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Method of purifiying nucleic acid using nonwoven fabric and detection method |
A method of separating and purifying nucleic acids from samples containing cells, such as blood and culture solutions. According to the method of the invention, a cell extract obtained by cell disruption is adsorbed by a filter made of a nonwoven fabric and the nucleic acid is eluted after washing the filter. Alkaline conditions of pH 12 or higher may be employed for elution of the nucleic acid, or the filter-adsorbed nucleic acid may be eluted by treatment with active oxygen or by using a surfactant. Nucleic acids separated and purified by the method of the invention can be used in nucleic acid amplification and nucleic acid sequence analysis techniques. |
1. A method of preparing cellular nucleic acid from a sample comprising cells, which method comprises: (a) disrupting the cells to prepare a cell extract; (b) contacting the cell extract with a nonwoven fabric to adsorb the nucleic acid in the cell extract onto the nonwoven fabric; and (c) eluting the nucleic acid from the nonwoven fabric. 2. The method of claim 1, wherein the nucleic acid adsorbed onto the nonwoven fabric is eluted by heat in a temperature range of 40° C. to 100° C. 3. The method of claim 1 or 2, wherein the nucleic acid adsorbed onto the nonwoven fabric is eluted by treatment with an alkali having a pH of 12 or higher. 4. The method of claim 1 or 2, wherein the nucleic acid adsorbed onto the nonwoven fabric is fragmenting said nucleic acid. 5. The method of claim 4, wherein the nucleic acid is fragmented by active oxygen. 6. The method of claim 5, wherein the active oxygen is generated by adding a divalent metal ion to a reducing sugar. 7. The method of claim 5, wherein the active oxygen is hydrogen peroxide. 8. The method of claim 5, wherein the active oxygen is generated by adding a divalent metal ion to hydrogen peroxide. 9. The method of claim 4, wherein the nucleic acid is fragmented by an enzyme. 10. The method of claim 1 or 2, wherein the nucleic acid adsorbed onto the nonwoven fabric is eluted by treatment with a surfactant. 11. The method of claim 10, wherein the surfactant is a non-ionic surfactant or an amphoteric surfactant. 12. A method of preparing and amplifying cellular nucleic acid from a sample comprising cells, which method comprises: (a) disrupting the cells to prepare a cell extract; (b) contacting the cell extract with a nonwoven fabric to adsorb the nucleic acid in the cell extract onto said nonwoven fabric; (c) adding a nucleic acid amplification solution to said nonwoven fabric, wherein the nucleic acid adsorbed onto said nonwoven fabric is a template for amplification of the nucleic acid; (d) amplifying the nucleic acid; and (e) recovering said reaction solution. 13. The method of claim 12, wherein the nucleic acid is amplified by Polymerase Chain Reaction (PCR) or Loop-Mediated Isothermal Amplification (LAMP). 14. A method of preparing cellular nucleic acid from a sample comprising cells and detecting a specific base sequence of said nucleic acid, which method comprises: (a) disrupting the cells to prepare a cell extract; (b) contacting the cell extract with a nonwoven fabric to adsorb the nucleic acid in the cell extract onto said nonwoven fabric; (c) adding a nucleic acid sequence detection solution to said nonwoven fabric; and (d) detecting a specific nucleic acid sequence by measuring the reaction solution. 15. A method of preparing cellular nucleic acid from a sample comprising cells and detecting a specific nucleic acid sequence, which method comprises: (a) disrupting the cells to prepare a cell extract; (b) contacting the cell extract with a nonwoven fabric to adsorb the nucleic acid in the cell extract onto said nonwoven fabric; (c) adding a base sequence detection solution to said nonwoven fabric; and (d) detecting the specific nucleic acid sequence by measuring the surface of said nonwoven fabric. 16. The method of claim 14 or 15, wherein the base sequence is detected by Polymerase Chain Reaction (PCR) or Loop-Mediated Isothermal Amplification (LAMP). 17. The method of claim 15, wherein said nucleic acid sequence detection solution comprises a probe which hybridizes to the specific nucleic acid sequence for detection. 18. The method of claim 14 or 15, wherein the specific nucleic acid sequence is detected by a nucleic acid extension reaction, wherein said reaction comprises primers and a nucleic acid synthetase. 19. The method of claim 18, wherein a labeled nucleotide is incorporated during the nucleic acid extension reaction. 20. The method of claim 18, wherein pyrophosphoric acid produced by the nucleic acid extension reaction is detected. 21. A method of preparing a cellular nucleic acid adsorbed onto a nonwoven fabric, which method comprises: (a) disrupting cells to prepare a cell extract; and (b) contacting the cell extract with a nonwoven fabric to adsorb the nucleic acid in the cell extract onto the nonwoven fabric. 22. A method of preparing cellular nucleic acid from a sample comprising cells and labeling the nucleic acid, which method comprises: (a) disrupting the cells to prepare a cell extract; (b) contacting the cell extract with a nonwoven fabric to adsorb the nucleic acid in the cell extract onto the nonwoven fabric; (c) adding a nucleic acid-labeling solution to said nonwoven fabric; and (d) labeling the nucleic acid. 23. The method of claim 22, wherein the nucleic acid is labeled with biotin, fluorescence or a hapten. 24. The method of claim 22 or 23, wherein the nucleic acid adsorbed onto the nonwoven fabric is itself labeled. 25. The method of any one of claims 1, 12, 14, 15, 21, and 22, wherein the nonwoven fabric comprises a material selected from the group consisting of polyester, polypropylene, and nylon. 26. The method of any one of claims 1, 12, 14, 15, 21, and 22, wherein the material is polyethylene terephthalate. 27. The method of any one of claims 1, 12, 14, 15, 21, and 22, wherein pore size of the nonwoven fabric is 2-150 μm. 28. The method of any one of claims 1, 12, 14, 15, 21, and 22, wherein fiber diameter of the nonwoven fabric is 0.3-20 μm. 29. The method of any one of claims 1, 12, 14, 15, 21, and 22, wherein the cell extract does not comprise a viscosity enhancer, chaotropic agent or alcohol. 30. The method of any one of claims 1, 12, 14, 15, 21, and 22, wherein the cell extract comprises salt. 31. The method of claim 30, wherein the salt is selected from the group consisting of sodium salt, potassium salt, magnesium salt, calcium salt, ammonium salt, phosphate, sulfate, and hydrochloride. 32. The method of claim 30, wherein the salt is sodium chloride having a concentration of from 10 mM to 1000 mM. 33. The method of claim 30, wherein the salt is magnesium chloride having a concentration of from 1 mM to 100 mM. 34. The method of claim 30, wherein the salt is sodium phosphate having a concentration of from 2 mM to 100 mM. 35. The method of claim 30, wherein the salt is ammonium sulfate having a concentration of from 20 mM to 1000 mM. 36. The method of any one of claims 1, 12, 14, 15, 21, and 22, wherein the cells are disrupted with a cytolytic solution containing an anionic surfactant, an amphoteric surfactant or a non-ionic surfactant. 37. The method of any one of claims 1, 12, 14, 15, 21, and 22, wherein the cells are disrupted in a temperature range of 80° C. to 110° C. 38. The method of any one of claims 1, 12, 14, 15, 21, and 22, wherein the cells are disrupted in the presence of a reducing agent. 39. The method of claim 38, wherein the reducing agent is a thiol group-containing compound. 40. The method of any one of claims 1, 12, 14, 15, 21, and 22, wherein the cell extract is contacted with the nonwoven fabric by filtration. 41. The method of any one of claims 1, 12, 14, 15, 21, and 22, further comprising a step of washing the nonwoven fabric with a washing solution after adsorbing the nucleic acid onto the nonwoven fabric. 42. The method of claim 41, wherein the washing solution does not contain a chaotropic agent or alcohol. 43. The method of claim 41, wherein the washing solution is cytolytic solution. 44. The method of claim 41, wherein the washing solution is a salt solution having a concentration range of 0.5 M to 2 M. 45. A kit for preparation of cellular nucleic acid from a sample comprising cells, which kit comprises: (a) a device incorporating a nonwoven fabric; and (b) a solution comprising a cytolytic solution or a nucleic acid eluent. 46. A kit for extraction of nucleic acid from cells and preparation of a nonwoven fabric having said nucleic acid adsorbed thereonto, which kit comprises: (a) a device incorporating a nonwoven fabric; and (b) a solution comprising a cytolytic solution or a washing solution. 47. A method of eluting nucleic acid adsorbed on a solid surface, comprising the step of treating said solid surface with an alkali having a pH of 12 or higher. 48. A method of eluting nucleic acid adsorbed on a solid surface, comprising the step of treating said solid surface with active oxygen. 49. A method of eluting nucleic acid adsorbed on a solid surface, comprising the step of treating said solid surface with a surfactant. 50. A nonwoven fabric comprising an adsorbed nucleic acid. |
<SOH> BACKGROUND ART <EOH>Nucleic acids, including DNA, are generally prepared from cells by treating a sample containing the cells with SDS or Proteinase K and then denaturing and removing the protein with phenol to purify the nucleic acid (Molecular Cloning 2nd Edition, 9.16-9.23, Cold Spring Harbor Laboratory Press, 1989). Because of the trouble and time required for this procedure, however, simpler methods are desired. An example of a simpler method employing silica is disclosed in EP0389063. In this method, the cells are first treated with a chaotropic reagent to lyse the cells and release the nucleic acid. Next, the nucleic acid is adsorbed onto a nucleic acid-binding support composed of silica or a derivative thereof, and the support is centrifuged and rinsed with a chaotropic reagent or organic solvent. Finally, the nucleic acid is eluted with a low salt buffer. Although this method is simpler than the phenol method, it has a disadvantage of entailing numerous steps and requiring a centrifugation procedure. Furthermore, because it uses a chaotropic agent or ethanol which strongly inhibit PCR and other enzyme reactions, it has a disadvantage of a necessity of thoroughly removing such substances through complicated and time-consuming procedures. Japanese Examined Patent Publication HEI No. 8-24583 and Japanese Unexamined Patent Publication HEI No. 8-280384 disclose methods of purifying nucleic acids from peripheral blood leukocytes using cell adsorbing fiber aggregates such as leukocyte separation filters. According to the method described in Japanese Examined Patent Publication HEI No. 8-24583, blood is first passed through a leukocyte separation filter to adsorb the blood leukocytes onto the filter and separate them from the other blood components. The filter is then rinsed with TE Buffer (10 mM Tris; 1 mM EDTA; pH 7.6) to remove the hemoglobin and other protein. The separated and rinsed leukocytes are frozen on each filter at −80° C., for example, and then allowed to stand at room temperature for thawing of the leukocytes. Next, TE Buffer-mix (TE Buffer, 10 mM NaCl, 1.5 mM MgCl 2 , pH 7.5) is added to the fibrous material, and the leukocytes adsorbed onto the fibrous material of the filter are recovered from the fibrous material. However, extraction of the genomic DNA from the recovered leukocytes in the method of Japanese Examined Patent Publication HEI No. 8-24583 is accomplished by a prior art procedure. Specifically, a surfactant such as 10% sodium dodecyl sulfate (SDS) and a protease (Proteinase K) are added to the leukocytes, incubation is performed at 65° C. for 15 hours, and then an RNase (RNaseA) is added prior to further incubation at 37° C. for 1 hour. This is then treated with a phenol reagent and the DNA is precipitated with ethanol and purified. Japanese Unexamined Patent Publication HEI No. 8-280384 discloses a method in which adsorption of nucleated cells is followed by extraction of the nucleic acid or protein in the nucleated cells. The method of extracting the nucleic acid or protein is carried out by passing a cytolytic solution through cell-binding ultrafine fiber aggregates, for lysis or disruption of the cells. The advantage of this method is that the adsorbed cells can be directly disrupted or lysed. Moreover, since the adsorbed cells are disrupted or lysed without dissociating the adsorbed cells, the method can be carried out more easily than the method of dissociating the adsorbed cells. However the method described for purification of the nucleic acid after cell lysis is a prior art method, and a new method is not disclosed therein. That is, the cell-adsorbing ultrafine fiber aggregates are treated with purified water or a surfactant, and the nucleic acid is purified by the ordinary phenol-chloroform method from the cytolytic solution which has passed through the ultrafine fiber aggregates. Such a method of preparing nucleic acid from peripheral blood leukocytes using cell-adsorbing fiber aggregates as leukocyte separation filters has been known, but these methods had a disadvantage that after the disclosed filtering method employed up to the steps of leukocyte separation or nucleic acid extraction, the nucleic acid must be purified by existing nucleic acid purification methods, thereby complicating the procedure and requiring more time and trouble. Recently, a method of directly purifying nucleic acid from cells using a filter has been disclosed in WO00/21973. This method comprises the following steps: (1) The cell-containing sample is passed through a filter to adsorb the cells onto the filter; (2) The cells adsorbed on the filter are lysed; (3) Filtration is carried out using the filter; (4) The nucleic acid adsorbed onto the filter is rinsed; (5) The nucleic acid is eluted from the filter. The adsorbed nucleic acid elutes upon heating from 40° C. to 125° C., and the pH of the elution buffer is in the range of 5 to 11, with either a high or low salt concentration. The absorbance ratio A 260 /A 280 of the purified nucleic acid is 1.8 and it may be used as a template for PCR. WO00/21973 mentions Whatman GF/D variant filters as filters that can be used for purification of nucleic acid, and Whatman GF/C filters as examples of unusable filters. It is stated that characteristic features of the filters suitable for this method are that it is impossible to capture a purified DNA when it passes through the filters, and that when lysed cells are passed through filters, the DNA yield is reduced by 80% and thus it is not practical. Furthermore, when nucleic acid is prepared from blood by this method, the experimenter must hemolyze the erythrocytes before lysing the leukocytes. Such methods wherein purification is carried out after adsorbing the cells onto the filter also have a drawback that the filter must be selected according to the type of cell. U.S. Pat. No. 5,187,083 and U.S. Pat. No. 5,234,824 disclose methods of purifying DNA by lysing blood cells with a surfactant and then passing them through a filter with a pore size of 0.2 to 0.8 μm. The method claimed in U.S. Pat. No. 5,234,824 is a method of rapidly preparing purified DNA from blood cells, and comprises the following steps. (1) The cells are gently lysed with a solution containing a surfactant and a viscosity reinforcer for a period of 2 to 40 minutes without a high shear force, to prepare a lysate containing the cellular DNA. The DNA must have a sufficiently large molecular weight in order to be trapped by the filter. (2) The extract is then filtered with a filter having a pore size of from 0.2 to 0.8 μm to trap the DNA in the filter. (3) The filter is heated in purified water at about 100° C. for 5 to 15 minutes to extract the DNA. The extract may also contain up to 100 mM magnesium or calcium. The viscosity reinforcer used in step (1) may be polyvinyl alcohol (molecular weight: 70,000-100,000), a water-soluble polymer, sugar, a polypeptide, gelatin or the like. Also, blood must be diluted at least 10-fold in order to purify the DNA from the blood by this method. Otherwise, the viscosity is too high to allow filtration with the filter. Japanese Patent Public Inspection No. 2001-520894 discloses a method of isolating nucleic acids through the steps of (1) loading the nucleic acids onto a surface from a prescribed direction, (2) immobilizing the nucleic acids on the surface, (3) releasing the immobilized nucleic acids from the surface and (4) removing the nucleic acids released from the surface, primarily in the loading direction. A membrane may be used as the surface, and the membrane may be either hydrophobic or hydrophilic. As membrane materials there may be used nylon, polysulfone, polyethersulfone, polycarbonate or polyacrylate, as well as acrylic acid copolymers, polyurethane, polyamide, polyvinyl chloride, polyfluorocarbonate, polytetrafluoroethylene, polyvinylidene fluoride, polyvinylidene difluoride, polyethylene-tetrafluoroethylene copolymer, polyethylene-chlorotrifluoroethylene copolymer, or polyphenylene sulfide. The pores of the membrane have a diameter of 0.001-50 μm, preferably 0.01-20 μm and most preferably 0.05-10 μm. For immobilization of the nucleic acids there may be used salts of mineral acids and alkali or alkaline earth metals, salts of alkali or alkaline earth metals and monobasic, polybasic or polyfunctional organic acids, hydroxyl derivatives of aliphatic or acyclic saturated or unsaturated hydrocarbons, phenols or polyphenols, or chaotropic agents. This method has a drawback that an alcohol or a chaotropic agent must be added during adsorption in order to increase the yield of the nucleic acid. For example, when adsorbing RNA onto Hydrolon (Pall) which is a hydrophobic nylon membrane with a pore size of 1.2 μm, using 1 M NaCl as the binding solution gives a yield of 0.15 μg, whereas using 1 M NaCl containing 36% ethanol gives a 10-fold yield of 1.55 μg. Also, using 500 mM guanidium isothiocyanate containing 36% ethanol or 1 M guanidinium hydrochloride containing 36% ethanol as the binding solution gives yields of 2.3 μg or 6.7 μg, respectively. Using a chaotropic agent requires subsequent thorough washing with an alcohol-containing buffer, followed by drying. Japanese Patent Public Inspection No. 11-501504 discloses a method of isolating nucleic acid from a sample, which method comprises contacting the sample with a surfactant and an immobilizing support to bind the soluble nucleic acid in the sample onto the support, and then separating the nucleic acid-binding support from the sample. Magnetic particles marketed under the name of DYNABEADS are mentioned as particularly suitable as the immobilizing support for this invention, and are used exclusively in the examples. The surfactant may be any type of surfactant, i.e., an anionic or cationic surfactant, or a non-ionic or amphoteric surfactant. When magnetic particles are used, the sample volume will usually be from several microliters to several tens of microliters. A larger sample volume therefore requires prior concentration of the cells by a procedure such as centrifugation, or concentration of the target cells after removing the unwanted cells and substances. For example, when purifying leukocytic DNA in 0.5 ml of blood, the procedure required includes hemolysis followed by centrifugation to prepare a leukocyte pellet. The DNA is collected as a gel-like DNA/DYNABEADS complex, which easily disintegrates by such manipulation as pipetting. Care must therefore be taken to carry out the washing in a gentle manner so as not to break up the complex and thereby notably to reduce the DNA yield. Elution of solid surface-immobilized nucleic acid with either active oxygen or an alkali has not been hitherto reported. With the rapid development of human genome analysis in recent years, attempts have been made to actively link the resulting genomic information with qualitative improvements in medical care. Processing of data on single nucleotide polymorphisms (SNPs) is expected to allow discrimination of drug effects between different individuals to determine optimum drugs and doses for each patient, thereby opening the door to so-called “order-made medical treatment”. In order to realize order-made medical treatment utilizing SNPs, it is essential to develop rapid, accurate and inexpensive SNP typing techniques. Here, SNP typing techniques encompass all of the techniques used from specimen processing to output of the final examination results, including genomic extraction and purification, nucleic acid amplification and nucleic acid sequence analysis. For amplification of specific nucleic acid sequences by nucleic acid amplifying reactions such as the Polymerase Chain Reaction (PCR), or for analysis of nucleic acid sequences by SNP typing techniques, the nucleic acid target of amplification or sequence analysis is usually supplied to the reaction system in a liquid state. In all of the references cited above (WO00/21973, U.S. Pat. No. 5,187,083, U.S. Pat. No. 5,234,824, Japanese Patent Public Inspection No. 2001-520894), the nucleic acid is collected as a solution. Nucleic acid preparation methods are commonly known, but elution is time consuming and requires setting of suitable elution conditions. The time necessary for examination can be notably shortened by reducing the time required for nucleic acid elution or by shortening the elution process itself and proceeding to the subsequent examination steps. U.S. Pat. No. 5,756,126 describes a method of directly analyzing nucleic acid adsorbed onto a filter. In this method, (1) a sample containing a nucleic acid substance is added to a dry solid medium onto which is adsorbed a weak base, a chelating agent, an anionic surfactant and a component necessary for analysis, (2) the sample is stored, (3) the protein or hemoglobin in the sample is removed and (4) the sample is analyzed. U.S. Pat. No. 5,972,386 describes a method of directly analyzing nucleic acid adsorbed on a filter, using a dry solid medium for nucleic acid storage comprising the following 3 elements: (a) a solid matrix having adsorbed thereto a protein denaturing agent, (b) a component necessary for analysis, and (c) a retaining agent for retaining the component on the matrix. Specifically, (1) a sample containing a nucleic acid substance is added to a dry solid medium, (2) the sample is stored, (3) the protein or hemoglobin in the sample is removed and (4) the sample is analyzed. In these two patents, cellulose paper having uric acid, Tris, EDTA and SDS adsorbed thereon is mentioned as an example of the dry solid medium. The analysis is conducted after placing 3 μl of blood on a 9 mm 2 piece of the cellulose paper, or 100 μl of blood on a 10.0 mm 2 piece, and drying and storing it. The analysis referred to in this case is PCR or electrophoresis based on Restriction Fragment Length Polymorphism (RFLP). These methods have drawbacks because they require labor for preparation of the dry solid medium and are limited in the volumes of samples that can be added to the dry solid medium, thereby reducing the density of nucleic acid that can be immobilized on the medium, while organic solvents such as phenol or alcohol must be used to remove the protein including hemoglobin, and the procedure is thus complicated and time consuming. EP 389,063 describes a method of separating nucleic acid by mixing a sample, a chaotropic agent and a nucleic acid-binding solid phase, binding the nucleic acid to the solid phase, and then separating and washing the solid phase. As examples of nucleic acid-binding solid phases there are mentioned silica beads, as well as filters of PVDF (MILLIPORE), Nitrocellulose (Schleicher and Schuell), Hybond-N (Amersham) or the like. A method of PCR using the nucleic acid-adsorbed filter directly as a template is also described. The nucleic acid solution is mixed with a guanidine thiocyanate solution and the filter to adsorb the nucleic acid onto the filter, and it is then washed with the chaotropic agent and 70% ethanol and dried at 56° C. The filter is directly added to a PCR reaction system to allow amplification of the target nucleic acid. The drawbacks of this method, however, include a necessity of using a chaotropic agent and organic solvent, the need to completely remove the chaotropic agent and ethanol, which strongly inhibit the PCR reaction, and the resulting complex and time consuming operations. In addition, because the filter is normally used for blotting, it is not suited for the purpose of purifying nucleic acid from biological substances such as blood. WO98/51693 describes a general method for detecting cells in a sample using nucleic acid, wherein (1) cells in a sample are bound to a solid, (2) the cells are lysed, (3) the nucleic acid freed from the cells is bound to the same solid as in (1) and (4) the nucleic acid of the target cells is detected. The disadvantages of this method are that because the cells must be bound first to the solid, the solid must therefore be selected to match the type of cell, and that there is a restriction on the flow rate for filtration, because the cells must also be bound without disrupting them. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 shows 0.7% agarose electrophoresis of nucleic acids purified from blood. Lane 1: 1 kb DNA ladder (GibcoBRL); Lane 2: Marker7GT (Nippon Gene); Lane 3: nucleic acid purified with HM-3-coated A040C01; Lane 4: A040C01; Lane 5: P020A (EL); Lane 6: P020C; Lane 7: P090D; Lane 8: N05070; Lane 9: E05070. FIG. 2 shows 2% agarose electrophoresis of a PCR amplification product. The G3PDH gene was amplified by PCR using nucleic acid purified from blood as a template. Lane 1: 100 bp DNA ladder (GibcoBRL); Lane 2: negative control; Lane 3: positive control; Lane 4: HM-3-coated A040C01; Lane 5: A040C01; Lane 6: P020A (EL); Lane 7: P020C; Lane 8: P090D; Lane 9: N05070; Lane 10: E05070. FIG. 3 shows 0.7% agarose electrophoresis of nucleic acid amplified from E. coli . Lane 1: 1 kb DNA ladder (GibcoBRL); Lane 2: Marker7GT (Nippon Gene); Lane 3: nucleic acid purified with HM-3-coated A040C01. FIG. 4 shows 3% agarose electrophoresis of a PCR amplification product. The ribosomal protein L25 gene was amplified by PCR using nucleic acid purified from E. coli as a template. Lane 1: BioMarker Low (BioVentures); Lane 2: negative control; Lane 3: positive control; Lane 4: nucleic acid purified with HM-3-coated A040C01. FIG. 5 shows blood treatment times. ▪: room temperature, +Proteinase K; □: room temperature, −Proteinase K; ●: 50° C., +Proteinase K; ◯: 50° C., −Proteinase K. FIG. 6 shows leukocyte adsorption rate on nonwoven fabrics, and corresponding DNA recovery yields. FIG. 7 shows DNA elution under alkaline conditions. A nucleic acid-adsorbed nonwoven fabric was immersed in TE Buffer, 0.2N NaOH or 0.05N NaOH and heated at 95° C. for 5-20 minutes, and the amount of eluted DNA was measured. ♦: TE Buffer; ▪: 0.2N NaOH; ▴: 0.05N NaOH. FIG. 8 shows 0.7% agarose electrophoresis of nucleic acid eluted under alkaline conditions. Lane 1: 1 kb DNA ladder (GibcoBRL); Lane 2: Marker7GT (Nippon Gene); Lane 3: TE Buffer, 95° C., 20 min; Lane 4: 0.2N NaOH, 95° C., 5 min; Lane 5: 0.2N NaOH, 95° C., 10 min; Lane 6: 0.2N NaOH, 95° C., 20 min; Lane 7: 0.05N NaOH, 95° C., 5 min; Lane 8: 0.05N NaOH, 95° C., 10 min; Lane 9: 0.05N NaOH, 95° C., 20 min. FIG. 9 shows 2% agarose electrophoresis of a PCR amplification product. The G3PDH gene was amplified by PCR using nucleic acid eluted under alkaline conditions as a template. Lane 1: 100 bp DNA ladder (GibcoBRL); Lane 2: negative control; Lane 3: positive control; Lane 4: TE Buffer, 95° C., 20 min; Lane 5: 0.2N NaOH, 95° C., 5 min; Lane 6: 0.2N NaOH, 95° C., 10 min; Lane 7: 0.2N NaOH, 95° C., 20 min; Lane 8: 0.05N NaOH, 95° C., 5 min; Lane 9: 0.05N NaOH, 95° C., 10 min; Lane 10: 0.05N NaOH, 95° C., 20 min. FIG. 10 shows the effect of temperature on alkaline elution. □: TE Buffer; ▪: 0.2N NaOH; : 0.05N NaOH. FIG. 11 shows 0.7% agarose electrophoresis of nucleic acid eluted under acidic conditions. Lane 1: 1 kb DNA ladder (GibcoBRL); Lane 2: Marker7GT (Nippon Gene); Lane 3: TE Buffer, 95° C., 20 min; Lane 4: 10 mM Citrate (pH 4.5)/1 mM EDTA, 95° C., 10 min. FIG. 12 shows 2% agarose electrophoresis of a PCR amplification product. The G3PDH gene was amplified by PCR using nucleic acid eluted under acidic conditions as a template. Lane 1: 100 bp DNA ladder (GibcoBRL); Lane 2: negative control; Lane 3: positive control; Lane 4: TE Buffer, 95° C., 20 min; Lane 5: 10 mM Citrate (pH 4.5)/1 mM EDTA, 95° C., 10 min. FIG. 13 shows the effects of alkaline elution on a DNA probe. A DIG-labeled DNA probe was treated with 0.05N NaOH for 5 minutes at 95° C. (spots 1,2) and untreated (3,4). The amounts of immobilized Lambda DNA were 10 ng (spots 1,3) and 1 ng (spots 2,4). FIG. 14 shows 0.7% agarose electrophoresis of nucleic acid eluted with hydrogen peroxide. Elution was performed with 3% hydrogen peroxide containing 0.1 mM CuCl 2 . Lane 1: 1 kb DNA ladder (GibcoBRL); Lane 2: hydrogen peroxide treatment, room temperature, 1 min; Lane 3: hydrogen peroxide treatment, room temperature, 2 min; Lane 4: hydrogen peroxide treatment, room temperature, 3 min; Lane 5: hydrogen peroxide treatment, room temperature, 5 min. FIG. 15 shows 2% agarose electrophoresis of a PCR amplification product using hydrogen peroxide-eluted nucleic acid as a template. A human G3PDH partial sequence was amplified by PCR using nucleic acid eluted with 3% hydrogen peroxide containing 0.1 mM CuCl 2 as a template. Lane 1: 100 bp DNA ladder (GibcoBRL); Lane 2: hydrogen peroxide treatment, room temperature, 1 min; Lane 3: hydrogen peroxide treatment, room temperature, 2 min; Lane 4: hydrogen peroxide treatment, room temperature, 3 min; Lane 5: hydrogen peroxide treatment, room temperature, 5 min. FIG. 16 shows 0.7% agarose electrophoresis of nucleic acid eluted with a reducing sugar and metal ion. Elution was performed with an active oxygen solution containing 100 mM D-ribose 5-phosphate and 0.1 mM CuCl 2 . Lane 1: 1 kb DNA ladder (GibcoBRL); Lane 2: 50° C., 1 min; Lane 3: 50° C., 3 min; Lane 4: 50° C., 5 min; Lane 5: 50° C., 10 min; Lane 6: 50° C., 30 min; Lane 7: Elution with TE Buffer, 95° C., 20 min (control). FIG. 17 shows 2% agarose electrophoresis of a PCR amplification product obtained using nucleic acid eluted with a reducing sugar as a template. A human G3PDH partial sequence was amplified by PCR using nucleic acid eluted with an active oxygen solution containing 100 mM D-ribose 5-phosphate and 0.1 mM CuCl 2 as a template. Lane 1: 100 bp DNA ladder (GibcoBRL); Lane 2: 50° C., 1 min; Lane 3: 50° C., 3 min; Lane 4: 50° C., 5 min; Lane 5: 100 bp DNA ladder; Lane 6: 50° C., 10 min; Lane 7: 50° C., 30 min. FIG. 18 shows 0.7% agarose electrophoresis of nucleic acid eluted from a nonwoven fabric with a restriction enzyme. Lane 1: 1 kb DNA ladder (GibcoBRL); Lane 2: room temperature, 5 min; Lane 3: room temperature, 5 min (column purification); Lane 4: room temperature, 10 min; Lane 5: room temperature, 10 min (column purification); Lane 6: room temperature, 30 min; Lane 7: room temperature, 30 min (column purification); Lane 8: 37° C., 5 min; Lane 9: 37° C., 5 min (column purification); Lane 10: 37° C., 10 min; Lane 11: 37° C., 10 min (column purification); Lane 12: 37° C., 30 min; Lane 13: 37° C., 30 min (column purification); Lane 14: 1 kb DNA ladder (GibcoBRL). FIG. 19 shows the effect of active oxygen treatment on hybridization. Hybridization was performed using a DIG-labeled probe treated with final concentrations of 50 mM D-ribose 5-phophate and 25 μM CuCl 2 , and DNA immobilized on a nylon membrane. DIG-labeled DNA probes without active oxygen treatment or treated in TE Buffer at 95° C. for 20 minutes were used as controls. FIG. 20 shows PCR performed using nonwoven