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A fuel cell system (1) comprises at least one fuel cell (2) and at least one gas generation system (6) in which at least one component requires air during operation. A compression device (11) is provided to supply the oxygen-containing gas, for example air, to the cathode chamber (4) of the fuel cell. At least one branch line (13) also supplies oxygen-containing gas to the gas generation system component(s) that requires air during operation thereof. Exhaust gases from the fuel cell cathode chamber anode chamber are combined downstream of the fuel cell. |
1. An apparatus for supplying an oxygen-containing gas to a fuel cell system, the fuel cell system comprising at least one fuel cell and at least one gas generation system having at least one component requiring oxygen during operation, and a compression device to supply oxygen-containing gas to a cathode chamber of the fuel cell, the apparatus comprising: a first supply line connected to the compression device and the cathode chamber; at least one branch line connected to the first supply line for supplying oxygen-containing gas to the at least one component of the gas generation system; and a junction point downstream of the fuel cell at which cathode exhaust from the cathode chamber and anode exhaust from an anode chamber of the fuel cell are combined. 2. The apparatus of claim 1, further comprising a heat exchanger between the cathode chamber and the junction point. 3. The apparatus of claim 1, further comprising a throttle element disposed within the at least one branch line. 4. The apparatus of claim 1, wherein the at least one branch line comprises a first branch line for supplying oxygen-containing gas to a reformer, a second branch line for supplying oxygen-containing gas to a selective oxidizer, and a third branch line for supplying oxygen-containing gas to the anode chamber of the fuel cell. 5. The apparatus of claim 4, further comprising an anode side throttle element disposed in at least two of the three branch lines. 6. The apparatus of claim 5, wherein the anode-side throttle elements are fixed annular diaphragms. 7. The apparatus of claim 6, further comprising a cathode-side throttle device between the cathode chamber and the junction point. 8. The apparatus of claim 7, wherein the cathode-side throttle device comprises at least one discrete flow cross-section. 9. The apparatus of claim 1, further comprising a pressure-maintaining valve located downstream of the junction point. 10. The apparatus of claim 1, wherein the at least one branch line comprises at least one fan. 11. The apparatus of claim 10, wherein the fan is located upstream of the at least one branch line. 12. The apparatus of claim 5, wherein at least one of the branch lines contains a fan. 13. A fuel cell system comprising: at least one fuel cell having a cathode chamber and an anode chamber; a gas generation system for producing a hydrogen-containing gas from a process stream containing carbon and hydrogen and supplying the hydrogen-containing gas to the anode chamber of the fuel cell, the gas generating system having at least one component requiring oxygen during operation; a compression device for supplying an oxygen-containing gas to the cathode chamber of the fuel cell; a first supply line connected to the compression device and the cathode chamber; at least one branch line connected to the first supply line for supplying the oxygen-containing gas to the at least one component of the gas generation system; and a junction point downstream of the fuel cell for combining a cathode exhaust from the cathode chamber and an anode exhaust from an anode chamber of the fuel cell. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The invention concerns apparatus for supplying an oxygen containing gas to a fuel cell system, and fuel cell systems comprising such apparatus. 2. Description of the Related Art Fuel cell systems with a gas generation system and at least one fuel cell, which for example can be a solid polymer (PEM) fuel cell, are known in the art. The gas generation system produces a hydrogen containing gas for the fuel cell from a is process stream, which contains carbon and hydrogen. Suitable process streams are hydrocarbons; such as for example natural gas or similar substances, or hydrocarbon derivatives such as alcohols, or higher hydrocarbons, such as benzene or similar substances. Liquid hydrocarbon derivatives such as methanol or ethanol, in particular are contemplated for use in motor vehicles, due to their high energy density and ease of transport. The supply of the fuel cell system with an oxygen containing gas, typically air, is normally implemented by means of a compression device, e.g. a compressor, which supplies air to the cathode side of the fuel cell. Should the gas generation system also require an oxygen containing gas such as