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1. A method for identifying a modulator of a Rhomboid polypeptide, which method comprises: (a) bringing into contact an Rhomboid polypeptide and a polypeptide substrate in the presence of a test compound; and (b) determining proteolytic cleavage of the polypeptide substrate. 2. A method according to claim 1 wherein the Rhomboid polypeptide has a sequence shown in Table 1. 3. A method according to claim 2 wherein the Rhomboid polypeptide is selected from the group consisting of Drosophila Rhomboid 1, Drosophila Rhomboid 2, Drosophila Rhomboid 3, Drosophila Rhomboid 4, Human RHBDL-1, Human RHBDL-2 and Human RHBDL-3, E. coli glgG, B. subtilis ypqP, P. stuartii A55862 gene product, P. aeruginosa B83259 gene product, S. cervisiae YGR101w and S. cervisiae YPL246c. 4. A method according any one of the preceding claims wherein the Rhomboid polypeptide comprises an ER (endoplasmic reticulum) retention signal. 5. A method according any one of the preceding claims wherein the polypeptide substrate is an EGFR ligand. 6. A method according to claim 5 wherein the polypeptide substrate has a sequence shown in Table 2. 7. A method according to any one of the preceding claims wherein polypeptide substrate comprises a detectable label. 8. A method according to any one of the preceding claims comprising identifying said test compound as a modulator of Rhomboid protease activity. 9. A method according to claim 8 further comprising determining the ability of said test compound to inhibit the infectivity of a microbial pathogen. 10. A method according to claim 8 or claim 9 comprising isolating said test compound. 11. A method according to claim 10 comprising formulating said test compound in a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier. 12. A modulator of Rhomboid protease activity obtained by a method of any one of claims 1 to 10. 13. A modulator according to claim 12 comprising a peptide fragment of a Rhomboid polypeptide. 14. An isolated nucleic acid encoding a Rhomboid polypeptide which comprises the amino acid sequence shown in FIG. 8, FIG. 11 or a fragment of one of these sequences which comprises the residues R152, G215, S217 and H281. 15. An isolated nucleic acid according to claim 14 comprising the nucleic acid sequence of FIG. 7 or FIG. 10 or a fragment thereof. 16. An isolated Rhomboid polypeptide encoded by a nucleic acid sequence according to claim 14 or claim 15. 17. An isolated Rhomboid polypeptide having greater than about 70% sequence identity with the amino acid sequence of Human RHBDL3 shown in FIG. 8 or Zebrafish RHBDL2 shown in FIG. 11. 18. An isolated nucleic acid encoding a Rhomboid polypeptide according to claim 17 and having greater than about 55% sequence identity with the nucleic acid sequence of Human RHBDL3 as shown in FIG. 7 or Zebrafish RHBDL shown in FIG. 10. 19. An isolated nucleic acid encoding a Rhomboid polypeptide according to claim 17 that hybridizes with the nucleic acid sequence shown in FIG. 7 or FIG. 10 under stringent conditions. 20. A Rhomboid polypeptide fragment consisting of 325 amino acids or less which proteolytically cleaves a polypeptide substrate. 21. A Rhomboid polypeptide fragment according to claim 20 wherein the polypeptide substrate is an EGFR ligand shown in Table 2. 22. A Rhomboid polypeptide fragment according to claim 20 or claim 21 comprising amino acid residues R152, G215, S217 and H291 of the Drosophila Rhomboid-1 sequence. 23. A Rhomboid polypeptide fragment according to claim 22 wherein the Rhomboid polypeptide is selected from the group consisting of Drosophila Rhomboid 1, Drosophila Rhomboid 2, Drosophila Rhomboid 3, Drosophila Rhomboid 4, Human RHBDL-1, Human RHBDL-2 and Human RHBDL-3, E. coli glgG, B. subtilis ypqP, P. stuartii A55862 gene product, P. aeruginosa 883259 gene product, S. cervisiae YGR101w and S. cervisiae YPL246c. 24. A Rhomboid polypeptide which proteolytically cleaves a polypeptide substrate and which comprises a heterogeneous endoplasmic reticulum retention signal. 25. A Rhomboid polypeptide according to claim 24 wherein the endoplasmic reticulum retention signal is KDEL. 26. A Rhomboid polypeptide according to claim 24 or claim 25 wherein the polypeptide substrate is an EGFR ligand shown in Table 2. 27. A Rhomboid polypeptide according to any one of claims 24 to 26 comprising amino acid residues R152, G215, S217 and H281 of the Drosophila Rhomboid-1 sequence. 28. A Rhomboid polypeptide according to claim 27 wherein the Rhomboid polypeptide is selected from the group consisting of Drosophila Rhomboid 1, Drosophila Rhomboid 2, Drosophila Rhomboid 3, Drosophila Rhomboid 4, Human RHBDL-1, Human RHBDL-2 and Human RHBDL-3, E. coli glgG, B. subtilis ypqp, P. stuartii A55862 gene product, P. aeruginosa B83259, gene product, S. cervisiae YGR101w and S. cervisiae YPL246c. 29. An isolated nucleic acid encoding a Rhomboid polypeptide according to any one of claims 24 to 28. 30. A recombinant vector comprising a nucleic acid according to any one of claims 14, 15, 18, 19 or 29. 31. A host cell comprising a recombinant vector according to claim 30. 32. A method of producing a Rhomboid polypeptide comprising: (a) causing expression from nucleic acid which encodes a Rhomboid polypeptide in a suitable expression system to produce the polypeptide recombinantly; (b) testing the recombinantly produced polypeptide for Rhomboid protease activity. 33. A method of obtaining a substrate for a Rhomboid polypeptide comprising, (a) providing a test polypeptide, (b) bringing into contact an Rhomboid polypeptide and the test polypeptide under conditions in which the Rhomboid polypeptide normally catalyses proteolytic cleavage of a substrate; and, (c) determining cleavage of the test polypeptide. 34. A method according to claim 33 wherein the test polypeptide comprises the amino acid sequence IASGA. 35. A method according to claim 34 wherein the test polypeptide comprises the amino acid sequence ASIASGA. 36. A method for proteolytically cleaving the transmembrane domain of a polypeptide comprising; contacting the polypeptide with a Rhomboid polypeptide; and, determining the proteolytic cleavage of said polypeptide by said Rhomboid polypeptide. 37. Use of a Rhomboid polypeptide for the proteolytic cleavage of the transmembrane domain of a polypeptide substrate. 38. A pharmaceutical composition comprising a polypeptide according to claim 16 or claim 17, a polypeptide fragment according to any one of claims 20 to 23 or a modulator according to claim 12 and a pharmaceutically acceptable excipient, vehicle or carrier. 39. Use of a polypeptide according to claim 16 or claim 17, a polypeptide fragment according to any one of claims 20 to 23 or a modulator according to claim 12 in the manufacture of a composition for treatment of a condition associated with aberrant ErbB or EGF receptor activity or a pathogen infection. 40. A method comprising administration of a composition according to claim 38 to a patient for treatment of a condition associated with aberrant ErbB or EGF receptor activity or a pathogen infection. 41. A method according to claim 40 wherein the condition associated with aberrant ErbB or EGF receptor activity is cancer, coronary atherosclerosis, psoriasis, wound healing, infant prematurity or a peripheral nerve injury/neuropathy. |
Cosmetic or dermatological preparations containing one or more ketohexoses |
Cosmetic or dermatological preparations with a content of one or more ketohexoses. |
1.-8. (canceled) 9. A cosmetic or dermatological composition, wherein the composition comprises at least one compound which is a ketohexose or a derivative thereof. 10. The composition of claim 9, wherein the ketohexose comprises a 2-ketohexose. 11. The composition of claim 10, wherein the 2-ketohexose comprises a D-ketohexose. 12. The composition of claim 9, wherein the composition comprises at least one of psicose, fructose, sorbose, tagatose and derivatives thereof. 13. The composition of claim 9, wherein the composition comprises at least one of psicose and polyacylated psicose. 14. The composition of claim 9, wherein the composition comprises at least one of sorbose and polyacylated sorbose. 15. The composition of claim 9, wherein the composition comprises at least one of tagatose, glucosyltagatose and polyacylated tagatose. 16. The composition of claim 9, wherein the composition comprises said at least one compound in a total amount of from 0.001% to 10% by weight. 17. The composition of claim 16, wherein the amount is from 0.01% to 1% by weight. 18. The composition of claim 16, wherein the amount is from 0.1% to 2.5% by weight. 19. The composition of claim 10, wherein the composition further comprises one or more antioxidants. 20. The composition of claim 19, wherein the one or more antioxidants are present in an amount of from 0.05% to 20% by weight. 21. The composition of claim 20, wherein the amount is from 1% to 10% by weight. 22. The composition of claim 10, wherein the composition further comprises at least one of a UV-A and a UV-B filter substance. 23. The composition of claim 22, wherein the UV-A and UV-B filter substances are present in a total amount of from 0.5% to 20% by weight. 24. The composition of claim 23, wherein the total amount is from 1% to 15% by weight. 25. The composition of claim 9, wherein the composition further comprises at least one pigment. 26. The composition of claim 25, wherein the at least one pigment comprises an inorganic pigment. 27. The composition of claim 25, wherein the at least one pigment comprises an organic pigment. 28. An emulsion which comprises the composition of claim 9. 29. A hydrodispersion which comprises the composition of claim 9. 30. A gel which comprises the composition of claim 9. 31. A solid stick which comprises the composition of claim 9. 32. An aerosol which comprises the composition of claim 9. 33. A cosmetic or dermatological composition, wherein the composition comprises one or more of psicose, sorbose, tagatose and derivatives thereof in a total amount of from 0.001% to 10% by weight. 34. The composition of claim 33, wherein the composition comprises tagatose in an amount of from 0.1% to 2.5% by weight. 35. The composition of claim 33, wherein the composition comprises psicose in an amount of from 0.1% to 2.5% by weight. 36. The composition of claim 33, wherein the composition comprises sorbose in an amount of from 0.1% to 2.5% by weight. 37. The composition of claim 33, wherein the composition further comprises at least one of an antioxidant, a UV-A filter substance, and a UV-B filter substance. 38. The composition of claim 37, wherein the composition comprises one or more antioxidants in an amount of from 1% to 10% by weight. 39. The composition of claim 37, wherein the composition comprises at least one of a UV-A and a UV-B filter substance in a total amount of from 1% to 15% by weight. 40. A method for at least one of the treatment and the prophylaxis of symptoms of skin aging, wherein the method comprises applying to the skin the composition of claim 9. 41. A method for at least one of the treatment and the prophylaxis of inflammatory skin conditions, wherein the method comprises applying to the skin the composition of claim 9. 42. A method for protecting sensitive skin, wherein the method comprises applying to the skin the composition of claim 9. 43. A method for at least one of the treatment and the prophylaxis of skin pigmentation disorders, wherein the method comprises applying to the skin the composition of claim 9. 44. A method for at least one of the treatment and the prophylaxis of harmful effects of UV radiation on skin, wherein the method comprises applying to the skin the composition of claim 9. 45. A method of increasing ceramide biosynthesis in skin, wherein the method comprises applying to the skin the composition of claim 9. 46. A method of enhancing the barrier function of skin, wherein the method comprises applying to the skin the composition of claim 9. 47. A composition which comprises one or more of D-psicose, D-sorbose, D-tagatose, D-fructose and derivatives thereof in a total amount of from 0.001% to 10% by weight, at least one inorganic pigment and at least one antioxidant. |
Modulation of cd200 receptors |
The present invention relates to CD200 receptor isoforms and modulators thereof and their use in methods of immune modulation and pharmaceutical compositions. |
1. An isolated CD200R2a nucleic acid molecule encoding a protein having the amino acid sequence shown in FIG. 4 (SEQ ID NO:2) or a homolog or analog thereof. 2. An isolated CD200R2a nucleic acid molecule wherein the nucleic acid sequence comprises: (a) a nucleic acid sequence as shown in FIG. 1 (SEQ ID NO:1), wherein T can also be U; (b) a nucleic acid sequence that is complimentary to a nucleic acid sequence of (a); (c) a nucleic acid sequence that has substantial sequence homology to a nucleic acid sequence of (a) or (b); (d) a nucleic acid sequence that is an analog of a nucleic acid sequence of (a), (b) or (c); or (e) a nucleic acid sequence that hybridizes to a nucleic acid sequence of (a), (b), (c) or (d) under stringent hybridization conditions. 3. An isolated CD200R2b nucleic acid molecule encoding a protein having the amino acid sequence shown in FIG. 5 (SEQ ID NO:4) or a homolog or analog thereof. 4. An isolated CD200R2b nucleic acid molecule wherein the nucleic acid sequence comprises: (a) a nucleic acid sequence as shown in FIG. 2 (SEQ ID NO:3), wherein T can also be U; (b) a nucleic acid sequence that is complimentary to a nucleic acid sequence of (a); (c) a nucleic acid sequence that has substantial sequence homology to a nucleic acid sequence of (a) or (b); (d) a nucleic acid sequence that is an analog of a nucleic acid sequence of (a), (b) or (c); or (e) a nucleic acid sequence that hybridizes to a nucleic acid sequence of (a), (b), (c) or (d) under stringent hybridization conditions. 5. An isolated CD200R3a nucleic acid molecule encoding a protein having the amino acid sequence shown in FIG. 6 (SEQ ID NO:6) or a homolog or analog thereof. 6. An isolated CD200R3a nucleic acid molecule wherein the nucleic acid sequence comprises: (a) a nucleic acid sequence as shown in FIG. 3 (SEQ ID NO:5), wherein T can also be U; (b) a nucleic acid sequence that is complimentary to a nucleic acid sequence of (a); (c) a nucleic acid sequence that has substantial sequence homology to a nucleic acid sequence of (a) or (b); (d) a nucleic acid sequence that is an analog of a nucleic acid sequence of (a), (b) or (c); or (e) a nucleic acid sequence that hybridizes to a nucleic acid sequence of (a), (b), (c) or (d) under stringent hybridization conditions. 7. An isolated CD200R2a protein having an amino acid sequence shown in FIG. 4 (SEQ ID NO:2) or an analog, homolog or fragment thereof. 8. An isolated CD200R2b protein having an amino acid sequence shown in FIG. 5 (SEQ ID NO:4) or an analog, homolog or fragment thereof. 9. An isolated CD200R3a protein having an amino acid sequence shown in FIG. 6 (SEQ ID NO:6) or an analog, homolog or fragment thereof. 10. An antibody that binds to an isolated protein according to claim 7. 11. An antibody according to claim 10 which is a monoclonal antibody. 12. (canceled) 13. (canceled) 14. A pharmaceutical composition for use in modulating an immune response comprising an effective amount of a protein according to claim 7 in admixture with a suitable diluent or carrier. 15-35. (canceled) 36. A method of preparing suppressive antigen presenting cells comprising culturing a starting cell population in the presence of an effective amount of CD200 receptor agonist. 37. A method according to claim 36 wherein the agonist is an antibody that crosslinks a CD200 receptor. 38. A method according to claim 36 wherein the antigen presenting cell is a dendritic cell. 39. A method according to claim 36 wherein the starting cell population contains bone marrow cells. 40. (canceled) 41. A method of preparing suppressive T cells comprising culturing a cell population containing T cells or precursors thereof in the presence of suppressive antigen presenting cells prepared according to the method of claim 36. 42. (canceled) 43. A method of identifying substances which bind with a CD200 receptor, comprising the steps of: (a) reacting the CD200 receptor and a test substance, under conditions which allow for formation of a complex, and (b) assaying for complexes of the CD200 receptor and the test substance, for free substance, and for non-complexed CD200 receptor, wherein the presence of complexes indicates that the test substance is capable of binding the CD200 receptor. 44. A method according to claim 43 wherein the CD200 receptor is an isolated CD200R2a nucleic acid molecule encoding a protein having the amino acid sequence shown in FIG. 4 (SEQ ID NO:2) or a homolog or analog thereof. 45. A method for identifying a compound that modulates a CD200 receptor comprising: (a) incubating a test compound with a CD200 receptor protein or a nucleic acid encoding a CD200 receptor protein; and (b) determining an amount of CD200 receptor protein activity or expression and comparing with a control, wherein a change in the CD200 receptor protein activity or expression as compared to the control indicates that the test compound modulates a CD200 receptor. 46. A method of identifying a CD200 receptor agonist comprising the steps of: (a) incubating a test substance with a CD200 receptor; and (b) determining whether or not the test substance stimulates the CD200 receptor. 47. A method of identifying a CD200 receptor antagonist comprising the steps of: (a) incubating a test substance with a CD200 receptor; and (b) determining whether or not the test substance inhibits the CD200 receptor. 48. A method according to claim 45 wherein the CD200 receptor is an isolated CD200R2a nucleic acid molecule encoding a protein having the amino acid sequence shown in FIG. 4 (SEQ ID NO:2) or a homolog or analog thereof. 49. A method according to claim 45 wherein the CD200 receptor is expressed on the surface of a cell. 50. A CD200 receptor modulator identified according to the method of claim 45. 51. A method of preparing a pharmaceutical composition for use in modulating an immune response comprising mixing a modulator of a CD200 receptor identified according to the method of claim 45 with a suitable diluent or carrier. 52. A method of modulating an immune response comprising administering an effective amount of a protein according to claim 7 to an animal in need thereof. 53. A method according to claim 52 comprising modulating an immune response involved in graft rejection, fetal loss, autoimmunity, allergy, inflammatory conditions, skin conditions or cancer. 54. A method of suppressing an immune response comprising administering an effective amount of a CD200 receptor agonist to an animal in need thereof. 55. A method according to claim 54 wherein said agonist is an antibody, small molecule, peptide mimetic or peptide. 56. A method according to claim 54 wherein the agonist is an antibody that crosslinks a CD200 receptor. 57. A method according to claim 56 wherein the antibody is a whole immunoglobulin that binds to a CD200 receptor. 58. A method according to claim 56 wherein said antibody binds to a CD200R3a receptor. 59. A method according to claim 58 wherein the antibody binds to a CD200R3a receptor which has the sequence shown in FIG. 6 (SEQ ID NO:6) or a homolog or analog thereof. 60. A method according to claim 54 for preventing or treating transplant rejection, fetal loss, allergy, inflammatory conditions or skin conditions. 61. A method according to claim 54 for preventing or treating an autoimmune disease. 62. A method according to claim 61 for preventing or treating arthritis. 63. A method according to claim 61 for preventing or treating diabetes. 64. A method according to claim 54 wherein said agonist is administered with an additional immune suppressant. 65. A method according to claim 64 wherein said immune suppressant is selected from CD200 or fragment thereof, immune suppressive cytokines and/or anti-inflammatory agents. 66. A method of inhibiting immune suppression comprising administering an effective amount of CD200 receptor antagonist to an animal in need thereof. 67. A method according to claim 66 wherein said antagonist is an antibody, antibody fragment, small molecule, peptide mimetic, peptide or an antisense oligonucleotide. 68. A method according to claim 66 wherein the antagonist is an antibody that binds to the CD200 receptor but does not cause activation thereof. 69. A method according to claim 68 wherein the antibody is an F(ab′)2 or Fab fragment. 70. A method according to claim 69 wherein said antibody binds to a CD200R3a receptor. 71. A method according to claim 70 wherein the antibody binds to a CD200R3a receptor which has the sequence shown in FIG. 6 (SEQ ID NO:6) or a homolog or analog thereof. 72. A method according to claim 66 for the treatment or prevention of viral, bacterial or fungal infections or cancer. 73. A method according to claim 66 wherein said antagonist is administered with an immune stimulant. 74. A method according to claim 73 wherein said immune stimulant is a cytokine. 75. A method of suppressing an immune response comprising administering an effective amount of a population of suppressive antigen presenting cells prepared according to the method of claim 36 to an animal in need thereof. 76. A method of suppressing an immune response comprising administering an effective amount of a population of suppressive T cells prepared according to the method of claim 41 to an animal in need thereof. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The immune system protects the body from infectious agents and disease and is critical to our survival. However, in certain instances, the immune system can be the cause of illness. One example is in autoimmune disease wherein the immune system attacks its own host tissues, in many instances causing debilitating illness and sometimes resulting in death. Examples of autoimmune diseases include multiple sclerosis, type 1 insulin-dependent diabetes mellitus, lupus erythematosus and arthritis. A second example where the immune system can cause illness is during tissue or organ transplantation. Except in the cases of genetically identical animals, such as monozygotic twins, tissue and organ transplants are rejected by the recipient's immune system as foreign. The immune reaction against transplants is even more pronounced in transplantation across species or xenotransplantation. A third example where the immune system harms the host is during an allergic reaction where the immune system is activated by a generally innocuous antigen causing inflammation and in some cases tissue damage. A fourth example where the immune system is involved is in fetal loss. In order to inhibit the detrimental immune reactions during transplantation, autoimmune disease and allergic reactions, immunosuppressive drugs (such as cyclosporin A, tacrolimas, and corticosteroids) or antibody therapies (such as anti-T cell antibodies) are generally administered. Unfortunately, these non-specific modes of immunosuppression generally have undesirable side effects. For example, cyclosporin may cause decreased renal function, hypertension, toxicity and it must be administered for the life of the patient. Corticosteroids may cause decreased resistance to infection, painful arthritis, osteoporosis and cataracts. The anti-T cell antibodies may cause fever, hypertension, diarrhea or sterile meningitis and are quite expensive. In view of the problems associated with immunosuppression, there has been an interest in developing methods or therapies that induce unresponsiveness or tolerance in the host to a transplant, to “self” tissues in autoimmune disease and to harmless antigens associated with allergies. The inventors have been studying the mechanisms involved in transplant rejection and have developed methods for inducing a state of antigen-specific immunological tolerance in transplantation. In particular, in animal allograft models, the inventors have demonstrated that graft survival, such as renal and skin allograft survival, can be increased if the recipient animal is given a pre-transplant infusion via the portal vein of irradiated spleen cells from the donor animal (2,3). In contrast, a pre-transplant infusion via the tail vein does not prolong graft survival. Increased graft survival was further shown to be in turn associated with increased expression of a number of distinct mRNAs (4), one of which encodes CD200 (previously called OX2), a molecule expressed on the surface of dendritic cells (5). The CD200 protein (also known as OX2) has a high degree of homology to molecules of the immunoglobulin gene family, which includes molecules important in lymphocyte antigen recognition and cell-cell interaction (e.g. CD4, CD8, ICAMs, VCAMs), as well as adhesion receptor molecules (NCAMs) in the nervous system. However, prior to the present inventors, the function of the CD200 protein was largely unknown. The present inventors showed subsequently that infusion of anti-CD200 monoclonal antibodies from the time of transplantation blocks the protective effect of pv immunization in mice receiving renal allografts (4) and rats receiving SIT (6), and the polarization to type-2 cytokine production seen in these models (4, 6). A soluble immunoadhesin, in which the extracellular domain of CD200 was linked to a murine IgG2aFc region, inhibited T-cell allostimulation and type-1 cytokine production (IL-2, IFNγ) in vitro and in vivo (1). Since the intracellular domain of CD200 lacks signalling motifs, or any docking sites for adapter molecules which might engage an intracellular signalling cascade, the present inventors suggest that these and other data (4,7,25) are consistent with the idea that engagement of a receptor for CD200 (CD200R) by CD200 may deliver key immunoregulatory signals (36). T-cells are activated after concomitant engagement of TCRs with antigen presented on APC in association with MHC molecules and the delivery of costimulatory signals resulting from the interaction of several ligand:coreceptor complexes (8-11). Major positive costimulatory interactions include the following: CD40L with CD40, and CD28 with CD80/CD86; CTLA4 interactions with CD80/CD86 may deliver a negative signal (12-17). While positive costimulatory signals are clearly important in T-cell triggering, blocking this costimulation alone, and/or facilitating signalling via CTLA4, has not reproducibly induced tolerance. This may reflect the need for other molecules (such as CD200) in active immunoregulation (4). In recent studies the inventors reported that dendritic cells (DC) expressing CD200, triggered an immunoregulatory function leading to increased allograft survival. Moreover, these cells were physically distinguishable from those DC with optimal allostimulatory capacity (7). Early attempts to characterize CD200R by Preston et al. (18) were performed by constructing a soluble chimeric protein with the extracellular domains of CD200 engineered onto domains 3+4 of rat CD4 antigen. In order to detect weak interactions, the chimeric protein was coupled to fluorescent covaspheres to ensure an avid display of CD200. These CD200 covaspheres were reported to bind to macrophages but not other cell types. The specificity of the interaction was documented by inhibition studies using Fab fragments of the CD200 monoclonal antibody (mAb). Using site-directed mutagenesis this group further reported results suggesting that the ligand-binding domain of CD200 was in the NH 2 -terminal domain of the extracellular region of CD200. Recently, Barclay et al. reported several forms of the CD200R (WO 00/70045, published Nov. 23, 2000). |
<SOH> SUMMARY OF THE INVENTION <EOH>The present inventors have studied the CD200 receptor (CD200R) and revealed evidence for a family of CD200Rs. The present inventors have now determined the full length sequence for three isoforms of the murine CD200 receptor called CD200R2a, CD200R2b and CD200R3a. Accordingly, in one aspect, the present invention provides an isolated CD200R2a, CD200R2b or CD200R3a or a homolog or analog thereof. In one embodiment, the present invention provides an isolated CD200R2a having the nucleic acid shown in FIG. 1 (SEQ ID NO:1) or a homolog or analog thereof. In another embodiment, the present invention provides an isolated CD200R2b having the nucleic acid sequence found in FIG. 2 (SEQ ID NO:3) or a homolog or analog thereof. In a further embodiment, the present invention provides an isolated CD200R3a having the nucleic acid shown in FIG. 3 (SEQ ID NO:5) or a homolog or analog thereof. In another aspect, the invention includes a method of immune modulation comprising administering an effective amount of an CD200R2a, CD200R2b or CD200R3a molecule to a cell or animal in need thereof. In one embodiment, the CD200 receptor may be co-administered with a CD200 peptide or a nucleic acid sequence coding for a CD200 peptide. Preferably, a CD200 peptide is administered and more preferably, the CD200 peptide is a soluble fusion protein, such as CD200:Fc. The inventors have also prepared antibodies to the different isoforms of CD200R. Accordingly, the present invention includes an antibody that binds to CD200R2a, CD200R2b or CD200R3a. The present inventors have also shown that administering cross-linking antibodies to a CD200 receptor enhances immune suppression as seen by prolonged graft survival and the prevention of autoimmune disease. Accordingly, the present invention provides a method of suppressing an immune response comprising administering an effective amount of a CD200 receptor agonist to a cell or animal in need thereof. In one embodiment, the agonist is an antibody that crosslinks a CD200 receptor such as a whole anti-CD200 receptor Ig. Accordingly, the present invention provides a method of suppressing an immune response comprising administering an effective amount of an antibody that crosslinks a CD200 receptor to a cell or animal in need thereof. In a specific embodiment, the antibody binds a CD200R selected from CD200R2a, CD200R2b or CD200R3a. The inventors have also shown that administering antibody fragments (e.g. Fab or F(ab′) 2 fragments) that bind to a CD200 receptor inhibits the immune suppression caused by CD200. Accordingly, in another aspect, the present invention provides a method of inhibiting immune suppression by administering an effective amount of a CD200 receptor antagonist to a cell or animal in need thereof. Preferably, the antagonist is an agent that inhibits the interaction of the CD200 receptor with CD200. An agent that inhibits the interaction of the CD200 receptor and CD200 may be an antibody that binds to the CD200 receptor. Accordingly, the invention includes a method of inhibiting immune suppression comprising administering an effective amount of an antibody that binds to an CD200 receptor to a cell or animal in need thereof. The antibody is preferably an antibody fragment such as an F(ab′) 2 or Fab fragment. In yet another aspect, the present invention includes screening methods for identifying substances which are capable of modulating CD200 receptors. In particular, the methods may be used to identify substances which are capable of binding to and augmenting or attenuating the effects of CD200 or the CD200 receptors (i.e. agonists). Alternatively, the methods may be used to identify substances which are capable of binding to CD200 receptor and which inhibit the effects of CD200 or a CD200 receptor (i.e. antagonists). Accordingly, the invention provides a method of identifying substances which bind with a CD200 receptor, comprising the steps of: (a) reacting the CD200 receptor and a test substance, under conditions which allow for formation of a complex, and (b) assaying for complexes of the CD200 receptor and the test substance, for free substance, and for non-complexed CD200 receptor, wherein the presence of complexes indicates that the test substance is capable of binding the CD200 receptor. In a specific embodiment the CD200R is selected from CD200R2a, CD200R2b or CD200R3a. The invention also includes screening assays for identifying agonists or antagonists of a CD200R comprising the steps of: (a) incubating a test substance with a CD200R; and (b) determining whether the test substance activates or inhibits the function of the CD200R. In another embodiment, agonists and/or antagonists of the binding of CD200 to its receptor can be identified. Therefore the invention also contemplates a method for assaying for an agonist or antagonist of the binding of CD200 with its receptor or other CD200 ligands such as antibodies to CD200. The agonist or antagonist may be an endogenous physiological substance or it may be a natural or synthetic substance. Accordingly the invention provides a method for identifying an antagonist or agonist of CD200 binding comprising the steps of: (a) reacting CD200, a known binding target, preferably an CD200 receptor, and a potential antagonist or agonist; and (b) determining the amount of CD200 bound to the binding target and comparing this with a control in the absence of the antagonist or agonist. The present invention also includes the pharmaceutical compositions comprising any of the above molecules that modulate CD200 reeptors for use in immune modulation. The pharmaceutical compositions can further comprise an CD200 peptide, preferably CD200:Fc, or nucleic acid encoding a CD200 peptide. The pharmaceutical compositions can further comprise a suitable diluent or carrier. Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. |
Retinoic acid metabolizing cytochrome p450 |
The present invention provides a novel retinoic acid metabolizing cytochrome P450, P450RAI-3, that is predominantly expressed in the adrenal gland. Methods for and uses of the new polynucleotide, polypeptide, fragments thereof and modulators thereof, include the treatment of cancer. |
1-72. (Cancelled) 73. An isolated nucleic acid molecule comprising a polynucleotide comprising a sequence selected from the group consisting of: (a) SEQ. ID. NO. 10 or encoding the amino acid sequence of SEQ. ID. NO. 11; (b) a polynucleotide of (a) wherein T can also be U: (c) a polynucleotide having a nucleic acid sequence which differs from any of the nucleic acid molecules of (a) to (c) in codon due to the degeneracy of the genetic code; and (d) a polynucleotide that is a variant, allelic variant or encodes a species homologue of any one of the polynucleotides of (a) to (c). 74. An isolated nucleic acid molecule comprising a fragment of the nucleic acid molecule of claim 73 and which will hybridize thereto under stringent hybridization conditions wherein the polynucleotide is at least 15 bases and does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues. 75. An isolated nucleic acid molecule of claim 74, wherein the polynucleotide fragment comprises a nucleotide sequence selected from SEQ. ID NOS. 12, 14, 16, 18, 20 and 22 or encoding the amino acid sequence selected from SEQ. ID. NOS. 13, 15, 17, 19, 21 and 23 or variant thereof. 76. An isolated nucleic acid molecule having a nucleic acid sequence complementary to the nucleic acid molecule of claim 73. 77. An isolated nucleic acid molecule of claim 73 encoding a cytochrome P450 retinoid metabolizing protein, comprising SEQ. ID. NO. 10 or a nucleic acid molecule encoding the amino acid sequence of SEQ. ID. NO. 11 or a variant thereof or biologically active fragment thereof. 78. The isolated nucleic acid molecule of claim 77, wherein the retinoid is all trans retinoic acid(ATRA) or 9-cis-retinoic acid. 79. An isolated nucleic acid molecule of that is 90% homologous to that of claim 77. 80. The isolated nucleic acid molecule of claim 73, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus. 81. A recombinant vector comprising an isolated nucleic acid molecule of claim 73. 82. A recombinant host cell comprising an isolated nucleic acid molecule of claim 73. 83. The recombinant host cell of claim 82 wherein the isolated nucleic acid molecule is operatively linked to a regulatory sequence to allow expression of a peptide encoded by said nucleic acid sequence. 84. An isolated polypeptide comprising an amino acid sequence at least 95% homologous to a sequence selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence of SEQ. ID. NO. 11; (b) a polypeptide encoded by any of the polynucleotides selected from the group consisting of: i. a polynucleotide encoding a polypeptide comprising amino acid sequence of SEQ. ID. NO. 13, 15, 17, 19, or 21; ii. a polynucleotide consisting of the nucleotide sequence of SEQ. ID. NO. 10: iii. a polynucleotide of SEQ. ID. NO. 10 wherein T can also be U; iv. a polynucleotide that is a variant, allelic variant or encodes a species homologue of any one of the polynucleotides of (i) to (iii). (c) a mature form, variant, allelic variant or species homologue to any of the polypeptides of (a) or (b); (d) fragment of any of the polypeptides of (a) to (c). 85. The isolated polypeptide of claim 84 that is a cytochrome P450 retinoid metabolizing polypeptide comprising amino acid sequence, SEQ. ID. NO. 11, a variant thereof or a biologically active fragment thereof. 86. The isolated polypeptide of claim 85, wherein the retinoid is all-trans retinoic acid or 9-cis retinoic acid. 87. The isolated polypeptide of claim 85 consisting of the amino acid sequence of SEQ. ID. NO. 11 (FIG. 3). 88. An isolated antibody that binds specifically to the isolated polypeptide of claim 84 or immunogenic or antigenic portion thereof. 89. A microsome comprising the isolated polypeptide of claim 84. 90. A method of making an isolated polypeptide of claim 84 comprising: (e) culturing a recombinant host cell comprising a nucleic acid molecule encoding said polypeptide operatively linked to a regulatory sequence to enable expression of said polypeptide, under conditions such that said polypeptide is expressed; and (f) recovering said polypeptide. 91. A pharmaceutical composition comprising the isolated polypeptide of claim 85, and/or optionally a modulator of P450RAI-3 activity in combination with a pharmaceutically acceptable carrier. 92. The pharmaceutical composition of claim 91, further comprising an adjuvant. 93. A method for preventing, treating or ameliorating a medical condition related to P450RAI-3 expression which comprises administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim 85 or a polynucleotide encoding for said polypeptide and/or a modulator of P450RAI-3. 94. The method of claim 93, wherein the modulator of P450RAI-3 is an inhibitor and the inhibitor is administered with a P450RAI-3 substrate. 95. The method of claim 94, wherein the substrate is all trans retinoic acid and/or 9-cis retinoic acid. 96. A method of diagnosis of a P450RAI-3-related condition or a predisposition to a P450RAI-3-related condition comprising detecting a polymorphism in a P450RAI-3 gene, wherein detection of said polymorphism is indicative of the occurrence of said condition or a predisposition thereto. 97. The method of claim 96 wherein the condition is related to vitamin A or retinoic acid metabolism. 98. A diagnostic Kit for identification of polymorphisms in the P450RAI-3 gene, comprising a nucleic acid molecule of claim 73 or a nucleic acid molecule complimentary thereto, and optionally directions for the method comprising screening the P450RAI-3 gene from a human for polymorphisms, wherein detection of said polymorphisms is indicative of the occurrence of a P450RAI-3-related condition or a predisposition thereto. 99. A method of treating a disease or condition related to vitamin A or retinoic acid metabolism in a patient comprising administering to the patient in need thereof, an effective amount of a therapeutically effective polypeptide of claim 85 and/or an agonist or antagonist thereof. 100. A method of identifying modulators of P450RAI-3 activity in a biological assay, wherein the method comprises: [a]expressing P450RAI-3 in a cell or obtaining P450RAI-3 in a microsome or in vitro: [b] adding a substrate under conditions that enable P450RAI-3 activity; and [c] detecting activity of P450RAI-3 on said substrate in the presence or absence of a modulator. 101. The method of claim 100, wherein the substrate is ATRA and/or 9-cis RA. 102. The method of claim 101, wherein the substrate comprises radio-labeled ATRA or 9-cis-RA. 103. A method of determining the ATRA and/or 9-cis-RA metabolizing activity of a polypeptide of claim 84, comprising: expressing the polypeptide in a host cell, adding ATRA and/or 9-cis-RA to the cell, and determining the amount anchor rate of ATRA and/or 9-cis-RA metabolism. 