fabric-adsorbed human genomic DNA as a template. Blood-extracted nucleic acid was adsorbed onto a nonwoven fabric and used for amplification of the G3PDH gene by PCR. Lane 1: 100 bp DNA ladder (GibcoBRL); Lane 2: negative control; Lane 3: positive control; Lane 4: HM-3-coated A040C01; Lane 5: A040C01; Lane 6: P020A (EL); Lane 7: P090D; Lane 8: N05070; Lane 9: E05070. FIG. 21 shows PCR performed using nonwoven fabric-adsorbed E. coli genomic DNA as a template. Nucleic acid extracted from E. coli was adsorbed onto HM-3-coated A040C01 and used for amplification of the ribosomal protein L25 gene by PCR. Lane 1: BioMarker Low (BioVentures); Lane 2: negative control; Lane 3: positive control; Lane 4: HM-3-coated A040C01. FIG. 22 shows detection of nonwoven fabric-adsorbed human genomic DNA. A radioisotope-labeled DNA probe was hybridized onto two different nonwoven fabrics HM-3-coated A040C01 and A040C01, onto which were adsorbed human genomic DNA extracted from 0.25 ml of blood. Hybond-N+ nylon membranes (Amersham Pharmacia Biotech) immobilizing 1 μg of human genomic DNA (hgDNA) and 1 μg of Lambda DNA (λDNA) were used as a control. The legend indicates the mixing proportions of the human G3PDH probe and Lambda DNA probe. All the mixtures had a total radioactivity of 448,000 cpm. FIG. 23 shows scanning electron micrographs of nonwoven fabric-adsorbed nucleic acid. A: Nonwoven fabric P03050 (control), B: Blood nucleic acid-adsorbed nonwoven fabric P03050. FIG. 24 shows the examination of surfactants used in cytolytic solutions. X-114 (Triton X-100), TOC (Nissan Dispanol TOC), X-100 (Triton X-100), CA630 (Igepal CA630), NS-208.5 (Nissan Nonion NS-208.5), HPC (hexadecylpyridinium chloride), HPB (hexadecylpyridinium bromide), HTAC (hexadecyltrimethylammonium chloride), HTAB (hexadecyltrimethylammonium bromide), CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate), CHAPSO (3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate), SDS (sodium dodecyl sulfate). FIG. 25 shows the dependency on NaCl concentration of purified genomic DNA adsorption. ♦ A040C01; ▴ A066A; ▪ A040B FIG. 26 shows the dependency on MgCl 2 concentration of purified genomic DNA adsorption. ♦ A040C01; ▴ A066A; ▪ A040B; ● E01030 FIG. 27 shows the dependency on HPO 4 2− concentration of purified genomic DNA adsorption. ♦ A040C01; ▴ A066A; ▪ A040B; ● A040C01/HM-3 FIG. 28 shows the dependency on (NH 4 ) 2 SO 4 concentration of purified genomic DNA adsorption. ♦ A040C01; ▪ A066A; ▴ E01030 FIG. 29 shows the effect of ethanol on purified genomic DNA adsorption. Ethanol was added to 10 mM phosphoric acid buffer (pH 7.4) and the effect on purified genomic DNA adsorption was examined. ▪ 10 mM phosphoric acid (pH 7.4); 10 mM phosphoric acid (pH 7.4)/10% ethanol; 10 mM phosphoric acid (pH 7.4)/20% ethanol; 10 mM phosphoric acid (pH 7.4)/40% ethanol. FIG. 30 shows shaking adsorption of purified genomic DNA (1). ▪ 10 mM Tris (pH 8)/1 mM EDTA/50 mM NaCl; 10 mM Tris (pH 8)/2 mM MgCl 2 ; 50 mM Na 2 HPO 4 /NaH 2 PO 4 (pH 7.4). FIG. 31 shows shaking adsorption of purified genomic DNA (2) ▪ 10 mM phosphoric acid (pH 7.4); 10 mM phosphoric acid (pH 7.4)/0.2M ammonium sulfate; □ 10 mM phosphoric acid (pH 7.4)/0.5M ammonium sulfate; □ 10 mM phosphoric acid (pH 7.4)/1.0M ammonium sulfate. FIG. 32 shows screening of nonwoven fabrics by DNA yields. Fresh blood was used as specimens. Gr1 to Gr5 represent different blood donors of the blood used. FIG. 33 shows screening of nonwoven fabrics by DNA purity (A 260 /A 280 ). The same samples as in FIG. 31 were used for measurement of the absorbance ratio A 260 /A 280 . FIG. 34 shows nucleic acid purification from E. coli -added sputum by heat treatment (1). These are the results of detecting the E. coli -derived nucleic acid of Example 27. PCR amplification was followed by analysis by agarose gel electrophoresis. Lane 1: DNA molecular weight marker; Lane 2: PCR amplification product of nucleic acid extract obtained from 10 5 E. coli -added sputum specimen; Lane 3: PCR amplification product of nucleic acid extract obtained from 10 4 E. coli -added sputum specimen; Lane 4: PCR amplification product of nucleic acid extract obtained from 10 3 E. coli -added sputum specimen; Lane 5: PCR amplification product of nucleic acid extract obtained from E. coli -free sputum specimen. FIG. 35 shows nucleic acid purification from E. coli -added sputum by heat treatment (2). These are the results of detecting the human-derived nucleic acid of Example 27. PCR amplification was followed by analysis by agarose gel electrophoresis. Lane 1: DNA molecular weight marker; Lane 2: PCR amplification product of nucleic acid extract obtained from 10 5 E. coli -added sputum specimen; Lane 3: PCR amplification product of nucleic acid extract obtained from 10 4 E. coli -added sputum specimen; Lane 4: PCR amplification product of nucleic acid extract obtained from 10 3 E. coli -added sputum specimen; Lane 5: PCR amplification product of nucleic acid extract obtained from E. coli -free sputum specimen. FIG. 36 shows nucleic acid purification from E. coli -added sputum by reducing agent treatment (1). These are the results of detecting the E. coli -derived nucleic acid of Example 28. PCR amplification was followed by analysis by agarose gel electrophoresis. Lane 1: DNA molecular weight marker; Lane 2: PCR amplification product of nucleic acid extract obtained from 10 5 E. coli -added sputum specimen; Lane 3: PCR amplification product of nucleic acid extract obtained from 10 4 E. coli -added sputum specimen; Lane 4: PCR amplification product of nucleic acid extract obtained from 10 3 E. coli -added sputum specimen; Lane 5: PCR amplification product of nucleic acid extract obtained from E. coli -free sputum specimen. FIG. 37 shows nucleic acid purification from E. coli -added sputum by reducing agent treatment (2). These are the results of detecting the human-derived nucleic acid of Example 28. PCR amplification was followed by analysis by agarose gel electrophoresis. Lane 1: DNA molecular weight marker; Lane 2: PCR amplification product of nucleic acid extract obtained from 10 5 E. coli -added sputum specimen; Lane 3: PCR amplification product of nucleic acid extract obtained from 10 4 E. coli -added sputum specimen; Lane 4: PCR amplification product of nucleic acid extract obtained from 10 3 E. coli -added sputum specimen; Lane 5: PCR amplification product of nucleic acid extract obtained from E. coli -free sputum specimen. FIG. 38 shows nucleic acid extension reaction on a nonwoven fabric. The two primers bACT1 and bACT2 were hybridized to a Klenow Large Fragment enzyme reaction solution in amounts of 10 μg, 100 ng, 1 ng and 0 ng, respectively and an extension reaction was conducted at 37° C. FIG. 39 shows amplification and detection of nucleic acid on a nonwoven fabric by the LAMP method. Bovine genomic DNA amplified with a Loopamp was subjected to 1% agarose gel electrophoresis. Lane 1: molecular weight marker; Lane 2: human genomic DNA-adsorbed nonwoven fabric as a template; Lane 3: bovine genomic DNA-adsorbed nonwoven fabric as a template; Lane 4: supernatant of bovine genomic DNA-adsorbed nonwoven fabric heat eluted in TE Buffer as a template; Lane 5: no template; Lane 6: kit-supplied control bovine genomic DNA as a template. FIG. 40 shows hybridization of genomic DNA purified with nonwoven fabric (1). The test was conducted by the TE Buffer elution method and alkaline elution method. A 1 μl portion of DNA solution purified with a nonwoven fabric was dotted on a Hybond N+ membrane and used as the target for hybridization. FIG. 41 shows hybridization of genomic DNA purified with nonwoven fabric (2). The test was conducted by the TE Buffer elution method and hydrogen peroxide elution method. A 1 μl portion of DNA solution purified with a nonwoven fabric was dotted on a Hybond N+ membrane and used as the target for hybridization. FIG. 42 shows hybridization of genomic DNA purified with nonwoven fabric (3). The test was conducted by the TE Buffer elution method, alkaline elution method and hydrogen peroxide elution method, using labeled DNA purified with a nonwoven fabric as the hybridization probe. FIG. 43 is a graph showing elution of nucleic acid using a surfactant. A 500 μl portion of an aqueous solution or TE Buffer containing 0.5% of a surfactant was added, and heating was carried out at 80° C. for 20 minutes for elution of the nucleic acid adsorbed on the nonwoven fabric. The amount of nucleic acid eluted upon heating in TE Buffer at 95° C. for 20 minutes was defined as 100%. FIG. 44 shows electrophoresis of nucleic acid eluted with a surfactant. The eluted sample of FIG. 43 was concentrated and purified with a NucleoSpin column and subjected to 0.7% agarose gel electrophoresis. FIG. 45 shows electrophoresis of a PCR amplification product of nucleic acid eluted with a surfactant. The eluted sample of FIG. 43 was used as the template for PCR amplification of the G3PDH gene. FIG. 46 shows electrophoresis of a PCR product. The surfactant used for genome elution was added to a PCR reaction system for PCR amplification of the G3PDH gene. A 1 kb ladder (GibcoBRL) was used as the DNA marker. FIG. 47 shows hybridization using a Biotin Chem-Link-labeled nucleic acid probe. A Biotin Chem-Link-labeled nucleic acid probe 1 or 2 was hybridized to a membrane dotted with human genomic DNA or Lambda DNA. detailed-description description="Detailed Description" end="lead"? |
Process for the preparation of hydroxylammonium |
The invention relates to a process for the preparation of hydroxylammonium, said process comprising the steps of: a) feeding gaseous hydrogen to a reaction mixture, said reaction mixture comprising an aqueous reaction medium and a gaseous phase; b) catalytically reducing, in said reaction mixture, nitrate or nitrogen oxide with hydrogen to form the hydroxylammonium; c) withdrawing a gas mixture from the reaction mixture, said gas mixture comprising gaseous hydrogen and gaseous non-hydrogen compounds; d) separating at least part of the gaseous non-hydrogen compounds from the gas mixture to obtain a hydrogen-enriched gas; and e) passing the hydrogen-enriched gas to a hydrogenation zone. |
1. Process for the preparation of hydroxylammonium, said process comprising: a) feeding gaseous hydrogen to a reaction mixture, said reaction mixture comprising an aqueous reaction medium and a gaseous phase; b) catalytically reducing, in said reaction mixture, nitrate with hydrogen to form the hydroxylammonium; c) withdrawing a gas mixture from the reaction mixture, said gas mixture comprising gaseous hydrogen and gaseous non-hydrogen compounds; d) separating at least part of the gaseous non-hydrogen compounds from the gas mixture, resulting in a hydrogen-enriched gas; and e) passing the hydrogen-enriched gas to a hydrogenation zone. 2. Process according to claim 1, wherein said process comprises passing the hydrogen-enriched gas to the reaction mixture. 3. Process according to claim 1, wherein said process comprises separating said at least part of the non-hydrogen compounds from said gas mixture by using a membrane. 4. Process according to claim 1, wherein said process comprises separating said at least part of the non-hydrogen compounds from said gas mixture by using pressure swing adsorption. 5. Process according to claim 1, wherein said process comprises separating said at least part of the non-hydrogen compounds from said gas mixture by using cryogene distillation. 6. Process according to claim 1, wherein the molar fraction of hydrogen in the gas mixture is higher than 0.4. 7. Process according to claim 6, wherein the molar fraction of hydrogen in the gas mixture is higher than 0.5. 8. Process according to claim 1, wherein the hydrogen partial pressure in the reaction mixture is higher than 1.0 MPa. 9. Process according to claim 8, wherein the hydrogen partial pressure in the reaction mixture is higher than 1.3 MPa. 10. Process according to claim 1, wherein said at least part of the gaseous non-hydrogen compounds include N2. 11. Process according to claim 1, wherein said at least part of the gaseous non-hydrogen compounds include CH4. 12. Process according to claim 1, wherein said at least part of the gaseous non-hydrogen compounds include N2O. 13. Process according to, wherein said process is a continuous process. |
Information processing apparatus and method |
This invention relates to an information processing device and method that enable generation of an unlearned new pattern. Data xt corresponding to a predetermined time series pattern is inputted to an input layer (11) of a recurrent neural network (1), and a prediction value x*t+1 is acquired from an output layer 13. A difference between teacher data xt+1 and the prediction value x*t+1 is learned by a back propagation method, and a weighting coefficient of an intermediate layer 12 is set at a predetermined value. After the recurrent neural network is caused to learn plural time series patterns, a parameter having a different value from the value in learning is inputted to parametric bias nodes (11-2), and an unlearned time series pattern corresponding to the parameter is generated from the output layer (13). This invention can be applied to a robot. |
1. An information processing device for outputting a time series pattern, comprising: input means for inputting a time series pattern; model decision means for deciding a model based on a common nonlinear dynamic system having one or more feature parameters that can be operated from outside with respect to each of plural time series patterns inputted by the input means; arithmetic means for calculating a value of the feature parameter on the basis of the decided model; and output means for setting a value that is different from the value calculated by the arithmetic means, as the feature parameter, and performing inverse operation of the calculation of the value of the feature parameter, thereby outputting a new time series pattern. 2. The information processing device as claimed in claim 1, wherein the nonlinear dynamic system is a recurrent neural network with an operating parameter. 3. The information processing device as claimed in claim 1, wherein the feature parameter indicates a dynamic structure of the time series pattern in the nonlinear dynamic system. 4. The information processing device as claimed in claim 3, wherein the output means outputs a new time series pattern having a dynamic structure that is shareable with plural inputted time series patterns. 5. An information processing method adapted for an information processing device for outputting a time series pattern, the method comprising; an input step of inputting a time series pattern; a model decision step of deciding a model based on a common nonlinear dynamic system having one or more feature parameters that can be operated from outside with respect to each of plural time series patterns inputted by the processing of the input step; an arithmetic step of calculating a value of the feature parameter on the basis of the decided model; and an output step of setting a value that is different from the value calculated by the processing of the arithmetic step, as the feature parameter, and performing inverse operation of the calculation of the value of the feature parameter, thereby outputting a new time series pattern. 6. A program storage medium having a computer-readable program stored therein, the program being adapted for an information processing device for outputting a time series pattern, the program comprising: an input step of inputting a time series pattern; a model decision step of deciding a model based on a common nonlinear dynamic system having one or more feature parameters that can be operated from outside with respect to each of plural time series patterns inputted by the processing of the input step; an arithmetic step of calculating a value of the feature parameter on the basis of the decided model; and an output step of setting a value that is different from the value calculated by the processing of the arithmetic step, as the feature parameter, and performing inverse operation of the calculation of the value of the feature parameter, thereby outputting a new time series pattern. 7. A computer program for controlling an information processing device that outputs a time series pattern, the program comprising: an input step of inputting a time series pattern; a model decision step of deciding a model based on a common nonlinear dynamic system having one or more feature parameters that can be operated from outside with respect to each of plural time series patterns inputted by the processing of the input step; an arithmetic step of calculating a value of the feature parameter on the basis of the decided model; and an output step of setting a value that is different from the value calculated by the processing of the arithmetic step, as the feature parameter, and performing inverse operation of the calculation of the value of the feature parameter, thereby outputting a new time series pattern. |
<SOH> BACKGROUND ART <EOH>Recently, various studies on human and animal brains have been made. It is known that a neural network can be used as a brain model. In a neural network, as a predetermined pattern is learned, the learned pattern can be identified. However, there is a problem that the neural network cannot generate a new pattern. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a view showing the structure of a recurrent neural network to which the present invention is applied. FIG. 2 is a flowchart for explaining learning processing of the recurrent neural network of FIG. 1 . FIG. 3 is a flowchart for explaining coefficient setting processing of the recurrent neural network of FIG. 1 . FIG. 4A is a view showing an exemplary time series pattern having different amplitude and the same cycle. FIG. 4B is a view showing an exemplary time series pattern having different amplitude and the same cycle. FIG. 4C is a view showing an exemplary time series pattern having different amplitude and the same cycle. FIG. 5A is a view showing an exemplary time series pattern having a different cycle and the same amplitude. FIG. 5B is a view showing an exemplary time series pattern having a different cycle and the same amplitude. FIG. 5C is a view showing an exemplary time series pattern having a different cycle and the same amplitude. FIG. 6 is a view showing an exemplary learned pattern. FIG. 7 is a view showing an exemplary learned pattern. FIG. 8 is a flowchart for explaining time series pattern generation processing of the recurrent neural network of FIG. 1 . FIG. 9 is a view showing an exemplary time series pattern to be generated. FIG. 10 is a view showing change of error in the case of causing the neural network to learn a first pattern. FIG. 11 is a view showing a target in the case of causing the neural network to learn the first pattern. FIG. 12 is a view showing an output in the case of causing the neural network to learn the first pattern. FIG. 13 is a view showing change of parametric bias in the case of causing the neural network to learn the first pattern. FIG. 14 is a view showing change of error in the case of causing the neural network to learn a second pattern. FIG. 15 is a view showing a target in the case of causing the neural network to learn the second pattern. FIG. 16 is a view showing an output in the case of causing the neural network to learn the second pattern. FIG. 17 is a view showing change of parametric bias in the case of causing the neural network to learn the second pattern. FIG. 18 is a view showing change of error in the case of causing the neural network to learn a third pattern. FIG. 19 is a view showing a target in the case of causing the neural network to learn the third pattern. FIG. 20 is a view showing an output in the case of causing the neural network to learn the third pattern. FIG. 21 is a view showing change of parametric bias in the case of causing the neural network to learn the third pattern. FIG. 22 is a view showing an example of generated pattern. FIG. 23 is a view showing another example of generated pattern. FIG. 24 is a view showing an output corresponding to FIG. 12 . FIG. 25 is a view showing an output corresponding to FIG. 16 . FIG. 26 is a view showing an output corresponding to FIG. 20 . FIG. 27 is a view showing the relation between change of the cycle of the patterns of FIGS. 22 to 26 and the parametric bias. FIG. 28 is a view showing the relation between change of the amplitude of the patterns of FIGS. 22 to 26 and the parametric bias. FIG. 29 is a view showing an exemplary pattern to be learned. FIG. 30 is a view showing another exemplary pattern to be learned. FIG. 31 is a view showing a pattern generated when the patterns shown in FIGS. 29 and 30 are learned. FIG. 32 is a view showing change of error in the case of causing the neural network to learn a fourth pattern. FIG. 33 is a view showing a target in the case of causing the neural network to learn the fourth pattern. FIG. 34 is a view showing an output in the case of causing the neural network to learn the fourth pattern. FIG. 35 is a view showing change of parametric bias in the case of causing the neural network to learn the fourth pattern. FIG. 36 is a view showing change of error in the case of causing the neural network to learn a fifth pattern. FIG. 37 is a view showing a target in the case of causing the neural network to learn the fifth pattern. FIG. 38 is a view showing an output in the case of causing the neural network to learn the fifth pattern. FIG. 39 is a view showing change of parametric bias in the case of causing the neural network to learn the fifth pattern. FIG. 40 is a view showing an example of generated pattern. FIG. 41 is a view showing an example of generated pattern. FIG. 42 is a view showing an example of generated pattern. FIG. 43 is a view showing an output corresponding to FIG. 34 . FIG. 44 is a view showing an output corresponding to FIG. 38 . FIG. 45 is a view showing the relation between the cycle of the patterns of FIGS. 40 to 44 and the parametric bias. FIG. 46 is a view showing the relation between the amplitude of the patterns of FIGS. 40 to 44 and the parametric bias. FIG. 47 is a block diagram showing an exemplary structure of a personal computer to which the present invention is applied. detailed-description description="Detailed Description" end="lead"? |
Compounds and methods for the inhibition of compounds cruzi |
The present invention relates to compounds according to the formula (I): Where RA is a C1-C10 substituted or unsubstituted linear, branch-chained or cyclic alkyl or alkenyl group or a phenyl group according to the formula (II): RB is a C1-C10 substituted or unsubstituted linear, branch-chained or cyclic alkyl or alkenyl group or a phenyl group of the formula (III): R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are each independently selected from H, C1-C10 (preferably a C1-C4) alkyl or alkenyl group, CF3, F, Cl, Br, I, CN, NO2, NH2, NHR, NRR, COR (acyl group), OR (hydroxyl or ether group), CO2R (carboxylic acid or ester group), or COSR (thioester group) where R is H or a C1-C10 (preferably a C1-C4) alkyl or alkenyl group, an unsubstituted or substituted aryl (preferably, phenyl) or heterocycle group, or a (IV) group, where R3 is H, a C1-C10 (preferably a C1-C4) alkyl, alkenyl, ether or a thioether group; and R11 and R12 are independently selected from H or a C1-C3 alkyl or alkenyl group, or a pharmaceutically acceptable salt thereof and methods for treating infections caused by protozoal, fungal and/or bacterial agents such as Trypanosoma cruzi, Mycobacterium spp., Leishmania spp., Cryptococcus spp., Aspergillus spp., Histoplasma spp., Candida spp., especially Candida albicans, Pneumocystis carinii, Trichophyton spp., Microsporum spp., Malassezia spp., Rhizopus spp., Pseudallescheria spp., Blastomyces dermatitidis and Coccidiodes spp., among others. |
1. A compound according to formula I: Where RA is a C1-C10 substituted or unsubstituted linear, branch-chained or cyclic alkyl or alkenyl group or a phenyl group according to the formula: RB is a C1-C10 substituted or unsubstituted linear, branch-chained or cyclic alkyl or alkenyl group or a phenyl group of the formula: R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are each independently selected from H, a C1-C10 alkyl or alkenyl group, CF3, F, Cl, Br, I, CN, NO2, NH2, NHR, NRR, COR, OR, CO2R, or COSR, where R is H or a C1-C10 alkyl or alkenyl group, an unsubstituted or substituted aryl or heterocycle group, or a group, where R3 is H, a C1-C10 alkyl, alkenyl, ether or a thioether group; and R11 and R12 are independently selected from H or a C1-C3 alkyl or alkenyl group, or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1 wherein RA and RB are substituted phenyl groups, R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are independently H or a C1-C3 alkyl group, CO2R, OR, CN, CF3, Cl, Br, NRR, NO2, unsubstituted phenyl and R is H or CH3 . 3. The compound according to claim 1 wherein RA and RB are substituted phenyl groups, R1, R2 and R3 are independently selected from H, Phenyl, CN, CF3, Cl, Br, OR, NRR, NO2 and CH3, R4, R5, R6, R9, R10, R11 and R12 are each H, R7 is phenyl or substituted phenyl, R8 is selected from H, CN, CF3, Cl, Br, OR, NRR, NO2, CH3 and COOR and R is H or C1-C3 alkyl. 4. The compound according to claim 3 wherein when one of R1, R2 and R3 is other than H, the other of R1, R2 or R3 are H and R is H or CH3. 5. The compound according to claim 4 wherein R1 is NH2, CN or Br, R7 is phenyl and R8 is COOR. 6. The compound according to claim 5 wherein R2 and R3 are both H. 7. The compound according to 6 wherein R1 is NH2 and R is CH3. 8. The compound according to claim 3 wherein R7 is phenyl, R8 is COOR and R is a C1-C3 alkyl. 9. The compound according to claim 8 wherein R1 and R2 are H, R3 is H, CN, phenyl, CH3, OCH3, Br or Cl and R is CH3. 10. The compound according to claim 9 wherein R3 is phenyl, Cl or CH3. 11. The compound according to claim 10 wherein R3 is phenyl. 12. The compound according to claim 3 wherein R1 and R3 are H, R2 is CN, NO2, CH3, Cl, Br or CF3, R7 is phenyl, and R is C1-C3 alkyl. 13. The compound according to claim 12 wherein R2 is CH3, Cl or Br and R is CH3. 14. The compound according to claim 3 wherein R1 and R2 are H, R3 is CN, NO2, CH3, Cl, Phenyl, Br or CF3, R7 is phenyl and R8 is H. 15. The compound according to claim 14 wherein R3 is CN or phenyl. 16. The compound according to claim 14 wherein R3 is phenyl. 17. A pharmaceutical composition comprising an effective amount of a compound according to formula I: Where RA is a C1-C10 substituted or unsubstituted linear, branch-chained or cyclic alkyl or alkenyl group or a phenyl group according to the formula: RB is a C1-C10 substituted or unsubstituted linear, branch-chained or cyclic alkyl or alkenyl group or a phenyl group of the formula: R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are each independently selected from H, C1-C10 alkyl or alkenyl group, CF3, F, Cl, Br, I, CN, NO2, NH2, NHR, NRR, COR, OR, CO2R, or COSR, where R is H or a C1-C10 alkyl or alkenyl group, an unsubstituted or substituted aryl or heterocycle group, or a group, where R3 is H, a C1-C10 alkyl, alkenyl, ether or a thioether group; and R11 and R2 are independently selected from H or a C1-C3 alkyl or alkenyl group, or a pharmaceutically acceptable salt thereof, optionally in combination with a pharmaceutically acceptable additive carrier or excipient. 18. The composition according to claim 17 wherein RA and RB are substituted phenyl groups, R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are independently H or a C1-C3 alkyl group, CO2R, OR, CN, CF3, Cl, Br, NRR, NO2, unsubstituted phenyl and R is H or CH3 . 19. The composition according to claim 17 wherein RA and RB are substituted phenyl groups, R1, R2 and R3 are independently selected from H, Phenyl, CN, CF3, Cl, Br, OR, NRR, NO2 and CH3, R4, R5,R6, R9, R10, R11 and R12 are each H, R7 is phenyl or substituted phenyl, R8 is selected from H, CN, CF3, Cl, Br, OR, NRR, NO2, CH3 and COOR and R is H or C1-C3 alkyl. 20. The composition according to claim 19 wherein when one of R1, R2 and R3 is other than H, the other of R1, R2 or R3 are H and R is H or CH3. 21. The composition according to claim 20 wherein R1 is NH2, CN or Br, R7 is phenyl and R8 is COOR. 22. The composition according to claim 21 wherein R2 and R3 are both H. 23. The composition according to 22 wherein R1 is NH2 and R is CH3. 24. The composition according to claim 19 wherein R7 is phenyl, R8 is COOR and R is a C1 to C3 alkyl. 25. The composition according to claim 24 wherein R1 and R2 are H, R3 is H, CN, phenyl, CH3, OCH3, Br or Cl and R is CH3. 26. The composition according to claim 25 wherein R3 is phenyl, Cl or CH3. 27. The composition according to claim 26 wherein R3 is phenyl. 28. The composition according to claim 19 wherein R1 and R3 are H, R2 is CN, NO2, CH3, Cl, Br or CF3, R7 is phenyl, and R is C1-C3 alkyl. 29. The composition according to claim 28 wherein R2 is CH3, Cl or Br and R is CH3. 30. The composition according to claim 19 wherein R1 and R2 are H, R3 is CN, NO2, CH3, Cl, Phenyl, Br or CF3, R7 is phenyl and R8 is H. 31. The composition according to claim 30 wherein R3 is CN or phenyl. 32. The composition according to claim 31 wherein R3 is phenyl. 33. A method of treating an infection in a patient caused by an agent selected from the group consisting of Trypanosoma cruzi, Mycobacterium spp., Leishmania spp., Cryptococcus spp., Aspergillus spp., Histoplasma spp., Candida spp., Pneumocystis carnii, Trichophyton spp., Microsporum spp. Malassezia spp., Rhizopus spp., Pseudallescheria spp., Blastomyces dermatitidis and Coccidiodes spp. comprising administering to said patient in need thereof an effective amount of a compound according to formula I: Where RA is a C1-C10 substituted or unsubstituted linear, branch-chained or cyclic alkyl or alkenyl group or a phenyl group according to the formula: RB is a C1-C10 substituted or unsubstituted linear, branch-chained or cyclic alkyl or alkenyl group or a phenyl group of the formula: R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are each independently selected from H, C1-C10 (preferably a C1-C4) alkyl or alkenyl group, CF3, F, Cl, Br, I, CN, NO2, NH2, NHR, NRR, COR, OR, CO2R, or COSR, where R is H or a C1-C10 (preferably a C1-C4) alkyl or alkenyl group, an unsubstituted or substituted aryl or heterocycle group, or a group, where R3 is H, a C1-C10 (preferably a C1-C4) alkyl, alkenyl, ether or a thioether group; and R11 and R12 are independently selected from H or a C1-C3 alkyl or alkenyl group, or a pharmaceutically acceptable salt thereof, optionally in combination with a pharmaceutically acceptable additive carrier or excipient. 34. The method according to claim 33 wherein RA and RB are substituted phenyl groups, R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are independently H or a C1-C3 alkyl group, CO2R, OR, CN, CF3, Cl, Br, NRR, NO2, unsubstituted phenyl and R is H or CH3. 35. The method according to claim 33 wherein RA and RB are substituted phenyl groups, R1, R2 and R3 are independently selected from H, Phenyl, CN, CF3, Cl, Br, OR, NRR, NO2 and CH3, R4, R5, R6, R9, R10, R11 and R12 are each H, R7 is phenyl or substituted phenyl, R8 is selected from H, CN, CF3, Cl, Br, OR, NRR, NO2, CH3 and COOR and R is H or C1-C3 alkyl. 36. The method according to claim 35 wherein when one of R1, R2 and R3 is other than H, the other of R1, R2 or R3 are H and R is H or CH3. 37. The method according to claim 36 wherein R1 is NH2, CN or Br, R7 is phenyl and R8 is COOR. 38. The method according to claim 37 wherein R2 and R3 are both H. 39. The method according to 38 wherein R1 is NH2 and R is CH3. 40. The method according to claim 35 wherein R7 is phenyl, R8 is COOR and R is a C1 to C3 alkyl. 41. The method according to claim 40 wherein R1 and R2 are H, R3 is H, CN, phenyl, CH3, OCH3, Br or Cl and R is CH3. 42. The method according to claim 41 wherein R3 is phenyl, Cl or CH3. 