air, then this is typically supplied by means of a separate compressor in the gas generation system. The second compressor obtains its air either from the surroundings or draws in pre-compressed air from the air feed to the cathode side of the fuel cell. In general, the gas generation system requires air at least for selective oxidation devices, which oxidize residual carbon monoxide in the hydrogen containing gas to carbon dioxide, since carbon monoxide can damage the fuel cell catalyst material. A system of this type is known from DE 197 55 116 C1, wherein the exhaust gases from the cathode chamber of the fuel cell (air and H 2 O) and from the anode area of the fuel cell (H 2 and CO 2 ) are fed to a catalytic burner. The above mentioned design of apparatus to supply air to a fuel cell system has the disadvantage that it requires at least two compression devices. Each of the compression devices entails additional costs, weight, and volume, and creates noise, which are significant drawbacks in motor vehicle applications. In addition, the air supply to the gas generation system is very complicated in such systems, since it requires a suitable regulation of the second compressor, as well as metering of the oxygen containing gas by means of complicated nozzles, such as Laval nozzles or similar devices. Accordingly, there remains a need for apparatus for supplying an oxygen-containing gas to a fuel cell system, particularly apparatus embodying a simple, lightweight, and self regulating design. The present invention fulfils these needs and provides further related advantages. |
<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>In one embodiment of an apparatus for supply an oxygen containing gas to a fuel cell system, a compression device directly supplies an oxygen containing gas to both the gas generation system and to the cathode side of the fuel cell. Exhaust gases from the anode side and the cathode side of the fuel cell are combined down-stream of the fuel cell, possibly downstream of other intermediate components. In the area of this junction point the exhaust gases are at substantially the same pressure level. This may permit the metering of a set or desired dosage of oxygen-containing gas into the component(s), independent of the press individual components. In further embodiments, the design of the fuel cell system is such that the sum of the pressure drops in the components carrying oxygen- containing gas upstream of the exhaust gas junction on the cathode side is larger than the sum of the pressure drops in the components carrying oxygen containing gas in the gas generation system upstream of the exhaust gas junction on the anode side. In still another embodiment, the apparatus further comprises at least one main branch line for supplying an oxygen containing gas to the gas generation system, wherein the main branch line contains at least one fan. The fan may increase the pressure of the oxygen containing gas that is metered into the one or more components of the gas generation system by a small amount, for example by 50 to 300 mbar. These and other aspects will be evident upon reference to the attached Figures and following detailed description. |
Superconducting coil fabrication |
A method of fabricating a superconducting coil is disclosed. The method comprises the step of fabricating individual coil windings by depositing, shaping and texturing superconductive material in situ on a former which has a substantially curved surface. |
1. A method of fabricating a superconducting coil, the method comprising the step of fabricating individual coil windings by depositing, shaping and texturing superconductive material in situ on a former which has a substantially curved surface. 2. A method as claimed in claim 1, wherein the former defines a substantially cylindrical surface. 3. A method as claimed in claim 2, wherein the former defines a substantially right circular cylindrical surface. 4. A method as claimed in claim 1, further comprising the step of depositing and shaping buffer layers between successive coil windings. 5. A method as claimed in claim 4, wherein the superconductive material is deposited on the former by film deposition technique. 6. A method as claimed in claim 4, wherein the buffer layers are deposited by a film deposition technique. 7. A method as claimed in claim 6, further comprising an initial step of forming a spiral of textured buffer layers on the former. 8. A method as claimed in claim 7, wherein the spiral textured buffer layer is formed by helically positioning a flexible textured tape onto the former. 9. A method as claimed in claim 7, wherein the spiral of textured buffer layer is formed by a film deposition technique. 