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Dual circular polarization flat plate antenna that uses multilayer structure with meander line polarizer |
An apparatus performs dual circular polarization in a flat plate antenna simultaneously, and does not require more than two meander line polarization layers. A first linear polarization layer and a second linear polarization layer including a polarization power divider (2, 4) and a radiation panel (3, 5) positioned on the polarization power divider (2, 4), are provided to respectively perform first and second senses of linear polarization. Additionally, a first meander line polarizer layer (6) is positioned on the second linear polarization layer and a second meander line polarizer layer (7) is positioned on the first meander line polarizer layer. The first and second meander line polarizer layers (6, 7) convert linear polarization signals into a circular polarization signals. |
1. An apparatus for performing dual circular polarization in a flat plate antenna, comprising: a linear polarization structure configured to perform a first sense and a second sense of a linear polarization, and generate linear polarization outputs; a meander line polarizer positioned on said linear polarization structure and having a first layer stacked on a second layer, wherein said meander line polarizer generates circular polarization signals based on said linear polarization outputs. 2. The apparatus of claim 1, said linear polarization structure comprising: a first linear polarization layer configured to perform said first sense of said linear polarization; and a second linear polarization layer, positioned on said first linear polarization layer, and configured to perform said second sense of said linear polarization. 3. The apparatus of claim 2, further comprising at least one foam layer positioned between each of said first linear polarization layer, said second linear polarization layer and said first layer and said second layer of said meander line polarizer. 4. The apparatus of claim 2, wherein each of said first linear polarization layer and said second linear polarization layer comprises: a polarization power divider; and a radiation panel positioned on said polarization power divider. 5. The apparatus of claim 4, further comprising said at least one foam layer positioned between said polarization power divider and said radiation panel of each of said first linear polarization layer and said second linear polarization layer. 6. The apparatus of claim 1, further comprising a ground plane positioned on a surface of said linear polarization structure and opposite said meander line polarizer. 7. The apparatus of claim 1, wherein said first sense comprises a left hand circular polarization component, and said second sense comprises a right hand circular polarization component. 8. The apparatus of claim 1, said first layer and said second layer of said meander line polarizer each comprising at least one meander line constructive strip array positioned on a thin dielectric at a 45 degree angle to a direction of said linear polarization. 9. The apparatus of claim 1, wherein an axial ratio of said apparatus at a bandwidth greater than 500 MHz is 1 dB. 10. The apparatus of claim 1, wherein an axial ratio of said apparatus at a bandwidth greater than 2 GHz is 2 dB. 11. A flat plate antenna configured to perform dual circular polarization, comprising: an apparatus for performing dual circular polarization, including, a first linear polarization layer configured to perform a first sense of a linear polarization; a second linear polarization layer, positioned on said first linear polarization layer, configured to perform a second sense of said linear polarization; a first meander line polarizer layer positioned on said second linear polarization layer, and a second meander line polarizer layer positioned on said first meander line polarizer layer, wherein said first meander line polarizer layer and said second meander line polarizer layer convert linear polarization signals output from said first linear polarization layer and said second linear polarization layer into circular polarization signals. 12. A method of performing dual circular polarization, comprising the steps of: (a) performing a first sense of linear polarization to generate a first linearized output; (b) performing a second sense of linear polarization to generate a second linearized output; and (c) receiving said first linearized output and said second linearized output in a two-layer meander line polarizer to generate circular polarization signals. 13. The method of claim 12, said (a) comprising: performing said first sense of said linear polarization in a first linear polarization layer; and performing said second sense of said linear polarization in a second linear polarization layer that is positioned on said first linear polarization layer. 14. The method of claim 13, wherein at least one foam layer is positioned between each of said first linear polarization layer, said second linear polarization layer and said first layer and said second layer of said meander line polarizer. 15. The method of claim 12, wherein a ground plane is positioned on a surface of said linear polarizer and opposite said meander line polarizer. 16. The method of claim 12, wherein said (a) comprises generating a left hand circular polarization component, and said (b) comprises generating a right hand circular polarization component. 17. The method of claim 12, wherein said first layer and said second layer of said meander line polarizer each comprise at least one meander line constructive strip array positioned on a thin dielectric at a 45 degree angle to a direction of said linear polarization. 18. The method of claim 12, wherein an axial ratio of said apparatus at a bandwidth greater than 500 MHz is 1 dB. 19. The method of claim 12, wherein an axial ratio of said apparatus at a bandwidth greater than 2 GHz is 2 dB. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention disclosure relates to a low-cost flat plate antenna for direct broadcasting systems (DBS) and other low cost applications, and more specifically, a two-layer meander-line polarizer is used to simultaneously produce two senses (i.e., components) of orthogonal circular polarizations. 2. Background of the Invention In the related art, two orthogonal senses of linear polarization in a multilayer printed circuit structure can be produced, as well as a single circular polarization using a multilayer printed circuit structure. The related art single circular polarization implementation includes a special radiating element with perturbation segments and a single point feeding or a linear polarization antenna with at least 3 to 4 layers of a meander line polarizer. However, the related art does not disclose or suggest use of dual linear polarization antenna with a meander line polarizer. More specifically, the use of two meander line layers to convert the linear polarization into a circular polarization for a single or dual senses of circular polarization has not been demonstrated as achievable in the related art. Thus, the aforementioned related art structure has at least the disadvantage of requiring extra layers in the printed circuit antenna, which results in an increased cost, if production of an output having two orthogonal senses is desired. |
<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to overcome at least the aforementioned problems and disadvantages of the related art system. It is another object of the present invention to minimize a number of layers present in a multilayer structure of a flat plate antenna, thus minimizing cost and size of the flat plate antenna. To achieve at least the above objects, an apparatus for performing dual circular polarization in a flat plate antenna is provided, comprising a linear polarizer configured to perform a first sense and a second sense of a linear polarization, and generate linear polarization outputs, and a meander line polarizer positioned on the linear polarization structure and having a first layer stacked on a second layer. In this apparatus, the meander line polarizer generates circular polarization signals based on the linear polarization outputs. Additionally, a method of performing dual circular polarization is provided, comprising the steps of (a) performing a first sense of linear polarization to generate a first linearized output, and (b) performing a second sense of linear polarization to generate a second linearized output The method further comprises the step of (c) receiving the first linearized output and the second linearized output in a two-layer meander line polarizer to generate circular polarization signals. Further, a flat plate antenna configured to perform dual circular polarization is provided, comprising an apparatus for performing dual circular polarization. The apparatus includes a first linear polarization layer configured to perform a first sense of a linear polarization, a second linear polarization layer, positioned on the first linear polarization layer, configured to perform a second sense of the linear polarization, a first meander line polarizer layer positioned on the second linear polarization layer, and a second meander line polarizer layer positioned on the first meander line polarizer layer. The first meander line polarizer layer and the second meander line polarizer layer convert linear polarization signal outputs from the first linear polarization layer and the second linear polarization layer into circular polarization signals. |
Two-layer wide-band meander-line polarizer |
A two-layer polarizer (6, 7) comprising a first substrate (10) having formed on a major surface thereof a first conductive meander line array (2) and a second substrate (12) having formed on a major surface thereof a second conductive meander line array (3). The first and second substrates are disposed as adjacent layers and are separated by a dielectric space (8). At least a portion of the first meander line array and a portion of said second meander line array are overlapping. |
1. A two-layer polarizer comprising: a first substrate having formed on a major surface thereof a first conductive meander line array; a second substrate having formed on a major surface thereof a second conductive meander line array, said first and second substrates being disposed as adjacent layers and separated by a dielectric space, at least a portion of said first meander line array and a portion of said second meander line array overlapping. 2. A two-layer polarizer as claimed in claim 1, wherein said dielectric space is less than ¼ λ, where λ is the wavelength of an incoming wave. 3. A two-layer polarizer as claimed in claim 2, wherein the dielectric space is approximately 0.15 λ. 4. A two-layer polarizer as claimed in claim 1, wherein said dielectric space comprises an insulating material. 5. A two-layer polarizer as claimed in claim 1, wherein said first meander line array and said second meander line array comprises a plurality of conductive strips, each having a square wave shape. 6. A two-layer polarizer as claimed in claim 5, wherein said square wave shape comprises a period A, a height B, a horizontal width W1 and a vertical width W2. 7. A two-layer polarizer as claimed in claim 6, wherein the operating frequency and the bandwidth of the polarizer are determined by the values of B, W1 and W2. 8. A two-layer polarizer as claimed in claim 1, wherein said meander line is extended at approximately 45° with respect to a polarization direction of a linearly polarized wave. 9. A two-layer polarizer as claimed in claim 9, wherein said wherein said substrate comprises Mylar. 10. A two-layer polarizer as claimed in claim 9 wherein said dielectric space is filled with low loss polyfoam. 11. A two-layer polarizer as claimed in claim 1, wherein said polarizer exhibits an axial ratio of 2 db at 2 Ghz. 12. A two-layer polarizer as claimed in claim 1, wherein said polarizer exhibits an axial ratio of approximately 1 dB at a bandwidth of approximately 500 Mhz. 13. A two-layer polarizer as claimed in claim 7, wherein said parameters are scaled to different frequencies 14. A two-layer polarizer as claimed in claim 1, wherein said axial ratio of 2 dB is for a signal at approximately 11-13 Ghz. 15. In combination, an antenna having at least one aperture, and a two-layer polarizer disposed over said at least one aperture and comprising: a first substrate having formed on a major surface thereof a first conductive meander line array; a second substrate having formed on a major surface thereof a second conductive meander line array, said first and second substrates being disposed as adjacent layers and separated by a dielectric space, at least a portion of said first meander line array and a portion of said second meander line array overlapping. 16. A combination as claimed in claim 15 in which said two-layer polarizer dielectric space is less than ¼ λ, where λ is the wavelength of an incoming wave. 17. A combination as claimed in claim 16, wherein the dielectric space is approximately 0.15 λ. 18. In combination, an antenna having at least one aperture, and a two-layer polarizer means disposed over said at least one aperture for transferring a linear polarization of propagation waves into a circular polarization. 19. The combination as set forth in claim 18, wherein said two layer polarizer means is operative to introduce phase shifts and signal decomposition, which leads to two orthogonal linear polarizations at phase quadrature to produce circular polarization. 20. The combination as set forth in claim 18, wherein said combination provides an axial ratio less than or equal to 2 dB. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention disclosure relates to a meander-line polarizer, particularly a polarizer having only two printed layers and operative to provide wide-band performance with a low axial ratio. The polarizer is especially useable in aperture-type antennas, particularly antennas operative to convert electromagnetic field polarization from linear to circular and from circular to linear. 2. Background of the Invention In the related art, two orthogonal senses of linear polarization in a multilayer printed circuit structure can be produced, as well as a single circular polarization using a multilayer printed circuit structure having a meander line conductor. Previous designs of meander line polarizers used 3 to 4 layers of printed circuits to achieve the required axial ratio for the circular polarization across the band. The printed layers are separated by supporting dielectric substrate layers that are quarter-wavelength-thick. Such meander-line polarizers are expensive to manufacture and are undesirably thick. However, the related art does not disclose or suggest use of a two-layer meander line polarizer, particularly one with low axial ratio over a wide bandwidth. More specifically, the use of only two meander line layers to convert a linear polarization into a circular polarization for a single or dual senses of circular polarization has not been demonstrated as achievable in the related art. Thus, the aforementioned related art structure has at least the disadvantage of requiring extra layers in the printed circuit polarizer, which results in an increased cost, if production of an output having a single sense or two orthogonal senses is desired. Even more specifically, the prior art does not disclose an antenna in combination with a two layer meander line polarizer. |
<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to overcome at least the aforementioned problems and disadvantages of the related art system. It is another object of the present invention to minimize a number of layers present in a multilayer structure of a polarizer, thus minimizing cost and size of the polarizer. Accordingly, a first feature of the invention involves a two-layer polarizer comprising a first substrate having formed on a major surface thereof a first conductive meander line array and a second substrate having formed on a major surface thereof a second conductive meander line array. The first and second substrates are stacked with their major surfaces in parallel as adjacent layers that are separated by a dielectric space. The separation of the two layers is less than quarter wavelength. A second feature of the invention involves a combination of an antenna having at least one aperture, and a two layer polarizer disposed over the at least one aperture. The two-layer polarizer includes a first substrate having formed on a major surface thereof a first conductive meander line array; and a second substrate having formed on a major surface thereof a second conductive meander line array. The first and second substrates are disposed with their major surfaces in parallel as adjacent layers that are separated by a dielectric space. The separation of the two layers is less than quarter wavelength. |
Ltcc-based modular mems phased array |
The present invention uses low temperature co-fired ceramic (LTCC) technology for fabrication of micro-electro-mechnical systems (MEMS) based phase shifters. As a result, low-cost subarray modules can be constructed using LTCC, and the subarray modules (10) can be used as building blocks for large antenna arrays. In an exemplary description of the present invention, all layers of the multilayer subarray modules, including MEMS, are in LTCC. As a result, a significant cost reduction for satellite terminals using highly integrated LTCC subarray modules. |
1. A method of fabricating at least one subarray of at least one phased array, comprising the steps of: (a) forming a first layered substrate including at least one first device; (b) forming a micromachined device layer on said first layered substrate; and (c) forming a second layered substrate including at least one second device on said micromachined device layer, wherein a low temperature co-fired ceramic (LTCC) process is employed in said (a), said (b) and said (c). 2. The method of claim 1, wherein said (a) comprises forming a polarizer on a power divider. 3. The method of claim 1, wherein a micromachined device in said micromachined device layer comprises a phase shifter. 4. The method of claim 1, wherein said (c) comprises forming a radiating element on said micromachined device layer. 5. The method of claim 1, wherein said (a), said (b) and said (c) are performed substantially simultaneously. 6. The method of claim 1, further comprising forming an interconnection between at least one of said first layered substrate, said micromachined device layer and said second layered substrate by a LTCC via formation process. 7. The method of claim 1, further comprising combining ones of said at least one subarray to form said at least one phased array in an antenna array system. 8. The method of claim 7, further comprising installing said antenna array system in a satellite terminal. 9. The method of claim 1, wherein at least one amplifier is coupled between corresponding ones of said at least one subarray and a distribution network. 10. The method of claim 9, wherein said at least one amplifier forms a power dividing circuit if said at least one phased array is a transmit array, and said at least one amplifier forms a power combining circuit if said at least one phased array is a receive array. 11. The method of claim 1, wherein a transmission line loss for said at least one phased array is 0.2 dB/cm at 30 GHz. 12. The method of claim 1, further comprising applying a flip chip bonding process to attach said first layered substrate to said micromachined device layer. 13. The method of claim 1, wherein said at least one subarray comprises a number of subarrays or elements being simultaneously fabricated. 14. A phased array device including at least one subarray said at least one subarray comprising: (a) a first layered substrate including at least one first device; (b) a micromachined device layer on said first layered substrate; and (c) a second layered substrate including at least one second device on said micromachined device layer wherein said first layered substrate, said micromachined device layer and said second layered substrate are formed by a low temperature co-fired ceramic (LTCC) process. 15. The device of claim 14, wherein said (a) comprises a polarizer formed on a power divider. 16. The device of claim 14, wherein a micromachined device in said micromachined device layer comprises a phase shifter. 17. The device of claim 14, wherein said second layered substrate is a radiating element. 18. The device of claim 14, wherein said first layered substrate, said micromachined device layer and said second layered substrate are formed substantially simultaneously. 19. The device of claim 14, further comprising interconnections between at least one of said first layered substrate, said micromachined device layer and said second layered substrate formed by a LTCC via formation process. 20. The device of claim 14, wherein ones of said at least one subarray are configured to be combined to form said at least one phased array in an antenna array system. 21. The device of claim 20, wherein said antenna array system is positioned in a satellite terminal. 22. The device of claim 14, wherein at least one amplifier is coupled between corresponding ones of said at least one subarray and a distribution network. 23. The device of claim 22, wherein said at least one amplifier comprises a power dividing circuit if said at least one phased array is a transmit array, and said at least one amplifier comprises a power combining circuit if said at least one phased array is a receive array. 24. The device of claim 14, wherein a transmission line loss for said at least one phased array is 0.2 dB/cm at 30 GHz. 25. The device of claim 14, wherein said at least one subarray comprises a number of subarrays or elements that are simultaneously fabricated. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates to a method of efficiently fabricating a low-cost phased array. More specifically, the present invention relates to a multilayer phased array and method of fabrication thereof, using low temperature co-fired ceramic (LTCC)— based layers, some of which are micro-electromechanical system (MEMS) layers. 2. Background of the Invention In the related art, micro-electromechanical system (MEMS) technology integrates the silicon substrate of printed-circuit and computer technologies with tiny mechanical devices. For example, but not by way of limitation, MEMS technology may include sensors, valves, gears, mirrors, switches or actuators embedded in semiconductor devices. While the related art electronics of MEMS devices are fabricated via an Integrated Circuit (IC) process, the micromechanical MEMS components are fabricated by a micromachining process that selectively etches or adds layers to form the MEMS device. Once produced, the micromechanical components form sensory and mechanical parts of a MEMS device, while the electronics part forms the “brain” of a MEMS device. Accordingly, MEMS combines the computational ability of microelectronics with control capabilities of microsensors and microactuators. In related art communications systems, the combination of silicon and micromachining allows for very high bandwidth mechanical devices. Related art MEMS devices are manufactured using techniques that are substantially similar to the related art IC fabrication processes. For example, but not by way of limitation, a batch fabrication process may be used to make a MEMS device. The modular concept of multilayer active phased arrays is the subject of co-pending International Application No. PCT/US02/03379, filed Feb. 14, 2002, which discloses the contents of the integrated module that contains radiating elements, polarizing circuits, power dividing networks and filters. The radiating elements are electromagnetically coupled patches, and the polarizing circuits are hybrid couplers that provide the phase quadrature needed to produce circular polarizations. The phase shifters and other components can be implemented using MEMS technology. Although the invention disclosed in the above-mentioned International Application No. PCT/US02/03379 reduces the cost of making the active phased arrays due to the modularity of the design and the use of lower loss MEMS components, the production costs are still prohibitive for some applications. The aforementioned related art has various problems and disadvantages. For example, but not by way of limitation, it is a disadvantage of the related art MEMS fabrication process that IC packaging processes must be used for manufacture of MEMS devices. As noted above, there are substantial structural and functional differences between IC and MEMS, including the need for MEMS to be manufactured in substantially continuous and intimate contact with their environment. Thus, the related art MEMS fabrication process is not efficient Further, in the related art, a new, specialized package must be created each time a new MEMS device is developed, at an additional time and financial cost. As a result, the related art MEMS fabrication process has another disadvantage and is one of the most expensive aspects of MEMS product development, as there are no related art standardized packages from which one would be able to choose a MEMS package for a new application without substantially compromising performance. Also, the related art fabrication process has another disadvantage, due to its complexity, and requires a substantial research investment to generate a suitable fabrication sequence. Because the design is dependent on the fabrication, there is an additional cost in that process-independent design tools cannot be used by a MEMS designer. |
<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to overcome at least the aforementioned problems and disadvantages of the related art system. It is another object of the present invention to provide a low-cost, efficient method of fabricating a phased array with MEMS technology. It is still another object of the present invention to simplify the phased array fabrication process by using LTCC technology to manufacture substantially all layers of the phased array. The use of same or similar material for all the layers in the subarray structure reduces the thermal and mechanical stresses that may result from using dissimilar materials of different coefficients of thermal expansion. To achieve at least the foregoing objects, a method of fabricating at least one subarray of at least one phased array is provided, comprising the steps of (a) forming a first layered substrate including at least one first device, (b) forming a micromachined device layer on the first layered substrate, and (c) forming a second layered substrate including at least one second device on the micromachined device layer, wherein a low temperature co-fired ceramic (LTCC) process is employed in (a), (b) and (c). Additionally, a phased array device including at least one subarray the at least one subarray is provided, comprising. (a) a first layered substrate including at least one first device, (b) a micromachined device layer on the first layered substrate, and (c) a second layered substrate including at least one second device on the micromachined device layer, wherein said first layered substrate, said micromachined device layer and said second layered substrate are formed by a low temperature co-fired ceramic (LTCC) process. |
Antigens for raising an immune response against rickettsieae and ehrlichieae pathogens |
The present invention relates to the identification of polypeptides which are useful for raising an immune response against Ehrlichieae and Rickettsieae pathogens when administered into a subject. These polypeptides, and polynucleotides encoding therefor, can be used in various strategies for preventing or treating Ehrlichieae and Rickettsieae infections. |
1. A vaccine comprising at least one polypeptide selected from the group consisting of: a) a sequence provided in SEQ ID NO:1; b) a polypeptide which is at least 80% identical to (a); c) a sequence provided in SEQ ID NO:2; d) a polypeptide which is at least 80% identical to (c); e) a sequence provided in SEQ ID NO:3; f) a polypeptide which is at least 80% identical to (e); g) a sequence provided in SEQ ID NO:4; and h) a polypeptide which is at least 80% identical to (g); wherein the polypeptide raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when administered to a subject. 2.-4. (canceled) 5. The vaccine according to claim 1, wherein the vaccine comprises a pharmaceutically acceptable carrier. 6. The vaccine according to claim 1, wherein the vaccine comprises an adjuvant. 7. A DNA vaccine comprising at least one polynucleotide selected from the group consisting of: a) a sequence encoding a polypeptide provided in SEQ ID NO:1; b) a sequence encoding a polypeptide which is at least 80% identical to SEQ ID NO:1; c) a sequence encoding a polypeptide provided in SEQ ID NO:2; d) a sequence encoding a polypeptide which is at least 80% identical to SEQ ID NO:2; e) a sequence encoding a polypeptide provided in SEQ ID NO:3; f) a sequence encoding a polypeptide which is at least 80% identical to SEQ ID NO:3; g) a sequence encoding a polypeptide provided in SEQ ID NO:4; and h) a sequence encoding a polypeptide which is at least 80% identical to SEQ ID NO:4; wherein the polypeptide encoded by the polynucleotide raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when the DNA vaccine is administered to a subject. 8.-10. (canceled) 11. The DNA vaccine according to claim 7, wherein the vaccine comprises a pharmaceutically acceptable carrier. 12. The DNA vaccine according to claim 7, wherein the polynucleotide is contained in a vector. 13. (canceled) 14. A method for raising an immune response against an Ehrlichieae or Rickettsieae pathogen in a subject, the method comprising administering to the subject at least one vaccine according to claim 1. 15. A method of treating or preventing an Ehrlichieae or Rickettsieae infection in a subject, the method comprising administering to the subject at least one vaccine according to claim 1. 16.-17. (canceled) 18. The method according to claim 14, wherein the subject is a mammal selected from the group consisting of; cows, sheep, goats, dogs and horses. 19.-20. (canceled) 21. A transgenic plant which produces at least one polypeptide selected from the group consisting of: a) a sequence provided in SEQ ID NO:1; b) a polypeptide which is at least 80% identical to (a); c) a sequence provided in SEQ ID NO:2; d) a polypeptide which is at least 80% identical to (c); e) a sequence provided in SEQ ID NO:3; f) a polypeptide which is at least 80% identical to (e); g) a sequence provided in SEQ ID NO:4; and h) a polypeptide which is at least 80% identical to (g); wherein the polypeptide raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when the transgenic plant is orally administered to a subject. 22. A method for raising an immune response against an Ehrlichieae or Rickettsieae pathogen in a subject, the method comprising orally administering to the subject at least one transgenic plant according to claim 21. 23. A method of treating or preventing an Ehrlichieae or Rickettsieae infection in a subject, the method comprising orally administering to the subject at least one transgenic plant according to claim 21. 24. An antibody raised against a polypeptide selected from the group consisting of: a) a sequence provided in SEQ ID NO:1; b) a polypeptide which is at least 80% identical to (a); c) a sequence provided in SEQ ID NO:2; d) a polypeptide which is at least 80% identical to (c); e) a sequence provided in SEQ ID NO:3; f) a polypeptide which is at least 80% identical to (e); g) a sequence provided in SEQ ID NO:4; and h) a polypeptide which is at least 80% identical to (g); wherein the antibody provides immune response against Ehrlichieae and/or Rickettsieae pathogens when administered to a subject. 25. A method of treating or preventing an Ehrlichieae or Rickettsieae infection in a subject, the method comprising administering to the subject at least one antibody according to claim 24. 26. A substantially purified polypeptide which specifically binds to an antibody according to claim 24. 27. A substantially purified polypeptide, the polypeptide being selected from: (i) a polypeptide comprising the sequence provided as SEQ ID NO:1; and (ii) a polypeptide which is at least 80% identical to (i); wherein the polypeptide raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when administered to a subject. 28. (canceled) 29. A substantially purified polypeptide, the polypeptide being selected from: (i) a polypeptide comprising the sequence provided as SEQ ID NO:2; and (ii) a polypeptide which is at least 80% identical to (i); wherein the polypeptide raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when administered to a subject. 30. A substantially purified polypeptide, the polypeptide being selected from: (i) a polypeptide comprising the sequence provided as SEQ ID NO:3; and (ii) a polypeptide which is at least 80% identical to (i); wherein the polypeptide raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when administered to a subject. 31. A substantially purified polypeptide, the polypeptide being selected from: (i) a polypeptide comprising the sequence provided as SEQ ID NO:4; and (ii) an antigenic fragment of (i), wherein the polypeptide, or antigenic fragment thereof, raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when administered to a subject. 32.-34. (canceled) 35. A fusion protein comprising a polypeptide according to claim 27 fused to at least one heterologous polypeptide sequence. 36. An isolated polynucleotide, the polynucleotide having a sequence selected from: (i) a sequence of nucleotides shown in SEQ ID NO:5; (ii) a sequence of nucleotides shown in SEQ ID NO:6; (iii) a sequence of nucleotides shown in SEQ ID NO:7; (iv) a sequence of nucleotides shown in SEQ ID NO:57; (v) a sequence of nucleotides shown in SEQ ID NO:8; (vi) a sequence of nucleotides shown in SEQ ID NO:56; (vii) a sequence encoding a polypeptide according to claim 16; (viii) a sequence capable of selectively hybridizing to any one of (i) to (iv) under high stringency; and (ix) a sequence of nucleotides which is at least 80% identical to any one of (i) to (iv), wherein the polynucleotide encodes a polypeptide that raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when administered to a subject. 37. A vector comprising at least one polynucleotide according to claim 36. 38. (canceled) 39. A host cell comprising the vector of claim 37. 40. (canceled) 41. A process for preparing a polypeptide according to claim 27, the process comprising cultivating a host cell according to claim 39 under conditions which allow expression of the polynucleotide encoding the polypeptide, and recovering the expressed polypeptide. 42. (canceled) |