43. The method according to claim 42 wherein R3 is phenyl. 44. The method according to claim 35 wherein R1 and R3 are H, R2 is CN, NO2, CH3, Cl, Br or CF3, R7 is phenyl, and R is C1-C3 alkyl. 45. The method according to claim 43 wherein R2 is CH3, Cl or Br and R is CH3. 46. The method according to claim 35 wherein R1 and R2 are H, R3 is CN, NO2, CH3, Cl, Phenyl, Br or CF3 R7 is phenyl and R8 is H. 47. The method according to claim 46 wherein R3 is CN or phenyl. 48. The method according to claim 47 wherein R3 is phenyl. 49. The method according to claim 33 wherein said agent is Trypanosoma cruzi. 50. The method according to claim 35 wherein said agent is Trypanosoma cruzi. 51. The method according to claim 37 wherein said agent is Trypanosoma cruzi. 52. The method according to claim 43 wherein said agent is Trypanosoma cruzi. 53. The method according to claim 46 wherein said agent is Trypanosoma cruzi. 54. The method according to claim 33 wherein said agent is Candida albicans. 55. The method according to claim 54 wherein R1, R2, R4, R5, R6, R8, R9, R10 R11 and R12 are H, R7 is phenyl and R3 is CH3. 56. A method of reducing the likelihood that a patient will contract an infection caused by an agent selected from the group consisting of Trypanosoma cruzi, Mycobacterium spp., Leishmania spp., Cryptococcus spp., Aspergillus spp., Histoplasma spp., Candida spp., Pneumocystis carinii, Trichophyton spp., Microsporum spp., Malassezia spp., Rhizopus spp., Pseudallescheria spp., Blastomyces dermatitidis and Coccidiodes spp., said method comprising administering to said patient in need thereof an effective amount of a compound according to formula I: Where RA is a C1-C10 substituted or unsubstituted linear, branch-chained or cyclic alkyl or alkenyl group or a phenyl group according to the formula: RB is a C1-C10 substituted or unsubstituted linear, branch-chained or cyclic alkyl or alkenyl group or a phenyl group of the formula: R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are each independently selected from H, C1-C10 (preferably a C1-C4) alkyl or alkenyl group, CF3, F, Cl, Br, I, CN, NO2, NH2, NHR, NRR, COR, OR, CO2R, or COSR, where R is H or a C1-C10 (preferably a C1-C4) alkyl or alkenyl group, an unsubstituted or substituted aryl or heterocycle group, or a group, where R3 is H, a C1-C10 (preferably a C1-C4) alkyl, alkenyl, ether or a thioether group; and R11 and R12 are independently selected from H or a C1-C3 alkyl or alkenyl group, or a pharmaceutically acceptable salt thereof, optionally in combination with a pharmaceutically acceptable additive carrier or excipient. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Chagas Disease and Trypanosoma cruzi Chagas Disease was discovered in 1909 by Carlos Chagas. It causes the third largest parasitic disease burden in the world and the largest in the Western hemisphere, currently affecting 16-18 million people throughout Central and South America with over 100 million people living in endemic regions and at risk of infection. The disease is caused by the Trypanosoma cruzi parasite, a zooflagellate protozoon similar to that which causes African sleeping sickness. The parasite is transmitted by a variety of vectors, most notably the large, crawling insect Triatoma infestans , which is known in much of South America as the vinchucha. The insects frequently live in the thatched roofs and cracked adobe walls of the houses common to the affected region; their move into populated regions, which has fueled if not caused the epidemic, was precipitated by the destruction of their natural habitat, the forest, by railroads and other development. 1 They feed on both human and animal blood, and inject a small dose of anesthetic into their victims, which allows them to feed unimpeded for up to thirty minutes. The parasite is not transmitted through the bite, but rather through the insect's fecal matter. The vinchucha defecates shortly after eating, leaving its feces close to the bite irritation; when the victim scratches the bite, fecal matter is rubbed into the open sore causing infection. The life cycle of T. cruzi involves three primary forms of the parasite: amastigote, trypomastigote, and epimastigote. 2 Structurally, the three varieties are most easily distinguished from one another through the location of their flagella. The trypomastigote has the origin of its flagellum at its posterior tip, the epimastigote near its center, and the amastigote is lacking an external flagellum. The non-infective epimastigote form lives within the gut of the vinchucha. It multiplies rapidly and serves to maintain the parasite level within the insect. In response to nutritional stress it is transported from the midgut to the rectum of the vinchucha, at which time it differentiates into the metacyclic trypomastigote. The trypomastigote, although non-proliferative, is the infectious form of the parasite in both animals and humans. It has been suggested that the urine of the transmitting vector, which can also cause parasite transmission, induces differentiation from epimastigotes into trypomastigotes. 3 Once they are transmitted into humans, the trypomastigotes invade a number of cell types, especially the muscle and nerve cells of the heart and gastrointestinal tract, and transform into the amastigote form. This differentiation takes places after a lag period of 20-30 hours and has been shown to be thermosensitive, although the temperature at which transformation occurs varies among parasitic strains. 3 The amastigotes, which are formed when the trypomastigotes are released from their phagolysosomal vacuoles, are the form of the parasite which causes the symptoms of Chagas disease: the amastigotes cluster to form cysts which through their repeated reproduction burst the host cells. The amastigotes also differentiate into trypomastigotes, which are the primary active form of the parasite within the blood. It is these trypomastigotes which are taken up by vinchuchas to repeat the parasitic cycle. Infection by T. cruzi is generally followed by an 8-10 day incubation period and then by the onset of the acute phase of the disease. Only a small percentage of infected individuals experience symptoms of the acute phase, and the phase is generally only fatal for young children and those with weakened immune systems. 4 Diagnosis of acute Chagas disease is difficult because the symptoms are common to a variety of common diseases: fever, enlargement of lymph nodes, and myocarditis. 5 The symptoms which are most associated with Chagas disease is Romaña's Sign, swelling of both the upper and lower eyelids on one eye, and chagomas, painful sores which occur at both the bite site and elsewhere on the body. Rassi, et. al. reports that nearly 75% of patients possess one of these two classic symptoms, but others report percentages of below 25%. 1 The acute stage is the point at which currently available drug therapies function, although these treatments are not very effective. The acute stage generally ends after 1-2 months, and on occasion the disease is spontaneously cured during this phase. The acute phase can be followed by a rapid onset of cardiopathy, a stage known as the sub-acute phase and which quickly leads to death, but is generally followed by a latent period which can last for decades. This latent period, called the indeterminant period by Carlos Chagas, is defined by the presence of parasitic infection but the absence of symptoms. The indeterminant phase is the terminal stage of the disease for up to 40% of infected individuals; the remainder develop chronic Chagas disease. The chronic phase has two principal symptomatic pathways, those of benign and malignant evolutions. 4 Benign evolution is the slow onset of cardiac or digestive symptoms, and can persist without catastrophic consequences for decades. It eventually, however, progresses to malignant chronic Chagas disease, which also can evolve directly from the indeterminant stage of the disease. The malignant form of the disease has two principal components 5 : cardiac and digestive Chagas disease. The cardiac symptoms of Chagas disease have their basis in disruptions of the electronic conduction system of the heart. This degeneration of the heart's conduction system, which is caused by lesions stemming from amastigotic cyst formation within the area, can lead to arrhythmia and bradycardia. These disruptions eventually leads to cardiac failure. Enlargement of the heart is also common, and is occasionally observed in other stages of the disease and can be used as a diagnostic tool. Digestive decay is slightly less common than cardiac symptoms of Chagas disease, but is more dramatic in its outward symptoms. Nerve damage caused by amastigotic cysts in either the colon or the esophagus diminishes peristalsis, the ability of smooth muscle to dilate and contract in order to move food along the digestive tract. This loss of activity causes muscle hypertrophy which leads to a loss of rigidity and a dramatic enlargement of the affected area. Megaesophagus and megacolon can lead to death due to malnutrition, and furthermore megacolon prevents bowel movements which eventually leads to further digestive failure and eventually results in death. Megaesophagus usually precedes colonic and cardiac symptoms. and is, for unknown reasons, more common among males than females. Sterol Biosynthesis in Trypanosoma cruzi Sterol biosynthesis is a complex enzymatic pathway which produces membrane lipids for all eukaryotic organisms. Mammals produce cholesterol as their primary sterol, whereas fungi and trypanosomes produce ergosterol, a similar molecule lacking cholesterol's Δ 5(6) double bond and containing a methyl group at C24. 6 Both cholesterol and ergosterol go through the common intermediate sterol lanosterol, which is formed in several steps from acetyl-CoA. The first of the post-lanosterol processing steps is the removal of a methyl group at C14 and the introduction of a C14-C15 double bond. The enzyme which catalyses this former transformation is lanosterol-C14α-demethylase. C-14α-demethylase has been extensively studied and characterized in fungal systems. It is a cytochrome P-450 enzyme, and consequently is know as P-450 14DM . P-450 14DM was first isolated from Saccharomyces cerevisiae. 7 The enzyme was found to catalyze the removal of the 14α methyl carbon (C32) in the presence of molecular oxygen and NADPH. 8 The removal of the 14α methyl group proceeds through a series of three successive monooxidations. 32-Hydroxylanosterol was confirmed as an intermediate in the reaction pathway, as it was found to both be a substrate for the enzyme and to bind to the enzyme with greater affinity than lanosterol itself. 9 Similar results were found for the second intermediate: 32-formyllanosterol 10 The mechanism of the final deformylation is not known conclusively, but both a Baeyer-Villiger rearrangement 11 and a radical mechanism 12 have been proposed. 14-Methyl sterols cannot function within cell membranes and consequently the inhibition of P-450 14DM is an active area of antifungal research. Azole compounds have been shown to form stochiometric complexes with fungal P-450 14DM . A paradigm of inhibitor design has been developed where a potent inhibitor would have to contain both a group capable of binding to the heme iron and a group which can interact with the hydrophobic cavity adjacent to the heme. 13 Likewise, inhibitors must contain a sterically accessible lone pair and a hydrophobic substituent at N-1 position on the azole ring. 14 It has been suggested that other substitution of the azole ring will not be tolerated. More recently, crystal structures of the P-450 14DM from Mycobacterium tuberulosis bound to 4-phenylimidazole and fluconazole were solved. 15 The results offered more precise evidence of the previous predictions of azole inhibitor binding to the enzyme: the imidazole ring binds perpendicularly to the heme and the aromatic region of the inhibitors participate in hydrophobic interactions with surrounding residues. This research was supported by NIH grants CA67771 and CA52874. Consequently, the United States government has retained certain rights in the invention. |
<SOH> BRIEF DESCRIPTION OF THE FIGURES <EOH>FIGS. 1-2 represent certain preferred chemical compounds according to the present invention. FIGS. 3A and B represent mouse data for compound JJ121. FIG. 3A represents Parasite levels and FIG. 3B represents Survival for treated and control mice. Mice were dosed orally at 50 mg/kg twice daily on days 1-10. They were infected with T. cruzi trypomastigotes (Tulahuen) 2×10 3 SQ at day 0. Parasitemia was quantified microscopically on a small drop of tail blood at 400×. detailed-description description="Detailed Description" end="lead"? |
Automatic loading extractor |
The present invention provides an industrial extractor (10) comprising a chassis (12) to which a cylindrical drum (14) is rotatably mounted. A pair of sliding doors (24, 26) are incorporated into the drum's (14) cylindrical structure thereby providing access to the drum (14) through its periphery. To load the extractor (10) with laundry, the sliding doors (24, 26) am actuated to provide an opening at the top of the drum (14). The doors (24, 26) are actuated via in interconnected series of levers (36, 38), plates (28, 30) and linkages (48, 50). The laundry is then dropped from a conveyor or chute into the drum (14) without having to tilt or pivot the drum (14). Once loaded, the doors (24, 26) are automatically closed and a locking assembly (60) engages the door (24, 26) to prevent them from inadvertently opening. Once the items are laundered and the drum (14) stops rotating, the drum (14) is pivotally rotated in a forward direction such that its axis (x) of rotation extends diagonally as opposed to horizontally. An unloading door (18) positioned on the front of the extractor (10) is then automatically opened and the items are permitted to fall out of the drum (14) via gravity and manual assistance. Once emptied, the drum (14) is pivotally rotated back to its operating position where it may then be re-loaded and run through another cleaning cycle. |
1. An industrial extractor, comprising: a. a chassis; b. a drum interconnected to said chassis for rotation about a first longitudinal axis; and c. a door for providing access to said drum, extending in a first plane that is parallel to said first longitudinal axis. 2. The industrial extractor of claim 1, further comprising means for moving said door between open and closed positions. 3. The industrial extractor of claim 2, wherein said means for moving said door comprises: a. a first plate attached to said door and extending in a second plane that is perpendicular to said first plane in which said door extends; b. a first rod extending along a second longitudinal axis that is parallel to said first longitudinal axis and having first and second ends, and attached at said first end to said first plate and extending perpendicularly outward therefrom; c. a lever attached to said second end of said first rod; and d. means for pivotally moving said lever about said second longitudinal axis. 4. The industrial extractor of claim 3, wherein said means for pivotally moving said lever comprises: a. a second plate extending in a third plane that is parallel to said second plane; b. a second rod extending along a third longitudinal axis and interconnecting said second plate to said lever; c. first and second knobs attached to said second plate and extending perpendicularly outward therefrom and positioned in straddling relation to said lever; d. a piston and cylinder member, said cylinder being selectively, longitudinally movable along a fourth longitudinal axis and interconnected to said second plate, whereby said selective longitudinal movement of said cylinder effects rotational movement of said second plate about said third longitudinal axis, thereby causing either of said first and second knobs to engage and rotate said lever about said second longitudinal axis, thereby effecting rotation of said first rod about its second longitudinal axis and movement of said door in a corresponding rotational direction. 5. The industrial extractor of claim 1, further comprising means for locking said door in a closed position. 6. The industrial extractor of claim 5, wherein said means for locking said door comprise: a. a pin extending along a second longitudinal axis that is parallel to said first longitudinal axis, and selectively moveable into and out of engagement with said door; and b. a piston and cylinder member interconnected to said pin and being selectively actuable to move said pin into and out of engagement with said door. 7. A method for loading an industrial extractor having a chassis and a drum that is rotatable about a first longitudinal axis, comprising the steps of: a. positioning said extractor in its operating position, wherein said first longitudinal axis extends horizontally; b. opening a first door in a plane that extends parallel to said first longitudinal axis; and c. dropping items to be laundered through the opening created by the opening of said door. |
<SOH> BACKGROUND OF TH INVENTION <EOH>1. Field of the Invention The present invention relates generally to industrial washers and dryers, and more particularly to the loading structure and process for such washers and dryers. 2. Description of Prior Art Industrial washers or extractors are large machines used in industrial operations that have a need for frequent washing of large quantities of clothes, linens, or other fabrics, such as a hotel. The extractors generally comprise a chassis to which a rotating drum is mounted. The drum is generally on the magnitude of 65 inches in diameter, and the extractor units are about 88 inches wide, 95 inches deep, and 105 inches high, and weigh on the magnitude of 21,000 pounds. Thus, these units require a large amount of square footage for operation. The drums are designed to rotate about a horizontally extending axis, and include a door which extends in a vertical plane when the unit is in its operating position. The units are typically loaded and unloaded through this door. To load the units, the drum is generally pivoted rearwardly via pneumatic or hydraulic pistons and cylinders such that the door is facing upwardly. A conveyor, chute, or other loading device is then actuated to drop the clothes (or other fabrics to be washed) through the upwardly facing opening. The door is then automatically closed and locked, and the unit pivoted back to its operating position. To unload a unit, it is pivoted via the pneumatic/hydraulic pistons and cylinders to a position in which the door is facing downwardly. A basket or other container is positioned beneath the door which is then opened, thereby permitting the clothes (or other fabrics) to fall out of the drum via gravity and into the container. Once emptied, the unit is pivoted back to its operating position. Due to the large size of these units and the fact that they must be pivoted both in a forward and a rearward direction to be loaded and unloaded, respectively, they must be mounted in a space that provides enough room for this full range of motion. In addition, the processing time for a load of laundry is increased as a consequence of the unit having to go through its pivoting operations in order to be loaded and unloaded. Moreover, many industrial operations do not have sufficient space for a suitable extractor to operate. Therefore, the operation must either implement a smaller extractor or contract with a service provider for the washing services. 3. Objects and Advantages It is therefore a principal object and advantage of the present invention to provide an industrial extractor that uses a minimal amount of space to operate. It is a further object and advantage of the present invention to provide an industrial extractor that decreases processing time. Other objects and advantages of the present invention will in part be obvious, and in part appear hereinafter. |
<SOH> SUMMARY OF THE INVENTION <EOH>In accordance with the forgoing objects and advantages, the present invention provides an industrial extractor comprising a chassis to which a cylindrical drum is rotatably mounted. A pair of sliding doors are incorporated into the drum's cylindrical wall structure, thereby providing access to the drum through its periphery. To load the extractor with laundry, the sliding doors are actuated to provide an opening at the top of the drum. The laundry is then dropped from a conveyor or chute into the drum without having to tilt or pivot the drum. To actuate the doors, a series of levers and linkages are employed. Each door includes a plate fixedly mounted thereto and extending perpendicularly downward therefrom. A rod fixedly extends between the door's plate and a first lever positioned outside the drum. The first lever is pivotally movable about an axis that extends through an actuating assembly. The actuating assembly comprises a pneumatic or hydraulic piston and cylinder arrangement wherein the end of the piston is fixedly attached to one end of a second lever. The opposite end of the second lever is fixedly secured to an intermediate plate which moves in response to actuation of the piston. Second and third rods are attached to the intermediate plate and move therewith. The second and third rods are positioned on opposite side of the first lever and one of them engages the first lever when the second plate is moved via the piston and cylinder arrangement. The first lever, in turn, pivots about its axis in response to the movement transferred thereto through the second or third rod (and associated actuating assembly). The pivotal motion of the first lever is then translated to the doors, thereby either opening or closing the doors to the drum. |
Images incorporating microstructures |
A method of generating an image incorporating a microstructure includes the steps of obtaining an original image, generating a microstructure, and rendering a region or the whole said original image with said microstructure. The operation of generating the microstructure includes an automatic synthesis of microstructure elements from original microstructure shapes. |
1-40. (canceled). 41. Method of generating an image incorporating a microstructure, including obtaining an original image; generating a microstructure; and rendering a region or the whole said original image with said microstructure; wherein the operation of generating the microstructure includes an automatic synthesis of microstructure elements as a microstructure dither matrix from original microstructure shapes. 42. Method according to claim 41 wherein the microstructure includes a low frequency microstructure with low frequency microstructure elements generated from the original microstructure shapes, and a high frequency microstructure with high frequency microstructure elements, whereby the low frequency microstructure elements are larger than the high frequency microstructure elements. 43. Method according to claim 41 wherein the synthesis of the dither matrix includes applying mathematical morphology operators to the microstructure shapes. 44. Method according to claim 43 wherein the applied mathematical morphology operators comprise a shape thinning operator for the bitmap shape foreground and an operator selected from a set of alternated dilation and dual bitmap thinning for the bitmap shape background. 45. Method according to claim 41 wherein the synthesized microstructure elements are visible at both high and low intensities after rendering with the original image. 46. Method according to claim 41 wherein the original microstructure shapes are bitmap elements. 47. Method according to claim 41 wherein the visibility of the microstructure elements is tuned by a mask whose values represent relative weights of the original image halftoned with conventional methods and the original image synthesized with the microstructure. 48. Method according to claim 41 further including applying a parametrized transformation to warp the microstructure incorporated in the image. 49. Method according to claim 48 wherein several image instances are successively generated by modifying parameters of the parametrized transformation, said set of image instances forming a displayable image animation. 50. Method according to claim 49 wherein said parameters are modified smoothly as a function of time to yield a smoothly evolving animated microstructure. 51. Method according to claim 41 wherein the rendering of the microstructure and original image includes a standard or multicolor dithering operation. 52. Method according to claim 41 further including applying a mask specifying a region of the original image to be rendered with the microstructure. 53. Method according to claim 41 further including applying a multi-valued mask expressing weights of original image colors and weights of the selected basic colors for generating the image. 54. Method according to claim 41 wherein the microstructure includes information personal to a user of the image. 55. Method according to claim 41 wherein the microstructure includes information identifying and specific to a particular event or transaction, such as date, venue, seating, destination, time. 56. Method according to claim 41 wherein the microstructure elements include alphanumerical characters provided at a size in relation to the image that allow their reading at a personal document reading distance. 57. Method according to claim 42 wherein the high-frequency microstructure elements are placed at locations corresponding to the background of the low frequency microstructure elements. 58. Method according to claim 57 wherein the comparison yields a deltamap which is dithered by a high frequency dither array, the resulting dithered deltamap being composed with the dithered image. 59. Method according to claim 41 wherein a mask whose shape expresses a visual message specifies the part of the microstructure that is to be printed with special inks that enable, under certain observation conditions, the mask shape to remain hidden within the image and under other observation conditions, the mask shape to be clearly revealed. 60. Method according to claim 59 wherein parts of the image specified by the mask are printed with a special ink selected from a group of metallic and iridescent inks, whereby the mask shape is hidden at a certain observation angle and is visible at a different observation angle. 61. Method according to claim 60 wherein parts of the the image specified by the mask are printed with a special ink invisible in daylight and visible in light at selected frequencies, such as Ultraviolet light. 62. Method according to claim 41 further including defining color information used for rendering the target image; defining parameters of a parametrized transformation; traversing a target image positions (x,y) pixel by pixel and row by row, determining corresponding positions in the original image (x′,y′) and, according to the parametrized transformation, corresponding positions in the microstructure (x″,y″); obtaining from the original image positions (x′,y′) the color Cr to be reproduced and from the microstructure positions (x″,y″) rendering information; rendering the target image by making use of the rendering information. 63. Method of generating a security document comprising producing an image incorporating a microstructure according to a method set forth in claim 41, wherein the original image rendered with the microstructure comprises information relevant to the purpose of the document and intended to be protected against counterfeiting. 64. Method of generating a security document for printing or display, including the steps of: selecting or retrieving an original image; selecting or retrieving information specific to a person, an event or transaction to which said security document relates; generating a microstructure comprising readable microstructure elements providing information on said person, event or transaction; rendering said original image with said microstructure image using the method according to claim 41. 65. Method according to claim 64 wherein the image is printed or displayed on a support selected from a group including any of paper, plastics, polymers, product packages, optical disks, and optical devices comprising holograms, kinegrams and diffractive elements. 66. Method according to claim 64 wherein the security document is a commercial instrument bearing value or relating to a commercial transaction. 67. Method according to claim 64 wherein the security document is an certificate, title or deed. 68. Method according to claim 64 wherein the security document includes information identifying a person or entity. 69. Method according to claim 64, wherein the image incorporating a microstructure is generated in a server system, said server system preparing and packaging a displayable file for printing said security document on a standard printer or for display on an electronic screen, such as a screen of a portable electronic device. 70. Method according to claim 69, wherein said security document is printed on said standard printer at a customer site remote from said server system and accessible to said server system via a communications network such as the internet. 71. Method according to claim 64 wherein some or all information included in the original image is retrieved from one or more databases via a global communications network such as the internet. 72. Method according to claim 64 wherein the original image comprises a portrait of a bearer selected from a customer database on the basis of information identifying said bearer. 73. An image incorporating a microstructure generated by a method according to claim 41 wherein the microstructure elements comprise information specific to a particular event, transaction, or person. 74. A security document comprising an image according to claim 73. 75. A computing system for synthesizing a security document, comprising: an interface operable for receiving a request for synthesizing the security document, a preparation software module operable for preparing data files according to document related information received with the request, where the preparation of data files comprises the generation of an original document image, the generation of microstructure shapes and the generation of transformation parameters, and a production software module operable for producing the security document, where producing the security document comprises the synthesis of a microstructure and the synthesis of a security document with that microstructure. 76. Computing system according to claim 75, wherein microstructure shapes are generated by producing a bitmap incorporating the microstructure shapes, where the microstructure is embodied in a dither array synthesized from said bitmap by applying to it mathematical morphology operations and where the security document is synthesized by dithering the original document image with the synthesized dither array. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention relates generally to images incorporating information both at the global level and at the microstructure level and to a method of generating such images. The information at the microstructure level offers, in particular, protection against counterfeiting and may be used as a security feature in documents. The invention also relates to documents comprising security features, and to a method of generating such documents, which may include for example, value bearing commercial instruments, certificates, coupons and personal identification instruments. The term ‘images’ used herein shall be understood in the broad sense to mean any visual representation of matter that may be printed or displayed on a display, for example text, pictures, photographs, drawings and so on. A microstructure may comprise microstructure elements such as a text, a logo, an ornament, a symbol or any other microstructure shape. When seen from a certain distance, mainly the global image is visible. When seen from nearby, mainly the microstructure is visible. At intermediate distances, both the microstructure and the global image are visible. Several attempts have already been made in the prior art to generate images incorporating information at the microstructure level, where from far away mainly the global image is visible and from nearby mainly the microstructure is visible. A prior art method hereinafter called “Artistic Screening” was disclosed in U.S. Pat. No. 6,198,545 and in the article by V. Ostromoukhov, R. D. Hersch, “Artistic Screening”, Siggraph95, Computer Graphics Proceedings, Annual Conference Series, 1995, pp. 219-228. This method requires however significant efforts by graphic designers in order to create the microstructure and is limited to bi-level images, i.e. images in black-white or a single color and white. A prior art method for incorporating a microstructure into an image by computing color differences is disclosed in European Patent application 99 114 740.6. This method does not modify the thickness of the microstructure according to the local intensity of the image. Another method hereinafter called “Multicolor Dithering” is disclosed in the article by V. Ostromoukhov, R. D. Hersch, “Multi-Color and Artistic Dithering”, Siggraph'99, Computer Graphics Proceedings, Annual Conference Series, 1999, pp. 425-432. The method allows to synthesize color images incorporating as screen dots a fine microstructure capable of representing various shapes such as characters, logos, and symbols and provides therefore strong anti-counterfeiting features. The publication also presents an iterative technique for equilibrating a dither array, which is however slow and cumbersome and does not always converge to yield a satisfying result. A disadvantage of the aforementioned and other known methods is the significant effort required to synthesize dither matrices incorporating the desired microstructure shapes. These efforts require the skills of a computer scientist for building 3D functions, discretizing them, renumbering the resulting dither values and applying to them an equilibration process. An additional method for creating microstructures within an image relies on a large dither matrix whose successive threshold levels represent the microstructure and uses standard dithering to render the final image (see Oleg Veryovka and John Buchanan, Halftoning with Image-Based Dither Screens, Graphics Interface Proceedings, years 1988-1999, Ed. Scott MacKenzie and James Stewart, Morgan Kaufmann Publ. or http://www.graphicsinterface.org/proceedings/1999/106/). In this paper, the authors show how to build a dither matrix from an arbitrary grayscale texture or grayscale image. They mainly apply histogram equilibration to ensure a uniform distribution of dither threshold levels. Texture control is obtained by error-diffusion. However, while their method allows to incorporate text within the microstructure, the typographic character shapes do not vary according to intensity, i.e. the character shapes do not become thin or fat depending on the local intensity. Their method is restricted to black-white or single color target images. The authors do not provide a method to construct a dither matrix starting from a bi-level bitmap incorporating the microstructure shapes. A further method of embedding a microstructure within an image is described in provisional U.S. patent application No. 60/312,170 (filed Aug. 14, 2001, inventor Huver Hu, available at Web site http://www.amgraf.com/), which teaches how to transform a grayscale seed image or a bi-level seed image into an array of dot ranking values (similar to a dither matrix) to be used by a PostScript Interpreter for synthesizing the final image incorporating the microstructure. This method is however limited to black-white or to single color output images (bi-level images). In addition, the seed image is preferably a grayscale image (FIG. 10 of patent application No. 60/312,170). With bi-level seed images, the generated microstructure is limited to rather simple shapes (FIG. 13 of patent application No. 60/312,170), since shapes grow at increasing darkness levels from a user specified growth center to the shape given by the bi-level seed image. The shape does not grow beyond 60% darkness: darker levels are produced by the growth of a separate superimposed geometric mask (e.g. a triangle, visible on all dark parts of the wedges in FIGS. 2, 12 and 13 of patent application No. 60/312,170). Furthermore, a manual interactive intervention is required to transform a seed image into an array of dot ranking values. Another approach for embedding information within a color image relies on the modification of brightness levels at locations specified by a mask representing the information to embed, while preserving the chromaticity of the image (see U.S. Pat. No. 5,530,759). However, since the embedded information is not really used to construct the global image, it cannot be considered a microstructure. If the embedded information incorporates large uniform surfaces, the global image may be subject to significant changes and the embedded information may become visible from a large distance. In addition, the mask is fixed, i.e. its shape does not vary as a function of the local intensity or color. One further related invention disclosed in U.S. Pat. No. 5,995,638 teaches a method for authenticating documents comprising a basic screen made of microstructures and a revealing screen for creating moire intensity profiles of verifiable shapes. U.S. patent application Ser. No. 09/902,445 describes a similar method, where however the basic screen and the revealing screen may undergo geometric transformations, yielding screens of varying frequencies. The incorporation of microstructures in images has applications not only in the field of generation of artistic images, but also in the field of generation of documents that require protection against counterfeiting. It is known to incorporate microstructures as a security feature in certain printed commercial instruments, such as bank notes, using professional printers and printing techniques on special substrates. A primary consideration in the generation of printed commercial instruments, such as bank notes, vouchers, transportation tickets, entertainment event tickets and other tickets, coupons or receipts bearing or representing a commercial value, is to provide sufficient safeguards against forgery. The required degree of difficulty in producing a forgery will depend above all on the value, the duration of validity and the generality of the commercial instrument. For example, bank notes which are not related to any specific event and remain valid for many years, require security features that are extremely difficult to reproduce. On the other hand, tickets of relatively limited duration, for example transportation tickets, such as train tickets valid on a certain day for a certain destination, or theatre tickets for a specific show, require lower level security features, as long as they ensure that the instrument is difficult to reproduce in the remaining time to the event or requires excessive technical means or human effort in comparison to the value of the commercial instrument. Verification of the authenticity of many commercial instruments is often based on a visual control. Although it is easy to provide commercial instruments with unique security features, such as encrypted bar codes or other codes, their verification entails the use of electronic processing means that are unpractical or inefficient in many situations. In commercial instruments relying primarily on a visual control of authenticity, a common security feature is the provision of special substrates that are difficult or too costly to reproduce for a potential forger in relation to the underlying value of the commercial instrument. A disadvantage of the use of special substrates or special printing techniques is that they do not allow the generation of commercial instruments at sites that are not under the issuer's control, whether directly or indirectly. In view of the wide-spread use of communications networks, such as the internet or local area networks, there is a demand for enabling the generation of visually verifiable printed commercial instruments, such as transportation tickets and entertainment event tickets, at the buyer's site, for example at home with a PC and standard printer. In international patent application WO 00/67192, a method of generating a commercial instrument with certain visually verifiable security features for printing on a standard printer is described. In the aforementioned application, data relevant to the commercial instrument are manipulated in accordance with predetermined rules to generate a pattern which is visually recognizable to an informed person. The security against forgery of an instrument generated according to the latter method relies on the potential forger's ignorance of the predetermined rules. Reliance on predetermined rules has a number of disadvantages. Firstly, the rules must be communicated to persons responsible for controlling authenticity, which becomes impractical where many controllers are involved. Secondly, the rules must result in features that are visually recognizable, with the consequence that a potential forger could, on the basis of a number of commercial instruments, be able to deduce the rules with a sufficient degree of approximation to generate forgeries using different data. In this regard, it should be noted that the relatively sophisticated image creation and editing software widely available and for use on PC provide the forger with fairly powerful tools to reproduce images and text manipulated in order to emulate visually recognizable patterns provided on authentic commercial instruments on the basis of predetermined rules as described in international application WO 00/67192. |
<SOH> SUMMARY OF THE INVENTION <EOH>An object of this invention is to provide images incorporating a microstructure that may be generated efficiently. Another object of this invention for certain applications is to provide images that are difficult to counterfeit, in particular for use in documents as a security feature. It is advantageous in certain applications to provide images incorporating a microstructure that may be rapidly generated. It is advantageous in certain applications to provide images incorporating a microstructure that have a high resolution or a high visual quality. It is advantageous in certain applications to provide images incorporating a microstructure, that can be animated. It is also an object of this invention to provide a method of generating such images, and a method of generating documents comprising such images. It is also an object of this invention to provide a computer system to generate such images. Another object of this invention is to provide a security document, such as a commercial instrument or certificate, and a method of generation thereof, that is difficult to forge yet enables visual verification of the authenticity thereof, and that can be printed with non-professional printing systems, such as standard PC printers, or displayed on an electronic display. It is advantageous to provide a security document with security features that are easy to verify visually by a verifying person, without the need for providing such person with restricted information on hidden or coded security features or other information unavailable to uninformed persons. It is advantageous to provide a method for generating security documents that is able to generate personal and/or event specific instruments rapidly, for example comprising information relating to a specific person, destination or event. It is further advantageous to provide a method that enables the printing, or downloading for display on a portable device screen, of secure commercial instruments by a customer with access to data processing and database means through a communications network such as the internet. Objects of this invention have been achieved by providing a method of generating an image incorporating a microstructure according to claim 1 . Disclosed herein is a method of generating an image incorporating a microstructure, including obtaining an original image; generating a microstructure; and rendering a region or the whole said original image with said microstructure; wherein the operation of generating the microstructure includes an automatic synthesis of microstructure elements from original microstructure shapes. The microstructure shapes are in an embodiment described originally in the form of a bi-level bitmap. The automatic synthesis from bitmaps enables very efficient creation of images on the fly, which may incorporate different microstructure shapes, for example based on information specific to the content of a document in which the image is used. In addition, thanks to a parametrized transformation carried out at microstructure image rendering time, different instances of the same microstructure image can be rendered on the fly. An important advantage of the presented automatic dither array synthesis method is its ability to ensure that the microstructure incorporated into an image or a security document remains visible at nearly all intensity levels (from 10% to 90% darkness in most cases). A high quality and secure image incorporating a microstructure can thus be generated. Objects of this invention have been achieved by providing a method of generating an image incorporating a microstructure according to claim 3 . Also disclosed herein is a method of generating an image incorporating a microstructure, including obtaining an original image; generating a microstructure; and rendering said original image with said microstructure; wherein the microstructure includes a low frequency microstructure generated from low frequency microstructure elements, and a high frequency microstructure generated from high frequency microstructure elements, whereby the low frequency microstructure elements are larger than the high frequency microstructure elements. The two levels of microstructure advantageously provides an image that is very difficult to forge. It is also possible to have further levels of microstructure incorporated in the image. The microstructure may be composed of text, graphic elements and symbols. The microstructure whose shapes vary according to intensity and color protects the security document's elements such as text, photographs, graphics, images, and possibly a background motif. Since the security document is built on top of the microstructure, document elements and microstructure elements cannot be erased or modified without introducing discontinuities in the security document. Furthermore, thanks to transformations having the effect of warping the microstructure into different orientations and sizes across the security document, individual microstructure elements cannot be simply copied and inserted elsewhere. The present disclosure also teaches how to equilibrate an image incorporating a microstructure (hereinafter also called: “microstructure image”) or a security document with the help of a high-frequency dither array. This high-frequency dither array may incorporate a second level microstructure providing an additional level of protection. Further disclosed herein are microstructure images and security documents with a microstructure rendered in black/white, color, or possibly rendered partly with non-standard inks, or special inks such as fluorescent inks, phosphorescent inks, metallic inks, iridescent inks or ultra-violet inks. A mask whose shape expresses a visual message (e.g. a bold text string or a symbol) may specify the part of the target document to be rendered with a special ink. Under given observation conditions (e.g. type of light, viewing angle), the special ink is hidden. Under other observation conditions, the special ink has the effect of making the mask shape (e.g. the text or symbol) clearly visible. For example at a certain viewing angle, the part covered by the special ink is hidden and when seen from another angle, it becomes apparent. Further disclosed herein is an animated microstructure image formed by a microstructure evolving over time, where from far away mainly the image is visible and from nearby mainly the evolving microstructure is visible. Such an animated microstructure image is displayed as a succession of image instances, each image instance differing from previous image instances by the microstructure-evolution. This microstructure evolution is determined by a parametrized transformation, whose parameters change smoothly as a function of time. Further disclosed herein is a method allowing to combine an original image, respectively a conventionally halftoned original image with a microstructure image, thereby providing within the target image more or less weight to the microstructure. This allows to create target images, where thanks to a multi-valued mask, the relative weight of the microstructure may at certain places, slowly reduce and disappear. In the case of an animated microstructure image, the mask specifies the part of the image to be rendered with an animated microstructure and the part which is being left without microstructure. With a multi-valued mask, the appearance of the microstructure can be tuned to be strong or on the contrary at the limit of what can be perceived by a human eye at a normal observation distance. In addition, mask values evolving over time yield apparent changes in the embedded microstructure appearance properties such as the visibility, location or spatial extension of the embedded microstructure within the image. In a preferred embodiment, original microstructure shapes are embedded within a bilevel bitmap, and the microstructure is embodied by a dither array. Starting from the bitmap incorporating the microstructure shapes, the dither array can be automatically generated. A black-white or color target image (or security document) is synthesized by dithering an original image with the dither array and by possibly equilibrating the resulting dithered original image. Also disclosed herein is a computing system for synthesizing security documents comprising a an interface operable for receiving a request for synthesizing a security document, a software preparation module operable for preparing data files from document information and a document production module operable for producing the security document. The preparation of data files may comprise the generation of an original document image, of microstructure shapes and possibly of transformation parameters. Producing the security document system comprises the synthesis of a microstructure and the synthesis of the security document with that microstructure. Further disclosed herein is a computing system for synthesizing images comprising an interface operable for receiving a request for synthesizing a microstructure image and comprising a software production module operable for producing the microstructure image. The request comprises an original image and microstructure shapes. The microstructure image is produced by the production module by first synthesizing a microstructure and then by synthesizing the microstructure image incorporating that microstructure. Further disclosed herein is a computing system capable of displaying a target image with an embedded microstructure evolving over time, where from far away mainly the image is visible and from nearby mainly the evolving microstructure is visible. The computing system comprises a server computing system and a client computing and display system. The client computing and display system receives from the server computing system as input data an original color image, microstructure data and microstructure evolution parameters. The client computing and display system synthesizes and displays the target image with the embedded microstructure on the fly. Other objects of this invention have been achieved by providing a method of generating a security document according to claims 34 or 35 . Disclosed herein is a method of generating a security document for printing or display, including the steps of: selecting, retrieving or composing an original image; selecting or retrieving information specific to a person, an event or transaction to which said security document relates; generating a microstructure comprising readable microstructure elements providing information on said person, event or transaction; rendering said original image with said microstructure image. The microstructure may advantageously be generated as a dither matrix, automatically synthesized from microstructure shapes such as bitmap elements. The microstructure may be rendered with the image by rendering methods described above or by a halftoning process, whereby the pixels of the dither matrix are compared with the pixels of the background image and, for example, if the pixel of the background image has a grey level greater than the inverse grey level of the dither matrix, then the pixel is printed as white, otherwise it is printed as black. The rendering of the microstructure and image may further comprise a step of balancing the halftoned image. Advantageously, in view of the rendering process, the event or transaction specific information is extremely difficult to separate out of the background or original image and is therefore difficult to replace with other information in view of producing forgeries. The microstructure dither matrix may advantageously comprise letters and/or numbers, such that the event or transaction specific information may be provided in the form of words or numbers. This enables information, such as the date, the price, the destination, the seat number, personal identification, credit card number, ticket transaction number or any other information specific to the event or transaction to form part of the microstructure image. The microstructure dither matrix may also comprise other characters, graphical elements, logos and other special designs. The original image may advantageously comprise a photographic representation or portrait of the customer, in addition to a background image that may be changed from time to time, the images being merged or superposed. The original image may further comprise written ticket transaction information. Further objects and advantageous aspects of this invention will be apparent from the following detailed description of embodiments of this invention with reference to the accompanying figures, in which: |
Method for controlling particular insect pest by applying anthranilamide compounds |
This invention pertains to a method for controlling lepidopteran, homopteran, hemipteran, thysanopteran and coleopteran insect pests comprising contacting the insects or their environment with an arthropodicidally effective amount of a compound of Formula I, its N-oxide or an agriculturally suitable salt thereof wherein A and B and R1 through R8 are as defined in the disclosure. This invention further relates to a bezoxazinone compound of Formula 10 wherein R4 through R8 are as defined in the disclosure, useful for preparation of a compound of Formula I. |
1. A method for controlling for controlling lepidopteran, homopteran, hemipteran, thysanopteran and coleopteran insect pests, comprising: contacting the insects or their environment with an arthropodicidally effective amount of a compound of Formula I, an N-oxide or an agriculturally suitable salt thereof wherein A and B are independently O or S; R1 is H, C1-C6 alkyl, C2-C6 alkoxycarbonyl or C2-C6 alkylcarbonyl; R2 is H or C1-C6 alkyl; R3 is H; C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or C3-C6 cycloalkyl, each optionally substituted with one or more substituents selected from the group consisting of halogen, CN, NO2, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, C1-C4 alkylthio, C1-C4 alkylsulfinyl, C1-C4 alkylsulfonyl, C2-C6 alkoxycarbonyl, C2-C6 alkylcarbonyl, C3-C6 trialkylsilyl, phenyl, phenoxy, 5-membered heteroaromatic rings, and 6-membered heteroaromatic rings; each phenyl, phenoxy, 5-membered heteroaromatic ring, and 6-membered heteroaromatic ring optionally substituted with one to three substituents independently selected from the group consisting of C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C2-C4 haloalkenyl, C2-C4 haloalkynyl, C3-C6 halocycloalkyl, halogen, CN, NO2, C1-C4 alkoxy, C1-C4 haloalkoxy, C1-C4 alkylthio, C1-C4 alkylsulfinyl, C1-C4 alkylsulfonyl, C1-C4 alkylamino, C2-C8 dialkylamino, C3-C6 cycloalkylamino, C4-C8 (alkyl)(cycloalkyl)amino, C2-C4 alkylcarbonyl, C2-C6 alkoxycarbonyl, C2-C6 alkylaminocarbonyl, C3-C8 dialkylaminocarbonyl and C3-C6 trialkylsilyl; C1-C4 alkoxy; C1-C4 alkylamino; C2-C8 dialkylamino; C3-C6 cycloalkylamino; C2-C6 alkoxycarbonyl or C2-C6 alkylcarbonyl; R4 is H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C1-C6 haloalkyl, CN, halogen, C1-C4 alkoxy, C1-C4 haloalkoxy or NO2; R5 is H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C4 alkoxyalkyl, C1-C4 hydroxyalkyl, C(O)R10, CO2R10, C(O)NR10R11, halogen, C1-C4 alkoxy, C1-C4 haloalkoxy, NR10R11, N(R11)C(O)R10, N(R1′)CO2R10 or S(O)nR12; R6 is H, C1-C6 alkyl, C1-C6 haloalkyl, halogen, CN, C1-C4 alkoxy or C1-C4 haloalkoxy; R7 is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C1-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl or C3-C6 halocycloalkyl; or R7 is a phenyl ring, a benzyl ring, a 5- or 6-membered heteroaromatic ring, a naphthyl ring system or an aromatic 8-, 9- or 10-membered fused heterobicyclic ring system, each ring or ring system optionally substituted with one to three substituents independently selected from R9; R8 is H, C1-C6 alkyl, C1-C6 haloalkyl, halogen, C1-C4 alkoxy or C1-C4 haloalkoxy; each R9 is independently C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C2-C4 haloalkenyl, C2-C4 haloalkynyl, C3-C6 halocycloalkyl, halogen, CN, NO2, C1-C4 alkoxy, C1-C4 haloalkoxy, C1-C4 alkylthio, C1-C4 alkylsulfinyl, C1-C4 alkylsulfonyl, C1-C4 alkylamino, C2-C8 dialkylamino, C3-C6 cycloalkylamino, C4-C8 (alkyl)(cycloalkyl)amino, C2-C4 alkylcarbonyl, C2-C6 alkoxycarbonyl, C2-C6 alkylaminocarbonyl, C3-C8 dialkylaminocarbonyl or C3-C6 trialkylsilyl; R10 is H, C1-C4 alkyl or C1-C4 haloalkyl; R11 is H or C1-C4 alkyl; R12 is C1-C4 alkyl or C1-C4 haloalkyl; and n is 0, 1 or 2. 2. The method of claim 1 wherein A and B are both 0; R7 is a phenyl ring or a 5- or 6-membered heteroaromatic ring selected from the group consisting of each ring optionally substituted with one to three substituents independently selected from R9; Q is O, S, NH or NR9; and W, X, Y and Z are independently N, CH or CR9, provided that in J-3 and J-4 at least one of W, X, Y or Z is N. 3. The method of claim 2 wherein R1, R2 and R8 are all H; R3 is C1-C4 alkyl optionally substituted with halogen, CN, OCH3 or S(O)pCH3; R4 group is attached at position 2; R4 is CH3, CF3, OCF3, OCHF2, CN or halogen; R5 is H, CH3 or halogen; R6 is CH3, CF3 or halogen; R7 is phenyl or 2-pyridinyl, each optionally substituted; and p is 0, 1 or 2. 4. The method of claim 3 wherein R3 is C1-C4 alkyl and R6 is CF3. 5. The method of claim 3 wherein R3 is C1-C4 alkyl and R6 is Cl or Br. 6. The method of claim 1 wherein the compound of Formula I is included in a composition further comprising an effective amount of at least one additional biologically active compound or agent. 7. The method of claim 6 wherein the at least one additional biologically active compound or agent is selected from arthropodicides of the group consisting of pyrethroids, carbamates, neonicotinoids, neuronal sodium channel blockers, insecticidal macrocyclic lactones, γ-aminobutyric acid (GABA) antagonists, insecticidal ureas and juvenile hormone mimics. 8. The method of claim 6 wherein the at least one additional biologically active compound or agent is selected from the group consisting of abamectin, acephate, acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin, azinphos-methyl, bifenthrin, binfenazate, buprofezin, carbofuran, chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl, chromafenozide, clothianidin, cyfluthrin, beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin, diafenthiuron, diazinon, diflubenzuron, dimethoate, diofenolan, emamectin, endosulfan, esfenvalerate, ethiprole, fenothicarb, fenoxycarb, fenpropathrin, fenproximate, fenvalerate, fipronil, flonicamid, flucythrinate, tau-fluvalinate, flufenerim (UR-50701), flufenoxuron, fonophos, halofenozide, hexaflumuron, imidacloprid, indoxacarb, isofenphos, lufenuron, malathion, metaldehyde, methamidophos, methidathion, methomyl, methoprene, methoxychlor, monocrotophos, methoxyfenozide, nithiazin, novaluron, noviflumuron (XDE-007), oxamyl, parathion, parathion-methyl, permethrin, phorate, phosalone, phosmet, phosphamidon, pirimicarb, profenofos, pymetrozine, pyridalyl, pyriproxyfen, rotenone, spinosad, spiromesifin (BSN 2060), sulprofos, tebufenozide, teflubenzuron, tefluthrin, terbufos, tetrachlorvinphos, thiacloprid, thiamethoxam, thiodicarb, thiosultap-sodium, tralomethrin, trichlorfon and triflumuron, aldicarb, oxamyl, fenamiphos, amitraz, chinomethionat, chlorobenzilate, cyhexatin, dicofol, dienochlor, etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin, fenpyroximate, hexythiazox, propargite, pyridaben, tebufenpyrad; and biological agents such as Bacillus thuringiensis including ssp. aizawai and kurstaki, Bacillus thuringiensis delta endotoxin, baculovirus, and entomopathogenic bacteria, virus and fungi. 9. The method of claim 6 wherein the at least one additional biologically active compound or agent is selected from the group consisting of cypermethrin, cyhalothrin, cyfluthrin and beta-cyfluthrin, esfenvalerate, fenvalerate, tralomethrin, fenothicarb, methomyl, oxamyl, thiodicarb, clothianidin, imidacloprid, thiacloprid, indoxacarb, spinosad, abamectin, avermectin, emamectin, endosulfan, ethiprole, fipronil, flufenoxuron, triflumuron, diofenolan, pyriproxyfen, pymetrozine, amitraz, Bacillus thuringiensis, Bacillus thuringiensis delta endotoxin and entomophagous fungi. 