text
stringlengths 0
1.67M
|
|---|
<SOH> SUMMARY OF THE INVENTION <EOH>The membranes of U.S. Pat. No. 6,258,276 have the crosslinked polyelectrolyte or hydrogel evenly distributed across the thickness of the membranes, i.e., the membranes are symmetrical. It has now been found that if the polyelectrolyte gel or hydrogel is distributed unevenly, with a greater concentration of the gel at or near one surface of the membrane than the other, then the membrane displays superior properties, particularly higher flux, as compared with, for example membranes of U.S. Pat. No. 6,258,276 and other commercially available membranes, and can be operated at ultra low pressure. Such membranes having uneven distribution of gel in the membrane cross-section are referred to herein as asymmetric membranes. Accordingly, in one aspect the present invention provides a membrane comprising a microporous substrate and a crosslinked gel, preferably a hydrogel or a polyelectrolyte gel, located in pores of the substrate, wherein the crosslinked gel is distributed unevenly across the thickness of the membrane so that there is a gradient of gel distribution from one major surface of the membrane towards the other major surface of the membrane. Preferably, the density of the crosslinked gel is substantially greater at or adjacent to one major surface or the membrane than the density at or adjacent to the other major surface of the membrane. It will be appreciated that the membranes of the invention have a continuous or substantially continuous band or layer of the gel in two dimensions of the microporous substrate that are parallel to a major surface of the membrane. The gradient in gel density is in the third, or thickness, dimension of the membrane. In another aspect the invention provides a process for preparing a membrane of the invention as described above, which process comprises forming a crosslinked gel, preferably a hydrogel or a polyelectrolyte gel, in pores of a microporous substrate, the formation being carried out in such a manner that the crosslinked gel is distributed unevenly across the thickness of the substrate so that there is a gradient of gel distribution from one major surface of the membrane towards the other major surface of the membrane. In yet another aspect the invention provides a process for preparing an asymmetric membrane wherein the pores of the microporous substrate are filled with a solution of a polymer, or a polymerizable monomer, whereafter some of the solvent is allowed to evaporate and crosslinking or polymerization and crosslinking is allowed to proceed in the pores to form the asymmetric membrane. In yet another aspect of the invention, the polymer or polymerizable monomer, or cross-linking agent bear functional groups that are ionically charged or groups that can be rendered ionically charged. In yet another aspect the invention provides a process for preparing an asymmetric membrane which comprises filling the pores of the microporous substrate with a solution of a polymerizable monomer, a crosslinking agent, a photoinitiator for polymerization and a photoblocker, and irradiating to cause polymerization to form the asymmetric membrane. In yet another aspect the invention provides a process for removal of matter from a liquid, usually water, which comprises passing the liquid through a membrane of the invention. A variety of different types of asymmetry are possible including variation in the density of the gel. Further, asymmetry in the host microporous substrate can also be used to assist in providing asymmetry in the gel loading. Membranes of the invention display high fluxes at pressures normally used in water softening applications using commercial membranes. In many instances they can be operated at lower pressures and still retain separation, so the productivity of commercial membranes can be achieved at lower operating pressures. This means that separations can be achieved with lower energy, lower capital costs and also lower fouling tendencies. |
Crystallized glucocorticoid receptor ligand binding domain polypeptide and screening methods employing same |
A method of modifying a test nuclear receptor (NR) polypeptide is disclosed. The method provides a test NR polypeptide sequence having a characteristic that is targeted for modification; aligning the test NR polypeptide sequence with at least one reference NR polypeptide sequence for which an X-ray structure is available; building a three-dimensional model for the test NR polypeptide using the three-dimensional coordinates of the X-ray structure(s) of at least one reference polypeptide and its sequence alignment with the test NR polypeptide sequence; examining the three-dimensional model of the test NR polypeptide sequence for characteristic differences with the reference polypeptide; and mutating at least one amino acid residue in the test NR polypeptide sequence at a characteristic difference, whereby the test NR polypeptide is modified. |
1. A method of modifying a test NR polypeptide, the method comprising: (a) providing a test NR polypeptide sequence having a characteristic that is targeted for modification; (b) aligning the test NR polypeptide sequence with at least one reference NR polypeptide sequence for which an X-ray structure is available, wherein the at least one reference NR polypeptide sequence has a characteristic that is desired for the test NR polypeptide; (c) building a three-dimensional model for the test NR polypeptide using the three-dimensional coordinates of the X-ray structure(s) of the at least one reference polypeptide and its sequence alignment with the test NR polypeptide sequence; (d) examining the three-dimensional model of the test NR polypeptide for a difference in an amino acid residue as compared to the at least one reference polypeptide, wherein the residues are associated with the desired characteristic; and (c) mutating an amino acid residue in the test NR polypeptide sequence located at a difference identified in step (d) to a residue associated with the desired characteristic, whereby the test NR polypeptide is modified. 2. The method of claim 1, wherein the reference NR polypeptide sequence is a PR sequence, and wherein the test polypeptide sequence is a GR polypeptide sequence. 3. The method of claim 1, wherein the polypeptide of a crystalline GR LBD is used as the reference polypeptide sequence. 4. The method of claim 1, wherein the method is carried out in a bacterial expression system. 5. The method of claim 1, wherein the bacteria is E. coli. 6. A method for modifying a test NR polypeptide to improve the solubility, stability in solution and other solution behavior, to alter and preferably improve the folding and stability of the folded structure, to alter and preferably improve the ability to form ordered crystals, or combination thereof, the method comprising: (a) providing a test NR polypeptide sequence for which the solubility, stability in solution, other solution behavior, tendency to fold properly, ability to form ordered crystals, or combination thereof is different from that desired; (b) aligning the test NR polypeptide sequence with the sequences of one or more reference NR polypeptides for which the X-ray structure is available and for which the solution properties, folding behavior and crystallization properties are closer to those desired; (c) building a three-dimensional model for the test NR polypeptide using the three-dimensional coordinates of the X-ray structure(s) of the one or more of reference polypeptides and their sequence alignment with the test NR polypetide sequence; (d) examining the three-dimensional model of the test NR polypeptide for lipophilic side-chains that are exposed to solvent, for clusters of two or more lipophilic side-chains exposed to solvent, for lipophilic pockets and clefts on the surface of the protein model, for sites on the surface of the protein model that are more lipophilic than the corresponding sites on the structure(s) of the reference NR polypeptide(s), or combinations thereof; (e) for each residue identified in step (d), mutating the amino acid to an amino acid with different hydrophilicity, whereby the exposed lipophilic sites are reduced, and the solution properties improved; (f) examining the three-dimensional model at each site where the amino acid in the test NR polypeptide is different from the amino acid at the corresponding position in the reference NR polypeptide, and checking whether the amino acid in the test NR polypeptide makes favorable interactions with the atoms that lie around it in the three-dimensional model, considering the side-chain conformations predicted in step (c), considering alternative conformations of the side-chains, considering the presence of water molecules, or combinations thereof; (g) for each residue identified in step (f) as not making favorable interactions with the atoms that lie around it, mutating the residue to another amino acid that makes favorable interactions with the atoms that lie around it, thereby promoting the tendency for the test NR polypeptide to fold into a stable structure with improved solution properties, less tendency to unfold, and greater tendency to form ordered crystals; (h) examining the three-dimensional model at each residue position where the amino acid in the test NR polypeptide is different from the amino acid at the corresponding position in the reference NR polypeptide, and checking whether the steric packing, hydrogen bonding and other energetic interactions could be improved by mutating that residue or any one or more of the surrounding residues lying within 8 angstroms in the three-dimensional model; (i) for each residue position identified in step (h) as potentially allowing an improvement in the packing, hydrogen bonding and energetic interactions, mutating those residues individually or in combination to residues that improve the packing, hydrogen bonding, energetic interactions, and combinations thereof, thereby promoting the tendency for the test NR polypeptide to fold into a stable structure with improved solution properties, less tendency to unfold, and greater tendency to form ordered crystals. 7. The method of claim 6, further comprising optimizing the side-chain conformations in the three-dimensional model of the test NR polypeptide by generating many alternative side-chain conformations, refining by energy minimization, and selecting side-chain conformations with lower energy. 8. The method of claim 6, wherein the mutating of step (e) further comprises a mutation to a more hydrophilic amino acid. 9. The method of claim 6, wherein the reference NR polypeptide is PR, and wherein the test NR polypeptide is GRα. 10. The method of claim 6, wherein the reference NR polypeptide is GRα, and wherein the test NR polypeptide is GRβ or MR. 11. The method of claim 6, wherein the method is carried out in a bacterial expression system. 12. The method of claim 6, wherein the bacteria is E. coli. 13. An isolated GR polypeptide comprising a mutation in a ligand binding domain, wherein the mutation alters the solubility of the ligand binding domain. 14. An isolated GR polypeptide, or functional portion thereof, having one or more mutations comprising a substitution of a hydrophobic amino acid residue by a hydrophilic amino acid residue in a ligand binding domain. 15. The isolated polypeptide of claim 13, wherein the mutation is at a residue selected from the group consisting of V552, W557, F602, L636, Y648, W712, L741, L535, V538, C638, M691, V702, Y648, Y660, L685, M691, V702, W712, L733, Y764 and combinations thereof. 16. The isolated polypeptide of claim 13, wherein the mutation is selected from the group consisting of V552K, W557S, F602S, F602D, F602E, F602Y, F602T, F602N, F602C., L636E, Y648Q, W712S, L741R, L535T, V538S, C638S, M691T, V702T, W712T and combinations thereof. 17. An isolated GR LBD polypeptide, or functional portion thereof, having a F602S mutation or a F602D mutation, or a phenylalanine to serine or phenylalanine to aspartic acid mutation at an analogous position in the sequence in any polypeptide based on sequence alignment to GRα. 18. The isolated polypeptide of claim 17, wherein the polypeptide has the sequence of SEQ ID NO:12 or 14. 19. An isolated nucleic acid molecule encoding a GR polypeptide of claim 13. 20. A chimeric gene, comprising the nucleic acid molecule of claim 19 operably linked to a heterologous promoter. 21. A vector comprising the chimeric gene of claim 20. 22. A host cell comprising the chimeric gene of claim 20. 23. A method of detecting a nucleic acid molecule that encodes a GR polypeptide, the method comprising: (a) procuring a biological sample comprising nucleic acid material; (b) hybridizing the nucleic acid molecule of claim 19 under stringent hybridization conditions to the biological sample of (a), thereby forming a duplex structure between the nucleic acid of claim 19 and a nucleic acid within the biological sample; and (c) detecting the duplex structure of (b), whereby a GR encoding nucleic acid molecule is detected. 24. An antibody that specifically recognizes a GR polypeptide of claim 13. 25. A method for producing an antibody that specifically recognizes a GR polypeptide, the method comprising: (a) recombinantly or synthetically producing a GR polypeptide of claim 13, or portion thereof; (b) formulating the polypeptide of (a) whereby it is an effective immunogen; (c) administering to an animal the formulation of (b) to generate an immune response in the animal comprising production of antibodies, wherein antibodies are present in the blood serum of the animal; and (d) collecting the blood serum from the animal of (c), the blood serum comprising antibodies that specifically recognize a GR polypeptide. 26. A method for detecting a level of GR polypeptide, the method comprising: (a) obtaining a biological sample comprising peptidic material; and (b) detecting a GR polypeptide in the biological sample of (a) by immunochemical reaction with the antibody of claim 24, whereby an amount of GR polypeptide in a sample is determined. 27. A method for identifying a substance that modulates GR LBD function, the method comprising: (a) isolating a GR LBD polypeptide of claim 13; (b) exposing the isolated GR polypeptide to a plurality of substances; (c) assaying binding of a substance to the isolated GR polypeptide; and (d) selecting a substance that demonstrates specific binding to the isolated GR LBD polypeptide. 28. A substantially pure GR ligand binding domain polypeptide in crystalline form. 29. The polypeptide of claim 28, wherein the crystalline form comprises lattice constants of a=b=126.014 Å, c=86.312 Å, a=90°, β=90°, γ=120°. 30. The polypeptide of claim 28, wherein the crystalline form is a hexagonal crystalline form. 31. The polypeptide of claim 28, wherein the crystalline form has a space group of P61. 32. The polypeptide of claim 28, wherein the GRα ligand binding domain polypeptide has the amino acid sequence shown in any one of SEQ ID NOs:12, 14, 16 and 31. 33. The polypeptide of claim 28, wherein the GR ligand binding domain polypeptide is in complex with a ligand. 34. The polypeptide of claim 33, wherein the ligand is a steroid. 35. The polypeptide of claim 34, wherein the steroid is dexamethasone. 36. The polypeptide of claim 28, wherein the GR ligand binding domain polypeptide is in complex with a ligand and a peptide. 37. The polypeptide of claim 36, wherein the ligand is a steroid. 38. The polypeptide of claim 37, wherein the steroid is dexamethasone. 39. The polypeptide of claim 38, wherein the ligand is a steroid and the peptide is a fragment of a co-activator. 40. The polypeptide of claim 36, wherein the ligand is a steroid and the peptide is a fragment of a co-repressor. 41. The polypeptide of claim 36, wherein the ligand is dexamethasone and the peptide comprises an LXXLL (SEQ ID NO:18) motif. 42. The polypeptide of claim 36, wherein the peptide is a fragment of a TIF2 protein. 43. The polypeptide of claim 42, wherein the ligand is dexamethasone and the peptide has the amino acid sequence shown in any one of SEQ ID NO:17. 44. The polypeptide of claim 28, wherein the GR ligand binding domain has a crystalline structure further characterized by the atomic structure coordinates shown in Table 4. 45. The polypeptide of claim 28, wherein the crystalline form contains two GRα ligand binding domain polypeptide in the asymmetric unit. 46. The polypeptide of claim 28, wherein the crystalline form is such that the three-dimensional structure of the crystallized GR ligand binding domain polypeptide can be determined to a resolution of about 2.8 Å or better. 47. The polypeptide of claim 28, wherein the crystalline form contains one or more atoms having a molecular weight of 40 grams/mol or greater. 48. A method for determining the three-dimensional structure of a crystallized GR ligand binding domain polypeptide to a resolution of about 2.8 Å or better, the method comprising: (a) crystallizing a GR ligand binding domain polypeptide; and (b) analyzing the GR ligand binding domain polypeptide to determine the three-dimensional structure of the crystallized GR ligand binding domain polypeptide, whereby the three-dimensional structure of a crystallized GR ligand binding domain polypeptide is determined to a resolution of about 2.8 Å or better. 49. The method of claim 48, wherein the analyzing is by X-ray diffraction. 50. The method of claim 48, wherein the crystallization is accomplished by the hanging drop method, and wherein the GR ligand binding domain is mixed with a reservoir. 51. The method of claim 50, wherein the reservoir comprises 50mM HEPES, pH 7.5-8.5, and 1.7-2.3M ammonium formate. 52. The method of claim 48, wherein the crystallizing further comprises crystallizing the GRα ligand binding domain with a ligand and a peptide. 53. The method of claim 52, wherein the ligand is a steroid. 54. The method of claim 53, wherein the ligand is dexamethasone. 55. The method of claim 52, wherein the ligand is a steroid and the peptide is a fragment of a co-activator. 56. The method of claim 52, wherein the ligand is a steroid and the peptide is a fragment of a co-repressor. 57. The method of claim 52, wherein the ligand is dexamethasone and the peptide comprises an LXXLL (SEQ ID NO:18) motif. 58. The method of claim 52, wherein the peptide is a fragment of a TIF2 protein. 59. The method of claim 52, wherein the ligand is dexamethasone and the peptide has the amino acid sequence shown in SEQ ID NO:17. 60. A method of generating a crystallized GR ligand binding domain polypeptide, the method comprising: (a) incubating a solution comprising a GR ligand binding domain with a reservoir; and (b) crystallizing the GR ligand binding domain polypeptide using the hanging drop method, whereby a crystallized GR ligand binding domain polypeptide is generated. 61. The method of claim 60, wherein the incubating further comprises incubating the GR ligand binding domain with a ligand and a peptide. 62. The method of claim 61, wherein the ligand is a steroid. 63. The method of claim 62, wherein the steroid is dexamethasone. 64. The method of claim 61, wherein the ligand is a steroid and the peptide is a fragment of a co-activator. 65. The method of claim 61, wherein the ligand is a steroid and the peptide is a fragment of a co-repressor. 66. The method of claim 61, wherein the ligand is dexamethasone and the peptide comprises an LXXLL (SEQ ID NO:18) motif. 67. The method of claim 61, wherein the peptide is a fragment of a TIF2 protein. 68. A crystallized GRα ligand binding domain polypeptide produced by the method of claim 60. 69. A method of designing a modulator of a nuclear receptor, the method comprising: (a) designing a potential modulator of a nuclear receptor that will make interactions with amino acids in the ligand binding site of the nuclear receptor based upon the atomic structure coordinates of a GR ligand binding domain polypeptide; (b) synthesizing the modulator; and (c) determining whether the potential modulator modulates the activity of the nuclear receptor, whereby a modulator of a nuclear receptor is designed. 70. The method of claim 69, wherein the atomic structure coordinates further comprises a ligand and a peptide bound to the GR ligand binding domain polypeptide. 71. The method of claim 69, wherein the atomic structure coordinates are the atomic structural coordinates shown in Table 3. 72. The method of claim 70, wherein the ligand is a steroid. 73. The method of claim 72, wherein the steroid is dexamethasone. 74. The method of claim 70, wherein the ligand is a steroid and the peptide is a fragment of a co-activator. 75. The method of claim 70, wherein the ligand is a steroid and the peptide is a fragment of a co-repressor. 76. The method of claim 70, wherein the ligand is dexamethasone and the peptide comprises an LXXLL (SEQ ID NO:18) motif. 77. The method of claim 70, wherein the peptide is a fragment of a TIF2 protein. 78. A method of designing a modulator that selectively modulates the activity of a GRα polypeptide the method comprising: (a) obtaining a crystalline form of a GRα ligand binding domain polypeptide; (b) determining the three-dimensional structure of the crystalline form of the GRα ligand binding domain polypeptide; and (c) synthesizing a modulator based on the three-dimensional structure of the crystalline form of the GRα ligand binding domain polypeptide, whereby a modulator that selectively modulates the activity of a GRα polypeptide is designed. 79. The method of claim 78, wherein the method further comprises contacting a GRα ligand binding domain polypeptide with the potential modulator; and assaying the GRα ligand binding domain polypeptide for binding of the potential modulator, for a change in activity of the GRα ligand binding domain polypeptide, or both. 80. The method of claim 78, wherein the crystalline form is a hexagonal form. 81. The method of claim 80, wherein the crystals are such that the three-dimensional structure of the crystallized GRα ligand binding domain polypeptide can be determined to a resolution of about 2.8 Å or better. 82. The method of claim 78, wherein the crystalline form comprises a GR□ ligand binding domain with a ligand and a peptide. 83. The method of claim 82, wherein the ligand is a steroid. 84. The method of claim 83, wherein the steroid is dexamethasone. 85. The method of claim 82, wherein the ligand is a steroid and the peptide is a fragment of a co-activator. 86. The method of claim 82, wherein the ligand is a steroid and the peptide is a fragment of a co-repressor. 87. The method of claim 82, wherein the ligand is dexamethasone and the peptide comprises an LXXLL (SEQ ID NO:18) motif. 88. The method of claim 82, wherein the peptide is a fragment of a TIF2 protein. 89. The method of claim 78, wherein the three-dimensional structure of the crystalline form of the GRα ligand binding domain polypeptide is described by the atomic coordinates shown in Table 4. 90. A method of screening a plurality of compounds for a modulator of a GR ligand binding domain polypeptide, the method comprising: (a) providing a library of test samples; (b) contacting a GR ligand binding domain polypeptide with each test sample; (c) detecting an interaction between a test sample and the GR ligand binding domain polypeptide; (d) identifying a test sample that interacts with the GR ligand binding domain polypeptide; and (e) isolating a test sample that interacts with the GR ligand binding domain polypeptide, whereby a plurality of compounds is screened for a modulator of a GR ligand binding domain polypeptide. 91. The method of claim 90, wherein the test samples are bound to a substrate. 92. The method of claim 90, wherein the test samples are synthesized directly on a substrate. 93. A method for identifying a GR modulator, the method comprising: (a) providing atomic coordinates of a GR ligand binding domain to a computerized modeling system; and (b) modeling ligands that fit spatially into the binding pocket of the GR ligand binding domain to thereby identify a GR modulator, whereby a GR modulator is identified. 94. The method of claim 93, wherein the method further comprises identifying in an assay for GR-mediated activity a modeled ligand that increases or decreases the activity of the GR. 95. The method of claim 93, wherein the atomic coordinates are the atomic coordinates shown in Table 4. 96. A method of identifying modulator that selectively modulates the activity of a GRα polypeptide compared to other GR polypeptides, the method comprising: (a) providing atomic coordinates of a GRα ligand binding domain to a computerized modeling system; and (b) modeling a ligand that fits into the binding pocket of a GRα ligand binding domain and that interacts with conformationally constrained residues of a GRα conserved among GR subtypes, whereby a modulator that selectively modulates the activity of a GRα polypeptide compared to other polypeptides is identified. 97. The method of claim 96, wherein the method further comprises identifying in a biological assay for GR activity a modeled ligand that selectively binds to GRα and increases or decreases the activity of said GRα. 98. The method of claim 96, wherein the atomic coordinates are the atomic coordinates shown in Table 4. 99. A method of designing a modulator of a GR polypeptide, the method comprising: (a) selecting a candidate GR ligand; (b) determining which amino acid or amino acids of a GR polypeptide interact with the ligand using a three-dimensional model of a crystallized protein comprising a GRα LBD; (c) identifying in a biological assay for GR activity a degree to which the ligand modulates the activity of the GR polypeptide; (d) selecting a chemical modification of the ligand wherein the interaction between the amino acids of the GR polypeptide and the ligand is predicted to be modulated by the chemical modification; (e) synthesizing a chemical compound with the selected chemical modification to form a modified ligand; (f) contacting the modified ligand with the GR polypeptide; (g) identifying in a biological assay for GR activity a degree to which the modified ligand modulates the biological activity of the GR polypeptide; and (h) comparing the biological activity of the GR polypeptide in the presence of modified ligand with the biological activity of the GR polypeptide in the presence of the unmodified ligand, whereby a modulator of a GR polypeptide is designed. 100. The method of claim 99, wherein the GR polypeptide is a GRα polypeptide. 101. The method of claim 99, wherein the three-dimensional model of a crystallized protein is a GRα ligand binding domain with a ligand and a peptide. 102. The method of claim 101, wherein the ligand is a steroid. 103. The method of claim 101, wherein the steroid is dexamethasone. 104. The method of claim 101, wherein the ligand is a steroid and the peptide is a fragment of a co-activator. 105. The method of claim 101, wherein the ligand is a steroid and the peptide is a fragment of a co-repressor. 106. The method of claim 101, wherein the ligand is dexamethasone and the peptide comprises an LXXLL (SEQ ID NO:18) motif. 107. The method of claim 101, wherein the peptide is a fragment of a TIF2 protein. 108. The method of claim 99, wherein the three-dimensional model is represented by the three dimensional coordinates shown in Table 4. 109. The method of claim 99, wherein the method further comprises repeating steps (a) through (f), if the biological activity of the GR polypeptide in the presence of the modified ligand varies from the biological activity of the GR polypeptide in the presence of the unmodified ligand. 110. An assay method for identifying a compound that inhibits binding of a ligand to a GR polypeptide, the assay method comprising: (a) designing a test inhibitor compound based on the three dimensional atomic coordinates of GR; (b) incubating a GR polypeptide with a ligand in the presence of a test inhibitor compound; (c) determining an amount of ligand that is bound to the GR polypeptide, wherein decreased binding of ligand to the GR protein in the presence of the test inhibitor compound relative to binding of ligand in the absence of the test inhibitor compound is indicative of inhibition; and (d) identifying the test compound as an inhibitor of ligand binding if decreased ligand binding is observed, whereby a compound that inhibits binding of a ligand to a GR polypeptide is identified. 111. The method of claim 110, wherein the ligand is a steroid. 112. The method of claim 111, wherein the steroid is dexamethasone. 113. The method of claim 110, wherein the three dimensional coordinates are the three dimensional coordinates shown in Table 4. 114. A method of identifying a NR modulator that selectively modulates the biological activity of one NR compared to GRα, the method comprising: (a) providing an atomic structure coordinate set describing a GRα ligand binding domain structure and at least one other atomic structure coordinate set describing a NR ligand binding domain, each ligand binding domain comprising a ligand binding site; (b) comparing the atomic structure coordinate sets to identify at least one diference between the sets; (c) designing a candidate ligand predicted to interact with the difference of step (b); (d) synthesizing the candidate ligand; and (e) testing the synthesized candidate ligand for an ability to selectively modulate a NR as compared to GRα, whereby a NR modulator that selectively modulates the biological activity NR compared to GRα is identified. 115. The method of claim 114, wherein the GRα atomic structure coordinate set is the atomic structure coordinate set shown in Table 4. 116. The method of claim 114, wherein the NR is selected from the group consisting of MR, PR, AR, GRβ and isoforms thereof that have ligands that also bind GRα. |
