPA(n-6) or eicosatetraenoic acid in a form consumable or usable by humans or animals. 23. A dietetic, pharmaceutical or cosmetic composition comprising a polar lipid-rich fraction selected from the group consisting of: a) a polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6) eicosatetraenoic acid or ARA from genetically modified seeds or marine animals, and b ) a polar lipid-rich fraction enriched with at least one of EPA, DPA(n-3). DPA(n-6) or eicosatetraenoic acid from microbes genetically modified seeds or marine animals. 24. A dietetic, pharmaceutical or cosmetic composition comprising the fat blend of claim 16. 25. A dietetic, pharmaceutical or cosmetic composition comprising the blend of polar lipids of claim 18. 26. A dietetic, pharmaceutical or cosmetic composition comprising the purified phospholipids of claim 20. 27. The method of claim 1, wherein said marine animals are fish, squid, mollusks or shrimp. 28. The method of claim 1, wherein said marine animals are fish or fish eggs from the group including salmon, tuna, haddock, sardines, mackerel, or menhaden. 29. The method of claim 3, wherein said microbes are selected from fungi, microalgae, protozoa or bacteria. 30. The method of claim 3, wherein said microbes are selected from the group consisting of Stramenopiles, Thraustochytriales, Chrysophyceae, Xanthophyceae, Bacillariophyceae, Dinophyceae, Phaeophyceae, Rhodophyceae, Chlorophyceae, Euglenophyceae, Cryptophyceae, Oomycetes, Chytridomycetes, and Zygomycetes. 31. The method of claim 3, wherein said microbes are from a genus selected from the group consisting of Mortierella, Mucor, Phycomyces, Rhizopus, Pythium, Ochromonas, Nitzschia, Phaeodactylum, Skeletonema, Fucus, Laminaria, Platymonas, Achyla, Phytophera, Schizochytrium, Thraustochytrium, and Crypthecodinium. 32. The method claim 1, wherein said at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid, or ARA comprises at least two weight percent of the total fatty acids of the polar lipid fraction. 33. The method of claim 1, wherein said at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid, or ARA comprises at least five weight percent of the total fatty acids of the polar lipid fraction. 34. The method of claim 3, wherein said seeds or microbes have been genetically modified to increase their n-3 or n-6 HUFA content. 35. The method of claim 3, wherein said seeds or microbes have been genetically modified to increase the production of at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA. 36. The method of claim 1, wherein said seeds are from a plant selected from the group consisting of canola, rape seed, linseed, flaxseed, sunflower, safflower, peanut, soybean and corn. 37. The method of claim 3, wherein said polar lipid-rich fraction is extracted from said seeds or microbes using alcohol. 38. The method of claim 1, wherein said polar lipid-rich fraction is derived as a by-product of oil extraction from said seeds using hexane or other non-polar solvent. 39. The method of claim 3, wherein said polar lipid-rich fraction is extracted from said seeds or microbes by use of gravity or centrifugal extraction technology. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Highly unsaturated fatty acids of the omega-6 and omega-3 series represent a special class of bioactive lipids in that they are important structurally in membranes in the body, but also participate directly and indirectly in communication between cells through the eicosanoid pathways and by their influence of these fatty acids on gene expression. Six of these fatty acids, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA(n-3) or DPA(n-6)), arachidonic acid (ARA), and eicosatetraenoic acid (C20:4n-3) have been shown to be effective in preventing/treating cardiovascular disease, inflammatory disease, immune function imbalances, fertility and some of these fatty acids (ARA, DHA DPA(n-6) are important structurally in the brain and nervous system. Recent evidence indicates that some highly unsaturated fatty acids may be more bioavailable when supplied in a phospholipid form than in a triglyceride form. EPA, DHA, and ARA have historically been supplied to the nutritional supplement markets in the form of oil extracted from algae or fish. However recent evidence indicates that some polyunsaturated fatty acids may be more bioavailable in a phospholipid form rather than in a triglyceride form. This may be because of the bipolar nature of phospholipids, making them readily solubilizable in the gut and available for digestion and uptake. This same bipolar property of phospholipids additionally would make these fatty acids more functional in topical applications such as creams and lotions or more soluble in aqueous-based applications such as beverages because of there ability to participate in emulsification processes. We propose that there may be important advantages in supplying these omega-3 and omega-6 HUFAs in the form of phospholipids and improved processes for recovering polar lipids enriched in these fatty acids are also needed. Examples of polar lipids include phospholipids (e.g. phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidyl-glycerol, diphosphatidylglycerols), cephalins, sphingolipids (sphingomyelins and glycosphingolipids), and glycoglycerolipids. Phospholipids are composed of the following major structural units: fatty acids, glycerol, phosphoric acid, and amino alcohols. They are generally considered to be structural lipids, playing important roles in the structure of the membranes of plants, microbes and animals. Because of their chemical structure, polar lipids exhibit a bipolar nature, exhibiting solubility or partial solubility in both polar and non-polar solvents. The term polar lipid within the present description is not limited to natural polar lipids but also includes chemically modified polar lipids. Although the term oil has various meanings, as used herein, it will refer to the triacylglycerol fraction. One of the important characteristics of polar lipids, and especially phospholipids, is that they commonly contain polyunsaturated fatty acids (PUFAs: fatty acids with 2 or more unsaturated bonds). In many plant, microbial and animal systems, they are especially enriched in the highly unsaturated fatty acids (HUFAs: fatty acids with 4 or more unsaturated bonds) of the omega-3 and omega-6 series. Although these highly unsaturated fatty acids are considered unstable in triacylglycerol form, they exhibit enhanced stability when incorporated in phospholipids. The primary sources of commercial PUFA-rich phospholipids are soybeans and canola seeds. These biomaterials do not contain any appreciable amounts of highly unsaturated fatty acids unless they have been genetically modified. The phospholipids (commonly called lecithins) are routinely recovered from these oilseeds as a by-product of the vegetable oil extraction process. For example, in the production of soybean or canola oil, the beans (seeds) are first heat-treated and then crushed, ground, and/or flaked, followed by extraction with a non-polar solvent such as hexane. Hexane removes the triacylglycerol-rich fraction from the seeds together with a varying amount of polar lipids (lecithins). The extracted oil is then de-gummed (lecithin removal) either physically or chemically as a part of the normal oil refining process and the precipitated lecithins recovered. This process however has two disadvantages: (1) the seeds must be heat-treated before extraction with hexane, increasing the processing cost, and increasing undesirable oxidation reactions and denaturing the protein fraction, thereby decreasing its value as a by-product; and (2) the use of the non-polar solvents such as hexane also presents toxicity and flammability problems that must be dealt with. The crude lecithin extracted in the “de-gumming” process can contain up to about 33% oil (triacylglycerols) along with sterols and glucosides. One preferred method for separating this oil from the crude lecithin is by extraction with acetone. The oil (triacylglycerols) is soluble in acetone and the lecithin is not. The acetone solution is separated from the precipitate (lecithin) by centrifugation and the precipitate dried under first a fluidized bed drier and then a vacuum drying oven to recover the residual acetone as the product is dried. Drying temperatures of 50-70° C. are commonly used. The resulting dried lecithins contain approximately 2-4% by weight of oil (inacylglycerols). Process temperatures above 70° C. can lead to thermal decomposition of the phospholipids. However, even at temperatures below 70° C. the presence of acetone leads to the formation of products that can impair the organoleptic quality of the phospholipids. These by-products can impart musty odors to the product and also a pungent aftertaste. What is needed is an improved process for effectively recovering polar lipids and phospholipids rich in omega-3 and omega-6 HUFAs from biomaterials that enables the use of these fatty acid in food, nutritional supplement, pharmaceutical and cosmetic applications. Furthermore the fractions are needed as an ingredient in feed for companion animals and in aquaculture. |
<SOH> SUMMARY OF THE INVENTION <EOH>In accordance with the present invention, an improved process is provided for recovering polar lipids enriched in omega-3 and/or omega-6 HUFAs from native biomaterials such as seeds and microorganisms and the use thereof. In one embodiment of the present invention, a method is provided for providing a human, animal or aquaculture organism diet supplement enriched with at least one of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (n-3)(DPAn-3), eicosatetraenoic acid (n-3), docosapentaenoic acid (n-6)(DPAn-6) or arachidonic acid (ARA). The method includes the steps of producing a polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA from genetically modified seeds or marine animals; and providing the polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA in a form consumable or usable by humans or animals. Preferably, the animal is a companion animal. In another embodiment of the present invention, a method is provided for treating a deficiency in at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA. The method includes the steps of producing a polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA from microbes, genetically modified seeds or marine animals; and providing the polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA to treat the deficiency. The deficiency can result in an inflammatory condition, an immune system imbalance, a cardiovascular disease, a developmental deficit related to nervous system development, a woman's health condition or an infant's health condition. In another embodiment of the present invention, a method is provided for treating a chronic inflammatory disease state of the lung. The method includes the steps of producing a purified phospholipid fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA from microbes, genetically modified seeds or marine animals; blending the phospholipid fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA with at least one of EPA-, GLA- or SDA-rich oils; and producing an aerosol comprising the blend for the treatment of the disease states. The chronic inflammatory disease state of the lung can result in chronic obstructive pulmonary disease (COPD), asthma or cystic fibrosis. In another embodiment of the present invention, a method is provided for the treatment of skin lesions, induced bum, UV-irradiation or other skin disorders. The method includes the steps of producing a purified phospholipid fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA from microbes, genetically modified seeds or marine animals; blending the phospholipid fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA with at least one EPA-, GLA- or SDA-rich oil; and producing a lotion or cream for the treatment of the skin disorders. In another embodiment of the present invention, a method is provided for treating cachexia and severe fat malabsorption. The method includes the steps of producing a purified phospholipid enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA; blending the purified phospholipid enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA with at least one other purified phospholipid; blending the purified phospholipid enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA with at least one DHA, EPA, GLA- or SDA-rich oil; and producing a liquid or dried dietetic product for the treatment of the disease states. The cachexia or severe fat malabsorption can be a result of cancer or Crohn's disease. Preferably, the at least one other purified phospholipid is obtained from the group consisting of soy, rape seed, evening primrose, safflower, sunflower, canola, peanut, egg and mixtures thereof. In another embodiment of the present invention, a method is provided for the treatment of H. pylori -infection of gastrointestinal tract. The method includes the steps of producing a purified phospholipid fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA from microbes, genetically modified seeds or marine animals; blending the phospholipid fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA with at least one EPA-, GLA- or SDA-rich oil; and producing a fat emulsion or a dietetic product for the treatment of the disease. In another embodiment of the present invention, a method is provided for providing a fat blend enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA. The method includes the steps of extracting a polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA from microbes, genetically modified seeds or marine animals; and mixing the polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA with another oil. Preferably, the another oil is selected from the group consisting of fish oil, microbial oil, vegetable oil, GLA-containing oil, SDA-containing oil or mixtures thereof. In another embodiment of the present invention, a method is provided for providing a blend of polar lipids enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA. The method includes the steps of extracting a polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA from microbes, genetically modified seeds or marine animals; and mixing the polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA with another polar lipid. Preferably, the another polar lipid is selected from the group consisting of soy polar lipids, rapeseed polar lipids, sunflower polar lipids, safflower polar lipids, canola polar lipids, peanut polar lipids or egg yolk polar lipids and mixtures thereof. In another embodiment of the present invention, a fat blend is provided enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA. The fat blend includes a polar lipid-rich fraction enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid and ARA from microbes, genetically modified seeds or marine animals; and another oil. Preferably, the another oil is selected from the group consisting of fish oil, microbial oil, vegetable oil, GLA-containing oil, SDA-containing oil and mixtures thereof. In another embodiment of the present invention, a method is provided for producing a blend of polar lipids enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA. The method includes the steps of extracting an EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA-enriched polar lipid-rich fraction from microbes, seeds or marine animals; and mixing the EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA-enriched polar lipid-rich fraction with another polar lipid. Preferably, the another polar lipid is selected from the group consisting of soy polar lipids, rapeseed polar lipids, sunflower polar lipids, safflower polar lipids, canola polar lipids, peanut polar lipids or egg yolk polar lipids and mixtures thereof. In another embodiment of the present invention, purified phospholipids enriched with at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA derived from polar lipid-rich fraction extracted from genetically modified seeds or marine animals are provided. Preferably, the EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA-enriched phospholipid-fraction is in a form consumable or usable by humans or animals. In another embodiment of the present invention, a method is provided for providing a human, animal or aquaculture organism diet supplement enriched with at least one of eicosapentaenoic acid (EPA), docosapentaenoic acid (n-3)(DPAn-3), eicosatetraenoic acid (n-3) or docosapentaenoic acid (n-6)(DPAn-6). The method includes the steps of producing a polar lipid-rich fraction enriched with at least one of EPA, DPA(n-3), DPA(n-6) and eicosatetraenoic acid from microbes, genetically modified seeds or marine animals; and providing the polar lipid-rich fraction enriched with at least one of EPA, DPA(n-3), DPA(n-6) and eicosatetraenoic acid in a form consumable or usable by humans or animals. The polar lipid-rich fraction of the methods or products of can be provided as an ingredient of dietetic, pharmaceutical and cosmetic applications. The fat blend of the methods or products of the present invention can be provided as an ingredient of dietetic, pharmaceutical and cosmetic applications. The blend of polar lipids of the methods or products of the present invention can be provided as an ingredient of dietetic, pharmaceutical and cosmetic applications. The purified phospholipids of the methods or products of the present invention can be provided as an ingredient of dietetic, pharmaceutical and cosmetic applications. Preferably, the marine animals of the methods and products of the present invention are fish, squid, mollusks or shrimp. Preferably, the marine animals are fish or fish eggs from the group including salmon, tuna, haddock, sardines, mackerel, or menhaden. Preferably, the microbes of the methods and products of the present invention are selected from fungi, microalgae, protozoa or bacteria. More preferably, microbes are selected from the Stramenopiles, Thraustochytriales, Chrysophyceae, Xanthophyceae, Bacillariophyceae, Dinophyceae, Phaeophyceae, Rhodophyceae, Chlorophyceae, Euglenophyceae, Cryptophyceae, Oomycetes, Chytridomycetes, or Zygomycetes. More preferably, the microbes are selected from the group of genera consisting of Mortierella, Mucor, Phycomyces, Rhizopus, Pythium, Ochromonas, Nitzschia, Phaeodactylum, Skeletonema, Fucus, Laminaria, Platymonas, Achyla, Phytophera, Schizochytrium, Thraustochytrium, or Crypthecodinium. Preferably, the EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid, ARA or mixture thereof employed in the methods and products of the present invention makes up at least two weight percent of the total fatty acids of the polar lipid fraction. Preferably, the EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid, ARA or mixture thereof employed in the methods and products at the present invention makes up at least five weight percent of the total fatty acids of the polar lipid fraction. Preferably, the plant seeds or microbes employed in the methods and products to the present invention have been genetically modified to increase their n-3 or n-6 HUFA content. Preferably, the seeds or microbes used in the methods and products of the present invention have been genetically modified to increase the production of at least one of EPA, DHA, DPA(n-3), DPA(n-6), eicosatetraenoic acid or ARA. Preferably, the seeds employed in the methods and products of the present invention are selected from the group consisting of canola, rapeseed, linseed, flaxseed, sunflower, safflower, peanuts, soybeans or corn. Preferably, the polar lipid-rich fraction is extracted from the seeds and microbes using alcohol. In an alternative embodiment of the present invention, the polar lipid-rich fraction is derived as a by-product (e.g., by de-gumming) of oil extraction from the seeds using hexane and other non-polar solvents. Preferably, the polar lipid-rich fraction used in the methods and products of the present invention is extracted from the seeds and microbes by use of gravity or centrifugal extraction technology. detailed-description description="Detailed Description" end="lead"? |
Method of producing solid electrolytic capacitor |
A manufacturing method according to the present invention of a solid electrolytic capacitor includes: a step of immersing a anode body on which the dielectrics film layer is formed in a solution that includes from 0.7 to 10% by weight of hydrogen peroxide, from 0.3 to 3% by weight of sulfuric acid and water as a main solvent, followed by, after pulling up, exposing to vapor of pyrrole or a pyrrole derivative, and thereby forming, on the dielectrics film layer, a first conductive polymer layer made of polypyrrole or a polypyrrole derivative; and a step of immersing the anode body on which the dielectrics film layer and the first conductive polymer layer are formed in a solution that includes a polymerizing monomer and a supporting electrolyte to electrolytically polymerize the polymerizing monomer, and thereby forming a second conductive polymer layer on the first conductive polymer layer. |
1. A method of manufacturing a solid electrolytic capacitor, in a method of manufacturing a solid electrolytic capacitor in which on a surface of a anode body that is made of niobium or an alloy primarily made of niobium, a dielectrics film layer, a first conductive polymer layer and a second conductive polymer layer are sequentially formed, comprising: a step of forming, on the dielectrics film layer, a first conductive polymer layer made of polypyrrole or a polypyrrole derivative by immersing a anode body on which the dielectrics film layer is formed in a solution that includes from 0.7 to 10% by weight of hydrogen peroxide, from 0.3 to 3% by weight of sulfuric acid and water as a main solvent, followed by, after pulling up, exposing to vapor of pyrrole or a pyrrole derivative; and a step of forming a second conductive polymer layer on the first conductive polymer layer by immersing the anode body on which the dielectrics film layer and the first conductive polymer layer are formed in a solution that includes a polymerizing monomer and a supporting electrolyte, followed by energizing the solution to electrolytically polymerize the polymerizing monomer. 2. A method of manufacturing a solid electrolytic capacitor as set forth in claim 1, wherein the anode body on which the dielectrics film layer is formed is immersed in a solution that includes from 2 to 7% by weight of hydrogen peroxide, from 0.5 to 2% by weight of sulfuric acid and water as a main solvent, followed by, after pulling up, exposing to vapor of pyrrole or a pyrrole derivative, and thereby a first conductive polymer layer is formed. 3. A method of manufacturing a solid electrolytic capacitor as set forth in claim 1, wherein the anode body on which the dielectrics film layer is formed is immersed in a solution that includes hydrogen peroxide, sulfuric acid, water and a water-soluble organic solvent, followed by, after pulling up, exposing to vapor of pyrrole or a pyrrole derivative, and thereby a first conductive polymer layer is formed. 4. A method of manufacturing a solid electrolytic capacitor as set forth in claim 1, wherein prior to forming the second conductive polymer layer, a step of forming the first conductive polymer layer is repeated a plurality of times. 5. A method of manufacturing a solid electrolytic capacitor as set forth in claim 1, wherein the dielectrics film layer is formed by immersing the anode body in a solution that includes an acid or a salt thereof and water as a main solvent to apply a chemical conversion (anodic oxidation) process; wherein a temperature of the solution in the chemical conversion step is set at a solidifying point of the solution or more and substantially 40 degree centigrade or less. |
<SOH> BACKGROUND ART <EOH>As a anode body of an electrolytic capacitor, a wound body of surface-roughened aluminum foil, a single layer body or multi-layered body of surface-roughened aluminum thin plate, a porous sintered body of tantalum powder and so on are much used. As a material of the anode body, niobium is also gathering attention. Niobium that, similarly to tantalum, belongs to 5A group in the periodic table is a metal close in the physical properties to tantalum and has various advantages such as, in comparison with tantalum, the specific gravity being smaller, the reserves being more abundant, the price per kg being less expensive and so on. Accordingly, various attempts have been carried out to utilize niobium as the anode material. However, there are many problems in that in an electrolytic capacitor that uses niobium as the anode material, the leakage current is likely to increase, the aging treatment (an operation to make defect portions of a dielectrics film insulative by applying a direct current voltage in accordance with the polarity of the capacitor for a long period of time) to reduce the leakage current is difficult to demonstrate desired effects, a value of electrostatic capacitance is likely to fluctuate according to a direct current bias voltage and so on. These problems cannot be overcome by simply applying the technologies that are used in the tantalum anode body. As far as the present inventors know, the niobium anode body is not yet commercialized. On the other hand, as a cathode material of an electrolytic capacitor, in place of a conventional electrolyte solution, a solid electrolyte such as a conductive oxide such as manganese dioxide and so on, and an organic semiconductor such as TCNQ complex and so on has become to be frequently used. Furthermore, recently, conductive polymers such as polypyrrole, poly-thiophene and so on have been also put into practical use. Anyhow, the anode material is limited only to aluminum or tantalum. As means for forming the conductive polymer layer as the cathode material with a certain degree of thickness, (1) a method of repeating chemical polymerization several times, (2) a method of forming, on a thin conductive polymer layer formed by a chemical polymerization process, by means of electrolytic polymerization, a relatively thick conductive polymer layer (JP-B-04-74853) and so on are known. Here, the chemical polymerization means to oxidation-polymerize a polymerizing monomer without using energizing means but using an action of an oxidant. The electrolytic polymerization means to oxidation-polymerize a polymerizing monomar by use of the energizing means. The methods of forming a conductive polymer layer according to the (1) and (2), respectively, have merits and demerits depending on combinations of materials of anode body and constitutions thereof and so on. However, when taking the easiness in forming a dense conductive polymer layer, the easiness of controlling a polymerization reaction, the long pot-life of a polymerization solution and so on into consideration, the later (2) (a method that uses the chemical polymerization and the electrolytic polymerization in combination) is more advantageous. In recent years, technology as to niobium powder for capacitors has demonstrated dramatic improvements. That is, the CV product (a product of electrostatic capacitance C per unit mass obtained by forming a dielectrics film and chemical conversion (anodic oxidation) voltage V for forming the dielectrics film) was improved and proved to be hardly different from that of the tantalum powder; and, the purity of the niobium powder was remarkably improved such as that an absorbed oxygen concentration was reduced from the conventional several tens thousands ppm to several thousands ppm and so on. In this connection, researches toward the practical applications of niobium electrolytic capacitors are in boom. In particular, since niobium hates high temperatures, combinations with conductive polymer cathodes that allow forming by processing and operating at relatively low temperatures are targets of flourishing researches because these are considered the most shortest crosscut. However, as the researches of the niobium electrolytic capacitors progress as mentioned above, the differences from the case where the tantalum anode body is used are gradually revealed, and it has become obvious that simple transfer of the conventional technology of the tantalum electrolytic capacitors is far from manufacturing practically applicable ones. Problems that the present invention is to solve are cited as follows. Firstly, when a dielectrics film layer and a conductive polymer cathode layer are formed on a surface of a anode element that is made of a niobium sintered body, even when the conditions the same as that in the case where a tantalum sintered element is used are applied to form the respective layers, the leakage current becomes very large, that is, an almost short-circuited state is caused, resulting in being incapable of, in many cases, carrying out the aging treatment. Furthermore, granted that dielectrics film layer formation conditions and conductive polymer layer formation conditions that allow applying the aging to some extent are found, the electrostatic capacitance occurrence rate (a ratio of the electrostatic capacitance measured after the formation of the conductive polymer layer to that measured in a conductive aqueous solution before the formation of the conductive polymer layer) is low. That is, different from the case where the tantalum sintered body is used, it is very difficult to form the conductive polymer layer to a center portion of the niobium sintered body. As to the capacitance occurrence rate, in the case of the tantalum sintered body being used, whether the conductive polymer layer is formed according to the chemical polymerization method or to a method that combines the electrolytic polymerization method therewith, substantially 85% or so can be relatively easily achieved. On the other hand, in the case where the niobium sintered body is used, it is not easy to conquer a wall of from 20 to 30%. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a sectional view of a solid electrolytic capacitor. FIG. 2 is a diagram showing experimental results that become basis of the present invention. FIG. 3 is a diagram showing experimental results that become basis of the present invention. detailed-description description="Detailed Description" end="lead"? |
