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Transformed silkworm producing human collagen |
The present invention provides a transformed silkworm which has a polynucleotide encoding human collagen within the genomic DNA and produces recombinant human collagen as a part of proteins in the cocoon or the silk gland; a process for producing recombinant human collagen by using this transformed silkworm; and a recombinant vector for use in the generation of the transformed silkworm. Since human collagen is collected from the cocoon discharged by this transformed silkworm or its silk gland, highly pure human collagen can be conveniently obtained in a large amount. Moreover, because the recombinant human collagen produced by the transformed silkworm is safe collagen which is free from any fear of the contamination with pathogens such as viruses or prions and exhibits no antigenicity toward humans. Thus, it can be utilized in various industrial fields including medicines, foods, cosmetics and the like. |
1-24. (canceled) 25. A process for generating a transformed silkworm which produces recombinant human collagen as a part of proteins in the cocoon or the silk gland, said process comprising the steps of: (a) a step of constructing a recombinant plasmid vector having a fusion polynucleotide including a polynucleotide encoding human collagen ligated under the control of an expression regulatory sequence of a silkworm silk protein gene, in a region sandwiched between a pair of inverted terminal repeats of DNA transposon derived from an insect; (b) a step of injecting a recombinant plasmid vector having a polynucleotide encoding transposase of said transposon, and the recombinant plasmid vector of the step (a) into a silkworm egg; (c) a step of allowing development to a silkworm from the silkworm egg having said recombinant plasmid vectors injected respectively; and (d) a step of identifying the silkworm having the fusion polynucleotide of the recombinant plasmid vector constructed in the step (a) incorporated into the genomic DNA. 26. The process according to claim 25, wherein the silkworm identified in the step (d) has an exogenous polynucleotide encoding prolyl hydroxylase incorporated into its genomic DNA. 27. The process according to claim 26, wherein the exogenous polynucleotide encoding prolyl hydroxylase is at least a coding region of a DNA fragment having a base sequence of SEQ ID NO: 1. 28. A process for generating a transformed silkworm which produces a fusion protein of recombinant human collagen with a silkworm silk protein as a part of proteins in the cocoon or the silk gland, said process comprising the steps of: (a) a step of constructing a recombinant plasmid vector having a fusion polynucleotide including a polynucleotide encoding a silkworm silk protein and a polynucleotide encoding human collagen ligated under the control of an expression regulatory sequence of a silkworm silk protein gene, in a region sandwiched between a pair of inverted terminal repeats of DNA transposon derived from an insect; (b) a step of injecting a recombinant plasmid vector having a polynucleotide encoding transposase of said transposon and the recombinant plasmid vector of the step (a) into a silkworm egg; (c) a step of allowing development to a silkworm from the silkworm egg having said recombinant plasmid vectors injected respectively; and (d) a step of isolating the silkworm having the fusion polynucleotide of the recombinant plasmid vector constructed in the step (a) incorporated into the genomic DNA. 29. The process according to claim 28, wherein the silkworm identified in the step (d) has an exogenous polynucleotide encoding prolyl hydroxylase incorporated into its genomic DNA. 30. The process according to claim 29, wherein the exogenous polynucleotide encoding prolyl hydroxylase is at least a coding region of a DNA fragment having a base sequence of SEQ ID NO: 1. 31. A transformed silkworm generated by the process according to claim 25, said silkworm comprising a fusion polynucleotide including a polynucleotide encoding human collagen ligated under the control of an expression regulatory sequence of a silkworm silk protein gene within the genomic DNA, and producing recombinant human collagen as a part of proteins in the cocoon or the silk gland. 32. The transformed silkworm of claim 31 which is generated by the process according to claim 26, said silkworm producing recombinant human collagen as a part of proteins in the cocoon or the silk gland, and producing prolyl hydroxylase in at least the silk gland. 33. The transformed silkworm of claim 32, wherein the prolyl hydroxylase has an amino acid sequence of SEQ ID NO: 2. 34. A transformed silkworm generated by the process according to claim 28, said silkworm comprising a fusion polynucleotide including a polynucleotide encoding a silkworm silk protein and a polynucleotide encoding human collagen ligated under the control of an expression regulatory sequence of a silkworm silk protein gene within the genomic DNA, and producing a fusion protein of recombinant human collagen with a silkworm silk protein as a part of proteins in the cocoon or the silk gland. 35. The transformed silkworm of claim 34 which is generated by the process according to claim 29, said silkworm producing a fusion protein of recombinant human collagen with a silkworm silk protein as a part of proteins in the cocoon or the silk gland, and producing prolyl hydroxylase in at least the silk gland. 36. The transformed silkworm of claim 35, wherein the prolyl hydroxylase has an amino acid sequence set out in SEQ ID NO: 2. 37. A process for producing recombinant human collagen, which comprises isolating and purifying recombinant human collagen from the cocoon or the silk gland of the transformed silkworm of claim 31, 32 or 33. 38. A process for producing recombinant human collagen, which comprises isolating a fusion protein of recombinant human collagen with a silkworm silk protein from the cocoon or the silk gland of the transformed silkworm of claim 34, 35 or 36, and isolating and purifying recombinant human collagen from the fusion protein. 39. A fusion protein of recombinant human collagen with a silkworm silk protein produced by the transformed silkworm according to claim 34, 35 or 36. 40. A recombinant plasmid vector having a fusion polynucleotide including a polynucleotide encoding human collagen ligated under the control of an expression regulatory sequence of a silkworm silk protein gene, in a region sandwiched between a pair of inverted terminal repeats of DNA transposon derived from an insect. 41. A recombinant plasmid vector having a fusion polynucleotide including a polynucleotide encoding a silkworm silk protein and a polynucleotide encoding human collagen ligated under the control of an expression regulatory sequence of a silkworm silk protein gene, in a region sandwiched between a pair of inverted terminal repeats of DNA transposon derived from an insect. 42. A recombinant plasmid vector having an exogenous polynucleotide encoding prolyl hydroxylase in a region sandwiched between a pair of inverted terminal repeats of DNA transposon derived from an insect. 43. The recombinant plasmid vector of claim 42, wherein the exogenous polynucleotide encoding prolyl hydroxylase is at least a coding region of a DNA fragment having a base sequence of SEQ ID NO: 1. 44. A set of vectors, which comprises a recombinant plasmid vector of claim 40 or 41, and a recombinant plasmid vector having a polynucleotide encoding transposase of transposon. 45. A set of vectors, which comprises a recombinant plasmid vector of claim 42 or 43, and a recombinant plasmid vector having a polynucleotide encoding transposase of transposon. 46. A polynucleotide encoding the a subunit of silkworm prolyl hydroxylase having an amino acid sequence of SEQ ID NO: 2. 47. The polynucleotide of claim 46, which is a DNA fragment having a base sequence of SEQ ID NO: 1. |
<SOH> BACKGROUND ART <EOH>Collagen is a major protein which constitutes extracellular matrices, and has various physiological functions controlling cell proliferation, differentiation, migration and the like in addition to mechanical functions to maintain the structure of a living organism by serving as a scaffold of cells. Therefore, collagen has been widely utilized in a medical field as a biomaterial for repairing injury of a living organism (J. Surg. Res. 10: 485-491, 1970), or as a carrier for sustained release of a certain type of drugs (J. Controlled Release 33: 307-315, 1995). However, most collagen which is used at present is derived from an animal tissue such as one from a cattle or a pig. It is known that an allergic response occurs in patients accounting for about 3% when such collagen is transplanted to a human (J. Immunol. 136: 877-882, 1986; Biomaterials 11: 176-180, 1990). Furthermore, dangers of the contamination with pathogens such as viruses or prions in collagen derived from animal tissues have been big problems in recent years. Thus, it has been desired to develop a system for producing recombinant human collagen without antigenicity and dangerous contamination of pathogen. Therefore, the inventors of this application achieved an invention and filed a patent application of a process for producing recombinant human collagen having a triple helical structure which is equivalent to that in a human living body through infecting a recombinant virus having an inserted cDNA encoding human collagen to insect cells (JP-A-8-23979). Moreover, a process for producing human collagen by using mammalian cells or yeast has been also proposed (JP-T-7-501937). As described above, processes in which insect cells, mammalian cells, or yeast are used have been proposed as the process for producing recombinant human collagen. However, according to the process in which insect cells or mammalian cells are used, to achieve high amount of the production is difficult which allows for utilization in a medical field. Further, according to the process in which yeast is used, the recombinant product is produced in the fungus bodies, therefore, purification of recombinant human collagen is not necessarily easy. The present invention was accomplished in light of the circumstances as described above, and an object of the invention is to solve the problems in the conventional art and to provide a process for producing recombinant human collagen with high productivity and the easiness in purification as well as the genetic engineering materials for the same. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 is a restriction enzyme map of a transfer vector pMOSRA- 1. FIG. 2 is a restriction enzyme map of mini collagen genes incorporated in a piggyBac plasmid vector (pMOSRA-4B, pMOSRA-5 and pMOSRA-6). FIG. 3 is an electrophoresis images of PCR products amplified with genomic DNAs extracted from positive F1 adult silkworms as a template. The number above images, e.g., 1 and 2 show numbers of positive silkworms. The lane M is for a 100 bp ladder marker. FIG. 4 is an electrophoresis images of RT-PCR products amplified using an RNA extracted from the silk bland of positive F1 larval silkworms of the fifth-instar stage. The electrophoresis was conducted: in the lanes 1 and 2 for RT-PCR products from positive silkworms; and in the lane C for RT-PCR products from a wild type silkworm. The lane M indicates a 100 bp ladder marker. detailed-description description="Detailed Description" end="lead"? |
2-iminoimidazole derivatives (2) |
A 2-iminoimidazole derivative represented by the formula: {wherein R1, R2 and R3 represent hydrogen, optionally substituted C1-6 alkyl, etc.; R6 represents hydrogen, C1-6 alkyl, C1-6 alkyloxycarbonyl, etc.; Y1 represents a single bond, —CH2—, etc.; Y2 represents a single bond, —CO—, etc.; and Ar represents hydrogen, a group represented by the formula: [wherein R10-R14 represent hydrogen, C1-6 alkyl, hydroxyl, C1-6 alkoxy, etc., and R11 and R12 or R12 and R13 may bond together to form a 5- to 8-membered heterocycle], etc.} or salt thereof. |
1. A compound represented by the formula: {wherein R1, R2 and R3 may be the same or different and each independently represents (1) hydrogen, (2) cyano, (3) halogen or (4) a group selected from Substituent Group A below, and R1 and R2 may bond together to form a 5-membered ring; R6 represents (1) hydrogen, (2) C1-6 alkyl, (3) acyl, (4) carbamoyl, (5) hydroxyl, (6) C1-6 alkoxy, (7) C1-6 alkyloxycarbonyloxy, (8) C3-8 cycloalkyl, (9) C1-6 alkyloxycarbonyl optionally substituted with acyloxy or (10) a C6-14 aromatic hydrocarbon ring group or a 5- to 14-membered aromatic heterocyclic group (each of the foregoing members being optionally substituted with at least one group selected from Substituent Group E below); Y1 represents a single bond, —(CH2)m—, —CR8—, —CR8R9—, —CH2CO—, —NR8—, —SO—, —O2—, —CO—, —CONR8— or —SO2NR8— [wherein m represents an integer of 1 to 3, and R8 and R9 are the same or different and each independently represents hydrogen, halogen, C1-6 alkyl, carboxyl or C1-6 alkoxycarbonyl]; Y2 represents a single bond, O, N, —(CH2)m—, —CR8—, CR8R9—, —CO—, —SO—, —SO2— or —C(═N—OR8)— [wherein m, R8 and R9 have the same definitions given above]; and Ar represents (1) hydrogen, (2) a group represented by the formula: [wherein R10, R11, R12, R13 and R14 are the same or different and each independently represents (1) hydrogen, (2) cyano, (3) halogen, (4) nitro or (5) a group selected from Substituent Group B below, and R11 and R12 or R12 and R13 may bond together to form a 5- to 8-membered heterocycle optionally having 1 to 4 hetero atoms selected from N, S and O and optionally substituted with at least one group selected from Substituent Group F below] or (3) a 5- to 14-membered aromatic heterocyclic group optionally substituted with at least one group selected from Substituent Group G below; wherein <Substituent Group A> represents moieties selected from the group consisting of C1-6 alkyl, alkylidene, C2-6 alkenyl, C2-6 alkynyl, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, sulfonylamino, sulfonyl, sulfamoyl, C3-9 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group, each of the foregoing members being optionally substituted with at least one group selected from Substituent Group A′ below; <Substituent Group A′> represents moieties selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, sulfonylamino, sulfonyl, sulfamoyl, halogen, C3-8 cycloalkyl, a heterocyclic alkyl group, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group, wherein the C6-14 aromatic hydrocarbon ring group and the 5- to 14-membered aromatic heterocyclic group may be substituted with at least one group selected from the group consisting of C1-6 alkyl, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, nitro, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, sulfonylamino, sulfonyl, sulfamoyl, halogen and C3-8 cycloalkyl; <Substituent Group B> represents moieties selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, amino, C1-6 aminoalkyl, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, sulfonylamino, sulfonyl, sulfamoyl, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group, each of the foregoing members being optionally substituted with at least one group selected from Substituent Group B′ below; <Substituent Group B′> represents moieties selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, oxo, cyano, C1-6 cyanoacyl, C2-7 acyl, C1-6 alkanoyl, benzoyl, aralkanoyl, C1-6 alkoxyalkylcarbonyl, C1-6 hydroxyalkylcarbonyl, carboxyl, C1-6 carboxyalkyl, C1-6 carboxyalkyloxy, carbamoyl, carbamoylalkyloxy, C1-6 alkoxycarbonyl, C1-10 alkoxycarbonyl-C1-6 alkyl, C1-10 alkoxycarbonyl-C1-6 alkyloxy, C1-6 monoalkylaminocarbonyl, C2-6 dialkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C1-10 alkoxyalkyl, C1-10 aralkyloxyalkyl, C1-6 hydroxyalkyl, C3-9 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, C1-6 alkylsulfonylamino, phenylsulfonylamino, C1-6 alkylsulfonyl, phenylsulfonyl, C1-6 monoalkylaminosulfonyl, C2-6 dialkylaminosulfonyl, sulfamoyl, halogeno, C3-9 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group, a 5- to 14-membered aromatic heterocyclic group, a heterocyclic aminocarbonyl group, a heterocyclic aminosulfonyl group and isoxazolinyl, wherein the 5- to 14-membered non-aromatic heterocyclic group, the C6-14 aromatic hydrocarbon ring group, the 5- to 14-membered aromatic heterocyclic group and isoxazolinyl may be independently substituted with at least one group selected from the group consisting of C1-6 alkyl, oxo, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, nitro, amino, C1-6 aminoalkyl, C1-6 alkylamino, C1-6 dialkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, alkylsulfonylamino, alkylsulfonyl, sulfamoyl, halogeno and C3-8 cycloalkyl; <Substituent Group E> represents moieties selected from the group consisting of C1-6 alkyl, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, sulfonylamino, sulfonyl, sulfamoyl, halogen and C3-8 cycloalkyl; <Substituent Group F> represents moieties selected from the group consisting of (1) hydrogen, (2) cyano, (3) halogen, (4) oxo and (5) C1-6 alkyl, alkenyl, alkynyl, acyl, C1-6 alkanoyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, amino, imino, C1-6 aminoalkyl, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, sulfonylamino, sulfonyl, sulfamoyl, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group (each of the foregoing members being optionally substituted with at least one group selected from Substituent Group F′ below); <Substituent Group F′> represents moieties selected from the group consisting of C1-6 alkyl, oxo, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, benzyloxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-9 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, C1-6 alkylsulfonylamino, C1-6 alkylsulfonyl, sulfamoyl, halogeno, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group; <Substituent Group G> represents moieties selected from the group consisting of C1-6 alkyl, cyano, acyl, carboxyl, carbamoyl, C1-6-alkoxycarbonyl, C1-6 alkylaminocarbonyl, sulfonyl, sulfamoyl, halogeno and C3-8 cycloalkyl.} or salt thereof. 2. A compound according to claim 1 or salt thereof, wherein R1, R2 and R3 may be the same or different and each independently represents C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy, each of the foregoing members being optionally substituted with at least one group selected from Substituent Group A″ below: <Substituent Group A″> represents moieties selected from the group consisting of C1-6 alkyl, acyl, carboxyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C1-6 alkylamino, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group, wherein the C6-14 aromatic hydrocarbon ring group and the 5- to 14-membered aromatic heterocyclic group may be substituted with at least one group selected from the group consisting of C1-6 alkyl, carboxyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, nitro, C1-6 alkylamino, acylamino, sulfonylamino and halogen; R6 represents a group selected from the group consisting of hydrogen, C1-6 alkyl and C1-6 alkyloxycarbonyl optionally substituted with acyloxy; Y′ represents a single bond or —(CH2)m— [wherein m represents an integer of 1 to 3]; Y2 represents a single bond or —CO—; and Ar represents hydrogen or a group represented by the formula: [wherein R10, R11, R12, R13 and R14 are the same or different and each independently represents a group selected from the group consisting of hydrogen, C1-6 alkyl, hydroxyl, C1-6 alkoxy, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, a 5- to 14-membered non-aromatic heterocyclic group and C1-6 alkyloxycarbonyloxy, and R11 and R12 or R12 and R13 may bond together to form a 5- to 8-membered heterocycle (i) optionally having 1 to 4 hetero atoms selected from N, S and O and (ii) optionally substituted with at least one group selected from the group consisting of cyano, oxo, and C1-6 alkyl, acyl, C1-6 alkanoyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, sulfonyl and a 5- to 14-membered non-aromatic heterocyclic group (each of the foregoing members being optionally substituted with at least one group selected from Substituent Group F″ below; wherein <Substituent Group F″> represents moieties selected from the group consisting of C1-6 alkyl, oxo, cyano, acyl, carboxyl and C1-6 alkoxy)]. 3. A compound according to claim 1 or salt thereof, wherein Y1 is —CH2—. 4. A compound according to claim 1 or salt thereof, wherein Y2 is —CO—. 5. A compound according to claim 1 or salt thereof, wherein Y1 is —CH2— and Y2 is —CO—. 6. A compound according to claim 1 or a salt salt thereof, wherein Y1 is a single bond, Y2 is a single bond and Ar is hydrogen. 7. A compound according to claim 1 or salt thereof, wherein Ar is a group represented by the formula: [wherein R10, R11, R12, R13 and R14 are as defined in claim 1]. 8. A compound according to claim 7 or salt thereof, wherein R10 and R14 are hydrogen. 9. A compound according to claim 1 or salt thereof, wherein Ar is (1) a group represented by the formula: [wherein R10, R11, R12, R13 and R14 have the same definitions given above are as defined in claim 1] or (2) a 5- to 14-membered aromatic heterocyclic group optionally substituted with at least one group selected from Substituent Group G; wherein Substituent Group G is as defined in claim 1. 10. A compound according to claim 9 or salt thereof, wherein R10 and R14 are hydrogen. 11. A compound according to claim 1 or salt thereof, wherein Ar is a group represented by the formula: [wherein R11 and R13 are as defined in claim 1, and R15 represents (1) hydrogen or (2) a group selected from Substituent Group H below, and R11 and R15 may bond together to form a 5- to 8-membered heterocycle optionally substituted with at least one group selected from Substituent Group F above and optionally having 1 or 2 hetero atoms selected from N, S and O; wherein Substituent Group F is as defined in claim 1; <Substituent Group H> represents moieties selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, acyl, C1-6 alkoxycarbonyl, aminocarbonyl, C1-6 alkylaminocarbonyl, C3-8 cycloalkyl, C1-6 aminoalkyl, sulfonyl, C3-8 cycloalkylamino, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group, each of the foregoing members being optionally substituted with at least one group selected from Substituent Group H′ below; <Substituent Group H′> represents moieties selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, oxo, cyano, C1-6 cyanoalkyl, C2-7 acyl, C1-6 alkanoyl, benzoyl, aralkanoyl, C1-6 alkoxyalkylcarbonyl, C1-6 hydroxyalkylcarbonyl, carboxyl, C1-6 carboxyalkyl, C1-6 carboxyalkyloxy, carbamoyl, carbamoylalkyloxy, C1-6 alkoxycarbonyl, C1-10 alkoxycarbonyl-C1-6 alkyl, C1-10 alkoxycarbonyl-C1-6 alkyloxy, C1-6 monoalkylaminocarbonyl, C2-6 dialkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C1-10 alkoxyalkyl, C1-10 aralkyloxyalkyl, C1-6 hydroxyalkyl, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, C1-6 alkylsulfonylamino, phenylsulfonylamino, C1-6 alkylsulfonyl, phenylsulfonyl, C1-6 monoalkylaminosulfonyl, C2-6 dialkylaminosulfonyl, sulfamoyl, halogeno, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group, a 5- to 14-membered aromatic heterocyclic group, a heterocyclic aminocarbonyl group, a heterocyclic aminosulfonyl group and isoxazolinyl, wherein the 5- to 14-membered non-aromatic heterocyclic group, the C6-14 aromatic hydrocarbon ring group, the 5- to 14-membered aromatic heterocyclic group and isoxazolinyl may be independently substituted with at least one group selected from the group consisting of C1-6 alkyl, oxo, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-9 cycloalkyloxy, nitro, amino, C1-6 aminoalkyl, C1-6 alkylamino, C1-6 dialkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, alkylsulfonylamino, alkylsulfonyl, sulfamoyl, halogeno and C3-8 cycloalkyl]. 12. A compound according to claim 1 or salt thereof, wherein Ar is a group represented by the formula: [wherein R11 and R15 are as defined in claim 1, and R16 represents (1) hydrogen or (2) a group selected from Substituent Group H, and R11 and R15 or R15 and R16 may bond together to form a 5- to 6-membered heterocycle optionally substituted with at least one group selected from Substituent Group F and optionally having 1 or 2 hetero atoms selected from N, S and O]; wherein Substituent Groups F and H are as defined in claim 1. 13. A compound according to claim 1 or salt thereof, wherein Ar is a group represented by the formula: [wherein R11 and R15 are as defined in claim 1, and R17 and R18 are the same or different and each independently represents (1) hydrogen or (2) a group selected from Substituent Group I below, and R11 and R15, R15 and R17, R15 and R18 or R17 and R18 may bond together to form a 5- to 8-membered heterocycle optionally substituted with at least one group selected from Substituent Group F above and optionally having 1 or 2 hetero atoms selected from N, S and O, wherein Substituent Group F is as defined in claim 1; <Substituent Group I> represents moieties selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, acyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, C1-6 aminoalkyl, sulfonyl, sulfamoyl, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group, each of the foregoing members being optionally substituted with at least one group selected from Substituent Group I′ below; <Substituent Group I′> represents moieties selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, oxo, cyano, C1-6 cyanoalkyl, C2-7 acyl, C1-6 alkanoyl, benzoyl, aralkanoyl, C1-6 alkoxyalkylcarbonyl, C1-6 hydroxyalkylcarbonyl, carboxyl, C1-6 carboxyalkyl, C1-6 carboxyalkyloxy, carbamoyl, carbamoylalkyloxy, C1-6 alkoxycarbonyl, C1-10 alkoxycarbonyl-C1-6 alkyl, C1-10 alkoxycarbonyl-C1-6 alkyloxy, C1-6 monoalkylaminocarbonyl, C2-6 dialkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C1-10 alkoxyalkyl, C1-10 aralkyloxyalkyl, C1-6 hydroxyalkyl, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, C1-6 alkylsulfonylamino, phenylsulfonylamino, C1-6 alkylsulfonyl, phenylsulfonyl, C1-6 monoalkylaminosulfonyl, C2-6 dialkylaminosulfonyl, sulfamoyl, halogeno, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group, a 5- to 14-membered aromatic heterocyclic group, a heterocyclic aminocarbonyl group, a heterocyclic aminosulfonyl group and isoxazolinyl, wherein the 5- to 14-membered non-aromatic heterocyclic group, the C6-14 aromatic hydrocarbon ring group, the 5- to 14-membered aromatic heterocyclic group and isoxazolinyl may be independently substituted with at least one group selected from the group consisting of C1-6 alkyl, oxo, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, nitro, amino, CI6 aminoalkyl, C1-6 alkylamino, C1-6 dialkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, alkylsulfonylamino, alkylsulfonyl, sulfamoyl, halogeno and C3-8 cycloalkyl]. 14. A pharmaceutical composition comprising a compound according to claim 1 or asalt salt thereof. 15. A composition according to claim 14, wherein the composition is useful as a thrombin receptor antagonist. 16. A composition according to claim 14, wherein the composition is useful as a thrombin receptor PAR1 antagonist. 17. A composition according to claim 14, wherein the composition is useful as a platelet aggregation inhibitor. 18. A composition according to claim 14, wherein the composition is useful as a proliferation inhibitor for smooth muscle cells. 19. A composition according to claim 14, wherein the composition is useful as a proliferation inhibitor for endothelial cells, fibroblasts, nephrocytes, osteosarcoma cells, muscle cells, cancer cells and/or glia cells. 20. A composition according to claim 14, wherein the composition is useful as a therapeutic or prophylactic agent for thrombosis, vascular restenosis, deep venous thrombosis, pulmonary embolism, cerebral infarction, heart disease, disseminated intravascular coagulation, hypertension, inflammatory disease, rheumatism, asthma, glomerulonephritis, osteoporosis, neurological disease and/or malignant tumor. 21. Use of a compound according to claim 1 or salt thereof for the manufacture of a thrombin receptor antagonist. 22. Use according to claim 21, wherein the thrombin receptor antagonist is a PAR1 receptor antagonist. 23. Use of a compound according to claim 1 or salt thereof for the manufacture of a platelet aggregation inhibitor. 24. A therapeutic method for treating or preventing a disease associated with thrombin receptors, comprising administering to a patient suffering from the disease, a therapeutically effective dose of a compound according to claim 1 or salt thereof. 25. A therapeutic method for treating or preventing a proliferative disease of endothelial cells, fibroblasts, nephrocytes, osteosarcoma cells, muscle cells, cancer cells and/or glia cells, comprising administering to a patient suffering from the disease, a therapeutically effective dose of a compound according to claim 1 or salt thereof. |
<SOH> BACKGROUND OF THE INVENTION <EOH>A recent approach for thrombosis has involved inhibiting thrombin enzyme activity, and compounds used for this purpose have included heparin, low molecular weight heparin, hirudin, argatroban, hirulog and the like. All such compounds inhibit the enzyme activity of thrombin, and work by inhibiting fibrin blood clot formation without specifically inhibiting the effect of thrombin on cells. Bleeding tendency is therefore a common side effect encountered in the clinic. The role of thrombin in thrombosis is not limited to its blood clotting activity, as it is believed to also participate in platelet aggregation at sites of vascular injury occurring as a result of the activation of platelet thrombin receptor. Another approach for thrombosis has been the use of intravenous injection agents such as Abciximab, Eptifibatide and Tirofiban, as GPIIb/IIIa receptor antagonists. These compounds, while exhibiting powerful anti-thrombotic effects by suppressing platelet aggregation induced by various stimulation such as thrombin, ADP, collagen, PAF or the like, also produce a bleeding tendency as a side effect similarly to thrombin enzyme activity inhibitors. For this reason, no such compounds have yet been marketed, although their development as oral agents continues to progress. Restenosis is a vascular hyperproliferative response to vascular wall injury induced by invasive treatment such as coronary angioplasty, and this phenomenon may be provoked by the direct or indirect effect of thrombin on cells. Platelets adhere to injured blood vessels, leading to release of growth factors and eliciting proliferation of smooth muscle cells. Smooth muscle cells may also be affected indirectly by the action of thrombin on endothelial cells. Also, platelet adhesion occurs and procoagulant activity increases at sites of vascular injury. Smooth muscle cells can undergo further direct stimulation due to the high local thrombin concentration which is produced at such sites. While recent studies using the powerful thrombin inhibitor hirudin have suggested that thrombin induces cell proliferation during the process of restenosis, it has not yet been determined whether the thrombin effect is direct or indirect (Sarembock et al., Circulation 1992, 84: 232-243). Despite the implication of the cellular effects of thrombin in a variety of pathological symptoms, no therapeutically active substance is known which specifically blocks such effects. The thrombin receptor (PAR1) has recently been cloned (Vu et al., Cell, 1991, 64: 1057-1068), opening an important door to development of substances which target cellular thrombin receptors. Detailed examination of the amino acid sequence of this thrombin receptor has revealed a thrombin binding site and hydrolysis site located in the 100 residue amino terminal domain of the receptor. Later research by amino acid mutation in the receptor has established that limited hydrolysis of this portion of the thrombin receptor by thrombin is necessary for receptor activation (Vu et al., Nature, 1991, 353: 674-677). A synthetic peptide corresponding to the amino acid sequence newly generated by hydrolysis of the thrombin receptor (the synthetic peptide is known as “thrombin receptor activating peptide”, or TRAP) can activate receptors which have not been hydrolyzed by thrombin. This suggests that the cleavage of the receptor, the new amino acid sequence generated at the amino terminal (known as the “tethered ligand peptide”) functions as the ligand and interacts with the distal binding site. Further studies of TRAP have confirmed homology of the thrombin receptors present in platelet, endothelial cell, fibroblast and smooth muscle cell (Hung et al., J. Cell. Biol. 1992, 116: 827-832; and Ngaiza, Jaffe, Biochem. Biophys. Res. Commun. 1991, 179: 1656-1661). Research on the structure activity relationship of TRAP suggests that the pentapeptide Phe-Leu-Leu-Arg-Asn is a weak antagonist for platelet thrombin receptors activated by either thrombin or TRAP (Vassallo. et al., J. Biol. Chem., 1992, 267: 6081-6085 (1992)). Different approaches to receptor antagonism have also been examined by other groups. One of these approaches has been an attempt to prepare antibodies for the thrombin binding domain of the thrombin receptor. Such antibodies specifically and effectively suppress activation of platelets by thrombin, and act as thrombin receptor antagonists (Hung et al., J. Clin. Invest. 1992, 89: 1350-1353). Another approach has been the development of peptide derivatives from TRAP (Steven M. S., J. Med. Chem. 1996, 39: 4879-4887; William J. H., Bioorg. Med. Chem. Lett. 1998, 8: 1649-1654; and David F. M., Bioorg. Med. Chem. Lett. 1999, 9: 255-260). Yet another approach has been the development of low molecular weight compounds discovered by high throughput screening using various assay systems such as receptor binding (Andrew W. S. et al., Bioorg. Med Chem. Lett. 1999, 9: 2073-2078; Scherig Plough WO99/26943; and Halord S. et al., ACS meeting in October 2001). detailed-description description="Detailed Description" end="lead"? |