104. The method of claim 103, wherein the ATRA comprises radio-labeled ATRA and the 9-cis-RA comprises radio-labeled 9-cis-RA. 105. The method of claim 103, wherein the amount and/or rate of ATRA and 9-cis-RA metabolism is determined by measuring the amount and/or rate of production of hydoxy-metabolites and oxo-metabolites. 106. The method of claim 103, wherein prior to the step adding ATRA and/or 9-cis-RA to the cell, a candidate activator or inhibitor of the polypeptide is added to the cell. 107. A method of identifying the substrate of a polypeptide of claim 85, comprising: expressing the polypeptide in a host cell, adding a candidate substrate, and determining if the substrate is metabolized, wherein metabolization indicates that the candidate substrate is a substrate of the polypeptide. 108. The method of claim 107, wherein a plurality of candidate substrates are added to the cell to determine the amount and rate of metabolism of each substrate. 109. The method of claim 108, wherein, prior to the step adding the candidate substrates to the cell, a candidate activator or inhibitor of the polypeptide is added to the cell. 110. A method of determining the binding activity of a substrate to a polypeptide of claims 85, comprising expressing the polypeptide in a host cell, adding a candidate substrate, and determining a Kd value. 111. The method of claim 110, wherein prior to the step adding the substrate to the cell, a candidate activator or inhibitor of the polypeptide is added to the cell. 112. A method for conducting a drug discovery and/or a pharmaceutical business; (k) providing one or more assay systems for identifying agents by their ability to modulate P450RAI-3 activity or expression or retinoic acid metabolism; (l) conducting therapeutic profiling of agents identified in step (a), or further analogs thereof, for efficacy and toxicity in animals; and (m)formulating a pharmaceutical preparation including one or more agents identified in step (b) as having an acceptable therapeutic profile. 113. The method of claim 112, including a step of establishing a distribution system for distributing the pharmaceutical preparation for sale. 114. The method of claim 113, including establishing a sales group for marketing the pharmaceutical preparation. 115. A method of conducting a target discovery business comprising. i. providing one or more assay systems for identifying agents by their ability to modulate P450RAI-3 and/or retinoic acid metabolism; ii. (optionally) conducting therapeutic profiling of agents identified in step (a) for efficacy and toxicity in animals; and iii. licensing, to a third party, the rights for further drug development and/or sales for agents identified in step (a), or analogs thereof. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Cytochrome P450s The cytochromes P450 comprise a large gene superfamily that encodes over 500 distinct heme-thiolate proteins that catalyze the oxidation of drugs and numerous other compounds in the body [Nelson et al., (1996); Guengerich (1991)]. Since there are at least 500 different cytochrome P450 enzymes, it is of considerable interest in the pharmaceutical and other fields to identify which of these enzymes are most important in the metabolism of individual compounds. There are now numerous examples of adverse drug-drug interactions and side effects that can now be understood in terms of the cytochrome P450 enzymes. P450 proteins are ubiquitous in living organisms, and have been identified in bacteria, yeast, plants and animals [Nelson et al (1996); and Nelson, (1999a)]. The P450 enzymes catalyze the metabolism of a wide variety of drugs, xenobiotics, carcinogens, mutagens, and pesticides, and are responsible for the bioactivation of numerous endogenous compounds including steroids, prostaglandins, bile acids and fatty acids body [Nelson et al., (1996); Guengerich (1991); Nebert et al., (1989)]. Cytochrome P450 metabolism of xenobiotics can result in detoxification of toxic compounds by their conjugation into excretable forms or can result in activation of compounds into metabolites that are toxic, mutagenic, or carcinogenic. Many steroids are deactivated by cytochrome P450-catalyzed oxidation. Microsomal cytochromes occur on the membrane of the ER and require NADPH cytochrome P450 reductase and a flavoprotein for activity, whereas mitochondrial cytochromes occur on the inner mitochondrial membrane and require ferredoxin and NADPH ferredoxin reductase for activity (Beckman, M., and DeLuca, H. (1997) Methods in Enzymol. 282, 200-223; Armbrecht, H. J., Okuda, K., Wongsurawat, N., Nemani, R., Chen, M., and Boltz, M. (1992) J. Steroid Biochem. Molec. Biol. 43, 1073-1081.) Vitamin A and Retinoic Acid Vitamin A metabolism gives rise to several active forms of retinoic acid (RA), which are involved in regulating gene expression during development, regeneration, and in the growth and differentiation of epithelial tissues. [Maden, 1992; Chambon, 1995; Mangelsdorf, 1995; Gudas, 1994; Lotan, 1995; Morriss-Kay, 1996] RA has been linked to apoptosis, or programmed cell death in a number of cell types; and to have anticarcinogenic and antitumoral properties [Lotan, 1996]. Early studies of retinol deficiency indicated a correlation between vitamin A depletion and a higher incidence of cancer and increased susceptibility to chemical carcinogenesis [Chytil, 1984]. Several animal models have been used to demonstrate the effectiveness of retinoids in suppressing carcinogenesis in a variety of tissues including skin, mammary epithelia, oral cavity, aerodigestive tract, liver, bladder and prostate [Moon, 1994]. These studies have led to the preventative use of retinoids to treat premalignant lesions including actinic keratosis and oral leukoplakia, as well as in the prevention of secondary tumors of the head and neck and the recurrence of non-small cell lung carcinomas, and basal cell carcinomas [Hong, 1994; Lippman, 1995]. RA itself has been found to be useful therapeutically, notably in the treatment of cancers, including acute promyelocytic leukemia (APL), tumors of the head and neck, and skin cancer, as well as in the treatment of skin disorders such as the premalignancy associated actinic keratoses, acne, psoriasis and ichthyosis. There is evidence that the effectiveness of RA as an anti-tumor agent is at least partially due to induction of cellular differentiation and/or inhibition of proliferation [Lotan, 1996]. Studies over the past several years indicate that a high proportion of patients with acute promyelocytic leukemia (APL) achieve complete remission after a short period of treatment with all-trans RA. Unfortunately, this high rate of remission is in most cases brief. Following relapse, patients are clinically resistant to further treatment with RA [Warrell, 1994; Warrell, et al., 1994; Chomienne, 1996; Muindi, 1992]. The nature of this resistance is unknown. Interestingly, leukemic cells taken from patients exhibiting clinical resistance to RA have been shown to be sensitive to the differentiating action of RA when grown in vitro [Muindi, 1992; Muindi, 1994]. This suggests that pharmacokinetic mechanisms may account for the acquired resistance to RA. This possibility is supported by studies showing that peak plasma concentrations of RA were much higher in patients after initial administration than in patients treated following relapse. This decrease in peak plasma RA concentration was accompanied by a 10-fold increase in urinary 4-oxo-retinoic acid concentration. In addition, ketoconazole, a broad spectrum inhibitor of cytochrome P450 function was shown to modulate RA pharmacokinetics in vivo [Muindi, 1992; Muindi, 1994]. It is therefore likely that RA increases the rate of its own metabolism, which in turn results in the inability to sustain effective therapeutic doses of RA. Therapeutic administration of RA can result in a variety of undesirable side effects and it is therefore important to establish and maintain the minimal requisite doses of RA in treatment. For example, RA treatments during pregnancy can lead to severe teratogenic effects on the fetus. Adverse reactions to RA treatment also include headache, nausea, chelitis, facial dermatitis, conjunctivitis, and dryness of nasal mucosa. Prolonged exposure to RA can cause major elevations in serum triglycerides and can lead to severe abnormalities of liver function, including hepatomegaly, cirrhosis and portal hypertension. RA metabolism may also account for the lack of response of certain tumors to RA treatment. For example, recent studies have shown that cytochrome P450 inhibitors that block RA metabolism, resulting in increased tissue levels of RA, may be useful therapeutic agents in the treatment of prostate cancer [Wouters, 1992; De Coster, 1996]. Thus RA metabolizing cytochrome P450s may be useful targets for the treatment of a number of different types of cancer. The classical view of vitamin A metabolism holds that all trans-RA, the most active metabolite is derived from conversion of retinol to retinaldehyde to RA through two oxidation steps and that RA is further metabolized to the polar derivatives 4-OH RA and 4-oxo RA [Blaner, 1994; Napoli, 1995; Formelli, 1996; Napoli, 1996]. It is unknown whether the 4-oxo- and 4-OH— metabolites are simply intermediates in the RA catabolic pathway or whether they can also have specific activities which differ from those of all-trans RA and 9-cis RA. Pijnappel et al. [Pijnappel, 1993] have shown that, in Xenopus, 4-oxo-RA can efficiently modulate positional specification in early embryos and exhibits a more potent ability to regulate Hoxb-9 and Hoxb-4 gene expression than all-trans RA. 4-oxo-RA has been found to bind to retinoic acid receptor-β (RAR-β) with affinity comparable to all-trans RA [Pijnappel, 1993] but poorly to RAR-γ [Reddy, 1992], suggesting that this metabolite exhibits some receptor selectivity. 4-oxo-RA also binds to cellular retinoic acid binding protein (CRABP) but with an affinity slightly lower than that of all-trans RA [Fiorella, 1993]. Takatsuka et al. [Takatsuka, 1996] have shown that growth inhibitory effects of RA correlate with RA metabolic activity but it is unknown whether there is a causal relationship between production of RA metabolites and growth inhibition. The asymmetric distribution of these metabolites in developing embryos suggests that they may be preferentially sequestered or generated by tissue specific isomerases [Creech Kraft, 1994]. The normal balance of these metabolites is dependent upon rate of formation from metabolic precursors, retinol and retinaldehyde [Leo, 1989], and rate of catabolism. Little is presently known about the enzymes involved in this metabolic scheme, in particular the catabolism of RA. The catabolism of RA is thought to be initiated by hydroxylation either at the C4-, or C18-position of the β-ionone ring of RA [Napoli, 1996]. The C4-hydroxylation step is mediated by cytochrome P450 activity, as judged by the ability of broad spectrum P450 inhibitors such as ketoconazole and liarazole to block 4-hydroxylation [Williams, 1987, Van Wauwe, 1988; Van Wauwe, 1990, Van Wauwe, 1992, Wouters, 1992]. In certain tissues, including testis, skin and lung and in numerous cell lines, such as NIH3T3 fibroblasts, HL 60 myelomonocytic leukemic cells, F9 and P19 murine embryonal carcinoma cell lines and MCF7, RA metabolism can be induced by RA pretreatment [Frolik, 1979, Roberts, 1979a and b; Duell, 1992; Wouters, 1992]. Studies involving targeted disruption of RAR genes in F9 cells suggest that RAR-α and RAR-γ isoforms may play a role in regulating the enzymes responsible for this increased metabolism [Boylan, 1995]. The glucuronidation of RA is a significant metabolic step in the inactivation of RA [Blaner, 1994; Formelli, 1996]. The elimination of RA may require oxidation to 4-oxo, followed by conjugation to form the 4-oxo all-trans RA glucuronide. This is supported by studies in both primates and humans showing that the 4-oxo RA glucuronide is the only retinoid conjugate found in urine [Muindi, 1992; Muindi, 1994]. The fact that following RA therapy, 4-oxo RA is not present or barely detectable in serum, suggests that oxidation may be the rate limiting step in this process. It has recently been shown that 4-oxoretinol (4-oxo-ROL) can have greater biological activity than retinol. The 4-oxo-ROL is inducible by RA in F9 and P19 mouse teratocarcinoma cells [Blumberg et al., 1995; Achkar et al., 1996]. It is known that zebrafish fins regenerate through an RA sensitive process, which utilizes many gene regulatory pathways involved in early vertebrate development [White, 1994; Akimenko, 1995a & b]. Cytochome P450s and Retinoic Acid Metabolism In 1979, Roberts et al., [Roberts (1979a)] first postulated that the catabolism of retinoic acid (RA) was mediated by a cytochrome P450 enzyme. Several P450s have since been shown to metabolize RA, including P450 proteins from human, zebrafish and mouse. For example, human P450RAI, which is induced by RA, metabolizes RA to more poplar derivatives including 4-hydroxy retinoic acid (4-OHRA) and 4-oxo retinoic acid (4-oxo RA) [White et al. (1996a)]. Since RA is useful as an antitumor agent, it is desirable to maintain high tissue levels of RA. Thus, cytochrome P450 inhibitors that block RA metabolism, resulting in increased tissue levels of RA, may be useful therapeutic agents in the treatment of cancers, such as prostate cancer [Wouters et al., (1992); and De Coster et al., (1996)]. International Patent Publication No. WO 97/49815, published Dec. 31, 1997, describes a family of retinoid metabolizing proteins, CYP26A, including proteins from human, zebrafish and mouse and their coding sequences. This earlier publication is incorporated herein in its entirety. cDNAs encoding a cytochrome P450-dependent enzyme (P450RAI) which is induced by RA have been cloned and characterized from zebrafish and the protein metabolizes RA to more polar derivatives including 4-hydroxy retinoic acid (4-OH RA) and 4-oxo retinoic acid (4-oxo RA) [White et al., 1996a]. The identification of P450RAI gene is an important step in the understanding of RA signaling but its presence has been known since Roberts et al. (1979a) first postulated that the catabolism of RA was mediated by a P450 enzyme [Frolik et al., 1979; Roberts et al., 1979a]. More recently, the isolation of cDNAs which encode the full-length human and mouse P450RAI orthologs whose expression, like that of the fish cytochrome, is highly inducible by RA has been achieved [Fujii et al., 1997; Ray et al., 1997]. Human and mouse genomic P450RAI-1 sequences and the mouse cDNA sequence encoding P450RAI-1 I have been identified. The human cDNA and amino acid sequence of P450RAI-1 is identified herein as SEQ. ID. NOS. 1 and 2, respectively (also see FIG. 6A ). Homologs have also been isolated from human, mouse, chick and xenopus all exhibiting a high degree of sequence conservation [Abu-Abed et al., 1998; Hollermann et al., 1998; White et al., 1997]. There is extensive identity between the human and fish P450RAI genes which overall is 68% at the amino acid level (over 90% between mouse and human). MCF7 cells have been shown to have RA inducible RA metabolism [Butler and Fontana, 1992; Wouters et al., 1992]. The expression of P450RAI in these cells is dependent on the continuous presence of RA [White et al., 1997]. This suggests that P450RAI regulation by RA forms an autoregulatory feedback loop that functions to limit local concentrations of RA, such that when normal physiological levels of RA are exceeded, induction of P450RAI acts to normalize RA levels. The inducible expression of P450RAI in mouse embryos also suggests that a similar autoregulatory mechanism may limit exposure to RA sensitive tissues during development [lulianella et al., 1999]. A second retinoic acid metabolizing cytochrome P450, P450RAI-2 has also recently been identified in human, rat, mouse and zebrafish. The human cDNA and amino acid sequence are identified herein as SEQ. ID. NOS. 3 and 4, respectively. Retinoic Acid, Cytochrome p450 and Embryonic Development All-trans-RA is a critical regulator of gene expression during embryonic development and in the maintenance of adult epithelial tissues [Gudas, et al. (1994).; Lotan, R. M. (1995); Lotan, R. (1996); Morriss-Kay, G. M. (1996)]. The effects of all-trans-RA are mediated by heterodimers of nuclear receptors for retinoic acid (RARs) and retinoid-X-receptors, which are regulated by the 9-cis isomer of RA. Three different subtypes exist for each of these receptors (RARα, β and γ; RXR RAR α, β and γ), which individually are expressed in a tissue specific manner but collectively can be found in essentially all cell types, both during embryonic development and in the adult [Chambon, P. (1995).]. The activity of RA in these tissues is controlled, to a large extent, by enzymes involved in its synthesis from retinaldehyde (ALDH-1 and RALDH-2) and its catabolism to 4-OH, 4-oxo and 18-OH products (P450RAI) [White J. A., et al. (1997); lulianella, A. et al. (1999); McCaffery P. et al., (1999) Niederreither, K. et al. (1999) Swindell E., et al. (1999)]. It has been shown that P450RAI-1 (CYP26A) from zebrafish, mouse, human, chick and xenopus is responsible for the metabolism of active all-trans-RA to inactive polar metabolites including 4-OH-RA, 4-oxo-RA and 18-OH-RA [White J., et al. (1997); Swindell E., et al. (1999); White, J. & Petkovich, M. (1996); Abu-Abed, et al. (1998); Fujii, H. et al. (1997); Ray, W. et al. (1997); Hollermann, T et al. (1998)]. P450RAI-1 expression can be induced by all-trans-RA pre-treatment in multiple tissues, and cell types, and this expression is concomitant with increased all-trans-RA catabolism. In MCF7 cells, all all-trans-RA suggesting a feedback-loop mechanism is dependent on the continued presence of all-trans-RA suggesting a feedback-loop mechanism for the regulation of all-trans-RA levels [White J., et al. (1997)]. Inducible expression of P450RAI-1 has also been observed in vivo in zebrafish, chick, xenopus and mouse embryos suggesting that this autoregulatory feedback-loop plays an important role in balancing all-trans-RA levels in certain developing tissues. Studies from several groups show that tissues such as neural folds in chick embryos [Swindell E., et al. (1999)], caudal neuroepithelia [lulianella, A et al. (1999); Fujii, H. et al. (1997)] and developing retina [McCaffery P. et al. (1999)] from mouse express P450RAI-1 constitutively thus forming a barrier to all-trans-RA exposure. Comparison of the expression patterns of RALDH-2 and P450RAI-1 in these models suggests that these enzymes act together to form regions of RA synthesis and activity (where RALDH-2 is expressed). RALDH-2 expressing tissues have been shown to contain retinoid activity as measured by both retinoid responsive reporter gene activity and direct measurement of RA levels from tissue extracts; by similar analyses, P450RAI-1 expressing tissues do not [lulianella, A et al. (1999); McCaffery P. et al. (1999)]. In addition, over expression of P450RAI-1 in xenopus embryos has been shown to abrogate the teratogenic effects of exogenously applied RA, consistent with a catabolic role for its enzyme [Hollermann, T et al. (1998)]. The Adrenal Glands The adrenal glands comprise an inner part (the medulla) that secretes hormones such as adrenaline (epinephrine) that affect blood pressure, heart rate, sweating, and other activities also regulated by the sympathetic nervous system. The outer part (the cortex) secretes many different hormones, including corticosteroids, androgens and minerlocorticoids, which control blood pressure and the levels of salt and potassium in the body. The adrenal glands are part of a complex system that produces interacting hormones. The hypothalamus produces corticotropin-releasing hormone, triggering the pituitary gland to secrete corticotropin, which regulates the production of orticosteroids by the adrenal glands. Adrenal glands may stop functioning when either the pituitary or hypothalamus fails to produce sufficient amounts of the appropriate hormones. Underproduction or overproduction of any adrenal hormones can lead to serious illness. Diseases associated with the adrenal gland include Addison's disease, Cushing's syndrome, pheochromocytoma, adenoma, hyperaldosteronism, high blood pressure, weakness, paralysis, darkening of the skin, osteoporosis, and fat accumulation. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is directed to a novel cytochrome P450 that is part of the retinoic acid metabolizing family of cytochrome P450s. In another aspect, the novel cytochrome P450 is preferentially expressed in the adrenal gland. In another embodiment the novel cytochrome metabolized 13-all-trans-retnoic acid. In yet another embodiment the novel cytochrome metabolized 9-cis-retnoic acid. The present inventors have characterized for the first time human cytochrome P450RAI-3 [hereinafter “P450RAI-3 or “CYP26C”]. In one embodiment the P450RAI-3 is a microsomal cytochrome. In one embodiment the P450RAI-3 is isolated from adrenal tissue. These findings have important implications in terms of increased understanding of cytochrome P450s and the retinoic acid pathway and the application to various disease states, such as those noted above, i.e. cancer, adenoma, high blood pressure, muscle weakness, skin discolouration, osteoporosis, fat accumulation, pheochromocytoma, Addison's disease, Cushing's syndrome. Although, the P450RAI-3 and encoding nucleic acid sequence of the invention can be isolated and characterized from any tissue, it is preferably isolated and characterized from adrenal tissue. Accordingly, the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding a P450RAI-3, preferably a human P450RAI-3 and to variants, homologs, analogs thereof and to fragments thereof. Complimentary (or antisense) polynucleotide sequences to the polynucleotides of the invention are also encompassed within the scope of the invention. In a preferred embodiment, an isolated polynucleotide is provided comprising a nucleic acid sequence as shown in SEQ. ID. NOS 9 ( FIG. 1 ) or 10 ( FIG. 2 ). Most preferably, the purified and isolated polynucleotide comprises: (a) a nucleic acid sequence as shown in SEQ. ID. NOS. 9 ( FIG. 1 ) or 10 ( FIG. 2 ) or a nucleic acid sequence encoding the amino acid sequence of SEQ. ID. NO. 3 ( FIG. 3 ), wherein T can also be U; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which are homologous to (a) or (b); or, (d) a fragment of (a) to (c) that will hybridize to (a) to (c) under stringent hybridization conditions. Preferably the fragment is 10 or more, preferably at least 15 bases, most preferably 20 to 30 bases. In another embodiment, the isolated poynucleotide of the invention comprises a sequence encoding any one or more of exons 1 to 6 of P450RAI-3 as depicted in SEQ. ID. NOS 13, 15, 17, 19, 21, or 23 (See FIG. 5 for amino acid regions) or Sequences 12, 14, 16, 18, 20 pr 22 (See FIG. 5 ). In a further embodiment, the invention provides polynucleotides that consist of the isolated polynucleotides noted herein. The present invention also includes the P450RAI-3 polypeptide. In one embodiment, the invention provides a polypeptide having an amino acid sequence as shown in SEQ. ID. NO. 11 ( FIG. 3 ) and to variants, homologs, and analogs, insertions, deletions, substitutions and mutations thereto. The invention also comprises polypeptides comprising fragments of the amino acid sequence of SEQ. ID. NO. 11 ( FIG. 3 ) or to their respective variants, homologs, analogs, insertions, deletions, substitutions and mutations. In another embodiment the fragments preferably comprise 14 or more amino acid residues and are most preferably antigenic or immunogenic. In another embodiment the invention provides polypeptides encoded by a polynucleotide having the sequence of SEQ. ID. NO. 10 ( FIG. 2 ), or to variants, homologs, analogs or fragments thereof. In another embodiment the polypeptide of the invention comprises or consists of any one or more of the amino acid sequences of exons 1 to 6 of P450RAI-3 as depicted in SEQ. ID. NOS. 13, 15, 17, 19, 21 or 23 (see FIG. 5 ). Accordingly, in one embodiment the invention relates to vectors, host cells comprising the polynucleotides of the invention or that can express the polypeptides of the invention. Antibodies to the polypeptides of the invention are also encompassed within the scope of this invention. The invention further provides recombinant methods for producing P450RAI-3 polypeptides and polynucleotides of the invention. In one embodiment, the invention provides a polynucleotide of the invention operationally linked to an expression control sequence in a suitable expression vector. In another embodiment, the expression vector comprising a polynucleotide of the invention is capable of being activated to express the peptide, which is encoded by the polynucleotide and is capable of being transformed or transfected into a suitable host cell. Such transformed or transfected cells are also encompassed with the scope of this invention. The invention also provides a method of preparing a polypeptide of the invention utilizing a polynucleotide of the invention. In one embodiment, a method for preparing the polypeptide, preferably P450RAI-3 is provided comprising: transforming a host cell with a recombinant expression vector comprising a polynucleotide of the invention; (b) selecting transformed host cells from untransformed host cells; (c) culturing a selected transformed host cell under conditions which allow expression of the protein; and (d) isolating the protein. In yet another embodiment, the invention also includes diagnostic methods for detecting and screening for disorders related to P450RAI-3 gene expression and polypeptides and to therapeutic methods for treating such disorders. As such, the invention also includes a method for detecting a P450RAI-3 related condition in an animal. A P450RAI-3 related condition includes but is not limited to diseases associated with vitamin A or retinoic acid metabolism. The method comprises assaying for P450RAI-3 from a sample, such as a biopsy, or other cellular or tissue sample, from an animal susceptible of having such a condition. In one embodiment, the method comprises contacting the sample with an antibody of the invention, which binds P450RAI-3, and measuring the amount of antibody bound to P450RAI-3 in the sample, or unreacted antibody. In another embodiment, the method involves detecting the presence of a nucleic acid molecule having a sequence encoding a P450RAI-3, comprising contacting the sample with a nucleotide probe which hybridizes with the nucleic acid molecule, preferably mRNA or cDNA to form a hybridization product under conditions which permit the formation of the hybridization product, and assaying for the hybridization product. The invention further includes a kit for detecting a P450RAI-3 related condition from a sample comprising an antibody of the invention, preferably a monoclonal antibody. Preferably directions for its use are also provided. The kit may also contain reagents, which are required for binding of the antibody to a P450RAI-3 protein in the sample. The invention also provides a kit for detecting the presence of a polypeptide having a sequence encoding a polypeptide of, related to or analogous to a polypeptide of the invention, comprising a nucleotide probe which hybridizes with the nucleic acid molecule, reagents required for hybridization of the nucleotide probe with the nucleic acid molecule, and directions for its use. The invention also includes screening methods for identifying binding partners of P450RAI-3. In addition, the invention relates to screening methods for identifying modulators, such as agonists and antagonists, of P450RAI-3 activity. In one embodiment such modulators of P450RAI-3 activity or expression can include antibodies to P450RAI-3 and antisense polynucleotides to the P450RAI-3 gene or fragment thereof. The invention further provides a method of treating or preventing a disease associated with P450RAI-3 expression comprising administering an effective amount of an agent that activates, simulates or inhibits P450RAI-3 expression, as the situation requires, to an animal in need thereof. In a preferred embodiment, P450RAI-3, a therapeutically active fragment thereof, or an agent, which activates or simulates P450RAI-3 expression is administered to the animal in need thereof to treat a disease or condition associated with too much retinoic acid. In another embodiment the disease is associated with over expression of P450RAI-3 or retinoic acid deficiency (i.e. not enough retinoic acid or desire to maintain retinoic acid levels) and the method of treatment comprises administration of an effective amount of an agent that inhibits P450RAI-3 expression such as an antagonist of P450RAI-3, an antibody to P450RAI-3, a mutation thereof, or an antisense nucleic acid molecule to all or part of the P450RAI-3 gene. In another embodiment the invention provides pharmaceutical compositions comprising a modulator of P450RAI-3 activity and a pharmaceutical acceptable carrier. In another embodiment, the pharmaceutical composition of the invention comprises P450RAI-3 (preferably a soluble form thereof) or a therapeutically effective fragment thereof and a pharmaceutically acceptable carrier. In another embodiment the pharmaceutical compositions of the invention comprise both a modulator of P450RAI-3 activity and P450RAI-3 (preferably a soluble form thereof) or a therapeutically effective fragment thereof. In a further embodiment the pharmaceutical compositions of the invention can further comprise a modulator of P450RAI-3 and any one or more of: (a) retinoic acid, (b) a ligand of P450RAI-3; a substrate of P450RAI-3. The invention also includes a method of identifying a modulator of P450RAI-3 activity comprising: [a]incubating P450RAI-3 or a cell expressing P450RAI-3 with a test compound under conditions that promote P450RAI-3 expression or activity; [b] detecting the activity or expression, as the case may be, of P450RAI-3 in the presence of said test compound, a decrease in said activity or expression being indicative that the test compound is an inhibitor of P450RAI-3 expresssion or activity, while an increase in said expression or activity is indicative that the test compound is a P450RAI-3 agonist. Another aspect of the invention relates to a method of identifying a substate of P450RAI-3 comprising: [a]incubating P450RAI-3 with a test substrate under conditions that promote P450RAI-3/substate complex formation or interaction; [b] determining P450RAI-3/substrate complex formation or interaction. The incubation step optionally further comprises a known modulator of P450RAI-3. Step [b] can be determined by comparing the effect on P450RAI-3 in the absence and presence of the test substrate. Another aspect of the invention is a method for identifying a substance which associates with a protein of the invention comprising (a) reacting the protein with at least one substance which potentially can associate with the protein, under conditions which permit the association between the substance and protein, and (b) removing or detecting protein associated with the substance, wherein detection of associated protein and substance indicates the substance associates with the protein. Another embodiment of the invention relates to a method for evaluating a compound for its ability to modulate the biological activity of a protein of the invenition comprising providing the protein with a substance which associates with the protein and a test compound under conditions which permit the formation of complexes between the substance and protein, and removing and/or detecting complexes. The invention also relates to a method for identifying inhibitors of a P450RAI-3 Protein interaction, comprising (a) providing a reaction mixture including the P450RAI-3 Related Protein and a substance that binds to the P450RAI-3 Related Protein, or at least a portion of each which interact; (b) contacting the reaction mixture with one or more test compounds; (c) identifying compounds which inhibit the interaction of the P450RAI-3 Related Protein and substance. The invention also includes a method for detecting a nucleic acid molecule encoding a protein comprising an amino acid sequence of SEQ. ID. NO. 40 in a biological sample comprising the steps of: (a) hybridizing a nucleic acid molecule of claim 1 to nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex wherein the presence of the hybridization complex correlates with the presence of a nucleic acid molecule encoding the protein in the biological sample. The invention also includes a method as claimed in claim 50 wherein nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step. (a) The invention also includes a composition comprising one or more of a nucleic acid molecule or protein claimed in any of the preceding claims or a substance or compound identified using a method, and a pharmaceutically acceptable carrier, excipient or diluent. The invention also includes a nucleic acid molecule or protein of the invention, or a substance or compound identified using a method of the invention in the preparation of a pharmaceutical composition for treating a condition mediated by a protein of the invention, or a nucleic acid molecule of the invention. The invention includes compounds identified with methods of the invention. Another aspect of the invention includes a vaccine for stimulating or enhancing in a subject to whom the vaccine is administered production of antibodies directed against a protein of the invention. The invention also includes a method for stimulating or enhancing in a subject production of antibodies directed against a protein. The invention includes a method involving administering to the subject a vaccine of the invention in a dose effective for stimulating or enhancing production of the antibodies. The invention includes the use of the isolated polypeptide of the invention, and optionally a modulator of P450RAI-3 activity for preparation of a pharmaceutical substance. The invention also includes the use of a therapeutically effective amount of the polypeptide of the invention or of the polynucleotide of the invention and/or a modulator of P450RAI-3 for preventing, treating or ameliorating a medical condition related to P450RAI-3 expression. The invention includes the use of a therapeutically effective polypeptide of the invention and/or an agonist thereof for treating a disease or condition related to vitamin A or retinoic acid metabolism in a patient. The invention also includes the use of a P450RAI-3 inhibitor and a P450RAI-3 substrate for preventing, treating or ameliorating a medical condition related to P450RAI-3 expression or for preparation of a pharmaceutical substance. Another embodiment of the invention relates to a method of determining the ATRA and/or 9-cis-RA metabolizing activity of a polypeptide of the invention, comprising: expressing the polypeptide in a host cell, adding ATRA and/or 9-cis-RA to the cell, and determining the amount and/or rate of ATRA and/or 9-cis-RA metabolism. Another embodiment of the invention relates to a method of determining the substrate of a polypeptide of the invention, comprising: expressing the polypeptide in a host cell, adding a candidate substrate, and determining if the substrate is metabolized, wherein metabolization indicates that the candidate substrate is a substrate of the polypeptide. The invention also relates to a method of determining the binding activity of a substrate to a polypeptide of the invention, comprising expressing the polypeptide in a host cell, adding a candidate substrate, and determining a Kd value. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. |
Pyrimidine derivatives useful as selective cox-2 inhibitors |
The invention provides the compounds of formula (I) in which: R1 is selected from the group consisting of H, C1-6alkyl, C1-2alkyl substituted by one to five fluorine atoms, C3-6alkenyl, C3-10cycloalkylC0-6alkyl, C4-12bridged cycloalkyl, A(CR4R5)n and b(CR4R5)n; R2 is C1-2alkyl substituted by one to five fluorine atoms; R3 is selected from the group consisting of C1-6alkyl, NH2 and R7CONH; R4 and R5 are independently selected from H or C1-6alkyl; A is an unsubstituted 5- or 6-membered heteroaryl or an unsubstituted 6-membered aryl, or a 5- or 6-membered heteroaryl or a 6-membered aryl substituted by one or more R6; R6 is selected from the group consisting of halogen, C1-6alkyl, C1-6alkyl substituted by one or more fluorine atoms, C1-6alkoxy, C1-6alkoxy substituted by one or more F, NH2SO2 and C1-6alkylSO2; B is selected from the group consisting of Formula (i) and (ii) and where (iv) defines the point of attachment of the ring; R7 is selected from the group consisting of H, C1-6alkyl, C1-6alkoxy, C1-6alkylOC1-6alkyl, phenyl, HO2CC1-6alkyl, C1-6alkylOCOC1-6alkyl, C1-6alkylOCO, H2C1-6alkyl, C1-4alkylOCONHC1-6alkyl and C1-6alkylCONHC1-6alkyl; and n is 0 to 4. Compounds of formula (I) are potent and selective inhibitors of COX-2 and are of use in treatment of the pain, fever and inflammation of variety of conditions and diseases. |
1. A compound of formula (I) in which: R1 is selected from the group consisting of H, C1-6alkyl, C1-2alkyl substituted by one to five fluorine atoms, C3-6alkenyl, C3-6alkynyl, C3-10cycloalkylC0-6alkyl, C4-12bridged cycloalkyl, A(CR4R5)n and B(CR4R5)n; R2 is C1-2alkyl substituted by one to five fluorine atoms; R3 is selected from the group consisting of C1-6alkyl, NH2 and R7CONH; R4 and R5 are independently selected from H or C1-6alkyl; A is an unsubstituted 5- or 6-membered heteroaryl or an unsubstituted 6-membered aryl, or a 5- or 6-membered heteroaryl or a 6-membered aryl substituted by one or more R6; R6 is selected from the group consisting of halogen, C1-6alkyl, C1-6alkyl substituted by one more fluorine atoms, C1-6alkoxy, C1-6alkoxy substituted by one or more F, NH2SO2 and C1-6alkylSO2; B is selected from the group consisting of where defines the point of attachment of the ring; R7 is selected from the group consisting of H, C1-6alkyl, C1-6alkoxy, C1-6alkylOC1-6alkyl, phenyl, HO2CC1-6alkyl, C1-6alkylOCOC1-6alkyl, C1-6alkylOCO, H2C1-6alkyl, C1-6alkylOCONHC1-6alkyl and C1-6alkylCONHC1-6alkyl; and n is 0 to 4. 2. The compound as claimed in claim 1 wherein R1 is selected from the group consisting of C1-6alkyl, C1-2alkyl substituted by one to five fluorine atoms, C3-10cycloalkylC0-6alkyl and A(CR4R5)n. 3. The compound as claimed in claim 1 wherein R2 is CHF2, CH2F or CF3. 4. The compound as claimed in claim 1 wherein R3 is C1-6alkyl. 5. The compound as claimed in claim 1 wherein R4 and R5 are independently selected from H or methyl. 6. The compound as claimed in claim 1 wherein A is selected from the group consisting of where defines the point of attachment of the ring and A is unsubstituted or substituted by one or two R6. 7. The compound as claimed in claim 1 wherein R6 is selected from the group consisting of halogen, C1-3alkyl, C1-3alkyl substituted by one to three fluorine atoms, and C1-3alkoxy. 8. The compound as claimed in claim 1 wherein R7 is selected from the group consisting of C1-6alkyl, phenyl and aminomethyl. 9. The compound as claimed in claim 1 wherein n is 0 to 2. 10. The compound as claimed in claim 1 wherein R1 is C1-6alkyl; R2 is CF3; and R3 is C1-6alkyl. 11. The compound as claimed in claim 1 wherein R1 is C3-10cycloalkylC0-6alkyl; R2 is CF3; and R3 is C1-6alkyl. 12. The compound as claimed in claim 1 wherein R1 is C3-10cycloalkylmethyl; R2 is CF3; and R3 is C1-6alkyl. 13. The compound as claimed in claim 1 wherein R1 is A(CR4R5)n; R2 is CF3; R3 is methyl; R4 and R5 are both H; A is selected from the group consisting of and A is unsubstituted or substituted by one or two R6; R6 is selected from the group consisting of fluorine, chlorine, methyl, CF3 and methoxy; and n is 0 or 1. 14. (Canceled.) 15. 2-Butoxy-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)pyrimidine. 16. A process for the preparation of a compound as defined in claim 1, which comprises: (A), reacting an alcohol R1OH of formula (II) or a protected derivative thereof with a compound of formula (III) and thereafter and if necessary, (B), interconverting a compound of formula (I) into another compound of formula (I); and/or (C), deprotecting a protected derivative of compound of formula (I). 17. A pharmaceutical composition comprising a compound as defined in claim 1 in admixture with one or more physiologically acceptable carriers or excipients. 18. (Canceled) 19. A method of treating a subject suffering from a condition which is mediated by COX-2 which comprises administering to said subject an effective amount of a compound as defined in claim 1. 20. A method of treating a subject suffering from an inflammatory disorder, which method comprises administering to said subject an effective amount of a compound as defined claim 1. 21-22. (Canceled) 23. A compound selected from 2-(4-fluorophenoxy)-4-[4-(methylsulfonyl)phenyl]-6](trifluoromethyl)pyrimidine; 2-(4-methoxyphenoxy)-4-[4-(methylsulfonyl)phenyl]-6-trifluoromethyl)pyrimidine; 2-butoxy-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)pyrimidine; 2-[(5-chloropyridin-3-yl)oxy]-4-[4-(methylsulfony)phenyl]-6-(trifluoromethyl)pyrimidine; and 2-(cyclohexyloxy)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)pyrimidine. 24. The method according to claim 19, wherein said subject is a human. 25. The method according to claim 20, wherein said subject is a human. 26. The compound according to claim 1, wherein R1 is C1-6alkyl; R2 is CF3; and R3 is C1-3alkyl. 27. The compound as claimed in claim 1 wherein R1 is C3-10cycloalkyl; R2 is CF3; and R3 is C1-3alkyl. 28. The compound as claimed in claim 1 wherein R1 is C3-7cycloalkylmethyl; R2 is CF3; and R3 is C1-3alkyl. |