10. A method as claimed in claim 7, further comprising the steps of depositing a superconductive layer over the spiral buffer layer to form a first coil winding, depositing a second buffer layer onto the superconductive layer, and depositing a second superconductive layer onto the second buffer layer, thereby forming a second winding of the coil, and repeating the buffer layer and superconductive layer depositions to form as many coil windings as required, each deposition process being such as the transfer the texture of the underlying layer to the newly-deposited layer. 11. A method as claimed in claim 10, wherein the spiral of textured buffer layer is written onto the former using the IBAD technique using a fixed ion beam and rotating and translating the former. 12. A method as claimed in claim 10, wherein each of the superconductive layers is a YBCO layer. 13. A method as claimed in claim 12, wherein after the addition of a predetermined number of coil windings, a reinforcement shell is formed on the coil, a textured spiral is formed on the surface of the reinforcement shell to define a further buffer layer using the same process as used on the original buffer layer, and then further coil windings are deposited by the addition of sequential superconducting and buffer layers. 14. A method as claimed in claim 13, further comprising providing connecting links to connect the ends of adjacent coil windings. 15. A method as claimed in claim 14, wherein the connecting links are provided within the former. 16. A method as claimed in claim 14, wherein each of the connecting links includes a fault current limiter. 17. A method as claimed in claim 13, wherein the deposition steps take place in a film deposition chamber. 18. A method as claimed in claim 17, wherein deposition of the buffer layers occurs from one side of the chamber, and deposition of the superconductive layers occurs from the opposite side of the chamber, and the former is rotated during the deposition processes. 19. A method as claimed in claim 18, wherein the two sides of the deposition chamber are separated by baffles. 20. A method as claimed in claim 19, wherein the former is provided with a textured cylindrical surface, and with a spirally-wound heater winding wire, the turns of which are spaced by a means of spacing, a first buffer layer is deposited to form a spiral buffer layer track between the turns of the heater winding, an superconductive layer is deposited over the first buffer layer, and a further buffer layer is deposited on top of the superconductive layer to form a first coil winding, a second heater winding wire is wound between the turns of the first heater winding wire, the first heater winding is removed, and a second coil winding is formed, the second coil winding being constituted by a first deposited buffer layer, a deposited superconductive layer and a second deposited buffer layer, and the process is repeated to form additional coil windings as required, each deposition process being such as to transfer the texture of the underlying layer to the newly-deposited layer. 21. A method as claimed in claim 20, wherein the means for spacing the turns of the spirally-wound heater winding wire is the diameter of the wire. 22. A method as claimed in claim 20, wherein the means for spacing the turns of the spirally-wound heater winding wire is a means for spacing the turns by grooves in the former. 23. A method as claimed in claim 20, further comprising the step of circulating coolant within the former. 24. A method as claimed in claim 20, further comprising the step of testing, in situ, each coil winding in terms of texture or superconducting performance. 25. A method as claimed in claim 1, wherein the fabrication of making a multilayered textured superconducting coil, the method step includes the steps of fabricating superconductor coil windings and insulating layers by film deposition means, whereby the films are patterned by masking or machining operations before or after film deposition, in-situ, and/or by subsequently patterning using lithographic techniques allowing a tailoring of coil properties by controlling the geometry (width, thickness, spacing, or pitch) of the superconducting paths at every point on the surface. 26. A method of fabricating a superconducting coil, the method comprising the step of helically positioning a flexible textured tape onto a substantially cylindrical former. 27. A method as claimed in claim 26, wherein the tape is constituted by an insulating buffer layer on a flexible tape substrate and a coating of superconductive material. 28. A method as claimed in claim 26, wherein YBCO constitutes the superconductive material. 29. A method as claimed in claim 26, wherein the tape is a textured substrate made, for example, by the RABiTS process or by the IBAD process. 30. A method as claimed in claim 27, wherein the former is generally barrel-shaped, with tapered portions at each end of a cylindrical control main portion. 31. A method as claimed in claim 26, wherein the former is itself the textured substrate made directly to the RABiTS process or the IBAD process. |