Apparatus and method for authentication of computer-readable medium |
An apparatus and method for authentication of a computer-readable medium (120) provides advantages of automating the authentication process, and further provides redundancy in processes that may be used by a customer for authentication. It enables downloading of files and/or licenses from a central server (104), and the local use of an authentication program running on the client (102), who reduces communications and processing demands on the server (104). Further advantages include the flexibility to customize the authentication approach by varying the local criteria checked during authentication. Accordingly, downloading and further copying and distribution of software or content is effectively controlled, making piracy and other unauthorized copying more difficult. |
1. A method of authenticating an article of digital media having a digital work provided thereon, comprising steps of: identifying criteria on the article of digital media; and comparing the criteria to corresponding criteria that is known to be present on an original master version of the digital work. 2. A copy-protected digital storage medium, comprising: a digital storage medium; a digital work encoded on said digital storage medium; and a computer program also encoded on said digital storage medium, said computer program constructed and arranged to be executable on a personal computer in order to thwart access to said digital work by said personal computer. 3. A copy-protected digital storage medium according to claim 2, wherein said digital storage medium is a compact disc. 4. A copy-protected digital storage medium according to claim 3, wherein said digital work comprises an audio work containing audio files. 5. A copy-protected digital storage medium according to claim 4, wherein said audio files are located on a first session of the compact disc. 6. A copy-protected digital storage medium according to claim 5, wherein said computer program is located on a second session of the compact disc. 7. A copy-protected digital storage medium according to claim 4, further comprising an auto-run information file on said compact disc. 8. A copy-protected digital storage medium according to claim 7, wherein said auto-run information file is configured to instruct a personal computer to execute said computer program. 9. A copy-protected digital storage medium according to claim 1, further comprising an auto-run information file on said article of digital media. 10. A copy-protected digital storage medium according to claim 9, wherein said auto-run information file is configured to instruct a personal computer to execute said computer program. 11. A copy-protected digital storage medium according to claim 1, wherein said digital storage medium comprises a standard audio compact disc that is playable by a standard audio compact disc player. 12. A copy-protected digital storage medium according to claim 11, wherein said computer program is constructed and arranged so as not to interfere with playability of said compact disc in a standard audio compact disc player. 13. A copy-protected digital storage medium according to claim 1, wherein said computer program is constructed and arranged to control a computer user's ability to digitally extract the digital work from the digital media. 14. A copy-protected digital storage medium according to claim 1, wherein said computer program is constructed and arranged to inspect low-level content from a hardware device that is provided on the personal computer for reading said digital media. 15. A method of operating a personal computer, comprising steps of: connecting a digital storage medium bearing a digital work and a computer program to a personal computer; and executing the computer program on the personal computer to thwart access to the digital work. 16. A method of operating a personal computer according to claim 15, wherein said digital storage medium is a compact disc. 17. A method of operating a personal computer according to claim 16, wherein said digital work comprises an audio work containing audio files. 18. A method of operating a personal computer according to claim 17, wherein said audio files are located on a first session of the compact disc. 19. A method of operating a personal computer according to claim 18, wherein said computer program is located on a second session of the compact disc. 20. A method of operating a personal computer according to claim 19, further comprising an auto-run information file on said compact disc. 21. A method of operating a personal computer according to claim 20, wherein said auto-run information file is configured to instruct a personal computer to execute said computer program. 22. A method of operating a personal computer according to claim 15, further comprising an auto-run information file on said article of digital media. 23. A method of operating a personal computer according to claim 22, wherein said step of executing the computer program comprises using said auto-run information file to instruct the personal computer to execute said computer program. 24. A method of operating a personal computer according to claim 15, wherein said digital storage medium comprises a standard audio compact disc that is playable by a standard audio compact disc player. 25. A method of operating a personal computer according to claim 24, wherein said computer program is constructed and arranged so as not to interfere with playability of said compact disc in a standard audio compact disc player. 26. A method of operating a personal computer according to claim 15, wherein said step of executing the computer program comprises controlling a computer user's ability to digitally extract the digital work from the digital media. 27. A method of operating a personal computer according to claim 15, wherein said step of executing the computer program comprises inspecting low-level content from a hardware device that is provided on the personal computer for reading said digital media. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates in general to authentication in computer systems, and, more specifically, to authentication of a computer-readable medium containing valuable informational content. 2. Description of the Related Technology The software and entertainment industries have a strong interest in protecting valuable business software and other types of software, such as recreational game software, and music, movie and other entertainment content from unauthorized copying and distribution. The widespread use of personal computers, Internet access, and portable devices such as MP3 players has permitted extensive unauthorized distribution of software and entertainment content. As the software and entertainment industries are increasingly using the Internet for distribution of software and content to businesses and consumers, it has become important to limit this distribution to authorized customers who have properly paid for or otherwise are entitled to receive this software and content. The providing of software updates and additional entertainment content or related services through Internet distribution, for example as may be provided under subscription-based distribution models, further increases the need to control distribution to authorized customers. Also, purchasers of software often desire to interact with other users of compatible software, for example Internet-based games software, and do so through a central server computer that enables this interaction. Prior approaches to limiting distribution to authorized customers have included efforts to authenticate the customer prior to permitting the customer to download software and/or entertainment content. These approaches include establishing an Internet connection between a client computer and a server computer and the manual entry of authenticating data by the user. Such authenticating data may include a password provided by a software or content vendor at the time of sale or specific text that is located by the user from a manual or other paper guide provided as part of the customer's purchase. A limitation of these manual approaches is the manual effort required by the customer, which may find the locating of information in a manual or typing in of a password more time-consuming or difficult than is offered in competing products. Thus, it would be desirable to have an authentication approach that is automated and does not require manual action by the customer. Another limitation of prior manual authentication approaches is that they are susceptible to piracy because the password or other authenticating data provided to an original customer may be copied and distributed along with pirated copies of software or entertainment content. More complicated manual approaches have required the entering of additional authenticating data by the customer that varies with time or other events associated with the customer's use of a purchased computer product or on-line service, but such approaches only increase customer effort and frustration. It would be preferable for any use of such additional authentication criteria to be automated and handled without additional customer interaction. Yet another limitation of prior manual authentication approaches is that they do not provide a convenient alternative authentication approach if the primary authentication approach fails. The typical back-up alternative requires live communication with a vendor. It would be preferred to have an automatic authentication approach with redundancy that permits at least a semi-automatic authentication approach in case the primary approach fails. Hence, there is a need for an authentication process for controlling distribution of software and content to customers that is automated, provides redundancy, and permits more extensive checking of multiple authentication criteria without additional manual involvement by customers. |
<SOH> SUMMARY OF THE INVENTION <EOH>Accordingly, it is an object of the invention to provide an authentication process for controlling distribution of software and content to customers that is automated, provides redundancy, and permits more extensive checking of multiple authentication criteria without additional manual involvement by customers. In order to achieve the above and other objects of the invention, a method of authenticating an article of digital media having a digital work provided thereon includes identifying criteria on the article of digital media; and comparing the criteria to corresponding criteria that is know to be present on an original master version of the digital work. These and various other advantages and features of novelty that characterize the invention are pointed out with particularity in the claims annexed hereto and forming a part hereof. However, for a better understanding of the invention, its advantages, and the objects obtained by its use, reference should be made to the drawings which form a further part hereof, and to the accompanying descriptive matter, in which there is illustrated and described a preferred embodiment of the invention. |
Method and device for calculating a price for using a specific link in a network |
The invention relates to a method for calculating in a network that comprises links, a price for using a specific link in the network. The method comprises the following steps: a) a comparison step for determining a price difference between the price for using the specific link and the price for using instead of the specific link an alternative path in the network, which does not comprise the specific link, b) a change-calculation step for determining a link-price change in the price for using the specific link, and a link-price change in the price for using the links in the alternative path, in response to the determined price difference, c) a combination step for combining for the specific link the determined link-price changes on the price for using the specific link from all links in the network, to determine a total price-change for the specific link, d) a merging step for merging the determined total price-change with a market-induced price change in the price for using the specific link, to calculate the price for using the specific link, wherein the market-induced price change is being driven by at least one random variable. |
1. A method of calculating for a network commodity, a new market price for one unit of a constituent commodity, the unit of the constituent commodity comprises a specific quantity of the commodity, where the network commodity is defined by the property that constituent commodities are represented by links of a network, the links having nodes at their end points, and the constituent commodities are usable to construct further network commodities provided that the constituent commodities share a common node, the constituent commodities comprise commodities such that units of these commodities are indistinguishable provided that they have the same end nodes and that there is a continuous path between the nodes, where a path is defined as a series of links with common nodes, said method comprising: determining a price difference between a market price for one unit of a first constituent commodity defined by a specific link in the network representing the networked commodity and market prices for one unit of each of the alternative constituent commodities, defined by links in the network, where the alternative constituent commodities form an alternative path between the same nodes as the first constituent commodity and excluding the first constituent commodity; determining a link-price change for the market price for one unit of the first constituent commodity defined by the specific link, and a link-price change for the market prices for one unit of each of the alternative constituent commodities defined by the links in said alternative path, in response to said determined price difference; combining, for the market price for one unit of the first constituent commodity defined by the specific link, the determined link-price changes from the market prices for one unit of the first constituent commodity defined by the specific link and for one unit of each of the alternative constituent commodities defined by the links of the network commodity, to determine a total link-price-change for one unit of the first constituent commodity defined by the specific link, and merging said determined total link-price-change, for one unit of the first constituent commodity defined by the specific link, with a market-induced price change in the market price for one unit of the first constituent commodity, to calculate the new market price (p′ab) for one unit of the first constituent commodity, said market-induced price change being driven by at least one random variable. 2. The method according to claim 1, wherein said market-induced price change comprises one or more functions of: Brownian price-change, Poisson price-change semi-Markov price-change, Levy price-change or Ito price-change, and the previous price. 3. The method according to claim, wherein the market-induced price change is modeled to comprise a function for price-spikes and/or price-jumps. 4. The method according to claim 1, wherein the determination of the link-price change is modeled using one or more of: a term (for the quantification of market liquidity, an arbitrage-correction function, and the previous price. 5. The method according to claim one of, wherein the link-price change on the specific link and the link-price change in the links of the alternative path are all based on a common demand change. 6. The method according to claim 1, wherein the determination of the link-price comprises: determining an amount of an existing transport capacity demand that is to be shifted from said specific link to said alternative path, in response to said determined price difference; and determining the link-price change effected by the determined transport capacity demand that is to be shifted, on the link-price for using said specific link and on the link-price for using the links in said alternative path. 7. The method according to claim 6, wherein the transport capacity demand is modeled to have a price elasticity. 8. The method according to claim 6, wherein the transport capacity demand is approximated with a linear function. 9. The method according to claim, wherein, on the links, a transport capacity supply is modeled as having a constant elasticity. 10. The method according to claim 6, wherein the amount of demand to be shifted from the specific link to the alternative path is positive. 11. The method according to claim 1, wherein the method is carried out for each of said links in said network. 12. The method according to claim 1, wherein said network comprises a communication network. 13. The method according to claim 1, further comprising deciding in view of the calculated link-price, whether to increase, decrease or maintain the transport capacity demand for transporting a unit, over said specific link or said alternative path. 14. The method according to claim one of 1, further comprising changing the transport capacity demand for transporting the unit over said specific link or a said alternative path in response to the calculated link-price. 15. The method according to claim 1, further comprising one of a buy, hold, or sell action for transportation bandwidth on said network in response to the calculated link-price. 16. The method according to claim 1, wherein the method is executed several times sequentially to provide a link-price series comprising several of said subsequent calculated link-prices. 17. The method according to claim 16, wherein the method is being executed several times to provide several of said link-price series. 18. The method according to claim 17, further comprising; a discounting the link-prices within said link-price series back to their present-value, thereby providing a discounted price series; and integrating said discounted price series for the network or a sub-network thereof, to obtain a network present-value for the network or a sub-network thereof. 19. The method according to claim 18, further comprising one of a buy/hold/sell action for said network or subnetwork. 20. The method according to claim 19, further comprising changing all adaptation step wherein the transport capacity of the specific link or a different of said links in said network in response to the calculated link-price. 21. A computer program product comprising program code means for performing the method of claim 1, 22. The computer program product according to claim 21, wherein the program code means is stored on a computer-readable medium. 23. A network-pricing device comprising; the computer program product according to claim 22; and a processor for executing the program code means, wherein said processor has access to network information comprising the link-price and the price for using, instead of the specific link, an alternative path. 24. The network-pricing device according to claim 23, further comprising a first network interface via which the processor has the access to the network information. 25. The network-pricing device according to claim 23, further comprising a second network interface via which the transport capacity demand for transporting the unit over the specific link or the alternative path can be decreased or increased in response to the calculated link-price and/or via which the buy, hold, or sell action for transportation bandwidth on the network or for the network or subnetwork can be effected. 26. The network-pricing device according to claim 23, further comprising a controller via which the transport capacity of the specific link or a different of said links in said network is changeable. |
<SOH> TECHNICAL FIELD AND BACKGROUND OF THE INVENTION <EOH>Bandwidth is becoming commoditized and markets are starting to appear. Potential behaviors of these markets are not understood because these markets are still in the early stages of development. This is reflected in the lack of current research on the structure and dynamics of network commodity market prices. A method is presented for constructing telecom commodity spot price processes. Bandwidth, like electricity, is not storable, so inspiration is drawn from electricity prices and models. However, unique network features of telecommunications require specific inclusion. These are: geographical substitution, referred to as arbitrage; quality of service (QoS); and the continuing pace of technological development. Developing liquidity acts as a further complication. Liquidity refers to the ease with which partners for trades at a given price can be found. Geographical arbitrage means that spot price development on point-to-point links cannot be understood in isolation from price development on alternative paths with equivalent QoS. This implies some form of price modification derived from load-balancing across appropriately specified QoS limited alternative paths. Technological development continually pushes prices down as new equipment is installed by competitors. So, unlike other commodities, the prices revert towards a mean whose drift is strongly structurally downwards. Market liquidity is quantified as the extent to which geographical arbitrage modifies link-prices. Thus price development is modeled as a combination of link-price processes modified by prices for equivalent QoS paths. The presented model covers the existence and value of arbitrage opportunities together with its effect on price development and network present-value (NPV). Application of this work ranges from network design to infrastructure valuation and construction of real options. 1998, McGraw-Hill, New York, a pricing model for energy markets is described which is very similar to a model for general commodities which was exactly equivalent to an earlier model under a linear transformation of parameters, described in “The stochastic behavior of commodity prices: implications for valuing and hedging”, E. Schwartz. 1997 , J. Finance 52, pp. 923-973, The observations of congestion on the Internet suggest however, that even for single links, these models are insufficient because they do not include spikes or jumps in prices. Price spikes are particular features of electricity prices and some modelling has been done there as described in “Stochastic models of energy commodity prices and their applications: mean-reversion with jumps and spikes”, S. Deng, 1998. PSERC working paper 98 - 28 , readable at http://www.pserc.wisc.edu/. The international publication WO 00/54198A2 refers to a system, method, software, and portfolios for managing risk in markets relating to a commodity delivered over a network, in which a market participant constructs portfolios of preferably liquid price risk instruments in proportions that eliminate the Spatial Price Risk for the market participant's underlying position. Techniques are also disclosed for constructing and evaluating new price risk instruments and other sets of positions, as well as identifying arbitrage opportunities in those markets. Apart from link-price processes in isolation there is the question of network effects in the sense of interactions. The Most important network effect here is geographical arbitrage, i.e. the existence of many prices for end-to-end capacity at equivalent QoS. This has been observed in the market. Detection of such arbitrage opportunities is in general an NP complete problem based on shortest path algorithms with side constraints. A variety of pseudo-polynomial time algorithms exist that result from quantification of these constraints as is to be expected under commoditization. Geographical arbitrage opportunities could exist in the forward market even with a no-arbitrage situation in the spot market. There is vast computer science literature on price-setting for network resources in order to achieve some aim, e.g. social welfare maximization, cost-allocation, congestion control, etc. Here price dynamics are approached from a completely different direction in that it is started from modelling the price process rather than modelling supply and demand and then solving for the best price in some sense relative to a given network. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention is directed to a method for calculating in a network that comprises links, a price for using a specific link in the network. The method comprises the following steps: a) a comparison step for determining a price difference between the price for using the specific link and the price for using instead of the specific link an alternative path in the network, which does not comprise the specific link, b) a change-calculation step for determining a link-price change in the price for using the specific link, and a link-price change in the price for using the links in the alternative path, in response to the determined price difference, c) a combination step for combining for the specific link the determined link-price changes on the price for using the specific link from all links in the network, to determine a total price-change for the specific link, d) a merging step for merging the determined total price-change with a market-induced price change in the price for using the specific link, to calculate the price for using the specific link, wherein the market-induced price change is being driven by at least one random variable. |
2-Iminopyrrolidine derivatives |
A 2-iminopyrrolidine derivative represented by the formula: {wherein ring B represents a benzene ring, pyridine ring, etc.; R101-R103 represent hydrogen, halogen, C1-6 alkyl, etc.; R5 represents hydrogen, C1-6 alkyl, C1-6 alkoxy-C1-6 alkyl, etc.; R6 represents hydrogen, C1-6 alkyl, C1-6 alkyloxycarbonyl, etc.; Y1 represents a single bond, —CH2—, etc.; Y2 represents a single bond, —CO—, etc.; and Ar represents hydrogen, a group represented by the formula: [wherein R10—R14 represent hydrogen, C1-6 alkyl, hydroxyl, C1-6 alkoxy, etc.; and R11 and R12 or R12 and R13 may bond together to form a 5- to 8-membered heterocyclic ring], etc.}, or a salt thereof. |
