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1. A system establishing a communication channel on a connection between a client machine and a service application present on a communicating device dependent on a terminal comprising a representative of the service application on the client machine, a formatting module situated at the client machine, downstream of said representative, for formatting the messages of the client application in a form understandable by the service application; and a gateway situated at the terminal for taking delivery of said messages understandable by the communicating device and transmitting them to the service application. 2. A system according to claim 1, wherein the communicating device is a smart card, and the service application is a card application (12) resident on the smart card. 3. A system according to claim 1, further including means for making the messages secure at least by the level of the formatting module in order to enable secure messages issuing from the formatting module to pass in secure form through the gateway and as far as the communicating device. 4. A system according to claim 1, wherein said connection comprises a network connecting the client machine and the terminal, and said gateway is a gateway of the network/communicating device type. 5. A system according to claim 4, wherein the formatting module is connected to the network via a concatenation of modules successively comprising: a representative of said gateway, for receiving the formatting commands from the formatting module and in response producing formatting commands from these commands according to a format understandable by said gateway; a network formatting module for formatting said commands from the gateway representative as messages in the network format; and a transport module for adapting said messages to the standards of the network; and wherein said gateway is connected to the network via a concatenation of modules successively comprising: a module complementary with the transport module, and connected to the network for retrieving said messages issuing from the network; a module complementary with the network formatting module for retrieving the commands as issuing from the representative of said gateway; and a network command driver for transmitting the messages coming from the preceding module to said gateway. 6. A system according to claim 4, wherein: the formatting module is connected to the network via a transport module to directly receive said messages from the formatting module and format them to the standards of the network, and the network/communicating device gateway is connected to the network via a module complementary with the transport module for directly retrieving therefrom said messages and retransmitting them directly to said gateway. 7. A system according to claim 1, wherein said client machine and said terminal are combined in the same physical medium and connected by an internal connection, and wherein the formatting module directly transmits on said internal connection the formatted client application messages to said gateway. 8. A terminal for the system of claim 1, comprising a gateway of the network/communicating device type, for receiving and processing messages from a client application in network format delivered by internal connection or by network. 9. A method of establishing a communication channel on a connection between a client application on a client machine and a service application, present on a communicating device that is dependent on a terminal, the client machine comprising a representative of the service application, comprising the following steps: a formatting messages from the client application at the client machine, downstream of said representative in a form understandable by the service application; and receiving said messages understandable by the card at a gateway and transmitting them to the service application. 10. A method according to claim 9, wherein the communicating device is of the smart card type, and the service application is a card application. 11. A method according to claim 9, further including the step of encrypting the messages during or before the formatting to enable encrypted messages issuing from the formatting module to pass in encrypted form through the gateway and as far as the communicating device. 12. A method according to claim 9, wherein the client machine is connected to the terminal by a network, and the formatting step is followed, at the client machine, successively by the steps of: receiving formatting commands from the formatting step at a representative of said gateway and in response producing formatting commands from these commands according to a format understandable by said gateway; formatting the messages issuing from said representative of the gateway as messages in the network format; and adapting said messages to the standards of the network (R); and wherein the messages received from the network at the terminal are processed successively by steps of: retrieving said messages from the messages issuing from the network; retrieving the commands as issuing from the representative of said gateway; and transmitting the messages coming from the preceding step to said gateway. 13. A method according to claim 9, wherein: the formatting step is followed directly by a step of adapting said messages to the standards of the network, and processing the adapted messages at the terminal to retrieve said messages issuing from the formatting step and retransmit them directly to said gateway. 14. Use of a gateway of the network/communicating device type for receiving messages issuing from a client application and transmitting said messages directly to said gateway by an internal connection, said messages being formatted as network messages. |
Methods of screening, determining, developing and utilizing negative cross resistance toxins |
A method is provided for evaluating the efficacy of a molecule against a target population, the target population, including a strain resistant to a first toxin. The method comprising determining a susceptible strain in the target population. The susceptible strain being susceptible to the first toxin. The method further comprising selecting for a resistant strain in the target population. The resistant strain being resistant to the first toxin. The method further comprising evaluating the efficacy of the resistant strain with a plurality of molecules to determine a second toxin that is more toxic to the resistant strain than to the susceptible strain. Wherein the target population is at least one of an insect, a fungi, a plant, and a nematode. Methods and systems are provided for determining and developing negative cross resistance factors. The method includes evolving a strengthened NCR toxin from an initial NCR toxin by obtaining an initial NCR compound, selectively increasing the toxicity of the NCR compound, and testing the evolved compound to determine if the evolved compound is a stronger NCR compound than the initial NCR toxin. Methods and systems are also provided for deployment and commercial use of NCR toxins. |
1. A method of evaluating the efficacy of a molecule against a target population, the target population including a pest strain resistant to a first toxin, said method comprising: determining a susceptible pest strain, the susceptible strain being susceptible to the first toxin; selecting for the resistant strain, the resistant strain being resistant to the first toxin; and evaluating the efficacy of the resistant strain with a plurality of molecules to determine a second toxin that is more toxic to the resistant strain than to the susceptible strain; wherein the resistant and susceptible pest strains coexist in the target population. 2. A method in accordance with claim 1 wherein the target population is an insect population. 3. A method in accordance with claim 1 wherein the plurality of molecules are evaluated against only the resistant strain. 4. A method in accordance with claim 1 wherein the plurality of molecules are evaluated against the resistant strain and a subset of molecules are evaluated against the susceptible strains. 5. A method in accordance with claim 1 wherein the plurality of molecules are evaluated against only the heterozygous resistant strain. 6. A method in accordance with claim 1 wherein the plurality of molecules are evaluated against the heterozygous resistant strain and homozygous susceptible strain. 7. A method in accordance with claim 1 wherein the plurality of molecules are evaluated against the heterozygous resistant strain and homozygous resistant strain. 8. A method in accordance with claim 1 wherein the target population is at least one of a mammalian population, a plant population, an animal population, and a virus population. 9. A method in accordance with claim 1 wherein said method further comprises assigning a priority rating to the second toxin if applications of the first toxin and the second toxin are at least as toxic to the bacterial strain containing at least one copy of the resistance gene as to the susceptible bacterial strain. 10. A method for controlling a host species that includes a bacteria existing in a symbiotic relationship with the host species, said method comprising: determining a susceptible strain of the bacteria that is susceptible to a first toxin; determining a resistant strain of the bacteria that is resistant to the first toxin; determining a second toxin that is more toxic to the resistant strain than to the susceptible strain; and applying the first toxin and the second toxin to the host species such that the host species is adversely impacted. 11. A method in accordance with claim 11 wherein the host species is an insect population. 12. A method in accordance with claim 10 wherein the host species is a mammalian population. 13. A method in accordance with claim 10 wherein application of the first and second toxin has an adverse impact on the bacteria. 14. A method for generating a resistant organism to be used in developing NCR toxins, said method comprising creating genomic changes in the organism to create a resistance trait, wherein the resistant organism is generated for the purpose of developing NCR toxins. 15. A method in accordance with claim 14 further comprising generating a first resistance allele to a first toxin. 16. A method in accordance with claim 15 wherein the resistance allele to the first toxin is generated through at least one of creation of point mutations in genes, genomic changes that involve up-regulation or down regulation of existing genes, mutations that result in a loss of function of the target gene, and genomic changes that cause duplication of genes that result in a resistant trait that will ultimately be used in the discovery of NCR toxins. 17. A method in accordance with claim 14 further comprising generating a dually resistant allele. 18. A method in accordance with claim 17 wherein the dually resistant allele is generated through at least one of creation of point mutations in genes, genomic changes that involve up-regulation or down regulation of existing genes, and genomic changes that cause duplication of genes that result in a resistant trait that will ultimately be used in the discovery of further NCR toxins. 19. A method in accordance with claim 14 wherein the genomic changes occur through at least one of EMS, X-rays, Gamma-rays, radioactive materials, P-elements, mobile genetic elements, ‘jumping genes’, and other genomic technology that result in genomic changes in an organism. 20. A method in accordance with claim 14 wherein the resistant organism is at least one of a naturally occurring insect population, a naturally occurring plant population, a naturally occurring fungi population, a naturally occurring bacterial population, a maintained field, a maintained laboratory, a maintained freezer, and a stock of an organism. 21. A method in accordance with claim 15 further comprising delivering, either externally or internally, to the organism the toxins to be used for selection of the first resistance allele. 22. A method in accordance with claim 17 further comprising delivering, either externally or internally, to the organism the materials to be used for selection of the dually-resistant allele. 23. A method in accordance with claim 14 wherein the resistant organism makes its own toxin. 24. A method in accordance with claim 14 further comprising feeding a toxin to the organism. 25. A method in accordance with claim 14 further comprising delivering the toxin to the organism through at least one of an external membrane and a surface of the organism. 26. A method in accordance with claim 25 further comprising delivering the toxin to the organism using at least one of an organic solvent and an inorganic solvent. 27. A method in accordance with claim 14 wherein the organism produces the toxin internally through transgenic means. 28. A method in accordance with claim 14 wherein the toxin is delivered internally to the target organism by injection. 29. A method in accordance with claim 14 wherein the toxin is delivered by organisms that are symbiotically associated with the resistant organism. 30. A method in accordance with claim 29 wherein the delivery organisms are transgenic organisms. 31. A method in accordance with claim 14 wherein the toxin is delivered by organisms that live in association with the target organism. 32. A method in accordance with claim 14 wherein the target genes for the toxins include, but are not limited to, Cytochrome P450 enzymes, Esterase enzymes, ion pumps, glutathione-S-transferases, voltage-gated sodium channels, sodium channels, calcium channels, membrane transport proteins, GABA receptors, GABA-gated chloride channel, nicotinic acetylcholine receptors, calcium channels, amino-peptidases, proteases, a-amylases, lipases, and allelic variants. 33. A method in accordance with claim 14 wherein the toxins used to select the resistance trait include, but are not limited to, target genes in insects of the Bacillus thuringiensis insecticidal toxins, target genes in insects of the Photorhabdus luminescens insecticidal toxins, target genes in insects of the Xenorhabdus nematophilus insecticidal toxins, target genes in insects of Spinosad, target genes in insects of Spinosyn, target genes in insects of imidacloprid, and allelic variants. 34. A method in accordance with claim 14 wherein the toxins used to select the resistance trait include, but are not limited to, DDT, pyrethroids, AaIT scorpion toxin, chlorinated hydrocarbons, organophosphates, and carbamates. 35. A method in accordance with claim 14 wherein a target site for the development of NCR toxins is at least one gene that metabolizes a first toxin. 36. A method in accordance with claim 14 wherein the resistant organism is associated with resistance to toxins through at least one of reduced penetration by the toxin and resistance to toxins through sequestration of the toxin. 37. A method in accordance with claim 15 wherein the resistant allele is obtained in a screen for allelic variants of a gene causing resistance using an in vitro system. 38. A method in accordance with claim 17 wherein the dually resistant allele is obtained in a screen for allelic variants of a gene causing resistance using an in vitro system. 39. A method of screening for negative cross resistance with respect to a target allele, said method comprising: evaluating whether a first toxin that was originally effective against a susceptible line of organism is effective against a susceptible line of organism; and if the toxin is no longer effective against the susceptible line of organism, evaluating whether the toxin is effective against a resistant line of organism. 40. A method in accordance with claim 39 wherein the toxin is evaluated against a second line of organism homozygous for the target allele to identify high levels of toxicity. 41. A method in accordance with claim 39 wherein the first toxin is evaluated against organisms heterozygous for second alleles considered to be resistant to the first toxin and susceptible to a second toxin and wherein the second allele is resistant to the second toxin and is susceptible to the first toxin. 42. A method in accordance with claim 39 wherein homozygous insects that are resistant to a second toxin are evaluated to be susceptible to the first toxin. 43. A method in accordance with claim 39 further comprising delivering the first toxin to an external surface of the organism. 44. A method in accordance with claim 39 wherein the toxin is delivered using at least one of an organic solvent and an inorganic solvent. 45. A method in accordance with claim 39 further comprising feeding the toxin to the organism. 46. A method in accordance with claim 39 further comprising delivering the toxin by organisms that are symbiotically associated with the susceptible organism. 47. A method in accordance with claim 39 further comprising delivering the toxin by transgenic organisms that are symbiotically associated with the susceptible organism. 48. A method in accordance with claim 39 further comprising delivering toxin internally to the organism using at least one of an inorganic solvent, an organic solvent, transgenic techniques, transgenic organisms, P-element transformation of the target organism with the toxin, mobile genetic elements, agrobacterium transformation of the target organism with the toxin, and particle gun transformation of the target organism with the toxin. 49. A method in accordance with claim 39 wherein the toxin is a second-generation insecticide. 50. A method in accordance with claim 39 wherein the toxin includes at least one of Bacillus thuringiensis insecticidal toxins, Photorhabdus luminescens insecticidal toxins, Xenorhabdus nematophilus insecticidal toxins, Spinosad, Spinosyn, imidacloprid and derivatives thereof. 51. A method in accordance with claim 39 wherein the toxin includes at least one of DDT, pyrethroids, AaIT scorpion toxin, chlorinated hydrocarbons, organophosphates, and carbamates. 52. A method in accordance with claim 50 wherein the Bacillus thuringiensis insecticidal toxin is altered such that it is no longer toxic to susceptible lines of the insects and the toxin is subsequently used in a bioassay to determine that the toxin kills the resistant organisms. 53. A method in accordance with claim 50 wherein the Photorhabdus luminescens insecticidal toxin is subsequently used in a bioassay to determine that the toxin kills the resistant organisms. 54. A method in accordance with claim 50 wherein the Xenorhabdus nematophilus insecticidal toxin is altered such that it is no longer toxic to susceptible lines of insect and is subsequently evaluated to determine that the toxin kills the resistant insects. 55. A method in accordance with claim 50 wherein the Spinosyn or Spinosad compounds are altered such that they are no longer toxic to the susceptible line of organisms and are subsequently used in a bioassay to determine that the toxin kills the resistant organisms. 56. A method in accordance with claim 50 wherein the imidicloprid compounds are altered such that they are no longer toxic to susceptible lines of organisms and are subsequently used in a bioassay to determine that the toxin kills the resistant organisms. 57. A method in accordance with claim 50 wherein the AaIT scorpion toxins are altered such that they are no longer toxic to susceptible lines of the organism and are subsequently used in a bioassay to determine that the toxin kills the resistant organisms. 58. A method in accordance with claim 51 wherein second generation toxins are altered such that they are no longer toxic to susceptible lines of the organism and the toxins are subsequently used in a bioassay to determine that the toxin kills the resistant organisms. 59. A method in accordance with claim 39 wherein the toxins are altered by changing coding sequence in a toxin gene. 60. A method of screening compounds for negative cross resistance activity comprising: altering compounds originally toxic to susceptible insects such that they are no longer toxic to susceptible lines of insects; and using the altered compounds in a bioassay to determine whether the toxin will kill resistant organisms. 61 A method of evaluating the efficacy of a molecule against a target population, the target population including a strain resistant to a first toxin, said method comprising: determining a susceptible strain in the target population, the susceptible strain being susceptible to the first toxin; selecting for the resistant strain in the target population, the resistant strain being resistant to the first toxin; and evaluating the efficacy of the resistant strain with a plurality of molecules to determine a second toxin that is more toxic to the resistant strain than to the susceptible strain; 62. A method in accordance with claim 61 wherein the target population is at least one of a fungi, a plant, and a nematode. 63. A method in accordance with claim 61 further comprising: evaluating the efficacy of a heterozygous strain of the target population with separate applications of the first toxin and the second toxin; and assigning a priority rating to the second toxin if the separate applications of the first toxin and the second toxin are at least as toxic to the heterozygous strain as to the susceptible strain. 64. A method in accordance with claim 61 further comprising screening the heterozygous strain with the first toxin and the second toxin applied at the same time. 65. A method in accordance with claim 63 further comprising assigning a priority rating to the second toxin if the application of the first toxin and the second toxin at the same or substantially the same time are at least as toxic to the heterozygous strain as to the susceptible strain. 66. A method in accordance with claim 61 further comprising determining whether both the first toxin and the second toxin can be applied to the target population at the same or substantially the same time at an acceptable rate. 67. A method in accordance with claim 66 further comprising assigning a priority rating to the second toxin if the first toxin and the second toxin can be applied to the target population at the same or substantially the same time at an acceptable rate. 68. A method in accordance with claim 61 wherein selecting for the resistant strain in the target population comprises selecting for a homozygous resistant strain in the target population. 69. A method in accordance with claim 61 wherein selecting for the resistant strain in the target population comprises selecting for a resistant strain in the target population using at least one of a field collected line and an EMS-mutagenized line. 70. A method in accordance with claim 61 wherein evaluating the efficacy of the resistant strain comprises evaluating the efficacy of the resistant strain with between about 10 and 109 molecules. 71. A method of testing for negative cross resistance in a target population, said method comprising: determining a susceptible strain (S/S) in the target population, the susceptible strain (S/S) susceptible to a first toxin; selecting for a resistant strain (R/R) in the target population, the resistant strain (R/R) resistant to the first toxin; and evaluating the efficacy of the resistant strain (R/R) with between about 10 and 109 molecules to determine a second toxin that is more toxic to the resistant strain (R/R) than to the susceptible strain (S/S); 72. A method in accordance with claim 71 wherein the target population is at least one of a fungi, a plant, and a nematode. 73. A method in accordance with claim 71 further comprising: evaluating the efficacy of a heterozygous strain (R/S) of the target population with separate applications of the first toxin and the second toxin to determine if separate applications of the first toxin and the second toxin are at least as toxic to the heterozygous strain (R/S) as to the susceptible strain (S/S); and assigning a high negative cross resistance priority to the second toxin if the separate applications of the first toxin and the second toxin are at least as toxic to the heterozygous strain (R/S) as to the susceptible strain (S/S). 74. A method in accordance with claim 71 further comprising evaluating the efficacy of the heterozygous strain (R/S) with the first toxin and the second toxin applied at the same or substantially the same time to determine if the application of the first toxin and the second toxin at the same or substantially the same time is at least as toxic to the heterozygous strain (R/S) as to the susceptible strain (S/S). 75. A method in accordance with claim 73 further comprising determining whether both the first toxin and the second toxin can be applied to the target population at the same or substantially the same time at an economically acceptable rate. 76. A method in accordance with claim 74 further comprising assigning a high negative cross resistance priority to the second toxin if the first toxin and the second toxin can be applied to the target population at the same or substantially the same time at an economically acceptable rate. 77. A method in accordance with claim 71 wherein selecting for a resistant strain (R/R) in the target population comprises selecting for a resistant strain (R/R) in the target population using at least one of a field collected line and an EMS-mutagenized line. 78. A method of using a first negative cross-resistance (NCR) toxin and a second NCR toxin against a pest population in a refuge to selectively kill heterozygotes and homozygotes carrying resistance alleles to the first NCR toxin, wherein the first NCR toxin is used in a main field and the second NCR toxin is used in the refuge. 79. A method in accordance with claim 78 wherein the second toxin comprises at least one of a Bacillus thuringiensis protein toxin, a lectin protein toxin, a Saccharopolyspora spinosa protein toxin, a Photorhabdus luminescens protein toxin, a Xenorhabdus nematophilus protein toxin, a imidacloprid toxin, a Cysteine protease inhibitors protein toxin, a Bowman-Birk Inhibitors protein toxin, a Kunitz inhibitors protein toxin, and an alpha-amylase inhibitor protein toxin. 80. A method in accordance with claim 78 wherein a target site for the second NCR toxin is at least one of a Cadherin gene protein and a truncated Cadherin gene protein. 81. A method in accordance with claim 78 further comprising using the second toxin to slow the rate at which resistance enters the pest population in the field. 82. A method in accordance with claim 78 further comprising using the second toxin to slow the rate at which the resistance allele enters the pest population in the field. 83. A method in accordance with claim 78 further comprising using the second toxin to decrease the level of resistance in the pest population in the field. 84. A method in accordance with claim 78 further comprising using the second toxin to decrease the levels of the resistance allele in the pest population in the field. 85. A method in accordance with claim 78 further comprising using the second toxin to maintain the level of resistance in the pest population in the field. 86. A method in accordance with claim 78 further comprising using the second toxin to maintain the levels of the resistance allele in the pest population in the field. 87. A method in accordance with claim 78 wherein the second toxin is carried by plants in the refuge, the plants containing the second toxin are planted separate from the transgenic plants containing other toxins. 88. A method in accordance with claim 78 wherein the second toxin is carried by plants in the refuge, the plants containing the second toxin are planted within or near the region where the transgenic plants containing other toxins are planted. 89. A method in accordance with claim 78 further comprising delivering the first toxin through a transgenic plant. 90. A method in accordance with claim 78 where the pest population is one of an insect population, a nematode population, a plant population, a fungi population, and a bacterial population. 91. A method in accordance with claim 78 further comprising delivering the second toxin through a transgenic plant. 92. A method in accordance with claim 91 wherein the transgenic plant is the same or substantially the same species as a crop grown in the main field. 93. A method in accordance with claim 91 wherein the transgenic plant is a different species from a crop grown in the main field. 94. A method in accordance with claim 78 further comprising delivering at least one of the first toxin and the second toxin as a spray. 95. A method in accordance with claim 78 further comprising delivering the second toxin in a transgenic plant in the refuge field in as few as 1/10,000,000 of the total plants in the main field. 96. A method in accordance with claim 78 further comprising delivering the second toxin in a transgenic plant in the refuge field that is up to 9,999,999/10,00,000 of the total plants in the main field. 97. A method of using a first negative cross-resistance (NCR) toxin and a second NCR toxin against a pest population in a refuge to selectively kill heterozygotes that carry resistance alleles to the first NCR toxin which is used in the main field. 98. A method in accordance with claim 97 wherein the first toxin is one of a Bacillus thuringiensis protein toxin, a Saccharopolyspora spinosa protein toxin, a Photorhabdus luminescens protein toxin, a Xenorhabdus nematophilus protein toxin, a imidacloprid toxin, a lectin protein toxin, a Cysteine protease inhibitors protein toxin, a Bowman-Birk Inhibitors protein toxin, a Kunitz inhibitors protein toxin, and a 1 alpha-amylase inhibitor protein toxin. 99. A method in accordance with claim 97 further comprising delivering at least one of the first toxin and the second toxin in a transgenic plant. 100. A method in accordance with claim 97 where the target site for the first NCR toxin is at least one of a Cadherin gene and pseudogene. 101. A method in accordance with claim 97 wherein the target population is one of an insect population, a nematode population, a plant population, a fungi population, and a bacterial population. 102. A method in accordance with claim 97 further comprising delivering the second toxin through a plant that is the same or substantially the same species as a crop grown in a main field. 103. A method in accordance with claim 97 further comprising delivering the second toxin through a plant that is a different species from a crop grown in a main field. 104. A method in accordance with claim 97 further comprising delivering at least one of the first toxin and the second toxin as a spray. 105. A method in accordance with claim 97 further comprising delivering the second toxin in a transgenic plant in the refuge field in as few as 1/10,000,000 of the total plants in the main field. 106. A method in accordance With claim 97 further comprising delivering the second toxin in a transgenic plant in the refuge field that is up to 9,999,999/10,00,000 of the total plants in the field. 107. A method in accordance with claim 97 further comprising planting refuge plants containing the NCR toxin separate from the transgenic plants containing other toxins. 108. A method in accordance with claim 97 further comprising planting refuge plants containing the NCR toxin within the region where transgenic plants containing other toxins are planted. 109. A method in accordance with claim 97 further comprising utilizing the second toxin to slow the rate at which resistance enters the pest population in the main field. 110. A method in accordance with claim 97 further comprising utilizing the second toxin to slow the rate at which the resistance allele enters the pest population in the main field. 111. A method in accordance with claim 97 further comprising utilizing the second toxin to decrease the level of resistance in the pest population in the main field. 112. A method in accordance with claim 97 further comprising utilizing the second toxin to decrease the levels of the resistance allele in the pest population in the main field. 113. A method in accordance with claim 97 further comprising utilizing the second toxin to maintain the level of resistance in the pest population in the main field. 114. A method in accordance with claim 97 further comprising utilizing the second toxin to maintain the levels of the resistance allele in the pest population in the main field. 115. A method of evaluating the efficacy of a molecule against a target population, the target population including a strain resistant to a Bacillus thuringiensis insecticidal toxin, said method comprising: determining a susceptible strain susceptible to a Bacillus thuringiensis insecticidal toxin; selecting for the resistant strain, the resistant strain being resistant to the Bacillus thuringiensis insecticidal toxin; and evaluating the efficacy of the resistant strain with a plurality of molecules to determine a second toxin that is more toxic to the resistant strain than to the susceptible strain. 116. A method in accordance with claim 115 wherein the target population is an insect population. 117. A method in accordance with claim 115 wherein the target population is a nematode population. 118. A method in accordance with claim 115 further comprising assigning a priority rating to the second toxin if applications of the Bacillus thuringiensis insecticidal toxin and the second toxin are at least as toxic to the heterozygous insect strain as to the susceptible bacterial strain. 119. A method in accordance with claim 115 further comprising assigning a priority rating to the second toxin if applications of the Bacillus thuringiensis insecticidal toxin and the second toxin are at least as toxic to the heterozygous nematode strain as to the susceptible bacterial strain. 120. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets a cadherin gene protein in insects. 121. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets a truncated cadherin gene protein in insects. 122. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets a non-glycosolated cadherin gene protein in insects. 123. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets a glycosolated cadherin gene protein in insects. 124. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets the cadherin gene protein in Heliothis virescens. 125. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets a non-functional cadherin gene protein in Heliothis virescens. 126. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets a truncated cadherin gene protein in Heliothis virescens. 127. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets the r1 Bacillus thuringiensis allele in Heliothis virescens. 128. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets the beta-1,3-galactosyltransferase gene in Heliothis virescens. 129. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets a cadherin gene protein in nematodes. 130. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets a truncated cadherin gene protein in nematodes. 131. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets a non-glycosolated cadherin gene protein in nematodes. 132. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets a glycosolated cadherin gene protein in nematodes. 133. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets the cadherin gene protein in nematodes. 134. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets a non-functional cadherin gene protein in nematodes. 135. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets a truncated cadherin gene protein in nematodes. 136. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets the bre-5 Bacillus thuringiensis resistant allele in nematodes. 137. A method in accordance with claim 115 wherein the negative cross-resistance toxin targets the beta-1,3-galactosyltransferase gene in nematodes. 138. A method in accordance with claim 115 wherein the genomic changes are naturally occurring and are selected for in at least one of the laboratory and in the field. 139. A method for evolving a strengthened NCR toxin from an initial NCR toxin, said method comprising the steps of obtaining an initial NCR compound; selectively increasing the toxicity of the NCR compound; and testing the evolved compound to determine if the evolved compound is a stronger NCR compound than the initial NCR toxin. 140. A method in accordance with claim 139 further comprising using a laboratory evolved negative cross-resistance toxin in a refuge to selectively kill at least one of heterozygotes and homozygotes carrying resistance alleles to the toxin used in the main field. 141. A method in accordance with claim 139 wherein the toxin used in the main field is the Bacillus thuringiensis protein toxin,.a lectin protein toxin, the Saccharopolyspora spinosa protein toxin and the Photorhabdus luminescens protein toxin. 142. A method in accordance with claim 139 wherein the target site for the NCR toxin is at least one of a Cadherin gene protein and truncated Cadherin gene protein. 143. A method in accordance with claim 139 wherein the toxin used in the refuge is used to slow the rate at which resistance enters the pest population in the field. 144. A method in accordance with claim 139 wherein the toxin used in the refuge is used to slow the rate at which at least one resistance allele enters the pest population in the field. 145. A method in accordance with claim 139 wherein the toxin used in the refuge is used to decrease the level of resistance in the pest population in the field. 146. A method in accordance with claim 139 wherein the toxin used in the refuge is used to decrease the levels of the resistance allele or alleles in the pest population in the field. 147. A method in accordance with claim 139 wherein the toxin used in the refuge is used to maintain the level of resistance in the pest population in the field. 148. A method in accordance with claim 139 wherein the toxin used in the refuge is used to maintain the levels of at least one resistance allele in the pest population in the field. 149. A method in accordance with claim 139 wherein the refuge plants containing the NCR toxin are planted separate from the transgenic plants containing other toxins are planted. 150. A method in accordance with claim 139 wherein the refuge plants containing the NCR toxin are planted within or near the region where the transgenic plants containing other toxins are planted. 151. A method in accordance with claim 139 wherein the toxin used in the main field is delivered in a transgenic plant. 152. A method in accordance with claim 139 wherein the target population is one of an insect population, nematode population, plant population, fungi population, and bacterial population. 153. A method in accordance with claim 139 wherein the NCR toxin used in the refuge field is delivered in a transgenic plant. 154. A method in accordance with claim 139 wherein the NCR toxin used in the refuge field is delivered in a transgenic plant that is the same or substantially the same species as the crop grown in the main field. 155. A method in accordance with claim 139 wherein the NCR toxin used in the refuge field is delivered in a transgenic plant that is a different species from the crop grown in the main field. 156. A method in accordance with claim 139 wherein the toxin used in the main field is delivered as a spray. 157. A method in accordance with claim 139 wherein the toxin is delivered to an organism through at least one of a vaccine, a pill, a gel tablet, an injection, a syrup, a powder, or mixed in a food or drink product. 158. A method in accordance with claim 139 wherein the NCR 5 toxin used in the refuge field is delivered as a spray. 159. A method in accordance with claim 139 wherein the NCR toxin used in the refuge field is delivered in a transgenic plant in the field that is as few as 0.00001 percent of the total plants in the field. 160. A method in accordance with claim 139 wherein the NCR toxin used in the refuge field is delivered in a transgenic plant in the field that is up to 99.99999 percent of the total plants in the field. 161. A method in accordance with claim 139 wherein the NCR toxin used in the refuge field is delivered in a transgenic plant in the field is as few as 0.00001 percent or is up to 99.99999 percent or inclusive of these two extremes of the total plants in the field. 162. A method in accordance with claim 70 further comprising delivering the second toxin in a transgenic plant in the refuge field that is up to 99,999/100,000 of the total plants in the main field. 163. A method in accordance with claim 70 further comprising delivering the second toxin in a transgenic plant in the refuge field in as few as 1/10,000 of the total plants in the main field. 164. A method in accordance with claim 70 further comprising delivering the second toxin in a transgenic plant in the refuge field that is up to 9,999/10,000 of the total plants in the main field. 165. A method in accordance with claim 70 further comprising delivering the second toxin in a transgenic plant in the refuge field in as few as 1/1,000 of the total plants in the main field. 166. A method in accordance with claim 70 further comprising delivering the second toxin in a transgenic plant in the refuge field that is up to 999/10,000 of the total plants in the main field. |
<SOH> BACKGROUND OF THE INVENTION <EOH>This invention relates generally to negative cross resistance, and more particularly, to methods and systems for evaluating, determining, developing, and utilizing negative cross resistance to extend and increase the efficacy of molecules to kill unwanted resistant organisms. Two of the most important scientific events of the twentieth century are the green revolution and the development of pesticides and antibiotics. The green revolution, with the large-scale use of pesticides, i.e., insecticides, herbicides, and fungicides, brought about dramatic increases in quantity and quality of food for an ever-growing human population, allowing for reliable food sources for billions of people on this planet. In addition, antibiotics dramatically reduced the mortality rates of the human population to bacterial diseases, virtually wiping out some bacterial epidemics. However, (pesticide and antibiotic) resistance has evolved due to the large scale use of the pesticides and antibiotics. Although efforts have been made to slow the development of such resistance, the evolution of resistance is generally considered inevitable. Once widespread resistance develops which is unfortunately inheritable, the chemical (or chemical-class) that resistance has developed against is typically abandoned thus causing loss of capability of using such pesticides and antibiotics, the incurring of significant expense to develop new ones and needless interruption in field and management of land practices. The subsequent focus in the research and industrial community is to identify novel pesticides and antibiotics with different modes of action, where positive cross-resistance to previously used biocides does not occur. One alternative to the seemingly endless treadmill practice of discarding old and existing compounds and continually seeking new compounds is the development of negative cross-resistance strategies to control organisms containing the resistance allele. Negative cross-resistance (NCR) as a strategy for pesticide resistance management encompasses a scenario where organisms tolerant to one compound are highly sensitive to another compound and vice versa. For example, if a pest population is treated with a toxin such as pesticide ‘A’, the number of pests carrying alleles resistant to pesticide ‘A’ will increase in frequency. After numerous generations, pests carrying the resistance allele will comprise the majority of the population. At this time a second toxin that preferentially kills those pests tolerant to the first toxin is used to treat the pest population. Use of the second toxin changes the frequency of the alleles such that the first toxin can again be used to control the pests' population for one to several generations. By alternately deploying the two toxins, a NCR strategy is used to maintain effective control of the pest while ‘managing’ the resistance alleles in the pest population. Negative cross-resistance has been shown to occur with a wide variety of organisms. It has been observed with pesticides used to control German cockroaches, diamondback moths, mosquitoes, spider mites, and horn flies. Additionally, this phenomenon has been observed in weeds resistant to herbicides, fungal strains resistant to fungicides, and in bacteria resistant to antibiotics. Negative cross-resistance has been associated, in some instances, with a single amino acid change in the targeted allele. For example, negative cross-resistance in Ustilago maydis between the fungicides benzimidazole and diethofencarb was reportedly due to a mutation at a single locus. Even where practically applicable toxin pairs exist, there is typically little understanding of how such toxins affect the frequency of resistance alleles. Although negative cross-resistance occurs across a wide array of toxins and organisms, the use of a pair of second generation pesticides in a NCR strategy has rarely been commercially applied. One of the few practically applicable NCR strategies is with N-methylcarbamate and N-propylcarbamate to green rice leafhopper Nephotittix cincticeps Uhler, whose populations contain both mutant and wild-type acetylcholinesterases. Use of N-propylcarbamate on the green rice leafhopper population resulted in a population more susceptible to N-methylcarbamate and vice versa. Researchers have been able to shift the resistance level back and forth by alternating between the two aforementioned carbamates. One reason put forth for such little commercial applicability is because much of the discovery effort in industrial entomology has focused on finding toxins with novel modes of action and improving the efficacy and spectrum of action of already discovered toxins. One of the limitations to developing NCR toxins as with any type of biocide is that determining highly effective toxins requires screening a large number of test compounds. A methodology to reduce the numbers of lead toxins that need to be creened will greatly increase the efficiency of development of practical NCR toxins. Another limitation to developing NCR toxins is that there is currently no use for NCR toxins that will cause only moderate mortality rates in the homozygous and heterozygous resistant organisms. A commercial use for moderately toxic NCR factors greatly increases the need for the development of practical NCR toxins. Another limitation to developing NCR toxins is that even if moderately effective NCR toxins are determined through screening and there is currently no way to evolve these toxins into highly effective NCR toxins. A methodology for evolving moderately effective NCR toxins into highly effective NCR toxins will greatly increase the efficiency of developing practical NCR toxins. Another limitation to developing NCR toxins is that currently no business model provides a significant market advantage to those companies that develop these toxins for use with a biocide that has been previously developed. |
<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>In one aspect of the invention, a method is provided for evaluating the efficacy of a molecule against a target population. The target population includes a bacterial strain resistant to a first toxin. The method comprises determining a susceptible bacterial strain, the susceptible strain being susceptible to the first toxin. The method further comprises selecting for the resistant strain, the resistant strain being resistant to the first toxin. The method further comprises evaluating the efficacy of the resistant strain with a plurality of molecules to determine a second toxin that is more toxic to the resistant strain than to the susceptible strain, wherein the resistant and susceptible bacterial strains coexist in the target population. In another aspect of the invention, a method is provided for controlling a host species that includes a bacteria existing in a symbiotic relationship with the host species. The method comprises determining a susceptible strain of the bacteria that is susceptible to a first toxin, determining a resistant strain of the bacteria that is resistant to the first toxin, determining a second toxin that is more toxic to the resistant strain than to the susceptible strain, and applying the first toxin and the second toxin to the host species such that the host species is adversely impacted. In another aspect of the invention, a method is provided for generating a resistant organism to be used in developing NCR toxins. The method comprising creating genomic changes in the organism to create a resistance trait, wherein the resistant organism is generated for the purpose of developing NCR toxins. In another aspect of the invention, a method is provided for screening for negative cross resistance with respect to a target allele. The method comprising evaluating whether a first toxin that was originally effective against a susceptible line of organism is effective against a susceptible line of organism, and if the toxin is no longer effective against the susceptible line of organism, evaluating whether the toxin is effective against a resistant line of organism. In another aspect of the invention, a method is provided for evaluating the efficacy of a molecule against a target population, the target population including a strain resistant to a first toxin. The method comprises determining a susceptible strain in the target population, the susceptible strain being susceptible to the first toxin. The method further comprises selecting for the resistant strain in the target population, the resistant strain being resistant to the first toxin. The method further comprises evaluating the efficacy of the resistant strain with a plurality of molecules to determine a second toxin that is more toxic to the resistant strain than to the susceptible strain. In one embodiment, only the resistant strains are evaluated, wherein the target population is at least one of a fungi, a plant, and a nematode. In another aspect of the invention, a method is provided for testing for negative cross resistance in a target population, the method comprising determining a susceptible strain (S/S) in the target population, the susceptible strain (S/S) susceptible to a first toxin. The method further comprises selecting for a resistant strain (R/R) in the target population, the resistant strain (R/R) resistant to the first toxin. The method further comprising evaluating the efficacy of the resistant strain (R/R) with between about 10 and about 10 9 molecules to determine a second toxin that is more toxic to the resistant strain (R/R) than to the susceptible strain (S/S), wherein the target population is at least one of a fungi, a plant, and a nematode. In another aspect of the invention, a method is provided for using a first negative cross-resistance (NCR) toxin and a second NCR toxin against a pest population in a refuge to selectively kill heterozygotes and homozygotes carrying resistance alleles to the first NCR toxin, wherein the first NCR toxin is used in a main field and the second NCR toxin is used in the refuge. The current refuge strategy involves a high dose approach to controlling the pest insects. By high dose is meant an expression level of the toxin in the transgenic plant that will kill 90% or greater of the homozygous or heterozygous susceptible insects. In another aspect of the invention, a method is provided for using a first negative cross-resistance (NCR) toxin and a second NCR toxin against a pest population in a refuge to selectively kill heterozygotes that carry resistance alleles to the first NCR toxin, which is used in a main field. The “main field” includes the region or locus where the transgenic plants containing the initial toxin are located. In a passive refuge strategy the main (Agricultural) field is where the transgenic plants are located and the refuge contains the non-transgenic plants. In the active refuge the main field is where the plants containing the initial transgenic pesticide are located and the refuge is where the plants containing or being treated with the NCR product are located. In one illustrative example, the main field has transgenic maize, which expresses a Bacillus thuringiensis insecticidal toxin. In another aspect of the invention, a method is provided for evaluating the efficacy of a molecule against a target population, the target population including a strain resistant to a Bacillus thuringiensis insecticidal toxin (Bt). The method comprises determining a susceptible strain susceptible to a Bacillus thuringiensis insecticidal toxin, selecting for the resistant strain, the resistant strain being resistant to the Bacillus thuringiensis insecticidal toxin, and evaluating the efficacy of the resistant strain with a plurality of molecules to determine a second toxin that is more toxic to the resistant strain than to the susceptible strain. In another aspect of the invention, a method is provided for evolving NCR toxins from weak toxins to strong toxins. The evolution can occur either with a known NCR compound or with newly determined or identified NCR compounds. The evolution can occur with weak NCR compounds, moderate NCR compounds, and strong NCR compounds. In one embodiment, the evolution-occurs utilizing phage-display technology. In another aspect of the invention, a method is provided for use of NCR toxins in the commercial market place for companies to add value to another product which is the first toxin. |
Processes for the production of amino-protected derivatives of 4-aminomethylene-pyrrolidin-3-one and/or 4-aminomethylene-pyrrolidin-3-alkoxyimino derivatives and/or gemifloxacin or a salt thereof |
The invention provides a process for the production of a compound of formula (I): wherein P1 and P2, which may be the same or different, are amino protecting groups, which comprises protection of a compound of formula (II) in solution phase continuous operation mode. This confers advantages over batch mode operation. The process is usually conducted in reaction equipment adapted for use in continuous processing mode, for example comprising one or more static mixers or a plug flow reactor. Preferably, the plug flow reactor comprises a jacketed tubular reactor fitted inside with internal mixing elements which continually split and remix the reaction streams promoting mass and heat transfer, whereby a uniform plug flow profile with turbulent fluid flow is achieved. The invention also provides a process for production of the antibacterial compound gemifloxacin or a pharmaceutically acceptable salt and/or hydrate thereof, comprising converting a compound of formula (I). The invention also provides a process for the production of a compound of formula (VIIIa). |
1. A process for the production of a compound of formula (I): wherein P1 and P2, which may be the same or different, are amino protecting groups, which comprises protection of a compound of formula (II): in solution phase continuous operation mode. 2. A process as claimed in claim 1 wherein protection of the compound of formula (I) is performed by reacting the compound of formula (I) with a reagent for introducing the amino-protecting group P2, in the presence of a base. 3. A process as claimed in claim 2 wherein the reagent for introducing the amino-protecting group is t-butoxycarbonyl anhydride. 4. A process as claimed in claim 2, wherein the reagent for introducing the amino-protecting group is an isopropanol/water solution. 5. A process as claimed in claim 1, wherein P1 and P2 are amino protecting groups removable by acidic conditions. 6. A process as claimed in claim 1, wherein the protecting group P1 and/or the protecting group P2 is formyl, acetyl, trifluoroacetyl, benzoyl, para-toluenesulfonyl, methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, para-methylbenzyloxycarbonyl, trichloroethoxycarbonyl, beta-iodoethoxycarbonyl, benzyl, para-methoxybenzyl, trityl, tetrahydropyranyl or pivaloyl. 7. A process as claimed in claim 1 wherein P1 and P2 are both t-butyloxycarbonyl. 8. A process as claimed in claim 2 wherein the base is aqueous potassium hydroxide solution. 9. A process as claimed in claim 8 wherein the base is 30% w/w to 50% aqueous potassium hydroxide solution. 10. A process as claimed in claim 1 which is carried out using isopropanol/water as solvent. 11. A process as claimed in claim 2, wherein in the continuous operation mode, a stream of aqueous base and a stream of a solution of the 1-(N-protected)-4-aminomethylene-pyrrolidin-3-one of formula (II) containing the reagent for introducing the amino-protecting group are brought together prior to quenching in acid. 12. A process as claimed in claim 1, which is conducted at a temperature of less than 20° C. 13. A process according to claim 1 wherein the process is conducted in reaction equipment adapted for use in continuous processing mode and comprising a substantially tubular reactor to form the product of formula (I) and in which or upstream of which two or more streams of the reagents/starting materials are brought together. 14. A process according to claim 1 wherein the process is conducted in reaction equipment adapted for use in continuous processing mode, and wherein the mixing time in the reaction equipment is shorter than the reaction half life (the time required to reach 50% conversion). 15. A process according to claim 1 wherein the process is conducted in reaction equipment adapted for use in continuous processing mode and being a non back-mixed system. 16. A process according to claim 1 wherein the process is conducted in reaction equipment adapted for use in continuous processing mode and in which mixing is induced passively through the fluid flow. 17. A process according to claim 16 wherein the reaction equipment comprises a plug flow reactor. 18. A process according to claim 17 wherein the plug flow reactor comprises a jacketed tubular reactor fitted inside with internal mixing elements which continually split and remix the reaction streams promoting mass and heat transfer, whereby a uniform plug flow profile with turbulent fluid flow is achieved. 19. A process as claimed in claim 16 wherein the reaction equipment comprises one or more static mixers. 20. A process as claimed in claim 17, wherein one or more of the static mixers or the plug flow reactor comprises one or more static mixing elements, wherein some, most or all of the mixing elements are adapted to split and mix a fluid stream flowing therethrough substantially in one dimension transverse to the fluid flow. 21. A process as claimed in claim 17, wherein one or more of the static mixers or the plug flow reactor comprises one or more static mixing elements, wherein some, most or all of the mixing elements comprise a plurality of stacked corrugated sheets, wherein the grooves of each corrugated sheet are transverse to the grooves of any directly adjacent corrugated sheet. 22. A process as claimed in claim 21, wherein a plurality of the mixing elements are substantially axially aligned within a or the substantially tubular reactor, with the corrugated sheets arranged so as to allow fluid flow longitudinally along the tubular reactor. 23. A process as claimed in claim 22, wherein some or all of the mixer elements in the tubular reactor are statically arranged so as to be in a rotated orientation about the longitudinal axis relative to one or both of the neighbouring mixing elements. 24. A process as claimed in claim 19, wherein one or more of the static mixers comprise one or more mixing elements arranged substantially axially within and in substantially static relation to a substantially tubular reactor, and wherein the mixing elements define at least one substantially helical fluid flow path in the axial direction between the mixing element and the inner circumferential walls of the tubular reactor. 25. A process as claimed in claim 24 wherein two substantially helical fluid flow paths are defined by the mixing elements. 26. A process as claimed in claim 24 wherein the mixing elements are helically-twisted about their axis so as to define the at least one substantially helical fluid flow path. 27. A process as claimed in claim 26, wherein wherein one or more (e.g. all) of the static mixers comprise a series of alternating right- and left-handed helically-twisted mixing elements, i.e. having alternating rotational directions. 28. A process as claimed in any of claims 24, wherein each or most mixing element(s) is/are a plate and a leading edge of each element is located substantially perpendicularly to a trailing edge of the following element. 29. A process as claimed in claim 1, wherein the residence time is about 5 to about 60 seconds. 30. A process as claimed in claim 29, wherein the residence time is 10 to 30 seconds. 31. A process as claimed in claim 24 wherein protection of the compound of formula (1) is performed by reacting the compound of formula (I) with a reagent for introducing the amino-protecting group P2, in the presence of a base and wherein the base is used at a stoichiometry of ≧6 equivalents relative to the compound of formula (II). 32. A process as claimed in claim 24 wherein protection of the compound of formula (I) is performed by reacting the compound of formula (I) with a reagent for introducing the amino-protecting group P2, in the presence of a base and wherein the base is used at a stoichiometry of 5 to 12 equivalents relative to the compound of formula (II). 33. A process as claimed in claim 1 wherein two, three or more streams of the reagents/starting materials are brought together upstream of a or the reactor (a) by a direct junction which contains substantially no mixing chamber at the junction, or (b) by a stream-mixing means comprising a substantial mixing chamber having two, three or more reagent inlets in fluid communication with the stored reagents and an outlet in fluid communication with the reactor. 34. A process as claimed in claim 33 wherein the stream-mixing means comprises a substantial mixing chamber and is a vortex mixer adapted to mix at least partly the reagent streams in use by generation of a vortex in the mixing chamber and/or in the outlet. 35. A process as claimed in claim 33 wherein the stream-mixing means is at a temperature of about 0° C. to about −15° C., preferably about −5° C. to about −15° C. 36. A process for the production of a compound of formula (III), or a pharmaceutically acceptable salt and/or hydrate thereof: which comprises: producing a compound of formula (I) according to a process as claimed in claim 1; and converting a compound of formula (I) to a compound of formula (IV): or a salt thereof, followed by reaction of the compound of formula (IV) or a salt thereof, with a compound of formula (V): wherein X is a leaving group; and (V) optionally forming a pharmaceutically acceptable salt and/or hydrate thereof. 37. A process as claimed in claim 36 wherein the reaction of the compound of formula (IV) and the compound of formula (V) is conducted in a solvent in the presence of a base. 38. A process as claimed in claim 36 wherein the compound of formula (III) is (R,S)-7-(3-aminomethyl-4-syn-methoxyimino-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate or a hydrate thereof. 39. A process as claimed in claim 38 wherein the compound of formula (III) is (R,S)-7-(3-aminomethyl-4-syn-methoxyimino-pyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate sesquihydrate. 40. A process for the production of a compound of formula (IVa): or a salt thereof, wherein R is C1-10 alkyl optionally fluorinated; C2-6alkenyl; C3-10 cycloalkyl with a 3-8-membered carbocyclic ring; C6-14aryl; C2-13 monocyclic, bicyclic or tricyclic heteroaryl; or C6-14aryl C1-4alkyl; wherein any aryl or heteroaryl moieties are optionally substituted by one, two or three of: C1-3 alkyl optionally fluorinated, a halogen atom, C1-2alkoxy optionally fluorinated, or NR1R2 where R1 and R2 independently are C1-2alkyl or H; the process comprising the steps of: (i) optionally reducing a compound of formula (I): , wherein P1 and P2 are amino protecting groups as defined in claim 1, to a compound of formula (VII): , wherein P1 and P2 are as defined in formula (I), in the presence of a first organic solvent; (ii) reacting the compound of formula (VII) with a compound of formula (VIa): R—ONH2 (VIa) or an acid addition salt thereof, wherein R is as defined in formula (IVa), in the presence of a second organic solvent, to form a compound of formula (Villa): , wherein P1 and P2 are as defined in formula (I) and R is as defined in formula (IVa); followed by (iii) removing the amino protecting groups P1 and P2 of the compound of formula (VIIIa) in the presence of a third organic solvent and a deprotecting agent capable of performing this removal, to form the compound of formula (IVa) or the salt thereof; characterised in that, after formation of the compound of formula (VIIIa) in step (ii), part or all of the second organic solvent is removed and replaced by the third organic solvent without isolation of the compound of formula (Villa). 41. A process as claimed in claim 40 wherein R is C1-6 alkyl optionally fluorinated; C2-4alkenyl; C3-8 cycloalkyl with a 3-6-membered carbocyclic ring; or phenyl, naphthyl, benzyl or phenylethyl in which the aryl moiety is optionally substituted by one or two of methyl, ethyl, trifluoromethyl, F, Cl, Br, I, methoxy, trifluoromethoxy or NMe2. 42. A process for the production of a compound of formula (IV): or a salt thereof, comprising the steps of: (i) optionally reducing a compound of formula (I): , wherein P1 and P2 are amino protecting groups as defined in claim 1, to a compound of formula (VII): , wherein P1 and P2 are as defined in formula (I), in the presence of a first organic solvent; (ii) reacting the compound of formula (VII) with a compound of formula (VI): Me-ONH2 (VI) or an acid addition salt thereof in the presence of a second organic solvent, to form a compound of formula (VIII): , wherein P1 and P2 are as defined in formula (I); followed by (iii) removing the amino protecting groups P1 and P2 of the compound of formula (VIII) in the presence of a third organic solvent and a deprotecting agent capable of performing this removal, to form the compound of formula (IV) or the salt thereof, characterised in that, after formation of the compound of formula (VIII) in step (ii), part or all of the second organic solvent is removed and replaced by the third organic solvent without isolation of the compound of formula (VIII). 43. A process as claimed in claim 40, wherein the compound of formula (IV) or (IVa) or the salt thereof is the dimethanesulfonate salt thereof. 44. A process as claimed in claim 40, wherein both P1 and P2 are t-butoxycarbonyl. 45. A process as claimed in any of claims 40, wherein optional step (i) is carried out, and steps (i) and (ii) are carried out without isolation of the compound of formula (VII). 46. A process as claimed in claim 45, wherein the second organic solvent is or includes some or all of the first organic solvent. 47. A process as claimed in claim 40, wherein the reaction of step (ii) is carried out in the presence of water and the pH is adjusted to a pH of about 3 to about 6. 48. A process as claimed in claim 40, wherein the second organic solvent is a substantially water-immiscible organic solvent. 49. A process as claimed in claim 40, wherein the first organic solvent is a substantially water-immiscible organic solvent which does not consist essentially of dichloromethane. 50. A process as claimed in claim 48, wherein the first and/or second substantially water-immiscible organic solvent comprises, consists essentially of or is: toluene; xylene; a C1-4alkyl C1-4alkyl ether; and/or a C1-4alkyl C2-4alkanoate. 51. A process as claimed in claim 40, wherein the second organic solvent is an antisolvent for the compound of formula (IV) or (IVa) or the salt thereof which is formed in step (iii); and in step (iii) the reaction solution contains no more of the antisolvent than that amount which would lead to early crystallisation of the compound of formula (IV) or the salt thereof and/or which would increase the E/Z-oxime ratio in the compound (IV) or salt formed, compared to the crystallisation time and/or E/Z-oxime ratio obtainable with no antisolvent present. 52. A process as claimed in claim 40, wherein the second organic solvent is an antisolvent for the compound of formula (IV) or (IVa) or the salt thereof which is formed in step (iii); and in step (iii) the reaction solution contains ≦1% of the antisolvent by volume of pure solvent or by volume of solution (including the possibility that no antisolvent is present). 53. A process as claimed in claim 48, wherein the second organic solvent which is substantially water-immiscible and/or which is an antisolvent comprises, consists essentially of or is ethyl acetate. 54. A process as claimed in claim 40, wherein: (a) the second organic solvent is different from and forms an azeotropic mixture with the third organic solvent allowing azeotropic removal of the second organic solvent from such a mixture, and/or (b) the second organic solvent is different from and has a lower boiling point than the third organic solvent; and after formation of the compound of formula (VIII) or (VIIIa) in step (ii), the second organic solvent is mixed with part or all of the third organic solvent, and part or all of the second organic solvent is removed by distillation or evacuation of the solvent mixture; followed by optionally adding a further portion of the third organic solvent before or during step (iii). 55. A process for the production of a compound of formula (III), or a pharmaceutically acceptable salt and/or hydrate thereof: which comprises: (1) optionally producing a compound of formula (I) as defined herein according to a process described in the first aspect of the invention; and (2) converting a compound of formula (I) as defined herein to a compound of formula (IV): , or a salt thereof, as defined in claim 40; followed by (3) reaction of the compound of formula (IV) or the salt thereof, with a compound of formula (V): wherein X is a leaving group; and (4) optionally forming a pharmaceutically acceptable salt and/or hydrate thereof. |
Taci and br3 polypeptides and uses thereof |
Novel, receptors, referred to herein as “TACIs” and “BR3”, agonists and antagonists thereof, and method of using TACIs and BR3, as well as agonists and antagonists thereof, to modulate, for example, activity of tumor necrosis factor (TNF) and TNFR-related molecules, including members of the TNF and TNFR families referred to as TALL-1, APRIL, TACI, and BCMA, are provided. Methods for in vitro, in situ, and/or in vivo diagnosis and/or treatment of mammalian cells or pathological conditions associated with such TNF and TNFR-related molecules are further provided. |