10. The method of claim 1 wherein at least one of the insect pests controlled is selected from the group consisting of Alabama argillacea Huibner (cotton leaf worm), Archips argyrospila Walker (fruit tree leaf roller), A. rosana Linnaeus (European leaf roller) and other Archips species, Chilo suppressalis Walker (rice stem borer), Cnaphalocrosis medinalis Guenee (rice leaf roller), Crambus caliginosellus Clemens (corn root webworm), Crambus teterrellus Zincken (bluegrass webworm), Cydia pomonella Linnaeus (codling moth), Earias insulana Boisduval (spiny bollworm), Earias vittella Fabricius (spotted bollworm), Helicoverpa armigera Huibner (American bollworm), Helicoverpa zea Boddie (corn earworm), Heliothis virescens Fabricius (tobacco budworm), Herpetogramma licarsisalis Walker (sod webworm), Lobesia botrana Denis & Schiffermutller (grape berry moth), Pectinophora gossypiella Saunders (pink bollworm), Phyllocnistis citrella Stainton (citrus leafminer), Pieris brassicae Linnaeus (large white butterfly), Pieris rapae Linnaeus (small white butterfly), Plutella xylostella Linnaeus (diamondback moth), Spodoptera exigua Huibner (beet armyworm), Spodoptera litura Fabricius (tobacco cutworm, cluster caterpillar), Spodoptera frugiperda J. E. Smith (fall armyworm), Trichoplusia ni Htibner (cabbage looper) and Tuta absoluta Meyrick (tomato leafminer), Acyrthisi phonpisum Harris (pea aphid), Aphis craccivora Koch (cowpea aphid), Aphis fabae Scopoli (black bean aphid), Aphis gossypii Glover (cotton aphid, melon aphid), Aphis pomi De Geer (apple aphid), Aphis spiraecola Patch (spirea aphid), Aulacorthum solani Kaltenbach (foxglove aphid), Chaetosiphon fragaefolii Cockerell (strawberry aphid), Diuraphis noxia Kurdjumov/Mordvilko (Russian wheat aphid), Dysaphis plantaginea Paaserini (rosy apple aphid), Eriosoma lanigerum Hausmann (woolly apple aphid), Hyalopterus pruni Geoffroy (mealy plum aphid), Lipaphis erysimi Kaltenbach (turnip aphid), Metopolophium dirrhodum Walker (cereal aphid), Macrosipum euphorbiae Thomas (potato aphid), Myzus persicae Sulzer (peach-potato aphid, green peach aphid), Nasonovia ribisnigri Mosley (lettuce aphid), Pemphigus spp. (root aphids and gall aphids), Rhopalosiphum maidis Fitch (corn leaf aphid), Rhopalosiphum padi Linnaeus (bird cherry-oat aphid), Schizaphis graminum Rondani (greenbug), Sitobion avenae Fabricius (English grain aphid), Therioaphis maculata Buckton (spotted alfalfa aphid), Toxoptera aurantii Boyer de Fonscolombe (black citrus aphid), and Toxoptera citricida Kirkaldy (brown citrus aphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande (pecan phylloxera); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotato whitefly), Bemisia argentifolii Bellows & Perring (silverleaf whitefly), Dialeurodes citri Ashmead (citrus whitefly) and Trialeurodes vaporariorum Westwood (greenhouse whitefly); Empoasca fabae Harris (potato leafhopper), Laodelphax striatellus Fallen (smaller brown planthopper), Macrolestes quadrilineatus Forbes (aster leafhopper), Nephotettix cinticeps Uhler (green leafhopper), Nephotettix nigropictus Stal (rice leafhopper), Nilaparvata lugens Stal (brown planthopper), Peregrinus maidis Ashmead (corn planthopper), Sogatella furcifera Horvath (white-backed planthopper), Sogatodes orizicola Muir (rice delphacid), Typhlocyba pomaria McAtee white apple leafhopper, Erythroneoura spp. (grape leaffioppers); Magicidada septendecim Linnaeus (periodical cicada); Icerya purchasi Maskell (cottony cushion scale), Quadraspidiotus perniciosus Comstock (San Jose scale); Planococcus citri Risso (citrus mealybug); Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster (pear psylla), Trioza diospyri Ashmead (persimmon psylla), Acrosternum hilare Say (green stink bug), Anasa tristis De Geer (squash bug), Blissus leucopterus leucopterus Say (chinch bug), Corythuca gossypii Fabricius (cotton lace bug), Cyrtopeltis modesta Distant (tomato bug), Dysdercus suturellus Herrich-Schaffer (cotton stainer), Euchistus servus Say (brown stink bug), Euchistus variolarius Palisot de Beauvois (one-spotted stink bug), Graptosthetus spp. (complex of seed bugs), Leptoglossus corculus Say (leaf-footed pine seed bug), Lygus lineolaris Palisot de Beauvois (tarnished plant bug), Nezara viridula Linnaeus (southern green stink bug), Oebalus pugnax Fabricius (rice stink bug), Oncopeltus fasciatus Dallas (large milkweed bug), Pseudatomoscelis seriatus Reuter (cotton fleahopper), Frankliniella occidentalis Pergande (western flower thrip), Scirthothrips citri Moulton (citrus thrip), Sericothrips variabilis Beach (soybean thrip), and Thrips tabaci Lindeman (onion thrip), Leptinotarsa decemlineata Say (Colorado potato beetle), Epilachna varivestis Mulsant (Mexican bean beetle) and wireworms of the genera Agriotes, Athous or Limonius). 11. A benzoxazinone compound of Formula 10 wherein R4 is H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C1-C6 haloalkyl, CN, halogen, C1-C4 alkoxy, C1-C4 haloalkoxy or NO2; R5 is H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C4 alkoxyalkyl, C1-C4 hydroxyalkyl, C(O)R10, CO2R10, C(O)NR10R11, halogen, C1-C4 alkoxy, C1-C4 haloalkoxy, NR10R11, N(R11)C(O)R10, N(R11)CO2R10 or S(O)nR12; R6 is H, C1-C6 alkyl, C1-C6 haloalkyl, halogen, CN, C1-C4 alkoxy or C1-C4 haloalkoxy; R7 is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C1-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl or C3-C6 halocycloalkyl; or R7 is a phenyl ring, a benzyl ring, a 5- or 6-membered heteroaromatic ring, a naphthyl ring system or an aromatic 8-, 9- or 10-membered fused heterobicyclic ring system, each ring or ring system optionally substituted with one to three substituents independently selected from R9; R8 is H, C1-C6 alkyl, C1-C6 haloalkyl, halogen, C1-C4 alkoxy or C1-C4 haloalkoxy; each R9 is independently C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C2-C4 haloalkenyl, C2-C4 haloalkynyl, C3-C6 halocycloalkyl, halogen, CN, NO2, C1-C4 alkoxy, C1-C4 haloalkoxy, C1-C4 alkylthio, C1-C4 alkylsulfinyl, C1-C4 alkylsulfonyl, C1-C4 alkylamino, C2-C8 dialkylamino, C3-C6 cycloalkylamino, C4-C8 (alkyl)(cycloalkyl)amino, C2-C4 alkylcarbonyl, C2-C6 alkoxycarbonyl, C2-C6 alkylaminocarbonyl, C3-C8 dialkylaminocarbonyl or C3-C6 trialkylsilyl; R10 is H, C1-C4 alkyl or C1-C4 haloalkyl; R11 is H or C1-C4 alkyl; R12 is C1-C4 alkyl or C1-C4 haloalkyl; and n is 0, 1 or 2. 12. The compound of claim 11 wherein R7 is a phenyl ring or a 5- or 6-membered heteroaromatic ring selected from the group consisting of each ring optionally substituted with one to three substituents independently selected from R9; Q is O, S, NH or NR9; and W, X, Y and Z are independently N, CH or CR9, provided that in J-3 and J-4 at least one of W, X, Y or Z is N. 13. The compound of claim 12 wherein R8 is H; R4 group is attached at position 2; R4 is CH3, CF3, OCF3, OCHF2, CN or halogen; R5 is H, CH3 or halogen; R6 is CH3, CF3 or halogen; and R7 is phenyl or 2-pyridinyl, each optionally substituted. 14. The compound of claim 13 wherein R6 is CF3. 15. The compound of claim 13 wherein R6 is Cl or Br. 16. The compounds of claim 11 wherein R4 is at the 2 position and is CH3, Cl or Br; R5 is at the 4 position and is F, Cl, Br, I or CF3; R6 is CF3, Cl or Br; R7 is 3-C1-2-pyridinyl or 3-Br-2-pyridinyl; and R8 is H. 17. The method of claim 1 wherein a plant is contacted by a composition comprising the compound of Formula I, the N-oxide, or the agriculturally suitable salt thereof, applied as a soil drench of a liquid formulation. |
<SOH> BACKGROUND OF THE INVENTION <EOH>This invention relates to a method of use for controlling invertebrate pests in both agronomic and nonagronomic environments of certain anthranilamides, their N-oxides, agriculturally suitable salts and compositions. The control of invertebrate pests is extremely important in achieving high crop efficiency. Damage by invertebrate pests to growing and stored agronomic crops can cause significant reduction in productivity and thereby result in increased costs to the consumer. The control of invertebrate pests in forestry, greenhouse crops, ornamentals, nursery crops, stored food and fiber products, livestock, household, and public and animal health is also important. Many products are commercially available for these purposes, but the need continues for new compounds that are more effective, less costly, less toxic, environmentally safer or have different modes of action. NL 9202078 discloses NV-acyl anthranilic acid derivatives of Formula i as insecticides wherein, inter alia, X is a direct bond; Y is H or C 1 -C 6 alkyl; Z is NH 2 , NH(C 1 -C 3 alkyl) or N(C 1 -C 3 alkyl) 2 ; and R 1 through R 9 are independently H, halogen, C 1 -C 6 alkyl phenyl, hydroxy, C 1 -C 6 alkoxy or C 1 -C 7 acyloxy. |
<SOH> SUMMARY OF THE INVENTION <EOH>This invention pertains to a method for controlling lepidopteran, homopteran, hemipteran, thysanopteran and coleopteran insect pests comprising contacting the insects or their environment with an arthropodicidally effective amount of a compound of Formula I, its N-oxide or an agriculturally suitable salt thereof wherein A and B are independently O or S; R 1 is H, C 1 -C 6 alkyl, C 2 -C 6 alkoxycarbonyl or C 2 -C 6 alkylcarbonyl; R 2 is H or C 1 -C 6 alkyl; R 3 is H; C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, or C 3 -C 6 cycloalkyl, each optionally substituted with one or more substituents selected from the group consisting of halogen, CN, NO 2 , hydroxy, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 haloalkoxy, C 1 -C 4 alkylthio, C 1 -C 4 alkylsulfinyl, C 1 -C 4 alkylsulfonyl, C 2 -C 6 alkoxycarbonyl, C 2 -C 6 alkylcarbonyl, C 3 -C 6 tialkylsilyl, phenyl, phenoxy, 5-membered heteroaromatic rings, and 6-membered heteroaromatic rings; each phenyl, phenoxy, 5-membered heteroaromatic ring, and 6-membered heteroaromatic ring optionally substituted with one to three substituents independently selected from the group consisting of C 1 -C 4 alkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl, C 1 -C 4 haloalkyl, C 2 -C 4 haloalkenyl, C 2 -C 4 haloalknyl, C 3 -C 6 halocycloalkyl, halogen, CN, NO 2 , C 1 -C 4 alkoxy, C 1 -C 4 haloalkoxy, C 1 -C 4 alkylthio, C 1 -C 4 alkylsulfinyl, C 1 -C 4 alkylsulfonyl, C 1 -C 4 alkylamino, C 2 -C 8 dialkylamino, C 3 -C 6 cycloalkylamino, C 4 -C 8 (alkyl)(cycloalkyl)amino, C 2 -C 4 alkylcarbonyl, C 2 -C 6 alkoxycarbonyl, C 2 -C 6 alkylaminocarbonyl, C 3 -C 8 dialkylaminocarbonyl and C 3 -C 6 trialkylsilyl; C 1 -C 4 alkoxy; C 1 -C 4 alkylamino; C 2 -C 8 dialkylamino; C 3 -C 6 cycloalkylamino; C 2 -C 6 alkoxycarbonyl or C 2 -C 6 alkylcarbonyl; R 4 is H, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 6 cycloalkyl, C 1 -C 6 haloalkyl, CN, halogen, C 1 -C 4 alkoxy, C 1 -C 4 haloalkoxy or NO 2 ; R 5 is H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 4 alkoxyalkyl, C 1 -C 4 hydroxyalkyl, C(O)R 10 , CO 2 R 10 , C(O)NR 10 R 11 , halogen, C 1 -C 4 alkoxy, C 1 -C 4 haloalkoxy, NR 10 R 11 , N(R 11 )C(O)R 10 , N(R 11 )CO 2 R 10 or S(O) n R 12 ; R 6 is H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, halogen, CN, C 1 -C 4 alkoxy or C 1 -C 4 haloalkoxy; R 7 is C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 6 cycloalkyl, C 1 -C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloalkynyl or C 3 -C 6 halocycloalkyl; or R 7 is a phenyl ring, a benzyl ring, a 5- or 6-membered heteroaromatic ring, a naphthyl ring system or an aromatic 8-, 9- or 10-membered fusedheterobicyclic ring system, each ring or ring system optionally substituted with one to three substituents independently selected from R 9 ; R 8 is H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, halogen, C 1 -C 4 alkoxy or C 1 -C 4 haloalkoxy; each R 9 is independently C 1 -C 4 alkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl, C 3 -C 6 cycloalkyl, C 1 -C 4 haloalkyl, C 2 -C 4 haloalkenyl, C 2 C 4 haloayyl, C 3-6 halocycloalkyl, halogen, CN, NO 2 , C 1 -C 4 alkoxy, C 1 -C 4 haloalkoxy, C 1 -C 4 alkylthio, C 1 -C 4 alkylsulfinyl, C 1 -C 4 alkylsulfonyl, C 1 -C 4 alkylamino, C 2 -C 8 dialkylamino, C 3 -C 6 cycloalkylamino, C 4 -C 8 (alkyl)(cycloalkyl)amino, C 2 -C 4 alkylcarbonyl, C 2 -C 6 alkoxycarbonyl, C 2 -C 6 alkylaminocarbonyl, C 3 -C 8 dialkylaminocarbonyl or C 3 -C 6 trialkylsilyl; R 10 is H, C 1 -C 4 alkyl or C 1 -C 4 haloalkyl; R 11 is H or C 1 -C 4 alkyl; R 12 is C 1-4 alkyl or C 1 -C 4 haloalkyl; and n is 0, 1 or 2. This invention also relates to such a method wherein an invertebrate pest or its environment is contacted with a composition comprising a biologically effective amount of a compound of Formula I or a composition comprising a compound of Formula I and a biologically effective amount of at least one additional biologically active compound. This invention further relates to a benzoxazinone compound of Formula 10 wherein R 4 , R 5 , R 6 , R 7 and R 8 are defined as aboved in Formula I. The compound of Formula 10 is useful as a synthetic intermediate for preparing a compound of Formula I. detailed-description description="Detailed Description" end="lead"? |
Chain transport system with add-on components |
The invention relates to a chain-transport system containing chain links (10, 20, 30, 40, 50, . . . ) which are strung together and adjacent chain links (10-20, 20-30, 30-40, 40-50, . . . ) being rotatably joined together about an axis of rotation (11, 21, 31, 41, 51) common to one of the two chain links and all of the axes of rotation (11, 21, 31, 41, 51, . . . ) in the chain-transport system running parallel to each other. At least some of the chain links (10, 50, 90, . . . ) are adapted (10-12, 50-52, 90-92, . . . ) in such a way that respectively one add-on component (13, 53, 93) can be attached thereto. According to the invention, attachment of the respective component (13, 53, 93, . . . ) to the respective adapted chain link (10-12, 50-52, 90-92, . . . ) occurs in the form of a positive fit connection between a connecting area (13a, 13b, 13c, . . . ) of the add-on component (13, . . . ) and a connecting area (12a, 12b, 12c, . . . ) of the adapted chain link (10-12, . . . ). The invention also relates to a system for securing add-on components to a transport chain, whereby an add-on component (13, 53, 93, . . . ) is secured to each respectively selected chain link (10, 50, 90, . . . ). According to the invention, the respective add-on component (13, 53, 93, . . . ) is secured to the respective selected chain link (10, 50, 90, . . . ) with the aid of an adapter (12, 52, 92, . . . ). |
1. A chain transporter system which includes sequentially arranged chain links (10, 20, 30, 40, 50, . . . ) and in which adjacent chain links (10-20, 20-30, 30-40, 40-50, . . . ) are rotatably attached to one another about a rotational axis common to both chain links (11, 21, 31, 41, 51, . . . ), and all rotational axes (11, 21, 31, 41, 51) of the chain transporter system are extend parallel to one other, wherein at least some of the chain links (10, 50, 90, . . . ) are adapted (10-12, 50-52, 90-92, . . . ) in such manner that a mounting part (13, 53, 93) is attachable to each, characterised in that the attachment of the respective mounting part (13, 53, 93, . . . ) to the respective adapted chain link (10-12, 50-52, 90-92, . . . ) is created by means of a positive locking connection between a connection area (13a, 13b, 13c, . . . ) of mounting part (13, . . . ) and a connection area (12a, 12b, 12c, . . . ) of the adapted chain link (10-12, . . . ). 2. The chain transporter system according to claim 1, characterised in that one mounting part is attached to each of the at least some chain links (10, 50, 90). 3. The chain transporter system according to claim 1 or 2, characterised in that the connection area (12a, 12b, 12c, . . . ) of the adapted chain link (10-12, . . . ) is a counterpart recess for the connection area (13a, 13b, 13c, . . . ) of the mounting part (13, . . . ). 4. The chain transporter system according to any of claims 1 to 3, characterised in that the recess is a groove-type depression in the surface (12d, 12e) of the adapted chain link (10-12, . . . ) along a groove direction (N), wherein opposing walls of the groove, each furnished with an undercut (12a, 12b, . . . ) parallel to the surface (12d, 12e) of the adapted chain link (10-12, . . . ) and having a depth (t) perpendicular to the groove direction (N), extend in complementary fashion into the corresponding socket-like extensions (13a, 13b, . . . ) in the connection area of the mounting part (13, . . . ). 5. The chain transporter system according to claim 4, characterised in that at least one of the two undercuts (12a) has a undercut depth (t) perpendicular to the groove direction (N) that is less than the maximum flaring (Δa) of the groove's clearance (a) that can be attained by elastic distortion of the groove perpendicular to the groove direction (N). 6. The chain transporter system according to claim 4 or 5, characterised in that at least one of the undercuts (12a) has the form of a concave rounding (12a) in a section perpendicular to the groove direction (N) and the surface area of the adapted chain link (10-12) has the form of a convex rounding (12f) above the undercut (12a) in a section perpendicular to the groove direction (N), wherein the transition area (12g) between the convex rounding (12f) and the concave rounding (12a) is the area of the groove wall projecting farthest into the groove opening perpendicularly to the groove direction (N). 7. The chain transporter system according to claims 4 to 6, characterised in that at least one knob-like projection (12c) and/or depression is provided in the floor of the recess that fits into and/or into which a corresponding counterpart depression (13c) and/or stop boss fits in the connection area of the mounting piece (13). 8. The chain transporter system according to claims 4 to 7, characterised in that at least one bulging projection and/or one depression is provided in the floor of the recess which extends in a direction not parallel to the groove direction (N). 9. The chain transporter system according to any of the preceding claims, characterised in that the adapted chain links (10-12, 50-52, 90-92, . . . ) each consist of a regular chain link (10, 50, 90) and an adapter element (12, 52, 92, . . . ) positively engaged therewith, which adapter element is conformed such that the mounting part (13, 53, 93) is attachable thereto. 10. The chain transporter system according to claim 9, characterised in that the adapter element (12, 52, 92, . . . ) is positively engaged with pins (14, 15) projecting on both sides of the respective adapted chain link (10-12, 50-52, 90-92, . . . ) of the chain. 11. The chain transporter system according to claim 10, characterised in that perpendicularly to the longitudinal direction (K) of the chain, the adapter element (12) has an essentially U-shaped cross-section with a crosspiece (12h) and two legs (12i, 12j) angled essentially perpendicularly therefrom, wherein the U-shaped adapter element (12) clasps the respective chain link (10) in positive locking engagement. 12. The chain transporter system according to claim 11, characterised in that the legs (12i, 12j) have at least one recess or hole (12k) on the insides thereof facing the chain link (10), in which the at least one projecting pin (14, 15) of the respective chain link is form-fitted. 13. The chain transporter system according to claim 12, characterised in that the end area of the legs (12i, 12j) of the adapter element (12) are tapered on the inside facing the chain link by a surface (12l) inclined towards the leg extremity. 14. The chain transporter system according to any of the preceding claims, characterised in that the mounting part (13) is an angle element in which the first angled leg includes the connection area (13a, 13b, 13c) of the mounting part (13), and the second angled leg (13d) extends at an angle (γ) to the first angled leg. 15. The chain transporter system according to claim 14, characterised in that the first angled leg engages positively with the adapted chain link (10-12) in such manner that the inside surface (13e) of the first angled leg is flush with the surface (12d, 12e) of the adapted chain link (10-12). 16. A system for securing mounting parts to a transporter chain, wherein one mounting part (13, 53, 93, . . . ) each is attached to a selected chain link (10, 50, 90, . . . ), characterised in that each mounting part (13, 53, 93, . . . ) is attached to the respective selected chain link (10, 50, 90, . . . ) using an adapter element (12, 52, 92, . . . ). 17. The system according to claim 16, characterised in that the mounting part (13, 53, 93, . . . ) is in positive engagement with pins (14, 15) projecting on both sides of the respective chain link (10, 50, 90, . . . ). 18. The system according to claim 16 or 17, characterised in that the adapter element (12, 52, 92) has an essentially U-shaped cross-section perpendicularly to the chain's longitudinal direction (K), with a crosspiece (12h) and two legs (12i, 12j) angled essentially perpendicularly to the crosspiece, wherein the U-shaped adapter element (12, 52, 92) clasps the respective chain link (10, 50, 90) in positive engagement. 19. The system according to any of claims 16 to 18, characterised in that the legs (12i, 12j) have at least one recess or hole (12k) on the insides thereof facing the chain link (10), in which the at least one projecting pin (14, 15) of the respective chain link (10) is form-fitted. 20. The system according to any of claims 16 to 19, characterised in that at the end area thereof, the legs (12i, 12j) are tapered on an inside surface facing the chain link by a surface (12l) inclined towards the leg extremity. 21. The system according to any of claims 16 to 20, characterised in that the mounting part (13) is connected to adapter element (12) via a positive locking engagement between a connection area (13a, 13b, 13c) of the mounting part (13) and a connection area (12a, 12b, 12c) of the adapter element (12). 22. The system according to claims 16 to 21, characterised in that the connection area (12a, 12b, 12c) of the adapter element (12) has a recess matching the connection area (13a, 13b, 13c) of the mounting part (13). 23. The system according to any of claims 16 to 22, characterised in that the recess is a groove type depression in the surface (12d, 12e), of the adapter element (12) along a groove direction (N), wherein opposing groove walls are each furnished with an undercut (12a, 12b) parallel to the surface (12d, 12e) of the adapter element (12) and having a depth (t) perpendicular to the groove direction (N), in which corresponding socket-type enlargements (13a, 13b) in the connection area of the mounting part (13) engage mutually. 24. The system according to any of claims 16 to 23, characterised in that at least one of the two undercuts (12a) has an undercut depth (t) perpendicular to the groove direction (N) that is less than the maximum flaring (Δa) of the groove's clearance (a) that can be attained by elastic distortion of the groove perpendicular to the groove direction (N). 25. The system according to any of claims 16 to 24, characterised in that at least one of the undercuts (12a) has the form of a concave rounding (12a) in a section perpendicular to the groove direction (N) and the surface area of the adapter element (12) above the undercut (12a) has a convex rounding (12f) in a section perpendicular to the groove direction (N), wherein the transition area (g) between the convex rounding (f) and the concave rounding (a) is the area of the groove wall projecting farthest into the groove opening perpendicularly to the groove direction (N). 26. The system according to any of claims 16 to 25, characterised in that at least one projection (12c) and/or one depression is provided in the floor of the recess of the adapter element (12), which fits into or is fitted by a corresponding matching depression (13c) or projection and in the connection area of the mounting part (13). 27. The system according to any of claims 16 to 25, characterised in that at least one knob-type projection and/or a depression is provided in the floor of the recess of the adapter element (12), which extends in a direction not parallel to the groove direction (N). 28. The system according to any of the preceding claims, characterised in that the mounting part (13) is an angled element, in which the first angled leg is the connection area (13a, 13b, 13c) of the mounting part (13) and the second angled leg (13d) extends at an angle (γ) to the first angled leg (13a, 13b, 13c). 29. The system according to claim 28, characterised in that the first angled leg engages positively with the adapter element (12), such that the inside surface (13e) of the first angled leg is flush with the upper surface (12d, 12e) of the adapter element (12). 30. The chain transporter system according to any of claims 1 to 15, characterised in that it is usable in chocolate processing, particularly in the area of cooling lines and/or heating lines. 31. The system for securing mounting parts to a transporter chain according to any of claims 16 to 29, characterised in that it is usable in chocolate-processing, particularly in the area of cooling lines and/or heating lines. |
Integrated adjustable lumbar support and trim attachment system |
An adjustable lumbar support (10) adapted to be integrated into new or existing seat structures. Adjustable lumbar support (10) includes a floating plate (15) supported by a support linkage. In one embodiment, the support linkage is defined by first and second spring members (25) and (26) which engage the seat frame. In an alternate embodiment, the support linkage is defined by first and second spring members (25) and (26) engaged with first and second linkage members (52), respectively, and includes a rotary power mechanism (50) for applying a rotating motion to a shaft (54) threadably engaged with first and second linkage members (52). The degree of support provided by floating plate (15) is adjusted by adjusting the effective length of the support linkage. Lumbar support mechanism (10) can be mounted in a seat back alone or in conjunction with an integral trim attachment array (70). Trim attachment array (70) is adapted to replace the trim wires molded into the cushion foam of a standard seat. |
1. An adjustable lumbar support for seats, said adjustable lumbar support comprising: a floating support plate for providing support to a lumbar region of a seat, said support plate having an front surface and a rear surface; a support linkage for supporting said floating support plate, wherein said support linkage is adapted for engaging a seat frame, said support linkage having an effective length and including at least one spring member disposed between said floating support plate and the seat frame, said support linkage supporting said floating support plate in a pivotal manner; and an actuator mechanism for adjusting said effective length of said support linkage, whereby an amount of lumbar support provided by said floating support plate is increased as said effective length of said support linkage is decreased. 2. The adjustable lumbar support of claim 1 wherein said floating support plate has a curved front surface. 3. The adjustable lumbar support of claim 1 wherein said actuator mechanism is manually activated. 4. The adjustable lumbar support of claim 1 wherein said actuator mechanism is electrically powered. 5. The adjustable lumbar support of claim 4 wherein said actuator mechanism applies rotating motion to a shaft having first and second ends wherein said first and second ends are oppositely threaded. 6. The adjustable lumbar support of claim 1 wherein said floating support plate is constructed of a substantially rigid material. 7. The adjustable lumbar support of claim 1 wherein said support linkage is defined by a cable member and first and second spring members, wherein said spring members are adapted for engaging a seat frame, wherein said cable member has a first end secured to said actuator mechanism and a second end secured to one of said spring members, said cable member slides in a cable housing secured to said rear surface of said floating support plate. 8. The adjustable lumbar support of claim 1 wherein said floating support plate includes a projection disposed from said rear surface of said floating support plate. 9. The adjustable lumbar support of claim 7 wherein said cable housing is integral with said floating support plate. 10. The adjustable lumbar support for seats of claim 1 wherein said support linkage is defined by first and second spring members engaged with first and second linkage members respectively, wherein said first and second spring members are adapted to further engage a seat frame and further wherein said first and second linkage members include threaded fasteners, wherein said actuator mechanism is a powered mechanism for providing rotary motion to a shaft having first and second ends, said first and second ends of said shaft being oppositely threaded and said threaded fastener of said first linkage member being threadably engaged with said first end of said shaft and said threaded fastener of said second linkage member being threadably engaged with said second end of said shaft such that rotation of said shaft in a first direction draws said first and second linkage members inward thereby shortening said effective length of said support linkage, and rotation of said shaft in a second direction moves said first and second linkage members outward thereby increasing said effective length of said support linkage. 11. The adjustable lumbar support of claim 1 wherein said adjustable lumbar support is in active cooperation with an integral trim attachment array comprising a trim ring having integrated pinch clips disposed along a length of said trim ring. 12. The adjustable lumbar support of claim 11 wherein said trim ring further includes a plurality of repair hole members adapted for receiving a repair pinch clip, wherein said repair hole member includes at least one keyway and said repair pinch clip includes at least one key wherein said key and said keyway coact to substantially prevent rotation of said repair pinch clip within said repair hole member. 13. The adjustable lumbar support of claim 1 wherein said floating support plate is molded in a seatback foam cushion. 14. The lumbar support of claim 1 wherein said floating plate includes a plurality of apertures disposed in said front surface, each of said apertures including at least one inward projecting tab, said inward projecting tabs being adapted for receiving a projection molded in a foam cushion whereby said floating plate can be fixed to the foam cushion. 15. An adjustable lumbar support for seats, said adjustable lumbar support comprising: a floating support plate for providing support to a lumbar region of a seat, said support plate having a curved front surface and a rear surface, said floating support plate further having a projection disposed from said rear surface of said floating support plate; a support linkage for supporting said floating support plate, wherein said support linkage is adapted for engaging a seat frame, said support linkage having an effective length and including at least one spring member disposed between said floating support plate and the seat frame, said support linkage supporting said floating support plate in a pivotal manner; and an actuator mechanism for adjusting said effective length of said support linkage, whereby an amount of lumbar support provided by said floating support plate is increased as said effective length of said support linkage is decreased; wherein said adjustable lumbar support is in active cooperation with an integral trim attachment array comprising a trim ring having integrated pinch clips disposed along a length of said trim ring. 16. The adjustable lumbar support of claim 15 wherein said actuator mechanism is manually activated. 17. The adjustable lumbar support of claim 15 wherein said actuator mechanism is electrically powered. 18. The adjustable lumbar support of claim 17 wherein said actuator mechanism applies rotating motion to a shaft having first and second ends wherein said first and second ends are oppositely threaded. 19. The adjustable lumbar support of claim 15 wherein said support linkage is defined by a cable member and first and second spring members, wherein said spring members are adapted for engaging a seat frame, wherein said cable member has a first end secured to said actuator mechanism and a second end secured to one of said spring members, said cable member slides in a cable housing secured to said rear surface of said floating support plate. 20. The adjustable lumbar support of claim 19 wherein said cable housing is integral with said floating support plate. 21. The adjustable lumbar support for seats of claim 15 wherein said support linkage is defined by first and second spring members engaged with first and second linkage members respectively, wherein said first and second spring members are adapted to further engage a seat frame and further wherein said first and second linkage members include threaded fasteners, wherein said actuator mechanism is a powered mechanism for providing rotary motion to a shaft having first and second ends, said first and second ends of said shaft being oppositely threaded and said threaded fastener of said first linkage member being threadably engaged with said first end of said shaft and said threaded fastener of said second linkage member being threadably engaged with said second end of said shaft such that rotation of said shaft in a first direction draws said first and second linkage members inward thereby shortening said effective length of said support linkage, and rotation of said shaft in a second direction moves said first and second linkage members outward thereby increasing said effective length of said support linkage. 22. The adjustable lumbar support of claim 15 wherein said trim ring further includes a plurality of repair hole members adapted for receiving a repair pinch clip, wherein said repair hole member includes at least one keyway and said repair pinch clip includes at least one key wherein said key and said keyway coact to substantially prevent rotation of said repair pinch clip within said repair hole member. 23. The lumbar support of claim 15 wherein said floating plate includes a plurality of apertures disposed in said front surface, each of said apertures including at least one inward projecting tab, said inward projecting tabs being adapted for receiving a projection molded in a foam cushion whereby said floating plate can be fixed to the foam cushion. 24. The adjustable lumbar support of claim 15 wherein said floating support plate is molded in a seatback foam cushion. 25. The adjustable lumbar support of claim 15 wherein said floating support plate is constructed of a substantially rigid material. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of Invention The present invention relates generally to seats, including vehicular seats. More particularly, the present invention relates to an insert for seats that provides adjustable lumbar support to a seat occupant. 2. Description of the Related Art It is well-recognized that a person sitting in a seat for an extended time period may desire additional support to obtain greater seat comfort and/or alleviate seat discomfort. Vehicle operators, vehicle passengers, and people whose jobs require them to be seated for extended periods of time are typically chief among those individuals who seek such additional support The support is typically desired to provide greater comfort in the lower or the lumbar region of the back. As used herein, the term “seat” applies to a variety of seat structures, including chairs and vehicular seats. The discomfort problem has been addressed in the prior art in several ways. A simple, although not complete, solution is to utilize a pillow placed low in the seat against the back thereof. By configuring the pillow into different shapes, a variation in lumbar support can be achieved. More permanent solutions to lumbar support problems have also been addressed, for example, in U.S. Pat. No. 5,076,643 issued to A. Colasanti, et al., on Dec. 31, 1993; and U.S. Pat. No. 5,190,348 issued to A. Colasanti on Mar. 2, 1993. Both of the devices described in the '643 and '348 patents are of complex construction and require substantial time and cost for fabrication and installation. The requirements for the installation of a mounting plate, as well as the inter-securement of the bladder and plate elements, are primary reasons for the high cost, complexity of construction and the need of substantial time for the manufacture and installation of each device. Other devices are also known in the art. Among these other devices are those disclosed in U.S. Pat. No. 4,567,615 issued to H. Fanti on Feb. 4, 1986, and U.S. Pat. No. 6,056,360, issued on May 2, 2000, to the predecessor in interest of the assignee of the present application, discloses a lumbar support system comprising unitary and divided leaf members. This device proved to be complicated and costly to manufacture. It is also known, in the art of vehicular seats, that seat trim is attached to standard trim wires, which are typically molded in the seat back and cushion foam, which provide trim cover attachment points via mechanical fasteners such as hog rings. What is needed, and is missing from the art, is a lumbar support which is integral with a trim attachment array. Accordingly, it is an object of the present invention to provide an adjustable lumbar support for seats that can be installed with a minimum of labor. It is also an object of the present invention to provide an adjustable lumbar support that utilizes a floating plate that “self centers” or “self aligns” offering firm yet compliant support and that “moves” with the occupant. It is another object of the present invention to provide an adjustable lumbar support which is regulable using different regulation mechanisms. Further, it is an object of the present invention to provide an adjustable lumbar support which can be installed alone or in conjunction with a trim attachment array. Still another object of the present invention is to provide a lumbar support that can be integral with a trim attachment array. Yet another object of the present invention is to provide a lumbar support that is integral with a seat suspension. These and other objects of the present invention will become apparent upon a consideration of the drawings referred to hereinafter, and a complete description thereof. |