Secreted proteins |
Various embodiments of the invention provide human secreted proteins(SECP) and polynucleotides which identify and encode SECP. Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing disorders associated with aberrant expression of SECP. |
1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-33, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, SEQ ID NO:4-13, SEQ ID NO: 15-19, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:28-29, and SEQ ID NO:31, c) a polypeptide comprising a naturally occurring amino acid sequence at least 93% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:23 and SEQ ID NO:25, d) a polypeptide comprising a naturally occurring amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:22, and SEQ ID NO:27, e) a polypeptide comprising a naturally occurring amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO:30, f) a polypeptide comprising a naturally occurring amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:33, g) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-33, and h) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-33. 2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-33. 3. An isolated polynucleotide encoding a polypeptide of claim 1. 4. An isolated polynucleotide encoding a polypeptide of claim 2. 5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:34-66. 6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3. 7. A cell transformed with a recombinant polynucleotide of claim 6. 8. (CANCELLED) 9. A method of producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed. 10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-33. 11. An isolated antibody which specifically binds to a polypeptide of claim 1. 12. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:34-66, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:34-56 and SEQ ID NO:58-66, c) a polynucleotide complementary to a polynucleotide of a), d) a polynucleotide complementary to a polynucleotide of b), and e) an RNA equivalent of a)-d). 13. (CANCELLED) 14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof. 15. A method of claim 14, wherein the probe comprises at least 60 contiguous nucleotides. 16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof. 17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient. 18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-33. 19. (CANCELLED) 20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample. 21-22. (CANCELLED) 23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample. 24-25. (CANCELLED) 26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1. 27. (CANCELLED) 28. A method of screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising: a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. 29. A method of assessing toxicity of a test compound, the method comprising: a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. 30.-112. (CANCELLED) |
<SOH> BACKGROUND OF THE INVENTION <EOH>Protein transport and secretion are essential for cellular function. Protein transport is mediated by a signal peptide located at the amino terminus of the protein to be transported or secreted. The signal peptide is comprised of about ten to twenty hydrophobic amino acids which target the nascent protein from the ribosome to a particular membrane bound compartment such as the endoplasmic reticulum (ER). Proteins targeted to the ER may either proceed through the secretory pathway or remain in any of the secretory organelles such as the ER, Golgi apparatus, or lysosomes. Proteins that transit through the secretory pathway are either secreted into the extracellular space or retained in the plasma membrane. Proteins that are retained in the plasma membrane contain one or more transmembrane domains, each comprised of about 20 hydrophobic amino acid residues. Secreted proteins are generally synthesized as inactive precursors that are activated by post-translational processing events during transit through the secretory pathway. Such events include glycosylation, proteolysis, and removal of the signal peptide by a signal peptidase. Other events that may occur during protein transport include chaperone-dependent unfolding and folding of the nascent protein and interaction of the protein with a receptor or pore complex. Examples of secreted proteins with amino terminal signal peptides are discussed below and include proteins with important roles in cell-to-cell signaling. Such proteins include transmembrane receptors and cell surface markers, extracellular matrix molecules, cytokines, hormones, growth and differentiation factors, enzymes, neuropeptides, vasomediators, cell surface markers, and antigen recognition molecules. (Reviewed in Alberts, B. et al. (1994) Molecular Biology of The Cell , Garland Publishing, New York, N.Y., pp. 557-560, 582-592.) Cell surface markers include cell surface antigens identified on leukocytic cells of the immune system These antigens have been identified using systematic, monoclonal antibody (mAb)-based “shot gun” techniques. These techniques have resulted in the production of hundreds of mAbs directed against unknown cell surface leukocytic antigens. These antigens have been grouped into “clusters of differentiation” based on common immunocytochemical localization patterns in various differentiated and undifferentiated leukocytic cell types. Antigens in a given cluster are presumed to identify a single cell surface protein and are assigned a “cluster of differentiation” or “CD” designation. Some of the genes encoding proteins identified by CD antigens have been cloned and verified by standard molecular biology techniques. CD antigens have been characterized as both transmembrane proteins and cell surface proteins anchored to the plasma membrane via covalent attachment to fatty acid-containing glycolipids such as glycosylphosphatidylinositol (GPI). (Reviewed in Barclay, A. N. et al. (1995) The Leucocyte Antigen Facts Book , Academic Press, San Diego, Calif., pp. 17-20.) Matrix proteins (MPs) are transmembrane and extracellular proteins which function in formation, growth, remodeling, and maintenance of tissues and as important mediators and regulators of the inflammatory response. The expression and balance of MPs may be perturbed by biochemical changes that result from congenital, epigenetic, or infectious diseases. In addition, MPs affect leukocyte migration, proliferation, differentiation, and activation in the immune response. MPs are frequently characterized by the presence of one or more domains which may include collagen-like domains, EGF-like domains, immunoglobulin-like domains, and fibronectin-like domains. In addition, MPs may be heavily glycosylated and may contain an Arginine-Glycine-Aspartate (RGD) tripeptide motif which may play a role in adhesive interactions. MPs include extracellular proteins such as fibronectin, collagen, galectin, vitronectin and its proteolytic derivative somatomedin B; and cell adhesion receptors such as cell adhesion molecules (CAMs), cadherins, and integrins. (Reviewed in Ayad, S. et al. (1994) The Extracellular Matrix Facts Book , Academic Press, San Diego, Calif., pp. 2-16; Ruoslahti, E. (1997) Kidney Int. 51:1413-1417; Sjaastad, M. D. and W. J. Nelson (1997) BioEssays 19:47-55.) Mucins are highly glycosylated glycoproteins that are the major structural component of the mucus gel. The physiological functions of mucins are cytoprotection, mechanical protection, maintenance of viscosity in secretions, and cellular recognition. MUC6 is a human gastric mucin that is also found in gall bladder, pancreas, seminal vesicles, and female reproductive tract (Toribara, N. W. et al. (1997) J. Biol. Chem 272:16398-16403). The MUC6 gene has been mapped to human chromosome 11 (Toribara, N. W. et al. (1993) J. Biol. Chem 268:5879-5885). Hemomucin is a novel Drosophila surface mucin that may be involved in the induction of antibacterial effector molecules (Theopold, U. et al. (1996) J. Biol. Chem. 217:12708-12715). Tuftelins are one of four different enamel matrix proteins that have been identified so far. The other three known enamel matrix proteins are the amelogenins, enamelin and ameloblastin. Assembly of the enamel extracellular matrix from these component proteins is believed to be critical in producing a matrix competent to undergo mineral replacement (Paine, C. T. et al. (1998) Connect Tissue Res. 38:257-267). Tuftelin mRNA has been found to be expressed in human ameloblastoma tumor, a non-mineralized odontogenic tumor (Deutsch, D. et al. (1998) Connect. Tissue Res. 39:177-184). Olfactomedin-related proteins are extracellular matrix, secreted glycoproteins with conserved C-terminal motifs. They are expressed in a wide variety of tissues and in a broad range of species, from Caenorhabditis elegans to Homo sapiens . Olfactomedin-related proteins comprise a gene family with at least 5 family members in humans. One of the five, TIGR/myocilin protein, is expressed in the eye and is associated with the pathogenesis of glaucoma (Kulkarni, N. H. et al. (2000) Genet. Res. 76:41-50). Research by Yokoyama, M. et al. (1996; DNA Res. 3:311-320) found a 135-amino acid protein, termed AMY, having 96% sequence identity with rat neuronal olfactomedin-releated ER localized protein in a neuroblastoma cell line cDNA library, suggesting an essential role for AMY in nerve tissue. Neuron-specific olfactomedin-related glycoproteins isolated from rat brain cDNA libraries show strong sequence similarity with olfactomedin. This similarity is suggestive of a matrix-related function of these glycoproteins in neurons and neurosecretory cells (Danielson, P. E. et al. (1994) J. Neurosci. Res. 38:468-478). Mac-2 binding protein is a 90-kD serum protein (90 K), a secreted glycoprotein isolated from both the human breast carcinoma cell line SK-BR-3, and human breast milk. It specifically binds to a human macrophage-associated lectin, Mac-2. Structurally, the mature protein is 567 amino acids in length and is proceeded by an 18-amino acid leader. There are 16 cysteines and seven potential N-linked glycosylation sites. The first 106 amino acids represent a domain very similar to an ancient protein superfamily defined by a macrophage scavenger receptor cysteine-rich domain (Koths, K. et al. (1993) J. Biol. Chem. 268:14245-14249). 90 K is elevated in the serum of subpopulations of AIDS patients and is expressed at varying levels in primary tumor samples and tumor cell lines. Ullrich, A. et al. (1994; J. Biol. Chem 269:18401-18407) have demonstrated that 90 K stimulates host defense systems and can induce interleukin-2 secretion. This immune stimulation is proposed to be a result of oncogenic transformation, viral infection or pathogenic invasion (Ullrich et al., supra). Semaphorins are a large group of axonal guidance molecules consisting of at least 30 different members and are found in vertebrates, invertebrates, and even certain viruses. All semaphorins contain the sema domain which is approximately 500 amino acids in length. Neuropilin, a semaphorin receptor, has been shown to promote neurite outgrowth in vitro. The extracellular region of neuropilins consists of three different domains: CUB, discoidin, and MAM domains. The CUB and the MAM motifs of neuropilin have been suggested to have roles in protein-protein interactions and are thought to be involved in the binding of semaphorins through the sema and the C-terminal domains (reviewed in Raper, J. A. (2000) Curr. Opin. Neurobiol. 10:88-94). Plexins are neuronal cell surface molecules that mediate cell adhesion via a homophilic binding mechanism in the presence of calcium ions. Plexins have been shown to be expressed in the receptors and neurons of particular sensory systems (Ohta, K. et al. (1995) Cell 14:1189-1199). There is evidence that suggests that some plexins function to control motor and CNS axon guidance in the developing nervous system. Plexins, which themselves contain complete semaphorin domains, may be both the ancestors of classical semaphorins and binding partners for semaphorins (Winberg, ML. et al (1998) Cell 95:903-916). Human pregnancy-specific beta 1-glycoprotein (PSG) is a family of closely related glycoproteins of molecular weights of 72 KDa, 64 KDa, 62 KDa, and 54 KDa. Together with the carcinoembryonic antigen, they comprise a subfamily within the immunoglobulin superfamily (Plouzek, C. A. and J. Y. Chou, (1991) Endocrinology 129:950-958) Different subpopulations of PSG have been found to be produced by the trophoblasts of the human placenta, and the amnionic and chorionic membranes (Plouzek, C. A. et al. (1993) Placenta 14:277-285). Torsion dystonia is an autosomal dominant movement disorder consisting of involuntary muscular contractions. The disorder has been linked to a 3-base pair mutation in the DYT-1 gene, which encodes torsin A (Ozelius, L. J. et al. (1997) Nat. Genet. 17:4048). Torsin A bears significant homology to the Hsp100/Clp family of ATPase chaperones, which are conserved in humans, rats, mice, and C. elegans . Strong expression of DYT-1 in neuronal processes indicates a potential role for torsins in synaptic communication (Kustedjo, K. et al. (2000) J. Biol. Chem. 275:27933-27939 and Konakova M. et al. (2001) Arch. Neurol. 58:921-927). Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration; therefore identification of the signaling pathway coupled with it has critical importance. Autocrine motility factor receptor (AMFR) expression has been found to be associated with tumor progression in thymoma (Ohta Y. et al. (2000) Int. J. Oncol. 17:259-264). AMFR is a cell surface glycoprotein of molecular weight 78 KDa Hormones are secreted molecules that travel through the circulation and bind to specific receptors on the surface of, or within, target cells. Although they have diverse biochemical compositions and mechanisms of action, hormones can be grouped into two categories. One category includes small lipophilic hormones that diffuse through the plasma membrane of target cells, bind to cytosolic or nuclear receptors, and form a complex that alters gene expression. Examples of these molecules include retinoic acid, thyroxine, and the cholesterol-derived steroid hormones such as progesterone, estrogen, testosterone, cortisol, and aldosterone. The second category includes hydrophilic hormones that function by binding to cell surface receptors that transduce signals across the plasma membrane. Examples of such hormones include amino acid derivatives such as catecholamines (epinephrine, norepinephrine) and histamine, and peptide hormones such as glucagon, insulin, gastrin, secretin, cholecystokinin, adrenocorticotropic hormone, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, and vasopressin. (See, for example, Lodish et al. (1995) Molecular Cell Biology , Scientific American Books Inc., New York, N.Y., pp. 856-864.) Pro-opiomelanocortin (POMC) is the precursor polypeptide of corticotropin (ACTH), a hormone synthesized by the anterior pituitary gland, which functions in the stimulation of the adrenal cortex. POMC is also the precursor polypeptide of the hormone beta-lipotropin (beta-LPH). Each hormone includes smaller peptides with distinct biological activities: alpha-melanotropin (alpha-MSH) and corticotropin-like intermediate lobe peptide (CLIP) are formed from ACTH; gamma-lipotropin (gamma-LPH) and beta-endorphin are peptide components of beta-LPH; while beta-MSH is contained within gamma-LPH. Adrenal insufficiency due to ACTH deficiency, resulting from a genetic mutation in exons 2 and 3 of POMC results in an endocrine disorder characterized by early-onset obesity, adrenal insufficiency, and red hair pigmentation (Chretien, M. et al. (1979) Can. J. Biochem. 57:1111-1121; Krude, H. et al. (1998) Nat. Genet. 19:155-157; Online Mendelian Inheritance in Man (OMIM) 176830). Growth and differentiation factors are secreted proteins which function in intercellular communication. Some factors require oligomerization or association with membrane proteins for activity. Complex interactions among these factors and their receptors trigger intracellular signal transduction pathways that stimulate or inhibit cell division, cell differentiation, cell signaling, and cell motility. Most growth and differentiation factors act on cells in their local environment (paracrine signaling). There are three broad classes of growth and differentiation factors. The first class includes the large polypeptide growth factors such as epidermal growth factor, fibroblast growth factor, transforming growth factor, insulin-like growth factor, and platelet-derived growth factor. The second class includes the hematopoietic growth factors such as the colony stimulating factors (CSFs). Hematopoietic growth factors stimulate the proliferation and differentiation of blood cells such as B-lymphocytes, T-lymphocytes, erythrocytes, platelets, eosinophils, basophils, neutrophils, macrophages, and their stem cell precursors. The third class includes small peptide factors such as bombesin, vasopressin, oxytocin, endothelin, transferrin, angiotensin II, vasoactive intestinal peptide, and bradykinin, which function as hormones to regulate cellular functions other than proliferation. Growth and differentiation factors play critical roles in neoplastic transformation of cells in vitro and in tumor progression in vivo. Inappropriate expression of growth factors by tumor cells may contribute to vascularization and metastasis of tumors. During hematopoiesis, growth factor misregulation can result in anemias, leukemias, and lymphomas. Certain growth factors such as interferon are cytotoxic to tumor cells both in vivo and in vitro. Moreover, some growth factors and growth factor receptors are related both structurally and functionally to oncoproteins. In addition, growth factors affect transcriptional regulation of both proto-oncogenes and oncosuppressor genes. (Reviewed in Pimentel, B. (1994) Handbook of Growth Factors , CRC Press, Ann Arbor, Mich., pp. 1-9.) The Slit protein, first identified in Drosophila , is critical in central nervous system midline formation and potentially in nervous tissue histogenesis and axonal pathfinding. Itoh et al. (1998; Brain Res. Mol. Brain Res. 62:175-186) have identified mammalian homologues of the slit gene (human Slit-1, Slit-2, Slit-3 and rat Slit-1). The encoded proteins are putative secreted proteins containing EGF-like motifs and leucine-rich repeats, both of which are conserved protein-protein interaction domains. Slit-1, -2, and -3 mRNAs are expressed in the brain, spinal cord, and thyroid, respectively (Itoh et al., supra). The Slit family of proteins are indicated to be functional ligands of glypican-1 in nervous tissue and it is suggested that their interactions may be critical in certain stages during central nervous system histogenesis (Liang, Y. et al. (1999) J. Biol. Chem. 274:17885-17892). Neuropeptides and vasomediators (NP/VM) comprise a large family of endogenous signaling molecules. Included in this family are neuropeptides and neuropeptide hormones such as bombesin, neuropeptide Y, neurotensin, neuromedin N, melanocortins, opioids, galanin, somatostatin, tachykinins, urotensin II and related peptides involved in smooth muscle stimulation, vasopressin, vasoactive intestinal peptide, and circulatory system-borne signaling molecules such as angiotensin, complement, calcitonin, endothelins, formyl-methionyl peptides, glucagon, cholecystokinin and gastrin. NP/VMs can transduce signals directly, modulate the activity or release of other neurotransmitters and hormones, and act as catalytic enzymes in cascades. The effects of NP/VMs range from extremely brief to long-lasting. (Reviewed in Martin, C. R. et al. (1985) Endocrine Physiology , Oxford University Press, New York, N.Y., pp. 57-62.) NP/VMs are involved in numerous neurological and cardiovascular disorders. For example, neuropeptide Y is involved in hypertension, congestive heart failure, affective disorders, and appetite regulation. Somatostatin inhibits secretion of growth hormone and prolactin in the anterior pituitary, as well as inhibiting secretion in intestine, pancreatic acinar cells, and pancreatic beta-cells. A reduction in somatostatin levels has been reported in Alzheimer's disease and Parkinson's disease. Vasopressin acts in the kidney to increase water and sodium absorption, and in higher concentrations stimulates contraction of vascular smooth muscle, platelet activation, and glycogen breakdown in the liver. Vasopressin and its analogues are used clinically to treat diabetes insipidus. Endothelin and angiotensin are involved in hypertension, and drugs, such as captopril, which reduce plasma levels of angiotensin, are used to reduce blood pressure (Watson, S. and S. Arkinstall (1994) The G - protein Linked Receptor Facts Book , Academic Press, San Diego Calif., pp. 194; 252; 284; 55; 111). Neuropeptides have also been shown to have roles in nociception (pain). Vasoactive intestinal peptide appears to play an important role in chronic neuropathic pain. Nociceptin, an endogenous ligand for for the opioid receptor-like 1 receptor, is thought to have a predominantly anti-nociceptive effect, and has been shown to have analgesic properties in different animal models of tonic or chronic pain (Dickinson, T. and S. M. Fleetwood-Walker (1998) Trends Pharmacol. Sci. 19:346-348). Other proteins that contain signal peptides include secreted proteins with enzymatic activity. Such activity includes, for example, oxidoreductase/dehydrogenase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, or ligase activity. For example, matrix metalloproteinases are secreted hydrolytic enzymes that degrade the extracellular matrix and thus play an important role in tumor metastasis, tissue morphogenesis, and arthritis (Reponen, P. et al. (1995) Dev. Dyn. 202:388-396; Firestein, G. S. (1992) Curr. Opin. Rheumatol. 4:348-354; Ray, J. M. and W. G. Stetler-Stevenson (1994) Bur. Respir. J. 7:2062-2072; and Mignatti, P. and D. B. Riflin (1993) Physiol. Rev. 73:161-195). Additional examples are the acetyl-CoA synthetases which activate acetate for use in lipid synthesis or energy generation (Luong, A. et al. (2000) J. Biol. Chem. 275:26458-26466). The result of acetyl-CoA synthetase activity is the formation of acetyl-CoA from acetate and CoA. Acetyl-CoA sythetases share a region of sequence similarity identified as the AMP-binding domain signature. Acetyl-CoA synthetase has been shown to be associated with hypertension (Toh, H. (1991) Protein Seq. Data Anal. 4:111-117; and Iwai, N. et al. (1994) Hypertension 23:375-380). A number of isomerases catalyze steps in protein folding, phototransduction, and various anabolic and catabolic pathways. One class of isomerases is known as peptidyl-prolyl cis-trans isomerases (PPIases). PPIases catalyze the cis to trans isomerization of certain proline imidic bonds in proteins. Two families of PPIases are the FK506 binding proteins (FKBPs), and cyclophilins (CyPs). FKBPs bind the potent immunosuppressants FK506 and rapamycin, thereby inhibiting signaling pathways in T-cells. Specifically, the PPIase activity of FKBPs is inhibited by binding of FK506 or rapamycin. There are five members of the FKBP family which are named according to their calculated molecular masses (FKBP12, FKBP13, FKBP25, FKBP52, and FKBP65), and localized to different regions of the cell where they associate with different protein complexes (Coss, M. et al. (1995) J. Biol. Chem. 270:29336-29341; Schreiber, S. L. (1991) Science 251:283-287). The peptidyl-prolyl isomerase activity of CyP may be part of the signaling pathway that leads to T-cell activation. CyP isomerase activity is associated with protein folding and protein trafficking, and may also be involved in assembly/disassembly of protein complexes and regulation of protein activity. For example, in Drosophila , the CyP NinaA is required for correct localization of rhodopsins, while a mammalian CyP (Cyp40) is part of the Hsp90/Hsc70 complex that binds steroid receptors. The mammalian CypA has been shown to bind the gag protein from human immunodeficiency virus 1 (HIV-1), an interaction that can be inhibited by cyclosporin. Since cyclosporin has potent anti-HIV-1 activity, CypA may play an essential function in HIV-1 replication. Finally, Cyp40 has been shown to bind and inactivate the transcription factor c-Myb, an effect that is reversed by cyclosporin. This effect implicates CyPs in the regulation of transcription, transformation, and differentiation (Bergsma, D. J. et al (1991) J. Biol. Chem. 266:23204-23214; Hunter, T. (1998) Cell 92:141-143; and Leverson, J. D. and S. A. Ness, (1998) Mol. Cell. 1:203-211). Gamma-carboxyglutamic acid (Gla) proteins rich in proline (PRGPs) are members of a family of vitamin K-dependent single-pass integral membrane proteins. These proteins are characterized by an extracellular amino terminal domain of approximately 45 amino acids rich in Gla. The intracellular carboxyl terminal region contains one or two copies of the sequence PPXY, a motif present in a variety of proteins involved in such diverse cellular functions as signal transduction, cell cycle progression, and protein turnover (Kulman, J. D. et al. (2001) Proc. Natl. Acad. Sci. USA 98:13701375). The process of post-translational modification of glutamic residues to form Gla is Vitamin K-dependent carboxylation. Proteins which contain Gla include plasma proteins involved in blood coagulation. These proteins are prothrombin, proteins C, S, and Z, and coagulation factors VII, IX, and X. Osteocalcin (bone-Gla protein, BGP) and matrix Gla-protein (MGP) also contain Gla (Friedman, P. A. and C. T. Przysiecki (1987) Int. J. Biochem. 