1. An isolated nucleic acid comprising (a) DNA encoding a TACIs polypeptide comprising the sequence of amino acid residues 1 to 246 of SEQ ID NO:14, or (b) the complement of the DNA molecule of (a). 2. The nucleic acid of claim 1, wherein said DNA comprises the coding nucleotide sequence of SEQ ID NO:13. 3. The nucleic acid of claim 1, wherein said DNA consists of the coding nucleotide sequence of SEQ ID NO:13. 4. An isolated nucleic acid comprising DNA which has (a) at least 95% sequence identity to the coding sequence of nucleotides of SEQ ID NO:13 and. (b) encodes aTACIs polypeptide. 5. An isolated nucleic acid comprising DNA from the group consisting of: a) a DNA havinq at least 90% sequence identity to a DNA sequence encoding a TACIs polypeptide comprising amnio acid residues 1 to 246 of SEQ ID NO:14; b) a DNA sequence that hybridizes under stringent conditions to a DNA of a); c) a DNA sequence that, due to the degeneracy of the genetic code, encodes a TACIs polypeptide of a); and d) DNA fully complementary to the DNA of a), b), or c). 6. A vector comprising the nucleic acid of claim 5. 7. The vector of claim 6 operably linked to control sequences recognized by a host cell transformed with the vector. 8. A host cell which includes the vector of claim 6. 9. The host cell of claim 8, wherein said cell is a CHO cell. 10. The host cell of claim 8, wherein said cell is an E.coli. 11. The host cell of claim 8, wherein said cell is a yeast cell. 12. A process for producing a TACIs polypeptide comprising culturing the host cell of claim 8 under conditions suitable for expression of said TACIs polypeptide and recovering said TACIs polypeptide from the cell culture. 13. An isolated TACIs polypeptide comprising amino acid residues 1 to 246 of FIG. 5B (SEQ ED NO:14). 14. An isolated TACIs polypeptide comprising the sequence of contiguous amino acid residues 1 to 246 of FIG. 5B (SEQ ID NO:14). 15. An isolated soluble TACIs polypeptide comprising amino acid residues 1 to 119 of FIG. 5B (SEQ ID NO:14). 16. An isolated TACIs polypeptide comprising a polypeptide selected from the group consisting of: a) a TACIs polypeptide comprising amino acid residues 1 to 246 or 1 to 119 of FIG. 5B (SEQ ID NO:14) and b) a fragment of a), wherein said fragment is a biologically active polypeptide. 17. A chimeric molecule comprising the TACIs polypeptide of claim 16 fused to a heterologous amino acid sequence. 18. The chimeric molecule of claim 17, wherein said heterologous amino acid sequence is an epitope tag sequence. 19. The chimeric molecule of claim 17, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin. 20. An isolated monoclonal antibody which binds to the TACIs polypeptide of claim 16. 21. A composition comprising the TACIs polypeptide of claim 16 and a carrier. 22. The composition of claim 21 wherein said carrier is a pharmaceutically-acceptable carrier. 23. An isolated nucleic acid comprising (a) DNA encoding a BR3 polypeptide comprising the sequence of amino acid residues 1 to 184 of SEQ ID NO:16, or (b) the complement of the DNA molecule of (a). 24. The nucleic acid of claim 23, wherein said DNA comprises the coding nucleotide sequence of SEQ ID NO:15. 25. The nucleic acid of claim 24, wherein said DNA consists of coding nucleotide sequence of SEQ ID NO:15. 26. An isolated nucleic acid comprising DNA which has (a) at least 95% sequence identity to the coding sequence of nucleotides of SEQ ID NO:15 and (b) encodes a BR3 polypeptide. 27. An isolated nucleic acid comprising DNA from the group consisting of: a) a DNA having at least 90% sequence identity to a DNA sequence encoding a BR3 polypeptide comprising amino acid residues 1 to 184 of SEQ ID NO:16; b) a DNA sequence, that hybridizes under stringent conditions to a DNA of a); c) a DNA sequence that, due to the degeneracy of the genetic code, encodes a BR3 polypeptide of a); and d) DNA fully complementary to the DNA of a), b), or c). 28. A vector comprising the nucleic acid of claim 27. 29. The vector of claim 28 operably linked to control sequences recognized by a host cell transformed with the vector. 30. A host cell which includes the vector of claim 28. 31. The host cell of claim 30, wherein said cell is a CHO cell. 32. The host cell of claim 30, wherein said cell is an E. coli. 33. The host cell of claim 30, wherein said cell is a yeast cell. 34. A process for producing a BR3 polypeptide comprising culturing the host cell of claim 30 under conditions suitable for expression of said BR3 polypeptide and recovering said BR3 polypeptide from the cell culture. 35. An isolated BR3 polypeptide comprising amino acid residues 1 to 184 of FIG. 6B (SEQ ID NO:16). 36. An isolated BR3 polypeptide comprising the sequence of contiguous amino acid residues 1 to 184 of FIG. 6B (SEQ ID NO:16). 37. An isolated soluble BR3 polypeptide comprising amino acid residues 1 to 77 or 2 to 62 of FIG. 6B (SEQ ID NO:16). 38. An isolated BR3 polypeptide comprising a polypeptide selected from the group consisting of: a) a BR3 polypeptide comprising amino acid residues 1 to 184, 1 to 77, or 2 to 62 of FIG. 6B (SEQ ID NO:16) and b) a fragment of a), wherein said fragment is a biologically active polypeptide. 39. A chimeric molecule comprising the BR3 polypeptide of claim 38 fused to a heterologous amino acid sequence. 40. The chimeric molecule of claim 39, wherein said heterologous amino acid sequence is an epitope tag sequence. 41. The chimeric molecule of claim 39, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin. 42. An isolated monoclonal antibody which binds to the BR3 polypeptide of claim 38. 43. A composition comprising the BR3 polypeptide of claim 38 and a carrier. 44. The composition of claim 43 wherein said carrier is a pharmaceutically-acceptable carrier. 45. A method of inhibiting or neutralizing TALL-1 polypeptide biological activity in mammalian cells, comprising exposing said mammalian cells to an effective amount of TALL-1 polypeptide antagonist, wherein said TALL-1 polypeptide antagonist is selected from the group consisting of a) a TACIs receptor immunoadhesin; b) a BR3 receptor immunoadhesin; c) a TACIs receptor linked to a nonproteinaceous polymer selected from the group consisting of polyethylene glycol, polypropylene glycol, and polyoxyalkylene; d) a BR3 receptor linked to a nonproteinaceous polymer selected from the group consisting of polyethylene glycol, polypropylene glycol, and polyoxyalkylene; e) a TACIs receptor antibody; and f) a BR3 receptor antibody. 46. The method of claim 45 wherein said TACIs receptor immunoadhesin comprises a TACIs extracellular domain sequence fused to a Fc region of an immunoglobulin. 47. The method of claim 45 wherein said BR3 receptor immunoadhesin comprises a BR3 extracellular domain sequence fused to a Fc region of an immunoglobulin. 48. The method of claim 45 wherein said TALL-1 polypeptide antagonist comprises an antagonist molecule which inhibits or neutralizes both TALL-1 polypeptide and APRIL polypeptide biological activity in mammalian cells. 49. The method of claim 45 wherein said mammalian cells comprise white blood cells. 50. A method of inhibiting or neutralizing APRIL polypeptide biological activity in mammalian cells, comprising exposing said mammalian cells to an effective amount of APRIL polypeptide antagonist, wherein said April polypeptide antagonist is selected front the group consisting of a) a TACIs receptor immunoadhesin; b) a TACIs receptor linked to a nonproteinaceous polymer selected from the group consisting of polyethylene glycol, polypropylene glycol, and polyoxyalkylene; c) a TACIs receptor antibody. 51. The method of claim 50 wherein said TACIs receptor immunoadhesin comprises a TACIs extracellular domain sequence fused to a Fc region of an immunoglobulin. 52. The method of claim 50 wherein said APRIL polypeptide antagonist comprises an antagonist molecule which inhibits or neutralizes both TALL-1 polypeptide and APRIL polypeptide biological activity in mammalian cells. 53. The method of claim 50 wherein said mammalian cells comprise white blood cells. 54. A method of enhancing or stimulating TACI polypeptide activity in mammalian cells, comprising exposing said mammalian cells to an effective amount of TACIs polypeptide agonist, wherein said TACIs polypeptide agonist comprises an anti-TACIs agonist antibody. 55. A method of enhancing or stimulating BR3 polypeptide activity in mammalian cells, comprising exposing said mammalian cells to en effective amount of BR3 polypeptide agonist, wherein said BR3 polypeptide agonist comprises an anti-BR3 agonist antibody. 56. A method of treating systemic lupus erythmatosus in a mammal, comprising administering to said mammal an effective amount of BR3 receptor immunoadhesin which comprises a BR3 extracellular domain sequence fused to a Fc region of an immunoglobulin. 57. A method of conducting a screening assay to identify a candidate molecule which acts as an antagonist or agonist of TALL-1, TACI, TACIs, BCMA or BR3, comprising an assay using the TACIs DNA or polypeptide of claims 5 or 16, or the BR3 DNA or polypeptide of claims 27 or 38. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Various molecules, such as tumor necrosis factor-α(“TNF-α”), tumor necrosis factor-β (“TNF-α” or “lymphotoxin-α”), lymphotoxin-β (“LT-β”), CD30 ligand, CD27 ligand, CD40 ligand, OX-40 ligand, 4-1BB ligand, Apo-1 ligand (also referred to as Fas ligand or CD95 ligand), Apo-2 ligand (also referred to as TRAIL), Apo-3 ligand (also referred to as TWEAK), APRIL, OPG ligand (also referred to as RANK ligand, ODF, or TRANCE), and TALL-1 (also referred to as BlyS, BAFF or THANK) have been identified as members of the tumor necrosis factor (“TNF”) family of cytokines [See, e.g., Gruss and Dower, Blood, 85:3378-3404 (1995); Schmid et al., Proc. Natl. Acad. Sci., 83:1881 (1986); Dealtry et al., Eur. J. Immunol., 17:689 (1987); Pitti et al., J. Biol. Chem., 271:12687-12690 (1996); Wiley et al., Immunity, 3:673-682 (1995); Browning et al., Cell, 72:847-856 (1993); Armitage et al. Nature, 357:80-82 (1992), WO 97/01633 published Jan. 16, 1997; WO 97/25428 published Jul. 17, 1997; Marsters et al., Curr. Biol., 8:525-528 (1998); Chicheportiche et al., Biol. Chem., 272:32401-32410 (1997); Hahne et al., J. Exp. Med., 188:1185-1190 (1998); WO98/28426 published Jul. 2, 1998; WO98/46751 published Oct. 22, 1998; WO/98/18921 published May 7, 1998; Moore et al., Science, 285:260-263 (1999); Shu et al., J. Leukocyte Biol., 65:680 (1999); Schneider et al., J. Exp. Med., 189:1747-1756 (1999); Mukhopadhyay et al., J. Biol. Chem., 274:15978-15981 (1999)]. Among these molecules, TNF-α, TNF-β, CD30 ligand, 4-1BB ligand, Apo-1 ligand, Apo-2 ligand (Apo2L/TRAIL) and Apo-3 ligand (TWEAK) have been reported to be involved in apoptotic cell death. Various molecules in the TNF family also have purported role(s) in the function or development of the immune system [Gruss et al., Blood, 85:3378 (1995)]. Zheng et al. have reported that TNF-α is involved in post-stimulation apoptosis of CD8-positive T cells [Zheng et al., Nature, 377:348-351 (1995)]. Other investigators have reported that CD30 ligand may be involved in deletion of self-reactive T cells in the thymus [Amakawa et al., Cold Spring Harbor Laboratory Symposium on Programmed Cell Death, Abstr. No. 10, (1995)]. CD40 ligand activates many functions of B cells, including proliferation, immunoglobulin secretion, and survival [Renshaw et al., J. Exp. Med., 180:1889 (1994)]. Another recently identified TNF family cytokine, TALL-1 (BlyS), has been reported, under certain conditions, to induce B cell proliferation and immunoglobulin secretion. [Moore et al., supra; Schneider et al., supra; Mackay et al., J. Exp. Med., 190:1697 (1999); Shu et al., J. Leukocyte Biol., 65:680-683 (1999); Gross et al., Nature, 404:995-999 (2000)]. Mutations in the mouse Fas/Apo-1 receptor or ligand genes (called lpr and gld, respectively) have been associated with some autoimmune disorders, indicating that Apo-1 ligand may play a role in regulating the clonal deletion of self-reactive lymphocytes in the periphery [Krammer et al., Curr. Op. Immunol., 6:279-289 (1994); Nagata et al., Science, 267:1449-1456 (1995)]. Apo-1 ligand is also reported to induce post-stimulation apoptosis in CD4-positive T lymphocytes and in B lymphocytes, and may be involved in the elimination of activated lymphocytes when their function is no longer needed [Krammer et al., supra; Nagata et al., supra]. Agonist mouse monoclonal antibodies specifically binding to the Apo-1 receptor have been reported to exhibit cell killing activity that is comparable to or similar to that of TNF-α [Yonehara et al., J. Exp. Med., 169:1747-1756 (1989)]. The TNF-related ligand called OPG ligand (also referred to as RANK ligand, TRANCE, or ODF) has been reported in the literature to have some involvement in certain immunoregulatory activities. WO98/28426 published Jul. 2, 1998 describes the ligand (referred to therein as RANK ligand) as a Type 2 transmembrane protein, which in a soluble form, was found to induce maturation of dendritic cells, enhance CD1a+ dendritic cell allo-stimulatory capacity in a MLR, and enhance the number of viable human peripheral blood T cells in vitro in the presence of TGF-beta. [see also, Anderson et al., Nature, 390:175-179 (1997)]. The WO98/28426 reference also discloses that the ligand enhanced production of TNF-alpha by one macrophage tumor cell line (called RAW264.7; ATCC TIB71), but did not stimulate nitric oxide production by those tumor cells. The putative roles of OPG ligand/TRANCE/ODF in modulating dendritic cell activity [see, e.g., Wong et al., J. Exp. Med., 186:2075-2080 (1997); Wong et al:, J. Leukocyte Biol., 65:715-724 (1999); Josien et al., J. Immunol., 162:2562-2568 (1999); Josien et al., J. Exp. Med., 191495-501 (2000)] and in influencing T cell activation in an immune response [see, e.g., Bachmann et al., J. Exp. Med., 189:1025-1031 (1999); Green et al., J. Exp. Med., 189:1017-1020 (1999)] have been explored in the literature. Kong et al., Nature, 397:315-323 (1999) report that mice with a disrupted opgl gene showed severe osteoporosis, lacked osteoclasts, and exhibited defects in early differentiation of T and B lymphocytes. Kong et al. have further reported that systemic activation of T cells in vivo led to an OPGL-mediated increase in osteoclastogenesis and bone loss. [Kong et al., Nature, 402:304-308 (1999)]. Induction of various cellular responses mediated by such TNF family cytokines is believed to be initiated by their binding to specific cell receptors. Previously, two distinct TNF receptors of approximately 55-kDa (TNFR1) and 75-kDa (TNFR2) were identified [Hohman et al., J. Biol. Chem., 264:14927-14934 (1989); Brockhaus et al., Proc. Natl. Acad. Sci., 87:3127-3131 (1990); EP 417,563, published Mar. 20, 1991; Loetscher et al., Cell, 61:351 (1990); Schall et al., Cell, 61:361 (1990); Smith et al., Science, 248:1019-1023 (1990); Lewis et al., Proc. Natl. Acad. Sci., 88:2830-2834 (1991); Goodwin et al., Mol. Cell. Biol., 11:3020-3026 (1991)]. Those TNFRs were found to share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular regions. The extracellular portions of both receptors were found naturally also as soluble TNF-binding proteins [Nophar, Y. et al., EMBO J., 9:3269 (1990); and Kohno, T. et al., Proc. Natl. Acad. Sci. U.S.A., 87:8331 (1990); Hale et al., J. Cell. Biochem. Supplement 15F, 1991, p. 113 (P424)]. The extracellular portion of type 1 and type 2 TNFRs (TNFR1 and TNFR2) contains a repetitive amino acid sequence pattern of four cysteine-rich domains (CRDs) designated 1 through 4, starting from the NH 2 -terminus. [Schall et al., supra; Loetscher et al., supra; Smith et al., supra; Nophar et al., supra; Kohno et al., supra; Banner et al., Cell, 73:431-435 (1993)]. A similar repetitive pattern of CRDs exists in several other cell-surface proteins, including the p75 nerve growth factor receptor (NGFR) [Johnson et al., Cell, 47:545 (1986); Radeke et al., Nature, 325:593 (1987)], the B cell antigen CD40 [Stamenkovic et al., EMBO J., 8:1403 (1989)], the T cell antigen OX40 [Mallet et al., EMBO J., 9:1063 (1990)] and the Fas antigen [Yonehara et al., supra and Itoh et al., Cell, 66:233-243 (1991)]. CRDs are also found in the soluble TNFR (sTNFR)-like T2 proteins of the Shope and myxoma poxviruses [Upton et al., Virology, 160:20-29 (1987); Smith et al., Biochem. Biophys. Res. Commun., 176:335 (1991); Upton et al., Virology, 184:370 (1991)]. Optimal alignment of these sequences indicates that the positions of the cysteine residues are well conserved. These receptors are sometimes collectively referred to as members of the TNF/NGF receptor superfamily. The TNF family ligands identified to date, with the exception of lymphotoxin-α, are typically type II transmembrane proteins, whose C-terminus is extracellular. In contrast, most receptors in the TNF receptor (TNFR) family identified to date are typically type I transmembrane proteins. In both the TNF ligand and receptor families, however, homology identified between family members has been found mainly in the extracellular domain (“ECD”). Several of the TNF family cytokines, including TNF-α, Apo-1 ligand and CD40 ligand, are cleaved proteolytically at the cell surface; the resulting protein in each case typically forms a homotrimeric molecule that functions as a soluble cytokine. TNF receptor family proteins are also usually cleaved proteolytically to release soluble receptor ECDs that can function as inhibitors of the cognate cytokines. The TNFR family member, referred to as RANK, has been identified as a receptor for OPG ligand (see WO98/28426 published Jul. 2, 1998; Anderson et al., Nature, 390:175-179 (1997); Lacey et al., Cell, 93:165-176 (1998). Another TNFR-related molecule, called OPG (FDCR-1 or OCIF), has also been identified as a receptor for OPG ligand. [Simonet et al., Cell, 89:309 (1997); Yasuda et al., Endocrinology, 139:1329 (1998); Yun et al., J. Immunol., 161:6113-6121 (1998)]. Yun et al., supra, disclose that OPG/FDCR-1/OCIF is expressed in both a membrane-bound form and a secreted form and has a restricted expression pattern in cells of the immune system, including dendritic cells, EBV-transformed B cell lines and tonsillar B cells. Yun et al. also disclose that in B cells and dendritic cells, expression of OPG/FDCR-1/OCIF can be up-regulated by CD40, a molecule involved in B cell activation. However, Yun et al. acknowledge that how OPG/FDCR-1/OCIF functions in the regulation of the immune response is unknown. More recently, other members of the TNFR family have been identified. In von Bulow et al., Science, 278:138-141 (1997), investigators describe a plasma membrane receptor referred to as Transmembrane Activator and CAML-Interactor or “TACI”. The TACI receptor is reported to contain a cysteine-rich motif characteristic of the TNFR family. In an in vitro assay, cross linking of TACI on the surface of transfected Jurkat cells with TACI-specific antibodies led to activation of NF-KB. [see also, WO 98/39361 published Sep. 18, 1998]. TACI knockout mice have been reported to have hyperresponsive B cells, while BCMA null mice had no discernable phenotype [Yan et al., Nature Immunology, 2:638-643 (2001); von Bulow et al., Immunity, 14:573-582 (2001); Xu et al., Mol. Cell. Biology, 21:4067-4074 (2001)]. Laabi et al., EMBO J., 11:3897-3904 (1992) reported identifying a new gene called “BCM” whose expression was found to coincide with B cell terminal maturation. The open reading frame of the BCM normal cDNA predicted a 184 amino acid long polypeptide with a single transmembrane domain. These investigators later termed this gene “BCMA.” [Laabi et al., Nucleic Acids Res., 22:1147-1154 (1994)]. BCMA mRNA expression was reported to be absent in human malignant B cell lines which represent the pro-B lymphocyte stage, and thus, is believed to be linked to the stage of differentiation of lymphocytes [Gras et al., Int. Immunology, 7:1093-1106 (1995)]. In Madry et al., Int. Immunology, 10:1693-1702 (1998), the cloning of murine BCMA cDNA was described. The murine BCMA cDNA is reported to encode a 185 amino acid long polypeptide having 62% identity to the human BCMA polypeptide. Alignment of the murine and human BCMA protein sequences revealed a conserved motif of six cysteines in the N-terminal region, suggesting that the BCMA protein belongs to the TNFR superfamily [Madry et al., supra]. The Tall-1 (BlyS) ligand has been reported to bind the TACI and BCMA receptors [Gross et al., supra, (2000); Thompson et al., J. Exp. Med., 192:129-135 (2000); Yan et al., supra, (2000); Marsters et al., Curr. Biol., 10:785-758 (2000); WO 00/40716 published Jul. 13, 2000; WO 00/67034 published Nov. 9, 2000]. TACI and BCMA have likewise been reported to bind to the ligand known as April. In Marsters et al., Curr. Biol., 6:750 (1996), investigators describe a full length native sequence human polypeptide, called Apo-3, which exhibits similarity to the TNFR family in its extracellular cysteine-rich repeats and resembles TNFR1 and CD95 in that it contains a cytoplasmic death domain sequence [see also Marsters et al., Curr. Biol., 6:1669 (1996)]. Apo-3 has also been referred to by other investigators as DR3, wsl-1, TRAMP, and LARD [Chinnaiyan et al., Science, 274:990 (1996); Kitson et al., Nature, 384:372 (1996); Bodmer et al., Immunity, 6:79 (1997); Screaton et al., Proc. Natl. Acad. Sci., 94:4615-4619 (1997)]. Pan et al. have disclosed another TNF receptor family member referred to as “DR4” (Pan et al., Science, 276:111-113 (1997); see also WO98/32856 published Jul. 30, 1998]. The DR4 was reported to contain a cytoplasmic death domain capable of engaging the cell suicide apparatus. Pan et al. disclose that DR4 is believed to be a receptor for the ligand known as Apo2L/TRAIL. In Sheridan et al., Science, 277:818-821 (1997) and Pan et al., Science, 277:815-818 (1997), another molecule believed to be a receptor for Apo2L/TRAIL is described [see also, WO98/51793 published Nov. 19, 1998; WO98/41629 published Sep. 24, 1998]. That molecule is referred to as DR5 (it has also been alternatively referred to as Apo-2; TRAIL-R, TR6, Tango-63, hAPO8, TRICK2 or KILLER [Screaton et al., Curr. Biol., 7:693-696 (1997); Walczak et al., EMBO J., 16:5386-5387 (1997); Wu et al., Nature Genetics, 17:141-143 (1997); WO98/35986 published Aug. 20, 1998; EP870,827 published Oct. 14, 1998; WO98/46643 published Oct. 22, 1998; WO99/02653 published Jan. 21, 1999; WO99/09165 published Feb. 25, 1999; WO99/11791 published Mar. 11, 1999]. Like DR4, DR5 is reported to contain a cytoplasmic death domain and be capable of signaling apoptosis. The crystal structure of the complex formed between Apo-2L/TRAIL and DR5 is described in Hymowitz et al., Molecular Cell, 4:563-571 (1999). Yet another death domain-containing receptor, DR6, was recently identified [Pan et al., FEBS Letters, 431:351-356 (1998)]. Aside from containing four putative extracellular cysteine rich domains and a cytoplasmic death domain, DR6 is believed to contain a putative leucine-zipper sequence that overlaps with a proline-rich motif in the cytoplasmic region. The proline-rich motif resembles sequences that bind to src-homology-3 domains, which are found in many intracellular signal-transducing molecules. In contrast to other death domain-containing receptors referred to above, DR6 does not induce cell death in the apoptosis sensitive indicator cell line, MCF-7, suggesting an alternate function for this receptor. Consistent with this observation, DR6 is presently believed not to associate with death-domain containing adapter molecules, such as FADD, RAIDD and RIP, that mediate downstream signaling from activated death receptors [Pan et al., FEBS Lett., 431:351 (1998)]. A further group of recently identified receptors are referred to as “decoy receptors,” which are believed to function as inhibitors, rather than transducers of signaling. This group includes DCR1 (also referred to as TRID, LIT or TRAIL-R3) [Pan et al., Science, 276:111-113 (1997); Sheridan et al., Science, 277:818-821 (1997); McFarlane et al., J. Biol. Chem., 272:25417-25420 (1997); Schneider et al., FEBS Letters, 416:329-334 (1997); Degli-Esposti et al., J. Exp. Med., 186:1165-1170 (1997); and Mongkolsapaya et al., J. Immunol., 160:3-6 (1998)] and DCR2 (also called TRUNDD or TRAIL-R4) [Marsters et al., Curr. Biol., 7:1003-1006 (1997); Pan et al., FEBS Letters, 424:41-45 (1998); Degli-Esposti et al., Immunity, 7:813-820 (1997)], both cell surface molecules, as well as OPG [Simonet et al., supra; Emery et al., infra] and DCR3 [Pitti et al., Nature, 396:699-703 (1998)], both of which are secreted, soluble proteins. Additional newly identified members of the TNFR family include CAR1, HVEM, GITR, ZTNFR-5, NTR-1, and TNFL1 [Brojatsch et al., Cell, 87:845-855 (1996); Montgomery et al., Cell, 87:427-436 (1996); Marsters et al., J. Biol. Chem., 272:14029-14032 (1997); Nocentini et al., Proc. Natl. Acad. Sci. USA 94:6216-6221 (1997); Emery et al., J. Biol. Chem., 273:14363-14367 (1998); WO99/04001 published Jan. 28, 1999; WO99/07738 published Feb. 18, 1999; WO99/33980 published Jul. 8, 1999]. As reviewed recently by Tewari et al., TNFR1, TNFR2 and CD40 modulate the expression of proinflammatory and costimulatory cytokines, cytokine receptors, and cell adhesion molecules through activation of the transcription factor, NF-κB [Tewari et al., Curr. Op. Genet. Develop., 6:39-44 (1996)]. NF-κB is the prototype of a family of dimeric transcription factors whose subunits contain conserved Rel regions [Verma et al., Genes Develop., 9:2723-2735 (1996); Baldwin, Ann. Rev. Immunol., 14:649-681 (1996)]. In its latent form, NF-κB is complexed with members of the IκB inhibitor family; upon inactivation of the IκB in response to certain stimuli, released NF-κB translocates to the nucleus where it binds to specific DNA sequences and activates gene transcription. As described above, the TNFR members identified to date either include or lack an intracellular death domain region. Some TNFR molecules lacking a death domain, such as TNFR2, CD40, HVEM, and GITR, are capable of modulating NF-κB activity. [see, e.g., Lotz et al., J. Leukocyte Biol., 60:1-7 (1996)]. For a review of the TNF family of cytokines and their receptors, see Ashkenazi and Dixit, Science, 281:1305-1308 (1998); Golstein, Curr. Biol., 7:750-753 (1997); Gruss and Dower, supra, and Nagata, Cell, 88:355-365 (1997). |
<SOH> SUMMARY OF THE INVENTION <EOH>Applicants have identified novel molecules referred to as “TACIs” and “BR3”. TACIs polypeptide has been characterized as having a single cysteine-rich domain, in contrast to the full-length human TACI molecule described in von Bulow et al., supra, which includes two cysteine-rich domains. Likewise, BR3 polypeptide as described herein has been characterized as having a single cysteine-rich domain. Applicants have surprisingly found that the TNF family ligands referred to as TALL-1 and April bind to the TACIs receptor. Applicants have also surprisingly found that the TNF family ligand referred to as TALL-1 binds to BR3 receptor. In contrast to the TACI and BCMA receptors, BR3 does not appear to bind the ligand, April, and does not activate the NF-KB pathway. The present invention thus provides for novel methods of using antagonists or agonists of these TNF-related ligands and receptors. The antagonists and agonists described herein find utility for, among other things, in vitro, in situ, or in vivo diagnosis or treatment of mammalian cells or pathological conditions associated with the presence (or absence) of TALL-1, APRIL, TACI, BCMA, TACIs, or BR3. In one embodiment, the invention provides isolated nucleic acid molecules comprising DNA encoding a TACIs polypeptide. In certain aspects, the isolated nucleic acid comprises DNA encoding the TACIs polypeptide having amino acid residues 1 to 246 or 1 to 119 of FIG. 5B (SEQ ID NO:14), or is complementary to such encoding nucleic acid sequences, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. In another embodiment, the invention provides vectors comprising DNA encoding a TACIs polypeptide. A host cell comprising such a vector is also provided. By way of example, the host cells may be CHO cells, E. coli, or yeast. A process for producing TACIs polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of TACIs polypeptide and recovering TACIs polypeptide from the cell culture. In another embodiment, the invention provides isolated TACIs polypeptides. In particular, the invention provides isolated TACIs polypeptides which include an amino acid sequence comprising residues 1 to 246 of FIG. 5B (SEQ ID NO:14). Additional embodiments of the present invention are directed to isolated extracellular domain sequences of TACIs polypeptide comprising amino acids 1 to 119 of the amino acid sequence shown in FIG. 5B (SEQ ID NO:14), or fragments thereof, particularly biologically active fragments. In another embodiment, the invention provides chimeric molecules comprising TACIs polypeptide or extracellular domain sequence or other fragment thereof fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule comprises a TACIs polypeptide fused to an epitope tag sequence or a Fc region of an immunoglobulin. In another embodiment, the invention provides an antibody which specifically binds to a TACIs polypeptide or extracellular domain thereof. Optionally, the antibody is a monoclonal antibody. In a still further embodiment, the invention provides diagnostic and therapeutic methods using TACIs polypeptide or DNA encoding TACIs polypeptide. In another embodiment, the invention provides isolated nucleic acid molecules comprising DNA encoding a BR3 polypeptide. In certain aspects, the isolated nucleic acid comprises DNA encoding the BR3 polypeptide having amino acid residues 1 to 184, 1 to 77 or 2 to 62 of FIG. 6B (SEQ ID NO:16), or is complementary to such encoding nucleic acid sequences, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. In another embodiment, the invention provides vectors comprising DNA encoding a BR3 polypeptide. A host cell comprising such a vector is also provided. By way of example, the host cells may be CHO cells, E. coli, or yeast. A process for producing BR3 polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of BR3 polypeptide and recovering BR3 polypeptide from the cell culture. In another embodiment, the invention provides isolated BR3 polypeptides. In particular, the invention provides isolated BR3 polypeptides which include an amino acid sequence comprising residues 1 to 184, 1 to 77 or 2 to 62 of FIG. 6B (SEQ ID NO:16). Additional embodiments of the present invention are directed to isolated extracellular domain sequences of BR3 polypeptide comprising amino acids 1 to 77 or 2 to 62 of the amino acid sequence shown in FIG. 6B (SEQ ID NO:16), or fragments thereof. In another embodiment, the invention provides chimeric molecules comprising a BR3 polypeptide or extracellular domain sequence or other fragment thereof fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule comprises a BR3 polypeptide fused to an epitope tag sequence or a Fc region of an immunoglobulin. In another embodiment, the invention provides an antibody which specifically binds to a BR3 polypeptide or extracellular domain thereof. Optionally, the antibody is a monoclonal antibody. In a still further embodiment, the invention provides diagnostic and therapeutic methods using BR3 polypeptide or DNA encoding BR3 polypeptide. The methods of the invention include methods to treat pathological conditions or diseases in mammals associated with or resulting from increased or enhanced TALL-1 or APRIL expression and/or activity. In the methods of treatment, TALL-1 antagonists or APRIL antagonists may be administered to the mammal suffering from such pathological condition or disease. The TALL-1 antagonists and APRIL antagonists contemplated for use in the invention include TACIs receptor immunoadhesins or BR3 receptor immunoadhesins, as well as antibodies against the TACIs receptor or BR3 receptor, which preferably block or reduce the respective receptor binding or activation by TALL-1 ligand and/or APRIL ligand. For instance, TACIs receptor immunoadhesins may be employed to treat rheumatoid arthritis or multiple sclerosis. The TALL-1 antagonists and APRIL antagonists contemplated for use further include anti-TALL-1 antibodies or anti-APRIL antibodies which are capable of blocking or reducing binding of the respective ligands to the TACIs or BR3 receptors. Still further antagonist molecules include covalently modified forms, or fusion proteins, comprising TACIs or BR3. By way of example, such antagonists may include pegylated TACIs or BR3 and TACIs or BR3 fused to heterologous sequences such as epitope tags or leucine zippers. Optionally, the antagonist molecule(s) employed in the methods will be capable of blocking or neutralizing the activity of both TALL-1 and APRIL, e.g., a dual antagonist which blocks or neutralizes activity of both TALL-1 and APRIL. Optionally, the antagonist molecule(s) employed in the methods will be capable of blocking or neutralizing the activity of TALL-1 but not APRIL, e.g., an antagonist (such as a BR3 immunoadhesin) which blocks or neutralizes activity of TALL-1. For instance, a BR3 immunoadhesin may be employed to treat an autoimmune disorder such as lupus. The methods contemplate the use of a single type of antagonist molecule or a combination of two or more types of antagonist. In another embodiment of the invention, there are provided methods for the use of TALL-1 antagonists to block or neutralize the interaction between TALL-1 and TACIs and/or BR3. Such antagonists may also block or neutralize the interaction between TALL-1 and TACI and/or BCMA. For example, the invention provides a method comprising exposing a mammalian cell, such as a white blood cell (preferably a B cell), to one or more TALL-1 antagonists in an amount effective to decrease, neutralize or block activity of the TALL-1 ligand. The cell may be in cell culture or in a mammal, e.g. a mammal suffering from, for instance, an immune related disease or cancer. Thus, the invention includes a method for treating a mammal suffering from a pathological condition such as an immune related disease or cancer comprising administering an effective amount of one or more TALL-1 antagonists, as disclosed herein. In particular embodiments, the immune related disorder is an autoimmune disease such as arthritis or lupus. The invention also provides methods for the use of APRIL antagonists to block or neutralize the interaction between APRIL and TACIs. Such antagonists may also block or neutralize the interaction between APRIL and TACI and/or BCMA. For example, the invention provides a method comprising exposing a mammalian cell, such as a white blood cell (preferably a B cell), to one or more APRIL antagonists in an amount effective to decrease, neutralize or block activity of the APRIL ligand. The cell may be in cell culture or in a mammal, e.g. a mammal suffering from, for instance, an immune related disease or cancer. Thus, the invention includes a method for treating a mammal suffering from a pathological condition such as an immune related disease or cancer comprising administering an effective amount of one or more APRIL antagonists, as disclosed herein. The invention also provides compositions which comprise one or more TALL-1 antagonists or APRIL antagonists. Optionally, the compositions of the invention will include pharmaceutically acceptable carriers or diluents. Preferably, the compositions will include one or more TALL-1 antagonists or APRIL antagonists in an amount which is therapeutically effective to treat a pathological condition or disease. The invention also provides articles of manufacture and kits which include one or more TALL-1 antagonists or APRIL antagonists. In addition, the invention provides methods of using TACIs agonists or BR3 agonists to, for instance, stimulate or activate TACIs receptor or BR3 receptor. Such methods will be useful in treating pathological conditions characterized by or associated with insufficient TALL-1 or APRIL expression or activity such as immunodeficiency or cancer (such as by boosting the immune anti-cancer response). The TACIs agonists or BR3 agonists may comprise agonistic anti-TACIs or anti-BR3 antibodies. The agonistic activity of such TACIs agonists or BR3 agonists may comprise enhancing the activity of a native ligand for TACIs or BR3 or activity which is the same as or substantially the same as (i.e., mimics) the activity of a native ligand for TACIs or BR3. Thus, the invention also provides compositions which comprise one or more TACIs agonists or BR3 agonists. Optionally, the compositions of the invention will include pharmaceutically acceptable carriers or diluents. Preferably, the compositions will include one or more TACIs agonists or BR3 agonists in an amount which is therapeutically effective to stimulate signal transduction by TACIs or BR3. Further, the invention provides articles of manufacture and kits which include one or more TACIs agonists or BR3 agonists. The invention also provides methods of conducting screening assays to identify candidate molecules, such as small molecule compounds, polypeptides or antibodies, which act as agonists or antagonists with respect to the interaction between TALL-1 and TACIs or BR3, or to the interaction between APRIL and TACIs. |
