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Crystallized hnf4 gamma ligand binding domain polypeptide and screening methods employing same |
A solved three-dimensional crystal structure of an HNF4g ligand binding domain polypeptide is disclosed, along with a crystal form of the HNF4g ligand binding domain. Methods of designing modulators of the biological activity of HNF4g, and other HNF4 ligand binding domain polypeptides are also disclosed. |
1. A substantially pure HNF4γ ligand binding domain polypeptide in crystalline form. 2. The polypeptide of claim 1, wherein the crystalline form has lattice constants of a=152.71 Å, b=152.71 Å, c=93.42 Å, α=90°, β=90°, γ=90°. 3. The polypeptide of claim 1, wherein the crystalline form is a tetragonal crystalline form. 4. The polypeptide of claim 1, wherein the crystalline form has a space group of 14,22. 5. The polypeptide of claim 1, wherein the HNF4γ ligand binding domain polypeptide has the amino acid sequence shown in SEQ ID NO:4. 6. The polypeptide of claim 1, wherein the HNF4γ ligand binding domain polypeptide is in complex with a ligand. 7. The polypeptide of claim 6, wherein the ligand is a fatty acid. 8. The polypeptide of claim 7, wherein the fatty acid is selected from the group consisting of lauristic acid, myristic acid, palmitic acid, stearic acid, mono-unsaturated analogs of palmitic acid, mono-unsaturated analogs of stearic acid. 9. The polypeptide of claim 1, wherein the HNF4γ ligand binding domain has a crystalline structure further characterized by the coordinates corresponding to Table 2. 10. The polypeptide of claim 1, wherein the crystalline form contains one HNF4γ ligand binding domain polypeptide in the asymmetric unit. 11. The polypeptide of claim 1, wherein the crystalline form is such that the three-dimensional structure of the crystallized HNF4γ ligand binding domain polypeptide can be determined to a resolution of about 3 Å or better. 12. The polypeptide of claim 10, wherein the crystalline form contains one or more atoms having an atomic weight of 40 grams/mol or greater. 13. A method for determining the three-dimensional structure of a crystallized HNF4γ ligand binding domain polypeptide to a resolution of about 3 Å or better, the method comprising: (a) crystallizing an HNF4γ ligand binding domain polypeptide; and (b) analyzing the HNF4γ ligand binding domain polypeptide to determine the three-dimensional structure of the crystallized HNF4γ ligand binding domain polypeptide, whereby the three-dimensional structure of a crystallized HNF4γ ligand binding domain polypeptide is determined to a resolution of about 3 Å or better. 14. The method of claim 13, wherein the analyzing is by X-ray diffraction. 15. The method of claim 13, wherein the crystallization is accomplished by the hanging drop vapor diffusion method, and wherein the HNF4γ ligand binding domain is mixed with an equal volume of reservoir. 16. The method of claim 15, wherein the reservoir comprises 0.75 M ammonium phosphate pH=5.0-5.5 and 10 mM DTT. 17. The method of claim 15, wherein the reservoir comprises 0.7-1.0 M sodium or potassium phosphate pH 5.0-6.0. 18. A method of generating a crystallized HNF4γ ligand binding domain polypeptide, the method comprising: (a) incubating a solution comprising an HNF4γ ligand binding domain with an equal volume of reservoir; and (b) crystallizing the HNF4γ ligand binding domain polypeptide using the hanging drop method, whereby a crystallized HNF4γ ligand binding domain polypeptide is generated. 19. A crystallized HNF4γ ligand binding domain polypeptide produced by the method of claim 18. 20. A method of designing a modulator of an HNF4 polypeptide, the method comprising: (a) designing a potential modulator of an HNF4 polypeptide that will form bonds with amino acids in a substrate binding site based upon a crystalline structure of an HNF4γ ligand binding domain polypeptide; (b) synthesizing the modulator; and (c) determining whether the potential modulator modulates the activity of the HNF4 polypeptide, whereby a modulator of an HNF4 polypeptide is designed. 21. A method of designing a modulator that selectively modulates the activity of an HNF4 polypeptide, the method comprising: (a) obtaining a crystalline form of an HNF4γ ligand binding domain polypeptide; (b) evaluating the three-dimensional structure of the crystallized HNF4γ ligand binding domain polypeptide; and (c) synthesizing a potential modulator based on the three-dimensional crystal structure of the crystallized HNF4γ ligand binding domain polypeptide, whereby a modulator that selectively modulates the activity of an HNF4 polypeptide is designed. 22. The method of claim 21, wherein the method further comprises contacting an HNF4γ ligand binding domain polypeptide with the potential modulator; and assaying the HNF4γ ligand binding domain polypeptide for binding of the potential modulator, for a change in activity of the HNF4γ ligand binding domain polypeptide, or both. 23. The method of claim 21, wherein the crystalline form is in tetragonal form. 24. The method of claim 23, wherein the crystalline form is such that the three-dimensional structure of the crystallized HNF4γ ligand binding domain polypeptide can be determined to a resolution of about 3 Å or better. 25. A method for identifying an HNF4 modulator, the method comprising: (a) providing atomic coordinates of an HNF4γ ligand binding domain to a computerized modeling system; and (b) modeling ligands that fit spatially into the binding pocket of the HNF4γ ligand binding domain, whereby an HNF4 modulator is identified. 26. The method of claim 25, wherein the method further comprises identifying in an assay for HNF4-mediated activity a modeled ligand that increases or decreases the activity of the HNF4. 27. A method of identifying an HNF4γ modulator that selectively modulates the activity of an HNF4γ polypeptide compared to other polypeptides, the method comprising: (a) providing atomic coordinates of an HNF4γ ligand binding domain to a computerized modeling system; and (b) modeling a ligand that fits into the binding pocket of an HNF4γ ligand binding domain and that interacts with conformationally constrained residues of an HNF4γ that are conserved among HNF4 isoforms, whereby an HNF4γ modulator is identified. 28. The method of claim 27, wherein the method further comprises identifying in a biological assay for HNF4γ mediated activity a modeled ligand that selectively binds to the HNF4γ ligand binding domain and increases or decreases the activity of the HNF4γ. 29. A method of designing a modulator of an HNF4 polypeptide, the method comprising: (a) selecting a candidate HNF4 ligand; (b) determining which amino acid or amino acids of an HNF4 polypeptide interact with the ligand using a three-dimensional model of a crystallized protein comprising an HNF4γ LBD; (c) identifying in a biological assay for HNF4 activity a degree to which the ligand modulates the activity of the HNF4 polypeptide; (d) selecting a chemical modification of the ligand wherein the interaction between the amino acids of the HNF4 polypeptide and the ligand is predicted to be modulated by the chemical modification; (e) performing the chemical modification on the ligand to form a modified ligand; (f) contacting the modified ligand with the HNF4 polypeptide; (g) identifying in a biological assay for HNF4 activity a degree to which the modified ligand modulates the biological activity of the HNF4 polypeptide; and (h) comparing the biological activity of the HNF4 polypeptide in the presence of modified ligand with the biological activity of the HNF4 polypeptide in the presence of the unmodified ligand, whereby a modulator of an HNF4 polypeptide is designed. 30. The method of claim 29, wherein the HNF4 polypeptide is an HNF4γ polypeptide. 31. The method of claim 29, wherein the three-dimensional model of a crystallized protein is an HNF4γ LBD polypeptide with a bound ligand. 32. The method of claim 31, wherein the ligand is a fatty acid. 33. The method of claim 32, wherein the fatty acid is palmitic acid. 34. The method of claim 29, wherein the method further comprises repeating steps (a) through (f), if the biological activity of the HNF4 polypeptide in the presence of the modified ligand varies from the biological activity of the HNF4 polypeptide in the presence of the unmodified ligand. 35. An assay method for identifying a compound that inhibits binding of a ligand to an HNF4 polypeptide, the assay method comprising: (a) incubating an HNF4 polypeptide with a ligand in the presence of a test inhibitor compound; (b) determining an amount of ligand that is bound to the HNF4 polypeptide, wherein decreased binding of ligand to the HNF4 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 (c) 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 an HNF4 polypeptide is identified. 36. The method of claim 35, wherein the ligand is a fatty acid. 37. The method of claim 36, wherein the fatty acid is selected from the group consisting of lauristic acid, myristic acid, palmitic acid, stearic acid, mono-unsaturated analogs of palmitic acid, mono-unsaturated analogs of stearic acid. |
<SOH> BACKGROUND ART <EOH>Nuclear receptors 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 glucocorticoids, androgens, mineralcorticoids, progestins, estrogens, thyroid hormones, vitamin D retinoids, icosanoids and peroxisomes. Many nuclear receptors, identified by either sequence homology to known receptors (See, Drewes et al., (1996) Mol. Cell. Biol. 16:925-31) or based on their affinity for specific DNA binding sites in gene promoters (See, Sladek et al., Genes Dev. 4:2353-65), have unascertained ligands and are therefore termed “orphan receptors”. Hepatocyte Nuclear Factor 4 (HNF4) is an orphan nuclear receptor and two isoforms, HNF4α and HNF4γ, have currently been identified. HNF4α was originally identified based on its ability to bind promoter regions in the plasma transthyretin (TTR) and apoCIII genes. Sladek et al., Genes Dev. 4:2353-65. HNF4γ was identified based on its known homology to HNF4α. Drewes et al., (1996) Mol. Cell. Biol. 16:925-31. Nuclear receptors activate or repress transcription through partner proteins called co-activators or co-repressors, respectively. CREB-binding protein, or CBP, is a known co-activator for HNF4α (Wang et al., (1998) J. Biol. Chem. 273: 30847-50; Dell & Hadzopoulou-Cladaras, (1999) J. Biol. Chem. 274: 9013-21). Mutations in HNF4α have been linked to the metabolic disorder Mature Onset of Diabetes of the Young (MODY), type 1. Yamagata et al., (1996) Nature 384:458-60. HNF4α +/− subjects experience reduced serum levels of apolipoprotein AII, apolipoprotein CIII and lipoproitein(a), leading to reduced triglycerides. Shih et al., (2000) Diabetes 49:832-37. HNF4α regulation had previously been identified for these apolipoprotien genes (Mietus-Snyder et al., (1992) Mol. Cell. Biol. 12:1708-18; Chan et al., (1993) Nucleic Acid Res. 21:1205-11), as well as regulation of other factors involved in glucose metabolism and insulin secretion. Diaz Guerra et al., (1993) Mol. Cell. Biol. 13:7725-33; Miguerol et al., (1994) J. Biol. Chem. 269:8944-51; Stoffel & Duncan, (1997) Proc. Natl. Acad. Sci. U.S.A. 94:13209-14; Wang et al., (1998) J. Biol. Chem. 273:30847-50. Structurally, the HNF4 family of nuclear receptors, including HNF4α and HNF4γ, are generally characterized by two distinct structural elements. First, nuclear receptors comprise a central DNA binding domain which targets the receptor to specific DNA sequences, which are known as hormone response elements (HREs). The DNA binding domains of these receptors are related in structure and sequence, and are located within the middle of the receptor. Second, the C-terminal region of the HNF4 family of nuclear receptors encompasses the ligand binding domain (LBD). Upon binding a ligand, the receptor shifts to a transcriptionally active state. Almost all nuclear hormone receptors bind DNA, and the physiologically active complex of many is as a heterodimer with the retinoid X receptor (RXR). The HNF4 isoforms are unusual in that they are obligate homodimers and cannot dimerize with any other nuclear receptors. In fact, retinoid X receptor (RXR) heterodimer formation is actually prevented by LBD interactions. Jiang & Sladek, (1997) J. Biol. Chem. 272:1218-25. Polypeptides, including the ligand binding domain of HNF4γ, 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 in soluble or membrane bound forms. 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, N.J., 221-39). This technique relies on the ability of crystalline lattices to diffract X-rays or other forms of radiation. Diffraction 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 HNF4γ, as well as many other proteins of interest. Thus, high-quality diffracting crystals of the ligand binding domain of HNF4γ would greatly assist in the elucidation of HNF4γ's three-dimensional structure, and would provide insight into the ligand binding properties of HNF4γ. Clearly, the solved crystal structure of the HNF4γ ligand binding domain would be useful in the design of modulators of activity mediated by all HNF4 isoforms. Evaluation of the available sequence data has made it clear that HNF4α shares significant sequence homology with HNF4γ. Further, HNF4γ shows structural homology with the three-dimensional fold of other proteins. The solved HNF4γ-ligand crystal structure would provide structural details and insights necessary to design a modulator of HNF4γ that maximizes preferred requirements for any modulator, i.e. potency and specificity. By exploiting the structural details obtained from an HNF4γ-ligand crystal structure, it would be possible to design an HNF4 modulator that, despite HNF4γ's similarity with other proteins, exploits the unique structural features of HNF4γ. An HNF4 modulator developed using structure-assisted design would take advantage of heretofore unknown HNF4 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. An HNF4γ modulator designed using the structural coordinates of a crystalline form of HNF4γ would also provide a starting point for the development of modulators of other HNF4s. What is needed, therefore, is a crystallized form of an HNF4γ LBD polypeptide, preferably in complex with a ligand. Acquisition of crystals of the HNF4γ LBD polypeptide will permit the three dimensional structure of the HNF4γ LBD to be determined. Knowledge of this three dimensional structure will facilitate the design of modulators of HNF4γ activity. Such modulators can lead to therapeutic compounds to treat a wide range of conditions, including lipid homeostasis disorders and glucose homeostasis disorders. |
<SOH> SUMMARY OF THE INVENTION <EOH>A substantially pure HNF4γ ligand binding domain polypeptide in crystalline form is disclosed. Preferably, the crystalline form has lattice constants of a=152.71 Å, b=152.71 Å, c=93.42 Å, α=90°, β=90°, γ=90°. More preferably, the crystalline form is a tetragonal crystalline form. Even more preferably, the crystalline form has a space group of 14 1 22. Still more preferably, the HNF4γ ligand binding domain polypeptide has the amino acid sequence shown in SEQ ID NO:4. In a preferred embodiment, the HNF4γ ligand binding domain polypeptide is in complex with a ligand. More preferably, the ligand is a fatty acid. A method for determining the three-dimensional structure of a crystallized HNF4γ ligand binding domain polypeptide to a resolution of about 3.0 Å or better is also disclosed. The method comprises (a) crystallizing an HNF4γ ligand binding domain polypeptide; and (b) analyzing the HNF4γ ligand binding domain polypeptide to determine the three-dimensional structure of the crystallized HNF4γ ligand binding domain polypeptide, whereby the three-dimensional structure of a crystallized HNF4γ ligand binding domain polypeptide is determined to a resolution of about 3.0 Å or better. A method of designing a modulator of an HNF4 polypeptide is also disclosed. The method comprises (a) designing a potential modulator of an HNF4 polypeptide that will form bonds with amino acids in a substrate binding site based upon a crystalline structure of an HNF4γ ligand binding domain polypeptide; (b) synthesizing the modulator; and (c) determining whether the potential modulator modulates the activity of the HNF4 polypeptide, whereby a modulator of an HNF4 polypeptide is designed. In an alternative embodiment, a method of designing a modulator that selectively modulates the activity of an HNF4 polypeptide in accordance with the present invention comprises: (a) obtaining a crystalline form of an HNF4γ ligand binding domain polypeptide; (b) evaluating the three-dimensional structure of the crystallized HNF4γ ligand binding domain polypeptide; and (c) synthesizing a potential modulator based on the three-dimensional crystal structure of the crystallized HNF4γ ligand binding domain polypeptide, whereby a modulator that selectively modulates the activity of an HNF4 polypeptide is designed. Preferably, the method further comprises contacting an HNF4γ ligand binding domain polypeptide with the potential modulator; and assaying the HNF4γ ligand binding domain polypeptide for binding of the potential modulator, for a change in activity of the HNF4γ ligand binding domain polypeptide, or both. More preferably, the crystalline form is such that the three-dimensional structure of the crystallized HNF4γ ligand binding domain polypeptide can be determined to a resolution of about 3.0 Å or better. In yet another embodiment, a method of designing a modulator of an HNF4 polypeptide in accordance with the present invention comprises: (a) selecting a candidate HNF4 ligand; (b) determining which amino acid or amino acids of an HNF4 polypeptide interact with the ligand using a three-dimensional model of a crystallized protein comprising an HNF4γ LBD; (c) identifying in a biological assay for HNF4 activity a degree to which the ligand modulates the activity of the HNF4 polypeptide; (d) selecting a chemical modification of the ligand wherein the interaction between the amino acids of the HNF4 polypeptide and the ligand is predicted to be modulated by the chemical modification; (e) performing the chemical modification on the ligand to form a modified ligand; (f) contacting the modified ligand with the HNF4 polypeptide; (g) identifying in a biological assay for HNF4 activity a degree to which the modified ligand modulates the biological activity of the HNF4 polypeptide; and (h) comparing the biological activity of the HNF4 polypeptide in the presence of modified ligand with the biological activity of the HNF4 polypeptide in the presence of the unmodified ligand, whereby a modulator of an HNF4 polypeptide is designed. Preferably, the HNF4 polypeptide is an HNF4γ polypeptide. More preferably, the three-dimensional model of a crystallized protein is an HNF4γ LBD polypeptide with a bound ligand. Even more preferably, the method further comprises repeating steps (a) through (f), if the biological activity of the HNF4 polypeptide in the presence of the modified ligand varies from the biological activity of the HNF4 polypeptide in the presence of the unmodified ligand. A method for identifying an HNF4 modulator is also disclosed. The method comprises (a) providing atomic coordinates of an HNF4γ ligand binding domain to a computerized modeling system; and (b) modeling ligands that fit spatially into the binding pocket of the HNF4γ ligand binding domain to thereby identify an HNF4 modulator. Preferably, the method further comprises identifying in an assay for HNF4-mediated activity a modeled ligand that increases or decreases the activity of the HNF4. A method of identifying an HNF4γ modulator that selectively modulates the activity of an HNF4γ polypeptide compared to other polypeptides is disclosed. The method comprises (a) providing atomic coordinates of an HNF4γ ligand binding domain to a computerized modeling system; and (b) modeling a ligand that fits into the binding pocket of an HNF4γ ligand binding domain and that interacts with conformationally constrained residues of an HNF4γ that are conserved among HNF4 isoforms to thereby identify an HNF4γ modulator. Preferably, the method further comprises identifying in a biological assay for HNF4γ-mediated activity a modeled ligand that selectively binds to the HNF4γ ligand binding domain and increases or decreases the activity of the HNF4γ. An assay method for identifying a compound that inhibits binding of a ligand to an HNF4 polypeptide is disclosed. The assay method comprises (a) incubating an HNF4 polypeptide with a ligand in the presence of a test inhibitor compound; (b) determining an amount of ligand that is bound to the HNF4 polypeptide, wherein decreased binding of ligand to the HNF4 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 (c) identifying the test compound as an inhibitor of ligand binding if decreased ligand binding is observed. Preferably, the ligand is a fatty acid. Accordingly, it is an object of the present invention to provide a three dimensional structure of the ligand binding domain of HNF4γ. 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. |
Methods and compositions for modulating the immune system of animals |
Methods and compositions are disclosed for modulating the immune system of animals. Applicant has identified that oral administration of immunoglobulins purified from animal blood can modulate serum IgG, TNF-α or other nonspecific immunity components' levels for treatment of immune dysfunction disorders, potentiation of vaccination protocols, and improvement of overall health and weight gain in animals, including humans. |