<SOH> BACKGROUND OF THE INVENTION <EOH>Tick-borne diseases are a major problem of domestic livestock production in large areas of the world, though they are at their most acute in tropical and subtropical regions. The causative agents of tick fevers include Babesia bovis, Anaplasma marginale and Babesia bigemina, the first two being the most important and pathogenic. Anaplasma marginale , the major pathogen causing anaplasmosis, is an intra-erythrocytic rickettsia , which causes extra-vascular erythrocyte destruction. Pathology is manifested as an anaemia that, especially in non-immune adult cattle, causes serious morbidity and often mortality. Cattle that survive initial infection, although carriers of the pathogen, are resistant to clinical disease. Live attenuated vaccines for these diseases have been produced. A degree of immunity against Anaplasma marginale is conferred by vaccination of cattle with bovine blood infected with the less virulent Anaplasma centrale . However, like many such attenuated vaccines they have disadvantages. A significant level of pathology is induced on vaccination and in some animals, particularly older cattle, this can be severe. Secondly, despite careful quality control, there is always a significant risk that other disease organisms can be inadvertently transmitted with the vaccine. Thirdly, particularly in the case of Babesia bovis , there is a possibility of reversion to virulence following transmission by ticks of the partially attenuated vaccine organisms. This leads, at the very least, to a reluctance to use the vaccine in areas where disease incidence is not high while in some areas the vaccine cannot be used at all. Low coverage is also due, in part, to the use of Bos indicus cattle which are much more resistant to ticks and babesiosis than the more productive Bos taurus breeds. However, a recent study (Bock et al. 1997) has shown that Bos taurus and Bos indicus breeds are equally susceptible to anaplasmosis, thus supporting the use of a stand-alone non-living vaccine against A. marginale . Further, in some areas, the move towards ‘finishing’ cattle in feedlots is also significant for the future use of vaccines against A. marginale as under intensive conditions mechanical transmission is far more likely to occur. In the U.S. where the use of live A. centrale is precluded, A. marginale has been purified from infected blood on a commercial scale and used as a non-living vaccine (e.g. ‘Anaplaz’, Fort Dodge Laboratories, ‘Am-Vax’, Scheering Plough, ‘Plaz-Vax’, Mallinckrodt). Safety problems with these vaccines have occurred on several occasions in the past due to contamination of the product with host red cell antigens which results in mortality due to isoerythrolysis in suckling calves of vaccinated mothers. This same complex of diseases is of even greater importance over large areas of Central and South America and in parts of East Asia, for example the southern half of China. In most of these areas attenuated live vaccines are not available or their use is very restricted due, in part, to their relatively high cost. The great disincentive, however, is the difficulty of maintaining adequate quality control of the vaccine and ensuring its delivery in effective form in all areas. Despite the fact that protective vaccination with killed Anaplasma material has been repeatedly demonstrated (Montenegro-James et al., 1991) little is known of the protective antigens themselves. A major difficulty in identifying novel antigens is the presence of large amounts of immunodominant but poorly protective, known antigens which are, in most cases, highly variable. Work has focussed on a complex mixture of surface proteins labelled the Major Surface Protein complex (MSP) (e.g. Vidotto et al., 1994) and in particular on a neutralization-sensitive surface protein Am105 (Palmer et al., 1986). These proteins have been available for some time, but progress in generating a recombinant vaccine has been slow. The available evidence suggests the antigens are of inadequate efficacy. Anaplasma species which infect ruminants other than cattle include Anaplasma ovis which infects sheep. Closely related pathogens include; Cowdria ruminantium (also known as heartwater) which is a major problem in South Africa and the Carribean, numerous Ehrlichieae and Rickettsieae pathogens of domestic animals (including horses), as well as pathogens infecting humans such as Ehrlichia phagocytophila, Ehrlichia chaffeensis, Rickettsia prowazekii (which causes epidemic typhus), Rickettsia rickettsii and Rickettsia conorii (both of which cause spotted fever), and Ehrlichia sp. which cause human granulocytic ehrlichiosis. The present inventors have now identified and characterized polypeptides which can be used in a vaccine to provide immune protection against Ehrlichieae and Rickettsieae pathogens. |
<SOH> SUMMARY OF THE INVENTION <EOH>In one aspect, the present invention provides a vaccine comprising at least one polypeptide selected from the group consisting of: a) a sequence provided in SEQ ID NO:1; b) a polypeptide which is at least 50% identical to (a); c) a sequence provided in SEQ ID NO:2; d) a polypeptide which is at least 50% identical to (c); e) a sequence provided in SEQ ID NO:3; f) a polypeptide which is at least 50% identical to (e); g) a sequence provided in SEQ ID NO:4; and h) a polypeptide which is at least 50% identical to (g); wherein the polypeptide raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when administered to a subject. Preferably, the polypeptide is at least 60% identical, more preferably at least 70% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, and even more preferably at least 99% identical to (a), (c), (e) or (g). Preferably, the polypeptide has an N-terminal sequence as provided in SEQ ID NO:12 and has a molecular weight of approximately 17 kDa. Further, it is preferred that the polypeptide can be purified from a species of Ehrlichieae or Rickettsieae. Preferably, the polypeptide can be purified from a species selected from the group consisting of: Anaplasma sp., Ehrlichia sp., Rickettsia sp. and Cowdria sp. More preferably, the polypeptide can be purified from the group consisting of: Anaplasma marginale, Anaplasma centrale, Anaplasma ovis, Cowdria ruminantium, Ehrlichia equi, Ehrlichia phagocytophila, Ehrlichia chaffeensis, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia conorii , and Ehrlichia sp. which cause human granulocytic ehrlichiosis. Even more preferably, the polypeptide can be purified from Anaplasma marginale. Preferably, the vaccine comprises a pharmaceutically acceptable carrier. It is also preferred that the vaccine comprises an adjuvant. As is known in the art, an immune response can be provided through the use of DNA vaccines. Accordingly, in another aspect the present invention provides a DNA vaccine comprising at least one polynucleotide selected from the group consisting of: a) a sequence encoding a polypeptide provided in SEQ ID NO:1; b) a sequence encoding a polypeptide which is at least 50% identical to SEQ ID NO:1; c) a sequence encoding a polypeptide provided in SEQ ID NO:2; d) a sequence encoding a polypeptide which is at least 50% identical to SEQ ID NO:2; e) a sequence encoding a polypeptide provided in SEQ ID NO:3; f) a sequence encoding a polypeptide which is at least 50% identical to SEQ ID NO:3; g) a sequence encoding a polypeptide provided in SEQ ID NO:4; and h) a sequence encoding a polypeptide which is at least 50% identical to SEQ ID NO:4; wherein the polypeptide encoded by the polynucleotide raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when the DNA vaccine is administered to a subject. Preferably, the polynucleotide encodes a polypeptide which is at least 60% identical, more preferably at least 70% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, and even more preferably at least 99% identical to any one of SEQ ID NO's 1 to 4. Preferably, the polynucleotide encodes a polypeptide which has an N-terminal sequence as provided in SEQ ID NO:12 and has a molecular weight of approximately 17 kDa. Further, it is preferred that the polynucleotide can be isolated from a species of Ehrlichieae or Rickettsieae. Preferably, the polynucleotide can be isolated from a species selected from the group consisting of: Anaplasma sp., Ehrlichia sp., Rickettsia sp. and Cowdria sp. More preferably, the polynucleotide can be isolated from the group consisting of Anaplasma marginale, Anaplasma centrale, Anaplasma ovis, Cowdria ruminantium, Ehrlichia equi, Ehrlichia phagocytophila, Ehrlichia chaffeensis, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia conorii , and Ehrlichia sp. which cause human granulocytic ehrlichiosis. Even more preferably, the polynucleotide can be isolated from Anaplasma marginale. In another preferred embodiment, the polynucleotide is contained in a vector. More preferably, the vector is a viral vector. In a further aspect, the present invention provides a method for raising an immune response against an Ehrlichieae or Rickettsieae pathogen in a subject, the method comprising administering to the subject at least one vaccine according to the present invention. In yet another aspect, the present invention provides a method of treating or preventing an Ehrlichieae or Rickettsieae infection in a subject, the method comprising administering to the subject at least one vaccine according to the present invention. Preferably, the Ehrlichieae or Rickettsieae pathogen is selected from the group consisting of: Anaplasma sp., Ehrlichia sp., Rickettsia sp. and Cowdria sp. More preferably, the Ehrlichieae or Rickettsieae pathogen is selected from the group consisting of: Anaplasma marginale, Anaplasma centrale, Anaplasma ovis, Cowdria ruminantium, Ehrlichia equi, Ehrlichia phagocytophila, Ehrlichia chaffeensis, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia conorii , and Ehrlichia sp. which cause human granulocytic ehrlichiosis. Even more preferably, the Ehrlichieae or Rickettsieae pathogen is Anaplasma marginale. Preferably, the subject is a mammal. In one embodiment, the mammal is selected from the group consisting of; cows, sheep, goats, dogs and horses. In another embodiment, the mammal is a human. In a further aspect, the present invention provides for the use of a vaccine according to the present invention for the manufacture of a medicament for raising an immune response against an Ehrlichieae or Rickettsieae pathogen in a subject. It is also known in the art that an immune response can be provided by the consumption of a transgenic plant expressing an antigen. Thus, in a further aspect the present invention provides a transgenic plant which produces at least one polypeptide selected from the group consisting of: a) a sequence provided in SEQ ID NO:1; b) a polypeptide which is at least 50% identical to (a); c) a sequence provided in SEQ ID NO:2; d) a polypeptide which is at least 50% identical to (c); e) a sequence provided in SEQ ID NO:3; f) a polypeptide which is at least 50% identical to (e); g) a sequence provided in SEQ ID NO:4; and h) a polypeptide which is at least 50% identical to (g); wherein the polypeptide raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when the transgenic plant is orally administered to a subject. Preferably, the polypeptide is at least 60% identical, more preferably at least 70% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, and even more preferably at least 99% identical to (a), (c), (e) or (g). In yet another aspect, the present invention provides a method for raising an immune response against an Ehrlichieae or Rickettsieae pathogen in a subject, the method comprising orally administering to the subject at least one transgenic plant of the invention. In a further aspect, the present invention provides a method of treating or preventing an Ehrlichieae or Rickettsieae infection in a subject, the method comprising orally administering to the subject at least one transgenic plant of the invention. In another aspect, the present invention provides an antibody raised against a polypeptide selected from the group consisting of: a) a sequence provided in SEQ ID NO:1; b) a polypeptide which is at least 50% identical to (a); c) a sequence provided in SEQ ID NO:2; d) a polypeptide which is at least 50% identical to (c); e) a sequence provided in SEQ ID NO:3; f) a polypeptide which is at least 50% identical to (e); g) a sequence provided in SEQ ID NO:4; and h) a polypeptide which is at least 50% identical to (g); wherein the antibody provides immune protection against Ehrlichieae and/or Rickettsieae pathogens when administered to a subject. Preferably, the polypeptide is at least 60% identical, more preferably at least 70% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, and even more preferably at least 99% identical to (a), (c), (e) or (g). In a further aspect, the present invention provides a method of treating or preventing an Ehrlichieae or Rickettsieae infection in a subject, the method comprising administering to the subject at least one antibody according to the invention. In another aspect, the present invention provides a substantially purified polypeptide which specifically binds to an antibody according to the invention. In another aspect, the present invention provides a substantially purified polypeptide, the polypeptide being selected from: (i) a polypeptide comprising the sequence provided as SEQ ID NO:1; and (ii) a polypeptide which is at least 50% identical to (i); wherein the polypeptide raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when administered to a subject. Preferably, the polypeptide is at least 60% identical, more preferably at least 70% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, and even more preferably at least 99% identical to (i). Preferably, the polypeptide has an N-terminal sequence as provided in SEQ ID NO:12 and has a molecular weight of approximately 17 kDa. In another aspect, the present invention provides a substantially purified polypeptide, the polypeptide being selected from: (i) a polypeptide comprising the sequence provided as SEQ ID NO:2; and (ii) a polypeptide which is at least 50% identical to (i); wherein the polypeptide raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when administered to a subject. Preferably, the polypeptide is at least 60% identical, more preferably at least 70% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, and even more preferably at least 99% identical to (i). In another aspect, the present invention provides a substantially purified polypeptide, the polypeptide being selected from: (i) a polypeptide comprising the sequence provided as SEQ ID NO:3; and (ii) a polypeptide which is at least 50% identical to (i); wherein the polypeptide raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when administered to a subject. Preferably, the polypeptide is at least 60% identical, more preferably at least 70% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, and even more preferably at least 99% identical to (i). In another aspect, the present invention provides a substantially purified polypeptide, the polypeptide being selected from: (i) a polypeptide comprising the sequence provided as SEQ ID NO:4; and (ii) an antigenic fragment of (i), wherein the polypeptide, or antigenic fragment thereof, raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when administered to a subject. Further, it is preferred that the polypeptide can be purified from a species of Ehrlichieae or Rickettsieae. Preferably, the polypeptide can be purified from a species selected from the group consisting of: Anaplasma sp., Ehrlichia sp., Rickettsia sp. and Cowdria sp. More preferably, polypeptide can be purified from the group consisting of: Anaplasma marginale, Anaplasma centrale, Anaplasma ovis, Cowdria ruminantium, Ehrlichia equi, Ehrlichia phagocytophila, Ehrlichia chaffeensis, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia conorii , and Ehrlichia sp. which cause human granulocytic ehrlichiosis. Even more preferably, the polypeptide can be purified from Anaplasma marginale. It is preferred that the immune response is against Anaplasma marginale infection. In a further preferred embodiment, a polypeptide of the present invention is obtainable by (i) disrupting Anaplasma marginale in a sample to obtain a homogenate, (ii) centrifuging the homogenate from step (i) to obtain a pellet; (ii) extracting the pellet from step (ii) with a detergent to obtain a detergent soluble fraction and a detergent insoluble fraction; and (iii) subjecting the detergent soluble fraction to further purification steps. Anaplasma marginale in the sample may be disrupted by any suitable means such as sonication and/or enzymatic degradation. Preferably, the detergent is n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate. Further, it is preferred that the further purification steps include isoelectric focusing. In another aspect, the present invention provides a fusion protein comprising a polypeptide according to the invention fused to at least one heterologous polypeptide sequence. Preferably, the at least one heterologous polypeptide sequence is selected from the group consisting of: a polypeptide that enhances the stability of the polypeptide of the present invention, and a polypeptide that assists in the purification of the fusion protein. In yet another aspect, the present invention provides an isolated polynucleotide, the polynucleotide having a sequence selected from: (i) a sequence of nucleotides shown in SEQ ID NO:5; (ii) a sequence of nucleotides shown in SEQ ID NO:6; (iii) a sequence of nucleotides shown in SEQ ID NO:7; (iv) a sequence of nucleotides shown in SEQ ID NO:57; (v) a sequence of nucleotides shown in SEQ ID NO:8; (vi) a sequence of nucleotides shown in SEQ ID NO:56; (vii) a sequence encoding a polypeptide according to the present invention; (viii) a sequence capable of selectively hybridizing to any one of (i) to (iv) under high stringency; and (ix) a sequence of nucleotides which is at least 50% identical to any one of (i) to (iv), wherein the polynucleotide encodes a polypeptide that raises an immune response against Ehrlichieae and/or Rickettsieae pathogens when administered to a subject. Preferably, the polynucleotide is at least 60% identical, more preferably at least 70% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, and even more preferably at least 99% identical to any one of (i) to (iv). In a further aspect, the present invention provides a vector comprising at least one polynucleotide of the invention. The vectors may be, for example, plasmid, virus or phage vectors provided with an origin of replication, and preferably a promotor for the expression of the polynucleotide and optionally a regulator of the promotor. The vector may contain one or more selectable markers, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian expression vector. The vector may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell. Preferably, the vector is a viral vector. In another aspect, the present invention provides a host cell comprising a vector of the invention. Preferably, the host cell is mammalian cell. In a further aspect, the present invention provides a process for preparing a polypeptide according to the invention, the process comprising cultivating a host cell according to the invention under conditions which allow expression of the polynucleotide encoding the polypeptide, and recovering the expressed polypeptide. This process can be used for the production of commercially useful quantities of the encoded polypeptide. In a further aspect, the present invention provides a composition comprising a polypeptide according to the invention, and a pharmaceutically acceptable carrier. The invention will hereinafter be described by way of the following non-limiting Figures and Examples. |
Rotor system vibration absorber |
A rotor system vibration absorber for use with a helicopter of other rotocraft is disclosed in which spring forces are provided by a plurality of elongated rods (73) arranged in a selected pattern. The rods are coupled at one end to a fixed base (79) that is coupled to a rotor hub (55), and at the other end to a tuning weight (81). |
1. A rotorcraft comprising: a fuselage; a drive means carried by the fuselage; a rotor system including a rotor hub and rotor blades, the rotor system being coupled to the drive means; and a spring-mass vibration absorber comprising: a base member coupled to the rotor system; a tuning weight; and a plurality of elongated rods disposed between the base member and the tuning weight; wherein the rods serve as the spring and the tuning weight serves as the mass such that vibration from the rotor system is absorbed by the oscillatory deflection of the rods and the tuning weight. 2. The rotorcraft according to claim 1, wherein the spring rate of the vibration absorber is determined by selectively tailoring the number, location, size, and shape of the rods. 3. The rotorcraft according to claim 1, wherein the vibration absorber absorbs vibratory hub shear forces. 4. The rotorcraft according to claim 1, wherein the vibration absorber absorbs vibratory hub moments. 5. The rotorcraft according to claim 1, wherein the vibration absorber absorbs both vibratory hub shear forces and vibratory hub moments. 6. The rotorcraft according to claim 1, wherein the vibration is the principal blade-passage frequency. 7. The rotorcraft according to claim 1, wherein the rods are composite rods manufactured from a unidirectional composite fiber reinforced material. 8. The rotorcraft according to claim 7, wherein the composite rods are covered with a composite fabric to minimize delamination. 9. The rotorcraft according to claim 1, wherein the rods have a uniform cross-sectional geometry. 10. The rotorcraft according to claim 1, wherein the rods have a non-uniform cross-sectional geometry. 11. The rotorcraft according to claim 10, wherein the rods have a longitudinal profile in the shape of a non-linear function. 12. The rotorcraft according to claim 11, wherein the non-linear function is a cubic function. 13. The rotorcraft according to claim 1, wherein the base member is coupled to the rotor system above the rotor hub and the tuning weight is disposed above the rotor hub. 14. The rotorcraft according to claim 1, wherein the base member is coupled to the rotor system below the rotor hub and the tuning weight is disposed below the rotor hub. 15. The rotorcraft according to claim 1, wherein the base member is coupled to the rotor system above the rotor hub and the tuning weight is disposed below the rotor hub. 16. The rotorcraft according to claim 1, wherein the base member is coupled to the rotor system below the rotor hub and the tuning weight is disposed above the rotor hub. 17. The rotorcraft according to claim 1, further comprising: a second vibration absorber comprising: a second base member coupled to the rotor system; a second tuning weight; and a second plurality of elongated rods disposed between the second base member and the second tuning weight; wherein vibration from the rotor system is also absorbed by deflection of the second plurality of rods. 18. The rotorcraft according to claim 17, wherein the base member is coupled to the rotor system above the rotor hub and the tuning weight is disposed above the rotor hub; and wherein the second base member is coupled to the rotor system below the rotor hub and the second tuning weight is disposed below the rotor hub. 19. A vibration absorber for use on a rotorcraft having a rotor system including a drive means, a drive mast coupled to the drive means, a rotor hub coupled to the drive mast, and rotor blades pivotally coupled to the rotor hub, the vibration absorber comprising: a housing adapted for mounting to the underside of the rotor hub; a base member coupled to the housing; a tuning weight disposed above the rotor hub; and a plurality of rods, each rod being coupled at one end to the base member and coupled at the other end to the tuning weight; wherein the vibration absorber absorbs both vibratory hub shear forces and vibratory hub moments generated by the rotor system. 20. The vibration absorber according to claim 19, wherein one rod is disposed between each pair of adjacent rotor blades. 21. The vibration absorber according to claim 19, further comprising: an upper plate disposed between the rods and the tuning weight; wherein the upper ends of the rods are coupled to the upper plate, and the tuning weight is coupled to the upper plate. 22. The vibration absorber according to claim 21, further comprising: a travel stop means disposed on the upper plate and operably associated with the drive mast to prevent the vibration absorber from damaging the rotor system in the event of failure of the vibration absorber. 23. The vibration absorber according to claim 19, wherein the rods are composite rods manufactured from a unidirectional composite fiber reinforced material. 24. The vibration absorber according to claim 23, wherein the composite rods are covered with a composite fabric to minimize delamination. 25. The vibration absorber according to claim 19, further comprising: a canopy disposed over the vibration absorber to reduce aerodynamic drag generated by the vibration absorber. 26. The vibration absorber according to claim 19, wherein each rod comprises: an elongated body portion manufactured from a unidirectional fiber reinforced pultruded composite material; and a longitudinal profile in which each half of each rod is in the shape of a non-linear function, such that the ends have enlarged cross-sectional areas at the couplings to the base member and the tuning weight, and the smallest cross-sectional area is located at the longitudinal midpoint of each rod; whereby the fatigue life of each rod is increased and the greatest vibratory hub shear forces are located at the longitudinal midpoint of each rod. 27. The vibration absorber according to claim 26, wherein the non-linear function is a cubic function. 28. The vibration absorber according to claim 19, wherein the blade-passage frequency vibration is reduced. 29. A composite spring-mass assembly comprising: a fixed body; at least one elongated composite rod attached at one end to the fixed body, the composite rod being manufactured from a unidirectional fiber reinforced pultruded composite material; a movable mass attached to the other end of the composite rod, such that the movable mass is cantilevered relative to the fixed body; wherein the spring rate of the composite spring-mass system is determined by selectively tailoring the number, arrangement, and shape of the rods. 30. The composite spring-mass system according to claim 25, wherein each end of each elongated composite rod is tapered in the shape of a non-linear function, such that both ends of each composite rod have enlarged cross-sectional areas and the longitudinal midpoint has the smallest cross-sectional area. 31. The composite spring-mass system according to claim 30, wherein the non-linear function is a cubic function. |
<SOH> TECHNICAL FIELD <EOH>The present invention relates to vibration absorbers. In particular, the present invention relates to rotor hub vibration absorbers for helicopters and other rotorcraft. |
<SOH> SUMMARY OF THE INVENTION <EOH>There is a need for a rotor system vibration absorber for use on a helicopter or other rotorcraft that can be installed above and/or below the rotor hub for minimizing vibration due to both in-plane hub shear forces and out-of plane hub bending moments, and that requires little or no maintenance. Therefore, it is an object of the present invention to provide a weight-efficient rotor system vibration absorber for use on a helicopter or other rotorcraft that can be installed above and/or below the rotor hub for minimizing vibration due to both in-plane hub shear forces and out-of-plane hub bending moments, and that requires little or no maintenance. The above object is achieved by providing a rotor system vibration absorber having a simple, low-cost design in which a plurality of elongated rods are arranged in a selected pattern. Each rod is coupled at one end to the rotor hub, and at the opposing end to a tuning weight. The vibration absorber of the present invention provides the following significant advantages over the prior art. The vibration absorber according to the present invention has a simple, low-cost design having no moving parts. This feature significantly reduces wear and maintenance. Each rod provides an independent load path, thereby making the system fail safe. In the present invention, over 80% of the weight of the vibration absorber is utilized as a tuning weight, thereby eliminating the weight inefficiencies present in prior-art devices. The vibration absorber of the present invention can be installed above and/or below the rotor hub. This allows it to counteract not only in-plane hub shear forces, but out-of-plane hub bending moments, i.e., roll and pitch. The rotor system vibration absorber of the present invention is easily maintainable in the field because it is has a high level of reliability and failures are easily detectable. |