Yeast membrane protein expression system and its application in drug screening |
The invention relates to an in vitro cell based expression system for overexpressing heterologous pump proteins associated with drug resistance into the membrane of the host cell for drug screening applications. |
1. A protein expression system comprising: i) a host yeast cell; and ii) a vector comprising the coding sequence of a target heterologous membrane protein, said sequence being under the control of a promoter which, upon transformation of said host cell and chromosomal integration, causes over-expression of the functional target protein in the membrane of the host cell. 2. A protein expression system as claimed in claim 1, wherein the host cell is a yeast cell of the genus Saccharomyces. 3. A protein expression system as claimed in claim 1 or 2, wherein the host cell comprises a mutant strain deficient in one or more naturally occurring membrane proteins thereby enabling the target protein to be expressed prominently in the membrane of the host cell and to be accessible for drug screening applications. 4. A protein expression system as claimed in claim 3, wherein the host cell is deficient in drug efflux pump proteins. 5. A protein expression system as claimed in any one of claims 2 to 4, wherein the host cell is the Saccharomyces cerevisiae AD1-8u− strain. 6. A protein expression system as claimed in any preceding claim, wherein the host cell contains a mutation that leads to the formation of secretory vesicles whose ability to fuse normally with the plasma membrane is temperature sensitive. 7. A protein expression system as claimed in claim 6, wherein the host cell is a sec6-4 mutant of the AD1-8u− strain. 8. A protein expression system as claimed in claim 1, wherein the coding sequence of the target protein is incorporated into the host cell in a defined location in the genome. 9. A protein expression system as claimed in claim 1, wherein the coding sequence comprises the entire natural coding sequence of the heterologous target protein, or a functional fragment or variant thereof. 10. A protein expression system as claimed in claim 8 or 9, where the target heterologous membrane protein is a drug efflux pump protein selected from the group consisting of pump proteins involved in multidrug resistance in fungi, P-glycoprotein, cystic fibrosis transmembrane conductance regulator and any other human, animal, plant or microbial plasma membrane proteins that play a role in conferral of drug resistance. 11. A protein expression system as claimed in any one of claims 3 to 10 wherein the target heterologous membrane protein is of the same class of membrane proteins which have been deleted from the host cell. 12. A protein expression system as claimed in claim 11, where the target heterologous membrane proteins are selected from Candida albicans Cdr1p, Cdr2p and from Candida galbrata Cdr1p and Pdh1p. 13. A protein expression system as claimed in any one of claims 3 to 10 wherein the target heterologous membrane protein is of a different class to the class of membrane proteins which have been deleted from the host cell. 14. A protein expression system as claimed in claim 13 wherein the target heterologous membrane product is selected from the Candida albicans BenRP and Erg11p. 15. A protein expression system as claimed in claim 1, wherein the vector is a plasmid vector. 16. A protein expression system as claimed in claim 15, wherein the plasmid vector contains elements which allow replication in Escherichia coli. 17. A protein expression system as claimed in claim 15, wherein the plasmid vector is pABC. 18. A protein expression system as claimed in claim 1, wherein the promoter is a Saccharomyces cerevisiae promoter. 19. A protein expression system as claimed in claim 18, wherein the promoter is selected from the group comprising S. cerevisiae PDR5, PMA1, CTR3, ADH1, PGK and GAL and bacterial tet0 promoters and tet0::ScHOP1 controllable cassette. 20. A protein expression system as claimed in claim 19, wherein the promoter is the PDR5 promoter. 21. A protein expression system as claimed in any one of claims 18 to 20, wherein the Saccharomyces promoter is under the control of a transcriptional regulator so as to induce over-expression of the target protein coding sequence in the membrane of the host cell. 22. A protein expression system as claimed in claim 21, wherein the transcriptional regulator is the Pdr1-3p transcriptional regulator. 