<SOH> BACKGROUND ART <EOH>Nuclear receptors reside in either the cytoplasm or nucleus of eukaryotic cells and represent a superfamily of proteins that specifically bind a physiologically relevant small molecule, such as a hormone or vitamin. As a result of a molecule binding to a nuclear receptor, the nuclear receptor changes the ability of a cell to transcribe DNA, i.e. nuclear receptors modulate the transcription of DNA. However, they can also have transcription independent actions. Unlike integral membrane receptors and membrane-associated receptors, nuclear receptors reside in either the cytoplasm or nucleus of eukaryotic cells. Thus, nuclear receptors comprise a class of intracellular, soluble, ligand-regulated transcription factors. Nuclear receptors include but are not limited to receptors for androgens, mineralcorticoids, progestins, estrogens, thyroid hormones, vitamin D, retinoids, eicosanoids, peroxisome proliferators and, pertinently, glucocorticoids. Many nuclear receptors, identified by either sequence homology to known receptors (See, e.g., Drewes et al., (1996) Mol. Cell. Biol. 16:925-31) or based on their affinity for specific DNA binding sites in gene promoters (See, e.g., Sladek et al., Genes Dev. 4:2353-65), have unascertained ligands and are therefore commonly termed “orphan receptors”. Glucocorticoids are an example of a cellular molecule that has been associated with cellular proliferation. Glucocorticoids are known to induce growth arrest in the G1-phase of the cell cycle in a variety of cells, both in vivo and in vitro, and have been shown to be useful in the treatment of certain cancers. The glucocorticoid receptor (GR) belongs to an important class of transcription factors that alter the expression of target genes in response to a specific hormone signal. Accumulated evidence indicates that receptor associated proteins play key roles in regulating glucocorticoid signaling. The list of cellular proteins that can bind and co-purify with the GR is constantly expanding. Glucocorticoids are also used for their anti-inflammatory effect on the skin, joints, and tendons. They are important for treatment of disorders where inflammation is thought to be caused by immune system activity. Representative disorders of this sort include but are not limited to rheumatoid arthritis, inflammatory bowel disease, glomerulonephritis, and connective tissue diseases like systemic lupus erythmatosus. Glucocorticoids are also used to treat asthma and are widely used with other drugs to prevent the rejection of organ transplants. Some cancers of the blood (leukemias) and lymphatic system (lymphomas) can also respond to corticosteroid drugs. Glucocorticoids exert several effects in tissues that express receptors for them. They regulate the expression of several genes either positively or negatively and in a direct or indirect manner. They are also known to arrest the growth of certain lymphoid cells and in some cases cause cell death (Harmon et al., (1979) J. Cell Physiol. 98: 267-278; Yamamoto, (1985) Ann. Rev. Genet. 19: 209-252; Evans, (1988) Science 240:889-895; Beato, (1989) Cell 56:335-344; Thompson, (1989) Cancer Res. 49: 2259s-2265s.). Due in part to their ability to kill cells, glucocorticoids have been used for decades in the treatment of leukemias, lymphomas, breast cancer, solid tumors and other diseases involving irregular cell growth, e.g. psoriasis. The inclusion of glucocorticoids in chemotherapeutic regimens has contributed to a high rate of cure of certain leukemias and lymphomas which were formerly lethal (Homo-Delarche, (1984) Cancer Res. 44: 431-437). Although it is clear that glucocorticoids exert these effects after binding to their receptors, the mechanism of cell kill is not completely understood, although several hypotheses have been proposed. Among the more prominent hypotheses are: the deinduction of critical lymphokines, oncogenes and growth factors; the induction of supposed “lysis genes”; alterations in calcium ion influx; the induction of endonucleases; and the induction of a cyclic AMP-dependent protein kinase (McConkey et al., (1989) Arch. Biochem. Biophys. 269: 365-370; Cohen & Duke, (1984) J. Immunol. 152: 38-42; Eastman-Reks & Vedeckis, (1986) Cancer Res. 46: 2457-2462; Kelso & Munck, (1984) J. Immunol. 133:784-791; Gruol et al., (1989) Molec. Endocrinol. 3: 2119-2127; Yuh & Thompson, (1989) J. Biol. Chem. 264: 10904-10910). Polypeptides, including the glucocorticoid receptor ligand binding domain, have a three-dimensional structure determined by the primary amino acid sequence and the environment surrounding the polypeptide. This three-dimensional structure establishes the polypeptide's activity, stability, binding affinity, binding specificity, and other biochemical attributes. Thus, knowledge of a protein's three-dimensional structure can provide much guidance in designing agents that mimic, inhibit, or improve its biological activity. The three-dimensional structure of a polypeptide can be determined in a number of ways. Many of the most precise methods employ X-ray crystallography (See, e.g., Van Holde, (1971) Physical Biochemistry, Prentice-Hall, New Jersey, pp. 221-39). This technique relies on the ability of crystalline lattices to diffract X-rays or other forms of radiation. Dffraction experiments suitable for determining the three-dimensional structure of macromolecules typically require high-quality crystals. Unfortunately, such crystals have been unavailable for the ligand binding domain of a human glucocorticoid receptor, as well as many other proteins of interest. Thus, high-quality diffracting crystals of the ligand binding domain of a human glucocorticoid receptor in complex with a ligand and a peptide would greatly assist in the elucidation of its three-dimensional structure. Clearly, the solved crystal structure of the ligand binding domain of a glucocorticoid receptor polypeptide would be useful in the design of modulators of activity mediated by the glucocorticoid receptor. Evaluation of the available sequence data shows that GRα is particularly similar to MR, PR and AR. The GRα LBD has approximately 56%, 54% and 50% sequence identity to the MR, PR and AR LBDs, respectively. The GRβ amino acid sequence is identical to the GRα amino acid sequence for residues 1-727, but the remaining 15 residues in GRβ show no significant similarity to the remaining 50 residues in GRα. If no X-ray structure were available for GRα, then one could build a model for GRα using the available X-ray structures of PR and/or AR as templates. These theoretical models have some utility, but cannot be as accurate as a true X-ray structure, such as the X-ray structure disclosed here. Because of their limited accuracy, a model for GRα will generally be less useful than an X-ray structure for the design of agonists, antagonists and modulators of GRα. The solved GRα-ligand-co-activator crystal structure would provide structural details and insights necessary to design a modulator of GRα that maximizes preferred requirements for any modulator, i.e. potency and specificity. By exploiting the structural details obtained from a GRα-ligand-co-activator crystal structure, it would be possible to design a GRα modulator that, despite GRα's similarity with other steroid receptors and nuclear receptors, exploits the unique structural features of the ligand binding domain of human GRα. A GRα modulator developed using structure-assisted design would take advantage of heretofore unknown GRα structural considerations and thus be more effective than a modulator developed using homology-based design. Potential or existent homology models cannot provide the necessary degree of specificity. A GRα modulator designed using the structural coordinates of a crystalline form of the ligand binding domain of GRα in complex with a ligand and a co-activator would also provide a starting point for the development of modulators of other nuclear receptors. Although several journal articles have referred to GR mutants having “increased ligand efficacy” in cell-based assays, it has not been mentioned that such mutants could have improved solution properties so that they could provide a suitable reagent for purification, assay, and crystallization. See Garabedian & Yamamoto (1992) Mol. Biol. Cell 3: 1245-1257; Kralli, et al., (1995) Proc. Natl. Acad. Sci. 92: 4701-4705; Bohen (1995) J. Biol. Chem. 270: 29433-29438; Bohen (1998) Mol. Cell. Biol. 18: 3330-3339; Freeman et al., (2000) Genes Dev. 14: 422-434. Indeed, it is well documented that GR associates with molecular chaperones (such as hsp90, hsc70, and p23). In the past, it has been considered that GR would either not be active or soluble if purified away from these binding partners. In fact, it has even been mentioned that GR must be in complex with hsp90 in order to adopt a high affinity steroid binding conformation. See Xu et al. (1998) J. Biol. Chem. 273: 13918-13924; Rajapandi et al. (2000) J. Biol. Chem. 275: 22597-22604. Still other journal articles have reported E.coli expression of GST-GR, but also noted a failure to purify the purported polypeptide. See Ohara-Nemoto et al., (1990) J. Steroid Biochem. Molec. Biol. 37: 481-490; Caamano et al., (1994) Annal. NY Acad. Sci. 746: 68-77. What is needed, therefore, is a purified, soluble GRα LBD polypeptide for use in structural studies, as well as methods for making the same. Such methods would also find application in the preparation of modified NRs in general. What is also needed is a crystallized form of a GRα ligand binding domain, preferably in complex with a ligand and more preferably in complex with a ligand and a co-activator. Acquisition of crystals of the GRα ligand binding domain polypeptide permits the three-dimensional structure of a GRα ligand binding domain (LBD) polypeptide to be determined. Knowledge of the three dimensional structure can facilitate the design of modulators of GR-mediated activity. Such modulators can lead to therapeutic compounds to treat a wide range of conditions, including inflammation, tissue rejection, auto-immunity, malignancies such as leukemias and lymphomas, Cushing's syndrome, acute adrenal insufficiency, congenital adrenal hyperplasia, rheumatic fever, polyarteritis nodosa, granulomatous polyarteritis, inhibition of myeloid cell lines, immune proliferation/apoptosis, HPA axis suppression and regulation, hypercortisolemia, modulation of the TH1/TH2 cytokine balance, chronic kidney disease, stroke and spinal cord injury, hypercalcemia, hypergylcemia, acute adrenal insufficiency, chronic primary adrenal insufficiency, secondary adrenal insufficiency, congenital adrenal hyperplasia, cerebral edema, thrombocytopenia, Little's syndrome, inflammatory bowel disease, systemic lupus erythematosus, polyartitis nodosa, Wegener's granulomatosis, giant cell arteritis, rheumatoid arthritis, osteoarthritis, hay fever, allergic rhinitis, urticaria, angioneurotic edema, chronic obstructive pulmonary disease, asthma, tendonitis, bursitis, Crohn's disease, ulcerative colitis, autoimmune chronic active hepatitis, organ transplantation, hepatitis, cirrhosis, inflammatory scalp alopecia, panniculitis, psoriasis, discoid lupus erythematosus, inflamed cysts, atopic dermatitis, pyoderma gangrenosum, pemphigus vulgaris, bullous pemphigoid, systemic lupus erythematosus, dermatomyositis, herpes gestationis, eosinophilic fasciitis, relapsing polychondritis, inflammatory vasculitis, sarcoidosis, Sweet's disease, type 1 reactive leprosy, capillary hemangiomas, contact dermatitis, atopic dermatitis, lichen planus, exfoliative dermatitus, erythema nodosum, acne, hirsutism, toxic epidermal necrolysis, erythema multiform, cutaneous T-cell lymphoma. Other applications of a GR modulator developed in accordance with the present invention can be employed to treat Human Immunodeficiency Virus (HIV), cell apoptosis, and can be employed in treating cancerous conditions including, but not limited to, Kaposi's sarcoma, immune system activation and modulation, desensitization of inflammatory responses, IL-1 expression, natural killer cell development, lymphocytic leukemia, treatment of retinitis pigmentosa. Other applications for such a modulator comprise modulating cognitive performance, memory and learning enhancement, depression, addiction, mood disorders, chronic fatigue syndrome, schizophrenia, stroke, sleep disorders, anxiety, immunostimulants, repressors, wound healing and a role as a tissue repair agent or in anti-retroviral therapy. |
<SOH> SUMMARY OF THE INVENTION <EOH>A method of modifying a test NR polypeptide is disclosed. The method can comprise: providing a test NR polypeptide sequence having a characteristic that is targeted for modification; aligning the test NR polypeptide sequence with at least one reference NR polypeptide sequence for which an X-ray structure is available, wherein the at least one reference NR polypeptide sequence has a characteristic that is desired for the test NR polypeptide; building a three-dimensional model for the test NR polypeptide using the three-dimensional coordinates of the X-ray structure(s) of the at least one reference polypeptide and its sequence alignment with the test NR polypeptide sequence; examining the three-dimensional model of the test NR polypeptide for differences with the at least one reference polypeptide that are associated with the desired characteristic; and mutating at least one amino acid residue in the test NR polypeptide sequence located at a difference identified above to a residue associated with the desired characteristic, whereby the test NR polypeptide is modified. A method of altering the solubility of a test NR polypeptide is also disclosed in accordance with the present invention. In a preferred embodiment, the method comprises: (a) providing a reference NR polypeptide sequence and a test NR polypeptide sequence; (b) comparing the reference NR polypeptide sequence and the test NR polypeptide sequence to identify one or more residues in the test NR sequence that are more or less hydrophilic than a corresponding residue in the reference NR polypeptide sequence; and (c) mutating the residue in the test NR polypeptide sequence identified in step (b) to a residue having a different hydrophilicity, whereby the solubility of the test NR polypeptide is altered. Optionally, the reference NR polypeptide sequence is an AR or a PR sequence, and the test polypeptide sequence is a GR polypeptide sequence. Alternatively, the reference polypeptide sequence is a crystalline GR LBD. The comparing of step (b) is preferably by sequence alignment. An isolated GR polypeptide comprising a mutation in a ligand binding domain, wherein the mutation alters the solubility of the ligand binding domain, is also disclosed. An isolated GR polypeptide, or functional portion thereof, having one or more mutations comprising a substitution of a hydrophobic amino acid residue by a hydrophilic amino acid residue in a ligand binding domain is also disclosed. Preferably, in each case, the mutation can be at a residue selected from the group consisting of V552, W557, F602, L636, Y648, W712, L741, L535, V538, C638, M691, V702, Y648, Y660, L685, M691, V702, W712, L733, Y764 and combinations thereof. More preferably, the mutation is selected from the group consisting of V552K, W557S, F602S, F602D, F602E, L636E, Y648Q, W712S, L741R, L535T, V538S, C638S, M691T, V702T, W712T and combinations thereof. Antibodies against such polypeptides are also disclosed, as are methods of detecting such polypeptides and methods of identifying substances that modulate the biological activity of such polypeptides. An isolated nucleic acid molecule encoding a GR polypeptide comprising a mutation in a ligand binding domain, wherein the mutation alters the solubility of the ligand binding domain, or encoding a GR LBD polypeptide, or functional portion thereof, having one or more mutations comprising a substitution of a hydrophobic amino acid residue by a hydrophilic amino acid residue, is also disclosed. A chimeric gene, comprising the nucleic acid molecule operably linked to a heterologous promoter, a vector comprising the chimeric gene, and a host cell comprising the chimeric gene are also disclosed. Methods for detecting such a nucleic acid molecule are also disclosed. A substantially pure GRα ligand binding domain polypeptide in crystalline form is disclosed. Preferably, the crystalline form has lattice constants of a=b=126.014 A, c=86.312 Å, α=900, β=900, γ=120°. Preferably, the crystalline form is a hexagonal crystalline form. More preferably, the crystalline form has a space group of P6 1 . Even more preferably, the GRα ligand binding domain polypeptide has the F602S amino acid sequence shown in Example 2. Even more preferably, the GRα ligand binding domain has a crystalline structure further characterized by the coordinates corresponding to Table 4. Preferably, the GRα ligand binding domain polypeptide is in complex with a ligand. Optionally, the crystalline form contains two GRα ligand binding domain polypeptides in the asymmetric unit. Preferably, the crystalline form is such that the three-dimensional structure of the crystallized GRα ligand binding domain polypeptide can be determined to a resolution of about 2.8 Å or better. Even more preferably, the crystalline form contains one or more atoms having a molecular weight of 40 grams/mol or greater. A method for determining the three-dimensional structure of a crystallized GR ligand binding domain polypeptide to a resolution of about 2.8 Å or better, the method comprising: (a) crystallizing a GR ligand binding domain polypeptide; and (b) analyzing the GR ligand binding domain polypeptide to determine the three-dimensional structure of the crystallized GR ligand binding domain polypeptide, whereby the three-dimensional structure of a crystallized GR ligand binding domain polypeptide is determined to a resolution of about 2.8 Å or better. Preferably, the analyzing is by X-ray diffraction. More preferably, the crystallization is accomplished by the hanging drop method, and wherein the GRα ligand binding domain is mixed with a reservoir. A method of generating a crystallized GR ligand binding domain polypeptide, the method comprising: (a) incubating a solution comprising a GR ligand binding domain with a reservoir; and (b) crystallizing the GR ligand binding domain polypeptide using the hanging drop method, whereby a crystallized GR ligand binding domain polypeptide is generated. A method of designing a modulator of a nuclear receptor, the method comprising: (a) designing a potential modulator of a nuclear receptor that will make interactions with amino acids in the ligand binding site of the nuclear receptor based upon the atomic structure coordinates of a GR ligand binding domain polypeptide; (b) synthesizing the modulator; and (c) determining whether the potential modulator modulates the activity of the nuclear receptor, whereby a modulator of a nuclear receptor is designed. A method of designing a modulator that selectively modulates the activity of a GRα polypeptide the method comprising: (a) obtaining a crystalline form of a GRα ligand binding domain polypeptide; (b) determining the three-dimensional structure of the crystalline form of the GRα ligand binding domain polypeptide; and (c) synthesizing a modulator based on the three-dimensional structure of the crystalline form of the GRα ligand binding domain polypeptide, whereby a modulator that selectively modulates the activity of a GRα polypeptide is designed. Preferably, the method further comprises contacting a GRα ligand binding domain polypeptide with the potential modulator; and assaying the GRα ligand binding domain polypeptide for binding of the potential modulator, for a change in activity of the GRα ligand binding domain polypeptide, or both. More preferably, the crystalline form is in orthorhombic form. Even more preferably, the crystals are such that the three-dimensional structure of the crystallized GRα ligand binding domain polypeptide can be determined to a resolution of about 2.8 Å or better. A method of screening a plurality of compounds for a modulator of a GR ligand binding domain polypeptide, the method comprising: (a) providing a library of test samples; (b) contacting a GR ligand binding domain polypeptide with each test sample; (c) detecting an interaction between a test sample and the GR ligand binding domain polypeptide; (d) identifying a test sample that interacts with the GR ligand binding domain polypeptide; and (e) isolating a test sample that interacts with the GR ligand binding domain polypeptide, whereby a plurality of compounds is screened for a modulator of a GR ligand binding domain polypeptide. Preferably, the test samples are bound to a substrate, and more preferably, the test samples are synthesized directly on a substrate. The GR ligand binding domain polypeptide can be in soluble or crystalline form. A method for identifying a GR modulator is also disclosed. In a preferred embodiment, the method comprises: (a) providing atomic coordinates of a GR ligand binding domain to a computerized modeling system; and (b) modeling ligands that fit spatially into the binding pocket of the GR ligand binding domain to thereby identify a GR modulator, whereby a GR modulator is identified. Preferably, the method further comprises identifying in an assay for GR-mediated activity a modeled ligand that increases or decreases the activity of the GR. A method of identifying modulator that selectively modulates the activity of a GRα polypeptide compared to other GR polypeptides, the method comprising: (a) providing atomic coordinates of a GRα ligand binding domain to a computerized modeling system; and (b) modeling a ligand that fits into the binding pocket of a GRα ligand binding domain and that interacts with conformationally constrained residues of a GRα conserved among GR subtypes, whereby a modulator that selectively modulates the activity of a GRα polypeptide compared to other polypeptides is identified. Preferably, the method further comprises identifying in a biological assay for GRα activity a modeled ligand that selectively binds to GRα and increases or decreases the activity of said GRα. A method of designing a modulator of a GR polypeptide, the method comprising: (a) selecting a candidate GR ligand; (b) determining which amino acid or amino acids of a GR polypeptide interact with the ligand using a three-dimensional model of a crystallized protein comprising a GRα LBD; (c) identifying in a biological assay for GR activity a degree to which the ligand modulates the activity of the GR polypeptide; (d) selecting a chemical modification of the ligand wherein the interaction between the amino acids of the GR polypeptide and the ligand is predicted to be modulated by the chemical modification; (e) synthesizing a chemical compound with the selected chemical modification to form a modified ligand; (f) contacting the modified ligand with the GR polypeptide; (g) identifying in a biological assay for GR activity a degree to which the modified ligand modulates the biological activity of the GR polypeptide; and (h) comparing the biological activity of the GR polypeptide in the presence of modified ligand with the biological activity of the GR polypeptide in the presence of the unmodified ligand, whereby a modulator of a GR polypeptide is designed. Preferably, the GR polypeptide is a GRα polypeptide. More preferably, the three-dimensional model of a crystallized protein is a GRα LBD polypeptide with a bound ligand. Optionally, the method further comprises repeating steps (a) through (f) if the biological activity of the GR polypeptide in the presence of the modified ligand varies from the biological activity of the GR polypeptide in the presence of the unmodified ligand. An assay method for identifying a compound that inhibits binding of a ligand to a GR polypeptide, the assay method comprising: (a) designing a test inhibitor compound based on the three dimensional atomic coordinates of GR; (b) incubating a GR polypeptide with a ligand in the presence of a test inhibitor compound; (c) determining an amount of ligand that is bound to the GR polypeptide, wherein decreased binding of ligand to the GR protein in the presence of the test inhibitor compound relative to binding of ligand in the absence of the test inhibitor compound is indicative of inhibition; and (d) identifying the test compound as an inhibitor of ligand binding if decreased ligand binding is observed, whereby a compound that inhibits binding of a ligand to a GR polypeptide is identified. A method of identifying a NR modulator that selectively modulates the biological activity of one NR compared to GRα is also disclosed. The method comprises: (a) providing an atomic structure coordinate set describing a GRα ligand binding domain structure and at least one other atomic structure coordinate set describing a NR ligand binding domain, each ligand binding domain comprising a ligand binding site; (b) comparing the atomic structure coordinate sets to identify at least one diference between the sets; (c) designing a candidate ligand predicted to interact with the difference of step (b); (d) synthesizing the candidate ligand; and (e) testing the synthesized candidate ligand for an ability to selectively modulate a NR as compared to GRα, whereby a NR modulator that selectively modulates the biological activity NR compared to GRα is identified. Accordingly, it is an object of the present invention to provide a three dimensional structure of the ligand binding domain of a GR. The object is achieved in whole or in part by the present invention. An object of the invention having been stated hereinabove, other objects will be evident as the description proceeds, when taken in connection with the accompanying Drawings and Laboratory Examples as best described hereinbelow. |
Method and compositions relating to hpv-associated pre-cancerous and cancerous growths, including cin |
The present invention concerns the use of E6 and/or E7 peptides from human papilloma virus (HPV) to evaluate a cell-mediated response in a patient infected with HPV to determine the prognosis for that patient with respect to the development or recurrence of pre-cancerous or cancerous growths, including cervical intraepithelial neoplasia (CIN). |
1. A method for determining the possibility of recurrence of a pre-cancerous or cancerous growth in a patient infected with human papilloma virus (HPV) or suspected of being infected with HPV, wherein the patient also has or had a pre-cancerous or cancerous growth on or around the cervix comprising: a) incubating at least one E6 or E7 peptide of HPV with a sample from the patient; and b) assaying the sample for a cell-mediated immune response against the peptide. 2. The method of claim 1, wherein the sample is incubated with at least two E6 or at least two E7 peptides. 3. The method of claim 1, wherein the sample is incubated with an E6 peptide of HPV. 4. The method of claim 3, wherein the E6 peptide is K9L, E10L C10R, Q15L, V10C, P9L, P10I, Q20P, R16R, or G10S. 5. The method of claim 4, wherein the E6 peptide is K9L, E10I, C10R, Q15L, V10C, or a combination thereof. 6. The method of claim 1, wherein the sample is incubated with an E7 peptide of HPV. 7. The method of claim 6, wherein the E7 peptide is T10Q, M9T, D9L, Q19D, R9F, R9V, L9V, G10C, or D20C. 8. The method of claim 7, wherein the E7 peptide is Q19D, R9F, R9V, L9V, G10C, or a combination thereof. 9. The method of claim 1, wherein the sample is incubated with at least one E6 peptide and at least one E7 peptide. 10. The method of claim 1, wherein the patient is known to be infected with HPV. 11. The method of claim 1, further comprising determining whether the patient is infected with HPV. 12. The method of claim 1, wherein the patient has or had a pre-cancerous growth. 13. The method of claim 12, wherein the pre-cancerous growth is cervical intraepithelial neoplasia (CIN) 14. The method of claim 1, wherein the patient no longer has the pre-cancerous or cancerous growth. 15. The method of claim 14, wherein the patient no longer has a pre-cancerous growth. 16. The method of claim 15, wherein the pre-cancerous growth is CIN. 17. The method of claim 1, wherein the sample is blood. 18. The method of claim 1, wherein the sample is obtained by a vaginal swab, 19. The method of claim 1, wherein the sample comprises peripheral blood mononuclear cells. 20. The method of claim 1, further comprising incubating the sample in media after obtaining the sample. 21. The method of claim 1, wherein the assaying comprises contacting the sample with the peptide and measuring the sample for T-cell proliferation. 22. The method of claim 21, wherein T-cell proliferation is assayed by measuring incorporation of tritiated thymidine. 23. The method of claim 22, wherein the sample has an SI value of 2.0 or greater, indicating a cell-mediated immune response. 24. The method of claim 23, wherein the sample has an SI value of 3.0 or greater, indicating a cell-mediated immune response. 25. The method of claim 1, wherein the assaying comprises measuring an amount of a TH1 or TH2 cytokine. 26. The method of claim 25, wherein the amount of a TH1 cytokine is measured. 27. The method of claim 26, wherein the TH1 cytokine is IL-2, IFN-γ, TNF-α, or TNF-β. 28. The method of claim 25, wherein the amount of a TH2 cytokine is measured. 29. The method of claim 28, wherein the TH2 cytokine is IL-4, IL-5, IL-10, or IL-13. 30. The method of claim 25, wherein the TH1 or TH2 cytokine is measured with an immunoassay. 31. The method of claim 30, wherein the immunoassay is ELISA or a radioimmunoassay. 32. The method of claim 25, wherein the TH1 or TH2 cytokine is measured by flow-cytometry. 33. The method of claim 1, wherein the sample is assayed more than once. 34. The method of claim 33, wherein the sample is assayed using different assays. 35. The method of claim wherein, further comprising obtaining a second sample from the patient and assaying the second sample for a cell-mediated immune response against at least one E6 or E7 peptide of HPV. 36. The method of claim 1, wherein the sample is obtained from the patient at least one month after treatment for a pre-cancerous or cancerous growth. 37. The method of claim 1, wherein the patient has undergone ablative treatment of a pre-cancerous or cancerous growth in the genitourinary tract. 38. A method of identifying an HPV-infected patient at risk for recurrence of a pre-cancerous or cancerous growth comprising: a) incubating a blood sample from the patient with an E6 or E7 peptide; b) evaluating the sample for a cell-mediated immune response against the peptide. 39. The method of claim 38, wherein the E6 or E7 peptide has the amino acid sequence of K9L, E10I, C10R, Q15L, V10C, P9L, Q20P, P10I, R16R, G10S, T10Q, M9T, D9L, Q19D, R9F, R9V, L9V, G10C, or D20C. 40. The method of claim 1, wherein the human papilloma virus is a high grade type. 41. The method of claim 40, wherein the human papilloma virus is HPV 16. 42. A method for preventing recurrence of a pre-cancerous or cancerous growth in a patient infected with HPV and treated for the growth comprising: a) identifying a patient at risk for recurrence of HPV-associated pre-cancerous or cancerous growth; and b) administering to the patient an effective amount of at least one E6 or E7 HPV peptide to induce a cell-mediated immune response against the peptide. 43. A kit for determining the possibility of recurrence of a pre-cancerous or cancerous growth in a patient infected with HPV and treated for the growth comprising, in a suitable container means, at least one E6 or E7 HPV peptide, an antibody, and a detection reagent. 44. The kit of claim 43, wherein the kit comprises at least one E6 peptide. 45. The kit of claim 44, wherein the E6 peptide is K9L, E10I, C10R, Q15L, V10C, P9L, P10I, Q20P, R16R, or GS10S. 46. The kit of claim 43, wherein the kit comprises at least one E7 peptide. 47. The kit of claim 46, wherein the E7 peptide is T10Q, M9T, D9L, Q19D, R9F, R9V, L9V, G10C, or D20C. 48. The kit of claim 43, wherein the antibody is directed against a TH1 cytokine. 49. The kit of claim 43, wherein the antibody is directed against a TH1 cytokine receptor. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates generally to the fields of immunology, virology, and oncology. More particularly, it concerns diagnostic and therapeutic methods related to the development and recurrence of pre-cancerous and cancerous growths or lesions, including cervical intraepithelial neoplasia (CIN), caused by human papilloma virus (HPV). 2. Description of Related Art Cervical cancer is the second most common malignancy in women worldwide, accounting for 15% of all cancers diagnosed in women (Parkin et al., 1993). In the United States, cervical cancer is one of the most common neoplasm of the female genital tract. Laboratory and epidemiological research has focused on the etiological role of some types of human papilloma virus (HPV) in the pathogenesis of cervical neoplasia (Brinton, 1992; Munoz et al., 1992). Overall, HPV DNA has been detected in more than 79% of specimens of women with definite cervical disease. The most prevalent HPV type is HPV 16, which is detected in high-grade squamous intraepithelial lesions and cancer (Lörincz et al., 1992). Results from epidemiological studies support an association between cervical neoplasia and HPV, which is markedly stronger with HPV type 16 (Morrison et al., 1991; Koutsky et al., 1992; and Munoz et al., 1992). Neoplasia is characterized by abnormal growth of cells, which often results in the invasion of normal tissues, e.g., primary tumors or the spread to distant organs, e.g., metastasis. The E6 and E7 genes of HPV 16 are frequently co-expressed and are most abundant viral transcripts in biopsies from HPV 16 positive cervical carcinoma (Wettstein, 1990; Seedorf et al., 1987). There is a strong evidence that co-expression of both E6 and E7 open reading frames is necessary and sufficient for efficient malignant transformation of a variety of mammalian cells (Munger et al., 1989). Furthermore, continued expression of the E6 and E7 regions of the viral genome appears to be required to maintain the malignant phenotype (von Knebel Doeberitz et al., 1988). While some HPV infected patients develop cervical neoplasia, others do not. Also there is a high rate of spontaneous regression observed indicating the role of host immune responses. The induction of a cytotoxic T-lymphocyte (CTL) response constitutes a significant defense mechanism against viral infections; occasionally, a virus-specific. CTL response can render full protection without a concomitant antibody response (Sastry et al., 1992; Bevan, 1989; Lukacher, 1984). Based on reports in the literature describing a relation between increased prevalence of anti-HPV antibodies, in particular those directed against the E 7 oncoprotein, with severity of the cervical disease (Cason et al., 1992; Hamsikova et al., 1994; Jha et al., 1993), it has been suggested that HPV-specific humoral response may not play a protective role against HPV-associated cervical neoplasia (Nakagawa et al., 1996). On the other hand, it has been reported that individuals with defects in CMI have an increased prevalence of HPV-associated cervical neoplasia, indicating that T cells participate in the control of HPV-associated neoplasia in humans (Nakagawa et al., 1996; Tsukui et al.; 1996; Feltkamp et al., 1993 and Clerici et al., 1997). Decreased IL-2 production and proliferative responses to mitogens such as PHA and concanavalin-A have been observed in patients with invasive cervical carcinoma (Park et al., 1992). A number of in vitro and in vivo strategies have been described to identify peptides from HPV-16 E6, E7, and L1 proteins that induce T-cell activity in mice and humans (Feltkamp et al., 1993; Strang et al., 1990; Tindel et al., 1991; Shepherd et al., 1992; Stauss et al., 1992; Kast et al., 1993). Typically, induction of virus-specific CTLs can be effected by infection with a virus or recombinant virus that expresses a viral gene product. The viral gene product is processed and presented as a peptide on the surface of infected cells in association with an MHC class I molecule for recognition by the CTL (Unanue, 1989). Additionally, research efforts have concentrated on identifying and characterizing HIV peptides that elicit a viral-specific CTL response. Townsend et al. illustrated the concept of using T-cell epitopes in proteins as vaccine candidates when their group demonstrated the use of short synthetic peptides from influenza nucleoprotein as epitopes for CTL responses (Townsend et al., 1986). The inventors and others have reported using synthetic peptides to generate virus-specific CTLs in vivo (Kast et al, 1991; Aichele et al., 1990; Deres et al, 1989; Sastry et al., 1992; Sastry et al., 1994; Casement et al., 1995) against influenza, lymphocytic choriomeningitis, Sendai virus and HIV. Over 90% of cervical carcinomas express human papillomavirus (HPV) E6 and E7 proteins. These unique antigens are ideal targets for the development of cytotoxic T-lymphocytes (CTL) for antitumor immunotherapy. Synthetic peptides have been identified corresponding with the E6 and E7 oncoproteins of HPV-16 that were effective in including HPV-specific CTL responses in vivo (Sarkar et al., 1995). Recently, Nakagawa et al. reported that systemic T-cell proliferative responses and CTL responses to HPV-16 peptides and proteins were detectable in many virgin as well as sexually active women without cervical lesions but not in those with active disease (Nakagawa et al., 1997). Similarly, Tsukui et al. reported that TH lymphocyte response, particularly IL-2 production, to HPV antigens was greater among cytologically normal women than in women with different degrees of progressive cervical neoplasia (Tsukui et al., 1996). Also, Clerici et al observed that production of TH1 cytokines (IL-2 and IFN-γ) which potentially enhances CMI, to be defective in women with extensive HPV infection and that progression to CIN to be associated with a shift from TH1 to TH2 cytokine production (Clerici et al., 1997). Employing a long term in vitro stimulation protocol for determining the TH activity Kadish et al. reported that lymphoproliferative responses to specific HPV peptides were associated with HPV clearance and regression to CIN (Kadish et al., 1997). On the other hand, de Gruijil et al. reported that T-cell proliferative responses to HPV16 E7 peptides correlated with persistence of HPV infection, but antigen-specific IL-2 production was associated with both virus clearance as well as progression of cervical lesions (de Gruijil et al., 1996). A common clinical management strategy for CIN patients includes excisional or ablative treatment. However, follow-up studies indicate that a significant number of patients experience recurrence. At present no clear understanding exists regarding the development of pre-cancerous or cancerous growths, their recurrence, or disease-free status in the patients who have undergone ablative or excisional treatment for CIN. Better and improved strategies for effective diagnostics of HPV-associated pre-cancerous or cancerous growths and lesions is needed. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is based on the observation that a cell-mediated immune (CMI) response by patients infected with human papilloma virus (HPV) against E6 and/or E7 peptides of HPV is correlated with their prognosis. A cell-mediated immune response is indicative of a reduced risk for the development of pre-cancerous or cancerous growths in the genitourinary tract, particularly the cervix, than the risk for a patient who does not exhibit a cell-mediated immune response; in other words, a patient who exhibits a positive cell-mediate immune response to particular E6 and/or E7 peptides has a good prognosis with respect to the development of HPV-associated pre-cancerous or cancerous growths. Alternatively, a patient who exhibits no or a low CMI response to an E6 or E7 proteinaceous compound of HPV has a greater risk of a bad prognosis with respect to physiological effects as a result of HPV infection. Thus, the present invention encompasses compositions and methods for identifying patients at risk for HPV-related hyperproliferative conditions, including warts, CIN, and malignant growths or other pre-cancerous or cancerous growths; the invention is particularly suited to evaluating patients for recurrence of a hyperproliferative condition. As used herein, the terms “growth” and “lesion” are used interchangeably. Also, the term “pre-cancerous or cancerous growth” refers to HPV-associated growths. In addition to pre-cancerous or cancerous growths or lesions on the cervix, such growths or lesions may occur throughout the urogenitary tract and they include perineal, vulvar and penile growths or lesions. Patients for whom the methods may be applied include any mammals capable of HPV infection; in some embodiments, the patient is specifically contemplated to be a human, either male or female. In some embodiments the present invention encompasses methods for determining the possibility of the development or recurrence of a pre-cancerous or cancerous growth in a patient infected with human papilloma virus. In some cases, the patient has been treated for the growth. The methods involve employing the following steps: obtaining a sample from the patient; incubating the sample with at least one E6 or E7 peptide of HPV; and assaying the sample for a cell-mediated immune (CMI) response against the peptide. A cell-mediated immune response against an E6 or E7 peptide, or a combination thereof indicates a reduced risk of recurrence as compared to a person who does not exhibit such a response. A pre-cancerous growth frequently observed with the development of cervical cancer is cervical intraepithelial neoplasia or CIN. In some embodiments of the invention, the method of the invention is used with respect to patients who have or had CIN of any stage (CIN 1, CIN 2, or CIN 3 or squamous intraepithelial lesions (SIL), low grade-SIL (L-SIL) and high grade SIL (H-SIL)). Furthermore, in other embodiments, the methods may be implemented with patients who have or had more severe stages of hyperproliferative growth than CIN, such as a malignancy or cancerous growth. As used in this application, the term “recurrence” refers to the appearance of a pre-cancerous or cancerous growth, or a reappearance of the first growth, or evidence thereof, after a first pre-cancerous or cancerous growth was reduced, eliminated, or treated. As used herein, the term “incubating” refers to exposing or contacting the sample with a composition that includes a peptide. The claimed methods have applicability to human papilloma virus infections. The human papilloma virus may be a high grade or high risk type, such as HPV 16, 18, 31, 45, 56, or 58. In some embodiments, the human papilloma virus is HPV 16. In other embodiments, the human papilloma virus is a intermediate risk type, such as HPV 33, 35, 37, 51, 52, 59, 66, or 68. In still further embodiments the HPV is a low risk or low grade type associated with warts, such as type 6, 11, 26, 40, 42, 43, 44, 53, 55, 62, or 66. The sample will include cells that give rise to a cell-mediated immune response. In some embodiments, the sample is a blood sample or serum sample, while in other embodiments, the sample is obtained by lavage, smear, or swab of the area suspected of infection or known to be infected, such as in the vaginal, cervical, or penile area. Peripheral blood mononuclear cells (PBMC) can render a cell-mediated immune response and any sample containing such cells can also be employed in methods of the invention. In some embodiments, it is contemplated that cells from a sample are incubated in media after they have been obtained but before they are assayed. It is contemplated that the sample may be incubated up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more hours and up to 1, 2, 3, 4, 5, 6, or 7 days, and up to 1, 2, 3, 4, or 5 or more weeks and up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months in media prior to assaying. It is also contemplated that the cells may be incubated in media and/or stored under conditions of sub-zero degrees centrigrade prior to assaying. The cells themselves or the cell culture supernatant (media, not intact cells) may be used for subsequent assays for a cell-mediated immune response. In some embodiments, the cells are incubated between 2 and 8 hours—in some cases for 6 hours—in media prior to performing intracellular cytokine analysis by flow cytometry. In other embodiments, the cells are incubated in media between 2 days and 20 days—in some cases 15 days—in media prior to performing chromium release assays to determine cytotoxic T lymphocyte (CTL) activity. Methods of the present invention involve determining whether a patient exhibits a cell-mediated immune response against HPV peptides. In several embodiments, the peptides are E6 or E7 peptides meaning they have an amino acid sequence that is at least 90% identical over its length to a contiguous amino acid sequence in an E6 or E7 polypeptide. Specifically contemplated is the use of a peptide comprising 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more contiguous amino acids of SEQ ID NO:19 (E6 from HPV 16) or SEQ ID NO:20 (E7 from HPV 16). It is contemplated that in some embodiments that peptides of only one sequence are tested—for example, an E6 peptide or in another example, an E7 peptide—while in other embodiments, multiple sequences may be tested. In one embodiment, an E6 and an E7 peptide are employed in methods of the invention. In other embodiments, at least two E6 peptides (referring to at least two different E6 sequences), at least two E7 peptides (referring to at least two different E7 sequences), or both may be implemented. It is contemplated that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or more E6 or E7 peptides may be employed, as well as any combination of E6 or E7 peptides thereof. In still further embodiments the E6 peptide is K9L, E10I, C10R, Q15L, V10C, P9L, P10I, Q20P, R16R, or G10S, or a combination thereof. In specific embodiments, the following E6 peptides are employed individually or as a cocktail that includes one or more of the following peptides: K9L, E10I, C10R, Q15L, or V10C. While in other embodiments, an E7 peptide is T10Q, M9T, D9L, Q19D, R9F, R9V, L9V, G10C, or D20C, or a combination thereof. In certain embodiments, the following E7 peptides are employed individually or as a cocktail Q19D, R9F, R9V, L9V, G10C. Furthermore, a cocktail that includes at least one E6 peptide and one E7 peptide from the following is contemplated: K9L, E10I, C10R, Q15L, V10C, Q19D, R9F, R9V, L9V, or G10C. In some embodiments, it is specifically contemplated that one or more peptides in the cocktails described above be excluded. It is also specifically contemplated that compositions discussed with respect to diagnostic methods of the invention may also be applied to preventative or therapeutic methods of the invention. Methods of the invention concern a cell-mediated immune (CMI) response against human papilloma virus. There are different ways of identifying and evaluating a cell-mediated immune response (distinguished from a serum or antibody-mediated immune response). In one embodiment of the invention, T cell proliferation is measured. T-cell proliferation may be assayed by measuring incorporation of tritiated thymidine. A proliferative response of equal to or greater than 2.0 using an SI scale to at least one E6 or E7 peptide is considered positive and is indicative a patient with a reduced risk of recurrence of a pre-cancerous or cancerous growth or lesion. A proliferative response of equal to or greater than 3.0 using an SI scale to at least one E6 or E7 peptide is indicative of a cell mediated response, and thus, identifies a patient with a improved prognosis with respect to the development of pre-cancerous or cancerous growths. Alternatively, a patient who has an SI of less than 2.0, including an SI of zero, would be considered to have a low or no cell-mediated immune response to the E6 or E7 peptide(s) and would be considered as having an increased risk for the development or recurrence of pre-cancerous or cancerous growths. A cell mediated response can also be measured using non-radioactive means such as an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) dye or reduction assay, which is a calorimetric assay for live cells (T cell proliferation) (daCosta et al., 1999), or the alamar Blue assay, another colorimetric assay that measures IL-2-responding cells (Gloeckner et al, 2001; Kwack et al, 2000). In another embodiment of the invention, assaying for a cell-mediated immune response involves measuring TH1 or TH2 cytokine amounts. Even if a patient does not exhibit a CMI response by a T-cell proliferation assay, an increased risk of recurrence may be associated with the production of a TH2 cytokine, such as IL-10. A reduced risk of recurrence is observed with a patient who exhibits production of a TH1 cytokine such as IFN-γ and IL-2 in response to an E6 and/or E7 peptide. In some examples, the amount of a TH1 cytokine is measured, such as IL-2, interferon (IFN) γ, tumor necrosis factor (TNF) α, or TNF-β, IL-3, IL-12, IL-15, IL-16, IL-17, or IL-18. In a specific embodiment, the amount of IL-18 is measured. In additional examples, the amount of a TH2 cytokine is measured, such as IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-13 or IL-14. Measuring a CMI response may be accomplished by an immunoassay, such as ELISA or a radioimmunoassay, or by flow-cytometry. In embodiments of the invention, a sample may be assayed more than once either as duplicate samples or by different assays. In some embodiments, more than one sample is obtained from the patient. The multiple samples may be the same type, for example, multiple blood sample, or they may be different types, for example, a blood sample and a vaginal swab. Patients for whom the methods of the invention have applicability include patients not yet diagnosed with HPV but suspected of having HPV, patients once infected with HPV but no longer showing signs of HPV infection, patients known to be infected with HPV, patients with a pre-cancerous or cancerous growth on or around the cervix or other genitourinary area who may or may not know she is infected with HPV, patients whose pre-cancerous or cancerous growth(s) have been treated successfully or unsuccessfully, and patients who have had at least one recurrence of a pre-cancerous or cancerous growth(s). A pre-cancerous or cancerous growth or lesion refers to a hyperproliferative cells whose growth is not controlled, and includes pre-neoplasias, such as CIN and neoplasias—benign and malignant—involving squamous epithelial cells and atypical squamous cells of uncertain significance (ASCUS). It is contemplated that a patient have more than one growth or lesion. Treatment for any growths may involve surgery—ablative or excisional—as well as conventional cancer therapy and treatment against HPV. Such treatments include chemotherapy, radiation therapy, hormonal therapy, immunotherapy, administration of foscarnet, Thiovir, thiovir analogs (BioKeys), podofilox, podophyllin, trichloracetic acid (TCA), or 5-fluorouracil (5-FU), intralesional or intransal interferon, Imiquimid cream. Ablative techniques include the use of liquid nitrogen, electrocautery or electrodissection, surgical excision, or laser technology. A successful treatment refers to treatment that completely removes any signs of a growth, while a partially successful treatment refers to a treatment that affects the growth by reducing its size or growth rate, or preventing its enlargement, or reducing the number of growths if there is more than one. Patients once infected with HPV may at later times not exhibits signs of an HPV infection. However, it is believed such patients may still experience recurrence of a pre-cancerous or cancerous growth, like patients who have signs of continued HPV infection. In some methods of the invention, the patient is evaluated to determine whether he/she is infected with HPV. In further embodiments, a serotyping of HPV is also included or is part of the initial determination of infection. In still further embodiments, the patient is evaluated to determine whether she has a pre-cancerous or cancerous growth, and if it is cancerous, whether the growth is benign or malignant. Methods of the invention include embodiments in which the sample is obtained from the patient at least one month after treatment for a pre-cancerous or cancerous growth. The patient may have undergone treatment for at least one pre-cancerous or cancerous growth, such as by some form of ablation. The present invention also includes therapeutic methods that may be employed with the diagnostic methods of the invention. In some embodiments of the invention, a patient is identified as having an increased risk for the development of recurrence of pre-cancerous or cancerous growths. A course of action that was not previously considered prior to the patient being identified as having that increased risk may be undertaken. In some embodiments, a patient who would not otherwise be treated is administered preventative treatment against pre-cancerous or cancerous growths or examined more frequently, or both. Preventative treatments are treatments administered in the absence of physiological signs of pre-cancerous or cancerous growths; “therapeutic treatment” encompasses medical treatment of a physiological condition that the patient exhibits. These preventative treatments include the use of therapeutic treatments for both HPV infection and HPV-associated pre-cancerous and cancerous growths, as described above. In some embodiments, a preventative method to protect against or reduce the risk of the development of pre-cancerous and cancerous growths involves immunotherapy with HPV E6 and E7 peptides disclosed herein. If a patient is identified as having a low or no cell-mediated immune response against a particular E6 or E7 peptide, or against a combination of such peptides, a peptide or peptides of E6 or E7 sequence may be administered to the patient to elicit a CMI response. Such peptides include any E6 or E7 peptide, specifically including all or part of the peptides of Table 3. Also, peptides from an E6 or E7 polypeptide, such as those discussed with respect to diagnostic methods of the invention, may be employed in preventative methods as well. It is contemplated that the patient may be administered a composition containing one or more peptide sequences, and in some embodiments, with an adjuvant, liposome-based compound, or both. In further embodiments, the patient is administered peptides more than one time. In some embodiments, there is a method for preventing recurrence of a pre-cancerous or cancerous growth, such as CIN, in a patient infected with HPV and treated for the growth by identifying a patient at risk for recurrence of an HPV-associated growth using methods disclosed herein; and, treating the patient to prevent or treat any recurrence. Treatment options may involve surgery—ablative or excisional—as well as conventional cancer therapy and treatment against HPV, as described above. In some embodiments, the treatment is the immunotherapy treatment involving at least one E6 or E7 peptide from HPV described above. Furthermore, the present invention also encompasses kits for determining the possibility of recurrence of a pre-cancerous or cancer growth in a patient once infected with HPV and treated for the growth comprising, in a suitable container means, at least one E6 or E7 peptide from HPV and an antibody that allows the detection of a cell-mediated immune response against the peptide. In some embodiments, the antibody is attached to a non-reacting structure on which a sample can be applied, such as a plate with wells. In further embodiments, the non-reacting structure has membrane, which can be affixed or attached to the structure. In some embodiments, the kit can be used in an Enzyme-Linked Immunospot (ELISPOT) Assay to detect, and in some embodiments, quantitate, cytokine secreting cells. In still further embodiments, the kit includes an antibody against a TH1 or TH2 cytokine disclosed herein. Other embodiments include a detection reagent to detect the included antibody. A detection reagent is any compound that allows the detection of another compound, including reagents that allow detection visually, such as by a colorimetric detection reagent. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” 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 specific 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. |
Actuator system |
The aim of the invention is to produce an actuator system which also carries out combined adjusting movements, comprising linear and rotating displacement components. As a result, the actuators are connected together at the end points thereof by means of corner connections in such a manner that they form a closed, polygonal arrangement; the opposite corner connections of the actuator system are held together at a constant distance therebetween by means of rigid connections; the rigid connections can move freely with respect to each other and are connected in a flexible manner to the respective corner connections; the actuator system forms two outputs with the opposite corner connections. |
1-13. (Canceled) 14. An actuator system comprising: a plurality of interacting actuators; and a plurality of rigid connections; wherein said actuators are connected to one another at respective end points via corner connections such that said actuators form a closed polygonal arrangement, said corner connections opposite from one another are maintained at a constant distance from one another by said rigid connections, said rigid connections are freely movable relative to one another and are flexibly connected to respective corner connections, said actuator system forms two outputs with opposite corner connections. 15. The system of claim 14, wherein said actuators are connected electrically in series and are evenly subjected to a direct voltage source. 16. The system of claim 15, wherein, to realize combined adjusting motions, an alternating voltage is imposed upon each of said actuators for its individual control. 17. The system of claim 16, wherein said adjusting motions comprise linear and rotating motion components. 18. The system of claim 17, wherein electrical control of said actuators is designed according to a counter-player system. 19. The system of claim 17, wherein said corner connections are respectively replaced by a pair of corner connections, each of said pair of corner connections being at a rigid distance from the other. 20. The system of claim 17, wherein said corner connections are constructed as rigid or flexible angle arrangements. 21. The system of claim 19, wherein said corner connections are constructed as rigid or flexible angle arrangements. 22. The system of claim 17, wherein said rigid connections are constructed as rods and/or as frames. 23. The system of claim 17, wherein said actuators are constructed as piezoelectric stacks, as piezoelectric fiber actuators, and/or as pneumatic actuators. 24. The system of claim 17, wherein segments of said corner connections are respectively arranged at a same angle toward one another. 25. The system of claim 17, wherein opposite corner connections are executed as acute and obtuse angled angle arrangements. 26. The system of claim 17, wherein, in each case, two opposite actuators are constructed identically. 27. The system of claim 26, wherein each pair of actuators is constructed differently from another pair. 28. The system of claim 17, wherein said actuators are arranged rotation-symmetrically around an output axis. 29. The system of claim 28, wherein said direct voltage source is controllable. 30. The system of claim 29, wherein said system can be subjected to low offset voltage from time to time. 31. The system of claim 28, wherein electrical control of said actuators takes place with a primary alternating voltage source and two subordinate alternating voltage sources. 32. They system of claim 30, wherein electrical control of said actuators takes place with a primary alternating voltage source and two subordinate alternating voltage sources. 33. The system of claim 31, wherein a supply voltage of said subordinate alternating voltage sources is tapped by said primary alternating voltage source. |
Conformationally constrained parathyroid hormone (pth) analogs |
The present invention relates to conformationally constrained parathyroid hormone (PTH) analogs, and methods of preparing and using the PTH analogs. The invention provides novel PTH polypeptide derivatives containing amino acid substitutions at selected positions in the polypeptides. The invention provides derivatives of PTH (1-34), PTH(1-21), PTH(1-20), PTH(1-19), PTH(1-18), PTH(1-17), PTH(1-16), PTH(1-15), PTH(1-14), PTH(1-13), PTH(1-12), PTH(1-11) and PTH(1-1 0) polypeptides, wherein at least one residue in each polypeptide is a helix, preferably an a-helix, stabilizing residue. The invention also provides methods of making such peptides. Further, the invention encompasses compositions and methods for use in limiting undesired bone loss in a vertebrate at risk of such bone loss, in treating conditions that are characterized by undesired bone loss or by the need for bone growth, e.g. in treating fractures or cartilage disorders and for raising cAMP levels in cells where deemed necessary. |
1. A biologically active peptide consisting essentially of the formula selected from: (a) X01ValX02GluIleGlnLeuMetHisX03X04X05X05X06X07 (SEQ ID NO. 1); (b) fragments thereof, containing amino acids 1-11, 1-12 or 1-13; (c) pharmaceutically acceptable salts thereof; or (d) N- or C- derivatives thereof; wherein: X01 is an α-helix-stabilizing residue, desaminoGly, desaminoSer or desaminoAla; X02 is an α-helix-stabilizing residue, Ala, or Ser; X03 is Ala, Gln or Asn; X04 is Arg, Har or Leu; X05 is an α-helix-stabilizing residue, Ala or Gly; X06 is an α-helix-stabilizing residue or Lys; X07 is an α-helix-stabilizing residue, Trp or His; and wherein at least one of X01, X02, X05, X06 or X07 is an α-helix-stabilizing residue. 2. The peptide of claim 1, wherein said α-helix-stabilizing amino acid is selected from the group consisting of Aib, ACPC (1-aminocyclopropylcarboxylic acid), DEG (diethylglycine) and 1-aminocyclopentanecarboxylic acid. 3. The peptide of claim 1, wherein said peptide is selected from: (a) AibValSerGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 2); (b) fragments thereof, containing amino acids 1-11, 1-12 or 1-13; (c) pharmaceutically acceptable salts thereof; or (d) N- or C- derivatives thereof. 4. The peptide of claim 1, wherein said peptide is selected from: (a) desaminoAlaValAibGluIleGlnLeuMetHisAsnLeuGlyLys His (SEQ ID NO. 3); (b) fragments thereof, containing amino acids 1-11, 1-12 or 1-13; (c) pharmaceutically acceptable salts thereof; or (d) N- or C- derivatives thereof. 5. The peptide of claim 1, wherein said peptide is selected from: (a) desaminoSerValAibGluIleGlnLeuMetHisAsnLeuGlyLys His (SEQ ID NO. 4); (b) fragments thereof, containing amino acids 1-11, 1-12 or 1-13; (c) pharmaceutically acceptable salts thereof; or (d) N- or C- derivatives thereof. 6. The peptide of claim 1, wherein said peptide is selected from: (a) desaminoGlyValAibGluIleGlnLeuMetHisAsnLeuGlyLys His (SEQ ID NO. 5); (b) fragments thereof, containing amino acids 1-11, 1-12 or 1-13; (c) pharmaceutically acceptable salts thereof; or (d) N- or C- derivatives thereof. 7. The peptide of claim 1, wherein said peptide is selected from: (a) AibValAibGluIleGlnLeuMetHisGlnHarGlyLysTrp (SEQ ID NO. 6); (b) fragments thereof, containing amino acids 1-11, 1-12 or 1-13; (c) pharmaceutically acceptable salts thereof; or (d) N- or C- derivatives thereof. 8. The peptide of claim 1, said peptide selected from: (a) AibValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 7); (b) fragments thereof, containing amino acids 1-11, 1-12 or 1-13; (c) pharmaceutically acceptable salts thereof; or (d) N- or C- derivatives thereof. 9. The peptide of claim 1, said peptide selected from: (a) AibValAlaGluIleGlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 9); (b) fragments thereof, containing amino acids 1-11, 1-12 or 1-13; (c) pharmaceutically acceptable salts thereof; or (d) N- or C- derivatives thereof. 10. The peptide of claim 1, said peptide selected from: (a) AlaValAibGluIleGlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 10); (b) fragments thereof, containing amino acids 1-11, 1-12 or 1-13; (c) pharmaceutically acceptable salts thereof; or (d) N- or C- derivatives thereof. 11. The peptide of claim 1, said peptide selected from: (a) SerValAibGluIleGlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 11); (b) fragments thereof, containing amino acids 1-11, 1-12 or 1-13; (c) pharmaceutically acceptable salts thereof; or (d) N- or C- derivatives thereof. 12. A biologically active peptide consisting essentially of the formula selected from: (a) AibValAibGluIleGlnLeuNleHisGlnHarAlaLysTrpLeuAla SerValArgArgTyr (SEQ ID NO. 8); (b) fragments thereof, containing amino acids 1-20, 1-19, 1-18, 1-17, 1-16 or 1-15; (c) pharmaceutically acceptable salts thereof; or (d) N- or C- derivatives thereof. 13. The peptide of claim 1, wherein said peptide is AibValSerGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 2). 14. The peptide of claim 1, wherein said peptide is desaminoAlaValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 3). 15. The peptide of claim 1, wherein said peptide is desamninoSerValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 4). 16. The peptide of claim 1, wherein said peptide is desaminoGlyValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 5). 17. The peptide of claim 1, wherein said peptide is AibValAibGluIle GlnLeuMetHisGlnHarGlyLysTrp (SEQ ID NO. 6). 18. The peptide of claim 1, wherein said peptide is AibValAibGluIle GlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 7). 19. The peptide of claim 12, wherein said peptide is AibValAibGluIle GlnLeuNleHisGlnHarGlyLysTrpLeuAlaSerValArgArgTyr (SEQ ID NO. 8). 20. The peptide of claim 1, wherein said peptide is AibValAlaGluIle GlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 9). 21. The peptide of claim 1, wherein said peptide is AlaValAibGluIle GlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 10). 22. The peptide of claim 1, wherein said peptide is SerValAibGluIleGlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 11). 23. The peptide of claim 1 or 12, wherein said peptide is labeled. 24. The peptide of claim 23, wherein said peptide is labeled with a fluorescent label. 25. The peptide of claim 23, wherein said peptide is labeled with a chemiluminescent label. 26. The peptide of claim 23, wherein said peptide is labeled with a bioluminescent label. 27. The peptide of claim 23, wherein said peptide is labeled with a radioactive label. 28. The peptide of claim 27, wherein said peptide is labeled with 125I. 29. The peptide of claim 27, wherein said peptide is labeled with 99mTc. 30. A pharmaceutical composition comprising the biologically active peptide of claim 1 or 12, and a pharmaceutically acceptable carrier. 31. A method for treating mammalian conditions characterized by decreases in bone mass, said method comprising administering to a subject in need thereof an effective bone mass-increasing amount of a biologically active peptide of claim 1 or 12. 32. A method for treating mammalian conditions characterized by decreases in bone mass, said method comprising administering to a subject in need thereof an effective bone mass-increasing amount of a composition comprising a biologically active peptide of claim 1 or 12 and a pharmaceutically acceptable carrier. 33. A method for determining rates of bone reformation, bone resorption and/or bone remodeling comprising administering to a patient an effective amount of a peptide of claim 1 or 12 and determining the uptake of said peptide into the bone of said patient. 34. The method of claim 32, wherein said condition to be treated is osteoporosis. 35. The method of claim 32, wherein said condition to be treated is old age osteoporosis. 36. The method of claim 32, wherein said condition to be treated is post-menopausal osteoporosis. 37. The method of claim 32, wherein said effective amount of said peptide for increasing bone mass is from about 0.01 μg/kg/day to about 1.0 μg/kg/day. 38. The method of claim 32, wherein the method of administration is parenteral. 39. The method of claim 32, wherein the method of administration is subcutaneous. 40. The method of claim 32, wherein the method of administration is nasal insufflation. 41. A method of making the peptide of claim 1 or 12, wherein said peptide is synthesized by solid phase synthesis. 42. The method of making the peptide of claim 1 or 12, wherein said peptide is protected by FMOC. 43. The peptide of claim 2, wherein said α-helix-stabilizing amino acid is Aib. 44. A biologically active peptide consisting essentially of the formula selected from: (a) X01 ValX02GluIleGlnLeuX03HisX04X05X06X07X08LeuX09Ser X10X11ArgX12X13Trp LeuArgLysLysLeuGlnAspValHisAsn X14 (SEQ ID NO. 19); (b) pharmaceutically acceptable salts thereof; or (c) N- or C- derivatives thereof; wherein: X01 is an α-helix-stabilizing residue desaminoGly, desaminoSer or desaminoAla; X02 is an α-helix-stabilizing residue, Ala, or Ser; X03 is Met or Nle; X04 is Ala, Gln or Asn; X05 is Arg, Har or Leu; X06 is an α-helix-stabilizing residue, Ala or Gly; X07 is an α-helix-stabilizing residue or Lys; X08 is an α-helix-stabilizing residue, Trp or His; X09 is Ala or Asn; X10 is Met or Val; X11 is Arg or Glu; X12 is Met or Val; X13 is Gln or Glu; X14 is Tyr or Phe; and wherein at least one of X01, X02, X06, X07or X08 is an α-helix-stabilizing residue. 45. The peptide of claim 44, wherein said α-helix-stabilizing amino acid is selected from the group consisting of Aib, ACPC (1-aminocyclopropylcarboxylic acid), DEG (diethylglycine) and 1-aminocyclopentanecarboxylic acid. 46. The peptide of claim 44, wherein said peptide is selected from: (a) AibValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX09 SerX10X11ArgX12X13 Trp LeuArgLysLysLeuGlnAspValHis AsnX14 (SEQ ID NO. 20); (b) pharmaceutically acceptable salts thereof; or (c) N- or C- derivatives thereof. 47. The peptide of claim 44, wherein said peptide is selected from: (a) desaminoAlaValAibGluIleGlnLeuMetHisAsnLeuGlyLys HisLeuX09SerX10X11ArgX12X13TrpLeuArgLysLysLeuGln AspValHisAsnX14 (SEQ ID NO. 21); (b) pharmaceutically acceptable salts thereof; or (c) N- or C- derivatives thereof. 48. The peptide of claim 44, wherein said peptide is selected from: (a) desaminoSerValAibGluIleGlnLeuMetHisAsnLeuGlyLys HisLeuX09SerX10X11ArgX12X13TrpLeuArgLysLysLeuGln AspValHisAsnX14 (SEQ ID NO. 22); (b) pharmaceutically acceptable salts thereof; or (c) N- or C- derivatives thereof. 49. The peptide of claim 44, wherein said peptide is selected from: (a) desaminoGlyValAibGluIleGlnLeuMetHisAsnLeuGlyLys HisLeuX09SerX10X11ArgX12X13Trp LeuArgLysLysLeuGln AspValHisAsnX14 (SEQ ID NO. 23); (b) pharmaceutically acceptable salts thereof; or (c) N- or C- derivatives thereof. 50. The peptide of claim 44, wherein said peptide is selected from: (a) AibValAibGluIleGlnLeuMetHisGlnHarGlyLysTrpLeuX09 SerX10X11ArgX12X13Trp LeuArgLysLysLeuGlnAspValHis AsnX14 (SEQ ID NO. 24); (b) pharmaceutically acceptable salts thereof; or (c) N- or C- derivatives thereof. 51. The peptide of claim 44, said peptide selected from: (a) AibValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX09 SerX10X11ArgX12X13Trp LeuArgLysLysLeuGlnAspValHis AsnX14 (SEQ ID NO. 25); (b) pharmaceutically acceptable salts thereof; or (c) N- or C- derivatives thereof. 52. The peptide of claim 44, said peptide selected from: (a) AibValAlaGluIleGlnLeuMetHisGlnHarAlaLysTrpLeuX09SerX10X11ArgX12X13Trp LeuArgLysLysLeuGlnAspValHis AsnX14 (SEQ ID NO. 26); (b) pharmaceutically acceptable salts thereof; or (c) N- or C- derivatives thereof. 53. The peptide of claim 44, said peptide selected from: (a) AlaValAibGluIleGlnLeuMetHisGlnHarAlaLysTrpLeuX09 SerXX11ArgX12X13TrpLeuArgLysLysLeuGlnAspValHis AsnX14 (SEQ ID NO. 27); (b) pharmaceutically acceptable salts thereof; or (c) N- or C- derivatives thereof. 54. The peptide of claim 44, said peptide selected from: (a) SerValAibGluIleGlnLeuMetHisGlnHarAlaLysTrpLeuX09 SerX10X10ArgX12X13Trp LeuArgLysLysLeuGlnAspValHis AsnX14 (SEQ ID NO. 28); (b) pharmaceutically acceptable salts thereof; or (c) N- or C- derivatives thereof. 55. A biologically active peptide consisting essentially of the formula selected from: (a) AibValAibGluIleGlnLeuNleHisGlnHarAlaLysTrpLeuAla SerValArgArgX12X13Trp LeuArgLysLysLeuGlnAspValHis AsnX14 (SEQ ID NO. 29); (b) pharmaceutically acceptable salts thereof; or (c) N- or C- derivatives thereof; wherein X12 is Met or Val; X13 is Gln or Glu; and X14 is Tyr or Phe. 56. The peptide of claim 44, wherein said peptide is AibValSerGluIle GlnLeuMetHisAsnLeuGlyLysHisLeuX09SerX10X11ArgX12X13TrpLeuArgLysLys LeuGlnAspValHisAsnX14 (SEQ ID NO. 20). 57. The peptide of claim 44, wherein said peptide is desaminoAlaValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX09SerX10X11ArgX12X13Trp LeuArgLysLysLeuGlnAspValHisAsnX14 (SEQ ID NO. 21). 58. The peptide of claim 44, wherein said peptide is desaminoSerValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX09SerX10X11ArgX12X13Trp LeuArgLysLysLeuGlnAspValHisAsnX14 (SEQ ID NO. 22). 59. The peptide of claim 44, wherein said peptide is desaminoGlyValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX09Ser10X11ArgX12X13 Trp LeuArgLysLysLeuGlnAspValHisAsnX14 (SEQ ID NO. 23). 60. The peptide of claim 44, wherein said peptide is AibValAibGluIle GlnLeuMetHisGlnHarGlyLysTrpLeuX09SerX10X11ArgX12X13TrpLeuArgLysLys LeuGlnAspValHisAsnX14 (SEQ ID NO. 24). 61. The peptide of claim 44, wherein said peptide is AibValAibGluIle GlnLeuMetHisAsnLeuGlyLysHisLeuX09SerX10X11ArgX12X13Trp LeuArgLysLys LeuGlnAspValHisAsnX14 (SEQ ID NO. 25). 62. The peptide of claim 44, wherein said peptide is AibValAlaGluIle GlnLeuMetHisGlnHarAlaLysTrpLeuX09SerX10X11ArgX12X13Trp LeuArgLysLys LeuGlnAspValHisAsnX14 (SEQ ID NO. 26). 63. The peptide of claim 44, wherein said peptide is AlaValAibGluIle GlnLeuMetHisGlnHarAlaLysTrpLeuX09SerX10X11ArgX12X13Trp LeuArgLysLys LeuGlnAspValHisAsnX14 (SEQ ID NO. 27). 64. The peptide of claim 44, wherein said peptide is SerValAibGluIle GlnLeuMetHisGlnHarAlaLysTrpLeuX09SerX10X11ArgX12X13Trp LeuArgLysLys LeuGlnAspValHisAsnX14 (SEQ ID NO. 28). 65. The peptide of claim 55, wherein said peptide is AibValAibGluIle GlnLeuNleHisGlnHarGlyLysTrpLeuAlaSerValArgArgX12X13Trp LeuArgLysLys LeuGlnAspValHisAsnX14 (SEQ ID NO. 29). 66. The peptide of claim 44 or 55, wherein said peptide is labeled. 67. The peptide of claim 66, wherein said peptide is labeled with a fluorescent label. 68. The peptide of claim 66, wherein said peptide is labeled with a chemiluminescent label. 69. The peptide of claim 66, wherein said peptide is labeled with a bioluminescent label. 70. The peptide of claim 66, wherein said peptide is labeled with a radioactive label. 71. The peptide of claim 70, wherein said peptide is labeled with 125I. 72. The peptide of claim 70, wherein said peptide is labeled with 99mTc. 73. A pharmaceutical composition comprising the biologically active peptide of claim 44 or 55, and a pharmaceutically acceptable carrier. 74. A method for treating mammalian conditions characterized by decreases in bone mass, said method comprising administering to a subject in need thereof an effective bone mass-increasing amount of a biologically active peptide of claim 44 or 55. 75. A method for treating mammalian conditions characterized by decreases in bone mass, said method comprising administering to a subject in need thereof an effective bone mass-increasing amount of a composition comprising a biologically active peptide of claim 44 or 55 and a pharmaceutically acceptable carrier. 76. A method for determining rates of bone reformation, bone resorption and/or bone remodeling comprising administering to a patient an effective amount of a peptide of claim 44 or 55 and determining the uptake of said peptide into the bone of said patient. 77. The method of claim 75, wherein said condition to be treated is osteoporosis. 78. The method of claim 75, wherein said condition to be treated is old age osteoporosis. 79. The method of claim 75, wherein said condition to be treated is post-menopausal osteoporosis. 80. The method of claim 75, wherein said effective amount of said peptide for increasing bone mass is from about 0.01 μg/kg/day to about 1.0 μg/kg/day. 81. The method of claim 75, wherein the method of administration is parenteral. 82. The method of claim 75, wherein the method of administration is subcutaneous. 83. The method of claim 75, wherein the method of administration is nasal insufflation. 84. A method of making the peptide of claim 44 or 55, wherein said peptide is synthesized by solid phase synthesis. 85. The method of making the peptide of claim 44 or 55, wherein said peptide is protected by FMOC. 86. The peptide of claim 45, wherein said α-helix-stabilizing amino acid is Aib. |