19:1-80; Vermeer, C. (1990) Biochem. J. 266:625-636). Immunoglobulins Antigen recognition molecules are key players in the sophisticated and complex immune systems which all vertebrates have developed to provide protection from viral, bacterial, fungal, and parasitic infections. A key feature of the immune system is its ability to distinguish foreign molecules, or antigens, from “self” molecules. This ability is mediated primarily by secreted and transmembrane proteins expressed by leukocytes (white blood cells) such as lymphocytes, granulocytes, and monocytes. Most of these proteins belong to the immunoglobulin (Ig) superfamily, members of which contain one or more repeats of a conserved structural domain. This Ig domain is comprised of antiparallel β sheets joined by a disulfide bond in an arrangement called the Ig fold. The criteria for a protein to be a member of the Ig superfamily is to have one or more Ig domains, which are regions of 70-110 amino acid residues in length homologous to either Ig variable-like (V) or Ig constant-like (C) domains. Members of the Ig superfamily include antibodies (Ab), T cell receptors (TCRs), class I and II major histocompatibility (MHC) proteins and immune cell-specific surface markers such as the “cluster of differentiation” or CD antigens, CD2, CD3, CD4, CD8, poly-Ig receptors, Fc receptors, neural cell-adhesion molecule (NCAM) and platelet-derived growth factor receptor (PDGFR). Ig domains (V and C) are regions of conserved amino acid residues that give a polypeptide a globular tertiary structure called an immunoglobulin (or antibody) fold, which consists of two approximately parallel layers of β-sheets. Conserved cysteine residues form an intrachain disulfide-bonded loop, 55-75 amino acid residues in length, which connects the two layers of β-sheets. Each β-sheet has three or four anti-parallel β-strands of 5-10 amino acid residues. Hydrophobic and hydrophilic interactions of amino acid residues within the β-strands stabilize the Ig fold (hydrophobic on inward facing amino acid residues and hydrophilic on the amino acid residues in the outward facing portion of the strands). A V domain consists of a longer polypeptide than a C domain, with an additional pair of β-strands in the Ig fold. A consistent feature of Ig superfamily genes is that each sequence of an Ig domain is encoded by a single exon. It is possible that the superfamily evolved from a gene coding for a single Ig domain involved in mediating cell-cell interactions. New members of the superfamily then arose by exon and gene duplications. Modern Ig superfamily proteins contain different numbers of V and/or C domains. Another evolutionary feature of this superfamily is the ability to undergo DNA rearrangements, a unique feature retained by the antigen receptor members of the family. Many members of the Ig superfamily are integral plasma membrane proteins with extracellular Ig domains. The hydrophobic amino acid residues of their transmembrane domains and their cytoplasmic tails are very diverse, with little or no homology among Ig family members or to known signal-transducing structures. There are exceptions to this general superfamily description. For example, the cytoplasmic tail of PDGFR has tyrosine kinase activity. In addition Thy-1 is a glycoprotein found on thymocytes and T cells. This protein has no cytoplasmic tail, but is instead attached to the plasma membrane by a covalent glycophosphatidylinositol linkage. Another common feature of many Ig superfamily proteins is the interactions between Ig domains which are essential for the function of these molecules. Interactions between Ig domains of a multimeric protein can be either homophilic or heterophilic (i.e., between the same or different Ig domains). Antibodies are multimeric proteins which have both homophilic and heterophilic interactions between Ig domains. Pairing of constant regions of heavy chains forms the Fc region of an antibody and pairing of variable regions of light and heavy chains form the antigen binding site of an antibody. Heterophilic interactions also occur between Ig domains of different molecules. These interactions provide adhesion between cells for significant cell-cell interactions in the immune system and in the developing and mature nervous system. (Reviewed in Abbas, A. K. et al. (1991) Cellular and Molecular Immunology , W. B. Saunders Company, Philadelphia, Pa., pp. 142-145.) Antibodies MHC proteins are cell surface markers that bind to and present foreign antigens to T cells. MHC molecules are classified as either class I or class II. Class I MHC molecules (MHC I) are expressed on the surface of almost all cells and are involved in the presentation of antigen to cytotoxic T cells. For example, a cell infected with virus will degrade intracellular viral proteins and express the protein fragments bound to MHC I molecules on the cell surface. The MHC I/antigen complex is recognized by cytotoxic T-cells which destroy the infected cell and the virus within. Class II MHC molecules are expressed primarily on specialized antigen-presenting cells of the immune system, such as B-cells and macrophages. These cells ingest foreign proteins from the extracellular fluid and express MHC II/antigen complex on the cell surface. This complex activates helper T-cells, which then secrete cytokines and other factors that stimulate the immune response. MHC molecules also play an important role in organ rejection following transplantation. Rejection occurs when the recipient's T-cells respond to foreign MHC molecules on the transplanted organ in the same way as to self MHC molecules bound to foreign antigen. (Reviewed in Alberts et al., supra, pp. 1229-1246.) Antibodies are multimeric members of the Ig superfamily which are either expressed on the surface of B-cells or secreted by B-cells into the circulation. Antibodies bind and neutralize foreign antigens in the blood and other extracellular fluids. The prototypical antibody is a tetramer consisting of two identical heavy polypeptide chains (H-chains) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds. This arrangement confers the characteristic Y-shape to antibody molecules. Antibodies are classified based on their H-chain composition. The five antibody classes, IgA, IgD, IgE, IgG and IgM, are defined by the α, δ, ε, γ, and μ H-chain types. There are two types of L-chains, κ and λ, either of which may associate as a pair with any H-chain pair. IgG, the most common class of antibody found in the circulation, is tetrameric, while the other classes of antibodies are generally variants or multimers of this basic structure. H-chains and L-chains each contain an N-terminal variable region and a C-terminal constant region. The constant region consists of about 110 amino acids in L-chains and about 330 or 440 amino acids in H-chains. The amino acid sequence of the constant region is nearly identical among H- or L-chains of a particular class. The variable region consists of about 110 amino acids in both H- and L-chains. However, the amino acid sequence of the variable region differs among H- or L-chains of a particular class. Within each H- or L-chain variable region are three hypervariable regions of extensive sequence diversity, each consisting of about 5 to 10 amino acids. In the antibody molecule, the H- and L-chain hypervariable regions come together to form the antigen recognition site. (Reviewed in Alberts et al. supra, pp. 1206-1213; 1216-1217.) Both H-chains and L-chains contain the repeated Ig domains of members of the Ig superfamily. For example, a typical H-chain contains four Ig domains, three of which occur within the constant region and one of which occurs within the variable region and contributes to the formation of the antigen recognition site. Likewise, a typical L-chain contains two Ig domains, one of which occurs within the constant region and one of which occurs within the variable region. The immune system is capable of recognizing and responding to any foreign molecule that enters the body. Therefore, the immune system must be armed with a full repertoire of antibodies against all potential antigens. Such antibody diversity is generated by somatic rearrangement of gene segments encoding variable and constant regions. These gene segments are joined together by site-specific recombination which occurs between highly conserved DNA sequences that flank each gene segment. Because there are hundreds of different gene segments, millions of unique genes can be generated combinatorially. In addition, imprecise joining of these segments and an unusually high rate of somatic mutation within these segments further contribute to the generation of a diverse antibody population. Expression Profiling Microarrays are analytical tools used in bioanalysis. A microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry. One area in particular in which microarrays find use is in gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder. Steroids are a class of lipid-soluble molecules, including cholesterol, bile acids, vitamin D, and hormones, that share a common four-ring structure based on cyclopentanoperhydrophenanthrene and that carrry out a wide variety of functions. Cholesterol, for example, is a component of cell membranes that controls membrane fluidity. It is also a precursor for bile acids which solubilize lipids and facilitate absorption in the small intestine during digestion. Vitamin D regulates the absorption of calcium in the small intestine and controls the concentration of calcium in plasma. Steroid hormones, produced by the adrenal cortex, ovaries, and testes, include glucocorticoids, mineralocorticoids, androgens, and estrogens. They control various biological processes by binding to intracellular receptors that regulate transcription of specific genes in the nucleus. Glucocorticoids, for example, increase blood glucose concentrations by regulation of gluconeogenesis in the liver, increase blood concentrations of fatty acids by promoting lipolysis in adipose tissues, modulate sensitivity to catcholamines in the central nervous system, and reduce inflammation. The principal mineralocorticoid, aldosterone, is produced by the adrenal cortex and acts on cells of the distal tubules of the kidney to enhance sodium ion reabsorption. Androgens, produced by the interstitial cells of Leydig in the testis, include the male sex hormone testosterone, which triggers changes at puberty, the production of sperm and maintenance of secondary sexual characteristics. Female sex hormones, estrogen and progesterone, are produced by the ovaries and also by the placenta and adrenal cortex of the fetus during pregnancy. Estrogen regulates female reproductive processes and secondary sexual characteristics. Progesterone regulates changes in the endometrium during the menstrual cycle and pregnancy. Steroid hormones are widely used for fertility control and in anti-inflammatory treatments for physical injuries and diseases such as arthritis, asthma, and auto-immune disorders. Progesterone, a naturally occurring progestin, is primarily used to treat amenorrhea, abnormal uterine bleeding, or as a contraceptive. Endogenous progesterone is responsible for inducing secretory activity in the endometrium of the estrogen-primed uterus in preparation for the implantation of a fertilized egg and for the maintenance of pregnancy. It is secreted from the corpus luteum in response to luteinizing hormone (LH). The primary contraceptive effect of exogenous progestins involves the suppression of the midcycle surge of LH. At the cellular level, progestins diffuse freely into target cells and bind to the progesterone receptor. Target cells include the female reproductive tract, the mammary gland, the hypothalamus, and the pituitary. Once bound to the receptor, progestins slow the frequency of release of gonadotropin releasing hormone from the hypothalamus and blunt the pre-ovulatory LH surge, thereby preventing follicular maturation and ovulation. Progesterone has minimal estrogenic and androgenic activity. Progesterone is metabolized hepatically to pregnanediol and conjugated with glucuronic acid. Medroxyprogesterone (MAH), also known as 6α-methyl-17-hydroxyprogesterone, is a synthetic progestin with a pharmacological activity about 15 times greater than progesterone. MAH is used for the treatment of renal and endometrial carcinomas, amenorrhea, abnormal uterine bleeding, and endometriosis associated with hormonal imbalance. MAH has a stimulatory effect on respiratory centers and has been used in cases of low blood oxygenation caused by sleep apnea, chronic obstructive pulmonary disease, or hypercapnia. Mifepristone, also known as RU486, is an antiprogesterone drug that blocks receptors of progesterone. It counteracts the effects of progesterone, which is needed to sustain pregnancy. Mifepristone induces spontaneous abortion when administered in early pregnancy followed by treatment with the prostaglandin, misoprostol. Further, studies show that mifepristone at a substantially lower dose can be highly effective as a postcoital contraceptive when administered within five days after unprotected intercourse, thus providing women with a “morning-after pill” in case of contraceptive failure or sexual assault. Mifepristone also has potential uses in the treatment of breast and ovarian cancers in cases in which tumors are progesterone-dependent. It interferes with steroid-dependent growth of brain meningiomas, and may be useful in treatment of endometriosis where it blocks the estrogen-dependent growth of endometrial tissues. It may also be useful in treatment of uterine fibroid tumors and Cushing's Syndrome. Mifepristone binds to glucocorticoid receptors and interferes with cortisol binding. Mifepristone also may act as an anti-glucocorticoid and be effective for treating conditions where cortisol levels are elevated such as AIDS, anorexia nervosa, ulcers, diabetes, Parkinson's disease, multiple sclerosis, and Alzheimer's disease. Danazol is a synthetic steroid derived from ethinyl testosterone. Danazol indirectly reduces estrogen production by lowering pituitary synthesis of follicle-stimulating hormone and LH. Danazol also binds to sex hormone receptors in target tissues, thereby exhibiting anabolic, antiestrognic, and weakly androgenic activity. Danazol does not possess any progestogenic activity, and does not suppress normal pituitary release of corticotropin or release of cortisol by the adrenal glands. Danazol is used in the treatment of endometriosis to relieve pain and inhibit endometrial cell growth. It is also used to treat fibrocystic breast disease and hereditary angioedema. Corticosteroids are used to relieve inflammation and to suppress the immune response. They inhibit eosinophil, basophil, and airway epithelial cell function by regulation of cytokines that mediate the inflammatory response. They inhibit leukocyte infiltration at the site of inflammation, interfere in the function of mediators of the inflammatory response, and suppress the humoral immune response. Corticosteroids are used to treat allergies, asthma, arthritis, and skin conditions. Beclomethasone is a synthetic glucocorticoid that is used to treat steroid-dependent asthma, to relieve symptoms associated with allergic or nonallergic (vasomotor) rhinitis, or to prevent recurrent nasal polyps following surgical removal. The anti-inflammatory and vasoconstrictive effects of intranasal beclomethasone are 5000 times greater than those produced by hydrocortisone. Budesonide is a corticosteroid used to control symptoms associated with allergic rhinitis or asthma. Budesonide has high topical anti-inflammatory activity but low systemic activity. Dexamethasone is a synthetic glucocorticoid used in anti-inflammatory or immunosuppressive compositions. It is also used in inhalants to prevent symptoms of asthma. Due to its greater ability to reach the central nervous system, dexamethasone is usually the treatment of choice to control cerebral edema. Dexamethasone is approximately 20-30 times more potent than hydrocortisone and 5-7 times more potent than prednisone. Prednisone is metabolized in the liver to its active form, prednisolone, a glucocorticoid with anti-inflammatory properties. Prednisone is approximately 4 times more potent than hydrocortisone and the duration of action of prednisone is intermediate between hydrocortisone and dexamethasone. Prednisone is used to treat allograft rejection, asthma, systemic lupus erythematosus, arthritis, ulcerative colitis, and other inflammatory conditions. Betamethasone is a synthetic glucocorticoid with antiinflammatory and immunosuppressive activity and is used to treat psoriasis and fungal infections, such as athlete's foot and ringworm. The anti-inflammatory actions of corticosteroids are thought to involve phospholipase A 2 inhibitory proteins, collectively called lipocortins. Lipocortins, in turn, control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes by inhibiting the release of the precursor molecule arachidonic acid. Proposed mechanisms of action include decreased IgE synthesis, increased number of β-adrenergic receptors on leukocytes, and decreased arachidonic acid metabolism. During an immediate allergic reaction, such as in chronic bronchial asthma, allergens bridge the IgE antibodies on the surface of mast cells, which triggers these cells to release chemotactic substances. Mast cell influx and activation, therefore, is partially responsible for the inflammation and hyperirritability of the oral mucosa in asthmatic patients. This inflammation can be retarded by administration of corticosteroids. Histological and molecular evaluation of breast tumors reveals that the development of breast cancer evolves through a multi-step process whereby pre-malignant mammary epithelial cells undergo a relatively defined sequence of events leading to tumor formation. An early event in tumor development is ductal hyperplasia. Cells undergoing rapid neoplastic growth gradually progress to invasive carcinoma and become metastatic to the lung, bone, and potentially other organs. Several variables that may influence the process of tumor progression and malignant transformation include genetic factors, environmental factors, growth factors, and hormones. Based on the complexity of this process, it is critical to study a population of human mammary epithelial cells undergoing the process of malignant transformation, and to associate specific stages of progression with phenotypic and molecular characteristics. We have compared primary breast epithelial cells (HMECs) to breast carcinoma lines at various stages of tumor progression. HMEC is a primary breast epithelial cell line isolated from a normal donor. MCF-10A is a breast mammary gland (luminal ductal characteristics) cell line that was isolated from a 36 year-old woman with fibrocystic breast disease. MCF-10A expresses cytoplasmic keratins, epithelial sialomucins, and milkfat globule antigens. This cell lines exhibits three-dimensional growth in collagen and forms domes in confluent culture. MCF7 is a nonmalignant breast adenocarcinoma cell line isolated from the pleural effusion of a 69-year-old female. MCF7 has retained characteristics of the mammary epithelium such as the ability to process estradiol via cytoplasmic estrogen receptors and the capacity to form domes in culture. T-47D is a breast carcinoma cell line isolated from a pleural effusion obtained from a 54-year-old female with an infiltrating ductal carcinoma of the breast. Sk-BR-3 is a breast adenocarcinoma cell line isolated from a malignant pleural effusion of a 43-year-old female. It forms poorly differentiated adenocarcinoma when injected into nude mice. BT-20 is a breast carcinoma cell line derived in vitro from cells emigrating out of thin slices of the tumor mass isolated from a 74-year-old female. MDA-mb-231 is a breast tumor cell line isolated from the pleural effusion of a 51-year old female. It forms poorly differentiated adenocarcinoma in nude mice and ALS treated BALB/c mice. It also expresses the Wnt3 oncogene, EGF, and TGF-α. MDA-mb-435S is a spindle shaped strain that evolved from the parent line (435) as isolated in 1976 by R. Cailleau from the pleural effusion of a 31-year-old female with metastatic, ductal adenocarcinoma of the breast. Osteosarcoma is the most common malignant bone tumor in children. Approximately 80% of patients present with non-metastatic disease. After the diagnosis is made by an initial biopsy, treatment involves the use of 34 courses of neoadjuvant chemotherapy before definitive surgery, followed by post-operative chemotherapy. With currently available treatment regimens, approximately 30-40% of patients with non-metastatic disease relapse after therapy. Currently, prognostic factor exists that can be used at the time of initial diagnosis to predict which patients will have a high risk of relapse. The only significant prognostic factor predicting the outcome in a patient with non-metastatic osteosarcoma is the histopathologic response of the primary tumor resected at the time of definitive surgery. The degree of necrosis in the primary tumor is a reflection of the tumor response to neoadjuvant chemotherapy. A higher degree of necrosis (good or favorable response) is associated with a lower risk of relapse and a better outcome. Patients with a lower degree of necrosis (poor or unfavorable response) have a much higher risk of relapse and poor outcome even after complete resection of the primary tumor. Unfortunately, poor outcome cannot be altered despite modification of post-operative chemotherapy to account for the resistance of the primary tumor to neoadjuvant chemotherapy. Thus, there is an urgent need to identify prognostic factors that can be used at the time of diagnosis to recognize the subtypes of osteosarcomas that have various risks of relapse, so that more appropriate chemotherapy can be used at the outset to improve the outcome. The most important function of adipose tissue is its ability to store and release fat during periods of feeding and fasting. White adipose tissue is the major energy reserve in periods of excess energy use, and its primary purpose is mobilization during energy deprivation. Understanding how the various molecules regulate adiposity and energy balance in physiological and pathophysiological situations may lead to the development of novel therapeutics for human obesity. Adipose tissue is also one of the important target tissues for insulin. Adipogenesis and insulin resistance in type II diabetes are linked and present intriguing relations. Most patients with type II diabetes are obese and obesity in turn causes insulin resistance. Thiazolidinedione (TZD), a family of drugs of peroxisome proliferation-activated receptor gamma (PPAR-γ) agonists, are a new class of antidiabetic agents that improve insulin sensitivity and reduce plasma glucose and blood pressure in subjects with type II diabetes. TZD is also able to induce preadipocytes to differentiate into mature fat cells. The majority of research in adipocyte biology to date has been done using transformed mouse preadipocyte cell lines. It has been demonstrated that the culture condition, which stimulates mouse preadipocyte differentiation is different from that for inducing human primary preadipocyte differentiation. In addition, primary cells are diploid and may therefore reflect the in vivo context better than aneuploid cell lines. Colon cancer is causally related to both genes and the environment. Several molecular pathways have been linked to the development of colon cancer, and the expression of key genes in any of these pathways may be lost by inherited or acquired mutation or by hypermethylation. There is a particular need to identify genes for which changes in expression may provide an early indicator of colon cancer or a predisposition for the development of colon cancer. For example, it is well known that abnormal patterns of DNA methylation occur consistently in human tumors and include, simultaneously, widespread genomic hypomethylation and localized areas of increased methylation. In colon cancer in particular, it has been found that these changes occur early in tumor progression such as in premalignant polyps that precede colon cancer. Indeed, DNA methyltransferase, the enzyme that performs DNA methylation, is significantly increased in histologically normal mucosa from patients with colon cancer or the benign polyps that precede cancer, and this increase continues during the progression of colonic neoplasms (Wafik, S. et al. (1991) Proc. Natl. Acad. Sci. USA 88:3470-3474). Increased DNA methylation occurs in G+C rich areas of genomic DNA termed “CpG islands” that are important for maintenance of an “open” transcriptional conformation around genes, and hypermethylation of these regions results in a “closed” conformation that silences gene transcription. It has been suggested that the silencing or downregulation of differentiation genes by such abnormal methylation of CpG islands may prevent differentiation in immortalized cells (Antequera, F. et al. (1990) Cell 62:503-514). Familial Adenomatous Polyposis (FAP) is a rare autosomal dominant syndrome that precedes colon cancer and is caused by an inherited mutation in the adenomatous polyposis coli (APC) gene. FAP is characterized by the early development of multiple colorectal adenomas that progress to cancer at a mean age of 44 years. The APC gene is a part of the APC-β-catenin-Tcf (T-cell factor) pathway. Impairment of this pathway results in the loss of orderly replication, adhesion, and migration of colonic epithelial cells that results in the growth of polyps. A series of other genetic changes follow activation of the APC-β-catenin-Tcf pathway and accompanies the transition from normal colonic mucosa to metastatic carcinoma. These changes include mutation of the K-Ras proto-oncogene, changes in methylation patterns, and mutation or loss of the tumor suppressor genes p53 and Smad4/DPC4. While the inheritance of a mutated APC gene is a rare event, the loss or mutation of APC and the consequent effects on the APC-β-catenin-Tcf pathway is believed to be central to the majority of colon cancers in the general population. Hereditary nonpolyposis Colorectal Cancer (HNPCC) is another inherited autosomal dominant syndrome with a less well defined phenotype than FAP. HNPCC, which accounts for about 2% of colorectal cancer cases, is distinguished by the tendency to early onset of cancer and the development of other cancers, particularly those involving the endometrium, urinary tract, stomach and biliary system. HNPCC results from the mutation of one or more genes in the DNA mis-match repair (MMR) pathway. Mutations in two human MMR genes, MSH2 and MLH1, are found in a large majority of HNPCC families identified to date. The DNA MMR pathway identifies and repairs errors that result from the activity of DNA polymerase during replication. Furthermore, loss of MMR activity contributes to cancer progression through accumulation of other gene mutations and deletions, such as loss of the BAX gene which controls apoptosis, and the TGFβ receptor II gene which controls cell growth. Because of the potential for irreparable damage to DNA in an individual with a DNA MMR defect, progression to carcinoma is more rapid than usual. Although ulcerative colitis is a minor contributor to colon cancer, affected individuals have about a 20-fold increase in risk for developing cancer. Progression is characterized by loss of the p53 gene which may occur early, appearing even in histologically normal tissue. The progression of the disease from ulcerative colitis to dysplasia/carcinoma without an intermediate polyp state suggests a high degree of mutagenic activity resulting from the exposure of proliferating cells in the colonic mucosa to the colonic contents. Almost all colon cancers arise from cells in which the estrogen receptor (ER) gene has been silenced. The silencing of ER gene transcription is age related and linked to hypermethylation of the ER gene (Issa, J-P. J. et al. (1994) Nature Genetics 7:536-540). Introduction of an exogenous ER gene into cultured colon carcinoma cells results in marked growth suppression. The connection between loss of the ER protein in colonic epithelial cells and the consequent development of cancer has not been established. Clearly there are a number of genetic alterations associated with colon cancer and with the development and progression of the disease, particularly the downregulation or deletion of genes, that potentially provide early indicators of cancer development, and which may also be used to monitor disease progression or provide possible therapeutic targets. The specific genes affected in a given case of colon cancer depend on the molecular progression of the disease. Identification of additional genes associated with colon cancer and the precancerous state would provide more reliable diagnostic patterns associated with the development and progression of the disease. Prostate cancer is a common malignancy in men over the age of 50, and the incidence increases with age. In the US, there are approximately 132,000 newly diagnosed cases of prostate cancer and more than 33,000 deaths from the disorder each year. Once cancer cells arise in the prostate, they are stimulated by testosterone to a more rapid growth. Thus, removal of the testes can indirectly reduce both rapid growth and metastasis of the cancer. Over 95 percent of prostatic cancers are adenocarcinomas which originate in the prostatic acini. The remaining 5 percent are divided between squamous cell and transitional cell carcinomas, both of which arise in the prostatic ducts or other parts of the prostate gland. As with most cancers, prostate cancer develops through a multistage progression ultimately resulting in an aggressive, metastatic phenotype. The initial step in tumor progression involves the hyperproliferation of normal luminal and/or basal epithelial cells that become hyperplastic and evolve into early-stage tumors. The early-stage tumors are localized in the prostate but eventually may metastasize, particularly to the bone, brain or lung. About 80% of these tumors remain responsive to androgen treatment, an important hormone controlling the growth of prostate epithelial cells. However, in its most advanced state, cancer growth becomes androgen-independent and there is currently no known treatment for this condition. A primary diagnostic marker for prostate cancer is prostate specific antigen (PSA). PSA is a tissue-specific serine protease almost exclusively produced by prostatic epithelial cells. The quantity of PSA correlates with the number and volume of the prostatic epithelial cells, and consequently, the levels of PSA are an excellent indicator of abnormal prostate growth. Men with prostate cancer exhibit an early linear increase in PSA levels followed by an exponential increase prior to diagnosis. However, since PSA levels are also influenced by factors such as inflammation, androgen and other growth factors, some scientists maintain that changes in PSA levels are not useful in detecting individual cases of prostate cancer. Current areas of cancer research provide additional prospects for markers as well as potential therapeutic targets for prostate cancer. Several growth factors have been shown to play a critical role in tumor development, growth, and progression. The growth factors Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), and Tumor Growth Factor alpha (TGFα) are important in the growth of normal as well as hyperproliferative prostate epithelial cells, particularly at early stages of tumor development and progression, and affect signaling pathways in these cells in various ways (Lin J et al. (1999) Cancer Res. 59:2891-2897; Putz T et al. (1999) Cancer Res 59:227-233). The TGF-β family of growth factors are generally expressed at increased levels in human cancers and the high expression levels in many cases correlates with advanced stages of malignancy and poor survival (Gold LI (1999) Crit Rev Oncog 10:303-360). Finally, there are human cell lines representing both the androgen-dependent stage of prostate cancer (LNCaP) as well as the androgen-independent, hormone refractory stage of the disease (PC3 and DU-145) that have proven useful in studying gene expression patterns associated with the progression of prostate cancer, and the effects of cell treatments on these expressed genes (Chung T D (1999) Prostate 15:199-207). Alzheimer's disease is a progressive neurodegenerative disorder that is characterized by the formation of senile plaques and neurofibrillary tangles containing amyloid beta peptide. These plaques are found in limbic and association cortices of the brain, including hippocampus, temporal cortices, cingulate cortex, amygdala, nucleus basalis and locus caeruleus. Early in Alzheimer's pathology, physiological changes are visible in the cingulate cortex (Minoshima, S. et al. (1997) Annals of Neurology 42:85-94). In subjects with advanced Alzheimer's disease, accumulating plaques damage the neuronal architecture in limbic areas and eventually cripple the memory process. Leukemias can be classified into four major categories, and all involve malignant transformation of pluripotent stem cells. Acute leukemias, both lymphoblastic (ALL) and myeloid (AML) types, are characterized by the presence of immature cells in the blood. Chronic leukemias, both lymphocytic (CLL) and myelocytic (CML), are associated with mature, differentiated cells, but proportions of each cell type are abnormal. For example, CLL patients usually have clonal expansion of B cell lymphocytes. CML patients often have granulocytes of all stages of maturity present in blood, bone marrow, and other organs. Monoclonal antibodies specific for B- and T-cells are helpful diagnostic tools, in addition to histological analysis. Disease progresses as normal hematopoietic bone marrow is displaced by malignant cells. Cause has been determined to be genetic in some cases, and chemical or radiation-induced in others. There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of cell proliferative, autoimmune/inflammatory, cardiovascular, neurological, and developmental disorders. |