Olefin polymerization catalysts containing a pyrrole bisimine ligand |
Polydentate substituted pyrrole based chelants, metal complexes containing the same, olefin polymerization catalyst compositions, and polymerization processes using the same. |
1. A metal complex comprising a multidentate chelating ligand, said metal complexes corresponding to the formula: where M is a metal from one of Groups 3 to 13 of the Periodic Table of the Elements, the lanthanides or actinides; T is nitrogen or phosphorus; RA independently each occurrence is hydrogen, RB or T′RBj, RB independently each occurrence is a group having from 1 to 80 atoms not counting hydrogen, which is hydrocarbyl, hydrocarbylsilyl, halo-substituted hydrocarbyl, hydrocarbyloxy-substituted hydrocarbyl, hydrocarbylamino-substituted hydrocarbyl, or hydrocarbylsilyl-substituted hydrocarbyl, and optionally the RB and RA groups bonded to the same T=C grouping may be joined together to form a divalent ligand group; j is 1 or 2, and when j is 1, T′ is oxygen or sulfur and when j is 2, T′ is nitrogen or phosphorus, RC independently each occurrence is hydrogen or a group having from 1 to 80 atoms not counting hydrogen, which is hydrocarbyl, hydrocarbylsilyl, halo-substituted hydrocarbyl, hydrocarbyloxy-substituted hydrocarbyl, hydrocarbylamino-substituted hydrocarbyl, or hydrocarbylsilyl-substituted hydrocarbyl, or two RC groups are joined together forming a divalent ligand group; X is an anionic ligand group having up to 60 atoms (excluding ligands that are cyclic, delocalized, π-bound ligand groups), and optionally two X groups together form a divalent ligand group; X′ independently each occurrence is a Lewis base ligand having up to 20 atoms; x is a number from 0 to 5; and x′ is zero, 1 or 2: 2. The complex of claim 1, wherein M is a metal of Groups 4-8; T is nitrogen; X is chloride or C1-10 hydrocarbyl; and x′ is zero. 3. The complex of claim 2 wherein independently each occurrence RA is hydrogen, methyl or phenyl, RB is aryl or alkyl substituted aryl, and RC is hydrogen. 4. The complex of claim 1 corresponding to the formula: wherein RA′ independently each occurrence is C1-4 alkyl, most preferably methyl, isopropyl, or t-butyl, A′ is 0, 1 or 2; RA is hydrogen, or C1-10 hydrocarbyl, M is zirconium, vanadium or chromium; especially zirconium, X is halide or C1-10 hydrocarbyl, and x is 1 or 2. 5. A metal complex according to claim 1 which is [(2-(2,6-diisopropylphenyl)-iminomethyl)-5-(2,6-diisopropylphenyl)amido(benzyl)methyl)pyrrol-1-yl]Zr dibenzyl. 6. A catalyst composition for olefin polymerization comprising: (A) the metal complex of any one of claims 1-5; and (B) an activating cocatalyst wherein the molar ratio of (A) to (B) is from 1:10,000 to 100:1. 7. A process for polymerizing olefins comprising contacting one or more C2-20 α-olefin under polymerization conditions with a catalyst composition according to claim 6. 8. A process for preparing isotactic polypropylene comprising contacting propylene under polymerization conditions with a catalyst composition according to claim 6. |
Diagnostic assay for measuring a cell mediated immune response |
The present invention relates generally to a diagnostic assay and, more particularly, an assay for measuring cell-mediated immune reactivity. Even more particularly, the present invention provides an assay and a kit for measuring a cell-mediated response to an antigen using whole blood or other suitable biological sample. The assay may be conducted using ligands to immune effector molecules or at the nucleic acid level, screening for expression of genes encoding the immune effector molecules. The assay is useful in therapeutic and diagnostic protocols for human, livestock and veterinary and wild life applications. |
1. A method for measuring a CMI response in a subject, said method comprising collecting a sample from said subject wherein said sample comprises cells of the immune system which are capable of producing immune effector molecules following stimulation by an antigen, incubating said sample with an antigen and then measuring the presence of or elevation in the level of an immune effector molecule wherein the presence or level of said immune effector molecule is indicative of the capacity of said subject to mount a cell-mediated immune response. 2. The method of claim 1 wherein the subject is a human. 3. The method of claim 1 or 2 wherein the sample is whole blood. 4. The method of claim 3 wherein the whole blood is collected in a tube comprising antigen. 5. The method of claim 3 wherein the whole blood is collected in a tube comprising heparin. 6. The method of claim 4 wherein the title further comprises heparin. 7. The method of claim 1 wherein the sample is incubated with the antigen for from about 5 to about 50 hours. 8. The method of claim 3 wherein the whole blood is incubated with antigen for from about 5 to about 50 hours. 9. The method of claim 1 wherein the immune effector molecule is a cytokine. 10. The method of claim 9 wherein the cytokine is IFN-γ. 11. The method of claim 9 wherein the cytokine is GM-CSF. 12. The method of claim 9 wherein the cytokine is an interleukin. 13. The method of claim 9 wherein the cytokine is a TNFα. 14. The method of claim 1 or 2 wherein the subject is infected by a pathogenic agent. 15. The method of claim 14 wherein the pathogenic agent is HBV. 16. The method of claim 14 wherein the pathogenic agent is HIV. 17. The method of claim 1 wherein the immune cells are selected from NK cells, T-cells, B-cells, dendritic cells, macrophages or monocytes. 18. The method of claim 17 wherein the immune cells are T-cells. 19. The method of claim 7 or 8 wherein the incubation occurs in the presence of a simple sugar. 20. The method of claim 19 wherein the simple sugar is dextrose. 21. The method of claim 1 wherein the antigen is a peptide. 22. The method of claim 1 wherein the antigen is a polypeptide. 23. The method of claim 1 wherein the antigen is a protein. 24. The method of claim 1 wherein the antigen is a glycoprotein. 25. The method of claim 1 wherein the antigen is a carbohydrate. 26. The method of claim 1 wherein the antigen is selected from a phospholipid, phosphoprotein and a phospholipoprotein. 27. The method of claim 1 wherein the antigen is a TB-specific antigen. 28. The method of claim 27 wherein the TB-specific antigen is an overlapping peptide for ESAT-6. 29. The method of claim 27 wherein the TB-specific antigen is an overlapping peptide for CFP-10. 30. The method of claim 27 wherein the TB-specific antigen is an overlapping peptide for TB7. 31. The method of claim 1 wherein the antigen is PPD from Mycobacterium tuberculosis. 32. The method of claim 1 wherein the antigen is PPD from Mycobacterium avium. 33. The method of claim 1 wherein the antigen is phytohemagglutinin. 34. The method of claim 1 wherein the antigen is tetanus toxoid. 35. The method of claim 1 wherein the immune effectors are detected with antibodies specific for same. 36. The method of claim 35 wherein the immune effectors are detected using ELISA. 37. The method of claim 45 wherein the immune effectors are detected using ELISpot. 38. A method of treatment of a subject having a pathogenic infection, an autoimmune disorder or cancer or a propensity for developing such a disorder, said method comprising assessing the ability for said subject to mount a cell mediated immune response by the method of measuring a CMI response in a subject, said method comprising collecting a sample from said subject wherein said sample comprises cells of the immune system which are capable of producing immune effector molecules following stimulation by an antigen, incubating said sample with an antigen and then measuring the presence of or elevation in the level of an immune effector molecule wherein the presence or level of said immune effector molecule is indicative of the capacity of said subject to mount a cell-mediated immune response and then selecting a suitable therapeutic protocol. 39. The method of claim 38 wherein the subject is a human. 40. The method of claim 38 or 39 wherein the sample is whole blood. 41. The method of claim 40 wherein the whole blood is collected in a tube comprising antigen. 42. The method of claim 40 wherein the whole blood is collected in a tube comprising heparin. 43. The method of claim 41 wherein the title further comprises heparin. 44. The method of claim 38 wherein the sample is incubated with the antigen for from about 5 to about 50 hours. 45. The method of claim 40 wherein the whole blood is incubated with antigen for from about 5 to about 50 hours. 46. The method of claim 38 wherein the immune effector molecule is a cytokine. 47. The method of claim 46 wherein the cytokine is IFN-γ. 48. The method of claim 46 wherein the cytokine is GM-CSF. 49. The method of claim 46 wherein the cytokine is an interleukin. 50. The method of claim 46 wherein the cytokine is a TNFα. 51. The method of claim 38 wherein the immune cells are selected from NK cells, T-cells, B-cells, dendritic cells, macrophages or monocytes. 52. The method of claim 51 wherein the immune cells are T-cells. 53. The method of claim 44 or 45 wherein the incubation occurs in the presence of a simple sugar. 54. The method of claim 53 wherein the simple sugar is dextrose. 55. The method of claim 38 wherein the antigen is selected from a peptide. 56. The method of claim 38 wherein the antigen is selected from a polypeptide. 57. The method of claim 38 wherein the antigen is selected from a protein. 58. The method of claim 38 wherein the antigen is selected from a glycoprotein. 59. The method of claim 38 wherein the antigen is selected from a carbohydrate. 60. The method of claim 38 wherein the antigen is selected from a phospholipid, phosphoprotein and phospholipoprotein. 61. The method of claim 38 wherein the antigen is a TB-specific antigen. 62. The method of claim 61 wherein the TB-specific antigen is an overlapping peptide for ESAT-6. 63. The method of claim 61 wherein the TB-specific antigen is an overlapping peptide for CFP-10. 64. The method of claim 61 wherein the TB-specific antigen is an overlapping peptide for TB7. 65. The method of claim 38 wherein the antigen is PPD from Mycobacterium tuberculosis. 66. The method of claim 38 wherein the antigen is PPD for Mycobacterium avium. 67. The method of claim 38 wherein the antigen is phytohemagglutinin. 68. The method of claim 38 wherein the antigen is tetanus toxoid. 69. The method of claim 38 wherein the immune effectors are detected with antibodies specific for same. 70. The method of claim 69 wherein the immune effectors are detected using ELISA. 71. The method of claim 70 wherein the immune effectors are detected using ELISpot. 72. A kit for measuring a CM1 response in a subject said kit being in multicomponent form wherein a first component comprises a multiplicity of blood collection tubes, a second component comprises an antibody-based detection means for an immune effector molecule and a third component comprises a set of instructions which instructions comprise the following: (i) collect blood in the blood collection tubes; (ii) mix the tubes; (iii) incubate the tubes upright; (iv) centrifuge the tubes and collect the plasma; and (v) detect immune effector molecules in plasma. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates generally to a diagnostic assay and, more particularly, an assay for measuring cell-mediated immune reactivity. Even more particularly, the present invention provides an assay and a kit for measuring a cell-mediated response to an antigen using whole blood or other suitable biological sample. The assay may be conducted using ligands to immune effector molecules or at the nucleic acid level, screening for expression of genes encoding the immune effector molecules. The assay is useful in therapeutic and diagnostic protocols for human, livestock and veterinary and wild life applications. 2. Description of the Prior Art Bibliographic details of references provided in the subject specification are listed at the end of the specification. Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country. Measurement of cell-mediated immune (CMI) responses is important for immune diagnosis of many infectious and autoimmune diseases, as a marker for immunocompetence, and for detection of T-cell responses to endogenous and exogenous antigens (i.e. vaccines). Current methods for detecting CMI responses include skin tests measuring both immediate and delayed type hypersensitivity, lymphocyte proliferation assays and measurement of cytokines produced by purified mononuclear cells cultured with antigen. Most in vitro methods for detecting CMI responses involve the purification of lymphocytes from whole blood, culturing these lymphocytes with an antigen for periods from 12 hours to 6 days and then detecting T-cell reactivity to the antigen. Older, established methods, such as the proliferation assay, use the uptake of radioactive isotopes by dividing T-cells as a marker for CMI reactivity. More recently, techniques such as a single cell assay (ELISpot) have been used to detect the number of T-cells producing certain cytokines in response to the antigenic stimulation. Despite the existence of the previously used assays for measuring CMI responsiveness, the practical limitations of easily and accurately measuring CMI responses have hitherto precluded large scale adoption of these assays in standard and routine medical practice in the diagnosis of infectious disease, autoimmune disease and oncology. |
<SOH> SUMMARY OF THE INVENTION <EOH>Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. The present invention provides a method for measuring CMI in a subject by incubating a sample from the subject which comprises T-cells or other cells of the immune system with an antigen. Production of IFN-γ or other cytokine or immune effector molecule(s) is then detected. The presence or level of immune effector is then indicative of the level of cell mediated responsiveness of the subject. Preferably, the sample is whole blood which is collected in a suitable container comprising the antigen. Optionally, a simple sugar such as dextrose is added to the incubation mixture. Accordingly, one aspect of the present invention contemplates a method for measuring a CMI response in a subject, said method comprising collecting a sample from said subject wherein said sample comprises cells of the immune system which are capable of producing immune effector molecules following stimulation by an antigen, incubating said sample with an antigen and then measuring the presence of or elevation in the level of an immune effector molecule wherein the presence or level of said immune effector molecule is indicative of the capacity of said subject to mount a cell-mediated immune response. Preferably, the subject is a human. The present invention contemplates, however, primates, livestock animals, laboratory test animals, companion animals and avian species as well as non-mammalian animals such as reptiles and amphibians. The assay has applications, therefore, in human, livestock, veterinary and wild life therapy and diagnosis. In a preferred embodiment, the sample is whole blood which is collected in collection tubes containing the antigen or to which the antigen is added. Generally, blood is maintained in the presence of heparin. Heparin may be in the tube when blood is added or is added subsequently. The use of blood collection tubes is compatible with standard automated laboratory systems and these are amenable to analysis in large-scale and random access sampling. Blood collection tubes also minimize handling costs and reduces laboratory exposure to whole blood and plasma and, hence, reduces the risk of laboratory personnel from contracting a pathogenic agent such as human immunodeficiency virus HIV or hepatitis B virus (HBV). Furthermore, use of the collection tubes to conduct the incubation renders the assay far more sensitive than the previously used 24 well culture well plates. The present invention provides an enhanced CMI assay, therefore, comprising use of a collection tube, optionally a simple sugar such as dextrose and the incubation step. The incubation step is preferably from 5 to 50 hours. The immune effector molecule is preferably a cytokine such as but not limited to IFN-γ. The presence or level of immune effector may be determined at the level of the molecule itself or to the extent to which a gene is expressed. The present invention further provides kits comprising reagents and compartments required to perform the assay. Generally, the kit further comprises a set of instructions. The assay may also be automated or semi-automated and the automated aspects may be controlled by computer software. The present invention still further contemplates a method of treatment of a pathological condition such as infection by a microorganism, parasite or virus, an autoimmune condition or cancer. The method comprises assessing the capacity of a subject to mount a cell-mediated immune response and then selecting a therapeutic protocol such as inducing CMI against a target antigen alone or in combination with other therapy. A list of abbreviations used herein is provided in Table 1. TABLE 1 Abbreviations ABBREVIATION DESCRIPTION CMI cell-mediated immunity CSF colony stimulating factor ELISA enzyme linked immunosorbent assay ELISpot single cell ELISA G-CSF granulocyte-CSF GM-CSF granulocyte, macrophage-CSF HBV hepatitis B virus HIV human immunodeficiency virus IFN-γ interferon-gamma PPD Mycobacterium-tuberculosis purified protein derivative TNF-α tumor necrosis factor |
Restration adjuser and registration adjusting method |
The present invention provides an apparatus for adjusting registration to correct distortion and color shift of images on a display screen which comprises RAMs (13) and (15) for storing correction data for deflection used to correct scanning positions of image signals for respective plural adjustment points arranged on the display screen along the horizontal direction and the vertical direction, and an Interpolation calculation block (18) for determining the number of interpolation scanning lines being the number of scanning lines which are scanned between the adjustment points corresponding to input image signals as well as for performing interpolation calculation based on the number of interpolation scanning lines for correction waveforms corresponding to display screen position induced from the correction data of the respective adjustment points to generate current signals to be applied to deflection yokes. |
1. An apparatus for adjusting registration, comprising: storage means for storing correction data for deflection used to correct scanning positions of image signals for respective plural adjustment points arranged on a display screen along the horizontal direction and the vertical direction; interpolation scanning line number determination means for determining the number of scanning lines which are scanned between the adjustment points corresponding to input image signals; and correction waveform signal generation means for inducing correction waveforms corresponding to display screen position based on the correction data read out from the storage means, and performing interpolation calculation based on the number of scanning lines determined by the interpolation scanning line number determination means to generate current signals to be applied to deflection yokes. 2. The apparatus for adjusting registration as set forth in claim 1, wherein the storage means stores correction data for coarse adjustment used to correct scanning positions of image signals over the whole display screen and correction data for fine adjustment used to correct scanning positions of image signals partially, and further comprises correction waveform superimposition means for superimposing correction waveforms obtained from the correction data for coarse adjustment and correction waveforms obtained from the correction data for fine adjustment. 3. The apparatus for adjusting registration as set forth in claim 1, wherein the interpolation scanning line number determination means periodically changes the number of scanning lines between the adjustment points. 4. A method for adjusting registration, comprising the steps of storing correction data for deflection used to correct scanning positions of image signals for respective plural adjustment points arranged on a display screen along the horizontal direction and the vertical direction; determining the number of scanning lines which are scanned between the adjustment points corresponding to input image signals; and specifying correction waveforms corresponding to display screen position based on the correction data stored for the respective adjustment points, and performing interpolation calculation based on the number of scanning lines between the adjustment points to generate current signals to be applied to deflection yokes. 5. The method for adjusting registration as set forth in claim 4, further comprising the steps of: storing correction data for coarse adjustment used to correct scanning positions of image signals over the whole display screen for the respective adjustment points; storing correction data for fine adjustment used to correct scanning positions of image signals partially for the respective adjustment points; and superimposing correction waveforms obtained from the correction data for coarse adjustment and correction waveforms obtained from the correction data for fine adjustment to induce correction waveforms. 6. The method for adjusting registration as set forth in claim 4, wherein the number of scanning lines between the adjustment points are periodically changed. |
<SOH> BACKGROUND ART <EOH>There is known a triple-tube type CRT projector using three Cathode-Ray Tubes (CRTs) or a CRT 30R, a CRT 30G, and a CRT 30B which project three primary color images of R signals, G signals, and B signals respectively to form composite images of the R, G, B signals on a screen S, as shown in FIG. 1 . In forming the composite images using the triple-tube type CRT projector, since projection positions of images of the R, G, B signals projected respectively from the CRTs 30R, 30G, 30B onto the screen S are different from each other, there is raised a problem that thus; formed images are caused to be subject to distortion and color shift. So as to correct such distortion and color shift of images, the triple-tube type CRT projector is provided with a registration apparatus. The registration apparatus is an apparatus adapted for correcting distortion and color shift of images by generating correction waveform signals and providing predetermined deflection yokes for registration of the respective CRTs with deflecting currents corresponding to thus generated correction waveform signals. In the triple-tube type CRT projector, registration is adjusted under the process of a flow chart shown in FIG. 2 . Firstly, in step S 21 , main deflection adjustment is performed to cause the respective CRTs to scan images based on horizontal synchronizing signals and vertical synchronizing signals. Then in step S 22 , coarse adjustment (mode for performing coarse adjustment is referred to as coarse adjustment mode) is performed to adjust distortion and color shift of whole images projected onto the screen S, and then in step S 23 , fine adjustment (mode for performing fine adjustment is referred to as fine adjustment mode) is performed to adjust distortion and color shift of images independently at plural adjustment points arranged on the screen S, by the registration apparatus respectively. Thus, as the registration adjustment or adjustment of registration, there are the coarse adjustment mode and the fine adjustment mode. The triple-tube type CRT projector can process image signals supplied in various input video modes such as the NTSC (National Television System Committee) the PAL (Phase-Alternation Line), and the HDTV (High-Definition Television), and can also process image signals supplied in various image display modes, in which images can be displayed in different display configuration, such as the Full mode, the Zoom mode which enlarges predetermined parts of images, and the V (vertical) compression mode which displays images with their vertical components alone compressed. In case correction waveform signals which are effective in performing registration adjustment in the Full mode of the NTSC are used in the V compression mode, being in synchronization with horizontal synchronizing signals and vertical synchronizing signals of image signals, the correction waveform signals are compressed along the vertical direction similar to the image signals and waveforms of the correction waveform signals corresponding to the CRT tube surface position are undesirably changed, as shown in FIG. 3A and FIG. 3B . When considering the state based on time base, since a period of time required in scanning one field of the CRT tube surface by image signals is equal in the Full mode and in the V compression mode (scan time of 16.67 ms), the correction waveform signals themselves are the same. Thus, a triple-tube type CRT projector provided with a conventional registration apparatus requires different correction waveform signals corresponding to the respective input modes, and the user has to perform registration adjustment under manual operation for each input mode, which undesirably requires a long period of time in performing registration adjustment. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 shows a schematic view for explaining a conventional triple-tube type CRT projector. FIG. 2 shows a flow chart for explaining the processing of registration adjustment using the triple-tube type CRT projector. FIG. 3A and FIG. 3B show correction waveforms to be used in registration adjustment in the triple-tube type CRT projector. FIG. 4 shows a block diagram for explaining the configuration of a triple-tube type CRT projector of the present invention. FIG. 5 shows a block diagram for explaining the configuration of a system IC of the triple-tube type CRT projector. FIG. 6 shows correction waveform data for coarse adjustment stored in a coarse adjustment RAM of the triple-tube type CRT projector. FIG. 7 shows correction waveform data for coarse adjustment stored in the coarse adjustment RAM of the triple-tube type CRT projector. FIG. 8 shows a view for explaining adjustment points in the fine adjustment mode. FIG. 9 shows a view for explaining storage areas of a fine adjustment RAM of the triple-tube type CRT projector. FIG. 10A and FIG. 10B show correction waveforms to be used in registration adjustment in the triple-tube type CRT projector. FIG. 11 shows a view for explaining the relation between the CRT tube surface and image signals projected onto a screen by the triple-tube type CRT projector. FIG. 12 shows a view for explaining the processing of changing the number of interpolation lines between adjustment points in the triple-tube type CRT projector. FIG. 13A and FIG. 13B show views for explaining the number of interpolation lines in different modes in the triple-tube type CRT projector. FIG. 14 shows a view for explaining the difference of the number of interpolation lines between adjustment points on the CRT tube surface in the Full mode and in the V compression mode in the triple-tube type CRT projector. FIG. 15 shows a flow chart for explaining the processing of registration adjustment using the triple-tube type CRT projector. detailed-description description="Detailed Description" end="lead"? |
Aircraft cabin module |
An aircraft cabin module of an elongated rectangular shape and that includes two large walls connected by two small walls, a service module arranged along a wall separating the module from an aisle, a door mounted in an opening implemented in a small wall, and at least one seat having at least one configuration in which it is positioned longitudinally in relation to the direction of movement of the airplane. Each module includes elements configured to form a bedding surface for at least one passenger. |
1-28. (Canceled). 29. An aircraft cabin module of elongated rectangular shape comprising: two first walls connected by two second walls smaller than the first walls, a door mounted in an opening implemented in a wall separating the module from an aisle; at least one seat having at least one configuration in which it is positioned longitudinally in relation to a direction of movement of the aircraft; each module comprising elements configured to form a bedding surface for at least one passenger. 30. A module according to claim 29, further comprising a service module arranged along one of the second walls. 31. A module according to claim 30, wherein the service module is arranged beside the door. 32. A module according to claim 29, wherein the at least one seat is of a convertible type and constitutes the elements configured to form the bedding surface. 33. A module according to claim 32, wherein the at least one seat is set up pivoting around a vertical axis. 34. A module according to claim 29, wherein the elements configured to form a bedding surface comprise a panel set up pivoting between a more or less vertical turned-up position against the service module and a more or less horizontal turned-down position. 35. A module according to claim 34, wherein the elements configured to form a bedding surface further comprise a more or less horizontal support surface arranged along the opposite small wall and cooperating with the pivoting panel when the pivoting panel is in the turned-down position to form the bedding surface. 36. A module according to claim 35, wherein the support surface forms a seat equipped with a back. 37. A module according to claim 29, further comprising a second seat provided in the cabin module. 38. A module according to claim 37, further comprising a corner seat that connects the at least one seat and the second seat thus forming a corner banquette. 39. A module according to claim 29, wherein at least one wall has a transparent portion. 40. A module according to claim 39, wherein the transparent portion is arranged on a wall transverse in relation to a direction of movement of the aircraft. 41. A module according to claim 39, wherein the transparent portion is configured to be obscured. 42. A module according to claim 41, wherein the transparent portion comprises a liquid crystal layer combined with means for subjecting the liquid crystal layer to a difference of electric potential, the layer being opaque or translucent depending on the difference of electric potential applied. 43. A module according to claim 39, wherein the transparent portion is equipped with a shading element. 44. A module according to claim 30, wherein the service module is a bathroom facility having at least one water source. 45. A module according to claim 44, wherein the bathroom facility comprises a washbasin set up movable between a retracted position inside the bathroom facility and an extended position outside the bathroom facility. 46. A module according to claim 45, wherein the bathroom facility has an access door, and opening of the access door controls changeover of the washbasin from its retracted to its extended position. 47. A module according to claim 30, wherein the service module is a storage space. 48. A module according to claim 30, wherein the service module is a medical-care module. 49. A module according to claim 30, wherein the service module is a pantry unit equipped for passengers' refreshment. 50. A module according to claim 29, wherein the walls other than the cabin wall, the service module, and the seat at least one in number are each equipped with fastening means for their attachment on longitudinal attachment rails arranged on the floor of the cabin. 51. A module according to claim 29, wherein the module has a length in a longitudinal direction ranging between 1.9 m and 3.0 m and a width on the floor, in a transverse direction, ranging between 1.5 m and 2.1 m. 52. A series of modules according to claim 29 aligned one besides another along an aisle, wherein each large module wall, possibly except for a large wall located at one end of the series of modules, comprises a transparent portion, and wherein the transparent portions are aligned. 53. A series according to claim 52, wherein the seat of each module of the series has a configuration in which the seat is positioned parallel to a direction of movement of the aircraft and is located more or less in alignment with the transparent portions. 54. A section of an aircraft cabin, wherein the section comprises at least one module according to claim 29. 55. A section of an aircraft cabin according to claim 54, wherein the section comprises a central aisle on either side of which is at least one module. 56. An aircraft, comprising at least one module according to claim 29. |
Aircraft cabin module for passengers |
This aircraft cabin module comprises: two transverse walls (12) extending from a longitudinal cabin wall (4) up to a longitudinal aisle (8), at least one of these transverse walls (12) comprising a transparent portion (44), a longitudinal wall separating the cabin module from the aisle (8), at least one service module (14) arranged (5) between the two transverse walls (12), more or less symmetrically in relation to a transverse median plane, defining with the walls of the module two contiguous personal spaces, an access (16) from the aisle to each personal space, at least one seat (18) in each personal space, each seat having at least one configuration in which it is positioned parallel to the cabin wall (4), each personal space comprising elements (24, 28) capable of forming a bedding surface for at least one passenger. |
1-38. (Canceled). 39. Aircraft cabin module comprising: two transverse walls extending from a longitudinal cabin wall up to a longitudinal aisle; a longitudinal wall separating the cabin module from the aisle; at least one service module arranged between the two transverse walls, more or less symmetrically in relation to a transverse median plane, defining with the walls of the module two contiguous personal spaces; an access from the aisle to each personal space; and at least one seat in each personal space, each seat having at least one configuration in which it is positioned parallel to the cabin wall; wherein each personal space comprising elements configured to form a bedding surface for at least one passenger. 40. Module according to claim 39, wherein at least one of the two transverse walls comprises a transparent portion. 41. Module according to claim 39, wherein the at least one service module is arranged along the aisle, being separated from each transverse wall by an access from the aisle to the module. 42. Module according to claim 39, wherein one seat of each personal space is of a convertible type and constitutes the elements configured to form the bedding surface. 43. Module according to claim 42, wherein the one seat is set up pivoting around a vertical axis such that the bedding surface can be positioned crosswise. 44. Module according to claim 39, wherein the elements configured to form the bedding surface comprise a panel set up pivoting between a more or less vertical position turned up against the service module and a more or less horizontal turned-down position. 45. Module according to claim 44, wherein the elements configured to form the bedding surface further comprise a more or less horizontal support surface arranged along the cabin wall and cooperating with the pivoting panel when the pivoting panel is in the turned-down position to form the bedding surface. 46. Module according to claim 45, wherein the support surface forms a seat possibly equipped with a back. 47. Module according to claim 39, wherein a second seat is provided in each personal space. 48. Module according to claim 47, wherein the second seat is a foldaway seat. 49. Module according to claim 47, wherein the second seat is positioned perpendicular to the cabin wall against the cabin wall. 50. Module according to claim 49, wherein a corner seat connects the first and second seats thus forming a corner banquette. 51. Module according to claim 39, further comprising a movable or removable partition to separate the two personal spaces at will, extending between the cabin wall and the opposite longitudinal wall. 52. Module according to claim 51, wherein the partition is made of plural telescopic sections extensible transversely. 53. Module according to claim 52, wherein the service module is centered in relation to the transverse walls and is arranged along a longitudinal wall, and wherein the telescopic sections fold up toward the service module. 54. Module according to claim 53, wherein the telescopic sections are housed in folded position in the service module. 55. Module according to claim 54, wherein the service module comprises a transverse separation wall having a housing to accommodate the telescopic sections of the movable partition. 56. Module according to claim 51, wherein the movable partition comprises a transparent portion. 57. Module according to claim 40, wherein the transparent portions implemented in the at least one of the two transverse walls can be obscured. 58. Module according to claim 57, wherein at least one transparent portion comprises a liquid crystal layer to be subjected to a difference of electric potential, the liquid crystal layer being opaque or translucent depending on the difference of potential applied. 59. Module according to claim 57, wherein at least one transparent portion is equipped with a shading element. 60. Module according to claim 39, wherein the service module is a bathroom facility having at least one water source. 61. Module according to claim 60, wherein the bathroom facility is equipped with a central washbasin. 62. Module according to claim 60, wherein the bathroom facility is equipped with two access doors, each personal space comprising a door for access to the bathroom facility. 63. Module according to claim 62, wherein the bathroom facility comprises a washbasin for each personal space. 64. Module according to claim 63, wherein each washbasin is movable between a retracted position inside the bathroom facility and an extended position outside the bathroom facility. 65. Module according to claim 64, wherein opening of the access door for access to the bathroom facility controls changeover of the washbasin from its retracted position to its extended position. 66. Module according to claim 60, wherein the bathroom facility is equipped with a shower. 67. Module according to claim 39, wherein the service module is a storage space. 68. Module according to claim 39, wherein the service module is a medical care module. 69. Module according to claim 39, wherein the service module is a pantry unit equipped for passengers' refreshment. 70. Module according to claim 39, wherein the two transverse walls, the service module, and the at least one seat are each equipped with means for their attachment on longitudinal attachment rails arranged on a floor of the cabin. 71. Module according to claim 39, wherein the module has a length in the longitudinal direction ranging between 2.0 m and 4.0 m, and a width on the floor, in the transverse direction, ranging between 2.3 m and 3.5 m. 72. Series of modules according to claim 39, aligned one beside another along a longitudinal aisle, wherein each transverse module wall, possibly except for a transverse wall located at one end of the series of modules, comprises a transparent portion, and wherein the transparent portions are aligned. 73. Series according to claim 72, wherein the seat of each module of the series of modules has a configuration is positioned parallel to the aisle and located more or less in alignment with the transparent portions. 74. Section of an aircraft cabin, comprising at least one module according to claim 39. 75. Section of an aircraft cabin according to claim 74, comprising a central aisle on either side of which is at least one module. 76. Aircraft, comprising at least one module according to claim 39. |