Host apparatus, electronic device, and transmission system control method |
To provide a method for controlling a transmission system with quick response in which plural electronic devices are connected to one host apparatus through a common data line. The method for controlling the transmission system of the present invention comprises: a step of transmitting a command signal for releasing the data line and suspending the processing through a command signal line to the electronic device occupying the data line in the state where one of the plural electronic devices is executing the processing that occupies the data line so as to make the electronic device release the data line and suspend the processing and to perform data transmission between the host apparatus and the other one of the electronic devices; and a step of resuming the suspended processing after the data transmission between the host apparatus and the other electronic device. |
1. A host apparatus comprising: an input/output part having a command signal line for transmitting a command signal from the host apparatus to an electronic device and a response signal from said electronic device to the host apparatus in response to said command signal, and a data line, at least a part of which is commonly used by plural electronic devices and connected to the plural electronic devices, for transmitting, at least, data in connection with the command signal from the host apparatus to said electronic device; and a control part having a saving register for bus release, wherein data is transmitted conforming to a protocol that the host apparatus serves as a master and said electronic devices serve as slaves, the host apparatus transmits a command signal to said electronic device and then said electronic device that received the command signal returns a response signal to the host apparatus and further, if necessary, data is transmitted from the host apparatus to the electronic device or from the electronic device to the host apparatus; and said control part in the state where one of said electronic devices is executing the processing that occupies said data line, when data transmission between the host apparatus and the other one of said electronic devices is intended to be executed, transmits a command signal for releasing said data line and suspending said processing to the electronic device that occupies said data line through said command signal line; stores an identifier of the electronic device that occupies said data line and the information on the current stage of said processing in said saving register for bus release; after completion of data transmission between the host apparatus and said other electronic device, reads out the identifier of the electronic device that had occupied said data line and the information on the current stage of said processing from said saving register for bus release; and transmits a command signal for returning to said suspended processing to the electronic device that had occupied said data line through said command signal line. 2. A host apparatus comprising: an input/output part having a command signal line for transmitting a command signal from the host apparatus to an electronic device and a response signal from said electronic device to the host apparatus in response to said command signal, and a data line, at least a part of which is commonly used by plural electronic devices and connected to the plural electronic devices, for transmitting, at least, data in connection with the command signal from the host apparatus to said electronic device; and a control part having a saving register for bus release, wherein data is transmitted conforming to a protocol that the host apparatus serves as a master and said electronic devices serve as slaves, the host apparatus transmits a command signal to said electronic device and then said electronic device that received the command signal returns a response signal to the host apparatus and further, if necessary, data is transmitted from the host apparatus to the electronic device or from the electronic device to the host apparatus; and said control part in the state where one of said electronic devices is executing the processing that occupies said data line, when data transmission between the host apparatus and the other one of said electronic devices is intended to be executed, transmits a command signal for releasing said data line and suspending said processing to said plural electronic devices connected to said data line through said command signal line; stores the information that said data line has been released and the information on the current stage of said processing in said saving register for bus release; after completion of data transmission between the host apparatus and said other electronic device, reads out the information that said data line has been released and the information on the current stage of said processing from said saving register for bus release; and transmits a command signal for returning to said suspended processing to said plural electronic devices connected to said data line through said command signal line. 3. A host apparatus in accordance with claim 1, wherein said input/output part further has an interrupt signal line for transmitting an interrupt signal from said electronic devices to the host apparatus; said control part further has an interrupt priority determination part; in the case where the host apparatus receives the interrupt signal for requesting data transmission from the other one of said electronic devices through said interrupt signal line in the state where one of said electronic devices is executing the processing that occupies said data line; and said interrupt priority determination part determines that said data transmission has a higher priority than the processing that occupies said data line, there is a case where data transmission between the host apparatus and said other electronic device is intended to be executed in the state where one of said electronic devices is executing the processing that occupies said data line. 4. An electronic device comprising: an input/output part having a command signal line for transmitting a command signal from the host apparatus to an electronic device and a response signal from said electronic device to the host apparatus in response to said command signal, and a data line, at least a part of which is commonly used by plural electronic devices and connected to the plural electronic devices, for transmitting, at least, data in connection with the command signal from the host apparatus to said electronic device; and a control part having a saving register for bus release, wherein data is transmitted conforming to a protocol that said host apparatus serves as a master and the electronic devices serve as slaves, said host apparatus transmits a command signal to the electronic device and then the electronic device that received the command signal returns a response signal to the host apparatus and further, if necessary, data is transmitted from said host apparatus to the electronic device or from the electronic device to said host apparatus; and said control part in the state where the electronic device is executing the processing that occupies said data line, when receiving a command signal for releasing said data line and suspending said processing from said host apparatus, stores the information on the current stage of said processing in said saving register for bus release, suspends said processing and transmits a response signal for notifying that said processing has been suspended and said data line has been released to said host apparatus through said command signal line; when receiving a command signal for returning to said suspended processing from said host apparatus through said command signal line, transmits a response signal including the information that return of said processing is possible and the information as to whether the data line need to be reoccupied or not to said host apparatus through said comma signal line, reads out the information on the current signal of said processing from said saving register for bus release, resumes said processing from said stage, and if there is need to reoccupy said data line, transmits data through said data line. 5. A method for controlling a transmission system comprising a host apparatus, plural electronic devices, a command signal line for transmitting a command signal from the host apparatus to an electronic device and a response signal from said electronic device to the host apparatus in response to said command signal, and a data line, at least a part of which is commonly used by plural electronic devices and connected to the plural electronic devices, for transmitting, at least, data in connection with the command signal from the host apparatus to said electronic device; data is transmitted conforming to a protocol that said host apparatus serves as a master and said electronic devices serve as slaves, said host apparatus transmits a command signal to said electronic device and then said electronic device that received the command signal returns a response signal to said host apparatus and further, if necessary, data is transmitted from said host apparatus to the electronic device or from the electronic device to said host apparatus, wherein said method comprises the steps of: in the state where one of said plural electronic devices is executing the processing that occupies said data line, transmitting an command signal for releasing said data line and suspending said processing from the host apparatus to said electronic device that occupies said data line through said command signal line in order to execute data transmission between said host apparatus and the other one of said electronic devices; transmitting a response signal for notifying that said processing has been suspended and said data line has been released from the electronic device that occupies said data line to said host apparatus through said command signal line; after completion of data transmission between said host apparatus and said other electronic device, transmitting an command signal for returning to said suspended processing from the host apparatus to the electronic device that had occupied said data line through said command signal line; transmitting a response signal including the information that return of said processing is possible and the information as to whether the data line need to be reoccupied or not from the electronic device that had occupied said data line to said host apparatus through said command signal line; and if the electronic device that had occupied said data line need to reoccupy said data line, transmitting data through said data line and resuming said processing. 6. A method for controlling a transmission system comprising a host apparatus, plural electronic devices, a command signal line for transmitting a command signal from the host apparatus to an electronic device and a response signal from said electronic device to the host apparatus in response to said command signal, and a data line, at least a part of which is commonly used by plural electronic devices and connected to the plural electronic devices, for transmitting, at least, data in connection with the command signal from the host apparatus to said electronic device; data is transmitted conforming to a protocol that said host apparatus serves as a master and said electronic devices serve as slaves, said host apparatus transmits a command signal to said electronic device and then said electronic device that received the command signal returns a response signal to said host apparatus and further, if necessary, data is transmitted from said host apparatus to the electronic device or from the electronic device to said host apparatus, wherein said method comprises the steps of: in the state where one of said plural electronic devices is executing the processing that occupies said data line, transmitting an command signal for releasing said data line and suspending said processing from the host apparatus to said plural electronic devices connected to said data line through said command signal line in order to execute data transmission between said host apparatus and the other one of said electronic devices; transmitting a response signal for notifying that said processing has been suspended and said data line has been released from the electronic device that occupies said data line to said host apparatus through said command signal line; after completion of data transmission between said host apparatus and said other electronic device, transmitting an command signal for returning to said suspended processing from the host apparatus to said plural electronic devices connected to said data line through said command signal line; transmitting a response signal including the information that return of said processing is possible and the information as to whether the data line need to be reoccupied or not from the electronic devices had occupied said data line to said host apparatus through said command signal line; and if the electronic device that had occupied said data line need to reoccupy said data line, transmitting data through said data line and resuming said processing. 7. A method for controlling a transmission system in accordance with claim 5, further comprising an interrupt signal line for transmitting an interrupt signal from said electronic device to the host apparatus, wherein said method comprises the steps of: in the state where one of said electronic devices is executing the processing that occupies said data line, said host apparatus receiving an interrupt signal for requesting data transmission from the other one of said electronic devices through said interrupt signal line; and determining that said data transmission has a higher priority than the processing that occupies said data line; and based on the result of said determination, there is a case where data transmission between said host apparatus and said other electronic device is intended to be executed in the state wherein one of said electronic devices is executing the processing that occupies said data line. 8. (Canceled) 9. A host apparatus comprising: an input/output part having a communication line for transmitting a command signal from the host apparatus to an electronic device and a response signal from said electronic device to the host apparatus in response to said command signal; and a control part having a saving register, wherein data is transmitted conforming to a protocol that the host apparatus serves as a master and said electronic devices serve as slaves, the host apparatus transmits a command signal to said electronic device and then said electronic device that received the command signal returns a response signal to the host apparatus and further, if necessary, data is transmitted from the host apparatus to the electronic device or from the electronic device to the host apparatus; and said control part in the state of performing communication processing with one of said electronic devices, when the host apparatus intends to communicate with the other one of said electronic devices, transmits a command signal for suspending said communication processing to said electronic device through said communication line; stores an identifier of said electronic device and the information on the current stage of said communication processing in said saving register; after completion of communication between the host apparatus and said other electronic device, reads out the identifier of said electronic device and the information on the current stage of said communication processing from said saving register; and transmits a command signal for returning to said suspended communication processing to said electronic device through said communication line. 10. A transmission system comprising: a host apparatus having a register for bus release; plural electronic devices, each having a register for bus release; a command signal line for transmitting a command signal from said host apparatus to said electronic device and a response signal from said electronic device to said host apparatus in response to said command signal; and a data line, via which said plural electronic devices are connected to said host apparatus and at least a part of which is commonly used by plural electronic devices for transmitting, at least, data in connection with the command signal from the host apparatus to said electronic device, wherein data is transmitted conforming to a protocol that said host apparatus serves as a master and said electronic devices serve as slaves, said host apparatus transmits a command signal to said electronic device and then said electronic device that received the command signal returns a response signal to said host apparatus and further, if necessary, data is transmitted from said host apparatus to the electronic device or from the electronic device to said host apparatus, wherein said host apparatus, in the state where one of said plural electronic devices is executing the processing that occupies said data line, transmits a command signal for releasing said data line and suspending said processing to said electronic device that occupies said data line through said command signal line so that data transmission is executed between said host apparatus and the other one of said electronic devices, and stores the information that said data line has been released and the information on the current stage of said processing in said register for bus release; said electronic device that has occupied said data line transmits a response signal informing that said processing is suspended and said data line is released to said host apparatus through said command signal line, and stores the information that said data line is released and the information on the current stage of said processing in said register for bus release; after completion of data transmission between said host apparatus and said other electronic device, said suspended processing between said host apparatus and the electronic device that had occupied said data line is made return, and said processing is resumed from the suspended stage based on the information read out from said registers for bus release respectively. |
<SOH> BACKGROUND ART <EOH>There is a transmission system in which a host apparatus (master apparatus) is connected to plural electronic devices (slave devices) through a common data line and data is transmitted between the host apparatus and the plural electronic devices. In such transmission system, generally, the host apparatus permits a specific electronic device to occupy a data line and then data transmission between the host apparatus and the electronic device (data transmission from the host apparatus to the electronic device or data transmission from the electronic device to the host apparatus) is executed. When the data transmission is completed, the host apparatus permits another electronic device to occupy the data line and then data transmission between the host apparatus and the electronic device is executed. However, in the conventional transmission system in which plural electronic devices are connected to one host apparatus through the common data line (for example, data bus), while one electronic device is occupying the data line, data transmission between other electronic device and the host apparatus cannot be executed. The other electronic device is obliged to wait until the initial electronic device has finished processing (including data transmission) and then execute data transmission. To solve the above-mentioned conventional problem, the present invention intends to provide a transmission system with quick response, wherein even while one electronic device is occupying a data line, data transmission between the other electronic device and the host apparatus can be performed as necessary and plural electronic devices are connected to one host apparatus through a common data line, a method for controlling the transmission system, and a host apparatus and electronic devices constituting the transmission system. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a block diagram showing the configuration of a transmission system in accordance with a first embodiment of the present invention. FIG. 2 is a time chart of the transmission system in accordance with the first embodiment of the present invention. FIG. 3 is a block diagram showing the configuration of a transmission system in accordance with a second embodiment of the present invention. FIG. 4 is a time chart of the transmission system in accordance with the second embodiment of the present invention. FIG. 5 is a block diagram showing the configuration of a transmission system in accordance with a third embodiment of the present invention. FIG. 6 is a time chart of the transmission system in accordance with the third embodiment of the present invention. FIG. 7 is a block diagram showing the configuration of a transmission system in accordance with a fourth embodiment of the present invention. FIG. 8 is a block diagram showing the configuration of a transmission system in accordance with a fifth embodiment of the present invention. FIG. 9 is a time chart of the transmission system in accordance with the fifth embodiment of the present invention. detailed-description description="Detailed Description" end="lead"? Part or All of the drawings are drawn schematically for diagrammatic representation and it should be considered that they do not necessarily reflect relative size and position of components shown therein. |
Monitoring device and method using echo signals |
An echo-signal monitoring device having a plurality of transceiver units for transmitting signals and receiving the echoes reflected by an external object and an analyzer unit for estimating the distance from the monitoring device to the external object on the basis of the received echoes is characterized in that the device has an operating mode for calculating the relative position of the transceiver units from one another on the basis of the reflected echoes. |
1-15. (Canceled). 16. An echo-signal monitoring device comprising: transceiver units, each for transmitting a signal and receiving an echo reflected back by an external object; and an analyzer unit for estimating a distance from the monitoring device to the external object based on received echoes; wherein the monitoring device includes an operating mode for calculating a relative position of the transceiver units to one another based on a reflected echo. 17. The echo-signal monitoring device of claim 16, wherein the monitoring device checks whether propagation times of the received echoes meet predetermined conditions, and if so, the monitoring device switches to the operating mode for calculating the relative position of the transceiver units to one another. 18. The echo-signal monitoring device of claim 17, wherein the monitoring device includes a signal input for a speed signal and it switches to the operating mode for calculating the relative position of the transceiver units to one another only when the speed signal applied to the signal input indicates a speed below a predetermined limiting value. 19. The echo-signal monitoring device of claim 17, wherein the monitoring device is operable to estimate a speed of the external object associated with the echoes based on a change in the propagation times of successively received echoes over time, and it switches to the operating mode for calculating the relative position of the transceiver units to one another only when the estimated speed is below a predetermined limiting value. 20. The echo-signal monitoring device of claim 16, wherein the each of the transceiver units include a detection threshold which an amplitude of a received signal must exceed to be detected by the transceiver units, the detection threshold dropping gradually from a high value to a low value immediately after transmission of the signal by a corresponding one of the transceiver units. 21. The echo-signal monitoring device of claim 16, further comprising: a control unit to determine points in time for transmitting a pulse-shaped signal for the transceiver units according to a stochastic model. 22. The echo-signal monitoring device of claim 16, further comprising: adaptive filters for the transceiver units. 23. A method for determining a relative position of transceiver units of an echo-signal monitoring device, the method comprising: measuring direct echo propagation times from a pair of the transceiver units and back to the same transceiver units and a cross-echo propagation time from one of the transceiver units to another one of the transceiver units of the pair being measured for at least one pair of the transceiver units, the distance being determined based on measured direct echo propagation times. 24. The method of claim 23, wherein the direct echo propagation times are measured repeatedly and calculation of a distance includes averaging over repeated ones of the measured times. 25. The method of claim 24, wherein a geometry quality measure is determined based on the measurements, and is used to increase an accuracy of an estimate of a sensor position. 26. The method of claim 24, wherein the repeated ones of the measurements are performed during a movement of the echo-signal monitoring device. 27. The method of claim 23, wherein the direct echo propagation times are measured for a plurality of pairs of the transceiver units. 28. The method of claim 27, wherein the plurality of pairs is selected so that redundant measurements are performed, and positions of the transceiver units are obtained from the redundant measurements with an error minimization process. 29. The method of claim 28, wherein a reliability of a measurement of an echo propagation time is evaluated based on a fluctuation in the measured echo propagation time in the repeated measurements, and the measurement is considered in the error minimization process using a weighting that is greater the smaller the fluctuation. 30. The method of claim 28, wherein a reliability of a measurement of a cross-echo propagation time is evaluated based on its relationship to the direct echo propagation times of the transceiver units involved in the measurement of the cross-echo propagation time, the measurement being considered in the error minimization process using a weighting that is greater the longer the cross-echo propagation time is in relation to the direct echo propagation times. |
<SOH> BACKGROUND INFORMATION <EOH>Devices and methods for monitoring the environment with the help of echo signals are used in particular in motor vehicles, where obstacles in the environment of the vehicle are detected with the help of transceiver units, in particular for ultrasound, radar, and infrared signals. A plurality of such transceiver units may be fixedly mounted on the vehicle body, and if necessary a warning signal is generated to warn the driver of the vehicle of a potentially hazardous approach to an obstacle in complex traffic situations such as parking. Such a monitoring device and a method for operating same are referred to in DE 40 23 538 A1, for example. The accuracy with which such devices function depends to a great extent on the accuracy with which the relative positions of the plurality of transceiver units of such a monitoring device are known in relation to one another and can be taken into account in calculating the distance from an obstacle on the basis of the received echo signals. In particular, a rough classification of the obstacle (wall, point, inside corner, . . . ) according to the mathematical equations referred to in DE 40 23 538 A1 requires accurate knowledge of the relative positions. In addition, in the case of an obstacle at a great distance, i.e., an obstacle whose distance from the vehicle is much greater than the distances between the transceiver units, the direct echoes detected by the individual transceiver units have propagation times that differ only slightly, and these propagation times constitute a usable approximate value for the actual distance, but in the case of a near point obstacle such as a post whose distance from the vehicle is less than the distance between the transceiver units, neither a direct echo nor a cross-echo is a usable approximation for the actual distance. For example, if the vehicle is approaching a post so that it could strike the post at a point between two transceiver units, then with progressive approach to the post, the propagation time of the cross-echo reflected back from it does not approach zero but instead approaches the propagation time corresponding to the distance between the two transceiver units. The accuracy with which this latter distance is known is therefore crucial for the lower limit up to which the monitoring device is still capable of estimating a distance from an obstacle with a usable accuracy. This problem has resulted in echo-signal monitoring devices for motor vehicles being installed so far practically only in new vehicles by vehicle manufacturers. In retrofitting a vehicle with such devices, it may be very difficult to ensure the accuracy required for reliable measurements in positioning the transceiver units. This may be the case in particular when the retrofitting is performed by the vehicle owner himself. |
<SOH> SUMMARY OF THE INVENTION <EOH>The exemplary embodiment and/or exemplary method of the present invention provides an echo-signal monitoring device and a method for determining the relative position of transceiver units in an echo-signal monitoring device which permit a distance measurement with a high accuracy, which is virtually independent of the precision with which the transceiver units were positioned at the time of installation. With the exemplary embodiment and/or exemplary method of the present invention, the echo signals may be used in measuring not only the distance of the transceiver units from an obstacle in the case of a known distance between the transceiver units but also to measure their mutual spacing with the help of redundant measurements (measurement of the distances between transceiver units is also referred to below as calibration.) It is therefore sufficient if predetermined installation positions are only approximately achieved when installing the exemplary device according to the present invention. By subsequent measurement of the distances between the transceiver units and by using these measured distances in the analysis of echo propagation times measured in normal operation, an accurate determination of the distance from an obstacle may be achieved. This property also allows for the use of the same model of the monitoring device on different types of motor vehicles having different installation positions for the transceiver units. Specific adaptation of the device to the geometric characteristics of a vehicle is performed automatically after installation. Of course, measurement of the distances may be performed not only at the time of installation but also repeatedly at any later point in time—automatically or on input of a command by a user. For example, the device may recognize an ambient situation suitable for performing a calibration on the basis of the echo propagation times detected—by a method to be described in greater detail below—and to perform a calibration in such a situation. To simplify the analysis of the received echo signals, the transceiver units expediently have a detection threshold which the amplitude of a received echo must exceed to be detected by the transceiver unit and which drops gradually from a high level directly after transmission of a signal by the transceiver unit to a low level. This detection threshold is defined so that it always allows reliable detection of the direct echo. This simplifies the analysis of the echo signal at the first unit. Use of the detection threshold may result in a cross-echo of a second transceiver unit which transmitted at an earlier point in time than a first transceiver unit, not being detected by the first unit. Conversely, however, the second unit detects a cross-echo signal coming from the first unit because the detection threshold used by it is lower at the time of arrival of the cross-echo than that of the first. Since the cross-echo propagation time to be measured is the same on both paths, no distance information that could be derived from the echoes is lost. The points in time when the transceiver units each transmitting pulse-shaped signals may be determined by a control unit according to a stochastic model. This permits a simple differentiation of cross-echoes from direct echoes, so that the signals may be transmitted interlaced in time and thus multiple measurements may be performed within a short period of time. Adaptive filters are suitable in particular for analyzing these echo signals. Their use in an echo-signal monitoring device is known from DE 198 02 724 A1. To determine the relative position of a plurality of transceiver units of an echo-signal monitoring device, it is in principle sufficient in the case of a pair of transceiver units to measure the direct echo propagation times from a transceiver unit to a linear obstacle and back to the same transceiver unit and the cross-echo propagation time from one of the transceiver units via the obstacle back to the other transceiver unit of the pair in order to calculate the distance between the two units on the basis of the resulting propagation times. However, to improve accuracy, it is advisable to measure the echo propagation times repeatedly and to average the results over the repeated measurements in the calculation. The echo-signal monitoring device may be moved slowly while these measurements are being performed. To perform these calibration measurements, it is advisable to define a plurality of pairs among the multiple transceiver units of the echo-signal monitoring device and to measure the echo propagation times for this plurality of pairs. By using a sufficient number of pairs, redundant measurements may be obtained in this way, permitting a conclusion to be drawn as to the reliability of the individual measurements. Analysis of such redundant measurements may be performed using an error minimization method, in particular the least error squares method. If an echo propagation time is measured repeatedly in succession, noise components in the received echo signal cause fluctuation of the measured echo propagation time. The greater this fluctuation, the lower the signal-to-noise ratio of the received echo signal may be assumed to be and the lower is the reliability of the measurement. Therefore, a measurement is expediently taken into account in error minimization using weighting, which is the greater the smaller the fluctuation is. The relative positions of the transceiver units and an obstacle supplying an echo in relation to one another have an influence on the reliability with which the distance between transceiver units may be deduced from a measurement. For a given configuration of sensor systems and obstacles, a geometry quality measure may be defined, which may be used for weighting the individual measurements. The geometry quality measure is an error amplification factor, i.e., at a high geometry quality, the sensitivity of the sensor distance estimate to measurement errors is low and vice versa. For example, the closer the obstacle is to the vehicle, i.e., the more obtuse the angle is at which it reflects a cross-echo, the greater is the influence of the distance between the transceiver units on the cross-echo propagation time. At the same time, propagation times of the cross-echoes which are much greater than those of the direct echoes are obtained in reflection on a linear obstacle in such a case. It is therefore advantageous to evaluate the reliability of a measurement of a cross-echo propagation time on the basis of its ratio to the direct echo propagation times of the transceiver units involved in measurement of the cross-echo propagation time and to take into account the measurement using a weighting which is greater as the cross-echo propagation time is longer in relation to the direct echo propagation times. |