<SOH> Background of the Invention <EOH>1. Field of the Invention The present invention relates to conformationally constrained parathyroid hormone (PTH) analogs, and methods of preparing and using the PTH analogs. 2. Background Art Parathyroid Hormone Parathyroid hormone (PTH), an 84 amino acid peptide, is the principal regulator of ionized blood calcium in the human body (Kronenberg, H. M., et al., In Handbook of Experimental Pharmacology, Mundy, G. R., and Martin, T. J., (eds), pp. 185-201, Springer-Verlag, Heidelberg (1993)). Regulation of calcium concentration is necessary for the normal function of the gastrointestinal, skeletal, neurologic, neuromuscular, and cardiovascular systems. PTH synthesis and release are controlled principally by the serum calcium level; a low level stimulates and a high level suppresses both hormone synthesis and release. PTH, in turn, maintains the serum calcium level by directly or indirectly promoting calcium entry into the blood at three sites of calcium exchange: gut, bone, and kidney. PTH contributes to net gastrointestinal absorption of calcium by favoring the renal synthesis of the active form of vitamin D. PTH promotes calcium resorption from bone indirectly by stimulating differentiation of the bone-resorbing cells, osteoclasts. It also mediates at least three main effects on the kidney: stimulation of tubular calcium reabsorption, enhancement of phosphate clearance, and promotion of an increase in the enzyme that completes synthesis of the active form of vitamin D. PTH is thought to exert these effects primarily through receptor-mediated activation of adenylate cyclase and/or phospholipase C. Disruption of calcium homeostasis may produce many clinical disorders (e.g., severe bone disease, anemia, renal impairment, ulcers, myopathy, and neuropathy) and usually results from conditions that produce an alteration in the level of parathyroid hormone. Hypercalcemia is a condition that is characterized by an elevation in the serum calcium level. It is often associated with primary hyperparathyroidism in which an excess of PTH production occurs as a result of a parathyroid gland lesion (e.g., adenoma, hyperplasia, or carcinoma). Another type of hypercalcemia, humoral hypercalcemia of malignancy (HHM) is the most common paraneoplastic syndrome. It appears to result in most instances from the production by tumors (e.g., squamous, renal, ovarian, or bladder carcinomas) of a class of protein hormone which shares amino acid homology with PTH. These PTH-related proteins (PTHrP) appear to mimic certain of the renal and skeletal actions of PTH and are believed to interact with the PTH receptor in these tissues. Osteoporosis Osteoporosis is a potentially crippling skeletal disease observed in a substantial portion of the senior adult population, in pregnant women and even in juveniles. The term osteoporosis refers to a heterogeneous group of disorders. Clinically, osteoporosis is separated into type I and type II. Type I osteoporosis occurs predominantly in middle aged women and is associated with estrogen loss at menopause, while osteoporosis type II is associated with advancing age. Patients with osteoporosis would benefit from new therapies designed to promote fracture repair, or from therapies designed t6 prevent or lessen the fractures associated with the disease. The disease is marked by diminished bone mass, decreased bone mineral density (BMD), decreased bone strength and an increased risk of bone fracture. At present, there is no effective cure for osteoporosis, though estrogen, calcitonin and the bisphosphonates, etidronate and alendronate are used to treat the disease with varying levels of success. These agents act to decrease bone resorption. Since parathyroid hormone regulates blood calcium and the phosphate levels, and has potent anabolic (bone-forming) effects on the skeleton, in animals (Shen, V., et al., Calcif Tissue Int. 50:214-220 (1992); Whitefild, J. F., et al., Calcif Tissue Int. 56:227-231 (1995) and Whitfield, J. F., et al., Calcif Tissue Int. 60:26-29 (1997)) and humans (Slovik, D. M., et al., J. Bone Miner. Res. 1:377-381 (1986); Dempster, D. W., et al., Endocr. Rev. 14:690-709 (1993) and Dempster, D. W., et al., Endocr. Rev. 15:261 (1994)) when administered intermittently, PTH, or PTH derivatives, are prime candidates for new and effective therapies for osteoporosis. PTH Derivatives PTH derivatives include polypeptides that have amino acid substitutions or are truncated relative to the full length molecule. Both a 14 and a 34 amino acid amino-terminal truncated form of PTH, as well as a C-terminal truncated form have been studied. Additionally, amino acid substitutions within the truncated polypeptides have also been investigated. Synthetic PTH(1-34) exhibits full bioactivity in most cell-based assay systems, has potent anabolic effects on bone mass in animals and has recently been shown to reduce the risk of bone fracture in postmenopausal osteoporotic women (Neer, R. M., et al., N.E.J.M. 344:1434-1441 (2001); Dempster, D. W., et al., Endocr Rev 14:690-709 (1993)). PTH acts on the PTH/PTHrP receptor (P1R), a class II G protein-coupled heptahelical receptor that couples to the adenylyl cyclase/CAMP and phospolipase C/inositol phosphate (IP) signaling pathway (Rippner, H., et al., Science 254:1024-1026 (1991)). Deletion analysis studies have shown that the amino-terminal residues of PTH play a crucial role in stimulating the P1 R to activate the cAMP and IP signaling pathways (Tregear, G. W., et al., Endocrinology 93:1349-1353 (1973); Takasu, H., et al., Biochemistry 38:13453-13460(1999)). Crosslinking and receptor mutagenesis studies have indicated that residues in the amino-terminal portion of PTH interact with the extracellular loops and extracellular ends of the seven transmembrane helices, which reside within the juxtamembrane region of the receptor (Bergwitz, C., et al., J. Biol. Chem. 271:26469-26472 (1996); Hoare, S. R. J., et al., J. Biol. Chem 276:7741-7753 (2001); Behar, V., et al., J. Biol. Chem. 275:9-17 (1999); Shimizu, M., et al., J. Biol. Chem. 275:19456-19460(2000); Luck, M. D., et al., Molecular Endocrinology 13:670-680 (1999)). |
<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>The invention provides novel PTH polypeptide derivatives containing amino acid substitutions at selected positions in the polypeptides. The derivatives function as full, or nearly full, agonists of the PTH-1 receptor. Because of their unique properties, these polypeptides have a utility as drugs for treating human diseases of the skeleton, such as osteoporosis. The invention provides derivatives of PTH(1-21), PTH(1-20), PTH(1-19), PTH(1-18),PTH(1-17), PTH(1-16), PTH(1-15), PTH(1-14), PH(1-13), PTH(1-12), PTH(1-11) and PTH(1-10) polypeptides, wherein at least one residue in each polypeptide is a helix, preferably an α-helix, stabilizing residue. The invention also provides methods of making such peptides. Further, the invention encompasses compositions and methods for use in limiting undesired bone loss in a vertebrate at risk of such bone loss, in treating conditions that are characterized by undesired bone loss or by the need for bone growth, e.g. in treating fractures or cartilage disorders and for raising cAMP levels in cells where deemed necessary. In one aspect, the invention is directed to a biologically active peptide consisting essentially of X 01 ValX 02 GluIleGlnLeuMetHisX 03 X 04 X 05 X 06 X 07 (SEQ ID NO: 1), wherein X 01 is an α-helix-stabilizing residue, desaminoGly, desaminoSer or desaminoAla; X 02 is an α-helix-stabilizing residue, Ala, or Ser; X 03 is Ala, Gln or Asn; X 04 is Arg, Har or Leu; X 05 is an α-helix-stabilizing residue, Ala or Gly; X 06 is an α-helix-stabilizing residue or Lys; and X 07 is an α-helix-stabilizing residue, Trp or His: and wherein at least one of X 01 , X 02 , X 03 , X 04 , X 05 , X 06 or X 07 is an α-helix-stabilizing residue. In another aspect, the invention relates to SEQ ID NO: 1, wherein the α-helix-stabilizing amino acid is selected from the group consisting of Aib, ACPC (1-aminocyclopropylcarboxylic acid), DEG (diethylglycine) and 1-aminocyclopentanecarboxylic acid. In another aspect, the invention relates to SEQ ID NO: 1, wherein the α-helix-stabilizing amino acid is Aib. The invention is further drawn to fragments ofthe peptide of SEQ ID NO: 1, in particular X 01 ValX 02 GluIleGlnLeuMetHisX 03 X 04 X 05 X 06 (SEQ ID NO: 12), X 01 ValX 02 GluIleGlnLeuMetHisX 03 X 04 X 05 (SEQ ID NO: 13), X 01 ValX 02 GluIleGlnLeuMetHisX 03 X 04 (SEQ ID NO: 14) and X 01 ValX 02 GluIleGlnLeuMetHisX 03 (SEQ ID NO: 15). The invention further encompasses pharmaceutically acceptable salts of the above-described peptides, and N- or C-derivatives of the peptides. A preferable embodiment of the invention is drawn to any of the above recited polypeptides, wherein the polypeptide contains a C-terminal amnide. In addition, the invention is drawn to a biologically active polypeptide consisting essentially of AibValAibGluIleGlnLeuNleHisGlnHarAlaLysTrpLeu-AlaSerValArgArtTyr (SEQ ID NO. 8); fragments thereof, containing amino acids 1-20, 1-19, 1-18, 1-17, 1-16 or 1-15; pharmaceutically acceptable salts thereof; or N- or C-derivatives thereof. The invention is further drawn to any of the above polypeptides labeled with a label selected from the group consisting of: a radiolabel, a flourescent label, a bioluminescent label, or a chemiluminescent label. In a preferable embodiment the radiolabel is 125 I or 99m Tc. Preferred embodiments of the biologically active peptide include: AibValSerGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 2); desamino-AlaValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 3); desamino-SerValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 4); desamino-GlyValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 5); AibValAibGluIleGlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 6); AibValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO. 7); AibValAlaGluIleGlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 9); AlaValAibGluIleGlnLeuMetHisGlnHarAlaLysTrp (SEQ ID NO. 10); SerValAibGluIleGffi uMetHisGlnHarAlaLysTrp (SEQ ID NO. 11); and AibValAibGluIleGlnLeuMetHisGlnHar (SEQ ID NO. 16). It is contemplated that fragments of the above mentioned peptides, containing amino acids 1-10, 1-11, 1-12 or 1-13, are also embodiments of the present invention. The invention further encompasses pharmaceutically acceptable salts of the above-described peptides, and N- or C-derivatives of the peptides. Other constrained amino acids that are substituted for Aib are ACPC (1-aminocyclopropylcarboxylic acid), DEG (diethylglycine) and 1-aminocyclopentanecarboxylic acid. In accordance with yet a further aspect of the invention, this invention also provides pharmaceutical compositions comprising a PTH derivative and a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable solution such as saline or a physiologically buffered solution. This invention also provides a method for treating mammalian conditions characterized by decreases in bone mass, which method comprises administering to a subject in need thereof an effective bone mass-increasing amount of a biologically active PTH polypeptide. A preferable embodiment of the invention is drawn to conditions such as osteoporosis. The types of osteoporosis include, but are not limited to old age osteoporosis and postmenopausal osteoporosis. Additional preferable embodiments include using an effective amounts of the polypeptide of about 0.01 μg/kg/day to about 1.0 μg/kg/day wherein the polypeptide is administered parenterally, subcutaneously or by nasal insufflation. In accordance with yet a further aspect of the invention, this invention also provides a method for determining rates of bone reformation, bone resorption and/or bone remodeling comprising administering to a patient an effective amount of a labeled PTH polypeptide, such as for example, SEQ ID NO: 1 or a derivatives thereof and determining the uptake of the peptide into the bone of the patient. The peptide is labeled with a label selected from the group consisting of: radiolabel, flourescent label, bioluminescent label, or chemiluminescent label. An example of a suitable radiolabel is 99m Tc. The invention is further related to a method of increasing cAMP in a mammalian cell having PTH-1 receptors, the method comprising contacting the cell with a sufficient amount of the polypeptide of the invention to increase cAMP. The invention also provides derivatives of rat PTH(1-34) (rPTH(1-34)) given by AlaValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuAlaSerValGluArg MetGlnTrpLeuArgLysLysLeuGlnAspValHisAsnPhe (SEQ ID NO: 30), and of human PTH(1-34) (hPTH(1-34)) given by SerValSerGluIleGlnLeuMetHisAsn LeuGlyLysHisLeuAsnSerMetGluArgValGluTrpLeuArgLysLysLeuGlnAspVal HisAsnPhe (SEQ ID NO: 31). In another aspect, the invention relates to a biologically active peptide consisting essentially ofthe formula X 01 ValX 02 GluIleGlnLeuX 03 HisX 04 X 05 X 06 X 07 X 08 LeuX 09 SerX 10 X 11 ArgX 12 X 13 TrpLeuArgLysLysLeuGlnAspValHisAsnX 14 (SEQ ID NO: 19) wherein X 01 is an α-helix-stabilizing residue, desaminoGly, desaminoSer or desaminoAla; X 02 is an α-helix-stabilizing residue, Ala, or Ser; X 03 is Met or Nle; X 04 is Ala, Gln or Asn; X 05 is Arg, Har or Leu; X 06 is an α-helix-stabilizing residue, Ala or Gly; X 07 is an α-helix-stabilizing residue or Lys; X 08 is an α-helix-stabilizing residue, Trp or His; X 09 is Ala or Asn; X 10 is Met or Val; X 11 is Arg or Glu; X 12 is Met or Val; X 13 is Gln or Glu; X 14 is Tyr or Phe; and wherein at least one of X 01 , X 02 , X 06 , X 7 , or X 08 is an α-helix-stabilizing residue. The invention also relates to fragments thereof, containing amino acids 1-33, 1-32, 1-31, 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, or 1-11. The invention also relates to pharmaceutically acceptable salts and N- or C-derivatives of SEQ ID NO: 19 or the above described fragments. In another aspect, the invention relates to SEQ ID NO: 19, wherein the α-helix-stabilizing amino acid is selected from the group consisting of Aib, ACPC (1-aminocyclopropylcarboxylic acid), DEG (diethylglycine) and 1-aminocyclopropylcarboxylic acid. In another aspect, the invention relates to SEQ ID NO: 19, wherein the α-helix-stabilizing amino acid is Aib. In another aspect, the invention relates specifically to the following peptides: AibValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX 09 Ser 10 X 11 Arg X 12 X 13 TrpLeuArgLysLysLeuGlnAspValHisAsnX 14 (SEQ ID NO. 20); desaminoAlaValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis LeuX 09 SerX 10 X 11 ArgX 12 X 13 Trp LeuArgLysLysLeuGlnAspValHisAsnX 14 (SEQ ID NO. 21); desaminoSerValAibGluIleGlnLeuMetHisAsnLeuGlyLysHis LeuX 09 SerX 10 X 11 ArgX 12 X 13 Trp LeuArgLysLysLeuGlnAspValHisAsnX 14 (SEQ ID NO. 22); desaminoGlyValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeu X 09 SerX 10 X 11 ArgX 12 X 13 Trp LeuArgLysLysLeuGlnAspValHisAsnX 14 (SEQ ID NO. 23); AibValAibGluIleGlnLeuMetHisGlnHarGlyLysTrpLeuX 09 Ser X 10 X 11 ArgX 12 X 13 Trp LeuArgLysLysLeuGlnAspValHisAsnX 14 (SEQ ID NO. 24); AibValAibGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuX 09 Ser X 10 X 11 ArgX 12 X 13 Trp LeuArgLysLysLeuGlnAspValHisAsnX 14 (SEQ ID NO. 25); AibValAlaGluIleGlnLeuMetHisGlnHarAlaLysTrpLeuX 09 SerX 10 X 11 ArgX 12 X 13 Trp LeuArgLysLysLeuGlnAspValHisAsnX 14 (SEQ ID NO. 26); AlaValAibGluIleGlnLeuMetHisGlnHarAlaLysTrpLeuX 09 SerX 10 X 11 ArgX 12 X 13 TrpLeuArgLysLysLeuGlnAspValHisAsn X 14 (SEQ ID NO. 27); and SerValAibGluIleGlnLeuMetHisGlnHarAlaLysTrpLeu X 09 Ser X 10 X 11 ArgX 12 X 13 Trp LeuArgLysLysLeuGlnAspValHisAsnX 14 (SEQ ID NO. 28). X 09 , X 10 , X 11 , X 12 , X 13 and X 14 have the same meaning as defined for SEQ ID NO: 19. The invention also relates to pharmaceutically acceptable salts or N- or C-derivatives of the above peptides. The invention also relates to a biologically active peptide consisting essentially of the formula AibValAibGluIleGlnLeuNleHisGlnHarAlaLysTrpLeu AlaSerValArgArgX 12 X 13 TrpLeuArgLysLysLeuGlnAspValHisAsnX 14 (SEQ ID NO: 29) wherein X 12 is Met or Val; X 13 is Gln or Glu; and X 14 is Tyr or Phe. The invention also relates to pharmaceutically acceptable salts or N- or C-derivatives of SEQ ID NO: 29. The invention also relates to fragments thereof, containing amino acids 1-33, 1-32, 1-31, 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, or 1-11. In another aspect of the invention, SEQ ID NO: 19, SEQ ID NO: 29 or any of the above peptides are labeled. In another aspect of the invention,SEQ ID NO: 19, SEQ ID NO: 29 or any of the above peptides are labeled with a fluorescent label, a chemiluminescent label; a bioluminescent label; a radioactive label; 125 I; or 99m Tc. In another aspect, the invention is directed to a pharmaceutical composition comprising the biologically active peptide SEQ ID NO: 19, SEQ ID NO: 29 or any of the above peptides, and a pharmaceutically acceptable carrier. In another aspcet, the invention is directed to a method for treating mammalian conditions characterized by decreases in bone mass, the method comprising administering to a subject in need thereof an effective bone mass-increasing amount of a biologically active peptide of SEQ ID NO: 19, SEQ ID NO: 29 or any of the above peptides. In another aspcet, the invention is directed to a method for treating mammalian conditions characterized by decreases in bone mass, the method comprising administering to a subject in need thereof an effective bone mass-increasing amount of a composition comprising a biologically active peptide of SEQ ID NO: 19, SEQ ID NO: 29 or any of the above peptides and a pharmaceutically acceptable carrier. In another aspect of the invention, the condition to be treated is osteoporosis, old age osteoporosis, or post-menopausal osteoporosis. In another aspect of the invention, the effective amount of SEQ ID NO: 19, SEQ ID NO: 29 or any of the above peptides for increasing bone mass is from about 0.01 μg/kg/day to about 1.0 μg/kg/day. In another aspect of the invention, the method of administration is parenteral, subcutaneous or nasal insufflation. In another aspcet, the invention is directed to a method for determining rates of bone reformation, bone resorption and/or bone remodeling comprising administering to a patient an effective amount of SEQ ID NO: 19, SEQ ID NO: 29 or any of the above peptides and determining the uptake of the peptide into the bone of the patient. In another aspcet, the invention is directed to a method of making SEQ ID NO: 19, SEQ ID NO: 29 or any of the above peptides, wherein the peptide is synthesized by solid phase synthesis. in another aspcet, the invention is directed to a method of making SEQ ID NO: 19, SEQ ID NO: 29 or any of the above peptides, wherein the peptide is protected by FMOC. |
Allelic variants of grp50 |
The present invention provides isolated polynucleotides encoding a receptor gene called GPR50 having at least one polymorphic sites. It furthermore provides a method for analysing polimorphic sites in said receptor gene. Certain of these polynucleotides having a polymorphic site (allelic variants) are found to be more prevalent in a population of patients with clinical Bipolar Depression compared to a control population. A method for the genetic testing of Bipolar Depression is a further embodiment of the present invention. Furthermore, polynucleotides encompassing these polymorphic sites, the invariant distal or proximal to the polymorphic site localized polynucleotides as well as the polynucleotides encoding GPR50 are part of the invention. The present invention also provides a recombinant cell line expressing these novel receptors at appropriate levels such that novel compounds active at these receptors may be identified for therapeutic use. |
1. An isolated polynucleotide encoding a GPR50 receptor protein, wherein said polynucleotide has at least one polymorphic site. 2. The isolated polynucleotide of claim 1, wherein said polymorphic site is localised at position 1503-1504, 1582 or 1804 of SEQ ID NO.: 1. 3. The isolated polynucleotide of claim 1, wherein said polynucleotide comprises any one of SEQ ID NO.: 2 to 8. 4. The isolated polynucleotide of claim 1, wherein said polynucleotide encodes a polypeptide comprising any one of SEQ ID NO.: 10 to 16. 5. The isolated polynucleotide of claim 1, wherein said polynucleotide has an A at position 1582 and/or 1804, or an insertion at position 1503-1504 in combination with a G at position 1582 and/or 1804. 6. A recombinant expression vector comprising the polynucleotide according to claim 1. 7. Polypeptide encoded by the polynucleotide according to claim 1. 8. A cell transfected with the polynucleotide according to claim 1. 9. The cell according to claim 8 which is a stable transfected cell which expresses the polypeptide according to claim 7. 10. (canceled) 11. (canceled) 12. (canceled) 13. A method for determining binding of ligands of GPR50 protein or a polymorphic variant thereof according to claim 7, to prepare a medicament for a psychiatric disorder, preferably BPAD or UP, said method comprising the steps of: a) introducing into a suitable host cell a polynucleotide according to any of claims 1-5 or a recombinant expression vector according to claim 6; b) culturing the host cells under conditions to allow expression of the introduced polynucleotide; c) optionally isolating the expression product; bringing the expression product from step c or the host cell from step b into contact with potential ligands; establishing the amount of binding of the ligand to the expressed protein or its signal transduction capacity; and optionally, isolating the ligand. 14. A method for the formulation of a pharmaceutical composition comprising the method of claims 14 and mixing the compound identified with a pharmaceutically acceptable carrier. 15. A method for identifying an increased risk for clinical Bipolar Depression or UP in a human comprising analyzing a biological sample of an individual for the presence of a polynucleotide, said polynucleotide encoding the GPR50 receptor having an A at position 1804, and/or an absence of an insertion at position 1503-1504 of SEQ ID NO.: 1; and identifying said gene as having polymorphism associated with BPAD or UP when the presence of said polynucleotide is detected in said biological sample. 16. A diagnostic assay for detection of a psychiatric disorder comprising the steps of detecting mutations in the nucleic acid sequences encoding the gene of claim 1. 17. The assay of claim 16 wherein the step of detecting mutations is performed by a method selected from the group consisting of PCR, specific hybridization, and detection of relative levels of RNA. 18. The assay of claim 16 wherein the step of detecting mutations is detecting the presence and/or the levels of the GPR50. 19. The assay of claim 18 wherein the step of detecting the presence and/or levels of the GPR50 is performed by a method selected from the group consisting of immunological technologies and binding of specific antibodies. |
Occlusion device for gambing machine display elements |
It consists of an opaque blind (4) susceptible to covering part of the display (1) carrying the images (2) that are visible through the screen of the machine at the end of each play, for the purpose of temporarily hiding said images (2) to substitute them with others existing on the outer face of the blind (4). This blind (4) is associated to an arm (5) finished off with a ring (6) assembled with rotational freedom on a support (10), said disc ring (6) having a gearing (7) through which it receives the movement from a micromotor (9) which makes the blind (4) rock between the operating and non-operating limit positions, duly defined by a pair of end stops (11-11′) or by the electronic control of a “stepper” type drive motor (9). |
1. Stopper for displays for recreational machines, specifically for recreational machines provided with various displays capable of sequentially showing different figures through the screen of the machine, determining prize winning combinations, characterized in that it consists of an opaque blind (4) capable of covering the part of the display (1) appearing through the screen of the machine and which at the end of each play is susceptible to rocking or rotating to enable freely and directly visualizing said sector of the display, so that after the displays have stabilized, the figure of some of them participating in the winning line is kept hidden, and therefore the resulting combination is only partially visible, until the player makes a decision on the play to that respect. 2. Stopper for displays for recreational machines according to claim 1, characterized in that the blind (4) is associated to a rocking arm (5) finished off with a ring on its other end, assembled with rotational freedom on a support (10) preferably associated to the support of the main motor (3) of the display, a ring (6) provided with a geared sector (7) through which it receives the movement from an auxiliary micromotor (9) by means of a suitable set of reducing gears (8) established on said support (10) and capable of supplying said arm (5) with an angular movement suitable for the foreseen shift for the blind (4) between the stopping and non-stopping limit positions. 3. Stopper for displays for recreational machines according to previous claims, characterized in that when the auxiliary micromotor (9) is a conventional micromotor, two fixed end stops (11-11′), preferably microswitches, are established on said support, collaborating with the microswitches there is a stop (12) associated to the arm (5) of the blind (4) in order to limit the rocking movement of said arm in both directions. 4. Stopper for displays for recreational machines according to claims 1 and 2, characterized in that the micromotor (9) is a “stepper” motor which enables controlling its drive pulses and, therefore, the amplitude in the angular shift of the arm (5) associated to the blind (4). 5. Stopper for displays for recreational machines according to previous claims, characterized in that on its outer surface the blind (4′) incorporates additional figures (2′) that are complementary to the figures (2) present on the drum (1) or roller, as well as a window (13) which permits visualizing said figures of the drum (1) or roller, the number of said figures and windows being various in number and position. 6. Stopper for displays for recreational machines according to claim 1, characterized in that the blind (24) is associated to a rocking arm (25) finished off with a disc ring (26) with an inner gearing (27) upon which a drive motor (29) of the blind (4) acts, said motor (29) as well as its complementary group of reducing gears (28) housed on a support (30) like a circular shield, of a diameter coinciding with that of the main motor (23) and which, together with the latter, is housed in a support (31) materialized as a cylindrical capsule of an inner diameter coinciding with the outer diameter of the main motor (23) and of an axial height that is suitable for simultaneously receiving both elements, the output pinion (28) of the reducing group radially and partially emerging from this assembly, which meshes with the inner gearing (27) of the ring (26) finishing of the arm (25) of the blind (24). 7. Stopper for displays for recreational machines according to claim 6, characterized in that the output pinion (28) of the reducing group emerges to the outside of the support (30) through a slit (32) on its side wall, which operatively faces another slit (33) existing on the cylindrical capsule (31). 8. Stopper for displays for recreational machines according to claims 6 and 7, characterized in that the geared ring (26), assembled with rotational freedom on the cylindrical capsule (31) constitutive of the main support, is immobilized in an axial direction with regard to said capsule (31) with the collaboration of two washers (34-35) located on both sides of the geared ring (26) and duly fixed to the capsule (31), one of said washers (35) incorporating the end stops (36) for controlling the electric drive micromotor (29) of the blind (24), upon which a stop (37) acts that is established on the support arm (25) of the blind (24). 9. Stopper for displays for recreational machines according to claim 1, characterized in that the blind (41) is joined to the periphery of a flywheel (44) having a slightly over dimensioned diameter with regard to the roller or display (42) and intended to be laterally adapted to the latter in a coaxial arrangement, the shaft (45) of said flywheel being assembled with rotational freedom on a support (47) in turn parallel to and opposite to the support (48) of the complementary roller or display (42), provided with an orthogonal bracing (49) on one of its edges for the fixation thereof by means of screwing it to the structure of the machine or to the display (1) to which it serves. 10. Stopper for displays for recreational machines according to claim 9, characterized in that the flywheel (44) incorporates a perimetral gearing (51) through which it receives the movement from a micromotor (52), with or without interposing a reducing transmission (53), such that the shift of the blind (41) can reach 360°, said motor (52), its reducing transmission (53) and its control circuit (55) being assembled on the same support (47) as the flywheel (44), the latter incorporating suitably placed holes (56) on its periphery for assembling the end stops controlling the angular shift of the flywheel (44) and of the stopper blind (41) associated to it. 11. Stopper for displays for recreational machines according to claim 10, characterized in that the micromotor (52) is the “stepper” type. |