1-44. (canceled) 45. A method of regulating the immune response in an animal, comprising orally administering an immunoglobulin composition to an animal so that the oral administration of the immunoglobulin composition causes the serum TNF-α level to be lowered, so that when the animal is subjected to an immunological challenge, the immune response of the animal is increased a greater magnitude than the immune response in a corresponding challenged animal that has not been subjected to oral administration of the immunoglobulin composition. 46. The method of claim 45, wherein the animal is a human. 47. The method of claim 45, wherein the animal is a pig. 48. The method of claim 45, wherein the animal is in the poultry family. 49. The method of claim 48, wherein the animal is a turkey. 50. The method of claim 45, wherein the immunoglobulin composition is derived from an animal source. 51. The method of claim 50, wherein the animal source is a pig, bovine, poultry, equine or goat species. 52. The method of claim 45, wherein the immunoglobulin composition is derived from animal blood and/or fractions thereof. 53. The method of claim 45, wherein the immunoglobulin composition is derived from egg and/or fractions thereof. 54. The method of claim 45, wherein the immunoglobulin composition is derived from milk and/or fractions thereof. 55. The method of claim 45, wherein the source of the immunoglobulin composition is an animal that is a different species than the animal to which the treatment is administered. 56. The method of claim 45, wherein the source of the immunoglobulin composition is a cross-species source. 57. A method of lowering the immune response of an animal during a vaccine protocol, comprising orally administering to said animal an amount of an immunoglobulin composition effective to lower the immune response of said animal when exposed to the vaccine protocol. 58. The method of claim 57, wherein the animal is a human. 59. The method of claim 57, wherein the animal is a pig. 60. The method of claim 57, wherein the animal is in the poultry family. 61. The method of claim 60, wherein the animal is a turkey. 62. The method of claim 57, wherein the immunoglobulin composition is derived from an animal source. 63. The method of claim 62, wherein the animal source is a pig, bovine, ovine, poultry, equine or goat species. 64. The method of claim 57, wherein the immunoglobulin composition is derived from animal blood and/or fractions thereof. 65. The method of claim 57, wherein the immunoglobulin composition is derived from egg and/or fractions thereof. 66. The method of claim 57, wherein the immunoglobulin composition is derived from milk and/or fractions thereof. 67. The method of claim 57, wherein the source of the immunoglobulin composition is an animal that is a different species than the animal to which the treatment is given. 68. The method of claim 57, wherein the source of the immunoglobulin composition is a cross-species source. 69. The method of claim 57, wherein the immunoglobulin composition is administered prior to the administration of the vaccine. 70. The method of claim 57, wherein the immunoglobulin composition is administered simultaneously with the vaccine. 71. The method of claim 57, wherein the immunoglobulin composition is administered immediately following administration of the vaccine. 72. The method of claim 57, wherein the immunoglobulin composition is administered via the animal's water supply. 73. The method of claim 57, wherein the vaccine is a Rotavirus vaccine. 74. The method of claim 57, wherein the vaccine is a PRRS vaccine. 75. A method of increasing the survival rate of a disease challenged animal, comprising orally administering to said animal an amount of an immunoglobulin composition effective to increase the survival rate of said animal, wherein the disease is a respiratory disease. 76. The method of claim 75, wherein the animal is a human. 77. The method of claim 75, wherein the animal is a pig. 78. The method of claim 75, wherein the animal is in the poultry family. 79. The method of claim 78, wherein the animal is a turkey. 80. The method of claim 75, wherein the immunoglobulin composition is derived from an animal source. 81. The method of claim 80, wherein the animal source is a pig, bovine, ovine, poultry, equine or goat species. 82. The method of claim 75, wherein the immunoglobulin composition is derived from animal blood and/or fractions thereof. 83. The method of claim 75, wherein the immunoglobulin composition is derived from egg and/or fractions thereof. 84. The method of claim 75, wherein the immunoglobulin composition is derived from milk and/or fractions thereof. 85. The method of claim 75, wherein the source of the immunoglobulin composition is an animal that is a different species than the animal to which the treatment is given. 86. The method of claim 75, wherein the source of the immunoglobulin composition is a cross-species source. 87. The method of claim 75, wherein the immunoglobulin composition is administered via the animal's water supply. 88. The method of claim 75, wherein the respiratory disease is selected from the group consisting of influenza, chronic respiratory disease, infectious sinusitis, pneumonia, fowl cholera, and infectious synovitis. 89. The method of claim 75, wherein the animal is a starting animal. 90. A method of increasing the survival rate of starting poultry, comprising orally administering to starting poultry an amount of an immunoglobulin composition effective to increase the survival rate of the starting poultry. 91. The method of claim 90, wherein the poultry is turkey. 92. The method of claim 90, wherein the immunoglobulin composition is derived from an animal source. 93. The method of claim 92, wherein the animal source is a pig, bovine, ovine, poultry, equine or goat species. 94. The method of claim 90, wherein the immunoglobulin composition is derived from animal blood and/or fractions thereof. 95. The method of claim 90, wherein the immunoglobulin composition is derived from egg and/or fractions thereof. 96. The method of claim 90, wherein the immunoglobulin composition is derived from milk and/or fractions thereof. 97. The method of claim 90, wherein the source of the immunoglobulin composition is an animal that is a different species than the animal to which the treatment is given. 98. The method of claim 90, wherein the source of the immunoglobulin composition is a cross-species source. 99. The method of claim 90, wherein the immunoglobulin composition is administered via the animal's water supply. 100. The method of claim 90, wherein the poultry are disease challenged starting poultry. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The primary source of nutrients for the body is blood, which is composed of highly functional proteins including immunoglobulin, albumin, fibrinogen and hemoglobin. Immunoglobulins are products of mature B cells (plasma cells) and there are five distinct immunoglobulins referred to as classes: M, D, E, A, and G. IgG is the main immunoglobulin class in blood. Intravenous administration of immunoglobulin products has long been used to attempt to regulate or enhance the immune system. Most evidence regarding the effects of intravenous IgG on the immune system suggests the constant fraction (Fc) portion of the molecule plays a regulatory function. The specific antigen binding properties of an individual IgG molecule are conferred by a three dimensional steric arrangement inherent in the amino acid sequences of the variable regions of two light and two heavy chains of the molecule. The constant region can be separated from the variable region if the intact molecule is cleaved by a proteolytic enzyme such as papain. Such treatment yields two fractions with antibody specificity (Fab fractions) and one relatively constant fraction (Fc). Numerous cells in the body have distinct membrane receptors for the Fc portion of an IgG molecule (Fcr). Although some Fcr receptors bind free IgG, most bind it more efficiently if an antigen is bound to the antibody molecule. Binding an antigen results in a configurational change in the Fc region that facilitates binding to the receptor. A complex interplay of signals provides balance and appropriateness to an immune response generated at any given time in response to an antigen. Antigen specific responses are initiated when specialized antigen presenting cells introduce antigen, forming a complex with the major histocompatibility complex molecules to the receptors of a specific helper inducer T-cells capable of recognizing that complex. IgG appears to be involved in the regulation of both allergic and autoimmune reactions. Intravenous immunoglobulin for immune manipulation has long been proposed but has achieved mixed results in treatment of disease states. A detailed review of the use of intravenous immunoglobulin as drug therapy for manipulating the immune system is described in Vol. 326, No. 2, pages 107-116, New England Journal of Medicine Dwyer, John M., the disclosure of which is hereby incorporated by reference. There is a continuing effort and need in the art for improved compositions and methods for immune modulation of animals. Appropriate immunomodulation is essential to improve response to pathogens, vaccinations, for increasing weight gain and improving feed efficiency, for improved survival upon disease challenge, improved health and for treatment of immune dysfunction disease states. It is an object of the present invention to provide methods and pharmaceutical compositions for treating animals with immune dysfunction disease states. It is yet another object of the invention to provide methods and compositions for immunomodulation of animals including humans for optimizing the response to antigens presented in vaccination protocols. It is yet another object of the invention to provide methods and compositions for immunomodulation of animals including humans for an optimal immune system response when disease challenged. It is yet another object of the invention to increase weight gain, improve overall health and improve feed efficiency of animals by appropriately modulating the immune system of said animals. It is yet another object of the invention to provide a novel pharmaceutical composition comprising purified plasma, components or derivatives thereof, which may be orally administered to create a serum IgG or TNF-α response. These and other objects of the invention will become apparent from the detailed description of the invention which follows. |
<SOH> SUMMARY OF THE INVENTION <EOH>According to the invention, applicants have identified purified and isolated plasma, components, and derivatives thereof, which are useful as a pharmaceutical composition for immune modulation of animals including humans. According to the invention, a plasma composition comprising immunoglobulin, when administered orally, regulates and lowers nonspecific immunity responses and induces a lowering and regulation of serum IgG levels and TNF-α levels relative to animals not orally fed immunoglobulin or plasma fractions. An orally administered plasma composition comprising immunoglobulin affects the animals overall immune status when exposed to an antigen, vaccination protocols, and for treatment of immune dysfunction disease states. Applicants have unexpectedly shown that oral administration of plasma protein can induce a change in serum immunoglobulin amd TNF-α as well as other non-specific immunity responses. This is unexpected as traditionally it was thought that plasma proteins such as immunoglobulins, must be introduced intravenously to affect circulating IgG, TNF-α, or other components of nonspecific immunity. In contrast, applicants have demonstrated that oral globulin is able to impact circulating serum IgG or TNF-α levels. Further this effect may be observed in as little as 14 days. This greatly simplifies the administration of immunomodulating compositions such as immunoglobulin as these compositions, according to the invention, can now be simply added to feedstuff or even water to modulate vaccination, to modulate disease challenge, or to treat animals with immune dysfunction disease states. Also according to the invention, applicants have demonstrated that modulation of serum IgG and TNF-α impacts the immune system response to stimulation as in vaccination protocols or to immune dysfunction disorders. Modulation of serum IgG and TNF-α, according to the invention allows the animals' immune system to more effectively respond to challenge by allowing a more significant up regulation response in the presence of a disease state or antigen presentation. Further this immune regulation impacts rate and efficiency of gain, as the bio-energetic cost associated with heightened immune function requires significant amounts of energy and nutrients which is diverted from such things as cellular growth and weight gain. Modulation of the immune system allows energy and nutrients to be used for other productive functions such as growth or lactation. See, Buttgerut et al., “Bioenergetics of Immune Functions: Fundamental and Therapeutic Aspects”, Immunology Today , April 2000, Vol. 21, No. 4, pp. 192-199. Applicants have further identified that by oral consumption, the Fc region of the globulin composition is essential for communication and/or subsequent modulation of systemic serum IgG. This is unique, as this is the non-specific immune portion of the molecule which after oral consumption modulates systemic serum IgG without intravenous administration as previously noted (Dwyer, 1992). The antibody specific fractions produced less of a response without the Fc tertiary structure. Additionally, the globulin portion with intact confirmation gave a better reaction than the heavy and light chains when separated therefrom. |
Delivery of biologically active agents |
A method of delivering a biologically active agent to the cervix, the method comprising using a needleless injector. Typically, the biologically active agent is a cervical ripening agent. The needleless injector may be a powder injector or a liquid injector. |
1. A method of delivering a biologically active agent to the cervix, the method comprising using a needleless injector. 2. A method according to claim 1 wherein the needleless injector is a liquid injector. 3. A method according to claim 1 wherein the needless injector is a powder injector. 4. A method according to claim 1 wherein the biologically active agent is a cervical ripening agent. 5. A method according to claim 4 wherein the cervical ripening agent is any one or more of a prostaglandin, MCP-1 and IL-8. 6. A method according to claim 1 wherein the biologically active agent is a vasodilator or precursor thereof, a chemokine or a cytokine which stimulates monocyte or granulocyte entry into cervical tissue. 7. A method of ripening the female cervix, the method comprising administering a cervical ripening agent to the cervix using a needleless injector. 8. A system for delivering a biologically active agent to the cervix comprising an agent which is biologically active on the cervix and a needleless injector. 9. A system according to claim 8 wherein the biologically active agent is a cervical ripening agent. 10. A system according to claim 8 wherein the needleless injector is a liquid injector. 11. A system according to claim 8 wherein the needleless injector is a powder injector. 12. A needleless injector loaded for injection with an agent which is biologically active on the cervix. 13. A needleless injector according to claim 12 wherein the biologically active agent is a cervical ripening agent. 14. A needleless injector according to claim 12 which is a liquid injector. 15. A needleless injector according to claim 12 which is a powder injector. 16. A vial for insertion into, and containing an agent for delivery by, a needleless injector wherein the agent is an agent which is biologically active on the cervix. 17. A vial according to claim 16 wherein the agent is a cervical ripening agent. 18. A method of preparing a needleless injector for use in delivering a biologically active agent to the cervix, the method comprising loading the injector with the biologically active agent. 19. A method according to claim 18 wherein the agent is loaded in a vial disposed for insertion into the needleless injector. 20. A pharmaceutical formulation comprising an agent for delivery to the cervix and a carrier suitable for use in a needleless injector. 21. A pharmaceutical formulation according to claim 20 wherein the formulation contains particles of a density between about 0.1 and about 25 g/cm3 and of a size between 0.1 and 250 μm which particles comprise the said agent. 22. A pharmaceutical formulation according to claim 20 comprising an agent which permeabilises a mucosal surface, such as dimethylsulphoxide. 23. Use of a cervical ripening agent in the manufacture of a medicament for treating a female in need of a cervical ripening agent wherein the cervical ripening agent is for delivery using a needleless injector. 24-26. (canceled). |
System and method for fabricating bragg gratings |
A novel method and apparatus for fabrication of blazed and slanted fiber Bragg gratings is disclosed. The method comprises the step of simultaneously exposing the fiber with two mutually coherent light beams so as to create an interference pattern along a longitudinal axis of the fiber, wherein each one of said beams is brought into a line focus, which coincides with the core of the fiber. Further, the plane comprising the beams is rotated to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements. |
1. A method for photo-inducing a blazed grating in an optical fiber, comprising: simultaneously exposing the fiber with two mutually coherent light beams which intersects with a first angle in a plane comprising the beams and which interfere in a predetermined region of the fiber so as to create an interference pattern along a longitudinal axis of the fiber, wherein each one of said beams is brought into a line focus, which coincides with the core of the fiber, and wherein said plane comprising the beams, at least in the vicinity of the fiber, is rotated to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements. 2. The method according to claim 1, wherein the beams are focused on the fiber by at least one cylindrical lens and the rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said lens. 3. The method according to claim 2, wherein the beam incidence position is displaced in a direction essentially perpendicular to the fiber direction. 4. The method according to claim 2, wherein the incidence positions of both beams are displaced. 5. The method according to claim 4, wherein the incidence positions are displaced essentially symmetrically. 6. The method according to claim 1, wherein the beams are focused on the fiber by at least one curved mirror and rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said mirror. 7. The method according to claim 6, wherein the beam incidence position is displaced in a direction essentially perpendicular to the fiber direction. 8. The method according to claim 6, wherein the incidence positions of both beams are displaced. 9. The method according to claim 8, wherein the incidence positions are displaced essentially symmetrically. 10. The method according to claim 1, wherein the fiber is translated through the exposure area where the beams intersect. 11. An apparatus for photo-inducing a blazed grating in an optical fiber, comprising: a source for emitting light; a beam splitter for forming two mutually coherent light beams; a fiber holder for holding the fiber during exposure; and a projection system for making the beams intersect with a first angle in the exposure area and thereby to interfere in a predetermined region of the fiber so as to create an interference pattern along the longitudinal axis of the fiber, wherein the projection system further comprises means for focusing the beams so that each one of said beams is brought into a line focus, which coincides with the core of the fiber, said means for focusing the beams further comprising means for rotating the plane comprising the two light beams relative to the fiber, at least in the vicinity of the fiber, to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements. 12. The apparatus according to claim 11, wherein the means for focusing the beams comprises at least one lens for focusing the beams on the fiber, the means for rotating the beam plane comprising means for displacing the beam incidence position on said lens for at least one of the beams. 13. The apparatus according to claim 12, wherein the means for displacing the beam incidence position comprises at least one reflecting mirror, which is at least one of displaceable and rotatable, arranged in the beam path for said beam. 14. The apparatus according to claim 12, wherein the means for displacing the beam incidence position on said lens is adapted to displace the incidence positions of both beams. 15. The apparatus according to claim 12, wherein the means for displacing the beam incidence comprises means for parallel displacement of the beams. 16. The apparatus according to claim 11, wherein the means for focusing the beams comprises at least one curved mirror, and means for rotating the beam plane comprising means for displacing the beam incidence position on said mirror for at least one of the beams. 17. The apparatus according to claim 16, wherein the means for displacing the beam incidence position is adapted to displace the incidence position in a direction essentially perpendicular to the fiber direction. 18. The apparatus according to claim 16, wherein the means for displacing the incidence positions is adapted to displace the incidence position of both beams. 19. The apparatus according to claim 18, wherein the means for displacing the incidence positions displaces the incidence positions of the beams essentially symmetrically. 20. The apparatus according to claim 11, further comprising: means for moving the fiber essentially in the direction of a longitudinal axis of the fiber through the exposing area where the beams intersect. 21. The method according to claim 3, wherein the incidence positions of both beams are displaced. 22. The method according to claim 7, wherein the incidence positions of both beams are displaced. 23. The apparatus according to claim 13, wherein the means for displacing the beam incidence position on the lens is adapted to displace the incidence positions of both beams. 24. The apparatus according to claim 13, wherein the means for displacing the beam incidence includes means for parallel displacement of the beams. 25. The apparatus according to claim 14, wherein the means for displacing the beam incidence includes means for parallel displacement of the beams. 26. The apparatus according to claim 17, wherein the means for displacing the incidence positions is adapted to displace the incidence position of both beams. |