Medicine dispenser and method |
A dispensing device (10) for dispensing medication includes a habitually used mechanism (12) for being used habitually by a patient and a medication dispensing mechanism (14) for dispensing medication at the times of the use of the habitually used device. |
1. A medication dispensing device including habitual use means for being used habitually by a patient and medication dispensing means for dispensing medication at the times of use of the said habitual use means. 2. The device of claim 1, wherein said habitual use means is selected from the group consisting of a toothbrush dispenser, vitamin dispenser, soap dispenser, car key holder, silverware dispenser, remote control holder, and sugar dispenser. 3. A device of claim 2, including a base, said habitual use means including at least one pocket portion in said base for containing a habitually used object. 4. The device of claim 1, wherein said medication dispensing means is selected from the group consisting of inhalers, pill bottles, spray bottles, injectors, heaters, and inoculators. 5. The device of claim 1, including date indicating means for indicating the date of use of said habitual use means and said medication dispensing means to insure dispensing of said medication dispensing means. 6. The device of claim 4, including software operatively connected to said date indicating means for tracking use of said medication dispensing means. 7. A device of claim 6, wherein said medication dispensing means includes a dispenser device removable from a pocket portion of a body of said device, said date indicator means including sensor means for sensing removal of said dispenser device from said pocket portion, thereby indicating use of said medication dispensing means. 8. A device of claim 7, including online communication means operatively interconnecting said software to an offsite monitor for offsite monitoring of use of said medication dispensing means. 9. A device of claim 1, including audible reminder means for providing verbal reminders to the patient to use the medication after using said habitual use means. 10. A device of claim 9, wherein said audible reminder means includes sensor means for sensing use of said habitual use means and triggering an audible message to disseminate from said device reminding the patient to also use said medication dispensing means. 11. A device of claim 1, including visual reminder means for providing a visual reminder to the patient to use the medication after using said habitual use means. 12. A device of claim 11, wherein said visual reminder means includes sensor means for sensing use of said habitual use means and triggering a visual message to disseminate from said device reminding the patient to also use said medication dispensing means. 13. A method of dispensing a medication by steps of dispensing medication from a device that also dispenses a habitually used device to remind a user to dispense and use medication while using the habitually used device. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Medical treatment that requires regular ongoing administration of medicaments is plagued by poor compliance. For example, asthma affects up to thirty percent (30%) of the world population (GINA workshop report 1995) although the prevalence of the disease is variable in different societies. The currently accepted guidelines for the management of asthma recommend prophylactic (preventative) treatment in all but the mildest of cases. Most of these types of treatment must be taken at least twice daily, usually by an inhaler. Such treatment may also include administering tablets or other types of medication. However, compliance with such treatment regimens can be difficult, especially for older patients or patients suffering from cognitive diseases. Various alarm mechanisms are used by patients to remind them to take their medication. Such simple alarms are physically dissociated from the medication. People can hear or see the alarm and not remember what it relates to or forget to take the medication in between seeing the alarm and finding the medication. Hence, there are practical problems associated with the physical separation of the alarm from the medication. The problem can be as simple as the patient forgetting to carry the alarm with them. Accordingly, it would be beneficial to combine a medication dispenser with some means for reminding a patient to take the medication treatment wherein the alarm is not forgotten. |
<SOH> SUMMARY OF THE INVENTION <EOH>In accordance with the present invention, there is provided a medication dispensing device including a habitually used mechanism for being used habitually by a patient and a medication dispensing device for dispensing medication at the time of use of the habitually used mechanism. Accordingly, the patient would be reminded of taking the medication at each time the habitually used mechanism is used. The present invention further provides a method of dispensing the medication by the steps of dispensing medication from the device, which also dispenses a habitually used device to remind the user to take the medication while using the habitually used device. |
Device for analysing a sample in particular by flow cytometry |
The invention concerns a device for analysing a sample comprising a sample receptacle (8) and a mirror (38), the mirror (38) comprising a break (40) such that a light beam (42) can pass through the mirror to reach the receptacle (8). The invention also concerns an assembly for analysing a sample comprising a device (2) and an equipment having a removable housing for receiving the device and a light source, the source being adapted to emit a light beam (42) passing through the mirror (38) to reach the receptacle (8) when the device is in the housing. |
1.-26. (Cancel). 27. Assembly for the analysis of a sample by means of a light ray comprising: a device comprising a sample receptacle and a mirror; and an apparatus with a housing for removably receiving the device and a light source, wherein the mirror has a discontinuity such that the light source can emit a light ray that passes through the mirror to reach the receptacle when the device is in the housing. 28. Assembly according to claim 1, wherein the mirror (38, 138, 238) is fixed to the receptacle. 29. Assembly according to either of claims 1 or 2, wherein the device comprises an external wall (16, 116) forming the mirror. 30. Assembly according to either of claims 1 or 2, wherein the mirror is fixed on an external wall of the device. 31. Assembly according to either of claims 1 or 2, wherein the shape of the device is essentially flat. 32. Assembly according to either of claims 1 or 2, wherein the receptacle (8) extends parallel to a main face (34, 134) of the device, the mirror (38, 138, 238) extending adjacent to the receptacle along a thickness (e) of the device. 33. Assembly according to either of claims 1 or 2, wherein a discontinuity (40, 140) is adjacent to the receptacle along a thickness (e) of the device. 34. Assembly according to either of claims 1 or 2, wherein a discontinuity (240) is arranged such that the light ray (42) is inclined with respect to a thickness (e) of the device. 35. Assembly according to claim 34, wherein a fluid is capable of flowing in the receptacle along a predetermined direction (72), the discontinuity (240) being located in the half of the mirror (238) upstream with reference to the flow direction. 36. Assembly according to either of claims 1 or 2, wherein the device comprises a substrate (16) on which one face (38) forms the mirror, this face being in contact with a medium different from the substrate medium and extending between the substrate (16) and the receptacle (8). 37. Assembly according to claim 36, wherein the face (38) forms an internal cavity (39) in the device. 38. Assembly according to either of claims 1 or 2, wherein the device comprises a substrate with a face (138, 238) forming the mirror and extending in contact with a medium different from the substrate medium, the substrate (116) extending between the face (138, 238) and the receptacle (8). 39. Assembly according to claim 36, wherein the medium is a gas. 40. Assembly according to either of claims 1 or 2, wherein the mirror (38, 138, 238) is in the shape of a portion of a sphere. 41. Assembly according to either of claims 1 or 2, wherein a ray (42) can emerge from the device on the side of the receptacle (8) opposite the mirror (38, 138, 238). 42. Assembly according to either of claims 1 or 2, wherein the device also includes an optical means (48) fixed to the receptacle. 43. Assembly according to claim 42, wherein the receptacle (8) extends between the mirror (38, 138, 238) and the optical means. 44. Assembly according to claim 43, wherein the device comprises an external wall (18) forming the optical means (48). 45. Assembly according to either of claims 1 or 2, wherein the shape of the receptacle (8) is elongated. 46. Assembly according to either of claims 1 or 2, wherein the device comprises a fluid reservoir (6). 47. Assembly according to either of claims 1 or 2, wherein the sample comprises a fluid. 48. Assembly according to either of claims 1 or 2, wherein the device is an analysis device based on flow cytometry. 49. Assembly according to either of claims 1 or 2, wherein the device is a microfluidic device. 50. Assembly according to claim 49, wherein the apparatus comprises analysis means (30, 52) of the ray (42) arranged such that the device (2) extends between the source (28) and the analysis means when the device is in the housing (24). 51. Assembly according to claim 50, which includes means for analyzing radiation arriving directly or indirectly from the source so as to determine if the device is received at a predetermined position in the housing. |
Pregnancy-related enzyme activity |
This invention relates to the role of the enzyme proprotein convertase 5/6 in pregnancy, and in particular to the detection or modulation of proprotein convertase 5/6 and its isoforms in the uterus. This enzyme is useful in the control of fertility, the monitoring of early pregnancy, and for the detection of uterine receptivity. The invention also relates to methods of screening for compounds which have the ability to modulate the activity or expression of proprotein convertase 5/6, which may be useful in regulating fertility in mammals. Novel forms of proprotein convertase 5/6 are also disclosed and claimed. |
1-29. (canceled) 30. A nucleic acid molecule encoding an isoform of the protein proprotein convertase (PC) 5/6, which nucleic acid molecule: (a) encodes a uterus-specific RNA transcript of about 5.5 kb; (b) is expressed during the implantation period at implantation sites in a pregnant uterus but is not detectably expressed in intestine or other tissues where PC 5/6 protein may be present; (c) under at least moderately stringent conditions, is able to hybridize to DNA of sequence SEQ ID NO:4 but not to DNA of sequence SEQ ID NO:5 and SEQ ID NO:6. 31. A nucleic acid molecule according to claim 30 which is in the anti-sense orientation. 32. A nucleic acid molecule according to claim 30, which is a cDNA molecule, a genomic DNA molecule, or an RNA molecule. 33. A nucleic acid molecule according to claim 32, wherein the nucleic acid molecule is a cDNA molecule. 34. A nucleic acid molecule according to claim 30, which: (a) comprises the sequence SEQ. ID. NO: 1 or SEQ ID NO:2; or (b) is able to hybridize under at least moderately stringent conditions to DNA having the sequence SEQ ID NO:1 or SEQ ID NO:2; or (c) has at least 75% sequence identity to SEQ ID NO:1 or SEQ ID NO: 2. 35. A nucleic acid molecule according to claim 34, which is able to hybridize under stringent conditions to DNA with the sequence SEQ. ID. NO:1 or SEQ ID NO: 2. 36. A nucleic acid molecule according to claim 34, which has at least 80%, sequence identity to SEQ ID NO:1 or SEQ ID NO:2. 37. An isolated protein having PC 5/6 activity which is encoded by the nucleic acid molecule of claim 30. 38. A pharmaceutical composition comprising the PC 5/6 protein of claim 37, together with a pharmaceutically acceptable carrier. 39. A method of promoting fertility in a female mammalian subject in need of such fertility promotion, comprising the step of stimulating the activity of proprotein convertase (PC) 5/6 in the uterus of a said subject. 40. A method according to claim 39, wherein the activity is stimulated by administering a PC 5/6 polypeptide, or an agonist of PC 5/6. 41. A method according to claim 39, wherein the PC 5/6 is an isoform of the form of PC 5/6 that is specifically expressed in implantation sites of the uterus during embryo implantation. 42. A method according to claim 39, wherein the uterus of the subject is converted from a non-receptive state to a receptive state. 43. A method according to claim 39, wherein the stimulating promotes implantation of a fertilized egg in the uterus. 44. A method according to claim 39, wherein the subject suffers from a fertility-related condition. 45. A method according to claim 44, wherein the fertility-related condition is luteal phase defect, failure of implantation, pre-eclampsia, early abortion, intrauterine growth restriction, abnormal uterine bleeding, endometriosis or early parturition. 46. A method according to claim 39, wherein the subject is to be implanted with one of her own fertilized eggs. 47. A method according to claim 39, wherein the subject is to be implanted with a donor's fertilized egg. 48. A method of promoting fertility in a female mammalian subject in need of such fertility promotion, comprising the step of stimulating, in the uterus of a said subject, the activity of a PC 5/6 isoform that is encoded by the nucleic acid molecule of claim 19. 49. A method of detecting whether a female mammalian subject is in a fertile state or period, whether the uterus of said subject is in a receptive state, or whether the subject is in an early stage of pregnancy, comprising obtaining a first biological sample from the subject and detecting the presence or measuring the activity of PC 5/6 in said sample, and, optionally, comparing the result to a second or greater biological sample (i) obtained during a nonfertile period, or (ii) obtained at a stage of the estrous cycle when the uterus is not receptive, or (iii) obtained from a nonpregnant subject, wherein: (a) the presence of PC 5/6, or an increase in the activity of PC 5/6 in the first sample relative to the activity in the second sample obtained during a nonfertile period, indicates that the subject is in a fertile state or period; (b) the presence of PC 5/6 or an increased amount of PC 5/6 in the first sample compared with the PC 5/6 from the second sample from a nonreceptive stage of the cycle indicates that the uterus is in the receptive state; and (c) the presence of PC 5/6 activity in the first sample or an increase of PC 5/6 in said first sample compared to the level in the second sample from a non-pregnant subject indicates said early stage of pregnancy. 50. A method according to claim 49, wherein the biological sample is uterine tissue, washings from a uterine cavity, blood, plasma, serum or saliva. 51. A method of inhibiting fertility in a female mammalian subject in need or desirous thereof, comprising the step of administering an antagonist of PC 5/6 to said subject. 52. A method according to claim 51, wherein the antagonist inhibits conversion of a uterine non-receptive state to a receptive state. 53. A method according to claim 51, wherein the antagonist is an anti-PC 5/6 antibody or an anti-sense nucleic acid that specifically hybridizes in vivo with a nucleic acid encoding PC 5/6. 54. A method according to claim 51, wherein the antagonist inhibits embryo implantation. 55. A method of (i) screening a sample or collection of compounds for a compound that modulates the enzymatic activity of PC 5/6 protein, (ii) testing a candidate compound for said modulating activity, or (iii) testing a candidate compound for PC 5/6 enzymatic activity, the method comprising: (a) providing a composition comprising said PC 5/6 protein and assessing the ability of said sample or collection, or said candidate compound, to increase or decrease the enzymatic activity of said protein relative to the activity of said PC 5/6 protein in the absence of said sample, collection or candidate compound; or (b) testing the candidate compound for it ability to catalyze the conversion of protein precursors into mature proteins, which protein precursors are ones that are cleaved by PC 5/6 to become mature proteins. |
<SOH> BACKGROUND OF THE INVENTION <EOH>All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. Embryo implantation, the process by which the blastocyst attaches to and implants in the uterus, leads to the establishment of an intimate relationship between the embryo and the endometrium. Implantation is one of the most important limiting factors in establishing a successful pregnancy. It is a complex process involving active interactions between the blastocyst and the uterus. The uterus must undergo dramatic morphological and physiological changes to transform itself from a non-receptive to a receptive state. This differentiation process is primarily mediated by the coordinated effects of the ovarian-hormones, which act through their intracellular receptors to regulate gene expression, and hence to influence cellular proliferation and differentiation. While the details of the exact molecular events occurring in the uterus during this differentiation process towards receptivity are still unknown, in principle it can be predicted that a unique set of genes is up- or down-regulated in a temporally and spatially specific manner. Indeed, induction of specific genes in the uterus during the peri-implantation period, including those encoding some growth factors and cytokines, has been reported (Huet-Hudson et al, 1990; Stewart et al, 1992; Zhu et al, 1998; Robb et al, 1998; Das et al, 1999). However, given the complexity and the as-yet imprecisely defined molecular mechanisms of the process, many other molecules critical for implantation are still unidentified. We have used the mouse as a model in a search for hitherto unrecognised molecules which are important in the early stages of implantation. In the mouse on day 4.5 of pregnancy, the uterus undergoes dramatic morphological changes in association with cell proliferation and differentiation, leading to the acquisition of a receptive state (Finn & McLaren, 1967; Abrahamsohn & Zorn, 1993). This uterine remodelling is associated with an increase in vascular permeability at implantation sites (Psychoyos, 1973). We hypothesised that the proliferation and differentiation of endometrial cells at this time is associated with up- or down-regulation of a number of genes, many of which are still unknown (Nie et al, 1997). To identify uterine genes which are potentially critical for uterine receptivity, we used the technique of RNA differential display (DDPCR) (Liang & Pardee, 1992 & 1993), and compared the mRNA expression patterns of implantation and interimplantation sites on day 4.5 of pregnancy (Nie et al, 2000a & b). We found that one of the genes which was differently regulated between the two sites was that encoding proprotein (prohormone) convertase 5/6 (PC 5/6), a member of a serine proteinase family which is responsible for processing of precursor proteins to their active forms by selective proteolysis. PC 5/6 has not been previously detected in the uterus, and no role for this enzyme during embryo implantation has been suggested. A cDNA encoding PC5, as well as the complete amino acid and nucleotide sequence of human PC5, is disclosed in PCT/CA97/00535. PC5 is described as being a prorenin-processing enzyme which is overexpressed in artherosclerotic coronary arteries. Inhibition of smooth muscle cell proliferation by antisense oligonucleotides directed against PC5 is described, as well as the use of such oligonucleotides in the prevention of restenosis. PC5 RNA was also detected in the post-partum placental cotyledon and in the testes. Miranda et al. (1996) describes the presence of two isoforms of human PC6 protease in human T cells and its role in HIV-1 gp160 processing in CD4+ T cells. Its distribution in the intestine, adrenal glands, lung, ovaries, testes, brain, spleen, kidney and liver is described. There is no mention of the presence of PC6 in the uterus. Only the role of PC5 in the cleaving of human prorenin is discussed. There is no disclosure or suggestion in either of these documents that PC5 might be present in the uterus, or that it might play a role in fertility. PC 5/6 has been detected in the thyroid, parathyroid, gut, adrenals, nervous system, and the cardiovascular system, but has not been reported as being present in any gonadal tissue or other reproductive organ. (Seidah & Chretien, 1999). In another paper (Rancourt & Rancourt, 1997) there is a mention of PC6 expression detected by in situ hybridisation in decidua and trophoblast cells, but no additional data is provided. We have examined the uterine expression of PC 5/6 during early pregnancy in the mouse, and have demonstrated for the first time that this proprotein convertase is specifically upregulated in the mouse uterus at the sites of embryo attachment, and that its expression is restricted to the decidualized stromal cells. We have also identified a novel isoform of PC 5/6, which appears to be specifically expressed in implantation sites of the uterus during the embryo's implantation period. We have also detected PC 5/6 expression in human endometrium. |
<SOH> SUMMARY OF THE INVENTION <EOH>Proprotein convertase 5/6 is believed to be useful in promoting the implantation of the fertilized egg in the uterus, development of the embryo and maintenance of pregnancy. Therefore modulation of the activity of this enzyme may be used to promote or to inhibit fertility. It is contemplated that the fertility-promoting methods of the invention will be particularly useful in assisted reproduction programs, including but not limited to in vitro fertilisation and gamete intrafallopian transfer methodologies. It is also contemplated that modulation of PC 5/6 activity could be used to prevent the establishment of a pregnancy, ie that this could be used as a contraceptive. In a first aspect the invention provides a method of converting the uterus of a female mammal from a non-receptive to a receptive state, comprising the step of administering an effective amount of proprotein convertase 5/6 (PC 5/6), or an agonist thereof, to a female mammal in need of such treatment. In a second aspect, the invention provides a method of promoting the implantation of a fertilised egg in a host uterus, comprising the step of administering a proprotein convertase 5/6, or an agonist thereof, to a female mammal in need of such treatment. In a third aspect, the invention provides a method of detecting the fertile period in a female mammal, comprising the step of measuring the activity of or detecting the presence of proprotein convertase 5/6 in a biological sample from the mammal. The biological sample may be uterine tissue, washings from the uterine cavity, or another biological fluid, such as blood, plasma, serum or saliva. In a fourth aspect, the invention provides a method of promoting fertility of a female mammal, comprising the step of stimulating the activity of proprotein convertase 5/6, in the uterus of a female mammal in need of such treatment. In these first, second and fourth aspects of the invention it will be clearly understood that the method of the invention may be used to treat either a female mammal who is to receive one of her own fertilised eggs, or one who is to receive a fertilised egg from a donor female. In a fifth aspect, the invention provides a method of detecting whether the uterus of a female mammal is in a receptive state, comprising the step of detecting the presence or absence of PC 5/6, or its presence in increased amounts at a particular stage of the cycle compared with another stage, whereby the presence of or an increase in PC 5/6 means that the uterus is in a receptive state. The presence or absence of PC 5/6 may be assessed by detecting the activity of the enzyme itself, eg. using a specific antibody directed against the enzyme, or by detecting nucleic acid, encoding the enzyme, for example using PCR or in in situ hybridization. In a sixth aspect, the invention provides a method of detecting an early pregnancy, comprising the step of detecting the presence of PC 5/6 or an increase of PC 5/6 above the level in the non-pregnant state. In a seventh aspect, the invention provides a method of inhibiting the conversion of a non-receptive state of the uterus of a female mammal to a receptive state, comprising the step of administering an antagonist of PC 5/6 to a female mammal in need of such treatment. In an eighth aspect, the invention provides a method of inhibiting fertility, including but not limited to inhibiting uterine receptivity and/or embryo implantation, in a female mammal, comprising the step of administering an antagonist of PC 5/6 to a female mammal in need of such treatment. In a ninth aspect, the invention provides a method of screening for compounds which have the ability to modulate the activity of proprotein convertase 5/6, comprising the step of assessing the ability of a candidate compound to increase or decrease proprotein convertase 5/6 activity. The person skilled in the art will appreciate that a wide range of activities can be measured, for example stimulation of gene expression, stimulation of activation of PC 5/6 or inhibition of degradation by PC 5/6. In a tenth aspect, the invention provides a method of identifying molecules necessary for implantation, comprising the step of testing a candidate molecule for the ability to promote the conversion of protein precursors cleavable by PC 5/6 into mature proteins. In an eleventh aspect, the invention provides a nucleic acid molecule encoding an isoform of PC 5/6 which: (a) is present at the implantation sites in pregnant uterus during the implantation period but not in intestine or other tissues known to contain PC 5/6; (b) encodes an RNA transcript of about 5.5 kb; and (c) is able to hybridize under at least moderately stringent conditions to the sequence: 1 GAAGTTAGTT GTGCGCGCTG CTTAGCGCGC GAGCCAGCGG GCGGGCGAAG SEQ ID NO: 4 51 GCGGCGAAGC GTCGGGACCA TGGACTGGGA CTGGGGGAAC CGCTGCAGCC 101 GCCCGGGACG GCGGGACCTG CTGTGCGTGC TGGCACTGCT CGCCGGCTGT 151 CTGCTCCCGG TATGCCGGAC GCGCGTCTAC ACCAACCACT GGGCAGTGAA 201 GATCGCCGGC GGCTTCGCGG AGGCAGATCG CATAGCCAGC AAGTACGGAT 251 TCATCAACGT AGGACAGATC GGTGCACTGA AGGACTACTA TCACTTCTAC 301 CATAGTAGGA CCATTAAAAG GTCTGTTCTC TCGAGCAGAG GAACCCACAG 351 TTTCATTTCA ATGGAACCAA AGGTGGAGTG GATCCAACAG CAAGTGGTGA 401 AAAAAAGAAC CAAGAGGGAT TATGACCTCA GCCATGCCCA GTCAACCTAC 451 TTCAATGATC CCAAGTGGCC AAGTATGTGG TACATGCACT GCAGTGACAA 501 CACCCATCCT TGCCAGTCAG ACATGAATAT CGAAGGAGCC TGGAAGAGAG 551 GCTACACGGG GAAGAATATC GTGGTGACTA TCCTGGATGA CGGCATCGAG 601 AGAACCCACC CAGATCTGAT GCAAAACTAC GATGCTCTGG CAAGTTGCGA 651 TGTGAATGGG AATGACTTGG ACCCGATGCC TCGTTATGAT GCAAGCAATG 701 AGAACAAGCA TGGGACCCGC TGTGCCGGAG AAGTGGCAGC CACTGCAAAC 751 AACTCTCACT GCACTGTCGG GATCGCTTTC AACGCCAAGA TTGGAGGTGT 801 GAGAATGCTG GATGGTGATG TCACTGACAT GGTGGAGGCA AAGTCTGTCA 851 GCTACAACCC ACAGCATGTG CACATCTACA GTGCCAGCTG GGGACCAGAT 901 GACGATGGCA AGACTGTGGA TGGGCCAGCT CCCCTCACCC GGCAAGCCTT 951 TGAGAATGGC GTGAGAATGG GGCGGAGAGG CCTTGGATCT GTGTTTGTGT 1001 GGGCATCTGG CAATGGTGGA CGGAGCAAGG ATCACTGTTC TTGTGATGGC 1051 TATACCAACA GCATCTATAC CATCTCCATC AGCAGTACGG CCGAAAGTGG 1101 AAAGAAACCT TGGTACTTGG AAGAGTGTTC ATCTACACTG GCTACAACCT 1151 ACAGCAGTGG AGAATCCTAT GATAAGAAAA TAATCACTAC TGATCTAAGG 1201 CAGCGATGCA CAGACAATCA CACTGGAACG TCAGCCTCAG CCCCCATGGC 1251 TGCTGGAATC ATTGCCCTGG CCCTAGAAGC CAATCCGTTT CTGACCTGGA 1301 GAGACGTGCA GCATGTTATT GTCAGGACTT CCCGTGCGGG ACATTTGAAC 1351 GCTAATGACT GGAAAACCAA TGCTGCTGGT TTTAAGGTGA GCCATCTCTA 1401 TGGATTTGGA CTGATGGATG CCGAAGCCAT GGTGATGGAA GCAGAGAAGT 1451 GGACAACTGT TCCTCAGCAG CACGTGTGTG TGGAAAGCAC AGACCGACAA 1501 ATCAAGACCA TTCGACCAAA CAGTGCAGTG CGCTCCATCT ACAAAGCCTC 1551 AGGCTGCTCG GATAATCCCA ACCATCACGT CAATTACCTG GAGCATGTAG 1601 TTGTGCGTAT TACCATCACA CACCCACGGA GGGGAGACCT GGCCATCTAT 1651 CTGACATCAC CCTCAGGAAC CAGATCCCAG CTCTTGGCCA ACAGGCTCTT 1701 TGATCATTCC ATGGAAGGGT TTAAGAACTG GGAGTTCATG ACTATTCATT 1751 GCTGGGGAGA ACGGGCTGCT GGGGACTGGG TCCTCGAAGT TTATGATACG 1801 CCATCTCAGC TGAGGAACTT CAAGACTCCA GGTAAATTGA AAGAATGGTC 1851 CTTAGTCCTC TATGGCACGT CCGTACAGCC ATACTCCCCA ACCAACGAGT 1901 TTCCCAAAGT GGAACGCTTC CGCTACAGCC GAGTGGAAGA CCCCACAGAT 1951 GACTACGGTG CTGAAGATTA TGCAGGTCCC TGTGACCCTG AATGCAGTGA 2001 GGTTGGATGT GACGGGCCAG GACCAGATCA CTGCAGTGAC TGCTTACACT 2051 ACTACTACAA GCTGAAAAAT AACACCAGAA TCTGTGTCTC CAGCTGCCCT 2101 CCTGGCCACT ACCATGCTGA CAAGAAGAGG TGCCGGAAGT GTGCCCCAAA 2151 CTGCGAGTCC TGCTTTGGCA GCCATGGTGA TCAGTGCCTC TCCTGTAAAT 2201 ATGGCTACTT CCTGAATGAA GAAACTAGCA GCTGTGTTAC TCAGTGCCCT 2251 GATGGATCAT ACGAGGATAT CAAGAAAAAT GTCTGTGGGA AATGCAGTGA 2301 GAACTGCAAG GCATGCATTG GATTTCACAA CTGCACAGAG TGCAAGGGCG 2351 GGTTAAGTCT TCAGGGATCC CGCTGTTCGG TCACCTGCGA GGATGGACAG 2401 TTCTTCAATG GTCACGACTG CCAGCCCTGC CATCGCTTCT GTGCTACTTG 2451 CTCTGGGGCC GGAGCAGATG GATGTATTAA CTGCACCGAG GGGTATGTCA 2501 TGGAAGAGGG AAGGTGTGTA CAAAGTTGTA GTGTGAGCTA CTACTTGGAC 2551 CACTCTTCAG AGGGTGGCTA CAAATCCTGC AAGAGATGTG ATAACAGCTG 2601 TTTGACATGC AATGGGCCAG GATTCAAGAA CTGTTCCAGC TGCCCCAGTG 2651 GATATCTTTT AGACTTAGGA ACGTGTCAGA TGGGAGCCAT CTGCAAGGAT 2701 GCTACGGAAG AGTCCTGGGC AGAAGGAGGC TTCT but is not able to hybridize under at least moderately stringent conditions to the sequences: 1 TGGAAGGCAA GGACTGGAAT GAAGCCGTGC CCACTGAAAA GCCATCTTTG SEQ ID NO: 5 51 GTGAGGAGTC TGCTGCAGGA TCGACGAAAG TGGAAAGTTC AAATCAAAAG 101 AGATGCAACG AGCCAGAATC AACCTTGTCA CTCTTCTTGT AAAACCTGCA 151 ATGGATCTCT CTGCGCTTCA TGTCCCACAG GTATGTACCT GTGGCTGCAG 201 GCCTGTGTTC CTTCCTGTCC CCAAGGCACT TGGCCATCAG TCACCAGTGG 251 CAGCTGTGAA AAGTGTTCCG AGGACTGTGT CTCCTGCTCC GGTGCCGACC 301 TTTGCCAACA GTGCCTGAGC CAGCCGGACA ACACTCTGCT TCTCCATGAG 351 GGCAGGTGCT ACCACAGTTG CCCAGAG; or 1 AGGGAGGCTG AGTTTTACGA GCACACCAAG ACTGCTCTGC TAGTGACCTC SEQ ID NO: 6 51 TGGCGCCATG CTGTTGCTGC TGCTGGGGGC TGCTGCGGTC GTGTGGCGGA 101 AGTCTCGAAG CAGACCTGTG GCAAAGGGGC GGTACGAAAA GCTGGCAGAA 151 CCTACCGTGT CATACTCCTC CTACAGGAGC AGCTATCTTG ACGAGGACCA 201 GGTGATTGAG TACAGGGACC GGGACTACGA TGAGGACGAT GAGGACGACA 251 TCGTCTATAT GGGCCAAGAT GGCACTGTCT ACCGGAAGTT CAAGTATGGG 301 CTGCTGGATG AGACGGAAGA TGATGAGTTG GAGTACGATG ATGAGAGCTA 351 CTCTTACCAA TAAACAGAGC CCCCTCCCAT CTCAAACCCA CCACCCAC. At the protein level, this 5.5 kb transcript encodes a PC 5/6 protein which has a different C-terminal sequence for those of the known isoforms of PC6A or PC6B. The nucleic acid may be a cDNA, a genomic DNA, or an RNA and may be in the sense or the anti-sense orientation. Preferably the nucleic acid molecule is a cDNA. Preferably the nucleic acid molecule comprises a sequence selected from the group consisting of: (a) a cDNA molecule having the sequence set out in SEQ. ID. NO: 1 or SEQ ID NO: 2, or its human homologue (SEQ. ID. NO: 3); (b) a nucleic acid molecule which is able to hybridise under at least moderately stringent conditions to (a); and (c) a nucleic acid molecule which has at least 75% sequence identity to (a). More preferably in (b) the nucleic acid molecule is able to hybridise under stringent conditions to the molecule of (a). More preferably in (c) the nucleic acid molecule has at least 80%, even more preferably at least 90% sequence identity to the molecule of (a). In a twelfth aspect, the invention provides a protein having proprotein convertase 5/6 activity, which is encoded by a nucleic acid according to the invention. It will be clearly understood that for the purposes of the invention different isoforms of PC 5/6 may be suitable. Preferably in all aspects of the invention, an isoform of PC 5/6 which is specifically expressed in implantation sites of the uterus during embryo implantation is used. Defining appropriate hybridisation conditions is within the skill of the art. See e.g. Maniatis et al. DNA cloning volumes 1 and 11. Nucleic Acid Hybridisation. Briefly, “moderately stringent conditions” for hybridisation or annealing of nucleic acid molecules are those which (1) employ moderate ionic strength and moderate temperature for washing, for example 0.3 M NaCl/0.03 M sodium citrate/0.1% sodium dodecyl sulfate (SDS) at 42(C, or (2) employ during hybridisation a denaturing agent such as formamide, for example 40% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 40(C. Another example is use of 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 (g/mL), 0.1% SDS, and 10% dextran sulfate at 42(C, with washes at 42(C in 0.2×SSC and 0.1% SDS. In a thirteenth aspect, the invention provides a composition comprising a protein according to the invention, together with a pharmaceutically acceptable carrier. Thus the invention provides a method of identifying agonists and antagonists of PC 5/6. In view of the crucial role in implantation indicated by the results reported herein, it is contemplated that antagonists of PC 5/6 will be useful as contraceptives, and that agonists of PC 5/6 will be useful as agents for promoting fertility or for supporting at least the early phases of pregnancy. It is further contemplated that suitable antagonists of PC 5/6 include, but are not limited to, antibodies and anti-sense nucleic acids. For example, an inhibitory antisense 17mer oligonucleotide is disclosed in International Patent Application No. PCT/CA97/00535 (WO98/04686) by Day et al. As used herein, the term “receptive” refers to a state of the uterine lining which will allow an embryo to implant, whereas a “non-receptive” state refers to a state in which attachment and implantation are impaired. This can include a state known as “pre-receptive”. The term “PC 5/6 antagonist” or “antagonist” refers to a substance which opposes or interferes with a functional activity of PC 5/6. For the purposes of this specification it will be clearly understood that the word “comprising” means “including but not limited to”, and that the word “comprises” has a corresponding meaning. |