<SOH> SUMMARY OF THE INVENTION <EOH>Various embodiments of the invention provide purified polypeptides, secreted proteins, referred to collectively as “SECP” and individually as “SECP-1,” “SECP-2,” “SECP-3,” “SECP-4,” “SECP-5,” “SECP-6,” “SECP-7,” “SECP-8,” “SECP-9,” “SECP-10,” “SECP-11,” “SECP-12,” “SECP-13,” “SECP-14,” “SECP-15,” “SECP-16,” “SECP-17,” “SECP-18,” “SECP-19,” “SECP-20,” “SECP-21,” “SECP-22,” “SECP-23,” “SECP-24,” “SECP-25,” “SECP-26,” “SECP-27,” “SECP-28,” “SECP-29,” “SECP-30,” “SECP-31,” “SECP-32,”. “SECP-33,” “SECP-34,” “SECP-35,” “SECP-36,” “SECP-37,” “SECP-38,” “SECP-39,” “SECP-40,” “SECP-41,” “SECP-42,” “SECP-43,” “SECP-44,” “SECP-45,” “SECP-46,” “SECP-47,” “SECP-48,” “SECP-49,” “SECP-50,” “SECP-51,” “SECP-52,” “SECP-53,” “SECP-54,” “SECP-55,” “SECP-56,” “SECP-57,” “SECP-58,” “SECP-59,” “SECP-60,” “SECP-61,” “SECP-62,” “SECP-63,” “SECP-64,” “SECP-65,” “SECP-66,” “SECP-67,” “SECP-68,” “SECP-69,” “SECP-70,” “SECP-71,” “SECP-72,” “SECP-73,” “SECP-74,” “SECP-75,” “SECP-76,” “SECP-77,” “SECP-78,” “SECP-79,” and “SECP-80,” and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions. Embodiments also provide methods for utilizing the purified secreted proteins and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology. Related embodiments provide methods for utilizing the purified secreted proteins and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions. An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 1-80. Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-80. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:81-160. Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80. Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide. Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed. Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80. Still yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:81-160, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:81-160, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). In other embodiments, the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides. Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:81-160, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:81-160, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex. In a related embodiment, the method can include detecting the amount of the hybridization complex. In still other embodiments, the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides. Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:81-160, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:81-160, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof. In a related embodiment, the method can include detecting the amount of the amplified target polynucleotide or fragment thereof. Another embodiment provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, and a pharmaceutically acceptable excipient. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional SECP, comprising administering to a patient in need of such treatment the composition. Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional SECP, comprising administering to a patient in need of such treatment the composition. Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-80, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional SECP, comprising administering to a patient in need of such treatment the composition. Another embodiment provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-80, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide. Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-80. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide. Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:81-160, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:81-160, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:81-160, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:81-160, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:81-160, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. |
Examination of harmful bacterium in food or drink |
The present invention relates to a method capable of detecting proliferative bacteria in food and beverage, particularly acid or neutral beverage at low cost within a short time, a detection kit which enables simple measurement, and a technology for examination of harmful bacteria in food and beverage with respect to a method for rapid determination of proliferation properties of bacteria capable of producing guaiacol in food and beverage. There is disclosed a method for detecting bacteria capable of producing guaiacol, which comprises culturing or incubating a culture solution containing a specimen for a predetermined time in the presence of vanillic acid, and measuring the produced guaiacol by qualitative or quantitative analysis. Also there is disclosed a kit for detection of bacteria capable of producing guaiacol, comprising a container, and an acid or neutral culture medium containing a vanillic acid substrate for production of guaiacol, which is contained in the container and also sterilized and sealed. Furthermore there is disclosed a method for rapid determination of proliferation properties of bacteria capable of producing guaiacol in food and beverage, which comprises incubating a sporophyte of spore-forming bacteria capable of producing guaiacol for a predetermined time in the food and beverage in the presence of vanillic acid, and measuring the produced guaiacol by qualitative or quantitative analysis. |
1. A method for detecting bacteria capable of producing guaiacol, which comprises culturing or incubating a culture solution containing a specimen for a predetermined time in the presence of vanillic acid, and measuring the produced guaiacol by qualitative or quantitative analysis. 2. The method according to claim 1, wherein said produced guaiacol is measured by GC-MS analysis or color reaction. 3. The method according to claim 1 or 2, wherein the color reaction for guaiacol employs an oxidation reaction using peroxidase as a catalyst and is detected by spectrophotometry or visual observation. 4. The method according to any one of claims 1 to 3, wherein said bacteria capable of producing guaiacol to be detected are thermotolerant acidophilic bacteria and are cultured or incubated under an acid condition. 5. The method according to any one of claims 1 to 3, wherein said bacteria capable of producing guaiacol to be detected are bacteria belonging to the genus Bacillus and are cultured or incubated under a neutral condition. 6. The method according to claim 4, wherein said thermotolerant acidophilic bacteria are of A. acidoterrestris. 7. A kit for detection of bacteria capable of producing guaiacol, comprising a container, and an acid or neutral culture medium containing a vanillic acid substrate for production of guaiacol, which is contained in the container and also sterilized and sealed. 8. A kit for detection of bacteria capable of producing guaiacol, comprising a combination of the kit according to claim 7 and a means for detection of guaiacol in combination. 9. The kit according to claim 7 or 8, wherein said means for detection of guaiacol is based on color reaction and includes a combination of at least hydrogen peroxide water, a peroxidase enzyme and a buffer for enzymatic reaction. 10. The kit for detection of bacteria capable of producing guaiacol according to claim 9, comprising all or any combination of the kit according to claim 7, a vanillic acid substrate, hydrogen peroxide water, peroxidase, a buffer solution, a filter for filtrating a culture solution, a manual pump for filtrating a culture solution, a transparent cell containing a culture filtrate and a color sample plate. 11. The kit according to any one of claims 7 to 10, wherein said bacteria capable of producing guaiacol to be detected are thermotolerant acidophilic bacteria and an acid culture medium is contained therein. 12. The kit according to any one of claims 7 to 10, wherein said bacteria capable of producing guaiacol to be detected are bacteria belonging to the genus Bacillus and a neutral culture medium is contained therein. 13. The kit according to claim 11, wherein said thermotolerant acidophilic bacteria are of A. acidoterrestris. 14. A method for rapid determination of proliferation properties of bacteria capable of producing guaiacol in food and beverage, which comprises incubating a sporophyte of spore-forming bacteria capable of producing guaiacol for a predetermined time in the food and beverage in the presence of vanillic acid, and measuring the produced guaiacol by qualitative or quantitative analysis. 15. The method according to claim 14, wherein said spore-forming bacteria capable of producing guaiacol are thermotolerant acidophilic bacteria. 16. The method according to claim 15, wherein said thermotolerant acidophilic bacteria are of A. acidoterrestris. |
<SOH> BACKGROUND ART <EOH>Microorganisms having proliferation properties have recently been detected from acidic foods and beverages, particularly acidic beverage such as raw fruit juice and fruit juice, resulting in a problem. In general, bacteria hardly generate in an acid range like fruit juice and fruit juice beverage. However, thermotolerant acidophilic bacteria having thermotolerant spores among acidophilic bacteria which is fond of the acid range hardly die out under the conditions of a sterilization temperature of the beverage (usually about 93° C.), thus constituting a large problem. Typical thermotolerant acidophilic bacteria include bacteria of the genus Alicyclobacillus . It has been reported that two species ( A. acidoterrestris and A. acidiphilus ) among bacteria of the genus Alicyclobacillus contaminate fruit juice and fruit juice beverage to cause proliferation. Also these bacteria constitute a large problem on quality because a nasty smell component, guaiacol, is produced during proliferation. Particularly, A. acidoterrestris is isolated from fruit juice with high frequency and is considered as an indicator bacterium in control of microorganism of fruit juice and fruit juice-containing beverage. As a method for detecting such bacteria, there have been developed, e.g., a culturing method and a method for detecting a specific gene by PCR, which are described in Patent Documents described hereinafter. However, the culturing method, which requires no expensive devise, require the culture or detection time of about 48 hours or more after pre-culture and a simple method for detection within a short time has never been known. Japanese Patent No. 3177367 discloses a method for detecting acidophilic spore-forming bacteria using an acid culture medium containing aldehyde and a detection kit. It also discloses that, since acidophilic spore-forming bacteria converts vanillin into guaiacol and guaiacol emits a strong odor, the presence of acidophilic spore-forming bacteria in a test sample can be detected within a short time without confirming the formation of colony by adding a test sample such as fruit juice in a vanillin-containing medium. Japanese Patent Laid-Open Publication No. 8-140696 discloses, in a method for detecting thermotolerant acidophilic bacteria having proliferation properties in acid fruit juice or acidic beverage, the presence of the bacteria can be evaluated by the presence or absence of ω-cyclohexane fatty acid using a GC-MS method and the like. Japanese Patent Laid-Open Publication No. 10-234376 discloses a method for detecting thermotolerant acidophilic bacteria having proliferation properties in acid fruit juice or acidic beverage, which comprises detecting and identifying microorganisms belonging to the genus Alicyclobacillus by subjecting a nucleic acid coding for an enzyme involving in bio-synthesis of ω-cyclohexane fatty acid to the PCR reaction using a nucleic acid primer having a specific base sequence. Although various dairy products with vanilla flavor are commercially available, the production of a nasty smell component, guaiacol, associated with proliferation of specific Bacillus may cause deterioration of flavor, if the conditions are satisfied. However, harmfulness of microorganisms belonging to the genus Bacillus has little been noticed, and thus a method for detecting harmful microorganisms has scarcely been reported. The present inventors have hitherto carried out a challenge test (indicator bacteria proliferation test method) using Alicyclobacillus acidoterrestris as a means for understanding proliferation properties of bacteria in a specimen. In this test method, bactericidal conditions are determined by inoculating 10 1−3 /ml of spores (or vegetative cells) into a specimen such as beverage, standing at 35° C. for 2 to 3 weeks, measuring an increase or decrease of bacteria with a lapse of time using a plating culture method (requiring additional 3 to 5 days), and performing risk analysis due to the thermotolerant acidophilic bacteria in the beverage. Therefore, the determination requires at least a half-month. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a graph showing the results of comparison in a guaiacol production rate between the case of the addition of vanillic acid and the case of the addition of vanillin in Example 1. FIG. 2 is a graph showing the results of comparison in a guaiacol-producing rate between the case of the addition of vanillic acid and the case of the addition of vanillin in Example 2. FIG. 3 is a graph showing the measurement results of the guaiacol concentration and OD 470 using a potassium phthalate buffer solution (pH 4.5). FIG. 4 is a graph showing the measurement results of the guaiacol concentration and OD 470 using a sodium phosphate buffer solution (pH 6.8). FIG. 5 is an explanatory scheme showing a typical kit for detection of bacteria capable of producing guaiacol according to the present invention. detailed-description description="Detailed Description" end="lead"? |
Premeabilisation of cells |
Provided is a method for permeabilising a viable cell having a cell wall, comprising: (a) pressurising a fluid or gel in contact with a surface of the cell; and (b) depressurising the fluid or gel; to form at least one hole in a surface of the cell. |
1. A method for permeabilising a viable cell having a cell wall, comprising: (a) pressurising a fluid or gel in contact with a surface of the cell; and (b) depressurising the fluid or gel; to form at least one hole in a surface of the cell. 2. A method according to claim 1, wherein depressurising the fluid or gel generates bubbles of gas which are capable of forming at least one hole in a surface of the cell. 3. A method according to claim 1, wherein the reduction in pressure in step (b) is 2 MPa (20 Bar) or more. 4. A method according to claim 3, wherein the reduction in pressure in step (b) is from 2-11 MPa (20 Bar to 110 Bar). 5. A method according to claim 4, wherein the reduction in pressure in step (b) is from 5-11 MPa (50 Bar to 110 Bar). 6. A method according to claim 1, wherein the fluid or gel is depressurised in step (b) to substantially atmospheric pressure (about 1 Bar). 7. A method according to claim 1, wherein the hole in the surface of the cell comprises a hole in the cell membrane. 8. A method according to claim 1, wherein the pressure is reduced in step (b) over an interval of less than 10 seconds. 9. A method according to claim 1, wherein the fluid or gel is pressurised in step (a) for a period of 10 mins or more. 10. A method according to claim 9, wherein the fluid or gel is pressurised in step (a) for a period of 10-20 mins. 11. A method according to claim 10, wherein the fluid or gel is pressurised in step (a) for a period of about 15 mins. 12. A method according to claim 1, wherein the fluid or gel comprises an aqueous liquid. 13. A method according to claim 12, wherein the fluid or gel comprises a buffer or a cell culture medium. 14. A method according to claim 1, wherein a gas in contact with the fluid or gel which is subject to the pressurising has a solubility in the fluid or gel of 1.0×10−4 mol/l atm or more. 15. A method according to claim 14, wherein the gas has a solubility of 6.0×10−4 mol/l atm or more. 16. A method according to claim 14, wherein the gas comprises a gas selected from air, oxygen, nitrogen, carbon dioxide, methane, helium, neon, and argon. 17. A method according to claim 1, which method consists of a single pressurising and depressurising cycle, or multiple pressuring and depressurising cycles. 18. A method according to claim 1, wherein the cell is a plant cell, a-fungal cell or a bacterial cell. 19. A method according to claim 18, wherein the cell is a cell from a crop plant. 20. A method according to claim 19, wherein the crop plant is selected from a cereal or pulse, maize, wheat, potato, tapioca, rice, sorghum, millet, cassava, barley, pea, and another root, tuber, or seed crop. 21. A method according to claim 20, wherein the seed crop is selected from oil-seed rape, sugar beet, maize, sunflower, soybean, and sorghum. 22. A method according to claim 18, wherein the plant cell is a cell from a horticultural plant. 23. A method according to claim 22, wherein the horticultural plant is selected from lettuce, endive, vegetable brassicas including cabbage broccoli and cauliflower, carnation, geranium, tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper, chrysanthemum, poplar, eucalyptus, and pine. 24. A method according to claim 18, wherein the cell is from a seed producing plant selected from oil-seed plants, cereal seed producing plants and leguminous plants. 25. A method according to claim 24, wherein the oil seed plant is selected from cotton, soybean, safflower, sunflower, oil-seed rape, maize, alfalfa, palm, and coconut. 26. A method according to claim 24 wherein the cereal seed producing plant is selected from corn, wheat, barley, rice, sorghum, and rye, and other grain seed producing plants. 27. A method according to claim 24, wherein the leguminous plant is selected from peas and beans, including guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, and chickpea. 28. A method according to claim 18, wherein the cell is a cell from a plant selected from corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea Americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables, ornamentals, and conifers. 29. A method according to claim 18 wherein the bacterial cell is a gram-positive or gram-negative bacterium. 30. A method according to claim 29, wherein the cell is a cell selected from E. coil, B. subtilis, S. cerevisiae, F. graminearum, S. pombe, Z. mays, and N. tabacum. 31. A method according to claim 1, wherein the cell forms part of a cluster of cells. 32. A method according to claim 31, wherein the cluster is an embryogenic cluster. 33. A method according to claim 1, wherein the cell is a microspore. 34. A method according to claim 33, wherein the cell is a pollen microspore. 35. A method according to claim 1, wherein the temperature of the fluid or gel is up to 37° C. 36. A method according to claim 35, wherein the temperature is from 15-30° C. 37. A method for introducing a substance into a cell having a cell wall, comprising a method according to claim 1, and wherein the at least one hole facilitates entry of the substance into the cell. 38. A method according to claim 37, wherein the fluid or gel comprises the substance. 39. A method according to claim 37, wherein the substance is selected from a biological molecule or a macromolecule. 40. A method according to claim 39, wherein the substance is selected from a nucleic acid including DNA, cDNA, RNA or mRNA 41. A method according to claim 40, wherein the nucleic acid comprises a gene, a plasmid, a chromosome, an oligonucleotide, a nucleotide sequence, a ribozyme or a fragment thereof, or an expression vector. 42. A method according to claim 39, wherein the substance comprises a bio-active molecule, including a protein, a polypeptide, a peptide, an amino acid, a hormone, a polysaccharide, a dye, and a pharmaceutical agent such as drug. 43. A method according to claim 37, wherein the substance has a molecular weight of 100 Daltons or more. 44. A permeabilised cell having a cell wall obtainable by a method as defined in claim 1, wherein the surface of the cell comprises at least one hole which is capable of facilitating the entry of a substance into the cell. 45. A permeabilised cell according to claim 44, wherein the hole comprises a hole in the cell membrane. 46. A permeabilised cell according to claim 44, wherein the cell wall of the cell is substantially intact. 47. Use of a depressurisation means to permeabilise a cell and/or to introduce a substance into a cell, wherein the cell has a cell wall, and the depressurisation means is used to reduce the pressure applied to a fluid or gel comprising the cell by a step of 2 MPa (20 Bar) or more. 48. An apparatus for introducing a substance into a cell having a cell wall, using a method as defined in claim 1, which apparatus comprises: (a) an inlet for introducing a gas; (b) a pressure chamber into which the inlet feeds, which chamber is of substantially geometrical cross section; (c) a compartment within the pressure chamber for containing the cell in a fluid or gel; (d) optionally a pressure gauge for monitoring the pressure in the pressure chamber; and (e) an outlet for releasing gas from the pressure chamber; wherein both the inlet and the outlet comprise a valve for isolating the pressure chamber during pressurisation. 49. An apparatus according to claim 48, wherein the inlet and outlet comprise inlet and outlet tubes. 50. An apparatus according to claim 49 wherein the diameter of the inlet tube and/or the outlet tube is from 2-4 mm. 51. An apparatus according to claim 48, wherein the geometrical cross section of the pressurisation chamber is substantially cylindrical. 52. An apparatus according to claim 48, wherein the compartment for containing the cell in a fluid or gel comprises substantially the entire internal surface of the pressure chamber. 53. An apparatus according to claim 52, wherein the internal surface of the pressure chamber comprises a physiologically acceptable coating. 54. An apparatus according to claim 48, wherein the compartment for containing the cell in a fluid or gel comprises a receptacle positioned adjacent to an internal surface of the pressure chamber. 55. An apparatus according to claim 54, wherein the receptacle is supported by the internal surface of the pressure chamber. 56. An apparatus according to claim 54, wherein the internal surface of the receptacle comprises a physiologically acceptable coating. 57. An apparatus according to claim 48, wherein the valve in the inlet and/or the outlet comprises a needle valve. |
Process for the detection of oxidative stress and kit for its implementation |
The present invention relates to a process for detecting oxidative stress in a sample and to a kit for this implementation. According to one embodiment the present invention provides a method for the detection of oxidative stress in an individual carrying a risk factor for oxidative stress comprising determining the risk factor for oxidative stress of said individual; selecting at least two oxidative stress markers being increased or decreased for said risk factor relative to healthy individuals; and measuring the amount of said at least two oxidative stress markers in a sample obtained from said individual. |
1. A method for determining oxidative stress markers in a group of individuals comprising the steps of: a. determining the risk factor for oxidative stress in said group; b. measuring the amount of at least 10 different oxidative stress markers in a sample obtained from each of said group of individuals; and c. comparing the amount of each of said oxidative stress markers with the amount of each of said oxidative stress markers measured in a group of healthy individuals thereby determining the oxidative stress markers being increased or decreased in said group of individuals carrying a risk factor for oxidative stress relative to healthy individuals. 2. A method for the detection of oxidative stress in an individual carrying a risk factor for oxidative stress comprising: a. determining the risk factor for oxidative stress of said individual; b. selecting at least two oxidative stress markers being increased or decreased for said risk factor relative to healthy individuals; and c. measuring the amount of said at least two oxidative markers in a sample obtained from said individual. 3. The method according to claim 2, further comprising the step of evaluating the result in the context of said risk factor. 4. The method according to claim 2 wherein not more than 22 different oxidative stress markers are selected. 5. The method according to claim 1, wherein the risk factor is selected from: unbalanced diet, smoking habits, exposure to toxic environment, medical surgery, intense physical exercise, and diseases affecting the kidneys, lungs, heart, skin, brain, joints, gastrointestinal tract, eyes, blood vessels, red blood cells, liver and multiple organs. 6. The method according to claim 5, wherein the diseases are selected from transplantation, glomerular nephritis, respiratory distress syndrome, asthma, coronary thrombosis, burns, sunlight exposure, psoriasis, dermatosis, trauma, Parkinson's disease, neurotoxins, dementia, rheumatoid arthritis, diabetes, pancreatitis, endotoxemia, intestinal Ischaemia, cataract, retinopathy, retinal degeneration, atherosclerosis, Fanconi's anemia, malaria, inflammation, ischaemia-reperfusion, drug toxicity, iron overload, nutritional deficiency, alcohol, radiation, cancer, aging, HCV infection and AIDS. 7. The method according to claim 1, wherein the oxidative stress marker is selected from the group consisting of: antioxidants, trace elements, indicators of oxidative stress, iron metabolism markers, homocysteine, enzymes having antioxidant functions, enzymes having pro-oxidant functions, enzymes for DNA repair, enzymes of the glutathione metabolism, stress proteins, proteins implied in apoptosis, transcription factors, cytokines and chemokines. 8. The method according to claim 7, wherein the antioxidant is selected from: vitamin A, vitamin C, vitamin E, reduced glutathione (GSH)/oxidized glutathione (GSSG), protein thiols, glutathione peroxidase and superoxide dismutase. 9. The method according to claim 7, wherein the trace element is selected from selenium, copper and zinc. 10. The method according to claim 7, wherein the indicator of oxidative stress is selected from antibodies against oxidized LDL, 8-hydroxy-2′-deoxyguanosine, myeloperoxidase, glucose and glyoxal. 11. The method according to claim 7, wherein the iron metabolism marker is selected from transferrin, ferritin and ceruloplasmin. 12. The method according to claim 7, wherein the enzymes having antioxidant function are selected from catalase, Mn-containing superoxide dismutase (SOD), Copper and zinc containing SOD, thioredoxine-1, thioredoxine reductase-1, peroxyredoxine-1, metallothioneine-1, L-ferritine, H-ferritine and transferrine receptor, anti-oxidant protein 2, ceruloplasmin, lactoferrin, selenoprotein P, selenoprotein W, frataxin, serum paraoxonase/arylesterase 1, serum paraoxonase/arylesterase 2, and serum paraoxonase/arylesterase 3. 13. The method according to claim 7, wherein the enzymes having pro-oxidant functions are selected from cyclooxygenase-2, 5-lipoxygenase, c-phospholipase A2, phospholipase A alpha, phospholipase D-1, myeloperoxidase, nitric oxide synthetase, C reactive protein, elastase, haptoglobin, NADH-cytochrome b5 reductase and diaphorase A1. 14. The method according to claim 7, wherein the enzyme for DNA repair is 8-oxoguanine DNA glycosylase. 15. The method according to claim 7, wherein the glutathione metabolism enzyme is selected from glutathione peroxidase, non-Se glutathione phospholipid hydroperoxide, phospholipid, gamma-glutamyl cysteine synthetase, and glucose 6-phosphate dehydrogenase, extracellular glutathione peroxidase, glutathione peroxidase, glutathione peroxidase 2, glutathione peroxidase 4, glutathione reductase, glutathione S-transferase, glutathione synthetase, peroxiredoxin 1, peroxiredoxin 2, peroxiredoxin 3, peroxiredoxin 5, and thioredoxin 2. 16. The method according to claim 7, wherein the stress protein is a heat shock protein (HSP), heme-oxygenase-1, heme-oxygenase-2, 150 kDa oxygen-regulated protein (ORP)150, 27 kDa HSP27, HSP90A, HSP17, HSP40 or HSP110. 17. The method according to claim 7, wherein the protein implied in apoptosis is FasL, CD95, tumor necrosis factor (TNF) receptor 1, Bcl-2, GADD153, GADD45, RAD50, RAD51 B, RAD52, RAD54, p53 or Fas ligand. 18. The method according to claim 7, wherein the transcription factor is selected from NfκB-α, c-Fos, C-jun, IκB-α, monoamine oxidase A, monoamine oxidase B, and peroxisome proliferative-activated receptor α. 19. The method according to claim 7, wherein the cytokine or chemokine is selected from: IL-1, IL-6, IL-8, IL-1beta, IL-2 and TNF1 receptor associated protein. 