<SOH> BACKGROUND OF THE INVENTION <EOH>There is a rapidly growing demand for high-quality optical Bragg gratings with arbitrary phase and index profiles, as these gratings are key elements in many components that are used in WDM networks. Over the past few years, several methods that improve the quality and the flexibility in the grating fabrication process have been developed. A straightforward approach is to scan a UV beam over a long phase mask in a fixed relative position to the fiber. Non-uniform profiles can in this case be fabricated either by post processing the illuminated region or by using a phase mask that contains the appropriate structure. Complex grating structures can also be synthesized by moving the fiber slightly relative to the phase mask during the scan. In 1995, a novel versatile sequential technique for venting long and complex fiber gratings was demonstrated by R. Stubbe, B. Sahlgren, S. Sandgren and A. Asseh, in “Novel technique for writing long superstructured fiber Bragg gratings”, in Photosensitivity and Quadratic Nonlinearity in Glass Waveguides (Fundamentals and Applications), Portland, PD1 (1995) and by A. Asseh, H. Stormy, B. E. Sahlgren, S. Sandgren and R. A. H. Stubbe, in “A writing technique for long fiber Bragg gratings with complex reflectivity profiles”, J. Lightw. Techn. 15, 1419-1423 (1997). The idea was to expose a large number of small partially overlapping subgratings—each containing a few hundred periods or less—in sequence; where advanced properties such as chirp, phase shifts and apodization were introduced by adjusting the phase offset and pitch of the subgratings. In the setup that was used in the above-mentioned references, each subgrating was created by exposing the fiber with a short UV pulse while the fiber itself was translated at a constant speed. The UV pulses were triggered by the position of the fiber relative the UV beams, which was measured by a standard helium-neon laser interferometer. Bragg gratings normally have their grating elements aligned normal to the waveguide axis. However, there is an increasing interest in producing gratings which have their elements at an angle to the waveguide axis, known as blazed Bragg gratings. Such blazed Bragg gratings are difficult to fabricate efficiently and with appropriate precision with previously know methods and apparatuses. For example, U.S. Pat. No. 5,730,888 and U.S. Pat. No. 5,042,897 both relates to methods and apparatuses to photo-induce blazed gratings in optical fibers. The blazed gratings are formed by tilting the projection system relative to the fiber. However, there are several problems with these known methods. To be able to tilt the projection system relative to the fiber the equipment becomes complex and costly. Further, it is difficult to achieve an adequate focus on the fiber in the whole exposure area. Hereby, the known methods becomes slow and inefficient, with a low through-put. Still further, with the known methods it is only possible to produce blazed gratings with a limited blazing angle, whereas blazed gratings with larger angles of inclination is very complicated to produce, or even not possible to produce at all. |
<SOH> SUMMARY OF THE INVENTION <EOH>It is therefore an object of the present invention to provide a method and an apparatus for photo-inducing a blazed grating in an optical fiber, which alleviates the above-mentioned problems of the prior art. This object is achieved with an apparatus and a method as defined in the appended claims. According to the invention there is provided a method for photo-inducing a blazed grating in an optical fiber comprising the step of simultaneously exposing the fiber with two mutually coherent light beams, which intersects with an first angle in a plane comprising the beams and interfere in a predetermined region of the fiber so as to create an interference pattern along a longitudinal axis of the fiber. Further, each one of said beams is brought into a line focus, which coincides with the core of the fiber. The invention presents a novel method for fabrication of advanced blazed fiber Bragg gratings. As opposed to prior art methods, the method according to the invention does not tilt the projection system relative to the fiber, but provides a blazed interference pattern in line focus with the fiber. Especially, the method could be used when said plane comprising the beams, at least in the vicinity of the fiber, is rotated to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements. According to the inventive method, a blazing angle could be chosen arbitrarily, without any equipment restrictions. Further, the quality and precision of the photo-induced pattern is improved, since the line focus coincides with the fiber even at large blazing angles. Still further, the control of the fabrication process becomes simplified, rendering the fabrication more efficient and less costly, and with a shortening of fabrication times for complex gratings. Light is in the context of the application not limited to mean visible light, but a wide range of wavelengths from infrared (IR) to extreme UV. Further, with optical fiber is in the meaning of this application to be understood any kind of optical waveguide made of a material which has a refractive index that can be permanently changed by exposure to light of at least one predetermined wavelength. By photo-induction is to be understood the process of exposing the optical fiber of the above-mentioned type with light of the predetermined wavelength so as to form a permanent refractive index variation in the fiber. According to one embodiment of the invention, the beams are focused on the fiber by means of at least one lens and the rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said lens. The beam incidence position could preferably be displaced in a direction essentially perpendicular to the fiber direction. Hereby, the beams could be displaced on the lens, and still be focused on the fiber. Hence, the angle of incidence relative to the fiber could be varied without affecting the focus. In an alternative embodiment, the beams are focused on the fiber by means of at least one curved mirror and rotation of the beam plane is achieved by displacement of at least one of the beam incidence positions on said mirror. The beam incidence position is preferably displaced in a direction essentially perpendicular to the fiber direction. Hereby, the beams could be displaced on the mirror, and still be focused on the fiber. Hence, the angle of incidence relative to the fiber could be varied without affecting the focus even in this embodiment. The invention also relates to an apparatus for photo-inducing a blazed grating in an optical fiber comprising a source for emitting light; a beam splitter for forming two mutually coherent light beams; a fiber holder for holding the fiber during exposure; and a projection system for making the beams intersect with a first angle in the exposure area and thereby to interfere in a predetermined region of the fiber so as to create an interference pattern along the longitudinal axis of the fiber. The projection system further comprises means for focusing the beams so that each one of said beams is brought into a line focus, which coincides with the core of the fiber. With this apparatus, the method discussed above could be executed. Accordingly, a novel apparatus is presented for fabrication of advanced blazed fiber Bragg gratings. As opposed to prior art equipment, the apparatus according to the invention does not tilt the projection system relative to the fiber, but provides a blazed interference pattern in line focus with the fiber. Especially, the means for focussing the beams could further comprise means for rotating the plane comprising the two light beams relative to the fiber, at least in the vicinity of the fiber, to provide a second angle relative to the fiber direction, said rotation giving rise to a blazing angle of the photo-induced grating elements. According to the invention, a blazing angle could be chosen arbitrarily, without any equipment restrictions. Further, the quality and precision of the photo-induced pattern is improved, since the line focus coincides with the fiber even at large blazing angles. Still further, the control of the fabrication process becomes simplified, rendering the fabrication more efficient and less costly, and with a shortening of fabrication times for complex gratings. In one embodiment, the means for focussing the beams comprises at least one lens for focusing the beams on the fiber, the means for rotating the beam plane comprising means for displacing the beam incidence position on said lens for at least one of the beams. The beam incidence position is preferably displaced in a direction essentially perpendicular to the fiber direction. Hereby, the beams could be displaced on the lens, and still be focused on the fiber. Hence, the angle of incidence relative to the fiber could be varied without affecting the focus. The means for displacing the beam incidence position could comprise at least one displaceable or rotatable reflecting mirror arranged in the beam path for said beam, and preferably allowing parallax displacement of the beams. In an alternative embodiment, the means for focusing the beams comprises at least one curved mirror, and means for rotating the beam plane comprising means for displacing the beam incidence position on said mirror for at least one of the beams. The beam incidence position is preferably displaced in a direction essentially perpendicular to the fiber direction. Hereby, the beams could be displaced on the mirror, and still be focused on the fiber. Hence, the angle of incidence relative to the fiber could be varied without affecting the focus even in this embodiment. |
Non-isothermal method for fabricating hollow composite parts |
A process for making hollow composite structures or vessels which includes the steps of: A) heating a mixture of thermoplastic matrix and reinforcing fibres wrapped over a rigid or semi-rigid thermoplastic liner or bladder above the melting point of the thermoplastic composite matrix outside of a moulding tool; B) transferring the heated assembly to a mould that is maintained below the melting temperature of the thermoplastic matrix of the composite; C) closure of the mould and application of internal fluid pressure to the liner or bladder to apply pressure to the thermoplastic matrix and reinforcing fibres; D) optionally the use of a special coupling system for rapid connection of the internal pressure; E) cooling of the liner or bladder and thermoplastic matrix and reinforcing fibre assembly in contact with the cold or warm mould while consolidation of the assembly occurs; F) opening of the mould and removal of the finished assembly. Suitable thermoplastic materials for the liner/bladder and thermoplastic composite matrix material include: polypropylene, polyamide, polyethylene, cross-linked polyethylene, polybutylene terephthalate, polyethylene terephthalate, polyoxymethylene, polyphenylene sulfide and polyetheretherketone. |
1. A non-isothermal process for fabricating a hollow composite structure, comprising the steps of: preforming at least one thermoplastic liner; over-wrapping the at least one thermoplastic liner with thermoplastic composite material to form at least one over-wrapped liner; heating the at least one over-wrapped liner in an oven to a first temperature above a melting temperature (Tm) of the thermoplastic composite material to form a heated over-wrapped liner; placing the heated over-wrapped liner into a moulding tool that is maintained below the melting temperature (Tm) of the thermoplastic composite material; applying an internal pressure to an interior of the heated over-wrapped liner while the moulding tool is closed to consolidate the composite material and a bladder against a geometry defined by the mould; maintaining the internal pressure to the interior of over-wrapped liner until a second temperature of the thermoplastic composite material is below the thermoplastic composite melting temperature (Tm); and removing the over-wrapped liner from the moulding tool. 2. The process according to claim 1, wherein a plurality of over-wrapping phase inserts are positioned on the thermoplastic liner after the step of preforming the thermoplastic liner, and the over-wrapping phase inserts are consolidated with the over-wrapped thermoplastic comnposite material before the step of heating the over-wrapped thermoplastic liner. 3. The process according to claim 2, wherein the over-wrapping phase inserts have an operating temperature such that critical dimensions of the over-wrapping phase inserts are not distorted by the first temperature during the step of heating. 4. The process according to claim 2, wherein the thermoplastic composite material is locally consolidated onto the over-wrapping phase inserts. 5. The process according to claim 1, wherein a plurality of the thermoplastic liners are included during the step of overwrapping the at least one thermoplastic liner to form a continuous process. 6. The process according to claim 1, wherein the step of applying an internal pressure includes a rapid coupling system. 7. The process according to claim 6, wherein the rapid coupling system includes a rapid gas connection system having an integral pneumatic cylinder comprising a conical member having an axial hole that is pushed into the heated over-wrapped liner against a block having an internally profiled axial hole to create a seal against the heated over-wrapped liner. 8. The process according to claim 1, wherein at least one of the thermoplastic liner and the thermoplastic composite material is selected from a group comprising polypropylene, polyamide, particularly polyamide 12, polyethylene, cross-linked polyethylene, polybutylene terephthalate, polyethylene terephthalate, polyoxymethylene, polyphenylene sulfide, and polyetheretherketone. 9. A The process according to claim 1, wherein the step of preforming includes at least one of extrusion blow moulding and rotary casting. 10. The process according to claim 1, wherein the step of preforming includes high molecular weight polymers to increase a melt strength of the thermoplastic liner. 11. The process according to claim 1, wherein the step of preforming includes discontinuous reinforcing fibres to increase a melt strength of the thermoplastic liner. 12. The process according to claim 1, wherein the step of preforming includes discontinuous reinforcing fibres to perform a load bearing function of the hollow composite structure. 13. The process according to claim 1, wherein the step of over-wrapping includes one of filament winding, braiding, tailored braiding, 3-D braiding, and over-wrapping of one of fabrics and wide tapes. 14. The process according to claim 1, wherein the step of over-wrapping includes a final layer of material to tailor surface properties. 15. The process according to claim 1, further comprising a step of heating the at least one over-wrapped liner to a third temperature above the first temperature before the step of heating the at least one over-wrapped liner to the first temperature. 16. The process according to claim 1, wherein the step of heating the at least one over-wrapped liner includes inducing a temperature gradient such that a third temperature of an outer layer of the thermoplastic composite material is above the melting temperature (Tm) of the thermoplastic composite material and the over-wrapped liner is maintained at a fourth temperature above the melting point of the thermoplastic liner. 17. The process according to claim 1, wherein the step of heating the at least one over-wrapped liner includes an oven system comprising a combined forced hot gas oven and an infra red oven. 18. The process according to claim 1, wherein the internal pressure is about 2 bar to maintain a shape of the at least one over-wrapped liner. 19. The process according to claim 1, wherein the step of heating the at least one over-wrapped liner includes maintaining the liner at a geometry using a frame that enables heat transfer into the moulding tool. 20. The process according to claim 1, wherein during the step of applying an internal pressure the heated over-wrapped liner is located between two mould halves of the moulding tool such that contact of both mould halves occurs substantially simultaneously. 21. The process according to claim 1, wherein during the step of placing the heated over-wrapped liner the over-wrapped liner is heated to a third temperature above the melting temperature (Tm) of the thermoplastic composite material such that partial impregnation of the thermoplastic liner occurs and the bulk of the thermoplastic composite material is reduced. 22. The process according to claim 1, further comprising at least one step of connecting external features to the hollow composite structure. 23. The process according to claim 1, wherein the hollow composite structure includes an integral thermoplastic liner. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Hollow composite structures, or pressure vessels, here referred to as ‘vessels’, such as used to store fluids and solids, particularly under pressure, such as pressurized gas tanks, or more generally in space frame or tubular, load bearing, assemblies have traditionally been fabricated from metals such as steel or aluminium. However, in recent years, the use of composite vessels has become more prevalent. Such vessels are manufactured by a variety of processes, which include filament winding, resin transfer moulding and bladder-assisted moulding. The technology of filament winding is the process of impregnating dry reinforcing fibres, such as fibreglass or carbon strands, with an activated resin prior to application to a mandrel. Preimpregnated materials may also be used. The mixture of reinforcing fibres and matrix is then applied to the mandrel. In the case of thermoset based matrices, a cure cycle follows, while for thermoplastic matrices the polymer is softened (melted) before application to the mandrel where fusion bonding then occurs between layers of the wound material that forms the structure. Either the cure cycle with thermosets or cooling of the material as it is wound with thermoplastics produces a hollow structure or shell that is then removed from the mandrel to give the component. To produce hollow structures or vessels, the technology of resin transfer moulding uses a mandrel and an outer mould, where a mixture of activated matrix resin is injected in the cavity between the mandrel and the outer mould where reinforcing fibres of typically glass, carbon or Kevlar have been placed. Impregnation of the reinforcing fibres then occurs, with cross-linking of thermoset polymers or polymerisation of thermoplastic monomer forming the matrix polymer and the finished shell-like or hollow component. The mandrel could then be removed. The technology of bladder-assisted moulding, for the case of thermoplastic matrices, uses an outer mould and inner bladder to define the shape of the part. To melt the thermoplastic matrix and hence impregnate the reinforcing fibres, of typically glass, carbon or Kevlar, the thermoplastic is heated above its melting temperature and a pressure is then applied to the bladder to consolidate the material. The thermoplastic material must then be cooled, while under pressure, to form the hard shell-like or hollow composite component. Bladder materials comprising rubber-like materials, typically silicon rubber, are used to apply the compaction force, whereby the bladder stretches or elongates under the influence of the applied internal pressure. Alternate bladders consist of in-situ extrusion blow moulded polymeric materials. The thermoplastic matrix is typically heated by heating the outer mould, often metallic, above the matrix melting temperature before the pressure is applied for the required period, after which the mould is cooled sufficiently below the matrix melting temperature for solidification and crystallisation to occur. Alternately, additional or separate heating may be applied by passing hot oil through the bladder or by using embedded electric heaters in the bladder, to heat the composite from the inside, thus eliminating the need to cycle the mould temperature. Conductive reinforcing materials, such as carbon fibre based composites or composites containing a proportion of conductive fibres such as carbon or steel, may be heated in the mould through induction heating or electrical resistance heating. In-situ extrusion blow moulding has been previously claimed to provide heat to aid the melting of the thermoplastic matrix composite, but in practice additional temperature cycling of the mould above and below the matrix melting temperature is also needed. The technology of bladder-assisted moulding, for the case of thermoset based matrices, uses an outer mould and inner bladder to define the shape of the part. A preimpregnated mixture of reinforcing fibres and activated matrix are placed over the bladder and the bladder with the surrounding preimpregnated material is placed into the outer mould. The mould temperature must be high enough to induce cure of the thermosetting matrix and after cure, cooled to allow part release. The bladder may also be heated, either through the passage of a hot fluid, or by embedded electrical heaters, to apply the heat necessary for cure of the impregnated material. The technology also consists of bladder-assisted resin transfer moulding where reinforcement substantially without matrix is placed over the bladder and the bladder coated with reinforcement is then placed into the mould. Activated resin is then applied that impregnates the reinforcing fibres with the application of internal pressure via the bladder, thereby aiding impregnation. Such techniques for fabricating hollow structures or vessels, particularly if a rapid cycle time is required (which is the case for many applications of hollow composite structures or vessels as it reduces the cost of the component) have well known drawbacks which include: for thermoplastic filament winding based processes, the thermoplastic matrix based reinforcement must be heated prior to the application to a mandrel to ensure the thermal conditions necessary for fusion bonding. Hence high processing speeds, notably the application of the thermoplastic matrix based reinforcement to the mandrel, creates a limit on the speed of the process and hence the cycle time. High drag or friction forces induced by the placement of thermoplastic matrix based reinforcement create further quality problems whereby segregation of the thermoplastic matrix and reinforcing fibres may occur, creating an unfavourable microstructure. Additionally, the geometry of the component is limited where there is a need to remove the mandrel after processing. Where the mandrel remains in the component after processing, the mandrel must be compatible with the thermoplastic matrix based reinforcement and be able to sustain the high radial crush forces induced by hot fibre and matrix mixtures applied under high tension forces. The sequential application of composite layers to the mandrel is known to create high internal stresses in the component, affecting dimensional stability and the eventual durability of the component. The filament winding process also places restrictions on geometries that can be produced, where concave curvature requires in-situ bonding of the thermoplastic based composite material to the mandrel or preceding layer of composite; for thermoset based filament winding processes, the thermoset matrix must be cured onto the mandrel requiring a heating cycle either directly as the thermoset matrix based reinforcement is placed, or subsequent heating of the placed material and the mandrel. The process cycle time depends on the winding speed and hence this creates a limit