Method and composition for determining resistance to an acetohydroxy-acid synthase inhibitor |
The invention describes a method for determining whether a plant is resistant to an acetohydroxyacid synthase (“AHAS”) inhibitor. The method comprises: (a) treating a part taken from the plant with a acetohydroxyacid synthase inhibitor, a ketol-acid reductoisomerase inhibitor and an external supplement that is either a compound or combination of compounds selected from the group consisting of alanine, ammonium hydroxide, asparagine, and pyruvic acid or a salt thereof; and (b) measuring the amounts of acetohydroxybutyrate and/or acetolactate. |
1. A method for determining whether a plant is resistant to an acetohydroxyacid synthase inhibitor comprising: treating a first part taken from the plant with a acetohydroxyacid synthase inhibitor, a ketol-acid reductoisomerase inhibitor and a first external supplement comprising alanine, ammonium hydroxide, asparagine, pyruvic acid or a salt thereof or a combination of any of the preceding compounds; and measuring a first amount of acetohydroxybutyrate or acetolactate accumulated by said first part. 2. The method of claim 1 further comprising: treating a second part taken from the plant with a ketol-acid reductoisomerase inhibitor and a second external supplement comprising alanine, ammonium hydroxide, asparagine, pyruvic acid or a salt thereof, or a combination of any of the proceeding compounds; and determining a second amount of acetohydroxybutyrate or acetolactate accumulated by said second part; and comparing the first amount accumulated by said first part with the second amount accumulated by said second part. 3. A method for determining whether a test-compound inhibits acetohydroxyacid synthase comprising: treating a first part taken from the plant with a test-compound, a ketol-acid reductoisomerase inhibitor and an external supplement comprising alanine, ammonium hydroxide, asparagine, and pyruvic acid or a salt thereof, or a combination of any of the preceding compounds; and measuring a first amount of acetohydroxybutyrate acetolactate accumulated by said first part. 4. The method to of claim 3 further comprising: treating a second part taken from the plant with a ketol-acid reductoisomerase inhibitor and a second external supplement comprising alanine, ammonium hydroxide, asparagine, and pyruvic acid or a salt thereof, or a combination of any of the preceding compounds; and determining a second amount of acetohydroxybutyrate or acetolactate accumulated by said second part; and comparing the first amount of accumulated by said first part with the second amount accumulated by said second part. 5. The method of claim 1, wherein the first part is selected from an actively growing region of said plant. 6. The method of claim 1, wherein the first part is taken from a basipetal region of a blade of the plant. 7. The method of claim 2, wherein the plant has a blade with a first portion and a second portion separated by a midvein, wherein the first part is taken from the first portion and the second part is taken from the second portion. 8. The method of claim 1, wherein the first external supplement is selected from the group consisting of alanine, ammonium hydroxide, pyruvic acid, a salt of alanine, a salt of pyruvic acid and mixtures thereof. 9. The method of claim 8, wherein the first external supplement is alanine and pyruvic acid or a salt thereof. 10. The method of claim 9, wherein the alanine and pyruvic acid or salt thereof are present at a ratio of about 8:1 to 1:50. 11. The method of claim 8, wherein the first external supplement is ammonium hydroxide and pyruvic acid or a salt thereof. 12. The method of claim 11, wherein the ammonium hydroxide and pyruvic acid or a salt thereof are present at a ratio of about 8:1 to 1:50. 13. The method of claim 1, wherein the first external supplement for treating said first part is present at about 0.2 to less than 5 percent by weight of a sum of the weights of the first part the acetohydroxyacid synthase inhibitor, the ketol-acid reductoisomerase inhibitor and the first external supplement. 14. The method of claim 1, wherein the ketol-acid reductoisomerase inhibitor is selected from the group consisting of (dimethylphosphinyl) glycolic acid, 2-(dimethylphosphinoyl)-2-hydroxyacetic acid, a sodium N-hydroxy-N-(C1-C6 alkyl or C3-C7 cycloalkyl) oxamate, sodium N-hydroxy-N-aralkyloxamate, 2-methylphosphinoyl-2-hydroxyacetic acid, N-hydroxy-N-isopropyloxamate, a monomethyl and monoethyl ester of 1,1-cyclopropanedicarboxylic acid, ethylenemalonic acid or a mono or disalt thereof, and combinations thereof. 15. The method of claim 14, wherein the sodium-N-hydroxy-N-(C1-C6 alkyl) oxamate is sodium N-hydroxy-N-isopropyloxamate. 16. The method of claim 14, wherein the sodium N-hydroxy-N-aralkyloxamate is sodium N-hydroxy-N-benzyloxamate. 17. The method of claim 2, wherein the second part is selected from an actively growing region of said plant. 18. The method of claim 2, wherein the second part is taken from a basipetal region of a blade of the plant. 19. The method of claim 7, wherein the first and second portions are taken from approximately equal and opposite locations of said blade. 20. The method of claim 2, wherein the second external supplement is selected from the group consisting of alanine, ammonium hydroxide, pyruvic acid, a salt of alanine, a salt of pyruvic acid and mixtures thereof. 21. The method of claim 20, wherein the second external supplement is alanine and pyruvic acid or a salt thereof. 22. The method of claim 21, wherein the alanine and pyruvic acid or salt thereof are present at a ratio of about 8:1 to 1:50. 23. The method of claim 2, wherein the second external supplement is ammonium hydroxide and pyruvic acid or a salt thereof. 24. The method of claim 23, wherein the ammonium hydroxide and pyruvic acid or a salt thereof are present at a ratio of about 8:1 to 1:50. 25. The method of claim 2, wherein the second external supplement for treating said second part is present in an amount of about 0.2 to less than 5 percent by weight of a sum of the weights of the second part, the acetohydroxyacid synthase inhibitor, the ketol-acid reductoisomerase inhibitor and the second external supplement. 26. The method of claim 25, wherein the ketol-acid reductoisomerase inhibitor is selected from the group consisting of (dimethylphosphinyl) glycolic acid, 2-(dimethylphosphinoyl)-2-hydroxyacetic acid, a sodium N-hydroxy-N-(C1-C6 alkyl or C3-C7 cycloalkyl) oxamate, sodium N-hydroxy-N-aralkyloxamate, 2-methylphosphinoyl-2-hydroxyacetic acid, N-hydroxy-N-isopropyloxamate, a monomethyl or monoethyl ester of 1,1-cyclopropanedicarboxylic acid, an ethylenemalonic acid or a mono or disalt thereof, and combinations thereof. 27. The method of claim 26, wherein the sodium-N-hydroxy-N-(C1-C6 alkyl) oxamate is sodium N-hydroxy-N-isopropyloxamate. 28. The method of claim 26, wherein the sodium N-hydroxy-N-aralkyloxamate is sodium N-hydroxy-N-benzyloxamate. 29. The method of claim 2, wherein the first external supplement and the second external supplement are the same. 30. The method of claim 2, wherein the first external supplement and the second external supplement are different. 31. The method of claim 3, wherein the first part is selected from an actively growing region of said plant. 32. The method of claim 4, wherein the second part is selected from an actively growing region of said plant. 33. The method of claim 3, wherein the first part is taken from a basipetal region of a blade of the plant. 34. The method of claim 4, wherein the second part is taken from a basipetal region of a blade of the plant. 35. The method of claim 4, wherein the plant has a blade with a first portion and a second portion separated by a midvein, wherein the first part is taken from the first portion and the second part is taken from the second portion. 36. The method of claim 35, wherein the first and second portions are taken from approximately equal and opposite locations of said blade. 37. The method of claim 3, wherein the first external supplement is selected from the group consisting of alanine, ammonium hydroxide, pyruvic acid, a salt of alanine, a salt of pyruvic acid and mixtures thereof. 38. The method of claim 37, wherein the first external supplement is alanine and pyruvic acid or a salt thereof. 39. The method of claim 38, wherein the alanine and pyruvic acid or salt thereof are present at a ratio of about 8:1 to 1:50. 40. The method of claim 37, wherein the first external supplement is ammonium hydroxide and pyruvic acid or a salt thereof. 41. The method of claim 40, wherein the ammonium hydroxide and pyruvic acid or a salt thereof are present at a ratio of about 8:1 to 1:50. 42. The method of claim 3, wherein the first external supplement for treating said first part is present at about 0.2 to less than 5 percent by weight of a sum of the weights of the first part, the acetohydroxyacid synthase inhibitor, the ketol-acid reductoisomerase inhibitor and the first external supplement. 43. The method of claim 3, wherein the ketol-acid reductoisomerase inhibitor is selected from the group consisting of (dimethylphosphinyl) glycolic acid, 2-(dimethylphosphinoyl)-2-hydroxyacetic acid, a sodium N-hydroxy-N-(C1-C6 alkyl or C3-C7 cycloalkyl) oxamate, sodium N-hydroxy-N-aralkyloxamate, 2-methylphosphinoyl-2-hydroxyacetic acid, N-hydroxy-N-isopropyloxamate, a monomethyl and monoethyl ester of 1,1-cyclopropanedicarboxylic acid, ethylenemalonic acid or a mono or disalt thereof, and combinations thereof. 44. The method of claim 43, wherein the sodium-N-hydroxy-N-(C1-C6 alkyl) oxamate is sodium N-hydroxy-N-isopropyloxamate. 45. The method of claim 43, wherein the sodium N-hydroxy-N-aralkyloxamate is sodium N-hydroxy-N-benzyloxamate. 46. The method of claim 4, wherein the second external supplement is selected from the group consisting of alanine, ammonium hydroxide, pyruvic acid, a salt of alanine, a salt of pyruvic acid and mixtures thereof. 47. The method of claim 46, wherein the second external supplement is alanine and pyruvic acid or a salt thereof. 48. The method of claim 47, wherein the alanine and pyruvic acid or salt thereof are present at a ratio of about 8:1 to 1:50. 49. The method of claim 46, wherein the second external supplement is ammonium hydroxide and pyruvic acid or a salt thereof. 50. The method of claim 49, wherein the ammonium hydroxide and pyruvic acid or a salt thereof are present at a ratio of about 8:1 to 1:50. 51. The method of claim 4, wherein the second external supplement for treating said second part is present in an amount of about 0.2 to less than 5 percent by weight of a sum of the weights of the second part, the acetohydroxyacid synthase inhibitor, the ketol-acid reductoisomerase inhibitor and the second external supplement. 52. The method of claim 51, wherein the ketol-acid reductoisomerase inhibitor is selected from the group consisting of (dimethylphosphinyl) glycolic acid, 2-(dimethylphosphinoyl)-2-hydroxyacetic acid, a sodium N-hydroxy-N-(C1-C6 alkyl or C3-C7 cycloalkyl) oxamate, sodium N-hydroxy-N-aralkyloxamate, 2-methylphosphinoyl-2-hydroxyacetic acid, N-hydroxy-N-isopropyloxamate, a monomethyl or monoethyl ester of 1,1-cyclopropanedicarboxylic acid, an ethylenemalonic acid or a mono or disalt thereof, and combinations thereof. 53. The method of claim 52, wherein the sodium-N-hydroxy-N-(C1-C6 alkyl) oxamate is sodium N-hydroxy-N-isopropyloxamate. 54. The method of claim 52, wherein the sodium N-hydroxy-N-aralkyloxamate is sodium N-hydroxy-N-benzyloxamate. 55. The method of claim 4, wherein the first external supplement and the second external supplement are the same. 56. The method of claim 4, wherein the first external supplement and the second external supplement are different. 57. The method of claim 4, further comprising treating said second part with said test compound. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Certain classes of herbicides (e.g., an imidazolinone, sulfonylurea, triazolopyrimidine and pyrimidyl salicylic acid) destroy plants by inhibiting acetohydroxyacid synthase (AHAS), which is also known as acetolactate synthase. AHAS is the first enzyme in the branched chain amino acid pathway. Crop varieties have been developed that are resistant to AHAS inhibitor herbicides. In addition, overuse of these classes of herbicides has propagated selected weed populations that are resistant to AHAS inhibition. A need has thus developed to rapidly detect both resistant crops and weed populations. This need led to the discovery of various in vivo AHAS assay methods, e.g. as described in U.S. reissue Pat. No. 36,175 reissued Mar. 30, 1999, U.S. Pat. No. 5,356,789 issued Oct. 18, 1994 and U.S. Pat. No. 5,932,434 issued Aug. 3, 1999, both of which are incorporated herein by reference. The assays in these references are based on an inhibitor of ketoacid reductoisomerase (KARI), which is the next enzyme after AHAS in the branched chain amino acid pathway. Inhibition of KARI results in the accumulation of acetolactate in the plant. Acetolactate is the product of AHAS. Thus, the in vivo activity of AHAS can be measured by combining a KARI inhibitor with an AHAS inhibitor and measuring the amount of acetolactate in the plant. These prior art assays have the advantage of being able to obtain an indication of AHAS resistance, without the need for elaborate biochemical expertise or equipment. The disadvantage is that it requires a substantial amount of plant tissue, in order to accumulate enough acetolactate for the detection and quantification of AHAS resistance. Imidazolinone resistant crop varieties have been developed in several crops including maize, canola, wheat, rice and sunflowers. E.g., please see generally European patent 508,161 granted May 10, 2000 and European patent application 965,265 published Dec. 12, 1999, both of which are incorporated herein by reference. The herbicide resistance is due to a single base pair change in the gene encoding for the AHAS enzyme, so that it no longer binds the herbicide. In many crops, there is more than one gene that encodes for AHAS. For example, in canola and wheat, the commercially available imidazolinone resistant varieties have two genes. If the AHAS is encoded by only one gene, the level of herbicide resistance at the whole plant level is insufficient for commercial use. The in vivo AHAS assays can be used to differentiate between plant lines containing various levels of a resistant AHAS enzyme. However, the assays need to be done on relatively large plants, in order to obtain enough plant material to run the assay. Also, the throughput time is relatively long. One way to do this is to spray the plant with a high rate of an imidazolinone. Although this method is very accurate, it requires a relatively large greenhouse and length of time, both of which increase the expense of developing new imidazolinone resistant crop varieties. Also, it limits the amount of plant material that can be handled. Another way is to conduct in vitro AHAS assays, which tends to be very accurate, but requires access to a fully equipped biochemical laboratory and also takes a considerable amount of time. For all of the reasons, above, a plant breeder needs a rapid and nondestructive method to determine the level of herbicide resistance in plant breeding lines. This felt need has thus led to this invention, which is a high throughput, in vivo assay method and composition for detecting AHAS resistance. The method and composition described in the invention increases the rate of accumulation of acetolactate. Thus, the plant tissue requirement is reduced. Also, the assay can take advantage of the microtiter plate system that is currently used for high throughput assays in the pharmaceutical industry. The objective of this assay is a method for increasing the rate of accumulation of acetolactate in plant material in the presence of a KARI inhibitor, and thus reduce the amount of plant material needed to run the assay to the point where the assay could be run in a microtiter plate. The objective is accomplished by determining that external supplementation of the in vivo AHAS assay medium greatly increases the rate of accumulation of acetolactate in plant tissue. In one embodiment of the invention, the method for determining whether a plant is resistant to an acetohydroxyacid synthase inhibitor comprises the following steps; (a) treating a part taken from the plant with an effective amount of the acetohydroxyacid synthase inhibitor, a ketol-acid reductoisomerase inhibitor and an external supplement that is either a compound or combination of compounds selected from the group consisting of alanine, ammonium hydroxide, asparagine, and pyruvic acid or a salt thereof; and (b) measuring the amounts of acetohydroxybutyrate and/or acetolactate. In a preferred embodiment, the method according for determining whether a plant is resistant to an acetohydroxyacid synthase inhibitor comprises the following steps: (a) treating a first part taken from the plant with an effective amount of the acetohydroxyacid synthase inhibitor, a ketol-acid reductoisomerase inhibitor, and an external supplement that is either a compound or combination of compounds selected from the group consisting of alanine, ammonium hydroxide, asparagine, and pyruvic acid or a salt thereof; and (b) treating a second part taken from the plant with an external supplement that is either a compound or combination of compounds selected from the group consisting of alanine, ammonium hydroxide, asparagine, and pyruvic acid or a salt thereof; or (c) treating a second part taken from the plant with an effective amount of the ketol-acid reductoisomerase inhibitor and with an external supplement that is either a compound or combination of compounds selected from the group consisting of alanine, ammonium hydroxide, asparagine, and pyruvic acid or a salt thereof; and (d) determining the amounts of acetohydroxybutyrate and/or acetolactate accumulated in the part of the plant from step a) and in the part of the plant from step b) or c); and (e) comparing the amount of acetohydroxybutyrate and/or acetolactate from step a) with step b) or c). The part taken from the plant in the above-mentioned methods can be taken from a blade with a first and second portion that is separated by a midvein. The first part of the plant used in step a) is taken from the first portion of the blade. The second part of the blade used in step b) or c) can be taken from the second portion at an approximately equal and opposite location from the midvein in the blade. In yet another embodiment, the method according to the present invention for determining whether a population of a plant species is resistant to an acetohydroxyacid synthase inhibitor, the plant species has a blade with a first and second portion that is separated by a midvein. The method comprises: (a) treating a first part taken from the first portion, with an effective amount of the acetohydroxyacid synthase inhibitor, a ketol-acid reductoisomerase inhibitor, and either a compound or combination of compounds selected from the group consisting of alanine, ammonium hydroxide, asparagine, and pyruvic acid or a salt thereof; (b) treating a second part taken from the second portion at an approximately equal and opposite location from the midvein in the blade, with an effective amount of the ketol-acid reductoisomerase inhibitor in step (a); (c) obtaining a first indication in the first part and a second indication in the second part of either or both acetolactate and acetohydroxybutyrate; and (d) contrasting the first and second indication. If the method is done in a High Through-Put Screening it is preferable to use only small amounts of the plant tissue. For example, each of the first and second part or of the part of the plant of step a) can be even less than about 50 mg, but also less than about 20 mg and even less than about 10 mg. In a specific embodiment, the first and second portion is within a basipetal region of the blade. In a more specific embodiment, the plant species comprises a leaf having a blade and a petiole. In a yet more specific embodiment, the basipetal region is adjacent to the petiole. In a further embodiment, the invention is a method for determining whether a test compound inhibits acetohydroxyacid synthase in a plant. All of the above-mentioned embodiments of the above-mentioned methods can be used for this method except that the acetohydroxyacid synthase inhibitor is replaced with a test-compound. In detail, said methods comprises: (a) treating a part taken from the plant with a test-compound, a ketol-acid reductoisomerase inhibitor and an external supplement that is either a compound or combination of compounds selected from the group consisting of alanine, ammonium hydroxide, asparagine, and pyruvic acid or a salt thereof; and (b) measuring the amounts of acetohydroxybutyrate and/or acetolactate. Numerous salts of pyruvic acid are known by the skilled artisan e.g. the natrium, kalium or potassium salt. In yet another embodiment the method for determining whether a test-compound inhibits acetohydroxyacid synthase comprises (a) treating a first part taken from the plant with a test-compound, a ketol-acid reductoisomerase inhibitor, and an external supplement that is either a compound or combination of compounds selected from the group consisting of alanine, ammonium hydroxide, asparagine, and pyruvic acid or a salt thereof; and (b) treating a second part taken from the plant with an external supplement that is either a compound or combination of compounds selected from the group consisting of alanine, ammonium hydroxide, asparagine, and pyruvic acid or a salt thereof; or (c) treating a second part taken from the plant with a ketol-acid reductoisomerase inhibitor and with an external supplement that is either a compound or combination of compounds selected from the group consisting of alanine, ammonium hydroxide, asparagine, and pyruvic acid or a salt thereof; and (d) determining the amounts of acetohydroxybutyrate and/or acetolactate accumulated in the part of the plant from step a) and in the part of the plant from step b) or c); and (e) comparing the amount of acetohydroxybutyrate and/or acetolactate from step a) with step b) or c). The part taken from the plant in the above-mentioned methods can be taken from a blade with a first and second portion that is separated by a midvein. The first part of the plant used in step a) is taken from the first portion of the blade. The second part of the blade used in step b) or c) can be taken from the second portion at an approximately equal and opposite location from the midvein in the blade. All of the above-mentioned methods are herein below termed “methods according to the invention”. In a preferred embodiment, the part of the plant used in a methods according to the invention is selected from an actively growing part of the plant. Activly growing means that the tissue should be in the logarithmic stage of growth for maximum activity. Furthermore, it is preferred that the part of the plant used in the methods of the present invention is taken from the basipetal region of a blade from the plant. The amounts of acetohydroxybutyrate and/or acetolactate can be determined as described U.S. Pat. No. 5,356,789 based on the use creatine and naphthol. The activity can be either determined by measuring OD 530 or by comparing the intensity of colour visually. The acetohydroxyacid synthase inhibitor can be selected from certain classes of herbicides that inhibit acetohydroxyacid synthase (e.g., an imidazolinone, sulfonylurea, triazolopyrimidine and pyrimidyl salicylic acid). Numerous examples of these herbicides are well known by the skilled artisian and e.g. given in the Pesticide manual, 12th Edition, 2000 British Crop Protection Councel). The amount of the acetohydroxyacid synthase inhibitor, a ketol-acid reductoisomerase inhibitor (herein further also referred to as “effective amount”) can be determined easily by the skilled artisian. As a rule, if the plant part has a weight of 50 mg, the effective amount of the acetohydroxyacid synthase inhibitor, a ketol-acid reductoisomerase inhibitor is between 100-500 μM. The external supplement is either a compound or combination of compounds selected from the group consisting of alanine, ammonium hydroxide, asparagine, and pyruvic acid (or a salt thereof), preferably alanine alone, alanine combined with pyruvic acid (or a salt thereof) or ammonium hydroxide and pyruvic acid (or a salt thereof). It is preferred to use small leaf discs in the method of the present invention. This is because the smaller the leaf discs, the greater the ratio of edge to total area and the better is the entrance of chemicals into the leaf. Preferably, the external supplement in step a) of the methods according to the invention, has an amount of about 0.2 to less than 5 percent by weight of a sum consisting of the part taken from the plant, the effective amount of the acetohydroxyacid synthase inhibitor, the ketol-acid reductoisomerase inhibitor and the external supplement of step a). Furthermore, it has been found that certain combinations of the external supplements show a synergistic increase in accumulation of acetolactate in the assays according to the invention. In a preferred embodiment, the supplement used in methods according to the invention is the combination of compounds alanine and pyruvic acid or a salt thereof. In a specific embodiment, the alanine and pyruvic acid or salt is in a ratio of about 8:1 to 1:50. In another specific embodiment, the alanine, and pyruvic acid or salt has an amount of about 0.2 to less than 5 percent by weight of a sum consisting of the first part, the acetohydroxyacid synthase and ketol-acid reductoisomerase inhibitor, and the alanine, and pyruvic acid or salt. In a more specific embodiment, the alanine is L-alanine. In still another preferred embodiment, the supplement used in methods mentioned above the combination of compound ammonium hydroxide, and pyruvic acid or a salt thereof. In a specific embodiment, the ammonium hydroxide and pyruvic acid or salt is in a mole to mole ratio of about 8:1 to about 1:50. In another specific embodiment, the ammonium hydroxide, and pyruvic acid or salt has an amount of about 0.2 to less than 5 percent by weight of a sum in step (a) consisting of the first part, the acetohydroxyacid synthase and ketol-acid reductoisomerase inhibitor, and the ammonium hydroxide, and pyruvic acid or salt. In yet another preferred embodiment, the method for determining whether a plant is resistant to a herbicide comprises: (a) combining in an aqueous medium a fresh sample of a tissue from the plant, the fresh sample is an amount of less than about 50 mg, an effective amount of the herbicide, an effective amount of a ketol-acid reductoisomerase inhibitor, and either a compound or a combination of compounds selected from the group consisting of alanine, ammonium hydroxide, asparagine, and pyruvic acid or a salt thereof, the compound or combination of compounds is an amount of about 0.2 to less than 5 percent by weight of a sum of the fresh sample and herbicide, the ketol-acid reductoisomerase inhibitor, and the compound or combination of compounds; and (b) identifying an accumulation of acetolactate. In yet another embodiment, the invention is a composition comprising alanine, and pyruvic acid or a salt thereof. In a specific embodiment, the alanine, and pyruvic acid or salt are in a mole to mole ratio of 8:1 to about 1:50. In another specific embodiment, the composition is in an aqueous medium. In still yet another embodiment, the invention is a composition comprising ammonium hydroxide, and pyruvic acid or a salt thereof. In a specific embodiment, the ammonium hydroxide, and pyruvic acid or salt are in a mole to mole ratio of 8:1 to about 1:50. In another specific embodiment, the composition is in an aqueous medium. In a further embodiment, the imethod for determining whether a material inhibits acetohydroxyacid synthase in a plant comprises: (a) treating a first part taken from the first portion, with an effective amount of the material, a ketol-acid reductoisomerase inhibitor, and either a compound or combination of compounds selected from the group consisting of alanine, ammonium hydroxide, asparagine, and pyruvic acid or a salt thereof; (b) treating a second part taken from the second portion at an approximately equal and opposite location from the midvein in the blade, with an effective amount of the ketol-acid reductoisomerase inhibitor in step (a); and (c) obtaining a first indication in the first part and a second indication in the second part of either or both acetolactate and acetohydroxybutyrate; and (d) contrasting the first and second indication, to determine if an amount of either or both acetolactate and acetohydroxybutyrate present in the first indication is less than an amount of either or both acetolactate and acetohydroxybutyrate present in the second indication. The term “indication” means determination of the level of either or both acetolactate and acetohydroxybutyrate. Methods how to determine this indication are disclosed above. In a more specific embodiment, each of the first and second part is taken from a basipetal region of the blade. In a still further embodiment, the invention is a method for determining whether a herbicide is capable of inhibiting acetolactate synthesis in a plant. The method comprises: (a) combining in an aqueous medium a fresh sample of a tissue from the plant, the fresh sample being an amount of less than about 50 mg, and an effective amount of the herbicide, a ketol-acid reductoisomerase inhibitor, and either a compound or a combination of compounds selected from the group consisting of alanine, ammonium hydroxide, asparagine, and pyruvic acid or a salt thereof, the compound or combination of compounds being from about 0.2 to less than 5 percent by weight of a sum of the fresh sample and herbicide, the ketol-acid reductoisomerase inhibitor, and the compound or combination of compounds; and (b) determining if there is an accumulation of acetolactate from step (a). A ketol-acid reductoisomerase inhibitor described in all of the embodiments above, is disclosed in the prior art. In one embodiment, the ketol-acid reductoisomerase inhibitor is selected from the group consisting of (dimethylphosphinyl) glycolic acid, 2-(dimethylphosphinoyl)-2-hydroxyacetic acid, a sodium N-hydroxy-N-(C 1 -C 6 alkyl or C 3 -C 7 cycloalkyl)oxamate, sodium N-hydroxy-N-aralkyloxamate, 2-methylphosphinoyl-2-hydroxyacetic acid, N-hydroxy-N-isopropyloxamate, the monomethyl and monoethyl ester of 1,1-cyclopropanedicarboxylic acid, and ethylenemalonic acid and the mono and disalt therof. In a specific embodiment, the sodium N-hydroxy-N-(C 1 -C 6 alkyl) oxamate is sodium N-hydroxy-N-isopropyloxamate. In another specific embodiment, the sodium N-hydroxy-N-aralkyloxamate is sodium N-hydroxy-N-benzyloxamate. The increased accumulation of acetolactate was used to reduce the plant material to amounts that leads to small samples enabeling high throughput screening. For example, a 5 mm diameter leaf disc that could easily fit in a 96-well microtiter plate well can be used in the method according to the present invention. Using high throughput screening, many discrete compounds can be tested n parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well, 384-well and 1536-well microtiter plates. As mentioned above, the 96-well formate is preferred in the above-mentioned applicaiton. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the respective well format. In addition, a method was developed for cutting leaf discs from plant material and for extracting the acetolactate from the leaf discs in situ in the microtiter plate so that the assay can easily be run on over 1000 plants per week. Furthermore, if freeze thawing is used for tissue extraction, it is also possible that the plates can be stored frozen for an extended period of time before complementing the rest of the reaction. detailed-description description="Detailed Description" end="lead"? |
Tricyclic imidazopyridines |
The invention provides compounds of the formula (I), in which the substituents and symbols are as defined in the description. The compounds inhibit the secretion of gastric acid. |