23. A method of screening for drugs useful as a pharmaceutical or agrochemical comprising the steps of: i) transforming the chromosomal DNA of a host yeast cell with DNA comprising the coding sequence of a target heterologous membrane protein, said sequence being under the control of a host promoter leading to over-expression of the functional target protein in the membrane of the host cell; ii) introducing at least one candidate compound to said host cell environment or the environment of a plasma membrane fraction derived from the transformed host strain; and iii) measuring the effect, if any, of the candidate compound on the host cell growth and/or viability and/or specific biochemical or physiological functions mediated by the target membrane protein; and/or measuring the binding of the candidate compound to the target membrane protein. 24. A method as claimed in claim 23, wherein the host cell is a yeast cell of the genus Saccharomyces. 25. A method as claimed in claim 24, wherein the host cell is a Saccharomyces cell which has been genetically altered to be depleted in one or more natural membrane proteins. 26. A method as claimed in claim 25, wherein the host cell is the Saccharomyces cerevisiae AD 1-8u− strain. 27. A method as claimed in claim 25 or 26, wherein the host cell is a sec6-4 mutant of the AD1-8u− strain. 28. A method as claimed in claim 23, wherein the target heterologous membrane protein comprises a drug efflux pump protein. 29. A method as claimed in claim 23, wherein the drug efflux drug pump protein is selected from the group consisting of pump proteins involved in multidrug resistance in fungi, the P-glycoprotein, the cystic fibrosis transmembrane conductance regulator and other human, animal, plant and microbial plasma membrane proteins that play a role in the conferral of drug resistance. 30. A method as claimed in any one of claims 23 to 29, wherein the target membrane protein is a drug efflux pump protein and the candidate compound is an efflux pump inhibitor. 31. A method as claimed in claim 23, wherein said coding sequence and promoter are introduced into said host cell via a plasmid vector. 32. A method as claimed in claim 31, wherein the plasmid vector contains elements which allow replication in Escherichia coli. 33. A method as claimed in claim 31, wherein the plasmid vector is pABC3. 34. A method as claimed in any one of claims 31 to 33, wherein said promoter is a Saccharomyces cerevisiae promoter. 35. A method as claimed in claim 34, wherein the promoter is selected from the group consisting of S. cerevisiae PDR5, PMA1, CTR3, ADH1, PGK and GAL and bacterial tet0 promoters and the tet0::ScHOP1 controllable cassette. 36. A method as claimed in claim 34 or 35, wherein the promoter is the PDR5 promoter. 37. A method as claimed in any one of claims 34 to 36, wherein Saccharomyces promoter is under the control of a transcriptional regulator so as to induce over-expression of the target protein coding sequence in the membranes of the host cell. 38. A method as claimed in claim 37, wherein the transcriptional regulator is the Pdr1-3p transcriptional regulator. 39. A vector suitable for use in the overexpression of a target heterologous membrane protein in a yeast host cell comprising pABC3. 40. A bioactive, pharmaceutical or agrochemical compound identified using the method of any one of claims 23 to 38 or the protein expression system of any one of claims 1 to 22. 41. A compound as claimed in claim 40, wherein said compound was obtained from compound libraries. 42. A compound as claimed in claim 40 or 41, comprising KN20 as defined herein. 43. A compound comprising KN20 as defined herein suitable for use as an antifungal. 44. An antifungal composition comprising compound KN20 as defined herein together with a suitable carrier or diluent. 45. An antifungal composition comprising compound KN20 as defined herein in a mixture with a known antifungal compound selected from the group consisting of fluconazole and other xenobiotics that are transported by multidrug efflux mechanisms. 46. A purified membrane protein produced by the method of any one of claims 23 to 38 or protein expression system of any one of claims 1 to 22. 47. A kit for screening for drugs useful as a pharmaceutical or agrochemical comprising: (i) a host cell; (ii) a vector containing the coding sequence of a target heterologous membrane protein, said sequence being under the control of a promoter which, upon transformation of said host cell and chromosomal integration, causes over-expression of the functional target protein in the membrane of the host cell; and (iii) instructions to carry out said transformation and drug screening procedures. 48. A kit as claimed in claim 47 wherein said host cell comprises S. crevisiae AD1-8u− and said vector is pABC3 and comprises a PDR5 promoter and a coding sequence of a heterologous drug efflux pump protein. 49. A kit as claimed in claim 47 or 48, wherein said pump protein is selected from the group comprising C. albicans Cdr1p, Cdr2p, BenRp, Erg11p and C. galbrata Cdr1p and Pdh1p. |