<SOH> BACKGROUND OF THE INVENTION <EOH>In the practical field of application of the invention, that of recreational gambling machines with cash prizes, displays having a plurality of different images or figures are used, which sequentially appear on the screen of the machine in the development of each play, such that at the end of the play, the images corresponding to the different displays, established on a winning line, produce a determined combination that is susceptible to a prize according to a wining plan also suitable established on the screen of the machine. These displays adopt multiple shapes and different types of actuation in practice, but they have the common denominator of showing only a portion of them through the screen of the machine, which are normally comprised by three of the previously mentioned figures, and occasionally part of the immediately prior and subsequent figures, such that in the end phase of the play, when the displays are stabilized, a series of figures are seen through them which regard those which can establish one or more horizontal lines immediately prior and subsequent to said figure, and also diagonal lines on occasion, so that when the figures of the valid winning line coincide at all times with one of the winning plan combinations, the machine gives the player the corresponding cash prize. To date, the only possibility of increasing the chances of playing on this main group of machine displays consisted of establishing an auxiliary display, which in turn shows a figure and which, in determined circumstances, can be moved to any one of the displays of the winning line in order to substitute the figure of the latter, or to perform “advances” in the displays in order to equally modify the final combination. |
Display device and double drum for gambling machines |
The invention is structured on the basis of two flywheels (14) and (17) which are coaxial and having different diameters, upon which respective annular bands (15) and (18) are assembled, which are concentric and consequently parallel, bearing their respective alignments of images or figures that are collated with empty and transparent areas permitting the visualization of the inner band through the outer band, said bands having independent actuation by means of respective motors (13) and (19), in turn controlled by respective optic regulators (23) and (24), such that the movement of one of the annular and image-bearing bands (25) is totally independent from the other (28). |
1.—display for roulettes or drums in recreational machines, of the type incorporating a mobile plate closed in on itself bearing a plurality of figures, a plate that is partially visible through the screen of the machine, showing at least one image at the end of each play that can be combined with that of the other displays in order to obtain a prize winning combination, displays wherein behind the area of the screen of the machine through which the figures are visible, there is a reflective screen having recesses that are dimensionally and positionally in coincidence with the visible figures on the mobile plate, at the bottom of which there are lights for enhancing the visualization of said figures, characterized in that at least one of said lights (5) incorporates a complementary signaling device (6) of any suitable, conventional type, such as a display, which is unnoticed when inoperative and which provides complementary information through the mobile plate (1) when operative, information that is a number or any other type of figure modifying the conditions of the game, changing the value of the prize obtained in the winning line or permitting access to an auxiliary game. 2.—Display for roulettes or drums in recreational machines according to claim 1, characterized in that the mobile plate (1) bearing the Figures (2) that are sequentially visible through the screen of the machine incorporates a transparent or less opaque area in correspondence with each one of said Figures (2), which is formally and dimensionally suitable for permitting better visualization of the information provided by the subsequent signaling device when it is operative. 3.—Display for roulettes or drums in recreational machines according to claim 1, characterized in that it comprises two coaxial flywheels (14) and (17) but with independent actuation, having slightly different diameters, each one of which constitutes a support and mobilizer means for an annular band (15-18) bearing a plurality of uniformly distributed images (28-30) which are intercalated with transparent sectors (29-31), such that after the double drum stops and through the corresponding outer screen or window, the images of the band (15-18) adopting the outer position are visible, and where applicable and through the transparent sectors (29-31) thereof, images of the band (15-18) adopting the inner position. 4.—Display for roulettes or drums in recreational machines according to claim 3, characterized in that the main flywheel (14) is joined to the output shaft (16) of an electric motor (13) assembled in a cylindrical support casing (12) joined to a support chassis (11) for the entire assembly, said motor (13) being coaxial to the drum, whereas the secondary flywheel (17) is assembled with rotational freedom on the cylindrical support (12) and receives the movement through a toothed crown (22) from a second motor (19) that is independent from the motor (13), assembled on a radial extension (20) of the support (12) and whose output pinion (21) meshes with said crown (22). 5.—Display for roulettes or drums in recreational machines according to claims 3 and 4, characterized in that the optic regulator (23) is established on the radial extension (20) for the secondary motor (19) in order to control the main flywheel (14), whereas the optic regulator (24) for the secondary flywheel (17) is established on another radial extension of the cylindrical support (12). 6.—Display for roulettes or drums in recreational machines according to claims 3, 4, and 5, characterized in that the main band (15) associated to the main flywheel (14) can be placed inside or outside of the secondary band (18) associated to the secondary flywheel (17). 7.—Display for roulettes or drums in recreational machines according to claims 3, 4, 5 and 6, characterized in that the radial extension (25) constituting the support for the multiple screen (16) containing the lighting lights (27) is placed opposite the extension (20). |
<SOH> BACKGROUND OF THE INVENTION <EOH>In the practical field of application of the invention, that of recreational machines with cash prizes, displays are used that bear a plurality of different images or figures sequentially appearing on the screen of the machine in the development of each play, such that at the end of the play, the images corresponding to the different displays, established on a winning line, produce a determined combination that is susceptible to a prize according to a winning plan also suitably established on the screen of the machine. These displays adopt multiple shapes and different types of actuation in practice, but they have the common denominator of showing only a portion of them through the screen of the machine, they are normally comprised of three of the previously mentioned figures, and occasionally part of the immediately prior and subsequent figures, such that in the end phase of the play, when the displays are stabilized, a series of figures are seen through which can establish one or more winning lines, such as an intermediate horizontal line, the intermediate horizontal lines immediately prior and subsequent to that which has been mentioned, and also diagonal lines on occasion, such that when the figures of the valid winning line coincide at all times with one of the winning plan combinations, the machine gives the player the corresponding cash prize. Regardless of the general structure of the display, which may vary as previously stated, it is normal that behind the participating figure-bearing mobile plate, specifically in the area that can be visualized through the screen of the machine, a type of reflective screen is established, generally with three frontally opened recesses corresponding to the three figures which normally and simultaneously appear on the screen of the machine within each display, respective lights being established at the bottom of said recesses which can light up separately or all together, and whose evident purpose is to highlight the figure participating in the winning line or to enhance visualization of all the figures appearing through the screen of the machine. In any case, these displays are only capable of showing part of the figures participating in the game, such that the game alternatives offered do not go beyond the possible combinations to be carried out with the figures from the different displays. The use of an auxiliary display is known for trying to increase the possibilities of the game, which in turn shows a figure and which in certain circumstances can be moved to any one of the displays of the winning line in order to replace the figure in the latter. |
Modification of human variable domains |
The present invention relates to a method for the optimization of isolated human immunoglobulin variable heavy (VH) and light (VL) constructs. |
1. An isolated polypeptide comprising a VH domain selected from the group consisting of (i) a VH domain belonging to the VH1a subclass, wherein said VH domain comprises an amino acid residue F at position 29 and/or L at position 89; (ii) a VH domain belonging to the VH1b subclass, wherein said VH domain comprises the amino acid residue L at position 89; (iii) a VH domain belonging to the VH2 subclass, wherein said VH domain comprises at least one amino acid residue selected from the group consisting of G at position 16, V at position 44, A at position 47, G at position 76, F at position 78, Y at position 90, R at position 97, E at position 99, wherein if R is at position 97, then E is at position 99; (iv) a VH domain belonging to the VH4 subclass, wherein said VH domain comprises at least one amino acid residue selected from the group consisting of G at position 16, A at position 47, F at position 78, Y at position 90, R at position 97, and E at position 99, wherein if R is at position 97, then E is at position 99; (v) a VH domain belonging to the VH5 subclass, wherein said VH domain comprises at least one amino acid residue selected from the group consisting of L at position 89, R at position 97, and E at position 99, wherein if R is at position 97, then E is at position 99; and (vi) a VH domain belonging to the VH6 subclass, wherein said VH domain comprises at least one amino acid residue selected from the group consisting of V at position 5, G at position 16, I at position 58, F at position 78, Y at position 90 and R at position 97, and E at position 99, wherein if R is at position 97, then E is at position 99. 2. An isolated polypeptide according to claim 1, comprising a VH domain belonging to the VH1a subclass, wherein said VH domain comprises an amino acid residue F at position 29 and/or L at position 89. 3. An isolated polypeptide according to claim 1, comprising a VH domain belonging to the VH1b subclass, wherein said VH domain comprises the amino acid residue L at position 89. 4. An isolated polypeptide according to claim 1, comprising a VH domain belonging to the VH2 subclass, wherein said VH domain comprises at least one amino acid residue selected from the group consisting of G at position 16, V at position 44, A at position 47, G at position 76, F at position 78, Y at position 90, R at position 97, E at position 99, wherein if R is at position 97, then E is at position 99. 5. An isolated polypeptide according to claim 1, comprising a VH domain belonging to the VH4 subclass, wherein said VH domain comprises at least one amino acid residue selected from the group consisting of G at position 16, A at position 47, F at position 78, Y at position 90, R at position 97, and E at position 99, wherein if R is at position 97, then E is at position 99. 6. An isolated polypeptide according to claim 1, comprising a VH domain belonging to the VH5 subclass, wherein said VH domain comprises at least one amino acid residue selected from the group consisting of L at position 89, R at position 97, and E at position 99, wherein if R is at position 97, then E is at position 99. 7. An isolated polypeptide according to claim 1, comprising a VH domain belonging to the VH6 subclass, wherein said VH domain comprises at least one amino acid residue selected from the group consisting of V at position 5, G at position 16, I at position 58, F at position 78, Y at position 90 and R at position 97, and E at position 99, wherein if R is at position 97, then E is at position 99. 8. An antibody or functional fragment thereof comprising a VH domain according to claim 1. 9. A library of antibodies or functional fragments thereof comprising one or more antibodies or functional fragments thereof according to claim 8. 10. An isolated nucleic acid sequence encoding a polypeptide selected from the group consisting of (i) a polypeptide comprising a VH domain belonging to the VH1a subclass, wherein said VH domain comprises an amino acid residue F at position 29 and/or L at position 89; (ii) a polypeptide comprising a VH domain belonging to the VH1b subclass, wherein said VH domain comprises the amino acid residue L at position 89; (iii) a polypeptide comprising a VH domain belonging to the VH2 subclass, wherein said VH domain comprises at least one amino acid residue selected from the group consisting of G at position 16, V at position 44, A at position 47, G at position 76, F at position 78, Y at position 90, R at position 97, E at position 99, wherein if R is at position 97, then E is at position 99; (iv) a polypeptide comprising a VH domain belonging to the VH4 subclass, wherein said VH domain comprises at least one amino acid residue selected from the group consisting of G at position 16, A at position 47, F at position 78, Y at position 90, R at position 97, and E at position 99, wherein if R is at position 97, then E is at position 99; (v) a polypeptide comprising a VH domain belonging to the VH5 subclass, wherein said VH domain comprises at least one amino acid residue selected from the group consisting of L at position 89, R at position 97, and E at position 99, wherein if R is at position 97, then E is at position 99; and (vi) a polypeptide comprising a VH domain belonging to the VH6 subclass, wherein said VH domain comprises at least one amino acid residue selected from the group consisting of V at position 5, G at position 16, I at position 58, F at position 78, Y at position 90 and R at position 97, and E at position 99, wherein if R is at position 97, then E is at position 99. 11. A vector comprising a nucleic acid sequence corresponding to the nucleic acid sequence according to claim 10. 12. A host cell harboring a nucleic acid sequence corresponding to the nucleic acid sequence according to claim 10. 13. A method for producing a VH domain or an antibody or a functional fragment thereof comprising the step of expressing an isolated nucleic acid sequence according to claim 10. 14. A method for obtaining an isolated nucleic acid sequence, comprising the step of (i) substituting, in a nucleic acid sequence that encodes a VH1a subclass domain, at least one codon that encodes an amino acid residue selected from the group consisting of F at position 29 and L at position 89; or (ii) substituting, in a nucleic acid sequence that encodes a VH1b subclass domain, a codon that encodes the amino acid residue L at position 89; or (iii) substituting, in a nucleic acid sequence that encodes a VH2 subclass domain, at least one codon that encodes an amino acid residue selected from the group consisting of G at position 16, V at position 44, A at position 47, G at position 76, F at position 78, R at position 97, and E at position 99, wherein if R is at position 97, then E is at position 99; or (iv) substituting, in a nucleic acid sequence that encodes a VH2 subclass domain, a codon that encodes the amino acid residue Y at position 90; or (v) substituting, in a nucleic acid sequence that encodes a VH4 subclass domain, at least one codon that encodes an amino acid residue selected from the group consisting of G at position 16, V at position 44, A at position 47, G at position 76, F at position 78, R at position 97, and E at position 99, wherein if R is at position 97, then E is at position 99; or (vi) substituting, in a nucleic acid sequence that encodes a VH4 subclass domain, a codon that encodes the amino acid residue Y at position 90; or (vii) substituting, in a nucleic acid sequence that encodes a VH5 subclass domain, at least one codon that encodes an amino acid residue selected from the group consisting of R at position 77, L at position 89, R at position 97, and E at position 99, wherein if R is at position 97, then E is at position 99; or (viii) substituting, in a nucleic acid sequence that encodes a VH6 subclass domain, at least one codon that encodes an amino acid residue selected from the group consisting of V at position 5, G at position 16, V at position 44, I at position 58, D at position 72, G at position 76, F at position 78, R at position 97, and E is at position 99, wherein if R is at position 97, then E is at position 99; or (ix) substituting, in a VH6 subclass domain, a codon that encodes the amino acid residue Y at position 90. 15. A method according to claim 14, wherein 2 or more codons are substituted in said nucleic acid sequence. 16. A method according to claim 14, further comprising the steps of: (i) identifying for said domain the corresponding amino acid consensus sequence selected from the group of VH consensus sequences consisting of VH1a, VH1b, VH2, VH4, VH5, and VH6; (ii) substituting one or more codons corresponding to amino acid residues of said consensus sequence into a corresponding position(s) in said nucleic acid sequence of said domain. 17. A method of obtaining a polypeptide, comprising the step of expressing a nucleic acid sequence according to claim 14. 18. A method for constructing a library of antibodies or functional fragments thereof comprising the steps of: (i) obtaining at least one nucleic acid sequence according to claim 14; and (ii) diversifying said obtained nucleic acid sequence to generate a population of diversified nucleic acid sequences, wherein said diversified nucleic acid sequences can be expressed for generating and screening of antibody libraries comprising diversified VH domains. 19. An isolated polypeptide comprising a VL domain selected from the group consisting of (i) a VL domain belonging to the VLκ2 subclass, wherein said VL domain comprises the amino acid residue R at position 18, and wherein if R is at position 18, then T is at position 92; and (ii) a VL domain belonging to the VLλ1 subclass, wherein said VL domain comprises the amino acid residue K at position 47. 20. An isolated polypeptide according to claim 19, comprising a VL domain belonging to the VLκ2 subclass, wherein said VL domain comprises the amino acid residue R at position 18, and wherein if R is at position 18, then T is at position 92. 21. An isolated polypeptide according to claim 19, comprising a VL domain belonging to the VLλ1 subclass, wherein said VL domain comprises the amino acid residue K at position 47. 22. An antibody or a functional fragment thereof comprising a VL domain according to claim 19. 23. A library of antibodies or functional fragments thereof comprising one or more antibodies or functional fragments thereof according to claim 22. 24. An isolated nucleic acid molecule encoding a polypeptide selected from the group consisting of (i) a polypeptide comprising a VL domain belonging to the VLκ2 subclass, wherein said VL domain comprises the amino acid residue R at position 18, and wherein R is at position 18, then T is at position 92; and (ii) a polypeptide comprising a VL domain belonging to the VLλ1 subclass, wherein said VL domain comprises the amino acid residue K at position 47. 25. A vector comprising a nucleic acid sequence corresponding to the nucleic acid sequence according to claim 24. 26. A host cell harbouring a nucleic acid sequence molecule corresponding to the nucleic acid sequence according to claim 24. 27. A method for producing a VL domain or an antibody or a functional fragment thereof comprising the step of expressing an isolated nucleic acid sequence according to claim 24. 28. A method for obtaining a nucleic acid sequence, comprising the step of (i) substituting, in a nucleic acid sequence that encodes a VLκ2 subclass domain, at least one codon that encodes an amino acid residue selected from the group consisting of S at position 12, Q at position 45, and R at position 18, and wherein if R is at position 18, then T is at position 92; or (ii) substituting, in a nucleic acid sequence that encodes a VLλ1 subclass domain, at least one codon that encodes the amino acid residue K at position 47; or (iii) substituting, in a nucleic acid sequence that encodes a VLλ1 domain, at least three codons that encode the amino acid residues S at position 7, P at position 8, and S at position 9, respectively; or (iv) substituting, in a nucleic acid sequence that encodes a VLλ2 domain, at least three codons that encode the amino acid residues S at position 7, P at position 8, and S at position 9, respectively; or (v) substituting, in a nucleic acid sequence that encodes a VLλ3 domain, at least three codons that encode the amino acid residues S at position 7, P at position 8, and S at position 9, respectively. 29. A method according to claim 28, wherein 2 or more codons are substituted in said nucleic acid sequence. 30. A method according to claim 28, further comprising the steps of: (i) identifying for said domain the corresponding amino acid consensus sequence selected from the group of VL consensus sequences consisting of VLλ2, VLλ1 VLλ2, and VLλ3; and (ii) substituting one or more codons corresponding to amino acid residues of said consensus sequence into a corresponding position(s) in said nucleic acid sequence of said domain. 31. A method of obtaining a polypeptide, comprising the step of expressing a nucleic acid sequence according to claim 24. 32. A method for constructing a library of antibodies or functional fragments thereof, comprising the steps of: (i) obtaining at least one nucleic acid sequence according to claim 24; and (ii) diversifying said obtained nucleic acid sequence to generate a population of diversified nucleic acid sequences, wherein said diversified nucleic acid sequences can be expressed for generating and screening of antibody libraries comprising said diversified VH domains. 33. An antibody or a functional fragment thereof comprising a polypeptide of claim 1 and a polypeptide comprising a VL domain selected from the group consisting of (i) a VL domain belonging to the VLκ2 subclass, wherein said VL domain comprises the amino acid R at position 18, and wherein if R is at position 18, then T is at position 92; and (ii) a VL domain belonging to the VLλ1 subclass, wherein said VL domain comprises the amino acid residue K at position 47. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Because of their high degree of specificity and broad target range, antibodies have found numerous applications in a variety of settings in basic research, clinical and industrial use, where they serve as tools to selectively recognize virtually any kind of substrate. However, despite their versatility there are intrinsic limitations in the use of antibody molecules for some important applications. For example, therapeutic or in vivo diagnostic antibody fragments require a long serum half-life in human patients to accumulate at the desired target, and they must, therefore, be resistant to precipitation and degradation by proteases (Willuda et al., 1999). Industrial applications often demand antibodies, that can function in organic solvents, surfactants or at high temperatures—all of which pose severe challenges to the stability of these molecules (Dooley et al., 1998; Harris et al., 1994). There is also a size consideration, especially in clinical applications. Enhanced tumor penetration favors smaller molecules, thus making the large size of whole antibodies a potential liability in some treatment regimens. Furthermore, the high demand for, and the increasing number of, applications of antibodies require more efficient methods for their high-level production. Single-chain Fv (scFv) fragments are one antibody format designed to circumvent some of these limitations (Bird et al., 1988; Huston et al., 1988). The size of these molecules is reduced to the antigen binding part of an antibody, and they contain the variable domains of the heavy and light chain connected via a flexible linker. Most scFv fragments can be easily obtained from recombinant expression in E. coli in sufficient amounts (Glockshuber et al., 1992; Plückthun et al., 1996). As production yields of these fragments are influenced by their stability, as well as solubility and folding efficiency, considerable efforts have been made to identify positions in scFv fragments critical for influencing their expression behavior (Knappik & Plückthun, 1995; Forsberg et al., 1997; Kipriyanov et al., 1997; Nieba et al., 1997). The factors influencing the stability of antibody molecules have been studied mostly with scFv fragments (Wörn & Plückthun, 2001). The overall stability of scFv fragments depends on the intrinsic structural stability of V L and V H as well as on the extrinsic stabilization provided by their interaction (Wörn & Plückthun, 1999). For some scFvs, the stabilities of isolated V H and V L domains, as well as of the whole scFv fragment, have been measured and compared recently (Jäger et al., 2001; Jäger & Plückthun, 1999a; Wörn & Plückthun, 1999). The V H domain of the anti-HER2 scFv hu4D5-8, which was generated by loop grafting on a human V H 3 consensus framework (Carter et al., 1992; Rodrigues et al., 1992), shows a free energy of unfolding of 14.4 kJ/mol −1 l (Jäger et al., 2001). This low thermodynamic stability is surprising at first glance, but there are several differences in framework residues of the V H 3 consensus sequence introduced after the loop grafting to increase affinity to HER2 (Carter et al., 1992). The V H domain IcaH-01 of a catalytic antibody (Ohage et al., 1999) was engineered for stability by converting it to the consensus sequence (Steipe et al., 1994). Because of the frequent usage of V H 3 domains, this overall consensus is heavily biased towards the V H 3 consensus. Seven positions were identified and separately exchanged (Wirtz & Steipe, 1999). ScFv fragments, as well as complete human antibodies against a broad variety of tailored antigens, can now be obtained from several antibody libraries (Griffiths et al., 1994; Vaughan et al., 1996; Knappik et al., 2000). The libraries are enriched by panning for antibody fragments that bind the desired target molecule, but the selection procedure is biased for additional factors such as expression behavior, toxicity of the expressed antibody construct to the bacterial host, protease sensitivity, folding efficiency, and stability. There are two conceivable solutions to make a diverse library of stable frameworks. The first is to use a single stable framework (Holt et al., 2000; Pini et al., 1998; Söderlind et al., 2000). These libraries use the germ line gene DP47 (Tomlinson et al., 1992) as the master framework for the V H domain, since this gene is well expressed in bacterial systems (Griffiths et al., 1994) and most frequently expressed in vivo in human individuals (de Wildt et al., 1999). The Griffiths library is built from a germline V H bank using in vitro generated CDR3 and FR4 sequences (Griffiths et al., 1994). The diversity has been reached by introducing various point mutations in the CDRs (Holt et al., 2000; Pini et al., 1998) or sampled CDRs from in vivo-processed gene sequences (Söderlind et al., 2000). The second possibility to achieve a structurally diverse library of stable frameworks is to optimize the human consensus antibody frameworks further. Different frameworks with conformational changes for framework 1 conformations (Honegger & Plückthun, 2001 a; Jung et al., 2001; Saul & Poljak, 1993) may access a different range of CDR2 conformations (Saul & Poljak, 1993), while different framework 4 sequences affect CDR3 conformation. The Human Combinatorial Antibody Library (HuCAL, Knappik et al., 2000) consists of combinations of seven V H and seven V L synthetic consensus frameworks connected via a linker region forming 49 master genes (Knappik et al., 2000). The basis for this library is a set of consensus sequences of the framework regions of the major V H - and V L -subfamilies (V H 1, V H 2, V H 3, V H 4, V H 5, and V H 6, Vκ1, Vκ2, Vκ3, Vκ4, Vλ1, Vλ2 and Vλ3). These subfamilies were identified from known germline sequences (VBASE, Cook & Tomlinson, 1995) with the V H 1 subfamily further divided into V H 1a and V H 1b because of different CDR-H2 conformations. For each of the subfamilies, a consensus sequence for the framework regions was calculated from a database of all known rearranged antibody sequences belonging to that subfamily. These 14 consensus sequences ideally represent the structural repertoire of human variable domain frameworks. These consensus sequences containing germline CDR1 and CDR2 sequences of the corresponding germline variable domain and identical CDR3s were used for expression studies (Knappik et al., 2000). Thus, it could be shown that the individual VH and VL domains are well expressed and stable in E. coli . However, these studies, and studies on their individual perfomance in recombinant libraries (Hanes et al., 2000) showed that nevertheless there are striking differences between the individual variable domains when compared to each other. Enhanced overall expression and stability of antibodies or fragments thereof is highly desirable for most applications of antibody libraries. Thus, the technical problem of the present invention is to improve the relative stability, overall expression and solubility of antibodies or fragments thereof. The solution to the above mentioned technical problem is achieved by providing the embodiments characterized in the claims and disclosed hereinafter. The technical approach of the present invention i.e. modifying one or more framework residues in a human variable heavy or light chain antibody domain of a particular subclass with reference to a V H or a V L domain, respectively, of another subclass, is neither provided nor suggested by the prior art. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides antibodies having, inter alia, a modified framework region, using methods described and contemplated herein. Methods for mutating nucleic acid sequences are well known to the practitioner skilled in the art, including but not limited to cassette mutagenesis, site-directed mutagenesis, mutagenesis by PCR (see for example Sambrook et al., 1989; Ausubel et al., 1999). In one aspect, the present invention provides isolated polypeptides (and isolated nucleic acid sequences encoding the same) that contain a V H domain selected from the group consisting of (i) a V H domain belonging to the V H 1a subclass, wherein the V H domain contains an amino acid residue F at position 29 and/or L at position 89; (ii) a V H domain belonging to the V H 1b subclass, wherein the V H domain contains the amino acid residue L at position 89; (iii) a V H domain belonging to the V H 2 subclass, wherein the V H domain contains at least one amino acid residue selected from the group consisting of G at position 16, V at position 44, A at position 47, G at position 76, F at position 78, Y at position 90, R at position 97, E at position 99, wherein if R is at position 97, then E is at position 99; (iv) a V H domain belonging to the V H 4 subclass, wherein the V H domain contains at least one amino acid residue selected from the group consisting of G at position 16, A at position 47, F at position 78, Y at position 90, R at position 97, and E at position 99, wherein if R is at position 97, then E is at position 99; (v) a V H domain belonging to the V H 5 subclass, wherein the V H domain contains at least one amino acid residue selected from the group consisting of L at position 89, R at position 97, and E at position 99, wherein if R is at position 97, then E is at position 99; and (vi) a V H domain belonging to the V H 6 subclass, wherein the V H domain contains at least one amino acid residue selected from the group consisting of V at position 5, G at position 16, I at position 58, F at position 78, Y at position 90 and R at position 97, and E at position 99, wherein if R is at position 97, then E is at position 99. The present invention also provides isolated polypeptides (and isolated nucleic acid sequences encoding the same) that contain a V L domain selected from the group consisting of (i) a V L domain belonging to the V L κ2 subclass, wherein the V L domain contains the amino acid residue R at position 18, and wherein if R is at position 18, then T is at position 92; and (ii) a V L domain belonging to the V L λ1 subclass, wherein the V L domain contains the amino acid residue K at position 47. The nucleic acid sequences encoding the polypeptides of the invention can be used, e.g., for the construction of libraries of antibodies or fragments thereof. Libraries of antibodies or fragments thereof have been described in various publications (see, e.g., Vaughan et al., 1996; Knappik et al., 2000; U.S. Pat. No. 6,300,064, which are incorporated by reference in their entirety), and are well-known to one of ordinary skill in the art. In the context of the present invention, the term “V H domain” refers to the variable part of the heavy chain of an immunoglobulin molecule. The term “V H . . . subclass” includes the subclass defined by the corresponding “V H . . . ” consensus sequence taken from the HuCAL (V H 1a, V H 1b, V H 2, V H 3, V H 4, V H 5, and V H 6 (Knappik et al., 2000) generated as described above. In this context, the term “subclass” refers to a group of variable domains sharing a high degree of identity and similarity represented by a consensus sequence of the major V H -subfamilies, wherein the term “subfamily” is used as a synonym for “subclass.” In the context of the present invention, the term “consensus sequence” refers to the HuCAL consensus genes. The determination whether a given V H domain is “belonging to a V H subclass” is made by alignment of the V H domain with all known human V H germline segments (VBASE, Cook & Tomlinson, 1995) and determination of the highest degree of homology using a homology search matrix such as BLOSUM (Henikoff & Henikoff, 1992). Methods for determining homologies and grouping of sequences according to homologies are well known to one of ordinary skill in the art. The grouping of the individual germline sequences into subclasses is done according to Knappik et al., (2000). In the context of the present invention the term “V L domain” refers to the variable part of the light chain of an immunoglobulin molecule. The term “V L . . . subclass” refers to the subclass defined by the corresponding V L . . . consensus sequence taken from the HuCAL (Vκ1, Vκ2, Vκ3 and Vκ4 as well as Vλ1, Vλ2 and Vλ3; Knappik et al., 2000) generated as described above. In this library, a consensus sequence for each of the major V L -subfamilies was generated from known antibody sequences (VBASE, Cook & Tomlinson, 1995). In the context of the present invention, the numbering of the amino acid residues is according to the structurally adjusted scheme of Honegger & Plückthun (2001b). In the context or the present invention, the term “antibody” is used as a synonym for “immunoglobulin”. Antibodies or fragments thereof according to the present invention may be Fv (Skerra & Plückthun, 1988), scFv (Bird et al., 1988; Huston et al., 1988), disulfide-linked Fv (Glockshuber et al., 1992; Brinkmann et al., 1993), Fab, (Fab′) 2 fragments, single V H domains or other fragments well-known to the practitioner skilled in the art, which comprise at least one variable domain of an immunoglobulin or immunoglobulin fragment and have the ability to bind to a target. |