1. A compound of the formula 1, where R1 is hydrogen, 1-4C-alkyl, 3-7C-cycloalkyl, 3-7C-cycloalkyl-1-4C-alkyl, 1-4C-alkoxy, 1-4C-alkoxy-1-4C-alkyl, 1-4C-alkoxycarbonyl, 2-4C-alkenyl, 2-4C-alkynyl, fluoro-1-4C-alkyl or hydroxy-1-4C-alkyl, R2 is hydrogen, 1-4C-alkyl, 3-7C-cycloalkyl, 3-7C-cycloalkyl-1-4C-alkyl, 1-4C-alkoxycarbonyl, hydroxy-1-4C-alkyl, halogen, 2-4C-alkenyl, 2-4C-alkynyl, fluoro-1-4C-alkyl or cyanomethyl, R3 is hydroxy-1-4C-alkyl, 1-4C-alkoxy-1-4C-alkyl, 1-4C-alkoxy-1-4C-alkoxy-1-4C-alkyl, 1-4C-alkoxycarbonyl, fluoro-1-4C-alkoxy-1-4C-alkyl or the radical —CO—NR31R32, where R31 is hydrogen, 1-7C-alkyl, hydroxy-1-4C-alkyl or 1-4C-alkoxy-1-4C-alkyl and R32 is hydrogen, 1-7C-alkyl, hydroxy-1-4C-alkyl or 1-4C-alkoxy-1-4C-alkyl, or where R31 and R32 together and including the nitrogen atom to which they are attached form a pyrrolidino, piperidino or morpholino radical, Arom is a R4-, R5-, R6 and R7-substituted mono- or bicyclic aromatic radical selected from the group consisting of phenyl, naphthyl, pyrrolyl, pyrazolyl, imidazolyl, 1,2,3-triazolyl, indolyl, benzimidazolyl, furanyl (furyl), benzofuranyl (benzofuryl), thiophenyl (thienyl), benzothiophenyl (benzothienyl), thiazolyl, isoxazolyl, pyridinyl, pyrimidinyl, quinolinyl and isoquinolinyl, where R4 is hydrogen, 1-4C-alkyl, hydroxy-1-4C-alkyl, 1-4C-alkoxy, 2-4C-alkenyloxy, 1-4C-alkylcarbonyl, carboxyl, 1-4C-alkoxycarbonyl, carboxy-1-4C-alkyl, 1-4C-alkoxycarbonyl-1-4C-alkyl, halogen, hydroxyl, aryl, aryl-1-4C-alkyl, aryloxy, aryl-1-4C-alkoxy, trifluoromethyl, nitro, amino, mono- or di-1-4C-alkylamino, 1-4C-alkylcarbonylamino, 1-4C-alkoxycarbonylamino, 1-4C-alkoxy-1-4C-alkoxycarbonylamino or sulfonyl, R5 is hydrogen, 1-4C-alkyl, 1-4C-alkoxy, 1C-alkoxycarbonyl, halogen, trifluoromethyl or hydroxyl, R6 is hydrogen, 1-4C-alkyl or halogen and R7 is hydrogen, 1-4C-alkyl or halogen, where aryl is phenyl or substituted phenyl having one, two or three identical or different substituents form the group consisting of 1-4C-alkyl, 1-4C-alkoxy, carboxyl, 1-4C-alkoxycarbonyl, halogen, trifluoromethyl, nitro, trifluoromethoxy, hydroxyl and cyano, X is O (oxygen) or NH, and its salts. 2. A compound of the formula 1 as claimed in claim 1, where R1 is hydrogen, 1-4C-alkyl, 3-7C-cycloalkyl, 1-4C-alkoxy-1-4C-alkyl, 2-4C-alkynyl or fluoro-1-4C-alkyl, R2 is hydrogen, 1-4C-alkyl, halogen, 2-4C-alkenyl, 2-4C-alkynyl or fluoro-1-4C-alkyl, R3 is hydroxy-1-4C-alkyl, 1-4C-alkoxy-1-4C-alkyl, 1-4C-alkoxy-1-4C-alkoxy-1-4C-alkyl, 1-4C-alkoxycarbonyl or the radical —CO—NR31R32, where R31 is hydrogen, 1-7C-alkyl, hydroxy-1-4C-alkyl or 1-4C-alkoxy-1-4C-alkyl and R32 is hydrogen, 1-7C-alkyl, hydroxy-1-4C-alkyl or 1-4C-alkoxy-1-4C-alkyl, or where R31 and R32 together and including the hydrogen atom to which they are attached are a pyrrolidino, piperidino or morpholino radical, Arom is a R4-, R5-, R6- and R7-substituted mono- or bicyclic aromatic radical selected from the group consisting of phenyl, furanyl (furyl) and thiophenyl (thienyl), where R4 is hydrogen, 1-4C-alkyl, hydroxy-1-4C-alkyl, 1-4C-alkoxy, 1-4C-alkylcarbonyl, carboxyl, 1-4C-alkoxycarbonyl, halogen, hydroxyl, trifluoromethyl, 1-4C-alkylcarbonylamino, 1-4C-alkoxycarbonylamino, 1-4C-alkoxy-1-4C-alkoxycarbonylamino or sulfonyl, R5 is hydrogen, 1-4C-alkyl, 1-4C-alkoxy, 1-4C-alkoxycarbonyl, halogen, trifluoromethyl or hydroxyl, R6 is hydrogen or 1-4C-alkyl and R7 is hydrogen, X is O (oxygen) or NH, and its salts. 3. A compound of the formula 1 as claimed in claim 1, where R1 is 1-4C-alkyl, R2 is 1-4C-alkyl, halogen or fluoro-1-4C-alkyl, R3 is hydroxy-1-4C-alkyl, 1-4C-alkoxy-1-4C-alkyl, 1-4C-alkoxy-1-4C-alkoxy-1-4C-alkyl, 1-4C-alkoxycarbonyl or the radical —CO—NR31R32, where R31 is hydrogen, 1-4C-alkyl, hydroxy-1-4C-alkyl or 1-4C-alkoxy-1-4C-alkyl and R32 is 1-4C-alkyl, hydroxy-1-4C-alkyl or 1-4C-alkoxy-1-4C-alkyl, or where R31 and R32 together and including the nitrogen atom to which they are attached are a pyrrolidino, piperidino or morpholino radical, Arom is R4-, R5-, R6- and R7-substituted phenyl, where R4 is hydrogen or 1-4C-alkyl, R5 is hydrogen or 1-4C-alkyl, R6 is hydrogen or 1-4C-alkyl and R7 is hydrogen, X is O (oxygen) or NH, and its salts. 4. A compound of the formula 1 as claimed in claim 1, where R1 is is methyl, R2 is methyl, chlorine or difluoromethyl, R3 is hydroxymethyl, methoxymethyl, methoxyethoxymethyl, 1-4C-alkoxycarbonyl or the radical —CO—NR31R32, where R31 is methyl or ethyl and R32 is methyl or ethyl, or where R31 and R32 together and including the nitrogen atom to which they are attached are a morpholino radical, Arom is phenyl, 2-methylphenyl, 2-isopropylphenyl, 2,6-dimethylphenyl, 2,6-diisopropylphenyl, 2,4,6-trimethylphenyl or 2,4,6-triisopropylphenyl and X is O (oxygen) or NH, and its salts. 5. A compound of the formula 1 as claimed in claim 1, where R1 is 1-4C-alkyl, R2 is 1-4C-alkyl, R3 is 1-4C-alkoxycarbonyl or the radical —CO—NR31R32, where R31 is 1-4C-alkyl and R32 is 1-4C-alkyl, Arom is phenyl and X is O (oxygen), and its salts. 6. A compound of the formula 1 as claimed in claim 1, where R1 is 1-4C-alkyl, R2 is 1-4C-alkyl, R3 is 1 AC-alkoxycarbonyl or the radical —CO—NR31R32, where R31 is 1-4C-alkyl and R32 is 1-4C-alkyl. Arom is phenyl or 2-methyl-6-ethylphenyl and X is O (oxygen), and its salts. 7. A medicament comprising a compound as claimed in claim 1 and/or a pharmacologically acceptable salt thereof together with customary pharmaceutical auxiliaries and/or excipients. 8. The use of a compound as claimed in claim 1 and its pharmacologically acceptable salts for the prevention and treatment of gastrointestinal disorders. |
<SOH> TECHNICAL FIELD <EOH>The invention relates to novel compounds which are used in the pharmaceutical industry as active compounds for preparing medicaments. |
Use of a polyfunctional active substance mixture as an antagonist against harmful substances contained in tobacco smoke |
The invention relates to the use of a polyfunctional active substance mixture with anti-inflammatory, spasmolytic and anti-stress action comprised of a fraction of specific peptides with molar weights of up to 10,000 dalton and/or of a fraction of essential and non-essential amino acids, and the polyfunctional active substance mixture is obtained from a multifactorial immune-modulator mixture serving as an antagonist against the harmful substances contained in tobacco smoke with a health-protecting function during the smoking of tobacco products. It is preferably applied in the form of an inhalant (adhesion principle, atomization) and leads to the amelioration, elimination and prevention of inflammations of the mucous membranes, of regulatory disorders of the organ systems with smooth muscles (blood vessels, bronchial system, esophagus, bladder, stomach, intestines, etc.), of the myocardial function as well as of the central and peripheral nerve systems that are induced by the harmful substances contained in tobacco smoke. |
1. Use of a polyfunctional active substance mixture with anti-inflammatory, spasmolytic and anti-stress action as an antagonist against the harmful substances contained in tobacco smoke comprised of a fraction of specific peptides with molar weights of up to 10,000 dalton and/or of a fraction of essential and non-essential amino acids, and said polyfunctional active substance mixture was obtained from a multifactorial immune-modulator mixture: by way of incubation at the temperatures that are typical for the original species of 20 to 39° C. for a time period of 2 to 8 days; followed by lysis; harvested together with the conservation medium; and, separation of cell components which have a molar mass of over 10,000 dalton by way of high-speed centrifuging at a running time of 24 hours; and, gaining the fraction of specific peptides with molar weights of up to 10,000 dalton and/or of the fraction of essential and non-essential amino acids. 2. Use as claimed in claim 1 wherein a fraction of specific peptides with molar weights of between 200 and 6,000 dalton and/or a fraction of essential and non-essential amino acids are applied. 3. Use as claimed in claim 1 wherein mixtures of peptides, as well as neuro-peptides contained therein, are applied that regulate homeostasis, act upon the smooth muscles and regulate stress. 4. Use as claimed in one of the claims 1 to 3 wherein the mixture is applied at low concentrations, preferably at a dosage of 10−6 to 10−9 g/kg of body weight and wherein its action occurs primarily in the form of a bioactive triggering function. 5. Use as claimed in one of the claims 1 to 4 wherein the dose-action relationship follows the principle of a hyperbolic curve whereby an overdose is impossible. 6. Use as claimed in one of the claims 1 to 5 wherein the mixture is applied in a specific filter (specific mouth tip) of the tobacco product (cigarette, cigar, cigarillo, tobacco pipe, etc.) and wherein the mixture is incorporated into the tobacco product at the time of the manufacture of the tobacco product and inhaled during smoking. 7. Use as claimed in one of the claims 1 to 6 wherein it [the filter] itself is not harmful to the smoker's health, nor does it constitute a risk for addiction, and it does not change the smoking enjoyment. 8. Use as claimed in one of the claims 1 to 7 wherein the polyfunctional active substance mixture is applied for ameliorating, eliminating and preventing: inflammations of the mucous membranes; regulatory disorders; [impairment] of the function of the organ systems with smooth muscles (bronchial system, blood vessels, esophagus, bladder, stomach, intestines, etc.); [impairment] of the myocardial functions; and, [impairment] of the functions of the central and peripheral nerve system induced by the harmful substances contained in tobacco smoke. 9. Specific filters for tobacco products wherein these filters comprise a polyfunctional active substance mixture as claimed in one of the claims 1 to 4 featuring a health-protecting function during the smoking of tobacco. |
Antigenic polypeptides |
A method for the identification of antigenic polypeptides, typically opsonic antigens, expressed by pathogenic microbes; vaccines comprising said antigens; and therapeutic antibodies directed to said antigenic polypeptides. |
1. An antigenic polypeptide, or part thereof, encoded by an isolated DNA molecule selected from the group consisting of: (i) DNA molecules represented by the DNA sequences in Table 7 or 9; (ii) DNA molecules which hybridize to the sequences identified in (i) which encode a polypeptide expressed by a pathogenic organism; and (iii) DNA molecules which are degenerate as a result of the genetic code to the DNA sequences defined in (i) and (ii), for use as a vaccine. 2. An antigenic polypeptide according to claim 1 wherein said DNA molecule is genomic DNA. 3. An antigenic polypeptide according to claim 1 or 2 wherein said DNA molecule hybridizes to the the sequences in Tables 7 or 9 under stringent hybridization conditions. 4. An antigenic polypeptide according to any of claims 1-3 wherein said polypeptide (s) are represented by the amino acid sequences in Tables 8 or 10. 5. An antigenic polypeptide according to any of claims 1-4 wherein said polypeptide is derived from a bacterial genus/species selected from the group consisting of: Staphylococcus spp.; Staphylococcus aureus; Staphylococcus epidermidis; Enterococcus faecalis; Mycobacterium tuberculsis; Streptococcus group B; Streptoccocus pneumoniae; Helicobacter pylori; Neisseria gonorrhea; Streptococcus group A; Borrelia burgdorferi; Coccidiodes immitis; Histoplasma sapsulatum; Neisseria meningitidis type B; Shigella flexneri; Escherichia coli; Haemophilus influenzae: 6. An antigenic polypeptide according to claim 5 wherein said polypeptide is derived from the genus Staphylococcus spp. 7. An antigenic polypeptide according to claim 6 wherein said polypeptide is derived from the species Staphylococcus aureus. 8. An antigenic polypeptide according to claim 6 wherein said polypeptide is derived from the species Staphylococcus epidermidis. 9. An antigenic polypeptide according to any of claims 1-8 wherein said polypeptide is an opsonin. 10. A vaccine composition comprising at least one antigenic polypeptide according to any of claims 1-9. 11. A vaccine composition according to claim 10 wherein said composition further comprises a carrier and/or an adjuvant. 12. A method to immunize an animal against a disease or condition caused by a pathogenic microbe comprising administering to said animal at least one antigenic polypeptide according to any of claims 1-9 or a vaccine composition according to claim 10 or 11. 13. A method according to claim 12 wherein said animal is human. 14. A method according to claim 12 or 13 wherein said disease or condition is selected from the group consisting of: bacterimia; septic shock; organ infection; skin infection; bacterial nasal colonisation; bacterial eye infections; septicaemia; tuberculosis; bacteria-associated food poisoning; blood infections; peritonitis; endocarditis; sepsis; meningitis; pneumonia; stomach ulcers; gonorrhoea; strep throat; streptococcal-associated toxic shock; necrotizing fasciitis; impetigo; histoplasmosis; Lyme disease; gastro-enteritis; dysentery; shigellosis; Staphylococcus aureus-associated septicaemia, food-poisoning or skin disorders; Staphylococcus epidermidis-associated septicaemia, peritonitis or endocarditis. 15. A method according to claim 14 wherein said disease or condition is the result of a Staphylococcus spp infection. 16. A method according to Claim 15 wherein said disease or condition is Staphylococcus aureus-associated septicaemia, food-poisoning or skin disorders. 17. A method according to claim 15 wherein said disease or condition is Staphylococcus epidermidis-associated septicaemia, peritonitis or endocarditis. 18. An antibody, or binding part thereof, obtainable by the method according to any of claims 12-17. 19. An antibody according to claim 18 wherein said antibody is a monoclonal antibody. 20. An antibody according to claim 18 or 19 wherein said antibody is a chimeric antibody. 21. An antibody according to claim 18 or 19 wherein said antibody is a humanized antibody. 22. An antibody according to any of claims 18-21 wherein said antibody is an opsonic antibody. 23. An antibody according to any of claims 18-22 wherein said antibody is a therapeutic antibody or a diagnostic antibody. 24. A method for preparing a hybridoma cell-line producing monoclonal antibodies according to claim 19 comprising the steps of: immunising an immunocompetent mammal with an immunogen comprising at least one polypeptide having the amino acid sequence as represented in Tables 8 or 10, or polypeptide fragments thereof; ii) fusing lymphocytes of the immunised immunocompetent mammal with myeloma cells to form hybridoma cells; iii) screening monoclonal antibodies produced by the hybridoma cells of step (ii) for binding activity to the amino acid sequences of (i); iv) culturing the hybridoma cells to proliferate and/or to secrete said monoclonal antibody; and optionally v) recovering the monoclonal antibody from the culture supernatant. 25. A method according to claim 24 wherein said hybridoma cell-line produces opsonic antibodies. 26. A hybridoma cell-line produced by the method of claim 24 or 25. 27. A method to identify opsonic antigens expressed by a pathogenic microbe comprising: i) providing a host cell transformed with a DNA library encoding genes, or partial gene sequences, of a pathogenic microbe; ii) providing conditions conducive to the expression of said transformed genes or partial sequences; iii) contacting the antigens expressed by said gene sequences with autologous antisera derived from an animal infected with, or has been infected with, said pathogenic microbe; iv) purifying the DNA encoding antigenic polypeptides binding to said autologous antisera; and v) testing the opsonic activity of a polypeptide encoded by said DNA molecule. |
Modulation of insulin-regulated aminopeptidase (irap)/angiotensin iv (at4) receptor activity |
The invention relates to modulators of insulin-regulated aminopeptidase (IRAP)/Angiotensin IV receptor (AT4) activity, which have the ability to (a) increase local concentration of a wide range of peptides, thereby enhancing the activity of these peptides; (b) regulate glucose uptake into cells and tissues, thereby altering the metabolic or energy status of the cells or tissues; (c) trigger second messenger or signalling pathways associated with IRAP/AT4 function; and/or (d) increase cell surface expression of IRAP/AT4. The modulators may enhance learning and memory in both normal subjects and those with memory disorders. They also stimulate cellular proliferation while reducing apoptosis, thus stimulating tissue growth development, and affect angiogenesis. Thus they are also useful in tissue repair and regeneration. The invention also relates to methods of identifying modulators of insulin-regulated aminopeptidase activity. |
1-34 (canceled) 35. A method for modulating the expression, production, or activity of AT4 receptor/IRAP in a subject, comprising the step of administering an effective amount of a compound which modulates AT4 receptor/IRAP expression, production or activity, in which the compound is (a) a ligand of AT4 receptor/IRAP, (b) an oligonucleotide molecule which is antisense to a nucleic acid encoding AT4 receptor/IRAP, or (c) a compound which is able to modulate the cellular localization of AT4 receptor/IRAP to the subject. 36. A method of screening for compounds which have the ability to modulate the activity of AT4 receptor/IRAP, comprising the steps of (a) assessing the ability of a candidate compound to bind to AT4 receptor/IRAP, and one or more of (b) assessing the ability of the compound to modulate the enzymatic activity of AT4 receptor/IRAP; (c) assessing the effect of the compound on the interaction between AT4 receptor/IRAP and a protein which interacts with AT4 receptor/IRAP; (d) assessing the ability of the candidate compound to modulate one or more signalling or intracellular pathways activated by binding of the candidate compound to AT4 receptor/IRAP; (e) assessing the ability of the candidate compound to modulate an activity of AT4 receptor/IRAP; (e) assessing the ability of the candidate compound to translocate AT4 receptor/IRAP to the cell surface. 37. A method according to claim 36, comprising the steps of assessing the effect of the candidate compound on a) binding to AT4 receptor/IRAP or to a protein which interacts with AT4 receptor/IRAP; b) altering the ability of a protein to interact with AT4 receptor/IRAP; and c) modifying the enzymatic or signalling activities of AT4 receptor/IRAP. 38. A method according to claim 36 for identifying compounds which translocate AT4 receptor/IRAP to the cell surface, comprising the steps of (a) exposing cells of a stably transfected cell line expressing AT4 receptor/IRAP to a candidate compound, and (b) assessing the amount of AT4 receptor/IRAP expressed on the cell surface. 39. A method according to claim 36, in which the compound is an intracellular or extracellular protein. 40. A method according to claim 37, in which the protein which interacts with AT4 receptor/IRAP is tankyrase. 41. A method according to claim 35, in which the compound is a specific, high affinity ligand of AT4 receptor/IRAP which modulates AT4 receptor/IRAP levels or AT4 receptor/IRAP activity. 42. A method of modulating the in vivo concentration of a neuropeptide or a polypeptide, comprising the step of administering an effective amount of a compound which can modulate an effect mediated by AT4 receptor/IRAP to a subject in need of such treatment. 43. A method according to claim 42, in which the neuropeptide is selected from the group consisting of arginine-vasopressin, oxytocin, somatostatin, angiotensin III, Lys-bradykinin, neurotensin, met-enkephalin, dynorphin A, neurokinin A, neuromedin B, and cholecystokinin 8. 44. A method according to claim 42, in which the polypeptide is selected from the group consisting of insulin, insulin-like growth factor I and insulin-like growth factor II. 45. A method of prevention or treatment of a disease or condition associated with altered activity of AT4 receptor/IRAP, comprising the step of administering an effective amount of a compound which has the ability to modulate an effect mediated by AT4 receptor/IRAP to a subject in need of such treatment. 46. A method according to claim 45, in which the disease or condition is (a) a disorder of the central nervous system (CNS)associated with dementia or memory loss; (b) a disorder of the CNS involving motor and sensory systems; (c) a disorder of the CNS resulting from trauma or stroke; (d) a disorder of the cardiovascular system; (e) a disorder of development or growth; (f) a disorder of glucose and fat metabolism; (g) a disorder of the reproductive tract; (h) a disorder associated with pregnancy; or (i) a cancer. 47. A method according to claim 45, in which the modulator of an effect mediated by AT4 receptor/IRAP is used for promotion of neuroregeneration and repair following CNS damage or injury, for promotion of tissue regeneration and repair following damage or injury, for promotion of wound healing, for inhibition of angiogenesis, or for treatment of premature or delayed labor. 48. A method according to claim 45, in which the condition is selected from the group consisting of (a) a neurodegenerative condition selected from the group consisting of motor neurone disease (amyotrophic lateral sclerosis), progressive spinal muscular atrophy, infantile muscular atrophy, Charcot-Marie-Tooth disease, Parkinson's Disease, Parkinson-Plus syndrome, Guamanian Parkinsonian dementia complex, progressive bulbar atrophy, and Alzheimer's disease; (b) a neurodegenerative condition arising from ischaemia, hypoxia, neural injury, surgery, or exposure to neurotoxins; (c) a peripheral sensory neuropathy resulting from exposure to drugs or toxins; and (d) a peripheral sensory neuropathy resulting from diabetes. 49. A method according to claim 45, in which the condition is characterised by neuronal deficit or neuronal death, comprising the step of administering an effective amount of a compound which has the ability to modulate an effect mediated by AT4 receptor/IRAP to a subject in need of such treatment. 50. A method according to claim 45, of stimulation of growth in an animal, comprising the step of administering an effective amount of a compound which can modulate an effect mediated by AT4 receptor/IRAP to the animal. 51. A method according to claim 45, in which the mammal is a farm animal. 52. A method according to claim 51, in which the farm mammal is selected from sheep, cattle, pigs, goats and poultry. 53. A method according to claim 50, in which the in vivo uptake of glucose into cells or tissues is modulated, comprising the step of administering an effective amount of a compound which can modulate an effect mediated by AT4 receptor/IRAP to a subject in need of such treatment. 54. A method according to claim 48, of treating memory disorders or of reversing memory loss. 55. A method of enhancing memory or learning in a normal subject, comprising the step of administering an effective amount of a compound which can modulate an effect mediated by AT4 receptor/IRAP to a subject in need of such treatment. 56. A method according to claim 44, in which the compound which modulates an effect mediated by AT4 receptor/IRAP is able to decrease or increase the enzymatic activity of AT4 receptor/IRAP, thereby increasing the serum and tissue levels of at least one biologically-active substance selected from the group consisting of arginine-vasopressin, oxytocin, somatostatin, angiotensin III, Lys-bradykinin, neurotensin, met-enkephalin, dynorphin A, neurokinin A, neuromedin B, and cholecystokinin 8. 57. A method of monitoring the activity of AT4 receptor/IRAP, comprising the step of assessing the aminopeptidase activity of AT4 receptor/IRAP in a biological sample. 58. A method of diagnosing an AT4 receptor/IRAP-related condition, comprising the step of measuring the level of activity of AT4 receptor/IRAP in a subject suspected of having such a condition, and comparing the level to that found in a normal individual, in which any difference between the levels is indicative of the condition. 59. A therapeutic or prophylactic agent for a disease or condition associated with altered activity of AT4 receptor/IRAP, comprising (a) a peptide which has an amino acid sequence selected from the group consisting of LVVYPWTQRF (SEQ ID NO:2), KYLPGPLQ (SEQ ID NO:3) and VYIHPF (SEQ ID NO:1), or an analogue or peptidomimetic thereof, which is able to modulate an effect mediated by AT4 receptor/IRAP, (b) an inhibitor of leucine aminopeptidase activity which has the ability to inhibit IRAP, or (c) an antisense AT4 receptor/IRAP nucleic acid molecule or a fragment thereof which has the ability to inhibit expression of AT4 receptor/IRAP, and optionally (d) a pharmaceutically acceptable carrier. 60. An agent according to claim 59, in which the inhibitor of leucine aminopeptidase activity is L-leucinethiol, L-leucinal, L-leucinol, or a derivative thereof which has the ability to inhibit IRAP. 61. An agent according to claim 60, in which the peptide is KYLPGPLQ (SEQ ID NO:3), or an analogue or peptidomimetic thereof, which is able to modulate an effect mediated by AT4 receptor/IRAP. 62. A method according to claim 35, comprising the step of administering to a patient in need thereof an effective amount of an antisense nucleic acid molecule directed against nucleic acid encoding AT4 receptor/IRAP, or a fragment thereof which has the ability to inhibit expression of AT4 receptor/IRAP. 63. A method according to claim 62, in which the antisense nucleic acid molecule directed against nucleic acid encoding AT4 receptor/IRAP has a nucleotide sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28. 64. An oligonucleotide molecule which is antisense to a nucleic acid encoding AT4 receptor/IRAP, which is a) an oligonucleotide molecule which has a nucleotide sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28; b) a fragment of a) which has the ability to inhibit expression of AT4 receptor/IRAP; c) a nucleic acid molecule which has at least 75% sequence homology to a sequence in a) or b); or d) a nucleic acid molecule which is capable of hybridizing to a sequence in a) or b) under stringent conditions; optionally operatively linked to a vector or present in a transfected host cell. 65. A construct for in vivo delivery of an effective amount of antisense AT4 receptor/IRAP to a subject, comprising: a) an oligonucleotide according to claim 64; and b) a vector comprising a control sequence, wherein the control sequence is capable of controlling the expression of a nucleotide sequence of a), which in turns modulates the activity of AT4 receptor/IRAP. 66. A construct according to claim 65, operatively linked to a vector or present in a transfected host cell. |
<SOH> BACKGROUND OF THE INVENTION <EOH>All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. There are a number of conditions which have no known treatment, or for which the treatments that are available are not effective. For example, disorders of the central nervous system, including Alzheimer's disease (AD) and other forms of dementia and memory loss, motor neurone diseases, disorders of the cardiovascular system, including cardiac hypertrophy, congestive heart failure, hypertension, and atherosclerosis, and disorders of development and growth, including disorders of glucose and fat metabolism, are all serious conditions for which effective treatment is currently unavailable. Given the importance of these conditions, it is readily apparent that effective treatments are urgently needed. Alzheimer's disease (AD), for example, is one of the major health care problems facing first world countries. In 1995, 130,000 Australians aged over 65 years had moderate to severe dementia, and it is estimated that by 2041 the number of people with dementia in Australia will have increased by 254% (Henderson and Jorm, 1998). Despite a dramatic increase in the understanding of the neuropathogenic and neurochemical alterations accompanying AD, physicians have as yet no pharmacological agent that can be prescribed safely, either to arrest or reverse this condition. Some of the other conditions identified above are also increasing in prevalence, and will become increasingly economically and socially burdensome. Accordingly, there is a need for new forms of treatment and new therapeutic agents which can provide sufferers of these diseases with effective treatment. Angiotensin IV (Ang IV) is a hexapeptide with the sequence VYIHPF (SEQ ID NO:1), which has been shown in vitro to be produced by the consecutive actions of aminopeptidases A and N on angiotensin II. This hexapeptide was initially thought to be inactive, but has subsequently been shown to elicit actions in the brain and blood vessels. Ang IV acts via a specific binding site, designated the Angiotensin IV receptor (AT 4 receptor). The AT 4 receptor is defined as a high affinity binding site which selectively binds Ang IV with an affinity ranging from 1-10 nM (Swanson et al, 1992). The AT 4 receptor isolated from bovine adrenal membranes is a glycoprotein of 165 kDa, with a single transmembrane domain, a large extracellular domain, and a small intracellular domain (Zhang et al, 1998). In the brain, the AT 4 receptor occurs as a 150 kDa glycoprotein (Zhang et al, 1999). We have shown that a decapeptide, LVVYPWTQRF (SEQ ID NO:2) (Moeller et al, 1996) binds with nanomolar affinity to the AT 4 receptor, and can mimic the actions of Ang IV, and that Nle′-Ang IV (Nle′-YIHPF) (SEQ ID NO:4) binds with sub-nanomolar affinity to AT 4 receptor (Zhang et al, 1999). AT 4 receptors are widely distributed in the brains of guinea-pig (Miller-Wing et al, 1993), rat (Roberts et al, 1995), sheep (Moeller et al, 1996), monkey (Moeller et al, 1996), and human (Chai et al, 2000), and the distributions of the receptors are highly conserved throughout these species. The receptor sites occur in high abundance in the basal nucleus of Meynert, in the CA2 and dentate gyrus of the hippocampus, and throughout the neocortex, a distribution which closely resembles those of cholinergic neurones and their projections, and is consistent with the memory-enhancing effects of the receptor. High levels of the receptors are also found in most brain regions involved in motor control, including the motor cortex, ventral lateral thalamic nucleus, cerebellum and the cranial nerve motor nuclei. Many sensory regions also contain moderate levels of the AT 4 receptors, suggesting a more widespread role for this receptor in the brain. Central infusions of Ang IV, Nle-Ang IV and Norleucinal Ang IV, which are agonists of the AT 4 receptor, were found to facilitate memory retention and retrieval in rats in the passive avoidance and Morris water maze paradigms (Braszko et al, 1988, Wright et al, 1993, Wright et al, 1999). In two rat models of amnesia, respectively induced by scopolamine or perforant pathway lesion, the AT 4 receptor agonists reversed the performance deficits detected in the Morris water maze paradigm (Pederson et al, 1998; Wright et al, 1999). Ang IV was also reported to improve long-term memory in crabs (Delorenzi et al, 1999). Insulin-regulated aminopeptidase (IRAP) is a constituent of vesicles containing the glucose transporter molecule GLUT4. See U.S. Pat. No. 5,968,784 and U.S. Pat. No. 5,972,600, both by Knowles et al. It is known that the distribution and recycling of both IRAP and GLUT4 between cellular vesicles and the cell membrane is regulated by insulin signalling, and thereby modulates glucose, fat and energy metabolism in tissues such as muscle, adipose tissue, and cardiac tissue (Waters et al, 1997, Ross et al, 1998, Garza and Birnbaum, 2000). However, it has never to our knowledge previously been suggested that IRAP might have any role in central nervous system function, and more specifically it has not been suggested that this protein has any role in memory or learning. We have now isolated the AT 4 receptor by protein purification, and have determined its amino acid sequence. We surprisingly discovered that the AT 4 receptor has a span of 18 amino acid residues which has a 95% sequence homology with the deduced amino acid sequence of human oxytocinase (TrEMBL accession #O00769), which is known to be a homologue of insulin-regulated aminopeptidase (IRAP) (Rasmussen et al, 2000). This enabled us to demonstrate that IRAP is the AT 4 receptor, and to hypothesise that modulation of the activity of IRAP by peptides such as Ang IV, Nle 1 -Ang IV, LVVYPWTQRF (SEQ ID NO:2) and KYLPGPLQ (SEQ ID NO:3) will activate neurones in the central nervous system to enhance learning and memory. Moreover, the identification of IRAP as the AT 4 receptor means that screening assays can be developed to identify compounds which have the ability to modulate IRAP enzymatic activity. Such agents can be used to increase serum and tissue levels of biologically active compounds such as arginine-vasopressin, oxytocin, somatostatin, angiotensin III, Lys-bradykinin, neurotensin, met-enkephalin, dynorphin A, neurokinin A, neuromedin B, cholecystokinin 8, insulin, insulin-like growth factor 1 or insulin-like growth factor 2 which may assist in preventing or treating conditions associated with deficient activity of these compounds. |