20. The method according to claim 1, when the risk factor of said individual is hemodialysis, and the oxidative marker is selected from the group of catalase, glucose 6 phosphate dehydrogenase, HSP70, 5-lipoxygenase, vitamin C, glutathione peroxidase, SOD, Se, lipid peroxide, oxidized LDL and homocysteine. 21. The method according to claim 1, when the risk factor of said individual in cardiac surgery, and the oxidative marker is selected from the group of superoxide dismutase containing manganese, c-phospholipase A2, H-ferritin, IL-8, nitric oxide synthase 2 (NOS2), vitamin C, vitamin E/cholesterol, glutathione peroxidase (GPx), antibodies against LDL and homocysteine. 22. The method according to claim 1, when the risk factor of said individual is intense physical exercise, the oxidative marker is selected from the group of vitamin E, vitamin E/cholesterol, GSH, GSH/GSSG ratio, zinc, GPx, GSSG, copper/zinc ratio, antibodies against oxidized LDL and oxidized proteins. 23. The method according to claim 1, when the risk factor of said individual is exhaustion due to physical exercise and injuries, the oxidative marker is selected from the group of vitamin E, vitamin E/cholesterol, GSH, GSH/GSSG ratio, zinc, GPx, GSSG; copper/zinc ratio, antibodies against oxidized LDL and oxidized proteins. 24. The method according to claim 1, when the risk factor of said individual is smoking, the oxidative marker is selected from the group of vitamin C, Se, GPx, antibodies against oxidized LDL and homocysteine. 25. The method according to claim 1, wherein the amount of the oxidative stress marker is determined by measuring the concentration of the oxidative marker. 26. The method according to claim 1, wherein the amount is determined by measuring the concentration of the gene transcript/mRNA encoding the oxidative marker. 27. The method according to claim 26, wherein the amount of at least two oxidative stress markers is determined in parallel. 28. The method according to claim 27, wherein the amount of oxidative stress marker is measured by using a DNA chip. 29. (canceled) 30. (canceled) 31. (canceled) 32. (canceled) 33. A process of detecting oxidative stress in a blood sample comprising cells, which method comprises: a. extracting mRNA from cells from the blood sample, b. reverse transcribing said mRNA into cDNA, with labeling of these cDNA compounds, and c. bringing these cDNA into contact with a population of several synthetic DNA fragments, selected in a way to realize a molecular hybridization between the cDNA and said synthetic DNA fragments, in case of expression of oxidative stress in the cells, and simultaneous detection of signals of said hybridization, which correspond to some expressed genes. 34. The process according to claim 33, wherein said blood sample is a preparation of lymphocytes. 35. The process according to claim 33, characterized in that step c. is realized on a DNA chip array, which bears said synthetic DNA fragments according to a specific topography. 36. The process according to claim 33, characterized in that the population of synthetic DNA fragments is composed of oligonucleotides having a size of between 25 to 100 b. 37. The process according to claim 33, characterized in that the population of the synthetic DNA fragments is composed of in vitro polymerase chain reaction (PCR) enzymatic amplification products. 38. The process according to claim 33, characterized in that the population of synthetic DNA fragments is composed of oligonucleotides having a size of between 25 and 100 b, and of products coming from in vitro PCR enzymatic amplification. 39. The process according to claim 33, characterized in that the population of synthetic DNA fragments comprises at least some gene fragments belonging to a family of genes chosen from the group constituted by the one coding for: a. enzymes with antioxidant functions, enzymes with pro-oxidant functions, b. enzymes for the DNA repair, c. enzymes of the glutathion metabolism, d. stress proteins, e. proteins implied in apoptosis, f. transcription factors, g. cytokines, or h. chemokines. 40. The process according to claim 39, characterized in that the population of synthetic DNA fragments comprises at least two genes, each of which belongs to one of the families of genes a-h. 41. The process according to claim 33, characterized in that it comprises, in addition, before said mRNA extraction, an in vitro exposition of the cells of the blood sample to factors generating oxidative stress. 42. The process according to claim 33, characterized in that it comprises in parallel a quantification of blood markers of oxidative stress. 43. (canceled) 44. (canceled) 45. (canceled) 46. (canceled) 47. (canceled) 48. The method according to claim 4, wherein not more than 15 different oxidative stress markers are selected. 49. The method according to claim 48, wherein not more than 10 different oxidative stress markers are selected. 50. The method according to claim 49, wherein not more than 5 different oxidative stress markers are selected. 51. The method according to claim 3, wherein not more than 22 different oxidative stress markers are selected. 52. The method according to claim 51, wherein not more than 15 different oxidative stress markers are selected. 53. The method according to claim 52, wherein not more than 10 different oxidative stress markers are selected. 54. The method according to claim 53, wherein not more than 5 different oxidative stress markers are selected. 55. The method according to claim 2, wherein the amount of the oxidative stress marker is determined by measuring the concentration of the oxidative marker. 56. The method according to claim 3, wherein the amount of the oxidative stress marker is determined by measuring the concentration of the oxidative marker. |
Intracellular signaling molecules |
Various embodiments of the invention provide human intracellular signaling molecules (INTSIG) and polynucleotides which identify and encode INTSIG. Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing disorders associated with aberrant expression of INTSIG. |
1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-3, SEQ ID NO:6-8, SEQ ID NO:10, SEQ ID NO:12-15, SEQ ID NO:17-22, SEQ ID NO:25-28, SEQ ID NO:31, SEQ ID NO:36-38, and SEQ ID NO:40-43, c) a polypeptide comprising a naturally occurring amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:33-34, d) a polypeptide comprising a naturally occurring amino acid sequence at least 98% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:29-30, SEQ ID NO:32, SEQ ID NO:39, and SEQ ID NO:45, e) a polypeptide comprising a naturally occurring amino acid sequence at least 94% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:16, and SEQ ID NO:44, f) a polypeptide comprising a naturally occurring amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO:11, g) a polypeptide comprising a naturally occurring amino acid sequence at least 91% identical to the amino acid sequence of SEQ ID NO:23, h) a polypeptide comprising a naturally occurring amino acid sequence at least 92% identical to the amino acid sequence of SEQ ID NO:24, i) a polypeptide comprising a naturally occurring amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO:35, j) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, and k) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45. 2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-45. 3. An isolated polynucleotide encoding a polypeptide of claim 1. 4. An isolated polynucleotide encoding a polypeptide of claim 2. 5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:46-90. 6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3. 7. A cell transformed with a recombinant polynucleotide of claim 6. 8. (canceled) 9. A method of producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed. 10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-45. 11. An isolated antibody which specifically binds to a polypeptide of claim 1. 12. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:46-90, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:46-55 and SEQ ID NO:57-89, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO:56, d) a polynucleotide consisting essentially of a naturally occurring polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO:90, e) a polynucleotide complementary to a polynucleotide of a), f) a polynucleotide complementary to a polynucleotide of b), g) a polynucleotide complementary to a polynucleotide of c), h) a polynucleotide complementary to a polynucleotide of d), and i) an RNA equivalent of a)-h). 13. (canceled) 14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof. 15. (canceled) 16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof. 17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient. 18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-45. 19. (canceled) 20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample. 21. (canceled) 22. (canceled) 23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample. 24. (canceled) 25. (canceled) 26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1. 27. (canceled) 28. A method of screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising: a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. 29. A method of assessing toxicity of a test compound, the method comprising: a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. 30-145. (canceled) |
<SOH> BACKGROUND OF THE INVENTION <EOH>Cell-cell communication is essential for the growth, development, and survival of multicellular organisms. Cells communicate by sending and receiving molecular signals. An example of a molecular signal is a growth factor, which binds and activates a specific transmembrane receptor on the surface of a target cell. The activated receptor transduces the signal intracelularly, thus initiating a cascade of biochemical reactions that ultimately affect gene transcription and cell cycle progression in the target cell. Intracellular signaling is the process by which cells respond to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc.) through a cascade of biochemical reactions that begins with the binding of a signaling molecule to a cell membrane receptor and ends with the activation of an intracellular target molecule. Intermediate steps in the process involve the activation of various cytoplasmic proteins by phosphorylation via protein kinases, and their deactivation by protein phosphatases, and the eventual translocation of some of these activated proteins to the cell nucleus where the transcription of specific genes is triggered. The intracellular signaling process regulates all types of cell functions including cell proliferation, cell differentiation, and gene transcription, and involves a diversity of molecules including protein kinases and phosphatases, and second messenger molecules such as cyclic nucleotides, calcium-calmodulin, inositol and various mitogens that regulate protein phosphorylation. A distinctive class of signal transduction molecules are involved in odorant detection. The process of odorant detection involves specific recognition by odorant receptors. The olfactory mucosa also appears to possess an additional group of odorant-binding proteins which recognize and bind separate classes of odorants. For example, cDNA clones from rat have been isolated which correspond to mRNAs highly expressed in olfactory mucosa but not detected in other tissues. The proteins encoded by these clones are homologous to proteins that bind lipopolysaccharides or polychlorinated biphenyls, and the different proteins appear to be expressed in specific areas of the mucosal tissue. These proteins are believed to interact with odorants before or after specific recognition by odorant receptors, perhaps acting as selective signal filters (Dear, T. N. et al. (1991) EMBO J. 10:2813-2819; Vogt, R. G. et al (1991) J. Neurobiol. 22:74-84). Cells also respond to changing conditions by switching off signals. Many signal transduction proteins are short-lived and rapidly targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Cells also maintain mechanisms to monitor changes in the concentration of denatured or unfolded proteins in membrane-bound extracytoplasmic compartments, including a transmembrane receptor that monitors the concentration of available chaperone molecules in the endoplasmic reticulum and transmits a signal to the cytosol to activate the transcription of nuclear genes encoding chaperones in the endoplasmic reticulum. Certain proteins in intracellular signaling pathways serve to link or cluster other proteins involved in the signaling cascade. These proteins are referred to as scaffold, anchoring, or adaptor proteins. (For review, see Pawson, T. and J. D. Scott (1997) Science 278:2075-2080.) As many intracellular signaling proteins such as protein kinases and phosphatases have relatively broad substrate specificities, the adaptors help to organize the component signaling proteins into specific biochemical pathways. Many of the above signaling molecules are characterized by the presence of particular domains that promote protein-protein interactions. A sampling of these domains is discussed below, along with other important intracellular messengers. Intracellular Signaling Second Messenger Molecules Protein Phosphorylation Protein kinases and phosphatases play a key role in the intracellular signaling process by controlling the phosphorylation and activation of various signaling proteins. The high energy phosphate for this reaction is generally transferred from the adenosine triphosphate molecule (ATP) to a particular protein by a protein kinase and removed from that protein by a protein phosphatase. Protein kinases are roughly divided into two groups: those that phosphorylate serine or threonine residues (serine/threonine kinases, STK) and those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK). A few protein kinases have dual specificity for serine/threonine and tyrosine residues. Almost all kinases contain a conserved 250-300 amino acid catalytic domain containing specific residues and sequence motifs characteristic of the kinase family (Hardie, G. and S. Hanks (1995) The Protein Kinase Facts Books, Vol I:7-20, Academic Press, San Diego, Calif.). STKs include the second messenger dependent protein kinases such as the cyclic-AMP dependent protein kinases (PKA), involved in mediating hormone-induced cellular responses; calcium-calmodulin (CaM) dependent protein kinases, involved in regulation of smooth muscle contraction, glycogen breakdown, and neurotransmission; and the mitogen-activated protein kinases (MAP kinases) which mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades. Altered PKA expression is implicated in a variety of disorders and diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease (Isseroacher, K. J. et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New York, N.Y.; pp. 416-431, 1887). PTKs are divided into transmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs. Transmembrane PTKs are receptors for most growth factors. Non-receptor PTKs lack transmembrane regions and, instead, form complexes with the intracellular regions of cell surface receptors. Receptors that function through non-receptor PTKs include those for cytokines and hormones (growth hormone and prolactin) and antigen-specific receptors on T and B lymphocytes. Many of these PTKs were first identified as the products of mutant oncogenes in cancer cells in which their activation was no longer subject to normal cellular controls. In fact, about one third of the known oncogenes encode PTKs, and it is well known that cellular transformation (oncogenesis) is often accompanied by increased tyrosine phosphorylation activity (Charbonneau H. and N. K. Tonks (1992) Annu. Rev. Cell Biol. 8:463-493). An additional family of protein kinases previously thought to exist only in prokaryotes is the histidine protein kinase family (HPK). HPKs bear little homology with mammalian STKs or PTKs but have distinctive sequence motifs of their own (Davie, J. R et al. (1995) J. Biol. Chem. 270:19861-19867). A histidine residue in the N-terminal half of the molecule (region I) is an autophosphorylation site. Three additional motifs located in the C-terminal half of the molecule include an invariant asparagine residue in region II and two glycine-rich loops characteristic of nucleotide binding domains in regions III and IV. Recently a branched chain alpha-ketoacid dehydrogenase kinase has been found with characteristics of HPK in rat (Davie et al., supra). Protein phosphatases regulate the effects of protein kinases by removing phosphate groups from molecules previously activated by kinases. The two principal categories of protein phosphatases are the protein (serine/threonine) phosphatases (PPs) and the protein tyrosine phosphatases (PTPs). PPs dephosphorylate phosphoserine/threonine residues and are important regulators of many cAMP-mediated hormone responses (Cohen, P. (1989) Annu. Rev. Biochem 58:453-508). PTPs reverse the effects of protein tyrosine kinases and play a significant role in cell cycle and cell signaling processes (Charbonneau and Tonks, supra). As previously noted, many PTKs are encoded by oncogenes, and oncogenesis is often accompanied by increased tyrosine phosphorylation activity. It is therefore possible that PTPs may prevent or reverse cell transformation and the growth of various cancers by controlling the levels of tyrosine phosphorylation in cells. This hypothesis is supported by studies showing that overexpression of PTPs can suppress transformation in cells, and that specific inhibition of PTPs can enhance cell transformation (Charbonneau and Tonks, supra). Phospholipid and Inositol-Phosphate Signaling Inositol phospholipids (phosphoinositides) are involved in an intracellular signaling pathway that begins with binding of a signaling molecule to a G-protein linked receptor in the plasma membrane. This leads to the phosphorylation of phosphatidylinositol (PI) residues on the inner side of the plasma membrane to the biphosphate state (PIP 2 ) by inositol kinases. Simultaneously, the G-protein linked receptor binding stimulates a trimeric G-protein which in turn activates a phosphoinositide-specific phospholipase C-β. Phospholipase C-β then cleaves PIP 2 into two products, inositol triphosphate (IP 3 ) and diacylglycerol. These two products act as mediators for separate signaling events. IP 3 diffuses through the plasma membrane to induce calcium release from the endoplasmic reticulum (ER), while diacylglycerol remains in the membrane and helps activate protein kinase C, a serine-threonine kinase that phosphorylates selected proteins in the target cell. The calcium response initiated by IP 3 is terminated by the dephosphorylation of IP 3 by specific inositol phosphatases. Cellular responses that are mediated by this pathway are glycogen breakdown in the liver in response to vasopressin, smooth muscle contraction in response to acetylcholine, and thrombin-induced platelet aggregation. Inositol-phosphate signaling controls tubby, a membrane bound transcriptional regulator that serves as an intracellular messenger of Gα q -coupled receptors (Santagata et al (2001) Science 292:2041-2050). Members of the tubby family contain a C-terminal tubby domain of about 260 amino acids that binds to double-stranded DNA and an N-terminal transcriptional activation domain. Tubby binds to phosphatidylinositol 4,5-bisphosphate, which localizes tubby to the plasma membrane. Activation of the G-protein α q leads to activation of phospholipase C-β and hydrolysis of phosphoinositide. Loss of phosphatidylinositol 4,5-bisphosphate causes tubby to dissociate from the plasma membrane and to translocate to the nucleus where tubby regulates transcription of its target genes. Defects in the tubby gene are associated with obesity, retinal degeneration, and hearing loss (Boggon, T. J. et al. (1999) Science 286:2119-2125). Cyclic Nucleotide Signaling Cyclic nucleotides (cAMP and cGMP) function as intracellular second messengers to transduce a variety of extracellular signals including hormones, light, and neurotransmitters. In particular, cyclic-AMP dependent protein kinases (PKA) are thought to account for all of the effects of cAMP in most mammalian cells, including various hormone-induced cellular responses. Visual excitation and the phototransmission of light signals in the eye is controlled by cyclic-GMP regulated, Ca 2+ -specific channels. Because of the importance of cellular levels of cyclic nucleotides in mediating these various responses, regulating the synthesis and breakdown of cyclic nucleotides is an important matter. Thus adenylyl cyclase, which synthesizes cAMP from AMP, is activated to increase cAMP levels in muscle by binding of adrenaline to β-adrenergic receptors, while activation of guanylate cyclase and increased cGMP levels in photoreceptors leads to reopening of the Ca 2+ -specific channels and recovery of the dark state in the eye. There are nine known transmembrane isoforms of mammalian adenylyl cyclase, as well as a soluble form preferentially expressed in testis. Soluble adenylyl cyclase contains a P-loop, or nucleotide binding domain, and may be involved in male fertility (Buck, J. et al. (1999) Proc. Natl. Acad. Sci. USA 96:79-84). In contrast, hydrolysis of cyclic nucleotides by cAMP and cGMP-specific phosphodiesterases (PDEs) produces the opposite of these and other effects mediated by increased cyclic nucleotide levels. PDEs appear to be particularly important in the regulation of cyclic nucleotides, considering the diversity found in this family of proteins. At least seven families of mammalian PDEs (PDE1-7) have been identified based on substrate specificity and affinity, sensitivity to cofactors, and sensitivity to inhibitory drugs (Beavo, J. A. (1995) Physiol. Rev. 75:725-748). PDE inhibitors have been found to be particularly useful in treating various clinical disorders. Rolipram, a specific inhibitor of PDE4, has been used in the treatment of depression, and similar inhibitors are undergoing evaluation as anti-inflammatory agents. Theophylline is a nonspecific PDE inhibitor used in the treatment of bronchial asthma and other respiratory diseases (Banner, K. H. and C. P. Page (1995) Eur. Respir. J. 8:996-1000). Calcium Signaling Molecules Ca 2+ is another second messenger molecule that is even more widely used as an intracellular mediator than cAMP. Ca 2+ can enter the cytosol by two pathways, in response to extracellular signals. One pathway acts primarily in nerve signal transduction where Ca 2+ enters a nerve terminal through a voltage-gated Ca 2+ channel The second is a more ubiquitous pathway in which Ca 2+ is released from the ER into the cytosol in response to binding of an extracellular signaling molecule to a receptor. Ca 2+ directly activates regulatory enzymes, such as protein kinase C, which trigger signal transduction pathways. Ca 2+ also binds to specific Ca 2+ -binding proteins (CBPs) such as calmodulin (CaM) which then activate multiple target proteins in the cell including enzymes, membrane transport pumps, and ion channels. CaM interactions are involved in a multitude of cellular processes including, but not limited to, gene regulation, DNA synthesis, cell cycle progression, mitosis, cytokinesis, cytoskeletal organization, muscle contraction, signal transduction, ion homeostasis, exocytosis, and metabolic regulation (Celio, M. R. et al (1996) Guidebook to Calcium - binding Proteins, Oxford University Press, Oxford, UK, pp. 15-20). Some Ca 2+ binding proteins are characterized by the presence of one or more EF-hand Ca 2+ binding motifs, which are comprised of 12 amino acids flanked by α-helices (Celio, supra). The regulation of CBPs has implications for the control of a variety of disorders. Calcineurin, a CaM-regulated protein phosphatase, is a target for inhibition by the immunosuppressive agents cyclosporin and FK506. This indicates the importance of calcineurin and CaM in the immune response and immune disorders (Schwaninger M. et al. (1993) J. Biol Chem. 268:23111-23115). The level of CaM is increased several-fold in tumors and tumor-derived cell lines for various types of cancer (Rasmussen, C. D. and A. R. Means (1989) Trends Neurosci. 12:433-438). The annexins are a family of calcium-binding proteins that associate with the cell membrane (Towle, C. A. and B. V. Treadwell (1992) J. Biol. Chem. 267:5416-5423). Annexins reversibly bind to negatively charged phospholipids (phosphatidylcholine and phosphatidylserine) in a calcium dependent manner. Annexins participate in various processes pertaining to signal transduction at the plasma membrane, including membrane-cytoskeleton interactions, phospholipase inhibition, anticoagulation, and membrane fusion. Annexins contain four to eight repeated segments of about 60 residues. Each repeat folds into five alpha helices wound into a right-handed superhelix. G-Protein Signaling Guanine nucleotide binding proteins (G-proteins) are critical mediators of signal transduction between a particular class of extracellular receptors, the G-protein coupled receptors (GPCRs), and intracellular second messengers such as cAMP and Ca 2+ . G-proteins are linked to the cytosolic side of a GPCR such that activation of the GPCR by ligand binding stimulates binding of the G-protein to GTP, inducing an “active” state in the G-protein. In the active state, the G-protein acts as a signal to trigger other events in the cell such as the increase of cAMP levels or the release of Ca 2+ into the cytosol from the ER, which, in turn, regulate phosphorylation and activation of other intracellular proteins. Recycling of the G-protein to the inactive state involves hydrolysis of the bound GTP to GDP by a GTPase activity in the G-protein. (See Alberts, B. et al. (1994) Molecular Biology of the Cell Garland Publishing, Inc. New York, N.Y., pp.734-759.) The superfamily of G-proteins consists of several families which maybe grouped as translational factors, heterotrimeric G-proteins involved in transmembrane signaling processes, and low molecular weight (LMW) G-proteins including the proto-oncogene Ras proteins and products of rab, rap, rho, rac, smg21, smg25, YPT, SEC4, and ARF genes, and tubulins (Kaziro, Y. et al (1991) Annu. Rev. Biochem. 60:349-400). In all cases, the GTPase activity is regulated through interactions with other proteins. G protein activity is triggered by seven-transmembrane cell surface receptors (G-protein coupled receptors) which respond to lipid analogs, amino acids and their derivatives, peptides, cytokines, and specialized stimuli such as light, taste, and odor. Activation of the receptor by its stimulus causes the replacement of the G protein-bound GDP with GTP. Gα-GTP dissociates from the receptor/βγ complex, and each of these separated components can interact with and regulate downstream effectors. The signaling stops when Gα hydrolyzes its bound GTP to GDP and reassociates with the βγ complex (Neer, supra). Ras proteins are membrane-associated molecular switches that bind GTP and GDP and slowly hydrolyze GTP to GDP. This intrinsic GTase activity of ras is stimulated by a family of proteins collectively known as ‘GAP’ or GTPase-activating proteins. Since the GTP bound form of ras is active, ras-GAP proteins down-regulate ras. ras Gap is an alpha-helical domain that accelerates the GTPase activity of Ras, thereby “switching” it into an “off” position (Wittinghofer, A. et al. (1997) FEBS Lett. 410:63-67) Guanine nucleotide binding proteins (GTP-binding proteins) participate in a wide range of regulatory functions in all eukaryotic cells, including metabolism, cellular growth, differentiation, signal transduction, cytoskeletal organization, and intracellular vesicle transport and secretion. In higher organisms they are involved in signaling that regulates such processes as the immune response (Aussel, C. et al. (1988) J. Immunol. 140:215-220), apoptosis, differentiation, and cell proliferation including oncogenesis (Dhanasekaran, N. et al. (1998) Oncogene 17:1383-1394). Exchange of bound GDP for GTP followed by hydrolysis of GTP to GDP provides the energy that enables GTP-binding proteins to alter their conformation and interact with other cellular components. The superfamily of GTP-binding proteins consists of several families and may be grouped as translational factors, heterotrimeric GTP-binding proteins involved in transmembrane signaling processes (also called G-proteins), and low molecular weight (LMW) GTP-binding proteins including the proto-oncogene Ras proteins and products of rab, rap, rho, rac, smg21, smg25, YPT, SEC4, and ARF genes, and tubulins (Kaziro, Y. et al. (1991) Annu. Rev. Biochem. 