on the speed of the process and thereby the cycle time. High drag or friction forces induced by the placement of thermoset matrix based reinforcement create further quality problems whereby segregation of the thermosetting matrix and reinforcing fibres may occur, creating an unfavourable microstructure. Additionally, the geometry of the component is limited where there is a need to remove the mandrel after processing. Where the mandrel remains in the component after processing, the mandrel must be compatible with the thermoset matrix based reinforcement and be able to sustaining the high radial crush forces induced by the fibre and matrix mixtures applied under high tension forces; for the resin transfer moulding based process, as used with thermoset based reinforced material, a preform must be assembled over the mandrel and resin injected between the mould and the mandrel. The cycle time is limited by the need to inject the resin into this space, where pressures are limited where a permanent hollow mandrel is used such that the pressure of the incoming fluid, here the activated resin, does not distort the geometry of the mandrel, hence limiting processes to lower rate and pressure injection rather than the shorter cycle time, high pressure process of structural reaction injection moulding. Furthermore, the cure reaction of the thermoset matrix either limits the cycle time or, where tailored to occur at a higher speed, induces high stresses in the composite. Unless complex and expensive preforms of reinforcement are prefabricated, parts of a constant wall thickness must be produced unless a local decrease in the reinforcing fibre fraction can be tolerated. Where the mandrel needs to be removed, expensive technologies, such as low melting temperature alloys, must be used where part geometries are not to be impaired. for the resin transfer moulding based process, for the case of thermoplastic based reinforced material, a preform must be assembled over the mandrel and activated monomer injected between the mould and the mandrel. The cycle time is limited by the need to inject the resin into this space, where pressures are limited where a permanent hollow mandrel is used such that the pressure of the incoming fluid, here the activated resin, does not distort the geometry of the mandrel, hence limiting processes to lower rate and pressure injection rather than the shorter cycle time, high pressure process of structural reaction injection moulding. Furthermore, the polymerisation reaction of the monomer either limits the cycle time or, where tailored to occur at a higher speed, places limitations on bladder materials. Thermal cycling above the monomer melting temperature for polymerisation to occur most rapidly and cooling after polymerisation to below the polymer melting temperature are also often required, requiring cycling of the mould temperature and/or mandrel temperatures for certain thermoplastic resin transfer moulding material systems. Unless complex and expensive preforms of reinforcement are prefabricated, parts of a constant wall thickness must be produced unless a local decrease in the reinforcing fibre fraction can be tolerated. Where the mandrel needs to be removed, expensive technologies, such as low melting temperature alloys, must be used where part geometries are not to be impaired. for the bladder assisted moulding process, for the case of thermoset based reinforced material, the need to cure the thermoset matrix material creates problems with chemical attack with rubber-like bladder materials, typically styrene with silicon rubbers, and hence bladder life is reduced. Bladder materials are generally removable due to the difficulty of achieving a bladder that is flexible enough to enable compaction of the impregnated reinforcing fibres prior and during cure while forming a reliable bond between the bladder and cured composite with the lack of high internal stresses in the finished component. Heating of the impregnated reinforcement is also required that is accomplished either through cycling of the mould temperature or heating the bladder material. Cycling the mould temperature is energy inefficient and time consuming, limiting the potential to reduce cycle times, and the fabrication of heated bladder systems is uneconomic for components made in large quantities while also being fragile and of limited life span. Additional internal inspection is also required in the case of removable bladders to ensure that small particles of the bladder material do not remain on the inside wall of the hollow composite or vessel and that the bladder integrity has been maintained such that it is safely usable for further moulding trials. for the bladder assisted moulding process, for the case of thermoplastic based reinforced material, the thermoplastic composite must be heated above the melt temperature of the matrix and cooled below the melt temperature before demoulding. This occurs either by heating the outer mould temperature above the matrix melting temperature and then cooling the mould below the matrix melting temperature, whereby the thermoplastic matrix composite is also heated above and below the thermoplastic composite matrix melting temperature during which time a compaction force is applied by the bladder, or by heating the bladder material such that the thermoplastic matrix is heated sufficiently above the melting temperature of the matrix for adequate impregnation to occur, followed by cooling of the composite structure sufficiently below the matrix melting temperature for stable component release from the mould. The currently available techniques outlined above have inherent limitations that reduce the potential of the process for applications where a minimum cycle time is required. Heating the (often metallic) outer mould is both time consuming and requires large amounts of energy. The use of embedded electrical heaters in a silicon rubber bladder creates a bladder system that is both fragile and difficult to extract from the cooled hollow composite structure or vessel, thereby limiting the geometry of applications. Furthermore, at the higher processing temperatures required for certain thermoplastics, for example PA12 and PET, the life span of the often costly silicon rubber bladders is limited. Previously disclosed inventions have claimed that in-situ extrusion blow moulding can form a bladder material that renders the thermoplastic composite matrix fluid, but where additional heating of the mould is required to ensure full melting. Where the outer layers of the thermoplastic composite are not heated sufficiently above the melting temperature of the thermoplastic matrix, inadequate impregnation occurs, and hence an unfavourable set of mechanical properties results and surface quality is diminished. Thermosetting materials have additional disadvantages compared with thermoplastic based materials, notably for higher rate production, whereby a limited temperature capability results, unsatisfactory finished product aesthetics, lack of extended durability, lack of appropriateness for recycling and manufacturing related issues such as downtime due to clean-up and material handling costs and sub-ambient storage. Further, there are environmental concerns arising from worker exposure to vapour, overspray, emissions, etc., encountered during the fabrication process. Some engineered thermoset resins improve material performance through higher temperature capacity, but unacceptable material costs are associated with them. Thermoplastic based materials have overcome many of the above problems but the processing techniques available are limited in cycle time due to complex heating and cooling cycles, either of the mould or of expensive and fragile bladder materials and bladders based assemblies with a heating ability. Additionally, high energy costs are associated with processes that require a cycling of the mould temperature above and below the matrix melting temperature or complex bladder based heating systems. |
<SOH> SUMMARY OF THE INVENTION <EOH>It is therefore the object of the present invention to provide an improved process for the fabrication of hollow composite parts and vessels. This object is achieved by a process as outlined in claim 1 . This non-isothermal process for fabricating hollow composite structures or vessels comprises the steps of: a) preforming of a thermoplastic liner; b) over-wrapping the thermoplastic liner with thermoplastic composite material; c) heating the over-wrapped liner in an oven above the melting temperature of the thermoplastic composite material; d) placing the heated over-wrapped liner into a mould that is maintained below the melting temperature of the thermoplastic composite material; e) applying an internal pressure inside the heated over-wrapped liner while the moulding tool is closed to consolidate the composite material and bladder against a geometry defined by the mould; f) maintaining the internal pressure inside the cooling over-wrapped liner until the thermoplastic composite material is sufficiently below the thermoplastic composite melting temperature; and g) removing the formed hollow composite structure or vessel from the mould. Such an improved process enjoys advantages including, as opposed to prior art processes of fabricating hollow composite parts and vessels: reduced cycle times due to the method of assembling the fibre, matrix and bladder prior to insertion into the mould, reduced cycle times due to the method of assembling the fibre, matrix and bladder in the mould, a reduction in the number of manufacturing steps, faster changeover times, faster start-ups, potential labour saving due to less material handling, floor space reduction, adaptability to automation, simplification of material storage and handling, a safer environment for employees, lower training costs, recyclable scrap materials, reduced cycle times from the thermal conditions required for moulding, reduced energy usage from the thermal conditions required for moulding, better distributions of fibers and matrix in the composite, the ability to mould in metallic or polymeric based inserts for connection of additional items to the vessel, the ability to have an integral liner of the same or different polymer to the matrix material of the composite, the ability to tailor barrier properties at the inside of the vessel, the ability to tailor the reinforcing fibre content through the wall thickness of the hollow or vessel component, etc. The resulting hollow composite parts or vessels can be used for many applications, as typical diameters of these vessels could be 25 cm or larger. These applications include for example the use as containers for compressed air e.g. for brake systems of cars and trucks, breathing apparatus, water storage, hollow automotive or aerospace space frame structures, and generally everywhere where lightweight is of advantage. According to another preferential mode of the invention, after step a) before the over-wrapping phase inserts are positioned on the liner, and before step c) the over-wrapped composite is consolidated onto the positioned inserts. Even more preferentially, it is possible to choose the inserts to have an operating temperature such that the critical dimensions are not distorted by the temperature of the heating phase during step c). Additionally, the over-wrapped composite can be locally consolidated onto the positioned inserts. According to a further preferential mode of the invention, during step b) one or more liners are placed on the same over-wrapping line to form a continuous process and/or in step e) a rapid coupling device is used for rapid connection of the internal pressure. Additional modes of realization are described in the dependent claims. Material forms that may be considered for this invention combine either or both discontinuous and continuous reinforcing fibres with a thermoplastic matrix material with different reinforcing fibre architectures including both random and different degrees of reinforcing fibre alignment. Suitable arrangements of materials with a suitable mingling quality of reinforcement and matrix that may be used include tows or yarns, tapes, braids, weaves, knits, fabrics and other textile processes suitable for manipulating or combining reinforcing fibre and matrix. Materials may be used in either or both an unconsolidated state, a partially consolidated state or a fully consolidated state where the material becomes flexible for the moulding process when the combination of matrix and reinforcing fibre material is heated using the principle of this invention to a forming temperature. Suitable thermoplastic materials include: polypropylene, polyamide, in particular polyamide 12, polyethylene, cross-linked polyethylene, polybutylene terephthalate, polyethylene terephthalate and polyoxymethylene. Suitable reinforcing fibres include: glass, carbon, Kevlar, ultra high molecular weight polyethylene, ultra high molecular weight polypropylene and steel. Techniques for combining reinforcing fibres and thermoplastic matrix materials, in both continuous and discontinuous fibre lengths, which are applicable for use with the above process include: melt impregnation, film stacking, solvent impregnation, commingling, powder impregnating using both aqueous slurries and electrostatic methods of coating, powder impregnated fibres with an unmelted sheath around the powder impregnated bundle for textile processing, standard fabrics impregnated by powder to form a semifinished product, the utilization of existing textile processing techniques applied to the warp knitting of split polymer films with weft insertion of reinforcement fibres, weft-inserted multi-axial warp knitting of reinforcing fibres and thermoplastic matrix ribbons, co-weaving yarns of the two fibres into a form possessing good drape characteristics, using a polymer fibre woven around a bundle of reinforcement fibres in the plied matrix technique, reinforcing and matrix yarns inter-dispersed on line during fabric formation to produce drapable stitch bonded fabrics, heating of fibres impregnated with monomers that polymerize in-situ to form the composite, wire coating processes adapted to coating reinforcing fibres or bundles of fibres with matrix, pultrusion of fibres and matrix materials together and any other such process that achieves the required combination of fibre and matrix in a form between fully consolidated and unconsolidated forms. Commingled materials, which are a preferred material for this moulding process, can be formed either directly as described in U.S. Pat. No. 5,011,523 and U.S. Pat. No. 5,316,561 where the reinforcing fibers are drawn from the molten state through a die where the reinforcing glass fibers are combined with a drawing head where the thermoplastic matrix is extruded and drawn mechanically onto the glass fibres or indirectly by separating fibre and matrix fibre yarns (U.S. Pat. No. 5,241,731) into open ribbons where the matrix fibres are electrically charged and then drawn into a flat open bundle via a ribboning bar to the commingling bar where the Carbon fibre yarn is opened with an air curtain into an open flat ribbon that is then combined with the PEEK ribbon at the commingling bar or in the example shown herein of the material used to illustrate this invention where stretch broken Carbon fibres (80 mm average length) are blended with polyamide 12 (PA12) staple fibres using a textile spinning technique (U.S. Pat. No. 4,825,635) with the addition of a wrapping filament (U.S. Pat. No. 5,910,361). |
Methods and devices for treating and/or processing data |
At the inputs and/or outputs, memories are assigned to a reconfigurable module to achieve decoupling of internal data processing and in particular decoupling of the reconfiguration cycles from the external data streams (to/from peripherals, memories, etc.). |
1-9. (canceled) 10. A method for decoupling data streams in a reconfigurable module, comprising: decoupling module-internal data transfers from external data transfers via at least one FIFO memory. 11. A method for decoupling data streams in a reconfigurable module, comprising: providing multiple independent address generators. 12. The method as recited in claim 11, further comprising: configuring the address generators independently of data processing. 13. A method for programming a reconfigurable module, comprising: breaking down, by a compiler, address computations into multiple configurations. 14. A device for data processing, comprising: a field of reconfigurable modules; and an interface module configured for an exchange of data to be processed between the field of reconfigurable modules and an external circuit configuration, wherein the interface module includes at least one FIFO memory in which data may be stored and from which data may be retrieved. 15. The device as recited in claim 14, further comprising: a circuit configuration to determine data which will be needed in a future configuration and to preloading the determined data into the interface configuration. 16. The device according to claim 15, wherein the circuit configuration includes a memory area addressable rapidly via the interface module. 17. The device as recited in claim 16 wherein the rapidly addressable memory area includes at least one of a cache memory, an external RAM, a FIFO memory, and a register. 18. The device as recited in claim 14, further comprising: means for backing up data of a configuration which has previously been executed and is at least partially no longer active at the time of data retrieval, the configuration data being stored in an interface memory, the configuration data already canceled by reconfiguration of participating field elements. 19. The device as recited in claim 14, further comprising: means for backing up data of a configuration which has previously been executed and is at least partially no longer active at the time of data retrieval, for at least one of preventing deletion, and overwriting of data already sent once but not yet known to have been satisfactorily received. 20. The device as recited in claim 14, further comprising: at least one of multiple field elements and field elements grouped into configurations of addressable memories to store data at least one of non-locally and temporarily. 21. The device as recited in claim 20, wherein the addressable memory are RAM cells. |
<SOH> BACKGROUND INFORMATION <EOH>The present invention relates to reconfigurable components in general, and in particular but not exclusively the decoupling of data processing within the reconfigurable component and/or within parts of the reconfigurable component and data streams, specifically both within the reconfigurable component and also to and from peripherals, mass memories, host processors, and the like (see, e.g., German Patent Application Nos. DE 101 10 530.4 and DE 102 02 044.2). Memories are assigned to a reconfigurable module (VPU) at the inputs and/or outputs to achieve decoupling of internal data processing, the reconfiguration cycles in particular, from the external data streams (to/from peripherals, memories, etc.). Reconfigurable architecture includes modules (VPUs) having a configurable function and/or interconnection, in particular integrated modules having a plurality of unidimensionally or multidimensionally positioned arithmetic and/or logic and/or analog and/or storage and/or internally/externally interconnecting modules, which are interconnected directly or via a bus system. These generic modules include in particular systolic arrays, neural networks, multiprocessor systems, processors having a plurality of arithmetic units and/or logic cells and/or communication/peripheral cells (IO), interconnecting and networking modules such as crossbar switches, as well as conventional modules including FPGA, DPGA, Chameleon, XPUTER, etc. Reference is also made in particular in this context to the following patents and patent applications of the same applicant: P 44 16 881.0-53, DE 197 81 412.3, DE 197 81 483.2, DE 196 54 846.2-53, DE 196 54 593.5-53, DE 197 04 044.6-53, DE 198 80 129.7, DE 198 61 088.2-53, DE 199 80 312.9, PCT/DE 00/01869, DE 100 36 627.9-33, DE 100 28 397.7, DE 101 10 530.4, DE 101 11 014.6, PCT/EP 00/10516, EP 01 102 674.7, DE 196 51 075.9, DE 196 54 846.2, DE 196 54 593.5, DE 197 04 728.9, DE 198 07 872.2, DE 101 39 170.6, DE 199 26 538.0, DE 101 42 904.5, DE 101 10 530.4, DE 102 02 044.2, DE 102 06 857.7, DE 101 35 210.7, EP 02 001 331.4, EP 01 129 923.7 as well as the particular parallel patent applications thereto. The entire disclosure of these documents are incorporated herein by reference. The above-mentioned architecture is used as an example to illustrate the present invention and is referred to hereinafter as VPU. The architecture includes an arbitrary number of arithmetic, logic (including memory) and/or memory cells and/or networking cells and/or communication/peripheral (IO) cells (PAEs—Processing Array Elements), which may be positioned to form a unidimensional or multidimensional matrix (PA); the matrix may have different cells of any desired configuration. Bus systems are also understood here as cells. A configuration unit (CT) which affects the interconnection and function of the PA is assigned to the entire matrix or parts thereof. Memory access methods for reconfigurable modules which operate according to a DMA principle are described in German Patent No. P 44 16 881.0, where one or more DMAs are formed by configuration. In German Patent Application No. 196 54 595.1, DMAs are fixedly implemented in the interface modules and may be triggered by the PA or the CT. German Patent Application No. DE 196 54 846.2 describes how internal memories are written by external data streams and data is read out of the memory back into external units. German Patent Application No. DE 199 26 538.0 describes expanded memory concepts according to DE 196 54 846.2 for achieving more efficient and easier-to-program data transmission. U.S. Pat. No. 6,347,346 describes a memory system which corresponds in all essential points to German Patent Application No. DE 196 54 846.2, having an explicit bus (global system port) to a global memory. U.S. Pat. No. 6,341,318 describes a method for decoupling external data streams from internal data processing by using a double-buffer method, in which one buffer records/reads out the external data while another buffer records/reads out the internal data; as soon as the buffers are full/empty, depending on their function, the buffers are switched, i.e., the buffer formerly responsible for the internal data now sends its data to the periphery (or reads new data from the periphery) and the buffer formerly responsible for the external data now sends its data to the PA (reads new data from the PA). These double buffers are used in the application to buffer a cohesive data area. Such double-buffer configurations have enormous disadvantages in the data-stream area in particular, i.e., in data streaming, in which large volumes of data streaming successively into a processor field or the like must always be processed in the same way. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 shows an example reconfigurable processor. FIG. 2 a shows a direct FIFO to PA coupling. FIG. 2 b shows IO connected via RAM-PAEs. FIG. 2 c shows FIFOs connected upstream from the IOs. FIGS. 3 a - 3 e show an example data processing method in a VPU. FIGS. 4 a - 4 e show another example data processing method in a VPU. FIG. 5 shows an example embodiment of a PAE. FIG. 6 shows an example of a wiring connection of ALU-PAEs and RAM-PAEs via a bus system. FIG. 7 a shows a circuit for writing data. FIG. 7 b shows a circuit for reading data. FIG. 8 shows an example connection between interface modules and/or PAEs to numerous and/or other data streams. FIG. 9 shows an example sequence of a data read transfer via the circuit of FIG. 8 . detailed-description description="Detailed Description" end="lead"? |