1. A compound represented by the formula: (wherein ring B represents an optionally substituted (1) aromatic hydrocarbon ring or (2) aromatic heterocycle optionally having 1 or 2 nitrogen; R101, R102 and R103 are the same or different and each represents (1) hydrogen or (2) a group selected from Substituent Group C below; R5 represents (1) hydrogen, (2) cyano, (3) halogen or (4) a group selected from Substituent Group A below; R6 represents (1) hydrogen, (2) C1-6 alkyl, (3) acyl, (4) carbamoyl, (5) hydroxyl, (6) C1-6 alkoxy, (7) C1-6 alkyloxycarbonyloxy, (8) C3-8 cycloalkyl, (9) C1-6 alkyloxycarbonyl optionally substituted with acyloxy or (10) a C6-14 aromatic hydrocarbon ring group or 5- to 14-membered aromatic heterocyclic group (each of the foregoing members being optionally substituted with at least one group selected from Substituent Group E); Y1 represents a single bond, —(CH2)m—, —CR8—, —CR8R9—, —CH2CO—, —NR8—, —SO—, —SO2—, —CO—, —CONR8— or —SO2NR8— (wherein m represents an integer of 1 to 3, and R8 and R9 are the same or different and each represents hydrogen, halogen, C1-6 alkyl, carboxyl or C1-6 alkoxycarbonyl]; Y2 represents a single bond, O, N, —(CH2)m—, —CR8—, CR8R9—, —CO—, —SO—, —SO2— or —C(═N—OR8)-[wherein m, R8 and R9 are as defined above]; Ar represents (1) hydrogen, (2) a group represented by the formula: [wherein R10, R11, R12, R13 and R14 are the same or different and each represents (1) hydrogen, (2) cyano, (3) halogen, (4) nitro or (5) a group selected from Substituent Group B below, and R11 and R12 or R12 and R13 may bond together to form a 5- to 8-membered heterocycle optionally having 1 to 4 hetero atoms selected from N, S and O and also optionally substituted with at least one group selected from Substituent Group F] or (3) a 5- to 14-membered aromatic heterocyclic group optionally substituted with at least one group selected from Substituent Group G below. <Substituent Group A> The group consisting of C1-6 alkyl, alkylidene, C2-6 alkenyl, C2-6 alkynyl, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, sulfonylamino, sulfonyl, sulfamoyl, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group, each of the foregoing members being optionally substituted with at least one group selected from Substituent Group A′ below; <Substituent Group A′> The group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, sulfonylamino, sulfonyl, sulfamoyl, halogen, C3-8 cycloalkyl, a heterocyclic alkyl group, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group, wherein the C6-14 aromatic hydrocarbon ring group and the 5- to 14-membered aromatic heterocyclic group may be substituted with at least one group selected from the group consisting of C1-6 alkyl, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, nitro, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, sulfonylamino, sulfonyl, sulfamoyl, halogen and C3-8 cycloalkyl; <Substituent Group B> The group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, amino, C16 aminoalkyl, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, sulfonylamino, sulfonyl, sulfamoyl, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group, each of the foregoing members being optionally substituted with at least one group selected from Substituent Group B′ below; <Substituent Group B′> The group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, oxo, cyano, C1-6 cyanoacyl, C2-7 acyl, C1-6 alkanoyl, benzoyl, aralkanoyl, C1-6 alkoxyalkylcarbonyl, C1-6 hydroxyalkylcarbonyl, carboxyl, C1-6 carboxyalkyl, C1-6 carboxyalkyloxy, carbamoyl, carbamoylalkyloxy, C1-6 alkoxycarbonyl, C1-10 alkoxycarbonyl-C1-6 alkyl, C1-10 alkoxycarbonyl-C1-6 alkyloxy, C1-6 monoalkylaminocarbonyl, C2-6 dialkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C1-10 alkoxyalkyl, C1-10 aralkyloxyalkyl, C1-6 hydroxyalkyl, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, C1-6 alkylsulfonylamino, phenylsulfonylamino, C1-6 alkylsulfonyl, phenylsulfonyl, C1-6 monoalkylaminosulfonyl, C2-6 dialkylaminosulfonyl, sulfamoyl, halogeno, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group, a 5- to 14-membered aromatic heterocyclic group, a heterocyclic aminocarbonyl group, a heterocyclic aminosulfonyl group and isoxazolinyl, wherein the 5- to 14-membered non-aromatic heterocyclic group, the C6-14 aromatic hydrocarbon ring group, the 5- to 14-membered aromatic heterocyclic group and isoxazolinyl may be independently substituted with at least one group selected from the group consisting of C1-6 alkyl, oxo, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, nitro, amino, C1-6 aminoalkyl, C1-6 alkylamino, C1-6 dialkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, alkylsulfonylamino, alkylsulfonyl, sulfamoyl, halogeno and C3-8 cycloalkyl; <Substituent Group C> The group consisting of (1) cyano, (2) halogen and (3) C1-6 alkyl, C2-8 alkenyl, C2-8 alkynyl, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, sulfonylamino, sulfonyl, sulfamoyl, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and 5- to 14-membered aromatic heterocyclic group (each of the foregoing members being optionally substituted with at least one group selected from Substituent Group C′ below); <Substituent Group C′> The group consisting of C1-6 alkyl, C2-8 alkenyl, C2-8 alkynyl, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, sulfonylamino, sulfonyl, sulfamoyl, halogeno, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group; <Substituent Group E> The group consisting of C1-6 alkyl, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, sulfonylamino, sulfonyl, sulfamoyl, halogen and C3-8 cycloalkyl; <Substituent Group F> The group consisting of (1) hydrogen, (2) cyano, (3) halogen, (4) oxo and (5) C1-6 alkyl, alkenyl, alkynyl, acyl, C1-6 alkanoyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, amino, imino, C1-6 aminoalkyl, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, sulfonylamino, sulfonyl, sulfamoyl, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group (each of the foregoing members being optionally substituted with at least one group selected from Substituent Group F′ below); <Substituent Group F′> The group consisting of C1-6 alkyl, oxo, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, benzyloxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, C1-6 alkylsulfonylamino, C1-6 alkylsulfonyl, sulfamoyl, halogeno, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group; <Substituent Group G> The group consisting of C1-6 alkyl, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, sulfonyl, sulfamoyl, halogeno and C3-8 cycloalkyl.} or a salt thereof. 2. A compound according to claim 1 or a salt thereof, wherein ring B represents a further optionally substituted benzene ring or pyridine ring; R101, R102 and R103 are the same or different and each represents a group selected from the group consisting of hydrogen, halogen, C1-6 alkyl, C1-6 alkylaminocarbonyl, C1-6 alkoxy, C1-6 alkylamino and C3-8 cycloalkyl; R5 represents a group selected from the group consisting of hydrogen, C1-6 alkyl and C1-6 alkoxy-C1-6 alkyl; R6 represents a group selected from the group consisting of hydrogen, C1-6 alkyl and C1-6 alkyloxycarbonyl optionally substituted with acyloxy; Y1 represents a single bond or —(CH2)m— [wherein m represents an integer of 1 to 3]; Y2 represents a single bond or —CO—; and Ar represents hydrogen or a group represented by the formula: [wherein R10, R11, R12, R13 and R14 are the same or different and each represents a group selected from the group consisting of hydrogen, C1-6 alkyl, hydroxyl, C1-6 alkoxy, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, a 5- to 14-membered non-aromatic heterocyclic group and C1-6 alkyloxycarbonyloxy, and R11 and R12 or R12 and R13 may bond together to form a 5- to 8-membered heterocyclic ring (i) optionally having 1 to 4 hetero atoms selected from N, S and O and (ii) optionally substituted with at least one group selected from the group consisting of cyano, oxo, and C1-6 alkyl, acyl, C1-6 alkanoyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, sulfonyl and a 5- to 14-membered non-aromatic heterocyclic group (each of the foregoing member being optionally substituted with at least one group selected from Substituent Group F″ below)< <Substituent Group F″> The group consisting of C1-6 alkyl, oxo, cyano, acyl, carboxyl and C1-6 alkoxy). 3. A compound according to claim 1 or a salt thereof, wherein ring B is an optionally substituted benzene ring. 4. A compound according to claim 1 or a salt thereof, wherein Y1 is —CH2—. 5. A compound according to claim 1 or a salt thereof, wherein Y2 is —CO—. 6. A compound according to claim 1 or a salt thereof, wherein Y1 is —CH2— and Y2 is —CO—. 7. A compound according to claim 1 or a salt thereof, wherein Y1 is a single bond, Y2 is a single bond and Ar is hydrogen. 8. A compound according to claim 1 or a salt thereof, wherein Ar is a group represented by the formula: [wherein R10, R11, R12, R3 and R14 are as defined above]. 9. A compound according to claim 8 or a salt thereof, wherein R10 and R14 are hydrogen. 10. A compound according to claim 1 or a salt thereof, wherein Ar is (1) a group represented by the formula: [wherein R10, R1 R12 R13 and R14 are as defined above] or (2) a 5- to 14-membered aromatic heterocyclic group optionally substituted with at least one group selected from Substituent Group G above. 11. A compound according to claim 10 or a salt thereof, wherein R10 and R14 are hydrogen. 12. A compound according to claim 1 or a salt thereof, wherein Ar is a group represented by the formula: [wherein R11 and R13 are as defined above, R15 represents (1) hydrogen or (2) a group selected from Substituent Group H below, and R11 and R15 may bond together to form a 5- to 8-membered heterocycle optionally substituted with at least one group selected from Substituent Group F above and optionally having 1 or 2 hetero atoms selected from N, S and O]. <Substituent Group H> The group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, acyl, C1-6 alkoxycarbonyl, aminocarbonyl, C1-6 alkylaminocarbonyl, C3-8 cycloalkyl, C1-6 aminoalkyl, sulfonyl, C3-8 cycloalkylamino, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group, each of the foregoing members being optionally substituted with at least one group selected from Substituent Group H′ below; <Substituent Group H′> The group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, oxo, cyano, C1-6 cyanoalkyl, C2-7 acyl, C1-6 alkanoyl, benzoyl, aralkanoyl, C1-6 alkoxyalkylcarbonyl, C1-6 hydroxyalkylcarbonyl, carboxyl, C1-6 carboxyalkyl, C1-6 carboxyalkyloxy, carbamoyl, carbamoylalkyloxy, C1-6 alkoxycarbonyl, C1-10 alkoxycarbonyl-C1-6 alkyl, C1-10 alkoxycarbonyl-C1-6 alkyloxy, C1-6 monoalkylaminocarbonyl, C2-6 dialkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C1-10 alkoxyalkyl, C1-10 aralkyloxyalkyl, C1-6 hydroxyalkyl, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, C1-6 alkylsulfonylamino, phenylsulfonylamino, C1-6 alkylsulfonyl, phenylsulfonyl, C1-6 monoalkylaminosulfonyl, C2-6 dialkylaminosulfonyl, sulfamoyl, halogeno, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group, a 5- to 14-membered aromatic heterocyclic group, a heterocyclic aminocarbonyl group, a heterocyclic aminosulfonyl group and isoxazolinyl, wherein the 5- to 14-membered non-aromatic heterocyclic group, the C6-14 aromatic hydrocarbon ring group, the 5- to 14-membered aromatic heterocyclic group and isoxazolinyl may be independently substituted with at least one group selected from the group consisting of C1-6 alkyl, oxo, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, nitro, amino, C1-6 aminoalkyl, C1-6 alkylamino, C1-6 dialkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, alkylsulfonylamino, alkylsulfonyl, sulfamoyl, halogeno and C3-8 cycloalkyl). 13. A compound according to claim 1 or a salt thereof, wherein Ar is a group represented by the formula: [wherein R11 and R15 are as defined above, and R16 represents (1) hydrogen or (2) a group selected from Substituent Group H above, and R11 and R15 or R15 and R16 may bond together to form a 5- to 6-membered heterocycle optionally substituted with at least one group selected from Substituent Group F above and also optionally having 1 or 2 hetero atoms selected from N, S and O]. 14. A compound according to claim 1 or a salt thereof, wherein Ar is a group represented by the formula: (wherein R11 and R15 are as defined above, and R17 and R18 are the same or different and each represents (1) hydrogen or (2) a group selected from Substituent Group I below, and R11 and R15, R15 and R17, R15 and R18 or R17 and R18 may bond together to form a 5- to 8-membered heterocycle optionally substituted with at least one group selected from Substituent Group F above and also optionally having 1 or 2 hetero atoms selected from N, S and O. <Substituent Group I> The group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, acyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, C16 aminoalkyl, sulfonyl, sulfamoyl, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group and a 5- to 14-membered aromatic heterocyclic group, each of the foregoing members being optionally substituted with at least one group selected from Substituent Group I′ below; <Substituent Group I′> The group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, oxo, cyano, C1-6 cyanoalkyl, C2-7 acyl, C1-6 alkanoyl, benzoyl, aralkanoyl, C1-6 alkoxyalkylcarbonyl, C1-6 hydroxyalkylcarbonyl, carboxyl, C1-6 carboxyalkyl, C1-6 carboxyalkyloxy, carbamoyl, carbamoylalkyloxy, C1-6 alkoxycarbonyl, C1-10 alkoxycarbonyl-C1-6 alkyl, C1-10 alkoxycarbonyl-C1-6 alkyloxy, C1-6 monoalkylaminocarbonyl, C2-6 dialkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C1-10 alkoxyalkyl, Cβ1-o aralkyloxyalkyl, C1-6 hydroxyalkyl, C3-8 cycloalkyloxy, amino, C1-6 alkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, C1-6 alkylsulfonylamino, phenylsulfonylamino, C1-6 alkylsulfonyl, phenylsulfonyl, C1-6 monoalkylaminosulfonyl, C2-6 dialkylaminosulfonyl, sulfamoyl, halogeno, C3-8 cycloalkyl, a 5- to 14-membered non-aromatic heterocyclic group, a C6-14 aromatic hydrocarbon ring group, a 5- to 14-membered aromatic heterocyclic group, a heterocyclic aminocarbonyl group, a heterocyclic aminosulfonyl group and isoxazolinyl, wherein the 5- to 14-membered non-aromatic heterocyclic group, the C6-14 aromatic hydrocarbon ring group, the 5- to 14-membered aromatic heterocyclic group and isoxazolinyl may be independently substituted with at least one group selected from the group consisting of C1-6 alkyl, oxo, cyano, acyl, carboxyl, carbamoyl, C1-6 alkoxycarbonyl, C1-6 alkylaminocarbonyl, hydroxyl, C1-6 alkoxy, C3-8 cycloalkyloxy, nitro, amino, C16 aminoalkyl, C1-6 alkylamino, C1-6 dialkylamino, C3-8 cycloalkylamino, acylamino, ureido, ureylene, alkylsulfonylamino, alkylsulfonyl, sulfamoyl, halogeno and C3-8 cycloalkyl]. 15. A compound according to claim 1 or a salt thereof, wherein the compound is represented by the formula: [wherein the definitions of the symbols are the same as given above]. 16. A compound according to claim 1 or a salt thereof, wherein the compound is represented by the formula: [wherein R104 and R105 are the same or different and each represents hydrogen, C1-6 alkoxy, C1-6 alkyl or halogen, and R101, R102, R5, R6, Y1, Y2 and Ar are as defined above]. 17. A compound according to claim 1 or a salt thereof, wherein the compound is represented by the formula: [wherein U represents N or CH, V represents N or CR101, W represents N or CR102, Z represents N or CR105, one or two from U, V, W and Z are N, and R101, R102, R105, R5, R6, Y1, Y2 and Ar are as defined above]. 18. A compound according to claim 16 or 17 or a salt thereof, wherein Y1 is —CH2—. 19. A compound according to claim 16 or 17 or a salt thereof, wherein Y2 is —CO—. 20. A compound according to claim 17 or a salt thereof, wherein U is N and V is CR101 [where R101 are as defined above]. 21. A pharmaceutical composition comprising a compound according to claim 1 or a salt thereof. 22. A composition according to claim 21, wherein the composition is a thrombin receptor antagonist. 23. A composition according to claim 21, wherein the composition is a thrombin receptor PAR1 antagonist. 24. A composition according to claim 21, wherein the composition is a platelet aggregation inhibitor. 25. A composition according to claim 21, wherein the composition is a proliferation inhibitor for smooth muscle cells. 26. A composition according to claim 21, wherein the composition is a proliferation inhibitor for endothelial cells, fibroblasts, nephrocytes, osteosarcoma cells, muscle cells, cancer cells and/or glia cells. 27. A composition according to claim 21, wherein the composition is a therapeutic or preventive agent for thrombosis, vascular restenosis, deep venous thrombosis, pulmonary embolism, cerebral infarction, heart disease, disseminated intravascular coagulation, hypertension, inflammatory disease, rheumatism, asthma, glomerulonephritis, osteoporosis, neurological disease and/or malignant tumor. 28. Use of a compound according to claim 1 or a salt thereof for the manufacture of a thrombin receptor antagonist. 29. Use according to claim 28, wherein the thrombin receptor antagonist is a PAR1 receptor antagonist. 30. Use of a compound according to claim 1 or a salt thereof for the manufacture of a platelet aggregation inhibitor. 31. A method of treating a thrombin receptor mediated disease in a patient suffering from the disease, comprising administering to the patient, a therapeutically effective amount of a compound according to claim 1 or a salt thereof. 32. A method for treating a proliferative disease of endothelial cells, fibroblasts, nephrocytes, osteosarcoma cells, muscle cells, cancer cells and/or glia cells, in a patient suffering from the disease, comprising administering to the patient, a therapeutically effective amount of a compound according to claim 1 or a salt thereof. |
<SOH> BACKGROUND ART <EOH>A recent approach for thrombosis have involved inhibiting thrombin enzyme activity, and compounds used for this purpose have included heparin, low molecular weight heparin, hirudin, argatroban, hirulog and the like. All such compounds inhibit the enzyme activity of thrombin, and work by inhibiting fibrin blood clot formation without specifically inhibiting the effect of thrombin on cells. Bleeding tendency is therefore a common side effect encountered in the clinic. The role of thrombin in thrombosis is not limited to its blood clotting activity, as it is believed to also participate in platelet aggregation at sites of vascular injury occurring as a result of the activation of platelet thrombin receptor. Another approach for thrombosis has been the use of intravenous injection agents such as Abciximab, Eptifibatide and Tirofiban, as GPIIb/IIIa receptor antagonists. These compounds, while exhibiting powerful anti-thrombotic effects by suppressing platelet aggregation induced by various stimulation such as thrombin, ADP, collagen, PAF or the like, also produce a bleeding tendency as a side effect similarly to thrombin enzyme activity inhibitors. For this reason, no such compounds have yet been marketed, although their development as oral agents continues to progress. Restenosis is a vascular hypertrophic response to vascular wall injury induced by invasive treatment such as coronary angioplasty, and this phenomenon may be provoked by the direct or indirect effect of thrombin on cells. Platelets adhere to injured blood vessels, leading to release of growth factors and eliciting proliferation of smooth muscle cells. Smooth muscle cells may also be affected indirectly by the action of thrombin on endothelial cells. Also, platelet adhesion occurs and procoagulant activity increases at sites of vascular injury. Smooth muscle cells can undergo further direct stimulation due to the high local thrombin concentration which is produced at such sites. While recent studies using the powerful thrombin inhibitor hirudin have suggested that thrombin induces cell proliferation during the process of restenosis, it has not yet been determined whether the thrombin effect is direct or indirect (Sarembock et al., Circulation 1992, 84:232-243). Despite the implication of the cellular effects of thrombin in a variety of pathological symptoms, no therapeutically active substance is known which specifically blocks such effects. The thrombin receptor (PAR1) has recently been cloned (Vu et al., Cell, 1991, 64:1057-1068), opening an important door to development of substances which target cellular thrombin receptors. Detailed examination of the amino acid sequence of this thrombin receptor has revealed a thrombin binding site and hydrolysis site located in the 100 residue amino terminal domain of the receptor. Later research by amino acid mutation in the receptor has established that limited hydrolysis of this portion of the thrombin receptor by thrombin is necessary for receptor activation (Vu et al., Nature, 1991, 353:674-677). A synthetic peptide corresponding to the amino acid sequence newly generated by hydrolysis of the thrombin receptor (the synthetic peptide is known as “thrombin receptor activating peptide”, or TRAP) can activate receptors which have not been hydrolyzed by thrombin. This suggests that upon the cleavage of the receptor, the new amino acid sequence generated at the amino terminal (known as the “tethered ligand peptide”) functions as the ligand and interacts with the distal binding site. Further studies of TRAP have confirmed homology of the thrombin receptors present in platelet, endothelial cell, fibroblast and smooth muscle cell (Hung et al., J. Cell. Biol. 1992, 116:827-832, Ngaiza, Jaffe, Biochem. Biophys. Res. Commun. 1991, 179:1656-1661). Research on the structure activity relationship of TRAP suggests that the pentapeptide Phe-Leu-Leu-Arg-Asn is a weak antagonist for platelet thrombin receptors activated by either thrombin or TRAP (Vassallo. et al., J. Biol. Chem.,1992, 267:6081-6085(1992)). Different approaches to receptor antagonism have also been examined by other groups. One of these approaches has been an attempt to prepare antibodies for the thrombin binding domain of the thrombin receptor. Such antibodies specifically and effectively suppress activation of platelets by thrombin, and act as thrombin receptor antagonists (Hung et al., J. Clin. Invest. 1992, 89:1350-1353). Another approach has been development of peptide derivatives from TRAP (Steven M. S., J. Med. Chem. 1996, 39:4879-4887; William J. H., Bioorg. Med. Chem. Lett. 1998, 8:1649-1654; David F. M., Bioorg. Med. Chem. Lett. 1999, 9:255-260). Yet another has been development of low molecular weight compounds discovered by high throughput screening using various assay systems such as receptor binding (Andrew W. S. et al., Bioorg. Med Chem. Lett. 1999, 9:2073-2078; Scherig Plough WO99/26943; Halord S. et al., ACS meeting in October 2001). |
Belt making apparatus and method |
An apparatus and method for making cable reinforced belts or bands and includes cable supply unit for supplying a plurality of reinforcing cables (12), clamping unit for clamping cables under predetermined tension in each of hte reinforcing cables (12). Each tensioning stand (16) includes tensioning units (32) with a tension sensor for monitoring tension in an assocaited cable (12), an actuator (66) for applying a force to the cable (12) to produce therein a predetermined tension and an enclosed feedback controller for the force controlling actuator (66) which controls as a function of the signal generated by a load cell (100) connected to the actuator (66). A cable hold back unit is upstream the tensioning stand (16). Each tensioning unit (32) includes a pair of spaced apart cable sheaves (56) having rotational axes fixed with respect to the respect to the tensioning unit (32). |
1. Apparatus for making cable reinforced belts, comprising: a) a cable supply unit for supplying a plurality of reinforcing cables; b) a clamping unit for clamping said reinforcing cables under predetermined operating conditions; c) at least one cable tensioning stand for maintaining a predetermined tension in each of said cables, said tensioning stand comprising a plurality of tensioning units, each tensioning unit including; i) a tension sensor for monitoring tension in an associated cable; ii) an actuator for applying a force to the associated cable in order to produce a predetermined tension in said cable; iii) an actuator controller for controlling the force applied by said actuator as a function of signal generated by said tension sensor. 2. The apparatus of claim 1, wherein said tension sensor is a load cell operatively connected to said actuator. 3. The apparatus of claim 2, wherein said controller includes a proportional valve for maintaining a predetermined fluid pressure in said actuator such that said actuator is controlled to produce a substantially constant tension in said associated cable. 4. The apparatus of claim 1 further including a cable hold back unit, located upstream of said tensioning stand which is operative to control tension in said reinforcing cables during advancement of said cables. 5. The apparatus of the claim 4, wherein said cable hold back unit includes a driven roll extending transversely to a direction of movement of said reinforcing cables and an idler roll extending parallel to said driven roll and in a confronting relationship therewith, said idler roll moveable towards and away from said driven roll. 6. The apparatus of claim 1 wherein said tensioning unit includes a pair of spaced apart cable sheaves having rotational axes fixed with respect to said tensioning unit and a moveable sheave operatively coupled to said actuator and moveable with respect to said fixed sheaves. 7. A method for making a cable reinforced belt, comprising the steps of: a) providing a plurality of reinforcing cables to form part of said belt; b) maintaining a predetermined tension on each of said cables by: i) applying a force to each cable by means of an associated fluid pressure operated actuator; ii) monitoring a load on said actuator as a result of applying forces to said associated cable; iii) generating a signal that is a function of the load on said actuator; iv) using said signal to calculate an actual tension in said associated cable; v) comparing said calculated tension with a tension data set point; vi) generating an error signal, if said tension data set point and calculated tension are not equal; and, vii) using said error signal to generate a signal for a proportional valve that is operative to control fluid pressure applied to said actuator in order to adjust the force applied to said associated cable until a desired tension is obtained. 8. The method of claim 7, further including the step of utilizing a cable hold back unit having a driven roll and an idler roll and adjusting a spacing between said rolls in order to control tension in said cables during an advancing step. 9. The method of claim 7, further including the step of providing a pair of spaced apart sheaves that are fixed with respect to said actuator and providing a movable sheave operatively connected to said actuator such that relative movement between said fixed sheaves and said movable sheave applies forces to said associated cable. 10. A cable tensioning stand for use in a cable reinforced belt making apparatus, comprising: a) a frame; b) a plurality of tensioning units mounted within said frame; c) each tensioning unit including: i) an actuator operatively connected to a moveable sheave; ii) a pair of spaced apart sheaves located on either side of a path of movement for said movable sheave; iii) a load cell for monitoring a force exerted by said actuator on an associated cable; iv) a controller responsive to signals produced by said load cell and operative to compare said monitored force with a desired force; and, v) said controller generating a signal for a proportional valve that is a function of the difference between said monitored force and said desired force; vi) said proportional valve responsive to said generated signal and operative to control fluid pressure applied to said actuator in order to control the force applied by said actuator to its associated cable. 11. The tension stand of claim 10, including a manifold for feeding pressurized fluid to all of said actuators in order to move said movable sheave to a cable loading position. |
<SOH> BACKGROUND ART <EOH>Cable reinforced belts are used in many applications that require goods, people or other material to be moved from one location to another. These types of belts are generally made using a vulcanizing process in which elastomeric material is bonded to reinforcing cables which may comprise steel wire. There currently exists, machinery for manufacturing these types of belts. In general, these belts are generally formed in successive sections as the belt is intermittently advanced through the processing line. For many applications, the belt must be of very high quality so that it has a very long service life. In general, the manufacture and replacement of these types of conveyor belts can be very costly. It has been found that premature failure of these types of belts can arise due to relative movement of the reinforcing cables within the vulcanized material. The failure producing movement in the reinforcing cables can sometimes be traced to the manufacturing process. In particular, if the tension in the reinforcing cables is not carefully controlled during the belt making process, premature failure in the belt due to movement in the reinforcing cables relative to the belt material, can occur. There currently exists equipment for tensioning the reinforcing cables during the belt manufacturing process. An example of prior art cable tensioning apparatus is illustrated in U.S. Pat. No. 3,502,535. It has been found however there exists a need for an apparatus capable of producing higher quality belts and bands than can be produced with existing equipment. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a side elevational view, shown somewhat schematically, of an apparatus for making reinforced belts or bands such as conveyor belts; FIG. 2 is a side elevational view of a portion of the apparatus shown in FIG. 1 , showing a hold back device and tensioning devices, constructed in accordance with the preferred embodiment of the invention; FIG. 3 is a side view of one of the tensioning devices shown in FIG. 2 as seen from the plane indicated by the line 3 - 3 in FIG. 2 ; FIG. 4 is a side elevational view of a tensioning unit forming part of the tensioning device as seen from the plane indicated by the line 4 - 4 in FIG. 3 ; FIG. 5 is a fragmentary view of the tensioning unit shown in FIG. 4 as seen from the plane indicated by the line 5 - 5 in FIG. 3 ; FIG. 6 is a fragmentary view of a portion of the tensioning device, indicated by the circle marked “ FIG. 6 ” in FIG. 3 ; FIG. 7 is a side view of a cable hold back device constructed in accordance with the preferred embodiment of the invention; FIG. 8 is a schematic representation of a control arrangement for controlling an actuator forming part of the tensioning unit shown in FIG. 4 ; and, FIG. 9 is a flow chart illustrating the modes of operation and functions that are controlled by a controller during the making of a belt. detailed-description description="Detailed Description" end="lead"? |
Pulse oximetry device and method |
In one embodiment, an anal pulse oximeter device is characterized by an anal canal surface; and a rectal-vault cuff having a leading portion and a trailing portion, the trailing portion substantially proximate to said anal canal surface. In another embodiment, a method of using a pulse oximeter device is characterized by positioning an insertion end of the pulse oximeter device against an anus; advancing the pulse oximeter device until a rectal-vault cuff of the pulse oximeter device substantially clears an internal anal sphincter; and withdrawing the pulse oximeter device until the rectal-vault cuff of the pulse oximeter device contacts a rectal vault. In another embodiment, a method of manufacturing a pulse oximeter device is characterized by attaching a rectal-vault cuff, the rectal-vault cuff having a leading portion and a trailing portion, such that the trailing portion is substantially proximate to an anal canal surface. In various embodiments the anal canal surface is an anatomical anal canal surface, while in other embodiments the anal canal surface is a surgical anal canal surface. |