Ceiling suspension with cable pathway |
Combined ceiling support device and cable pathway, comprising a plurality of spaced apart junctions (3) arranged in a predetermined array above an area of a building, means (5, 15) for supporting said junctions (3), support means (6) connected to said junctions for forming a grid over said area, said support means adapted to support a multiplicity of ceiling tiles to form a ceiling for said area, and a multiplicity of cable ducts (1a, 1b, 2) extending between at least some of said junctions (3) for routing cable over said area. |
1. A combined ceiling support device and cable pathway, comprising a plurality of spaced apart junctions arranged in a predetermined array above an area of a building, means for supporting said junctions, support means connected to said junctions for forming a grid over said area, said support means adapted to support a multiplicity of ceiling tiles to form a ceiling for said area, and a multiplicity of cable ducts extending between at least some of said junctions for routing cable over said area. 2. A combined ceiling support device and cable pathway according to claim 1, wherein each of said junctions includes a first member for supporting said support means and a second member for supporting said ducts. 3. A combined ceiling support device and cable pathway according to claim 2, further including junction links connected between adjacent junctions beneath said ducts. 4. A combined ceiling support device and cable pathway according to claim 3, wherein said second member is a plate and said ducts and junction links are attached to the plates of said junctions. 5. A combined ceiling support device and cable pathway according to claim 2, wherein said ducts and support means are connected to each other. 6. A combined ceiling support device and cable pathway according to claim 5, wherein said support means comprises rails including downwardly facing U-shaped sections for receiving the upper ends of said ducts. 7. A combined ceiling support device and cable pathway according to claim 3, wherein a pair of junction links extend between adjacent junctions. 8. A combined ceiling support device and cable pathway according to claim 1, wherein wall panels are provided between selected junctions. 9. A combined ceiling support device and cable pathway according to claim 1, including a post extending downwardly from at least some of said junctions to the floor of the building. 10. A combined ceiling support device and cable pathway according to claim 9, wherein wall panels are provided between selected posts, and wherein at least some of said wall panels are adapted to receive cabling from said cable ducts and include terminals for connection to the cabling. 11. A combined ceiling support device and cable pathway according to claim 7, wherein said ducts comprise two vertical plates attached to respective junction links of a pair of junction links. 12. A combined ceiling support device and cable pathway according to claim 11, wherein the ducts further include bottom plates extending between at least some of the junctions. 13. A combined ceiling support device and cable pathway, comprising a plurality of spaced apart junctions arranged in a predetermined array above an area of a building, means for supporting said junctions on the superstructure of the building, a plurality of posts connected to at least some of said junctions and extending to the floor, first and second pairs of transverse rails connected to said junctions and forming a grid over said area, said rails adapted to support a multiplicity of ceiling tiles to form a ceiling for said area, and a multiplicity of cable ducts extending between at least some of said junctions beneath said transverse rails for routing cable over said area. 14. A combined ceiling support device and cable pathway according to claim 13, wherein each of said junctions includes an upper member for supporting said pairs of rails and a lower member for supporting said ducts. 15. A combined ceiling support device and cable pathway according to claim 14, further including junction links connected between adjacent junctions beneath said ducts. 16. A combined ceiling support device and cable pathway according to claim 15, wherein a pair of junction links extend between adjacent junctions and said ducts are attached to the upper portions of said junction links. 17. A combined ceiling support device and cable pathway according to claim 16, wherein said lower member is a plate and said ducts and junction links are attached to the plates of said junctions. 18. A combined ceiling support device and cable pathway according to claim 14, wherein said ducts and rails are connected to each other. 19. A combined ceiling support device and cable pathway according to claim 18, wherein said rails include downwardly facing U-shaped sections for receiving the upper ends of said ducts. 20. A combined ceiling support device and cable pathway according to claim 16, wherein wall panels are provided between selected junctions, the tops of said panels being positioned between the two junction links extending between said selected junctions. 21. A combined ceiling support device and cable pathway according to claim 13, wherein a screw extends downwardly from said junctions to which a post is attached into threaded engagement with the upper portions of said posts whereby the posts can be extended toward the floor by rotation. 22. A combined ceiling support device and cable pathway according to claim 16, wherein said ducts comprise two plates removably attached to respective junction links of a pair of junction links. 23. A combined ceiling support device and cable pathway according to claim 22, wherein the ducts further include bottom plates extending between at least some of the junctions. 24. A combined ceiling support device, cable pathway and room divider, comprising a plurality of spaced apart junctions arranged in a predetermined array above an area of a building, means for supporting said junctions, support means connected to said junctions for forming a grid over said area, said support means adapted to support a multiplicity of ceiling tiles to form a ceiling for said area, a multiplicity of cable ducts extending between at least some of said junctions for routing cable over said area, and a plurality of wall panels secured between selected junctions. 25. A combined ceiling support device, cable pathway and room divider according to claim 24, including a post extending downwardly from at least some of said junctions to the floor of the building, said wall panels being secured to said posts. 26. A combined ceiling support device, cable pathway and room divider according to claim 25, wherein a pair of junction links extend between adjacent junctions, and wherein said wall panels are each connected to a pair of junction links. 27. A combined ceiling support device, cable pathway and room divider according to claim 26, wherein said wall panels include means for receiving cabling from said cable ducts and terminals connected to said cabling for enabling users to connect devices to said cabling. a plurality of spaced apart junctions arranged in a predetermined array above in area of a building. The junctions are supported on the superstructure of the building and posts connected to the junctions extend to the floor. First and second paris of transverse rails are connected to the junctions and form a grid over the area, the rails being adapted to support the ceiling tiles which form the ceiling for the area. A multiplicity of cable ducts extend between the junctions beneath the transvese rails for routing cabling over the area. Wall panels, supported between adjacent posts, can receive cabling from the overhead ducts for connection to outlet terminals on the panels. |
<SOH> BACKGROUND <EOH>Ceilings within a typical commercial office environment are generally referred to as dropped ceilings and form a barrier between the lower office space and the upper area. Standard sized ceiling tiles, usually 2′×2′ or 2′×4,′ are supported on a matrix of inverted “T” grids that are suspended from wires attached to a building's superstructure. Cables and wiring (hereafter referred to as cabling) are ordinarily installed above hung ceilings and are used for a variety of purposes, such as electric, voice, and data transmission. Cabling installed above a hung ceiling may result in a number of undesirable circumstances. With digital signals, there is a potential for electrical interference from lighting fixtures, motors and other sources. Also, an installed cabling configuration above a dropped ceiling is cumbersome to update and troubleshoot. Due to the difficulty in removing obsolete cabling, updated cabling installations are typically added on to the existing cabling runs resulting in an ever-increasing level of disorganization between the current and obsolete cable installations. This leads to increasing complexity and confusion and a considerable disruption of service whenever updates or repairs are necessary. Furthermore, computer server rooms and data processing centers typically require cabling to be routed underneath raised floors. When cabling in other areas is installed above the ceiling, the overall configuration of the cabling system is convoluted. To facilitate the transition from above the ceiling to below the floor, special rooms (closets) are built. Cabling running above the overhead ceiling is routed down a closet wall and then through the bottom of that wall into the space below the adjacent raised floor area. The cabling is then routed up through openings in the floor to and from racks of equipment. Many different techniques have been proposed to inconspicuously hide or at least minimize the appearance of cabling which emanates from above the ceiling. In some cases the cabling is merely tacked to the wall or installed within metal or plastic surface conduits routed to the work areas. In more expensive installations, walls are constructed to accommodate the separation of office spaces and the concealment of the cabling. Cabling may also be routed down from the ceiling through vertical pipes to offices or cubicles that are isolated in the center of open areas by corridors. In this case, the installation of electric outlets at desired locations requires extensive under-floor installation work, unsightly surface and floor mounted conduits, or cumbersome rubber “thresholds.” In all cases the great expense of the cabling is further increased by the disruption and/or displacement caused by a major installation or upgrade when an area is already occupied. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention avoids the drawbacks mentioned above by providing a matrix of ducts, which are readily accessible from beneath the ceiling and serve as a support mechanism for conventional ceiling tiles. Cabling of any description can be easily routed and/or rerouted in an orderly fashion to virtually any location within an open office area and away from interference (e.g. fluorescence and motors.) In the preferred embodiment, the design of the ceiling results in a physical structure that is stable in all directions and thus capable of supporting building elements, such as wall panels and doorways. In addition to the cabling being easily directed from overhead, spare cabling for future expansion can be readily stored within the wall panels so that an initial cabling configuration can endure for an extended period of time. Based upon reasonable estimates of future business needs, additional cabling is already installed when reconfigurations are needed making the rerouting of cabling relatively simple. During installation planning all cabling can be catalogued by type or other designation. Because the invention provides a matrix of ductwork, a cable may be assigned to a particular pathway and thus be tracked by a simple coordinate system. Since all cabling is routed overhead, including server rooms and the like, there is no need to transition the cable from the ceiling to a space beneath the floor. As a result, there is less need for closets and cabling from server racks, telephone switching equipment, and electrical sources can all be oriented overhead using the overhead duct system. The invention thus provides for an aesthetically pleasing and standardized way to design and build office environments that are durable and secure as well as easy to install, re-design, and reconfigure. |
Method and apparatus for carrying out non-destructive testing of materials |
A method of testing a material sample for defects, comprising the steps of applying a test signal to the material sample, receiving a return signal from the material sample and analysing the return signal to determine whether the material sample is defective. In one aspect of the present invention the steps of receiving a return signal and analysing it include the step of selecting a frequency range to reduce the effect of potentially interfering signals. In another aspect of the present invention a reference signal obtained from a non-defective sample is subtracted from the return signal to give a difference result and the difference result is indicative of whether the material sample is defective. Apparatus for carrying out the above-defined methods are also provided. |
1. A method of testing a material sample for defects, comprising the steps of applying a test signal to the material sample, receiving a return signal from the material sample and analysing the waveform of the return signal to determine where the material sample is defective, and wherein the steps of receiving a return signal and analysing it include the step of selecting a frequency range to reduce the effect of potentially interfering signals. 2. A method in accordance with claim 1, wherein the potentially interfering signals are resonance signals caused by a test probe. 3. A method in accordance with claim 1, wherein the frequency range selected is between 1 and 22 kHz. 4. A method in accordance with claim 3, wherein the frequency range selected is between 3 and 15 kHz. 5. A method in accordance with claim 3, wherein the frequency range selected is between 18 and 22 kHz. 6. A method in accordance with claim 1, wherein the step of selecting a frequency is implemented by filtering the return signal. 7. A method in accordance with claim 1, comprising the further step of taking a reference signal which has been obtained from a non-defective sample, subtracting the reference signal from the return signal, to give a difference result. 8. A method in accordance with claim 7, wherein the difference result is provided as a difference number which is calculated by summing the absolute value of the point by point difference over the whole or some user selected part of the return signal versus the reference signal. 9. A method in accordance with claim 8, comprising the further step of utilising the difference result to produce a display of the difference result in relation to position on the test sample. 10. An apparatus for testing material samples for defects, comprising a means for applying a test signal to the material sample, receiving means for receiving a return signal from the material sample, and analysis means for analysing the return signal to determine whether the material sample is defective, and further including a selection means for selecting a frequency range to reduce the effect of potentially interfering signals. 11. An apparatus in accordance with claim 10, wherein the potentially interfering signals may be caused by resonance signals caused by the means for applying the test signal. 12. An apparatus in accordance with claim 10, wherein the selection means is arranged to select a frequency of between 1 and 22 kHz. 13. An apparatus in accordance with claim 12, wherein the selection means is arranged to select a frequency of between 3 and 15 kHz. 14. An apparatus in accordance with claim 12, wherein the selection means is arranged to select a frequency of between 18 and 22 kHz. 15. An apparatus in accordance with claim 10, wherein the analysis means includes a means arranged to take a reference signal obtained from an undefected material sample and to subtract the reference signal from the return signal, to give a difference result. 16. An apparatus in accordance with accordance with claim 10, further comprising a display means for displaying the difference result. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Non-destructive testing (NDT) may be used to test composite materials, such as aeroplane panels, for mechanical defects. Defects can be caused by stress on materials or mechanical damage from the impact of objects on panels or defective manufacture. The ability to test these materials in situ is very important. Regular testing is obviously required for safety, particularly in the aeroplane industry, and it is impractical to disassemble aircraft or parts of aircraft to carry out testing. One preferred method of NDT is to utilise a probe to excite the test material or structure with radiation (generally, but not exclusively, acoustic and near acoustic frequencies are used for NDT) and detect a response. The response can be indicative of whether the test sample is faulty or not “defected”. Usually the response is compared with a response from similar excited radiation of a reference material or structure, which will usually be an undefected composite of the same type as the test material, but may also be a composite having reference defects. Determination of whether there is a defect in the test material usually involves merely the comparison of the output waveform provided by the detection electronics with the response signal waveform of the reference material (which may in some cases, for example, be an undefected area of the same aircraft—where it is an aircraft panel which is being tested). Acoustic or Ultrasound frequencies used to excite the materials are typically in the range of 5 to 70 kHz. One of the most popular known NDT systems utilises the pitch/catch impulse test. A typical pitch/catch probe comprises 2 spring-loaded (or otherwise resiliently mounted) contact tips set approximately 10 mm apart which are held in contact with the test sample. These are equipped with 2 actuators, one of which generates a mechanical vibration within the above frequency range and the other of which detects the response. The drive signal is generally a short wave train, up to 6 cycles of sinusoidal impulse (or similar). The detector measures the response of the test sample at its contact point. The propagation of the disturbance from the driver to the detector is influenced by the nature of the intervening structure and in particular, by any damage or anomaly in this region. It is therefore (in theory) possible to obtain information concerning mechanical defects in a non-destructive manner. With composite materials in particular defects may also include failure of adhesion i.e. a disbond or delamination. NDT is used to detect any defect, including disbonds and delaminations and other damages of the material itself or of rivets and joints etc. To initially calibrate the apparatus and select drive frequency, the drive frequency is chosen so as to optimise the difference in received signal between a faulty sample and an undefected sample. The frequency is usually selected to provide the maximum output from the detector electronics. It has been found that the performance of presently available NDT systems of this type is not good. Often it is difficult to determine with accuracy whether a defect exists or not in a test sample. Further, the present systems comprise complex and expensive hardware. They also require a relatively high level of skill to be able to operate them and interpret the results. It would be desirable to be able to provide a method and apparatus which provides a more reliable detection of defects in test samples than in the prior art, which generally improves and simplifies the analysis of the NDT data, and in which the NDT apparatus is relatively simple and inexpensive. |
<SOH> SUMMARY OF THE INVENTION <EOH>In accordance with a first aspect of the present invention, there is provided a method of testing a material sample for defects, comprising the steps of applying a test signal to the material sample, receiving a return signal from the material sample and analysing the waveform of the return signal to determine whether the material sample is defective, and wherein the steps of receiving a return signal and analysing it include the step of selecting a frequency range to reduce the effect of potentially interfering signals. The applicants have discovered by experiment with prior art NDT systems that the available pitch/catch probes have internal structural resonances associated with the dimensions of the driver and receive transducers, the contact tips and the housing (some other types of prior art probes may also have similar problems). These resonances occur throughout the range 15 to 60 kHz. In other prior art probes, however, it is possible that resonances may occur at other frequencies. Resonances may also occur below 3 kHz in present probes. Further, the applicants have undertaken experiments which show that for most composite panels used as test samples there is a strong local vibration response over a defect in the range of 5 to 50 kHz. Typical materials are carbon or glass fibre reinforced plastics in a sandwich construction with skins of these materials and a core of Nomex or foam. Skin thicknesses can be in the range from fractions of millimetres to several millimetres and core thickness is in the range of 5 to 50 mm or even thicker. In the prior art, therefore, the frequency range of defected sample responses and the frequency range containing the pitch/catch probe resonances substantially overlap. In addition, the applicants have found that the output generated by the probe resonances is considerably larger (by a factor usually 10 to 20 times) than the output generated by the defect. In the prior art, the drive frequency is chosen, according to manufacturers instructions, so as to optimise the difference in received signal between undefected and defected panels. As a consequence the set up process biases the users strongly towards frequencies in the range 20 to 30 kHz. It is believed by the applicant, therefore, that the probe resonances of the frequencies utilised in prior art systems lead to difficulty in determining from signals whether defects actually do or do not exist in the samples. The present invention includes the novel step of selecting a frequency range which at least reduces the effect of these potentially interfering signals on the return signal. The prior art methods appear to have been unaware of these potentially interfering signals. A frequency range of between 1 and 20 kHz preferably is selected and, more preferably, between 1 and 22 kHz. For a typical composite material, most preferably a range of between 3 and 15 kHz is selected. Note that other ranges may be selected, however, where interfering signals occur in other frequency ranges than in typical probes. In general, there appears to be the tendency that materials that are of lower density have a higher frequency range within which interfering signals are substantially reduced. For example, for aluminium it has been observed that within a frequency range of 18 to 22 kHz interfering signals are substantially reduced and for this material this frequency range therefore most preferably is selected. The present applicant, by selecting the above discussed frequency range for the received signal, leaves a range where there is a minimum overlap of defect and probe response and existence of defects can be determined accurately. Preferably, the test signal is also selected to be within the range 1 to 20 kHz and more preferably within the range ol to 22 kHz. For example for a typical composite material the test signal most preferably is selected within the range of 3 to 15 kHz and for aluminium the test signal most preferably is selected within the range of 18 to 22 kHz. In a preferred embodiment, the test signal is generally chosen to be approximately 2 cycles at approximately 10 kHz. This is a fairly broadband excitation which ensures that plenty of energy is supplied within the most preferred frequency ranges. In order to select the preferred range of return signal processing, the return signal is preferably bandpass filtered between 3 and 15 kHz or between 18 and 22 kHz with a very high Q filter (8 pole). This preferably eliminates both low frequency noise from environmental structural sources and all probe resonances above the upper limit of the frequency range. It leaves a range where there is a minimum overlap of defect and probe response (note that this range may be varied depending upon where the interfering frequencies are occurring). One of the other problems of the prior art is presentation of the information once the signal has been processed. Preferably, in the present invention the method comprises the further step of taking a reference signal which has been obtained from a non-defective sample and subtracting the reference signal from the return signal, to give a difference result. Preferably, the difference result is displayed. This step has the advantage that a user is easily able to determine from the difference result that a defect exists, and also is preferably able to determine the relative extent of the defect (depending upon the size of the difference result). Preferably, the reference signal and return signal are digitised before the subtraction operation. The subtraction operation preferably is performed in the frequency domain and the signal may also be digitally filtered. A computer routine most preferably is used to perform the subtraction. Preferably, the difference result is provided as a difference number which is calculated by summing the absolute value of the point by point difference over the whole or some user selected part of the return signal versus reference signal. This yields a single number for each sample position on the test piece, which is known here as the “damage index”. An electronic library of saved response signals may be used to facilitate identification of a defect. Preferably, utilising the damage index, a map across the test piece is presented on a display for viewing by the user. Preferably, the method of the present invention is implemented by software utilising a computing system. Input to the system is from a pitch/catch probe. The computing system may be a standard computing system such as a personal computer or laptop, adapted by any additional signal processing hardware that may be required. This is may be cheaper and more convenient than the complex and expensive hardware required for prior art systems. In accordance with a second aspect of the present invention there is provided a method of testing a material sample for defects comprising the steps of applying a test signal to the material sample, receiving a return signal from the material sample, taking a reference signal obtained from a non-defective sample and subtracting the reference signal from the return signal to give a difference result, whereby the difference result is indicative of whether the material sample is defective. In accordance with a third aspect of the present invention, there is provided an apparatus for testing material samples for defects, comprising means for applying a test signal to the material sample, receiving means for receiving the return signal from the material sample, and analysis means for analysing the return signal to determine whether the material sample is defective, and further including selection means for selecting a frequency range to reduce the effect of potentially interfering signals. Preferably, the selection means is arranged to select a frequency within the range 1 to 20 kHz and more preferably between 1 and 22 kHz. The selection means most preferably is arranged to select a frequency between 3 and 15 kHz or 18 and 22 kHz. Preferably, the selection means includes a bandpass filter, preferably having a very high Q. Preferably, the analysis means includes a means arranged to take a reference signal obtained from an undefected material sample and to subtract the reference signal from the return signal, to give a difference result. Preferably, the difference result is provided to a display for viewing by a user. Preferably, the analysis means is arranged to digitise the reference signal and the return signal and subtract the return signal from the reference signal to give a difference quantity. It is preferably arranged to provide difference quantities for a plurality of areas across the test sample. These difference quantities, known as “damage index” are preferably utilised to produce a map on the display of the damage index across the area of the test sample. The analysis means may also be arranged to filter the return signal. The analysis means preferably is arranged to process signals digitally. The analysis means preferably is equipped with an electronic library of response signals. In accordance with a fourth aspect of the present invention, there is provided an apparatus for testing a material sample for defects, comprising means for applying a test signal to the material sample, a receiving means for receiving a return signal from the material sample, an analysis means for taking a reference signal obtained from a non-defective sample and subtracting the reference signal from the return signal, to give a difference result, whereby the difference result is indicative of whether the material sample is defective. |
Transporter and ion channels |
The invention provides human transporters and ion channels (TRICH) and polynucleotides which identify and encode TRICH. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of TRICH. |