<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>An adjustable lumbar support mechanism, constructed in accordance with the present invention, is provided which provides advantages over the prior art. The adjustable lumbar support can be easily integrated into a seat structure either alone or in conjunction with an integral trim attachment array. The adjustable lumbar support includes a floating plate that “self centers” or “self aligns” offering firm yet compliant support and that “moves” with the occupant and is fully sprung, so as not to interfere with state of the art vibration insulation aspects of a seat, especially a vehicle seat. The floating support plate includes a curved front surface used to achieve lumbar support for a seat. The floating support plate is supported by a support linkage which includes first and second spring members that are engaged with the seat frame. The degree of lumbar support is adjusted by altering the effective length of the support linkage. A manual adjustment is provided for as well as a power adjustment. The powered embodiment is fully adaptable with “memory seating”, wherein the memory function is provided by means of hall effect pulse counting of motor rotations or simple potentiometer based sensors as is readily known to those skilled in the art. The integral trim attachment array of the present invention is adapted to replace the standard trim wires which are molded into the seat back and cushion foam of a standard, state-of-the-art vehicle seat The integral trim attachment array is defined by a trim ring having integrated pinch clips. The trim ring has a rectilinear configuration adapted to conform to the outline of a selected seat back or cushion foam. Linkages are provided for linking a plurality of trim rings together or for linking the trim ring to the floating support plate. Additionally, the floating support plate could be fashioned with integrated pinch clips to provide additional points of attachment for the seat trim. The pinch clips are adapted to secure the listing wires in the trim assembly to the face of the seat. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS The above-mentioned features of the invention will become more clearly understood from the following detailed description of the invention read together with the drawings in which: FIG. 1 is a front elevation view of the manually adjustable lumbar support mechanism of the present invention. FIG. 2 is a top plan view of the manually adjustable lumbar support mechanism of the present invention showing the lumbar support mechanism in the retracted position, it being understood that the retracted position is the position that provides the least amount of lumbar support. FIG. 3 is a top plan view of the manually adjustable lumbar support mechanism of the present invention showing the lumbar support mechanism in the extended position, it being understood that the extended position is the position that provides the greatest degree of lumbar support. FIG. 4 is a rear elevation view of the manually adjustable lumbar support mechanism of the present invention. FIG. 5 is a front elevation view of an alternate embodiment of the adjustable lumbar support mechanism of the present invention which uses an electrically powered rotary mechanism for adjusting the lumbar support between the extended and retracted positions. FIG. 6 is a top plan view of the electrically powered embodiment of the lumbar support mechanism of the present invention showing the lumbar support mechanism in the retracted position. FIG. 7 is a rear elevation view of the electrically powered embodiment of the lumbar support mechanism of the present invention showing the lumbar support mechanism in the extended position. FIG. 8 is a rear elevation view of the electrically powered embodiment of the lumbar support mechanism of the present invention. FIG. 9 is a side elevation view of the lumbar support mechanism of the present invention, along with the integrated trim attachment system positioned within a typical seatback cushion (shown in phantom). FIG. 10 is a rear elevation view of the lumbar support mechanism and integrated trim attachment system shown in FIG. 9 . FIG. 11 is a rear elevation view illustrating the mounting of the lumbar support mechanism in a seat frame. FIGS. 12A and 12B show perspective detail views of a portion of the trim ring showing a repair clip and a receptor for the repair clip. FIGS. 13A and 13B show an alternate embodiment of the lumbar support mechanism of the present invention for mounting the lumbar support mechanism to the foam cushion of a seat back. detailed-description description="Detailed Description" end="lead"? |
Water-based pigment dispersions, the production thereof and the use of the same |
The invention relates to a pigment dispersion consisting essentially of a) at least one organic or inorganic pigment or a combination of the same, b) an alpha-methyl-omega-hydroxy-polyethylene glycol ether having an average molar mass of between 250 and 1000 g/mol, c) standard dispersing agents for producing aqueous pigment dispersions, d) water, and e) optionally other standard additives. |
1) A pigment dispersion consisting essentially of a) at least one organic or inorganic pigment or combination thereof, b) an alpha-methyl-omega-hydroxy-polyethylene glycol ether having an average molar mass of from 250 to 1000 g/mol, c) at least one dispersant, and d) water 2) A pigment dispersion as claimed in claim 1, consisting essentially of a) from 10 to 80% by weight of at least one organic or inorganic pigment; b) from 1 to 50% by weight of an alpha-methyl-omega-hydroxy-polyethylene glycol ether having an average molar mass of between 250 and 1000 g/mol; c) from 2 to 25% by weight of a usual dispersant, d) from 5 to 80% by weight of water; e) from 0 to 5% by weight of customary additivesat least one additive, the percentages by weight being based in each case on the total weight of the pigment dispersion. 3) A pigment dispersion as claimed in claim 1, consisting essentially of a) from 20 to 70% by weight of at least one organic or inorganic pigment; b) from 2 to 30% by weight of an alpha-methyl-omega-hydroxy-polyethylene glycol ether having an average molar mass of between 250 and 1000 g/mol; c) from 3 to 15% by weight of a usual dispersant, d) from 10 to 60% by weight of water; e) from 0 to 5% by weight of customary additivesat least one additive, the percentages by weight being based in each case on the total weight of the pigment dispersion. 4) A pigment dispersion as claimed in claim 3, comprising the alpha-methyl-omega-hydroxy-polyethylene glycol ether in an amount of from 4 to 20% by weight. 5) A pigment dispersion as claimed in claim 1, wherein the alpha-methyl-omega-hydroxy-polyethylene glycol ether has an average molar mass of between 400 and 600 g/mol. 6) A pigment dispersion as claimed in one claim 1, wherein the alpha-methyl-omega-hydroxy-polyethylene glycol ether has an average molar mass of between 470 and 530 g/mol. 7) A pigment dispersion as claimed in claim 1, wherein the organic pigment is a monoazo pigment, disazo pigment, laked azo pigment, triphenylmethane pigment, thioindigo pigment, thiazineindigo pigment, perylene pigment, perinone pigment, anthanthrone pigment, diketopyrrolopyrrole pigment, dioxazine pigment, quinacridone pigment, phthalocyanine pigment, isoindolinone pigment, isoindoline pigment, benzimidazolone pigment, naphthol pigment, and quinophthalone pigment, or an acidic to alkaline carbon black from the furnace black or gas black group. 8) A process for preparing a pigment dispersion as claimed in claim 1 comprising the step of dispersing component (a) in powder, granule or aqueous presscake form in the presence of water and of components (b) and (c) to form an aqueous pigment dispersion. 9) A process for coloring a natural or synthetic material comprising the step of pigmenting the material with a pigment dispersion as claimed in claim 1. 10) The process as claimed in claim 9, wherein the material is selected from the group consisting of paints, emulsion paints, dispersion varnishes, printing inks, wallpaper inks, water-thinnable coating materials, sausage casings, seed, glass bottles, plasters, wood stains, paper stocks, colored pencil leads, felttip pens, artist's inks, pastes for ballpoint pens, chalks, detergents and cleaning products, shoecare products, latex products, abrasives, plastics, electrophotographic toners and developers, powder coating materials, and inkjet inks. 11. A pigment dispersion as claimed in claim 1, further comprising at least one additive. 12. The process as claimed in claim 8, wherein the dispersing step further comprises adding at least one of water (d) and at least one additive (e). 13. The process as recited in claim 8, further comprising the step of diluting the aqueous pigment dispersion with water. 14. A pigment dispersion made in accordance with the process of claim 8. 15. A pigmented natural or synthetic material made in accordance with the process of claim 9. |
Electroluminescent materials and devices |
An electroluminescent material is a metal complex, preferably aluminium, of a substituted pyrazol-5-one. |
1. An electroluminescent compound which has the formula where M is a metal selected from metals other than a rare earth metals, a transition metals, a lanthanides or an and actinides; n is the valency of M; R1, R2 and R3, which may be the same or different are selected from hydrogen, hydrocarbyl groups, substituted and unsubstituted aliphatic groups, substituted and unsubstituted aromatic, heterocyclic and polycyclic ring structures, fluorocarbons such as trifluoryl methyl groups, halogens such as fluorine or thiophenyl groups or nitrile; R1 and R3 can also be form ring structures, and R1, R2 and R3 can be copolymerisable with a monomer. 2. A compound according to claim 1 in which M is aluminium and R3 is a phenyl or substituted phenyl group. 3. An electroluminescent device comprising (i) a first electrode, (ii) an electroluminescent layer consisting essentially of a layer of an electroluminescent compound according to claim 1, and (iii) a second electrode. 4. An electroluminescent device according to claim 3 in which M is aluminium and R3 is a phenyl or substituted phenyl group. 5. An electroluminescent device according to claim 3 in which R3 is selected from methyl, ethyl, propyl, butyl and pentyl groups. 6. A device according to claim 3 in which there is a layer of a hole transmitting material between the first electrode and the layer of the electroluminescent compound. 7. A device according to claim 3 in which there is a layer of an electron transmitting material between the second electrode and the layer of the electroluminescent compound. 8. An electroluminescent device which comprises (i) a first electrode, (ii) a layer of a hole transmitting material, (iii) an electroluminescent layer consisting essentially of an electroluminescent compound according to claim 1, (iv) a layer of an electron transmitting material, and (v) a second electrode. 9. An electroluminescent device according to claim 8 in which the hole transmitting layer is an aromatic amine complex. 10. An electroluminescent device according to claim 8 in which the hole transmitting layer is formed from a poly(vinylcarbazole), N,N′-diphenyl-N,N′-bis (3-methylphenyl)-1,1′-biphenyl-4,4′-diamine (TPD), polyaniline, or a substituted polyaniline. 11. An electroluminescent device according to claim 8 in which the hole transmitting layer has a formula (II) or (III) herein of wherein formula (II) is: where R is in the ortho- or meta-position and is hydrogen, C1-18 alkyl, C1-6 alkoxy, amino, chloro, bromo, hydroxy or the group where R is alky or aryl and R′ is hydrogen, C1-6 alkyl or aryl with at least one other monomer; and wherein formula (III) is where p is from 1 to 10 and n is from 1 to 20, R is as defined above and X is an anion, preferably selected from Cl, Br, SO4, BF4, PF6, H2PO3, H2PO4, arylsulphonate, arenedicarboxylate, polystyrenesulphonate, polyacrylate alkysulphonate, vinylsulphonate, vinylbenzene sulphonate, cellulosesulphonate, camphor sulphonates, cellulose sulphate or a perfluorinated polyanion. 12. An electroluminescent device according to claim 8 in which the hole transmitting layer is a conjugated polymer as herein specified. 13. An electroluminescent device according to claim 12 in which the hole transmitting layer is selected from poly (p-phenylenevinylene)-PPV and copolymers including PPV, poly(2,5 dialkoxyphenylene vinylene), poly (2-methoxy-5-(2-methoxypentyloxy-1,4-phenylene vinylene), poly(2-methoxypentyloxy)-1,4-phenylenevinylene), poly(2-methoxy-5-(2-dodecyloxy-1,4-phenylenevinylene) and other poly(2,5 dialkoxyphenylenevinylenes) with at least one of the alkoxy groups being a long chain solubilising alkoxy group, poly fluorenes and oligofluorenes, polyphenylenes and oligophenylenes, polyanthracenes and oligo anthracenes, polythiophenes and oligothiophenes. 14. An electroluminescent device according to claim 8 in which the hole transmitting material and the electroluminescent compound are mixed to form one layer in a proportion of 5 to 95% of the hole transmitting material to 95 to 5% of the electroluminescent compound. 15. An electroluminescent device according to claim 8 in which the electron transmitting material is a metal quinolate or aluminium or scandium dibenzoyl methane. 16. An electroluminescent device according to claim 15 in which the electron transmitting material is a metal quinolate selected from the group consisting of lithium, sodium, potassium, zinc, magnesium or aluminium quinolate. 17. An electroluminescent device according to claim 8 in which the electron transmitting material and the electroluminescent compound are mixed to form one layer in a proportion of 5 to 95% of the electron transmitting material to 95 to 5% of the electroluminescent compound. 18. An electroluminescent device according to claim 8 in which the anode and/or cathode is formed on a substrate of crystalline silicon and the surface of the substrate may be polished or smoothed to produce a flat surface prior to the deposition of electrode, or electroluminescent compound. 19. An electroluminescent device according to claim 8 in which the first and/or second electrode is formed on a substrate of a non-planarised silicon substrate. 20. An electroluminescent device according to claim 8 in which there is a copper phthalocyanine layer on the first electrode and a lithium fluoride layer on the second electrode. |
Method of genomic analysis |
The present invention relates to a method for identifying genomic regions comprising one or more genes that affect a biological phenotype such as the level of lymphocyte subpopulations. The present invention also relates to isolated genomic regions identified using the method of the present invention, one or more genes contained in the genomic regions and methods for detecting the presence of the one or more genes in an individual. |
1. A method for identifying genomic regions comprising one or more genes which affect a biological phenotype, comprising performing linkage analysis on one or more extended families wherein the total number of individuals is at least 50. 2. The method according to claim 1, wherein the biological phenotype is the level of lymphocyte subpopulations. 3. The method according to claim 2, wherein the biological phenotype is the level of CD4+ T cells, CD8+ T cells, B cells, NK cells or the ratio of CD4 to CD8+ T cells. 4. The method according to any one of the previous claims wherein the linkage analysis is quantitative trait linkage analysis. 5. The method according to claim 4, wherein the quantitative trait linkage analysis is performed using the SIBPAL2 program implemented in the SAGE package. 6. The method according to any one of the previous claims, wherein the linkage analysis is performed on at least 4 extended families. 7. The method according to any one of the previous claims, wherein the linkage analysis is performed on at least 17 extended families. 8. The method according to any one of the previous claims, wherein the extended families comprise at least 2 generations and at least 5 siblings in the youngest generation. 9. The method according to claim 8, wherein the extended families comprise at least 3 generations. 10. The method to any one of the previous claims, additionally comprising performing fine mapping techniques on the genomic region. 11. The method according to claim 10 where in the fine mapping technique is quantitative transmission disequilibrium test (QTDT) analysis 12. An isolated genomic region identified using the method of any one of claims 1 to 11. 13. An isolated genomic region according to claim 12 which is listed in Table 5. 14. An isolated genomic region from human chromosome 1, which is located at 197 to 218 cM on chromosome 1. 15. The isolated genomic region of claim 14, wherein the genomic region contains one or more genes that affect the level of CD8+ T cells in a human. 16. An isolated genomic region from human chromosome 4, which is flanked by markers D4S405 and D4S2363 on chromosome 4. 17. The isolated genomic region of claim 16 wherein the genomic region contains one or more genes that affect the ratio of CD4 to CD8+ T cells in a human. 18. An isolated genomic region from human chromosome 18, which is located at 90 to 110 cM on chromosome 18. 19. The isolated genomic region of claim 17, wherein the genomic region contains one or more genes that affect the level of CD4+ T cells in a human. 20. A gene contained in the genomic region according to any one of claims 10 to 17, wherein the gene affects the biological phenotype. 21. The gene of claim 20, wherein the gene is locayed within ±1 cM of a marker of the biological phenotype, wherein the marker has a p-value of less than 0.05 as calculated by QTDT analysis. 22. The gene of claim 21, wherein the marker is any one of the markers listed in Table 6. 23. The encoded product of the gene of any one of claims 20 to 22. 24. Use of a probe for the gene of any one of claims 20 to 22, in an assay for detecting an individual's risk of developing a disease, for diagnosing a particular disease, for prognosis of a particular disease or for determining the efficacy or toxicity of a particular treatment. 25. The use of claim 24, wherein the probe is a labelled nucleic acid molecule capable of specifically binding to gene. 26. Use of the gene of any one of claims 20 to 22 in an assay for identifying an agonist or antagonist of the gene. 27. Use of an agonist or antagonist identified by the use of claim 26 in the treatment or prophylaxis of diseases which are caused by the gene. 28. Use of a nucleic acid molecule comprising the gene of any one of claims 20 to 22 in the manufacture of a medicament for use in gene therapy. 29. Use of the encoded product of claim 23 in therapy. 30. Use of the gene of any one of claims 20 to 22 in an assay for identifying a biochemical pathway which is involved in the development or prevention of a disease. 31. A method of predicting the speed of development of AIDS in an individual infected with HIV comprising detecting the presence of one or more genes, which affect the ratio of CD4+ T cells to CD8+ T cells, in the genomic region of chromosome 4 that is flanked by markers D4S405 and D4S2363 in an individual. 32. The method according to claim 27, which comprises: taking a cell sample from the individual; and determining the presence of the one or more genes, which increase the ratio of CD4+ T cells to CD8+ T cells, wherein the presence of the one or more genes is indicative of a genetic predisposition to a high CD4:CDS ratio which reduces the speed of development of AIDS in the individual. |
Information processing apparatus and method |
This invention relates to an information processing device and method that enable classification of a new time series pattern. A time series pattern N of a curve L (21) is inputted to an output layer (13) of a recurrent neural network 1. An intermediate layer (12) has already learned a predetermined time series pattern, and a weighting coefficient corresponding to that time series pattern is held in its neurons. The intermediate layer (12) calculates a parameter corresponding to the time series pattern N on the basis of the weighting coefficient and outputs the calculated parameter from parametric bias nodes (11-2). A comparator unit (31) compares a parameter of a learned pattern stored in a storage unit (32) with the parameter of the time series pattern N and thus classifies the time series pattern N. This invention can be applied to a robot. |
1. An information processing device for classifying a time series pattern, comprising: input means for inputting a time series pattern to be classified; and modeling means for modeling each of plural said time series patterns inputted from the input means on the basis of a common nonlinear dynamic system having one or more feature parameters that can be operated from outside; wherein when a new time series pattern is inputted, said modeling is further performed, and a feature parameter obtained by the modeling and the already obtained feature parameters are compared with each other, thereby classifying the new time series pattern. 2. The information processing device as claimed in claim 1, wherein the nonlinear dynamic system is a recurrent neural network with an operating parameter. 3. The information processing device as claimed in claim 1, wherein the feature parameter indicates a dynamic structure of the time series pattern in the nonlinear dynamic system. 4. An information processing method for an information processing device for classifying a time series pattern, the method comprising: an input step of inputting a time series pattern to be classified; and a modeling step of modeling each of plural time series patterns inputted by the processing of the input step on the basis of a common nonlinear dynamic system having one or more feature parameters that can be operated from outside; wherein when a new time series pattern is inputted, said modeling is further performed, and a feature parameter obtained by the modeling and the already obtained feature parameters are compared with each other, thereby classifying the new time series pattern. 5. A program storage medium having a computer-readable program stored therein, the program being adapted for an information processing device for classifying a time series pattern, the program comprising: an input step of inputting a time series pattern to be classified; and a modeling step of modeling each of plural time series patterns inputted by the processing of the input step on the basis of a common nonlinear dynamic system having one or more feature parameters that can be operated from outside; wherein when a new time series pattern is inputted, said modeling is further performed, and a feature parameter obtained by the modeling and the already obtained feature parameters are compared with each other, thereby classifying the new time series pattern. 6. A computer program for controlling an information processing device for classifying a time series pattern, the program comprising: an input step of inputting a time series pattern to be classified; and a modeling step of modeling each of plural time series patterns inputted by the processing of the input step on the basis of a common nonlinear dynamic system having one or more feature parameters that can be operated from outside; wherein when a new time series pattern is inputted, said modeling is further performed, and a feature parameter obtained by the modeling and the already obtained feature parameters are compared with each other, thereby classifying the new time series pattern. |
<SOH> BACKGROUND ART <EOH>Recently, a neural network has been studies as a mode related to human and animal brains. In a neural network, as a predetermined pattern is learned in advance, whether inputted data corresponds to the learned pattern or not can be identified. Conventionally, in the case of classifying patterns using such a neural network, independent sub-modules are caused to learn the plural patterns. The outputs of the respective sub-modules are weighted at a predetermined rate and constitute the output of the entire module. If an unknown pattern is inputted, it is known to estimate a coefficient value for weighting the outputs of the respective sub-modules to generate a pattern that is most approximate to the inputted pattern, as the output of the entire module, and classify a newly provided pattern in accordance with the value. However, such a classifying method has a problem that a time series pattern as a classification target cannot be classified on the basis of the relation with already learned patterns. That is, only a pattern expressed by a linear sum of learned patterns can be classified and a pattern expressed by a nonlinear sum cannot be classified. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a view showing the structure of a recurrent neural network to which the present invention is applied. FIG. 2 is a flowchart for explaining learning processing of the recurrent neural network of FIG. 1 . FIG. 3 is a flowchart for explaining coefficient setting processing of the recurrent neural network of FIG. 1 . FIG. 4A is a view showing an exemplary time series pattern having different amplitude and the same cycle. FIG. 4B is a view showing an exemplary time series pattern having different amplitude and the same cycle. FIG. 4C is a view showing an exemplary time series pattern having different amplitude and the same cycle. FIG. 5A is a view showing an exemplary time series pattern having a different cycle and the same amplitude. FIG. 5B is a view showing an exemplary time series pattern having a different cycle and the same amplitude. FIG. 5C is a view showing an exemplary time series pattern having a different cycle and the same amplitude. FIG. 6 is a view showing an exemplary learned pattern. FIG. 7 is a view showing an exemplary learned pattern. FIG. 8 is a flowchart for explaining time series pattern generation processing of the recurrent neural network of FIG. 1 . FIG. 9 is a view showing an exemplary time series pattern to be generated. FIG. 10 is a view showing the structure of a recurrent neural network to which the present invention is applied. FIG. 11 is a view showing learned patterns. FIG. 12 is a flowchart for explaining classification processing in the recurrent neural network of FIG. 10 . FIG. 13 is a block diagram showing the structure of a personal computer to which the present invention is applied. detailed-description description="Detailed Description" end="lead"? |
Methods and apparatus for producing gender enriched sperm |
Sperm in semen are sorted by fluorescence-activated cell sorting into gender-enriched populations enriched in X-chromosome or Y-chromosome bearing sperm by use of a fluorescent quantitative DNA-binding vital stain. |