1. An anal pulse oximeter device comprising: an anal canal surface; and a rectal-vault cuff having a leading portion and a trailing portion, the trailing portion substantially proximate to said anal canal surface. 2. The anal pulse oximeter device of claim 1, wherein said anal canal surface comprises: an anatomical anal canal surface; or a surgical anal canal surface. 3. The anal pulse oximeter device of claim 1, wherein said anal canal surface comprises: a bowel-evacuation channel such that a patient's bowel may be evacuated. 4. The anal pulse oximeter device of claim 1, wherein said anal canal surface comprises: a cylindrically-shaped surface. 5. The anal pulse oximeter device of claim 1, wherein said anal canal surface comprises: said anal canal surface formed from a medical-grade polymeric material. 6. The anal pulse oximeter device of claim 1, wherein said anal canal surface comprises: either a light source or a light sensor substantially integral with said anal canal surface. 7. The anal pulse oximeter device of claim 6, wherein said light source or said light sensor substantially integral with said anal canal surface comprises: a pulse oximetry adapter/plug operably coupled with either said light source or said light sensor. 8. The anal pulse oximeter device of claim 6, wherein said light source or said light sensor substantially integral with said anal canal surface comprises: said light source or said light sensor either at, or movable to, a substantially non-zero angle relative to said anal canal surface. 9. The anal pulse oximeter device of claim 8, wherein said light source or said light sensor either at, or movable to, a substantially non-zero angle relative to said anal canal surface comprises: a member adapted to hold either said light source or said light sensor at the substantially non-zero angle relative to said anal canal surface. 10. The anal pulse oximeter device of claim 9, wherein said member adapted to hold either said light source or said light sensor at the substantially non-zero angle relative to said anal canal surface comprises: a member adapted to flex sufficient to hold either said light source or said light sensor at the substantially non-zero angle relative to said anal canal surface. 11. The anal pulse oximeter device of claim 9, wherein said member adapted to hold either said light source or said light sensor at the substantially non-zero angle relative to said anal canal surface comprises: a member having curvature sufficient to hold either said light source or said light sensor at the substantially non-zero angle relative to said anal canal surface. 12. The anal pulse oximeter device of claim 8, wherein said light source or said light sensor either at, or movable to, a substantially non-zero angle relative to said anal canal surface comprises: an external cuff adapted to hold either said light source or said light sensor at the substantially non-zero angle relative to said anal canal surface. 13. The anal pulse oximeter device of claim 1, wherein said anal canal surface comprises: a physiological sensor substantially integral with said anal canal surface. 14. The anal pulse oximeter device of claim 13, wherein said physiological sensor substantially integral with said anal canal surface comprises: a physiological sensor adapter/plug operably coupled with said physiological sensor. 15. The anal pulse oximeter device of claim 1, wherein said rectal-vault cuff comprises: an inflatable cuff. 16. The anal pulse oximeter device of claim 1, further comprising: an external cuff having a leading portion and a trailing portion, the leading portion substantially proximate to said anal canal surface. 17. The anal pulse oximeter device of claim 16, wherein said external cuff comprises: an inflatable cuff. 18. The anal pulse oximeter device of claim 1, comprising: rounded insertion end. 19. A method of using a pulse oximeter device comprising: positioning an insertion end of the pulse oximeter device against an anus; advancing the pulse oximeter device until a rectal-vault cuff of the pulse oximeter device substantially clears an internal anal sphincter; and withdrawing the pulse oximeter device until the rectal-vault cuff of the pulse oximeter device contacts a rectal vault. 20. The method of claim 19, wherein said advancing the pulse oximeter device until a rectal-vault cuff of the pulse oximeter device substantially clears an internal anal sphincter comprises: digitally verifying that the rectal-vault cuff of the pulse oximeter device has substantially cleared the patient's internal anal sphincter. 21. The method of claim 19, wherein said advancing the pulse oximeter device until a rectal-vault cuff of the pulse oximeter device substantially clears an internal anal sphincter comprises: advancing the pulse oximeter device until an external anus marking is substantially proximate to the anus. 22. The method of claim 19, wherein said withdrawing the pulse oximeter device until the rectal-vault cuff of the pulse oximeter device contacts a rectal vault comprises: withdrawing the pulse oximeter device until an increase in resistance is encountered. 23. The method of claim 19, wherein said withdrawing the pulse oximeter device until the rectal-vault cuff of the pulse oximeter device contacts a rectal vault comprises: withdrawing the pulse oximeter device until a patient reports an anal sensation of contact. 24. The method of claim 19, further comprising: detecting at least one physiological metric selected from the physiological-metric group including a pulse oximetry metric, a temperature metric, and a manometer metric. 25. The method of claim 19, further comprising: evacuating a bowel via a bowel-evacuation channel of the pulse oximeter device. 26. A method of manufacturing a pulse oximeter device comprising: attaching a rectal-vault cuff, the rectal-vault cuff having a leading portion and a trailing portion, such that the trailing portion is substantially proximate to an anal canal surface. 27. The method of claim 26, wherein the anal canal surface comprises: an anatomical anal canal surface; or a surgical anal canal surface. 28. The method of claim 26, wherein the anal canal surface comprises: a bowel-evacuation channel such that a patient's bowel may be evacuated. 29. The method of claim 26, wherein the anal canal surface comprises: a cylindrically-shaped surface. 30. The method of claim 26, wherein the anal canal surface comprises: the anal canal surface formed from a medical-grade polymeric material. 31. The method of claim 26, wherein the anal canal surface comprises: either a light source or a light sensor substantially integral with the anal canal surface. 32. The method of claim 31, wherein said light source or said light sensor substantially integral with said anal canal surface comprises: a pulse oximeter adapter/plug operably coupled with said light source or said light sensor. 33. The method of claim 31, wherein said light source or said light sensor substantially integral with said anal canal surface comprises: a member adapted to hold either said light source or said light sensor at a substantially non-zero angle relative to said anal canal surface. 34. The method of claim 31, wherein said light source or said light sensor substantially integral with said anal canal surface comprises: a member adapted to flex sufficient to hold either said light source or said light sensor at a substantially non-zero angle relative to said anal canal surface. 35. The method of claim 31, wherein said light source or said light sensor substantially integral with said anal canal surface comprises: a member having curvature sufficient to hold either said light source or said light sensor at a substantially non-zero angle relative to said anal canal surface. 36. The method of claim 31, wherein said light source or said light sensor substantially integral with said anal canal surface comprises: an external cuff adapted to hold either said light source or said light sensor at a substantially non-zero angle relative to said anal canal surface. 37. The method of claim 26, wherein said anal canal surface comprises: a physiological sensor substantially integral with said anal canal surface. 38. The method of claim 37, wherein said physiological sensor substantially integral with said anal canal surface comprises: a physiological sensor adapter/plug operably coupled with said physiological sensor. 39. The method of claim 26, further comprising: attaching an external cuff, the external cuff having a leading portion and a trailing portion, such that the leading portion is substantially proximate to said anal canal surface. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates, in general, to pulse oximetry devices and methods. 2. Description of the Related Art Pulse oximetry refers to the process of inferring the oxygen-hemoglobin saturation of a patient's blood via use of a photoelectric oximeter. It has long been known in the art how to correlate the reflectance or transmittance of certain wavelengths of light (e.g., light having wavelengths which constitute visible red light and/or light having wavelengths that constitute infrared light) with the oxygen content (or oxygen saturation) of pulsing blood. Consequently, in one type of pulse oximetry, known in the art as reflectance pulse oximetry, a light source, such as a light emitting diode (LED) and a light sensor, such as a photosensor (e.g., a photodiode), are positioned to one side of a portion a patient's circulatory system. Thereafter, the LED is activated, and the photosensor is monitored to collect data on light reflected from the portion of the patient's circulatory system. Using algorithms well known to those within the art, the amount of oxygen saturation of the patient's blood is then inferred based on the measured reflectance data. For example, in one implementation of reflectance pulse oximetry, both the LED and the photosensor are positioned against a patient's tympanic membrane (i.e., in the ear), and measured reflected light is used to infer the oxygen saturation of the patient. In another type of pulse oximetry, known in the art as transmittal pulse oximetry, a light emitting diode (LED) and a photosensor (e.g., a photodiode) are positioned on either side of a portion a patient's circulatory system. Thereafter, the LED is activated, and the photosensor is monitored to collect data on light transmitted through the portion of the patient's circulatory system. Using algorithms well known to those within the art, the amount of oxygen saturation of the patient's blood is then inferred based on the measured transmittance data. For example, in one well-known implementation of transmittal pulse oximetry, the LED and the photosensor are positioned on either side of a patient's finger via use of a finger clip, and measured transmitted light is used to infer the oxygen saturation of the patient. |
<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>The inventor named herein has devised a pulse oximetry device and method. In one embodiment, an anal pulse oximeter device is characterized by an anal canal surface; and a rectal-vault cuff having a leading portion and a trailing portion, the trailing portion substantially proximate to said anal canal surface. In another embodiment, a method of using a pulse oximeter device is characterized by positioning an insertion end of the pulse oximeter device against an anus; advancing the pulse oximeter device until a rectal-vault cuff of the pulse oximeter device substantially clears an internal anal sphincter; and withdrawing the pulse oximeter device until the rectal-vault cuff of the pulse oximeter device contacts a rectal vault. In another embodiment, a method of manufacturing a pulse oximeter device is characterized by attaching a rectal-vault cuff, the rectal-vault cuff having a leading portion and a trailing portion, such that the trailing portion is substantially proximate to an anal canal surface. In various embodiments the anal canal surface is an anatomical anal canal surface, while in other embodiments the anal canal surface is a surgical anal canal surface. The foregoing is a summary and thus contains, by necessity, simplifications, generalizations and omissions of detail; consequently, those skilled in the art will appreciate that the summary is illustrative only and is NOT intended to be in any way limiting. Other aspects, inventive features, and advantages of the devices and/or processes described herein, as defined solely by the claims, will become apparent in the non-limiting detailed description set forth herein. |
Compositions and methods for the identification of protein interactions in vertebrate cells |
The present invention relates to a two-hybrid system for studying protein-protein interactions in mammalian host cells. The invention provides methods, reagents and kits for carrying out the two-hybrid screen. Accordingly the invention provides a bait construct that is capable of targeting a bait protein to a specific subcellular locale within the host cell. The invention also provides a prey construct that contains a detection sequence fused to a prey protein. The bait and prey constructs are introduced into a host cell under conditions that promote expression of the bait and prey constructs. A positive bait/prey interaction can be detected by comparing the subcellular localization of the prey construct in relation to the bait construct. The invention also provides methods for screening for compounds capable of disrupting protein-protein interaction and methods for detecting interactions between proteins and small molecule compounds. |
1. A method for detecting protein interactions in a host cell, comprising: (ii) providing a host cell including: (a) a first nucleic acid coding sequence encoding a bait fusion protein comprising a bait polypeptide sequence fused to a targeting domain that targets the bait fusion protein to a subcellular location within the host cell; (b) a second nucleic acid coding sequence encoding a prey fusion protein comprising a prey polypeptide sequence fused to one or more detection domains that can be detected in a transcriptionally-independent manner in the host cell to determine if the prey fusion protein is associated in a complex with the bait fusion protein or not; (ii) detecting the subcellular location of the prey protein within the host cell; wherein accumulation of the prey fusion at the same subcellular pattern as the bait fusion indicates that the prey fusion protein is associated in a complex with the bait fusion protein. 2. A kit for detecting protein interactions, comprising: (iii) a first expression construct including a coding sequence for a targeting domain and a ligation site flanking an end of the targeting domain coding sequence for ligating a coding sequence of a bait polypeptide sequence in frame with said targeting domain coding sequence to produce a bait fusion protein, said first expression construct operably linked to a transcriptional regulatory element; and (iv) a second expression construct including a coding sequence for a detection domain and a ligation site flanking an end of the detection domain coding sequence for ligating a coding sequence of a prey polypeptide sequence in frame with said detection domain coding sequence to produce a prey fusion protein, said second expression construct operably linked to a transcriptional regulatory element, wherein, the targeting domain localizes the bait fusion protein to a subcellular location within a host cell, and the detection domain can be detected in a transcriptionally-independent manner in the host cell to determine if the prey fusion protein is associated in a complex with the bait fusion protein or not. 3. The method of claim 1 or kit of claim 2, wherein the targeting domain localizes the bait fusion protein to subcellular comparment or organelle selected from the group consisting of the nucleus, nucleoli, telomeres, kinetochores, nuclear envelope, chromosomes, chromatin, cytoplasm, endoplasmic reticulum, Golgi, centrosome, transgolgi network, cytoplasmic vesicles, mitochondria, secretory vesicles, lysosome, plasma membrane, intracellular membrane vesicles, nuclear membranes, synapses and basolaternal membranes. 4. The method of claim 1 or kit of claim 2, wherein the targeting domain localizes the bait fusion protein to a kinetochore structure. 5. The method or kit of claim 4, wherein the targeting domain includes an amino acid sequence form a protein selected from the group consisting of CENP-A, CENP-B, CENP-C, CENP-E, CENP-F, Bub1, Bub3, MAD3L and MAD2, or a homologous sequence thereto localizes the bait fusion protein to a kinetochore structure. 6. The method or kit of claim 5, wherein the targeting domain comprises at least amino acids 373-943 of the human CENP-C sequence or a sequence at least 80 percent identical thereto. 7. The method or kit of claim 4, wherein the targeting domain associates with the kinetochore structure with a dissociation constant (kd) of 1 mM or less. 8. The method of claim 1 or kit of claim 2, wherein the targeting domain localizes the bait fusion protein to the nuclear envelope. 9. The method or kit of claim 8, wherein the targeting domain comprises an amino acid sequence from a protein selected from the group consisting of lamin A, lamin B, lamin C, emerins and porins, or a portion thereof capable of targeting the bait fusion protein to the nuclear envelope. 10. The method of claim 1 or kit of claim 2, wherein the detection domain is a fluorescent polypeptide sequence or a luminescent polypeptide sequence. 11. The method or kit of claim 10, wherein the detection domain is all or a fluorescent portion of a green fluorescent protein sequence. 12. The method of claim 1 or kit of claim 2, wherein localization of the prey fusion proteins can be determined within 120 minutes of expression. 13. The method of claim 1 or kit of claim 2, wherein the prey fusion protein further includes an instability sequence which renders the prey fusion protein with a shorter intracellular half-life when not associated in complexes with the bait fusion protein relative to when it is. 14. The method or kit of claim 13, wherein the instability sequence comprises at least amino acids 249-323 of the human CENP-C sequence or a homologous sequence thereto. 15. The method of claim 1 or kit of claim 2, wherein either or both of the bait and prey fusion proteins includes a rescue sequence. 16. The method or kit of claim 15, wherein the rescue sequence is selected from the group consisting of His6 tag, myc tag, flu tag, lacZ, GST, Strep tag I and Step tag II. 17. The method of claim 1 or kit of claim 2, wherein the bait and/or prey fusion protein includes an oligomerization domain. 18. The method of claim 1 or kit of claim 2, wherein the coding sequences for said bait and prey fusion proteins are operably linked to the same transcriptional regulatory sequence. 19. The method of claim 1 or kit of claim 2, wherein the coding sequences for said bait and prey fusion proteins are operably linked to different transcriptional regulatory sequences. 20. The method of claim 1 or kit of claim 2, wherein the coding sequences for said bait and prey fusion proteins are provided on the same expression vector. 21. The method of claim 1 or kit of claim 2, wherein the coding sequences for said bait and prey fusion proteins are provided on different expression vectors. 22. The method of claim 1 or kit of claim 2, wherein at least one of the coding sequences are provided as part of an integrative vector. 23. The method of kit of claim 22, wherein the vector is a retroviral vector. 24. The method of claim 1 or kit of claim 2, wherein at least one of the coding sequences are provided as part of an episomal vector. 25. The method of claim 1 or kit of claim 2, wherein at least one of the coding sequences are provided as part of a vector which includes a recovery element. 26. The method of claim 1 or kit of claim 2, wherein the host cell is a mammalian cell. 27. The method or kit of claim 26, wherein the host cell is a human cell. 28. The method of claim 1, wherein the subcellular location of the prey protein is determined in the presence of a test agent contacted with the cell. 29. The method of claim 28, wherein the test agent is a small organic molecule. 30. The method of claim 28, carried out consecutively or simultaneously for a library of at least 100 different test agents. 31. The method of claim 28, wherein the test agent includes a portion which is predetermined to bind to one the bait or prey fusion protein, and a test portion which being tested for binding to the other fusion protein. 32. The method of claim 28, wherein the ability of the test compound to inhibit association of the prey fusion protein in a complex with the bait fusion protein is determined. 33. The method of claim 28, wherein the ability of the test compound to potentiate association of the prey fusion protein in a complex with the bait fusion protein is determined. 34. The method of claim 30, wherein the identity of test agents in the library which inhibit or potentiate association of the prey fusion protein in a complex with the bait fusion protein is determined. 35. The method of claim 31, carried out for a library of at least 100 different test agents having varied test portions amongst members of the library. 36. The method of claim 28, comprising the further step of formulating a pharmaceutical preparation including one or more compounds identified as inhibitors or potentiators of the association of the prey fusion protein in a complex with the bait fusion protein, or analogs thereof. 37. The method of claim 1, wherein the subcellular location of the prey protein is determined after induction of the host cell with an agent the causes post-translational modification of proteins in the host cell. 38. The method of claim 1, wherein the subcellular location of the prey protein is determined using flow cytometry analysis. 39. The method of claim 1, wherein the subcellular location of the prey protein is determined using microscopy. 40. A method for detecting protein interactions in a host cell, comprising: (i) providing a host cell culture, the cells of which include: (c) a first nucleic acid coding sequence encoding a bait fusion protein comprising a bait polypeptide sequence fused to a targeting domain that targets the bait fusion protein to a subcellular location within the host cell, and (d) a second nucleic acid coding sequence encoding a prey fusion protein comprising a prey polypeptide sequence fused to one or more detection domains that can be detected in a transcriptionally-independent manner in the host cell to determine if the prey fusion protein is associated in a complex with the bait fusion protein or not, wherein the culture is a variegated mixture of cells containing different prey polypeptide sequences and/or different bait polypeptide sequences; (ii) selecting cells from the culture in which the prey fusion protein is localized in the cell in the same subcellular pattern as the bait fusion protein; (iii) identifying the sequence of the bait and prey fusion proteins from the selected cells 41. The method of claim 40, wherein the culture includes at least 100 different bait and/or prey polypeptide sequences. 42. The method of claim 40, wherein only one of the bait or prey polypeptide sequences is variegated in the culture. 43. A method for conducting a drug discovery business, comprising: (vi) by the method of claim 1, identifying a protein complex for which an agent that inhibits or potentiates the formation or activity of the complex is desired; (vii) generating a drug screening assay for identifying agents that inhibit or potentiate the formation or activity of the complex; (viii) conducting animal toxicity profiles on a agent identified in step (ii), or an analog thereto; (ix) manufacturing a pharmaceutical preparation of an agent having a suitable animal toxicity profile; and (x) marketing the pharmaceutical preparation to healthcare providers. 44. A method for conducting a drug discovery business, comprising: (vi) by the method of claim 1, identifying a protein complex which is mediated by post-translational modification and for which an agent that inhibits or potentiates the post-translational modification is desired; (vii) generating a drug screening assay for identifying agents that inhibit or potentiate the post-translational modification and effect the formation of the protein complex; (viii) conducting animal toxicity profiles on a agent identified in step (ii), or an analog thereto; (ix) manufacturing a pharmaceutical preparation of an agent having a suitable animal toxicity profile; and (x) marketing the pharmaceutical preparation to healthcare providers. 45. A method for conducting a bioinformatics business, comprising: (iii) by the method of claim 1, identifying networks of protein complexes; (iv) generating a database including information identifying interactions of different proteins in a signal pathway and information identifying the proteins. 46. A system for analyzing protein complexes in cells, comprising a flow cytometer for analyzing cells and determining if a fluorescent signal is dispersed in a cell or localized to kinetochore structures. 47. The system of claim 46, including a microprocessor for comparing the flow spectra of cells and distinguishing between a diffuse pattern of fluorescence in the cells and a kinetochore-localized pattern. 48. A system for analyzing protein complexes in cells, comprising a microscope having a camera mounted therein for analyzing cells in a field of vision of the microscope, and a microprocessor for processing images obtained from said camera and determining if a fluorescent signal is dispersed in a cell or localized to kinetochore structures. 49. The system of claim 48, further comprising an cell picking robot which is controlled by said microprocessor and isolates cells which the microprocessor has determined have a fluorescent signal localized to kinetochore structures. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Protein-protein interactions are of paramount and fundamental interest in biological systems. These interactions are involved in a wide variety of important biological reactions, including the assembly of enzyme subunits, in antigen-antibody reactions, in supramolecular structures of ribosomes, filaments, and viruses, in recognition and transport, in transcription regulation, and in ligand-receptor interactions. In addition, the area of protein-protein interactions has received significant attention in the area of signal transduction and biochemical pathway analysis. Traditionally, protein-protein interactions were evaluated using biochemical techniques, including chemical cross-linking, co-immunoprecipitation and co-fractionation and -purification. Recently, genetic systems have been described to detect protein-protein interactions. The first work was done in yeast systems, and was termed the “yeast two-hybrid” system. The basic system requires a protein-protein interaction in order to turn on transcription of a reporter gene. Similar systems operating in bacteria and mammalian cells have also been developed. See Fields et al., Nature 340:245 (1989); Vasavada et al., PNAS USA 88:10686 (1991); Fearon et al., PNAS USA 89:7958 (1992); Dang et al., Mol. Cell. Biol. 11:954 (1991); Chien et al., PNAS USA 88:9578 (1991); and U.S. Pat. Nos. 5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463. (need patent for bacterial two hybrid if not here) However, while the yeast system works well, it is unsuitable for use in mammalian systems for a variety of reasons. Furthermore, the existing mammalian two-hybrid systems are neither suitable for a wide variety of cells, nor flexible, as they generally require quite highly specialized conditions. Finally, these systems tend to have high background signals from non-specific interactions, giving rise to unacceptable levels of “false positives”. A number of factors make a mammalian two-hybrid system highly desirable. First of all, post-translational modifications of proteins may contribute significantly to their ability to interact, and yet such modifications may not be supported in a yeast environment. Consequently, proteins that would interact with correct post-translational processing may not be identified in a yeast system. Certain post-translational modifications that influence protein-protein interactions only occur secondarily to activation of particular signaling cascades. As yeast lacks many of the cell surface receptors that trigger such cascades, discovery of signal-dependent modifications and protein-protein interactions is generally not feasible in a yeast cell. However, several decades of research have been devoted to initiating such signaling cascades in mammalian cells. A mammalian two-hybrid system operable in a variety of mammalian cell types would be highly desirable, since the regulation, induction, processing, etc. of specific proteins within a particular cell type can vary significantly, it would thus be a distinct advantage to assay for relevant protein-protein interactions in the relevant cell type. For example, proteins involved in a disease state could be tested in the relevant disease cells, resulting in a higher chance of identifying important protein interactions. Similarly, for testing of random proteins, assaying them under the relevant cellular conditions will give the highest chance of positive results. Furthermore, the mammalian cells can be tested under a variety of experimental conditions that may affect intracellular protein-protein interactions, such as in the presence of hormones, drugs, growth factors and cytokines, cellular and chemical stimuli, etc., that may contribute to conditions which can effect protein-protein interactions. Thus, a robust and adaptable mammalian two-hybrid system that can work in a wide variety of mammalian cell types is highly desirable. In certain embodiments, the two hybrid assay systems of the prior art have been modified to detect ligand-dependent interaction of proteins. In these “three hybrid assay” systems, the ability of another protein or small organic molecule to mediate interaction of two proteins is detected. Accordingly, it is an object of the invention to provide compositions and methods useful in two-hybrid assay systems that can be utilized reproducibly and stably in vertebrate cells, especially mammalian cells. Another object of the invention to provide compositions and methods useful in a three-hybrid systems that can be utilized reproducibly and stably in vertebrate cells, especially mammalian cells. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to, inter alia, compositions and methods for identification of protein-protein interactions and protein-compound interactions in mammalian cells. In one aspect, the invention provides a method for detecting protein interactions in a host cell, comprising: (i) providing a host cell including: (a) a nucleic acid encoding a bait fusion protein comprising a bait polypeptide sequence fused to a targeting domain that targets the bait fusion protein to a specific subcellular location within the host cell; (b) a nucleic acid sequence encoding a prey fusion protein comprising a prey polypeptide sequence fused to one or more detection domains that can be detected in a transcriptionally-independent manner in the host cell to determine if the prey fusion protein is associated in a complex with the bait fusion protein or not; (ii) detecting the subcellular location of the prey protein within the host cell; wherein accumulation of the prey fusion at the same subcellular pattern as the bait fusion indicates that the prey fusion protein is associated in a complex with the bait fusion protein. The subject method can be used to identify or measure direct or indirect protein-protein interactions in protein complexes, as well as identify or measure ligand-mediated protein-protein interactions. In either case, the assay can be derived to permit observation of the effects post-translational modifications, such as phosphorylation or the like, may have on the formation of protein complexes. In the case of ligand-mediated complexes, the assay can be used to identify or measure the ability of a ligand (such as a protein, peptide or small organic molecule) to directly bind both the bait and prey fusion proteins. It can also be used to identify or measure the ability of a ligand to act as an allosteric agent, e.g., binding one of the bait or prey fusion proteins and inducing a conformation change in the protein which effects the formation of bait-prey complexes. In still other embodiments, the subject method and system can be used to identify agents which can disrupt the formation of prey-bait complexes, e.g., either directly by competitive binding to the bait or prey polypeptide sequences, indirectly by allosterically modifying one of the bait or prey polypeptide sequences, or by inhibiting post-translational modification of one or both of the bait and prey polypeptide sequences. In one embodiment, the targeting domain is capable of targeting the bait fusion to an intracellular structure or organelle selected from the group consisting of the nucleus, nucleoli, telomeres, kinetochores, nuclear envelope, chromosomes, chromatin, cytoplasm, endoplasmic reticulum, Golgi, centrosome, transgolgi network, cytoplasmic vesicles, mitochondria, secretory vesicles, lysosome, plasma membrane, intracellular membrane vesicles, nuclear membranes, synapses or basolatemal membranes. Preferably the targeting domain causes localization of the prey fusion protein, should the bait and prey proteins form a complex, in a manner in which localization of the prey fusion protein can be discerned by microscopy or flow cytometry. In certain embodiments, the intracellular localization of the prey fusion protein is determined by direct visualization (including by automated processes) of the localization pattern of the prey fusion proteins, as opposed to a transcriptionally-dependent readout such as expression of a reporter gene. In preferred embodiments, the localization of the prey fusion proteins can be determined within minutes of expression of the bait and prey proteins in the host cells, e.g., preferably less 120 minutes, even more preferably less than 60, 45, 30, 15 or even 10 minutes. In certain preferred embodiments, the targeting domain is capable of targeting the bait fusion protein to the kinetochores. Preferred domains for targeting to the kinetochore are those derived from a CENP-A, CENP-B, CENP-C, CENP-E, CENP-F, Bub1, Bub3, MAD3L or MAD2 protein, or a portion thereof capable of targeting the bait fusion to the kinetochores. In a particularly preferred embodiment, the targeting domain comprises at least amino acids 373-943 of the human CENP-C sequence or a homolog thereof which retains the ability to be docked to the kinetochore. In certain preferred embodiments, the targeting domain associates with the kinetochore structure with a dissociation constant (k d ) of 1 mM or less, and even more preferably with a k d less than 10 μM, 1 μM, 100 nM, 10 n, or even 1 nM. In other preferred embodiments, the targeting domain is capable of targeting the bait fusion to the nuclear envelope. Preferred domains for targeting to the nuclear envelope are those derived from a lamin A, lamin B, lamin C, emerins or porins protein, or a portion thereof capable of targeting the bait fusion to the nuclear envelope. In certain embodiments, the detection domain fused to the prey protein is a fluorescent protein or a luminescent protein. Suitable detection domains include, for example, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Renilla Reniformis green fluorescent protein, GFPmut2, GFPuv4, enhanced yellow fluorescent protein (EYFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), citrine, red fluorescent protein from discosoma (dsRED), and variants thereof. Preferred detection sequences are the green fluorescent protein and the S26T/V 163A mutant of the green fluorescent protein. In certain embodiments, the bait and/or prey fusions include an instability sequence. Preferred instability sequences give the resulting fusion protein a shorter intracellular half-life (e.g., at least 50 percent shorter, even more preferably at least 75, 90, 95 or even 99 percent shorter) when not sequestered at the intracellular site to which the bait fusion protein is directed relative to when it is. In certain embodiments, the instability sequence includes a CENP-C instability domain, e.g., the fusion protein includes at least amino acids 249-323 of the human CENP-C sequence or a sequence homologous thereto and causes degradation of the protein if not docked at the kinetochore. In certain preferred embodiments, the prey fusion protein includes the instability sequence and the half life of the detection domain is shortened if the prey fusion protein is not localized with the bait fusion protein. In certain embodiments, the bait and/or prey fusions may also include one or more amino acid sequences selected from the group consisting of rescue sequences and/or oligomerization domains (such as a dimerization domain). Preferred rescue sequences are the His 6 tag, myc tag, flu tag, lacZ, GST, Strep tag I and Strep tag II. Preferred oligomerization domains comprise the dimerization domain from the yeast GCN4 protein or from p53. In certain embodiments, the nucleic acid sequences encoding the bait and prey fusions are each contained on a vector, which may be the same vector or separate vectors. Exemplary vectors include integrative as well as episomal vectors. In certain preferred embodiments, the vector is a retroviral vector. Preferred plasmids of the invention include pCEP4, pCI-NEO, pBI-EGF, pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg. The bait and prey fusions may be contained on the same or separate vectors and may be operably linked to the same or different transcriptional regulatory sequences. In certain embodiments, the the vector includes a recovery element, e.g., a nucleic acid sequence which binds to an agent which permits affinity purification of the vector from a cell lysate. An exemplary recovery element is a lacO sequence which binds to a lacZ protein. In certain embodiments, the vectors encoding the bait and/or prey fusions may also include one or more selection sequence. Preferred selection sequences are neomycin, blastocidin, bleomycin, puromycin and hygromycin, and the bait and prey fusions may contain the same or different selection sequences. In certain embodiments, the nucleic acid sequences encoding the bait and prey fusions further comprise a promoter sequence. The promoter may constitutive or inducible. Preferred promoters include the CMV, SV40, SRα, RSV, TK and beta-globin promoters. Preferred host cells of the invention include mammalian cells, such as primate, murine, porcine, rat cells and the like. Primate cells, particularly human cells are expecially preferred in certain embodiments. However, in the use of embodiments in which the bait fusion protein localizes to the kinetochore, any cell which includes chromosomal structures including kinetochores can be used. In certain embodiments, libraries of bait and/or prey fusion proteins (and the corresponding coding sequences) can be constructed by fusing a variegated library of coding sequences to a common targeting sequence (to form a library of bait proteins) or to a common detection sequence (to form a library of prey proteins). Identification of a protein complexes including the bait and prey fusion proteins may be carried out by screening a single bait fusion against a single prey fusion, by screening a single bait fusion against a library of prey fusions to identify prey fusions which are capable of interacting with the bait fusion, by screening a single prey fusion against a library of bait fusions to identify bait fusions which are capable of interacting with the prey fusion, or by screening a library of prey fusions against a library of bait fusions in order to identify bait and prey fusions capable of interacting. In a preferred embodiment, the method further comprises the step of determining the specific subcellular localization of the prey fusion within the host cell. Preferably, the expression of the prey fusion is detected using FACS analysis. In another aspect, the invention provides a method for detecting a protein-compound interaction in a mammalian cell, comprising: (i) constructing a nucleic acid encoding a bait fusion comprising a sequence capable of targeting the bait fusion to a specific subcellular location within a cell fused in frame with receptor protein; (ii) constructing a compound comprising a ligand for the receptor of the bait fusion fused to a compound; (iii) constructing a nucleic acid sequence encoding a prey fusion comprising a detection sequence capable of permitting determination of the subcellular localization of the prey fusion within a cell fused in frame with a prey protein; (iv) introducing the nucleic acids encoding the bait and prey fusions and the receptor-compound fusion into a host cell; (v) detecting the subcellular location of the prey protein within the host cell; wherein the ligand-compound fusion is localized to the same subcellular location to which the bait fusion was targeted via interaction of the ligand with the receptor portion of the bait fusion; and wherein accumulation of the prey fusion at the same subcellular location to which the bait fusion was targeted is indicative of an interaction between the compound and the prey fusion. In a preferred embodiment, a library of compounds is fused to the ligand and is screened against a particular prey fusion to find compounds which interact with the prey fusion. In another preferred embodiment, a single compound is fused to the ligand and is screened against a library of prey fusions to find a prey fusion which interacts with the compound. In a particularly preferred embodiment, the bait fusion comprises the ecdysone receptor, or a portion thereof which is capable of binding to the ecdysone ligand, and the ligand is ecdysone. In another aspect, the invention provides a method for screening for compounds which inhibit or potentiate a protein-protein interaction in a mammalian cell, comprising: (i) constructing a nucleic acid encoding a bait fusion comprising a subcellular localization domain and a bait protein; (ii) constructing a nucleic acid encoding a prey fusion comprising a reporter sequence and a prey protein; (iii) introducing the nucleic acids encoding the bait and prey fusions into a host cell; (iv) contacting the cell expressing the bait and prey fusions with a test compound; (iv) detecting the subcellular location of the prey protein within the host cell in the presence and absence of the test compound; wherein a change in the accumulation of the prey fusion at the same subcellular location to which the bait fusion was targeted in the presence of the test compound as compared to the accumulation in the absence of the test compound is indicative of a test compound capable of inhibiting or potentiating an interaction between the bait and prey fusions. In another aspect, the invention provides a kit for detecting protein interactions, comprising: (i) a first expression construct including a coding sequence for a targeting domain and a ligation site flanking an end of the targeting domain coding sequence for ligating a coding sequence of a bait polypeptide sequence in frame with said targeting domain coding sequence to produce a bait fusion protein, said first expression construct operably linked to a transcriptional regulatory element; and (ii) a second expression construct including a coding sequence for a detection domain and a ligation site flanking an end of the detection domain coding sequence for ligating a coding sequence of a prey polypeptide sequence in frame with said detection domain coding sequence to produce a prey fusion protein, said second expression construct operably linked to a transcriptional regulatory element, wherein, the targeting domain localizes the bait fusion protein to a subcellular location within a host cell, and the detection domain can be detected in a transcriptionally-independent manner in the host cell to determine if the prey fusion protein is associated in a complex with the bait fusion protein or not. In yet another aspect, the invention provides a method for detecting protein interactions in a host cell, comprising: (i) providing a host cell culture, the cells of which include: (a) a first nucleic acid coding sequence encoding a bait fusion protein comprising a bait polypeptide sequence fused to a targeting domain that targets the bait fusion protein to a subcellular location within the host cell, and (b) a second nucleic acid coding sequence encoding a prey fusion protein comprising a prey polypeptide sequence fused to one or more detection domains that can be detected in a transcriptionally-independent manner in the host cell to determine if the prey fusion protein is associated in a complex with the bait fusion protein or not, wherein the culture is a variegated mixture of cells containing different prey polypeptide sequences and/or different bait polypeptide sequences; (ii) selecting cells from the culture in which the prey fusion protein is localized in the cell in the same subcellular pattern as the bait fusion protein; (iii) identifying the sequence of the bait and prey fusion proteins from the selected cells In another aspect, the invention provides a method for conducting a drug discovery business, comprising: (i) using the methods of the invention to identify a protein complex for which an agent that inhibits or potentiates the formation or activity of the complex is desired; (ii) generating a drug screening assay for identifying agents that inhibit or potentiate the formation or activity of the complex; (iii) conducting animal toxicity profiles on a agent identified in step (ii), or an analog thereto; (iv) manufacturing a pharmaceutical preparation of an agent having a suitable animal toxicity profile; and (v) marketing the pharmaceutical preparation to healthcare providers. In another aspect, the invention provides a method for conducting a drug discovery business, comprising: (i) using the methods of the invention to identify a protein complex which is mediated by post-translational modification and for which an agent that inhibits or potentiates the post-translational modification is desired; (ii) generating a drug screening assay for identifying agents that inhibit or potentiate the post-translational modification and effect the formation of the protein complex; (iii) conducting animal toxicity profiles on a agent identified in step (ii), or an analog thereto; (iv) manufacturing a pharmaceutical preparation of an agent having a suitable animal toxicity profile; and (v) marketing the pharmaceutical preparation to healthcare providers. In aother aspect, the invention provides a method for conducting a bioinformatics business, comprising: (i) using the methods of the invention to identify networks of protein complexes; (ii) generating a database including information identifying interactions of different proteins in a signal pathway and information identifying the proteins. In aother aspect, the invention provides a system for analyzing protein complexes in cells, comprising a flow cytometer for analyzing cells and determining if a fluorescent signal is dispersed in a cell or localized to kinetochore structures. In certain embodiments, the invention may further comprise a microprocessor for comparing the flow spectra of cells and distinguishing between a diffuse pattern of fluorescence in the cells and a kinetochore-localized pattern. In aother aspect, the invention provides a system for analyzing protein complexes in cells, comprising a microscope having a camera mounted therein for analyzing cells in a field of vision of the microscope, and a microprocessor for processing images obtained from said camera and determining if a fluorescent signal is dispersed in a cell or localized to kinetochore structures. In certain embodiments, the invention may further comprise a cell picking robot which is controlled by said microprocessor and isolates cells which the microprocessor has determined have a fluorescent signal localized to kinetochore structures. |
Method and apparatus for access checking and access control |
A method and apparatus for checking and controlling access to mobile telephones, computers or related devices with regard to transactions and to the handling of information, respectively. The user is provided with a user-specific identification tag that communicates with the mobile telephone or computer via a radio wave transmitter/receive unit that has a short range. The identification tag exchanges with the telephone or the computer identity information carried by the identification tag to ratify identification of the user, and to thereby control access to the telephone or computer. |
1. A method for checking and controlling access to electronic devices such as mobile telephones, computers, or corresponding devices with regard to transactions and handling of information, respectively, said method comprising the steps of: providing a user with a user-specific identification tag having user identity information and including a radio wave transponder which retransmits an incoming radio signal back to a radio wave transmitter/receiver unit associated with the electronic device that transmitted said incoming signal, wherein a radio wave transmitter/receiver unit of the identification tag has a predetermined short transmitting range; communicating via Bluetooth wireless transmission between the identification tag and the electronic device in a predetermined communications area adjacent to the electronic device; and exchanging between the identification tag and the electronic device identity information carried by the identification tag to ratify identification of the user, wherein one or more functions of the electronic device can only be performed subsequent to a given identification tag having been identified and accepted by the electronic device giving access to said functions. 2. A method according to claim 1, including the step of ascertaining at least at uniform short time intervals whether or not the identification tag is situated within the predetermined communications area determined by said range. 3. A method according to claim 2, including the step of terminating access to the electronic device when the identification tag leaves said predetermined communications area. 4. A method according to claim 1, wherein the predetermined range is less than about 10 meters. 5. Apparatus for checking and controlling access to electronic devices such as mobile telephones, computers, or corresponding devices with regard to transactions and handling of information, respectively, said apparatus comprising: a user-specific identification tag having user identity information and including a transponder which retransmits an incoming radio signal back to a radio wave transmitter/receiver unit associated with the electronic device that transmitted said incoming signal, wherein the identification tag has a predetermined short transmitting range in that the identification tag communicates according to Bluetooth wireless transmission with said electronic device via radio waves in an area adjacent to the electronic device, wherein the identification tag exchanges with the electronic device information carried by the identification tag; and wherein the user is considered to have been identified by such information exchange and wherein one or more functions of the electronic device can only be carried out subsequent to a given identification tag having been identified and accepted by the electronic device giving access to said functions. 6. Apparatus according to claim 5, wherein the electronic device ascertains at least at uniform short time intervals whether or not the identification tag is situated within a the predetermined communications area determined by said range. 7. Apparatus according to claim 5, wherein access to the electronic device is terminated when the identification tag leaves said predetermined communications area. 8. Apparatus according to claim 7, wherein the predetermined range is less than about 10 metres. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention relates to a method of checking and controlling access to mobile telephones, computers or corresponding devices for transactions and handling of information, respectively, user is provided with a user-specific identification tag which communicates with the mobile telephone or with computers in an area in the vicinity of the telephone or the computer via radio waves. A transmitter/receiver unit of the identification tag has a short range with respect to radio waves. The identification tag exchanges with the telephone and the computer, respectively, information that includes identification of the identification tag, and such identification is considered to ratify the identification of the user. The invention also relates to apparatus for carrying out the method. |
<SOH> BRIEF DESCRIPTION OF THE DRAWING <EOH>The invention will now be described in more detail partly with reference to an exemplifying embodiment of the invention illustrated in the accompanying drawing, in which FIG. 1 shows an identification tag and a computer; and FIG. 2 shows an identification tag and a telephone. detailed-description description="Detailed Description" end="lead"? |
Device for assistance in the analysis of adventitious sounds |
The invention concerns a device for assistance in the analysis of adventitious sounds, such as wheezing or crackling sounds. The invention is characterised in that said adventitious sounds are located on time/frequency spectral representations and parameterised according to a set of coefficients characterising them. Said sets of coefficients are stored in a storage unit for establishing a database or compared to synthesized sounds, based on parameters retrieved from such a database. |
1. A device for assistance in the analysis of respiratory sounds, of the type comprising a working memory (2), capable of storing samples of a signal representing a respiratory sound, and a computing module (4, 3) capable of cooperating with the working memory in order to carry out a time and frequency conversion of the signal (114), characterized in that it furthermore includes means (4, 3, 120) for identifying, in a converted signal, at least one component relating to an adventitious sound (Si; FCi; Cci) and in that the computing module is designed to extract a set of parameters (Ai, ω0; H, L) characterizing this adventitious sound from the converted signal. 2. The device as claimed in claim 1, characterized in that said means are designed to identify the component that represents the adventitious sound in a frequency and time representation (FIG. 3, FIG. 4, FIG. 5) of the converted signal. 3. The device as claimed in claim 2, characterized in that it includes an acquisition member (92, 93), whereas the means for identifying the component are manual. 4. The device as claimed in either of claims 1 or 2, characterized in that the computing module is furthermore capable of comparing (120) successive intensity values of the converted signal in order to identify said component. 5. The device as claimed in claim 4, characterized in that the computing module (120) is designed to compare the intensity values by carrying out: a frequency scan of the intensities, for various successive instants, when the adventitious sound to be identified is a set of sibilant rhonchi; and/or a time scan of the intensities, for various frequencies, when the adventitious sound to be identified is a set of crackles. 6. The device as claimed in claim 1, characterized in that the parameters characterizing a set of crackles comprise the number of crackles identified, and also a temporal segment (L) and preferably a mean intensity in this temporal segment, for each crackle. 7. The device as claimed in claim 6, characterized in that the parameters furthermore include a frequency range (H) within which each identified crackle lies. 8. The device as claimed in claim 1, characterized in that the parameters characterizing a set of sibilant rhonchi comprise, for each sibilant rhonchus, a mean frequency over a stable phase of the sibilant rhonchus (Si2) and at least one coefficient of linear variation in frequency of the start (Sil) and/or of the end (Si3) of the sibilant rhonchus. 9. The device as claimed in claim 1, characterized in that the parameters characterizing a set of sibilant rhonchi comprise the number of fundamental sibilant rhonchi of lowest frequencies (S1, S4). 10. The device as claimed in claim 1, characterized in that the parameters characterizing a set of sibilant rhonchi comprise, for each fundamental sibilant rhonchus (S4) and its harmonic sibilant rhonchi (S2, S3), the number of harmonic sibilant rhonchi and a mean ratio (Ai) of the intensities of the harmonics to the intensities of the fundamental. 11. The device as claimed in claim 1, characterized in that the computing module is furthermore designed to compare said parameters with values prerecorded in an ordered memory (ROM), comprising data of sets of parameters, each set characterizing at least one adventitious sound, and in that the device furthermore includes a warning module suitable for being activated or not, depending on the comparison. 12. The device as claimed in claim 1, characterized in that the parameters characterizing an adventitious sound are stored in succession in an ordered memory (4). 13. The device as claimed in either of claims 11 or 12, characterized in that the ordered memory (4) is structured as a database. 14. The device as claimed in claim 13, characterized in that it furthermore includes a telecommunication module (MODEM) for remote consultation of the database. 15. A removable medium intended to cooperate with a reader (5) of a device as claimed in claim 1, characterized in that it includes data for programs for using the computing module. 16. The removable medium as claimed in claim 14, characterized in that it furthermore includes prerecorded data of parameters (Ai, ω0; H, L) characterizing adventitious sounds. |
Detection and quantification of prion isoforms in neurodegenerative diseases using mass spectrometry |
Disclosed are methods, compositions and kits for diagnosing prion-mediated pathological conditions and presence of aberrant prion protein in animal derived products, utilizing mass spectrometry. |
1. A method of detecting a prion-mediated pathological condition in a human or animal, comprising: (a) obtaining a fluid or cellular or tissue sample from the human or animal; (b) extracting prion proteins from the sample; (c) digesting the extracted prion proteins to produce a composition that contains peptide fragments of the extracted prion proteins, wherein the fragments include signature peptides at least one of which is differentially released from an aberrant prion protein compared to a normal prion protein; (d) analyzing the digested sample via mass spectrometry wherein the digested sample also contains for each signature peptide, a corresponding internal standard peptide; and (e) generating for each signature peptide, a normalized value obtained by comparing mass spectrometry signals generated by the signature peptides with mass spectrometry signals generated by the corresponding internal standard peptides, wherein a difference between the normalized value for the signature peptide that is differentially released and a normalized value for the signature peptide that is not differentially released, or wherein a difference between the normalized value for the signature peptide that is differentially released and a control, is indicative of a priommediated pathological condition. 2. The method of claim 1 wherein the control comprises a normalized value obtained by comparing mass spectrometry signals generated by signature peptides obtained from a healthy human or animal, compared to the corresponding internal standard peptides. 3. A method of detecting a prion-mediated pathological condition in a human or animal, comprising: (a) obtaining a fluid or cellular or tissue sample from said human or animal; (b) extracting prion proteins from the sample using a. chaotropic agent so as to produce denatured prion proteins; (c) digesting the denatured prion proteins to produce a composition that contains peptide fragments of the prion proteins, wherein the fragments include signature peptides; (d) analyzing via mass spectrometry the signature peptides and for each signature peptide, a corresponding internal standard peptide; and (e) generating for each signature peptide, a normalized value obtained by comparing mass spectrometry signals generated by the signature peptide with mass spectrometry signals generated by the corresponding internal standard peptide, wherein a difference in the nomialized value for at least one of the signature peptides compared to a control is indicative of a prion-mediated pathological condition. 4. The method of claim 3 wherein the control comprises a normalized value obtained by comparing mass spectrometry signals generated by the signature peptide obtained from a healthy human or animal, compared to the corresponding internal standard peptide. 5. The method of claim 1 wherein the sample is a fluid sample obtained from serum, cerebrospinal fluid, blood, saliva, tears, urine, semen, amniotic fluid, milk or lactation fluid. 6. The method of claim 1 wherein the sample is a cellular or tissue sample obtained from muscle, skin, eyelids, brain, spinal cord, lymphoid organs, spleen, kidney, bone marrow or tissue obtained from lymphoreticular system, peripheral nervous system, central nervous system, immune system, follicular dendritic cells, lymphocytes or leucocytes. 7. The method of claim 1 wherein said extracting comprises contacting said sample with a buffer. 8. The method of claim 7 wherein said buffer comprises a detergent. 9. The method of claim 8 wherein said detergent comprises SDS or sarkosyl. 10. The method of claim 2 wherein said digesting comprises (c1) contacting extracted proteins of (b) with a nonspecific proteinase under conditions to allow digestion of non-core prion peptides, followed by (c2) denaturing non-specific proteinase resistant core prion peptide in the presence of a denaturing agents followed by (c3) contacting denatured core peptide with a protease, and wherein in (e) the normalized value for the signature peptide that is differentially released is compared to a control. 11. The method of claim 10 wherein the control compzises a normalized value obtained by comparing mass spectrometry signals generated by the signature peptide that is differentially released and contained in a sample obtained from healthy humans or animnals, compared to the corresponding internal standard. 12. The method of claim 10 wherein the denaturing agent comprises guanidine hydrochloride, acetonitrile, urea or heat. 13. The method of claim 11 wherein the denaturing agent comprises guanidine hydrochloride in a concentration of from about 4 to about 6M. 14. The method of claim 11 wherein the denaturing agent comprises urea in a concentration of from about 4 to about 8M. 15. The method of claim 1 further comprising (f) concentrating the extracted prior proteins of (b), and wherein said digesting compnses producing peptide fiagments of the extracted and concentrated prion proteins. 16. The method of claim 15 wherein said concentrating comprises contacting the extracted proteins of (b) with a resin that adsorbs prion proteins or non-prion proteins. 17. The method of claim 16 wherein said concentrating further comprises filtering the extracted proteins of (b). 18. The method of claim 1 wherein said digesting comprises treating the extracted prion proteins with at least one protease. 19. The method of claim 18 wherein the protease comprises trypsin. 20. Trhe method of claim 1 wherein the composition further comprises a matrix and said analyzing comprises introducing the composition into a matrix assisted lascr desorption ionization (MALDI) lime-of-flight (TOF) analyzer. 21. The method of claim 20 wherein the matrix comprises alpha-cyano4-hydroxycinnamic acid. 22. The method of claim 1 wherein said analyzing comprises introducing the composition into an ion trap electrospray ionization apparatus (ESI). 23. The method of claim 1 further comprising (g) introducing the composition into a liquid chromatograph (LC) prior to said analyzing. 24. The method of claim 23 wherein the LC is a micro-LC. 25. The method of clain 23 wherein tne LC is a nano-LC. 26. The method of claim 23 wherein said analyzing comprises introducing the composition into an ion-trap ESI. 27. The method of claim 23 wherein said analyzing comprises introducing the composition into a MALDI-TOF analyzer. 28. The mnethod of claim 1 wherein said digesting comprises treating the extracted prion proteins with itypsin, wherein the signature peptides comprise at least one core signature peptide and at least one non-core signature peptide, wherein the internal standard peptides comprises mass-labeled reference peptides, and wherein said generating comprises detecting increased or decreased presence or amount of the core signature peptide relative to the non-core signature peptide. 