1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-2, SEQ ID NO:4-8, and SEQ ID NO:10-15, c) a polypeptide comprising a naturally occurring amino acid sequence at least 91% identical to an amino acid sequence selected from the group consisting of consisting of SEQ ID NO:3 and SEQ ID NO:9, d) a polypeptide comprising a naturally occurring amino acid sequence at least 94% identical to an amino acid sequence consisting of SEQ ID NO:16, e) a polypeptide comprising a naturally occurring amino acid sequence at least 93%. identical to an amino acid sequence consisting of SEQ ID NO:17, f) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and g) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. 2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. 3. An isolated polynucleotide encoding a polypeptide of claim 1. 4. An isolated polynucleotide encoding a polypeptide of claim 2. 5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34. 6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3. 7. A cell transformed with a recombinant polynucleotide of claim 6. 8. (canceled) 9. A method of producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed. 10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. 11. An isolated antibody which specifically binds to a polypeptide of claim 1. 12. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-19, SEQ ID NO:21-25, and SEQ ID NO:27-34, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 91% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:20 and SEQ ID NO:26, d) a polynucleotide complementary to a polynucleotide of a), e) a polynucleotide complementary to a polynucleotide of b), f) a polynucleotide complementary to a polynucleotide of c), and g) an RNA equivalent of a)-f). 13. (canceled) 14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof. 15. (canceled) 16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof. 17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient. 18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. 19. (canceled) 20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample. 21. (canceled) 22. (canceled) 23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample. 24. (canceled) 25. (canceled) 26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1. 27. (canceled) 28. A method of screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising: a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. 29. A method of assessing toxicity of a test compound, the method comprising: a) treating a biological sample containing nucleic acids with the test compound, b) hybridization nucleic acids of the treated biological same with a probe comprising at least 20 contagious nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. 30.-89. (canceled) |
<SOH> BACKGROUND OF THE INVENTION <EOH>Eukaryotic cells are surrounded and subdivided into functionally distinct organelles by hydrophobic lipid bilayer membranes which are highly impermeable to most polar molecules. Cells and organelles require transport proteins to import and export essential nutrients and metal ions including K + , NH 4 + , P i , SO 4 2− , sugars, and vitamins, as well as various metabolic waste products. Transport proteins also play roles in antibiotic resistance, toxin secretion, ion balance, synaptic neurotransmission, kidney function, intestinal absorption, tumor growth, and other diverse cell functions (Griffith, J. and C. Sansom (1998) The Transporter Facts Book, Academic Press, San Diego Calif., pp. 3-29). Transport can occur by a passive concentration-dependent mechanism, or can be linked to an energy source such as ATP hydrolysis or an ion gradient. Proteins that function in transport include carrier proteins, which bind to a specific solute and undergo a conformational change that translocates the bound solute across the membrane, and channel proteins, which form hydrophilic pores that allow specific solutes to diffuse through the membrane down an electrochemical solute gradient. Carrier proteins which transport a single solute from one side of the membrane to the other are called uniporters. In contrast, coupled transporters link the transfer of one solute with simultaneous or sequential transfer of a second solute, either in the same direction (symport) or in the opposite direction (antiport). For example, intestinal and kidney epithelium contains a variety of symporter systems driven by the sodium gradient that exists across the plasma membrane. Sodium moves into the cell down its electrochemical gradient and brings the solute into the cell with it. The sodium gradient that provides the driving force for solute uptake is maintained by the ubiquitous Na + /K + ATPase system. Sodium-coupled transporters include the mammalian glucose transporter (SGLT1), iodide transporter (NIS), and multivitamin transporter (SMVT). All three transporters have twelve putative transmembrane segments, extracellular glycosylation sites, and cytoplasmically-oriented N— and C-termini. NIS plays a crucial role in the evaluation, diagnosis, and treatment of various thyroid pathologies because it is the molecular basis for radioiodide thyroid-imaging techniques and for specific targeting of radioisotopes to the thyroid gland (Levy, O. et al. (1997) Proc. Natl. Acad. Sci. USA 94:5568-5573). SMVT is expressed in the intestinal mucosa, kidney, and placenta, and is implicated in the transport of the water-soluble vitamins, e.g., biotin and pantothenate (Prasad, P. D. et al. (1998) J. Biol. Chem. 273:7501-7506). One of the largest families of transporters is the major facilitator superfamily (MFS), also called the uniporter-symporter-antiporter family. MFS transporters are single polypeptide carriers that transport small solutes in response to ion gradients. Members of the MFS are found in all classes of living organisms, and include transporters for sugars, oligosaccharides, phosphates, nitrates, nucleosides, monocarboxylates, and drugs. MFS transporters found in eukaryotes all have a structure comprising 12 transmembrane segments (Pao, S. S. et al. (1998) Microbiol. Molec. Biol. Rev. 62:1-34). The largest family of MFS transporters is the sugar transporter family, which includes the seven glucose transporters (GLUT1-GLUT7) found in humans that are required for the transport of glucose and other hexose sugars. These glucose transport proteins have unique tissue distributions and physiological functions. GLUT1 provides many cell types with their basal glucose requirements and transports glucose across epithelial and endothelial barrier tissues; GLUT2 facilitates glucose uptake or efflux from the liver; GLUT3 regulates glucose supply to neurons; GLUT4 is responsible for insulin-regulated glucose disposal; and GLUT5 regulates fructose uptake into skeletal muscle. Defects in glucose transporters are involved in a recently identified neurological syndrome causing infantile seizures and developmental delay, as well as glycogen storage disease, Fanconi-Bickel syndrome, and non-insulin-dependent diabetes mellitus (Mueckler, M. (1994) Eur. J. Biochem. 219:713-725; Longo, N. and L. J. Elsas (1998) Adv. Pediatr. 45:293-313). Monocarboxylate anion transporters are proton-coupled symporters with a broad substrate specificity that includes L-lactate, pyruvate, and the ketone bodies acetate, acetoacetate, and beta-hydroxybutyrate. At least seven isoforms have been identified to date. The isoforms are predicted to have twelve transmembrane (TM) helical domains with a large intracellular loop between TM6 and TM7, and play a critical role in maintaining intracellular pH by removing the protons that are produced stoichiometrically with lactate during glycolysis. The best characterized H + -monocarboxylate transporter is that of the erythrocyte membrane, which transports L-lactate and a wide range of other aliphatic monocarboxylates. Other cells possess H + -linked monocarboxylate transporters with differing substrate and inhibitor selectivities. In particular, cardiac muscle and tumor cells have transporters that differ in their K m values for certain substrates, including stereoselectivity for L- over D-lactate, and in their sensitivity to inhibitors. There are Na + -monocarboxylate cotransporters on the luminal surface of intestinal and kidney epithelia, which allow the uptake of lactate, pyruvate, and ketone bodies in these tissues. In addition, there are specific and selective transporters for organic cations and organic anions in organs including the kidney, intestine and liver. Organic anion transporters are selective for hydrophobic, charged molecules with electron-attracting side groups. Organic cation transporters, such as the ammonium transporter, mediate the secretion of a variety of drugs and endogenous metabolites, and contribute to the maintenance of intercellular pH (Poole, R. C. and A. P. Halestrap (1993) Am. J. Physiol. 264:C761-C782; Price, N. T. et al. (1998) Biochem. J. 329:321-328; and Martinelle, K. and I. Haggstrom (1993) J. Biotechnol. 30:339-350). ATP-binding cassette (ABC) transporters are members of a superfamily of membrane proteins that transport substances ranging from small molecules such as ions, sugars, amino acids, peptides, and phospholipids, to lipopeptides, large proteins, and complex hydrophobic drugs. ABC transporters consist of four modules: two nucleotide-binding domains (NBD), which hydrolyze ATP to supply the energy required for transport, and two membrane-spanning domains (MSD), each containing six putative transmembrane segments. These four modules may be encoded by a single gene, as is the case for the cystic fibrosis transmembrane regulator (CFTR), or by separate genes. When encoded by separate genes, each gene product contains a single NBD and MSD. These “half-molecules” form homo- and heterodimers, such as Tap1 and Tap2, the endoplasmic reticulum-based major histocompatibility (MHC) peptide transport system. Several genetic diseases are attributed to defects in ABC transporters, such as the following diseases and their corresponding proteins: cystic fibrosis (CFTR, an ion channel), adrenoleukodystrophy (adrenoleukodystrophy protein, ALDP), Zellweger syndrome (peroxisomal membrane protein-70, PMP70), and hyperinsulinemic hypoglycemia (sulfonylurea receptor, SUR). Overexpression of the multidrug resistance (MDR) protein, another ABC transporter, in human cancer cells makes the cells resistant to a variety of cytotoxic drugs used in chemotherapy (Taglicht, D. and S. Michaelis (1998) Meth. Enzymol. 292:130-162). A number of metal ions such as iron, zinc, copper, cobalt, manganese, molybdenum, selenium, nickel, and chromium are important as cofactors for a number of enzymes. For example, copper is involved in hemoglobin synthesis, connective tissue metabolism, and bone development, by acting as a cofactor in oxidoreductases such as superoxide dismutase, ferroxidase (ceruloplasmin), and lysyl oxidase. Copper and other metal ions must be provided in the diet, and are absorbed by transporters in the gastrointestinal tract. Plasma proteins transport the metal ions to the liver and other target organs, where specific transporters move the ions into cells and cellular organelles as needed. Imbalances in metal ion metabolism have been associated with a number of disease states (Danks, D. M. (1986) J. Med. Genet. 23:99-106). Transport of fatty acids across the plasma membrane can occur by diffusion, a high capacity, low affinity process. However, under normal physiological conditions a significant fraction of fatty acid transport appears to occur via a high affinity, low capacity protein-mediated transport process. Fatty acid transport protein (FATP), an integral membrane protein with four transmembrane segments, is expressed in tissues exhibiting high levels of plasma membrane fatty acid flux, such as muscle, heart, and adipose. Expression of FATP is upregulated in 3T3-L1 cells during adipose conversion, and expression in COS7 fibroblasts elevates uptake of long-chain fatty acids (Hui, T. Y. et al. (1998) J. Biol. Chem. 273:27420-27429). Mitochondrial carrier proteins are transmembrane-spanning proteins which transport ions and charged metabolites between the cytosol and the mitochondrial matrix. Examples include the ADP, ATP carrier protein; the 2-oxoglutarate/malate carrier; the phosphate carrier protein; the pyruvate carrier; the dicarboxylate carrier which transports malate, succinate, fumarate, and phosphate; the tricarboxylate carrier which transports citrate and malate; and the Grave's disease carrier protein, a protein recognized by IgG in patients with active Grave's disease, an autoimmune disorder resulting in hyperthyroidism. Proteins in this family consist of three tandem repeats of an approximately 100 amino acid domain, each of which contains two transmembrane regions (Stryer, L. (1995) Biochemistry, W.H. Freeman and Company, New York N.Y., p. 551; PROSITE PDOC00189 Mitochondrial energy transfer proteins signature; Online Mendelian Inheritance in Man (OMIM) *275000 Graves Disease). This class of transporters also includes the mitochondrial uncoupling proteins, which create proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from ATP synthesis. The result is energy dissipation in the form of heat. Mitochondrial uncoupling proteins have been implicated as modulators of thermoregulation and metabolic rate, and have been proposed as potential targets for drugs against metabolic diseases such as obesity (Ricquier, D. et al. (1999) J. Int. Med. 245:637-642). Ion Channels The electrical potential of a cell is generated and maintained by controlling the movement of ions across the plasma membrane. The movement of ions requires ion channels, which form ion-selective pores within the membrane. There are two basic types of ion channels, ion transporters and gated ion channels. Ion transporters utilize the energy obtained from ATP hydrolysis to actively transport an ion against the ion's concentration gradient. Gated ion channels allow passive flow of an ion down the ion's electrochemical gradient under restricted conditions. Together, these types of ion channels generate, maintain, and utilize an electrochemical gradient that is used in 1) electrical impulse conduction down the axon of a nerve cell, 2) transport of molecules into cells against concentration gradients, 3) initiation of muscle contraction, and 4) endocrine cell secretion. Ion Transporters Ion transporters generate and maintain the resting electrical potential of a cell. Utilizing the energy derived from ATP hydrolysis, they transport ions against the ion's concentration gradient. These transmembrane ATPases are divided into three families. The phosphorylated (P) class ion transporters, including Na + —K + ATPase, Ca 2+ -ATPase, and H + -ATPase, are activated by a phosphorylation event. P-class ion transporters are responsible for maintaining resting potential distributions such that cytosolic concentrations of Na + and Ca 2+ are low and cytosolic concentration of K + is high. The vacuolar (V) class of ion transporters includes H + pumps on intracellular organelles, such as lysosomes and Golgi. V-class ion transporters are responsible for generating the low pH within the lumen of these organelles that is required for function. The coupling factor (F) class consists of H + pumps in the mitochondria. F-class ion transporters utilize a proton gradient to generate ATP from ADP and inorganic phosphate (P i ). The P-ATPases are hexamers of a 100 kD subunit with ten transmembrane domains and several large cytoplasmic regions that may play a role in ion binding (Scarborough, G. A. (1999) Curr. Opin. Cell Biol. 11:517-522). The P-type ATPases include three subfamilies: one involved in transport of heavy metal ions such as Cu 2+ or Cd 2+ across a bilayer, another that transports non-heavy metal ions such as H + , Na + , K + , or Ca 2+ ; and a third recently identified group responsible for transport of amphipathic molecules such as aminophospholipids. Most of these family members are highly expressed in the central nervous system, but have substantially different patterns of expression. One member of this family was identified as the gene mutated in two types of familial inherited cholestasis (Halleck, M. S. et al. (1999) Physiol. Genomics 1:139-150). The V-ATPases are composed of two functional domains: the V 1 domain, a peripheral complex responsible for ATP hydrolysis; and the V 0 domain, an integral complex responsible for proton translocation across the membrane. The F-ATPases are structurally and evolutionarily related to the V-ATPases. The F-ATPase F 0 domain contains 12 copies of the c subunit, a highly hydrophobic protein composed of two transmembrane domains and containing a single buried carboxyl group in TM2 that is essential for proton transport. The V-ATPase V 0 domain contains three types of homologous c subunits with four or five transmembrane domains and the essential carboxyl group in TM4 or TM3. Both types of complex also contain a single a subunit that may be involved in regulating the pH dependence of activity (Forgac, M. (1999) J. Biol. Chem. 274:12951-12954). The resting potential of the cell is utilized in many processes involving carrier proteins and gated ion channels. Carrier proteins utilize the resting potential to transport molecules into and out of the cell. Amino acid and glucose transport into many cells is linked to sodium ion co-transport (symport) so that the movement of Na + down an electrochemical gradient drives transport of the other molecule up a concentration gradient. Similarly, cardiac muscle links transfer of Ca 2+ out of the cell with transport of Na + into the cell (antiport). Gated Ion Channels Gated ion channels control ion flow by regulating the opening and closing of pores. The ability to control ion flux through various gating mechanisms allows ion channels to mediate such diverse signaling and homeostatic functions as neuronal and endocrine signaling, muscle contraction, fertilization, and regulation of ion and pH balance. Gated ion channels are categorized according to the manner of regulating the gating function. Mechanically-gated channels open their pores in response to mechanical stress; voltage-gated channels (e.g., Na + , K + , Ca 2+ , and Cl − channels) open their pores in response to changes in membrane potential; and ligand-gated channels (e.g., acetylcholine-, serotonin-, and glutamate-gated cation channels, and GABA- and glycine-gated chloride channels) open their pores in the presence of a specific ion, nucleotide, or neurotransmitter. The gating properties of a particular ion channel (i.e., its threshold for and duration of opening and closing) are sometimes modulated by association with auxiliary channel proteins and/or post translational modifications, such as phosphorylation. Mechanically-gated or mechanosensitive ion channels act as transducers for the senses of touch, hearing, and balance, and also play important roles in cell volume regulation, smooth muscle contraction, and cardiac rhythm generation. A stretch-inactivated channel (SIC) was recently cloned from rat kidney. The SIC channel belongs to a group of channels which are activated by pressure or stress on the cell membrane and conduct both Ca 2+ and Na + (Suzuki, M. et al. (1999) J. Biol. Chem. 274:6330-6335). The pore-forming subunits of the voltage-gated cation channels form a superfamily of ion channel proteins. The characteristic domain of these channel proteins comprises six transmembrane domains (S1-S6), a pore-forming region (P) located between S5 and S6, and intracellular amino and carboxy termini. In the Na + and Ca 2+ subfamilies, this domain is repeated four times, while in the K + channel subfamily, each channel is formed from a tetramer of either identical or dissimilar subunits. The P region contains information specifying the ion selectivity for the channel. In the case of K + channels, a GYG tripeptide is involved in this selectivity (Ishii, T. M. et al. (1997) Proc. Natl. Acad. Sci. USA 94:11651-11656). Voltage-gated Na + and K + channels are necessary for the function of electrically excitable cells, such as nerve and muscle cells. Action potentials, which lead to neurotransmitter release and muscle contraction, arise from large, transient changes in the permeability of the membrane to Na + and K + ions. Depolarization of the membrane beyond the threshold level opens voltage-gated Na + channels. Sodium ions flow into the cell, further depolarizing the membrane and opening more voltage-gated Na + channels, which propagates the depolarization down the length of the cell. Depolarization also opens voltage-gated potassium channels. Consequently, potassium ions flow outward, which leads to repolarization of the membrane. Voltage-gated channels utilize charged residues in the fourth transmembrane segment (S4) to sense voltage change. The open state lasts only about 1 millisecond, at which time the channel spontaneously converts into an inactive state that cannot be opened irrespective of the membrane potential. Inactivation is mediated by the channel's N-terminus, which acts as a plug that closes the pore. The transition from an inactive to a closed state requires a return to resting potential. Voltage-gated Na + channels are heterotrimeric complexes composed of a 260 kDa pore-forming α subunit that associates with two smaller auxiliary subunits, β1 and β2. The β2 subunit is a integral membrane glycoprotein that contains an extracellular Ig domain, and its association with α and β1 subunits correlates with increased functional expression of the channel, a change in its gating properties, as well as an increase an whole cell capacitance due to an increase in membrane surface area (Isom, L. L. et al. (1995) Cell 83:433-442). Non voltage-gated Na + channels include the members of the amiloride-sensitive Na + channel/degenerin (NaC/DEG) family. Channel subunits of this family are thought to consist of two transmembrane domains flanking a long extracellular loop, with the amino and carboxyl termini located within the cell. The NaC/DEG family includes the epithelial Na + channel (ENaC) involved in Na + reabsorption in epithelia including the airway, distal colon, cortical collecting duct of the kidney, and exocrine duct glands. Mutations in ENaC result in pseudohypoaldosteronism type 1 and Liddle's syndrome (pseudohyperaldosteronism). The NaC/DEG family also includes the recently characterized H + -gated cation channels or acid-sensing ion channels (ASIC). ASIC subunits are expressed in the brain and form heteromultimeric Na + -permeable channels. These channels require acid pH fluctuations for activation. ASIC subunits show homology to the degenerins, a family of mechanically-gated channels originally isolated from C. elegans. Mutations in the degenerins cause neurodegeneration. ASIC subunits may also have a role in neuronal function, or in pain perception, since tissue acidosis causes pain (Waldmann, R. and M. Lazdunski (1998) Curr. Opin. Neurobiol. 8:418-424; Eglen, R. M. et al. (1999) Trends Pharmacol. Sci. 20:337-342). K + channels are located in all cell types, and may be regulated by voltage, ATP concentration, or second messengers such as Ca 2+ and cAMP. In non-excitable tissue, K + channels are involved in protein synthesis, control of endocrine secretions, and the maintenance of osmotic equilibrium across membranes. In neurons and other excitable cells, in addition to regulating action potentials and repolarizing membranes, K + channels are responsible for setting resting membrane potential. The cytosol contains non-diffusible anions and, to balance this net negative charge, the cell contains a Na + —K + pump and ion channels that provide the redistribution of Na + , K + , and Cl − . The pump actively transports Na + out of the cell and K + into the cell in a 3:2 ratio. Ion channels in the plasma membrane allow K + and Cl − to flow by passive diffusion. Because of the high negative charge within the cytosol, Cl − flows out of the cell. The flow of K + is balanced by an electromotive force pulling K + into the cell, and a K + concentration gradient pushing K + out of the cell. Thus, the resting membrane potential is primarily regulated by K + flow (Salkoff, L. and T. Jegla (1995) Neuron 15:489-492). Potassium channel subunits of the Shaker-like superfamily all have the characteristic six transmembrane/1 pore domain structure. Four subunits combine as homo- or heterotetramers to form functional K channels. These pore-forming subunits also associate with various cytoplasmic b subunits that alter channel inactivation kinetics. The Shaker-like channel family includes the voltage-gated K + channels as well as the delayed rectifier type channels such as the human ether-a-go-go related gene (HERG) associated with long QT, a cardiac dysrythmia syndrome (Curran, M. E. (1998) Curr. Opin. Biotechnol. 9:565-572; Kaczorowski, G. J. and M. L. Garcia (1999) Curr. Opin. Chem. Biol. 3:448-458). A second superfamily of K + channels is composed of the inward rectifying channels (Kir). Kir channels have the property of preferentially conducting K + currents in the inward direction. These proteins consist of a single potassium selective pore domain and two transmembrane domains, which correspond to the fifth and sixth transmembrane domains of voltage-gated K + channels. Kir subunits also associate as tetramers. The Kir family includes ROMK1, mutations in which lead to Bartter syndrome, a renal tubular disorder. Kir channels are also involved in regulation of cardiac pacemaker activity, seizures and epilepsy, and insulin regulation (Doupnik, C. A. et al. (1995) Curr. Opin. Neurobiol. 5:268-277; Curran, supra). The recently recognized TWIK K + channel family includes the mammalian TWIK-1, TREK-1 and TASK proteins. Members of this family possess an overall structure with four transmembrane domains and two P domains. These proteins are probably involved in controlling the resting potential in a large set of cell types (Duprat, F. et al. (1997) EMBO J 16:5464-5471). The voltage-gated Ca 2+ channels have been classified into several subtypes based upon their electrophysiological and pharmacological characteristics. L-type Ca 2+ channels are predominantly expressed in heart and skeletal muscle where they play an essential role in excitation-contraction coupling. T-type channels are important for cardiac pacemaker activity, while N-type and P/Q-type channels are involved in the control of neurotransmitter release in the central and peripheral nervous system. The L-type and N-type voltage-gated Ca 2+ channels have been purified and, though their functions differ dramatically, they have similar subunit compositions. The channels are composed of three subunits. The α 1 subunit forms the membrane pore and voltage sensor, while the α 2 δ and β subunits modulate the voltage-dependence, gating properties, and the current amplitude of the channel. These subunits are encoded by at least six α 1 , one α 2 δ, and four β genes. A fourth subunit, γ, has been identified in skeletal muscle (Walker, D. et al. (1998) J. Biol. Chem. 273:2361-2367; McCleskey, E. W. (1994) Curr. Opin. Neurobiol. 4:304-312). The transient receptor family (Trp) of calcium ion channels are thought to mediate capacitative calcium entry (CCE). CCE is the Ca 2+ influx into cells to resupply Ca 2+ stores depleted by the action of inositol triphosphate (IP3) and other agents in response to numerous hormones and growth factors. Trp and Trp-like were first cloned from Drosophila and have similarity to voltage gated Ca2+ channels in the S3 through S6 regions. This suggests that Trp and/or related proteins may form mammalian CCC entry channels (Zhu, X. et al. (1996) Cell 85:661-671; Boulay, G. et al. (1997) J. Biol. Chem. 272:29672-29680). Melastatin is a gene isolated in both the mouse and human, and whose expression in melanoma cells is inversely correlated with melanoma aggressiveness in vivo. The human cDNA transcript corresponds to a 1533-amino acid protein having homology to members of the Trp family. It has been proposed that the combined use of malastatin mRNA expression status and tumor thickness might allow for the determination of subgroups of patients at both low and high risk for developing metastatic disease (Duncan, L. M. et al (2001) J. Clin. Oncol. 19:568-576). Chloride channels are necessary in endocrine secretion and in regulation of cytosolic and organelle pH. In secretory epithelial cells, Cl − enters the cell across a basolateral membrane through an Na + , K + /Cl − cotransporter, accumulating in the cell above its electrochemical equilibrium concentration. Secretion of Cl − from the apical surface, in response to hormonal stimulation, leads to flow of Na + and water into the secretory lumen. The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel encoded by the gene for cystic fibrosis, a common fatal genetic disorder in humans. CFTR is a member of the ABC transporter family, and is composed of two domains each consisting of six transmembrane domains followed by a nucleotide-binding site. Loss of CFTR function decreases transepithelial water secretion and, as a result, the layers of mucus that coat the respiratory tree, pancreatic ducts, and intestine are dehydrated and difficult to clear. The resulting blockage of these sites leads to pancreatic insufficiency, “meconium ileus”, and devastating “chronic obstructive pulmonary disease” (Al-Awqati, Q. et al. (1992) J. Exp. Biol. 172:245-266). The voltage-gated chloride channels (CLC) are characterized by 10-12 transmembrane domains, as well as two small globular domains known as CBS domains. The CLC subunits probably function as homotetramers. CLC proteins are involved in regulation of cell volume, membrane potential stabilization, signal transduction, and transepithelial transport. Mutations in CLC-1, expressed predominantly in skeletal muscle, are responsible for autosomal recessive generalized myotonia and autosomal dominant myotonia congenita, while mutations in the kidney channel CLC-5 lead to kidney stones (Jentsch, T. J. (1996) Curr. Opin. Neurobiol. 6:303-310). Ligand-gated channels open their pores when an extracellular or intracellular mediator binds to the channel. Neurotransmitter-gated channels are channels that open when a neurotransmitter binds to their extracellular domain. These channels exist in the postsynaptic membrane of nerve or muscle cells. There are two types of neurotransmitter-gated channels. Sodium channels open in response to excitatory neurotransmitters, such as acetylcholine, glutamate, and serotonin. This opening causes an influx of Na + and produces the initial localized depolarization that activates the voltage-gated channels and starts the action potential. Chloride channels open in response to inhibitory neurotransmitters, such as γ-aminobutyric acid (GABA) and glycine, leading to hyperpolarization of the membrane and the subsequent generation of an action potential. Neurotransmitter-gated ion channels have four transmembrane domains and probably function as pentamers (Jentsch, supra). Amino acids in the second transmembrane domain appear to be important in determining channel permeation and selectivity (Sather, W. A. et al. (1994) Curr. Opin. Neurobiol. 4:313-323). Ligand-gated channels can be regulated by intracellular second messengers. For example, calcium-activated K + channels are gated by internal calcium ions. In nerve cells, an influx of calcium during depolarization opens K + channels to modulate the magnitude of the action potential (Ishi et al., supra). The large conductance (BK) channel has been purified from brain and its subunit composition determined. The a subunit of the BK channel has seven rather than six transmembrane domains in contrast to voltage-gated K + channels. The extra transmembrane domain is located at the subunit N-terminus. A 28-amino-acid stretch in the C-terminal region of the subunit (the “calcium bowl” region) contains many negatively charged residues and is thought to be the region responsible for calcium binding. The b subunit consists of two transmembrane domains connected by a glycosylated extracellular loop, with intracellular N— and C-termini (Kaczorowski, supra; Vergara, C. et al. (1998) Curr. Opin. Neurobiol. 8:321-329). Cyclic nucleotide-gated (CNG) channels are gated by cytosolic cyclic nucleotides. The best examples of these are the cAMP-gated Na + channels involved in olfaction and the cGMP-gated cation channels involved in vision. Both systems involve ligand-mediated activation of a G-protein coupled receptor which then alters the level of cyclic nucleotide within the cell. CNG channels also represent a major pathway for Ca 2+ entry into neurons, and play roles in neuronal development and plasticity. CNG channels are tetramers containing at least two types of subunits, an a subunit which can form functional homomeric channels, and a b subunit, which modulates the channel properties. All CNG subunits have six transmembrane domains and a pore forming region between the fifth and sixth transmembrane domains, similar to voltage-gated K + channels. A large C-terminal domain contains a cyclic nucleotide binding domain, while the N-terminal domain confers variation among channel subtypes (Zufall, F. et al. (1997) Curr. Opin. Neurobiol. 