Lateral-opening rigid hinged-lid packet |
A rigid cigarette packet having a case (1) defined by a cup-shaped container (12) closed by a hinged, lateral-opening lid (11), and wherein the lid (11) incorporates at least part of a top wall (4) of the case (1); and the case (1) is formed from a blank (44) having two panels (4′, 4″) which are superimposed to form the top wall (4) of the case (1), and houses a tubular collar (18) closed at the top by a wall (20; 67; 74) adjacent to the top wall (4) of the case (1) and including a removable panel (27; 71; 80) located at the lid (18). |
1) A lateral-opening, rigid, hinged-lid packet, the packet (A; B; C) comprising a case (1) having four lateral walls (7, 8, 9) and a top wall (4) and in turn comprising a cup-shaped container (12) with a top opening (17; 63; 73), a collar (18) projecting partly from said cup-shaped container (12) through said top opening (17; 63; 73), and a lid (11) hinged to said cup-shaped container (12) along a lateral hinge (13) and movable about said hinge (13) to and from a closed position closing said top opening (17; 63; 73); said lid (11) incorporating at least part of said top wall (4); said case (1) being formed from a blank (44) having two substantially identical first panels (4′, 4″) which are superimposed to form said top wall (4) and a number of second panels (7′, 8′, 9′, 7″, 8″), which form said lateral walls (7, 8, 9) and are substantially identical to each other as regards form and dimensions; the packet (A; B; C) being characterized in that said blank (44) has three of said second panels (7′, 8′, 9′; 9′, 7″, 8″) arranged in a line so as to be side by side. 2) The packet of claim 1, wherein said collar (18) is a tubular collar (18) coaxial with said cup-shaped container (12); said tubular collar having a top closing wall (20; 67; 74) adjacent to said top wall (4) and having a passage (29; 71a; 81), for the cigarettes (2), facing said lid (11). 3) The packet of claim 2, wherein said passage (29; 71a; 81) is closed by a removable panel (27; 71; 80). 4) The packet of claim 1, wherein said blank (44) comprises a number of main panels (3′, 4′, 4″, 51, 52) arranged in a line so as to be side by side; three of said main panels (3′, 4′, 4″) being substantially identical, and comprising said two first panels (4′, 4″), and a further main panel (3′) defining a bottom wall (3) of said case (1). 5) The packet of claim 4, wherein said two first panels (4′, 4″) are located at opposite ends of said blank (44); said further main panel (3′) being an intermediate panel (3′) of said blank (44). 6) The packet of claim 2, wherein said case (1) and said tubular collar (18) have beveled longitudinal edges (6, 10). 7) The packet of claim 2, wherein said case (1) and said tubular collar (18) have rounded longitudinal edges (60, 61). 8) The packet of claim 2, wherein said case (1) and said tubular collar (18) have sharp longitudinal edges (72). 9) The packet of claim 2, wherein said passage (29; 71a; 81) extends over a lateral portion (20b) of said top closing wall (20; 67; 74) of said collar (18). 10) The packet of claim 9, wherein said passage (71a) extends over a lateral portion of said top closing wall (67) adjacent to said lid (11). 11) The packet of claim 9, wherein said top closing wall (20) comprises a fixed portion (20a); said passage (29) extending over a lateral portion (20b) of said top closing wall (20) located on the opposite side to said fixed portion (20a) with respect to said lid (11). 12) The packet of claim 3, wherein said removable panel (27; 71; 80) comprises a lateral pull tab (28). 13) The packet of claim 9, wherein said passage (81) extends over a central portion (74b) of said top closing wall (74). 14) The packet of claim 2, further comprising a foil inner wrapping (30); said inner wrapping comprising a further removable panel (31) located at said passage (29; 71a; 81). 15) The packet of claim 14, further comprising an extracting device (40) housed partly inside said collar (18) and between the collar (18) and said inner wrapping (30); part of said extracting device (40) being defined by a pull tab (39) projecting from said collar (18) through said passage (29; 71a; 81). 16) The packet of claim 2, further comprising a toggle strap (32) interposed between said lid (11) and said collar (18) to limit the angular travel of said lid (11) about said hinge (13). 17) The packet of claim 16, wherein said toggle strap (32) forms an integral part of said collar (18). 18) The packet of claim 16, wherein said toggle strap (32) forms an integral part of said blank (44). 19) The packet of claim 2, further comprising two lateral guide plates (88), each comprising a first portion (88a) integral with an inner lateral surface of said lid (11), and a second portion (88b) engaged in sliding manner between said case (1) and said collar (18). 20) A lateral-opening, rigid, hinged-lid packet, the packet (A; B; C) comprising a case (1) having a top wall (4) and in turn comprising a cup-shaped container (12) with a top opening (17; 63; 73), a collar (18) projecting partly from said cup-shaped container (12) through said top opening (17; 63; 73), and a lid (11) hinged to said cup-shaped container (12) along a lateral hinge (13) and movable about said hinge (13) to and from a closed position closing said top opening (17); said lid (11) incorporating at least part of said top wall (4); and the packet (A; B; C) being characterized in that said collar (18) is a tubular collar (18) coaxial with said cup-shaped container (12); said tubular collar (18) having a top closing wall (20, 67, 74) adjacent to said top wall (4) and having a passage (29; 71a; 81), for said cigarettes (2), facing said lid (11). 21) The packet of claim 20, wherein said passage (29; 71a; 81) is closed by a removable panel (27; 71; 80). 22) The packet of claim 20, wherein said case (1) is formed from a blank (44) having two substantially identical panels (4′; 4″) which are superimposed to form said top wall (4). 23) The packet of claim 22, wherein said blank (44) comprises a number of main panels (3′, 4′, 4″, 51, 52) arranged in a line so as to be side by side; three of said main panels (3′, 4′, 4″) being substantially identical, and comprising said two panels (4′, 4″), and a further main panel (3′) defining a bottom wall (3) of said case (1). 24) The packet of claim 23, wherein said two panels (4′, 4″) are located at opposite ends of said blank (44); said further main panel (3) being an intermediate panel of said blank (44). 25) The packet of claim 20, wherein said case (1) and said tubular collar (18) have beveled longitudinal edges (6, 10). 26) The packet of claim 20, wherein said case and said tubular collar have rounded longitudinal edges (60, 61). 27) The packet of claim 20, wherein said case and said tubular collar have sharp longitudinal edges (72). 28) The packet of claim 20, wherein said passage (29; 71a; 81) extends over a lateral portion (20b) of said top closing wall (20; 67; 74) of said collar (18). 29) The packet of claim 28, wherein said passage (71a) extends over a lateral portion of said top closing wall (67) adjacent to said lid (11). 30) The packet of claim 28, wherein said top closing wall (20) comprises a fixed portion (20a); said passage (29) extending over a lateral portion (20b) of said top closing wall (20) located on the opposite side to said fixed portion with respect to said lid (11). 31) The packet of claim 21, wherein said removable panel (27; 71; 80) comprises a lateral pull tab (28). 32) The packet of claim 28, wherein said passage (81) extends over a central portion (74b) of said top closing wall (74). 33) The packet of claim 20, further comprising a foil inner wrapping (30); said inner wrapping comprising a further removable panel (31) located at said passage (29; 71a; 81). 34) The packet of claim 33, further comprising an extracting device (40) housed partly inside said collar (18) and between the collar (18) and said inner wrapping (30); part of said extracting device (40) being defined by a pull tab (39) projecting from said collar (18) through said passage (29; 71a; 81). 35) The packet of claim 20, further comprising a toggle strap (32) interposed between said lid (11) and said collar (18) to limit the angular travel of said lid (11) about said hinge (13). 36) The packet of claim 35, wherein said toggle strap (32) forms an integral part of said collar (18). 37) The packet of claim 35, wherein said toggle strap (32) forms an integral part of said blank (44). 38) The packet of claim 20, further comprising two lateral guide plates (88), each comprising a first portion (88a) integral with an inner lateral surface of said lid (11), and a second portion (88b) engaged in sliding manner between said case (1) and said collar (18). 39) The packet of claim 20, wherein said tubular collar comprises at least one longitudinal lateral wall formed from the superimposition of at least two panels. 40) The packet of claim 35, wherein the toggle strap (32) comprises a longitudinal appendix (58) having a number of crease lines (59). |
<SOH> BACKGROUND ART <EOH>Packets of the aforementioned type are, for example disclosed in WO 01/1599. In particular, WO 01/1599 discloses a lateral-opening, rigid, hinged-lid cigarette packet comprising a case having two minor lateral walls and two major lateral walls, which are wider than the two minor lateral walls, and a top wall. The case in turn comprises a cup-shaped container with a top opening, a collar projecting partly from said cup-shaped container through said top opening, and a lid, which incorporates at least part of the top wall and is hinged to said cup-shaped container along a lateral hinge and movable about said hinge to and from a closed position closing said top opening. The lateral hinge is located on the top wall and is perpendicular to the major lateral walls. In known lateral-opening, rigid, hinged-lid packets of the above type, the hinge connecting the lid to the cup-shaped container is normally fairly short. This is inevitable in lateral-opening packets, and seriously impairs the shape stability of the packet, which, with use, tends to deform, so that the lid fails to close the cup-shaped container properly. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>A number of non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying drawings, in which: FIG. 1 shows a view in perspective of a first preferred embodiment of the packet according to the present invention in the closed position; FIG. 2 shows an exploded view in perspective of the FIG. 1 packet in the open position; FIG. 3 shows a partly sectioned side view of the FIG. 2 packet; FIGS. 4, 5 , 6 and 7 show plan views of respective elements of the FIG. 1 packet; FIGS. 6 a and 6 b show alternative embodiments of the element of FIG. 6 ; and FIG. 8 shows a view in perspective of a second preferred embodiment of the packet according to the present invention in the closed position; FIGS. 9, 10 and 11 show plan views of respective elements of the FIG. 8 packet; FIG. 12 shows a view in perspective of a third preferred embodiment of the packet according to the present invention in the closed position; FIG. 13 shows a partly exploded view in perspective of the FIG. 12 packet in the open position; FIGS. 14, 15 , 16 and 17 show plan views of respective elements of the FIG. 12 packet. detailed-description description="Detailed Description" end="lead"? |
Manufacturing process for the preparation of a, a-branched alkane carboxylic acids providing esters with an improved softness |
A manufacturing process for the preparation of a, a-branched alkane carboxylic acids, by reacting a mono-olefin or a precursor thereof, with carbon monoxide in the presence of a strong acid catalyst characterized in that the starting olefin is a dimmer C8 or trimer C12 derived from n-butene, which predominantly comprise 2-butene, that the acid catalyst is composed of BF3/H3PO4 in a molar ratio of BF3:H3PO4 in the range of from 0.5:1.0 to 5.0:1.0, or of CF3SO3H, that the weight ratio of the catalyst relative to the mono-olefin is in the range of from 1:1 to 10:1, that the reaction is carried out at a temperature in the range of from 60 to 140° C., and with an initial water content in the catalyst system in the range of from 8 to 25 wt %; and vinyl esters and glycidyl esters derived from said carboxylic acids. |
1. A manufacturing process for the preparation of α,α-branched alkane carboxylic acids, the process comprising reacting a mono-olefin feed comprising a dimer (C8) or trimer (C12) of n-butene, comprising predominantly 2-butene, with carbon monoxide in the presence of a strong acid catalyst, wherein the n-butene dimer or n-butene trimer olefin feed has not been fractionated, wherein the acid catalyst is composed of BF3/H3PO4 in a molar ratio of BF3:H3PO4 in the range of from 0.5:1.0 to 5.0:1.0, or of CF3SO3H, wherein a weight ratio of the catalyst relative to the mono-olefin is in the range of from 1:1 to 10:1, wherein the reaction is carried out at a temperature in the range of from 60 to 140° C. and wherein the catalyst has an initial water content in the range of from 8 to 25 wt %. 2. The manufacturing process of claim 1, wherein the catalyst is BF3/H3PO4 and wherein the molar ratio of BF3H3PO4 is in the range of from 1:1 to 3:1. 3. The manufacturing process of claim 1 wherein the carbon monoxide is at a pressure is in the range of from 20 to 100 bar. 4. The manufacturing process of claim 1 wherein the reaction temperature is in the range of from 80 to 140° C. and the carbon monoxide is at a pressure is in the range of from 30 to 90 bar. 5. The manufacturing process of claim 1 wherein the weight ratio of the catalyst relative to the mono-olefin is in the range of from 2:1 to 6:1. 6. The manufacturing process of claim 1 wherein the manufacturing process is a continuous process. 7. The manufacturing process of claim 1 wherein the process is a nickel catalyzed DIMERSOL process or an acid catalyzed Montmorillonite oligomerisation process. 8. The manufacturing process according to claim 7, wherein the process is the acid catalyzed Montmorillonite oligomerization process. 9. Glycidyl esters of α,α-branched saturated carboxylic acids obtained according to the process of claim 1. 10. Vinyl esters of α,α-branched saturated carboxylic acids obtained according to the process of claim 1, of which a homopolymer has a Tg lower than −3° C. in the case of C9 acids and lower than −13° C. in the case of C13 acids. |
Compounds derived from diaminopyrazoles substituted by an aminoalkyl or aminoalkenyl radical and their use in oxidation dyeing of keratinous fibres |
The invention concerns compounds derived from diaminopyrazole of formula (I), wherein: R1 is a linear or branched radical selected among C2, C3, C4 aminoalkyl radicals or C2, C3, C4 aminoalkenyl radicals, or one of the physiologically acceptable salts thereof. The invention also concerns compositions containing said compound for dyeing keratinous fibres and the method using said compositions. |
1-18.(cancelled) 19. A diaminopyrazole compound of formula (I): in which R1 is a linear or branched radical further defined as a C2, C3 or C4 aminoalkyl or C2, C3 or C4 aminoalkenyl radicals, or a physiologically acceptable salt thereof. 20. The compound of claim 19, wherein the aminoalkyl or aminoalkenyl radical is ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, or 2-methylpropyl substituted by 1, 2 or 3 amino groups. 21. The compound of claim 19, further defined as 2-(2-aminoethyl)-2-H-pyrazole-3,4-diamine, 2-(3-aminopropyl)-2H-pyrazole-3,4-diamine, 2-(2-aminopropyl)-2H-pyrazole-3,4-diamine, 2-(2,3-diamino-propyl)-2H-pyrazole-3,4-diamine, 2-(4-aminobutyl)-2H-pyrazole-3,4-diamine, 2-(3-aminobutyl)-2H-pyrazole-3,4-diamine, 2-(2-aminobutyl)-2H-pyrazole-3,4-diamine, 2-(3,4-diaminobutyl)-2H-pyrazole-3,4-diamine, 2-(2,4-diaminobutyl)-2H-pyrazole-3,4-diamine, 2-(2,3-diaaminobutyl)-2H-pyrazole-3,4-diamine, 4-(4,5-diaminopyrazol-1-yl)-butane-1,2,3-triamine, 2-(3-amino-1-methylpropyl)-2H-pyrazole-3,4-diamine, 2-(2-amino-1-methyl-propyl)-2H-pyrazole-3,4-diamine, 2-(1-aminomethylpropyl)-2H-pyrazole-3,4-diamine, 2-(3-amino-1-amino-methylpropyl)-2H-pyrazole-3,4-diamine, 2-(2-amino-1-aminomethylpropyl)-2H-pyrazole-3,4-diamine, 2-(2,3-diamino-1-aminomethylpropyl)-2H-pyrazole-3,4-diamine, 2-(3-amino-2-methylpropyl)-2H-pyrazole-3,4-diamine, 2-(3-amino-2-aminomethylpropyl)-2H-pyrazole-3,4-diamine, 2-(3-aminoprop-2-ene)-2H-pyrazole-3,4-diamine, 2-(2-aminoprop-2-ene)-2H-pyrazole-3,4-diamine, 2-(4-aminobut-2-ene)-2H-pyrazole-3,4-diamine, 2-(3-aminobut-3-ene)-2H-pyrazole-3,4-diamine, 2-(1-aminomethylprop-2-ene)-2H-pyrazole-3,4-diamine, or a physiologically acceptable acid salt of any of these. 22. The compound of claim 19, wherein the physiologically acceptable salt is a hydrochloride, hydrobromide, sulfate, tartrate, lactate, or acetate. 23. A composition for the oxidation dyeing of keratin fibers, comprising, as an oxidation base, a diaminopyrazole compound of formula (I): in which R1 is a linear or branched radical further defined as a C2, C3 or C4 aminoalkyl or C2, C3 or C4 aminoalkenyl radicals, or a physiologically acceptable salt thereof, in a medium suitable for dying. 24. The composition of claim 23, further defined as comprising from 0.001% to 10% by weight of at least one diaminopyrazole of formula (I) or salt thereof. 25. The composition of claim 23, wherein the medium that is suitable for dyeing comprises water or a mixture of water and at least one organic solvent further defined as a C1-C4 lower alkanol, polyol, polyol ether, or aromatic alcohol, or a mixture thereof. 26. The composition of claim 23, further defined as having a pH of between 3 and 12. 27. The composition of claim 23, comprising at least one additional oxidation base further defined as a para-phenylenediamine, bis(phenyl)alkylenediamine, para-aminophenol, ortho-aminophenol, or heterocyclic base other than the diaminopyrazole of formula (I), or an addition salt of one of these with an acid. 28. The composition of claim 27, wherein the additional oxidation base comprises from 0.0005% to 12% by weight relative to the total weight of the dye composition. 29. The composition of claim 23, further defined as comprising at least one direct dye. 30. The composition of claim 23, further defined as comprising at least one coupler. 31. The composition of claim 30, wherein the coupler is a meta-phenylenediamine, meta-aminophenol, meta-diphenol, monohydroxylated naphthalene derivative, polyhydroxylated naphthalene derivative, or heterocyclic coupler, or an addition salt of one of these with an acid. 32. The composition of claim 30, wherein the coupler represents from 0.0001% to 10% by weight relative to the total weight of the dye composition. 33. The composition of claim 23, further defined as comprising at least one direct dye and at least one coupler. 34. A process for dyeing keratin fibers, comprising applying to the fibers a composition comprising, as an oxidation base, a diaminopyrazole compound of formula (I): in which R1 is a linear or branched radical further defined as a C2, C3 or C4 aminoalkyl or C2, C3 or C4 aminoalkenyl radicals, or a physiologically acceptable salt thereof, in a medium suitable for dying, for a time that is sufficient to develop the desired coloration, either in air or using an oxidizing agent. 35. The process of claim 34, wherein the keratin fibers are further defined as human hair. 36. The process of claim 34, further comprising applying to the fibers an oxidation catalyst. 37. The process of claim 34, wherein no oxidizing agent is applied and coloration is revealed by contact with atmospheric oxygen. 38. The process of claim 34, further comprising an application of an oxidizing agent to the fibers. 39. The process of claim 38, wherein the oxidizing agent is added to the dye composition prior to application of the dye composition to the fibers. 40. The process of claim 38, wherein the oxidizing agent is comprised in an oxidizing composition that is applied simultaneously or sequentially with regard to the dyeing composition to the fibers. 41. The process of claim 38, wherein the oxidizing agent is hydrogen peroxide, urea peroxide, an alkali metal bromate, or a persalt. 42. A kit comprising: a first compartment containing a composition comprising a diaminopyrazole compound of formula (I): in which R1 is a linear or branched radical further defined as a C2, C3 or C4 aminoalkyl or C2, C3 or C4 aminoalkenyl radicals, or a physiologically acceptable salt thereof, in a medium suitable for dying; and a second compartment containing an oxidizing composition. |
Bioassay method, bioassay device, and bioassay substrate |
Disclosed is a bioassay method in which, by controlling the electric field formation in the reaction region where an interaction between substances, such as a hybridization, is performed, the efficiency of the interaction can be improved. Also disclosed is a bioassay apparatus in which the method can be favorably carried out. In the method, an interaction between substances is detected by a detecting element 1 (10), the detecting element including at least a detection surface S (S′) which is surface-treated for immobilizing a detecting substance D, a reaction region R (R′) which provides a field for interaction between the detecting substance D immobilized on the detection surface S (S′) and a target substance T, and an electric field-forming means E which forms an electric field in the reaction region R (R′) by applying a potential difference in the reaction region R (R′), and the method includes at least a step of turning on/off the electric field formation by the electric field-forming means E at a predetermined timing. |
1. A bioassay method comprising detecting an interaction between substances by a detecting element, the detecting element comprising at least: a detection surface which is surface-treated for immobilizing a detecting substance; a reaction region which provides a field for interaction between the detecting substance immobilized on the detection surface and a target substance; and an electric field-forming means which forms an electric field in the reaction region by applying a potential difference in the reaction region, wherein the method comprises at least a step of turning on/off the electric field formation by the electric field-forming means at a predetermined timing. 2. A bioassay method according to claim 1, wherein the detecting substance and the target substance are nucleotide chains, and the interaction is a hybridization. 3. A bioassay method according to claim 2, further comprising the steps of: forming an electric field so as to stretch the detecting nucleotide chain immobilized on the detection surface at the end; subsequently adding the target nucleotide chain into the reaction region; subsequently forming an electric field so as to relatively move the detecting nucleotide chain and the target nucleotide chain in the reaction region; and subsequently performing the hybridization with the electric field being removed. 4. A bioassay apparatus comprising a detecting element, the detecting element comprising at least: a detection surface which is surface-treated for immobilizing a detecting substance; a reaction region which provides a field for interaction between the detecting substance immobilized on the detection surface and a target substance; and an electric field-forming means which can form an electric field in the reaction region by applying a potential difference and which can turn on/off the electric field formation at a predetermined timing. 5. A bioassay substrate comprising a detecting element provided on a disc-shaped substrate from which recorded information can be optically read, the detecting element comprising at least: (1) a detection surface which is surface-treated for immobilizing an end of a detecting nucleotide chain; (2) positive and negative electrodes for forming an electric field to stretch the detecting nucleotide chain immobilized on the detection surface; and (3) a reaction region which provides a field for hybridization between the detecting nucleotide chain and a target nucleotide chain. 6. A bioassay substrate according to claim 5, wherein the detecting element is a cell-type detecting element in which the detection surface is placed on one surface, and a plurality of cell-type detecting elements are arrayed on the disc-shaped substrate. 7. A bioassay substrate according to claim 3 claim 6, wherein the cell-type detecting elements are arrayed radially, when viewed from above, on the disc-shaped substrate. 8. A bioassay substrate according to claim 7, wherein different detecting nucleotide chains are immobilized for each cell-type detecting element or for each group of cell-type detecting elements. 9. A bioassay substrate according to claim 5, wherein the reaction region of the detecting element is provided in each of the grooves radially extending on the disc-shaped substrate, and the detection surface is placed on an inner surface of the groove. 10. A bioassay substrate according to claim 9, wherein different detecting nucleotide chains are immobilized for each groove or for each group of grooves. 11. A bioassay substrate according to claim 5, further comprising a means for providing the positional information of the detection surface site and rotational synchronization information. 12. A bioassay substrate according to claim 11, wherein the means comprises a wobbled groove or address pits provided on the substrate. 13. A bioassay substrate according to claim 5, wherein the reaction region is filled with a material which can undergo a reversible phase change between gel and sol at between room temperature and the optimal temperature for the reaction. 14. A bioassay substrate according to claim 5, wherein the hybridization is detected by an intercalator. |
<SOH> BACKGROUND ART <EOH>The main conventional techniques which are related to the present invention will be described below. Currently, integrated substrates for bioassays, i.e., so-called DNA chips or DNA microarrays, (hereinafter referred to as “DNA chips”), in which selected DNA is microarrayed by microarray technology, have been used for the analysis of gene mutations, SNP (single nucleotide polymorphism) analysis, gene expression frequency analysis, etc., and have also started to be used extensively for the development of new drugs, clinical diagnosis, pharmacogenomics, forensic medicine, and other fields. The DNA chips are characterized in that it is possible to extensively analyze intermolecular interactions, such as hybridizations, because various types and a large number of DNA oligomer chains, cDNA (complementary DNA), etc., can be integrated on glass substrates or silicon substrates. One example of analytical methods using DNA chips will be briefly described below. That is, PCR amplification is performed in which mRNA extracted from cells, tissues, etc., is incorporated by reverse transcription-PCR or the like into DNA probes solid-phased on a glass substrate or silicon substrate, and hybridization is performed on the substrate. Fluorescent light measurement is then carried out with a suitable detector. DNA chips can be classified into two types. In the first type of chip, oligonucleotides are synthesized directly on a given substrate using photolithography by applying semiconductor exposure technology. A typical example is the one manufactured by Affymetrix, Inc., U.S.A. (for example, refer to U.S. Pat. No. 5,445,934). In this type of chip, although the integration degree is high, DNA synthesis on the substrate has limitations, and the length is limited to about several tens of bases. In the second type of chip, which is also referred to as “the Stanford method”, the chip is fabricated by dispensing and solid-phasing prepared DNA on a substrate using a split taper pin (for example, refer to U.S. Pat. No. 5,807,522). In this type of chip, although the integration degree is lower than that of the former type, it is possible to solid-phase about 1 kb of DNA fragments. Recently, biosensor technology has been advancing, in which a selected detecting substance is solid-phased on a fine detection surface site provided on a thin plate, which is referred to as a biosensor chip, such as a protein chip, and a microvolume of solution containing a target substance is allowed to flow toward the detecting substance, and then the interaction between the two substances is observed and analyzed based on the surface plasmon resonance principle, quartz crystal oscillator principle, or the like. This technology is becoming useful for analyzing interactions between substances, such as antibody-antigen reactions and hormone responses. In the conventional DNA chip technology and biosensor technology, however, interactions between substances, such as hybridization reactions and antibody-antigen reactions, are carried out by solid-phasing (immobilizing) detecting nucleotide chains, such as DNA probes, and proteins, etc., on two-dimensional, small detection regions of substrates. Consequently, the interactions are carried out mainly based on the Brownian movement of the reaction products under not necessarily favorable reaction conditions in which freedom of the reaction products is limited spatially and there is also a possibility that steric hindrance may occur during the reactions. Therefore, the conventional DNA chip technology and biosensor technology have technical problems, i.e., low interaction efficiency and long reaction time. Furthermore, in the known DNA chips, etc., sample solutions are only dripped onto predetermined spot sites (detection regions) on substrates, and no devices are used to relatively align the target substances contained in the sample solutions and the detecting substances immobilized on the spot sites. Accordingly, it is a principal object of the present invention to provide a bioassay method, a bioassay apparatus, and a bioassay substrate that can be advantageously used in the bioassay method and the bioassay apparatus, in which, by controlling the electric field formation in the reaction region where an interaction between substances, such as a hybridization, is carried out, the detecting substance and the target substance are relatively aligned with each other, and the structures of the substances are adjusted, thereby increasing the efficiency of the interaction. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a flowchart which illustrates preferred steps of a bioassay method of the present invention and a referred embodiment of a bioassay apparatus of the present invention. FIG. 2 is a waveform chart which illustrates an example of a step of applying/removing a voltage in the bioassay method or apparatus. FIG. 3 is a perspective view showing the appearance of a bioassay substrate viewed from above in a preferred embodiment of the present invention. FIG. 4 is an enlarged perspective view showing a cell-type detecting element provided on a substrate. FIG. 5 is an enlarged view showing the detection surface and its vicinity of the reaction region of the cell-type detecting element. FIGS. 6 (A) and 6 (B) are diagrams showing a bioassay substrate in another preferred embodiment of the present invention; FIG. 6 (A) is a top plan view of the substrate; and FIG. 6 (B) is an enlarged plan view of the section X in FIG. 6 (A). detailed-description description="Detailed Description" end="lead"? |
Ntb-a,a a surface molecule involved in natural killer cellss activity |
The present invention relates to a novel protein, termed NTB-A, nucleic acid molecules encoding the same and uses thereof. The invention also relates to methods of regulating Natural Killer cells activity by regulating the activity of NTBA-A in vitro, ex vivo or in vivo. The invention also comprises methods of screening active compounds using NTB-A or fragments thereof, or nucleic acid encoding the same, or recombinant host cells expressing said polypeptide. |