Integration of e-mail with instant messaging services |
A system for instant communication in real time is described wherein it is determined if a user with a given e-mail address is available for on-line messaging. The system takes in a group of e-mail addresses and automatically looks for screen names that are available for instant messaging. Once a screen name is available for online communication, an ion appears nest to the screen name. Instant messaging is achieved by pressing the icon. The screen names, the icons and the e-mail addresses all appear in the same window, thereby, integrating traditional e-mail addresses with instant messaging services making real time communication an effective tool. |
1. A computer implementation method for instant communication, comprising the steps of: receiving in a group of e-mail addresses; making a request for a list of screen names from an instant messaging service associated with said e-mail addresses; automatically verifying screen names that are available for real-time communication; correlating said screen names to said e-mail addresses; and establishing said real-time communication by selecting an icon associated with said screen name. 2. The method of claim 1, further comprising the step of manually verifying screen names associated with said e-mail addresses. 3. The method of claim 2, further comprising the steps of manually verifying screen names associated with said e-mail addresses that are available for real-time communication. 4. The method of claim 3, further comprising the step of making a list of said screen names associated with a particular e-mail address. 5. A computer implemented method for updating a recipient of available screen names for instant communication, comprising the steps of: receiving in a group of e-mail addresses; making a request for a list of screen names from an instant messaging service associated with said e-mail addresses; automatically verifying screen names that are available for real-time communication; correlating said screen names to said e-mail addresses; making a list of e-mail addresses with their associated screen names; updating said list periodically for establishing said real-time communication; and establishing said communication instantly by pressing an icon associated with a user's screen name. 6. The method of claim 5, further comprising the step of manually verifying screen names associated with said e-mail addresses for communication at a later time. 7. A method to start instant communication, comprising the steps of: receiving in a group of e-mail addresses; manually verifying screen names associated with said e-mail addresses that are available for real-time communication; sending an invitation for instant communication; obtaining screen names that are available for real-time communication; correlating said screen names to said e-mail addresses; and establishing said real-time communication by selecting an icon with said screen name. 8. The method of claim 7, further comprising the step of sending an invitation for instant communication via electronic mail. 9. An apparatus for enabling instant communication between users of a computer, comprising: a means for displaying a graphic window; a means for displaying e-mail addresses between said users of said computer for the purpose of online communication; a means for displaying screen names associated with said e-mail addresses for the purpose of said instant communication; and said graphic window including said e-mail addresses and said screen names in the same window for the purpose of enabling said instant communication between said users of said computer instantly. 10. An apparatus for enabling instant communication as in claim 9, further comprising a graphic icon situated next to said screen name of said graphic window for the purpose of indicating an availability of said screen name for instant messaging. 11. An apparatus for enabling instant communication as in claim 10, wherein said instant communication between users of said computer is enabled by activating said icon. |
<SOH> TECHNICAL FIELD <EOH>The invention relates generally to software solving communication problems. More particularly, the invention relates to an integration of traditional e-mail with instant messaging systems in real time. |
<SOH> SUMMARY OF THE INVENTION <EOH>This invention provides a mechanism for the automatic integration of traditional e-mail with instant messaging services such that instant communication is provided in real-time. In one embodiment of the invention, the system has a single generic graphics window with both e-mail addresses and on-line chat addresses next to it so that anyone with access to the e-mail address could start the AOL's free instant messaging service instantly. The active screen names that are available for instant messaging would have an icon next to the screen name, such that instant messaging is achieved by pressing the icon as it appears next to the screen name. In the particular embodiment of the invention, the system takes in a group of e-mail addresses and makes a request for a list of screen names from the instant messaging service associated with that e-mail address. The system then automatically verifies each screen name that is available for online communication. Once a particular screen name is available for instant communication, the system correlates that screen name to that particular e-mail address. An icon appears next to the screen name that is available for online instant messaging. Instant messaging is accomplished by pressing the icon as it appears next to the active screen name. In one embodiment, the system sets up a list of screen names for tracking their online presence into a buddy list. In yet another embodiment, the system is associated with a user interface (UI) that prevents others from knowing the person's screen name. In yet another embodiment, the system allows a manual search when the screen name is not currently used but still associated with the same e-mail addresses. Next, the system enquires of the user associated with that particular screen name for online communication. The recipient can choose to communicate with the user by clicking the icon associated with the screen name that is currently available for instant communication. Thus, the system establishes instant messaging in real time. |
Large touch-sensitive area with time-controlled and location-controlled emitter and receiver modules |
The invention enables the realization of an optical and electronic structure of a touch-sensitive area on any flat display surface such as a monitor. The optical structure does not require any additional optical components such as lenses. Laser diodes are likewise not required for the operation of a structure of this type. Large distances and, nevertheless, high sensitivity and high resolution of the touch-sensitive area are accomplished by a novel time-control and location-control of both the emitter diodes as well as the receiver diodes or receiver photo-transistors. If still or moving images are displayed on the surface, the invention can be used as a touch screen and can interactively control the display. A central module simulates a computer mouse and a keyboard. |
1. A touch-sensitive region on a display device for detecting a user input, comprising: emitter diodes for outputting light; receiver diodes/phototransistors for detecting the light, the emitter diodes and receiver diodes/phototransistors being arranged in vertical and horizontal emitter and receiver units set opposite to each other, one of the emitter diodes and one of the receiver diodes/phototransistors being optically set in a direct paired relationship to each other; and a central module; wherein the detection of a user input is realized through continuously repeating time-sequenced and position-sequenced on-and-off switching of individual emitter diodes and receiver diodes/phototransistors; wherein one of the emitter diodes and one of the receiver diodes/phototransistors are controlled in pairs; and wherein for evaluating and increasing the resolution, the signals of the emitter diodes are referenced, adjacent to the emitter diodes, and all electrical signals of the receiver diodes/phototransistors are assembled into the central module, and also the emitter diodes are controlled from the Central module, such that the emitter diodes output a plurality of pulses, whose integrative, quantitative evaluation is a measure of the position of the interruption of a light barrier during an evaluation period and provides information on the position of the interruption relative to the emitter diodes. 2. A touch-sensitive region according to claim 1, characterized in that the emitter diodes are operated during their short on-time with higher power than their nominal continuous power. 3. A touch-sensitive region according to claim 1, characterized in that the wavelength of the applied electromagnetic radiation is in the infrared range of the spectrum and the emitter diodes and receiver diodes/phototransistors are emitting and receiving semiconductor elements. 4. A touch-sensitive region according to claim 1, characterized in that the emitter diodes are operated with a burst signal during their on time, which means that the electrical alternating signal controlling them has a higher frequency f than the alternating frequency of the emitter diodes controlled one after the other; wherein for faster saturation, measures, such as increasing the start-up voltage amplitude/voltage rise time and necessary discharge of charge carriers from the emitter diodes after the saturation pulse, are used. 5. A touch-sensitive region according to claim 1, characterized in that the amplifier circuit of the receiver diodes or phototransistors has a large amplification at the burst frequency f and otherwise enables a high attenuation through an inductive operating resistor followed by an integrator. 6. A touch-sensitive region according to claim 1, characterized in that not only the emitter diodes arranged optically in pairs with a receiver diode or phototransistor are active during the on time of the receiver diode or phototransistor, but adjacent emitter diodes are also active. 7. A touch-sensitive region according to claim 1, characterized in that an optical aperture is arranged in front of the emitter diodes and receiver diodes or phototransistors. 8. A touch-sensitive region according to claim 1, characterized in that the optical paired relationship of emitter diodes and receiver diodes or phototransistors involves oppositely positioned emitter diodes and receiver diodes or phototransistors in opposing emitter and receiver units. 9. A touch-sensitive region according to claim 1, characterized in that the optically paired relationship of the emitter diodes and receiver diodes or phototransistors involves emitter diodes and receiver diodes or phototransistors positioned one next to the other in a mixed emitter and receiver unit and an oppositely positioned mirror surface. 10. A touch-sensitive region according to claim 1, characterized in that the optical relationship of the emitter diodes and receiver diodes or phototransistors involves emitter diodes and receiver diodes or phototransistors positioned one next to the other in a mixed emitter and receiver unit and an oppositely positioned absorbing surface. 11. A touch-sensitive region according to claim 1, characterized in that the total number of emitter diodes in the emitter units is exactly equal to or not equal to the number of receiver diodes or phototransistors in the receiver units. 12. A touch-sensitive region according to claim 1, characterized in that a microprocessor of the central module calculates the position of the user input from digital receiver module signals by means of a statistical analysis method. 13. A touch-sensitive region according to claim 1, characterized in that the central module can simulate any arbitrary interface relative to an attached computer. 14. A touch-sensitive region according to claim 2, characterized in that the wavelength of the applied electromagnetic radiation is in the infrared range of the spectrum and the emitter diodes and receiver diodes/phototransistors are emitting and receiving semiconductor elements. 15. A touch-sensitive region according to claim 2, characterized in that the emitter diodes are operated with a burst signal during their on time, which means that the electrical alternating signal controlling them has a higher frequency f than the alternating frequency of the emitter diodes controlled one after the other; wherein for faster saturation, measures, such as increasing the start-up voltage amplitude/voltage rise time and necessary discharge of charge carriers from the emitter diodes after the saturation pulse, are used. 16. A touch-sensitive region according to claim 3, characterized in that the emitter diodes are operated with a burst signal during their on time, which means that the electrical alternating signal controlling them has a higher frequency f than the alternating frequency of the emitter diodes controlled one after the other; wherein for faster saturation, measures, such as increasing the start-up voltage amplitude/voltage rise time and necessary discharge of charge carriers from the emitter diodes after the saturation pulse, are used. 17. A touch-sensitive region according to claim 2, characterized in that the amplifier circuit of the receiver diodes or phototransistors has a large amplification at the burst frequency f and otherwise enables a high attenuation through an inductive operating resistor followed by an integrator. 18. A touch-sensitive region according to claim 3, characterized in that the amplifier circuit of the receiver diodes or phototransistors has a large amplification at the burst frequency f and otherwise enables a high attenuation through an inductive operating resistor followed by an integrator. 19. A touch-sensitive region according to claim 4, characterized in that the amplifier circuit of the receiver diodes or phototransistors has a large amplification at the burst frequency f and otherwise enables a high attenuation through an inductive operating resistor followed by an integrator. 20. A touch-sensitive region according to claim 2, characterized in that not only the emitter diodes arranged optically in pairs with a receiver diode or phototransistor are active during the on time of the receiver diode or phototransistor, but adjacent emitter diodes are also active. |
Method for the amplification and detection of hbv dna using a transcription based amplification |
The present invention provides a method for the transcription based amplification of a target HBV nucleic acid sequence starting from HBV DNA optionally present in a sample, comprising the steps of,—incubating the sample, suspected to contain HBV, in an amplification buffer with one or more restriction enzymes capable of cleaving the HBV DNA at a selected restriction site, said restriction enzyme creating a defined 3′ end of the said HBV DNA strand(s), a promotor-primer, said promotor-primer having a 5′ region comprising the sequence of a promotor recognized by a DNA-dependent RNA polymerase and a 3′ region complementary to the define 3′ end of the DNA strand, a second or reverse primer, having the opposite polarity of the promotor-primer and comprising the 5′ end of the said target sequence, and in case of HBV ssDNA as the target sequence, a restriction primer,—maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for a digestion by the restriction enzyme to take place,—subjecting the sample thus obtained to a heat treatment at a temperature and time sufficient to inactivate the restriction enzyme and/or to render at least partially a double strand single stranded,—adding the following reagents to the sample: an enzyme having RNA dependent DNA polymerase activity, an enzyme having DNA dependent DNA polymerase activity, an enzyme having Rnase H activity, an enzyme having RNA polymerase activity, and—maintaining the thus created reaction mixture under the appropriate conditions to a sufficient amount of time for the amplification to take place. |
1. A method for the transcription based amplification of a target HBV nucleic acid sequence starting from HBV DNA optionally present in a sample, said method comprising: -a) incubating the sample, suspected to contain HBV, in an amplification buffer with i) one or more restriction enzymes capable of cleaving the HBV DNA at a selected restriction site, said restriction enzyme creating a defined 3′ end of the said HBV DNA strand(s), ii) a promoter-primer, said promoter-primer having a 5′ region comprising the sequence of a promoter recognized by a DNA-dependent RNA polymerase and a 3′ region complementary to the defined 3′ end of the DNA strand, iii) a second or reverse primer, having the opposite polarity of the promoter-primer and comprising the 5′ end of the said target sequence, and iv) in case of HBV ssDNA as the target sequence, a restriction primer; -b) maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for a digestion by the restriction enzyme to take place; -c) subjecting the sample thus obtained to a heat treatment at a temperature and time sufficient to inactivate the restriction enzyme and/or to render at least partially a double strand single stranded; -d) adding the following reagents to the sample: i) an enzyme having RNA dependent DNA polymerase activity ii) an enzyme having DNA dependent DNA polymerase activity iii) an enzyme having Rnase H activity iv) an enzyme having RNA polymerase activity; and e) maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for the amplification to take place. 2. The method according to claim 1, wherein the DNA is double stranded HBV DNA. 3. The method according to claim 1, wherein the DNA is single stranded and the promoter primer and the restriction primer are combined in using a combined promoter and restriction primer comprising a sequence complementary to the region including the restriction site of the target ssDNA and the sequence of a promoter recognized by a DNA-dependent RNA polymerase. 4. The method according to claim 1 in which nucleoside triphosphates are added to the initial incubation mixture prior to the heat treatment. 5. The method according to claim 1 in which a reverse transcriptase is used combining the activities of the enzyme having RNA dependent DNA polymerase activity and the enzyme having DNA dependent DNA activity. 6. The method according to claim 1, in which a reverse transcriptase is used having inherent RNase H activity replacing 3 enzymes, namely the enzyme having RNA dependent DNA polymerase activity, the enzyme having DNA dependent DNA activity as well as the enzyme having Rnase H activity. 7. The method according to claim 1, in which the incubation temperature is from about 35° C. to about 45° C. 8. The method according to claim 1 in which to the heating step is carried out at a temperature between about 92° C. and about 98° C. 9. The method according to claim 1, wherein a restriction enzyme is used that cuts the HBV DNA at a site that is conserved among the different genotypes of HBV. 10. The method according to claim 1, wherein the restriction site is located in the part of the HBV genome that encodes the surface antigen. 11. The method according to claim 10, wherein the restriction site is an XbaI site located at nucleotides 247-252 according to the EcoRI site, the BssSI site located at nucleotides 253-258 according to the EcoRI site or an AvrII site located at nucleotides 178-183 according to the EcoRI site, and the restriction enzyme used is the XbaI, BssSI or AvrII restriction enzyme. 12. The method according to claim 1, wherein the restriction primer is an oligonucleotide containing from about 10 to about 35 nucleotides which hybridize with the HBV target and comprise at least 10 nucleotides flanking the XbaI site located at nucleotides 247-252 according to the EcoRI site, the BssSI site located at nucleotides 253-258 according to the EcoRI site or the AvrII site located at nucleotides 178-183 according to the EcoRI site. 13. The method according to claim 12, wherein the restriction primer has the oligonucleotide sequence of SEQ ID No. 8 or SEQ ID No 9. 14. The method according to claim 1, wherein the promoter or forward primer is an oligonucleotide containing from about 10 to about 35 nucleotides which hybridize with the HBV target and comprising at least 10 nucleotides, counted from the cleavage site, of the nucleotide sequence of SEQ ID No 10 in combination with the restriction enzyme XbaI, or of SEQ ID No 11 in combination with BssSI or of SEQ ID No 12 in combination with AvrII, linked to a promoter sequence. 15. The method according to claim 14, wherein the promoter oligonucleotide has the nucleotide sequence of SEQ ID No 1, SEQ ID No 2, or SEQ ID No. 3. 16. The method according to claim 1, wherein the amplified HBV nucleic acid is additionally detected using a hybridization probe comprising an oligonucleotide sequence containing from about 10 to about 35 nucleotides which hybridize with the amplified HBV target and comprise at least 10 nucleotides of SEQ ID No. 13 or SEQ ID No 14. 17. The method according to claim 16, wherein the probe has the oligonucleotide sequence of SEQ ID No 6 or SEQ ID No 7. 18. The method according to claim 1 wherein the second or reverse primer is an oligonucleotide containing from about 10 to about 35 nucleotides and comprising at least 10 nucleotides of the nucleotide sequence of SEQ ID No. 4 or SEQ ID No. 5. 19. An oligonucleotide with an nucleotide sequence selected from the group consisting of SEQ ID No 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14. 