60:349-400). In all cases, the GTPase activity is regulated through interactions with other proteins. The low molecular weight ([MW) GTP-binding proteins regulate cell growth, cell cycle control, protein secretion, and intracellular vesicle interaction. These GTP-binding proteins respond to extracellular signals from receptors and activating proteins by transducing mitogenic signals (Tavitian, A. (1995) C. R. Seances Soc. Biol Fil. 189:7-12). Low molecular weight GTP-binding proteins consist of single polypeptides of 21-30 kD which are able to bind to and hydrolyze GTP, thus cycling from an inactive to an active state. Low molecular weight GTP-binding proteins play critical roles in cellular protein trafficking events, such as the translocation of proteins and soluble complexes from the cytosol to the membrane through an exchange of GDP for GTP (Ktistakis, N. T. (1998) BioEssays 20:495-504). In vesicle transport, the interaction between vesicle- and target-specific identifiers (v-SNAREs and tSNAREs) docks the vesicle to the acceptor membrane. The budding process is regulated by GTPases such as the closely related ADP ribosylation factors (ARFs) and SAR proteins, while GTPases such as Rab allow assembly of SNARE complexes and may play a role in removal of defective complexes (Rothman, J. E. and F. T. Wieland (1996) Science 272:227-234). The rab proteins control the translocation of vesicles to and from membranes for protein localization, protein processing, and secretion. The rho GTP-binding proteins control signal transduction pathways that link growth factor receptors to actin polymerization which is necessary for normal cellular growth and division. The ran GTP-binding proteins are located in the nucleus of cells and have a key role in nuclear protein import, the control of DNA synthesis, and cell-cycle progression (Hall, A. (1990) Science 249:635-640; Scheffzek, K. et al. (1995) Nature 374:378-381). The cycling of LMW GTP-binding proteins between the GTP-bound active form and the GDP-bound inactive form is regulated by additional proteins. Guanosine nucleotide exchange factors (GEFs) increase the rate of nucleotide dissociation by several orders of magnitude, thus facilitating release of GDP and loading with GTP. Certain Ras-family proteins are also regulated by guanine nucleotide dissociation inhibitors (GDIs), which inhibit GDP dissociation. The intrinsic rate of GTP hydrolysis of the LMW GTP-binding proteins is typically very slow, but it can be stimulated by several orders of magnitude by GTPase-activating proteins (GAPs) (Geyer, M. and Wittinghofer, A. (1997) Curr. Opin. Struct. Biol. 7:786-792). Heterotrimeric G-proteins are composed of 3 subunits, α, β, and γ, which in their inactive conformation associate as a trimer at the inner face of the plasma membrane. Gα binds GDP or GTP and contains the GTPase activity. The βγ complex enhances binding of Gα to a receptor. Gγ is necessary for the folding and activity of Gβ (Neer, E. J. et al. (1994) Nature 371:297-300). Multiple homologs of each subunit have been identified in mammalian tissues, and different combinations of subunits have specific functions and tissue specificities (Spiegel A. M. (1997) J. Inher. Metab. Dis. 20:113-121). The β subunits, also known as G-β proteins or β transducins, contain seven tandem repeats of the WD-repeat sequence motif, a motif found in many proteins with regulatory functions. Mutations and variant expression of β transducin proteins are linked with various disorders (Neer, E. J. et al. (1994) Nature 371:297-300; Margottin, F. et al. (1998) Mol. Cell. 1:565-574). The alpha subunits of heterotrimeric G-proteins can be divided into four distinct classes. The α-s class is sensitive to ADP-ribosylation by pertussis toxin which uncouples the receptor:G-protein interaction. This uncoupling blocks signal transduction to receptors that decrease cAMP levels which normally regulate ion channels and activate phospholipases. The inhibitory α-I class is also susceptible to modification by pertussis toxin which prevents α-I from lowering cAMP levels. Two novel classes of α subunits refractory to pertussis toxin modification are α-q, which activates phospholipase C, and α-12, which has sequence homology with the Drosophila gene concertina and may contribute to the regulation of embryonic development (Simon, M. I. (1991) Science 252:802-808). The mammalian Gβ and Gγ subunits, each about 340 amino acids long, share more than 80% homology. The Gβ subunit (also called transducin) contains seven repeating units, each about 43 amino acids long. The activity of both subunits may be regulated by other proteins such as calmodulin and phosducin or the neural protein GAP 43 (Clapham, D. and E. Neer (1993) Nature 365:403-406). The β and γ subunits are tightly associated. The β subunit sequences are highly conserved between species, implying that they perform a fundamentally important role in the organization and function of G-protein linked systems (Van der Voorn, L. (1992) FEBS Lett. 307:131-134). They contain seven tandem repeats of the WD-repeat sequence motif, a motif found in many proteins with regulatory functions. WD-repeat proteins contain from four to eight copies of a loosely conserved repeat of approximately 40 amino acids which participates in protein-protein interactions. Mutations and variant expression of β transducin proteins are linked with various disorders. Mutations in LIS1, a subunit of the human platelet activating factor acetylhydrolase, cause Miller-Dieker lissencephaly. RACK1 binds activated protein kinase C, and RbAp48 binds retinoblastoma protein. CstF is required for polyadenylation of mammalian pre-mRNA in vitro and associates with subunits of cleavage-stimulating factor. Defects in the regulation of β-catenin contribute to the neoplastic transformation of human cells. The WD40 repeats of the human F-box protein bTrCP mediate binding to β-catenin, thus regulating the targeted degradation of β-catenin by ubiquitin ligase (Neer et al., supra; Hart, M. et al. (1999) Curr. Biol 9:207-210). The γ subunit primary structures are more variable than those of the β subunits. They are often post-translationally modified by isoprenylation and carboxyl-methylation of a cysteine residue four amino acids from the C-terminus; this appears to be necessary for the interaction of the βγ subunit with the membrane and with other G-proteins. The βγ subunit has been shown to modulate the activity of isoforms of adenylyl cyclase, phospholipase C, and some ion channels. It is involved in receptor phosphorylation via specific kinases, and has been implicated in the p21ras-dependent activation of the MAP kinase cascade and the recognition of specific receptors by G-proteins (Clapham and Neer, supra). G-proteins interact with a variety of effectors including adenylyl cyclase (Clapham and Neer, supra). The signaling pathway mediated by cAMP is mitogenic in hormone-dependent endocrine tissues such as adrenal cortex, thyroid, ovary, pituitary, and testes. Cancers in these tissues have been related to a mutationally activated form of a Gα s known as the gsp (Gs protein) oncogene (Dhanasekaran, N. et al. (1998) Oncogene 17:1383-1394). Another effector is phosducin, a retinal phosphoprotein, which forms a specific complex with retinal Gβ and Gγ (Gβγ) and modulates the ability of Gβγ to interact with retinal Gα (Clapham and Neer, supra). Irregularities in the G-protein signaling cascade may result in abnormal activation of leukocytes and lymphocytes, leading to the tissue damage and destruction seen in many inflammatory and autoimmune diseases such as rheumatoid arthritis, binary cirrhosis, hemolytic anemia, lupus erythematosus, and thyroiditis. Abnormal cell proliferation, including cyclic AMP stimulation of brain, thyroid, adrenal, and gonadal tissue proliferation is regulated by G proteins. Mutations in Gα subunits have been found in growth-hormone-secreting pituitary somatotroph tumors, hyperfunctioning thyroid adenomas, and ovarian and adrenal neoplasms (Meij, J. T. A. (1996) Mol. Cell Biochem. 157:31-38; Aussel, C. et al. (1988) J. Immunol. 140:215-220). LMW G-proteins are GTPases which regulate cell growth, cell cycle control, protein secretion, and intracellular vesicle interaction. They consist of single polypeptides which, like the alpha subunit of the heterotrimeric G-proteins, are able to bind to and hydrolyze GTP, thus cycling between an inactive and an active state. LMW G-proteins respond to extracellular signals from receptors and activating proteins by transducing mitogenic signals involved in various cell functions. The binding and hydrolysis of GTP regulates the response of LMW G-proteins and acts as an energy source during this process (Bokoch, G. M. and C. J. Der (1993) FASEB J. 7:750-759). At least sixty members of the 1MW G-protein superfamily have been identified and are currently grouped into the ras, rho, arf, sar1, ran, and rab subfamilies. Activated ras genes were initially found in human cancers, and subsequent studies confirmed that ras function is critical in determining whether cells continue to grow or become differentiated. Ras1 and Ras2 proteins stimulate adenylate cyclase (Kaziro et al., supra), affecting a broad array of cellular processes. Stimulation of cell surface receptors activates Ras which, in turn, activates cytoplasmic kinases. These kinases translocate to the nucleus and activate key transcription factors that control gene expression and protein synthesis (Barbacid, M. (1987) Annu. Rev. Biochem. 56:779-827; Treisman, R (1994) Curr. Opi Genet. Dev. 4:96-98). Other members of the 1MW G-protein superfamily have roles in signal transduction that vary with the function of the activated genes and the locations of the G-proteins that initiate the activity. Rho G-proteins control signal transduction pathways that link growth factor receptors to actin polymerization, which is necessary for normal cellular growth and division. The rab, arf, and sar1 families of proteins control the translocation of vesicles to and from membranes for protein processing, localization, and secretion. Vesicle- and target-specific identifiers (v-SNAREs and t-SNAREs) bind to each other and dock the vesicle to the acceptor membrane. The budding process is regulated by the closely related ADP ribosylation factors (ARFs) and SAR proteins, while rab proteins allow assembly of SNARE complexes and may play a role in removal of defective complexes (Rothman, J. and F. Wieland (1996) Science 272:227-234). Ran G-proteins are located in the nucleus of cells and have a key role in nuclear protein import, the control of DNA synthesis, and cell-cycle progression (Hall, A. (1990) Science 249:635-640; Barbacid, supra; Ktistakis, N. (1998) BioEssays 20:495-504; and Sasaki, T. and Y. Takai (1998) Biochem. Biophys. Res. Commun. 245:641-645). The function of Rab proteins in vesicular transport requires the cooperation of many other proteins. Specifically, the membrane-targeting process is assisted by a series of escort proteins (Khosravi-Far, R. et al. (1991) Proc. Natl. Acad. Sci. USA 88:6264-6268). In the medial Golgi, it has been shown that GTP-bound Rab proteins initiate the binding of VAMP-like proteins of the transport vesicle to syntaxin-like proteins on the acceptor membrane, which subsequently triggers a cascade of protein-binding and membrane-fusion events. After transport, GTPase-activating proteins (GAPs) in the target membrane are responsible for converting the GTP-bound Rab proteins to their GDP-bound state. And finally, guanine-nucleotide dissociation inhibitor (GDI) recruits the GDP-bound proteins to their membrane of origin. The cycling of LMW G-proteins between the GTP-bound active form and the GDP-bound inactive form is regulated by a variety of proteins. Guanosine nucleotide exchange factors (GEFs) increase the rate of nucleotide dissociation by several orders of magnitude, thus facilitating release of GDP and loading with GTP. The best characterized is the mammalian homolog of the Drosophila Son-of-Sevenless protein. Certain Ras-family proteins are also regulated by guanine nucleotide dissociation inhibitors (GDIs), which inhibit GDP dissociation The intrinsic rate of GTP hydrolysis of the LMW G-proteins is typically very slow, but it can be stimulated by several orders of magnitude by GTPase-activating proteins (GAPs) (Geyer, M. and A. Wittinghofer (1997) Curr. Opin. Struct. Biol 7:786-792). Both GEF and GAP activity may be controlled in response to extracellular stimuli and modulated by accessory proteins such as RalBP1 and POB1. Mutant Ras-family proteins, which bind but cannot hydrolyze GTP, are permanently activated, and cause cell proliferation or cancer, as do GEFs that inappropriately activate LMW G-proteins, such as the human oncogene NET1, a Rho-GEF (Drivas, G. T. et al. (1990) Mol. Cell Biol. 10:1793-1798; Alberts, A. S. and P Treisman (1998) EMBO J. 14:4075-4085). A member of the ARM family of G-proteins is centaurin beta 1A, a regulator of membrane traffic and the actin cytoskeleton. The centaurin β family of GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors contain pleckstrin homology (PH) domains which are activated by phosphoinositides. PH domains bind phosphoinositides, implicating PH domains in signaling processes. Phosphoinositides have a role in converting Arf-GTP to Arf-GDP via the centaurin β family and a role in Arf activation (Kam, J. L. et al. (2000) J. Biol. Chem. 275:9653-9663). The rho GAP family is also implicated in the regulation of actin polymerization at the plasma membrane and in several cellular processes. The gene ARHGAP6 encodes GTPase-activating protein 6 isoform 4. Mutations in ARHGAP6, seen as a deletion of a 500 kb critical region in Xp22.3, causes the syndrome microphthalmia with linear skin defects (MLS). MLS is an X-linked dominant, male-lethal syndrome (Prakash, S. K. et al. (2000) Hum. Mol. Genet. 9:477-488). Rab proteins have a highly variable amino terminus containing membrane-specific signal information and a prenylated carboxy terminus which determines the target membrane to which the Rab proteins anchor. More than 30 Rab proteins have been identified in a variety of species, and each has a characteristic intracellular location and distinct transport function. In particular, Rab1 and Rab2 are important in ER-to-Golgi transport; Rab3 transports secretory vesicles to the extracellular membrane; Rab5 is localized to endosomes and regulates the fusion of early endosomes into late endosomes; Rab6 is specific to the Golgi apparatus and regulates intra-Golgi transport events; Rab7 and Rab9 stimulate the fusion of late endosomes and Golgi vesicles with lysosomes, respectively; and Rab10 mediates vesicle fusion from the medial Golgi to the trans Golgi. Mutant forms of Rab proteins are able to block protein transport along a given pathway or alter the sizes of entire organelles. Therefore, Rabs play key regulatory roles in membrane trafficking (Schimmöler, I. S. and S. R. Pfeffer (1998) J. Biol. Chem. 243:22161-22164). The function of Rab proteins in vesicular transport requires the cooperation of many other proteins. Specifically, the membrane-targeting process is assisted by a series of escort proteins (Khosravi-Far, R et al. (1991) Proc. Natl Acad. Sci. USA 88:6264-6268). In the medial Golgi, it has been shown that GTP-bound Rab proteins initiate the binding of VAMP-like proteins of the transport vesicle to syntaxin-like proteins on the acceptor membrane, which subsequently triggers a cascade of protein-binding and membrane-fusion events. After transport, GTPase-activating proteins (GAPs) in the target membrane are responsible for converting the GTP-bound Rab proteins to their GDP-bound state. Finally, guanine-nucleotide dissociation inhibitor (GDI) recruites the GDP-bound proteins to their membrane of origin. Other regulators of G-protein signaling (RGS) also exist that act primarily by negatively regulating the G-protein pathway by an unknown mechanism (Druey, K. M. et al (1996) Nature 379:742-746). Some 15 members of the RGS family have been identified. RGS family members are related structurally through similarities in an approximately 120 amino acid region termed the RGS domain and functionally by their ability to inhibit the interleukin (cytokine) induction of MAP kinase in cultured mammalian 293T cells (Druey et al., supra). A member of the Rho family of G-proteins is CDC42, a regulator of cytoskeletal rearrangements required for cell division. CDC42 is inactivated by a specific GAP (CDC42GAP) that strongly stimulates the GTPase activity of CDC42 while having a much lesser effect on other Rho family members. CDC42GAP also contains an SH3-binding domain that interacts with the SH3 domains of cell signaling proteins such as p85 alpha and c-Src, suggesting that CDC42GAP may serve as a link between CDC42 and other cell signaling pathways (Barfod, E. T. et al. (1993) J. Biol. Chem. 268:26059-26062). The Dbl proteins are a family of GEFs for the Rho and Ras G-proteins (Whitehead, I. P. et al. (1997) Biochim. Biophys. Acta 1332:F1-F23). All Dbl family members contain a Dbl homology (DH) domain of approximately 180 amino acids, as well as a pleckstrin homology (PH) domain located immediately C-terminal to the DH domain. Most Dbl proteins have oncogenic activity, as demonstrated by the ability to transform various cell lines, consistent with roles as regulators of Rho-mediated oncogenic signaling pathways. The kalirin proteins are neuron-specific members of the Dbl family, which are located to distinct subcellular regions of cultured neurons (Johnson, R. C. (2000) J. Cell Biol. 275:19324-19333). Other regulators of G-protein signaling (RGS) also exist that act primarily by negatively regulating the G-protein pathway by an unknown mechanism (Druey, K. M. et al. (1996) Nature 379:742-746). Some 15 members of the RGS family have been identified. RGS family members are related structurally through similarities in an approximately 120 amino acid region termed the RGS domain and functionally by their ability to inhibit the interleukin (cytokine) induction of MAP kinase in cultured mammalian 293T cells (Druey et al., supra). The Immuno-associated nucleotide (IAN) family of proteins has GTP-binding activity as indicated by the conserved ATP/GTP-binding site P-loop motif. The IAN family includes IAN-1, IAN-4, IAP38, and IAG-1. IAN-1 is expressed in the immune system, specifically in T cells and thymocytes. Its expression is induced during thymic events (Poirier, G. M. C. et al. (1999) J. Immunol. 163:4960-4969). IAP38 is expressed in B cells and macrophages and its expression is induced in splenocytes by pathogens. IAG-1, which is a plant molecule, is induced upon bacterial infection (Krucken, J. et al. (1997) Biochem. Biophys. Res. Commun 230:167-170). IAN-4 is a mitochondrial membrane protein which is preferentially expressed in hematopoietic precursor 32D cells transfected with wild-type versus mutant forms of the bcr/abl oncogene. The bcr/abl oncogene is known to be associated with chronic myelogenous leukemia, a clonal myelo-proliferative disorder, which is due to the translocation between the bcr gene on chromosome 22 and the abl gene on chromosome 9. Bcr is the breakpoint cluster region gene and abl is the cellular homolog of the transforming gene of the Abelson murine leukemia virus. Therefore, the IAN family of proteins appears to play a role in cell survival in immune responses and cellular transformation (Daheron, L. et al. (2001) Nucleic Acids Res. 29:1308-1316). Formin-related genes (FRL) comprise a large family of morphoregulatory genes and have been shown to play important roles in morphogenesis, embryogenesis, cell polarity, cell migration, and cytokinesis through their interaction with Rho family small GTPases. Formin was first identified in mouse limb deformity (ld) mutants where the distal bones and digits of all limbs are fused and reduced in size. FRL contains formin homology domains FH1, FH2, and FEB. The FH1 domain has been shown to bind the Src homology 3 (SH3) domain, WWP/WW domains, and profilin. The FH2 domain is conserved and was shown to be essential for formin function as disruption at the FM2 domain results in the characteristic ld phenotype. The FIB domain is located at the N-terminus of FRL, and is required for associating with Rac, a Rho family GTPase (Yayoshi-Yamamoto, S. et al. (2000) Mol. Cell. Biol. 20:6872-6881). Signaling Complex Protein Domains PDZ domains were named for three proteins in which this domain was initially discovered. These proteins include PSD-95 (postsynaptic density 95), Dlg ( Drosophila lethal(1)discs large-1), and ZO-1 (zonula occludens-1). These proteins play important roles in neuronal synaptic transmission, tumor suppression, and cell junction formation, respectively. Since the discovery of these proteins, over sixty additional PDZ-containing proteins have been identified in diverse prokaryotic and eukaryotic organisms. This domain has been implicated in receptor and ion channel clustering and in the targeting of multiprotein signaling complexes to specialized functional regions of the cytosolic face of the plasma membrane. (or a review of PDZ domain-containing proteins, see Ponting, C. P. et al (1997) Bioessays 19:469-479.) A large proportion of PDZ domains are found in the eukaryotic MAGUK (membrane-associated guanylate kinase) protein family, members of which bind to the intracellular domains of receptors and channels. However, PDZ domains are also found in diverse membrane-localized proteins such as protein tyrosine phosphatases, serine/threonine kinases, G-protein cofactors, and synapse-associated proteins such as syntrophins and neuronal nitric oxide synthase (nNOS). Generally, about one to three PDZ domains are found in a given protein, although up to nine PDZ domains have been identified in a single protein The glutamate receptor interacting protein (GRIP) contains seven PDZ domains. GRIP is an adaptor that links certain glutamate receptors to other proteins and may be responsible for the clustering of these receptors at excitatory synapses in the brain (Dong, H. et al. (1997) Nature 386:279-284). The Drosophila scribble (SCRIB) protein contains both multiple PDZ domains and leucine-rich repeats. SCRIB is located at the epithelial septate junction, which is analogous to the vertebrate tight junction, at the boundary of the apical and basolateral cell surface. SCRIB is involved in the distribution of apical proteins and correct placement of adherens junctions to the basolateral cell surface (Bilder, D. and N. Perrimon (2000) Nature 403:676-680). The PX domain is an example of a domain specialized for promoting protein-protein interactions. The PX domain is found in sorting nexins and in a variety of other proteins, including the PhoX components of NADPH oxidase and the Cpk class of phosphatidylinositol 3-kinase. Most PX domains contain a polyproline motif which is characteristic of SH3 domain-binding proteins (Ponting, C. P. (1996) Protein Sci. 5:2353-2357). SI3 domain-mediated interactions involving the PhoX components of NADPH oxidase play a role in the formation of the NADPH oxidase multi-protein complex (Leto, T. L. et al. (1994) Proc. Natl. Acad. Sci. USA 91:10650-10654; Wilson, L. et al. (1997) Inflamm. Res. 46:265-271). The SH3 domain is defined by homology to a region of the proto-oncogene c-Src, a cytoplasmic protein tyrosine kinase. SH3 is a small domain of 50 to 60 amino acids that interacts with proline-rich ligands. SH3 domains are found in a variety of eukaryotic proteins involved in signal transduction, cell polarization, and membrane-cytoskeleton interactions. In some cases, SH3 domain-containing proteins interact directly with receptor tyrosine kinases. For example, the SLAP-130 protein is a substrate of the T-cell receptor (TCR) stimulated protein kinase. SLAP-130 interacts via its SH3 domain with the protein SLP-76 to affect the TCR-induced expression of interleukin-2 (Musci, M. A. et al. (1997) J. Biol. Chem. 272:11674-11677). Another recently identified SH3 domain protein is macrophage actin-associated tyrosine-phosphorylated protein (MAYP) which is phosphorylated during the response of macrophages to colony stimulating factor-1 (CSF-1) and is likely to play a role in regulating the CSF-1-induced reorganization of the actin cytoskeleton (Yeung, Y.-G. et al. (1998) J. Biol. Chem. 273:30638-30642). The structure of the SH3 domain is characterized by two antiparallel beta sheets packed against each other at right angles. This packing forms a hydrophobic pocket lined with residues that are highly conserved between different 53 domains. This pocket makes critical hydrophobic contacts with proline residues in the ligand (Feng, S. et al. (1994) Science 266:1241-1247). A novel domain, called the WW domain, resembles the SH3 domain in its ability to bind proline-rich ligands. This domain was originally discovered in dystrophin, a cytoskeletal protein with direct involvement in Duchenne muscular dystrophy (Bork, P. and M. Sudol (1994) Trends Biochem. Sci. 19:531-533). WW domains have since been discovered in a variety of intracellular signaling molecules involved in development, cell differentiation, and cell proliferation. The structure of the WW domain is composed of beta strands grouped around four conserved aromatic residues, generally tryptophan. Lie SH3, the SH2 domain is defined by homology to a region of c-Src. SH2 domains interact directly with phospho-tyrosine residues, thus providing an immediate mechanism for the regulation and transduction of receptor tyrosine kinase-mediated signaling pathways. For example, as many as ten distinct SH2 domains are capable of binding to phosphorylated tyrosine residues in the activated PDGF receptor, thereby providing a highly coordinated and finely tuned response to ligand-mediated receptor activation. (Reviewed in Schaffhausen, B. (1995) Biochim Biophys. Acta. 1242:61-75.) The BLNK protein is a linker protein involved in B cell activation, that bridges B cell receptor-associated kinases with SH2 domain effectors that link to various signaling pathways (Fu, C. et al. (1998) Immunity 9:93-103). The pleckstrin homology PH) domain was originally identified in pleckstrin, the predominant substrate for protein kinase C in platelets. Since its discovery, this domain has been identified in over 90 proteins involved in intracellular signaling or cytoskeletal organization. Proteins containing the pleckstrin homology domain include a variety of kinases, phospholipase-C isoforms, guanine nucleotide release factors, and GTPase activating proteins. For example, members of the FGD1 family contain both Rho-guanine nucleotide exchange factor (GEF) and PH domains, as well as a FYVE zinc finger domain. FGD1 is the gene responsible for faciogenital dysplasia, an inherited skeletal dysplasia (Pasteris, N. G. and J. L. Gorski (1999) Genomics 60:57-66). Many PH domain proteins function in association with the plasma membrane, and this association appears to be mediated by the PH domain itself. PH domains share a common structure composed of two antiparallel beta sheets flanked by an amphipathic alpha helix. Variable loops connecting the component beta strands generally occur within a positively charged environment and may function as ligand binding sites (Lemmon, M. A. et al. (1996) Cell 85:621-624). Ankrin (ANK) repeats mediate protein-protein interactions associated with diverse intracellular signaling functions. For example, ANK repeats are found in proteins involved in cell proliferation such as kinases, kinase inhibitors, tumor suppressors, and cell cycle control proteins. (See, for example, Kalus, W. et al (1997) FEBS Lett. 401:127-132; Ferrante, A. W. et al. (1995) Proc. Natl. Acad. Sci. USA 92:1911-1915.) These proteins generally contain multiple ANK repeats, each composed of about 33 amino acids. Myotrophin is an ANK repeat protein that plays a key role in the development of cardiac hypertrophy, a contributing factor to many heart diseases. Structural studies show that the myotrophin ANK repeats, like other ANK repeats, each form a helix-turn-helix core preceded by a protruding “tip.” These tips are of variable sequence and may play a role in protein-protein interactions. The helix-turn-helix region of the ANK repeats stack on top of one another and are stabilized by hydrophobic interactions (Yang, Y. et al. (1998) Structure 6:619-626). Members of the ASB protein family contain a suppressor of cytokine signaling (SOCS) domain as well as multiple ankyrin repeats (Hilton, D. J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:114-119). The tetratricopeptide repeat (TPR) is a 34 amino acid repeated motif found in organisms from bacteria to humans. TPRs are predicted to form ampipathic helices, and appear to mediate protein-protein interactions. TPR domains are found in CDC16, CDC23, and CDC27, members of the anaphase promoting complex which targets proteins for degradation at the onset of anaphase. Other processes involving TPR proteins include cell cycle control, transcription repression, stress response, and protein kinase inhibition (Lamb, J. R. et al. (1995) Trends Biochem. Sci. 20:257-259). The armadillo/beta-catenin repeat is a 42 amino acid motif which forms a superhelix of alpha helices when tandemly repeated. The structure of the armadillo repeat region from beta-catenin revealed a shallow groove of positive charge on one face of the superhelix, which is a potential binding surface. The armadillo repeats of beta-catenin, plakoglobin, and p120 cas bind the cytoplasmic domains of cadherins. Betaatenin/cadherin complexes are targets of regulatory signals that govern cell adhesion and mobility (Huber, A. H. et al. (1997) Cell 90:871-882). Eight tandem repeats of about 40 residues (WD-40 repeats), each containing a central Trp-Asp motif, make up beta-transducin (G-beta), which is one of the three subunits (alpha, beta, and gamma) of the guanine nucleotide-binding proteins (G proteins). In higher eukaryotes G-beta exists as a small multigene family of highly conserved proteins of about 340 amino acid residues. WD repeats are also found in other protein families. For example, betaTRCP is a component of the ubiquitin ligase complex, which recruits specific proteins, including beta-catenin, to the ubiquitin-proteasome degradation pathway. BetaTRCP and its isoforms all contain seven WD repeats, as well as a characteristic “F-box” motif. (Koike, J. et al (2000) Biochem. Biophys. Res. Commun. 