<SOH> BACKGROUND <EOH>Proteins located in the plasma membrane or surface membranes of target cells are amongst the most prominent, accessible and attractive sites for intervention with small molecule drugs for pharmaceutical and agrochemical purposes. For example, drugs such as ouabain and the cardiac glycosides are effective therapeutics in the treatment of heart disease because of their activity against isoforms of the membrane protein Na + ,K + -ATPase of mammalian cells (Schwartz A, et al, 1982). Individual membrane proteins of interest that are located at the cell surface may be constitutively expressed cellular components found in a host or a pathogenic organism. Alternatively, the expression of these proteins may also be affected by mutation or by interactions between such cells and other organisms. These membrane proteins include transporters, channels, receptors and enzymes plus proteins with structural, regulatory or unknown roles. Various members of these classes of proteins are known to affect the growth, viability, and functional capacity of host organisms, tissues or cells. In particular, several classes of membrane proteins are known to be involved in drug resistance. These include the drug efflux pump proteins which act to increase the efflux of particular drugs, such as antibiotics and other xenobiotics, from the inside of a cell to the outside. This activity lowers the concentration of the drug at the intracellular target site to levels which are no longer effective. Yeast cell expression systems for testing drugs that inhibit drug efflux pump proteins are known. Decottignies et al 1998 describes a number of strains of Saccharomyces cerevisiea in which varying endogenous drug efflux pump proteins (ABC transporter proteins) have been deleted and a further endogenous membrane protein overexpressed in the cell membrane. Such a system also employs the use of regulators which aid in this overexpression. Examples of such regulators are described in Carjaval et al (1997). However, such a system is restrictive in its application as it may be species specific, ie it may only identify potential drugs useful in inhibiting drug resistance in Saccharomyces cerevisiea. As the problem of drug resistance is widely found in all fauna and flora, and not just in yeast, there exists a need to develop a simple in vitro cell based membrane protein expression system for testing potential inhibitors of drug efflux pump proteins, as well as other membrane proteins associated with drug resistance, from different species. In addition, as the number of potential test compounds, located mainly in compound libraries, is increasing in both size and complexity, there is a need for such a simple in vitro, cell based membrane protein expression system to screen for agonists or antagonists of putative membrane protein drug targets from a broad range of species and which can be adapted for high throughput formats. It is an object of the present invention to go some way towards providing for these needs and/or to provide the public with a useful choice. |
<SOH> SUMMARY OF INVENTION <EOH>The present invention provides a protein expression system comprising: i) a host yeast cell; and ii) a vector containing the coding sequence of a target heterologous membrane protein, said sequence being under the control of a promoter which, upon transformation of said host cell and chromosomal integration, causes over-expression of the functional target protein in the membrane of the host cell. The host yeast cell may comprise a strain of the genus Saccharomyces. The host yeast cell may comprise a mutant strain deficient in one or more naturally occurring membrane proteins, such as drug efflux pumps, so that the target protein expressed in the membrane of the host cell, is relatively prominent and accessible for drug screening applications. The preferred yeast strain is Saccharomyces cerevisiea AD1-8u − . In some applications, the host cell may contain a mutation that leads to the formation of secretory vesicles whose ability to fuse normally with the plasma membrane is temperature sensitive. A preferred mutated strain is the sec6-4 mutant of the AD1-8u − strain. The coding sequence of the target heterologous protein may be incorporated into the host cell in a defined location in the genome such as downstream of an endogenous promoter. The coding sequence of the target heterologous membrane protein may comprise the entire natural coding sequence of the target protein, or a functional fragment or variant thereof which, upon transformation and expression, will produce a functional membrane protein with a detectable phenotype. The target heterologous membrane protein of the invention may comprise a drug efflux pump protein such as those involved in multidrug resistance in fungi, but may also include other molecules such as the P-glycoprotein, the cystic fibrosis transmembrane conductance regulator and other human, animal, plant and microbial plasma membrane proteins that play a role in the conferral of resistance or sensitivity to xenobiotics, the etiology of disease or the modulation