Dwf12 and mutants thereof |
DWARF12(DWF12) sequences, mutants and methods of using the same are disclosed. The dwf12 polynucleotides can be used in the production of transgenic plants which display at least one dwf12 mutant phenotype, so that the resulting plants have altered biochemistry, structure or morphology. |
1-47. (Canceled) 48. An isolated polypeptide having at least 97% sequence identity to SEQ ID NO: 13. 49. An isolated polypeptide having the sequence set forth in SEQ ID NO: 13 and at least one mutation, said mutation comprising at least one non-conservative substitution, addition or deletion of an amino acid in a region spanning residues 46-69, residue 69, residues 70-77, residues 104-106, residues 157-172, residues 183-189, residues 187-189, residues 197-200, residues 199-204, residues 218-228, residues 220-223, residues 261-264, residues 310-312, residues 314-317, residues 316-327, residues 353-356, or residues 376-379. 50. The polypeptide of claim 49, wherein said mutation is in said region spanning residues 104-106, residues 220-223, residues 261-264, residues 314-317, or residues 353-356. 51. The polypeptide of claim 50, wherein said mutation comprises a substitution of a basic amino acid residue for an acidic amino acid residue. 52. The polypeptide of claim 51, wherein said mutation comprises a substitution of a Lys, Arg or His residue for an Asp or Glu residue. 53. The polypeptide of claim 50, wherein said mutation is in said region spanning residues 261-264. 54. The polypeptide of claim 53, wherein said mutation comprises substitution of a Lys residue at Glu-263 or Glu-264. 55. The polypeptide of claim 48, wherein said mutation is in said region spanning residues 187-189 or said region spanning residues 310-312. 56. The polypeptide of claim 55, wherein said mutation comprises a substitution of a basic amino acid residue for an acidic amino acid residue. 57. The polypeptide of claim 56, wherein said mutation comprises substitution of a Lys, Arg or His residue for an Asp or Glu residue. 58. An isolated polynucleotide comprising a coding sequence for the polypeptide of claim 48. 59. An isolated polynucleotide comprising a coding sequence for the polypeptide of claim 49. 60. The polynucleotide of claim 58, further comprising one or more tissue-specific or inducible control elements operably linked to said coding sequence. 61. The polynucleotide of claim 60, wherein said one or more control elements comprise a tissue-specific promoter. 62. The polynucleotide of claim 61, wherein said tissue-specific promoter is operably linked in sense orientation to said coding sequence. 63. The polynucleotide of claim 61, wherein said tissue-specific promoter is operably linked in antisense orientation to said coding sequence. 64. The polynucleotide of claim 59, further comprising one or more tissue-specific or inducible control elements operably linked to said coding sequence. 65. The polynucleotide of claim 64, wherein said one or more control elements comprise a tissue-specific promoter. 66. The polynucleotide of claim 65, wherein said tissue-specific promoter is operably linked in sense orientation to said coding sequence. 67. The polynucleotide of claim 65, wherein said tissue-specific promoter is operably linked in antisense orientation to said coding sequence. 68. A method of producing a polypeptide comprising: (a) providing a population of host cells comprising the polynucleotide of claim 60; and (b) culturing said host cells under conditions whereby said polypeptide encoded by said coding sequence is expressed. 69. A transgenic plant having a recombinant nucleic acid construct comprising the polynucleotide of claim 60. 70. A transgenic plant having a recombinant nucleic acid construct comprising the polynucleotide of claim 64. 71. A method of making a transgenic plant comprising introducing the polynucleotide of claim 62 into a plant cell to produce a transformed plant cell and producing a transgenic plant from said transformed plant cell, wherein said transgenic plant has an altered phenotype relative to a corresponding wild-type plant. 72. The method of claim 71, wherein altered phenotype is elongated leaf blades. 73. A method of making a transgenic plant comprising introducing the polynucleotide of claim 63 into a plant cell to produce a transformed plant cell and producing a transgenic plant from said transformed plant cell, wherein said transgenic plant has an altered phenotype relative to a corresponding wild-type plant. 74. The method of claim 71, wherein altered phenotype is reduced fertility. 75. A method of modulating a phenotype in a transgenic plant, said method comprising expressing the polynucleotide of claim 62 in a plant, said transgenic plant having an altered phenotype relative to a corresponding wild-type plant. 76. A method of modulating a phenotype in a transgenic plant, said method comprising expressing the polynucleotide of claim 63 in a plant, said transgenic plant having an altered phenotype relative to a corresponding wild-type plant. 77. A method of modulating a phenotype in a transgenic plant, said method comprising expressing the polynucleotide of claim 67 in a plant, said transgenic plant having an altered phenotype relative to a corresponding wild-type plant. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The brassinosteroids (BRs) are a group of plant steroid hormones that help regulate many different aspects of plant growth and development. BRs are known to stimulate cell elongation and division, and are also involved in vascular system differentiation, reproduction, and stress responses (Altmann, 1998; Clouse and Sasse, 1998). Recently, it has been shown that mutants defective either in BR biosynthesis or signaling, display altered developmental phenotypes including dwarfism, reduced fertility, and abnormal vasculature (Clouse and Feldmann, 1999). BR dwarf mutants can be divided into two classes. The first class of mutants is perturbed in BR signaling. For example, Clouse, et al., (1996) isolated a signaling mutant, brassinosteroid insensitive1 (bri1), that was resistant to exogenously applied BRs. BRI1 has been cloned and shown to encode a leucine-rich repeat receptor kinase, suggesting a role for BR perception at the cellular membrane (Li and Chory, 1997). Recently, it was demonstrated that bri1 mutants accumulate significant amounts of brassinolide (BL) and its precursors compared to wild-type controls, suggesting that perception is coupled to homeostasis of endogenous BR levels (Noguchi, et al., 1999b). The other class of BR mutants includes a large number of dwarfs that are defective in BR biosynthesis. Plants produce BRs using sterols as precursors, and the sterol biosynthetic pathway uses mevalonic acid as a precursor to synthesize sterols, such as sitosterol, stigmasterol, and campesterol. Sterols are modified by the BR-specific pathway to produce the end product, BL, and its congeners. Thus, mutants that are defective in either the sterol or BR-specific pathway display a typical BR dwarf phenotype, and can be rescued to a wild-type phenotype by exogenous application of BRs. The characteristic phenotype of BR dwarf mutants has been instrumental in isolating additional mutants, and their corresponding genes perturbed in the complex plant sterol biosynthesis network. dwf1 was the first mutant isolated to have this dwarf phenotype (Feldmann, et al., 1989). The dwf1 mutant is defective in C-24 reduction, and DWF1 encodes a FAD-binding oxidoreductase (Choe, et al., 1999a; Klahre, et al., 1998; Takahashi, et al., 1995). The pea lkb mutant is deficient in the same reaction as Arabidopsis dwf1 (Nomura, et al., 1999). Another sterol mutant, Arabidopsis dwf7/ste1, has been isolated and found to be defective in the Δ 7 sterol C-5 desaturase gene (Gachotte, et al., 1995; 1996, Husselstein, et al., 1999, Choe, et al., 1999b). Currently, little is known about the downstream events that occur in response to signals in the above pathways that ultimately control cell size. This is because the biochemical and cell biological processes involved have thus far been difficult to address. In addition, there is little information about the integration of regulatory signals converging at the cell from different signaling pathways and the ways they are coordinately controlled. In particular, the interaction of light and hormones in the control of cell elongation is not clear. Thus, there remains a need for the identification and characterization of additional mutants, and polypeptides encoded thereby, of enzymes involved in these pathways of plant growth. Eukaryotic protein kinases are an extensive family of enzymes, many of which mediate the response of eukaryotic cells to external stimuli. One type of protein kinase, known as “SHAGGY,” is widespread in the plant kingdom and is a serine/threonine protein kinase which is homologous to the mammalian glycogen synthase kinase-3 (GSK-3). Plant homologs of GSK-3 have been found in such divergent plant species as Arabidopsis (Bianchi et al., Mol. Gen. Genet. (1994) 242:337-345; Jonak et al., Plant. Mol. Biol. (1995) 27:217-221; Dornelas et al., Plant Physiol. (1997) 113:306) Medicago (Pay et al., Plant J. (1993) 3:847-856), Nicotiana (Elinzenberger et al., Biochem. Biophys. Acta (1995) 1260:315-319); and Petunia (Decroocq-Ferrant et al., Plant J. (1995) 7:897-911), among others. Despite the widespread occurrence of SHAGGY protein kinases in higher plants, very little is known about the role these proteins play in plant development. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is based on the discovery of various mutants in a BR signaling pathway featuring the kinase designated DWARF12 (DWF12) herein. The DWF12 protein has been characterized as a SHAGGY protein kinase. dwf12 mutants are phenotypically similar to other reported BR mutants, displaying phenotypes such as short stature, short round leaves, reduced fertility and fecundity, and abnormal de-etiolation. dwf12 mutants also have a unique phenotype, severe downward curling of leaves. Without being bound by a particular theory, DWF12 appears to act downstream of BRI in a phosphorylation cascade, ultimately leading to the activation of BR-dependent transcriptional events. Accordingly, in one embodiment, the present invention is directed to an isolated dwf12 mutant polypeptide with at least about 70% sequence identity to the polypeptide depicted at positions 38-326 or 1-380 of FIG. 3 , wherein said polypeptide comprises a mutation comprising at least one non-conservative substitution, addition or deletion of an amino acid in a region of the polypeptide depicted in FIG. 3 , wherein said region is one or more of (a) a casein kinase II phosphorylation domain spanning positions 104-106 of FIG. 3 ; (b) a casein kinase II phosphorylation domain spanning positions 220-223 of FIG. 3 ; (c) a casein kinase II phosphorylation domain spanning positions 261-264 of FIG. 3 ; (d) a casein kinase II phosphorylation domain spanning positions 314-317 of FIG. 3 ; (e) a casein kinase II phosphorylation domain spanning positions 353-356 (f) a protein kinase C phosphorylation domain spanning positions 104-106 of FIG. 3 ; (g) a protein kinase C phosphorylation domain spanning positions 187-189 of FIG. 3 ; (h) a protein kinase C phosphorylation domain spanning positions 310-312; (i) the active site lysine residue found at position 69 of FIG. 3 ; (j) a tyrosine phosphorylation domain spanning positions 70-77 of FIG. 3 ; (k) a protein kinase ATP-binding domain spanning positions 46-69 of FIG. 3 ; (l) a eukaryotic protein kinase ATP-binding domain spanning positions 157-172 of FIG. 3 ; (m) a eukaryotic protein kinase substrate-binding domain spanning positions 218-228 of FIG. 3 ; (n) a eukaryotic protein kinase signature sequence spanning positions 316-327 of FIG. 3 ; (o) a glycosylation domain spanning positions 197-200 of FIG. 3 ; or (p) a glycosylation domain spanning positions 376-379 of FIG. 3 . Additionally, the mutation may occur within the sequence CDFGSAK, found at positions 183-189 of FIG. 3 , as well as the sequence SYICSR, found at positions 199-204 of FIG. 3 . Alternatively, the isolated dwf12 mutant polypeptide has at least about 70% sequence identity to a polypeptide designated in Table 1, and has one or more mutations in a domain corresponding to the domains set forth above. In certain embodiments, the mutation comprises a change in a casein kinase II phosphorylation domain as specified above of an acidic amino acid residue such as Asp or Glu, to a basic amino acid residue, such as Lys, Arg or His. In other embodiments, the mutation comprises a change in a protein kinase C phosphorylation domain as specified above of a basic amino acid residue to an acidic amino acid residue. In additional embodiments, the mutation comprises a mutation to the sequence spanning positions 261-264 of FIG. 3 , such as a change of Glu-263, or Glu-264, to Lys. In another embodiment, the present invention is directed to an isolated dwf12 mutant polynucleotide which encodes a polypeptide as specified above. In certain embodiments, the polynucleotide is (a) a dwf12-1 polynucleotide comprising the dwf12-1 nucleotide sequence depicted in FIGS. 1A-1F ; or (b) a dwf12-2 polynucleotide comprising the dwf12-2 nucleotide sequence depicted in FIGS. 1A-1F . In certain embodiments, the isolated polynucleotide imparts at least one dwf12 mutant phenotype when expressed in a plant. The altered phenotype may be any microscopic or macroscopic change in structure or morphology, as well as biochemical differences, which are characteristic of a dwf12 plant, as compared to a progenitor, wild-type plant cultivated under the same conditions. Biochemical differences include reduced or increased SHAGGY kinase activity as compared to the wild-type polypeptide. Reduced activity, for example, may in turn result an accumulation of significant amounts of BRs, such as brassinolide. Generally, morphological differences include a short robust stature, short internodes, an increased number of inflorescences, and small dark-green, round leaves. Particularly unique to dwf12 plants is the presence of severe downward curling of leaves. Moreover, plants may have short siliques and be infertile. In a further embodiment, the invention is directed to a recombinant vector comprising (i) a polynucleotide as described above; and (ii) control elements operably linked to said polynucleotide whereby a coding sequence within said polynucleotide can be transcribed and translated in a host cell, as well as host cells comprising the vector. In still further embodiments, the invention is directed to a method of producing a recombinant polypeptide comprising: (a) providing a host cell as specified above; and (b) culturing said host cell under conditions whereby a recombinant polypeptide encoded by the coding sequence present in said recombinant vector is expressed. In another embodiment, the invention is directed to a transgenic plant comprising a dwf12 mutant polynucleotide, wherein said mutant polynucleotide encodes for a dwf12 mutant polypeptide having a mutation as described above. In certain embodiments, the dwf12 polynucleotide is dwf12-1 or dwf12-2, described above. In still another embodiment, the invention is directed to a method of producing a transgenic plant comprising: (a) introducing a dwf12 mutant polynucleotide, or a recombinant vector, as described above, into a plant cell to produce a transformed plant cell; and (b) producing a transgenic plant from the transformed plant cell. In another embodiment, the invention is directed to a method for producing a transgenic plant having an altered phenotype relative to the corresponding wild-type plant comprising: (a) introducing a dwf12 mutant polynucleotide, or recombinant vector as described above into a plant cell; and (b) producing a transgenic plant from the plant cell, said transgenic plant having an altered phenotype relative to the corresponding wild-type plant. In certain embodiments, the polynucleotide is operably linked to a promoter selected from the group consisting of a tissue-specific promoter, an inducible promoter and a constitutive promoter. Additionally, the polynucleotide may be overexpressed or may inhibit expression of DWF12. Additionally, the method may be one wherein at least first and second polynucleotides are introduced into the plant cell, said first and second polynucleotides operably linked to at least first and second tissue-specific promoters, wherein said first polynucleotide is overexpressed and said second polynucleotide inhibits expression of DWF12. In still further embodiments, the invention is directed to a method of modulating an endogenous DWF12 polypeptide in a transgenic plant, and hence SHAGGY kinase activity, said method comprising providing a polynucleotide as described above. The polynucleotide may be overexpressed or expression of the polynucleotide may be inhibited. In another embodiment the invention is directed a method for altering the biochemical activity, such as SHAGGY kinase activity, of a cell comprising: (a) introducing at least one polynucleotide as described above into the cell; and (b) causing expression of said polynucleotide such that the biochemical activity of the cell is altered. The polynucleotide may be introduced into the cell ex vivo or in vivo. Additionally, more than one dwf12 polynucleotide may be provided to the cell. In yet a further embodiment, the subject invention is directed to a method for modulating a trait in a plant, wherein said trait is selected from the group consisting of (a) biomass; (b) height; (c) tissue or organ size, such as but not limited to leaf, seed, root, flower, stem, petioles, internodes, hypocotyl size; (d) fertility; (e) fecundity; (f) leaf curling, including the rosette and/or cauline; (g) cell elongation; and (h) stress tolerance. In certain embodiments, the trait is modulated by modulating expression of an endogenous SHAGGY kinase by inserting a polynucleotide into a cell wherein said polynucleotide encodes for a SHAGGY kinase exhibiting reduced activity as compared to the corresponding endogenous SHAGGY kinase, such as a polynucleotide that encodes a mutant dwf12 polypeptide with reduced activity. Alternatively, the mutant dwf12 polypeptide may have enhanced activity, e.g., due to mutations in, for example, a phosphorylation site. The polynucleotide may be heterologous to the cell, i.e., it may be derived from a different plant species than the cell, and/or the polynucleotide may comprise multiple copies of the mutant SHAGGY kinase. The SHAGGY kinase modulated is preferably one involved in a BR signaling pathway, such as DWF12 or a homologue thereof. The polynucleotide may also encode a SHAGGY kinase exhibiting at least the same or enhanced activity as compared to the corresponding endogenous SHAGGY kinase, such as a polynucleotide that encodes a mutant dwf12 polypeptide with enhanced activity or a homologue thereof. Moreover, the polynucleotide may be one that is capable of hybridizing to the endogenous SHAGGY kinase mRNA. The polynucleotide may be a mutant dwf12 polynucleotide as specified above. These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein. |
Interior configuration for an aircraft cabin |
This layout comprises a set (18) of aircraft seats arranged in an aircraft cabin. Each set comprises two rows of seats (8) arranged transversely in relation to the longitudinal axis (2) of the cabin in which they are located and a single longitudinal aisle (16) allows access to the transverse rows. The two transverse rows are arranged opposite one another and a transverse aisle (20) separates the two rows of seats (8). In each row of seats, at least one seat (8) is located on one side of the longitudinal aisle (16) and at least two seats (8) are located on the other side of the longitudinal aisle (16). |
1-12. (Canceled). 13. A set of aircraft seats in an aircraft cabin comprising: two rows of seats arranged transversely in relation to a longitudinal axis of the cabin in which they are located, a single longitudinal aisle allowing access to the transverse rows, wherein the two transverse rows are arranged opposite one another, a transverse aisle separates the two rows of seats and in each row of seats, at least one seat is located on one side of the longitudinal aisle, and at least two seats are located on the other side of the longitudinal aisle. 14. A set of aircraft seats according to claim 13, wherein each seat is positioned parallel to an axis of the longitudinal aisle. 15. A set of aircraft seats according to claim 13, further comprising, combined with each seat, an appurtenance arranged facing and at a distance from the seat, with a lesser width than that of the seat. 16. A set of aircraft seats according to claim 15, wherein the appurtenance comprises a support surface arranged at a height more or less equal to that of a sitting surface of the seat, within about ten centimeters. 17. A set of aircraft seats according to claim 15, wherein each seat cooperates with the appurtenance arranged facing it to make a bedding surface. 18. A set of aircraft seats according to claim 13, wherein each seat comprises a sitting surface, a back and two armrests, and the seat is surrounded on its back and armrest side by a lateral wall serving as a divider with a neighboring seat. 19. A set of aircraft seats according to claim 15, wherein each seat comprises a sitting surface, a back, and two armrests, and a lateral wall surrounds each seat on its back and armrest side, as well as the appurtenance combined with the seat, and at least one opening is provided in the lateral wall for accessing the seat. 20. A set of aircraft seats according to claim 19, wherein each appurtenance is more or less centered on the longitudinal axis of the corresponding seat, and each lateral wall has an opening between the seat and the appurtenance on each of its sides located facing a neighboring seat. 21. A set of aircraft seats according to claim 20, wherein each lateral wall has two openings of identical width, one on each side of the corresponding seat, and a door is provided to close one of the two openings. 22. A set of aircraft seats according to claim 19, wherein the lateral wall comprises a first side more or less parallel to the longitudinal aisle, the seat and the appurtenance are arranged against this first side, wherein a second side of the wall, opposite the first side, widens out in relation to this first side starting from the appurtenance, and wherein the opening for accessing the seat is implemented in the second side. 23. A set of aircraft seats according to claim 22, comprising a first type of seat for which the first side of the lateral wall, that is more or less parallel to the longitudinal aisle, is to the right of a passenger sitting in the seat, and a second type of seat for which the first side of the wall is left of a passenger sitting in the seat, wherein the seats are arranged so that for the seats near a cabin wall, the first side of the corresponding wall faces the cabin wall and, in a transverse row of seats, between the cabin wall and the longitudinal aisle, the types of seats alternate from one seat to the other. 24. An aircraft, comprising at least one set of seats according to claim 13. |
Receptors and membrane-associated proteins |
The invention provides human receptors and membrane-associated proteins (REMAP) and polynucleotides which identify and encode REMAP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of REMAP. |
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:1-26, 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:1-6, SEQ ID NO:8-12, SEQ ID NO:15, and SEQ ID NO:17-25, c) a polypeptide comprising a naturally occurring amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:7, d) a polypeptide comprising a naturally occurring amino acid sequence at least 94% identical to the amino acid sequence of SEQ ID NO:13, e) a polypeptide comprising a naturally occurring amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:14, f) a polypeptide comprising a naturally occurring amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:16, g) a polypeptide consisting essentially of a naturally occurring amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:26, h) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26, and i) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26. 2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-26. 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:27-52. 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:1-26. 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:27-52, 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:27-32 and SEQ ID NO:34-52, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO:33, d) a polynucleotide complementary to a polynucleotide of a), e) a polynucleotide complementary to a polynucleotide of b), f) a polynucleotide complementary to a polynucleotide of c), and g) an RNA equivalent of a)-f). 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:1-26. 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. 30-107. (Canceled). |
<SOH> BACKGROUND OF THE INVENTION <EOH>Eukaryotic organisms are distinct from prokaryotes in possessing many intracellular membrane-bound compartments such as organelles and vesicles. Many of the metabolic reactions which distinguish eukaryotic biochemistry from prokaryotic biochemistry take place within these compartments. In particular, many cellular functions require very stringent reaction conditions, and the organelles and vesicles enable compartmentalization and isolation of reactions which might otherwise disrupt cytosolic metabolic processes. The organelles include mitochondria, smooth and rough endoplasmic reticula, sarcoplasmic reticulum, and the Golgi body. The vesicles include phagosomes, lysosomes, endosomes, peroxisomes, and secretory vesicles. Organelles and vesicles are bounded by single or double membranes. Signal transduction is the general process by which cells respond to extracellular signals. Signal transduction across the plasma membrane begins with the binding of a signal molecule, e.g., a hormone, neurotransmitter, or growth factor, to a cell membrane receptor. The receptor, thus activated, triggers an intracellular biochemical cascade that ends with the activation of an intracellular target molecule, such as a transcription factor. This process of signal transduction regulates all types of cell functions including cell proliferation, differentiation, and gene transcription. Biological membranes surround organelles, vesicles, and the cell itself. Membranes are highly selective permeability barriers made up of lipid bilayer sheets composed of phosphoglycerides, fatty acids, cholesterol, phospholipids, glycolipids, proteoglycans, and proteins. Membranes contain ion pumps, ion channels, and specific receptors for external stimuli which transmit biochemical signals across the membranes. These membranes also contain second messenger proteins which interact with these pumps, channels, and receptors to amplify and regulate transmission of these signals. Plasma Membrane Proteins Plasma membrane proteins (MPs) are divided into two groups based upon methods of protein extraction from the membrane. Extrinsic or peripheral membrane proteins can be released using extremes of ionic strength or pH, urea, or other disruptors of protein interactions. Intrinsic or integral membrane proteins are released only when the lipid bilayer of the membrane is dissolved by detergent. The majority of known integral membrane proteins are transmembrane proteins (TM) which are characterized by an extracellular, a transmembrane, and an intracellular domain. TM domains are typically comprised of 15 to 25 hydrophobic amino acids which are predicted to adopt an α-helical conformation. TM proteins are classified as bitopic (Types I and II) and polytopic (Types III and IV) (Singer, S. J. (1990) Annu. Rev. Cell Biol. 6:247-96). Bitopic proteins span the membrane once while polytopic proteins contain multiple membrane-spanning segments. TM proteins carry out a variety of important cellular functions, including acting as cell-surface receptor proteins involved in signal transduction. These functions are represented by growth and differentiation factor receptors, and receptor-interacting proteins such as Drosophila pecanex and frizzled proteins, LIV-1 protein, NF2 protein, and GNS1/SUR4 eukaryotic integral membrane proteins. TM proteins also act as transporters of ions or metabolites, such as gap junction channels (connexins), and ion channels, and as cell anchoring proteins, such as lectins, integrins, and fibronectins. TM proteins are found in vesicle organelle-forming molecules, such as caveolins; or cell recognition molecules, such as cluster of differentiation (CD) antigens, glycoproteins, and mucins. Many MPs contain amino acid sequence motifs that serve to localize proteins to specific subcellular sites. Examples of these motifs include PDZ domains, KDEL, RGD, NGR, and GSL sequence motifs, von Willebrand factor A (vWFA) domains, and EGF-like domains. RGD, NGR, and GSL motif-containing peptides have been used as drug delivery agents in targeted cancer treatment of tumor vasculature (Arap, W. et al. (1998) Science, 279:377-380). Furthermore, MPs may also contain amino acid sequence motifs that serve to interact with extracellular or intracellular molecules, such as carbohydrate recognition domains (CRD). Chemical modification of amino acid residue side chains alters the manner in which MPs interact with other molecules, for example, phospholipid membranes. Examples of such chemical modifications to amino acid residue side chains are covalent bond formation with glycosaminoglycans, oligosaccharides, phospholipids, acetyl and palmitoyl moieties, ADP-ribose, phosphate, and sulphate groups. RNA encoding membrane proteins may have alternative splice sites which give rise to proteins encoded by the same gene but with different messenger RNA and amino acid sequences. Splice variant membrane proteins may interact with other ligand and protein isoforms. Receptors The term receptor describes proteins that specifically recognize other molecules. The category is broad and includes proteins with a variety of functions. The bulk of receptors are cell surface proteins which bind extracellular ligands and produce cellular responses in the areas of growth, differentiation, endocytosis, and immune response. Other receptors facilitate the selective transport of proteins out of the endoplasmic reticulum and localize enzymes to particular locations in the cell. The term may also be applied to proteins which act as receptors for ligands with known or unknown chemical composition and which interact with other cellular components. For example, the steroid hormone receptors bind to and regulate transcription of DNA. Cell surface receptors are typically integral plasma membrane proteins. These receptors recognize hormones such as catecholamines; peptide hormones; growth and differentiation factors; small peptide factors such as thyrotropin-releasing hormone; galanin, somatostatin, and tachykinins; and circulatory system-borne signaling molecules. Cell surface receptors on immune system cells recognize antigens, antibodies, and major histocompatibility complex (MHC)-bound peptides. Other cell surface receptors bind ligands to be internalized by the cell. This receptor-mediated endocytosis functions in the uptake of low density lipoproteins (LDL), transferrin, glucose- or mannose-terminal glycoproteins, galactose-terminal glycoproteins, immunoglobulins, phosphovitellogenins, fibrin, proteinase-inhibitor complexes, plasminogen activators, and thrombospondin (Lodish, H. et al. (1995) Molecular Cell Biology, Scientific American Books, New York N.Y., p. 723; Mikhailenko, I. et al. (1997) J. Biol. Chem 272:6784-6791). Receptor Protein Kinases Many growth factor receptors, including receptors for epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, as well as the growth modulator α-thrombin, contain intrinsic protein kinase activities. When growth factor binds to the receptor, it triggers the autophosphorylation of a serine, threonine, or tyrosine residue on the receptor. These phosphorylated sites are recognition sites for the binding of other cytoplasmic signaling proteins. These proteins participate in signaling pathways that eventually link the initial receptor activation at the cell surface to the activation of a specific intracellular target molecule. In the case of tyrosine residue autophosphorylation, these signaling proteins contain a common domain referred to as a Src homology (SH) domain. SH2 domains and SH3 domains are found in phospholipase C-γ, PI-3-K p85 regulatory subunit, RasGTPase activating protein, and pp60 c-src (Lowenstein, E. J. et al. (1992) Cell 70:431-442). The cytoline family of receptors share a different common binding domain and include transmembrane receptors for growth hormone (GH), interleukins, erythropoietin, and prolactin. Other receptors and second messenger-binding proteins have intrinsic serine/threonine protein kinase activity. These include activin/TGF-β/BMW-superfamily receptors, calcium- and diacylglycerol-activated/phospholipid-dependant protein kinase (PK-C), and RNA-dependant protein kinase (PK-R). In addition, other serine/threonine protein kinases, including nematode Twitchin, have fibronectin-like, immunoglobulin C2-like domains. G-protein Coupled Receptors The G-protein coupled receptors (GPCRs), encoded by one of the largest families of genes yet identified, play a central role in the transduction of extracellular signals across the plasma membrane. GPCRs have a proven history of being successful therapeutic targets. GPCRs are integral membrane proteins characterized by the presence of seven hydrophobic transmembrane domains which together form a bundle of antiparallel alpha (α) helices. GPCRs range in size from under 400 to over 1000 amino acids (Strosberg, A. D. (1991) Eur. J. Biochem. 196: 1-10; Coughlin, S. R. (1994) Curr. Opin. Cell Biol. 6:191-197). The amino-terminus of a GPCR is extracellular, is of variable length, and is often glycosylated. The carboxy-terminus is cytoplasmic and generally phosphorylated. Extracellular loops alternate with intracellular loops and link the tnansmembrane domains. Cysteine disulfide bridges linking the second and third extracellular loops may interact with agonists and antagonists. The most conserved domains of GPCRs are the transmembrane domains and the first two cytoplasmic loops. The transmembrane domains account, in part, for structural and functional features of the receptor. In most cases, the bundle of a helices forms a ligand-binding pocket. The extracellular N-terminal segment, or one or more of the three extracellular loops, may also participate in ligand binding. Ligand binding activates the receptor by inducing a conformational change in intracellular portions of the receptor. In turn, the large, third intracellular loop of the activated receptor interacts with a heterotrimeric guanine nucleotide binding (G) protein complex which mediates further intracellular signaling activities, including the activation of second messengers such as cyclic AMP (cAMP), phospholipase C, and inositol triphosphate, and the interaction of the activated GPCR with ion channel proteins. (See, e.g., Watson, S. and S. Arkinstall (1994) The G - protein Linked Receptor Facts Book, Academic Press, San Diego Calif., pp. 2-6; Bolander, F. F. (1994) Molecular Endocrinology, Academic Press, San Diego Calif., pp. 162-176; Baldwin, J. M. (1994) Curr. Opin. Cell Biol. 6:180-190.) GPCRs include receptors for sensory signal mediators (e.g., light and olfactory stimulatory molecules); adenosine, γ-aminobutyric acid (GABA), hepatocyte growth factor, melanocortins, neuropeptide Y, opioid peptides, opsins, somatostatin, tachykinins, vasoactive intestinal polypeptide family, and vasopressin; biogenic amines (e.g., dopamine, epinephrine and norepinephrine, histamine, glutamate (metabotropic effect), acetylcholine (muscarinic effect), and serotonin); chemokines; lipid mediators of inflammation (e.g., prostaglandins and prostanoids, platelet activating factor, and leukotrienes); and peptide hormones (e.g., bombesin, bradykinin, calcitonin, C5a anaphylatoxin, endothelin, follicle-stimulating hormone (FSI), gonadotropic-releasing hormone (GnRH), neurokinin, and thyrotropin-releasing hormone (TRH), and oxytocin). GPCRs which act as receptors for stimuli that have yet to be identified are known as orphan receptors. The diversity of the GPCR family is further increased by alternative splicing. Many GPCR genes contain introns, and there are currently over 30 such receptors for which splice variants have been identified. The largest number of variations are at the protein C-terminus. N-terminal and cytoplasmic loop variants are also frequent, while variants in the extracellular loops or transmembrane domains are less common. Some receptors have more than one site at which variance can occur. The splicing variants appear to be functionally distinct, based upon observed differences in distribution, signaling, coupling, regulation, and ligand binding profiles (Kilpatrick, G. J. et al. (1999) Trends Pharmacol. Sci. 20:294-301). GPCRs can be divided into three major subfamilies: the rhodopsin-like, secretin-like, and metabotropic glutamate receptor subfamilies. Members of these GPCR subfamilies share similar functions and the characteristic seven transmembrane structure, but have divergent amino acid sequences. The largest family consists of the rhodopsin-like GPCRs, which transmit diverse extracellular signals including hormones, neurotransmitters, and light. Rhodopsin is a photosensitive GPCR found in animal retinas. In vertebrates, rhodopsin molecules are embedded in membranous stacks found in photoreceptor (rod) cells. Each rhodopsin molecule responds to a photon of light by triggering a decrease in cGMP levels which leads to the closure of plasma membrane sodium channels. In this manner, a visual signal is converted to a neural impulse. Other rhodopsin-like GPCRs are directly involved in responding to neurotransmitters. These GPCRs include the receptors for adrenaline (adrenergic receptors), acetylcholine (muscarinic receptors), adenosine, galanin, and glutamate (N-methyl-D-aspartate/NMDA receptors). (Reviewed in Watson, S. and S. Arkinstall (1994) The G - Protein Linked Receptor Facts Book, Academic Press, San Diego Calif., pp. 7-9, 19-22, 32-35, 130-131, 214-216, 221-222; Habert-Ortoli, E. et al. (1994) Proc. Natl. Acad. Sci. USA 91:9780-9783.) The galanin receptors mediate the activity of the neuroendocrine peptide galanin, which inhibits secretion of insulin, acetylcholine, serotonin and noradrenaline, and stimulates prolactin and growth hormone release. Galanin receptors are involved in feeding disorders, pain, depression, and Alzheimer's disease (Kask, K. et al. (1997) Life Sci. 60:1523-1533). Other nervous system rhodopsin-like GPCRs include a growing family of receptors for lysophosphatidic acid and other lysophospholipids, which appear to have roles in development and neuropathology (Chun, J. et al. (1999) Cell Biochem. Biophys. 30:213-242). The largest subfamily of GPCRS, the olfactory receptors, are also members of the rhodopsin-like GPCR family. These receptors function by transducing odorant signals. Numerous distinct olfactory receptors are required to distinguish different odors. Each olfactory sensory neuron expresses only one type of olfactory receptor, and distinct spatial zones of neurons expressing distinct receptors are found in nasal passages. For example, the RA1c receptor which was isolated from a rat brain library, has been shown to be limited in expression to very distinct regions of the brain and a defined zone of the olfactory epithelium (Raming, K. et al. (1998) Receptors Channels 6:141-151). However, the expression of olfactory-like receptors is not confined to olfactory tissues. For example, three rat genes encoding olfactory-like receptors having typical GPCR characteristics showed expression patterns not only in taste and olfactory tissue, but also in male reproductive tissue (Thomas, M. B. et al. (1996) Gene 178:1-5). GPCR mutations, which may cause loss of function or constitutive activation, have been associated with numerous human diseases (Coughlin, supra). For instance, retinitis pigmentosa may arise from mutations in the rhodopsin gene. Furthermore, somatic activating mutations in the thyrotropin receptor have been reported to cause hyperfunctioning thyroid adenomas, suggesting that certain GPCRs susceptible to constitutive activation may behave as protooncogenes (Parma, J. et al. (1993) Nature 365:649651). GPCR receptors for the following ligands also contain mutations associated with human disease: luteinizing hormone (precocious puberty); vasopressin V 2 (X-linked nephrogenic diabetes); glucagon (diabetes and hypertension); calcium (hyperparathyroidism, hypocalcuria, hypercalcemia); parathyroid hormone (short limbed dwarfism); β 3 -adrenoceptor (obesity, non-insulin-dependent diabetes meritus); growth hormone releasing hormone (dwarfism); and adrenocorticotropin (glucocorticoid deficiency) (Wilson, S. et al. (1998) Br. J. Pharmocol. 