269:103-109.) Signaling by Notch family receptors controls cell fate decisions during development (Frisen, J. and Lendabl, U. (2001) Bioessays 23:3-7). The Notch receptor signing pathway is involved in the morphogenesis and development of many organs and tissues in multicellular species. Notch receptors are large transmembrane proteins that contain extracellular regions made up of repeated EGF domains. Notchless was identified in a screen for molecules that modulate notch activity (Royet, J. et al. (1998) EMBO J. 17:7351-7360). Notchless, which contains nine WD40 repeats, binds to the cytoplasmic domain of Notch and inhibits Notch activity. Eps8 is a substrate for the intracellular epidermal growth factor receptors (EGR). Semaphorins are secreted, glycosylphosphatidylinositol (GPI) anchor and transmembrane glycoproteins. Semaphorins function as chemorepellants in various sensory and motor axons (Soker, S. (2001) Int. 3. Biochem. Cell Biol. 33:433437). Semaphorins constitute one type of ligand for the plexin receptor. Tumor necrosis factor receptor-associated factors (TRAFs) constitute a family of adaptor proteins that link the cytosolic domains of these receptors to downstream protein kinases or WD repeats are also found in other protein families. For example, betaTRCP is a component of the ubiquitin ligases. These proteins share a TRAP domain (TD), a distinctive region near the COOH terminus, that is responsible for mediating interactions between TRAFs and TNF receptors with other adaptor proteins and kinases. Expression Profiling Microarrays are analytical tools used in bioanalysis. A microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry. One area in particular in which microarrays find use is in gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder. Steroid Hormones Steroids are a class of lipid-soluble molecules, including cholesterol bile acids, vitamin D, and hormones, that share a common four-ring structure based on cyclopentanoperhydrophenanthrene and that carry out a wide variety of functions. Cholesterol, for example, is a component of cell membranes that controls membrane fluidity. It is also a precursor for bile acids which solubilize lipids and facilitate absorption in the small intestine during digestion. Vitamin D regulates the absorption of calcium in the small intestine and controls the concentration of calcium in plasma. Steroid hormones, produced by the adrenal cortex, ovaries, and testes, include glucocorticoids, mineralocorticoids, androgens, and estrogens. They control various biological processes by binding to intracellular receptors that regulate transcription of specific genes in the nucleus. Glucocorticoids, for example, increase blood glucose concentrations by regulation of gluconeogenesis in the liver, increase blood concentrations of fatty acids by promoting lipolysis in adipose tissues, modulate sensitivity to catcholamines in the central nervous system, and reduce inflammation. The principal mineralocorticoid, aldosterone, is produced by the adrenal cortex and acts on cells of the distal tubules of the kidney to enhance sodium ion reabsorption. Androgens, produced by the interstitial cells of Leydig in the testis, include the male sex hormone testosterone, which triggers changes at puberty, the production of sperm and maintenance of secondary sexual characteristics. Female sex hormones, estrogen and progesterone, are produced by the ovaries and also by the placenta and adrenal cortex of the fetus during pregnancy. Estrogen regulates female reproductive processes and secondary sexual characteristics. Progesterone regulates changes in the endometrium during the menstrual cycle and pregnancy. Steroid hormones are widely used for fertility control and in anti-inflammatory treatments for physical injuries and diseases such as arthritis, asthma, and auto-immune disorders. Progesterone, a naturally occurring progestin, is primarily used to treat amenorrhea, abnormal uterine bleeding, or as a contraceptive. Endogenous progesterone is responsible for inducing secretory activity in the endometrium of the estrogen-primed uterus in preparation for the implantation of a fertilized egg and for the maintenance of pregnancy. It is secreted from the corpus luteum in response to luteinizing hormone (LH). The primary contraceptive effect of exogenous progestins involves the suppression of the midcycle surge of LH. At the cellular level progestins diffuse freely into target cells and bind to the progesterone receptor. Target cells include the female reproductive tract, the mammary gland, the hypothalamus, and the pituitary. Once bound to the receptor, progestins slow the frequency of release of gonadotropin releasing hormone from the hypothalamus and blunt the pre-ovulatory LH surge, thereby preventing follicular maturation and ovulation Progesterone has minimal estrogenic and androgenic activity. Progesterone is metabolized hepatically to pregnanediol and conjugated with glucuronic acid. Medroxyprogesterone (MAH), also known as 6α-methyl-17-hydroxyprogesterone, is a synthetic progestin with a pharmacological activity about 15 times greater than progesterone. MAH is used for the treatment of renal and endometrial carcinomas, amenorrhea, abnormal uterine bleeding, and endometriosis associated with hormonal imbalance. MAH has a stimulatory effect on respiratory centers and has been used in cases of low blood oxygenation caused by sleep apnea, chronic obstructive pulmonary disease, or hypercapnia. Mifepristone, also known as RU-486, is an antiprogesterone drug that blocks receptors of progesterone. It counteracts the effects of progesterone, which is needed to sustain pregnancy. Mifepristone induces spontaneous abortion when administered in early pregnancy followed by treatment with the prostaglandin, misoprostol. Further, studies show that mifepristone at a substantially lower dose can be highly effective as a postcoital contraceptive when administered within five days after unprotected intercourse, thus providing women with a “morning-after pill” in case of contraceptive failure or sexual assault. Mifepristone also has potential uses in the treatment of breast and ovarian cancers in cases in which tumors are progesterone-dependent. It interferes with steroid-dependent growth of brain meningiomas, and may be useful in treatment of endometriosis where it blocks the estrogen-dependent growth of endometrial tissues. It may also be useful in treatment of uterine fibroid tumors and Cushing's Syndrome. Mifepristone binds to glucocorticoid receptors and interferes with cortisol binding. Mifepristone also may act as an anti-glucocorticoid and be effective for treating conditions where cortisol levels are elevated such as AIDS, anorexia nervosa, ulcers, diabetes, Parkinson's disease, multiple sclerosis, and Alzheimer's disease. Danazol is a synthetic steroid derived from ethinyl testosterone. Danazol indirectly reduces estrogen production by lowering pituitary synthesis of follicle-stimulating hormone and LH. Danazol also binds to sex hormone receptors in target tissues, thereby exhibting anabolic, antiestrognic, and weakly androgenic activity. Danazol does not possess any progestogenic activity, and does not suppress normal pituitary release of corticotropin or release of cortisol by the adrenal glands. Danazol is used in the treatment of endometriosis to relieve pain and inhibit endometrial cell growth. It is also used to treat fibrocystic breast disease and hereditary angioedema. Corticosteroids are used to relieve inflammation and to suppress the immune response. They inhibit eosinophil, basophil, and airway epithelial cell function by regulation of cytolines that mediate the inflammatory response. They inhibit leukocyte infiltration at the site of inflammation, interfere in the function of mediators of the inflammatory response, and suppress the humoral immune response. Corticosteroids are used to treat allergies, asthma, arthritis, and skin conditions. Beclomethasone is a synthetic glucocorticoid that is used to treat steroid-dependent asthma, to relieve symptoms associated with allergic or nonallergic (vasomotor) rhinitis, or to prevent recurrent nasal polyps following surgical removal The anti-inflammatory and vasoconstrictive effects of intranasal beclomethasone are 5000 times greater than those produced by hydrocortisone. Budesonide is a corticosteroid used to control symptoms associated with allergic rhinitis or asthma Budesonide has high topical anti-inflammatory activity but low systemic activity. Dexamethasone is a synthetic glucocorticoid used in anti-inflammatory or immunosuppressive compositions. It is also used in inhalants to prevent symptoms of asthma. Due to its greater ability to reach the central nervous system, dexamethasone is usually the treatment of choice to control cerebral edema. Dexamethasone is approximately 20-30 times more potent than hydrocortisone and 5-7 times more potent than prednisone. Prednisone is metabolized in the liver to its active form, prednisolone, a glucocorticoid with anti-inflammatory properties. Prednisone is approximately 4 times more potent than hydrocortisone and the duration of action of prednisone is intermediate between hydrocortisone and dexamethasone. Prednisone is used to treat allograft rejection, asthma, systemic lupus eryihematosus, arthritis, ulcerative colitis, and other inflammatory conditions. Betamethasone is a synthetic glucocorticoid with antiinflammatory and immunosuppressive activity and is used to treat psoriasis and fungal infections, such as athlete's foot and ringworm. The anti-inflammatory actions of corticosteroids are thought to involve phospholipase A 2 inhibitory proteins, collectively called lipocortins. Lipocortins, in turn, control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes by inhibiting the release of the precursor molecule arachidonic acid. Proposed mechanisms of action include decreased IgE synthesis, increased number of β-adrenergic receptors on leukocytes, and decreased arachidonic acid metabolism. During an immediate allergic reaction, such as in chronic bronchial asthma, allergens bridge the IgE antibodies on the surface of mast cells, which triggers these cells to release chemotactic substances. Mast cell influx and activation, therefore, is partially responsible for the inflammation and hyperirritability of the oral mucosa in asthmatic patients. This inflammation can be retarded by administration of corticosteroids. Immune Response Cells and Proteins Human peripheral blood mononuclear cells (PBMCs) contain B lymphocytes, T lymphocytes, NK cells, monocytes, dendritic cells and progenitor cells. Glucocorticoids are naturally occurring hormones that prevent or suppress inflammation and immune responses when administered at pharmacological doses. Unbound glucocorticoids readily cross cell membranes and bind with high affinity to specific cytoplasmic receptors. Subsequent to binding, transcription and protein synthesis are affected. The result can include inhibition of leukocyte infiltration at the site of inflammation, interference in the function of mediators of inflammatory response, and suppression of humoral immune responses. The anti-inflammatory actions of corticosteroids are thought to involve phospholipase A2 inhibitory proteins, collectively called lipocortins. Lipocortins, in turn, control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes by inhibiting the release of the precursor arachidonic molecule. Staphylococcal exotoxins specifically activate human T cells, expressing an appropriate TCR-Vbeta chain. Although polyclonal in nature, T cells activated by Staphylococcal exotoxins require antigen presenting cells (APCs) to present the exotoxin molecules to the T cells and deliver the costimulatory signals required for optimum T cell activation. Although Staphylococcal exotoxins must be presented to T cells by APCs, these molecules need not be processed by APC. Staphylococcal exotoxins directly bind to a non-polymorphic portion of the human MHC class II molecules, bypassing the need for capture, cleavage, and binding of the peptides to the polymorphic antigenic groove of the MHC class II molecules. Colon Cancer The potential application of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of cancers, such as colon cancer. Colon cancer evolves through a multi-step process whereby pre-malignant colonocytes undergo a relatively defined sequence of events leading to tumor formation. Several factors participate in the process of tumor progression and malignant transformation including genetic factors, mutations, and selection. To understand the nature of gene alterations in colorectal cancer, a number of studies have focused on the inherited syndromes. Familial adenomatous polyposis (FAP), is caused by mutations in the adenomatous polyposis coli gene (APC), resulting in truncated or inactive forms of the protein. This tumor suppressor gene has been mapped to chromosome 5q. Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations in mis-match repair genes. Although hereditary colon cancer syndromes occur in a small percentage of the population and most colorectal cancers are considered sporadic, knowledge from studies of the hereditary syndromes can be generally applied. For instance, somatic mutations in APC occur in at least 80% of sporadic colon tumors. APC mutations are thought to be the initiating event in the disease. Other mutations occur subsequently. Approximately 50% of colorectal cancers contain activating mutations in ras, while 85% contain inactivating mutations in p53. Changes in all of these genes lead to gene expression changes in colon cancer. There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of cell proliferative, endocrine, autoimmune/inflammatory, neurological, gastrointestinal, reproductive, developmental, and vesicle trafficking disorders. |
<SOH> SUMMARY OF THE INVENTION <EOH>Various embodiments of the invention provide purified polypeptides, intracellular signaling molecules, referred to collectively as ‘INTSIG’ and individually as ‘INTSIG-1,’ ‘INTSIG-2,’ ‘INTSIG-3,’ ‘INTSIG4,’ ‘INTSIG-5,’ ‘INTSIG-6,’ ‘INTSIG-7,’ ‘INTSIG-8,’ ‘INTSIG-9,’ ‘INTSIG-10,’ ‘INTSIG-11,’ ‘INTSIG-12,’ ‘INTSIG-13,’ ‘INTSIG-14,’ ‘INTSIG-15,’ ‘INTSIG-16,’ ‘INTSIG-17,’ ‘INTSIG-18,’ ‘INTSIG-19,’ ‘INTSIG-20,’ ‘INTSIG-21,’ ‘INTSIG-22,’ ‘INTSIG-23,’ ‘INTSIG-24,’ ‘INTSIG-25,’ ‘INTSIG-26,’ ‘INTSIG-27,’ ‘INTSIG-28,’ ‘INTSIG-29,’ ‘INTSIG-30,’ ‘INTSIG-31,’ ‘INTSIG-32,’ ‘INTSIG-33,’ ‘INTSIG-34,’ ‘INTSIG-35,’ ‘INTSIG-36,’ ‘INTSIG-37,’ ‘INTSIG-38,’ ‘INTSIG-39,’ ‘INTSIG-40,’ ‘INTSIG-41,’ ‘INTSIG-42,’ ‘INTSIG-43,’ INTSIG-44,’ and ‘INTSIG-45’ and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions. Embodiments also provide methods for utilizing the purified intracellular signaling molecules and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology. Related embodiments provide methods for utilizing the purified intracellular signaling molecules and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions. An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:1-45. Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:145, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-45. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:46-90. Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO:1-45. Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide. Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed. Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45. Still yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:46-90, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:46-90, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). In other embodiments, the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides. Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:46-90, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:46-90, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex. In a related embodiment, the method can include detecting the amount of the hybridization complex In still other embodiments, the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides. Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:46-90, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:46-90, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof. In a related embodiment the method can include detecting the amount of the amplified target polynucleotide or fragment thereof. Another embodiment provides a composition comprising an effective amount of a polypeptide elected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, and a pharmaceutically acceptable excipient. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:1-45. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional INTSIG, comprising administering to a patient in need of such treatment the composition Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional INTSIG, comprising administering to a patient in need of such treatment the composition. Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional INTSIG, comprising administering to a patient in need of such treatment the composition. Another embodiment provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide. Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-45. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide. Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:46-90, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:46-90, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:46-90, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:46-90, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:46-90, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. |
Mutated env gene, mutated env glycoprotein and the use thereof |
The invention relates to a mutated env gene which codes for a mutated envelope glycoprotein of virus HIV-1. In relation to the env gene of a reference or primary infection primary isolate, said gene presents at least two mutations at the glycosylation sites which are preserved from one primary isolate to another. Each mutation consists in the replacement of an AAC or AAT codon which codes for an asparagine with a CAG or CAA codon which codes for a glutamine, the two mutations at least being selected from the following: (a) at least two mutations in the part of the env gene that codes for region C3 of the env protein; (b) at least one mutation in the part that codes for region C3 and at least one mutation in the part that codes for region V3; (c) at least one mutation in the part that codes for region C2 and at least one mutation in the part that codes for region V3 chosen from the glycosylation sites at codons 862-864 and 970-972; (d) at least one mutation in the part that codes for region C2 selected from the glycosylation sites at codons 667-669, 679-681, 700-702,763-765, 844-846 and at least one mutation in the part that codes for region V3. Alternatively, the mutated env gene consists of any one of the following sequences: SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, or the complementary sequences thereof. The invention also relates to the mutated Env glycoprotein and the use of the gene and the glycoprotein in a pharmaceutical composition in order to produce antibodies and to evaluate a therapeutic agent. |
1. A mutated env gene encoding a mutated envelope glycoprotein of the HIV-1 virus, characterized in that said gene exhibits, compared to the env gene of a “reference” primary infection primary isolate, at least two mutations at the glycosylation sites conserved from one primary isolate to another, each mutation consisting of replacement of an AAC or AAT codon which encodes an asparagine with a CAG or CAA codon which encodes a glutamine, the two mutations at least being chosen from the following: (a) at least two mutations in the part of the env gene encoding the C3 region of the env protein, (b) at least one mutation in the part encoding the C3 region and at least one mutation in the part encoding the V3 region, (c) at least one mutation in the part encoding the C2 region and at least one mutation in the part encoding the V3 region chosen from the glycosylation sites at codons 862-864 and 970-972, (d) at least one mutation in the part encoding the C2 region chosen from the glycosylation sites at codons 667-669, 679-681, 700-702, 763-765 and 844-846, and at least one mutation in the part encoding the V3 region, it being possible for the position of at least any one of said codons to vary by three to twenty-four nucleotides, or the mutated env gene consists of any one of the sequences SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 or the sequences complementary thereto. 2. The gene as claimed in claim 1, characterized in that, according to the mutations (a), at least one mutation is effected at codons 976-978, 991-993 of the part encoding the C3 region and at least one mutation is effected at codons 1039-1041 or 1060-1062 of the part encoding the C3 region, it being possible for the position of at least any one of said codons to vary by three to twenty-four nucleotides. 3. The gene as claimed in claim 2, characterized in that its sequence consists of a sequence chosen from the sequences identified in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO:23, or the sequences complementary thereto. 4. The gene as claimed in claim 1, characterized in that, according to the mutations (b), at least one mutation is effected at codons 976-978, 991-993, 1.039-1041 or 1060-1062 of the part encoding the C3 region and at least one mutation is effected at codon 880-882 of the part encoding the V3 region, it being possible for the position of at least any one of said codons to vary by three to twenty-four nucleotides. 5. The gene as claimed in claim 4, characterized in that the mutations are effected at codons 976-978, 991-993 and 880-882, it being possible for the position of at least any one of said codons to vary by three to twenty-four nucleotides. 6. The gene as claimed in claim 5, characterized in that its sequence consists of a sequence chosen from the sequences identified in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or the sequences complementary thereto. 7. The gene as claimed in claim 4, characterized in that at least one mutation is effected at codon 976-978 or 991-993, at least one mutation is effected at codon 880-882, and at least one mutation is effected at codon 1039-1041 or 1060-1062, it being possible for the position of at least any one of said codons to vary by three to twenty-four nucleotides. 8. The gene as claimed in claim 7, characterized in that the mutations are effected at codons 976-978, 991-993, 880-882, 1039-1041 and 1060-1062. 9. The gene as claimed in claim 8, characterized in that its sequence consists of a sequence chosen from the sequences identified in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, or the sequences complementary thereto. 10. The gene as claimed in claim 1, characterized in that, according to the mutations (c), at least one mutation is effected at codon 805-807 of the part encoding the C2 region, it being possible for at least any one of said codons to vary by three to twenty-four nucleotides. 11. The gene as claimed in claim 1, characterized in that according to the mutations (d), at least one mutation is effected at codon 880-882 of the part encoding the V3 region, it being possible for the position of at least any one of said codons to vary by three to twenty-four nucleotides. 12. A mutated Env glycoprotein of the HIV-1 virus, characterized in that it exhibits, compared to a native Env protein of a “reference” primary infection primary isolate, at least two mutations at the glycosylation sites of said reference protein which are conserved from one primary isolate to another, each mutation consisting of replacement of an asparagine with a glutamine, the two mutations at least being chosen from the following: (a′) at least two mutations in the C3 region of the Env protein, (b′) at least one mutation in the C3 region and at least one mutation in the V3 region, (c′) at least one mutation in the C2 region and at least one mutation in the V3 region chosen from the glycosylation sites at amino acid 288 or 324, (d′) at least one mutation in the C2 region chosen from the glycosylation sites at any one of amino acids 223, 227, 234, 255 and 282, and at least one mutation in the V3 region, it being possible for the position of said glycosylation sites conserved from one primary infection primary isolate to another to vary by one to eight amino acids, or the mutated Env glycoprotein consists of any one of the sequences or the mutated env gene consists of any one of the sequences SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20. 13. The glycoprotein as claimed in claim 12, characterized in that, according to the mutations (a′), at least one mutation is effected at the glycosylation site at amino acid 326 or 331 and at least one mutation is effected at the glycosylation site at amino acid 347 or 354, it being possible for the position of said glycosylation sites conserved from one primary infection primary isolate to another to vary by one to eight amino acids. 14. The glycoprotein as claimed in claim 13, characterized in that its sequence consists of a sequence chosen from the sequences identified in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23. 15. The glycoprotein as claimed in claim 12, characterized in that, according to the mutations (b′), at least one mutation is effected at amino acid 326 or 331 and at least one mutation is effected at amino acid 294, it being possible for the position of at least any one of said glycosylation sites conserved from one primary infection primary isolate to another to vary by one to eight amino acids. 16. The glycoprotein as claimed in claim 15, characterized in that the mutations are effected at amino acids 326, 331 and 294, it being possible for the position of at least any one of said glycosylation sites conserved from one primary infection primary isolate to another to vary by one to eight amino acids. 17. The glycoprotein as claimed in claim 16, characterized in that its sequence consists of a sequence chosen from the sequences identified in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. 18. The glycoprotein as claimed in claim 12, characterized in that, according to the mutations (b′), at least one mutation is effected at amino acid 326 or 331, at least one mutation is effected at amino acid 347 or 354, and at least one mutation is effected at amino acid 294, it being possible for the position of at least any one of said glycosylation sites conserved from one primary infection primary isolate to another to vary by one to eight amino acids. 19. The glycoprotein as claimed in claim 18, characterized in that the mutations are effected at amino acids 326, 331, 294, 347 and 354, it being possible for the position of at least any one of said glycosylation sites conserved from one primary infection primary isolate to another to vary by one to eight amino acids. 20. The glycoprotein as claimed in claim 19, characterized in that its sequence consists of a sequence chosen from the sequences identified in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17. 21. The glycoprotein as claimed in claim 12, characterized in that, according to the mutations (c′), at least one mutation is effected at amino acid 269 of the C2 region, it being possible for the position of said glycosylation sites conserved from one primary infection primary isolate to another to vary by one to eight amino acids. 22. The glycoprotein as claimed in claim 12, characterized in that, according to the mutations (d′), at least one mutation is effected at amino acid 294 of the V3 region, it being possible for the position of said glycosylation sites conserved from one primary infection primary isolate to another to vary by one to eight amino acids. 23. A pharmaceutical composition comprising: (i) at least one mutated gene encoding a mutated envelope glycoprotein of the HIV-1 virus, said mutated gene being chosen from any one of the genes described in any one of claims 1 to 11 and being placed under the control of regulatory sequences which allow its expression in a host cell, a pharmaceutically acceptable vehicle, and optionally, an additional agent which facilitates the penetration of said mutated gene into said cell and/or which makes it possible to target said cell, or (ii) at least the gene identified in (i) cloned into a recombinant viral vector. 24. A pharmaceutical composition comprising: at least one mutated envelope glycoprotein of the HIV-1 virus, said mutated glycoprotein being chosen from any one of the glycoproteins described in any one of claims 12 to 22, a pharmaceutically acceptable vehicle and/or excipient and/or adjuvant and/or diluent. 25. A method for obtaining antibodies, according to which a mammalian animal, such as a mouse, rat or monkey, is immunized with at least one mutated gene encoding a mutated envelope glycoprotein of the HIV-1 virus, said mutated gene being chosen from any one of the genes described in any one of claims 1 to 11 and being placed under the control of regulatory sequences which allow its expression in vivo. 26. A method for obtaining antibodies, according to which a mammalian animal, such as a mouse, rat or monkey, is immunized with at least one mutated envelope glycoprotein of the HIV-1 virus, said mutated glycoprotein being chosen from any one of the glycoproteins described in any one of claims 12 to 22. 27. An antibody obtained according to the method of claim 25 or 26. 28. A pharmaceutical composition comprising at least one antibody as claimed in claim 27. 29. A method for evaluating a therapeutic agent, according to which at least one mutated gene encoding a mutated envelope glycoprotein of the HIV-1 virus as described in any one of claims 1 to 11 is administered to an animal, and the following are carried out: measurement of the titer of antibodies specific for the mutated glycoprotein, and/or measurement of the titer of neutralizing antibodies specific for the mutated or native glycoprotein, and/or measurement of the cellular immune response induced against the mutated glycoprotein, for example using an assay for the in vitro activation of T “helper” lymphocyte cells specific for the mutated glycoprotein. |