7:404-412). The activity of other types of ion channel proteins may also be modulated by a variety of intracellular signalling proteins. Many channels have sites for phosphorylation by one or more protein kinases including protein kinase A, protein kinase C, tyrosine kinase, and casein kinase II, all of which regulate ion channel activity in cells. Kir channels are activated by the binding of the Gbg subunits of heterotrimeric G-proteins (Reimann, F. and F. M. Ashcroft (1999) Curr. Opin. Cell. Biol. 11:503-508). Other proteins are involved in the localization of ion channels to specific sites in the cell membrane. Such proteins include the PDZ domain proteins known as MAGUKs (membrane-associated guanylate kinases) which regulate the clustering of ion channels at neuronal synapses (Craven, S. E. and D. S. Bredt (1998) Cell 93:495-498). Disease Correlation The etiology of numerous human diseases and disorders can be attributed to defects in the transport of molecules across membranes. Defects in the trafficking of membrane-bound transporters and ion channels are associated with several disorders, e.g., cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, von Gierke disease, and certain forms of diabetes mellitus. Single-gene defect diseases resulting in an inability to transport small molecules across membranes include, e.g., cystinuria, iminoglycinuria, Hartup disease, and Fanconi disease (van't Hoff, W. G. (1996) Exp. Nephrol. 4:253-262; Talente, G. M. et al. (1994) Ann. Intern. Med. 120:218-226; and Chillon, M. et al. (1995) New Engl. J. Med. 332:1475-1480). Human diseases caused by mutations in ion channel genes include disorders of skeletal muscle, cardiac muscle, and the central nervous system. Mutations in the pore-forming subunits of sodium and chloride channels cause myotonia, a muscle disorder in which relaxation after voluntary contraction is delayed. Sodium channel myotonias have been treated with channel blockers. Mutations in muscle sodium and calcium channels cause forms of periodic paralysis, while mutations in the sarcoplasmic calcium release channel, T-tubule calcium channel, and muscle sodium channel cause malignant hyperthermia. Cardiac arrythmia disorders such as the long QT syndromes and idiopathic ventricular fibrillation are caused by mutations in potassium and sodium channels (Cooper, E. C. and L. Y. Jan (1998) Proc. Natl. Acad. Sci. USA 96:4759-4766). All four known human idiopathic epilepsy genes code for ion channel proteins (Berkovic, S. F. and I. E. Scheffer (1999) Curr. Opin. Neurology 12:177-182). Other neurological disorders such as ataxias, hemiplegic migraine and hereditary deafness can also result from mutations in ion channel genes (Jen, J. (1999) Curr. Opin. Neurobiol. 9:274-280; Cooper, supra). Ion channels have been the target for many drug therapies. Neurotransmitter-gated channels have been targeted in therapies for treatment of insomnia, anxiety, depression, and schizophrenia. Voltage-gated channels have been targeted in therapies for arrythmia, ischemic stroke, head trauma, and neurodegenerative disease (Taylor, C. P. and L. S. Narasimhan (1997) Adv. Pharmacol. 39:47-98). Various classes of ion channels also play an important role in the perception of pain, and thus are potential targets for new analgesics. These include the vanilloid-gated ion channels, which are activated by the vanilloid capsaicin, as well as by noxious heat. Local anesthetics such as lidocaine and mexiletine which blockade voltage-gated Na+channels have been useful in the treatment of neuropathic pain (Eglen, supra). Ion channels in the immune system have recently been suggested as targets for immunomodulation. T-cell activation depends upon calcium signaling, and a diverse set of T-cell specific ion channels has been characterized that affect this signaling process. Channel blocking agents can inhibit secretion of lymphokines, cell proliferation, and killing of target cells. A peptide antagonist of the T-cell potassium channel Kv1.3 was found to suppress delayed-type hypersensitivity and allogenic responses in pigs, validating the idea of channel blockers as safe and efficacious immunosuppressants (Cahalan, M. D. and K. G. Chandy (1997) Curr. Opin. Biotechnol. 8:749-756). Expression Profiling Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder. The discovery of new transporters and ion channels, and the polynucleotides encoding them, satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of transport, muscle, autoimmune/inflammatory, infectious, immune deficiencies, metabolism, reproductive, neurological, cardiovascular, eye, and cell proliferative disorders, including cancer and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of transporters and ion channels. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention features purified polypeptides, transporters and ion channels, referred to collectively as “TRICH” and individually as “TRICH-1,” “TRICH-2,” “TRICH-3,” “TRICH-4,” “TRICH-5,” “TRICH-6,” “TRICH-7,” “TRICH-8,” “TRICH-9,” “TRICH-10,” “TRICH-11,” “TRICH-12,” “TRICH-13,” “TRICH-14,” “TRICH-15,” “TRICH-16,” and “TRICH-17.” In one aspect, the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1-17. The invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-17. In another alternative, the polynucleotide is selected from the group consisting of SEQ ED NO:18-34. Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide. The invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed. Additionally, the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. The invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). In one alternative, the polynucleotide comprises at least 60 contiguous nucleotides. Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof. In one alternative, the probe comprises at least 60 contiguous nucleotides. The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof. The invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional TRICH, comprising administering to a patient in need of such treatment the composition. The invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional TRICH, comprising administering to a patient in need of such treatment the composition. Additionally, the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. In one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional TRICH, comprising administering to a patient in need of such treatment the composition. The invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide. The invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide. The invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. The invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. |
Novel azoles having an insecticidal action |
The present application relates to novel heterocyclic compounds, to processes for their preparation, and to their use as crop protection agents, particularly for controlling animal pests. |
1-8. (canceled). 9. A compound of formula (I) in which A represents substituted aryl or hetaryl or heterocyclyl, R1 represents hydrogen or C1-C3-alkyl, R2 represents hydrogen or C1-C3-alkyl, R3 represents hydrogen or C1-C3-alkyl, R4 represents hydrogen or C1-C3-alkyl, X represents N or CH, Y represents CN or NO2, Z represents CH2, O, S, SO, SO2, or NR5, and R5 represents hydrogen or C1-C3-alkyl. 10. A compound of formula (I) according to claim 9 in which A represents optionally halogen-, cyano-, nitro-, C1-C4-alkyl-, C1-C4-haloalkyl-, C1-C4-alkoxy-, or C1-C4-haloalkoxy-substituted phenyl; represents pyrazolyl, 1,2,4-triazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, 1,2,5-thiadiazolyl, pyridyl, pyrazinyl, or pyrimidinyl, each of which is optionally substituted by fluorine, chlorine, bromine, cyano, nitro, C1-C2-alkyl that is optionally substituted by fluorine and/or chlorine, C1-C2-alkoxy that is optionally substituted by fluorine and/or chlorine, C1-C2-alkylthio that is optionally substituted by fluorine and/or chlorine, or C1-C2-alkylsulfonyl that is optionally substituted by fluorine and/or chlorine; or represents an optionally halogen- or C1-C3-alkyl-substituted saturated C5-C6-cycloalkyl radical in which one methylene group is replaced by O or S, R1 represents hydrogen, methyl, ethyl, n-propyl, or i-propyl, R2 represents hydrogen, methyl, ethyl, n-propyl, or i-propyl, R3 represents hydrogen, methyl, ethyl, n-propyl, or i-propyl, R4 represents hydrogen, methyl, ethyl, n-propyl, or i-propyl, X represents N or CH, Y represents CN or NO2, Z represents CH2, O, S, or NR5, and R5 represents hydrogen, methyl, ethyl, n-propyl, or i-propyl. 11. A compound of formula (I) according to claim 9 in which A represents thiazolyl or pyridyl, each of which is each optionally substituted by halogen or C1-C3-alkyl; or represents an optionally halogen- or C1-C3-alkyl-substituted tetrahydrofuryl radical, R1 represents hydrogen or methyl, R2 represents hydrogen or methyl or ethyl, R3 represents hydrogen or methyl, R4 represents hydrogen or methyl, X represents N or CH, Y represents CN or NO2, Z represents CH2, O, S, or NR5, and R5 represents hydrogen or methyl. 12. A compound of formula (I) according to claim 11 in which A represents thiazolyl or pyridyl substituted by chlorine or methyl, or represents an optionally chlorine- or methyl-substituted tetrahydrofuryl radical. 13. A compound of formula (I) according to claim 9 in which A represents one of the radicals R1 represents hydrogen or methyl, R2 represents methyl or ethyl, R3 represents hydrogen or methyl, R4 represents hydrogen or methyl, X represents N or CH, Y represents CN or NO2, Z represents CH2, O, S, or NR5, and R5 represents hydrogen or methyl. 14. A process for preparing a compound of formula (I) according to claim 9 comprising reacting a compound of formula (II) in which A, R1, R2, R3, R4, X, and Z have the meanings given for formula (I) in claim 9, with a cyanating agent or a nitrating agent. 15. A composition for controlling animal pests comprising one or more compounds of formula (I) according to claim 9. 16. A method for controlling animal pests comprising allowing a compound of formula (I) according to claim 9 to act on the animal pests and/or their habitat. 17. A method for preparing compositions for controlling animal pests comprising mixing a compound of formula (I) according to claim 9 with one or more diluents and/or surfactants. 18. A compound of formula (II) in which A represents substituted aryl or hetaryl or heterocyclyl, R1 represents hydrogen or C1-C3-alkyl, R2 represents hydrogen or C1-C3-alkyl, R3 represents hydrogen or C1-C3-alkyl, R4 represents hydrogen or C1-C3-alkyl, X represents N or CH, and Z represents CH2, O, S, SO, SO2, or NR5. 19. A compound of formula (IX) in which A represents substituted aryl or hetaryl or heterocyclyl, R1 represents hydrogen or C1-C3-alkyl, R2 represents hydrogen or C1-C3-alkyl, R3 represents hydrogen or C1-C3-alkyl, and R4 represents hydrogen or C1-C3-alkyl. |
Controllable oscillator |
Controllable oscillator circuit comprising a regenerative loop which incorporates a cascade circuit of first and second sections each having a controllable gain and a phase shift which is 90° at the oscillation frequency, the first and second sections comprising first and second transconductance amplifiers, respectively, outputs of which are coupled to third and fourth transconductance amplifiers, which are positively fed back from the output to the input, and via first and second capacitors to inputs of the second and first gain controlled amplifiers, said first and second capacitors being coupled in parallel to first and second load resistors, respectively, a tuning control current being supplied to control inputs of the first and second transconductance amplifiers, the output of at least one of the first and second transconductance amplifiers being coupled to an amplitude detection arrangement providing a gain control current for an automatic gain control to control inputs of the third and fourth transconductance amplifiers, said first and third transconductance amplifiers of the first section having a differential pair of first and second output terminals in common, said second and fourth transconductance amplifiers of the second section having a differential pair of third and fourth output terminals in common. In order to provide an oscillator capable of generating sinusoidal signals and tuneable at lower tuning frequencies and operating at lower supply voltages than the above conventional oscillator circuit, said first and second parallel RC filters are coupled between the first and second output terminals and the third and fourth output terminals, respectively, said first to fourth output terminals being respectively coupled to first to fourth DC current paths shunting at least a substantial part of said tuning and gain control currents to a supply voltage. |
1. Controllable oscillator circuit comprising a regenerative loop which incorporates a cascade circuit of first and second sections each having a controllable gain and a phase shift which is 90° at the oscillation frequency, the first and second sections comprising first and second transconductance amplifiers, respectively, outputs of which are coupled to third and fourth transconductance amplifiers, which are positively fed back from the output to the input, and via first and second capacitors to inputs of the second and first gain controlled amplifiers, said first and second capacitors being coupled in parallel to first and second load resistors, respectively, a tuning control current being supplied to control inputs of the first and second transconductance amplifiers, the output of at least one of the first and second transconductance amplifiers being coupled to an amplitude detection arrangement providing a gain control current for an automatic gain control to control inputs of the third and fourth transconductance amplifiers, said first and third transconductance amplifiers of the first section having a differential pair of first and second output terminals in common, said second and fourth transconductance amplifiers of the second section having a differential pair of third and fourth output terminals in common, characterized by said first and second load resistors being coupled between the first and second output terminals and the third and fourth output terminals, respectively, said first to fourth output terminals being respectively coupled to first to fourth DC current paths shunting at least a substantial part of said tuning and gain control currents to a supply voltage. 2. Controllable oscillator circuit according to claim 1, characterized by said first to fourth DC current paths comprising first to fourth controlled DC current sources, respectively, said first and second controlled DC current sources and said third and fourth controlled DC current sources providing DC currents corresponding to substantially half the summation of said tuning and gain control currents of each section. 3. Controllable oscillator circuit according to claim 1, characterized in that the first and second load resistors are constituted by a serial arrangement of a pair of mutually substantially equal resistances, the DC voltage level at the common node between the resistances of said first and second load resistors being negatively fed back to control inputs of the respective first to fourth controlled DC current sources. 4. Controllable oscillator circuit according to claim 3, characterized in that said common node is coupled to an input of a control signal generating device, comprising a comparator for comparing said DC voltage level with a set level to provide a current control signal to the respective first to fourth controlled DC current sources for a negative feedback of signal differences between said DC voltage level and said set level. 5. Controllable oscillator circuit according to claim 2, characterized by gain and tuning current mirror means, inputs thereof being supplied with said gain and tuning control currents, respectively, outputs thereof coupled through summation means to control inputs of said first to fourth controlled DC current sources, respectively. 6. Controllable oscillator circuit according to claim 5, characterized in that the first and second load resistors are provided by a serial arrangement of a pair of mutually substantially equal resistances, the common node between the pair of resistances of said first and second load resistors being coupled to a reference voltage. 7. Controllable oscillator circuit according to claim 4, characterized in that said set level is being supplied through a set voltage level terminal for setting the circuit working point voltage level. 8. Controllable oscillator circuit according to claim 2, characterized in that the first and second load resistors are constituted by a serial arrangement of a pair of mutually substantially equal resistances, the DC voltage level at the common node between the resistances of said first and second load resistors being negatively fed back to control inputs of the respective first to fourth controlled DC current sources. 9. Controllable oscillator circuit according to claim 8, characterized in that said common node is coupled to an input of a control signal generating device, comprising a comparator for comparing said DC voltage level with a set level to provide a current control signal to the respective first to fourth controlled DC current sources for a negative feedback of signal differences between said DC voltage level and said set level. 10. Controllable oscillator circuit according to claim 9, characterized in that said set level is being supplied through a set voltage level terminal for setting the circuit working point voltage level. 11. Controllable oscillator circuit according to claim 6, characterized in that said reference voltage is being supplied through a set voltage level terminal for setting the circuit working point voltage level. |
Filling method, filling machine and wrapping material therefor |
Even if a paper spliced portion is included in the web-shaped packaging material, in filling machine, seal insufficiency is not generated in longitudinal seal of the paper spliced portions, an interruption due to the broken part of continuous operation is prevented. When a using web-shaped packaging material rear end is spliced to a next web-shaped packaging material front end, in order to continuously operate filling machine for paper container, notches are provided on a corner portion of the paper spliced portion around metal layer. |
1. A filling method comprising steps of: splicing a front end portion of a web-shaped packaging material of next reel with a rear end portion of a web-shaped packaging material of a using reel to form a paper spliced portion, unwinding the web-shaped packaging material from the reel continually, longitudinal sealing the web packaging material including a metallic material layer having electromagnetic property to form the web into tube shape, filling liquid food, transversal sealing the tube packaging material to cut the seal and, folding the seal flap to form a paper container, characterized by that the web-shaped packaging material is longitudinal sealed in electromagnetic induction heating to be formed into the tube shape, notches are formed in the metallic material layer of a web edge corner portion heated with electromagnetic induction heating of the paper spliced portion. 2. A filling machine splicing a front end portion of a web-shaped packaging material of next reel with a rear end portion of a web-shaped packaging material of a using reel to form a paper spliced portion, unwinding the web-shaped packaging material from the reel continually, longitudinal sealing the web packaging material including a metallic material layer having electromagnetic property to form the web into tube shape, filling liquid food, transversal sealing the tube packaging material to cut the seal and, folding the seal flap to form a paper container, characterized by that a longitudinal seal apparatus to longitudinal seal the web-shaped packaging material and form into the tube shape with electromagnetic induction heating of one part or all of the metallic material layer is comprised and, a notch formation apparatus forming notches in the metallic material layer of a web edge corner portion heated with electromagnetic induction heating of the paper spliced portion is comprised. 3. Packaging material used by filling machine splicing a front end portion of a web-shaped packaging material of next reel with a rear end portion of a web-shaped packaging material of a using reel to form a paper spliced portion, unwinding the web-shaped packaging material from the reel continually, longitudinal sealing the web packaging material including a metallic material layer having an electromagnetic property to form the web into tube shape, filling liquid food, transversal sealing the tube packaging material to cut the seal and, folding the seal flap to form a paper container, characterized by that notches are formed in the metallic material layer of a web edge corner portion heated with electromagnetic induction heating of the paper spliced portion, 4. A filling method comprising steps of splicing a front end portion of a web-shaped packaging material of next reel with a rear end portion of a web-shaped packaging material of a using reel to form a paper spliced portion, unwinding the web-shaped packaging material from the reel continually, longitudinal sealing the web packaging material including a metallic material layer having an electromagnetic property to form the web into tube shape, filling liquid food, transversal sealing the tube packaging material to cut the seal and, folding the seal flap to form a paper container, characterized by that one part or all of the metallic material layer are heated with electromagnetic induction, an end of the web-shaped packaging material is protected in strip tape with seals, notches are formed in the metallic material layer of a web edge corner portion heated with electromagnetic induction heating of the paper spliced portion. |
<SOH> BACKGROUND ART <EOH>Drink paper container is provided, in filling machine, by unwinding a web-shaped packaging material from a roll or a reel continually, forming as tube shape by longitudinal sealing, filling drink content to seal it, cutting to individual container. Before the web of the roll- or reel-shaped packaging material is used up, the next roll is supplied. The forward packaging material and the backward packaging material are spliced. A paper-spliced portion which is spliced is disposed as a paper container including the paper spliced portion in the filling machine. However, seal insufficiency is generated in longitudinal seal corresponding to the paper-spliced portion and, the part is broken. An interruption of continuous running may be forced to. When the continuous running of filling machine is stopped, for a re-start, considerable time and cost are needed in re-setup of the packaging material and cleaning and sterilization. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 is a plane view showing the paper packaging material of an embodiment of the present invention that a notch is formed by an end portion of the paper spliced portion heated with electromagnetic induction. FIG. 2 is a perspective diagram of an example of paper container provided in accordance with an embodiment of the present invention. FIG. 3 is a schematic view of the filling machine of an embodiment of the present invention. FIG. 4 is an enlarged perspective diagram which explains a longitudinal seal part of a tube-shape web packaging material. FIG. 5 is a sectional view of an edge of a paper spliced portion of the paper packaging material that a notch was formed by an embodiment of the present invention. FIG. 6 is the perspective view which shows the paper packaging material feeding section which splices in the next paper packaging material reel in a paper packaging material reel using in filling machine of an embodiment of the present invention. FIG. 7 is an exploded perspective view showing a splice table to splice the next paper packaging material to a using paper packaging material in the paper packaging material feed portion of the filling machine of an embodiment of the present invention. FIG. 8 is the perspective view which shows a structure of slit device to form notches on a splice table to splice the next paper packaging material to the using paper packaging material in the paper packaging material feed portion of filling machine of an embodiment of the present invention. FIG. 9 is an exploded perspective diagram showing a structure of the slit device shown in FIG. 8 . detailed-description description="Detailed Description" end="lead"? |
Methods for identifying small molecules that bind specific rna structural motifs |
The present invention relates to a method for screening and identifying test compounds that bind to a preselected target ribonucleic acid (“RNA”). Direct, non-competitive binding assays are advantageously used to screen bead-based libraries of compounds for those that selectively bind to a preselected target RNA. Binding of target RNA molecules to a particular test compound is detected using any physical method that measures the altered physical property of the target RNA bound to a test compound. The structure of the test compound attached to the labeled RNA is also determined. The methods used will depend, in part, on the nature of the library screened. The methods of the present invention provide a simple, sensitive assay for high-throughput screening of libraries of compounds to identify pharmaceutical leads. |
1. A method for identifying a test compound that binds to a target RNA molecule, comprising the steps of: (a) contacting a detectably labeled target RNA molecule with a library of solid support-attached test compounds under conditions that permit direct binding of the labeled target RNA to a member of the library of solid support-attached test compounds so that a detectably labeled target RNA:support-attached test compound complex is formed; (b) separating the detectably labeled target RNA:support-attached test compound complex formed in step (a) from uncomplexed target RNA molecules and test compounds; and (c) determining a structure of the test compound of the RNA support-attached test compound complex. 2. The method of claim 1 in which the target RNA molecule contains an TAR element, internal ribosome entry site, “slippery site”, instability element, or ylate uridylate-rich element. 3. The method of claim 1 in which the RNA molecule is an element ed from the mRNA for tumor necrosis factor alpha (“TNF-α), granulocyte- ophage colony stimulating factor (“GM-CSF”), interleukin 2 (“IL-2”), interleukin 6 -6”), vascular endothelial growth factor (“VEGF”), human immunodeficiency virus I V-1”), hepatitis C virus (“HCV”—genotypes 1a & 1b), ribonuclease P RNA aseP”), X-linked inhibitor of apoptosis protein (“XIAP”), or survivin. 4. The method of claim 1 in which the detectably labeled RNA is ed with a fluorescent dye, phosphorescent dye, ultraviolet dye, infrared dye, visible radiolabel, enzyme, spectroscopic colorimetric label, affinity tag, or nanoparticle. 5. The method of claim 1 in which the test compound is selected from a binatorial library of solid support-attached test compounds comprising peptoids; om bio-oligomers; diversomers such as hydantoins, benzodiazepines and dipeptides; gous polypeptides; nonpeptidal peptidominetics; oligocarbamates; peptidyl honates; peptide nucleic acid libraries; antibody libraries; carbohydrate libraries; and organic molecule libraries. 6. The method of claim 5 in which the small organic molecule libraries braries of benzodiazepines, isoprenoids, thiazolidinones, metathiazanones, lidines, morpholino compounds, or diazepindiones. 7. The method of claim 1 in which screening a library of scid support- ed test compounds comprises contacting the test compound with the target nucleic n the presence of an aqueous solution wherein the aqueous solution comprises a buffer combination of salts. 8. The method of claim 7 wherein the aqueous solution approximates or cs physiologic conditions. 9. The method of claim 7 in which the aqueous solution optionally er comprises non-specific nucleic acids comprising DNA, yeast tRNA, salmon sperm , homoribopolymers, and nonspecific RNAs. 10. The method of claim 7 in which the aqueous solution further rises a buffer, a combination of salts, and optionally, a detergent or a surfactant. 11. The method of claim 10 in which the aqueous solution further rises a combination of salts, from about 0 mM to about 100 mM KCl, from about 0o about 1 M NaCl, and from about 0 mM to about 200 mM MgCl2. 12. The method of claim 11 wherein the combination of salts is about nM KCl, 500 mM NaCl, and 10 mM MgCl2. 13. The method of claim 10 wherein the solution optionally comprises about 0.01% to about 0.5% (w/v) of a detergent or a surfactant. 14. The method of claim 1 in which separating the detectably labeled RNA:support-attached test compound complex formed in step (a) from uncomplexed RNA and test compounds is by flow cytometry, affinity chromatography, manual mode separation, suspension of beads in electric fields, or microwave. 15. The method of claim 1 in which the library of solid support-attached mpounds are small organic molecule libraries. 16. The method of claim 15 in which the structure of the test compound rmined by mass spectroscopy, NMR, or vibration spectroscopy. 17. The method of claim 1 in which the library of solid support-attached mpounds are peptide or peptide-based libraries. 18. The method of claim 17 in which the structure of the test compound rmined by Edman degradation. |