of physiology, growth and development. Preferably, the target membrane protein is a drug efflux pump protein and the candidate compound is an efflux pump inhibitor. The vector used to integrate the coding sequence of the target heterologous membrane protein is preferably a plasmid vector which contains elements which allow replication in E. coli. The vector may also include a transcription terminator that is functional in the host cell. In addition, the vector may include a marker that confers a selectable phenotype on the cells after transformation. The promoter is selected from the group of promoters comprising constitutive S. cerevisiae PDR5 and PMA1 promoters, copper controllable CTR3, glucose inducible ADH1 and PGK promoters, the galactose inducible GAL promoter, the doxycycline controllable bacterial tet0 promoter and the tet0::ScHOP1 controllable cassette. The preferred promoter is PDR5. The preferred vector is pABC3. The yeast host strain may further comprise a mutated transcriptional regulator coding sequence that causes overexpression of the target coding sequence leading to abundant expression of the target protein in the membrane of the host cell. The mutated transcriptional mutator may be Pdrl-3p. The present invention further provides a method of screening for drugs useful as a pharmaceutical or agrochemical comprising the steps of: i) transforming the chromosomal DNA of a host yeast cell with DNA comprising the coding sequence of a target heterologous membrane protein, said sequence being under the control of a host promoter leading to over-expression of the functional target protein in the membrane of the host cell; ii) introducing at least one candidate compound to said host cell environment or the environment of a plasma membrane fraction derived from the transformed host strain; and iii) measuring the effect, if any, of the candidate compound on the host cell growth and/or viability and/or specific biochemical or physiological functions mediated by the target membrane protein; and/or measuring the binding of the candidate compound to the target membrane protein. The target heterologous membrane protein of the invention may comprise a drug efflux pump protein such as those involved in multidrug resistance in fungi, but may also include other molecules such as the P-glycoprotein, the cystic fibrosis transmembrane conductance regulator and other human, animal, plant and microbial plasma membrane proteins that play a role in the conferral of resistance or sensitivity to xenobiotics, the etiology of disease or the modulation of physiology, growth and development. Preferably, the target membrane protein is a drug efflux pump protein and the candidate compound is an efflux pump inhibitor. Preferably, the host yeast cell is of the genus Saccharomyces, and most preferably the host cell is a Saccharomyces cell which has been genetically altered to be depleted in one or more natural membrane proteins. A suitable host cell is the Saccharomyces cerevisiae AD1-8u − strain. In another embodiment a suitable host cell may be a sec6-4 mutant of the AD1-8u − strain. In a further embodiment the host strain may be a derivative of the AD1-8u − strain modified to select for a novel phenotype, such as prototrophy, an auxotrophic requirement or drug sensitivity. A transformation cassette derived from a plasmid vector may be used to transform the chromosomal DNA of the host cell. The vector may contain elements which allow replication in Escherichia coli, plus a promoter such as a Sacharomyces cerevisiae promoter and more preferably the PDR5 promoter. Activity of the Saccharomyces promoter is preferably under the additional control of a mutated transcriptional regulator causing over-expression of the target coding sequence, leading to abnormal expression of the target protein in the membrane of the host cell. The mutated transcriptional regulator Pdr1-3p is preferred and is located in the genome of the host cell. In some applications, a transcriptional terminator that is functional in yeast may be included in the vector. Either the natural terminator of the gene encoding the membrane protein or the yeast PGK1 terminator is preferred. In other applications immunological, affinity or fluorescent tags may be included in the vector. In some further applications, a selectable marker may also be included in the vector such as S. cerevisiae URA3 marker. In other applications a S. cerevisiae centromere or autonomously replicating sequence might be included in the vector. The vector is preferably pABC3. Compounds which are identified as useful bioactives, pharmaceuticals or agrochemicals using the method and system of the invention also form part of the present invention. These may include compounds obtained from compound libraries, such as NK20 as defined below. The method and system of the present invention may also find application for the over-expression