1. A method for sorting semen containing predominantly living, viable sperm into GES (gender enriched sperm) comprising: a) staining DNA in the sperm for an incubation period effective for staining nuclei of living sperm cells sufficient to distinguish chromosomal determinants of sex in individual sperm based on resulting fluorescence under a fluorescence stimulating light source using a quantitative DNA vital stain (QDVS) under conditions selected from the following and combinations thereof: (a) temperature in the range from about 18° C. to less than about 30° C., (b) pH in the range of about 7.1 to about 7.6 (c) incubation for an extended period at a lower temperature followed by a shorter period at higher temperature to enhance staining; and b) separating the thus-incubated sperm into GES using FACS (fluorescence-activated cell sorting and producing GES consisting predominantly of living sperm of which greater than 90% are of one sex. 2. The method of claim 1 wherein the temperature of staining is in the range of about 18° C. to about 25° C. and the pH of staining is in the range of about 7.3 to about 7.5. 3. The method of claim 1 wherein the QDVS is capable of fluorescence under visible light. 4. The method of claim 1 wherein the QDVS is selected from the group consisting of bisbenzimide and bisbenzimide labeled with a fluorophore capable of fluorescence under stimulation by visible light. 5. The method of claim 1 wherein the incubation period is in the range of about 1 to about 24 hours. 6. The method of claim 1 wherein the incubation period one hour or less. 7. The method of claim 1 wherein semen is contacted with QDVS shortly after semen collection and at least part of the incubation period occurs during transit from a semen collection facility to a semen sorting facility. 8. The method of claim 1 wherein incubation with QDVS at a temperature in the range of about 18° C. to less than about 30° C. is followed by flow cytometry at ambient temperatures. 9. The method of claim 2 wherein the semen is maintained at a pH less than about 7.1 prior to staining with QDVS at a pH in the range of about 7.1 to about 7.6. 10. The method of claim 2 wherein the semen are maintained during staining at a pH in the range of about 7.3 to about 7.5. 11. A process for producing GES (gender enriched semen) comprising: a) providing a suspension of viable sperm produced from collected semen ejaculate that is extended and transported to a sorting facility; b) staining the sperm using a QDVS (quantitative DNA vital stain) in the presence of a medium comprising a buffer system and further optionally including other components, the medium effective for maintaining viability of at least a portion of the semen; c) producing at least one of X-enriched and Y-enriched GES based on the extent of QDVS staining; d) collecting the resulting GES; e) wherein steps c) and d) occur in the presence of media comprising at least said buffer system, and f) partitioning the collected GES into dosage quantities for use or shipment. 12. The method of claim 11 wherein all of steps a) through completion of e) occur in the presence of media comprising at least said buffer system. 13. The method of claim 11 wherein step c) is conducted using a FACS (fluorescence-activated cell sorter) and the FACS utilizes a sheath fluid comprising said buffer system. 14. The method of claim 11 wherein said buffer system is selected based on efficacy of performance for staining and maintaining viability of sperm during staining of the sperm using the QDVS. 15. The method of claim 11 wherein said buffer system comprises a TEST (N[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid—tris-hydroxymethylaminomethane) buffer system. 16. The method of claim 11 wherein 11 wherein step c) is conducted using a FACS (fluorescence-activated cell sorter) and the FACS utilizes a sheath fluid comprising said buffer system and said buffer system comprises a TEST (N[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid—tris-hydroxymethylaminomethane) buffer system. 17. The method of claim 11 wherein all of steps a) through f) are conducted at a temperature between the between about the thermotropic phase transition temperature Tm of the membranes of the sperm being sorted up to less than about 39° C. and at an effective pH between about 6.8 and about 7.6. 18. The method of claim 11 wherein all of steps a) through f) are conducted at a temperature between the between about the thermotropic phase transition temperature Tm of the membranes of the sperm being sorted up to less than about 30° C. and at an effective pH between about 6.8 and about 7.6. 19. The method of claim 11 wherein all of steps a) through f) use media comprising said buffer system. 20. The method of claim 19 wherein all steps are conducted at a temperature between about the thermotropic phase transition temperature Tm of the membranes of the sperm being sorted up to less than about 39° C. and at an effective pH between about 7.3 and about 7.5. 21. A process for producing GES (gender enriched semen) comprising: a) providing a suspension of viable sperm produced from collected semen ejaculate that is extended and transported to a sorting facility; b) staining the sperm using a QDVS (quantitative DNA vital stain); c) based on the extent of QDVS staining producing at least one of X-enriched and Y-enriched GES; d) collecting the resulting GES; and e) partitioning the collected GES into dosage quantities for use or shipment; f) wherein from step a) starting after collection of the semen ejaculate until completion of step e) all steps occur at a temperature in a range from above the thermotropic phase transition temperature Tm of the membranes of the sperm being sorted up to less than about 30° C. and in the presence of a buffer system which is used at least for steps b), c) and d) and at an effective pH between about 6.8 and about 7.6. 22. The method of claim 21 wherein step c) is conducted using a FACS (fluorescence-activated cell sorter) and the FACS is operated at ambient temperature. 22. A process for producing GES (gender enriched semen) comprising: a) providing a suspension of viable sperm produced from collected semen ejaculate that is extended and transported to a sorting facility; b) staining the sperm using a QDVS (quantitative DNA vital stain) that fluoresces in response to visible light irradiation; c) based on the extent of QDVS staining producing at least one of X-enriched and Y-enriched GES: d) collecting the resulting GES; and e) partitioning the collected GES into dosage quantities for use or shipment; f) wherein from step a) starting after collection of the semen ejaculate until completion of step e) all steps occur (i) at a temperature in a range from above the lower semen viability temperature to less than the upper semen viability temperature and (ii) in the presence of sperm maintenance media effective for maintaining viability of at least a portion of the semen throughout the process. 23. The method of claim 21 wherein step c) is conducted using a FACS (fluorescence-activated cell sorter) and the FACS utilizes visible light irradiation for exciting fluorescence from stained sperm. 24. The method of claim 21 wherein the QDVS comprises a bisbenzimide modified by addition of a fluorophore that results in a fluorescence response by a resulting conjugate to excitation by visible light. 25. The method of claim 21 wherein the QDVS comprises a bisbenzimide-dipyrrometheneboron difluoride conjugate. 26. The method of claim 22 wherein stained sperm are irradiated with light at 488 nm. 27. The method of claim 21 wherein all steps occur at a temperature in a range from above the thermotropic phase transition temperature Tm of the membranes of the sperm being sorted up to less than about 30° C. 28. The method of claim 21 wherein all steps occur in the presence of a buffer system which is used at least for steps b), c) and d) and at an effective pH between bout 6.8 and about 7.6. 29. The method of claim 28 wherein said buffer system comprises a TEST N[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid—tris-hydroxymethylaminomethane) buffer system |
<SOH> BACKGROUND OF THE INVENTION <EOH>Artificial insemination is widely used in animal husbandry, for example, with economically important mammals such as cattle, pigs, horses, sheep, goats and other mammals. Likewise, in vitro fertilization and embryo transfer technology also have increasing application in species where the value of individual offspring is sufficiently high. Both of these techniques also have human applicability. It is frequently desired to produce offspring of a predetermined sex or sex ratio, for example, female bovines for milk production or breeding, male bovines and female porcines for meat production The simplest and most economically feasible way preferentially to produce offspring of a predetermined sex or sex ratio would be a high-throughput system for producing gender enriched sperm or semen (GES) which could then be used for artificial insemination (Al) or in vitro fertilization (IVF). Although there are reports that sperm may be distinguishable based on sex-specific surface antigens, it is generally considered that sperm nearly completely or perhaps completely lack any phenotypic sex-specific character. As a result, current efforts for producing GES in mammalian species rely on techniques responsive to the quantitatively different levels of DNA in male and female sperm in mammalian species. Since, for example, total DNA in mammalian Y-chromosome bearing sperm typically is 2.5 to 5% total DNA less than total DNA in mammalian X-chromosome bearing sperm, this difference has been used to separate sperm into GES using a DNA vital stain comprising a fluorochrome that readily permeates the cell membranes and relatively nonspecifically and uniformly binds to the DNA without unacceptably damaging the viability of the sperm (quantitative DNA vital binding stain or QDVS). The labeled sperm can then be sorted, for example, using ultraviolet laser based cell cytometry to distinguish the resulting quantitative differences in fluorescence between male and female chromosome bearing sperm and to produce GES. Exemplary of patent literature in this area are; Johnson et al., U.S. Pat. No. 5,135,759, and Rens et al., U.S. Pat. No. 5,985,216, which are hereby incorporated by reference for description of methods, compositions of matter and apparatus for producing GES known in the art. However, the methodology of Johnson et al. requires use of a bisbenzimide stain (Hoechst H 33342 fluorochrome (available from Calbiochem-Behring Co., La Jolla, Calif.), at relatively high temperatures to achieve relatively short staining times. According to Johnson et al., for example, incubation for 1 hr at 35° C. was found to be acceptable, and ranges of 30° C. to 39° C. were also stated to be effective requiring corresponding incubation times from 1.5 to 1 hour (the incubation period being less at higher temperatures). However, the use of temperatures in the range of 30° C. to 39° C. in the presence of a QDVS followed by ultraviolet laser based flow cytometry introduces a number of difficulties and disadvantages into the process which begins at semen collection and ends at fertilization which can reduce sperm viability and the efficiency (purity) of sorting sperm into GES. Prior to the work represented in the Johnson et al. and Rens et al. patents, other less successful or failed efforts had also been made. Some of these used dyes or stains which are only capable of entering permeabilized or dead cells and which are not effective vital stains for sperm, including acridine orange and derivatives thereof such as ethidium bromide, mithramycin or combinations thereof, and further including DAPI (4,6diamidino-2-phenylindole). Referring now to GB 2 145 112 A, that document purports to describe a method for staining sperm using Hoechst 33342 dye and then sorting the sperm ultimately into two populations AI and AII of motile sperm with the AD population having a fluorescence about 15% greater than that of the AI population. It is well known that the difference in fluorescence between two populations of sperm fully separated on the basis of sex should be on the order of about 3 to about 5% (3.0% for rabbit, 3.6% for boar, 3.8% for bull, and 4.2% for ram sperm). Perhaps for this reason, GB 2 145 112 A2 is able only to speculate on the significance of the difference in fluorescence between the two subpopulations: “The subpopulations (AI and AII) may reflect spermatozoa at distinct stages of late maturation or the difference between X- and Y-chromosome bearing spermatozoa.” For various reasons, however, it is clear to persons skilled in the art that in any event that GB 2 145 112 A did not accomplish separation into subpopulations of 90% or more X- OR Y-bearing sperm. In addition to the Johnson et al. and Rens et al. patents cited above, patent literature relevant to GES includes U.S. Pat. No. 6,263,745 B1, WO 01/37655, U.S. Pat. No. 6,149,867, U.S. Pat. No. 6,071,689, U.S. Pat. No. 4,362,246 and WO 99/33956. These patents and patent applications and those of Johnson et al. and Rens et al. are incorporated herein by reference as describing methods, compositions of matter and apparatus for handling and producing GES known to those skilled in the art. Notwithstanding the above-described systems, there remains an urgent need for new and improved GES production and handling methods and apparatus that results in GES having advantageous viability, motility and integrity. |
<SOH> SUMMARY OF THE INVENTION <EOH>If GES is to become widely used in animal husbandry, methodologies must be developed which take into account the effects on sperm of the entire sequence of collecting sperm and preparing and using GES. For example, sperm might be collected from a donor animal in a breeder herd maintained at a remote location, prepared for transport at the point of collection in a processing facility optionally with QDVS staining, transported under controlled conditions to a sorting facility, optionally with QDVS staining to occur at the sorting facility, sorted into GES, prepared optionally with freezing for shipping, shipped under controlled conditions to a breeding facility, thawed and used. At several or most of these steps, as practiced in the prior art, the sperm will be exposed to changes in temperature and to changes in the fluid environment including pH changes or other environmental conditions which will individually or cumulatively affect staining and separation efficiency and viability (motility) of the sperm. We have found in staining at a temperature in the range of about 17° C. to less than about 30° C. that the pH of the fluid environment to which the sperm are exposed during staining has a significant influence over the period of time required for uniform staining sufficient for production of GES. Accordingly, we have found that a prolonged period of staining, such as during transit from a collection facility to a sorting facility, can be used, at effective temperatures between about the thermotropic phase transition temperature T m of the membranes of the sperm being sorted up to less than about 30° C. and at an effective pH between about 6.8 and about 7.6, to reduce or eliminate the time required for higher temperature incubation with stain. The lower temperatures (compared to prior art techniques) are also believed to provide advantageous effects on sperm orientation during sorting. According to the invention herein, there are provided methods which avoid the high temperature QDVS stain incubation step of Johnson et al. and advantageously conduct all processing steps between collection and providing GES to the site of ultimate use within a narrower range of temperatures (from about 17° C., or even lower depending on species, to less than about 30° C.) that are advantageous and beneficial to high levels of viability (motility), and of separation efficiency (purity) of the resulting GES. According to an aspect of the invention, incubation with QDVS occurs at least in part at a pH in the range of about 7.1 to about 7.6, or according to another aspect in the range of about 6.8 to about 7.6. According to a further aspect of the invention, a QDVS is used which permits visible light-based flow cytometry (as compared to an ultraviolet-based flow cytometry system) to be used, further reducing damage to the sperm and reducing the costs of flow cytometry equipment. According to various other aspects, the invention relates to process and apparatus for producing GES (gender enriched semen) comprising providing a suspension of viable sperm produced from collected semen ejaculate that is extended and transported to a sorting facility, staining the sperm using a QDVS (quantitative DNA vital Stain), producing at least one of X-enriched and Y-enriched GES based on the extent of QDVS staining of DNA, collecting the resulting GES, and apportioning the collected GES into dosage quantities for use or shipment. In one aspect, all of the steps occur at a temperature between a lower temperature at which the sperm remain mostly viable and an upper temperature of less than about 30° C. According to other aspects, the upper temperature may range on upwards to less than about 39° C. and the staining, producing and collecting steps all occur in the presence of media comprising a buffer system and further optionally including other components effective for maintaining viability of at least a portion of the semen, wherein all of the media comprise the same or substantially the same buffer systems. According to a further aspect, even the providing and partitioning steps also occur in the presence of such media. According to yet further aspects of the invention, the step of producing involves the use of a Fluorescence-Activated Flow Sorter (FACS) to sort the sperm based on extent of DNA staining where the sheath fluid also is such a medium as previously described, or where the QVDS is a visible-light stimulated QVDS, or where the QVDS is a visible light excited QVDS and visible light irradiation is used for the producing step. The invention will be further described in detail and in terms of certain preferred embodiments; however, other uses, applications and embodiments will be apparent to, or readily developed without undue experimentation by, those skilled in the art from the following detailed description and the examples. |
Botulinum toxin in the treatment or prevention of acne |
Botulinum toxin may be used to inhibit the cascade of events leading to acne. Results in preliminary studies have been dramatic. Withoout wishing to be bound by this theory, it is believed that botulinum toxin achieves this result through parasympathetic effects, inhibiting sweat gland activity, stimulating keratinocyte locomotion, anti-inflammatory effects, and possibly anti-androgenic effects. Botulinum toxin can play an important role in decreasing and even preventing the formation of acne. |
1. A method for inhibiting acne vulgaris in a human who is susceptible to acne vulgaris, said method comprising delivering an effective amount of botulinum toxin A to one or more regions of the skin of the human that are susceptible to acne vulgaris. 2. A method as recited in claim 1, wherein said delivering comprises the intracutaneous injection of botulinum toxin A at multiple sites in the skin, wherein the sites of adjacent injections are separated by about 0.5 to 10 cm. 3. A method as recited in claim 1, wherein said delivering comprises the intracutaneous injection of botulinum toxin A at multiple sites in the skin, wherein the sites of adjacent injections are separated by about 1.5 to 3 cm. 4. A method as recited in claim 1, wherein said delivering comprises the topical application to susceptible regions of the skin of a composition comprising botulinum toxin A. 5. A method as recited in claim 1, wherein said delivering comprises the subcutaneous injection of the botulinum toxin A. 6. A method as recited in claim 1, wherein said delivering comprises the intramuscular injection of the botulinum toxin A. 7. A method as recited in claim 1, wherein said method is repeated periodically to inhibit the recurrence of acne vulgaris. 8. A method as recited in claim 1, wherein said method is repeated at intervals from about 3 months to about 6 months to inhibit the recurrence of acne vulgaris. 9. A method as recited in claim 1, wherein said method is repeated at intervals of about 4 months to inhibit the recurrence of acne vulgaris. 10. A method as recited in claim 1, wherein said delivering comprises the intracutaneous injection of botulinum toxin A at multiple sites in the skin, and wherein between about 1 U and about 20 U of botulinum toxin A is injected at each site. 11. A method as recited in claim 1, wherein said delivering comprises the intracutaneous injection of botulinum toxin A at multiple sites in the skin, and wherein between about 2 U and about 3 U of botulinum toxin A is injected at each site. 12. A method as recited in claim 1, additionally comprising the inhibition of at least one rhytid by said delivering of an effective amount of botulinum toxin A to one or more regions of the skin that are susceptible to acne vulgaris and that also contain at least one rhytid. 13. A method for inhibiting acne vulgaris in a human who is susceptible to acne vulgaris, said method comprising delivering an effective amount of botulinum toxin to one or more regions of the skin of the human that are susceptible to acne vulgaris. 14. A method as recited in claim 13, wherein said delivering comprises the intracutaneous injection of botulinum toxin at multiple sites in the skin, wherein the sites of adjacent injections are separated by about 0.5 to 10 cm. 15. A method as recited in claim 13, wherein said delivering comprises the intracutaneous injection of botulinum toxin at multiple sites in the skin, wherein the sites of adjacent injections are separated by about 1.5 to 3 cm. 16. A method as recited in claim 13, wherein said delivering comprises the topical application to susceptible regions of the skin of a composition comprising botulinum toxin. 17. A method as recited in claim 13, wherein said delivering comprises the subcutaneous injection of the botulinum toxin. 18. A method as recited in claim 13, wherein said delivering comprises the intramuscular injection of the botulinum toxin. 19. A method as recited in claim 13, wherein said method is repeated periodically to inhibit the recurrence of acne vulgaris. 20. A method as recited in claim 13, wherein said method is repeated at intervals from about 3 months to about 6 months to inhibit the recurrence of acne vulgaris. 21. A method as recited in claim 13, wherein said method is repeated at intervals of about 4 months to inhibit the recurrence of acne vulgaris. 22. A method as recited in claim 13, additionally comprising the inhibition of at least one rhytid by said delivering of an effective amount of botulinum toxin to one or more regions of the skin that are susceptible to acne vulgaris and that also contain at least one rhytid. 23. A method as recited in claim 13, wherein the botulinum toxin comprises botulinum toxin B. 24. A method as recited in claim 13, wherein the botulinum toxin comprises botulinum toxin C. 25. A method as recited in claim 13, wherein the botulinum toxin comprises botulinum toxin D. 26. A method as recited in claim 13, wherein the botulinum toxin comprises botulinum toxin E. 27. A method as recited in claim 13, wherein the botulinum toxin comprises botulinum toxin F. 28. A method as recited in claim 13, wherein the botulinum toxin comprises botulinum toxin G. 29. A method as recited in claim 13, additionally comprising the inhibition of at least one rhytid by said delivering of an effective amount of a botulinum toxin to one or more regions of the skin that are susceptible to acne vulgaris and that also contain at least one rhytid; wherein the botulinum toxin is selected from the group consisting of botulinum toxin B, botulinum toxin C, botulinum toxin D, botulinum toxin E, botulinum toxin F, and botulinum toxin G. |
<SOH> BACKGROUND ART <EOH>Acne Vulgaris While not life-threatening, acne vulgaris can cause significant problems for affected individuals. Depending on its severity and other factors, recalcitrant acne can be psychologically debilitating, and can impose significant financial and emotional costs on those whom it afflicts. Despite some recent successes in acne therapy, treatment failures are still common, especially in adult women. While many adults “outgrow” this disease, there are some who continue to be afflicted during much of adulthood, despite continued medical advances. Unfortunately, the most potent acne medication in current use is administered systemically via a treatment that is teratogenic, an important issue for many women. There is an unfilled need for a more localized and effective treatment for acne, one with minimal side effects. Acne, which most commonly occurs during adolescence, is influenced by several factors. The pathology centers on the pilosebaceous follicle, comprising the sebaceous gland, the follicle (pore), and the vellus hair. Factors that promote the formation of comedones (whiteheads or blackheads) include the following: (1) increased sebum production, (2) inflammation of the dermis and follicles by inflammatory mediators, (3) hyperkeratinization and obstruction of the upper region of the follicle, and (4) colonization of the follicle by the bacterium Propionibacterium acnes, Adolescence is marked by an increase in levels of circulating androgens, particularly dehydroepiandrosterone sulfate (DHEAS). The increased androgen levels are thought to cause sebaceous glands to enlarge and to increase sebum production. While most acne patients have normal hormone levels, there are reasons to conclude that increased sebum production plays an important role in acne. For example, there is a correlation between the rate of sebum production and the severity of acne. In addition, acne patients typically produce sebum that is deficient in linoleic acid, which is a potential cause of abnormal keratinization and follicular obstruction. In response to increased sebum levels, Propionibacterium acnes , a gram positive, anaerobic, diphtheroid bacterium, often colonizes the sebaceous follicles. P. acnes exacerbates acne by acting as a chemo-attractant for neutrophils (a type of white blood cells, also called polymorphonuclear leukocytes, or PMNs). The neutrophils ingest the P. acnes , and in doing so release various hydrolytic enzymes that damage the follicular wall. The released follicular contents then invade the dermis and cause an inflammatory reaction, manifesting as pustules, erythematous papules, or nodules. In a separate route, P. acnes can hydrolyze triglycerides to free fatty acids, which also increase inflammation and follicular obstruction. P. acnes may also activate the complement components of the immune system, which can also lead to follicular obstruction. The follicles are lined with squamous epithelium, a layer of cells that is contiguous with the skin surface. In an acne-prone individual, the shedding of cells from this lining is often impeded, perhaps due to an increased level of intracellular adhesion that promotes the retention of cells. The retained cells can obstruct the follicles, resulting in comedones. The exact cause of this inhibited shedding is unknown, but it may be related either to abnormalities in epidermal differentiation, or to abnormal sebum composition (e.g., a deficiency in linoleic acid). It has also been demonstrated that increased sebum levels can irritate keratinocytes, causing the release of interleukin-1, which in turn can cause follicular hyperkeratinization. The final common pathway in each of these acne-causing routes, which are not mutually exclusive, is follicular obstruction. Current Acne Therapies Currently-used acne therapies are directed at various aspects of the acne cascade. The most commonly used therapy is probably topical benzoyl peroxide, which has an antibacterial effect, and which may also decrease free fatty acids, resulting in a decrease in inflammation and follicular obstruction. Topical and systemic antibiotics have been used to target P. acnes . The antibiotics that have been used for this purpose include erythromycin, tetracycline, clindamycin, and doxycycline. Predictably, the prolonged use of antibiotics often leads to the development of resistant strains of P. acnes. Topical and systemic retinoids (derivatives of vitamin A) have been used to normalize the keratinization of the follicle, decreasing follicular obstruction and rupture. Systemic isotretinoin (Accutane™), which is highly effective for acne, unfortunately has serious adverse effects, most notably teratogenicity. Topical retinoids include tretinoin (Retin-A™), whose chemical structure is similar to that of isotretinoin. The current state of the art in treating acne is summarized, for example, in the following two review articles: D. Krowchuk, “Managing Acne in Adolescents,” Pediatric Dermatology , vol. 47, pp. 841-857 (2000); and B. Johnson et al., “Use of Systemic Agents in the Treatment of Acne Vulgaris,” American Family Physician , vol. 62, pp. 1823-1830, 1835-1836 (2000). Advances in acne therapy have followed better understanding of its multiple causes. Most treatments are directed at normalizing keratin production (e.g., through the use of retinoids), or at controlling bacterial colonization. To the inventors' knowledge, no prior treatments have sought to affect either sweat gland production or keratinocyte locomotion, both of which can be factors in the follicular occlusion that leads to acne. Botulinum Toxins and Pharmacology Clostridium botulinum , an anaerobic bacterium, produces seven toxins that have similar neurotoxic effects, but that are antigenically distinct: serotypes A through G. These toxins are potent neuroparalytic agents that inhibit the release of acetylcholine at neuromuscular junctions and at neuroglandular junctions. See M. F. Brin, “ Botulinum toxin: Chemistry, Pharmacology, Toxicity, and Immunology,” Muscle and Nerve Supp. 6, pp. S146-S168 (1997); and 1. Kinkelin et al., “Effective Treatment of Frontal Hyperhidrosis with botulinum toxin A,” Brit. J. Dermatol ., vol. 143, pp. 824-827 (2000). Neither the synthesis nor the storage of acetylcholine is affected by botulinum toxin. Therefore, its effects are temporary. Botulinum toxin penetrates the endosomal membrane into the cytosol, where the secretion of acetylcholine is blocked. Botulinum toxin also appears to possess anti-inflammatory effects. Studies of the effect of botulinum toxin on glandular function have observed that its effects occur at the presynaptic terminal of the parasympathetic cholinergic nerves. Botulinum toxin A (sold under the trademark Botox® by Allergan (Irvine, Calif.), and under the trademark Dysport® by Ipsen Limited (Maidenhead, Berkshire, United Kingdom)) has been used for certain therapeutic purposes at both neuroglandular and neuromuscular junctions. Its temporary neuromuscular blockade has found numerous previous uses, including the treatment of strabismus, migraines, achalasia, hemifacial spasm, rhytids, and spastic dysphonia. The injections have proven to be safe and effective for these purposes, with only minor side effects such as local swelling and transient weakness of nearby muscles. Botulinum toxins A and B have been reported to be well tolerated in patients who have used it. Intracutaneous botulinum toxin A has become a primary therapy for axillary, palmar, solar, and frontal hyperhidrosis (excessive sweating), as well as for gustatory sweating (Frey's Syndrome). It has also been shown to be an effective alternative to surgery in Hailey-Hailey disease, a benign familial pemphigus that affects primarily the groin and axillary folds (areas of high moisture). Studies have found no long-term effects of botulinum toxin A in human tissue. Histological studies have found no nerve fiber degeneration, and no sweat gland atrophy as a result of therapy. Antibodies to botulinum toxin A have been found in fewer than 5% of patients receiving botulinum toxin A injections. There have been no reported cases of anaphylaxis in response to injections of botulinum toxin A. Botulinum toxin B (sold under the trademark Myobloc™ and NeuroBloc™ by Elan Pharmaceuticals (Dublin, Ireland)) has been approved by FDA for use in treating patients with cervical dystonia. All seven botulinum toxin serotypes (A through G) are available commercially from Metabiologics, Inc. (Madison, Wis.). Neuromuscular Junction Blockade The first reported clinical use of botulinum toxin A was its use in the treatment of strabismus in the 1970's. Injecting the small extraocular muscles resulted in a realignment of the muscles, straightening of the globe, and improvement of visual alignment in some cases. Subsequently, botulinum toxin A has been used therapeutically for several other purposes, both functional and cosmetic. It has proven effective in treating focal dystonias, spasmodic dysphonia, blepharospasm, hemifacial spasm, torticollis, and cervical dystonia. It has also recently been used in facial plastic surgery, especially to treat rhytids (wrinkles). In 1990, the use of botulinum toxin A was reported in ambulatory and nonambulatory cerebral palsy patients, namely by injecting different muscle groups to treat spastic diplegia. For example, double-blinded, placebo-controlled studies on the effect of botulinum toxin A on limb dystonias and spasticity have found significant subjective as well as objective benefits. The American Academy of Ophthalmology, the American Academy of Neurology, the American Academy of Otolaryngology, and the National Institutes of Health have all released statements supporting the therapeutic efficacy of botulinum toxin A for a variety of clinical conditions. Among the conditions for which botulinum toxin A has been used are blepharospasm, strabismus, cervical dystonia, spasmodic torticollis, rhytids, hemifacial spasm, facial spasm, spasmodic dysphonia, focal hand dystonia, hyperfunctional facial wrinkles, Frey's syndrome, hyperhidrosis, adult spasticity, adjunctive treatment of spasticity in cerebral palsy, oromandibular dystonia, and other dystonias. Neuroglandular Junction Blockade The effects of botulinum toxin A as an anticholinergic agent at the neuroglandular junction have not been explored as extensively as those occurring at the neuromuscular junction. Clinical studies examining the effect of intracutaneous botulinum toxin for focal hyperhidrosis found complete abolition of sweating in the injected area within 3 to 7 days. No adverse effects were reported, and in a five month follow-up there were no clinical recurrences of the hyperhidrosis. See generally 1. Kinkelin et al., “Effective Treatment of Frontal Hyperhidrosis with botulinum toxin A,” Brit. J. Dermatol ., vol. 143, pp. 824-827 (2000). Gustatory sweating is another area of neuroglandular dysfunction in which botulinum toxin A has proven effective. Gustatory sweating (or Frey's syndrome) is a disabling disorder in which the cheek skin sweats profusely during eating. The syndrome often occurs after parotidectomy, and may be due to the misdirection of the regenerating parasympathetic fibers that enervate the sweat glands of the face. Intracutaneous botulinum toxin A has been reported to significantly decrease or prevent sweating for over six months, with no clinical evidence of facial weakness in any patients. Botulinum toxin A injected into the submandibular glands has been reported to significantly decrease salivation resulting from stimulation of the lingual nerves. The decreased salivation was temporary, and did not appear to be directly toxic to the acinar cells of the gland. See D. Suskind et al., “Clinical study of botulinum A toxin in the treatment of sialorrhea in children with cerebral palsy,” Laryngoscope , vol. 112, pp. 73-81 (2002). Canine studies have also shown that vasomotor rhinorhea, a parasympathetically controlled phenomenon, responds to topical botulinum toxin A. While the duration of botulinum toxin A's action at the neuromuscular junction appears to be about three months, there appears to be a longer-lasting effect at the glandular level. Botulinum toxin A has produced anhydrosis for over 12 months in patients with gustatory sweating. The reason for the difference in duration of action is uncertain. Hypotheses include a higher rate of re-synthesis of SNAP-25 (the protein cleaved by botulinum toxin) in neuromuscular synapses, and a higher area of axonal sprouting and consecutive reinervation of muscle fibers as compared to that in glandular tissue. To the inventors' knowledge, there have been no prior suggestions that botulinum toxin might be used in the treatment or prevention of acne. |