<SOH> 2. BACKGROUND OF THE INVENTION <EOH>Protein-nucleic acid interactions are involved in many cellular functions, including transcription, RNA splicing, mRNA decay, and mRNA translation. Readily accessible synthetic molecules that can bind with high affinity to specific sequences of single- or double-stranded nucleic acids have the potential to interfere with these interactions in a controllable way, making them attractive tools for molecular biology and medicine. Successful approaches for blocking function of target nucleic acids include using duplex-forming antisense oligonucleotides (Miller, 1996, Progress in Nucl. Acid Res. & Mol. Biol. 52:261-291; Ojwang & Rando, 1999, Achieving antisense inhibition by oligodeoxynucleotides containing N 7 modified 2′-deoxyguanosine using tumor necrosis factor receptor type 1, METHODS: A Companion to Methods in Enzymology 18:244-251) and peptide nucleic acids (“PNA”) (Nielsen, 1999, Current Opinion in Biotechnology 10:71-75), which bind to nucleic acids via Watson-Crick base-pairing. Triplex-forming anti-gene oligonucleotides can also be designed (Ping et al., 1997, RNA 3:850-860; Aggarwal et al., 1996, Cancer Res. 56:5156-5164; U.S. Pat. No. 5,650,316), as well as pyrrole-imidazole polyamide oligomers (Gottesfeld et al., 1997, Nature 387:202-205; White et al., 1998, Nature 391:468-471), which are specific for the major and minor grooves of a double helix, respectively. In addition to synthetic nucleic acids (i.e., antisense, ribozymes, and triplex-forming molecules), there are examples of natural products that interfere with deoxyribonucleic acid (“DNA”) or RNA processes such as transcription or translation. For example, certain carbohydrate-based host cell factors, calicheamicin oligosaccharides, interfere with the sequence-specific binding of transcription factors to DNA and inhibit transcription in vivo (Ho et al., 1994, Proc. Natl. Acad. Sci. USA 91:9203-9207; Liu et al., 1996, Proc. Natl. Acad. Sci. USA 93:940-944). Certain classes of known antibiotics have been characterized and were found to interact with RNA. For example, the antibiotic thiostreptone binds tightly to a 60-mer from ribosomal RNA (Cundliffe et al., 1990, in The Ribosome: Structure, Function & Evolution (Schlessinger et al., eds.) American Society for Microbiology, Washington, D.C. pp. 479-490). Bacterial resistance to various antibiotics often involves methylation at specific rRNA sites (Cundliffe, 1989, Ann. Rev. Microbiol. 43:207-233). Aminoglycosidic aminocyclitol (aminoglycoside) antibiotics and peptide antibiotics are known to inhibit group I intron splicing by binding to specific regions of the RNA (von Ahsen et al., 1991, Nature (London) 353:368-370). Some of these same aminoglycosides have also been found to inhibit hammerhead ribozyme function (Stage et al., 1995, RNA 1:95-101). In addition, certain aminoglycosides and other protein synthesis inhibitors have been found to interact with specific bases in 16S rRNA (Woodcock et al., 1991, EMBO J. 10:3099-3103). An oligonucleotide analog of the 16S rRNA has also been shown to interact with certain aminoglycosides (Purohit et al., 1994, Nature 370:659-662). A molecular basis for hypersensitivity to aminoglycosides has been found to be located in a single base change in mitochondrial rRNA (Hutchin et al., 1993, Nucleic Acids Res. 21:4174-4179). Aminoglycosides have also been shown to inhibit the interaction between specific structural RNA motifs and the corresponding RNA binding protein. Zapp et al. (Cell, 1993, 74:969-978) has demonstrated that the aminoglycosides neomycin B, lividomycin A, and tobramycin can block the binding of Rev, a viral regulatory protein required for viral gene expression, to its viral recognition element in the IIB (or RRE) region of HIV RNA. This blockage appears to be the result of competitive binding of the antibiotics directly to the RRE RNA structural motif. Single stranded sections of RNA can fold into complex tertiary structures consisting of local motifs such as loops, bulges, pseudoknots, guanosine quartets and turns (Chastain & Tinoco, 1991, Progress in Nucleic Acid Res. & Mol. Biol. 41:131-177; Chow & Bogdan, 1997, Chemical Reviews 97:1489-1514; Rando & Hogan, 1998, Biologic activity of guanosine quartet forming oligonucleotides in “Applied Antisense Oligonucleotide Technology” Stein. & Krieg (eds) John Wiley and Sons, New York, pages 335-352). Such structures can be critical to the activity of the nucleic acid and affect functions such as regulation of mRNA transcription, stability, or translation (Weeks & Crothers, 1993, Science 261:1574-1577). The dependence of these functions on the native three-dimensional structural motifs of single-stranded stretches of nucleic acids makes it difficult to identify or design synthetic agents that bind to these motifs using general, simple-to-use sequence-specific recognition rules for the formation of double- and triple-helical nucleic acids used in the design of antisense and ribozyme type molecules. Approaches to screening generally involve competitive assays designed to identify compounds that disrupt the interaction between a target RNA and a physiological, host cell factor(s) that had been previously identified to specifically interact with that particular target RNA. In general, such assays require the identification and characterization of the host cell factor(s) deemed to be required for the function of the target RNA. Both the target RNA and its preselected host cell binding partner are used in a competitive format to identify compounds that disrupt or interfere with the two components in the assay. Citation or identification of any reference in Section 2 of this application is not an admission that such reference is available as prior art to the present invention. |
<SOH> 3. SUMMARY OF THE INVENTION <EOH>The present invention relates to methods for identifying compounds that bind to preselected target elements of nucleic acids including, but not limited to, specific RNA sequences, RNA structural motifs, and/or RNA structural elements. The specific target RNA sequences, RNA structural motifs, and/or RNA structural elements are used as targets for screening small molecules and identifying those that directly bind these specific sequences, motifs, and/or structural elements. For example, methods are described in which a preselected target RNA having a detectable label is used to screen a library of test compounds, preferably under physiologic conditions. Any complexes formed between the target RNA and a member of the library are identified using methods that detect the labeled target RNA bound to a test compound. In particular, the present invention relates to methods for using a target RNA having a detectable label to screen a bead-based library of test compounds. Compounds in the bead-based library that bind to the labeled target RNA will form a bead-based detectably labeled complex, which can be separated from the unbound beads and unbound target RNA in the liquid phase by a number of physical means, including, but not limited to, flow cytometry, affinity chromatography, manual batch mode separation, suspension of beads in electric fields, and microwave of the bead-based detectably labeled complex. The detectably labeled complex can then be identified by the label on the target RNA and removed from the uncomplexed, unlabeled test compounds in the library. The structure of the test compound complexed with the labeled RNA is then ascertained by de novo structure determination of the test compounds using, for example, mass spectrometry or nuclear magnetic resonance (“NMR”). The test compounds identified are useful for any purpose to which a binding reaction may be put, for example in assay methods, diagnostic procedures, cell sorting, as inhibitors of target molecule function, as probes, as sequestering agents and the like. In addition, small organic molecules which interact specifically with target RNA molecules may be useful as lead compounds for the development of therapeutic agents. The methods described herein for the identification of compounds that directly bind to a particular preselected target RNA are well suited for high-throughput screening. The direct binding method of the invention offers advantages over drug screening systems for competitors that inhibit the formation of naturally-occurring RNA binding protein:target RNA complexes; i.e., competitive assays. The direct binding method of the invention is rapid and can be set up to be readily performed, e.g., by a technician, making it amenable to high throughput screening. The method of the invention also eliminates the bias inherent in the competitive drug screening systems, which require the use of a preselected host cell factor that may not have physiological relevance to the activity of the target RNA. Instead, the methods of the invention are used to identify any compound that can directly bind to specific target RNA sequences, RNA structural motifs, and/or RNA structural elements, preferably under physiologic conditions. As a result, the compounds so identified can inhibit the interaction of the target RNA with any one or more of the native host cell factors (whether known or unknown) required for activity of the RNA in vivo. The present invention may be understood more fully by reference to the detailed description and examples, which are intended to illustrate non-limiting embodiments of the invention. |
Textile strip curtain for car wash systems |
The invention relates to a textile strip curtain for car wash systems, comprising a support rod (3) which can be driven back and forth and on which adjacent strips (1) made of water absorbent material are suspended. In order to increase water absorption in the textile strip curtain without increasing costs, the strips (1) are made of a long pile plush textile fiber material, whereby said plush material has a pile length of more than 10 mm. |
1. Textile strip curtain for car wash systems, comprising a support rod which can be driven back and forth and on which adjacent strips made of a water absorbent material are suspended, characterized in that the strips consist of a long-pile plush made of absorbent textile fibers. 2. Textile strip curtain according to claim 1, characterized in that the absorbent textile fibers have a titer of 0.5 dtex to 5 dtex. 3. Textile strip curtain according to claim 1, characterized in that the absorbent textile fibers are made of viscose, cotton, microfibers, or mixtures of them. 4. Textile strip curtain according to claim 1, characterized in that the cleaning plush has a pile length of more than 10 mm. 5. Textile strip curtain according to claim 1, characterized in that the cleaning plush is knitted goods. |
System for monitoring medical parameters |
Described is a system for monitoring medical parameters of a being, in particular a human being, comprising medical functional means including at least one sensor section for detecting at least one predetermined medical parameter, a transmitting means for transmitting the medical parameter(s) detected by said sensor section, said transmitting means being adapted to be provided at the being, and a remote serving means for receiving and processing the medical parameter(s) from said transmitting means and providing instructions and/or data on the basis of the processed medical parameters. |
1. A system for monitoring medical parameters of a being, in particular a human being, comprising: medical functional means including at least one sensor section for detecting at least one predetermined medical parameter; a transmitting means for transmitting the at least one predetermined medical parameter detected by said at least one sensor section, said transmitting means being adapted to the provided at the being, and, a remote serving means for receiving and processing the at least one predetermined medical parameter from said transmitting means and providing at least one of instructions and data on the basis of the processed medical parameter; characterized in that, said medical functional means is adapted to create pre-alarm signals providing health qualification information, said medical functional means is adapted to transmit said pre-alarm signals, and said remote serving means is adapted to process said pre-alarm signals. 2. The system according to claim 1, characterized in that said remote serving means is adapted to give an alarm in case of a predetermined frequency of said pre-alarm signals. 3. The system according to claim 1, wherein said transmitting means is connected with said remote serving means via a network wireless connection. 4. The system according to claim 1, further comprising a receiving means for receiving the at least one of instructions and data from said remote serving means, said receiving means being adapted to be provided at the being. 5. The system according to claim 4, further comprising a communication device, wherein at least parts of said transmitting means and said receiving means form said communication device. 6. The system according to claim 5, wherein said transmitting means comprises an interface means connected with said communication device for receiving the at least one medical parameter from said medical functional means. 7. The system according to claim 6, wherein said receiving means comprises an interface means connected with said communication device for transmitting the at least one of instructions and data received from remote serving means to said medical functional means. 8. The system according to claim 4, wherein said receiving means is connected with said remote serving means via a network wireless connection. 9. The system according to claim 4, wherein said medical functional means includes a receiving section for wireless connection with said receiving means. 10. The system according to claim 1, wherein said medical functional means includes a transmitting section for wireless connection with said transmitting means. 11. The system according to claim 1, wherein said medical functional means includes a power source. 12. The system according to claim 1, wherein said medical functional means includes a passive telemetry power section for receiving external remote power. 13. The system according to claim 1, wherein said at least one sensor section comprises at least one of a blood gas pulse oximeter analyzer and metabolic parameters pulse oximeter analyzer. 14. The system according to claim 13, wherein said pulse oximeter analyzer locally processes and measures at least one of SO2, PO2, PCO2, PH, HCO3, N2 with at least one of multi wavelength measurements and multivariate analysis. 15. The system according to claim 13, wherein said pulse oximeter analyzer extracts at least one of heart arrhythmia information, beats per minute and relative blood pressure. 16. The system according to claim 1, wherein said at least one sensor section comprises at least one ECG monitor, in the form of two connecting parts, a Laplacian Electrode consumable pad, comprising two or more concentric cyclic electrodes having a shape of at least a portion of a cycle, connecting on top with non consumable self-powered electronic parts, comprising analog, digital, and PAN parts. 17. The system according to claim 16, wherein said digital part filters and locally analyses ECG waveforms, extracting at least one of medical useful information and symptoms, by at least one of standard signal processing, fuzzy logic and neural networks type, detecting known cardiac abnormalities, including at least one of arrhythmia types, early detection of ischemia and deterioration of ischemia. 18. The system according to claim 16, comprising a plurality of ECG monitors, wherein the corresponding waveforms are synchronized by real time clock reference using digital PAN, wherein a multi-waveform ECG is reconstructed and used for at least one of vector, ischemia and other analysis, at least part of which is done remotely by said remote serving means. 19. The system according to claim 1, wherein said at least one sensor section comprises an implantable ECG detector combined with a temperature sensor, getting power from external RFID signals and passively transmitting back at least one of signals and symptoms information processed on site, having internal wire connection to parts of the body. 20. The system according to claim 1, wherein said sensor section comprises a watch inflatable type pressure sensor, which has a wire connected wrist belt ECG electrode for at least one hand. 21. The system according to claim 1, wherein said at least one sensor section comprises an implantable passive telemetry pressure sensor. 22. The system according to claim 1, wherein said at least one sensor section comprises a respiration sensor of inductive plethysmograph type for detecting cardiac output. 23. The system according to claim 1, further comprising: a display means for displaying the at least one of instructions and data provided by said remote serving means. 24. The system according to claim 23, further comprising: a communication device, wherein at least parts of said transmitting means and said receiving means form said communication device, said communication device comprising said display means. 25. The system according to claim 1, wherein said medical functional means comprises an infusion pump, being adjusted so as to be controlled by the at least one of instructions and data provided by said remote serving means. 26. The system according to claim 13, wherein the at least one sensor section comprises a self contained finger ring with a light path to be located under a finger bone, the ring shape disabling rotation, and having light blocking flexible curtains at both sides. 27. The system according to claim 16, wherein the consumable pad is combined with at least one of a reflective type pulse oximeter, strain gauge for breath detection, and temperature sensor. |
Novel method for screening inhibitors of the linkage between the neuronal nitric oxide synthase associated protein and the protein inhibiting neuronal nitric oxide synthase |
The invention concerns a detection procedure for compounds modulating the complexation between neuronal nitric oxide synthase protein (nNOS) or one of its variants and the protein inhibitor of neuronal nitric oxide synthase (PIN), in which: a mixture of the said compound, the PIN and the nNOS or one of its variants is incubated, significant variation in the quantity of complex formed between the PIN and the nNOS or one of its variants with respect to a control value is detected, when there is significant variation as defined above, it is concluded that there is binding between the said compound and the PIN, or between the said compound and the nNOS or one of its variants, leading to modulation of the complexation between the PIN and nNOS or one of its variants. |
1-14. cancel. 15. A method of detecting compounds that modulate the complexation between neuronal nitric oxide synthase protein (nNOS), represented by the sequence SEQ ID NO:2, or one of its variants and the protein inhibitor of neuronal nitric oxide synthase (PIN), the modulation of this complexation causing a modification of the insulin response regulated by nNOS or one of its variants, in which: a mixture of the said compound, the PIN and the nNOS or one of its variants is incubated in conditions that enable the: formation of a complex between the PIN and nNOS or one of its variants, formation of a complex between the said compound and the PIN, or between the said compound and nNOS or one of its variants; any significant variation detected in the quantity of complex formed between the PIN and nNOS or one of its variants with respect to a control value corresponds to: the quantity of complex formed between the PIN and nNOS or one of its variants in the absence of the test compound, or the absence of a complex between the PIN and nNOS or one of its variants, resulting in the absence of PIN or the absence of nNOS or one of its variants, or the quantity of complex formed between the PIN and nNOS or one of its variants in the presence of a reference inhibitor; and when there is significant variation as defined above, it is concluded that there was binding between the said compound and the PIN or between the said compound and the nNOS or one of its variants, leading to modulation of the complexation between the PIN and nNOS or one of its variants. 16. The method of claim 15, wherein the compound does not substantially modify the catalytic activity of the nNOS or one of its variants. 17. A method for detecting compounds that decrease the complexation between neuronal nitric oxide synthase (nNOS) or one of its variants, and the protein inhibitor of neuronal nitric oxide synthase (PIN), the decrease in this complexation leading to a reduction in the insulin response regulated by the nNOS or one of its variants, in which: a mixture of the said compound, the PIN and the nNOS or one of its variants is incubated in conditions that enable the: formation of a complex between the PIN and nNOS or one of its variants, formation of a complex between the said compound and the PIN, or between the said compound and the nNOS or one of its variants; any significant decrease detected in the quantity of complex formed between the PIN and nNOS or one of its variants with respect to a control value corresponds to: the quantity of complex formed between the PIN and nNOS or one of its variants in the absence of the compound submitted to the detection procedure, or the absence of complex formed between the PIN and nNOS or one of its variants, resulting in the absence of PIN or the absence of nNOS or one of its variants, or the quantity of complex formed between the PIN and nNOS or one of its variants in the presence of a reference inhibitor; and when there is significant decrease as defined above, it is concluded that there was binding between the said compound and the PIN, or between the said compound and the nNOS or one of its variants, leading to reduction of the complexation between the PIN and nNOS or one of its variants. 18. The method of claim 15 or claim 17, in which variation is detected, specifically any significant decrease in the quantity of complex formed between the PIN and nNOS, with respect to a first, second, and third control value, one of these control values corresponding to the quantity of complex formed between the PIN and nNOS in the absence of the compound submitted to the detection procedure; another to the absence of complex between the PIN and nNOS, resulting in either the absence of PIN or the absence of nNOS; and another to the quantity of complex formed between the PIN and nNOS in the presence of a reference inhibitor. 19. The method of claim 15 or claim 17, in which the mixture of the PIN, nNOS, and the compound submitted to the detection procedure is prepared by: simultaneously adding the PIN, nNOS, and the compound submitted to the detection procedure, or consecutively adding the PIN, the compound submitted to the detection procedure, and the nNOS, or consecutively adding the nNOS, the compound submitted to the detection procedure, and the PIN, or adding the compound previously incubated with the PIN or the nNOS, to the nNOS protein or the PIN, respectively. 20. The method of claim 15 or claim 17, in which the nNOS protein is first fixed on a solid substrate. 21. The method of claim 15 or claim 17, in which the PIN is first fixed on a solid substrate. 22. The method of claim 15 or claim 17, in which the PIN and nNOS are in solution. 23. A protein characterized in that it contains or is constituted by the sequence of SEQ ID NO:2, or fragment of said protein comprising at least 100 amino acids, on condition the said fragment contains the amino acid in position (269). 24. A peptide having the sequence: Lys-Asp-Thr-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 3) Trp-Asp, Ile-Asp-Val-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 4) Trp-Asp, Cys-Asp-Thr-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 5) Arg-Asp, Ile-Asp-Thr-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 6) Arg-Asp, Val-Asp-Thr-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 7) Arg-Asp, Lys-Asp-Ala-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 8) Arg-Asp, Lys-Asp-Cys-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 9) Arg-Asp, Lys-Asp-Glu-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 10) Arg-Asp, Lys-Asp-Ile-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 11) Arg-Asp, Lys-Asp-Lys-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 12) Arg-Asp, Lys-Asp-Phe-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 13) Arg-Asp, Lys-Asp-Val-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 14) Arg-Asp, Lys-Asp-Thr-Gly-Ile-Gln-Thr-Asp- (SEQ ID NO: 15) Arg-Asp, Lys-Asp-Thr-Gly-Ile-Gln-Val-Cys- (SEQ ID NO: 16) Arg-Asp, Lys-Asp-Thr-Gly-Ile-Gln-Val-Asn- (SEQ ID NO: 17) Arg-Asp, Lys-Asp-Thr-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 18) Leu-Asp, Lys-Asp-Thr-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 19) Cys-Asp, Lys-Asp-Thr-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 20) Phe-Asp, Lys-Asp-Thr-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 21) Tyr-Asp, Lys-Asp-Thr-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 22) Arg-Phe, Lys-Asp-Thr-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 23) Arg-Trp, Val-Asp-Thr-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 24) Arg-Tyr, Ile-Asp-Val-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 25) Trp-Trp, Ile-Asp-Val-Gly-Ile-Gln-Thr-Asp- (SEQ ID NO: 26) Trp-Asp, Ile-Asp-Val-Gly-Ile-Gln-Thr-Asp- (SEQ ID NO: 27) Trp-Trp, Ile-Asp-Val-Gly-Ile-Gln-Thr-Cys- (SEQ ID NO: 28) Trp-Trp, Cys-Asp-Thr-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 29) Trp-Asp, Ile-Asp-Thr-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 30) Trp-Asp, Val-Asp-Thr-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 31) Trp-Asp, Lys-Asp-Val-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 32) Trp-Asp, Cys-Asp-Val-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 33) Trp-Asp, Val-Asp-Val-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 34) Trp-Asp, Cys-Asp-Ile-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 35) Trp-Asp, Ile-Asp-Ile-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 36) Trp-Asp, Val-Asp-Ile-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 37) Trp-Asp, Lys-Asp-Phe-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 38) Trp-Asp, Cys-Asp-Phe-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 39) Trp-Asp, Ile-Asp-Phe-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 40) Trp-Asp, Val-Asp-Phe-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 41) Trp-Asp, Cys-Asp-Glu-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 42) Trp-Asp, Ile-Asp-Glu-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 43) Trp-Asp, Val-Asp-Glu-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 44) Trp-Asp, Lys-Asp-Cys-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 45) Trp-Asp, Cys-Asp-Cys-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 46) Trp-Asp, Ile-Asp-Cys-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 47) Arg-Asp, Val-Asp-Cys-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 48) Arg-Asp, His-Asp-Val-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 49) Trp-Asp, Ser-Asp-Val-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 50) Trp-Asp, Thr-Asp-Val-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 51) Trp-Asp, Lys-Glu-Val-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 52) Trp-Asp, Lys-Asp-Ile-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 53) Trp-Asp, Lys-Asp-Glu-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 54) Trp-Asp, Lys-Asp-Gln-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 55) Trp-Asp, Lys-Asp-Val-Ala-Ile-Gln-Val-Asp- (SEQ ID NO: 56) Trp-Asp, Lys-Asp-Val-Gly-Val-Gln-Val-Asp- (SEQ ID NO: 57) Trp-Asp, Lys-Asp-Val-Gly-Thr-Gln-Val-Asp- (SEQ ID NO: 58) Trp-Asp, Lys-Asp-Val-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 59) Ile-Asp, Lys-Asp-Val-Gly-Ile-Gln-Val-Asp- (SEQ ID NO: 60) Trp-Glu, Ala-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 61) Leu-Asn, Arg-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 62) Leu-Asn, Asn-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 63) Leu-Asn, Asp-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 64) Leu-Asn, Gln-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 65) Leu-Asn, Gly-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 66) Leu-Asn, Pro-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 67) Leu-Asn, Ser-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 68) Leu-Asn, Thr-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 69) Leu-Asn, Glu-Phe-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 70) Leu-Asn, Glu-Ile-Asn-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 71) Leu-Asn, Glu-Ile-Asp-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 72) Leu-Asn, Glu-Ile-Cys-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 73) Leu-Asn, Glu-Ile-Gln-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 74) Leu-Asn, Glu-Ile-Glu-Ala-Val-Leu-Ser-Ile- (SEQ ID NO: 75) Leu-Asn, Glu-Ile-Glu-Arg-Val-Leu-Ser-Ile- (SEQ ID NO: 76) Leu-Asn, Glu-Ile-Glu-Asn-Val-Leu-Ser-Ile- (SEQ ID NO: 77) Leu-Asn, Glu-Ile-Glu-Asp-Val-Leu-Ser-Ile- (SEQ ID NO: 78) Leu-Asn, Glu-Ile-Glu-Gln-Val-Leu-Ser-Ile- (SEQ ID NO: 79) Leu-Asn, Glu-Ile-Glu-Glu-Val-Leu-Ser-Ile- (SEQ ID NO: 80) Leu-Asn, Glu-Ile-Glu-Gly-Val-Leu-Ser-Ile- (SEQ ID NO: 81) Leu-Asn, Glu-Ile-Glu-His-Val-Leu-Ser-Ile- (SEQ ID NO: 82) Leu-Asn, Glu-Ile-Glu-Lys-Val-Leu-Ser-Ile- (SEQ ID NO: 83) Leu-Asn, Glu-Ile-Glu-Met-Val-Leu-Ser-Ile- (SEQ ID NO: 84) Leu-Asn, Glu-Ile-Glu-Ser-Val-Leu-Ser-Ile- (SEQ ID NO: 85) Leu-Asn, Glu-Ile-Glu-Thr-Val-Leu-Ser-Ile- (SEQ ID NO: 86) Leu-Asn, Glu-Ile-Glu-Pro-Ile-Leu-Ser-Ile- (SEQ ID NO: 87) Leu-Asn, Glu-Ile-Glu-Pro-Val-Pro-Ser-Ile- (SEQ ID NO: 88) Leu-Asn, Glu-Ile-Glu-Pro-Val-Leu-Ala-Ile- (SEQ ID NO: 89) Leu-Asn, Glu-Ile-Glu-Pro-Val-Leu-Val-Ile- (SEQ ID NO: 90) Leu-Asn, Glu-Ile-Glu-Pro-Val-Leu-Ser-Leu- (SEQ ID NO: 91) Leu-Asn, Glu-Ile-Glu-Pro-Val-Leu-Ser-Phe- (SEQ ID NO: 92) Leu-Asn, Glu-Ile-Glu-Pro-Val-Leu-Ser-Trp- (SEQ ID NO: 93) Leu-Asn, Glu-Ile-Glu-Pro-Val-Leu-Ser-Tyr- (SEQ ID NO: 94) Leu-Asn, Glu-Ile-Glu-Pro-Val-Leu-Ser-Val- (SEQ ID NO: 95) Leu-Asn, Glu-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 96) Leu-Ala, Glu-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 97) Leu-Asp, Glu-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 98) Leu-Gln, Glu-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 99) Leu-Glu, Glu-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 100) Leu-Gly, Glu-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 101) Leu-His, Glu-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 102) Leu-Met, Glu-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 103) Leu-Pro, Glu-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 104) Leu-Ser, Glu-Ile-Glu-Pro-Val-Leu-Ser-Ile- (SEQ ID NO: 105) Leu-Thr, or Glu-Ile-Glu-Asp-Val-Leu-Ser-Phe- (SEQ ID NO: 106) Leu-Gly. 25. A nucleic acid molecule encoding a protein, a protein fragment, or a peptide of claim 23 or claim 24, said nucleic acid having the nucleotide sequence SEQ ID NO:1. 26. A pharmaceutical composition comprising a protein or protein fragment of claim 23, or a molecule with the following formula: in association with an acceptable pharmaceutical vehicle. 27. A method of treating prediabetes, hyperinsulinemia, or type II diabetes comprising administration of a protein or protein fragment of claim 23, or a molecule with the following formula: 28. A kit for detecting a compound that reduces complexation between the PIN and nNOS that contains the following: the pancreatic form of nNOS, PIN, media or buffers needed for dilution, materials needed for washing, as necessary media or buffers needed for the formation of a complex between the PIN and nNOS, and the formation of a complex between the PIN or the nNOS and the compound submitted to the detection procedure, and means to detect variation, specifically a decrease in the quantity of complex formed with the nNOS or with the PIN. 29. A method of preventing or treating prediabetes, hyperinsulinemia, or type II diabetes in a patient, said method comprising administering to the patient a compound that interferes with the binding of nNOS to PIN. |
Transgenic non-human animals for pharmacological and toxicological studies |
The present invention is directed to the production, breeding and use of transgenic non-human animals such as mice in which specific genes or portions of genes have been replaced by homologues from another animal to make the physiology of the animals so modified more like that of the other animal with respect to drug pharmacokinetics and metabolism. The invention also extends to the use of the genetically modified non-human animals of the invention for pharmacological and/or toxicological studies. |