1. an isolated nucleic acid molecule, wherein said nucleic acid molecule is selected from: a) a nucleic acid encoding a polypeptide comprising SEQ ID NO:2; b) a nucleic acid which hybridizses to the nucleic acid of a) or to a portion thereof, said portion comprising at least 30 contiguous nucleotides of a nucleic acid a); c) a complementary strand of a nucleic acid molecule of a) or b); or d) a nucleic acid molecule encoding a nolyleptide comprising at least 5 continuous amino acid residues of SEQ ID NO:2; e) a nucleic acid molecule that encodes a human NTB-A protein; or f) a fragment of a nucleic acid molecule of a), b), c), d) or e), said fragment comprising at least 9 contiguous nucleotides. 2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a sequence encoding a polypeptide comprising SEQ ID NO:2 or a polypeptide comprising at least 5 contiguous amino acid residues of SEQ ID NO:2. 3. (canceled) 4. A nucleic acid probe, wherein said probe is complementary and specifically hybridizes to a nucleic acid molecule of claim 1. 5. A pair of nucleic acid primers, wherein at least one primer of said pair is complementary and specifically hybridizes to a nucleic acid molecule of claim 1. 6. A vector comprising a nucleic acid molecule of claim 1. 7. A host cell comprising a vector of claim 6. 8. An isolated polypeptide comprising: a), an amino acid sequence encoded by a nucleic acid molecule of of claims 1; b) SEQ ID NO:2; or c) at least 5 contiguous amino acid residues of SEQ ID NO:2 9. (canceled) 10. The host cell of claim 7, wherein said host cell expresses a polypeptide of claim 8. 11. A method of preparing cells expressing a polypeptide, said method comprising introducing a nucleic acid molecule or vector into cells in vitro and selecting the cells which express the polypeptide or progeny of said cells, wherein said nucleic acid molecule or vector is: a) a nucleic acid encoding a polypeptide comprising SEQ ID NO:2; b) a nucleic acid which hybridizes to the nucleic acid of a) or to a portion thereof, said portion comprising at least 30 contiguous nucleotides of a nucleic acid of a); c) a complementary strand of a nucleic acid molecule of a) or b); d) a nucleic acid molecule encoding a polypeptide comprising at least 5 contiguous amino acid residues of SEQ ID NO:2; e) a nucleic acid molecule that encodes a human NTB-A protein; f) a fragment of a nucleic acid molecule of a), b) c), d) or e), said fragment comprising at least 9 contiguous nucleotides, or g) a vector comprising a nucleic acid molecule of a), b), c), d), e) or f. 12. An antibody that specifically binds to a polypeptide of claim 8. 13. A method of preparing an antibody, said method comprising injecting to a non-human mammal a polypeptide of claim 8 and collecting the antibody, serum or antibody-producing cells in said mammal. 14. A method of selecting, screening or characterizing a compound, said method comprising contacting a test compound with a polypeptide of claim 8 and determining the ability of said test compound to bind to said polypeptide. 15. A method of selecting, screening or characterizing a compound, said method comprising contacting a test compound with a host cell of claim 10 and determining the ability of said test compound to bind to said polypeptide. 16. A method of selecting, screening or characterizing a compound, said method comprising contacting a test compound with a NK cell in the presence of an antibody specific for NTB-A and determining the activity of said test compound by measuring the cytolytic activity of said NK cells. 17. A method of selecting, screening or characterizing a compound, said method comprising contacting a test compound with a NTB-A polypeptide in the presence of a binding partner thereof and assessing the capacity of said test compound to modulate the interaction between said NTB-A polypeptide and said binding partner. 18. A method of selecting, screening or characterizing a compound, said method comprising (i) determining the ability of a test compound to bind to a NTB-A polypeptide and (ii) determining the ability of a test compound selected in (i) to regulate NK cell-mediated target cell lysis. 19. A method of regulating the immune function in a subject comprising the administration of a compound that regulates the activity of a NTB-A polypeptide to a subject. 20. The method of claim 19, wherein the compound regulates the activity of NK cells in a subject. 21. A pharmaceutical composition comprising a compound that regulates the activity of NTB-A and a pharmaceutically acceptable vehicle or carrier. 22. The pharmaceutical composition of claim 21, further comprising a compound that regulates a 2B4 receptor, for combined, sequential or separated use. 23. The composition of claim 22, comprising an antibody that inhibits or reduces NTB-A activity and an antibody that inhibits or reduces 2B4 activity. 24. A method of detecting a dysfunction in a subject or the risk of a subject developing a dysfunction comprising determining, in a sample derived from said subject, the presence of a mutation or alteration in a NTB-A gene or RNA, or determining the presence or amount of a NTB-A polypeptide. |
Organo-phosphorous compounds for activating gamma/delta t cells |
The present invention describes organophosphorus compounds of general formula (I) their preparation and their uses in the activation of gamma/delta T-cells, in the screening of GcpE and LytB enzyme inhibitors and in the prophylaxis and treatment of diseases in humans and animals. |
1-41. (canceled) 42. A compound of formula (I): in which R1 is selected from the group consisting of a methyl radical, a formyl radical, substituted or unsubstituted hydroxymethyl radicals and C0H2R31, R31 being selected from the group consisting of OH, substituted or unsubstituted phosphate and substituted or unsubstituted pyrophosphate and it being impossible for R31 and R2 to be present in the molecule simultaneously, R33 is selected from the group consisting of hydrogen, OH, substituted or unsubstituted phosphate and substituted or unsubstituted pyrophosphate, R3 is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl having 1 to 26 carbon atoms, substituted or unsubstituted hydroxyalkyl having 1 to 26 carbon atoms, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted alkenyl having 1 to 26 carbon atoms, substituted or unsubstituted alkynyl having 1 to 26 carbon atoms, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic radicals, substituted or unsubstituted phosphate, a silyl, a nucleoside, a nucleoside monophosphate, diphosphate or triphosphate, a deoxynucleoside, a cation of an organic or inorganic base, a cation of an organic or inorganic base wherein the metal is from main group I, II or III of the Periodic Table, ammonium, substituted ammonium, ammonium compounds derived from ethylenediamine or amino acids, and OR34, wherein R34 is defined in the same way as R3, X2 is defined in the same way as X1 if a ring is formed between X2 and C1 of X2 is selected from the group consisting of —OR6, R7 and R8 being defined in the same way as R34, and R4 being defined in the same way as R3, Z1 being defined in the same way as X1, and X3 being defined in the same way as X1 if it forms a ring with C1 and, if it does not form a ring with C1, being a group R5 being defined in the same way as R3, and Z2 and X4, which forms a ring with C1, being defined in the same way as X1, R2 is selected from the group consisting of hydrogen, OH, alkoxy, phenoxy, benzyloxy, substituted or unsubstituted phosphate and substituted or unsubstituted pyrophosphate, and X1 can be oxygen or Y1 and Y2, which can be identical or different, being selected from the group consisting of H, OH, halogen, an amino radical, a C1-9-alkoxy radical and a C1-9-alkylthio radical, or together forming an oxo group, and it being possible for a double bond to be present between C0 and C1 or C1 and C2 or C2 and C3. 43. The compound according to claim 42, of formula (II): in which a single or double bond is present between C2 and C3, R1 is selected from the group consisting of a methyl radical, a formyl radical and substituted or unsubstituted hydroxymethyl-radicals, R2 is selected from the group consisting of hydrogen, hydroxyl, alkoxy, phenoxy and benzyloxy radicals, substituted or unsubstituted phosphate and substituted or unsubstituted pyrophosphate, X1 is oxygen or a group Y1 and Y2, which can be identical or different, being selected from the group consisting of H, OH, halogen, amino, a C1-9-alkoxy radical and a C1-9-alkylthio radical, or together forming an oxo group, R3 is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl having 1 to 26 carbon atoms, substituted or unsubstituted hydroxyalkyl having 1 to 26 carbon atoms, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted alkenyl having 1 to 26 carbon atoms, substituted or unsubstituted alkynyl having 1 to 26 carbon atoms, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic radicals, substituted or unsubstituted phosphate, a silyl, a nucleoside, a nucleoside monophosphate, diphosphate or triphosphate, a deoxynucleoside, a cation of an organic or inorganic base, especially a metal of main group I, II or III of the Periodic Table, ammonium, substituted ammonium and ammonium compounds derived from ethylenediamine or amino acids, X2 is defined in the same way as X1 if a ring is formed between X2 and C1, and otherwise is R4 being defined in the same way as R3, Z1 being defined in the same way as X1, and X3 being defined in the same way as X1 if it forms a ring with C, and, if it does not form a ring with C1, being a group R5 being defined in the same way as R3, and Z2 and X4, which forms a ring with C1, being defined in the same way as X1. 44. The compound according to claim 43, of formula (IIA): in which C2 and C3 are joined together by either a single bond or a double bond, R1 is a methyl group or a substituted or unsubstituted hydroxymethyl group, R2 is selected from the group consisting of hydrogen, OH, a substituted or unsubstituted phosphate and a substituted or unsubstituted pyrophosphate, X1 and X2 are selected from the group consisting of O, CHF, CHCl, CFCl, CH2, CF2 and CCl2, and R3 is selected from the group consisting of hydrogen, substituted or unsubstituted phosphate, a nucleoside, a nucleoside monophosphate, diphosphate or triphosphate, a deoxynucleoside, a cation of an organic or inorganic base, a cation of an organic or inorganic base wherein the metal is from main group I, II or III of the Periodic Table, ammonium, substituted ammonium and ammonium compounds derived from ethylenediamine or amino acids. 45. The compound according to claim 43, of formula (IIB): in which C2 and C3 are joined together by either a single bond or a double bond, R1 is a methyl group or a substituted or unsubstituted hydroxymethyl group, R2 is H if R1 is a substituted or unsubstituted hydroxymethyl, and is OH, a substituted or unsubstituted phosphate or a substituted or unsubstituted pyrophosphate if R1 is a methyl radical, X1, X2 and X3 are selected from the group consisting of O, CHF, CHCl, CFCl, CH2, CF2 and CCl2, and R3 and R4 are selected from the group consisting of hydrogen, substituted or unsubstituted phosphate, a nucleoside, a nucleoside monophosphate, diphosphate or triphosphate, a deoxynucleoside, a cation of an organic or inorganic base, a cation of an organic or inorganic base wherein the metal is from main group I, II or III of the Periodic Table, ammonium, substituted ammonium and ammonium compounds derived from ethylenediamine or amino acids. 46. The compound according to claim 43, of formula (IIC): in which there can be a single or double bond between C2 and C3, R1 is a methyl group or a substituted or unsubstituted hydroxymethyl group, R2 is H, OH, a substituted or unsubstituted phosphate or a substituted or unsubstituted pyrophosphate, X1, X2, X3 and X4 are selected from the group consisting of O, CHF, CHCl, CFCl, CH2, CF2 and CCl2, and R3, R4 and R5 are selected from the group consisting of hydrogen, substituted or unsubstituted phosphate, a nucleoside, a nucleoside monophosphate, diphosphate or triphosphate, a deoxynucleoside, a cation of an organic or inorganic base, a cation of an organic or inorganic base wherein the metal is from main group I, II or III of the Periodic Table, ammonium, substituted ammonium and ammonium compounds derived from ethylenediamine or amino acids. 47. The compound according to claim 44, wherein X1, X2, X3 and X4 are oxygen. 48. The compound according to claim 44, wherein R1 is a substituted or unsubstituted hydroxymethyl radical, hydroxymethyl, or a hydroxymethyl radical substituted by phosphate, diphosphate or nucleoside diphosphate, and R2 =H. 49. The compound according to claim 44, wherein R1 is a methyl radical and R2 is OH, a substituted or unsubstituted phosphate, a substituted pyrophosphate, or a nucleoside diphosphate. 50. The compound according to claim 47, of the following structural formulae: 51. The compound according to claim 42 of general formula (III): in which R31 and R2, which cannot be present in the molecule simultaneously, are selected from the group consisting of OH, substituted or unsubstituted phosphate and substituted or unsubstituted pyrophosphate, a double bond being formed between C1 and C2 if R31 is present in the molecule and a double bond being formed between C0 and C1 if R2 is present in the molecule, R33 is selected from the group consisting of hydrogen, OH, substituted or unsubstituted phosphate and substituted or unsubstituted pyrophosphate, R34 is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl having 1 to 26 carbon atoms, substituted or unsubstituted hydroxyalkyl having 1 to 26 carbon atoms, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted alkenyl having 1 to 26 carbon atoms, substituted or unsubstituted alkynyl having 1 to 26 carbon atoms, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic radicals, substituted or unsubstituted phosphate, a silyl, a nucleoside, a deoxynucleoside, a nucleoside monophosphate, diphosphate or triphosphate, a cation of an organic or inorganic base, a cation of an organic or inorganic base wherein the metal is from main group I, II or III of the Periodic Table, ammonium, substituted ammonium and ammonium compounds derived from ethylenediamine or amino acids, X2 is either —OR6, R6 being defined analogously to R34, or can be R7 and R8 being defined in the same way as R4, and X1, X32 and X33, which can be identical or different, can be oxygen or a group Y and Z, which can be identical or different, being selected from the group consisting of H, OH, halogen, amino, C1-9-alkoxy and C1-9-alkylthio, or together forming an oxo group. 52. The compound according to claim 51, of general formula (IIIA): in which R31 and R2, which cannot be present in the molecule simultaneously, are selected from the group consisting of OH, substituted or unsubstituted phosphate and substituted or unsubstituted pyrophosphate, a double bond being formed between C, and C2 if R31 is present in the molecule and a double bond being formed analogously between C0 and C1 if R2 is present in the molecule, R34, R7 and R8, which can be identical or different, are as defined in claim 42 and X1, X32 and X33, which can be identical or different, are likewise as defined in claim 51. 53. The compound according to claim 52 in which R31=OH and C0 and C1 are joined by a double bond. 54. The compound according to claim 52 in which R2=OH and C, and C2 are joined by a double bond. 55. The compound according to claim 51, wherein either R34 or R6 or R7 or R8 is a substituted or unsubstituted phosphate radical. 56. The compound according to claim 51, wherein either R31 or R2 or X2 is a substituted or unsubstituted phosphate radical. 57. The compound according to claim 51, wherein R34, R6, R7 and R8, which can be identical or different, are hydrogen, a cation of a metal of main group I, II or III of the Periodic Table, or substituted or unsubstituted ammonium. 58. The compound according to claim 51, wherein X1 and X32=O. 59. The compound according to claim 51, wherein X1=CYZ, X32=O and X33=CYZ, Y and Z being as defined in claim 51. 60. The compound according to claim 51, wherein X1=O, X32=CYZ and X33=O, Y and Z being as defined in claim 51. 61. The compound according to claim 51, wherein X1, X32 and X33, which can be identical or different, are selected from the group consisting of CH2, CHF, CHCl, CFCl, CCl2 and CF2. 62. The compound according to claim 51 selected from the following group: 63. A composition comprising a compound according to claim 42 and a pharmaceutically acceptable excipient. 64. A process for the preparation of an organophosphorous compound comprising the deletion, inactivation, or modification of the lytB gene in selected cells or organisms in order to increase the concentration of one of these compounds. 65. A method of activating gamma/delta T cells comprising contacting said T cells with a compound according to claim 42. 66. A method of performing enzyme inhibition tests and screening enzyme inhibitors comprising contacting a composition comprising enzymes with a compound according to claim 42. 67. The method of claim 66, wherein said enzymes are LytB. 68. The method of claim 66, wherein said enzymes are GcpE or LytB and GcpE. 69. A method for the prophylaxis or treatment of a human or animal disease comprising the administration of a composition according to claim 63. 70. The method according to claim 69, wherein said treatment or prophylaxis is of immune, autoimmune, respiratory diseases or allergies in humans. 71. The method according to claim 70, wherein the disease is selected from the group consisting of asthma, Crohn's disease, ulcerative colitis, multiple sclerosis, bone disease, especially osteoporosis, and chronic bronchitis, as well as rheumatoid arthritis, Hashimoto's thyroiditis, myasthenia gravis, lupus erythematosus, diabetes mellitus, primary biliary cirrhosis, active chronic hepatitis, adrenalitis/Addison's disease, polymyositis, dermatomyositis, autoimmune haemolytic anaemia, myocarditis and pericarditis, scleroderma, uveitis, phacouveitis, sympathetic ophthalmia, pemphigus vulgaris, pemphigoid, pernicious anaemia, autoimmune atrophic gastritis, bronchitis, diseases caused by viruses, bacteria and parasites, benign and malignant tumours, hepatitis C virus infections, and helicobacter eradication therapy on ulcers of the gastrointestinal tract. 72. The composition according to claim 63 wherein the compound is 4-hydroxy-3-methyl-2-butenyl 1-pyrophosphate. 73. The method according to claim 69, wherein said composition is provided by oral, inhalational, intravenous, parenteral, intracistemal, intravaginal, intraperitoneal, local or rectal administration. 74. The method according to claim 70, wherein said composition additionally contains a substance that can be recognized by the immune system as a foreign antigen or autoantigen. 75. The composition according to claim 63, further comprising at least one other pharmaceutical active ingredient or a substance recognized by the immune system as a foreign antigen or autoantigen. 76. A herbicide comprising at least one compound according to claim 42. |
Optical modulating device, display, and exposure device |
Subjects for the invention are to provide a light-modulating element having an elevated energy efficiency while preventing a decrease in contrast at low cost without using a display technique employing a waveguide or lightguide plate, and to provide a display element capable of producing high-quality display images and an exposure element capable of exposure treatment. In the invention, the light-modulating element comprises: a total-reflection optical member 2 having such a property that at least part of planar incident light introduced into the light-modulating element is totally reflected at an interface (total reflection plane) 22 of a layer constituted by the light-modulating element and the incident light does not substantially go out through the side opposite to the incident-light introduction side; and light-coupling elements 6 which are disposed on the total reflection plane 22 side of the total-reflection optical member 2 and serve to selectively couple with the incident light and take out the same from the total reflection plane 22. |
1. A light-modulating element having a flat shape, which comprises: a total-reflection optical member having such a property that at least part of incident light introduced into the light-modulating element is totally reflected at an total reflection plane provided at a side opposite to the incident-light introduction side and the incident light does not substantially go out through a side opposite to the incident-light introduction side; and a light-coupling element which is disposed on the total reflection plane side of the total-reflection optical member and serves to selectively couple with the incident light and take out the same from the total reflection plane. 2. The light-modulating element of claim 1, wherein the incident light is planar light. 3. The light-modulating element of claim 1, wherein the light-coupling element takes out the incident light by changing a condition of incident-light total reflection on the total reflection plane. 4. The light-modulating element of claim 1, wherein the light-coupling element comprises a flexible thin film supported so as to be capable of being brought near to the total reflection plane of the total-reflection optical member by an electromechanical motion. 5. The light-modulating element of claim 4, wherein the electromechanical motion is a motion caused by an electrostatic force. 6. The light-modulating element of claim 1, wherein the light-coupling element comprises a layer containing a liquid crystal which changes in optical property upon application of an electric field thereto. 7. The light-modulating element of claim 1, wherein the light-coupling elements have been arranged in one-dimensional array over the total-reflection optical member. 8. The light-modulating element of claim 1, wherein the light-coupling elements have been arranged in two-dimensional array over the total-reflection optical member. 9. The light-modulating element of claim 8, wherein the light-coupling element is connected to a passive-matrix operating means. 10. The light-modulating element of claim 1, wherein the light-coupling element has a light path deflector which changes the path of the light taken out. 11. The light-modulating element of claim 10, wherein the light path deflector changes, based on refraction, the path of the light taken out. 12. The light-modulating element of claim 11, wherein the light path deflector comprises a lens array, a prism array, or a graded-index lens body. 13. The light-modulating element of claim 10, wherein the light path deflector changes, based on diffraction, the path of the light taken out. 14. The light-modulating element of claim 13, wherein the light path deflector comprises a volume hologram, a phase-modulating diffraction grating, or an amplitude-modulating diffraction grating. 15. The light-modulating element of claim 10, wherein the light path deflector changes, based on light diffusion or light scattering, the path of the light taken out. 16. The light-modulating element of claim 15, wherein the light path deflector is a porous object, an object containing a substance with a different refractive index dispersed or distributed therein, or a light-diffusing or light-scattering object having irregularities on a surface thereof. 17. The light-modulating element of claim 1, wherein the light-coupling element has a specific-wavelength-component-absorbing means which absorbs and emits a specific-wavelength component of the light taken out. 18. The light-modulating element of claim 1, wherein the light-coupling element has a phosphor which shows excitation luminescence upon reception of the light taken out. 19. The light-modulating element of claim 1, which has phosphors which show excitation luminescence upon reception of the outgoing light taken out by the light-coupling elements. 20. The light-modulating element of claim 1, wherein the total-reflection optical member has, disposed therein, an optical element which changes a light path, and that at least part of the planar incident light introduced into the total-reflection optical member is introduced into the optical element changing a light path and substantially all the incident light thus introduced is reflected by total reflection at an interface of a layer constituted by the total-reflection optical member. 21. The light-modulating element of claim 1, wherein the total-reflection optical member has, disposed therein, an optical element which selects a light path, and that at least part of the planar incident light introduced into the total-reflection optical member is introduced into the optical element selecting a light path and substantially all the incident light thus introduced is reflected by total reflection at an interface of a layer constituted by the total-reflection optical member. 22. The light-modulating element of claim 1, wherein the total-reflection optical member has an optical element changing a light path and an optical element selecting a light path which are disposed in this order from the incident-light introduction side in the direction of the thickness of the total-reflection optical member, and that when planar incident light is introduced into the optical element changing a light path, then at least part of the incident light introduced is introduced into the optical element selecting a light path and substantially all the incident light thus introduced is reflected by total reflection at an interface of a layer constituted by the light-modulating element. 23. The light-modulating element of claim 22, wherein the optical element changing a light path and the optical element selecting a light path are in optical contact with each other. 24. The light-modulating element of claim 22, wherein the optical element changing a light path and the optical element selecting a light path are in optical contact with each other through a medium having a refractive index higher than 1. 25. The light-modulating element of claim 20, wherein the light-modulating element has a transparent medium constituting part of the total-reflection optical member and the optical element changing a light path has been disposed on the light-path front side of the transparent medium. 26. The light-modulating element of claim 21, wherein the light-modulating element has a transparent medium constituting part of the total-reflection optical member and the optical element selecting a light path has been disposed on the light-path front side of the transparent medium. 27. The light-modulating element of claim 22, wherein the light-modulating element has a transparent medium constituting part of the total-reflection optical member, and the optical element changing a light path and the optical element selecting a light path have been disposed in this order on the light-path front side of the transparent medium. 28. The light-modulating element of claim 20, wherein the optical element changing a light path forward outputs light comprising at least light components having an angle θt satisfying the requirement sin θt>nw/nt wherein nt is the average refractive index of the optical element changing a light path, nw is the refractive index of the medium disposed on the light-path front side of the total reflection plane, and θt is the angle of the light passing through the medium of the optical element changing a light path. 29. The light-modulating element of claim 20, wherein the optical element changing a light path is one which changes a light path based on refraction. 30. The light-modulating element of claim 29, wherein the optical element changing a light path is any of a lens array, a prism array, and a different-refractive-index distribution object in which different refractive indexes are distributed. 31. The light-modulating element of claim 20, wherein the optical element changing a light path is one which changes a light path based on diffraction. 32. The light-modulating element of claim 31, wherein the optical element changing, a light path is any of a volume hologram, a phase-modulating diffraction grating, and an amplitude-modulating diffraction grating. 33. The light-modulating, element of claim 20, wherein the optical element changing a light path is one which changes a light path based on light diffusion. 34. The light-modulating element of claim 33, wherein the optical element changing a light path is any of a porous object, an object containing a substance with a different refractive index dispersed or distributed therein, and a diffusing or scattering object having irregularities on a surface thereof. 35. The light-modulating element of claim 20, wherein the optical element changing a light path is one which changes a light path based on light reflection. 36. The light-modulating element of claim 21, wherein the optical element selecting a light path has such a property that substantially all the transmitted light emitted by the optical element has components having an angle larger than the critical total reflection angle at an interface of a layer disposed on the incident light light-path front side of the optical element selecting a light path or at the interface of the incident light light-path front side of the optical element selecting a light path and the incident light components having any other angle are selectively reflected and are not transmitted therethrough. 37. The light-modulating element of claim 21, wherein the optical element selecting a light path transmits substantially all light having an angle θs satisfying the requirement sin θs>nw/ns wherein ns is the average refractive index of the optical element selecting a light path, nw is the refractive index of the medium disposed on the light-path front side of the total reflection plane, and θs is the angle of the light passing through the medium of the optical element selecting a light path. 38. The light-modulating element of claim 21, wherein the optical element selecting a light path functions to reflect incident light selectively with respect to wavelength region, and that as the incidence angle of the light striking on the optical element selecting a light path with the plane of the optical element becomes smaller, the wavelength of the incident light selectively reflected shifts to the shorter-wavelength side. 39. The light-modulating element of claim 21, wherein when the incidence angel of the light striking on the optical element selecting a light path is regulated so that the angle of incidence on the total reflection plane on the incident light light-path front side is not larger than the critical total reflection angle, then the optical element selecting a light path selectively reflects substantially all the incident light. 40. The light-modulating element of claim 21, wherein the optical element selecting a light path is an optical interference filter comprising a dielectric multilayer film. 41. The light-modulating element of claim 21, wherein the optical element selecting a light path is a Bragg reflection filter comprising either a cholesteric liquid crystal or a volume hologram. 42. The light-modulating element of claim 1, wherein the total-reflection optical member has an optical element which introduces incident light into the light-modulating element and that when planar incident light is introduced into the incident-light-introducing optical element, then substantially all the incident light introduced is reflected by total reflection at an interface of a layer constituted by the light-modulating element. 43. The light-modulating element of claim 42, wherein the optical element introducing incident light is an array of prisms disposed in planar arrangement. 44. The light-modulating element of claim 1, wherein substantially all the incident light which has been totally reflected returns to the incident-light introduction side of the total-reflection optical member. 45. The light-modulating element of claim 1, wherein the layer constituting the total-reflection optical member shows substantially no absorption in the wavelength region for the incident light. 46. A display element comprising the light-modulating element of claim 1 and a light source for introducing incident light into the light-modulating element. 47. The display element of claim 46, wherein the incident light is collimated light having a specific incidence angle range. 48. The display element of claim 46, wherein the incident light is collimated light having two or more incidence angles. 49. The display element of claim 46, wherein the incident light is diffused light having arbitrary incidence angles. 50. The display element of claim 46, having a phosphor on the light-coupling elements or on the light-path front side thereof and further having, interposed between the total reflection plane of the total-reflection optical member and the light-coupling elements, an optical filter which reflects wavelength components of the luminescence of the phosphor and transmits wavelength components of the incident light. 51. The display element of claim 46, having the phosphor on the light-coupling elements or on the light-path front side thereof and further having, disposed on the light-path front side of the phosphor, an optical filter which transmits wavelength components of the luminescence of the phosphor and shuts off wavelength components of the incident light. 52. The display element of claim 46, having the phosphor on the light-coupling elements or on the light-path front side thereof and further having, on the light-path front side of the phosphor, an optical filter which absorbs light in the luminescence wavelength region. 53. The display element of claim 50, wherein the optical filter is an optical interference filter comprising a dielectric multilayer film. 54. The display element of claim 50, wherein the optical filter is a Bragg reflection filter comprising a cholesteric film. 55. The display element of claim 46, wherein the incident light has main wavelengths of from 350 nm to 400 nm. 56. The display element of claim 46, wherein the incident light has main wavelengths of from 400 nm to 500 nm. 57. The display element of claim 50, wherein the phosphor emits visible light. 58. The display element of claim 57, wherein the phosphor comprises luminescent materials which emit red, green, and blue lights. 59. The display element of claim 46, wherein the light source is a flat light source disposed within the total-reflection optical member and the incident light is the light emitted by the light source. 60. The display element of claim 46, wherein the incident light is one introduced from outside the total-reflection optical member. 61. The display element of claim 46, having a reflector which has been disposed so as to face the incident-light introduction side of the display element and by which the incident light which has been introduced into the display element and then reflected by the display element is directed again toward the display element. 62. An exposure element employing the display element of claim 46 and selectively emitting light toward a work with light modulation based on exposure data. |