20. A set of oligonucleotide primers suitable for use in the amplification of HBV nucleic acid according to the method of claim 1 comprising a promoter primer, wherein the promoter primer is an oligonucleotide containing from about 10 to about 35 nucleotides which hybridize with the HBV target and comprising at least 10 nucleotides, counted from the cleavage site, of the nucleotide sequence of SEQ ID No. 10 in combination with the restriction enzyme XbaI, or of SEQ ID No. 11 in combination with BssSI or of SEQ ID No. 12 in combination with AvrII, linked to a promoter sequence and a reverse primer, wherein the reverse primer is an oligonucleotide containing from about 10 to about 35 nucleotides and comprising at least 10 nucleotides of the nucleotide sequence of SEQ ID No. 4 or SEQ ID No. 5. 21. A test kit suitable for carrying out the transcription based amplification and detection of HBV DNA according to claim 1 comprising a restriction enzyme capable of cleaving the HBV DNA at a selected restriction site, said restriction enzyme creating a defined 3′ end of the said HBV DNA strand(s), a forward primer that corresponds with the cleavage site of the restriction enzyme chosen, said forward primer having a 5′ region comprising the sequence of a promoter recognized by a DNA-dependent RNA polymerase and a 3′ region complementary to the defined 3′ end of the DNA strand, and is provided with a promoter sequence, other reagents for carrying out a transcription based amplification reaction, and means for detecting the amplified HBV DNA. 22. The method according to claim 1, in which the incubation temperature is from about 37° C. to about 41° C. 23. The method according to claim 1, in which the heating step is carried out at a temperature of about 95° C. 24. The method according to claim 1, wherein the restriction primer is an oligonucleotide containing from about 15 to about 30 nucleotides which hybridize with the HBV target and comprise at least 15 nucleotides flanking the XbaI site located at nucleotides 247-252 according to the EcoRI site, the BssSI site located at nucleotides 253-258 according to the EcoRI site or the AvrII site located at nucleotides 178-183 according to the EcoRI site. 25. The method according to claim 1, wherein the promoter or forward primer is an oligonucleotide containing from about 15 to about 30 nucleotides which hybridize with the HBV target and comprising at least 15 nucleotides, counted from the cleavage site, of the nucleotide sequence of SEQ ID No. 10 in combination with the restriction enzyme XbaI, or of SEQ ID No. 11 in combination with BssSI or of SEQ ID No. 12 in combination with AvrII, linked to a promoter sequence. 26. The method according to claim 1, wherein the amplified HBV nucleic acid is additionally detected using a hybridization probe comprising an oligonucleotide sequence containing from about 15 to about 30 nucleotides which hybridize with the amplified HBV target and comprise at least 15 nucleotides of SEQ ID No. 13 or SEQ ID No. 14. 27. The method according to claim 1, wherein the second or reverse primer is an oligonucleotide containing from about 15 to about 30 nucleotides and comprising at least 15 nucleotides of the nucleotide sequence of SEQ ID No. 4 or SEQ ID No. 5. 28. A set of oligonucleotide primers comprising a promoter primer, wherein the promoter primer is an oligonucleotide containing from about 15 to about 30 nucleotides which hybridize with the HBV target and comprising at least 15 nucleotides, counted from the cleavage site, of the nucleotide sequence of SEQ ID No. 10 in combination with the restriction enzyme XbaI, or of SEQ ID No. 11 in combination with BssSI or of SEQ ID No. 12 in combination with AvrII, linked to a promoter sequence and a reverse primer, wherein the reverse primer is an oligonucleotide containing from about 15 to about 30 nucleotides and comprising at least 15 nucleotides of the nucleotide sequence of SEQ ID No. 4 or SEQ ID No. 5. |
<SOH> BRIEF DESCRIPTION OF THE FIGURES <EOH>FIG. 1 : Schematic presentation of DNA NASBA including restriction enzyme digestion. The restriction enzyme (arrow) is only active during the initiation phase of NASBA. After digestion, the forward primer is hybridized to the template. AMV RT will extend the 3′ end of the target strand (black) of the DNA, using the forward primer, including the T7 promoter sequence (dark grey) as template. The T7 DdRp will recognize the double stranded T7 promoter sequence and RNA amplicon (light grey) production will begin. The RNA amplicon sequence is complementary to the target DNA strand. During the cyclic phase, the RNA amplicon will be amplified and detected by molecular beacon technology. RNase H and the reverse primer are only required during the cyclic phase. FIG. 2 : NASBA of HBV DNA with and without digestion with XbaI in combination with forward primer S-p3.8. After digestion with XbaI, S-p3.8 can be used as template for the extension of target DNA. Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe. A sample without template (NT) is used as negative control. FIG. 3 : NASBA of HBV DNA with and without digestion with BssSI in combination with forward primer S-p3.10. After digestion with BssSI, S-p3.10 can be used as template for the extension of target DNA. Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe. A sample without template (NT) is used as negative control. FIG. 4 . NASBA of HBV DNA with and without digestion with XbaI in combination with forward primer S-p3.10. S-p3.10 can not be used as template for the extension of target DNA, after digestion with XbaI. Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe. A sample without template (NT) is used as negative control. FIG. 5 . NASBA of HBV DNA with and without digestion with BssSI in combination with forward primer S-p3.8. S-p3.8 can be used as template for the extension of target DNA, after digestion with BssSI. Primer S-p4.5 is used as reverse primer and molecular beacon S-WT2 as probe. A sample without template (NT) is used as negative control. FIG. 6 . NASBA of HBV DNA with and without digestion with AvrII in combination with forward primer S-p3.5. S-p3.5 can be used as template for the extension of target DNA, after digestion with AvrII. Primer S-p4.4 is used as reverse primer and molecular beacon S-WT4 as probe. A sample without template (NT) is used as negative control. The invention is further exemplified by the following Examples. detailed-description description="Detailed Description" end="lead"? |
Adenosine receptor selective modulators |
The invention relates to the compounds of formula (I), to a method for producing the same and to the use thereof as medicaments. |
1. A compound of the formula (I) in which R1, R2 and R3 independently of one another represent (C1-C8)-alkyl which may be substituted up to three times, independently of one another, by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C2-C4)-alkynyl, halogen or (C6-C10)-aryloxy, (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxy-carbonyl or mono- or di-(C1-C4)-alkylamino, (C1-C8)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C2-C4)-alkenyl, (C6-C10)-aryl, 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, (C6-C10)-aryloxy, halogen, cyano, (C1-C4)-alkoxycarbonyl, amino or mono- or di-(C1-C4)-alkylamino, hydrogen, hydroxyl, halogen, nitro, cyano, or —NH—C(O)—R7, in which R7 represents (C1-C4)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, or R1 and R2 are attached to adjacent phenyl ring atoms and, together with the two ring carbon atoms, form a 5- to 7-membered saturated or partially unsaturated heterocycle having one or two heteroatoms from the group consisting of N, O and/or S which may be substituted by (C1-C4)-alkyl or oxo, R4 represents (C1-C8)-alkyl which may be substituted by hydroxyl, —NH—CO—R8, (C1-C4)-alkoxy, (C3-C7)-cycloalkyl, (C6-C10)-aryl, 5- or 6-membered saturated or partially unsaturated heterocyclyl having up to three heteroatoms from the group consisting of N, O and/or S or 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, or (C3-C7)-cycloalkyl which may be substituted by hydroxyl or (C1-C8)-alkyl, in which R8 represents (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, R5 represents hydrogen or (C1-C4)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy or (C3-C7)-cycloalkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain one or two further heteroatoms from the group consisting of N, O and/or S in the ring and which may be mono- to trisubstituted, independently of one another, by oxo, fluorine, chlorine, bromine, hydroxyl, (C1-C6)-alkyl or (C1-C6)-alkoxy, and R6 represents (C3-C7)-cycloalkyl or (C1-C8)-alkyl, where alkyl may be substituted up to three times, independently of one another, by (C3-C7)-cyclo-alkyl, hydroxyl, —CO—NH—R9, (C1-C4)-alkoxy, (C2-C4)-alkenyl, (C6-C10)-aryl or 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where aryl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, and R9 represents hydrogen, (C1-C8)-alkyl which may be substituted by hydroxyl or (C1-C4)-alkoxy, (C3-C7)-cycloalkyl or (C6-C10)-aryl which may be substituted up to three times, independently of one another, by halogen, nitro, (C1-C4)-alkoxy, carboxyl, (C1-C4)-alkoxycarbonyl or mono- or di-(C1-C4)-alkylamino, or a salt, a hydrate, a hydrate of a salt or a solvate thereof. 2. A compound of the formula (I) in which R1, R2 and R3 independently of one another represent hydrogen, hydroxyl, (C1-C4)-alkyl, trifluoromethyl, trifluoromethoxy, fluorine, chlorine, (C1-C4)-alkoxy which may be substituted by hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl or (C3-C6)-cycloalkyl, —NH—C(O)—CH3 or —NH—C(O)—C2H5, or R1 and R2 are attached to adjacent phenyl ring atoms and represent a group —O—CH2—O— or —CH2—CH2—O—, R4 represents (C1-C6)-alkyl which may be substituted by hydroxyl, (C1-C4)— alkoxy, (C3-C6)-cycloalkyl, —NH—C(O)—CH3, phenyl, furyl, pyridyl, imidazolyl, thienyl or hexahydropyranyl, or (C3-C6)-cycloalkyl, R5 represents hydrogen or (C1-C4)-alkyl which may be substituted by hydroxyl, (C1-C4)-alkoxy or (C3-C6)-cycloalkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated or partially unsaturated heterocycle which may contain a further heteroatom from the group consisting of N; O or S in the ring and which may be mono- to trisubstituted, independently of one another, by hydroxyl, (C1-C4)-alkyl or (C1-C4)-alkoxy, and R6 represents (C3-C6)-cycloalkyl, (C1-C6)-alkyl which may be substituted up to two times, independently of one another, by (C3-C6)-cycloalkyl, —CO—NH—R9, hydroxyl, (C1-C4)-alkoxy, (C2-C4)-alkenyl, phenyl or 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and/or S, where phenyl and heteroaryl for their part may be substituted by halogen, (C1-C4)-alkyl, (C1-C4)-alkoxy, amino, mono- or di-(C1-C4)-alkylamino, nitro, cyano or hydroxyl, and R9 represents hydrogen or (C1-C4)-alkyl, or a salt, a hydrate, a hydrate of a salt or a solvate thereof. 3. A compound of the formula (I) in which R1 and R2 independently of one another represent hydrogen, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or —NH—C(O)—CH3, where the alkoxy radicals for their part may be substituted by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or cyclopropyl, or R1 and R2 are attached to adjacent phenyl ring atoms and represent a group —O—CH2—O—, R3 represents hydrogen, R4 represents methyl, ethyl, n-propyl, isopropyl, where the alkyl radicals for their part may be substituted by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, cyclopropyl, —NH—C(O)—CH3, furyl, pyridyl, imidazolyl or hexahydropyranyl, or cyclopropyl, R5 represents hydrogen or methyl, or R4 and R5 together with the nitrogen atom to which they are attached represent pyrrolidinyl, morpholinyl, piperidinyl or 4-hydroxypiperidinyl and R6 represents methyl, ethyl or n-propyl, where the alkyl radicals for their part may be substituted up to two times, independently of one another, by hydroxyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, imidazolyl, nitrofuranyl, pyridyl, phenyl which for its part may in turn be substituted by cyano, nitro, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or amino, —C(O)—NH2 or —C(O)—NH—CH3, or a salt, a hydrate, a hydrate of a salt or a solvate thereof. 4. A process for preparing compounds of the formula (I) as defined in claim 1, characterized in that compounds of the formula (II) in which R1, R2, R3, R4 and R5 are as defined in claim 1 are reacted with compounds of the formula (III) R6—X (III) in which R6 is as defined in claim 1 and X represents a leaving group. 5. A compound of the formula (I) as defined in claim 1 for the prophylaxis and/or treatment of disorders. 6. A composition, comprising at least one compounds of the formula (I) as defined in claim 1 and at least one further auxiliary. 7. The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of disorders of the cardiovascular system (cardiovascular disorders). 8. The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of disorders of the urogenital system and cancer. 9. The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of inflammatory and neuro-inflammatory disorders, neurodegenerative disorders and pain. 10. The use of compounds of the formula (I) as defined in claim 1 for preparing medicaments for the prophylaxis and/or treatment of disorders of the respiratory tract, of liver fibrosis and liver cirrhosis and diabetes. |
Helicomimetics and stabilized lxxll peptidomimetics |
This invention pertains to the design and synthesis of molecules that can act as protein mimics. In particular this disclosure teaches the preparation of short, cyclic peptide sequences that can adopt a helical conformation and display a particular arrangement of amino acid side chains oriented in a specific arrangement to serve as a pharmacophore. A ring, formed by a disulfide bridge between pairs of cysteine residues, maintains the helical structure. When the cysteines are arranged in a pattern of i to i+3 as illustrated in FIG. 1, and when the first cysteine is of the D-configuration, and the second cysteine is of the L-configuration, the helical arrangement is especially stabilized. A preferred version of this invention involves a pentapeptide sequence of general structure known as the NR Box, stabilized by a side chain to side chain disulfide bridge formed from the two cysteines. |
1. A helicomimetic compound for stabilizing the alpha helical structure of a protein fragment which can serve as an agonist or antagonist of protein-protein interactions, comprising: a compound consisting of the structure R1-(Xn)-D-Cys-Y-Y-L-Cys-(Xn)-R2, where R1 consists of H, an alkyl, aryl, acetyl, formyl, or other blocking or solubilizing group such as a polyethylene glycol (PEG) or other polyether moiety, linked to the N-terminal nitrogen through a carbon-nitrogen bond; said X comprises one or more natural or unusual amino acids, linked together in a chain from 0 to n in length; said Y comprises a selected natural or unnatural amino acid, usually of the L-configuration, and with two such amino acids that need not be identical, separating the pairs of cysteines to form an i to i+3 type of disulfide bridged unit; and R2 comprises a selected OH, NH2, NHR, or, OR other blocking or solubilizing group such as polyethylene glycol (peg) or other polyether moiety, linked to the c-terminal carbonyl through an oxygen or carbon or nitrogen linkage, such as an amide group. 2. A compound comprising a structure, wherein, R1 is (Xm)-D-Cys-Y-Y-L-Cys-(Xn)-R2, wherein Y-Y is selected from the group consisting of Ile and Leu; said Xm comprises one or more natural or unusual amino acids, linked together in a chain from 0 to n in length; and said Xn is selected from the group consisting of Leu-Leu, or Leu-Leu-Xm. 3. A compound comprising the structure R1-(Xm)-D-Aaa-Y-Y-L-Aaa-(Xn)-R2, wherein said Aaa comprises a cysteine, homocysteine, penicillamine, or other amino acid with a thiol side chain suitable for the formation of disulfide bridges; said Xm comprises one or more natural or unusual amino acids, linked together in a chain from 0 to n in length; said Y-Y is selected from the group consisting of Ile and Leu; said Xn is selected from the group consisting of Leu-Leu, Leu-Leu-Xm, and combinations thereof. 4. The compound of claim 1, wherein said compound is combined with a selected bioconjugate carrier to increase the the solubility, transport and delivery of said helicomimetic for use as a drug. 5. The compound of claim 2, wherein said compound is combined with a selected bioconjugate carrier to increase the the solubility, transport and delivery of said helicomimetic for use as a drug. 6. The compound of claim 3, wherein said compound is combined with a selected bioconjugate carrier to increase the the solubility, transport and delivery of said helicomimetic for use as a drug. 7. The compound of claim 1, wherein the compound is linked to a bioconjugate selected from a group consisting of a polyethylene glycol, an alkylated C-sugar, a transport peptide, and combinations thereof. 8. The compound of claim 2, wherein the compound is linked to a bioconjugate selected from a group consisting of a polyethylene glycol, an alkylated C-sugar, a transport peptide, and combinations thereof. 9. The compound of claim 3, wherein the compound is linked to a bioconjugate selected from a group consisting of a polyethylene glycol, an alkylated C-sugar, a transport peptide, and combinations thereof. 10. The compound of claim 1, said compound containing an amide bond replacement selected from a group consisting of a singly bonded carbon-carbon, double bonded carbon-carbon, a carbon-sulfur, a carbon-nitrogen between pairs of amino acids in order to render said compound more resistant to enyzmatic degradation. 11. The compound of claim 2, said compound containing an amide bond replacement selected from a group consisting of a singly bonded carbon-carbon, a double bonded carbon-carbon, a carbon-sulfur, a carbon-nitrogen between pairs of amino acids in order to render said compound more resistant to enyzinatic degradation. 12. The compound of claim 3, said compound containing an amide bond replacement selected from a group consisting of a singly bonded carbon-carbon, a double bonded carbon-carbon, a carbon-sulfur, a carbon-nitrogen between pairs of amino acids in order to render said compound more resistant to enyzmatic degradation. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates to the design and synthesis of a new class of stabilized peptide structures that are useful as mimics of the alpha helical structure ubiquitous in proteins. 2. Description of the Related Art Steroids, along with other lipophilic hormones such as the retinoids and vitamins, bind to members of the nuclear-receptor superfamily. These ligands modify the DNA-binding and transcriptional properties of these receptors, resulting in the activation or repression of target genes. Ligand binding induces conformational changes in nuclear receptors and promotes their association with a diverse group of nuclear proteins, including SRC-1/p160, TIF-2/GRIP-1 and CBP/p300 which function as co-activators of transcription. A short sequence motif LXXLL (where L is leucine and X is any amino acid) present in RIP-140, SRC-1 and CBP is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors. The ability of coactivators to bind the estrogen receptor and enhance its transcriptional activity is dependent upon the integrity of the LXXLL motifs and on key hydrophobic residues in a conserved helix (helix 12) of the estrogen receptor that are required for its ligand-induced activation function. Thus the LXXLL motif is a signature sequence that facilitates the interaction of different proteins with nuclear receptors, and is thus a defining feature of a new family of nuclear proteins (Heery, 1997). These compounds are intended as protein mimics and thus could find numerous applications, and especially for inhibition of protein-protein interactions where at least one of the proteins displays a helical segment as a prominent feature in terms of its tight binding to another protein. Where the protein-protein interaction is critical to a biological function, as is often the case, then by retarding or preventing this interaction through the intervention of the helicomimetic (helix protein mimic), this compound can serve as a useful drug candidate in the event of a pathologic process such as cancer or stroke, or other instances such as transcription mediated by nuclear receptors and their cognate macromolecules. |