Nucleic acids encoding recominant 56 and 82 kda antigens fromn gametocytes of eimeria maxima and their uses |
The present invention provides the recombinant cloning and sequencing of two of the major Eimeria maxima gametocyte antigens having molecular weights of 56 and 82 kDa and the expression of these recombinant antigens in an E. coli expression system using the plasmid pTrcHis. The subject invention also provides a vaccine against coccidiosis comprising the recombinant 56 kDa or 82 kDa antigen. The subject invention also provides two 30 kDa proteins and three 14 kDa proteins from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence described herein. The subject invention also provides a vaccine against coccidiosis comprising the recombinant 56 kDa or 82 kDa antigen and any of the aforementioned proteins. Additionally, the subject invention also provides a method of immunizing a subject against infection by Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis or Eimeria brunetti, or a microorganism expressing an immunologically cross-reactive antigen, comprising the step of administering to the subject any of the aforementioned vaccines. |
1-98. (Canceled) 99. An isolated nucleic acid comprising consecutive nucleotides having a nucleotide sequence encoding a 56 kDa polypeptide present in gametocytes of Eimeria maxima or a homolog of such polypeptide having the immunogenicity thereof. 100. The isolated nucleic acid of claim 99, wherein the nucleic acid encodes the 56 kDa polypeptide having the amino acid sequence shown as SEQ. ID. NO. 3. 101-106. (Canceled) 107. A vector comprising the nucleic acid of claim 99 operatively linked to a promoter of RNA transcription. 108-111. (Canceled) 112. A host cell comprising the vector of claim 107. 113-119. (Canceled) 120. An isolated 30 kDa protein present in Eimeria maxima gametocytes having at the N-terminal end of the protein consecutive amino acids, the sequence of which is shown as SEQ. ID NO. 35. 121. An isolated 30 kDa protein present in Eimeria maxima gametocytes having at the N-terminal end of the protein consecutive amino acids, the sequence of which is shown as SEQ. ID NO. 42. 122. An isolated 14 kDa protein present in Eimeria maxima gametocytes having at the N-terminal end of the protein consecutive amino acids, the sequence of which is shown as SEQ. ID NO. 37. 123. An isolated 14 kDa protein present in Eimeria maxima gametocytes having at the N-terminal end of the protein consecutive amino acids, the sequence of which is shown as SEQ. ID NO. 39. 124. An isolated 14 kDa protein present in Eimeria maxima gametocytes having at the N-terminal end of the protein consecutive amino acids, the sequence of which is shown as SEQ. ID NO. 41. 125. A vaccine against infection by an Eimeria species or a microorganism expressing an immunologically cross-reactive antigen comprising the plasmid of claim 109, or the 56 kDa polypeptide of claim 119. 126-134. (Canceled) 135. A method of immunizing a subject against infection by an Eimeria species or a microorganism expressing an immunologically cross-reactive antigen, comprising administering to the subject the vaccine of claim 125. 136-143. (Canceled) 144. A fertilized egg from an avian having an air sac wherein the air sac comprises the vaccine of claim 125, which vaccine is capable of inducing before, or immediately after, hatching of the egg in an embryo derived therefrom an immune response against a virulent form of an Eimeria species or a microorganism expressing an immunologically cross-reactive antigen. 145-146. (Canceled) 147. An isolated nucleic acid comprising consecutive nucleotides having a nucleotide sequence encoding an 82 kDa polypeptide present in gametocytes of Eimeria maxima or a homolog of such polypeptide having the immunogenicity thereof. 148. The isolated nucleic acid of claim 147, wherein the nucleic acid encodes the 82 kDa polypeptide having the amino acid sequence shown as SEQ. ID. NO. 6. 149-154. (Canceled) 155. A vector comprising the nucleic acid of claim 147 operatively linked to a promoter of RNA transcription. 156-159. (Canceled) 160. A host cell comprising the vector of claim 155. 161-167. (Canceled) 168. A vaccine against infection by an Eimeria species or a microorganism expressing an immunologically cross-reactive antigen comprising the plasmid of claim 157, or the 82 kDa polypeptide of claim 167. 169-171. (Canceled) 172. A method of immunizing a subject against infection by an Eimeria species or a microorganism expressing an immunologically cross-reactive antigen comprising administering to the subject the vaccine of claim 168. 173-180. (Canceled) 181. A fertilized egg from an avian having an air sac wherein the air sac comprises the vaccine of claim 168, which vaccine is capable of inducing before, or immediately after, hatching of the egg in an embryo derived therefrom an immune response against a virulent form of an Eimeria species or a microorganism expressing an immunologically cross-reactive antigen. 182-183. (Canceled) 184. A polypeptide comprising consecutive amino acids, the amino acid sequence of which is shown as SEQ. ID NO. 3. 185. A polypeptide comprising consecutive amino acids, the amino acid sequence of which is shown as SEQ. ID NO. 6. 186. A method of immunizing a subject against infection by an Eimeria species or a microorganism expressing an immunologically cross-reactive antigen, comprising administering to the subject the protein of any one of claims 120-124. 187. A method of conferring upon a newborn avian maternal immunity against an infection by an Eimeria species or a microorganism expressing an immunologically cross-reactive antigen comprising administering to the mother of the avian at a suitable time prior to the mother laying a fertilized egg from which the newborn avian is derived the vaccine of claim 125 or of claim 168 in order to thereby confer protection via maternal immunity against the infection. 188-189. (Canceled) 190. A method of reducing the output of Eimeria oocysts in feces from a newborn avian which comprises administering to the mother of the avian at a suitable time prior to the mother laying a fertilized egg from which the newborn avian is derived the vaccine of claim 125 or of claim 168 in order to induce an immune response and transmit maternal antibodies to the newborn avian so that the output of oocysts from the newborn avian is reduced. 191-192. (Canceled) |
<SOH> BACKGROUND OF THE INVENTION <EOH>The organisms which cause the disease known as “coccidiosis” in chickens belong to the phylum Apicomplexa, class Sporozoa, subclass Coccidia, order Eucoccidia, suborder Eimeriorina, family Eimeriidae, genus Eimeria . Within the Eimerian genus there are many species, several of which are pathogenic in chickens. The species of major concern to the chicken industry are Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix and Eimeria brunetti. Coccidiosis has become a major economic problem in the chicken industry over the past several decades, mainly due to the overcrowding of chicken houses and the development of drug resistance by the parasite. The rearing of chickens under crowded conditions on a litter floor provides optimal conditions for the growth and spread of Eimeria parasites. Under such circumstances, sanitary control is impossible and the farmer must rely on the effectiveness of coccidiostat drugs. However, drugs must be kept in the feed at all times, shuttle programs must be used to avoid the appearance of drug resistance strains of Eimeria , and certain drugs have costly side effects. Furthermore, these coccidiostats also have antibacterial effects and therefore are considered to be in-feed antibiotics. Recently the European Union has decided to ban the use of all in-feed antibiotics in the chicken industry including anticoccidial drugs. Thus, the only viable approach to the control of coccidiosis in the future is by vaccine development. The Eimeria parasite undergoes a complex life cycle in the mucosa of the intestinal tract. This life cycle is very similar to that of the other hemosporidian parasites (i.e. plasmodium, babesia, etc.) except for the lack of an arthropod vector. Oocysts sporulate on the litter floor producing four sporocysts, each containing two sporozoites (thus belonging to the class sporozoa). The oocysts are ingested by the chicken, and the sporocysts are released by the mechanical grinding of the gizzard. The sporozoites are then released from the sporocysts due to the digestion of the sporocyst wall by proteolytic enzymes in the intestine. Mobile sporozoites then invade lymphocytes and go on to invade epithelial cells where the asexual cycle begins. The parasite goes through 2-4 cycles of replication and division (each species having a defined number of divisions) leading to the production of large numbers of daughter merozoites. After the final cycle of merozoite production the sexual cycle begins with the production of the macrogametocyte (female) and microgametocyte (male). The macrogametocyte is characterized by the production of wall forming bodies, while microgametocytes contain the components involved in the formation of microgametes, which bud off from the surface of the intracellular parasite. Microgametes are flagellated and are responsible for the fertilization of the macrogamete. A zygote is formed which matures into the oocyst by fusion of the wall forming bodies and condensation of the nucleus. Oocysts are secreted in the feces, thus completing the cycle. Over the past several years, native antigens from the sexual (gametocyte) stages of Eimeria maxima have been used to immunize laying hens. Offspring chicks were consequently vaccinated via maternal immunity (protective maternal antibody). Three major protective antigens have previously been identified in E. maxima gametocytes having molecular weights of 250, 82 and 56 kDa (EP Patent No. 0 256 536, U.S. Pat. No. 5,496,550, and U.S. Pat. No. 5,932,225). EP Patent No. 0 256 536, U.S. Pat. No. 5,496,550, and U.S. Pat. No. 5,932,225 are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. It was shown that these antigens are well conserved amongst Eimeria species (Wallach 1995) and can cross protect against the 3 major species that cause coccidiosis in broiler chickens, E. maxima, E. tenella and E. acervulina . More recently, it was shown that in floor pen trials, chicks from hens vaccinated with these native gametocyte antigens were protected against Eimeria under field conditions (Wallach 1996). This protection acts to lower the peak in oocyst shedding to a level which does not cause any damaging effect on the performance of the broiler chicken. Based on the above results it was concluded that these antigens are effective against coccidiosis in chickens and also have the potential for use against coccidiosis in other domestic animals including turkeys, geese, sheep, cattle, pigs and fish. These three antigens were also characterized at the molecular level. Cell free translation experiments were carried out to identify the RNA molecules that encode them (Mencher er al.). cDNA molecules that encode these antigens were cloned by immunoscreening of a cDNA library made in the expression vector lambda zap (4, U.S. Pat. No. 5,932,225). By this approach, the gene encoding the 250 kDa antigen was cloned and sequenced.. The clone pEM 250/14 was partially sequenced in U.S. Pat. Nos. 5,932,225 and 5,496,550. FIG. 13a of the subject application reproduces FIG. 11 of U.S. Pat. Nos. 5,932,225 and 5,496,550, which portrays the DNA sequence of the first 293 nucelotides of clone pEM 250/14. FIG. 13b of the subject application reproduces FIG. 12 of U.S. Pat. Nos. 5,932,225 and 5,496,550, which shows the DNA sequence of the last 196 nucelotides of clone pEM 250/14. Also, in in U.S. Pat. Nos. 5,932,225 and 5,496,550, the putative genes encoding the 56 and 82 kDa antigens were cloned and sequenced. Subsequently, Fried et al. sequenced the entire pEM 250/14 clone and found that the antigen had a molecular weight of 230 kDa rather than 250 kDa as had been previously thought. Fried et al. found that the 230 kDa gene contains highly repetitive motifs and that these repeats are contained throughout the entire gene (Fried et al.). This clone was expressed in bacteria using the pATH plasmid vector and it was shown that it is recognized by convalescent chicken sera taken 14 days post infection with E. maxima . Finally, it was shown that this gene is expressed only in the macrogametocyte stage and by immunofluorescence was found to be located in the wall forming bodies of the macrogamete (Fried et al.). cDNA clones encoding the 56 and 82 kDa antigens were also obtained by screening the library with polyclonal antibodies as well as a monoclonal antibody against the 56 kDa antigen. This monoclonal antibody was previously shown to provide passive immunity to naive chicks (Wallach 1990). A few clones were obtained and analyzed. One of the clones was found to encode a small 10 kDa antigen and therefore was not the desired clone. Another clone was found to contain only a small part of the open reading frame (ORF) and by northern blotting was shown to hybridize with two mRNAs of about the expected size for the 56 and 82 kDa antigens. It was therefore concluded that this was the desired clone. Genomic libraries were then screened to obtain the full length clone. However, due to the highly repetitive GCA motifs in this clone, it was not possible to specifically isolate the full length clone. Attempts to clone the full length cDNA molecule were also not successful due to these repeats. Finally, attempts to express the partial cDNA clones in bacteria failed as well probably due to their unusual sequences and a reasonable level of gene expression was not obtained. It has previously been shown that the 56 and 82 kDa antigens are glycosylated (U.S. Pat. No. 5,932,225). This is based on their strong reactivity with Soybean lectin. Therefore, glycosylation may be required in order to obtain good expression of these genes and for proper conformation of the gene products. In addition to the 56, 82 and 230 kDa antigens, a 14 kDa antigen obtained from highly purified fractions of oocyst walls has been proposed as a possible candidate for vaccines against coccidiosis (Eschenbacher et al.). However, this hypothesis has not been explored. Several laboratories have been working on a subunit vaccine against coccidiosis. Most of these researchers have focused their efforts on the extracellular asexual stages of the life cycle, in particular the sporozoite and merozoite stages which are considered to be the most vulnerable to immune attack. In a previous study it was found that sporozoite extracts from E. tenella could induce in broilers protection against challenge infections against this parasite for up to 7 weeks of age (Karkhanis et al.). Work carried out using monoclonal antibodies against antigens from sporozoites of E. tenella led to the identification of a 25,000 molecular weight antigen which was cloned and sequenced (Eur. Patent publication No. 0 164 176, Dec. 11, 1985). Several other sporozoite genes were identified and their recombinant antigens or the transformed bacteria themselves were tested for protective immunity (Danforth et al.). The results indicated that these recombinants were only able to provide a relatively low level of protection against challenge infection with Eimeria and did not always prevent the appearance of significant lesions. A vaccine using antigens from the merozoite stage has also been tested (European patent publication No. 0 135 073). Using these antigens to immunize young broiler chicks, it was once again found that the protection afforded was relatively low (Danforth et al.). In 1993, it was found that there was a correlation between protective maternal immunity with the appearance of maternal antibodies against a 230 kDa merozoite (as opposed to gametocyte) antigen of Eimeria maxima (Smith et al.). This protection was often over 90% and was found to occur even when the maternal antibody level was relatively low (although reactivity with the 230 kDa protein remained strong). It was also found that a very small quantity of the native 230 kDa merozoite antigen cut out of an SDS-PAGE gel could induce a significant (60%) level of protective maternal immunity against infection with E. maxima in offspring chicks. Furthermore, Western blotting showed that this protein was expressed in both merozoites and sporozoites of E. maxima and is also well conserved between Eimeria species. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides the nucleic acid encoding two of the major Eimeria maxima gametocyte antigens having molecular weights of 56 and 82 kDa. The subject invention also provides a 30 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown by SEQ. ID NO. 35. The subject invention also provides a 30 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown by SEQ. ID NO. 42. The subject invention also provides a 14 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown by SEQ. ID NO. 37. The subject invention also provides a 14 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown by SEQ. ID NO. 39. The subject invention also provides a 14 kDa protein from Eimeria maxima gametocytes having at the N-terminal end the amino acid sequence shown by SEQ. ID NO. 41. The subject invention also provides a vaccine against coccidiosis comprising the recombinant 56 kDa antigen alone or in combination with any of the aforementioned proteins. The subject invention also provides a vaccine against coccidiosis comprising the recombinant 82 kDa antigen alone or in combination with any of the aforementioned proteins. The subject invention also provides a method of immunizing an subject against infection by Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix, Eimeria praecox, Eimeria mitis or Eimeria brunetti , or a microorganism expressing an immunologically cross-reactive antigen, comprising the step of administering to the subject any of the aforementioned vaccines. |
Arylheteroalkylamine derivatives and their use as inhibitors of nitric oxide synthase |
There are provided novel compounds of formula (I), wherein R1, R2, R3, T, U, X, Y, V and W are as defined in the specification, and pharmaceutically acceptable salts thereof, and enantiomers and racemates thereof; together with processes for their preparation, compositions containing them and their use in therapy. The compounds are inhibitors of nitric oxide synthase and are thereby particularly useful in the treatment or prophylaxis of inflammatory disease and pain. |
1. A compound of formula (I) wherein: X represents H, C1 to 4 alkyl, C1 to 4 alkoxy, halogen, OH, CN, C≡CH, NO2, CHO, COCH3 or NHCHO; said alkyl or alkoxy group being optionally further substituted by one or more fluorine atoms or by an OH group; Y represents C1 to 4 alkyl, C1 to 4 alkoxy, halogen, OH, CN, C═CH, NO2, CHO, COCH3 or NHCHO; said alkyl or alkoxy group being optionally further substituted by one or more fluorine atoms; Either one of T, U and W represents N and the other two independently represent CR4; or each of T, U and W represents CR4; and each R4 group independently represents H, F or CH3; V represents O or S(O)n; n represents an integer 0, 1 or 2; R1 represents C1 to 4 alkyl, C2 to 4 alkenyl, C2 to 4 alkynyl, C3 to 6 cycloalkyl or a 4 to 8 membered saturated heterocyclic ring incorporating one heteroatom selected from O, S and N; any of said groups being optionally further substituted by C 1 to 4 alkyl, C 1 to 4 alkoxy, C1 to 4 alkylthio, C3 to 6 cycloalkyl, one or more halogens or phenyl; said phenyl group being optionally further substituted by one or more substituents selected independently from halogen, C1 to 4 alkyl, C1 to 4 alkoxy, CF3, OCF3, CN or NO2; or R1 represents phenyl or a five or six membered aromatic heterocyclic ring containing 1 to 3 heteroatoms independently selected from O, S and N; said phenyl or aromatic heterocyclic ring being optionally substituted by one or more substituents selected independently from halogen, C1 to 4 alkyl, C 1 to 4 alkoxy, OH, CN, NO2 or NR5R6; said alkyl or alkoxy group being optionally further substituted by one or more fluorine atoms; R2 and R3 independently represent H, C1 to 4 alkyl or C3 to 6 cycloalkyl; said alkyl group being optionally substituted by one or more substituents selected independently from C 1 to 4 alkoxy, halogen, OH, NR7R8, ═NR7, phenyl or a five or six membered aromatic or saturated heterocyclic ring containing 1 to 3 heteroatoms independently selected from O, S and N; said phenyl or aromatic heterocyclic ring being optionally further substituted by halogen, C1 to 4 alkyl, C1 to 4 alkoxy, CF3, OCF3, CN or NO2; and R5. R6, R7 and R8 independently represent H or C1 to 4 alkyl; or a pharmaceutically acceptable salt thereof. 2. A compound of formula (I), according to claim 1, wherein V represents 0. 3. A compound of formula (I), according to claim 1, wherein V represents S(O)n and n represents 0. 4. A compound of formula (I), according to claim 1, wherein X and Y independently represent Br, Cl, CH3, CH2F, CHF2, CF3 or CN. 5. A compound according to claim 4 wherein Y represents CN. 6. A compound of formula (I), according to claim 1, which is: 2-{[(1S)-2-amino-1-phenylethyl]oxy}-4-chloro-5-fluorobenzonitrile; 2-[[(1S)-2-amino-1-phenylethyl]thio]-6-methyl-3-pyridinecarbonitrile; 2-[(2,5-dichlorophenyl)thio]-2-phenylethylamine; 2-[[(1S)-2-amino-1-phenylethyl]thio]-4-chlorobenzonitrile; N-[2-(5-chloro-2-cyano-4-fluorophenoxy)-2-phenylethyl]-ethanimidamide; N-[2-(5-chloro-2-cyano-4-fluorophenoxy)-2-phenylethyl]-2-hydroxyethanimidamide; 2-[[(1S)-2-amino-1-phenylethyl]thio]-6-(trifluoromethyl)-3-pyridinecarbonitrile; 2-[[(1S)-2-amino-1-phenylethyl]thio]-4-methoxybenzonitrile; 4-chloro-2-[[(1S)-2-(methylamino)-1-phenylethyl]thio]benzonitrile; 2-[[(1R)-2-amino-1-(3-isoxazolyl)ethyl]oxy]-4-chloro-5-fluorobenzonitrile; 2-[[2-amino-1-(3-pyridinyl)ethyl]thio]-4-chlorobenzonitrile; or a pharmaceutically acceptable salt thereof. 7. (canceled) 8. A pharmaceutical composition comprising a compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier. 9-17. (canceled) 18. A method of treating, or reducing the risk of, human diseases or conditions in which inhibition of nitric oxide synthase activity is beneficial which comprises administering a therapeutically effective amount of a compound of formula (I), as defined in claim 1, or a pharmaceutically acceptable salt thereof, to a person suffering from, or at increased risk of, such diseases or conditions. 19. A method of treating, or reducing the risk of, inflammatory disease in a person suffering from, or at risk of, said disease, wherein the method comprises administering to the person a therapeutically effective amount of a compound of formula (I), as defined in claim 1, or a pharmaceutically acceptable salt thereof. 20. A method of treating, or reducing the risk of, CNS disease in a person suffering from, or at risk of, said disease, wherein the method comprises administering to the person a therapeutically effective amount of a compound of formula (I), as defined in claim 1, or a pharmaceutically acceptable salt thereof. 21. A process for the preparation of a compound of formula (I), as defined in claim 1, or a pharmaceutically acceptable salt, enantiomer or racemate thereof, wherein the process comprises: (a) reaction of a compound of formula (II) wherein T, U, X, Y and W are as defined in claim 1 and L1 represents a leaving group, with a compound of formula (III) wherein R1, R2, R3 and V are as defined in claim 1; or (b) reaction of a compound of formula (IV) wherein T, U, W, X, Y and V are as defined in claim 1, with a compound of formula (V) wherein R1, R2 and R3 are as defined in claim 1 and L is a leaving group; or (c) reaction of a compound of formula (VI) wherein R1, T, U, W, X, Y and V are as defined in claim 1 and L is a leaving group, with a compound of formula (VII) R2R3NH (VII) wherein R2 and R3 are as defined in claim 1; or (d) reduction of a compound of formula (VIII) wherein R1, T, U, W, X, Y and V are as defined in claim 1 and Q represents azide (N3); or (e) hydrolysis of a compound of formula (VIII) wherein R1, T, U, W, X, Y and V are as defined in claim 1 and Q represents an imide group; and where desired or necessary converting the resultant compound of formula (I), or another salt thereof, into a pharmaceutically acceptable salt thereof; or converting one compound of formula (I) into another compound of formula (I); and where desired converting the resultant compound of formula (I) into an optical isomer thereof. 22. A method of treating, or reducing the risk of, inflammatory bowel disease in a person suffering from, or at risk of, said disease, wherein the method comprises administering to the person a therapeutically effective amount of a compound of formula (I), as defined in claim 1, or a pharmaceutically acceptable salt thereof. 23. A method of treating, or reducing the risk of, rheumatoid arthritis in a person suffering from, or at risk of, rheumatoid arthritis, wherein the method comprises administering to the person a therapeutically effective amount of a compound of formula (I), as defined in claim 1, or a pharmaceutically acceptable salt thereof. 24. A method of treating, or reducing the risk of, osteoarthritis in a person suffering from, or at risk of, osteoarthritis, wherein the method comprises administering to the person a therapeutically effective amount of a compound of formula (I), as defined in claim 1, or a pharmaceutically acceptable salt thereof. 25. A method for treatment or prophylaxis of pain in a person suffering from, or at risk of, pain, wherein the method comprises administering to the person a therapeutically effective amount of a compound of formula (I), as defined in claim 1, or a pharmaceutically acceptable salt thereof. 26. A method of treating, or reducing the risk of, migraine in a person suffering from, or at risk of, migraine, wherein the method comprises administering to the person a therapeutically effective amount of a compound of formula (I), as defined in claim 1, or a pharmaceutically acceptable salt thereof. 27. A composition comprising a compound of claim 1, in combination with a COX-2 inhibitor. 28. A method of treatment or prophylaxis of an inflammatory disease, the method comprising administering to a person identified as suffering from or at risk of said disease a therapeutically effective amount of the composition of claim 27. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Nitric oxide is produced in mammalian cells from L-arginine by the action of specific nitric oxide synthases (NOSs). These enzymes fall into two distinct classes—constitutive NOS (cNOS) and inducible NOS (iNOS). At the present time, two constitutive NOSs and one inducible NOS have been identified. Of the constitutive NOSs, an endothelial enzyme (eNOS) is involved with smooth muscle relaxation and the regulation of blood pressure and blood flow, whereas the neuronal enzyme (nNOS) appears to be involved in the regulation of various biological functions. Inducible NOS has been particularly implicated in the pathogenesis of inflammatory diseases. Regulation of these enzymes should therefore offer considerable potential in the treatment of a wide variety of disease states (J. E. Macdonald, Ann. Rep. Med. Chem., 1996, 31, 221-230). Considerable effort has been expended to identify compounds that act as specific inhibitors of one or more isoforms of the enzyme nitric oxide synthase. The use of such compounds in therapy has also been widely claimed. |
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