29. The method of claim 28 wherein the sample is obtained from a bovine, and wherein tile core signature peptides comprise peptides EHTVTTTTK, GENFTETDIK or VVEQMCITQYQR, or an equivalent, mutant or variant thereof having an amino acid substitution, deletion or addition, and the noncore signature peptides comprise RPKPGGGWNTGGSR, PGGWNTGGSR, YPGQGSPGGNR or ESQAYYQR or an equivalent, mutant or variant thereof having an amino acid substitution, deletion or addition. 30. The method of claim 28 wherein the sample is obtained from a human, and wherein the core signature peptide comprises peptides QHTVTTTTK, GENFTETDVK or VVEQMCITQYER, or an equivalent, mutant or variant thereof having an amino acid substitution, deletion or addition, and the non-core signature peptide comprises RPKPGGGWNTGGSR, PGGWNTGGSR, YPGQGSPGGNR or ESQAYYQR, or an equivalent, mutant or variant thereof having an amino acid substitution, deletion or addition. 31. The method of claim 28 wherein the signature peptides comprise more than one core prion proteins and more than one non-core prion protein. 32. The method of claim 1 wherein the prion-mediated pathological condition is transmissible spongifomi encephalopathy (TSE), Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy, scrapie, chronic wasting disease (CWD), transmissible mink encephalopathy (TME), or feline spongiform encephalopathy (FSE). 33. The method of claim 1 wherein the sample of (a) is a first portion of the sample, and wherein said method further comprises: (f) extracting the prion proteins from a second portion of the sample using a chaotropic agent so as to produce the prion proteins in denatured form; (g) digesting the denatured prion proteins to produce peptide fragnents of the denatured prion proteins. wherein the fragments include signattre peptides of the denatured and digested prion proteins; (h) analyzing via mass spectrometry the signature peptides of (g) and for each signature peptide, a corresponding internal standard peptide; and (i) generating for each signature peptide, a normalized value obtained by comparing mass spectrometry signals generated by the signature peptide with mass spectrometry signals generated by the corresponding internal standard peptide, wherein a difference in the normalized value for at least one of the signature poptides compared to a control is indicative of a prion-mediated pathological condition; and (j) comparing indication obtained from (h) with indication obtained from (e). 34. A method of detecting an aberrant prion protein in a product of human or animal origin, comprising: (a) obtaining a sample from a product of human or animal origin; (b) extracting prion proteins from the sample; (c) digesting the extracted prion proteins to produce peptide fragments of the extracted prion proteins, wherein the fragments include signature peptides at least one of which is differentially released from an aberrant prion protein compared to a normal prion protein: (d) analyzing the peptide fragments and for each of the signature peptides, a corresponding internal standard peptide, via mass spectrometry; and (e) generating for each signature peptide, a normalized value obtained by comparing mass spectrometry signals generated by the signature peptide with mass spectrometry signals generated by thc corresponding internal standard, wherein a difference between the normalized value for the signature peptide that is differentially released and a normalized value for the signature peptide that is not differentially released, or wherein a difference between the normalized value for the signature peptide that is differentially released and a control, is indicative of presence of an aberrant prion protein in the product. 35. he nethod of claim 34 wherein said digesting comprises (c1) contacting extracted proteins of (b) with a non-specific proteinase under conditions to allow digestion of non-core prion peptides, followed by (c2) denaturing non-specific proteinase resistant core prion peptide in the presence of a denaturing agent, followed by (c3) contacting denatured core peptide with a protease, and wherein in (e) the normalized value for the signature peptide that is differentially released is comnpared to a control. 36. A method of detecting an aberrant prion protein in a product of human or animal origin, comprising: (a) obtaining a sample from a product of human or animal origin; (b) extracting prion proteins from the sample using a chaotropic agent so as to produce denatured prion proteins; (c) digesting the denatured prion proteins to produce a composition that contains peptdde fragments of the prion proteins, wherein said fragments include signature peptides; (d) analyzing via mass spectrometry the signature peptides and for each signature peptide, a corresponding internal standard peptide; and (e) generating for each signature peptide, a normalized value obtained by comparing mass spectrometry signals generated by the signature pepuide with mass spectrometry signals generated by the corresponding internal standard peptide, wherein a difference in the normalized value for at least one of the signature peptides compared to a control is indicative of presence of the aberrant prion protein in the product. 37. The method of claim 34 or 36 wherein the product is blood or a blood-derived factor, a commercial food product or ingredient thereof, feed, or cosmetic, nutraceutical or pharmaceutical or an ingredient of said cosmetic, nutraceutical or pharmaceutical. |
<SOH> BACKGROUND <EOH>Bovine spongiform encephalopathy (BSE) is one of several documented prion neurodegenerative diseases, which includes Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, chronic wasting disease (CWD) in mule deer and elk, transmissible mink encephalopathy (TME), and feline spongiform encephalopathy (FSE) in cats (Aguzzi 2001). Recently, the occurrence of BSE in cows is becoming epidemic in Italy, France, Ireland, Portugal, Germany and other European countries, as it spreads from United Kingdom. Switzerland is second behind the United Kingdom for reported BSE cases. Similar to the transmission of TSE from sheep to cows, it has been reported that genetic evidence exists for the transmission of BSE to humans, as a “new variant” of CJD (nvCJD) (Scott 2000). The nature of the putative transmission to humans is unknown as well as the predisposition of an individual to nvCJD. An unfortunate aspect of TSE is that the prion neurodegenerative diseases are generally latent in onset, which may range from 2-8 years in cows and 3-5 years in sheep after the animal becomes infected. The latent period for humans is believed to be longer than that found in animals. Therefore, the extent of potential horizontal transmission remains largely unknown due to difficulties in the detection of nvCJD until several years after exposure. As expected, since the first reported cases of nvCJD in 1995 it has been rising, mirroring the early growth of BSE cases in the late 1980s. A more severe concern, similar to the AIDs virus, is the potential for rapid transmission of nvCJD through infected blood or tissue donors and bovine based products used in medical treatments and health supplements. Thus there is a pressing need for diagnostic tests that are sufficiently sensitive and reliable to be used to diagnose infected individuals before clinical symptoms develop. The precise mechanism for the onset of the disease is unknown, however no relationship has been observed between the disease and traditional infectious particles based on nucleic acids (Prusiner 1982a&b; Bolton 1982, Prusiner 1991). Rather, past studies have shown, although not unequivocally, that a specific class of proteins cause infection, denoted prions, and more specifically an aberrant isoform designated PrP SC , can induce the diseased state in laboratory animals and cell cultures. The PrP SC form is distinguishable from the normal cellular its form, denoted PrP C , by its relative resistance to proteases and low solubility. Upon protease treatment of PrP SC protein, the terminal amino acids are truncated leaving a large, resistant core referred to as PrP 27-30, which reflects its observed molecular size in kiloDaltons. It is believed that PrP SC can trigger or act to cascade the conversion of endogenous PrP C into the protease resistant isoform by some unknown mechanism, which accumulates, aggregates and leads to neurodegeneration. The conversion process is thought to facilitate a conformational change of PrP C from an α-helix to β-sheet protein structure. The clinical aspects of transmissible spongiform encephalopathies are named because of the microscopic or histopathological appearance of large vacuoles in the cortex and cerebellum of the brain in infected animals. The early diagnosis of TSE has been dependent upon the appearance of clinical signs, electroencephalography or invasive methods using brain biopsies. Postmortem histophathological evaluation of ruminant TSEs is based on the appearance of neuronal vacuolation, gliosis and astrocytosis, however these changes may not be realized until the late stages of infection. Other methods using post mortem diagnosis has included the use of immunohistochemical assays to improve the detection of the deposition of prion molecules in brain tissue. A modified method referred to as ID-Lelystad has been performed using immunocytochemistry on thin sections of brain biopsies, which can be completed within 6 hours. The test had 100% correlation with histopathology evaluations, however the method is qualitative and brain samples require the animal to be dead. Further, the nature of the detection protocol is quite laborious and not suitable for robust quantitative analysis. More recent diagnostic advances have focused on more rapid methods using a variety of other immunological applications that are also less laborious for the detection of TSE, however the single common element that exists with all immunological based assays is the development of a sensitive antibody. The immunological methods currently being used or developed include ELISA or immunometric systems, Western blots and capillary electrophoresis based detection. The preferred immunometric, or ELISA, quantification utilized an antibody sandwich assay method in conjunction with Protease K treatment to remove the PrP C isoforms (Grassi 2000). This method showed a good correlation with histopathological evaluations. The advantage of this technology is that is suitable for high throughput analysis, but false positives were reported. A modified ELISA employed the use of time-resolved fluorescence immunoassay in conjunction with two concentrations of guanidine hydrochloride to preferentially solubilize one PrP C isoforms relative to the PrP SC (Barnard 2000). The method scores prions in tissues as percentage insoluble prions with the higher ratio being more indicative of aberrant prions. The analysis provides a qualitative rather than a quantitative determination. A typical Western blot approach involves extracting brain tissue and subsequently subjecting the extract to polyacrylamide gels for separation of proteins followed by immunological probes for detection of prion protein. This type of analysis provides information on the relative molecular size of prion peptides and semi-verification of the result, thereby reducing false positive and negatives. However, polyacrylamide separation of proteins is not robust in determining accurate molecular sizes and has limited sensitivity. Further, the method is only somewhat applicable for low to moderate throughput and is relatively time constraining. In one study, referred to as Prionics Western blotting, it was shown that their results compared favorably to histopathological analysis, a small but significant number of samples tested either false negative (3 of 65) or positive (3 of 263) (Schaller 2000). This method is based on immunocompetition analysis using fluorescently tagged synthetic peptides (Schmerr 1998). Similar to the ELISA method, the sample is first treated with Protease K and subsequently assayed by capillary electrophoresis immunoassay. The study showed greater sensitivity over other methods and was the first method reported using blood samples rather than brain biopsies. The greater sensitivity of the assay facilitated the potential of performing non-invasive blood samples as opposed to biopsies from dead animals. Although this method has greater sensitivity over other immunological methods, it still suffers from the limitation of antibodies raised against a single epitope of a particular prion protein. The structural differences between the aberrant and native prion isoforms have provided an opportunity for the detection of BSE and other TSEs. Unfortunately, antibodies generated to date have failed to distinguish between the two forms. Thus immunological techniques rely on biochemical pre-protocols that preferentially remove the native isoforms from aberrant prion proteins on the basis of altered solubility or protease stability. Related problems with immunoassays have been the inability to recognize prions across animal species, distinguish between new variants, and have sufficient sensitivity and reliability to be applied to pre-mortem samples. In the late 1980's two mass spectrometries became available for the analysis of large biomolecules, namely, matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) and electrospray ionization (ESI). Requiring only a minute sample, mass spectrometry provides extremely detailed information about the molecules being analyzed, including high mass accuracy, and is easily automated. Both of these instruments are capable of mass analyzing biomolecules in complex biological solutions. MALDI-TOF MS involves laser pulses focused on a small sample plate comprising analyte molecules embedded in a low molecular weight, UV-absorbing matrix that enhances sample ionization. The matrix facilitates intact desorption and ionization of the sample. The laser pulses transfer energy to the matrix causing an ionization of the analyte molecules, producing a gaseous plume of intact, charged analyte. The ions generated by the laser pulses are accelerated to a fixed kinetic energy by a strong electric field and then pass through an electric field-free region in a vacuum in which the ions travel (drift) with a velocity corresponding to their respective mass-to-charge ratios (m/z). The lighter ions travel through the vacuum region faster than the heavier ions thereby causing a separation. At the end of the electric field-free region, the ions collide with a detector that generates a signal as each set of ions of a particular mass-to-charge ratio strikes the detector. Travel time is proportional to the square root of the mass as defined by the following equation t=(m/(2KE)z)½ where t=travel time, s=travel distance, m=mass, KE=kinetic energy, and z=number of charges on an ion. A calibration procedure using a reference standard of known mass can be used to establish an accurate relationship between flight time and the mass-to-charge ratio of the ion. Ions generated by MALDI exhibit a broad energy spread after acceleration in a stationary electric field. Forming ions in a field-free region, and then applying a high voltage pulse after a predetermined time delay (e.g. “delayed extraction™”) to accelerate the ions can minimize this energy spread, which improves resolution and mass accuracy. In a given assay, 50 to 100 mass spectra resulting from individual laser pulses are summed together to make a single composite mass spectrum with an improved signal-to-noise ratio. The entire process is completed in a matter of microseconds. In an automated apparatus, tens to hundreds of samples can be analyzed per minute. In addition to speed, MALDI-TOF technology has many advantages, which include: 1) mass range—where the mass range is limited by ionization ability, 2) complete mass spectrum can be obtained from a single ionization event (also referred to as multiplexing or parallel detection), 3) compatibility with buffers normally used in biological assays, 4) very high sensitivity; and 5) requires only femtomoles of sample. Thus, the performance of a mass spectrometer is measured by its sensitivity, mass resolution, and mass accuracy. In order for mass spectrometry to be a useful tool for detecting and quantifying proteins, several basic requirements need to be met. First, targeted proteins to be detected and quantified must be concentrated (e.g., enriched and/or fractionated) in order to minimize the effects of salt ions and other molecular contaminants that reduce the intensity and quality of the mass spectrometric signal to a point where either the signal is undetectable or unreliable, or the mass accuracy and/or resolution is below the value necessary to detect the target protein. Second, mass accuracy and resolution significantly degrade as the mass of the analyte increases. Thus, the size of the target protein or peptide must be within the range of the mass spectrometry device where there is the necessary mass resolution and accuracy. Third, to be able to quantify accurately, one would preferably resolve the masses of the peptides by at least six Daltons to increase quality assurance and to prevent ambiguities. Fourth, the mass spectrometric methods for protein detection and quantification diagnostic screening must be efficient and cost effective in order to screen a large number of samples in as few steps as possible. Mass spectrometry methods for the quantitation of proteins in complex mixtures have employed a system using protein reactive reagents comprised of three moieties that are linked covalently; an amino acid reactive group, an affinity group and an isotopically tagged linker group (Aebersold et al, 2000). This class of new chemical reagents is referred to as Isotope-Coded Affinity Tags (ICATs) (Gygi et al 1999). The reactive group embodied used sulfhydryl groups that react specifically with the amino acid cysteine. The presence of the affinity group facilitates the isolation of the specifically labeled proteins or peptides from a complex protein mixture. Selected affinity groups include strepavidin or avidin. Only those proteins containing these affinity groups may be isolated. The linker moiety may be isotopically labeled by a variety of isotopes that include 3 H, 13 C, 15 N, 17O, 18 O and 34 S. The use of differential isotopic ICATs provides a method for the quantitation of the relative concentration of peptides in different samples by mass spectrometry. The methods can be used to generate global protein expression profiles in cells and tissues exposed to a variety of conditions. In an analogous method, the N-terminal amino acids of proteins from two states are differentially labeled using different isotopically tagged nicotinyl-N-hydroxysuccinimide reagents (Munchbach et al, 2000). Unlike the ICAT system, proteins are first separated by two-dimensional SDS polyacrylamide gel electrophoresis before the analysis is performed The ratio of the isotope for each protein determined by mass spectrometry provides a relative concentration of each protein present in different physiological states. It is believed that the limitations of mass spectrometry methods employing either ICATs or N-succinylation isotopic tagging are inherently associated with the requirement that the protein from one sample is quantified relative to another state or sample rather than being quantified in absolute amounts. In the case of the ICAT method, it is a requirement that the protein or peptide being quantified contains at least one amino acid that is modified by the reactive group. A related requirement is that the reactive amino acid site on the protein in the two or more states or samples must be equivalently accessible to the reactive group on the ICATs. Similar to antibody methods, if the site is altered or conformationally obscured then the quantitation of the protein will be compromised. An additional limitation in the use of N-succinylation of proteins is that it requires the laborious task of two-dimensional SDS polyacrylamide gel electrophoresis prior to analysis. There remains a pressing need for easier, more reliable means to rapidly detect, quantify and characterize prion proteins from biological samples particularly complex samples. |
<SOH> SUMMARY OF THE INVENTION <EOH>One aspect of the present invention is directed to a method of detecting a prion-mediated pathological condition in a human or animal, comprising: (a) obtaining a fluid or cellular or tissue sample from the human or animal; (b) extracting prion proteins from the sample; (c) digesting the extracted prion proteins to produce a composition that contains peptide fragments of the extracted prion proteins, wherein the fragments include signature peptides at least one of which is differentially released from an aberrant prion protein compared to a normal prion protein; (d) analyzing the digested sample and for each signature peptide, a corresponding internal standard peptide, via mass spectrometry; and (e) generating for each signature peptide, a normalized value obtained by comparing mass spectrometry signals generated by the signature peptides with mass spectrometry signals generated by the corresponding internal standard peptides, wherein a difference between the normalized value for the signature peptide that is differentially released and a normalized value for the signature peptide that is not differentially released, or wherein a difference between the normalized value for the signature peptide that is differentially released and a control, is indicative of a prion-mediated pathological condition. In some embodiments, the digestion protocol entails treating the sample with a protease, preferably trypsin. In the case of a healthy sample, several signature peptides will be produced, all in roughly equal amounts. If on the other hand, the sample is obtained from a diseased human or animal, the digestion will yield signature prion peptides that are differentially released on account of the fact that the protease resistance of the core region of the disease-related prion protein will reduce the amount of core signature diagnostic peptide detected. Thus, in this case, the differential release is illustrated by a normalized ratio of core signature diagnostic peptides to non-core signature diagnostic peptides that is less than one (1). In other embodiments, the digestion protocol entails contacting extracted proteins of (b) with a non-specific proteinase under conditions to allow digestion of non-core prion peptides, followed by denaturing non-specific proteinase resistant core prion peptide in the presence of a denaturing agent, followed by contacting denatured core peptide with a protease that is more specific relative to the non-specific proteinase, and wherein in (e) the normalized value for the signature peptide that is differentially released is compared to a control. In this case, digestion of a sample obtained from a healthy or non-diseased animal will not result in the production of statistically significant signature peptide for purposes of the method. In contrast, this digestion of a sample obtained from diseased animal will yield signature peptides that would not otherwise be produced on account of the fact that the chaotropic agent renders the protease-resistant core of the prion protein susceptible to digestion by the specific protease e.g., trypsin. Thus, in this case, signature diagnostic peptides are differentially released and detected from disease-related prion protein because core signature diagnostic peptides from normal prion protein, and non-core signature diagnostic peptides from all prion proteins, will have been previously degraded by the initial treatment with the non-specific protease/proteinase. Thus, in this case, more than one signature peptide is said to be differentially released in that the corresponding peptides from a healthy sample are not present in statistically significant quantity. These two aspects of the invention can be used together to confirm results and thus provide even higher levels of confidence. Another related aspect of the present invention is directed to a method of detecting a prion-mediated pathological condition in a human or animal, comprising: (a) obtaining a fluid or cellular or tissue sample from said human or animal; (b) extracting prion proteins from the sample using a chaotropic agent so as to produce denatured prion proteins; (c) digesting the denatured prion proteins to produce a composition that contains peptide fragments of the prion proteins, wherein the fragments include signature peptides; (d) analyzing via mass spectrometry the signature peptides and for each signature peptide, a corresponding internal standard peptide; and (e) generating for each signature peptide, a normalized value obtained by comparing mass spectrometry signals generated by the signature peptide with mass spectrometry signals generated by the corresponding internal standard peptide, wherein a difference in the normalized value for at least one of the signature peptides compared to a control is indicative of a prion-mediated pathological condition. In this aspect, extraction with a chaotropic agent and digestion in either a healthy or diseased sample result in production of the same signature prion peptides but each in different amounts when comparing healthy to diseased samples. Denaturing disease-related prion protein allows release of signature diagnostic peptides from the core region that would otherwise be resistant to protease digestion. Thus, in this case, core peptides are differentially released when compared to methods that do not include a denaturing agent The mass spectrometry-based methods of the present invention are useful for diagnostic analysis of the family of TSE diseases which includes, but not limited to, Creutzfeldt-Jakob disease (CJD) in humans, BSE (bovine spongiform encephalopathy) in cattle, scrapie in sheep, chronic wasting disease (CWD) in mule deer and elk, transmissible mink encephalopathy (TME), and feline spongiform encephalopathy (FSE) in cats. The intended application of the method can be employed for the monitoring of biological samples that are amenable to non-invasive collection such as serum, saliva, tears, urine, stool, semen, lactation fluid and other biological fluids. The methods provides for the detection and quantitation of prion isoforms, native (PrP C ) and aberrant (PrP SC ), in uninfected and TSE infected animals. The mass spectrometry methods of this invention can be used for the improved detection of prion induced neurodegenerative diseases in animals and humans through quantitation and verification of aberrant prion isoforms in sera, body fluids and in tissues samples. They can also be applied to detecting prion proteins in products derived from animals, and not just animals afflicted with a prion-mediated disease. Hence, a further aspect of the present invention is directed to a method of detecting an aberrant prion protein in a product of human or animal origin, comprising: (a) obtaining a sample from a product of human or animal origin; (b) extracting prion proteins from the sample; (c) digesting the extracted prion proteins to produce peptide fragments of the extracted prion proteins, wherein the fragments include signature peptides at least one of which is differentially released from an aberrant prion protein compared to a normal prion protein; (d) analyzing the peptide fragments and for each of the signature peptides, a corresponding internal standard peptide, via mass spectrometry; and (e) generating for each signature peptide, a normalized value obtained by comparing mass spectrometry signals generated by the signature peptide with mass spectrometry signals generated by the corresponding internal standard, wherein a difference between the normalized value for the signature peptide that is differentially released and a normalized value for the signature peptide that is not differentially released, or wherein a difference between the normalized value for the signature peptide that is differentially released and a control, is indicative of presence of an aberrant prion protein in the product. In some embodiments, the digesting entails contacting extracted proteins of (b) with a non-specific proteinase under conditions to allow digestion of non-core prion peptides, followed by denaturing non-specific proteinase resistant core prion peptide in the presence of a denaturing agent, followed by contacting denatured core peptide with a protease, and wherein in (e) the normalized value for the signature peptide that is differentially released is compared to a control. In a related aspect, the present invention provides a method of detecting an aberrant prion protein in a product of human or animal origin, comprising: (a) obtaining a sample from a product of human or animal origin; (b) extracting prion proteins from the sample using a chaotropic agent so as to produce denatured prion proteins; (c) digesting the denatured prion proteins to produce a composition that contains peptide fragments of the prion proteins, wherein said fragments include signature peptides; (d) analyzing via mass spectrometry the signature peptides and for each signature peptide, a corresponding internal standard peptide; and (e) generating for each signature peptide, a normalized value obtained by comparing mass spectrometry signals generated by the signature peptide with mass spectrometry signals generated by the corresponding internal standard peptide, wherein a difference in the normalized value for at least one of the signature peptides compared to a control is indicative of presence of the aberrant prion protein in the product. The methods can be practiced on any product derived from humans or animals where there is risk of contamination with aberrant prion proteins. In some embodiments the sample is obtained from blood or a blood-derived factor, a commercial food product or ingredient thereof, feed, or cosmetic, nutraceutical or pharmaceutical or an ingredient of said cosmetic, nutraceutical or pharmaceutical. The present invention provides a relatively sensitive, reliable and verifiable detection and quantitation of diseased prion isoforms in diverse biological samples, with specific applications for non-invasive samples such as sera that may contain significantly lower concentrations of prion molecules. Unlike current immunological based protocols, the present invention does not require the lengthy and laborious production of antibodies, preparation and maintenance of a uniform antibody for kits nor suffer from false positive and negatives as a result of indirect measurement. The described invention provides for multiple, simultaneous, independent, high throughput analyses of the prion proteins, thereby significantly increasing the reliability of the diagnostic results obtained. The mass spectrometry method provides for the verification of prions, which reduces and can even eliminate false positives and negatives, particularly when testing samples that contain low concentrations of prion proteins and/or working near the limits of detection of analytical techniques. The technology is suitable for detection of prion proteins in different species as well as genetic variants that may arise in an animal population, particularly closely related variants. These advantages of the invention compared to existing immunological and other diagnostic methods are summarized in Table 1. TABLE 1 Comparison of Diagnostic Methods for Prions Detection Method Sensitivity Confidence Throughput Immunocytochemistry ng, qualitative high low ELISA (Two-Site) ng-pg, quantitative high high Prionics Western Blot ng, qualitative adequate moderate Capillary Peptide pg-fg, adequate moderate Competition semi-quantitative MS Diagnostics pg-fg, quantitative very high high Sensitivity: Order from best to lowest - fg > pg > ng A yet further aspect of the invention is directed to a kit for the detection or quantification of prion protein in specific sample types. It provides the user with reagents to analyze a particular prion target protein. Thus, in preferred embodiments, the kit contains extraction buffer(s), enrichment resin(s), protease(s), synthetic signature diagnostic peptide(s) and internal standard peptide(s) corresponding to the signature peptide(s), and precise instructions on their use. |