<SOH> SUMMARY OF THE INVENTION <EOH>This invention includes helix stabilized compounds that contain the so-called NR Box, found in a large number of Nuclear Receptor Coactivator Proteins. The NR Box sequence, consisting of Leu-Xxx-Yyy-Leu-Leu within a longer peptide, is found in both coactivator proteins and also in certain nuclear receptors themselves. In the case of the Androgen Receptor, this sequence is varied to include Phe-Xxx-Yyy-Leu-Phe and Phe-Xxx-Yyy-Leu-Trp, where Xxx and Yyy typically consist of two out of a rather large and diverse choice among the 20 common or natural amino acids. By preparing synthetic variants of relatively short peptides and peptide mimics that contain the crucial hydrophobic amino acids, e.g., leucines, that maintain contact with the nuclear receptor, it is possible to prevent binding of the coactivator proteins to the nuclear receptors. This intervention prevents the receptor from its normal subsequent step of binding to DNA and thus also prevents the transcription of the DNA segment known os the Estrogen Response Element. This intervention has a similar pattern with respect to the other types of nuclear receptors, such as the androgen, progestin, glucocorticoid, mineralocorticoid, and the growing number of orphan receptors that have been shown to function in a biochemically equivalent manner. Thus, by the prevention of the normal receptor-coactivator interactions, we will be able to control such effects as DNA transcription and the related downstream events that are affected, such as cell growth and division. Thus these molecules represent a novel approach to the control of such diseases as breast cancer and prostate cancer. These forms of cancer are currently treated with such agents as tamoxifen and raloxifene that function as estrogen antagonists. But tamoxifen and raloxifene have been shown to bind to the normal steroid binding site, and not to the site occupied by the helical peptide LXXLL. Thus our peptides represent a novel and distinct approach toward the treatment of cancer through a new form of inhibition of nuclear receptor action. In order for LXXLL peptides to bind to the receptor, it is believed essential that they do so in the form of an alpha helix conformation. Shorter linear peptides tend to adont random or β-sheet structures rather than helices. Various strategies have been used to induce helix folding including incorporation of α-alkyl amino acid residues such as Aib (aminoisobutyric acid) or Deg (diethylglycine). This approach may lead to unacceptably high hydrophobic character when matched with an LXXLL sequence. Other options include helix end capping and dipole stabilization, primarily useful for longer sequences. The peptides of the instant invention are preferable to LXXLL linear sequences for several reasons. The first is that we and others have shown that short, linear peptides are not able to inhibit coactivator binding, at least to the extent our bioassays reflect this activity. Second, by including pairs of cysteine residues within the sequence, we are able to enhance the helical character of these peptides. The preferred method for doing so involves the incorporation of one D-cysteine in the sequence and one L-cysteine. It is important to note that other workers have generally found that this type of side chain to side chain cyclization does not yield a strongly helical sequence. Our studies have also demonstrated this trend. But our work also demonstrates that the cysteine bridges do in fact help stabilize this helical tendency when the cyclic peptide comes into contact with the nuclear receptor. This has been shown most clearly by a study of the interaction of one of our peptides (that one known as PERM-1 for peptidomimetic estrogen receptor modulator −1) with the ligand binding domain of the estrogen receptor (see the Figure showing the X-ray crystal structure by N. Chirgadze and coworkers). This finding is significant since it shows the ability of our synthetic peptide to adopt a clear helical conformation in the presence of its partner and to form a strong and stable interaction with the receptor. Additionally, our peptides can easily be made selective to one or another of the multiple nuclear receptors by changing the structure of the amino acids in the flanking regions. Thus, as seen in Table I entitled “Peptide Analogs and their Ki values against ER alpha and ER beta”, it is apparent that by changes in amino acid composition, we are able to increase the binding toward ER alpha to a significant degree. Furthermore, this selectivity in preferred binding to a receptor is in most cases predictable through an examination of both the sequences of amino acids found in various naturally occurring coactivator proteins as well as by an examination of the receptor residues found in close proximity to the LXXLL binding sites. This is an important attribute of our cyclic peptide analogs since it means that we may retain the preferred small, cyclic helix-forming nature of our peptides and yet still embody the selectivity and specificity important to any useful drug. For instance a preferred embodiment of a compound of the present invention comprises of the structure R1-(Xn)-D-Cys-Y-Y-L-Cys-(Xn)-R2, where R1 consists of H, an alkyl, aryl, acetyl, formyl, or other blocking or solubilizing group such as a polyethylene glycol (PEG) or other polyether moiety, linked to the N-terminal nitrogen through a carbon-nitrogen bond. Moreover, X consists of one or more natural or unusual amino acids, linked together in a chain from 0 to n in length, and Y consists of any natural or unnatural amino acid, usually of the L-configuration, and with two such amino acids that need not be identical, separating the pairs of cysteines to form an i to i+3 type of disulfide bridged unit. R2 consists of an OH, NH2, NHR, OR, or other blocking or solubilizing group such as polyethylene glycol (PEG) or other polyether moiety linked to the C-terminal carbonyl through an oxygen or carbon or nitrogen linkage, such as an amide group. |
Novel fungicide compositions based on pyridylmethylbenzamide and propamocarb derivative |
1) Fungicidal compositions comprising: a) at least one pyridylmethylbenzamide derivative of formula (I): in which the various radicals are as defined in the description, and b) at least one compound (II), which is propamocarb. 2) Method of curatively or preventively combating phytopathogenic fungi in crops, characterized in that an effective, nonphytotoxic amount of one of these fungicidal compositions is applied to the aerial parts of the plants. |
1. Fungicidal compositions comprising: a) at least one pyridylmethylbenzamide derivative of formula (I) in which: R1 is selected from a hydrogen atom, an optionally substituted alkyl radical and an optionally substituted acyl radical; R2 is selected from a hydrogen atom and an optionally substituted alkyl radical; R3 and R4, which are identical or different, are selected independently from a halogen atom, a hydroxyl radical, a cyano radical, a nitro radical, an —SF5 radical, a trialkylsilyl radical, an optionally substituted amino radical, an acyl radical, and a group E, OE or SE in which E is selected from an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl and heterocyclyl radical, each of which can be optionally substituted; c represents 0, 1, 2, 3 or 4; q represents 0, 1, 2, 3 or 4; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field; and b) at least one compound (II), which is propamocarb. 2. Fungicidal compositions according to claim 1, characterized in that the compound (I) possesses one of the following characteristics, taken individually or in combination: R1 and R2, which are identical or different, are selected independently from a hydrogen atom and an optionally substituted alkyl radical; R3 and R4, which are identical or different, are selected independently from a halogen atom, a hydroxyl radical, a nitro radical, an optionally substituted amino radical, an acyl radical, and a group E, OE or SE in which E is selected from an alkyl, cycloalkyl, phenyl and heterocyclyl radical, each of which can be optionally substituted; c represents 0, 1, 2 or 3; q represents 0, 1, 2 or 3; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field. 3. Fungicidal compositions according to claim 1, characterized in that the compound (I) possesses one of the following characteristics, taken individually or in combination: R1 and R2, which are identical or different, are selected independently from a hydrogen atom and a methyl or ethyl radical; R3 and R4, which are identical or different, are selected independently from a halogen atom, a nitro radical, an optionally substituted amino radical and an alkyl, cycloalkyl, phenyl or heterocyclyl radical, each of which can be optionally substituted; c represents 1 or 2; q represents 1 or 2; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field. 4. Fungicidal compositions according to claim 1, characterized in that the compound (I) possesses one of the following characteristics: R1 and R2 each represent a hydrogen atom; R3 and R4, which are identical or different, are selected independently from a halogen atom, a nitro radical, an alkyl radical and a trifluoromethyl radical; c and q represent, independently of one another, 2; and their possible optical and/or geometric isomers, tautomers and addition salts with an acid or a base that are acceptable in the agricultural field. 5. Fungicidal compositions according to claim 1, characterized in that the compound of formula (I) is selected from: 2,6-dichloro-N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}benzamide, N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}-2-fluoro-6-nitrobenzamide, and N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}-2-methyl-6-nitrobenzamide, and their possible tautomers and addition salts with an acid or a base that are acceptable in the agricultural field. 6. Fungicidal compositions according to claim 1, characterized in that the compound (D is selected from 2,6-dichloro-N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}benzamide, N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}-2-fluoro-6-nitrobenzamide, and N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl} -2-methyl-6-nitrobenzamide and the compound (II) is propamocarb. 7. Fungicidal compositions according to claim 6, characterized in that the compound (I) is 2,6-dichloro-N-{[3-chloro-5-(trifluoromethyl)-2-pyridinyl]methyl}benzamide and in that the compound (II) is propamocarb. 8. Fungicidal compositions according to claim 1, characterized in that they comprise at least one compound of formula (I) and at least one compound (II), the compound (I)/compound (II) ratio being between 1/500 and 1/1. 9. Fungicidal compositions according to claim 8, characterized in that the compound (I)/compound (II) ratio is between 1/200 and 1/5. 10. Fungicidal compositions according to claim 9, characterized in that the compound (I)/compound (II) ratio is between 1/150 and 1/10. 11. Fungicidal compositions according to claim 1, characterized in that the compound (I)/compound (II) ratio is chosen so as to produce a synergistic effect. 12. Synergistic fungicidal compositions according to claim 11, characterized in that the compound (I)/compound (II) ratio is between 1/200 and 1/5. 13. Synergistic fungicidal compositions according to claim 12, characterized in that the compound (I)/compound (II) ratio is between 1/140 and 1/10. 14. Fungicidal compositions according to claim 1, characterized in that they comprise, in addition to compounds (I) and (II), an agriculturally suitable inert vehicle and optionally an agriculturally suitable surfactant. 15. Fungicidal compositions according to claim 1, characterized in that they contain from 0.5 to 99% of the combination of compound (I) and compound (II). 16. Method of curatively or preventively combating phytopathogenic fungi in crops, characterized in that an effective (agronomically effective), nonphytotoxic amount of a fungicidal composition according to claim 1 is applied to the soil in which the plants are growing or are liable to grow, to the leaves and/or the fruits of the plants or to the seeds of the plants. 17. Method according to claim 16, characterized in that the fungicidal composition is applied by spraying a liquid onto the aerial parts of the crops to be treated. 18. Method according to claim 16, characterized in that the amount of fungicidal composition corresponds to a dose of compound (I) and of compound (II) between approximately 1 g/ha and approximately 1 000 g/ha. 19. Method according to claim 16, characterized in that the treated crop is tomato, potato or grapevine. 20. Method according to claim 19, characterized in that the phytopathogenic fungus treated is downy mildew in tomatoes, potatoes and grapevines. 21. Product comprising a compound of formula (I) and a compound of formula (II) as a combined preparation for simultaneous, separate or sequential use in the control of phytopathogenic fungi in crops in one locus. 22. Method according to claim 17, characterized in that the amount of fungicidal composition corresponds to a dose of compound (I) and of compound (II) between approximately 1 g/ha and approximately 1 000 g/ha. |
Automated immunohistochemical and in situ hybridization assay formulations |
The present invention provides reagents for use in an automated environment for cell conditioning of biological samples wherein the cells or tissues are predisposed for access by reagent molecules for histochemical and cytochemical staining procedures. The reagents comprise components optimized to faciliate molecular access to cells and cell constituents within the biological sample. The present invention also provides reagents for use in an automated environment for removing or etching embedding media by exposing a biological sample to be stained in histochemical or cytochemical procedures without the dependence on organic solvents. The reagents comprise components optimized to facilitate removal or etching of the embedding media from the biological sample. |
1. An aqueous composition of matter, comprising citrate buffer, ethylene glycol, sodium metabisulfite and a surfactant. 2. A composition according to claim 1, wherein the composition is buffered from approximately pH 4 to approximately pH 9. 3. A composition according to claim 1, wherein composition is buffered at approximately pH 6. 4. A composition according to claim 1, wherein the composition comprises less than or equal to approximately 100 mM citrate buffer. 5. A composition according to claim 1, wherein the composition comprises approximately 10 mM citrate buffer. 6. A composition according to claim 1, wherein the composition comprises less than or equal to approximately 10% ethylene glycol. 7. A composition according to claim 1, wherein the composition comprises approximately 5% ethylene glycol. 8. A composition according to claim 1, wherein the composition comprises less than or equal to approximately 10 mM sodium metabisulfite. 9. A composition according to claim 1, wherein the composition comprises 1 mM sodium metabisulfite. 10. A composition according to claim 1, wherein the composition comprises less than or equal to approximately 10% SDS. 11. A composition according to claim 1, wherein the composition comprises approximately 0.3% SDS. 12. A composition according to claim 1, wherein the composition comprises approximately 10 mM citrate buffer at approximately pH 6, approximately 5% ethylene glycol, approximately 1 mM sodium metabisulfite and approximately 0.3% SDS. 13. An aqueous composition of matter comprising a buffer, a glycol, sodium metabisulfite and a surfactant. 14. A composition according to claim 13, wherein the buffer is a citrate buffer. 15. A composition according to claim 14, wherein the buffer is at approximately pH 6. 16. A composition according to claim 13, wherein the glycol is ethylene glycol. 17. A composition according to claim 13, wherein the concentration of the buffer is less than or equal to approximately 100 mM, the concentration of the glycol is less than or equal to approximately 10%, the concentration of the sodium metabisulfite is less than or equal to approximately 10 mM and the concentration of the SDS is less than or equal to approximately 10%. 18. A composition according to claim 17, wherein the buffer is citrate at a concentration of approximately 10 mM and the pH of the composition is approximately 6. 19. A composition according to claim 18, wherein the glycol is ethylene glycol and the concentration of ethylene glycol is approximately 5%, wherein the concentration of sodium metabisulfite is approximately 1 mM and wherein the concentration of SDS is approximately 0.3%. 20. An aqueous composition of matter comprising approximately 10 mM citrate buffer at approximately pH 6, approximately 5% ethylene glycol, approximately 1 mM sodium metabisulfite and approximately 0.3% surfactant. 21. An aqueous composition of matter according to claim 20, wherein the surfactant is SDS. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates to solutions methods, and instruments for conditioning cells or tissues so as to increase the accessibility of various molecules to their respective targets and generally to improve tissue and cell readability of biological samples on automated instruments prior to immunohistochemical (IHC), in situ hybridization (ISH) or other histochemical or cytochemical manipulations. 2. Summary of the Related Art All patents, patent applications and non-patent articles or references mentioned herein are hereby incorporated by reference to the extent that they are not contradictory. The diagnosis of disease based on the interpretation of tissue or cell samples taken from a diseased organism has expanded dramatically over the past few years. In addition to traditional histological staining techniques and immunohistochemical assays, ill situ techniques such as in situ hybridization and in situ polymerase chain reaction are now used to help diagnose disease states in humans. Thus, there are a variety of techniques that can assess not only cell morphology, but also the presence of specific macromolecules within cells and tissues. For example, the diagnosis of breast, ovarian and other carcinomas may be facilitated by the use of techniques designed to identify the presence or absence of the c-erb2/HER-2/neu protooncogene or the protein(s) expressed therefrom. The c-erb2/HER-2/neu protooncogene is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. Amplification and overexpression of the c-erb2/HER-2/neu protooncogene is found in about 30% of breast carcinomas, about 20% of ovarian carcinomas and others. (Andrechek, E. R. et al., Proc. Natl. Acad. Sci. USA , vol. 97, no. 7, pp. 3444-3449 (2000); Doherty, J. K. et al., Proc. Natl. Acad. Sci. USA , vol. 96, pp. 10869-10874 (1999); Oh, J. J. et al., Nucleic Acids Res ., vol. 27, no. 20, pp. 4008-4017 (1999); Klapper, L. N. et al., Proc. Natl. Acad. Sci. USA , vol. 96, pp. 4995-5000 (1999); and references found in each of the aforementioned articles.) Each of these techniques requires that sample cells or tissues undergo preparatory procedures that may include fixing the sample with chemicals such as an aldehyde (such as formaldehyde, glutaraldehyde), formalin substitutes, alcohol (such as ethanol, methanol, isopropanol) or embedding the sample in inert materials such as paraffin, celloidin, agars, polymers, resins, cryogenic media or a variety of plastic embedding media (such as epoxy resins and acrylics). Other sample tissue or cell preparations require physical manipulation such as freezing (frozen tissue section) or aspiration through a fine needle (fine needle aspiration (FNA)). Regardless of the tissue or cell sample or its method of preparation or preservation, the goal of the technologist is to obtain accurate, readable and reproducible results that permit the accurate interpretation of the data. One way to provide accurate, readable and reproducible data is to prepare the tissue or cells in a fashion that optimizes the results of the test regardless of the technique employed. In the case of immunohistochemistry and in situ techniques this means increasing the amount of signal obtained from the specific probe (e.g. antibody, DNA, RNA, etc.). In the case of histochemical staining it may mean increasing the intensity of the stain or increasing staining contrast. Without preservation, tissue samples rapidly deteriorate such that their use in diagnostics is compromised shortly after removal from their host. In 1893, Ferdinand Blum discovered that formaldehyde could be used to preserve or fix tissue so that it could be used in histochemical procedures. The exact mechanisms by which formaldehyde acts in fixing tissues are not fully established, but they involve cross-linking of reactive sites within the same protein and between different proteins via methylene bridges (Fox et al., J. Histochem. Cytochem. 33: 845-853 (1985)). Recent evidence suggests that calcium ions also play a role (Morgan et al., J. Path. 174: 301-307 (1994)). These links cause changes in the quaternary and tertiary structures of proteins, but the primary and secondary structures appear to be preserved (Mason et al., J. Histochem. Cytochem. 39: 225-229 (1991)). The extent to which the cross-linking reaction occurs depends on conditions such as the concentration of formalin, pH, temperature and length of fixation (Fox et al., J. Histochem. Cytochem. 33: 845-853 (1985)). Some antigens, such as gastrin, somatostatin and α-1-antitrypsin, may be detected after formalin fixation, but for many antigens, such as intermediate filaments and leukocyte markers, immunodetection after formalin treatment is lost or markedly reduced (McNicol & Richmond, Histopathology 32: 97-103 (1998)). Loss of antigen immunoreactivity is most noticeable at antigen epitopes that are discontinuous, i.e. amino acid sequences where the formation of the epitope depends on the confluence of portions of the protein sequence that are not contiguous. Antigen retrieval refers to the attempt to “undo” the structural changes that treatment of tissue with a cross-linking agent induces in the antigens resident within that tissue. Although there are several theories that attempt to describe the mechanism of antigen retrieval (e.g., loosening or breaking of crosslinkages formed by formalin fixation), it is clear that modification of protein structure by formalin is reversible under conditions such as high-temperature heating. It is also clear that several factors affect antigen retrieval: heating, pH, molarity and metal ions in solution (Shi et al., J. Histochem. Cytochem. 45: 327-343 (1997)). Microwave heating appears to be the most important factor for retrieval of antigens masked by formalin fixation. Microwave heating (100°±5° C.) generally yields better results in antigen retrieval immunohistochemistry (AR-IHC). Different heating methods have been described for antigen retrieval in IHC such as autoclaving (Pons et al, Appl. Immunohistochem. 3: 265-267 (1995); Bankfalvi et al., J. Path. 174: 223-228 (1994)); pressure cooking (Miller & Estran, Appl. Immunohistochem. 3: 190-193 (1995); Norton et al., J. Path. 173: 371-379 (1994)); water bath (Kawai et al., Path. Int. 44: 759-764 (1994)); microwaving plus plastic pressure cooking (U.S. Pat. No. 5,244,787; Pertschuk et al., J. Cell Biochem. 19(suppl.): 134-137 (1994)); and steam heating (Pasha et al., Lab. Invest. 72: 167A (1995); Taylor et al., CAP Today 9: 16-22 (1995)). Although some antigens yield satisfactory results when microwave heating is performed in distilled water, many antigens require the use of buffers during the heating process. Some antigens have particular pH requirements such that adequate results will only be achieved in a narrow pH range. Presently, most antigen retrieval solutions are used at a pH of approximately 6-8, but there is some indication that slightly more basic solutions may provide marginally superior results (Shi, et al., J. Histochem. Cytochem. 