125:1387-1392; Stadel, J. M. et al. (1997) Trends Pharmacol. Sci. 18:430-437). GPCRs are also involved in depression, schizophrenia, sleeplessness, hypertension, anxiety, stress, renal failure, and several cardiovascular disorders (Horn, F. and G. Vriend (1998) J. Mol. Med. 76:464-468). Mutations and changes in transcriptional activation of GPCR-encoding genes have been associated with neurological disorders such as schizophrenia, Parkinson's disease, Alzheimer's disease, drug addiction, and feeding disorders. The juvenile development and fertility-2 (jdf-2) locus, also called runty-jerky-sterile (ijs), is associated with deletions and point mutations in HBERC2, a gene encoding a guanine nucleotide exchange factor protein involved in vesicular trafficking (Walkowicz, M. et al. (1999) Mamm. Genome 10:870-878). In addition, within the past 20 years several hundred new drugs have been recognized that are directed towards activating or inhibiting GPCRs. The therapeutic targets of these drugs span a wide range of diseases and disorders, including cardiovascular, gastrointestinal, and central nervous system disorders as well as cancer, osteoporosis and endometriosis (Wilson, supra; Stadel, supra). For example, the dopamine agonist L-dopa is used to treat Parkinson's disease, while a dopamine antagonist is used to treat schizophrenia and the early stages of Huntington's disease. Agonists and antagonists of adrenoceptors have been used for the treatment of asthma, high blood pressure, other cardiovascular disorders, and anxiety; muscarinic agonists are used in the treatment of glaucoma and tachycardia; serotonin 5HT1D antagonists are used against migraine; and histamine H1 antagonists are used against allergic and anaphylactic reactions, hay fever, itching, and motion sickness (Horn, supra). Members of the secretin-like GPCR subfamily have as their ligands peptide hormones such as secretin, calcitonin, glucagon, growth hormone-releasing hormone, parathyroid hormone, and vasoactive intestinal peptide. For example, the secretin receptor responds to secretin, a peptide hormone that stimulates the secretion of enzymes and ions in the pancreas and small intestine (Watson, supra, pp. 278-283). Secretin receptors are about 450 amino acids in length and are found in the plasma membrane of gastrointestinal cells. Binding of secretin to its receptor stimulates the production of cAMP. Examples of secretin-like GPCRs implicated in inflammation and the immune response include the EGF module-containing, mucin-like hormone receptor (Emr1) and CD97 receptor proteins. CD97 is predominantly expressed in leukocytes and is markedly upregulated on activated B and T cells (McKnight, A. J. and S. Gordon (1998) J. Leukoc. Biol. 63:271-280). These GPCRs are members of the recently characterized EGF-TM7 receptors subfamily. These seven transmembrane hormone receptors exist as heterodimers in vivo and contain between three and seven potential calcium-binding EGF-like motifs. The EGF motif is about forty amino acid residues in length and includes six conserved cysteine residues, and a calcium-binding site near the N-terminus of the signature sequence. Post-translational hydroxylation of aspartic acid or asparagine residues has been associated with EGF-like domains in several proteins (Prosite PDOC00010 Aspartic acid and asparagine hydroxylation site). The EGF-TM7 family also includes the recently isolated EGF-TM7-latrophilin-related protein (ETL), which is expressed in cardiac myocytes and smooth muscle and is developmentally regulated in the heart. ETL may play a role in effecting the terminal differentiation of cardiac muscle. ETL may also be involved in coronary angiogenesis. (Nechiporuk, T. et al. (2001) J. Biol. Chem. 276:4150-4157). Another GPCR subfamily is the metabotropic glutamate receptor family. Glutamate is the major excitatory neurotransmitter in the central nervous system. The metabotropic glutamate receptors modulate the activity of intracellular effectors, and are involved in long-term potentiation (Watson, supra, p.130). The Ca 2+ -sensing receptor, which senses changes in the extracellular concentration of calcium ions, has a large extracellular domain including clusters of acidic amino acids which may be involved in calcium binding. The metabotropic glutamate receptor family also includes pheromone receptors, the GABAB receptors, and the taste receptors. Other subfamilies of GPCRs include two groups of chemoreceptor genes found in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae, which are distantly related to the mammalian olfactory receptor genes. The yeast pheromone receptors STE2 and STE3, involved in the response to mating factors on the cell membrane, have their own seven-transmembrane signature, as do the cAMP receptors from the slime mold Dictyostelium discoideum, which are thought to regulate the aggregation of individual cells and control the expression of numerous developmentally-regulated genes. Recent research suggests potential future therapeutic uses for GPCRs in the treatment of metabolic disorders including diabetes, obesity, and osteoporosis. For example, mutant V2 vasopressin receptors causing nephrogenic diabetes could be functionally rescued in vitro by co-expression of a C-terminal V2 receptor peptide spanning the region containing the mutations. This result suggests a possible novel strategy for disease treatment (Schöneberg, T. et al. (1996) EMBO J. 15:1283-1291). Mutations in melanocortin-4 receptor (MC4R) are implicated in human weight regulation and obesity. As with the vasopressin V2 receptor mutants, these MC4R mutants are defective in trafficking to the plasma membrane (Ho, G. and R. G. MacKenzie (1999) J. Biol. Chem. 274:35816-35822), and thus might be treated with a similar strategy. The type 1 receptor for parathyroid hormone (PTH) is a GPCR that mediates the PTH-dependent regulation of calcium homeostasis in the bloodstreaim Study of PTH/receptor interactions may enable the development of novel PTH receptor ligands for the treatment of osteoporosis (Mannstadt, M. et al. (1999) Am. J. Physiol. 277:F665-F675). The chemokine receptor group of GPCRs have potential therapeutic utility in inflammation and infectious disease. (For review, see Locati, M. and P. M. Murphy (1999) Annu. Rev. Med. 50:425-440.) Chemokines are small polypeptides that act as intracellular signals in the regulation of leukocyte trafficking, hematopoiesis, and angiogenesis. Targeted disruption of various chemokine receptors in mice indicates that these receptors play roles in pathologic inflammation and in autoimmune disorders such as multiple sclerosis. Chemokine receptors are also exploited by infectious agents, including herpesviruses and the human immunodeficiency virus (HIV-1) to facilitate infection. A truncated version of chemokine receptor CCR5, which acts as a coreceptor for infection of T-cells by HIV-1, results in resistance to AIDS, suggesting that CCR5 antagonists could be useful in preventing the development of AIDS. Interleukins (IL) mediate the interactions between immune and inflammatory cells. Several interleukins have been described; each has unique biological activities as well as some that overlap with the others. Macrophages produce IL-1 and IL-6, whereas T cells produce IL-2, IL-3, IL-4, IL-5 and IL-6 and bone marrow stromal cells produce IL 7. IL 1 and IL 6 not only play important roles in immune cell function, but also stimulate a spectrum of inflammatory cell types. The growth and differentiation of eosinophils is markedly enhanced by IL 5. IL 2 is a potent proliferative signal for T cells, natural killer cells, and lymphokine-activated killer cells. IL 1, IL 3, IL 4, and IL 7 enhance the development of a variety of hematopoietic precursors. IL 4-IL 6 also serve to enhance B cell proliferation and antibody production (Mizel, S. B. (1989) FASEB J. 3:2379-2388). Melatonin scavenges free radicals including the hydroxyl radical (.OH), peroxynitrite anion (ONOO—), and hypochlorous acid (HOCl), as well as preventing the translocation of nuclear factor-kappa B (NF-kappa B) to the nucleus and its binding to DNA, thereby reducing the upregulation of proinflammatory cytokines such as interleukins and tumor neurosis factor-alpha. Melatonin attenuates transendothelial cell migration and edema, which contribute to tissue damage (Reiter, R. J. et al. (2000) Ann. N.Y. Acad. Sci. 917:376-386). Activation of melatonin receptors enhances the release of T-helper cell cytokines, such as gamma-interferon and interleukin-2 (IL-2), as well as activation of opioid cytokines which crossreact immunologically with both interleukin-4 and dynorphin B. Hematopoiesis is influenced by melatonin-induced-opioids acting on kappa 1-opioid receptors present on bone marrow macrophages (Maestroni, G. J. (1999) Adv. Exp. Med. Biol. 467:217-226). Ligand-Gated Receptor Ion Channels Ligand-gated receptor ion channels fall into two categories. The first category, extracellular ligand-gated receptor ion channels (ELGs), rapidly transduce neurotransmitter-binding events into electrical signals, such as fast synaptic neurotransmission. ELG function is regulated by post-translational modification. The second category, intracellular ligand-gated receptor ion channels (ILGs), are activated by many intracellular second messengers and do not require post-translational modification(s) to effect a channel-opening response. ELGs depolarize excitable cells to the threshold of action potential generation. In non-excitable cells, ELGs permit a limited calcium ion-influx during the presence of agonist. ELGs include channels directly gated by neurotransmitters such as acetylcholine, L-glutamate, glycine, ATP, serotonin, GABA, and histamine. ELG genes encode proteins having strong structural and functional similarities. ILGs are encoded by distinct and unrelated gene families and include receptors for cAMP, cGMP, calcium ions, ATP, and metabolites of arachidonic acid. Macrophage Scavenger Receptors Macrophage scavenger receptors with broad ligand specificity may participate in the binding of low density lipoproteins (LDL) and foreign antigens. Scavenger receptors types I and II are trimeric membrane proteins with each subunit containing a small N-terminal intracellular domain, a transmembrane domain, a large extracellular domain, and a C-terminal cysteine-rich domain. The extracellular domain contains a short spacer domain, an α-helical coiled-coil domain, and a triple helical collagenous domain. These receptors have been shown to bind a spectrum of ligands, including chemically modified lipoproteins and albumin, polyribonucleotides, polysaccharides, phospholipids, and asbestos (Matsumoto, A. et al. (1990) Proc. Natl. Acad. Sci. USA 87:9133-9137; Elomaa, O. et al. (1995) Cell 80:603-609). The scavenger receptors are thought to play a key role in atherogenesis by mediating uptake of modified LDL in arterial walls, and in host defense by binding bacterial endotoxins, bacteria, and protozoa. T-Cell Receptors T cells play a dual role in the immune system as effectors and regulators, coupling antigen recognition with the transmission of signals that induce cell death in infected cells and stimulate proliferation of other immune cells. Although a population of T cells can recognize a wide range of different antigens, an individual T cell can only recognize a single antigen and only when it is presented to the T cell receptor (TCR) as a peptide complexed with a major histocompatibility molecule (MHC) on the surface of an antigen presenting cell. The TCR on most T cells consists of immunoglobulin-like integral membrane glycoproteins containing two polypeptide subunits, α and β, of similar molecular weight. Both TCR subunits have an extracellular domain containing both variable and constant regions, a transmembrane domain that traverses the membrane once, and a short intracellular domain (Saito, H. et al. (1984) Nature 309:757-762). The genes for the TCR subunits are constructed through somatic rearrangement of different gene segments. Interaction of antigen in the proper MHC context with the TCR initiates signaling cascades that induce the proliferation, maturation, and function of cellular components of the immune system (Weiss, A. (1991) Annu. Rev. Genet. 25: 487-510). Rearrangements in TCR genes and alterations in TCR expression have been noted in lymphomas, leukemias, autoimmune disorders, and immunodeficiency disorders (Aisenberg, A. C. et al. (1985) N. Engl. J. Med. 313:529-533; Weiss, supra). Netrin Receptors: The netrins are a family of molecules that function as diffusible attractants and repellants to guide migrating cells and axons to their targets within the developing nervous system. The netrin receptors include the C. elegans protein UNC-5, as well as homologues recently identified in vertebrates (Leonardo, E. D. et al. (1997) Nature 386:833-838). These receptors are members of the immunoglobulin superfamily, and also contain a characteristic domain called the ZU5 domain. Mutations in the mouse member of the netrin receptor family, Rcm (rostral cerebellar malformation) result in cerebellar and midbrain defects as an apparent result of abnormal neuronal migration (Ackerman, S. L. et al. (1997) Nature 386:838-842). VPS10 Domain Containing Receptors The members of the VPS10 domain containing receptor family all contain a domain with homology to the yeast vacuolar sorting protein 10 (VPS10) receptor. This family includes the mosaic receptor SorLA, the neurotensin receptor sortilin, and SorCS, which is expressed during mouse embryonal and early postnatal nervous system development (Hermey, G. et al. (1999) Biochem. Biophys. Res. Commun. 266:347-351; Hermey, G. et al. (2001) Neuroreport 12:29-32). A recently identified member of this family, SorCS2, is highly expressed in the developing and mature mouse central nervous system. Its main site of expression is the floor plate, and high levels are also detected transiently in brain regions including the dopaminergic brain nuclei and the dorsal thalamus (Rezgaoui, M. (2001) Mech. Dev. 100:335-338). Membrane-Associated Proteins Tetraspan Family Proteins The transmembrane 4 superfamily (TM4SF) or tetraspan family is a multigene family encoding type III integral membrane proteins (Wright, M. D. and Tomlinson, M. G. (1994) Immunol. Today 15:588-594). The TM4SF is comprised of membrane proteins which traverse the cell membrane four times. Members of the TM4SF include platelet and endothelial cell membrane proteins, melanoma-associated antigens, leukocyte surface glycoproteins, colonal carcinoma antigens, tumor-associated antigens, and surface proteins of the schistosome parasites (Jankowski, S. A. (1994) Oncogene 9:1205-1211). Members of the TM4SF share about 25-30% amino acid sequence identity with one another. A number of TM4SF members have been implicated in signal transduction, control of cell adhesion, regulation of cell growth and proliferation, including development and oncogenesis, and cell motility, including tumor cell metastasis. Expression of TM4SF proteins is associated with a variety of tumors and the level of expression may be altered when cells are growing or activated. Tumor Antigens Tumor antigens are surface molecules that are differentially expressed in tumor cells relative to normal cells. Tumor antigens distinguish tumor cells immunologically from normal cells and provide diagnostic and therapeutic targets for human cancers (Takagi, S. et al. (1995) Int. J. Cancer 61: 706-715; Liu, E. et al. (1992) Oncogene 7: 1027-1032). Ion Channels Ion channels are found in the plasma membranes of virtually every cell in the body. For example, chloride channels mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of ions across epithelial membranes. When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, chloride channels also regulate organelle pH. (See, e.g., Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.) Electrophysiological and pharmacological properties of chloride channels, including ion conductance, current-voltage relationships, and sensitivity to modulators, suggest that different chloride channels exist in muscles, neurons, fibroblasts, epithelial cells, and lymphocytes. Many channels have sites for phosphorylation by one or more protein kinases including protein kinase A, protein kinase C, tyrosine kinase, and casein kinase II, all of which regulate ion channel activity in cells. Inappropriate phosphorylation of proteins in cells has been linked to changes in cell cycle progression and cell differentiation. Changes in the cell cycle have been linked to induction of apoptosis or cancer. Changes in cell differentiation have been linked to diseases and disorders of the reproductive system, immune system, and skeletal muscle. Cerebellar granule neurons possess a non-inactivating potassium current which modulates firing frequency upon receptor stimulation by neurotransmitters and controls the resting membrane potential. Potassium channels that exhibit non-inactivating currents include the ether a go-go (EAG) channel. A membrane protein designated KCR1 specifically binds to rat EAG by means of its C-terminal region and regulates the cerebellar non-inactivating potassium current. KCR1 is predicted to contain 12 transmembrane domains, with intracellular amino and carboxyl termini. Structural characteristics of these transmembrane regions appear to be similar to those of the transporter superfamily, but no homology between KCR1 and known transporters was found, suggesting that KCR1 belongs to a novel class of transporters. KCR1 appears to be the regulatory component of non-inactivating potassium channels (Hoshi, N. et al. (1998) J. Biol. Chem. 273:23080-23085). Proton Pumps Proton ATPases are a large class of membrane proteins that use the energy of ATP hydrolysis to generate an electrochemical proton gradient across a membrane. The resultant gradient may be used to transport other ions across the membrane (Na + , K + , or Cl − ) or to maintain organelle pH. Proton ATPases are further subdivided into the mitochondrial F-ATPases, the plasma membrane ATPases, and the vacuolar ATPases. The vacuolar ATPases establish and maintain an acidic pH within various vesicles involved in the processes of endocytosis and exocytosis (Meilman, I. et al. (1986) Ann. Rev. Biochem. 55:663-700). Proton-coupled, 12 membrane-spanning domain transporters such as PEPT 1 and PEPT 2 are responsible for gastrointestinal absorption and for renal reabsorption of peptides using an electrochemical H + gradient as the driving force. Another type of peptide transporter, the TAP transporter, is a heterodimer consisting of TAP 1 and TAP 2 and is associated with antigen processing. Peptide antigens are transported across the membrane of the endoplasmic reticulum by TAP so they can be expressed on the cell surface in association with MHC molecules. Each TAP protein consists of multiple hydrophobic membrane spanning segments and a highly conserved ATP-binding cassette (Boll, M. et al. (1996) Proc. Natl. Acad. Sci. 93:284-289). Pathogenic microorganisms, such as herpes simplex virus, may encode inhibitors of TAP-mediated peptide transport in order to evade immune surveillance (Marusina, K. and Manaco, J. J. (1996) Curr. Opin. Hematol. 3:19-26). ABC Transporters ATP-binding cassette (ABC) transporters, also called the “traffic ATPases”, are a superfamily of membrane proteins that mediate transport and channel functions in prokaryotes and eukaryotes (Higgins, C. F. (1992) Annu. Rev. Cell Biol. 8:67-113). ABC proteins share a similar overall structure and significant sequence homology. All ABC proteins contain a conserved domain of approximately two hundred amino acid residues which includes one or more nucleotide binding domains. Mutations in ABC transporter genes are associated with various disorders, such as hyperbilirubinemia II/Dubin-Johnson syndrome, recessive Stargardt's disease, X-linked adrenoleukodystrophy, multidrug resistance, celiac disease, and cystic fibrosis. Semaphorins and Neuropilins 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). Membrane Proteins Associated with Intercellular Communication Intercellular communication is essential for the development and survival of multicellular organisms. Cells communicate with one another through the secretion and uptake of protein signaling molecules. The uptake of proteins into the cell is achieved by endocytosis, in which the interaction of signaling molecules with the plasma membrane surface, often via binding to specific receptors, results in the formation of plasma membrane-derived vesicles that enclose and transport the molecules into the cytosol. The secretion of proteins from the cell is achieved by exocytosis, in which molecules inside of the cell are packaged into membrane-bound transport vesicles derived from the trans Golgi network. These vesicles fuse with the plasma membrane and release their contents into the surrounding extracellular space. Endocytosis and exocytosis result in the removal and addition of plasma membrane components, and the recycling of these components is essential to maintain the integrity, identity, and functionality of both the plasma membrane and internal membrane-bound compartments. Synaptobrevins are synaptic vesicle-associated membrane proteins (VAMPs) which were first discovered in rat brain. These proteins were initially thought to be limited to neuronal cells and to function in the movement of vesicles from the plasmalemma of one cell, across the synapse, to the plasmalemma of another cell. Synaptobrevins are now known to occur and function in constitutive vesicle trafficking pathways involving receptor-mediated endocytotic and exocytotic pathways of many non-neuronal cell types. This regulated vesicle trafficking pathway may be blocked by the highly specific action of clostridial neurotoxins which cleave the synaptobrevin molecule. In vitro studies of various cellular membranes (Galli et al. (1994) J. Cell. Biol. 125:1015-24; Link et al. (1993) J. Biol. Chem. 268:18423-6) have shown that VAMPS are widely distributed. These important membrane trafficking proteins appear to participate in axon extension via exocytosis during development, in the release of neurotransmitters and modulatory peptides, and in endocytosis. Endocytotic vesicular transport includes such intracellular events as the fusions and fissions of the nuclear membrane, endoplasmic reticulum, Golgi apparatus, and various inclusion bodies such as peroxisomes or lysosomes. Endocytotic processes appear to be universal in eukaryotic cells as diverse as yeast, Caenorhabditis elegans, Drosophila, and mammals. VAMP-1B is involved in subcellular targeting and is an isoform of VAMP-1A (Isenmann, S. et al. (1998) Mol. Biol. Cell 9:1649-1660). Four additional splice variants (VAMP-1C to F) have recently been identified. Each variant has variable sequences only at the extreme C-terminus, suggesting that the C-terminus is important in vesicle targeting (Berglund, L. et al. (1999) Biochem Biophys. Res. Commun. 264:777-780). Nogo has been identified as a component of the central nervous system myelin that prevents axonal regeneration in adult vertebrates. Cleavage of the Nogo-66 receptor and other glycophosphatidylinositol-linked proteins from axonal surfaces renders neurons insensitive to Nogo-66, facilitating potential recovery from CNS damage (Fournier, A. E. et al. (2001) Nature 409:341-346). The slit proteins are extracellular matrix proteins expressed by cells at the ventral midline of the nervous system. Slit proteins are ligands for the repulsive guidance receptor Roundabout (Robo) and thus play a role in repulsive axon guidance (Brose, K. et al. (1999) Cell 96:795-806). Lysosomes are the site of degradation of intracellular material during autophagy and of extracellular molecules following endocytosis. Lysosomal enzymes are packaged into vesicles which bud from the trans-Golgi network. These vesicles fuse with endosomes to form the mature lysosome in which hydrolytic digestion of endocytosed material occurs. Lysosomes can fuse with autophagosomes to form a unique compartment in which the degradation of organelles and other intracellular components occurs. Protein sorting by transport vesicles, such as the endosome, has important consequences for a variety of physiological processes including cell surface growth, the biogenesis of distinct intracellular organelles, endocytosis, and the controlled secretion of hormones and neurotransmitters Rothman, J. E. and Wieland, F. T. (1996) Science 272:227-234). In particular, neurodegenerative disorders and other neuronal pathologies are associated with biochemical flaws during endosomal protein sorting or endosomal biogenesis (Mayer R. J. et al. (1996) Adv. Exp. Med. Biol. 389:261-269). Peroxisomes are organelles independent from the secretory pathway. They are the site of many peroxide-generating oxidative reactions in the cell Peroxisomes are unique among eukaryotic organelles in that their size, number, and enzyme content vary depending upon organism, cell type, and metabolic needs (Waterham, H. R. and Cregg, J. M. (1997) BioEssays 19:57-66). Genetic defects in peroxisome proteins which result in peroxisomal deficiencies have been linked to a number of human pathologies, including Zellweger syndrome, rhizomelic chonrodysplasia punctata, X-linked adrenoleukodystrophy, acyl-CoA oxidase deficiency, bifunctional enzyme deficiency, classical Refsum's disease, DHAP alkyl transferase deficiency, and acatalasemia (Moser, H. W. and Moser, A. B. (1996) Ann. NY Acad. Sci. 804:427-441). In addition, Gartner, J. et al. (1991; Pediatr. Res. 29:141-146) found a 22 kDa integral membrane protein associated with lower density peroxisome-like subcellular fractions in patients with Zellweger syndrome. Normal embryonic development and control of germ cell maturation is modulated by a number of secretory proteins which interact with their respective membrane-bound receptors. Cell fate during embryonic development is determined by members of the activin/TGF-β superfamily, cadherins, IGF-2, and other morphogens. In addition, proliferation, maturation, and redifferentiation of germ cell and reproductive tissues are regulated, for example, by IGF-2, inhibins, activins, and follistatins (Petraglia, P. (1997) Placenta 18:3-8; Mather, J. P. et al. (1997) Proc. Soc. Exp. Biol. Med. 215:209-222). Transforming growth factor beta (TGFbeta) signal transduction is mediated by two receptor Ser/Thr kinases acting in series, type II TGFbeta receptor and (TbetaR-II) phosphorylating type I TGFbeta receptor (TbetaR-I). TbetaR-I-associated protein-1 (TRECAP-1), which distinguishes between quiescent and activated forms of the type I transforming growth factor beta receptor, has been associated with TGFbeta signaling (Charng, M. J et al. (1998) J. Biol. Chem. 273:9365-9368). Retinoic acid receptor alpha (RAR alpha) mediates retinoic-acid induced maturation and has been implicated in myeloid development. Genes induced by retinoic acid during granulocytic differentiation include E3, a hematopoietic-specific gene that is an immediate target for the activated RAR alpha during myelopoiesis (Scott, L. M. et al. (1996) Blood 88:2517-2530). The μ-opioid receptor (MOR) mediates the actions of analgesic agents including morphine, codeine, methadone, and fentanyl as well as heroin. MOR is functionally coupled to a G-protein-activated potassium channel (Mestek A. et al. (1995) J. Neurosci. 15:2396-2406). A variety of MOR subtypes exist. Alternative splicing has been observed with MOR-1 as with a number of G protein-coupled receptors including somatostatin 2, dopamine D2, prostaglandin EP3, and serotonin receptor subtypes 5-hydroxytryptamine4 and 5-hydroxytryptamine7 (Pan, Y. X. et al. (1999) Mol. Pharm. 56:396-403). Peripheral and Anchored Membrane Proteins Some membrane proteins are not membrane-spanning but are attached to the plasma membrane via membrane anchors or interactions with integral membrane proteins. Membrane anchors are covalently joined to a protein post-translationally and include such moieties as prenyl, myristyl, and glycosylphosphatidyl inositol groups. Membrane localization of peripheral and anchored proteins is important for their function in processes such as receptor-mediated signal transduction. For example, prenylation of Ras is required for its localization to the plasma membrane and for its normal and oncogenic functions in signal transduction. Endoplasmic Reticulum Membrane Proteins The normal functioning of the eukaryotic cell requires that all newly synthesized proteins be correctly folded, modified, and delivered to specific intra- and extracellular sites. Newly synthesized membrane and secretory proteins enter a cellular sorting and distribution network during or immediately after synthesis and are routed to specific locations inside and outside of the cell. The initial compartment in this process is the endoplasmic reticulum (ER) where proteins undergo modifications such as glycosylation, disulfide bond formation, and oligomerization. The modified proteins are then transported through a series of membrane-bound compartments which include the various cistemae of the Golgi complex, where further carbohydrate modifications occur. Transport between compartments occurs by means of vesicle budding and fusion. Once within the secretory pathway, proteins do not have to cross a membrane to reach the cell surface. Although the majority of proteins processed through the ER are transported out of the organelle, some are retained. The signal for retention in the ER in mammalian cells consists of the tetrapeptide sequence, KDEL, located at the carboxyl terminus of resident ER membrane proteins (Munro, S. (1986) Cell 46:291-300). Proteins containing this sequence leave the ER but are quickly retrieved from the early Golgi cistemae and returned to the ER, while proteins lacking this signal continue through the secretory pathway. Disruptions in the cellular secretory pathway have been implicated in several human diseases. In familial hypercholesterolemia the low density lipoprotein receptors remain in the ER, rather than moving to the cell surface (Pathak, R. K. (1988) J. Cell Biol. 106:1831-1841). Altered transport and processing of the β-amyloid precursor protein (βAPP) involves the putative vesicle transport protein presenilin and may play a role in early-onset Alzheimer's disease (Levy-Lahad, E. et al. (1995) Science 269:973-977). Changes in ER-derived calcium homeostasis have been associated with diseases such as cardiomyopathy, cardiac hypertrophy, myotonic dystrophy, Brody disease, Smith-McCort dysplasia, and diabetes mellitus. Mitochondrial Membrane Proteins The mitochondrial electron transport (or respiratory) chain is a series of three enzyme complexes in the mitochondrial membrane that is responsible for the transport of electrons from NADH to oxygen and the coupling of this oxidation to the synthesis of ATP (oxidative phosphorylation). ATP then provides the primary source of energy for driving the many energy-requiring reactions of a cell. Most of the protein components of the mitochondrial respiratory chain are the products of nuclear encoded genes that are imported into the mitochondria, and the remainder are products of mitochondrial genes. Defects and altered expression of enzymes in the respiratory chain are associated with a variety of disease conditions in man, including, for example, neurodegenerative diseases, myopathies, and cancer. Lymphocyte and Leukocyte Membrane Proteins The B-cell response to antigens is an essential component of the normal immune system Mature B cells recognize foreign antigens through B cell receptors (BCR) which are membrane-bound, specific antibodies that bind foreign antigens. The antigen/receptor complex is internalized, and the antigen is proteolytically processed. To generate an efficient response to complex antigens, the BCR, BCR-associated proteins, and T cell response are all required. Proteolytic fragments of the antigen are complexed with major histocompatability complex-II (MHCII) molecules on the surface of the B cells where the complex can be recognized by T cells. In contrast, macrophages and other lymphoid cells present antigens in association with MHCI molecules to T cells. T cells recognize and are activated by the MHCI-antigen complex through interactions with the T cell receptor/CD3 complex, a T cell-surface multimeric protein located in the plasma membrane. T cells activated by antigen presentation secrete a variety of lymphokines that induce B cell maturation and T cell proliferation, and activate macrophages, which kill target cells. Leukocytes have a fundamental role in the inflammatory and immune response, and include monocytes/macrophages, mast cells, polymorphonucleoleukocytes, natural killer cells, neutrophils, eosinophils, basophils, and myeloid precursors. Leukocyte membrane proteins include members of the CD antigens, N-CAM, I-CAM, human leukocyte antigen (HLA) class I and HMA class II gene products, immunoglobulins, immunoglobulin receptors, complement, complement receptors, interferons, interferon receptors, interleukin receptors, and chemokine receptors. Abnormal lymphocyte and leukocyte activity has been associated with acute disorders such as AIDS, immune hypersensitivity, leukemias, leukopenia, systemic lupus, granulomatous disease, and eosinophilia. Apoptosis-Associated Membrane Proteins A variety of ligands, receptors, enzymes, tumor suppressors, viral gene products, pharmacological agents, and inorganic ions have important positive or negative roles in regulating and implementing the apoptotic destruction of a cell. Although some specific components of the apoptotic pathway have been identified and characterized, many interactions between the proteins involved are undefined, leaving major aspects of the pathway unknown. A requirement for calcium in apoptosis was previously suggested by studies showing the involvement of calcium levels in DNA cleavage and Fas-mediated cell death (Hewish, D. R. and L. A. Burgoyne (1973) Biochem. Biophys. Res. Comm. 52:504-510; Vignaux, F. et al. (1995) J. Exp. Med. 181:781-786; Oshimi, Y. and S. Miyazaki (1995) J. Immunol. 154:599-609). Other studies show that intracellular calcium concentrations increase when apoptosis is triggered in thymocytes by either T cell receptor cross-linking or by glucocorticoids, and cell death can be prevented by blocking this increase (McConkey, D. J. et al. (1989) J. Immunol. 143:1801-1806; McConkey, D. J. et al. (1989) Arch. Biochem. Biophys. 269:365-370). Therefore, membrane proteins such as calcium channels and the Fas receptor are important for the apoptotic response. Nuclear Hormone Receptors The nuclear hormone receptors, also known as the nuclear receptors or the intracellular receptors, constitute a protein superfamily whose members are both receptors and transcriptional regulators. Nuclear hormone receptors rely on both their receptor function and their transcriptional regulatory function to affect a broad array of biological processes, including development, homeostasis, cell proliferation, and cell differentiation. (Reviewed in Mangelsdorf, D. J. et al. (1995) Cell 83:835-840; Wen, D. X. and D. P. McDonnell (1995) Curr. Opin. Biotechnol. 