Fantasy lamp comprising a light-permeable, liquid-containing hollow chamber and a multiturn drive for said hollow chamber |
This invention relates to a fantasy lamp comprising a narrow, transparent hollow chamber (15) which receives at least two immiscible liquids and the back of which can be illuminated, wherein the hollow chamber comprises wall sections (13, 14) disposed at a slight distance from each other. The hollow chamber can be rotated about a substantially horizontal axis (10) when the wall sections are aligned substantially vertically. At least one of the liquids is coloured. An electric motor drive (4), which is mounted in a chassis (1), is provided for rotating the hollow chamber (9). The hollow chamber (15) forms a component of a cover (9), and the drive (4) cooperates with a radially outer region (18) of the cover (9). When the hollow chamber is slowly rotated about the substantially horizontal axis, particular aesthetic effects can be achieved due to the liquids by means of a fantasy lamp which is fashioned in this manner. The lamp is of simple construction and can be manufactured as a mass-produced product in an economically acceptable manner. |
1. A fantasy lamp comprising a narrow, transparent, hollow chamber (15) which contains at least two immiscible liquids (26, 27) and on the back of which a means of illumination (8) is disposed, wherein the hollow chamber (15) comprises wall sections (13, 14) disposed at a slight distance from each other, wherein when the wall sections (13, 14) are aligned substantially vertically the hollow chamber (15) can rotate about a substantially horizontal axis (10), and an electric motor drive (4) is provided for rotating the hollow chamber (15) and is mounted in a chassis (1), and at least one of the liquids is coloured, characterised in that the hollow chamber (15) forms a component of a cover (9) and the drive (4) cooperates a with radially outer region (18) of the cover (9). 2. A lamp according to claim 1, characterised in that the cover (9) is suspended in relation to the drive (4). 3. A cover according to claims 1 or 2, characterised in that the drive (4) cooperates with the cover (9) at a distance from the top vertex of the cover (9). 4. A lamp according to any one of claims 1 to 3, characterised in that at least one guide element (20) for the cover (9) is disposed at a distance from the drive (4) in a radially outer region (18) of the cover (9). 5. A lamp according to claim 4, characterised in that a plurality of guide elements (20) is provided spaced apart from each other in order to stabilise the cover (9), wherein at least one of the guide elements (20) can be moved from an active position with the cover (9). 6. A lamp according to any one of claims 1 to 5, characterised in that the electric motor drive (4) comprises gearing. 7. A lamp according to any one of claims I to 6, characterised in that a drive element (6) of the drive engages in a complementary element (19, 28, 31) which is attached to the cover (9). 8. A lamp according to claim 7, characterised in that the drive element is fashioned as a wheel, a roller, a gearwheel or the like, and the complementary element is a fashioned as a rail (19), a rack (31) of curved construction, a recess (28) in the cover or the like. 9. A lamp according to any one of claims 1 to 8, characterised in that the cover (9) is of rotationally symmetrical form. 10. A lamp according to claim 9, characterised in that the cover (9) of rotationally symmetrical form comprises an outer, substantially axial edge (18) which is provided with the encircling complementary element (19, 28, 31). 11. A lamp according to any one of claims 7 to 10, characterised in that in the region of its radially outer edge (18) the cover (9) comprises an encircling bulge (17) which is oriented away from the chassis (1) and through which the drive element (6) of the drive (4) passes. 12. A lamp according to any one of claims 1 to 11, characterised in that at least one means of illumination (8) is disposed inside the cover (9). 13. A lamp at according to any one of claims 1 to 12, characterised in that the wall sections (13, 14) are of curved construction and are parallel, and a film (22) is inserted in the hollow chamber (15), wherein the film (22) is planar before it is inserted between the wall sections. 14. A lamp according to claim 13, characterised in that the wall sections (13, 14) of the hollow chamber are constructed as a spherical shells. 15. A lamp according to claims 13 or 14, characterised in that the film (22) is circular before it is inserted between the wall sections (13). 16. A lamp according to any one of claims 13 to 15, characterised in that funnel-shaped voids (25) are formed between the film (22) and the wall sections (13, 14) of the hollow chamber (15) and diminish towards the centre of the hollow chamber (15). 17. A lamp according to any one of claims 1 to 16, characterised in that the cover (9) comprises a radially outer cover part (33) and a radially inner cover part (34), wherein the inner cover part (34) is substantially formed by the two wall sections (13, 14). 18. A lamp according to claim 17, characterised in that the inner cover part (34) constitutes a prefabricated component, which is filled with the liquids (26, 27) and/or which is provided with the film (22), and to which the outer cover part (33) is attached, particularly by welding or adhesive bonding. |
Lithium polymer secondary cell |
A lithium polymer secondary battery comprising a negative electrode, a positive electrode and an electrolyte layer between both electrodes, wherein the negative electrode and the positive electrode each has a solid electrolyte prepared by incorporating an organic electrolytic solution into a polymer, the polymer is obtained by solidifying a precursor solution containing vinylene carbonate and a content of vinylene carbonate in the precursor solution for the positive electrode is smaller than that in the precursor solution for the negative electrode. |
1. A lithium polymer secondary battery comprising a negative electrode, a positive electrode and an electrolyte layer between both electrodes, wherein the negative electrode and the positive electrode each has a solid electrolyte prepared by incorporating an organic electrolytic solution into a polymer, the polymer is obtained by solidifying a precursor solution containing vinylene carbonate and a content of vinylene carbonate in the precursor solution for the positive electrode is smaller than that in the precursor solution for the negative electrode. 2. A lithium polymer secondary battery according to claim 1, in which a content of vinylene carbonate is 7 wt % or less in the precursor solution for the positive electrode while a content of vinylene carbonate is 10 wt % or less in the precursor solution for the negative electrode. 3. A lithium polymer secondary battery according to claim 1, in which the positive electrode and the negative electrode are different from each other in respect of kind of the polymer or the organic electrolytic solution composing the solid electrolyte, or in respect of composition of the organic electrolytic solution. 4. A lithium polymer secondary battery according to claim 1, in which the solid electrolyte contains γ-butylolactone as an organic solvent. 5. A lithium polymer secondary battery according to claim 1, in which the negative electrode contains a graphite as an active material, the solid electrolyte of the negative electrode contains at least ethylene carbonate as an organic solvent. 6. A fabrication method for a lithium polymer secondary battery of claim 1 comprising the steps of separately solidifying precursor solutions of polymers included in solid electrolytes of a positive electrode and a negative electrode; and thereafter, sticking the positive electrode and the negative electrode to each other with an electrolyte layer interposed therebetween. 7. A fabrication method for a lithium polymer secondary battery of claim 1 comprising the steps of preliminary-solidifying a precursor solution of a polymer included in a solid electrolyte of one of a positive electrode and a negative electrode, or separately preliminary-solidifying precursor solutions of polymers included in the solid electrolytes of the positive electrode and the negative electrode, thereafter, sticking the positive electrode and the negative electrode to each other with an electrolyte layer interposed therebetween and furthermore, heat treating the composite of the electrodes with the electrolyte layer to thereby solidify the precursor solutions preliminary-solidified. |
<SOH> TECHNICAL FIELD <EOH>The present invention relates to a lithium polymer secondary battery. More particularly, the present invention relates to a lithium polymer secondary battery characterized by a content of vinylene carbonate in a precursor solution for use in obtaining a polymer included in a solid electrolyte, and had an excellent load characteristic, cycle characteristic and low temperature characteristic. Prior Art A secondary battery has been well used as a power supply for a portable device in consideration of cost effectiveness and others. Although various kinds of secondary batteries have been available, a nickel-cadmium battery is the most popular one at present and a nickel-hydrogen battery has also recently come to gain its popularity. Since a lithium secondary battery is higher in output voltage and energy density than the secondary batteries described above, it has been acquiring a place in the main stream. The lithium secondary battery employs: as a positive electrode active material, lithium cobalt oxide LiCoO 2 , lithium nickel oxide LiNiO 2 , a solid solution thereof. Li(Co 1-x Ni x )Q 2 , LiMn 2 O 4 of a spinel structure or the like; as a negative electrode active material, a carbon material such as graphite; and an organic electrolytic solution containing a liquid organic compound as a solvent and a lithium compound as a solute. A current version of the lithium secondary battery is perfectly sealed in a metal can or the like in order to prevent liquid leakage. In recent years, much of research activities have been directed to the lithium secondary battery using a solid electrolyte instead of an organic electrolytic solution. Since such a battery employs a solid electrolyte, no worry occurs of liquid leakage even by sealing with an easy-to-use resin film or the like, without sealing by using a metal can or the like to perfectness. Such a lithium secondary battery has a possibility of reduction of thickness in a battery as a feature. While there have been available some kinds of solid electrolytes described above, an attention is focused on gel solid electrolytes each retaining an electrolytic solution prepared by dissolving a lithium salt or the like into a non-aqueous solvent in a polymer, as a solid electrolyte meeting a performance required in a battery in recent years. The gel solid electrolytes are classified into two categories, in one of which a polymer such as a fluorine containing polymer represented by polyvinylidene fluoride (PVdF) or a polyacrylonitrile (PAN) is impregnated with an electrolytic solution prepared by dissolving a lithium salt or the like into an organic solvent to thereby create a gel state (a physical gel) and in the other of which polymerization is performed by giving energy such as heat or light to a solution prepared by mixing a monomer having at least one or more unsaturated double bonds in a molecule and an electrolytic solution prepared by dissolving a lithium salt or the like into a non-aqueous solvent together to thereby obtain a gel solid electrolyte (chemical gel). Since any of gel solid electrolytes is, however, lower in ionic conductivity, as compared with the electrolytic solution prepared by dissolving the lithium salt or the like into the organic solvent, there have been arisen problems that a load characteristic and a cycle characteristic of a battery are poor and a capacity thereof at low temperatures is low, as typical faults, in a case where gel solid electrolytes are used in batteries. Many of trials, in order to solve such problems, have been conducted about various additive agents in the electrolyte or the polymer. Among additive agents, vinylene carbonate has been generally known. As secondary batteries employing this additive agent, there have been known: batteries improved in an ionic conductivity and therefore, good in load characteristic (for example, Japanese Unexamined patent Publication No. HEI 10(1998)-223044, Japanese Unexamined patent Publication No. HEI 11(1999)-265616, Japanese Unexamined patent Publication No. 2000-82328, Japanese Unexamined patent Publication No. 2000-82496, Japanese Unexamined patent Publication No. 2000-67644 and others); batteries improved in cycle characteristic (for example, Japanese Unexamined patent Publication No. HEI 10(9998)-334946, Japanese Unexamined patent Publication No. 2000-67855 and others); and others. In any of secondary batteries of this kind, no satisfaction has yet been achieved simultaneously with respect to both performances of a cycle characteristic and a load characteristic. According to Japanese Unexamined patent Publication No. 2000-67855, it is described that improvement can be achieved simultaneously in both of performances of a cycle performance and a load characteristic. The number of cycles in the publication, however, is only on the order of 20 cycles, which cannot be said to be sufficient in consideration of a situation of practical use of a device. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a structural view shown a basic structure of a lithium polymer secondary battery of the present invention; and FIG. 2 is a model view of a structure of a lithium polymer secondary battery fabricated in Example 1. detailed-description description="Detailed Description" end="lead"? |
Dry etching method |
A tungsten silicide layer (104) is etched by plasma etching using Cl2+O2 gas as etching gas. When etching of the tungsten silicide layer (104) is ended substantially, etching gas is switched to Cl2+O2+NF3 and over etching is performed by plasma etching. Etching process is ended under a state where a polysilicon layer (103) formed beneath the tungsten silicide layer (104) is slightly etched uniformly. Residual quantity of the polysilicon layer (103) can be made uniform as compared with prior art and a high quality semiconductor device can be fabricated stably. |
1. An etching method, comprising the step of etching tungsten silicide formed directly on a silicon layer by employing an etching gas including Cl2 gas, O2 gas and NF3 gas, wherein the etching step is completed at a state where a part of the silicon layer remains. 2. An etching method, comprising the steps of etching tungsten silicide on silicon by employing an etching gas including Cl2 gas and O2 gas and thereafter etching the tungsten silicide on the silicon by employing an etching gas including Cl2 gas, O2 gas and NF3 gas. 3. The method of claim 2, wherein a ratio of a flow rate of O2 gas to that of Cl2 gas (a flow rate of O2 gas/a flow rate of Cl2 gas) is larger at the etching step employing the etching gas including Cl2 gas, O2 gas and NF3 gas than at the etching step employing the etching gas including Cl2 gas and O2 gas. 4. An etching method, comprising the step of etching tungsten silicide on silicon by employing an etching gas including Cl2 gas, O2 gas and NF3 gas, wherein a ratio of a flow rate of O2 gas to that of the whole etching gas is not less than 0.2 but not greater than 0.5 [0.2≦the flow rate of O2 gas/a flow rate of (Cl2 gas+O2 gas+NF3 gas)≦0.5]. 5. The method of claim 1, wherein the tungsten silicide is etched into a pattern having a dense pattern region where adjacent patterns are close to each other and a sparse pattern region where adjacent patterns are spaced apart from each other. 6. An etching method comprising the step of etching tungsten formed directly on a silicon layer by employing an etching gas including N2 gas and NF3 gas. 7. The method of claim 6, wherein the etching gas further includes O2 gas. 8. The method of claim 6, further comprising the step of etching the tungsten by employing an etching gas including Cl2 gas, O2 gas and NF3 gas, prior to the etching step employing the etching gas including N2 gas and NF3 gas. 9. An etching method comprising the step of etching tungsten formed directly on a silicon layer by employing an etching gas including O2 gas and NF3 gas. 10. The method of claim 6, wherein the tungsten is etched by using a silicon nitride layer as a mask. 11. An etching method, comprising the step of etching tungsten on silicon by employing an etching gas including Cl2 gas, O2 gas and NF3 gas, wherein a ratio of a flow rate of Cl2 gas to that of the whole etching gas is greater than 0 but not larger than 0.125 [0<the flow rate of Cl2 gas/the flow rate of (Cl2 gas+O2 gas+NF3 gas)≦0.125]. 12. The method of claim 11, further comprising, prior to the etching step, the preceding step of etching tungsten on silicon by employing an etchant gas, wherein a ratio of a Cl2 gas flow rate in the etchant gas to that of the whole etchant gas is greater than the ratio of the flow rate of Cl2 gas to that of the whole etching gas. 13. The method of claim 12, wherein the preceding step and the etching step are performed by using a parallel plate plasma etching apparatus including a lower electrode on which a substrate to be processed is mounted and to which a high frequency power is applied, the high frequency power applied to the lower electrode at the etching step is greater than that at the preceding step. 14. The method of claim 12, wherein a light having a wavelength of about 578 nm or about 542 nm is detected in plasma and the preceding step and the etching step are performed according to a change of the detected light. 15. The method of claim 6, wherein the tungsten is etched into a pattern having a dense pattern region where adjacent patterns are close to each other and a sparse pattern region where adjacent patterns are distant from each other. 16. The method of claim 11, wherein a barrier metal layer is placed between the silicon and the tungsten. 17. An etching method, comprising the step of etching tungsten and a barrier metal layer formed on a silicon layer by employing an etching gas including N2 gas and NF3 gas. 18. The method of claim 17, wherein the etching gas further includes O2 gas. 19. The method of claim 17, further comprising the step of etching the tungsten by employing an etching gas including Cl2 gas, O2 gas and NF3 gas, prior to the etching step employing the etching gas including N2 gas and NF3 gas. 20. An etching method, comprising the step of etching tungsten and a barrier metal layer formed on a silicon layer by employing an etching gas including O2 gas and NF3 gas. 21. The method of claim 17, wherein the tungsten is etched by using a silicon nitride layer as a mask. 22. The method of claim 17, wherein the tungsten is etched into a pattern having a dense pattern region where adjacent patterns are close to each other and a sparse pattern region where adjacent patterns are spaced apart from each other. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Recently, tungsten silicide and tungsten are widely used as, e.g., an electrode material of a semiconductor device. And, a polycide structure, in which metal silicide, e.g., tungsten silicide, is deposited on a polysilicon (polycrystalline silicon) layer, is widely used as a gate electrode of an MOS transistor in a semiconductor device. For the fabrication of such a gate electrode of the polycide structure, a gate oxide (SiO 2 ) film 202 , a polysilicon layer 203 , a tungsten silicide layer 204 are formed sequentially in that order on a silicon substrate 201 , and a patterned mask layer 205 made of, e.g., a silicon nitride film and a photoresist is formed on the tungsten silicide layer 204 , as shown in FIG. 10A . Then, the tungsten silicide layer 204 is patterned first by being etched through the mask layer 205 . A plasma etching employing an etching gas containing, e.g., Cl 2 +O 2 is conventionally used in such an etching process of the tungsten silicide layer 204 . Further, during the etching process of the tungsten silicide layer 204 , a certain degree of an overetching is generally carried out to remove stepped portions and a certain amount of surface portion of the polysilicon layer 203 is also etched by the overetching process as shown in FIG. 10B . And, the polysilicon layer 203 is etched after etching the tungsten silicide layer 204 , so that a polycide structure having a predetermined pattern can be obtained. As described above, a single step plasma etching process employing an etching gas containing, e.g., Cl 2 +O 2 is generally used during a conventional etching process of the tungsten silicide layer. In the above-mentioned conventional method, however, it is difficult to increase the selectivity of tungsten silicide with respect to polysilicon because a patterned shape is deteriorated and residues are generated when a condition is set for enhancing the selectivity of tungsten silicide with respect to polysilicon. Thus, a large amount of the underlying polysilicon layer is etched during the overetching process and there occurs a large variation in the residual amount of polysilicon layer remaining on a wafer surface after etching (designated by R in FIG. 10B ). The above-mentioned problem is aggravated especially in a case where a shape of the patterns thus formed has a dense pattern region in which adjacent patterns are close to each other and a sparse pattern region in which adjacent patterns are spaced apart from each other. That is to say, an etching rate of the tungsten silicide varies between the dense pattern region (a diameter of a patterned hole:the distance between adjacent patterned holes=1:0.8 to 1:1) and the sparse pattern region (a diameter of a patterned hole:the distance between adjacent patterned holes=1:10 to 1:10000). The variation of etching rate in turn causes a variation of time during which the underneath polysilicon layer is exposed. The polysilicon layer is etched more at a region where the polysilicon layer is exposed earlier, so that the residual amount of polysilicon layer is less thereat; and the polysilicon layer is etched less at a region where the polysilicon layer is exposed later, so that the residual amount of polysilicon layer is greater thereat, resulting in a large variation in the residual amount of polysilicon layer. If the residual amount of polysilicon layer is varied a lot as described above, during a subsequent step of etching the polysilicon layer, the underlying gate oxide film is exposed earlier at a region where the polysilicon layer is left in a smaller amount than at a region where the polysilicon layer remains in a larger amount. This causes a variation of time during which the gate oxide film is exposed. As a result, an earlier-exposed region of the gate oxide film is damaged to become less adequate to serve as the gate oxide film, lowering a production yield and quality. |
<SOH> SUMMARY OF THE INVENTION <EOH>It is, therefore, an object of the present invention to provide a dry etching method capable of leveling residual amounts of polysilicon layers compared with the cases in the prior art to thereby reliably fabricate high quality semiconductor devices. In accordance with the invention, there is provided an etching method including the step of etching tungsten silicide formed directly on a silicon layer by employing an etching gas containing Cl 2 gas, O 2 gas and NF 3 gas, wherein the etching step is completed at a state where a part of the silicon layer remains. Further, in accordance with the invention, there is provided an etching method including the steps of etching tungsten silicide on silicon by employing an etching gas containing Cl 2 gas and O 2 gas and thereafter etching the tungsten silicide on the silicon by employing an etching gas containing Cl 2 gas, O 2 gas and NF 3 gas. Still further, in accordance with the invention, there is provided an etching method, wherein a ratio of a flow rate of O 2 gas to that of Cl 2 gas (a flow rate of O 2 gas/a flow rate of Cl 2 gas) is larger at the etching step employing the etching gas containing Cl 2 gas, O 2 gas and NF 3 gas than at the etching step employing the etching gas containing Cl 2 gas and O 2 gas. Still further, in accordance with the invention, there is provided an etching method, wherein a ratio of the flow rate of O 2 gas to that of the whole etching gas of the etching step employing the etching gas containing Cl 2 gas, O 2 gas and NF 3 gas is not less than 0.2 but not greater than 0.5 [0.2≦the flow rate of O 2 gas/a flow rate of (Cl 2 gas+O 2 gas+NF 3 gas)≦0.5]. Still further, in accordance with the invention, there is provided an etching method, wherein the tungsten silicide is etched into a pattern having a dense pattern region where adjacent patterns are close to each other and a sparse pattern region where adjacent patterns are spaced apart from each other. Still further, in accordance with the invention, there is provided an etching method including the step of etching tungsten formed directly on a silicon layer by employing an etching gas containing N 2 gas and NF 3 gas. Still further, in accordance with the invention, there is provided an etching method including the step of etching tungsten formed on a silicon layer via a barrier metal layer by employing an etching gas including N 2 gas and NF 3 gas. Still further, in accordance with the invention, there is provided an etching method, wherein the etching gas further contains O 2 gas. Still further, in accordance with the invention, there is provided an etching method further including the step of etching tungsten on silicon by employing an etching gas containing Cl 2 gas, O 2 gas and NF 3 gas, prior to the etching step employing the etching gas containing N 2 gas and NF 3 gas. Still further, in accordance with the invention, there is provided an etching method including the step of etching tungsten formed directly on a silicon layer by employing an etching gas containing O 2 gas and NF 3 gas. Still further, in accordance with the invention, there is provided an etching method including the step of etching tungsten formed on a silicon layer via a barrier metal layer by employing an etching gas including O 2 gas and NF 3 gas. Still further, in accordance with the invention, there is provided an etching method, wherein the tungsten is etched by using a silicon nitride layer as a mask. Still further, in accordance with the invention, there is provided an etching method including the step of etching tungsten on silicon by employing an etching gas containing Cl 2 gas, O 2 gas and NF 3 gas, wherein a ratio of a flow rate of Cl 2 gas to that of the whole etching gas is greater than 0 but not larger than 0.125 [0<the flow rate of Cl 2 gas/the flow rate of (Cl 2 gas+O 2 gas+NF 3 gas)≦0.125]. Still further, in accordance with the invention, there is provided an etching method further including, prior to the etching step, the preceding step of etching tungsten on silicon by employing an etchant gas, wherein a ratio of a Cl 2 gas flow rate in the etchant gas to that of the whole etchant gas is greater than the ratio of the flow rate of Cl 2 gas to that of the whole etching gas. Still further, in accordance with the invention, there is provided an etching method, wherein the preceding step and the etching step are performed by using a parallel plate plasma etching apparatus having a lower electrode on which a substrate to be processed is mounted and to which a high frequency power is applied, the high frequency power applied to the lower electrode at the etching step is greater than that at the preceding step. Still further, in accordance with the invention, there is provided an etching method, wherein a light having a wavelength of about 578 nm or about 542 nm is detected in plasma and the preceding step and the etching step are performed according to a change of the detected light. Still further, in accordance with the invention, there is provided an etching method, wherein the tungsten is etched into a pattern having a dense pattern region where adjacent patterns are close to each other and a sparse pattern region where adjacent patterns are distant from each other. Still further, in accordance with the invention, there is provided an etching method, wherein a barrier metal layer is placed between the silicon and the tungsten. |
Process for the preparation of cefpodoxime acid |
The present invention relates to an improved and cost effective process for the industrial preparation of cefpodoxime acid of Formula (I) and a pharmaceutically acceptable ester thereof. |
1. A process for the preparation of cefpodoxime acid of Formula I, and a pharmaceutically acceptable ester thereof, comprising (i) reacting a compound of Formula VI, wherein R is hydrogen or a silyl group and R′ is a silyl group or COOR′ is a carboxylic acid salt, with a compound of Formula IV, or its reactive acid derivatives, wherein X is a halogen, to obtain a compound of Formula VII, wherein X and R′ are as defined above; (ii) desilylating or acidifying the compound of Formula VII to isolate the compound of formula V; and (iii) reacting the compound of Formula V with thiourea in aqueous medium in the presence of a weak base to obtain cefpodoxime acid of Formula I. 2. The process according to claim 1 wherein both R and R′ are trimethylsilyl in the compound of Formula VI. 3. The process according to claim 1 wherein X is chloro or bromo in the compound of Formula IV. 4. The process according to claim 1 wherein the reactive derivative of Formula IV is acid chloride. 5. The process according to claim 1 wherein the reaction of step (iii) in aqueous medium comprises reacting in water and a water-miscible organic solvent. 6. The process according to claim 5 wherein the water-miscible organic solvent is selected from the group consisting of ethanol, methanol, isopropanol, acetone, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, or a mixture thereof. 7. The process according to claim 5 wherein the reaction of step (iii) is carried out in water alone. 8. The process accounting to claim 1 wherein the weak base in step (iii) is selected from the group consisting of sodium acetate or sodium bicarbonate. 9. The process according to claim 1 wherein in step (iii), the compound of Formula V is added to an aqueous solution of sodium acetate or sodium bicarbonate at a temperature of about 0 to 50° C. 10. The process according to claim 1 wherein in step (iii), the thiourea is added at a temperature of about 0 to 10° C. 11. The process according to claim 1 wherein the reaction of step (iii) is carried out at a temperature of about 10 to 20° C. 12. The process according to claim 1 wherein the cefpodoxime acid is obtained at a pH of about 2.5 to 3.0. 13. The process according to claim 1 wherein the cefpodoxime acid of Formula I is reacted with 1 -iodoethylisopropyl carbonate in the presence of 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) in N,N-dimethylformamide to give cefpodoxime proxetil of Formula II |