<SOH> SUMMARY OF THE INVENTION <EOH>In a first aspect, the invention provides a method of screening for compounds which can modulate the activity of AT 4 receptor/insulin-regulated aminopeptidase (IRAP), comprising the step of a) assessing the ability of a candidate compound to bind to AT 4 receptor/IRAP and/or to increase or decrease the enzymatic activity of AT 4 receptor/IRAP in vitro, and optionally also comprising the step of b) assessing the effect of the candidate compound on the interaction between AT 4 receptor/IRAP and a protein which interacts with AT 4 receptor/IRAP, such as tankyrase. In preferred embodiment, this aspect provides: 1. A method comprising the steps of (a) assessing the ability of the candidate compound to bind to AT 4 receptor/IRAP, and (b) assessing the ability of the candidate compound to modulate enzymatic activity of AT 4 receptor/IRAP, thereby to identify compounds which bind to AT 4 receptor/IRAP with high affinity and modulate its enzymatic activity; 2. A method comprising the steps of (a) assessing the ability of the candidate compound to bind to AT 4 receptor/IRAP and (b) assessing the ability of the candidate compound to modulate one or more signalling or intracellular pathways activated by binding of the candidate compound to AT 4 receptor/IRAP, thereby to identify compounds which bind to AT 4 receptor/IRAP with high affinity and modulate its signalling/second messenger activity; 3. A method comprising the steps of (a) assessing the ability of the candidate compound to bind to AT 4 receptor/IRAP and (b) assessing the ability of the candidate compound to modulate activity of AT 4 receptor/IRAP, thereby to identify compounds which bind to AT 4 receptor/IRAP with high affinity and modulate other cellular activity associated with AT 4 receptor/IRAP; 4. A method comprising the steps of (a) identifying candidate proteins which interact with AT 4 receptor/IRAP and (b) assessing the ability of the proteins to modify the enzymatic or signalling activities of AT 4 receptor/IRAP, thereby to identify intracellular or extracellular proteins which interact with AT 4 receptor/IRAP and modulate its activity; and 5. A method comprising the steps of assessing the effect of the candidate compound on (a) binding to AT 4 receptor/IRAP or to a protein which interacts with AT 4 receptor/IRAP, or (b) altering the ability of proteins to interact with AT 4 receptor/IRAP, and on (c) modifying the enzymatic or signalling activities of AT 4 receptor/IRAP, thereby to identify compounds which bind to AT 4 receptor/IRAP or to a protein which interacts with AT 4 receptor/IRAP, and modifies the activity of AT 4 receptor/IRAP. In a second aspect, the invention provides a method for modulating the expression, production, or activity of AT 4 receptor/IRAP, comprising the step of administering an effective amount of a compound which alters AT 4 receptor/IRAP expression, production or activity to a subject in need of such treatment, in which the compound is a specific, high affinity ligand of AT 4 receptor/IRAP which modulates AT 4 receptor/IRAP levels or AT 4 receptor/IRAP activity. In a preferred embodiment, the invention provides a method of modulating the in vivo concentration of a neuropeptide and/or a polypeptide, comprising the step of administering an effective amount of a compound which can modulate the activity of AT 4 receptor/IRAP to a subject in need of such treatment. Preferably the neuropeptide is selected from the group consisting of arginine-vasopressin, oxytocin, somatostatin, angiotensin III, Lys-bradykinin, neurotensin, met-enkephalin, dynorphin A, neurokinin A, neuromedin B, and cholecystokinin 8. Preferably the polypeptide is selected from the group consisting of insulin, insulin-like growth factor I and insulin-like growth factor II. Cholecystokinin 8 is an 8-amino acid peptide derived from cholecystokinin, which is known to have activity in the brain. This aspect of the invention is generally directed to a method of prevention or treatment of a disease condition associated with altered activity of AT 4 receptor/IRAP, comprising the step of administering an effective amount of a compound which is able to modulate the activity of AT 4 receptor/IRAP to a subject in need of such treatment. Alternatively, this aspect is directed to a method for the prevention or treatment of a disease or condition by altering the activity of AT 4 receptor/IRAP, comprising the step of administration of an effective amount of a compound which has the ability to modulate the activity of AT 4 receptor/IRAP to a subject in need of such treatment. Compounds which modulate IRAP activity may be used to treat or prevent disorders of the central nervous system (CNS), including Alzheimer's disease and other forms of dementia and memory loss; other disorders of the CNS, particularly those involving motor and sensory systems; disorders of the CNS resulting from trauma or stroke; disorders of the cardiovascular system, including cardiac hypertrophy, congestive heart failure, vasospastic disorders, hypertension, and atherosclerosis; disorders of development and growth; disorders of the reproductive tract or disorders associated with pregnancy; disorders of glucose and fat metabolism including syndrome X, dyslipidaemia, and diabetes; and cancer; and for promotion of neuroregeneration and repair following CNS damage or injury, promotion of tissue regeneration and repair following damage or injury, promotion of wound healing, inhibition of angiogenesis, and for treatment of premature or delayed labour. It is therefore contemplated that the method of the invention is suitable for treatment of a variety of conditions, including but not limited to neurodegenerative diseases, such as motor neurone disease (amyotrophic lateral sclerosis), progressive spinal muscular atrophy, infantile muscular atrophy, Charcot-Marie-Tooth disease, Parkinson's Disease, Parkinson-Plus syndrome, Guamanian Parkinsonian dementia complex, progressive bulbar atrophy, Alzheimer's disease and the like; other neurodegenerative conditions, such as those arising from ischaemia, hypoxia, neural injury, surgery, or exposure to neurotoxins such as N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine); and peripheral sensory neuropathies, including those resulting from exposure to drugs such as cis-platin or toxins, and those resulting from diabetes, for example mononeuropathy multiplex. In one preferred embodiment, the invention provides a method of preventing or treating a condition characterised by neuronal deficit or neuronal death, comprising the step of administering an effective amount of a compound or agent of the invention to a subject in need of such treatment. In a second preferred embodiment, the invention provides a method of stimulation of growth in a mammal, comprising the step of administering an effective amount of a compound which can modulate the activity of AT 4 receptor/IRAP to the mammal. This embodiment is particularly applicable to farm mammals such as a sheep, cattle, pigs and goats. Preferably the compounds which modulate AT 4 receptor/IRAP activity are able to decrease or increase the enzymatic activity of AT 4 receptor/IRAP, thereby increasing the serum and tissue levels of biologically-active arginine-vasopressin, oxytocin, somatostatin, angiotensin III, Lys-bradykinin, neurotensin, met-enkephalin, dynorphin A, neurokinin A, neuromedin B, cholecystokinin 8, and/or insulin. Persons skilled in the art will appreciate that exogenous administration of a compound identified by the methods of this invention would have similar effects and uses to administration of exogenous arginine-vasopressin, oxytocin, somatostatin, angiotensin III, Lys-bradykinin, neurotensin, met-enkephalin, dynorphin A, neurokinin A, neuromedin B, cholecystokinin 8, insulin, IGF-I or IGF-II. In a third aspect, the invention provides a method of enhancing memory and/or learning in a normal subject, comprising the step of administering an effective amount of a compound which can modulate the activity of AT 4 receptor/IRAP to a subject in need of such treatment. Alternatively, this aspect provides a method of treating memory disorders and/or of reversing memory loss, comprising the step of administration of an effective amount of a compound which has the ability to modulate the activity of AT 4 receptor/IRAP to a subject in need of such treatment. In a fourth aspect, the present invention provides a method of monitoring the activity of AT 4 receptor/IRAP, comprising the step of assessing the aminopeptidase activity of AT 4 receptor/IRAP in a biological sample. The biological sample is preferably a biological fluid such as blood, plasma, cerebrospinal fluid, lymph, amniotic fluid or urine, but may also be a tissue sample or a sample of tissue or cell culture medium. In a fifth aspect, the invention provides a method of diagnosing an AT 4 receptor/IRAP-related condition, comprising the step of measuring the level of activity of AT 4 receptor/IRAP in a subject suspected of having such a condition, and comparing the level to that found in a normal individual, in which any difference between the levels is indicative of the condition. The activity of AT 4 receptor/IRAP may be measured in a sample of a biological fluid such as blood, serum, plasma, lymph, amniotic fluid or cerebrospinal fluid, or in a tissue or cell sample. In a sixth aspect, the invention provides a therapeutic or prophylactic agent comprising (a) a peptide which has an amino acid sequence selected from the group consisting of LVVYPWTQRF (SEQ ID NO:2), KYLPGPLQ (SEQ ID NO:3) and VYIHPF (SEQ ID NO:1), or an analogue or peptidomimetic thereof which is able to modulate the activity of AT 4 receptor/IRAP, (b) an inhibitor of leucine aminopeptidase activity, such as L-leucinethiol, L-leucinal, L-leucinol, or derivatives thereof which have the ability to inhibit IRAP, or (c) an antisense AT 4 receptor/IRAP nucleic acid molecule or a fragment thereof which has the ability to inhibit expression of AT 4 receptor/IRAP, and optionally (d) a pharmaceutically acceptable carrier. Variant sequences of these peptides, for example encompassing short deletions or conservative substitutions of amino acids, are within the scope of the invention, provided that the ability to modulate the activity of AT 4 receptor/IRAP is retained. It is to be clearly understood that the compounds of the invention include peptide analogues, including but not limited to the following: 1. Compounds in which one or more amino acids is replaced by its corresponding D-amino acid. The skilled person will be aware that retro-inverso amino acid sequences can be synthesised by standard methods. See for example Chorev and Goodman, 1993; 2 . Peptidomimetic compounds, in which the peptide bond is replaced by a structure more resistant to metabolic degradation. See for example Olson et al, 1993; or 3. Compounds in which individual amino acids are replaced by analogous structures, for example gem-diaminoalkyl groups or alkylmalonyl groups, with or without modified termini or alkyl, acyl or amine substitutions to modify their charge. The use of such alternative structures can provide significantly longer half-life in the body, since they are more resistant to breakdown under physiological conditions. Methods for combinatorial synthesis of peptide analogues and for screening of peptides and peptide analogues are well known in the art (see for example Gallop et al, 1994). It is particularly contemplated that the compounds of the invention are useful as templates for design and synthesis of compounds of improved activity, stability and bioavailability. Preferably where amino acid substitution is used, the substitution is conservative, i.e. an amino acid is replaced by one of similar size and with similar charge properties. In a seventh aspect, the invention provides a method of modulating the activity of AT 4 receptor/IRAP, comprising the step of administering to a subject in need thereof an effective amount of an antisense nucleic acid molecule directed against nucleic acid encoding AT 4 receptor/IRAP, or a fragment thereof which has the ability to inhibit expression of AT 4 receptor/IRAP. Preferably, but not exclusively, the antisense AT 4 receptor/IRAP nucleic acid molecule has a nucleotide sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. The antisense oligonucleotide may optionally be operatively linked to a vector and/or may be present in a transfected host cell. In an eighth aspect, the invention provides an oligonucleotide molecule which is antisense to a nucleic acid encoding AT 4 receptor/IRAP, which is a) a nucleotide sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23; b) a fragment of a) which has the ability to inhibit expression of AT 4 receptor/IRAP; c) a nucleic acid molecule which has at least 75% sequence homology to a sequence in a) or b); d) a nucleic acid molecule which is capable of hybridizing to a sequence in a) or b) under stringent conditions; or e) a nucleic acid molecule of homologous sequence derived from the species listed below. In one embodiment, the invention provides a construct for delivering in vivo an effective amount of an antisense nucleic acid molecule directed against nucleic acid encoding AT 4 receptor/IRAP, comprising: a) an oligonucleotide as in a) above; and b) a vector comprising a control sequence, wherein the control sequence is capable of controlling the expression of a nucleotide sequence of a), which in turns modulates the activity of AT 4 receptor/IRAP. Modified and variant forms of the constructs disclosed herein may be produced in vitro, by means of chemical or enzymatic treatment, or in vivo by means of recombinant DNA technology. Such constructs may differ from those disclosed, for example by virtue of one or more nucleotide substitutions, deletions or insertions, but substantially retain a biological activity of the construct or nucleic acid molecule of this invention. In a ninth aspect, the invention provides a composition comprising one or more agents or antisense oligonucleotides according to the invention, together with a pharmaceutically acceptable carrier. The compositions of the invention may be formulated so as to be suitable for a variety of routes of administration, for example intravenous, subcutaneous, intramuscular or intrathecal or intraventricular injection, or for oral or topical administration. The exact formulation will depend on the individual route of administration. Methods and pharmaceutical carriers for preparation of pharmaceutical compositions are well known in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 17 th Edition, Mack Publishing Company, Easton, Pa., USA. Pharmaceutically acceptable carriers include conventional carriers which are suitable for use with peptide-based drugs, including diluents, excipients, and preservatives and the like. For example, carriers such as dextrose, mannitol, sucrose, or lactose, buffer systems such as acetate, citrate and phosphate, and bulking agents such as serum albumin, preferably human serum albumin, may be used. Preferably the antisense oligonucleotide is presented as a composition, which may be in the form of a capsule or a cartridge, or as a kit provided with a delivery device and instructions for its use. The kit may also include other therapeutic agents, and ingredients for the composition. The dose required will depend on the nature and severity of the condition to be treated, and the route of administration, and will be at the discretion of the attending physician or surgeon. A suitable route, frequency of administration, and dosage can readily be established using conventional clinical trial methodology. While it is particularly contemplated that the methods and compounds of the invention are suitable for use in the medical treatment of humans, they are also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as non-human primates, felids, canids, bovids, and ungulates. Methods and pharmaceutical carriers for preparation of pharmaceutical compositions are well known in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Easton, Pa., USA. The compounds and compositions of the invention may be administered by any suitable route, and the person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated. Dosage will be at the discretion of the attendant physician or veterinarian, and will depend on the nature and state of the condition to be treated, the age and general state of health of the subject to be treated, the route of administration, and any previous treatment which may have been administered. The carrier or diluent, and other excipients, will depend on the route of administration, and again the person skilled in the art will readily be able to determine the most suitable formulation for each particular case. The methods and compositions of the invention may be used in conjunction with, ie before, during and after one or more other treatments for the condition concerned. The agent may be administered prophylactically or therapeutically, and may be used either alone or in conjunction with other therapies for conditions without known therapies, or as a substitute for therapies that have significant negative side effects. The composition of the invention may be administered by any suitable route, including oral, intracavitary, intranasal, intraanal, intravaginal, transdermal, intradermal, buccal, intravenous, subcutaneous, intramuscular, by inhalation, intraglandular, intraarterial, intravascular in general, into the ear, intracranial, intrathecal, intraorgan, by implantation, and intraocular administration. For the purposes of this specification it will be clearly understood that the word “comprising” means “including but not limited to”, and that the word “comprises” has a corresponding meaning. |
Method of determining the position of a target using transmitters of opportunity |
A method of determining the position of a target comprising the steps of: providing a transmitter or a plurality of transmitters to transmit a signal to the target, providing a receiver or a plurality of receivers to receive signals reflected from said target. Determining the time of arrival information of said reflected signal at the or each receiver; using information pertaining to the position of the or each receiver and/or the or each transmitter and with the information obtained determining the target position. Preferably the signal includes a modulated or coded portion unique to the transmitter; and said signal portion is associated with the or each particular transmitter by virtue of said signal portion. The signal maybe a mobile phone transmission, DAB, digital TV, digital Radio or digital satellite transmissions. |
1. In a communication system wherein pre-stored codes are used in transmissions as part of the communication protocol, a method of determining the position of a target comprising the steps of: a) providing a transmitter or to transmit a signal to the target, b) providing a plurality of receivers, in communication with each other, to receive signals reflected from said target, the receivers being time or phase synchronised; c) determining the time of arrival information of said code of reflected signal at each receiver by continuously correlating said received signal with the prestored codes in the receiver; d) using information pertaining to the position of each receiver and with the information obtained from step c), determining the target position. 2. In a communication system wherein pre-stored codes are used in transmissions as part of the communication protocol, a method of determining the position of a target comprising the steps of: a) providing a plurality of transmitters to transmit each a signal to the target, said transmitters being time or phase synchronized; b) providing a receiver to receive signals reflected from said target and wherein said signals are set out pre-set times known to the receiver; c) correlating said received signal with the pre-stored codes in each receiver to identify the transmitter of a particular signal; d) the receiver determining the time delay in receiving said signal by subtracting the time of arrival of a pre-stored code of each reflected signal from each transmitter from the known time the pre-stored code was sent, e) using information known to the receiver pertaining to the position of each transmitter and with the timing information obtained from step c), determining the target position. 3. A method as claimed in claim 1 wherein transmitted signals includes a modulated or coded portion unique to the transmitter which is used to identify the transmitter and hence its location for step (e). 4. A method as claimed in claim 1, wherein all the receivers communicate said time of arrival information and receiver identification to central unit. 5. A method as claimed in claim 4 wherein said central unit is one of the receivers. 6. A communication system having a plurality of transmitters and a plurality of receivers, using combined information obtained via the methods of claim 1 to determine the location of a target. 7. A method as claimed in claim 1, wherein said signal is a mobile phone transmission, DAB, digital TV, digital Radio or digital satellite transmission. 8. A method as claimed in claim 1, wherein said signal includes the mid-amble or a portion thereof or any unique sequence of data that is known to the receiver before transmission. |
Hybrid drive system and method for adjusting a hybrid drive system |
A hybrid drive system is for a vehicle and includes an intermediate circuit and an energy accumulator. The energy accumulator is connected to the intermediate circuit by way of a diode, whereby a transistor enabling current to flow in an opposite direction is connected parallel thereto. The transistor is placed in a blocking position in order to discharge the energy accumulator and the diode enables current to pass. If the energy accumulator is not used, the diode is also blocked by increasing the voltage of the intermediate circuit with the aid of the voltage on the energy accumulator. In order to charge the energy accumulator, the transistor is conductingly switched. The diode does not allow the current to pass in this direction. |
1. A method for controlling a hybrid drive system for a vehicle with an intermediate circuit and an energy accumulator, the energy accumulator being connected to the intermediate circuit via a switch, the switch including a diode with a forward direction toward the intermediate circuit and a transistor, adapted to be turned on and off, arranged parallel to the diode and including a forward direction toward the energy accumulator, wherein the transistor is turned off and the diode is forward-biased for discharging the energy accumulator and the transistor is turned on and the diode is reverse-biased for charging the energy accumulator, the method comprising: turning off the transistor; and increasing the voltage across the intermediate circuit until the voltage is greater than the voltage across the energy accumulator, to thereby disconnect the energy accumulator when it is neither to be charged nor discharged. 2. A method for controlling a hybrid drive system for a vehicle with an intermediate circuit and an energy accumulator, the energy accumulator being connected to the intermediate circuit via a switch, the switch including a diode with a forward direction toward the intermediate circuit and a transistor, adapted to be turned on and off, arranged parallel to the diode and including a forward direction toward the energy accumulator, wherein the transistor is turned off and the diode is forward-biased for discharging the energy accumulator and the transistor is turned on and the diode is reverse-biased for charging the energy accumulator, that the method comprising: turning the transistor off and blocking the diode, to disconnect the energy accumulator when it is neither to be charged nor discharged; and bringing the voltage across the intermediate circuit to a level of the voltage across the energy accumulator and then turning the transistor on to charge the energy accumulator. 3. The method as claimed in claim 1, wherein the voltage across the intermediate circuit is increased during a driving mode up to the operating point of the hybrid drive system. 4. The method as claimed in claim 1, wherein, to charge the energy accumulator, the voltage across the intermediate circuit is brought to the level of the voltage across the energy accumulator and then the transistor is turned on. 5. The method as claimed in claim 2, wherein controlling of the voltage across the intermediate circuit, to bring it to the level of the voltage across the energy accumulator, occurs at the same time as a transition from driving mode to a braking mode. 6. The method as claimed in claim 1, wherein the energy accumulator is an ultracapacitor. 7. The method as claimed in claim 3, wherein, to charge the energy accumulator, the voltage across the intermediate circuit is brought to the level of the voltage across the energy accumulator and then the transistor is turned on. 8. The method as claimed in claim 4, wherein controlling of the voltage across the intermediate circuit, to bring it to the level of the voltage across the energy accumulator, occurs at the same time as a transition from a driving mode to a braking mode. 9. The method as claimed in claim 7, wherein controlling of the voltage across the intermediate circuit, to bring it to the level of the voltage across the energy accumulator, occurs at the same time as a transition from the driving mode to a braking mode. 10. The method as claimed in claim 2, wherein the energy accumulator is an ultracapacitor. 11. The method as claimed in claim 3, wherein the energy accumulator is an ultracapacitor. 12. The method as claimed in claim 4, wherein the energy accumulator is an ultracapacitor. 13. The method as claimed in claim 5, wherein the energy accumulator is an ultracapacitor. 14. The method as claimed in claim 7, wherein the energy accumulator is an ultracapacitor. 15. The method as claimed in claim 8, wherein the energy accumulator is an ultracapacitor. 16. The method as claimed in claim 9, wherein the energy accumulator is an ultracapacitor. |
<SOH> BACKGROUND OF THE INVENTION <EOH>A known hybrid drive system is used both for road vehicles, for example passenger cars and buses, and for rail vehicles. It is distinguished by the fact that an internal combustion engine with a generator, for example a diesel engine with a generator, and an electric motor are present. The internal combustion engine is connected to the electric motor via the generator and an intermediate circuit. The intermediate circuit is assigned a battery or a capacitor as the energy accumulator. In comparison with a battery, a capacitor has a higher power density, a longer service life and is also maintenance-free. To be able to store an adequate amount of energy, special capacitors, known as ultracapacitors, are used. In the case of the vehicle, a distinction is made between stationary mode, starting mode, driving mode and braking mode. In stationary mode and during starting, energy is taken from the energy accumulator. In the case of urban buses, starting advantageously takes place exclusively electrically, in order that the energy stored in the energy accumulator is efficiently used and no exhaust gases are emitted, for example at a bus stop. During driving mode, the energy accumulator is neither discharged further nor charged. Only during braking mode is the kinetic energy of the vehicle to be stored in the energy accumulator. Owing to a relatively low energy density, in the case of a capacitor the voltage drops relatively quickly when discharging occurs. If the capacitor is operated directly on the intermediate circuit of the drive system, the voltage of the intermediate circuit, which is then also low, has a disadvantageous effect on the performance of the drive system, i.e. the drive components must be operated with low voltage. It is known to counteract this disadvantage of low voltage by generating a high current flow, which is possible only by way of overdimensioned components. It is also already known to connect the capacitor to the intermediate circuit via a converter, for example via a step-up/step-down converter. However, this converter includes complex and expensive electronics and also causes an appreciable additional weight. Both the overdimensioning of the components and the converter lead to a greater weight and higher costs. |
<SOH> SUMMARY OF THE INVENTION <EOH>An embodiment of the invention is based on an object of providing a hybrid drive system and a method for controlling the hybrid drive system which require neither overdimensioning of components nor additional, expensive electronic components. An object of providing a suitable hybrid drive system may be achieved according to an embodiment of the invention by the energy accumulator being connected to the intermediate circuit via a switch including a diode which has a forward direction toward the intermediate circuit and a transistor which can be turned on and off, is arranged parallel to the diode and has a forward direction toward the energy accumulator. This may achieve an advantage that, when it is not required during driving mode, the energy accumulator can be disconnected by a simple device, for example by a diode and a transistor, from the remaining drive system. As such, the low voltage of the energy accumulator in the state of partial discharge cannot have any disadvantageous effect on the drive. This also achieves the advantage that, since parallel connections comprising a diode and transistor are present in any case in the power converter to which the intermediate circuit belongs, no special circuits have to be set up. The energy accumulator is, for example, an ultracapacitor, which has a low weight with a high storage capacity. An object of providing a suitable method for controlling the hybrid drive is achieved according to an embodiment of the invention by the transistor being turned off and the diode forward-biased for discharging the energy accumulator, by the transistor being turned off and the diode also blocked for disconnecting the energy accumulator, when it is neither to be charged nor discharged, and/or by the transistor being turned on and the diode reverse-biased for charging the energy accumulator. The discharging consequently only takes place via the diode. As soon as energy is no longer required from the energy accumulator, the diode is also blocked, so that the low voltage across the partially discharged capacitor or the voltage of the battery cannot disturb the drive system. If, then, in braking mode, the energy accumulator is to be charged, the transistor is turned on. Charging of the energy accumulator via the diode is not possible, since it is reverse-biased toward the energy accumulator. The method achieves the advantage that a current flow from the energy accumulator to the drive system for discharging and a current flow in the reverse direction for charging the energy accumulator is made possible by simple devices, or otherwise the energy accumulator is disconnected from the drive system. A low voltage across the energy accumulator therefore cannot have any disadvantageous effect on the drive system. It therefore does not have to be overdimensioned. Furthermore, no complex electronics are necessary. During discharging, the diode is forward-biased, since the voltage across the intermediate circuit is less than the voltage across the energy accumulator by the amount of the diode voltage. When the energy accumulator has been discharged to a minimum level and its voltage has dropped, the diode is blocked and the transistor turned off in a specifically set way. For blocking the diode, for example the voltage across the intermediate circuit is specifically increased until it is greater than the voltage across the energy accumulator and, in addition, the drive system is thereby supplied with adequate voltage. For this purpose, an already present voltage regulator is used in power converters operating on the intermediate circuit. For charging the energy accumulator, for example the voltage across the intermediate circuit is brought in a specifically controlled manner to the level of the voltage across the energy accumulator before the transistor is turned on. This can achieve an advantage that, a short-circuit current, or at least an excessive equalizing current, cannot occur when the transistor is turned on. When the voltage across the intermediate circuit is increased, it can be set for example to an optimum operating point in order to operate the electrical machines that are operated by way of the power converters connected to the intermediate circuit with an optimum magnetic flux. |
Hard disk cartridge |
A hard disk cartridge, comprising an outer box and a hard disk body supported in the outer box through a cushion member, wherein a hard frame is installed on at least right and left side faces of the hard dish body and the cushion member is installed on the outer surfaces of the frames so that the upper and lower ends thereof are extruded from the upper and lower surfaces of the hard disk body to the outside, whereby external impacts applied to the hard disk body in lateral and vertical directions can be absorbed by the cushion member and, since the impact force can be relieved sufficiently even if the cushion member is thin, the size of the hard disk cartridge can be reduced accordingly. |
1. A hard disk cartridge in which a hard disk body is supported inside an external box via a cushion member, characterized in that a hard frame is attached on at least left and right side surfaces of the hard disk body, and the cushion member is attached on an external surface of the frame so that upper and lower edge portions of the cushion member protrude outwardly from the upper and lower surfaces of the hard disk body. 2. The hard disk cartridge as defined in claim 1, wherein bending edge portions are provided on front and rear edge portions of the frame in the state of being bent at a right angle so as to stay along front and rear side surfaces of the hard disk body, the bending edge portions being placed on the front and rear side surfaces of the hard disk body, and the cushion member is also attached on external surfaces of the bending edge portions of the frame so that the upper and lower edge portions of the cushion member protrude outwardly from the upper and lower surfaces of the hard disk body. 3. The hard disk cartridge as defined in claim 1, wherein the cushion member is formed to have an U-shape cross section having a recess portion that engages with the frame. 4. The hard disk cartridge as defined in claim 1, wherein the frame and the cushion member are molded integrally. 5. The hard disk cartridge as defined in claim 1, wherein an internal connector is provided on a front surface of the hard disk body, the internal connector being connected to an external connector provided on a front surface of the external box through a cable, and a bending edge portion is provided on the front edge portion of the frame in the state of being bent to the front side surface of the hard disk body, the bending edge portion preventing the internal connector from being pulled out forward. 6. The hard disk cartridge as defined in claim 5, wherein the bending edge portion of the frame receives a front side surface of the internal connector. 7. The hard disk cartridge as defined in claim 5, wherein a protrusion is provided on left and right side surfaces of the internal connector, and the bending edge portion of the frame receives a front side surface of the protrusion of the internal connector. |
<SOH> BACKGROUND ART <EOH>Hard disks are used as high-capacity external memory devices for storing data and programs. In recent years, as disclosed in Japanese Patent Laid-Open Publication No. 5-314745, there is known a hard disk cartridge in which a slim hard disk body having a small vertical thickness is housed in an external box so as to make it portable. The hard disk body of this hard disk cartridge is supported inside an external box via cushion members so as to prevent damages due to an external impact. Inside the hard disk body, there are provided a disk as a storage medium disposed in parallel to the upper and lower surfaces of the hard disk body, and a head that moves along the disk surface to read and write data. On the front surface of the external box of the hard disk cartridge, a connector for connecting to a personal computer and the like is provided. To the connector is connected the front end of a flexible cable that is disposed inside the external box. A substrate connector provided on the rear end of the flexible cable is inserted into a substrate pin provided on the front surface of the hard disk body. In the hard disk cartridge in the aforementioned Japanese Patent Laid-Open Publication No. 5-314745, the cushion members absorb an external impact applied to the external box so as to prevent the external impact from being exerted to the hard disk body. However, since the cushion members are disposed in the state of being scattered in pieces, each cushion member should be thicken for sufficiently absorbing the impact force. This imposes a problem that miniaturization of the external box is not achievable in the conventional hard disk cartridge. The upper and lower surfaces of the hard disk body are prone to curve, so that they are the part that highly requires the protection from an external impact. However, if the cushion members are disposed on the upper and lower surfaces of the hard disk body in the conventional way, the upper and lower surfaces of the hard disk body are pressed by elastic repulsive force when an impact is applied to the external box in the upper and lower directions, causing the upper and lower surfaces of the hard disk body to be curved. As a result, the head fails to appropriately access to the disk, causing an operation failure of the hard disk body. Further, there is a possibility that the substrate connector is pulled out from the hard disk body by an external impact. To solve this problem, it is considered, for example, to fix the substrate connecter on the hard disk body by means of screws, which, however, leads to increase of the overall manufacturing cost due to an additional process of screw-fixing. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a horizontal cross sectional view showing a hard disk cartridge according to a first embodiment of the present invention; FIG. 2 is a vertical cross sectional view showing the hard disk cartridge of FIG. 1 ; FIG. 3 is a fragmentary enlarged cross sectional view showing the hard disk cartridge of FIG. 1 ; FIG. 4A is a horizontal cross sectional view showing a modified example of the hard disk cartridge of FIG. 1 , in which a frame and a cushion member are integrated; FIG. 4B is a vertical cross sectional view of FIG. 4A ; FIG. 5 is a plane view showing another modified example of the hard disk cartridge of FIG. 1 ; FIG. 6 is a horizontal cross sectional view showing a hard disk cartridge according to a second embodiment of the present invention; FIG. 7 is a vertical cross sectional view showing the hard disk cartridge of FIG. 6 ; FIG. 8 is a horizontal cross sectional view showing a modified example of the hard disk cartridge of FIG. 6 ; and FIG. 9 is a plane view showing another modified example of the hard disk cartridge of FIG. 6 . detailed-description description="Detailed Description" end="lead"? |
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