6:582-589; Perlmann, T. and R. M. Evans (1997) Cell 90:391-397; Tenbaum, S. and A. Baniahmad (1997) Int. J. Biochem Cell Biol. 29:1325-1341; Moras, D. and H. Gronemeyer (1998) Curr. Opin. Cell Biol. 10:384-391; Willy, P. J. and D. J. Mangelsdorf (1998) in: Hormones and Signaling (ed: B. W. O'Malley) vol. 1, Academic Press, San Diego Calif., pp. 307-358; Weatherman, R. V. et al. (1999) Annu. Rev. Biocher 68:559-581.) Nuclear Hormone Receptors as Receptors Generally, the term receptor describes a protein that specifically recognizes other molecules. As receptors, nuclear hormone receptors specifically recognize and bind to their cognate ligands. Although nuclear hormone receptors are located intracellularly, many receptors are extracellular cell surface proteins which bind extracellular ligands. Such extracellular receptors produce cellular responses affecting growth, differentiation, endocytosis, and the immune response. Other receptors facilitate the selective transport of proteins out of the endoplasmic reticulum and localize enzymes to particular regions of the cell. Transcriptional regulation by nuclear hormone receptors, propagation of cellular signals by extracellular receptors, and transport and localization of proteins by other receptors, all rely upon specific interactions between the receptors and a variety of cellular components. In many cases, the identity of the cognate ligand to which a receptor binds is unknown. Such receptors are termed orphan receptors. This term also applies to those nuclear hormone receptors which carry out their transcriptional regulatory functions without binding any ligands. Nuclear Hormone Receptors as Transcriptional Regulators Multicellular organisms are comprised of diverse cell types that differ dramatically both in structure and function. The identity of a cell is determined by its characteristic pattern of gene expression, and different cell types express overlapping but distinctive sets of genes throughout development. Spatial and temporal regulation of gene expression is critical for the control of cell proliferation, cell differentiation, apoptosis, and other processes that contribute to organismal development. As transcriptional regulators, nuclear hormone receptors play key roles in controlling these fundamental biological processes. Other transcriptional regulators affect gene expression in response to extracellular signals that mediate cell-cell communication and that coordinate the activities of different cell types. In general, transcriptional regulators such as nuclear hormone receptors initiate, activate, repress, or terminate gene transcription by binding to the promoter, enhancer, and upstream regulatory regions of a gene in a sequence-specific manner. However, some transcriptional regulators bind regulatory elements within or downstream of a gene's coding region. Transcriptional regulatory proteins may bind to a specific region of DNA singly, or in a complex with other accessory factors. (Reviewed in Lewin, B. (1990) in: Genes IV, Oxford University Press, New York N.Y., and Cell Press, Cambridge Mass., pp. 554-570.) Mechanism of Nuclear Hormone Receptor Function In the unliganded state, a nuclear hormone receptor exists in association with a multiprotein complex of chaperones, including heat shock proteins such as hsp90 and immunophilins such as hsp56. These chaperones maintain the ligand-free receptor in an inactive state which is amenable to binding of free ligand, and prevent the ligand-free receptor from translocating to the nucleus. Upon activation by its cognate ligand, the receptor may form a homodimer or heterodimer which translocates to the nucleus, binds to specific DNA sequences, and exerts its transcriptional regulatory function. In order to effectively carry out its regulatory roles, an activated nuclear hormone receptor dissociates from a histone deacetylase-containing corepressor complex and associates with a histone acetyltransferase-containing coactivator complex (Xu, L. et al. (1999) Curr. Opin. Genet. Dev. 9:140-147). The association of the activated receptor with coactivator proteins results in remodeling of chromatin so that it adopts an open transcriptionally active state, providing access to the transcriptional regulatory elements of the activated nuclear receptor (Lemon, B. D. and L. P. Freedman (1999) Curr. Opin. Genet Dev. 9:499-504). Structure of Nuclear Hormone Receptors Nuclear hormone receptors function as signal transducers by converting hormonal signals into transcriptional responses. In general, nuclear hormone receptors consist of a variable amino-terminal domain, a highly conserved DNA-binding domain, and a conserved C-terminal ligand-binding domain. In the steroid-binding nuclear hormone receptors, the amino-terminal domain harbors a trans-activation element termed AF-1. Some nuclear hormone receptors also contain a trans-activation element in the ligand-binding domain termed AF-2. The DNA-binding and ligand-binding domains of nuclear hormone receptors may contain dimerization elements, and the DNA-binding domain may contain a nuclear localization signal (Weatherman, R. V. et al. (1999) Annu. Rev. Biochenm 68:559-581). The DNA-binding domain of nuclear hormone receptors is composed of two zinc finger motifs which mediate recognition of specific DNA sequences. A zinc finger motif contains periodically spaced cysteine and histidine residues which coordinate Zn +2 . Examples of this sequence pattern include the C2H2-type, C4-type, and C3HC4-type (“RING” finger) zinc fingers, and the PHD domain (Lewin, supra; Aasland, R. et al. (1995) Trends Biochem. Sci. 20:56-59). A zinc finger motif contains an a helix and an antiparallel B sheet whose proximity and conformation are maintained by the zinc ion. Contact with DNA is made by the arginine preceding the a helix and by the second, third, and sixth residues of the a helix. Zinc finger motifs may be repeated in a tandem array within a protein such that the a helix of each zinc finger in the protein makes contact with the major groove of the DNA double helix. This repeated contact between the protein and the DNA produces a strong and specific DNA-protein interaction. The strength and specificity of the interaction can be regulated by the number of zinc finger motifs within the protein. Although zinc fingers were originally identified in DNA-binding proteins as regions that interact directly with DNA, they have since been found in proteins that do not bind to DNA. (See, e.g., Lodish, H et al. (1995) Molecular Cell Biology, Scientific American Books, New York N.Y., pp. 447-451.) The ligand-binding domain of nuclear hormone receptors is responsible for binding to ligands, coactivator proteins, and corepressor proteins. This domain is composed of three layers of α helices, with the central layer consisting of two helices containing many hydrophobic side chains (Moras, D. and H. Gronemeyer (1998) Curr. Opin. Cell Biol. 10:384-391). These two central α helices thus create a hydrophobic pocket which is the site of ligand binding. A ligand bound in this hydrophobic ligand-binding site is completely buried inside the receptor protein and is not exposed to solvent. This suggests that large conformational changes in the ligand-binding domain would accompany binding of a ligand. One of the α helices of the ligand-binding domain provides many of the inter-subunit contacts in dimers of nuclear receptors. This a helix contacts the ligand when it is bound in the ligand-binding pocket, suggesting that ligand binding can affect formation of receptor dimers (Weatherman, R. V. et al. (1999) Annu. Rev. Biochenm 68:559-581). Classes of Nuclear Hormone Receptors and Associated Proteins Nuclear hormone receptors can be grouped into three broad classes: the steroid receptors, the RXR-heterodimeric receptors, and the orphan nuclear hormone receptors. The steroid receptors bind to steroid hormones, and this class includes the androgen receptor, mineralocorticoid receptor, estrogen receptor, glucocorticoid receptor, and progesterone receptor. The RXR-heterodimeric receptors bind to nonsteroid ligands, and this class includes the thyroid hormone receptor, retinoic acid receptor, vitamin D receptor, ecdysone receptor, and peroxisome prolifeiator activated receptor. The orphan nuclear hormone receptors include steroidogenic factor 1, nerve growth factor-induced receptor, and X-linked orphan receptor DAX-1. The steroid hormone receptors are activated upon binding to specific steroid hormones. The conformational change induced by ligand binding leads to dissociation of the receptor from heat shock proteins and formation of receptor homodimers which recognize specific palindromic DNA sequences called hormone response elements (HREs). Upon binding to an IRE, a steroid hormone receptor homodimer can regulate the transcription of target genes. For example, the progesterone receptor (PR) is a steroid hormone receptor which is activated by progesterone, a 4-pregnene-3,20-dione derived from cholesterol which is a critical oscillating component of the female reproductive cycle. These oscillations correlate with anatomical and morphological changes including menstruation and pregnancy. The activities of progesterone are mediated through PR. In the cytoplasm, PR associates with several other proteins and factors known as the PR heterocomplex. This heterocomplex includes heat shock proteins and immunophilins such as hsp70, hsp90, hsp27, p59 (hsp56), p48, and p23 (Johnson, J. L. et al. (1994) Mol. Cell. Biol. 14:1956-1963). Upon binding progesterone, activated PR translocates to the nucleus, binds to canonical DNA transcriptional elements, and regulates progesterone-regulated genes implicated in differentiation and the cell cycle (Moutsatsou, P and C. E. Sekeris (1997) Ann. N.Y. Acad. Sci. 816:99-115). The PR antagonist RU 486, which can be used to terminate a pregnancy, is an example of a commercial therapeutic targeted toward a steroid hormone receptor. The RXR-heterodimeric nuclear receptors are distinguished from the steroid hormone receptors in that members of the former group bind to their target DNA sequences upon formation of heterodimers with retinoid X receptors (RXRs) (Mangelsdorf, D. J. and R. M. Evans (1995) Cell 83:841-850). Three different isoforms of RXR have been identified (Minucci, S. and K. Ozato (1996) Curr. Opin. Genet. Dev. 6:567-574). The retinoic acid receptors (RARs) are examples of RXR-heterodimeric nuclear receptors. Retinoic acid (RA) is a biologically active metabolite of vitamin A (retinol), a fat-soluble vitamin found mainly in fish liver oils, liver, egg yolk, butter, and cream. While 9-cis-RA binds to RARs and RXRs, all-trans-RA binds only to RXRs. RAR/RXR heterodimers bind with high affinity to specific DNA sequences known as retinoic acid response elements (RAREs), thus acting as regulators of RA-dependent transcription. Peroxisome proliferator activated receptors (PPARs) are therapeutically important RXR-heterodimeric nuclear receptors which are induced by fatty acids and eicosanoids. There are three known isotypes of PPAR, each with specific expression patterns, and these PPARs are involved in the regulation of genes involved in systemic homeostatis of glucose and lipids (Kliewer, S. A. and T. M. Willson (1998) Curr. Opin. Genet. Dev. 8:576-581; Michallk, L. and W. Walli (1999) Curr. Opin. Biotechnol. 10:564-570). As such, PPARs are therapeutic targets for disorders such as diabetes, dyslipidemia, and obesity (Smith, S. A. (1996) Pharmacol. Rev. Commun. 8:57-64; Willson, T. M. and W. Wahli (1997) Curr. Opin. Chem. Biol. 1:235-241; Barroso, I. et al. (1999) Nature 402:880-883). The orphan nuclear receptors either have no known activating ligand, or can exert their transcriptional regulatory activities without benefit of ligand binding. For example, in Caenorhabditis elegans, the X-chromosome encoded nuclear hormone receptor homologue SEX-1 regulates transcription of the sex determination gene xol-1 (Carmi, I. et al. (1998) Nature 396:168-173). Rather than relying on ligand binding, SEX-1 acts as a transcriptional regulator in a dose-dependent manner, in effect controlling sexual differentiation through an X-chromosome-counting mechanism. Retinoid-related orphan receptor alpha (ROR alpha) is another member of the nuclear receptor superfamily. Mice carrying deletions in the ROR alpha gene demonstrate immune system abnormalities. ROR alpha1expression negatively affects the NF-kappaB signaling pathway apparently through the induction of IkappaB alpha, the major inhibitory protein of the NF-kappaB signaling pathway. These observations have suggested that ROR alpha1 is a target for treatment of chronic inflammatory diseases, including atherosclerosis and rheumatoid arthritis (Delerive, P. et al. (2001) EMBO 2:4248). NSD1 is a murine nuclear protein that interacts with the ligand-binding domains (LBDs) of several nuclear receptors. NSD1 contains a SET domain of the subtype represented by the proteins encoded by the Drosophila gene Ash1 and the S. cerevisiae gene YJQ8. SET domains are involved in chromatin organization and function and are found in a number of eukaryotic proteins. NSD1 also contains multiple zinc finger-like motifs known as PHD fingers or C4HC3 motifs. NSD1 contains two distinct nuclear receptor-interacting domains, designated NID −L and NID +L . NID −L interacts with the unliganded LBDs of retinoic acid receptors (RAR) and thyroid hormone receptors (TR). NID +L interacts with the liganded LBDs of RAR, TR, retinoid X receptor (RXR), and estrogen receptor (ER). It is therefore likely that NSD1 plays different roles with respect to transcriptional regulation depending on the presence of bound ligand in the LBDs of target nuclear receptors (Ningwu Huangl, N. et al. (1998) EMBO 17:3398-3412 and references within). Some nuclear hormone receptors lack the conventional DNA-binding domain typically associated with the nuclear hormone receptor family. DAX-1 is one such nuclear hormone receptor lacking the conventional DNA-binding domain, and mutations in DAX-1 have been shown to cause X-linked adrenal hypoplasia congenita (Zanaria, E. F. et al. (1994) Nature 372:635-641). DAX-1 is an orphan nuclear receptor which interacts directly with steroidogenic factor I (SF-1) (Ito, M. et al. (1997) Mol. Cell. Biol. 17:1476-1483), and DAX-1 is capable of modulating the action of SF-1 in sex-specific gene expression (Nachtigal, M. W. et al. (1998) Cell 445-454). SF-1 is an orphan nuclear receptor which acts as a transcription factor for several steroidogenic enzyme genes in the adrenal gland and gonads (Lala, D. S. et al. (1992) Mol. Endocrinol. 6:1249-1258; Lynch, J. P. et al. (1993) Mol. Endocrinol. 7:776-786; Clemens, J. W. et al. (1994) Endocrinology 134:1499-1508), and can also regulate several genes expressed in pituitary gonadotrope cells (Barnhart, K. M. and P. L. Mellon (1994) Mol. Endocrinol. 8:878-885; Ingraham, H. A. et al. (1994) Genes Dev. 8:2302-2312; Halvorson, L. M. et al. (1996) J. Biol. Chem. 271:6645-650; Keri, R. A. and J. H. Nilson (1996) J. Biol. Chera. 271:10782-10785). SF-1 also acts as a potent transactivator of small heterodimer partner (SHP; short heterodimer partner) (Lee, Y. K. et al. (1999) J. Biol. Chem. 274:20869-20873). SHP is another example of a nuclear hormone receptor lacking the conventional DNA-binding domain (Seol, W. et al. (1996) Science 272:1336-1339; Lee, H.-K. et al. (1998) J. Biol. Chem. 273:14398-14402). SHP interacts with many members of the nuclear hormone receptor family, including retinoid receptors, estrogen receptor, thyroid hormone receptor, and the orphan receptor CAR. SHP acts as an inhibitor of estrogen receptor-mediated transcriptional activation by competing with coactivators for binding to estrogen receptor (Johansson, L. et al. (1999) J. Biol. Chem. 274:345-353). SHP also inhibits transactivation by the orphan receptor hepatocyte nuclear factor 4, and by retinoid X receptor (Lee, Y. K. et al. (2000) Mol. Cell. Biol. 20:187-195). The human thyroid hormone receptor-associated protein (TRAP) complex is a coactivator for nuclear receptors. TRAP appears to be the equivalent of the yeast SRB- and MED-containing cofactor complex (SRB/MED, SMCC) capable of mediating activated transcription in vitro in the absence of TATA-binding (TBP)-associated factors TAFs). TAPs comprise the subunits of the TFIID that are distinct from TBP. SRB/ME comprises polypeptides identified genetically as suppressors of truncations of the carboxy-terminal repeat domain of the of largest subunit of RNA polymerase II (SRP) that mediate (MED) activated transcription in the absence of TAPs (Ito, M. et al. (1999) Mol. Cell 3:361-370; reviewed in Wei-Hua Wu, W-H. and Hampsey, M. (1999) Current Biology 9:R606-R609). Consequences of Defective Transcription Regulation Many neoplastic disorders in humans can be attributed to inappropriate gene expression. Malignant cell growth may result from either excessive expression of tumor promoting genes or insufficient expression of tumor suppressor genes (Cleary, M. L. (1992) Cancer Surv. 15:89-104). Chromosomal translocations may also produce chimeric loci which fuse the coding sequence of one gene with the regulatory regions of a second unrelated gene. Such an arrangement likely results in inappropriate gene transcription, potentially contributing to malignancy. In addition, the immune system responds to infection or trauma by activating a cascade of events that coordinate the progressive selection, amplification, and mobilization of cellular defense mechanisms. A complex and balanced program of gene activation and repression is involved in this process. However, hyperactivity of the immune system as a result of improper or insufficient regulation of gene expression may result in considerable tissue or organ damage. This damage is well documented in immunological responses associated with arthritis, allergens, heart attack, stroke, and infections. (See, e.g., Isselbacher et al. (1996) Harrison's Principles of Internal Medicine, 13/e, McGraw Hill, Inc. and Teton Data Systems Software.) Furthermore, the growth of multicellular organisms is based upon the induction and coordination of cell differentiation at the appropriate stages of development. Central to this process is differential gene expression, which confers the distinct identities of cells and tissues throughout the body. Failure to regulate gene expression during development could result in developmental disorders. T cell Activation Human T cells can be specifically activated by Staphyloccocal exotoxins, resulting in cytokine production and cell proliferation which can lead to septic shock (Muraille, E. et al. (1999) Int. Immunol. 11:1403-1410). Activation of T cells by Staphyloccocal exotoxins requires the presence of antigen presenting cells (APC) to present the exotoxin molecules to the T cells and to deliver the costimulatory signals required for optimum T cell activation. Although Staphyloccocal exotoxins must be presented to T cells by APC, these molecules do not require processing by APC. Instead, Staphyloccocal exotoxins directly bind to a non-polymorphic portion of the human major histocompatibility complex (MHC) class II molecules, thus bypassing the need for capture, cleavage, and binding of the peptides to the polymorphic antigenic groove of the MHC class II molecules. Expression Profiling 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. The discovery of new receptors and membrane-associated proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell proliferative, autoimmune/inflammatory, neurological, metabolic, developmental, endocrine, cardiovascular, reproductive, gastrointestinal, metabolic, genetic, and lipid metabolism disorders, cancer, and viral infections, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of receptors and membrane-associated proteins. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention features purified polypeptides, receptors and membrane-associated proteins, referred to collectively as “REMAP” and individually as “REMAP-1,” “REMA-2,” “REMAP-3,” “REMAP-4,” “REMAP-5,” “REMAP-6,” “REMAP-7,” “REMAP-8,” “REMAP-9,” “REMAP-10,” “REMAP-11,” “REMAP-12,” “REMAP-13,” “REMAP-14,” “REMAP-15,” “REMAP-16,” “REMAP-17,” “REMAP-18,” “REMAP-19,” “REMAP-20,” “REMAP-21,” “REMAP-22,” “REMAP-23,” “REMAP-24,” “REMAP-25,” and “REMAP-26.” In one aspect, the invention 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:1-26, 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:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1-26. The invention further 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:1-26, 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:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26. In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-26. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID NO:27-52. Additionally, the invention 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:1-26, 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:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide. The invention also 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:1-26, 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:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26. 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. Additionally, the invention 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:1-26, 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:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26. The invention further 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:27-52, 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:27-52, 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 one alternative, the polynucleotide comprises at least 60 contiguous nucleotides. Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:27-52, 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:27-52, 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, and optionally, if present, the amount thereof. In one alternative, the probe comprises at least 60 contiguous nucleotides. The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:27-52, 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:27-52, 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, and, optionally, if present, the amount thereof. The invention further 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:1-26, 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:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-26. The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional REMAP, comprising administering to a patient in need of such treatment the composition. The invention also 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:1-26, 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:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional REMAP, comprising administering to a patient in need of such treatment the composition. Additionally, the invention 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:1-26, 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:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. In one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional REMAP, comprising administering to a patient in need of such treatment the composition. The invention further 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:1-26, 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:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26. 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. The invention further 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:1-26, 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 D NO:1-26, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-26. 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. The invention further 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:27-52, 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. The invention further 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:27-52, ii) 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:27-52, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of if 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:27-52, ii) 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:27-52, 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 comprises a fragment of a polynucleotide sequence 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. |
Method of channel allocation for a mobile terminal moving in a cellular communication network |
The present invention proposes a method of channel allocation for a mobile terminal (MS) moving in a cellular communication network (NW), said method comprising the steps of: detecting (S21) the speed of said mobile terminal (MS) moving in said network, and dependent on said detected speed (S23, S25), allocating (S24, S26, S27) a channel of a specific type to said mobile terminal (MS). Accordingly, with the present invention implemented, delays in neighbor cell SCH decoding and signal level measurement are significantly reduced and may thus no longer result in incomplete data for inter-cell handover decision or even unsuccessfull handovers. Hence, communication network performance in particular in connection with handovers in a cellular network layout is improved. The present invention also concerns an accordingly adapted device for channel allocation. |
1. A method of channel allocation for a mobile terminal moving in a cellular communication network, said method comprising the steps of: detecting the speed of said mobile terminal moving in said network, and dependent on said detected speed, allocating a channel of a specific type to said mobile terminal. 2. A method according to claim 1, wherein said allocated channel of a specific type is a traffic channel, with the channel types being distinguishable by their transmission rate. 3. A method according to claim 1, wherein if said detected speed is below a first speed threshold, a first channel of a specific type is allocated to said mobile terminal. 4. A method according to claim 3, wherein if said detected speed is above said first speed threshold, another channel of a specific type different from said first channel is allocated to said mobile terminal. 5. A method according to claim 4, wherein if said detected speed is above said first but below a second speed threshold, a second channel of a specific type is allocated to said mobile terminal. 6. A method according to claim 4, wherein if said detected speed is above said first and above a second speed threshold, a third channel of a specific type is allocated to said mobile terminal. 7. A method according to claim 2, wherein said transmission rate differs by the number of idle frames in a multiframe of a traffic channel. 8. A method according to claim 7, further comprising a step of conducting measurements on cells neighboring a current cell in which the mobile terminal is located, during said idle frames. 9. A method according to claim 1, wherein the speed of said mobile terminal moving in said network is repeatedly detected. 10. A method according to claim 9, wherein said detecting is performed after a predetermined time interval has elapsed. 11. A method according to claim 1, wherein said cellular communication network is composed of plural cells each of which cell covering a small area such that the coverage area of said plural small cells may be comprised in the coverage area of a large cell. 12. A method according to claim 3, wherein said speed threshold is predetermined based on the cell radius of the cells constituting the network and the expected number of handovers occurring for a mobile terminal moving at a given speed via the cellular network. 13. A method according to claim 1, wherein allocating a channel of a specific type to said mobile terminal is implemented based on hysteresis. 14. A method according to claim 13, wherein hysteresis is implemented in case that the currently detected speed is different from an immediately preceding detected speed and differs by a certain amount from a speed threshold defined for being used in channel allocation. 15. A device adapted to allocate a channel to a mobile terminal moving in a cellular communication network, said device comprising: detecting means adapted to detect the speed of said mobile terminal moving in said network, and control means adapted to allocate a channel of a specific type to said mobile terminal dependent on said detected speed. 16. A device according to claim 15, wherein said allocated channel of a specific type is a traffic channel, with the channel types being distinguishable by their transmission rate. 17. A device according to claim 15, wherein said control means is adapted to allocate a first channel of a specific type to said mobile terminal, if said detected speed is below a first speed threshold. 18. A device according to claim 17, wherein said control means is adapted to allocate another channel of a specific type different from said first channel to said mobile terminal, if said detected speed is above said first speed threshold. 19. A device according to claim 18, wherein said control means is adapted to allocate a second channel of a specific type to said mobile terminal if said detected speed is above said first but below a second speed threshold. 20. A device according to claim 18, wherein said control means is adapted to allocate a third channel of a specific type to said mobile terminal, if said detected speed is above said first and above a second speed threshold. 21. A device according to claim 16, wherein said transmission rate differs by the number of idle frames in a multiframe of a traffic channel. 22. A device according to claim 15, wherein said detection means is adapted to repeatedly detect the speed of said mobile terminal moving in said network. 23. A device according to claim 22, wherein said detection means is adapted to perform said detection after a predetermined time interval has elapsed. 24. A device according to claim 15, wherein said control means is adapted to perform allocating a channel of a specific type to said mobile terminal (MS) based on hysteresis. 25. A device according to claim 24, wherein said control means is adapted to base the allocation on hysteresis in case that the currently detected speed is different from an immediately preceding detected speed and differs by a certain amount from a speed threshold defined for being used in channel allocation. 26. A device according to claim 15, wherein said control means and said detection means are located at a same network entity. 27. A device according to claim 15, wherein said control means and said detection means are located remotely from each other. 28. A device according to claim 27, wherein said detection means is located at said mobile terminal to which a channel is to be allocated. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Cellular communication networks such as the GSM network system have widely spread in recent years with the increase of the demand for mobile communication. FIG. 1 shows a rough outline of a part of a cellular communication network. Generally, the network area served by the network is composed of individual cells C 1 , C 2 , C 3 . . . , and/or c 1 , . . . c 7 . Each cell in turn is served by a respective base station BS or base transceiver station BTS (not shown in every cell). The coverage area of such a base station BS is defined by the cell radius R and/or r. The coverage area and cell radius are adjustable by the transmit power used by the transmitter of the base station BTS. Thus, dependent on the transmit power of the BTS used, the network may be composed of so-called macrocells meaning a cell covering a large area (with a cell radius R of for example up to 30 km, or even more. For example, GSM standard allows cells of radius 35 km, while with special well-known cell extension techniques, the radius may be extended—in areas with prevailing radio propagation condition which allow this—up to 120 km). Examples of such macrocells are illustrated in bold (solid and dashed) lines in FIG. 1 and labeled C 1 , C 2 , C 3 , respectively. On the other hand, a low set transmit power leads to a cellular network composed of microcells meaning a cell covering a small area only, e.g. cells c 1 to c 7 in FIG. 1 . (Note that in microcell network layouts the number of base stations per area—compared to macrocell layouts—needs to be increased so that there do not arise gaps between the coverage areas of the microcells.) Although typically microcells have a cell radius r not exceeding 500 m, this is not limiting for the present invention. Rather, a microcell in the present specification is to be understood as a cell covering a small area such that the coverage area of plural small cells (c 1 , . . . , c 4 ) is comprised in the coverage area of a large cell (C 1 ), as it is illustrated by way of example in FIG. 1 . Also, as illustrated in FIG. 1 , cellular networks may adopt a cellular structure in which a macrocell layout is overlaid to a microcell layout. Nevertheless, a microcell layout may be provided for without an overlaid macrocell layout (and vice versa). Microcell layouts are preferably used in “hot spots” of the network where a high demand for mobile communication services is expected to occur such as in shopping malls, airports, etc. A mobile terminal located in such a cellular communication network communicates with and/or via the network via an air interface between the terminal MS and the base station BS in a manner known as such in general and as for example set out in various GSM specifications, e.g. based on TDMA and/or CDMA etc. As the terminal MS is mobile it may move at different speeds within the cellular network area. Also, when moving, it may cross one or more borders of the cells shown in FIG. 1 . Upon crossing a cell border, the mobile terminal in most cases may require to be handed over to a new serving base station BS of the new cell to which it has moved. Such a handover is defined as a feature involving a change of physical channels, radio channels and/or terrestrial channels, involved in a call while maintaining a call. (A call being a logical association to/from the mobile terminal from/to a switch.) This change of channels might be required as caused by the movement of an active terminal (crossing a cell boundary) or caused by spectrum, user profile, capacity or network management issues. Data exchange between the base station and the mobile station via the air interface (sometimes referred to as U m interface) according to GSM adopts, e.g. a time divisional multiple access scheme TDMA. According to TDMA, data are transmitted in units of bursts during consecutive time slots TS. Eight time slots according to GSM form one frame. One frame according to GSM has a duration of 4.615 ms. It is, however, to be noted that the present specification refers to GSM specific features only for explanatory purposes and other TDMA methods (for example adopting another number of time slots per frame, or another time duration per frame) may likewise be used in connection with the present invention. For example, the present invention as to be described later is easily applicable to the American IS-54 digital cellular system adopting a TDMA scheme with 6 time slots per frame and a frame duration of 40 ms, or even to the Japanese digital cellular system having a 3 channel TDMA multiple access scheme (full rate). With regard to the GSM system again, individual frames are grouped in to multiframes. Dependent on the type of multiframes can be distinguished: 1) for traffic channels carrying/transmitting (mainly) user data, 26 frames form a 26-multiframe (duration 120 ms), while 2) for signaling channels carrying/transmitting (only) control signaling information, 51 frames form a 51-multiframe (duration 235.38 ms). Furthermore, 26*51 frames make up one superframe (duration 6.12 s), while 2048 times a superframe constitutes a hyperframe. FIG. 3 illustrates an example of a 26-multiframe for a traffic channel. The 26 frames are numbered from #0 to #25. In the first 12 frames (#0 to #11) user data traffic is carried, frame #12 carries the SACCH (slow associated control channel, an inband control channel assigned to the traffic channel TCH or the slow dedicated control channel SDCCH). Frames #13 to 24 carry again user data traffic, and frame #25 is an idle frame which is not used for transmission. Rather, the idle frame is required to be reserved for terminals for decoding SCH (synchronization channel) data transmitted in a 51-multiframe from the base station to the mobile terminal. More precisely, in GSM and/or GSM/EDGE networks (EDGE Enhanced Date rates for GSM Evolution, GSM=Global System for Mobile communications), as mentioned above, signaling information is carried in 51-multiframes. For example, in downlink direction (from BS to MS) in a combination of logical channels containing the SCH, the SCH is always transmitted in frames number #1, #11, #21, #31, and #41, respectively, i.e. five times per 51-multiframe. More precisely, the 51-multiframe is applied in the time slot 0 of the BCCH, or control channel, frequency. In GSM/EDGE networks, on the SCH, cell identity is transmitted, and as mentioned above it takes place in 5 frames in each 51-frame control channel multiframe. As the networks are typically non-synchronized, a full idle frame must be reserved for terminals for SCH data decoding purposes. A full idle frame is necessary even in a synchronized network, because one's call can take place in the time slot which coincides with time slot 0 of the target cell. Cell identities must be established in order to attach signal level measurements to a particular neighbor cell. The cell identity is transmitted as the base station identity code BSIC. The BSIC is an identifier for the BS although the BSIC does not uniquely identify a single BS, since it has to be reused several times per PLMN network (public land mobile network). The BSIC serves for identification and distinction among neighbor cells, even when neighbor cells use the same BCCH (broadcast control channel) frequency. Since the BSIC is broadcast from the BS, the mobile terminal does not even need to establish a connection to the BS in order to retrieve the BSIC. The BSIC in turn consists of the network color code NCC identifying the PLMN and the base station color code BCC (3 bit) used to distinguish among eight different training sequence codes that one BS may use and to distinguish between eight neighboring base stations without a need for the mobile terminal to register on any other BS. On full rate channels (FR), one frame in each 26-frame TCH multiframe is reserved for this purpose of SCH decoding, as seen from FIG. 3 . However, as the relative phases of TCH and control channel multiframes are random, in the worst case on a FR channel, one must attempt SCH decoding 11 times before it may be performed successful. The duration of this process is approximately 1.32 seconds. The reason therefore is that only after 286 frames (=11*26 multiframes) there occurs (for the first time) a coincidence and/or full overlap in time between a SCH frame in a 51-multiframe and an idle frame in a 26-multiframe. Thus, a delay in decoding of 286*4.615 ms=1319.89 ms≈1.32 s is caused. Thus, as set out above, in GSM/EDGE cellular networks there is a delay in decoding the SCH data from a new neighbor cell. In the worst case it can be about 11 traffic channel (TCH) multiframes, or 1.32 seconds. In preparation for a handover, however, one must decode SCH data from several neighbor cells and perform a number of signal level measurements on the neighbors. In cellular networks adopting e.g. a microcell network arrangement, fast moving mobiles may require frequent inter-cell handovers due to frequent cell border crossings. Just as a numeric example, assume a microcell cellular network of microcells having a radius r=500 m. A mobile terminal starting to move from approximately the center of a cell would encounter a need for handover after (radially) traveling a distance of about r=500 m. Assuming further a speed of 100 km/h (=27.7 m/s), the mobile terminal would reach the microcell border after about 18 seconds. Assuming further that 6 neighbor base stations are to be monitored, 6*1.32 s=7.92 s were required for decoding/measuring the SCH of the neighbor BS which, being about half the time the mobile terminal needs for traveling, is quite too long for taking a decision concerning handover Such delays in neighbor cell SCH decoding and level measurement may thus result in incomplete data for inter-cell handover decision or even unsuccessfull handovers. Previously, a common approach resided in locating fast moving cells in macrocells. This means that a fast moving mobile terminal was assigned to and handed over to base stations BS serving macrocells only (cells denoted with capital letter in FIG. 1 ). This, however, is not a feasible solution in networks or areas, where only the microcell network layout exists (cells denoted with lowercase letter in FIG. 1 ). |
<SOH> SUMMARY OF THE INVENTION <EOH>Hence, it is an object of the present invention to solve the above mentioned drawbacks even in a cellular communication network comprising only microcells. According to the present invention, this object is for example achieved by a method of channel allocation for a mobile terminal moving in a cellular communication network, said method comprising the steps of detecting the speed of said mobile terminal moving in said network, and dependent on said detected speed, allocating a channel of a specific type to said mobile terminal. According to advantageous further developments of the present invention as set out in the dependent claims, said allocated channel of a specific type is a traffic channel, with the channel types being distinguishable by their transmission rate, if said detected speed is below a first speed threshold, a first channel of a specific type is allocated to said mobile terminal, if said detected speed is above said first speed threshold, another channel of a specific type different from said first channel is allocated to said mobile terminal, if said detected speed is above said first but below a second speed threshold, a second channel of a specific type is allocated to said mobile terminal, if said detected speed is above said first and above a second speed threshold, a third channel of a specific type is allocated to said mobile terminal, said transmission rate differs by the number of idle frames in a multiframe of a traffic channel, measurements are conducted on cells neighboring a current cell in which the mobile terminal is located, during said idle frames, the speed of said mobile terminal moving in said network is repeatedly detected, said detecting is performed after a predetermined time interval has elapsed, said cellular communication network is composed of plural cells each of which cell covering a small area such that the coverage area of said plural small cells may be comprised in the coverage area of a large cell, said speed threshold is predetermined based on the cell radius of the cells constituting the network and the expected number of handovers occurring for a mobile terminal moving at a given speed via the cellular network, allocating a channel of a specific type to said mobile terminal is implemented based on hysteresis, and hysteresis is implemented in case that the currently detected speed is different from an immediately preceding detected speed and differs by a certain amount from a speed threshold defined for being used in channel allocation. Still further, according to the present invention this object is for example achieved by a device adapted to allocate a channel to a mobile terminal moving in a cellular communication network, said device comprising: detecting means adapted to detect the speed of said mobile terminal moving in said network, and control means adapted to allocate a channel of a specific type to said mobile terminal dependent on said detected speed. According to favorable refinements of said device, said allocated channel of a specific type is a traffic channel, with the channel types being distinguishable by their transmission rate; said control means is adapted to allocate a first channel of a specific type to said mobile terminal, if said detected speed is below a first speed threshold; said control means is adapted to allocate another channel of a specific type different from said first channel to said mobile terminal, if said detected speed is above said first speed threshold; said control means is adapted to allocate a second channel of a specific type to said mobile terminal if said detected speed is above said first but below a second speed threshold; said control means is adapted to allocate a third channel of a specific type to said mobile terminal, if said detected speed is above said first and above a second speed threshold; said transmission rate differs by the number of idle frames in a multiframe of a traffic channel; said detection means is adapted to repeatedly detect the speed of said mobile terminal moving in said network; said detection means is adapted to perform said detection after a predetermined time interval has elapsed; said control means is adapted to perform allocating a channel of a specific type to said mobile terminal (MS) based on hysteresis; said control means is adapted to base the allocation on hysteresis in case that the currently detected speed is different from an immediately preceding detected speed and differs by a certain amount from a speed threshold defined for being used in channel allocation; said control means and said detection means are located at a same network entity; said control means and said detection means are located remotely from each other; said detection means is located at said mobile terminal to which a channel is to be allocated. Advantageously, with the present invention implemented, delays in neighbor cell SCH decoding and signal level measurement are significantly reduced and may thus no longer result in incomplete data for inter-cell handover decision or even unsuccessful handovers. Hence, communication network performance in particular in connection with handovers in a cellular network layouts is improved. Thus, a continuous call connection even for fast moving mobile terminals in cellular network layouts (microcell and/or macrocell) due to successful handovers is enabled, while involving only a slight reduction of speech quality on half rate and/or quarter rate transmission channels assigned to the moving terminals as compared to full rate channels. Still further, a mobile terminal may perform more frequently signal level measurements concerning neighbor base stations, i.e. base stations of cells surrounding the current cell in which the mobile terminal is located. A rather rapid acquisition of signal level data and SCH data from new neighbor cells is enabled by more frequent measurements, which in turn results in a low delay experienced by a base station controller BSC receiving measurement data concerning new neighbor cells after an inter-cell handover occurred. Also, the method of the present invention may easily be implemented to the control algorithms at the base station controller, while no modifications to the protocols or the base station subsystem BSS and/or radio access network RAN are required. |