45: 327-343 (1997)). Although the chemical components of the antigen retrieval solution, including metal ions, may play a role as possible co-factors in the microwave heating procedure, thus far, no single chemical has been identified that is both essential and best for antigen retrieval. Many solutions and methods are used routinely for staining enhancements. These may include but are not limited to distilled water, EDTA, urea, Tris, glycine, saline and citrate buffer. Solutions containing a variety of detergents (ionic or non-ionic surfactants) may also facilitate staining enhancement under a wide range of temperatures (from ambient to in excess of 100° C.). In addition to cell surface molecules that may be present on the exterior portion of the cell, other molecules of interest in IHC, ISH and other histochemical and cytochemical manipulations are located within the cell, often on the nuclear envelope. Some of these molecules undergo molecular transformation when exposed to a fixative (coagulative or precipitive) such as formalin. Thus with respect to these molecules it is desirable to not only overcome the effects of fixation but also to increase the permeability of the cell in order to facilitate the interaction of organic and inorganic compounds with the cell. Other tissue samples may not have been subjected to cross-linking agents prior to testing, but improved results with respect to these tissues is also important. There are a variety of non-formalin methods for preserving and preparing cytological and histological samples. Examples of these methods include, but are not limited to: a) hematology smears, cytospins™, ThiinPreps™, touch preps, cell lines, Ficoll separations, etc. are routinely preserved in many ways which includes but are not limited to, air-drying, alcoholic fixation, spray fixatives and storage mediums such as sucrose/glycerin; b) tissues and cells (either fixed or unfixed) may be frozen and subsequently subjected to various stabilizing techniques which include, but are not limited to, preservation, fixation and desiccation; c) tissues and cells may be stabilized in a number of non-cross-linking aldehyde fixatives, non-aldehyde containing fixatives, alcoholic fixatives, oxidizing agents, heavy metal fixatives, organic acids and transport media. One way to improve testing results is to increase the signal obtained from a given sample. In a general sense, increased signal can be obtained by increasing the accessibility of a given molecule for its target. As in the case for antigens found within the cell, targets within the cell can be made more accessible by increasing the permeability of the cell thereby permitting a greater number of molecules entry into the cell, thereby increasing the probability that the molecule will “find” its target. Such increased permeability is especially important for techniques such as ISH, in situ PCR, IHC, histochemistry and cytochemistry. Tissues and cells are also embedded in a variety of inert media (paraffin, celloidin, OCT™, agar, plastics or acrylics etc.) to help preserve them for future analysis. Many of these inert materials are hydrophobic and the reagents used for histological and cytological applications are predominantly hydrophilic; therefore, the inert medium may need to be removed from the biological sample prior to testing. For example, paraffin embedded tissues sections are prepared for subsequent testing by removal of the paraffin from the tissue section by passing the slide through various organic solvents such as toluene, xylene, limonene or other suitable solvents. These organic solvents are very volatile causing a variety of problems including requiring special processing (e.g., deparaffinization is performed in ventilated hoods) and requires special waste disposal. The use of these organic solvents increases the cost of analysis and exposure risk associated with each tissue sample tested and has serious negative effects for the environment. Presently, there is no available technique for removing inert media from sample tissue by directly heating the slide in an automated fashion. Neither is it currently possible to remove inert media from sample tissue while conditioning the sample tissue or cell in a one-step automated staining process. The methods of the present invention permit a) automated removal of embedding media without the use of organic solvents, thus exposing the cells for staining and thereby reducing time, cost and safety hazards, b) automated cell conditioning without automated removal of embedding media from the sample cell or tissue, c) a multi-step automated process that exposes the cells, performs cell conditioning and increases permeability of the cytological or histological specimens, thereby increasing sample readability and improving interpretation of test data. The methods of the present invention can be used for improving the stainability and readability of most histological and cytological samples used in conjunction with cytological and histological staining techniques. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to solutions, methods and instruments for cell or tissue conditioning, which improves the accessibility of molecules in biological samples during histochemical or cytochemical assays. The conditioning may be performed at any point in a histochemical or cytochemical protocol. The present invention further relates to an automated method for exposing biological samples for use in histological or cytological testing procedures by removing the embedding media without the use of organic solvents. The present invention also relates to an automated method for the simultaneous exposing and cell conditioning of biological samples for histochemical and cytochemical applications. |
Tightening device for cables and similar |
The aim of the invention is to produce a tightening device for cables or similar for controlling the transmission of force. Said device comprises a clamping disk (6) and a deflector (10) engaging therebetween. As a result, the cable (4) is guided between the deflector (10) and a guiding element (20) arranged on the periphery of the clamping disk (6). |
1. A tightening apparatus for cables, comprising a clamping disk and a deflector engaging there between, characterized in that the cable is guided between the deflector and guiding element arranged on the circumference of the clamping disk, wherein the tightening apparatus is arranged at the base of a mast of a windsurfing sail, and wherein the guiding element is provided with a pipe-like or tubular configuration in which the loose part of the cable is guided. 2. A tightening apparatus as claimed in claim 1, characterized in that the guiding element is arranged in a fixed way, especially in the form of a spring steel tongue. 3. A tightening apparatus as claimed in claim 1, characterized in that the guiding element is arranged in a movable way. 4. A tightening apparatus as claimed in claim 3, characterized in that the guiding element is arranged in a swivelable way on a lever. 5. A tightening apparatus as claimed in claim 1, characterized in that the guiding element is arranged as a roller. 6. A tightening apparatus as claimed in claim 1, characterized in that the guiding element engages between wedge surfaces of the clamping disk and acts on the loose part of the cable. 7. (canceled) 8. A tightening apparatus as claimed in claim 1, characterized in that a ratchet lever, a crank or an electric drive is provided for driving the clamping disk. 9. A tightening apparatus as claimed in claim 1, characterized in that the clamping disk is provided on its wedge surfaces with a profiling. 10. (canceled) |
Quantitative analysis, visualization and movement correction in dynamic processes |
The invention relates to the quantitative analysis and/or visualization (virtual or real) of moving processes and to the detection, description, correction and the comparison of global movements inside the image space. The invention particularly relates to a method and device for precisely and, while being limited to few parameters, quantitatively describing the global and local movement occurring in the image space and for the single representation of the movement of the quantitative parameters with regard to the image space. A rough to fine recording and a detection of and compensation for global movements occurs whereby enabling a tracking and a corrected visualization. The detection of the movement, which corresponds as precisely as possible to the real movement occurring in the image space is effected by a splines a plaques minces recording technique that enables the number of movement parameters to be kept low. A fully automatic execution of all partial steps is made possible. |
1. A method for the quantitative and visual analysis of motion in a time sequence of images comprising the steps of: a) determination of the motion vectors for each point in the image space between images of directly or not directly subsequent time steps (i.e., the transformation of the corresponding image space) preferably by means of rigid, affine and/or “elastic” (including local deformations) overlapping/registration of images of an image sequence and calculation of the transformation parameter comprising the following further method steps: a1) for the non-rigid registration, local transformation types are given stepwise from coarse to fine, first global and then increasingly more local; a2) for more then two time steps, in order to obtain the transformation for not directly subsequent timesteps, the transformation is not only calculated in pairs for subsequent image pairs, but also a correction-registration step is further added thereto; a3) from the transformation result directly the local, defined for each point in the image/time space, quantities velocity, acceleration, warp, tension, deviation from the finest registration (i.e., distance between the point transformed by the selected transformation type and by the most local fineness level), etc. as well as the global quantities warp energy for the entire motion as well as for each “principal warp” separately (Bookstein 1989), entropy, “directedness” of the motion and the transformation parameters for various fineness levels of the transformation; b) reconstruction of the space-time-structure within the image space and visualization thereof by means of b1) (optionally interactive) determination of a point (or a region; or according to a regular scheme selected point in the space or on the surface of a region) for a selectable moment, the automatic calculation of its (their) position for all other moments by means of the under a) calculated transformation (for not directly subsequent time steps) as well as the interpolated representation as 3D rendered (surface rendering) path (or “tube” for regions); and/or b2) a 3D rendered reference grid, deformed according to the transformation calculated in a), animated (interpolation of intermediate time steps) or not (animation also continuously over several time steps), the image space overlapped (inserted) or not; in an image record having several color channels, the reference grids corresponding to the various separately treated color channels can be represented in different color but simultaneously; and/or b3) color/pattern encoding (including gray value encoding) of quantities (quantities having vector values by means of absolute values), which are assigned to a point in the image/time space as described under a), particularly for (i) all points, lying within an interactively selectable level of the image space, (ii) interactively (or regularly) selected points or surface points of interactively selected regions/shapes/surfaces (iii) the points of the under b1) determined path (or tubes) of the time space; and/or b4) a motion corrected representation of the paths (or tubes), corrected by a rigid, affine or a selected level of the non-global transformation, or by means of a motion corrected playing of the original image sequence, particularly the color encoding can be back projected onto the original image or any other given moment. 2. The method according to claim 1, which extracts in a pre-processing step a0) critical (characteristic) points of the image space or surface points of regions in the image space and uses these in the steps b1), b3) and b4) instead of interactively selected points, whereby in step b2) the (critical/surface) points of the original space can be used instead of the reference grid. 3. The method according to claim 2, which uses for the preprocessing the confinement tree technique (CTT), whereby a) for the visualization of a region (a confiner), the user clicks either on knot in the tree or on a point in the image, upon which the surface/shape of the smallest confiners (in the filtered tree) containing said point will be shown; and/or b) the registration is performed on the basis of the extracted points (confiner-surface points or critical points, e.g., confiner center points) by means of a point registration algorithm, whereby confiners corresponding to each other are pre-determined in the original and target image or an algorithm based on the minimization of an “iconic” similarity measure follow: and/or c) transformed confiner of the original image overlapped with non-transformed confiners of the target image can be represented whereby the confiner surfaces replace the parts of the reference grid in 1b2), and to the quantities from 1a3) the distance from one point to the next adjacent point extracted in the other image is added. 4. A method for registration by means of CTT, which either a) uses confiner center points (CS) or confiner density maxima (CD) as critical points for registration and by using the method of structural outlier determination (by means of relPos, see Mattes and Demongeot 2001) starting from an initial overlapping (optionally improved by a direct use of a point registration method, see page 6) ) first determines corresponding confiner pairs and thereby only maintains pairs with best relPos (optionally according to 6(i)), on the basis of this (CD, CS or confiner shapes of which) determines the best fitting (in the sense of the error functional, see 6) as well as Fieres et al. 2001) transformation, exerts on the data confiner and repeats this process iteratively, whereby the such determined landmarks can be used for the establishment of a “point distribution model”; or b) corresponding confiners are determined as follows: for the knots, which occur after a bifurcation in the CT of one of the images, the knots/confiner having the lowest relPos value in the CT of the other image are searched, the point outliers are thereby suppressed as in claim 6, then the relPos distribution for all confiner pairs are examined, and the confiner outlier are eliminated temporarily as described in 6)(i) for point outliers, finally a point registration method under consideration of corresponding confiners are used, by an optional usage of first highly cut-down CTs and then increasingly less highly cut-down CTs a coarse to fine effect can be achieved in a) and b), which can also be combined with/replaced by the following method(s) and the images are first highly (i.e., on great scales), (e.g., Gauss-) smoothed (or presented in a high pixel resolution), and in the proceeding of the method increasingly less highly smoothed (or represented in increasingly finer resolution). 5. The method according to claim 3 using the CTT as in claim 4. 6. The method according to claim 3, which instead of the pre-processing by means of CTT identifies objects in the image space in a segmentation step and calculates the surface and/or center point of which, the objects replace the confiners, in case only (non-coherent) points are identified on the object surface (e.g., by means of a Canny edge-detector), corresponding confiner/objects cannot be pre-determined according to 3b) and particularly for the segmented objects can also individual translations and rotation or deformations be determined and the object paths can individually corrected by selected transformation types. 7. A method for the registration of a data point (DP) set on a reference point (RP) set, which determines iteratively for a series of given transformation types, which allow an increasing number of local deformations, its free parameters in such a manner, that an error functional is minimized, which compares the positions of the transformed data points with those of the reference points, thereby selecting as transformation types first a rigid (rotation and translation), then an affine transformation and then transformation types, defined by a number of checkpoints, which can freely be displaced (the free parameter of the transformation) and between the displacement vectors of which with thin-plate splines is interpolated (Bookstein 1989) and, the number of checkpoints is iteratively increased (in order to allow an increasing number of local deformation), and the initial position of the checkpoints is adapted to the form of the data points and/or the reference points or the relative position thereof, particularly for the determination of the initial position of the data point set—whereby each point can be weight depending on its distance to the next reference point—a point-lustering-algorithm can be exerted und the cluster center points (or the point with the highest density within each cluster, etc.) can be selected as the initial position, particularly also the sum of the quadratic distances of each DP to the next RP (to this term the distances of each reference point to the next data point can be added and/or a regularization term which smoothes too high warp) can be selected as error functional, outliers can be suppressed in several ways: (i) starting from the distribution of the distances between the data points and the next reference point, all data points/reference points having higher distances as the median or average+standard deviation of the distribution are eliminated or (ii) instead of the quadratic distances another function of the distance is used (e.g., amount function or see Miesmator in Press et al. 1992). 8. The method according to claim 3, 4 and/or 6, using the method of claim 7 for point registration. 9. The method according to claim 1 to 3, 4, 5, 6 and/or 8, that automatically (also partially) compares the transformation determined in ia) (or corresponding) with idealized dynamic reference types (in time and space) as, for example, spatial linear dynamic, diffusion dynamic, Hamilton dynamic, elastic/harmonic vibration etc., whereby this is performed by the steps: 0 search for the best fitting reference dynamic und determination of its parameters and of the pointwise and absolute error with reference to the transformation; 1 optionally alternatively to 1b): color/pattern encoded representation of the (pointwise) error at the surface/in the image volume. 10. A method for the partition of the space into regions of different, internally homogeneous motion, whereby the dynamic parameters are separately determined for the various regions and for this purpose first for the point set of a series of time steps a “point distribution model” is established, for which the dominating main components of motion are selected, and then each point of the space is coordinated to the component which contributes the dominating part to its displacement during the time step under examination, the various region are represented by different colors. 11. A method for the continuous numerical description of the motion by means of statistic techniques, like clustering and histogram analysis, which simplifies the comparison of motions, application of these techniques on the pointwise determined local quantities (e.g. on the in 1b3) (i) selected level), in order to mark regions, in which a certain quantity occurs especially intensive or in which particularly those values occur, which are assumed especially often (clustering on the basis of the histogram). 12. Computer program comprising a program code means for performing a method according to any one of the preceding claims, if the computer program is executed by a computer. 13. A computer program product comprising a program code means which is stored on a computer readable medium, in order to perform a method according to any one of claims 1 to 11, if the program product is executed by a computer. 14. A data processing system particularly for performing the method according to any one of claims 1 to 11. |
Mechanical seal without elastomers |
A seal that provides sealing between a rotatable shaft and a housing has a stationary part for connection to the housing and a rotary part for rotation with the shaft. The rotary part includes a sleeve for mounting on the shaft. One end of the sleeve is provided with a non-elastometric sealing arrangement. The sealing arrangement has sealing surfaces for contacting outer circumferential surfaces of the sleeve and the shaft as well as means for effecting sealing engagement between each of said sleeve and said shaft and a respective sealing surface. |
1. A seal that provides sealing between a rotatable shaft and a housing, comprising: a stationary part connected to the housing; and a rotary part for rotation with the shaft, said rotary part comprising a sleeve mounted on the shaft, one end of the sleeve comprising a non-elastomeric sealing arrangement, said sealing arrangement comprising sealing surfaces that contact the outer circumferential surfaces of the sleeve and the shaft and means for effecting sealing engagement between each of said sleeve and said shaft and a respective sealing surface. 2. A seal according to claim 1, wherein the seal is a mechanical seal. 3. (canceled) 4. A seal according to claim 1, wherein said sealing arrangement is a single sealing member. 5. (canceled) 6. (canceled) 7. (canceled) 8. (canceled) 9. (canceled) 10. A seal according to claim 1, wherein the radial space cross section between the rotatable shaft and housing is at least 0.312″ (7.9 mm) or larger. 11. A seal according to claim 1, wherein the sleeve is radially supported at one or more points along its axial length. 12. A seal according to claim 1, wherein the sleeve is positively driven at one or more points along its axial length. 13. A seal according to claim 1, wherein a component of the seal has a radially disposed relief directly adjacent to a packing member. 14. A seal according to claim 1, wherein a component of the seal is axially secured to another component of the seal, a first one of said components having an axially extending protrusion which radially locates in an axially extending recess, said first component supporting the other component in the vicinity of a packing member. 15. A seal according to claim 14, wherein said components comprise different materials. 16. A seal according to claim 1, wherein one component of the seal, located in contact with process media, has a radially disposed groove to accommodate a sacrificial component. 17. (canceled) |
COOLING OF MILK IN AN AUTOMATIC MILKING SYSTEM |
The present invention relates to a method for cooling milk in a milk storage tank of an automatic milling system comprising the steps of (i) measuring an amount of extracted milk by means of a milk flow meter, (ii) determining a cooling need for milk stored or to be stored in said milk storage tank based on said amount of milk; (iii) measuring a quantity indicative of a temperature of an inner surface area of a bottom portion of the milk storage tank; and cooling said bottom portion of said milk storage tank in consecutive periods, such that each period of cooling (τ1, τ3) is followed by a respective period of non-cooling (τ2, τ4), wherein the duration of each period of cooling and/or noncooling is based on said measured quantity indicative of said inner surface area temperature, and said cooling need. The invention also relates to an arrangement for performing the method. |
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