Project modelling and management tool |
The invention concerns a project management tool comprising a plurality of terminals communicating with a central server, at least one of the terminals including: means (11) for defining project models and for inserting them in a model library (6a), each project model including a list of step models, one step representing a set of tasks contributing to the achievement of the project; means (15) for defining a scenario defining links between a source model and a target model, each link being associated with a condition concerning the properties of the source model of the link; means (21) for generating projects from project models and steps from step models; and means (30) for updating and display the state of the project steps, and for generating a new step from the target model of a scenario link, when the condition associated with the link is fulfilled. |
1. Project management tool comprising a series of terminals (2, 3, 4) communicating with a central server (1), characterised in that at least one of the terminals comprises: means (11) to define project templates and to store them in a template library (6a), accessible through the central server (1), each project template comprising a list of stage templates, each stage of a project representing a set of collective works participating in the execution of the project and achieving an intermediate result, with a state associated with each stage; means (15) to define at least a scenario for a project template, which is stored in the template library (6a) in relation with the corresponding project template, each scenario defining succession links (52, 54) between two nodes (51, 53) respectively associated with two stage templates, that is one node (51) at the origin of the link and one node (53) at the destination of the link, each link being associated with a condition expressed as a function of properties of the stage template associated with the node at the origin of the link; means (21) to generate a project from a project template selected in the template library; each terminal comprising: means (21) to generate stages from stage templates; and means (30) of project follow-up allowing to update and visualize the state of project stages that have been previously generated by the means to generate stages, and of triggering the generation of a new stage according to a stage template associated with the node at the destination of a scenario link when a condition associated with that link is satisfied by the properties of a stage associated with the node at the origin of the link. 2. Project management tool according to claim 1, characterised in that each stage template, from at least a part of the stage templates, comprises a list of task templates defining tasks necessary to the completion of a stage generated from this stage template, the project management tool comprising means (21) to generate tasks from task templates and means (32) for updating and following up tasks previously generated. 3. Project management tool according to claim 2, characterised in that it comprises means (15) to define at least one scenario for a stage template, which is stored in the template library in relation with the corresponding stage template, each scenario defining succession links between two nodes respectively associated to task templates, that is one node at the origin of the link and one node at the destination of the link, each link being associated with a condition expressed as a function of the properties of the task template associated with the node at the origin of the link. 4. Project management tool according to claim 1, characterised in that it comprises means (11) to define sub-project templates of a project template or another sub-project template, and means (15) to define scenarios comprising succession links between two templates among project templates and sub-project templates, that is one template at the origin of the link and one template at the destination of the link. 5. Project management tool according to claim 1, characterised in that it comprises means to define a terminal node in a scenario, which node is associated with an origin node in another scenario, and means to trigger a scenario whose origin node is associated with a terminal node of another scenario in progress, when a condition related to a template associated with the terminal node is validated. 6. Project management tool according to claim 1, characterised in that it comprises a stage template and this stage template comprises the definition of at least one planned date or one effective date, a stage according to such stage template including at least a corresponding planned date or effective date, each link of a scenario being associated with an interval and a date chosen among the planned dates and effective dates of a stage defined by its stage template and corresponding to an anchor node of the link. 7. Project management tool according to claim 6, characterised in that the anchor node of a link is the origin node of this link. 8. Project management tool according to claim 1, characterised in that the means to define a scenario comprise: means to insert in a screen display panel a graphical symbol for a node representing a stage template, means to insert in said panel a graphical symbol for a link between two node symbols, and representing a scenario link between two stage templates, and means to insert in said panel a graphical symbol for a recursive link having for origin and destination a same node symbol, indicating that the stage represented by the node at the origin and destination of the link should be executed several times at regular intervals. 9. Project management tool according to claim 1, characterised in that it comprises means (16) to associate a data entry form to a template, which data is specific to the object generated from this template, and means to convert a form defined by a user in a standard form description language into a form that can be used by the project management tool and associated with this template and to the objects generated from this template. 10. Project management tool according to claim 1, characterised in that during the execution of a scenario, the project management tool comprises means to detect the change of a property of a stage associated with a scenario node, means to find the node associated with that stage and the outbound links of this node, means to obtain the condition associated with each outbound link found, means to validate each of these conditions, means to obtain the destination node of a link associated with a validated condition and the stage template associated with the destination node obtained, and means to create a stage from the stage template thus obtained. |
Sulfated fucogalactan digesting enzyme gene |
A gene encoding a polypeptide having an activity of digesting sulfated fucogalactan which is useful in sugar chain engineering reagents, analyzing the structures of sulfated fucose-containing polysaccharides and preparing degraded products of the polysaccharides; a genetic engineering process for producing the polypeptide; and the polypeptide obtained by the process. |
1. A polypeptide having an activity of degrading a sulfated fucogalactan, which is selected from the group consisting of: (a) a polypeptide containing the amino acid sequence of SEQ ID NO:28 or 30, or a portion thereof; (b) a polypeptide having an amino acid sequence in which at least one amino acid residue is deleted, added, inserted or substituted in the amino acid sequence of SEQ ID NO:28 or 30; and (c) a polypeptide having an amino acid sequence that has a homology of at least 30% to the amino acid sequence of SEQ ID NO:28 or 30. 2. A nucleic acid encoding a polypeptide having an activity of degrading a sulfated fucogalactan, which is selected from the group consisting of: (a) a nucleic acid encoding a polypeptide that contains the amino acid sequence of SEQ. ID NO:28 or 30, or a portion-thereof; (b) a nucleic acid encoding a polypeptide that has an amino acid sequence in which at least one amino acid residue is deleted, added, inserted or substituted in the amino acid sequence of SEQ ID NO:28 or 30; (c) a nucleic acid containing a nucleotide sequence of SEQ ID NO:27 or 29; (d) a nucleic acid consisting of a nucleotide sequence in which at least one nucleotide is deleted, added, inserted or substituted in the nucleotide sequence of SEQ ID NO:27 or 29; (e) a nucleic acid capable of hybridizing to any one of the nucleic acids of (a) to (d) or a complementary strand thereof under stringent conditions; and (f) a nucleic acid having a nucleotide sequence that has a homology of at least 50% to the nucleotide sequence of SEQ ID NO:27 or 29. 3. The nucleic acid according to claim 2, wherein the polypeptide has an activity of converting a sulfated fucogalactan into a smaller molecule to release at least one selected from the compounds of the formulas (I), (II), (III) and (IV): wherein R is H or SO3H. 4. A recombinant DNA containing the nucleic acid defined by claim 2. 5. An expression vector for a microorganism, an animal cell or a plant cell as a host cell into which the recombinant DNA defined by claim 4 is inserted. 6. A transformant transformed with the recombinant DNA defined by claim 4 or an expression vector for a microorganism, an animal cell or a plant cell as a host cell into which the recombinant DNA is inserted. 7. A method for producing a polypeptide having an activity of degrading a sulfated fucogalactan, the method comprising: culturing the transformant defined by claim 6; and collecting a polypeptide having an activity of degrading a sulfated fucogalactan from the culture. 8. A polypeptide having an activity of degrading a sulfated fucogalactan which is obtainable by culturing Escherichia coli BL21(DE3)/pEA101 (FERM BP-8149) or Escherichia coli BL21(DE3)/pEB101 (FERM BP-8150). 9. A smaller molecule from a sulfated fucogalactan which is obtainable by allowing a polypeptide having an activity of degrading a sulfated fucogalactan selected from the group consisting of the following to act on a sulfated fucogalactan: (a) a polypeptide containing the amino acid sequence of SEQ ID NO:28 or 30, or a portion thereof; (b) a polypeptide having an amino acid sequence in which at least one amino acid residue is deleted, added, inserted or substituted in the amino acid sequence of SEQ ID NO:28 or 30; (c) a polypeptide having an amino acid sequence that has a homology of at least 30% to the amino acid sequence of SEQ ID NO:28 or 30; and (d) a polypeptide obtainable by culturing Escherichia coli BL21(DE3)/pEA101 (FERM BP-8149) or Escherichia coli BL21(DE3)/pEB101 (FERM BP-8150). 10. A method for producing a smaller molecule from a sulfated fucogalactan, the method comprising allowing a polypeptide having an activity of degrading a sulfated fucogalactan selected from the group consisting of the following to act on a sulfated fucogalactan: (a) a polypeptide containing the amino acid sequence of SEQ ID NO:28 or 30, or a portion thereof; (b) a polypeptide having an amino acid sequence in which at least one amino acid residue is deleted, added, inserted or substituted in the amino acid sequence of SEQ ID NO:28 or 30; (c) a polypeptide having an amino acid sequence that has a homology of at least 30% to the amino acid sequence of SEQ ID NO:28 or 30; and (d) a polypeptide obtainable by culturing Escherichia coli BL21(DE3)/pEA101 (FERM BP-8149) or Escherichia coli BL21(DE3)/pEB101 (FERM BP-8150). 11. The method according to claim 10, wherein the polypeptide having an activity of degrading a sulfated fucogalactan is allowed to act on a deacetylated sulfated fucogalactan. 12. A method for screening for a gene encoding a polypeptide having an activity of degrading a sulfated fucogalactan, the method comprising screening, using the gene defined by claim 2 or a portion thereof as a probe, for a gene encoding a polypeptide having an activity of degrading a sulfated fucogalactan. 13. A method for analyzing a structure of a polysaccharide, the method comprising allowing a polypeptide having an activity of degrading a sulfated fucogalactan selected from the group consisting of the following to act on a sulfated fucogalactan: (a) a polypeptide containing the amino acid sequence of SEQ ID NO:28 or 30, or a portion thereof; (b) a polypeptide having an amino acid sequence in which at least one amino acid residue is deleted, added, inserted or substituted in the amino acid sequence of SEQ ID NO:28 or 30; (c) a polypeptide having an amino acid sequence that has a homology of at least 30% to the amino acid sequence of SEQ ID NO:28 or 30; and (d) a polypeptide obtainable by culturing Escherichia coli BL21(DE3)/pEA101 (FERM BP-8149) or Escherichia coli BL21(DE3)/pEB101 (FERM BP-8150). |
<SOH> BACKGROUND ART <EOH>Brown algae contain a variety of sulfated fucose-containing polysaccharides. For example, sulfated fucose-containing polysaccharides such as (1) sulfated fucans consisting of fucose and sulfate groups; (2) sulfated fucoglucuronomannans containing glucuronic acid, mannose, fucose and sulfate groups, e.g., the sulfated fucose-containing polysaccharide-U as described in WO 97/26896 (approximate molar ratio of constituting saccharides, fucose:mannose:galactose:uronic acid:sulfate group=10:7:4:5:20; hereinafter referred to as U-fucoidan); and (3) sulfated fucogalactan consisting of fucose and galactose, e.g., the sulfated fucose-containing polysaccharide-F as described in WO 97/26896 (approximate molar ratio of constituting saccharides, fucose:galactose=10:1; hereinafter referred to as F-fucoidan), or the sulfated fucose-containing polysaccharide-G as described in WO 00/50464 (approximate molar ratio of constituting saccharides, galactose:fucose=2:1; hereinafter referred to as G-fucoidan) are known. Almost all of these sulfated fucose-containing polysaccharides are macromolecular anions. Therefore, they behave in a chemically and physically similar manner in various purification steps, making it difficult to separate them from each other. For this reason, biological activities of sulfated fucose-containing polysaccharides derived from brown algae have often been examined without separating them from each other. Therefore, it was difficult to identify the sulfated fucose-containing polysaccharide that was responsible for the observed biological activity. To date, correlation between an activity and a molecule is known for the sulfated fucan fraction as described in Agricultural and Biological Chemistry, 44(8): 1965-1966 (1980), which is responsible for anticoagulant activity, and U-fucoidan as described in WO 97/26896, which is responsible for an apoptosis-inducing activity against tumor cells. The use of the sulfated fucan fraction as an anticoagulant in place of heparin has been examined. However, it is required to obtain a highly pure sulfated fucan in order to use it as a pharmaceutical avoiding side effects due to unexpected activities. Thus, a method therefor has been desired. Regarding U-fucoidan, it is similarly required to conveniently obtain a highly pure sulfated fucose-containing polysaccharide-U in order to prepare a pharmaceutical utilizing the apoptosis-inducing activity against tumor cells. Thus, a method therefor has been desired. Generally, enzymatic degradation is the most efficient manner utilized for structural analyses of polysaccharides and production of oligosaccharides. Furthermore, only one polysaccharide can be readily removed from a mixture of polysaccharides which are hardly separated from each other as follows. The polysaccharide to be removed is converted into a smaller molecule using an enzyme that specifically degrades the polysaccharide. The mixture is then subjected to molecular weight fractionation such as ultrafiltration. It has been reported that abalones, scallops, sea urchins, marine microorganisms and the like produce enzymes that degrade sulfated fucose-containing polysaccharides. However, only a trace amount of such an enzyme is generally contained in an organism. In addition, since such an organism has plural sulfated fucose-containing polysaccharide-degrading enzymes, various purification steps are required for obtaining a single enzyme. For example, the sulfated fucogalactan-degrading enzyme as described in WO 00/50464 was separated using its activity as an index, although it is unknown if it was isolated as a single protein. As described above, it is difficult to purify and collect a large amount of a single naturally occurring sulfated fucogalactan-degrading enzyme. Furthermore, it is necessary to add a sulfated fucogalactan or sulfated fucose-containing polysaccharides including a sulfated fucogalactan to a culture in order to obtain a sulfated fucogalactan-degrading enzyme from a marine microorganism. Thus, there are problems that the cultivation procedure is made complicated and the cost is made high. There are further problems as follows. If a sufficient amount of a naturally occurring sulfated fucogalactan-degrading enzyme protein cannot be obtained as described above, it is almost impossible to obtain information about the amino acid sequence or the nucleotide sequence for the enzyme. Information about the N-terminal amino acid sequence might not be obtained due to blocking of the enzyme protein at the N-terminus even if a sufficient amount could be obtained. In addition, there may be unexpected problems as follows. Even if a gene encoding such an enzyme could be obtained, the gene might not be expressed or the expression level might be low due to the incompatibility between the gene and the expression promoter or the host. Alternatively, a recombinant enzyme retaining an enzymatic activity might not be obtained due to formation of inclusion bodies. |
<SOH> SUMMARY OF INVENTION <EOH>The present inventors have intensively carried out researches on a gene from a microorganism producing a sulfated fucogalactan-degrading enzyme in order to reveal an amino acid sequence and a nucleotide sequence for a polypeptide having a sulfated fucogalactan-degrading activity contained in a brown alga. As a result, the present inventors have revealed the existence of at least two genes that encode polypeptides each having an activity of degrading a sulfated fucogalactan derived from a bacterium of the genus Flavobacterium , and determined the nucleotide sequences of the genes. The present inventors have also revealed the amino acid sequences of the polypeptides. Furthermore, the present inventors have successfully developed an industrially advantageous method for producing polypeptides each having an activity of degrading a sulfated fucogalactan using the genes. Thus, the present invention has been completed. The first aspect of the present invention relates to a polypeptide having an activity of degrading a sulfated fucogalactan, which is selected from the group consisting of: (a) a polypeptide containing the amino acid sequence of SEQ ID NO:28 or 30, or a portion thereof; (b) a polypeptide having an amino acid sequence in which at least one amino acid residue is deleted, added, inserted or substituted in the amino acid sequence of SEQ ID NO:28 or 30; and (c) a polypeptide having an amino acid sequence that has a homology of at least 30% to the amino acid sequence of SEQ ID NO:28 or 30. The second aspect of the present invention relates to a nucleic acid encoding a polypeptide having an activity of degrading a sulfated fucogalactan, which is selected from the group consisting of: (a) a nucleic acid encoding a polypeptide that contains the amino acid sequence of SEQ ID NO:28 or 30, or a portion thereof; (b) a nucleic acid encoding a polypeptide that has an amino acid sequence in which at least one amino acid residue is deleted, added, inserted or substituted in the amino acid sequence of SEQ ID NO:28 or 30; (c) a nucleic acid containing a nucleotide sequence of SEQ ID NO:27 or 29; (d) a nucleic acid consisting of a nucleotide sequence in which at least one nucleotide is deleted, added, inserted or substituted in the nucleotide sequence of SEQ ID NO:27 or 29; (e) a nucleic acid capable of hybridizing to any one of the nucleic acids of (a) to (d) or a complementary strand thereof under stringent conditions; and (f) a nucleic acid having a nucleotide sequence that has a homology of at least 50% to the nucleotide sequence of SEQ ID NO:27 or 29. According to the second aspect, a polypeptide having an activity of converting a sulfated fucogalactan into a smaller molecule to release at least one selected from the compounds of the formulas (I), (II), (III) and (IV) is provided: wherein R is H or SO 3 H. The third aspect of the present invention relates to a recombinant DNA containing the nucleic acid of the second aspect. The fourth aspect of the present invention relates to an expression vector for a microorganism, an animal cell or a plant cell as a host cell into which the recombinant DNA of the third aspect is inserted. The fifth aspect of the present invention relates to a transformant transformed with the recombinant DNA of the third aspect or the expression vector of the fourth aspect. The sixth aspect of the present invention relates to a method for producing a polypeptide having an activity of degrading a sulfated fucogalactan, the method comprising: culturing the transformant of the fifth aspect; and collecting a polypeptide having an activity of degrading a sulfated fucogalactan from the culture. The seventh aspect of the present invention relates to a polypeptide having an activity of degrading a sulfated fucogalactan which is obtainable by culturing Escherichia coli BL21(DE3)/pEA101 (FERM BP-8149) or Escherichia coli BL21(DE3)/pEB101 (FERM BP-8150). The eighth aspect of the present invention relates to a smaller molecule from a sulfated fucogalactan which is obtainable by allowing the polypeptide having an activity of degrading a sulfated fucogalactan of the first or seventh aspect to act on a sulfated fucogalactan. The ninth aspect of the present invention relates to a method for producing a smaller molecule from a sulfated fucogalactan, the method comprising allowing the polypeptide having an activity of degrading a sulfated fucogalactan of the first or seventh aspect to act on a sulfated fucogalactan. According to the ninth aspect, a method in which the polypeptide having an activity of degrading a sulfated fucogalactan is allowed to act on a deacetylated sulfated fucogalactan is provided. The tenth aspect of the present invention relates to a method for screening for a gene encoding a polypeptide having an activity of degrading a sulfated fucogalactan, the method comprising screening, using the gene of the second aspect or a portion thereof as a probe, for a gene encoding a polypeptide having an activity of degrading a sulfated fucogalactan. The eleventh aspect of the present invention relates to a method for analyzing a structure of a polysaccharide, the method comprising allowing the polypeptide having an activity of degrading a sulfated fucogalactan of the first or seventh aspect to act on a sulfated fucogalactan. detailed-description description="Detailed Description" end="lead"? |
Device for extracting gas or liquid from microfluidid through-flow systems |
The invention concerns a device which is used to separate gas or liquid from microfluidic flow-through systems. The gas or liquid separation is achieved independently of the spatial orientation of the device. In addition the invention concerns a microfluidic flow-through system in which a device according to the invention enables bubble-free fluid transport. |
1-22. (cancelled) 23. A microfluidic device for separating liquid and gaseous phases in a microfluidic flow-through systems which can be operated independently of its orientation comprising: a hollow body connected to the microfluidic flow-through system such that the liquid to be transported is conveyed through the hollow body; wherein the hollow body comprises an inlet opening and outlet opening for feeding in and discharging the liquid; wherein the inlet opening and the feed outlet opening are connected to a feed tube, wherein the feed tube extends into a hollow space of the hollow body; and wherein the feed tube at the inlet opening protrudes into the interior space of the hollow body such that it prevents a direct flow from the inlet opening to the outlet opening. 24. The microfluidic device of claim 23, wherein the hollow body is filled with liquid before being put into operation. 25. The microfluidic device of claim 1, wherein the hollow body is filled with gas before being put into operation. 26. The microfluidic device of claim 23, wherein the cross-section of the hollow body in relation to the cross-section of the inlet opening or the cross-section of the feed tube is of such a magnitude that the flow rate decreases when the interior space of the liquid flows into the interior space of the hollow body. 27. The microfluidic device of claim 23, wherein the separated phase remains in the hollow space of the body and displaces the flowing phase. 28. The microfluidic device of claim 23, wherein the hollow space of the hollow body has a high degree of symmetry. 29. The microfluidic device of claim 23, wherein the hollow space of the hollow body is spherical. 30. The microfluidic device of claim 23, wherein the feed tube for the outlet opening extends to the centre of the hollow space of the hollow body. 31. The microfluidic device of claim 23, wherein the inlet opening has no feed tube extending into the hollow space of the hollow body. 32. The microfluidic device of claim 23, wherein the outlet opening is arranged relative to the inlet opening such that an imaginary connection between the inlet opening, the outlet opening and centre of the hollow space of the hollow body forms a rectangular triangle. 33. The microfluidic device of claim 23, wherein the feed tubes are arranged on the sides of the imaginary triangle. 34. The microfluidic device of claim 23, wherein the outlet opening is next to the inlet opening. 35. The microfluidic device of claim 23, further comprising a screen that prevents a direct flow from the inlet opening to the outlet opening. 36. The microfluidic device of claim 23,further comprising a connecting system connected to the hollow body for conveying substances. 37. The microfluidic device of claim 36, wherein the connecting system comprises a first connecting piece at the inlet opening of the hollow body to pass fluid into the hollow space and a second connecting piece at the outlet opening of the hollow body to convey the fluid forwards whereby the separated gas remains in the hollow space of the hollow body. 38. The microfluidic device of claim 36, wherein the connecting system has a pump to control the flow rate of the liquid to the inlet opening of the hollow body. 39. The microfluidic device of claim 36, further comprising a liquid reservoir, wherein the liquid reservoir is connected to the connecting system. 40. The microfluidic device of claim 39, wherein the liquid reservoir is connected to the first connecting piece at the inlet opening. 41. The microfluidic device of claim 23, further comprising a microdialysis system downstream from the hollow body, such that liquid reaching the hollow body is substantially free from gases. 42. The microfluidic device of claim 23, further comprising a microdialysis probe, such that the liquid passes from the outlet opening to the probe. 43. A microfluidic device for separating liquid and gaseous phases in a microfluidic flow-through system which can be operated independently of its orientation comprising: a hollow body connected to the microfluidic flow-through system such that the liquid to be transported is conveyed through the hollow body; wherein the hollow body comprises an inlet opening and outlet opening for feeding in and discharging the liquid; wherein the inlet opening and the feed outlet opening are connected to a feed tube, wherein the feed tube extends into a hollow space of the hollow body; wherein the feed tube at the inlet opening protrudes into the interior space of the hollow body such that it prevents a direct flow from the inlet opening to the outlet opening; and a connecting system connected to the hollow body for conveying substances. 44. The microfluidic device of claim 43, wherein the connecting system comprises a first connecting piece at the inlet opening of the hollow body to pass fluid into the hollow space and a second connecting piece at the outlet opening of the hollow body to convey the fluid forwards whereby the separated gas remains in the hollow space of the hollow body. 45. The microfluidic device of claim 43, wherein the connecting system has a pump to control the flow rate of the liquid to the inlet opening of the hollow body. 46. The microfluidic device of claim 43, further comprising a liquid reservoir, wherein the liquid reservoir is connected to the connecting system. 47. The microfluidic device of claim 46, wherein the liquid reservoir is connected to the first connecting piece at the inlet opening of the hollow body. 48. The microfluidic device of claim 43, further comprising a microdialysis system downstream from the hollow body, such that liquid reaching the hollow body is substantially free from gases. 49. The microfluidic device of claim 43, further comprising a microdialysis probe, such that the liquid passes from the outlet opening to the probe. |
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