<SOH> BACKGROUND OF THE INVENTION <EOH>Chemically, cefpodoxime acid is [(6R-[6α,7β(Z)]]-7-[2-(2-aminothiazol-4-yl)-2-methoxyimino) acetamido]-3-cephem-4-carboxylic acid having Formula I, and is known from U.S. Pat. No. 4,409,215. Although cefpodoxime acid is not suitable for oral administration, its ester derivative, 1-(isoproxycarbonyloxyl)ethyl ester i.e. cefpodoxime proxetil of Formula II, is a valuable orally administered antibiotic characterized by high broad spectrum activity against gram positive and gram negative microorganisms. A number of processes have been outlined in U.S. Pat. Nos. 4,409,215, 5,109,131, GB 2012276 and WO 00/63214 for the preparation of cepholosporin antibiotics. However, attempts to extend these processes for preparing cefpodoxime acid at an industrial scale did not give the desired results with respect to yield and quality. More particularly, the synthetic process comprising coupling of reactive acid derivative of compound of Formula III, with a reactive derivative of an open chain compound of Formula IV, wherein X is a halogen selected from chloro, bromo and iodo, to get a compound of Formula V, and its subsequent cyclization with thiourea to obtain cefpodoxime acid of Formula I, was found to be unsatisfactory at a commercial scale. Processes described in U.S. Pat. No. 4,409,215 and GB 2012276 require protection at the carboxylic position of the compound of Formula III followed by the steps of coupling, cyclization and hydrolysis to get cefpodoxime acid. The additional steps of protection and deprotection result in low yields and high costs. The processes described in PCT Application WO 00/63214 and U.S. Pat. No. 5,109,131 require formation of compound of Formula V and its subsequent cyclization with thiourea in a mixture of organic solvent and water to afford cefpodoxime acid. Cefpodoxime acid thus obtained is of poor quality and contains anti isomer of cefpodoxime acid as a major impurity. Accordingly, none of the processes described heretofore are completely satisfactory for various reasons. |
<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to provide a process for the preparation of cefpodoxime acid and a pharmaceutically acceptable ester thereof in good yields and high purity (99%) by HPLC. The process is simple and provides obvious benefits with respect to economics and convenience to operate at a commercial scale. Accordingly, the present invention provides a process for the preparation of cefpodoxime acid of Formula I and a pharmaceutically acceptable ester thereof comprising: (i) reacting a compound of Formula VI, wherein R is hydrogen or a silyl group and R′ is a silyl group or COOR′ is a carboxylic acid salt, with a compound of Formula IV, or its reactive acid derivatives, wherein X is a halogen, to obtain a compound of Formula VII, wherein X and R′ are as defined above; (ii) desilylating or acidifying the compound of Formula VII to isolate the compound of formula V; and (iii) reacting the compound of Formula V with thiourea in aqueous medium in the presence of a weak base to obtain cefpodoxime acid of Formula I. Cefpodoxime acid, so obtained may be converted into its ester such as cefpodoxime proxetil by methods known in the art. The carboxylic acid salts of Formula VI include salts with a metal such as sodium or potassium, or salt with an organic amine such as triethylamine, pyridine, diclyclohexylamine or N, N-dimethylaniline. R and R′ in the compound of Formula VI may be silyl groups which may be same or different. Suitable silyl groups are trialkyl silyl groups wherein the alkyl substitutents may be same or different. Preferred alkyl substituents are methyl, ethyl, isopropyl and tert-butyl. Preferred silyl groups are trimethylsilyl and tert-butyidimethylsilyl. X in the compounds of Formula IV, V and VIII is a halogen selected from chloro, bromo and iodo. X is preferably bromo. The reactive acid derivatives of Formula IV include the acid halides, the acid anhydride, mixed acid anhydrides, reactive esters, reactive amides and the acid azide. Preferred mixed acid anhydrides include anhydrides with lower alkanoic acids such as pivalic acid, trichloroacetic acid and anhydrides with a carbonic acid such as monomethylcarbonate. Preferred reactive esters include p-nitrophenylester, N-hydroxysuccinimido ester, N-hydroxyphthalimido ester, 2-mercaptobenzothioazolyl ester and 2-mercapto-5-methyl-1,3,4-thiadiazolyl ester. Among the reactive acid derivatives of Formula IV, acid halides are preferred. Where the compound of Formula IV is employed in the form of a free acid, the reaction step (i) is carried out in the presence of a condensing agent such as dicylohexylcarbodiimide, or a “Vilsmeier reagent” formed by the reaction of an amide compound such as dimethylformamide with a halogen compound such as phosphorous oxychloride. Where a reactive derivative of the acid of Formula IV is employed, the use of such a condensing agent is not required, however, it may be desirable to carry out the reaction in the presence of a base. Examples of suitable bases include alkali metal compound such as sodium bicarbonate, sodium carbonate and potassium carbonate or an organic amine such as triethylamine, lutidine or pyridine. The reaction of step (i) is usually carried out in a suitable solvent. When R, R′ or both are silyl in the compound of Formula VI, suitable solvents for the reaction include halogenated hydrocarbons such as methylene chloride, hydrocarbons such as toluene, ethers such as tetrahydrofuran or polar solvents such as dimethylformamide, or a mixture thereof. When R is hydrogen and COOR′ is a carboxylic acid salt in the compound of Formula VI, suitable solvents for the reaction include methanol, ethanol, acetonitrile, dimethylformamide, water, or a mixture thereof. The starting compounds of Formula VI wherein R, R′ or both are silyl may be obtained by silylating the corresponding 7-amino-3-methoxymethyl 3-cephem-4-carboxylic acid of Formula III with a suitable silylating agent. Appropriate silylating agents include halosilanes such as trimethylsilylchloride (TMCS), dimethyidichlorosilane (DMDCS), silylated amides such as N, 0-bistrimethylsilyl acetamide (BSA), silazanes such as 1,1,1,3,3,3-hexamethyidisilazane (HMDS), silylated ureas such as N, N′-bis-(trimethylsilyl) urea (BSU), or a mixture thereof Where COOR′ is a carboxylic acid salt in the compound of Formula VI, it may be obtained in a conventional manner, for example by treatment of a compound of Formula III with a base such as sodium bicarbonate, triethylamine etc. Compounds of Formula III and IV may be obtained by methods known in the art. The desilylation (step ii) of the compound of Formula VII (wherein R′ is a silyl group) may be carried out according to conventional methods such as treatment with methanol/water to isolate compound of Formula V. We believe that the isolation of the compound of Formula V plays a crucial role in obtaining the compound of Formula I in high yields and good quality. The reactions of steps (i) and (ii) result in the formation of impurities alongwith the desired product which are automatically removed during the isolation of compound of Formula V. The reaction of a compound of Formula V with thiourea is carried out in the presence of a weak base such as sodium acetate and sodium bicarbonate in an aqueous medium comprising water and a water-miscible organic solvent such as ethanol, methanol, isopropanol, acetone, tetrahydrofuran, acetonitrile, N, N-dimethylformamide, or a mixture thereof. The compound of Formula V is added to an aqueous solution of a weak base at a temperature of about 0 to 5° C. Thereafter, an aqueous solution of thiourea is added to the above mixture at a temperature of about 0 to 10° C. The reaction may then be carried out a temperature of about 0to 60° C., preferably at 0-25° C., more preferably at 10-20° C. Cefpodoxime acid of purity 99% is obtained by washing the reaction mixture with ethyl acetate and acidifying the aqueous layer to a pH of about 2.5 to 3. However, the reaction of compound of Formula V with thiourea is best carried out in water since a mixture of solvent and water may carryover impurities to the aqueous layer which may then precipitate along with the desired product. Also, lower yields are obtained as cefpodoxime acid is soluble in the water-miscible solvents mentioned above. Cefpodoxime acid so obtained may be converted to cefpodoxime proxetil by methods known in the art such as reaction with 1-iodoethylisopropyl carbonate in the presence of 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) in N, N-dimethylformamide. detailed-description description="Detailed Description" end="lead"? |
Sweet microwave popcorn product and method for production thereof |
A sweet microwave popcorn product is provided that can be prepared by the consumer in a single step. The product comprises a microwaveable container; a plurality of unpopped corn kernels in the container; and a plurality of sugar pellets in the container. Each sugar pellet comprises sugar in an amount of at least about 15% by weight, based on the total weight of the sugar pellet. The sugar pellets are substantially free of an emulsifying agent. |
1. A sweet popcorn product suitable for popping in a microwave oven, comprising: a microwaveable container having a bottom region, a middle region adjacent the bottom region, a top region adjacent the middle region; a plurality of unpopped corn kernels in the container; and a plurality of sugar pellets in the container, wherein each sugar pellet comprises sugar and corn syrup, each sugar pellet having sugar in an amount of at least about 15% by weight, based on the total weight of the sugar pellet, the sugar pellets are free of an emulsifying agent and not homogeneously mixed with the unpopped kernels, and a majority of the sugar pellets is contained within a portion of the middle region nearest one of the bottom region or the top region, and a majority of the unpopped corn kernels is contained within a portion of the middle region nearest the other of the regions. 2. (canceled) 3. (canceled) 4. (canceled) 5. A microwave popcorn product according to claim 4, wherein the ratio of sugar to corn syrup in each sugar pellet ranges from about 1:1 to about 5:1. 6. A microwave popcorn product according to claim 1, wherein the unpopped corn kernels are present in a total amount ranging from about 15% to about 95% by weight based on the total weight of the product excluding the container. 7. A microwave popcorn product according to claim 1, wherein the sugar pellets are present in a total amount ranging from about 1% to about 70% by weight based on the total weight of the product excluding the container. 8. A microwave popcorn product according to claim 1, wherein each sugar pellet has a moisture content no greater than about 3.5%. 9. A microwave popcorn product according to claim 1, wherein each sugar pellet has a moisture content no greater than about 2.5%. 10. A microwave popcorn product according to claim 1, wherein the sugar pellets are prepared according to the process comprising: admixing sugar and corn syrup to produce an admixture; reducing the moisture content of the admixture by heating the admixture to a temperature ranging from about 110° C. to about 170° C.; and forming the heated admixture into the sugar pellets. 11. A microwave popcorn product according to claim 10, wherein the process further comprises subjecting the heated admixture to a vacuum to further reduce the moisture content of the admixture. 12. A microwave popcorn product according to claim 1, wherein the product further comprises an oil component in the container, wherein the oil component is not homogeneously mixed with the sugar pellets. 13. A microwave popcorn product according to claim 12, wherein the oil component comprises partially hydrogenated soybean oil, salt, color, butter flavor and sucralose. 14. A microwave popcorn product according to claim 1, wherein the product further comprises salt in the container. 15. A microwave popcorn product according to claim 14, wherein the salt is present in a total amount up to about 10% by weight based on the total weight of the product excluding the container. 16. A microwave popcorn product according to claim 1, wherein the microwaveable container does not include a susceptor. 17. A microwave popcorn product according to claim 16, wherein the sugar pellets are present in a total amount ranging from about 10% to about 70% by weight based on the total weight of the product excluding the container. 18. A microwave popcorn product according to claim 1, wherein the microwaveable container includes a susceptor. 19. A microwave popcorn product according to claim 18, wherein the microwaveable container comprises a bag having a top surface and a bottom surface, and further wherein the susceptor is generally flat and attached to the bottom surface of the bag. 20. A microwave popcorn product according to claim 19, wherein the susceptor extends over at least about 40% of the length of the bag. 21. A microwave popcorn product according to claim 19, wherein the susceptor extends over at least about 50% of the length of the bag. 22. A microwave popcorn product according to claim 21, wherein the unpopped corn kernels are present in a total amount ranging from about 40% to about 95% by weight based on the total weight of the product excluding the container. 23. A microwave popcorn product according to claim 21, wherein the unpopped corn kernels are present in a total amount ranging from about 45% to about 75% by weight based on the total weight of the product excluding the container. 24. A microwave popcorn product according to claim 21, wherein the sugar pellets are present in a total amount ranging from about 1% to about 10% by weight based on the total weight of the product excluding the container. 25. A microwave popcorn product according to claim 21, wherein the sugar pellets are present in a total amount ranging from about 2% to about 4% by weight based on the total weight of the product excluding the container. 26. A microwave popcorn product according to claim 21, wherein the product further comprises an oil component present in a total amount ranging from about 10% to about 45% by weight based on the total weight of the product excluding the container. 27. A microwave popcorn product according to claim 21, wherein the product further comprises an oil component present in a total amount ranging from about 20% to about 40% by weight based on the total weight of the product excluding the container. 28. A microwave popcorn product according to claim 21, wherein: the unpopped corn kernels are present in a total amount ranging from about 40% to about 95% by weight based on the total weight of the product excluding the container; the sugar pellets are present in a total amount ranging from about 1% to about 10% by weight based on the total weight of the product excluding the container; and the product further comprises an oil component present in a total amount ranging from about 10% to about 45% by weight based on the total weight of the product excluding the container. 29. A microwave popcorn product according to claim 21, wherein: the unpopped corn kernels are present in a total amount ranging from about 45% to about 75% by weight based on the total weight of the product excluding the container; the sugar pellets are present in a total amount ranging from about 2% to about 4% by weight based on the total weight of the product excluding the container; and the product further comprises an oil component present in a total amount ranging from about 20% to about 40% by weight based on the total weight of the product excluding the container. 30. A microwave popcorn product according to claim 19, wherein the susceptor covers at least about 90% of the width of the bag. 31. A microwave popcorn product according to claim 19, wherein the susceptor covers from about 25% to about 50% of the length of the bag. 32. A microwave popcorn product according to claim 19, wherein the susceptor covers from about 30% to about 40% of the length of the bag. 33. A microwave popcorn product according to claim 19, wherein the susceptor covers from about 75% to about 85% of the width of the bag. 34. A microwave popcorn product according to claim 19, wherein the unpopped corn kernels are present in a total amount ranging from about 20% to about 80% by weight based on the total weight of the product excluding the container. 35. A microwave popcorn product according to claim 19, wherein the unpopped corn kernels are present in a total amount ranging from about 25% to about 55% by weight based on the total weight of the product excluding the container. 36. A microwave popcorn product according to claim 19, wherein the sugar pellets are present in a total amount ranging from about 5% to about 35% by weight based on the total weight of the product excluding the container. 37. A microwave popcorn product according to claim 19, wherein the sugar pellets are present in a total amount ranging from about 15% to about 25% by weight based on the total weight of the product excluding the container. 38. A microwave popcorn product according to claim 19, wherein the product further comprises an oil component present in a total amount ranging from about 20% to about 50% by weight based on the total weight of the product excluding the container. 39. A microwave popcorn product according to claim 19, wherein the product further comprises an oil component present in a total amount ranging from about 30% to about 50% by weight based on the total weight of the product excluding the container. 40. A microwave popcorn product according to claim 19, wherein: the unpopped corn kernels are present in a total amount ranging from about 25% to about 55% by weight based on the total weight of the product excluding the container; the sugar pellets are present in a total amount ranging from about 15% to about 25% by weight based on the total weight of the product excluding the container; and the product further comprises an oil component present in a total amount ranging from about 30% to about 50% by weight based on the total weight of the product excluding the container. 41. A microwave popcorn product according to claim 19, wherein the bag includes an opening in contact with the top region, wherein the bottom region, middle region and top region are approximately equal in length; and further wherein the bag is folded so that the bottom region and top region overlay the middle region. 42. A microwave popcorn product according to claim 41, further comprising an oil component wherein the unpopped corn kernels and sugar pellets, along with the oil component are contained substantially within the middle region. 43. A microwave popcorn product according to claim 42, wherein the majority of the sugar pellets is contained within a portion of the middle region nearest the bottom region or the top region, and further wherein the majority of the unpopped corn kernels and the majority of the oil component are contained within a portion of the middle region nearest the other of the bottom region or the top region. 44. A microwave popcorn product according to claim 43, wherein the susceptor covers substantially the entire length of the middle region. 45. A microwave popcorn product according to claim 44, wherein: the unpopped corn kernels are present in a total amount ranging from about 40% to about 95% by weight based on the total weight of the product excluding the container; the sugar pellets are present in a total amount ranging from about 1% to about 10% by weight based on the total weight of the product excluding the container; and the oil component is present in a total amount ranging from about 10% to about 45% by weight based on the total weight of the product excluding the container. 46. A microwave popcorn product according to claim 44, wherein: the unpopped corn kernels are present in a total amount ranging from about 45% to about 75% by weight based on the total weight of the product excluding the container; the sugar pellets are present in a total amount ranging from about 2% to about 4% by weight based on the total weight of the product excluding the container; and the product further comprises an oil component present in a total amount ranging from about 20% to about 40% by weight based on the total weight of the product excluding the container. 47. A microwave popcorn product according to claim 43, wherein the susceptor covers less than 90% of the length of the middle region, thereby creating a portion of the middle region not covered by the susceptor that corresponds to the portion of the middle region in which the majority of the sugar pellets is contained. 48. A microwave popcorn product according to claim 47, wherein: the unpopped corn kernels are present in a total amount ranging from about 25% to about 55% by weight based on the total weight of the product excluding the container; the sugar pellets are present in a total amount ranging from about 15% to about 25% by weight based on the total weight of the product excluding the container; and the oil component is present in a total amount ranging from about 30% to about 50% by weight based on the total weight of the product excluding the container. 49. A microwave popcorn product according to claim 1, wherein each sugar pellet comprises sugar in an amount ranging from about 30% to about 75% by weight, based on the weight of the sugar pellet. 50. A microwave popcorn product according to claim 1, wherein the sugar pellets each further comprise a polishing material. 51. A method for producing a microwave popcorn product comprising: providing a microwaveable container having a top region, a middle region, a bottom region, and an opening at the top of the container; introducing into the container sugar pellets and unpopped corn kernels, wherein each sugar pellet comprises sugar and corn syrup, wherein the sugar pellets are free of an emulsifying agent, and wherein the sugar pellets are not homogeneously mixed with the unpopped corn kernels; introducing into the container an oil component, wherein more of the oil component is maintained in the container with the unpopped corn kernels than is maintained with the sugar pellets, a majority of the sugar pellets is contained within the middle of the microwaveable container nearest one of the bottom or the top, and a majority of the unpopped corn kernels and a majority of the oil component are contained within a portion of the middle nearest the other of the regions, so that the corn kernels act as a buffer between the sugar pellets and the oil component. 52. A method according to claim 51, wherein the sugar pellets are introduced into the container before the unpopped kernels are introduced into the container. 53. A method according to 51, wherein the sugar pellets are introduced into the container before the oil component is introduced into the container. 54. A method according to 51, wherein the container includes a susceptor over at least a portion of the length of the container. 55. A method according to 51, wherein the microwaveable container comprises a bag having a top surface, a bottom surface, and a generally-flat susceptor attached to the bottom surface of the bag. 56. A method according to claim 55, wherein the susceptor extends over at least about 50% of the length of the bag. 57. A method according to claim 56, wherein the unpopped corn kernels are introduced in a total amount ranging from about 40% to about 95% by weight based on the total weight of the product excluding the container; the sugar pellets are introduced in a total amount ranging from about 1% to about 10% by weight based on the total weight of the product excluding the container; and the oil component is introduced in a total amount ranging from about 10% to about 45% by weight based on the total weight of the product excluding the container. 58. A method according to claim 55, wherein the susceptor covers at least about 90% of the width of the bag. 59. A method according to claim 58, wherein: the unpopped corn kernels are introduced in a total amount ranging from about 25% to about 55% by weight based on the total weight of the product excluding the container; the sugar pellets are introduced in a total amount ranging from about 15% to about 25% by weight based on the total weight of the product excluding the container; and the oil component is introduced in a total amount ranging from about 30% to about 50% by weight based on the total weight of the product excluding the container. 60. A method according to claim 54, wherein the bottom region, middle region and top region are approximately equal in length; and further wherein the bag is folded so that the bottom region and top region overlay the middle region. 61. (canceled) 62. (canceled) 63. A method according to claim 60, wherein the susceptor covers substantially the entire length of the middle region. 64. A method according to claim 63, wherein: the unpopped corn kernels are introduced in a total amount ranging from about 40% to about 95% by weight based on the total weight of the product excluding the container; the sugar pellets are introduced in a total amount ranging from about 1% to about 10% by weight based on the total weight of the product excluding the container; and the oil component is introduced in a total amount ranging from about 10% to about 45% by weight based on the total weight of the product excluding the container. 65. A method according to claim 63, wherein: the unpopped corn kernels are introduced in a total amount ranging from about 45% to about 75% by weight based on the total weight of the product excluding the container; the sugar pellets are introduced in a total amount ranging from about 2% to about 4% by weight based on the total weight of the product excluding the container; and the oil component is introduced in a total amount ranging from about 20% to about 40% by weight based on the total weight of the product excluding the container. 66. A method according to claim 55, wherein the susceptor covers less than 90% of the length of the middle region, thereby creating a portion of the middle region not covered by the susceptor that corresponds to the portion of the middle region in which the majority of the sugar pellets is contained. 67. A method according to claim 66, wherein: the unpopped corn kernels are introduced in a total amount ranging from about 25% to about 55% by weight based on the total weight of the product excluding the container; the sugar pellets are introduced in a total amount ranging from about 15% to about 25% by weight based on the total weight of the product excluding the container; and the oil component is introduced in a total amount ranging from about 30% to about 50% by weight based on the total weight of the product excluding the container. 68. A method for producing a microwave popcorn product comprising: providing a microwaveable container having a top, a middle, a bottom, and an opening at the top of the container; preparing sugar pellets having sugar and corn syrup, wherein each sugar pellet comprises sugar in an amount of at least about 15% by weight based on the total weight of the sugar pellet, and wherein the sugar pellets are free of an emulsifying agent; and introducing into the container the sugar pellets and unpopped corn kernels so that the sugar pellets and unpopped corn kernels are not homogeneously mixed, wherein a majority of the sugar pellets is contained within a portion of the middle of the microwaveable container nearest one of the bottom or the top, and a majority of the unpopped corn kernels is contained within a portion of the middle nearest the other of the regions. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Microwaveable popcorn is one of the most popular snack foods on the market today. Currently, there are several commercially available microwaveable popcorn products containing a sugar-based glaze or coating. In these products, the sugar-based glaze or coating is added separately to the popcorn after it has been popped. Manufacturers have experienced difficulties producing a product where the sugar-based coating is formed on the popcorn during popping (i.e., in a single step) due to technical difficulties in popping popcorn and heating a sugar-based composition simultaneously in a microwave oven. Specifically, at elevated temperatures and in the presence of moisture, simple sugars darken and polymerize in a process known as carmelization. Carmelization occurs at virtually the same temperature at which popcorn pops. Carmelization is enhanced by the presence of oil in the product. As a result, when a sugar-based glaze is heated in a microwave along with the popcorn, a food product is obtained that is not only visually undesirable, but also effectively inedible due to its burnt odor and flavor. Accordingly, a need exists for a single-step sweet microwave popcorn product that avoids these drawbacks. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention concerns a sweet microwave popcorn product that can be prepared by the consumer in a single step, as well as a method for its production. In one embodiment, the invention is directed to a sweet microwave popcorn product comprising a microwaveable container; a plurality of unpopped corn kernels in the container; and a plurality of sugar pellets in the container. Each sugar pellet comprises sugar in an amount of at least about 15% by weight, based on the total weight of the sugar pellet. The sugar pellets are substantially free of an emulsifying agent. In another embodiment, the invention is directed to a method for producing a microwave popcorn product. The method comprises providing a microwaveable container having a top, a bottom, and an opening at the top of the container. The method further comprises introducing into the container sugar pellets and unpopped corn kernels, wherein the sugar pellets and unpopped corn kernels are not homogeneously mixed; and introducing into the container an oil component, wherein more of the oil component is maintained in the container with the unpopped corn kernels than is maintained with the sugar pellets. In another embodiment, the invention is directed to a method for producing a microwave popcorn product. The method comprises providing a microwaveable container having a top, a bottom, and an opening at the top of the container. Sugar pellets and unpopped corn kernels are introduced into the container so that the sugar pellets and unpopped corn kernels are not homogeneously mixed. Each sugar pellet comprises sugar in an amount of at least about 15% by weight, based on the total weight of the sugar pellet, and is substantially free of an emulsifying agent. |
Diagnosis by sensing volatile components |
A state, particularly a disease state, associated with the production of volatiles is detected by passing a sample containing the volatiles to a single sensor. This may be a semiconductor gas sensor element or a surface acoustic wave device. This provides an output signal, e.g. in the form of a tailing peak. A plurality of characteristics of the signal (e.g. peak height and maximum positive gradient) are measured to characterise the sample and hence the underlying state. For example we can discriminate between urine samples which are (a) infected with proteus, (b) infected with E. coli or (c) uninfected. |
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