1.-43. (canceled) 44. A transgenic non-primate mammal for predicting the likely behaviour of a drug in a selected species of primate, the transgenic mammal expressing at least a portion of a foreign drug-binding polypeptide that is expressed naturally in the selected species of primate or in a primate of a different species, or that otherwise corresponds to the naturally expressed polypeptide, wherein the expression of an endogenous homologue of the foreign polypeptide in the transgenic mammal is abrogated or otherwise reduced. 45. The transgenic mammal of claim 44, wherein the drug-binding polypeptide is selected from a serum albumin and an a-acidic glycoprotein (AGP). 46. The transgenic mammal of claim 45, wherein the selected species of primate is human and the drug-binding polypeptide is human serum albumin. 47. The transgenic mammal of claim 45, wherein the selected species of primate is human and the drug-binding polypeptide is a human α-acidic glycoprotein (AGP) selected from the group consisting of: AGP-1, AGP-2 and AGP-3. 48. The transgenic mammal of claim 44, wherein the transgenic mammal further expresses at least a portion of at least one other foreign polypeptide that is associated with drug behaviour and/or metabolism and that is expressed naturally in the selected species of primate or in a primate of a different species, or that otherwise corresponds to the naturally expressed polypeptide, wherein the expression of a respective endogenous homologue of the corresponding other foreign polypeptide in the transgenic mammal is abrogated or otherwise reduced. 49. The transgenic mammal of claim 48, wherein the or each other foreign polypeptide is selected from a drug-binding polypeptide, a drug-metabolising polypeptide, a drug-binding and a drug-metabolising polypeptide, or a drug-transporting polypeptide. 50. The transgenic mammal of claim 48, wherein the or each other foreign polypeptide is selected from a serum albumin, an α-acidic glycoprotein (AGP), a cytochrome p450 (CYP), a uridine diphosphoglucuronosyl transferase (UGT), a multidrug-resistance (MDR) protein including multidrug-resistance-associated proteins (MRPs), an acetyl-transferase, a prenyl protein transferase, a peptidase, an esterase, an acetylase, a glucuronidase, a glutathione S-transferase, or a polypeptide that facilitates or catalyses a reaction selected from an oxidative reaction, a conjugation reaction, a hydrolytic reaction, a reductive metabolism or other catabolic or anabolic reaction involving a xenobiotic. 51. The transgenic mammal of claim 44, wherein the drug-binding polypeptide is selected from a serum albumin and an a-acidic glycoprotein (AGP) and wherein the transgenic mammal further expresses at least a portion of at least one other foreign polypeptide that is associated with drug behaviour and/or metabolism, that is expressed naturally in the selected species of primate or in a primate of a different species, or that otherwise corresponds to the naturally expressed polypeptide, and that is selected from a cytochrome p450 (CYP), a uridine diphosphoglucuronosyl transferase (UGT) or a multidrug-resistance protein, wherein the expression of a respective endogenous homologue of the corresponding other foreign polypeptide in the transgenic mammal is abrogated or otherwise reduced. 52. The transgenic mammal of claim 51, wherein the selected species of primate is human and at least one of the other foreign polyp eptides is a human cytochrome p450 (CYP) selected from the CYP 1, CYP 2, CYP 3 or CYP 4 families. 53. The transgenic mammal of claim 51, wherein the selected species of primate is human and at least one of the other foreign polypeptides is a human cytochrome p450 (CYP) selected from CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4A9 or CYP4A11. 54. The transgenic mammal of claim 51, wherein the selected species of primate is human and at least one of the other foreign polypeptides is a human uridine diphosphoglucuronosyl transferase (UGT). 55. The transgenic mammal of claim 51, wherein the selected species of primate is human and at least one of the other foreign polypeptides is human MDR1 (P-glycoprotein) or MRP1. 56. The transgenic mammal of claim 44, wherein the homologue is an orthologue. 57. The transgenic mammal of claim 44, wherein the homologue is a paralogue. 67. The transgenic mammal of claim 58, wherein the alteration comprises a disruption in an endogenous gene encoding the endogenous homologue, wherein the disruption comprises a deletion of the entire open reading frame encoding the endogenous homologue. 68. The transgenic mammal of claim 44, whose genome comprises a nucleotide sequence encoding the foreign polypeptide, which is operably linked to a regulatory polynucleotide. 69. The transgenic mammal of claim 68, wherein the regulatory polynucleotide is a polynucleotide that is naturally present in the transgenic mammal or in the selected species of primate. 70. The transgenic mammal of claim 68, wherein the regulatory polynucleotide is an endogenous polynucleotide of the transgenic mammal, or an ancestor thereof. 71. The transgenic mammal of claim 68, wherein the regulatory polynucleotide is an endogenous polynucleotide of the selected species of primate. 72. The transgenic mammal of claim 68, wherein the regulatory polynucleotide comprises a nucleotide sequence that is naturally operably connected to the coding sequence of an endogenous gene encoding an endogenous polypeptide that is a homologue of the foreign polypeptide. 73. The transgenic mammal of claim 68, wherein the regulatory polynucleotide comprises a nucleotide sequence that is naturally operably connected to the coding sequence of a gene encoding the foreign polypeptide. 74. The transgenic mammal of claim 68, wherein the regulatory polynucleotide is derived from an animal or source other than an animal selected from the transgenic mammal, an ancestor of the transgenic mammal or the selected species of primate. 75. The transgenic mammal of claim 68, wherein the regulatory polynucleotide comprises an inducible promoter. 76. The transgenic mammal of claim 58, wherein the alteration has been introduced into the genome of the transgenic mammal by homologous recombination, random integration or the use of a recombinase system, together with a nucleic acid construct, comprising a transgene that comprises a nucleotide sequence encoding the foreign polypeptide, in an embryonic stem cell such that the construct is stably integrated in the genome of the mammal. 77. The transgenic mammal of claim 58, wherein the transgenic mammal is heterozygous for a transgene that comprises a nucleotide sequence encoding at least a portion of the foreign polypeptide. 78. The transgenic mammal of claim 58, wherein the transgenic mammal is homozygous for a transgene that comprises a nucleotide sequence encoding at least a portion of the foreign polypeptide. 79. The transgenic mammal of claim 44, wherein the transgenic mammal is selected from the order Rodentia. 80. The transgenic mammal of claim 79, wherein the transgenic mammal is a mouse. 81. The transgenic mammal of claim 44, wherein the selected species of primate is human. 82. The transgenic mammal of claim 44, wherein the transgenic mammal is a mouse and the selected species of primate is human. 83. A method of predicting the likely behaviour of a drug in a selected species of primate, as part of a drug screening or evaluation process, comprising administering the drug to a transgenic non-primate mammal expressing at least a portion of a foreign polypeptide that is associated with drug behaviour and/or metabolism, and that is expressed naturally in the selected species of primate or in a primate of a different species, or that otherwise corresponds to the naturally expressed polypeptide, wherein the expression of an endogenous homologue of the foreign polypeptide in the transgenic mammal is abrogated or otherwise reduced, and wherein the foreign polypeptide is other than the intended target of the drug, and conducting analytical tests to determine the behaviour of the drug in the transgenic mammal, the results of which have a higher correlation to the behaviour of the drug in the selected species of primate than the results obtained from a mammal of the same species as the transgenic mammal, which expresses the endogenous homologue but which does not express the foreign polypeptide or portion thereof. 84. The method of claim 83, wherein the analytical test comprises assessing directly or indirectly, a concentration and/or distribution of the drug in the transgenic mammal to which it has been administered. 85. The method of claim 83, wherein the analytical test comprises assessing directly or indirectly, a efficacy of the drug in the transgenic mammal to which it has been administered. 86. The method of claim 83, wherein the analytical test comprises assessing directly or indirectly, a toxicity of the drug in the transgenic mammal to which it has been administered. 87. The method of claim 83, wherein the analytical test comprises assessing directly or indirectly, a half-life of the drug in the transgenic mammal to which it has been administered. 88. The method of claim 83, wherein the analytical test comprises assessing directly or indirectly, a pharmacodynamics of the drug in the transgenic mammal to which it has been administered. 89. The method of claim 83, wherein the analytical test comprises assessing directly or indirectly, a pharmacokinetics of the drug in the transgenic mammal to which it has been administered. 90. The method of claim 83, wherein the analytical test is at least part of a drug-screening process. 91. The method of claim 83, wherein the analytical test is at least part of a preclinical assessment of a drug. 92. The method of claim 83, wherein the analytical test is at least part of a drug-selection process. 93. The method of claim 83, wherein the foreign polypeptide is a drug-binding polypeptide. 94. The method of claim 93, wherein the drug-binding polypeptide is selected from a serum albumin and an a-acidic glycoprotein (AGP). 95. The method of claim 93, wherein the selected species of primate is human and the drug-binding polypeptide is human serum albumin. 96. The method of claim 95, wherein the selected species of primate is human and the drug-binding polypeptide is a human a-acidic glycoprotein (AGP) selected from AGP-1, AGP-2 and AGP-3. 97. The method of claim 83, wherein the foreign polypeptide is a drug-binding polypeptide selected from a serum albumin and an a-acidic glycoprotein (AGP) and the transgenic mammal further expresses at least a portion of at least one other foreign polypeptide that is associated with drug behaviour and/or metabolism and that is expressed naturally in the selected species of primate or in a primate of a different species, or that otherwise corresponds to the naturally expressed polypeptide, wherein the expression of a respective endogenous homologue of the corresponding other foreign polypeptide in the transgenic mammal is abrogated or otherwise reduced. 98. The method of claim 97, wherein the or each other foreign polypeptide is selected from another drug-binding polypeptide, a drug-metabolising polypeptide, a drug-binding and drug-metabolising polypeptide or a drug-transporting polypeptide. 99. The method of claim 97, wherein the or each other foreign polypeptide is selected from a serum albumin, an α-acidic glycoprotein (AGP), a cytochrome p450 (CYP), a uridine diphosphoglucuronosyl transferase (UGT), a multidrug-resistance (MDR) protein including multidrug-resistance-associated proteins (MRPs), an acetyltransferase, a prenyl protein transferase, a peptidase, an esterase, an acetylase, a glucuronidase, a glutathione S-transferase, or a polypeptide that facilitates or catalyses a reaction selected from an oxidative reaction, a conjugation reaction, a hydrolytic reaction, a reductive metabolism or other catabolic or anabolic reaction involving a xenobiotic. 100. The method of claim 83, wherein the foreign polypeptide is a drug-binding polypeptide and the transgenic mammal further expresses at least a portion of at least one other foreign polypeptide that is associated with drug behaviour and/or metabolism and that is expressed naturally in the selected species of primate or in a primate of a different species, or that otherwise corresponds the naturally expressed polypeptide, and that is selected from a cytochrome p450 (CYP), a uridine diphosphoglucuronosyl transferase (UGT) or a multidrug-resistance protein, wherein the expression of a respective endogenous homologue of the corresponding other foreign polypeptide in the transgenic mammal is abrogated or otherwise reduced. 101. The method of claim 100, wherein the selected species of primate is human and the drug-binding polypeptide is human serum albumin. 102. The method of claim 100, wherein the selected species of primate is human and the drug-binding polypeptide is a human a-acidic glycoprotein (AGP) selected from AGP-1, AGP-2 and AGP-3. 103. The method of claim 100, wherein the selected species of primate is human and at least one of the other foreign polypeptides is a human cytochrome p450 (CYP) selected from the CYP 1, CYP 2, CYP 3 or CYP 4 families. 104. The method of claim 103, wherein the selected species of primate is human and at least one of the other foreign polypeptides is a human cytochrome p450 (CYP) selected from CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4A9 or CYP4A11. 105. The method of claim 100, wherein the selected species of primate is human and at least one of foreign polypeptides is a human uridine diphosphoglucuronosyl transferase (UGT). 106. The method of claim 100, wherein the selected species of primate is human and at least one of the foreign polypeptides is human MDR1 (P-glycoprotein) or MRP1. 107. The method of claim 83, wherein the foreign polypeptide is a functional homologue of the endogenous polypeptide. 108. The method of claim 83, wherein the homologue is an orthologue. 109. The method of claim 83, wherein the homologue is a paralogue. 110. The method of claim 83, wherein the transgenic mammal comprises an alteration to its genome. 111. The method of claim 110, wherein the alteration comprises replacement of at least a portion of an endogenous gene encoding the endogenous homologue with a transgene comprising a nucleotide sequence encoding at least a portion of the foreign polypeptide. 112. The method of claim 100, wherein the alteration comprises a disruption in an endogenous gene encoding the endogenous homologue and insertion into the genome of a transgene comprising a nucleotide sequence encoding at least a portion of the foreign polypeptide. 113. The method of claim 110, wherein the alteration comprises a disruption in an endogenous gene encoding the endogenous homologue, wherein the disruption results in reduced expression levels of the endogenous homologue. 114. The method of claim 110, wherein the alteration comprises a disruption in an endogenous gene encoding the endogenous homologue, wherein the disruption results in abrogated expression levels of the endogenous homologue. 115. The method of claim 110, wherein the alteration comprises a disruption in an endogenous gene encoding the endogenous homologue, wherein the mammal lacks the ability to produce a functional endogenous homologue. 116. The method of claim 110, wherein the alteration comprises a disruption in an endogenous gene encoding the endogenous homologue, wherein the disruption comprises a deletion of at least a portion of the endogenous gene. 117. The method of claim 110, wherein the alteration comprises a disruption in an endogenous gene encoding the endogenous polypeptide, wherein the disruption comprises a deletion of nucleotide sequences encoding a region or domain of the endogenous homologue. 118. The method of claim 110, wherein the alteration comprises a disruption in an endogenous gene encoding the endogenous homologue, wherein the disruption comprises a deletion of a regulatory polynucleotide that controls at least in part the expression of the endogenous gene. 119. The method of claim 110, wherein the alteration comprises a disruption in an endogenous gene encoding the endogenous homologue, wherein the disruption comprises a deletion of the entire open reading frame encoding the endogenous homologue. 120. The method of claim 110, wherein the genome of the transgenic mammal comprises a nucleotide sequence encoding the foreign polypeptide, which is operably linked to a regulatory polynucleotide. 121. The method of claim 120, wherein the regulatory polynucleotide is a polynucleotide that is naturally present in the transgenic mammal or in the selected species of primate. 122. The method of claim 120, wherein the regulatory polynucleotide is an endogenous polynucleotide of the transgenic mammal, or ancestor thereof. 123. The method of claim 120, wherein the regulatory polynucleotide is an endogenous polynucleotide of the selected species of primate. 124. The method of claim 120, wherein the regulatory polynucleotide comprises a nucleotide sequence that is naturally operably connected to the coding sequence of an endogenous gene encoding an endogenous polypeptide that is a homologue of the foreign polypeptide. 125. The method of claim 120, wherein the regulatory polynucleotide comprises a nucleotide sequence that is naturally operably connected to the coding sequence of a gene encoding the foreign polypeptide. 126. The method of claim 120, wherein the regulatory polynucleotide is derived from an animal or source other than an animal selected from the transgenic mammal, an ancestor of the transgenic mammal or the selected species of primate. 127. The method of claim 120, wherein the regulatory polynucleotide comprises an inducible promoter. 128. The method of claim 110, wherein the alteration has been introduced into the genome of the transgenic mammal by homologous recombination, random integration or the use of a recombinase system, together with a nucleic acid construct, comprising a transgene that comprises a nucleotide sequence encoding the foreign polypeptide, in an embryonic stem cell such that the construct is stably integrated in the genome of the mammal. 129. The method of claim 128, wherein said recombinase system is a Cre-loxP or FLP-FRT system. 130. The method of claim 110, wherein the transgenic mammal is heterozygous for a transgene that comprises a nucleotide sequence encoding at least a portion of the foreign polypeptide. 131. The method of claim 110, wherein the transgenic mammal is homozygous for a transgene that comprises a nucleotide sequence encoding at least a portion of the foreign polypeptide. 132. The method of claim 83, wherein the transgenic mammal is selected from the order Rodentia. 133. The method of claim 132, wherein the transgenic mammal is a mouse. 134. The method of claim 83, wherein the selected species of primate is human. 135. The method of claim 83, wherein the transgenic mammal is a mouse and the selected species of primate is human. 136. The transgenic mammal of claim 76, wherein said recombinase system is a Cre-loxP or FLP-FRT system. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The cost of bringing a new drug to the market is extremely high. Typically, a pharmaceutical company will screen hundreds to hundreds of thousands of compounds, in order to choose a single drug for marketing. Initial screening is performed in vitro with the most promising compounds progressing to animal studies. It is on the basis of these animal studies that the best drug(s) is chosen for further development and clinical trials. Since a considerable amount of the cost associated with drug development occurs subsequent to the animal studies, the accuracy of the animal model at predicting a drug's behaviour in humans, is of obvious importance. During drug discovery and development, animal models are used in an iterative process of characterising the drug candidates. Initial animal studies determine the pharmacokinetics (the kinetics of drug absorption, distribution throughout the body and its eventual elimination from the body). Subsequent animal studies measure pharmacodynamics (mechanisms of drug action, and the relationship between drug concentration and effect). Typically, these studies also look at efficacy (e.g. does the compound block tumour growth, or is the compound effective in combating neurological disorders), short-term toxicity, optimal dosing and scheduling etc. Based on these animal studies, the most promising compound(s) is further developed. This stage involves continued animal studies (e.g. longer term toxicity studies, exhaustive metabolic studies, multiple-administration pharmacokinetic studies, expanded efficacy studies often including drug combination studies), chemical development (e.g. production) and pharmaceutical development (e.g. drug formulation and delivery). Compounds that successfully complete all of these stages are then tested in human patients (Phase I-III clinical trials). A successful Phase I trial would demonstrate good tolerability, suitable pharmacokinetics, and in some cases demonstrate the intended pharmacodynamic properties in humans. Such a drug would progress to Phase II and Phase III clinical trials for testing of the optimal dosing regime as well as efficacy in the treatment of disease. The development of a single drug often requires the testing of many different compounds in mice or other animal species. These animal studies determine the choice of compound for further development and clinical trials. The main reasons for drug failure at the clinical trial stage are inappropriate pharmacokinetics and toxicological effects (often both are related to drug metabolism), and to a lesser extent, lack of efficacy due to failed concept or lack of pharmacodynamic effect. That compounds progress to clinical trials and then fail at this late stage is usually due to the poor predictability of existing animal models. An incorrect decision, based on data from an animal model that did not accurately reflect drug behaviour in humans, can waste vital resources, many millions of dollars and years of labour. Moreover, the opportunities lost by not pursuing other candidate compounds could potentially cost billions in lost revenue. For example, current mouse models often (and unpredictably) do not accurately reflect human drug pharmacokinetics, metabolism and toxicology. Many drugs show promising results in mice but fail to work effectively in humans. Similarly, other drugs, which failed in mice and consequently were rejected, may have worked well in humans. Thus, the process of selecting candidate drugs for further development and clinical trials is currently based on data obtained from a flawed animal model. The consequences of this include; a) wastage of valuable resources pursuing drugs that will not work in humans; b) lost opportunities by not pursuing drugs which would work in humans and c) exposure of patients to unknown risks in Phase 1 clinical trials. An improved animal model, which more accurately predicts the behaviour (e.g. distribution, metabolism, efficacy and toxicity) of drugs in the animals of interest (e.g. humans and other mammals including livestock animals and companion animals) would provide enormous benefit to both the pharmaceutical and/or veterinary industries and to the treatment of diseases affecting those animals. |
<SOH> SUMMARY OF THE INVENTION <EOH>Accordingly, in one aspect of the present invention, there is provided the use of a transgenic non-primate mammal for predicting the likely behaviour of a drug in a selected species of primate, as part of a drug screening or evaluation process, the transgenic mammal expressing at least a portion of a foreign polypeptide that is associated with drug behaviour and/or metabolism, and that is expressed naturally in the selected species of primate or in a primate of a different species, or that otherwise corresponds to the naturally expressed polypeptide, wherein the expression of an endogenous homologue of the foreign polypeptide in the transgenic mammal is abrogated or otherwise reduced, and wherein the foreign polypeptide is other than the intended target of the drug. The foreign polypeptide is suitably selected from a drug-binding polypeptide, a drug-metabolising polypeptide, a drug-binding and drug-metabolising polypeptide or a drug-transporting polypeptide. Suitably, the transgenic mammal lacks the ability to produce a functional endogenous polypeptide or detectable levels of the endogenous polypeptide. The foreign polypeptide is preferably a functional homologue of the endogenous polypeptide. In one embodiment of this type, the foreign polypeptide is an orthologue of the endogenous polypeptide. In another embodiment of this type, the foreign polypeptide is a paralogue of the endogenous polypeptide. The transgenic mammal may comprise an alteration to its genome, wherein the alteration comprises replacement of the endogenous gene encoding the endogenous polypeptide with a transgene comprising a nucleotide sequence encoding the foreign polypeptide. Alternatively, the alteration may comprise a disruption in said endogenous gene. Suitably, the disruption results in reduced expression levels of the endogenous polypeptide. In a preferred embodiment, the disruption results in abrogated expression levels of the endogenous polypeptide. In another preferred embodiment, the mammal lacks the ability to produce a functional endogenous polypeptide. In another embodiment, the disruption comprises a deletion of at least a portion of the endogenous gene. Suitably said deletion comprises a deletion of nucleotide sequences encoding a region or domain of the endogenous polypeptide. Alternatively, the deletion comprises a deletion of a regulatory polynucleotide that controls at least in part the expression of the endogenous gene. In a preferred embodiment, the deletion comprises a deletion of the entire open reading frame encoding the endogenous polypeptide. Suitably, the nucleotide sequence, encoding the foreign polypeptide, is operably linked to a regulatory polynucleotide. The regulatory polynucleotide may comprise a nucleotide sequence of 1-10 kb. The regulatory polynucleotide is preferably a polynucleotide that is naturally present in the transgenic mammal or in the selected species of primate. In one embodiment, the regulatory polynucleotide is an endogenous polynucleotide of the transgenic mammal, or ancestor thereof. In another embodiment, the regulatory polynucleotide is an endogenous polynucleotide of the selected species of primate. In a preferred embodiment, the regulatory polynucleotide comprises a nucleotide sequence that is naturally located upstream of the coding sequence of the endogenous gene. In another preferred embodiment, the regulatory polynucleotide comprises a nucleotide sequence that is naturally located upstream of the coding sequence of a gene encoding the foreign polypeptide. In an alternative embodiment, the regulatory polynucleotide is derived from an animal or source other than an animal selected from said transgenic mammal, an ancestor of the transgenic mammal or the selected species of primate. In another embodiment, the regulatory polynucleotide comprises an inducible promoter (e.g. metallothionein promoter). Suitably, the alteration has been introduced into the genome of the transgenic mammal by homologous recombination, random integration or the use of a recombinase system (e.g. Cre-loxP or FLP-FRT system) with a nucleic acid construct, comprising the transgene, in an embryonic stem cell such that the construct is stably integrated in the genome of the mammal. The transgenic animal may be heterozygous, but is preferably homozygous, for the transgene. In one embodiment, the transgenic animal is selected from the order Rodentia. In a particularly preferred embodiment, the transgenic animal is a mouse. In a preferred embodiment, the selected species of primate is human. The foreign polypeptide may be selected from a serum albumin, an α-acidic glycoprotein (AGP), a cytochrome p450 (CYP), a uridine diphosphoglucuronosyl transferase (UGT), a multidrug-resistance (MDR) protein including multidrug-resistance-associated proteins (MRPs), an acetyl-transferase, a prenyl protein transferase, a peptidase, an esterase, an acetylase, a glucuronidase, a glutathione S-transferase, or a polypeptide that facilitates or catalyses a reaction selected from an oxidative reaction, a conjugation reaction, a hydrolytic reaction, a reductive metabolism or other catabolic or anabolic reaction involving a xenobiotic. In one embodiment, the foreign polypeptide is serum albumin, which is preferably but not exclusively human serum albumin. The human serum albumin preferably comprises the sequence set forth in SEQ ID NO: 2. In a preferred embodiment, the nucleotide sequence encoding the human serum albumin comprises the sequence set forth in any one of SEQ ID NO: 1 and 3. In another preferred embodiment, the expression of endogenous serum albumin is altered. Suitably, the endogenous serum albumin is a mouse serum albumin comprising the sequence set forth in SEQ ID NO: 6. Preferably, the endogenous gene for mouse serum albumin encodes a transcript comprising the sequence set forth in SEQ ID NO: 5. The regulatory polynucleotide suitably comprises a nucleotide sequence that is naturally located upstream of the coding sequence relating to the endogenous gene. Preferably, the regulatory polynucleotide comprises the sequence as set forth in SEQ ID NO: 7. In another embodiment, the foreign polypeptide is an alpha acidic glycoprotein (AGP). In a preferred embodiment of this type, the foreign polypeptide is a human AGP selected from AGP-1, AGP-2 and AGP-3. Suitably, the human AGP-1 (also known as orosomucoid (ORM)-1) comprises the sequence set forth in SEQ ID NO: 14. Preferably, the nucleotide sequence encoding the human AGP-1 comprises the sequence set forth in SEQ ID NO: 13. Suitably, the human AGP-2 (also known as ORM-2) comprises the sequence set forth in SEQ ID NO: 16. Preferably, the nucleotide sequence encoding the human AGP-2 comprises the sequence set forth in SEQ ID NO: 15. In another preferred embodiment, the expression of an endogenous AGP is altered. Suitably, the endogenous AGP is a mouse selected from AGP-1, AGP-2 and AGP-3. Suitably, the mouse AGP-1 comprises the sequence set forth in SEQ ID NO: 10. Preferably, the endogenous gene encoding the mouse AGP-1 comprises the sequence set forth in SEQ ID NO: 9. Suitably, the mouse AGP-3 comprises the sequence set forth in SEQ ID NO: 12. Preferably, the endogenous gene encoding the mouse AGP-3 comprises the sequence set forth in SEQ ID NO: 11. The regulatory polynucleotide, in this instance, suitably comprises a nucleotide sequence that is naturally located upstream of the coding sequence relating to the gene encoding the foreign polypeptide. Preferably, the regulatory polynucleotide comprises the sequence set forth in SEQ ID NO: 21 and/or 22, which correspond to regulatory polynucleotides located naturally upstream of the human AGP-1 and AGP-2 genes, respectively. In another aspect, the invention contemplates a transgenic non-primate mammal, or progeny thereof, for predicting the likely behaviour of a drug in a selected species of primate, the transgenic animal expressing at least a portion of a foreign drug-binding polypeptide that is expressed naturally in the selected species of primate or in a primate of a different species, or that otherwise corresponds to the naturally expressed polypeptide, wherein the expression of an endogenous homologue of the foreign polypeptide in the transgenic mammal is abrogated or otherwise reduced. In yet another aspect, the invention encompasses a transgenic non-primate mammal, or progeny thereof, for predicting the likely behaviour of a drug in a selected species of primate, the transgenic animal expressing at least a portion of a foreign drug-binding polypeptide selected from the group consisting of a serum albumin and an alpha acidic glycoprotein, wherein the drug-binding polypeptide is expressed naturally in the selected species of primate or in a primate of a different species, or that otherwise corresponds to the naturally expressed polypeptide, wherein the expression of an endogenous homologue of the foreign polypeptide in the transgenic mammal is abrogated or otherwise reduced. Preferably, the transgenic mammal further expresses at least a portion of at least one other foreign polypeptide that is associated with drug behaviour and/or