Polynucleotides and polypeptides linked to cancer and/or tumorigenesis |
The present invention is directed to novel TTYH2 polynucleotides whose expression is modulated in cancers or tumours and especially in renal cell carcinoma. More particularly, the invention is directed to isolated TTYH2 polynucleotides and the TTYH2 polypeptides encoded thereby. The invention is further directed to methods for detecting the presence or diagnosing the risk of a cancer by detecting aberrant expression of a gene selected from TTYH2 or a gene belonging to the same biosynthetic or regulatory pathway as TTYH2. Also disclosed is the use of the aforementioned polypeptides and polynucleotides in screening for agents that modulate the expression of a gene or the level and or functional activity of an expression product of that gene, wherein the gene is selected from TTYH2 or a gene belonging to the same biosynthetic or regulatory pathway as TTYH2. The invention also discloses the use of such agents for inhibiting or reducing tumorigenesis or for treating and/or preventing conditions that are associated with aberrant TTYH2 expression. Also disclosed are immunopotentiating compositions comprising TTYH2 polynucleotides or TTYH2 polypeptides for eliciting an immune response in a patient, including the production of elements which specifically bind a TTYH2 polypeptide and/or which provide a protective effect against tumorigenesis |
1. An isolated polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7, or to a variant or derivative thereof. 2. The polypeptide of claim 1, wherein said biologically active fragment comprises at least 6 contiguous amino acids contained within the sequence set forth in any one of SEQ ID NO: 2 and 7. 3. The polypeptide of claim 2, wherein said biologically active fragment is selected from residues 1-57, 109-216 or 259-391 of SEQ ID NO: 2 or 7. 4. The polypeptide of claim 2, wherein said biologically active fragment is selected from residues 58-74, 92-108, 217-233, 240-258 or 392-408 of SEQ ID NO: 2 or 7. 5. The polypeptide of claim 2, wherein said biologically active fragment is selected from residues 75-91, 234-239, 409-534 of SEQ ID NO: 2, or residues 409-532 of SEQ ID NO: 7. 6. The polypeptide of claim 1, wherein said variant has at least 50% sequence identity to said at least a biologically active fragment. 7. The polypeptide of claim 6, wherein said variant is distinguished from at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7 by the substitution of at least one amino acid residue. 8. The polypeptide of claim 6, wherein said variant is distinguished from at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7 by the substitution of at least one amino acid residue, which is a conservative substitution. 9. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7, or to a variant or derivative thereof. 10. An isolated polynucleotide comprising a nucleotide sequence that corresponds or is complementary to at least a portion of the sequence set forth in SEQ ID NO: 1, 3, 4, 6 or 8, or to a polynucleotide variant thereof. 11. The polynucleotide of claim 10, wherein said nucleotide sequence corresponds or is complementary to at least 18 contiguous nucleotides of the sequence set forth in SEQ ID NO: 1, 3, 4, 6 or 8. 12. The polynucleotide of claim 10, wherein said polynucleotide variant has at least 50% sequence identity to at least a portion of the sequence set forth in SEQ ID NO: 1, 3, 4, 6 or 8. 13. The polynucleotide of claim 10, wherein said variant is obtained from a mammal. 14. A vector comprising the polynucleotide of claim 10. 15. An expression vector comprising the polynucleotide of claim 10 in operable linkage with a regulatory polynucleotide. 16. A host cell containing the vector of claim 14 or the expression vector of claim 15. 17. A method of producing a recombinant polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7, or to a variant or derivative thereof, said method comprising: culturing a host cell containing the expression vector of claim 15 such that said recombinant polypeptide is expressed from said polynucleotide; and isolating the said recombinant polypeptide. 18. A method of producing a biologically active fragment of a polypeptide comprising the sequence set forth in SEQ ID NO: 2 or 7, comprising: introducing a fragment of the polypeptide or a polynucleotide from which said fragment can be translated into a cell; and detecting modulation of tumorigenesis, which indicates that said fragment is a biologically active fragment. 19. The method of claim 18, wherein said fragment is present in said cell at a level and/or functional activity that correlates with the presence or risk of a cancer or tumour. 20. he method of claim 19, wherein said level and/or functional activity corresponds to a level and/or functional activity of a polypeptide comprising the sequence set forth in SEQ ID NO: 2 or 7, which correlates with the presence or risk of said cancer or tumour. 21. The method of claim 18, wherein the cancer or tumour is associated with an organ selected from kidney, brain or testis. 22. The method of claim 18, wherein said cancer or tumour is a cancer or tumour of the kidney. 23. The method of claim 18, wherein said cancer or tumour is renal cell carcinoma. 24. A method of producing a polypeptide variant of a parent polypeptide comprising the sequence set forth in SEQ ID NO: 2 or 7, or a biologically active fragment thereof, said method comprising: providing a modified polypeptide whose sequence is distinguished from the parent polypeptide by the substitution, deletion or addition of at least one amino acid; introducing said modified polypeptide or a polynucleotide from which the modified polypeptide can be translated into a cell; and detecting modulation of tumorigenesis, which indicates that said modified polypeptide is a polypeptide variant. 25. The method of claim 24, wherein said variant is present in said cell at a level and/or functional activity that correlates with the presence or risk of a cancer or tumour. 26. he method of claim 24, wherein said level and/or functional activity corresponds to a level and/or functional activity of a polypeptide comprising the sequence set forth in SEQ ID NO: 2 or 7, which correlates with the presence or risk of said cancer or tumour. 27. The method of claim 24, wherein the cancer or tumour is associated with an organ selected from kidney, brain or testis. 28. The method of claim 24, wherein the cancer or tumour is a cancer or tumour of the kidney. 29. The method of claim 24, wherein said cancer or tumour is renal cell carcinoma. 30. A method of producing a polypeptide variant of a parent polypeptide comprising the sequence set forth in any one of SEQ ID NO: 2 and 7, or a biologically active fragment thereof, said method comprising: providing a modified polypeptide whose sequence is distinguished from the parent polypeptide or said biologically active fragment, by the substitution, deletion or addition of at least one amino acid; contacting the modified polypeptide with an antigen-binding molecule that is immuno-interactive with said parent polypeptide or said biologically active fragment; and detecting the presence of a complex comprising the antigen-binding molecule and the modified polypeptide, which indicates that said modified polypeptide is a variant. 31. A method of screening for an agent which modulates tumorigenesis, said method comprising: contacting a preparation comprising: (i) a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7, or to a variant or derivative thereof; or (ii) a polynucleotide comprising at least a portion of a genetic sequence that regulates said polypeptide, which is operably linked to a reporter gene, with a test agent; and detecting a change in the level and/or functional activity of said polypeptide, or an expression product of said reporter gene, relative to a normal or reference level and/or functional activity in the absence of said test agent. 32. The method of claim 31, wherein inhibits or otherwise reduces tumorigenesis. 33. The method of claim 32, further characterised by detecting an a reduction in the level and/or functional activity of said polypeptide, or an expression product of said reporter gene, relative to said normal or reference level and/or functional activity. 34. (canceled) 35. An antigen-binding molecule that is immuno-interactive with a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7, or to a variant or derivative thereof. 36. A method for detecting a specific polypeptide or polynucleotide sequence, comprising detecting a sequence of: SEQ ID NO: 2, or a fragment thereof at least 6 amino acids in length; or SEQ ID NO: 7, or a fragment thereof at least 6 amino acids in length; or SEQ ID NO: 1, or a fragment thereof at least 18 nucleotides in length; or SEQ ID NO: 3, or a fragment thereof at least 18 nucleotides in length; or SEQ ID NO: 4, or a fragment thereof at least 18 nucleotides in length; or SEQ ID NO: 6, or a fragment thereof at least 18 nucleotides in length; or SEQ ID NO: 8, or a fragment thereof at least 18 nucleotides in length. 37. A method for detecting a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7, or to a variant or derivative thereof, said method comprising: detecting expression in a cell of a polynucleotide comprising a nucleotide sequence encoding said polypeptide. 38. A method of detecting a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7, or to a variant or derivative thereof in a biological sample, said method comprising: contacting the sample with an antigen-binding molecule that is immuno-interactive with said polypeptide; and detecting the presence of a complex comprising said antigen-binding molecule and said polypeptide in said contacted sample. 39. A method for detecting the presence or diagnosing the risk of a cancer or tumour in a patient, comprising detecting aberrant expression of TTYH2 in a biological sample obtained from said patient. 40. The method of claim 39, wherein said aberrant expression is detected by detecting a level and/or functional activity of a TTYH2 expression product in said biological sample, which differs from a normal reference level and/or functional activity and which correlates with presence or risk of said cancer or tumour. 41. The method of claim 40, wherein said aberrant expression is detected by detecting a higher level and/or functional activity of said expression product than said normal reference level and/or functional activity. 42. The method of claim 40, wherein the level and/or functional activity of said expression product in said biological sample is at least 110% of that which is present in a corresponding biological sample obtained from a normal individual or from an individual who is not afflicted with said cancer or tumour. 43. The method of claim 39, wherein the cancer or tumour is associated with an organ selected from kidney, brain or testis. 44. The method of claim 39, wherein said cancer or tumour is a cancer or tumour of the kidney. 45. The method of claim 39, wherein said cancer or tumour is renal cell carcinoma. 46. A method for detecting the presence or diagnosing the risk of a cancer or tumour in a patient, comprising detecting in a biological sample obtained from said patient an aberrant level and/or functional activity of an expression product of a gene selected from TTYH2 or a gene relating to the same regulatory or biosynthetic pathway as TTYH2, wherein said aberrant level and/or functional activity correlates with the presence or risk of said cancer or tumour. 47. The method of claim 46, wherein said expression product is expressed at a higher level and/or functional activity than said normal reference level and/or functional activity. 48. The method of claim 46, wherein said aberrant level and/or functional activity is at least 110% of that which is present in a corresponding biological sample obtained from a normal individual or from an individual who is not afflicted with said cancer or tumour. 49. The method of claim 46, wherein the cancer or tumour is associated with an organ selected from kidney, brain or testis. 50. The method of claim 46, wherein said cancer or tumour is a cancer or tumour of the kidney. 51. The method of claim 46, wherein said cancer or tumour is renal cell carcinoma. 52. A method for diagnosing the progression of a cancer or tumour in a patient, comprising measuring aberrant TTYH2 expression in a biological sample obtained from said patient. 53. A method for prognostic assessment of a cancer or tumour in a patient, comprising detecting aberrant TTYH2 expression n a biological sample obtained from said patient. 54. A method for detecting the presence or diagnosing the risk of a cancer or tumour in a patient, comprising: providing a biological sample from said patient; and detecting relative to a normal reference value, an elevation in the level and/or functional activity of a member selected from the group consisting of a polypeptide comprising the sequence set forth in any one of SEQ ID NO: 2 and 7, or variant thereof, and a polynucleotide comprising the sequence set forth in any one of SEQ ID NO: 1, 3, 6 and 8, or variant thereof. 55. The method of claim 54, wherein said member is present in said biological sample at a higher level and/or functional activity than in a corresponding biological sample obtained from a normal individual or from an individual who is not afflicted with said cancer or tumour. 56. The method of claim 55, wherein said higher level and/or functional activity is at least 110% of that which is present in said corresponding biological sample. 57. The method of claim 54, wherein the cancer or tumour is associated with an organ selected from kidney, brain or testis. 58. The method of claim 54, wherein said cancer or tumour is a cancer or tumour of the kidney. 59. The method of claim 54, wherein said cancer or tumour is renal cell carcinoma. 60. A method for detecting the presence or diagnosing the risk of a cancer or tumour in a patient, comprising: providing a biological sample from said patient; and detecting aberrant expression of a TTYH2 polynucleotide or a TTYH2 polypeptide. 61. The method of claim 60, wherein said TTYH2 polynucleotide or said TTYH2 polypeptide is present in said biological sample at a higher level and/or functional activity than in a corresponding biological sample obtained from a normal individual or from an individual who is not afflicted with said cancer or tumour. 62. The method of claim 61, wherein said higher level and/or functional activity is at least 110% of that which is present in said corresponding biological sample. 63. The method of claim 60, wherein the cancer or tumour is associated with an organ selected from kidney, brain or testis. 64. The method of claim 60, wherein said cancer or tumour is a cancer or tumour of the kidney. 65. The method of claim 60, wherein said cancer or tumour is renal cell carcinoma. 66. A method for detecting the presence or diagnosing the risk of a cancer or tumour in a patient, comprising: contacting a biological sample obtained from said patient with an antigen-binding molecule that is immuno-interactive with a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7, or to a variant or derivative thereof, measuring the concentration of a complex comprising said antigen-binding molecule and a polypeptide comprising the sequence set forth in SEQ ID NO: 2 or 7, or a variant thereof, in said contacted sample; and relating said measured complex concentration to the concentration of said polypeptide in said sample, wherein the presence of an elevated concentration relative to a normal reference concentration is indicative of said cancer or tumour. 67. The method of claim 66, wherein said concentration of said polypeptide in said sample is at least 110% of that which is present in a corresponding biological sample obtained from a normal individual or from an individual who is not afflicted with said cancer or tumour. 68. The method of claim 67, wherein the cancer or tumour is associated with an organ selected from kidney, brain or testis. 69. The method of claim 67, wherein said cancer or tumour is a cancer or tumour of the kidney. 70. The method of claim 67, wherein said cancer or tumour is renal cell carcinoma. 71. A method for modulating tumorigenesis, said method comprising introducing into said cell an agent for a time and under conditions sufficient to modulate the level and/or functional activity of TTYH2 wherein said agent is identifiable by a screening method comprising: contacting a preparation comprising: (i) a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7, or to a variant or derivative thereof; or (ii) a polynucleotide comprising at least a portion of a genetic sequence that regulates said polypeptide, which is operably linked to a reporter gene, with a test agent; and detecting a change in the level and/or functional activity of said polypeptide, or an expression product of said reporter gene, relative to a normal or reference level and/or functional activity in the absence of said test agent. 72. The method of claim 71, wherein said agent decreases the level and/or functional activity of TTYH2. 73. The method of claim 71, wherein said agent is an antisense oligonucleotide or ribozyme that binds to, or otherwise interacts specifically with, a polynucleotide encoding TTYH2 or complement of thereof, or variant of these. 74. The method of claim 71, wherein said agent is an antigen-binding molecule that is immuno-interactive with TTYH2 or variant thereof. 75. A composition for delaying, repressing or otherwise inhibiting tumorigenesis, comprising an agent that reduces the level and/or functional activity of TTYH2, and optionally a pharmaceutically acceptable carrier. 76. A composition for treatment and/or prophylaxis of a cancer or tumour, comprising an agent that reduces the level and/or functional activity of TTYH2, an optionally a pharmaceutically acceptable carrier. 77. A method for treatment and/or prophylaxis of a cancer or tumour, said method comprising administering to a patient in need of such treatment an effective amount of an agent that reduces the level and/or functional activity of TTYH2, and optionally a pharmaceutically acceptable carrier wherein said agent is identifiable by a screening method comprising: contacting a preparation comprising: (i) a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7, or to a variant or derivative thereof; or (ii) a polynucleotide comprising at least a portion of a genetic sequence that regulates said polypeptide, which is operably linked to a reporter gene, with said agent; and detecting inhibition or reduction in the level and/or functional activity of said polypeptide, or an expression product of said reporter gene, relative to a normal or reference level and/or functional activity in the absence of said agent. 78. (canceled) 79. (canceled) 80. A non-human genetically modified animal model for TTYH2 function, wherein the genetically modified animal is characterised by having an altered TTYH2 gene. 81. The genetically modified animal of claim 80, comprising an alteration to its genome, wherein the alteration comprises replacement of an endogenous TTYH2 gene with a foreign TTYH2 gene. 82. The genetically modified animal of claim 80, comprising an alteration to its genome, wherein the alteration corresponds to a partial or complete loss of function in one or both alleles of the endogenous TTYH2 gene. 83. The genetically modified animal of claim 80, comprising a disruption in at least one allele of the endogenous TTYH2 gene. 84. A composition, comprising an immunopotentiating agent selected from the polypeptide of claim 1, or the polynucleotide of claim 10, or the vector of claim 14 or the expression vector of claim 15, together with a pharmaceutically acceptable carrier. 85. The composition of claim 84, further comprising an adjuvant. 86. A method for modulating an immune response against a cancer or tumour, comprising administering to a patient in need of such treatment an effective amount of an immunopotentiating agent selected from the polypeptide of claim 1, or the polynucleotide of claim 10, or the vector of claim 14 or the expression vector of claim 15. 87. The method of claim 86, wherein the cancer or tumour is associated with an organ selected from kidney, brain or testis. 88. The method of claim 86, wherein said cancer or tumour is a cancer or tumour of the kidney. 89. The method of claim 86, wherein said cancer or tumour is renal cell carcinoma. |
<SOH> BACKGROUND OF THE INVENTION <EOH>A limited number of genetic changes have been identified in renal cell carcinoma (RCC) based on studies of familial forms of this disease. Mutations in the von Hippel Lindau (VHL) gene, a tumour suppressor gene, (Latif et al., 1993; Maher et al., 1991) are associated with familial and many sporadic clear cell RCC, the most common form of RCC. Hereditary papillary RCC has been linked with the c-MET proto-oncogene (Schmidt et al., 1997) and increased expression is also associated with sporadic papillary RCC (Fleming et al., 1998). However, not all patients with RCC have mutations or alterations in the expression of these currently identified genes, as illustrated by other forms of familial RCC (Teh et al., 1997). Thus, it is reasonable to speculate that there are other, potentially functionally significant, genetic and/or molecular abnormalities involved in the initiation and progression of RCC that are yet to be identified. Tumorigenesis is the result of multiple genetic alterations, which act coordinately to contribute to the disease process. Identification of genes whose expression is dramatically altered in tumour versus normal cells will be invaluable in furthering our understanding of the molecular events underlying cancer development (Sager, 1997). Comparison of cellular gene expression profiles, using techniques such as differential display-polymerase chain reaction (DD-PCR) (Liang & Pardee, 1992), is a valuable tool for isolating disease-associated genes. DD-PCR has been used extensively to identify genes that are differentially expressed in cancers of the breast, prostate and ovary (Chen et al., 1998; Cole et al., 1998; Mok et al., 1998). In comparison, only a small number of studies have used this approach to examine RCC (Ivanov et al., 1998; Kocher et al., 1995; Stassar et al., 1999; Thrash-Bingham & Tartof, 1999). Although a number of genes associated with RCC were identified in these studies, their precise role in RCC tumorigenesis is yet to be elucidated. In work leading up to the present invention, the inventors sought to identify other genes that are differentially expressed in RCC, by performing DD-PCR using RNA derived from RCC and from normal kidney parenchyma obtained from the same individual. A novel partial gene sequence was identified whose expression was up-regulated in RCC. This gene was cloned and its genomic localisation, structure and tissue expression pattern determined. The predicted 534 amino acid protein shows homology to the human (48%) and mouse (49%) TTYH1 (tweety homologue 1) and Drosophila melanogaster tweety (29%) proteins and thus this novel gene was designated TTYH2 (tweety homologue 2). The mouse orthologue was also identified and shares 81% identity with the human TTYH2 protein. These two novel proteins have 5 transmembrane regions in the same arrangement to the other tweety-related proteins, indicating that they are members of a new family of putative membrane-spanning proteins. |
<SOH> SUMMARY OF THE INVENTION <EOH>Accordingly, in one aspect of the invention, there is provided an isolated polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7, or to a variant or derivative thereof. The biologically active fragment preferably comprises at least 6, and more preferably at least 8, contiguous amino acids contained within the sequence set forth in SEQ ID NO: 2 or 7. In one embodiment, the biologically active fragment is selected from residues 1-8, 9-16, 17-24, 25-32, 33-40, 41-48, 49-56, 57-64, 65-72, 73-80, 81-88, 89-96, 97-104, 105-112, 113-120, 121-128, 129-136, 137-144, 145-152, 153-160, 161-168, 169-176, 177-184, 185-192, 193-200, 201-208, 209-216, 217-224, 225-232, 223-240, 241-248, 249-256, 257-264, 265-272,273-280, 281-288, 289-296, 297-304, 305-312, 313-320, 321-328, 329-336, 337-344, 345-352, 353-360, 361-368, 369-376, 317-384, 385-392, 393-400, 401-408, 409-416, 417-424, 425-432, 423-440, 441-448, 449-456, 457-464, 465-472, 473-480,481-488, 489-496, 497-504, 505-512, 513-520, 521-528 and 527-534 of SEQ ID NO: 2. In another embodiment, the biologically active fragment is selected from residues 1-8, 9-16, 17-24, 25-32, 33-40, 41-48, 49-56, 57-64, 65-72, 73-80, 81-88, 89-96, 97-104, 105-112, 113-120, 121-128, 129-136, 137-144, 145-152, 153-160, 161-168, 169-176, 177-184, 185-192, 193-200, 201-208, 209-216, 217-224, 225-232, 223-240, 241-248, 249-256, 257-264, 265-272, 273-280, 281-288, 289-296, 297-304, 305-312, 313-320, 321-328, 329-336, 337-344, 345-352, 353-360, 361-368, 369-376, 377-384, 385-392, 393-400, 401-408, 409-416,417-424, 425-432, 423-440, 441-448, 449-456, 457-464, 465-472, 473-480, 481-488, 489-496, 497-504, 505-512, 513-520, 521-528 and 525-532 of SEQ ID NO: 7. In another embodiment, the biologically active fragment is selected from residues 1-57, 109-216 or 259-391 of SEQ ID NO: 2 or 7. In this instance, the biologically active fragment suitably comprises a predicted extracellular domain of TTYH2. In yet another embodiment, the biologically active fragment is selected from residues 58-74, 92-108, 217-233, 240-258 or 392-408 of SEQ ID NO: 2 or 7. In this instance, the biologically active fragment suitably comprises a predicted TTYH2 transmembrane domain. In yet another embodiment, the biologically active fragment is selected from residues 75-91, 234-239 or 409-534 of SEQ ID NO: 2, or residues 409-532 of SEQ ID NO: 7. In this instance, the biologically active fragment suitably comprises a predicted TTYH2 intracellular domain. Suitably, the variant has at least 50%, preferably at least 55%, more preferably at least 60%, even more preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90% and still even more preferably at least 95% sequence identity to the sequence set forth in any one of SEQ ID NO: 2 and 7 or biologically active fragment thereof. In a preferred embodiment, the variant is distinguished from at least a portion of the sequence set forth in SEQ ID NO: 2 or 7 by the substitution of at least one amino acid residue. In an especially preferred embodiment of this type, the substitution is a conservative substitution. In another aspect, the invention provides an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide as broadly described above. In a preferred embodiment, the polynucleotide comprises a nucleotide sequence that corresponds or is complementary to at least a portion of the sequence set forth in SEQ ID NO: 1, 3, 4, 6 or 8, or to a polynucleotide variant thereof. Preferred portions of the said sequence comprise at least 18, more preferably at least 24, contiguous nucleotides of the sequence set forth in SEQ ID NO: 1, 3, 4, 6 or 8. In one embodiment, the polynucleotide variant has at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80% and still more preferably at least 90% sequence identity to at least a portion of the sequence set forth in SEQ ID NO: 1, 3, 4, 6 or 8. The variant may be obtained from any suitable animal. Preferably, the variant is obtained from a mammal. In another aspect, the invention contemplates a vector comprising a polynucleotide as broadly described above. In yet another aspect, the invention features an expression vector comprising a polynucleotide as broadly described above wherein the polynucleotide is operably linked to a regulatory polynucleotide. In a further aspect, the invention provides a host cell containing a vector or expression vector as broadly described above. The invention also contemplates a method of producing a recombinant polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7, or to a variant or derivative thereof, said method comprising: culturing a host cell containing an expression vector as broadly described above such that said recombinant polypeptide is expressed from said polynucleotide; and isolating said recombinant polypeptide. In a further aspect, the invention provides a method of producing a biologically active fragment of a polypeptide comprising the sequence set forth in SEQ ID NO: 2 or 7, comprising: introducing a fragment of the polypeptide or a polynucleotide from which the fragment can be translated into a cell; and detecting modulation of tumorigenesis, which indicates that said fragment is a biologically active fragment. In a preferred embodiment, the fragment is present in said cell at a level and/or functional activity that correlates with the presence or risk of a cancer or tumour, which is preferably a cancer or tumour of the kidney and more preferably renal cell carcinoma. For example, that level and/or functional activity may correspond to a level and/or functional activity of a polypeptide comprising the sequence set forth in SEQ ID NO: 2 or 7, which correlates with the presence or risk of said cancer or tumour. In yet a further aspect, the invention provides a method of producing a polypeptide variant of a parent polypeptide comprising the sequence set forth in SEQ ID NO: 2 or 7, or a biologically active fragment thereof, comprising: providing a modified polypeptide whose sequence is distinguished from the parent polypeptide by the substitution, deletion or addition of at least one amino acid; introducing said modified polypeptide or a polynucleotide from which the modified polypeptide can be translated into a cell; and detecting modulation of tumorigenesis, which indicates that said modified polypeptide is a polypeptide variant. In yet a further aspect, the invention provides a method of producing a polypeptide variant of a parent polypeptide comprising the sequence set forth in any one of SEQ ID NO: 2 and 7, or a biologically active fragment thereof, comprising: providing a modified polypeptide whose sequence is distinguished from the parent polypeptide or said biologically active fragment, by the substitution, deletion or addition of at least one amino acid; contacting the modified polypeptide with an antigen-binding molecule that is immuno-interactive with said parent polypeptide or said biologically active fragment; and detecting the presence of a complex comprising the antigen-binding molecule and the modified polypeptide, which indicates that said modified polypeptide is a variant. The present inventors have determined that aberrant expression of TTYH2 is associated with modulation of tumorigenesis. Accordingly, the isolated polypeptides and polynucleotides as broadly described above can be used to provide both drug targets and regulators to promote or inhibit one or more of said activities and to provide diagnostic markers for cancers using, for example, detectable polypeptides and polynucleotides as broadly described above, or using detectable agents which interact specifically with those polypeptides or polynucleotides. Thus, in another aspect, the invention extends to a method of screening for an agent which modulates tumorigenesis, said method comprising: contacting a preparation comprising: (i) a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7, or to a variant or derivative thereof; or (ii) a polynucleotide comprising at least a portion of a genetic sequence that regulates said polypeptide, which is operably linked to a reporter gene, with a test agent; and detecting a change in the level and/or functional activity of said polypeptide, or an expression product of said reporter gene, relative to a normal or reference level and/or functional activity in the absence of said test agent. In a preferred embodiment, said agent inhibits or otherwise reduces tumorigenesis. In this instance, the method is further characterised by detecting an a reduction in the level and/or functional activity of said polypeptide, or an expression product of said reporter gene, relative to said normal or reference level and/or functional activity. In another aspect, the invention resides in the use of a polypeptide comprising an amino acid sequence that corresponds to at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7, or to a variant or derivative thereof, to produce an antigen-binding molecule that is immuno-interactive with said polypeptide. In yet another aspect, the invention provides antigen-binding molecules that are immuno-interactive with said polypeptide, fragment, variant or derivative. In another aspect, the invention envisions a method for detecting a specific polypeptide or polynucleotide sequence, comprising detecting a sequence of: SEQ ID NO: 2, or a fragment thereof at least 6 amino acids in length; or SEQ ID NO: 7, or a fragment thereof at least 6 amino acids in length; or SEQ ID NO: 1, or a fragment thereof at least 18 nucleotides in length; or SEQ ID NO: 3, or a fragment thereof at least 18 nucleotides in length, or SEQ ID NO: 4, or a fragment thereof at least 18 nucleotides in length; or SEQ ID NO: 6, or a fragment thereof at least 18 nucleotides in length; or SEQ ID NO: 8, or a fragment thereof at least 18 nucleotides in length. In yet another aspect, there is provided a method for detecting a polypeptide as broadly described above, comprising: detecting expression in a cell of a polynucleotide comprising a nucleotide sequence encoding said polypeptide. According to another aspect of the invention, there is provided a method of detecting a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of the sequence set forth in SEQ ID NO: 2 or 7, or to a variant or derivative thereof in a biological sample, method comprising: contacting the sample with an antigen-binding molecule as broadly described above; and detecting the presence of a complex comprising said antigen-binding molecule and said polypeptide, fragment, variant or derivative in said contacted sample. In another aspect of the invention, there is provided a method for detecting the presence or diagnosing the risk of a cancer or tumour in a patient, comprising detecting aberrant expression of TTYH2 in a biological sample obtained from said patient. Aberrant expression of a TTYH2 includes and encompasses (i) an aberrant TTYH2 expression product, which suitably comprises a substitution, deletion and/or addition of one or more subunits (e.g., nucleotides or amino acids) relative to a normal TTYH2 expression product; and (ii) a level and/or functional activity of an expression product of a gene selected from TTYH2 or a gene related to the same biosynthetic or regulatory pathway as TTYH2, which differs from a normal reference level and/or functional activity. In a preferred embodiment, the expression product, which is preferably a TTYH2 expression product is expressed at a higher level and/or functional activity than said normal reference level and/or functional activity. Thus, in another aspect of the present invention, there is provided a method for detecting the presence or diagnosing the risk of a cancer or tumour in a patient, comprising detecting in a biological sample obtained from said patient an aberrant level and/or functional activity of an expression product of a gene selected from TTYH2 or a gene related to the same regulatory or biosynthetic pathway as TTYH2, which correlates with the presence or risk of said cancer or tumour. In a preferred embodiment, the expression product is expressed at a higher level and/or functional activity than said normal reference level and/or functional activity. In another aspect, the invention provides a method for diagnosing the progression of a cancer or tumour in a patient, comprising measuring aberrant TTYH2 expression in a biological sample obtained from said patient. In yet another aspect, the invention contemplates a method for prognostic assessment of a cancer or tumour in a patient, comprising detecting aberrant TTYH2 expression n a biological sample obtained from said patient. In one embodiment, the method comprises detecting a change in the level and/or functional activity of a target molecule selected from an expression product of a gene selected from TTYH2 or a gene relating to the same regulatory or biosynthetic pathway as TTYH2, wherein the change is relative to a normal reference level and/or functional activity of said expression product. In a preferred embodiment, the method comprises detecting a change in the level and/or functional activity of an expression product of TTYH2 relative to a corresponding normal reference level and/or functional activity of said expression product. In yet another aspect, the invention encompasses a method for detecting the presence or diagnosing the risk of a cancer or tumour in a patient, comprising: providing a biological sample from said patient; and detecting relative to a normal reference value, an elevation in the level and/or functional activity of a member selected from the group consisting of a polypeptide comprising the sequence set forth in any one of SEQ ID NO: 2 and 7, or variant thereof, and a polynucleotide comprising the sequence set forth in any one of SEQ ID NO: 1, 3, 6 and 8, or variant thereof. In a further aspect, the invention envisions a method for detecting the presence or diagnosing the risk of a cancer or tumour in a patient, comprising: providing a biological sample from said patient; and detecting aberrant expression of a TTYH2 polynucleotide or a TTYH2 polypeptide. In yet another aspect, the invention encompasses a method for detecting the presence or diagnosing the risk of a cancer or tumour in a patient, comprising: contacting a biological sample obtained from said patient with an antigen-binding molecule as broadly described above, measuring the concentration of a complex comprising said antigen-binding molecule and a polypeptide comprising the sequence set forth in SEQ ID NO: 2 or 7, or a variant thereof, in said contacted sample; and relating said measured complex concentration to the concentration of said polypeptide in said sample, wherein the presence of an elevated concentration relative to a normal reference concentration is indicative of said cancer or tumour. The cancer or tumour is associated with an organ including, but not restricted to, kidney, brain and testis. In a preferred embodiment, the cancer or tumour is selected from a cancer or tumour of the kidney, more preferably renal cell carcinoma (RCC). In another aspect, the invention encompasses the use of at least a portion of a TTYH2 expression product as broadly described above, or the use of one or more antigen-binding molecules that are immuno-interactive with a TTYH2 expression product as broadly described above, in the manufacture of a kit for detecting a TTYH2 polynucleotide or a TTYH2 polypeptide or the aberrant expression TTYH2 expression product that correlates with the presence or risk of a cancer or tumour. In another aspect of the invention, there is provided a method for modulating tumorigenesis, said method comprising introducing into said cell an agent as broadly described above for a time and under conditions sufficient to modulate the level and/or functional activity of TTYH2. The agent preferably decreases the level and/or functional activity of TTYH2. In yet another aspect, the invention provides a composition for delaying, repressing or otherwise inhibiting tumorigenesis, comprising an agent that reduces the level and/or functional activity of TTYH2, and optionally a pharmaceutically acceptable carrier. In another aspect, the invention provides a composition for treatment and/or prophylaxis of a cancer or tumour, comprising an agent that reduces the level and/or functional activity of TTYH2, an optionally a pharmaceutically acceptable carrier. According to another aspect of the invention, there is provided a method for treatment and/or prophylaxis of a cancer or tumour, said method comprising administering to a patient in need of such treatment an effective amount of an agent that reduces the level and/or functional activity of TTYH2, and optionally a pharmaceutically acceptable carrier. The invention also encompasses the use of the polypeptide as broadly described above, the polynucleotide as broadly described above, the vectors as broadly described above or the modulatory agents as broadly described above in the study, and modulation of tumorigenesis. In yet another aspect, the invention contemplates the use of an agent as broadly described above in the manufacture of a medicament for restoring a normal level and/or functional activity of a TTYH2 expression product in a patient, wherein said agent is optionally formulated with a pharmaceutically acceptable carrier. In even yet another aspect, the invention contemplates the use of the polypeptide as broadly described above, the polynucleotide as broadly described above or the expression vector as broadly described above in the manufacture of a medicament for eliciting an immune response in a patient, including the production of elements which specifically bind said polypeptide and/or which provide a protective effect against tumorigenesis, wherein said agent is optionally formulated with a pharmaceutically acceptable carrier. In still another aspect, the invention extends to the use of an agent as broadly described above or the use of the polypeptide, fragment, variant or derivative as broadly described above or an expression vector as broadly described above in the manufacture of a medicament for the treatment and/or prophylaxis of a cancer or tumour in a patient, wherein said agent is optionally formulated with a pharmaceutically acceptable carrier. According to another aspect, the invention contemplates a composition, comprising an immunopotentiating agent selected from the polypeptide as broadly described above, the polynucleotide as broadly described above or the vector or expression vector as broadly described above, together with a pharmaceutically acceptable carrier. The composition may optionally comprise an adjuvant. In a further aspect, the invention encompasses a method for modulating an immune response, which response is preferably directed against a cancer or tumour, comprising administering to a patient in need of such treatment an effective amount of an immunopotentiating agent selected from the polypeptide as broadly described above, the polynucleotide as broadly described above or the vector or expression vector as broadly described above. In still another aspect, the invention encompasses a non-human genetically modified animal model for TTYH2 function, wherein the genetically modified animal is characterised by having an altered TTYH2 gene. The genetically modified animal may comprise an alteration to its genome, wherein the alteration comprises replacement of an endogenous TTYH2 gene with a foreign TTYH2 gene. Alternatively, the alteration may correspond to a partial or complete loss of function in one or both alleles of the endogenous TTYH2 gene. In a preferred embodiment, the genetically modified animal comprises a disruption in at least one allele of the endogenous TTYH2 gene. |