<SOH> BACKGROUND ART <EOH>Among devices in which incident light is regulated with respect to amplitude (intensity), phase, direction of traveling, or the like to process/exhibit an image or patterned data is a light-modulating element. In a light-modulating element, the refractive index of a substance which transmits light is changed by means of an external field applied to the substance and the intensity of the light which finally passes through or is reflected by this substance is controlled through an optical phenomenon such as refraction, diffraction, absorption, or scattering. Examples of this light-modulating element include liquid-crystal light-modulating elements which utilize an electrochemical effect of a liquid crystal. Such liquid-crystal light-modulating elements are being advantageously used in liquid-crystal displays, which are thin flat display elements. Known as light-emitting thin flat display elements are plasma displays, FEDs (field emission displays), and others. Liquid-crystal light-modulating elements are being advantageously used in liquid-crystal displays, which are thin flat display elements. A typical example of liquid-crystal displays has a structure formed by charging a nematic liquid crystal into the space between a pair of substrates having an electroconductive transparent film formed thereon so that the liquid crystal is oriented in parallel with each substrate and is twisted by 90° between both substrates, sealing the resultant package, and sandwiching this package with transverse polarizers. In this liquid-crystal display, when a voltage is applied between the electroconductive transparent films, the major axis of each liquid-crystal molecule is oriented so as to be perpendicular to the substrates and the transmittance of the light emitted by the backlight changes. Thus, an image is produced based on this change in transmittance. For imparting satisfactory suitability for dynamic images, an active-matrix liquid-crystal panel employing a TFT (thin-film transistor) is used. A plasma display has a structure comprising two glass plates between which a rare gas such as neon, helium, or xenon has been enclosed and many perpendicular electrodes regularly arranged between the two glass plates and corresponding to discharge electrodes. In this structure, the intersection of each pair of opposing electrodes serves as a pixel unit. In this plasma display, a voltage is applied selectively to the opposing electrodes corresponding to given intersections according to image information to thereby cause the intersections to discharge electricity and emit light. The resultant ultraviolet causes a phosphor to show excitation luminescence and thereby produce an image. An FED has a flat display tube structure comprising a pair of panels which have been disposed face to face at a minute distance and the periphery of which has been sealed. The viewing-side panel has a fluorescent film disposed on the inner surface thereof, while the back-side panel has field emission cathodes disposed for the respective unit luminescence regions. Typical field emission cathodes have field emission type microcathodes in a minute conically projecting shape called emitter tips. In this FED, electrons are taken out with emitter tips and are accelerated and caused to strike on a phosphor to thereby excite the phosphor. Thus, an image is produced. However, the existing flat display elements described above have the following problems. First, the liquid-crystal display has a problem that since the light emitted by the backlight is caused to pass through many layers comprising the polarizers, transparent electrodes, and color filter, the efficiency of light utilization is low. Other problems thereof include those characteristic of liquid crystals, i.e., deterioration in image quality due to viewing angle dependence and deterioration in dynamic-image quality due to a low response rate, and a cost problem in large displays employing a TFT. The plasma display has drawbacks that since partition walls for discharge should be formed for each pixel, it is difficult to obtain high brightness at a high efficiency when the resolution is high, and that the display is costly because it necessitates a high operating voltage. Furthermore, the FED has a drawback that the production cost is high as in the plasma display because the inside of the panels should be evacuated to an ultrahigh vacuum so as to enable a discharge to occur stably at a high efficiency. The FED has further had a disadvantage that a high voltage is necessary for accelerating electrons resulting from field emission and causing these to strike on the phosphor. A flat display element in which the position of a flexible thin film is changed by an electromechanical motion and the light emitted by a light source is modulated based on this position change to produce an image has recently been developed as a display which eliminates those various problems. There are various modes of producing an electromechanical motion, such as one utilizing a piezoelectric effect of voltage application and one utilizing an electromagnetic force caused by current application. However, the mode utilizing an electrostatic force, in particular, enables a short operating time of several microseconds or shorter at a low voltage with reduced power consumption as long as the positional change required of the flexible thin film for light modulation is up to about 1 μm. Furthermore, since the positional change with voltage shows hysteresis, passive-matrix operation with high contrast is possible in a two-dimensional array constitution and an active element such as a TFT is unnecessary. Consequently, a large flat display element can be produced at low cost. Examples of this kind of flat display element include those of the lightguide plate type described in the following documents. Large - Area Micromechanical Display IDRC 1997, p230-p233 U.S. Pat. No. 5,771,321 JP-T-2000-505911 (The term “JP-T” as used herein means a published Japanese translation of a PCT patent application.) FIG. 29 is a sectional view of part of a flat display element 80 of the lightguide plate type. This display element comprises a lightguide plate (or waveguide) 82 and a prism 84 optically connected to an edge of one side of the lightguide plate 82 . As shown in the figure, light is introduced from a light source (e.g., a white light source, LED, or laser) 86 through this prism 84 , and the light is led by means of total reflection within the lightguide plate 82 . This display element has flexible thin films 88 disposed over a surface of the lightguide plate 82 so as to be capable of separating from/contacting with the surface of the lightguide plate 82 . These flexible thin films 88 and the lightguide plate 82 each have an electrode layer 89 formed on a surface thereof. When a driving voltage is applied to the electrode layer 89 of a flexible thin film 88 , this thin film 88 comes into contact with the surface of the lightguide plate 82 , upon which a condition of total reflection on the surface of the lightguide plate 82 is disturbed and light is taken out of the lightguide plate 82 . On the other hand, the flexible thin films 88 to which no driving voltage is applied remain apart from the surface of the lightguide plate 82 and no light is released therethrough. By thus selectively applying a driving voltage to the electrode layers 89 of the flexible thin films 88 , a display image is produced on the surface of the lightguide plate 82 . Other examples of flat display elements include that described in the following document. Waveguide Panel Display Using Electromechanical Spatial Modulators, 1998 SID International Symposium Digest of Technical Papers, p.1022-p.1025. The flat display element described in the document shown above has a constitution which comprises, as shown in FIG. 30 , a front-side glass 91 , parallel lightguides 92 arranged over the glass 91 , and an LED (light-emitting diode) array 95 connected to an edge of the lightguides 92 through a light-transmitting material 94 having a microlens 93 . The LED array 95 comprises light-emitting parts arranged one-dimensionally, and the individual light-emitting parts correspond to the respective lightguides 92 . Flexible thin films (light switches) 96 spaced in parallel have been disposed over the lightguides 92 in a direction perpendicular to the lightguides 92 . A back-side glass 97 has been disposed over the flexible thin films 96 so as to be only partly in contact with the flexible thin films 96 . The back-side glass 97 supports these flexible thin films 96 in a manner capable of changing the positions thereof. In this flat display element 90 , which has such constitution, when a voltage is applied to the electrode on a given flexible thin film 96 , the flexible thin film 96 shifts its position toward the lightguides 92 by means of an electrostatic stress as shown in FIG. 31 . On the other hand, the LED array 95 emits light synchronously with the positional shifting according to image signals. As a result, the light which has proceeded through the lightguides 92 through total reflection is introduced into the flexible thin film 96 , reflected by a mirror 98 disposed in the flexible thin film 96 , and then injected again into the lightguides 92 in a direction nearly perpendicular thereto. The light injected into the lightguides 92 in a direction nearly perpendicular thereto cannot retain an incidence angle satisfying a condition of total reflection and, hence, passes through the lightguides 92 and is released from the front-side glass 91 . According to this flat display element 90 , the flexible thin films 96 can be operated at a high response rate because the positions of the flexible thin films are changed by means of an electrostatic stress. In addition, light does not pass through many layers unlike that in liquid-crystal displays, and this display element necessitates neither the formation of partition walls in discharge parts nor a high-voltage driving circuit unlike plasma displays. Consequently, a high-speed, inexpensive, flat display element can be realized. However, in the flat display elements 80 and 90 of the photowaveguide type described above, incident light is introduced by a method in which incident light is introduced through a prism connected to an edge of one side of the lightguide plate or through an edge of a lightguide plate/waveguide. However, the lightguide plate/waveguide of a thin flat shape has a small edge area available for incidence and tends to have an impaired efficiency of coupling with incident light. Moreover, since a further thickness reduction and a larger area are desired in lightguide plates and waveguides, the edge area available for incidence tends to decrease more and more and there is a fear of an impaired efficiency of coupling. Furthermore, there are limitations on the shape of incident light (light source) and on position for introduction. Namely, the size and number of light sources are limited and high-output light cannot be introduced. In addition, the incident light should have a beam/linear shape and this poses limitations on the kind of light sources or separately necessitates an optical system for forming such shape. As a result, there is a problem that the production process is complicated to increase the cost. In the flat display element 80 , when an element located upstream in the light path from the lightguide is brought into an ON state for image production, the light introduced into areas located downstream in the light path from this element attenuates to cause the so-called crosstalk and thereby impair image quality. Furthermore, there also is a problem that leakage light released from an ON-state element reduces the contrast of images around the element. On the other hand, in the flat display element 90 , since high-output light is injected into the thin lightguide, a loss of light coupling occurs in the lightguide, resulting in a reduced efficiency of light utilization. Furthermore, in case where the waveguide has even a slight defect in part thereof, leakage light is released from this part. Thus, the display element 90 has a constitution which is apt to suffer a decrease in image quality. An object of the invention, which has been achieved in view of the existing problems described above, is to provide a light-modulating element which eliminates the necessity of use of a display technique employing a waveguide or lightguide plate, enables use of any desired backlight, and has an elevated energy efficiency while preventing a decrease in contrast at low cost. Another object is to provide a display element capable of producing high-quality display images. Still another object is to provide an exposure element capable of exposure treatment. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a view illustrating the diagrammatic constitution of a display element having a light-modulating element according to the invention mounted thereon. FIG. 2 is a view showing an example of the constitution of a total-reflection optical member. FIG. 3 is views illustrating transmission type diffraction gratings: (a) is a volume hologram; (b) is a relief type diffraction grating; and (c) is refractive-index-modulating diffraction grating. FIG. 4 is views illustrating light-diffusing plates: (a) is a porous object; (b) is a different-refractive-index distribution/dispersion object, i.e., an object containing a substance with a different refractive index distributed/dispersed therein; and (c) is a light-diffusing or light-scattering object having irregularities on a surface thereof. FIG. 5 is a view illustrating the layer constitution of an optical interference filter. FIG. 6 is a view showing the relationship between the angle of incidence on each interface and the average refractive index of each medium in an optical element which comprises, disposed in this order, a light path-changing optical element, a light path-selecting optical element, a transparent medium u, a transparent medium v, and a transparent medium w on the front side of the total reflection plane. FIG. 7 is a view illustrating incidence angles of light striking on a light-selecting optical element. FIG. 8 is graphs showing the relationship between incident light wavelength and the spectral transmittance of a light path-selecting optical element, with respect to each of incidence angles. FIG. 9 is views illustrating light paths in and outside a light path-selecting optical element. FIG. 10 is views illustrating light-coupling elements which changes a light path based on refraction: (a) is a lens array; (b) is a prism array; and (c) is a photograph showing a graded-index lens body. FIG. 11 is views illustrating light-coupling elements which diffuse or scatter light taken out: (a) is a porous object; (b) is an object containing a substance with a different refractive index, e.g., high-refractive-index fine particles, dispersed or distributed therein; and (c) is a light-diffusing or light-scattering object having irregularities on a surface thereof. FIG. 12 is a plan view of a flat display element comprising two-dimensionally arranged display elements employing a flexible thin film. FIG. 13 is sectional views taken on C-C of FIG. 12 ; (a) is a view illustrating the OFF state and (b) is a view illustrating the ON state. FIG. 14 is a plan view of an excitation luminescence type flat display element comprising a flat display element and phosphors disposed on the viewing side thereof. FIG. 15 is sectional views taken on D-D of FIG. 14 ; (a) is a view illustrating the OFF state and (b) is a view illustrating the ON state. FIG. 16 is a sectional view illustrating part of an excitation luminescence type flat display element according to a fourth embodiment of the invention, which is an excitation luminescence type flat display element which includes phosphors disposed on a flexible thin film. FIG. 17 is views illustrating the conceptional constitution and light-modulating action of a light-coupling element according to a fifth embodiment of the invention. FIG. 18 is views illustrating the conceptional constitution and light-modulating action of a light-coupling element employing a PLDC. FIG. 19 is a view illustrating an example of a light path-selecting optical element comprising a liquid-crystal film. FIG. 20 is illustrations showing spectral transmittances in a light path-selecting optical element. FIG. 21 is views illustrating a total-reflection optical member according to a sixth embodiment of the invention, which comprises prisms. FIG. 22 is a view illustrating the sectional constitution of a total-reflection optical member obtained by bonding a microprism array to a transparent medium. FIG. 23 is a view illustrating the sectional constitution of a total-reflection optical member which comprises a structure obtained by bonding a microprism array to a transparent medium and, disposed on the light-path front side of the structure, an optical element which changes a light path to an angle according to an angle of incidence for the microprism array. FIG. 24 is views respectively illustrating other constitution examples of the total-reflection optical member. FIG. 25 is a view illustrating a specific constitution example of the total-reflection optical member. FIG. 26 is a graph showing the wavelength region of incident light. FIG. 27 is graphs showing changes of spectral transmittance T with wavelength λ with respect to each incidence angle θ. FIG. 28 is graphs showing the relationship between incidence angle θ and spectral transmittance T with respect to each wavelength λ. FIG. 29 is a sectional view of part of an existing flat display element of the lightguide plate type. FIG. 30 is a view illustrating the constitution of an existing flat display element. FIG. 31 is a view illustrating an action of the flat display element of FIG. 30 . detailed-description description="Detailed Description" end="lead"? In the figures, numerals 2 and 3 denote a total-reflection optical member, 4 a flat light source, 5 a flat UV source, 6 , 7 , and 8 a light-coupling element, 10 a light path-changing optical element, 12 and 13 a light path-selecting optical element, 14 a transparent medium, 16 a transparent medium (e.g., air), 20 a substance with a different refractive index, 22 , 52 , 66 , and 72 a total reflection plane, 26 a transparent electrode, 28 an alignment layer, 30 a cholesteric liquid-crystal layer, 32 a signal electrode, 34 a scanning electrode, 38 a light-diffusing layer, 40 a flexible thin film, 42 a, 42 b, and 42 c a phosphor, 44 a transparent substrate, 45 a front-side plate, 48 a black matrix, 50 a wavelength-selective reflecting film, 54 a liquid-crystal layer, 56 a PLDC layer, 60 a microcapsule, 64 a microprism array, 68 a prism, 70 a transparent medium, 100 a display element, 200 a flat display element, 300 and 400 an excitation luminescence type flat display element, θ 0 , θ 1 , Θ 2 , and θ 3 an incidence angle, θ c a critical total-reflection angle, and λ a wavelength. |
Method of detecting ps2v |
A method of detecting PS2V characterized by comprising reacting PS2V in a sample with a primary antibody which is bonded to a solid phase and specifically recognizes PA2V, then reacting with a secondary antibody recognizing PS2 or PS2V by any of the following procedures: (a) reacting with a secondary antibody having been enzyme-labeled and recognizing PS2 or PS2V; (b) reacting with a secondary antibody having been biotinylated and recognizing PS2 or PS2V and then reacting with an avidinylated or streptoavidinylated enzyme; (c) reacting with a secondary antibody having been biotinylated and recognizing PS2 or PS2V and then reacting with a biotinylated enzyme and avidin or streptoavidin; and (d) reacting with a secondary antibody recognizing PS2 or PS2V and then reacting with an antibody having been enzyme-labeled and recognizing the secondary antibody; then adding the substrate of the above enzyme and detecting the product formed by the enzyme reaction. |
1. A method of detecting PS2V characterized by comprising: reacting PS2V in a sample with a primary antibody which is immobilized to a solid phase; executing any one of the following steps (a) to (d): (a) reacting the PS2V with a secondary antibody which is marked with a label; (b) reacting the PS2V with a biotinylated secondary antibody, followed by reaction with an avidinated or streptoavidinated label; (c) reacting the PS2V with a biotinylated secondary antibody, followed by reaction with a biotinylated label and avidin or streptoavidin; and (d) reacting the PS2V with a secondary antibody, followed by reaction with an antibody which is marked with a label and recognizes the secondary antibody; and subsequently, determining the amount of the label captured by the solid phase. 2. A method of detecting PS2V as set forth in claim 1, wherein the combination of said antibodies is either one of the following sets (a) and (b): (a) the primary antibody that specifically recognizes PS2V and the secondary antibody which recognizes PS2 or PS2V. (b) the primary antibody which recognizes PS2 or PS2V and the secondary antibody which specifically recognizes PS2V. 3. A method of detecting PS2V characterized by comprising: executing any one of the following steps (a) to (d) in presence of PS2V derived from a sample and a certain amount of PS2V immobilized to a solid phase: (a) reacting PS2V with primary antibody which is marked with a label and specifically recognizes PS2V; (b) reacting PS2V with a biotinylated primary antibody which specifically recognizes PS2V, followed by reaction with an avidinated or streptoavidinated label; (c) reacting PS2V with a biotinylated primary antibody which specifically recognizes PS2V, followed by reaction with a biotinylated label and avidin or streptoavidin; and (d) reacting PS2V with a primary antibody which specifically recognizes PS2V, followed by reaction with a secondary antibody which is marked with a label and recognizes the primary antibody; and subsequently determining the amount of the label captured by the solid phase and detecting the PS2V derived from the sample by calculation from the certain amount of the primary antibody and the determined amount of the label. 4. A method of detecting PS2V characterized by comprising: reacting a certain amount of an antibody which is immobilized to a solid phase and specifically recognizes PS2V with PS2V derived from a sample and a certain amount of labeled PS2V in competition against each other; determining the amount of the label captured by the solid phase; and detecting the PS2V derived from the sample by calculation from the certain amount of the antibody and the determined amount of the label. 5. A method of detecting PS2V as set forth in claims 1, wherein the label is a radioisotope, an enzyme, fluorescent molecules, luminous molecules, chromophore or electroactive species. 6. A method of detecting PS2V characterized by comprising: reacting PS2V in a sample with a primary antibody which is immobilized to a solid phase and specifically recognizes 20 PS2V; executing any one of the following steps (a) to (d): (a) reacting the PS2V with a secondary antibody which is labeled with an enzyme and recognizes PS2 or PS2V; (b) reacting the PS2V with a biotinylated secondary antibody which recognizes PS2 or PS2V, followed by reaction with an avidinated or streptoavid inated enzyme; (c) reacting the PS2V with a biotinylated secondary antibody which recognizes PS2 or PS2V, followed by reaction with a biotinylated enzyme and avidin or streptoavidin; and (d) reacting the PS2V with a secondary antibody which recognizes PS2 or PS2V, followed by reaction with an antibody which is labeled with an enzyme and recognizes the secondary antibody; and subsequently, adding a substrate for the enzyme and detecting a product generated by enzymatic reaction. 7. A method of detecting PS2V as set forth in claim 5, wherein the enzyme is HRP, ALP, (3-D-galactosidase, glucose-6phosphate dehydrogenase or luciferase. 8. A method of detecting PS2V as set forth in claims 5, wherein the enzyme is ALP, and the substrate for the enzyme is a phosphate of a phenol derivative having an electron donative group at o-position and at p-position. 9. A method of detecting PS2V as set forth in claim 8, wherein the electron donative group is a lower alkoxy group. |
<SOH> BACKGROUND ART <EOH>With elderly population progressively increasing, a rapid growth of demential patients has become a serious social problem. Alzheimer's disease (AD) is a kind of dementia, and account for about 30% of senile dementia in Japan and more than half in Europe and U.S. AD is a kind of neurodegenerative disease, and its pathological characters include: (1) senile plaques in which A β is accumulated as a principle component are observed between neuronal cells; (2) abnormal phosphorylated tau protein aggregates in neuronal cells and fibrotic neurofibril are observed; (3) the cerebrum shrinks (deciduation of cerebral neocortex and neuronal cells of hippocampal). As the clinical characters, AD is a progressive dementia presenting hypergasia in general cognition, notably disorder of memory. AD is classified into familial Alzheimer's disease (FAD), which is small in the number of cases and shows autosomal dominant inheritance, and sporadic Alzheimer's disease (SAD), which apparently lacks family medical history and accounts for 90% of the total Alzheimer's disease. As genes causative of FAD, are identified amyloid precursor protein (APP) gene located on chromosome 21, presenilin-1 (PS1) gene found on chromosome 14 and presenilin-2 (PS2) gene located on chromosome 1. The mechanism of development of FAD has been gradually becoming clear. On the other hand, SAD which makes up a majority of AD cases presents the same neuropathological observations as FAD, but its development mechanism is unknown in a considerable number of aspects. For common clinical diagnosis of AD, cognition tests such as SM-IV, NINCDS-ADRDA and the like which are proposed in the United State are utilized. However, it is difficult to diagnose extremely slight cognition impediment in an early stage of AD as dementia using conventional cognition tests. The currently used definite diagnosis of AD consists of recognizing deposition of amyloid protein (senile plaques) and accumulation of tau protein (neurofibrillary tangles) in postmortem brain. Thus effective antemortem early diagnostic methods have not been established. In the present circumstances, it is too late when typical symptoms of AD (specific demential symptoms such as incapability of cognition and the like) are recognized. At present, several kinds of anti-dementia medicines are distributed in a lot of countries, and donepezil has been clinically used in Japan since 1999. These medicines often have a beneficial effect on early-stage cases. Since the medicines are expected to exhibit beneficial effect if AD is diagnosed early, there is a demand for development of a diagnostic marker effective for early treatment of AD. It has been reported that mRNA of a splicing variant (PS2V) of a PS2 gene with deletion of its fifth exon are observed manifesting frequently in about 70% of the encephala of SAD patients (J. Neurochem., Vol.72, No.6, 1999, 2498-2505). The mRNA of PS2V codes for a protein consisting of 124 amino acids having 5 amino acids (Ser-Ser-Met-Ala-Gly) added to 119 amino acid residues (Met1 to Leu 119) at the N terminal of PS2. Immunohistologic detection of PS2V using samples of CA1 regions of the hippocampi of the encephala of SAD patients have confirmed 100% manifestation of PS2V (J. Biol Chem,2001 Jan. 19; 276(3):2108-2114). In vitro analysis has shown that {circle over (1)} in human neuroblastoma SK-N—SH cells in which PS2V is forced to expressed, susceptibility to endoplasmic reticulum (ER) stress increases since the induction of stress responsive protein GRP78 is suppressed; that {circle over (2)} PS2V inhibits autophosphorylation of Ire1 protein (ER stress sensor) and inactivates ER stress response, thereby causing the suppression on the expression of GRP78; and that {circle over (3 )} in cells expressing PS2V, the production of both A β1-40 and A β1-42 is increased. From these three points, it is considered that in SAD, the expression of PS2V may possibly trigger neuronal death and increasing of A β production. Accordingly, it is considered that highly sensitive detection of PS2V leads to early diagnosis of AD since the expression of PS2V plays an important role in AD development. As the detection of PS2V, mention may be made of the detection of PS2V itself and the detection of mRNA of PS2V. For early diagnosis, it is impossible to collect a brain tissue of the encephalon of a patient. Therefore, it is necessary to carry out a test using a body fluid of a patient such as cerebrospinal fluid, blood, serum, urine or the like which can be collected relatively easily. However, mRNA is retained in cerebrospinal fluid or serum only for such an extremely short time and is decomposed so quickly that the diagnosis by detecting mRNA is impossible. On the other hand, PS2V is retained in cerebrospinal fluid or serum for a long time as compared with mRNA, and therefore, it is considered suitable for the diagnosis. However, since only a trace amount of PS2V is present in cerebrospinal fluid or serum, a method of detecting PS2V with high sensitivity is required. Japanese Unexamined Patent Application Publication NO. 2000-37192 has already disclosed a method of producing PS2V by inducing the expression of an abnormal splicing varient of the PS2 gene using oxidative stress loading and β-amyloid stimulation in a culture system of neuronal cells. However, highly sensitive detection of PS2V has not been found. Conventionally, alkaline phosphatase (referred to as ALP hereinafter) is often used as a labeling molecule in enzyme immunoassay and nucleic acid detection. Since ALP hydrolyzes a substrate which is a phosphate ester, the quantity of an object biomolecule conjugated with ALP can be determined by determining the quantity of a product obtained by hydrolysis with ALP. Depending upon different determination techniques for the product, the detection of the product is classified into four types, i.e., absorptiometric detection, chemoluminescent detection, fluorescent detection and electrochemical detection. Literature such as Analytica Chemica Acta 393 (1999) 95-102 and others have reported electrochemical detection using p-methoxyphenyl phosphoric acid as a substrate for ALP. However, methods of highly sensitive detection of PS2V using these substrates have not been found. Accordingly, there are desired a method of highly sensitive detection of PS2V which is applicable to early diagnosis of AD and a substrate for a conjugating enzyme usable for the method. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 shows the result of Example 1 of the present invention in which chemoluminescent was determined by sandwich method using rGST-PS2V as a sample; FIG. 2 shows the result of the present invention of determination of PS2V in cerebrospinal fluid of patients of subarachnoid hemorrhage, hydrocephalia and sporadic Alzheimer's disease; FIG. 3 shows the result of Example 1 of the present invention in which absorbance was determined by sandwich method using rGST-PS2V as a sample; FIG. 4 shows the construction of FIA-EC-DT used in electrochemical detection according to the present invention; FIG. 5 shows the construction of an FIA-EC-DT detector according to the present invention; FIG. 6 shows the result of Example 2 of the present invention in which electrochemical detection was carried out by sandwich method using rGST-PS2V as a sample (dependency upon the amount of an antigen, an average value of the detection repeated three times); FIG. 7 shows the result of Example 2 of the present invention in which electrochemical detection was carried out by sandwich method using rGST-PS2V as a sample (values are obtained by deducting a background value obtained with no antigen used); FIG. 8 shows the result of Example 3 of the present invention in which absorbance was determined by competitive assay using rGST-PS2V as a sample; FIG. 9 shows the results of Example 4 of the present invention in which electrochemical detection was carried out by competitive assay using rGST-PS2V as a sample. detailed-description description="Detailed Description" end="lead"? |
Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced succ or sucd gene |
The invention relates to a process for the preparation of L-amino acids, in particular L-threonine, in which the following steps are carried out: a) fermentation of microorganisms of the Enterobacteriaceae family which produce the desired L-amino acid and in which at least one or more of the genes chosen from the group consisting of sucC and sucD, or nucleotide sequences which code for these, is or are enhanced, in particular over-expressed, b) concentration of the desired L-amino acid in the medium or in the cells of the bacteria, and c) isolation of the desired L-amino acid. |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.