metabolism and that is expressed naturally in the selected species of primate or in a primate of a different species, or that otherwise corresponds to the naturally expressed polypeptide, wherein the expression of a respective endogenous homologue of the corresponding other foreign polypeptide in the transgenic mammal is abrogated or otherwise reduced. Suitably, the or each other foreign polypeptide is selected from a drug-binding polypeptide, a drug-metabolising polypeptide, a drug-binding and a drug-metabolising polypeptide or a drug-transporting polypeptide. In a preferred embodiment of this type, the or each other foreign polypeptide is selected from the group consisting of a serum albumin, an alpha acidic glycoprotein, a cytochrome p450 (CYP), which a preferably selected from selected from subfamily 3A, a uridine diphospho-glucuronosyl transferase (UGT) selected from subfamily 1A, a uridine diphospho-glucuronosyl transferase, and a multidrug-resistance protein. (MDR), including P-glycoprotein and multidrug-resistance-associated proteins (MRPs). In yet another aspect, the invention encompasses a nucleic acid construct, which is preferably but not exclusively a targeting construct, for use in producing a transgenic non-primate mammal for predicting the likely behaviour of a drug in a selected species of primate, the construct including a transgene comprising a nucleotide sequence encoding at least a portion of a foreign polypeptide that is associated with drug behaviour and/or metabolism, and that is expressed naturally in the selected species of primate or in a primate of a different species or that otherwise corresponds to the naturally expressed polypeptide. In one embodiment, the nucleic acid construct is a targeting construct comprising two regions flanking the transgene wherein the regions are sufficiently homologous with portions of the genome of the non-primate mammal to undergo homologous recombination with the portions. In a preferred embodiment of this type, the portions comprise a sequence flanking, or contained by, the endogenous gene that encodes a polypeptide of the non-primate mammal, which polypeptide is a homologue of the foreign polypeptide. The transgene preferably comprises a regulatory polynucleotide operably linked to the sequence encoding at least a portion of the foreign polypeptide. Suitably, the nucleic acid construct comprises a selectable marker gene. In a further aspect, the invention resides in a method of producing a transgenic non-primate mammal for predicting the likely behaviour of a drug in a selected species of primate, the method comprising: providing a transgene comprising a nucleotide sequence encoding at least a portion of a foreign polypeptide that is associated with drug behaviour and/or metabolism, and that is expressed naturally in the selected species of primate or in a primate of a different species or that otherwise corresponds to the naturally expressed polypeptide; and introducing the transgene into the genome of a non-primate mammal. Preferably, the introduction of the transgene into the genome includes producing a nucleic acid construct as broadly described above. Suitably, the introduction of the transgene into the genome includes functionally disrupting the endogenous gene, which is preferably achieved by disrupting the structure of the endogenous gene. Alternatively, the introduction of the transgene into the genome may include inserting the transgene at a site other than that of said endogenous gene. In one embodiment, the introduction of the transgene into the genome includes replacing the endogenous gene or portion thereof with the transgene. In a preferred embodiment, the function of the endogenous gene is disrupted using, for example, a suitable targeting construct. The method preferably further includes the step of introducing a selectable marker gene into the genome of the non-primate mammal. In a preferred embodiment of this type, the selectable marker gene is incorporated into a targeting construct, as for example, described above. In yet a further aspect, the invention resides in a method of producing a transgenic non-primate mammal for predicting the likely behaviour of a drug in a selected species of primate, the method comprising: providing a targeting construct including a transgene comprising a nucleotide sequence encoding at least a portion of a foreign polypeptide that is associated with drug behaviour and/or metabolism, and that is expressed naturally in the selected species of primate or in a primate of a different species or that otherwise corresponds to the naturally expressed polypeptide, and regions flanking the transgene wherein the regions are sufficiently homologous with portions of the genome of the non-primate mammal to undergo homologous recombination with the portions; and introducing the targeting construct into the genome of a non-primate cell under conditions sufficient for the transgene to homologously recombine into a region of the genome interposed between the portions. According to another aspect, the invention provides a method of producing a transgenic non-primate mammal for predicting the likely behaviour of a drug in a selected species of primate, the method comprising: providing a nucleic acid construct including a transgene comprising a nucleotide sequence encoding at least a portion of a foreign polypeptide that is associated with drug behaviour and/or metabolism, and that is expressed naturally in the selected species of primate or in a primate of a different species or that otherwise corresponds to the naturally expressed polypeptide; and introducing the construct into the genome of a non-primate cell under conditions such that the transgene is randomly integrated into the genome. In yet a further aspect, the invention resides in a method of producing a transgenic non-primate mammal for predicting the likely behaviour of a drug in a selected species of primate, the method comprising: providing a targeting construct including a transgene comprising a nucleotide sequence encoding at least a portion of a foreign polypeptide that is associated with drug behaviour and/or metabolism and that is expressed naturally in the selected species of primate or in a primate of a different species or that otherwise corresponds to the naturally expressed polypeptide, and regions flanking the transgene wherein the regions are sufficiently homologous with portions of the genome of the non-primate mammal to undergo homologous recombination with the portions, wherein the portions flank, or are contained within, the endogenous gene encoding at least a portion of a polypeptide of the non-primate mammal, which polypeptide is a homologue of the foreign polypeptide; and introducing the targeting construct into the genome of a non-primate cell under conditions sufficient for the transgene to homologously recombine into at least one of the alleles of the endogenous gene in the genome of the cell to thereby produce a cell containing at least one allele of the endogenous gene replaced, or disrupted, with the transgene. The present invention further resides in a method of producing a transgenic non-primate mammal for predicting the likely behaviour of a drug in a selected species of primate, the method comprising: providing a first targeting construct including a transgene comprising a nucleotide sequence encoding at least a portion of a foreign polypeptide that is associated with drug behaviour and/or metabolism and that is expressed naturally in the selected species of primate or in a primate of a different species or that otherwise corresponds to the naturally expressed polypeptide, wherein the transgene is flanked by portions of the genome of a non-primate cell; and providing a second targeting construct comprising: i) at least a portion of the endogenous gene encoding an endogenous polypeptide that is a homologue of the foreign polypeptide; and ii) a polynucleotide capable of disrupting the endogenous gene; introducing the first targeting construct into the non-primate cell under conditions sufficient for the transgene to homologously recombine into a region of the genome of the cell, corresponding to the portions; and introducing the second targeting construct into the cell under conditions sufficient for the polynucleotide to homologously recombine into at least one allele of the endogenous gene in the genome of the cell to thereby produce a cell containing at least one disrupted allele of the endogenous gene. The present invention further extends to a method of producing a transgenic non-primate mammal for predicting the likely behaviour of a drug in a selected species of primate, the method comprising: providing a nucleic acid construct including a transgene comprising a nucleotide sequence encoding at least a portion of a foreign polypeptide that is associated with drug behaviour and/or metabolism and that is expressed naturally in the selected species of primate or in a primate of a different species or that otherwise corresponds to the naturally expressed polypeptide; providing a targeting construct comprising: i) at least a portion of the endogenous gene encoding an endogenous polypeptide that is a homologue of the foreign polypeptide; and ii) a polynucleotide capable of disrupting the endogenous gene; introducing the nucleic acid construct into a non-primate cell under conditions sufficient for the transgene to randomly integrate into a region of the genome of the cell; and introducing the targeting construct into the cell under conditions sufficient for the polynucleotide to homologously recombine into at least one allele of the endogenous gene in the genome of the cell to thereby produce a cell containing at least one disrupted allele of the endogenous gene. The cell employed in the above production method is preferably an embryonic stem cell, preferably an embryonic stem cell from a mammal within the order Rodentia and most preferably a mouse embryonic stem cell. In a preferred embodiment, the method further comprises injecting the embryonic stem cell containing at least one transgene into the blastocyst or other early developmental stage of a non-human animal. In another preferred embodiment, the method further comprises introducing the injected blastocyst into a pseudo-pregnant non-human animal and permitting the pseudo-pregnant animal to deliver progeny containing at least one homologously recombined transgene. In yet another preferred embodiment, the progeny containing the at least one homologously recombined transgene is further characterised by expressing at least a portion of the foreign polypeptide at detectable levels. In another preferred embodiment, the progeny containing the at least one homologously recombined transgene is further characterised by expressing reduced or undetectable levels of the endogenous polypeptide. In an alternative preferred embodiment, the progeny lacks the ability to produce functional endogenous polypeptide. The method may further include the step of breeding a transgenic non-primate mammal produced by a method as broadly described above and producing progeny of that mammal. For example, mammals containing the same transgene can be inbred to produce mammals that are homozygous for the transgene. Alternatively or additionally, transgenic mammals containing different transgenes described in this invention can be interbred to produce mammals containing two or more different transgenes. Alternatively or additionally, any of these transgenic mammals can be crossbred with any other genetically modified, wild-type or mutant mammals of the same species in order to obtain mammals containing the transgene(s) described in the present invention as well as the desired genetic characteristics of the other mammals used in the crossbreeding strategy. When the transgenic mammal is a mouse, crossbreeding strategies may include crossbreeding the transgenic mouse with another mouse including, but not restricted to, a nude mouse, a SCID mouse, an inbred strain of mouse such as BALB/c, a mouse designed to mimic a specific human disease or a mouse with a useful reporter construct. The transgenic mammals and cells derived therefrom are useful for screening biologically active agents including drugs and for investigating their distribution, efficacy, metabolism and/or toxicity. These screening methods are of particular use for assessing with improved predictability the behaviour of a drug in the primate species of interest. Accordingly, in yet a further aspect, the invention features a method of assessing the behaviour of a drug in a selected species of primate, as part of a drug screening or evaluation process, comprising administering a drug to a transgenic non-primate mammal expressing at least a portion of a foreign polypeptide that is associated with drug behaviour and/or metabolism, and that is expressed naturally in the selected species of primate or in a primate of a different species or that otherwise corresponds to the naturally expressed polypeptide, and wherein the foreign polypeptide is other than the intended target of the drug, and conducting analytical tests to determine the behaviour of the drug in the transgenic mammal, the results of which have a higher correlation to the behaviour of the drug in the selected species of primate than the results obtained from a mammal of the same species as the transgenic mammal, which expresses the endogenous polypeptide but which does not express the foreign polypeptide or portion thereof. In one embodiment, the analytical test comprises assessing directly or indirectly, the concentration and/or distribution of the drug in the transgenic mammal to which it has been administered. In another embodiment, the analytical test comprises assessing directly or indirectly, the efficacy of the drug in the transgenic mammal to which it has been |
Medicine for inhibiting drug elimination pump |
A medicament for preventive and/or therapeutic treatment of a microbial infection which comprises as an active ingredient a compound represented by the following general formula (I): wherein, R1 and R2 represent hydrogen atom, a halogen atom, hydroxyl group or the like, W1 represents —CH═CH—, —CH2O—, —CH2CH2— or the like; R3 represents hydrogen atom, a halogen atom, hydroxyl group or an amino group; R4 represents hydrogen atom, a group of —OZ0-4R5 (Z0-4 represents an alkylene group, a fluorine-substituted alkylene group or a single bond, and R5 represents a cyclic alkyl group, an aryl group or the like); W2 represents a single bond or —C(R8)═C(R9)—(R8 and R9 represent hydrogen atom, a halogen atom, a lower alkyl group or the like, Q represents an acidic group, but W2 and Q may together form vinylidenethiazolidinedione or an equivalent heterocyclic ring; m and n represent an integer of 0 to 2, and q represents an integer of 0 to 3. |
1. A medicament for preventive and/or therapeutic treatment of a microbial infection, which comprises as an active ingredient a compound represented by the following general formula (I) or a physiologically acceptable salt thereof, or a hydrate thereof: wherein, R1 and R2 each independently represent hydrogen atom, a halogen atom, hydroxyl group, a group of OZ1-6 (the group of OZ1-6 represents an alkyl group having 1-6 carbon atoms or a fluoroalkyl group having 1-6 carbon atoms, which bonds via the oxygen atom), a group of S(O)nZ1-4 (Z1-4 represents an alkyl group having 1-4 carbon atoms or a fluoroalkyl group having 1-4 carbon atoms or an alkylene group derived therefrom), a group of N(R12)(R13) (R12 and R13 each independently represent hydrogen atom, an alkyl group having 1-4 carbon atoms or a fluoroalkyl group having 1-4 carbon atoms), a group of Z1-8 which may be substituted (Z1-8 represents an alkyl group having 1-8 carbon atoms or a fluoroalkyl group having 1-8 carbon atoms), a 5- to 7-membered cyclic alkyl group, an aryl group, a heteroaryl group, or a 4- to 7-membered saturated or partially saturated heterocyclic group (the cyclic alkyl group, aryl group, heteroaryl group and heterocyclic group may have one to three substituents selected from the group consisting of a halogen atom, hydroxyl group, a group of OZ1-4, a group of S(O)nZ1-4, a group of N(R12)(R13), a group of Z1-4, carboxyl group, a group of CO2Z1-4, group of CONH2, a group of CONH(Z1-4) and a group of CON(Z1-4)(Z1-4)); W1 represents a group selected from the group consisting of —CH═CH—, —N(R12)CO—, —CON(R12)—, —CH2O— and —CH2CH2— (each of the aforementioned groups binds to the thiazole ring at the left end); R3 represents hydrogen atom, a halogen atom, hydroxyl group or an amino group; R4 represents a group selected from the group consisting of hydrogen atom, a group of —OZ0-4R5 (Z0-4 represents an alkylene group having 1-4 carbon atoms, a fluorine-substituted alkylene group having 1-4 carbon atoms or a single bond, and R5 represents a 5- to 7-membered cyclic alkyl group, an aryl group, a heteroaryl group or a 4- to 7-membered saturated or partially saturated heterocyclic group (the cyclic alkyl group, aryl group, heteroaryl group and heterocyclic group may have one to three substituents selected from the group consisting of a halogen atom, hydroxyl group, a group of OZ1-4, a group of S(O)nZ1-4, a group of N(R12)(R13), a group of Z1-4, carboxyl group, a group of CO2Z1-4, group of CONH2, a group of CONH(Z1-4) and a group of CON(Z1-4)(Z1-4)), a group of —S(O)nZ0-4R5, a group of —N(R6)(R7) {R6 and R7 each independently represent hydrogen atom or Z1-4, or they may bind to each other to form a saturated or unsaturated 5- to 7-membered ring (the ring may contain one or two hetero atoms as ring constituting atoms), and R6 and R7 may have one to three substituents selected from the group consisting of a halogen atom, hydroxyl group, a group of OCON(R15)(R16), a group of CON(R15)(R16), a group of N(R12)CON(R15)(R16), a group of Z1-4, a group of OZ1-4, a group S(O)nZ1-4, group of CH2OH, a group of (CH2)mN(R12)(R13), a group of Z1-4CON(R15)(R16), a group of SO2N(R12)(R13), a group of OSO2N(R12)(R13), a group of OSO2R12, a group of NCOZ1-4R15 (in the formula, R15 and R16 independently represent hydrogen atom, a group of Z1-6R11, a group of Z1-4N(R12)(R13), a group of Z1-4OH, and a group of Z1-4OZ1-4), carboxyl group, cyano group, a group of COZ1-4R10, a group of CO-Z1-4(R10)—N(R12)(R13) (R10 is a substituent corresponding to a side chain on an amino acid carbon or a group of -Z1-4-R11 (R11 represents a substituent which forms a quaternary salt)) and a group of a 5- or 6-membered aryl group which may be substituted and a 5- or 6-membered unsaturated heterocyclic group which may be substituted; W2 represents a single bond or —C(R8)═C(R9)—(R8 and R9 each independently represent hydrogen atom, a halogen atom, a lower alkyl group, an alkoxy group, cyano group, carboxyl group, hydroxymethyl group, cyanomethyl group, vinyl group or a group of N(R12)(R13)), Q represents an acidic group, and W2 and Q may bind together to form vinylidenethiazolidinedione in E- or Z-configuration or an equivalent heterocyclic ring; m and n each independently represent an integer of 0 to 2, and q represents an integer of 0 to 3. 2. A medicament for eliminating resistance of a microorganism with acquired drug resistance, which comprises the compound represented by the aforementioned general formula (I) according to claim 1 or a physiologically acceptable salt thereof as an active ingredient. 3. A medicament for enhancing effect of an antimicrobial agent, which comprises a compound represented by the aforementioned general formula (I) according to claim 1 or a physiologically acceptable salt thereof as an active ingredient. 4. A pharmaceutical composition for preventive and/or therapeutic treatment of a microbial infection, which comprises a compound represented by the aforementioned general formula (I) according to claim 1 or a physiologically acceptable salt thereof together with an antimicrobial agent. 5. A medicament for preventive and/or therapeutic treatment of a microbial infection, which comprises as an active ingredient a compound represented by the following general formula (I) or a physiologically acceptable salt thereof, or hydrates thereof wherein, R1, R2, R3, R4, W1, W2 and Q have the same meanings as those defined above; R14 represents hydrogen atom, Z1-4, Z1-4R5 or Z1-4OR5; and X and Y each independently represent C—H or nitrogen atom. 6. A method for judging effectiveness of a drug efflux pump inhibitor against a microorganism, which comprises the steps of: (A1) spreading a microorganism to be tested on a surface of an agar medium, then providing an antibacterial agent as a spot on the surface of the agar medium and culturing the microorganism; (A2) determining a growth degree of the microorganism in a region of the agar medium into which the antibacterial agent has diffused during the culture period; (A3) determining a growth degree of the microorganism in a region of the agar medium in which the antibacterial agent that has diffused during the culture period and a drug efflux pump inhibitor contained in the agar medium coexist; and (A4) judging that the drug efflux pump inhibitor is effective against the microorganism when the growth degree of the microorganism determined in the step (A2) is significantly higher than the growth degree of the microorganism determined in the step (A3). 7. The method according to claim 6, wherein the antibacterial agent is provided as a spot on the agar medium surface by means of a disk. 8. The method according to claim 6, wherein the drug efflux pump inhibitor contained in the agar medium is the drug efflux pump inhibitor diffused from a disk provided as a spot on the agar medium surface. 9. The method according to claim 6, wherein the drug efflux pump inhibitor contained in the agar medium is the drug efflux pump inhibitor added beforehand to the agar medium during preparation of the agar medium. 10. The method according to claim 6, wherein the microorganism is Pseudomonas aeruginosa. 11. A method for identifying a drug efflux pump expressed in a microorganism, which comprises the steps of: (B1) spreading a microorganism to be tested on a surface of an agar medium, then providing an antibacterial agent that can be excreted by a particular drug efflux pump as a spot on the surface of the agar medium and culturing the microorganism; (B2) determining a growth degree of the microorganism in a region of the agar medium into which the antibacterial agent has diffused during culture period; (B3) determining a growth degree of the microorganism in a region of the agar medium in which the antibacterial agent that has diffused during the culture period and a drug efflux pump inhibitor contained in the agar medium coexist (provided that said drug efflux pump inhibitor is a specific inhibiter for the particular drug efflux pump); and (B4) judging that the microorganism expresses the drug efflux pump of the particular type when the growth degree of the microorganism measured in the step (B2) is significantly higher than the growth degree of the microorganism determined in the step (B3). 12. The method according to claim 11, wherein the antibacterial agent is provided as a spot on the agar medium surface by using a disk. 13. The method according to claim 11, wherein the drug efflux pump inhibitor contained in the agar medium is the drug efflux pump inhibitor diffused from a disk provided as a spot on the agar medium surface. 14. The method according to claim 11, wherein the drug efflux pump inhibitor contained in the agar medium is the drug efflux pump inhibitor added beforehand to the agar medium during preparation of the agar medium. 15. The method according to claim 11, wherein the microorganism is Pseudomonas aeruginosa. 16. The method according to claim 11, wherein the particular drug efflux pump is a MexAB-OprM pump. 17. The method according to claim 11, wherein the antibacterial agent is a β-lactam antibiotic. 18. The method according to claim 17, wherein the antibacterial agent is Aztreonam. 19. A method for verifying expression of two or more kinds of drug efflux pumps in a microorganism, which comprises the steps of: (C1) spreading a microorganism to be tested on a surface of an agar medium, then providing two or more kinds of antibacterial agents (provided that each of the two or more kinds of the antibacterial agents has different effluxing specificity by the two or more kinds of drug efflux pumps, and one of the two or more kinds of the antibacterial agents (hereinafter referred to as “Antibacterial agent (1)”) has a property of being excreted by only one of the two or more kinds of the drug efflux pumps (hereinafter referred to as “Drug efflux pump (1)”), whilst the other antibacterial agent or agents have a property of being excreted by Drug efflux pump (1) and the other drug efflux pump or pumps); (C2) determining a growth degree of the microorganism in a region of the agar medium into which each antibacterial agent has solely diffused during culture period; (C3) determining a growth degree of the microorganism in a region of the agar medium in which each antibacterial agent that has solely diffused during the culture period and a drug efflux pump inhibitor contained in the agar medium coexist (provided that the drug efflux pump inhibitor is a specific inhibiter for Drug efflux pump (1)); and (C4) judging that the microorganism expresses Drug efflux pump (1) and one or more kinds of other drug efflux pumps when the growth degree of the microorganism determined in the step (C2) is significantly higher than the growth degree of the microorganism determined in the step (C3) for Antibacterial agent (1) and the growth degree of the microorganism determined in the step (C2) is significantly lower than the growth degree of the microorganism determined in the step (C3) for the other antibacterial agent or agents. 20. The method according to claim 19, wherein each of the antibacterial agents is provided as a spot on the agar medium surface each by using a disk. 21. The method according to claim 19, wherein the drug efflux pump inhibitor contained in the agar medium is the drug efflux pump inhibitor diffused from a disk provided as a spot on the agar medium surface. 22. The method according to claim 19, wherein the drug efflux pump inhibitor contained in the agar medium is the drug efflux pump inhibitor added beforehand to the agar medium during preparation of the agar medium. 23. The method according to claim 19, wherein the microorganism is Pseudomonas aeruginosa. 24. The method according to claim 19, wherein one of the two or more kinds of drug efflux pumps is a MexAB-OprM pump. 25. The method according to claim 19, wherein the two or more kinds of antibacterial agents include a combination of a β-lactam antibiotic and a quinolone antibacterial agent. 26. The method according to claim 25, the antibacterial agents are Aztreonam and Levofloxacin. 27. The method according to claim 19, wherein the drug efflux pump inhibitor is a specific inhibitor for a MexAB-OprM pump. 28. The method according to claim 19, wherein the drug efflux pump inhibitor is a compound represented by the following formula. |
<SOH> BACKGROUND ART <EOH>For preventive or therapeutic treatment of infectious diseases caused by microorganisms, various antibacterial agents have so far been developed, and drugs such as β-lactam antibiotics (penicillins, cephems, monobactams, carbapenems, and penems), aminoglycosides, quinolones, macrolides, tetracyclines, rifamycins, chloramphenicols, and phosphomycins have been practically used. However, with the increase of clinically used amount of antibacterial agents, remarkable numbers of resistant bacterial strains to these antibacterial agents have emerged, which becomes a serious problem in the treatment of infectious diseases. Examples of problematic bacteria, which cause particularly intractable or serious infectious diseases among those caused by resistant bacteria, include Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA). Antibacterial agents effective against these bacteria have been limited so far, and it is not certain whether or not therapeutic efficacy of the currently available drugs will be expected in the future. In particular, no drug is available at present by which specifically high efficacy against resistant Pseudomonas aeruginosa can be achieved. With the increase of aged population and the popularization of sophisticated medical technologies including human organ transplantation and anti-cancer treatments, infections frequently occurring particularly in patients with reduced immunity, i.e., so-called opportunistic infections, have become an extremely serious problem in the clinical field, and under the circumstances, early developments of measures against the resistant bacteria are desired. Recently, the presence of drug efflux pumps has recognized as a bacterial excretion mechanism of drugs through researches on resistance acquiring mechanisms of resistant bacteria. In earlier researches, a pump that specifically excretes a tetracycline antibacterial agent from bacterial cells was identified in 1980 by the group of Levy, and the discovery was noted as a major factor of the resistance to tetracycline (L. McMurry, Proc. Natl. Acad. Sci. U.S.A., 77, 3974, 1980). Furthermore, based on recent researches, the presence of multidrug-excreting drug efflux pumps was reported in Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus bacteria, Diplococcus pneumoniae, and Neisseria gonorrhoeae. Four multidrug efflux pumps have so far been reported as homological drug efflux pumps deriving from Pseudomonas aeruginosa, and they have been considered as a cause of low drug sensitivity inherent to Pseudomonas aeruginosa (K. Poole et al., J. Bacteriol., 175, 7363, 1993; K. Poole et al., M. Microbiol., 21, 713, 1996; T. Kohler et al., M. Microbiol., 23, 345, 1997; T. Mine et al., Antimicrob. Agents Chemother., 43, 415, 1999). The drug efflux pumps of Pseudomonas aeruginosa excrete various drugs including β-lactams, tetracyclines, chloramphenicols, and quinolones, to which the drug resistance of Pseudomonas aeruginosa is attributable. In order to overcome the problem, it will be effective to invent an antibacterial agent that has a novel structure, by which resistance acquisition due to a drug efflux pump, one of factors of resistance acquisition, can be avoided, or develop an agent for a combinational use with currently available antibacterial agents that can restore their efficacy by inhibiting functions of drug efflux pumps. As one of the latter means, drug efflux pump inhibitors have been known (WO 01/30757). Under the circumstances, if information as to whether or not an etiologic bacterium expresses a drug efflux pump or information as to what kind of drug efflux pump is expressed by the microorganism can be obtained in a convenient manner, it is believed that more effective chemotherapy will become possible. For example, when a drug efflux pump inhibitor is used in combination which restores efficacy of an existing antibacterial agent by inhibiting the function of drug efflux pump, it is expected that judgment of appropriateness of application of a drug efflux pump inhibitor as well as selection of combinational therapy utilizing an inhibitor effective to a particular drug efflux pump and an antibacterial agent can be made by obtaining the aforementioned information. However, any method to conveniently obtain the aforementioned information has not been known so far. In particular, any means to conveniently identify a kind of drug efflux pump expressed in a microorganism or any means to conveniently know whether or not two or more kinds of drug efflux pumps are expressed in a microorganism has not yet been known to date. |
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