Conveyor |
A conveyance device, particularly relating to a shaking pocket star wheel for conveying containers, with a carrier, a reception element and a reception opening for the conveyed material. The reception element is fixed against the force of a spring in a manner so it can be withdrawn and swiveled about an axis of rotation. To simplify the construction of the conveyance device, and to make the design more universal and operationally reliable, a spring is arranged in such a manner that the force is applied substantially at a right angle to the conveyance direction. |
1. Conveyance device such as for use with a star wheel for conveying containers or performs along a conveyance direction, comprising a carrier, at least one reception element which is provided with a reception opening for conveyed material, the at least one reception element fixed against the force of a spring in a manner so it can be withdrawn and swiveled about an axis of rotation, and the force of the spring (13, 29) being substantially applied at a right angle with respect to the conveyance direction (F). 2. Conveyance device according to claim 1, wherein the spring (13, 29) is applied between a point of attack (12, 27) on the reception element (4a, 4b) and a point of attack (14, 26) on the carrier (2). 3. Conveyance device according to claim 2, wherein the point of attack (12, 27) of the spring (13, 29) on the reception element (4a, 4b), in the conveyance direction (F), presents a separation from the axis of rotation (9, 25a, 25b). 4. Conveyance device according to claim 1, and a stop (16, 17, 28a, 28b) for the swivel motion (S) is provided. 5. Conveyance device according to claim 1, and a bearing provided as a pivot shift bearing (8, 23), whereby the reception element (4a, 4b) can be both swiveled and also shifted against the force of the spring (13, 29). 6. Conveyance device according to claim 1, wherein the axis of rotation (9, 25a, 25b) is received in an opening (10, 24a, 24b) whose dimension is larger than the diameter of the axis of rotation (9, 25a, 25b). 7. Conveyance device according to claim 6, wherein the opening (10) is designed as a longitudinal hole. 8. Conveyance device according to claim 6, and a guide piece (11) is arranged between the opening (10) and the axis of rotation (9). 9. Conveyance device according to claim 8, wherein the guide piece (11) can be swiveled about the axis of rotation (9) and is one of applied with two opposite sliding surfaces (11a) on associated counter surfaces (10a) in the opening (10) and provided with two abutments (11b, 10b) to limit the shift (V). 10. Conveyance device according to claim 6, wherein the axis of rotation (9) is fixed on the carrier (2) and the opening (10) is provided in the reception element (4a). 11. Conveyance device according to claim 1, wherein for swiveling the reception element (4a), both in the conveyance direction (F) and also in the opposite conveyance direction (F), two springs (13a, 13b) are provided, which engage, with separation in the conveyance direction (F), on both sides of the axis of rotation (9) with the reception element (4a). 12. Conveyance device according to claim 1, wherein the spring (13) is a pressure spring. 13. Conveyance device according to claim 5, wherein the bearing (23) presents two axes of rotations (25a, 25b) which are separated in the conveyance direction (F) and two open seat recesses (24a, 24b) for the axes of rotations (25a, 25b). 14. Conveyance device according to claim 13, wherein the spring (29) engages between, and at a distance from, the axes of rotations (25a, 25b). 15. Conveyance device according to claim 13 wherein the spring (29) is a bolt spring. 16. Conveyance device according to claim 13, wherein the axes of rotations (25a, 25b) are received with clearance in the seat recesses (24a, 24b). 17. Conveyance device according to claim 13, wherein the seat recesses (24a, 24b) have a shape which is approximately semicircular and the axes of rotations (25a, 25b) present an approximately round cross section. 18. Conveyance device according to claim 13, wherein the seat recesses (24a, 24b) are connected with a carrier (2). 19. Conveyance device according to claim 13, wherein the seat recesses (24a, 24b) are arranged on a mounting plate (20) which is arranged on the carrier (2) in a manner so it can be moved. 20. Conveyance device according to claim 13, wherein the spring (29) presses the axis of rotation (25a, 25b) elastically against the seat recesses (24a, 24b). |
Method for communication and/or machine resource sharing among plurality of members of a community in a communication network |
The invention concerns a method for communication and/or machine resource sharing, in a communication network, among a plurality of members of a community, whereby each member is considered as active or passive depending on whether he is connected or not to the community. The invention is characterised in that the method comprises a step which consists in managing, through at least one central server, a graph of connections between the active members. Said management step itself comprises a step whereby, when one of the passive members (called future active member) wishes to be connected to the community, he sets up a temporary connection with the central server, so that the latter can provide connection instructions to the future active member and to active member(s) to whom the latter has to be connected. Then, the future active member sets up a permanent peer-to-peer connection with each active member indicated by the central server. The inventive technique can be said to be hybrid peer-to-peer, since it combines centralisation of the connection graph among active members, with decentralisation of exchanges between among active members. |
1. Process for communication and/or machine resource sharing, within a communication network, between a plurality of members of a community, each of the members being an active member or passive member depending on whether it is connected or not to the community, characterized in that the process includes a step of managing, via at least one central server, the graph of connections between the active community members, the management step itself including the following steps, when one of the passive members wishes to be connected to the community as a future active member: the future active member establishes a temporary connection with the central server, so as to inform the central server of its wish to connect to the community; the central server calculates, and/or causes to be calculated, to which active member(s) the future active member is to be connected, and generates corresponding connection directives; via the temporary connection, the central server provides connection directives to the future active member; the central server establishes a temporary connection with each active member with whom the future active member is to be connected, in order to provide it with connection directives; the future active member establishes a permanent connection, peer to peer, with each active member indicated to it by the central server. 2. Process according to claim 1, characterized in that the step of managing the graph of connections between active community members additionally includes the following steps, when one of the active members is disconnected from the community: the central server is informed of this disconnection, by the member who has become passive and/or by at least one of the active members, and determines any active member(s) who finds itself (find themselves) detached from the community by virtue of this disconnection; the central server calculates, and/or causes to be calculated, to which active member(s) each active member detached from the community is to be connected, and generates corresponding connection directives; the central server establishes a temporary connection with each active member detached from the community and with each active member to whom they are to be connected, so as to provide them with connection directives; each active member detached from the community establishes a permanent connection, peer to peer, with each active member indicated to it by the central server. 3. Process according to claim 1, characterized in that the step of managing the graph of connections between active community members additionally includes the following step: at at least one moment, the central server calculates, and/or causes to be calculated, an at least partial reorganization of the connection graph, affecting at least some active members, and generates corresponding disconnection/reconnection directives; the central server establishes a temporary connection with each of the active members affected by the reorganization, so as to provide them with disconnection/reconnection directives; the active members affected by the reorganization establish permanent connections between them, peer to peer, according to indications from the central server. 4. Process according to claim 1, characterized in that the connection directives and/or the disconnection/reconnection directives are calculated according to at least one algorithm taking into account at least one criterion for optimizing the graph of connections between active community members. 5. Process according to claim 4, characterized in that the at least one connection graph optimization criterion belongs to the group including: reducing the redundancy of messages exchanged between active members; cutting the time for transferring a message from one active member to another; optitimizing the geographic distribution of the active members; increasing the robustness of the structure in respect of the disconnection of one of the active members; cutting the average number of active members to whom the active member is directly connected. 6. Process according to claim 1, characterized in that the connection directives and/or the disconnection/reconnection directives are calculated, in a centralized way, in the central server. 7. Process according to claim 1, characterized in that the connection directives and/or the disconnection/reconnection directives are calculated, in a decentralized way, by the active community members. 8. Process according to claim 1, characterized in that each peer-to-peer connection between two active members supports data traffic that allows at least one of the following functionalities to be delivered: point to point message transmission; point-multipoint broadcast message transmission; specific content search, within disk storage resources pooled by one of the two active members; direct exploration, within disk storage resources pooled by one of the two active members; file downloads. 9. Process according to claim 1, characterized in that the communication network is of the IP type. 10. Process according to claim 1, characterized in that, for data transmission within the community, each active member uses a proprietary exchange protocol. 11. Process according to claim 1, characterized in that, for data transmission within the community, each active member uses an exchange protocol parameterized by a key, and in that the step of managing the graph of connections between active community members additionally includes the following step: at at least one moment, the central server invites each active member to modify his exchange protocol parameterization key, in such a way that the exchange protocol is at least partially modified. 12. Process according to claim 1, characterized in that the at least one central server includes: at least one connection graph management module; possibly, at least one module delivering at least one particular functionality. 13. Process according to claim 12, characterized in that the at least one module delivering at least one particular functionality belongs to the group including: modules authenticating passive members wishing to connect to the community; modules managing community security; modules drawing up reports about the activity of active members; modules controlling the content exchanged between active members; calculating unit resource sharing modules; storage unit resource sharing modules; text mode communication modules (chat, chatroom); video mode (videoconferencing) communication modules; multimedia authoring modules; modules for games and/or interactivity between active members. 14. Process according to claim 12, characterized in that the step of managing the graph of connections between active community members additionally includes the following step: each module type is duplicated into a number of copies depending on its load, relating to the temporary connections of active members and/or of future active members. 15. Process according to claim 1, characterized in that each community member includes a piece of client software itself including: a component for temporary connection with the central server, with a view to connecting to the community of active members; possibly, at least one component delivering at least one particular functionality. 16. Process according to claim 15, characterized in that the at least one component delivering at least one particular functionality belongs to the group including: calculating unit resource sharing components; storage unit resource sharing components; text mode communication components (chat, chatroom); video mode (videoconferencing) communication components; instant messaging components; deferred message delivery components; multimedia authoring components; components for games and/or interactivity between active members. 17. Process according to claim 1, characterized in that the at least one central server is pooled, so as to be able to manage at least two communities. 18. Process according to claim 1, characterized in that it additionally includes a step of controlling, via the central server, the content exchanged between active members. 19. Process according to claim 18, characterized in that the step of controlling the content exchanged between active members includes the following steps: at least one spy module, hosted by the at least one central server and behaving like an active member, is connected to at least one active community member; at least one processing and/or decision module, hosted by the at least one central server, receives from the at least one spy module information relating to at least some of the data exchanged between active community members. 20. Process according to claim 19, characterized in that the information relating to at least some of the data exchanged between active community members belongs to the group including: raw data exchanged between active community members; data resulting from a pre-processing, carried out by at least one of the active members and/or the at least one spy module, of raw data exchanged between active community members. 21. Process according to claim 1, characterized in that it additionally includes a step of managing the list of community members, and in that the step of managing the graph of connections between active community members additionally includes the following step: authentication, by the central server, of the future active member wishing to be connected to the community, the authentication consisting in verifying that the future active member belongs to the list of community members. 22. Computer program, characterized in that it includes sequences of instructions adapted to implementing a process according to claim 1 when the program is run on a computer. 23. Central server of a system for communication and/or machine resource sharing, within a communication network, between a plurality of members of a community, each of the members being an active member or passive member depending on whether it is connected or not to the community, characterized in that the central server includes means of managing the graph of connections between active community members, the management means themselves including the following means: means of establishing a temporary connection with one of the passive members who wishes to inform the central server of its wish to be connected to the community as a future active member; calculation means, so as to determine to which active member(s) the future active member is to be connected; means of generating corresponding connection directives; means of providing the connection directives, via the temporary connection, to the future active member; means of establishing a temporary connection with each active member with whom the future active member is to be connected, so as to provide it with connection directives; in such a way that the future active member establishes a permanent connection, peer to peer, with the active member indicated to it by the central server. |
<SOH> FIELD OF THE INVENTION <EOH>The field of the invention is that of communication networks, particularly, but not exclusively, of the IP type (Internet networks type). More exactly, the invention relates to the communication and/or machine resource sharing, within such a communication network, between a plurality of members of a community. By “communication between members” is understood the possibility given to the members of a community of communicating with each other, in different modes (between two or more members, in real-time or in deferred time, etc.). The concept of “sharing machine resources between members” covers the pooling of disk storage resources and/or computer resources for the machines available to members. By “community” (or “tribe”), is understood a structure allowing a number of people (called “members” or “adherents”) who have at least one common centre of interest and/or one common point to be put in contact with each other. In other words, in order to form a community, users unite by affinity, by age, by place of residence, by company, etc. A community can also be a work group. Within a community, a distinction is made between on-line members (i.e. connected members), known as active (or “resident”) members, and off-line members (i.e. not connected members), known as passive members. An active member becomes a passive member (again) as soon as he is disconnected from the community. Conversely, a passive member becomes an active member (again) as soon as he is connected to the community. Merely in the interests of simplification, in the remainder of the description, by “community member” is understood not only the natural person, but also the hardware available to that person, namely a machine (typically a microcomputer) and a piece of software (known as client software) run on this machine. The client software allows a member to be connected to at least one other active member of the community (following the example of “Internet Explorer” (trademark) software for browsing on the Web). |
<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>The particular objective of the invention is to overcome these different drawbacks of the prior art. More exactly, one of the objectives of the present invention is to provide a process for communication and/or machine resource sharing between a plurality of members of a community (in the aforementioned sense), which allows optimised management of the graph of connections between active members. Another objective of the invention is to provide a process of this kind that allows community members to communicate with each other in real-time, and/or to share machine resources between them in real-time. Yet another objective of the invention is to provide a process of this kind that does not require exchanges between active members to be centralised. Another objective of the invention is to provide a process of this kind that allows both the interests of the “hosts” (also called CAP, for “community access providers”) and the interests of community members (users) to be guaranteed. For “community access providers”, the following particularly must be guaranteed: the qualification of the active members (or residents), the control of community access and the supervision of the content exchanged between community members. For community members, the interests to be guaranteed are particularly: exchange security, data security and service quality. These different objectives, together with others which will emerge subsequently, are met according to the invention using a process for communication and/or machine resource sharing, within a communication network, between a plurality of community members, each of said members being known as an active member or passive member depending on whether he is connected or not to the community. According to the invention, said process includes a step of managing, via at least one central server, the graph of connections between active community members. This management step itself includes the following steps, when one of the passive members, known as a future active member, wishes to be connected to the community: the future active member establishes a temporary connection with the central server, so as to inform it of his wish to connect to the community; the central server calculates, and/or causes to be calculated, to which active member(s) the future active member is to be connected, and generates corresponding connection directives; via said temporary connection, the central server provides connection directives to the future active member; the central server establishes a temporary connection with each active member with whom the future active member is to be connected, in order to provide him with connection directives; the future active member establishes a permanent connection, peer to peer, with each active member indicated to him by the central server. The general principle of the invention therefore consists in combining centralisation of the management of the graph of connections between active members, with decentralisation of exchanges between active members (“peer-to-peer” connections between active members). The technique according to the present invention can therefore be termed “hybrid peer-to-peer”. It allows the advantages in terms of centralisation and those in terms of decentralisation to be added together, while avoiding their respective drawbacks. In the present case, the centralised aspect allows management of the graph of connections between active community members. The connections between active community members are thus permanent (unlike the Napster solution where they are temporary) and organised intelligently (unlike the Gnutella solution, where they are totally free). As discussed in detail in what follows, the centralised aspect also allows, possibly, community security management, control over the content of exchanges between members, the drawing up of reports (statistics for example) on the activity of active members, the authentication of passive members wishing to connect to the community, etc. The decentralised aspect, a fundamental difference from the customer/server model, for its part allows a reduction in the load induced on the central servers, an improvement in the capacities of the communities for autonomy, the distributed storage of the community “content” (in other words files that the community members can exchange between them), operation in real-time, etc. Preferentially, said step of managing the graph of connections between the active community members additionally includes the following steps, when one of the active members is disconnected from the community: the central server is informed of this disconnection by the member who has again become passive and/or by at least one of the active members, and determines any active member(s) who finds himself (find themselves) detached from the community by virtue of this disconnection; the central server calculates, and/or causes to be calculated, to which active member(s) each active member detached from the community is to be connected, and generates corresponding connection directives; the central server establishes a temporary connection with each active member detached from the community and with each active member to whom they are to be connected, so as to provide them with connection directives; each active member detached from the community establishes a permanent connection, peer to peer, with each active member indicated to him by the central server. To advantage, said step of managing the graph of connections between the active community members additionally includes the following step: at at least one moment, the central server calculates, and/or causes to be calculated, an at least partial reorganisation of the connection graph, affecting at least some active members, and generates corresponding disconnection/reconnection directives; the central server establishes a temporary connection with each of the active members affected by the reorganisation, so as to provide them with disconnection/reconnection directives; the active members affected by the reorganisation establish permanent connections between them, peer to peer, according to indications from the central server. Indeed, the connection graph is the result of a succession of additions of new active members or the removal of active members who are disconnected. A partial or total reorganisation of the connection graph may therefore sometimes be necessary. To advantage, the connection directives and/or the disconnection/reconnection directives are calculated according to at least one algorithm taking into account at least one criterion for optimising the graph of connections between active community members. Generally speaking, it is a matter particularly of increasing the quality of service in respect of the data exchanges between active community members (while preventing bottleneck phenomena), and maintaining the connective nature of the connection graph (while preventing the appearance, within the graph, of independent sub-units). Preferentially, said at least one connection graph optimisation criterion belongs to the group including: reducing the redundancy of messages exchanged between active members; cutting the time for transferring a message from one active member to another; optimising the geographic distribution of the active members; increasing the robustness of the structure in respect of the disconnection of one of the active members; cutting the average number of active members to whom the active member is directly connected. In a first advantageous embodiment of the invention, the connection directives and/or the disconnection/reconnection directives are calculated, in a centralised way, in the central server. In a second advantageous embodiment of the invention, the connection directives and/or the disconnection/reconnection directives are calculated, in a decentralised way, by the active community members. Preferentially, each peer-to-peer connection between two active members supports data traffic that allows at least one of the following functionalities to be delivered: point to point message transmission; point-multipoint broadcast message transmission; specific content search, within disk storage resources pooled by one of the two active members; direct exploration, within disk storage resources pooled by one of the two active members; file downloads. In one particular embodiment of the invention, the communication network is of the IP type. It is clear however that the present invention is not restricted to Internet networks. Preferentially, for data transmission within the community, each active member uses a proprietary exchange protocol. Knowledge of this proprietary exchange protocol thus has an important value, since it gives control over what can pass between active community members. To advantage, in respect of data transmission within the community, each active member uses an exchange protocol parameterised by a key. Said step of managing the graph of connections between the active community members additionally includes the following step: at at least one moment, the central server invites each active member to modify his exchange protocol parameterisation key, in such a way that said exchange protocol is at least partially modified. The invention thus proposes a dynamic defence, in the event of attack, through an open-ended modification of the exchange protocol between active members. It is possible to talk about a “mutant protocol”. Preferentially, said at least one central server includes at least one connection graph management module and, possibly, at least one module delivering at least one particular functionality. In other words, a low level architecture is developed, above which is positioned a set of modules (and components, as explained in detail hereinafter). Enhancing the central server with a new functionality amounts to adding a new module to it. It is thus possible to improve an existing module without modifying the other modules. It should be noted that in the present description, there is a semantic distinction between a module, which is found on the central server, and a component, which is found in the client software of one of the members. Apart from this distinction, the module and the component both denote a program part that is isolatable and delivers a particular functionality. To advantage, said at least one module delivering at least one particular functionality belongs to the group including: modules authenticating passive members wishing to connect to the community; modules managing community security; modules drawing up reports about the activity of active members; modules controlling the content exchanged between active members; calculating unit resource sharing modules; storage unit resource sharing modules; text mode communication modules (chat, chatroom); video mode (videoconferencing) communication modules; multimedia authoring modules; modules for games and/or interactivity between active members. In an advantageous way, said step of managing the graph of connections between active community members additionally includes the following step: each module type is duplicated into a number of copies depending on its load, relating to the temporary connections of active members and/or of future active members. This duplication characteristic is aimed particularly at the event of a slow variation in load. Indeed, it allows the central server to retain, for a given centralised functionality, the same quality of service to the active community members, during a slow rise in load of the module concerned by this centralised functionality. Load balancers included in the central server then allow the load to be balanced between the different modules arising out of the duplication of the initial module. Conversely, when the load is tending to drop slowly, the operation consists in deleting the modules that are not needed and/or in bringing the modules together as far as possible on the server. In the event of a rapid increase in load, module duplication seems more difficult to conceive and it then appears difficult to retain the same quality of service in respect of the community. In terms of the member (client software), the deterioration of the service may consist in arbitrarily refusing to route certain messages that are considered to be of less importance. In terms of the central server, this deterioration occurs in an open-ended way, particularly within the connection graph management module. The latter may be led to comply less rigorously with the criteria that normally determine the graph. This allows a reduction in calculation time during connections and reorganisation. In this event, all that is left is the concern to ensure community connectivity at all costs. Generally speaking, a loss in quality in respect of the centralised functionalities is then noticeable. Conversely, in the event of a rapid drop in load, the connection graph management module naturally returns to its optimum operation. Preferentially, each member of the community includes a piece of client software itself including: a component for temporary connection with the central server, with a view to connecting to the community of active members; possibly, at least one component delivering at least one particular functionality. As for the central server modules, the components of the client software have something of the nature of the aforementioned low-level architecture specific to the invention. Enhancing the client software with a new functionality amounts to adding a new component to it. It is possible to offer the user a range of client software, including for example a basic version that is free of charge and more advanced versions that have to be paid for. To advantage, said at least one component delivering at least one particular functionality belongs to the group including: calculating unit resource sharing components; storage unit resource sharing components; text mode communication components (chat, chatroom); video mode (videoconferencing) communication components; instant messaging components; deferred message delivery components; multimedia authoring components; components for games and/or interactivity between active members. It should be noted that some functionalities can be centralised (i.e. hosted by a module of the central server) and/or decentralised (i.e. hosted by a component of the client software of the community member). In a particular embodiment of the invention, said at least one central server is pooled, so as to be able manage at least two communities. It should be noted that one and the same user may be a member of several communities at the same time. Preferentially, said process additionally includes a step of controlling, by the central server, the content exchanged between active members. There is a synergy between this functionality of controlling the content exchanged and the aforementioned functionality of managing the connection graph. It will be noted that it is the centralised aspect that allows these two functionalities to be delivered. This functionality of controlling the content exchanged allows particularly for the law on Intellectual Property (particularly respect for original work protected by Copyright) to be upheld. It is for example the community access provider who decides the exact way in which he wishes to apply the content control. In an advantageous way, said step of controlling the content exchanged between active members includes the following steps: at least one spy module, hosted by said at least one central server and behaving like an active member, is connected to at least one active community member; at least one processing and/or decision module, hosted by said at least one central server, receives from said at least one spy module information relating to at least some of the data exchanged between active community members. Through the spy module, the central server is thus indirectly itself part of the community. In other words, it can “see the community from the inside”. To advantage, said information relating to at least some of the data exchanged between active community members belongs to the group including: raw data exchanged between active community members; data resulting from a pre-processing, carried out by at least one of the active members and/or said at least one spy module, of raw data exchanged between active community members. Preferentially, said process additionally includes a step of managing the list of community members, said step of managing the graph of connections between active community members additionally includes the following step: authentication, by this central server, of the future active member wishing to be connected to the community, said authentication consisting in verifying that the future active member belongs to said list of community members. The list of community members can be managed by the central server or by a piece of equipment remote from it (for example a specific Web site, dedicated to registering new members). The invention also relates to a computer program including sequences of instructions adapted to implementing the process described above, when said program is run on a computer. The invention also relates to a central server including means for managing the graph of connections between active community members, said management means themselves including the following means: means of establishing a temporary connection with one of the passive members, known as a future active member, who wishes to inform it of his wish to be connected to the community; calculation mans, so as to determine to which active member(s) the future active member is to be connected; means of generating corresponding connection directives; means of providing said connection directives, via said temporary connection, to the future active member; means of establishing a temporary connection with each active member with whom the future active member is to be connected, so as to provide him with connection directives; in such a way that the future active member establishes a permanent connection, peer to peer, with the active member indicated to him by the central server. |
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