of yeast and heterologous target membrane proteins for the purposes of physiological study, biochemical analysis, enzyme purification and structural analysis of said target membrane proteins. Purified membrane proteins produced by the method and system of the invention also form part of the present invention. In a further embodiment, the present invention provides a kit for screening for drugs useful as a pharmaceutical or agrochemical comprising the protein expression system of the present invention together with suitable instructions. In a further form of the invention the target membrane protein may be required for viability or virulence of a pathogen or the progression of a disease. For example, the target protein may be required for the attachment or uptake of viruses or other pathogens. In such cases, the effect, if any, of a compound on the function of the target membrane protein may be measured. Although the invention is broadly as defined above, it is not limited thereto and also includes embodiments of which the following description provides non-limiting examples. |
Method for regenerating an exhaust gas filtering device for diesel engine and device therefor |
The invention concerns a method for regenerating a device filtering exhaust gases produced by a diesel engine, said method being of the type wherein particles, retained on a filtering means (22) of the filtering device, are burnt by the action of a combustion catalyst system. Said method consists essentially in retaining the hot exhaust gases around the filtering means (22) so as to feed to the particles, at least part of the heat energy required for their combustion and in burning said particles to regenerate the filtering device. The invention also concerns a device for implementing the method, comprising a means for producing a combustion catalyst (20), a filtering means (22) of exhaust gases and a diesel oil injecting means, the means for producing a combustion catalyst and filtering means being contained in a reaction chamber (18). |
1-19. (Canceled) 20. A method for regeneration of a device for filtering the exhaust gases produced by a diesel engine in which particles retained on a filtration means of said filtration device are burnt through the action of a combustion catalyst, comprising retaining the hot exhaust gases around the filtration means in order to provide the particles with at least some of the heat energy necessary for their combustion, and burning said particles with at least some of the heat energy necessary for their combustion, so as to regenerate the filtration device. 21. The method of claim 20, further comprising: employing a combustion catalyst production means in the filtration device, measuring a temperature θm in the vicinity of the combustion catalyst production means, comparing θm with a temperature θr corresponding to the temperature at which the combustion of diesel fuel in the presence of the combustion catalyst is complete, if θm is greater than or equal to θr, initiating post-injection of diesel fuel through an injection means into the filtration device for a determined period of time, so as to cause a temperature rise of the particles in order to permit their combustion. 22. The method of claim 21, further comprising: measuring a pressure Pm in the vicinity of the combustion catalyst production means, said pressure Pm reflecting the degree of obstruction of the filtration means by the particles, measuring the temperature θm, comparing said pressure Pm with a reference pressure Pr corresponding to the maximum acceptable degree of obstruction, comparing θm with θr if Pm is greater than or equal to the pressure Pr, initiating the post-injection of diesel fuel if θm is greater than or equal to θr. 23. A device for carrying out the regeneration method of claim 20, comprising a combustion catalyst production means, a means for filtration of said exhaust gases downstream of said combustion catalyst production means, and a means for injection of diesel fuel upstream of said combustion catalyst production means, said combustion catalyst production means and filtration means being contained in a reaction enclosure along the flow path of the exhaust gases produced by an engine. 24. The device of claim 23, wherein said filtration means, comprising a set of filtering units and is exposed in a chamber for receiving the exhaust gases, with said exhaust gases heating said filtering units. 25. The devise of claim 24, wherein said filtration means comprises at least two particle filters having a body, formed by the filtering units which are connected together by a joint, and a metal casing. 26. The device of claim 23, wherein said means for injection of diesel fuel communicates with a line for discharging the exhaust gases. 27. The device of claim 23, wherein said means for injection comprises a diesel fuel reservoir and a chamber for injecting the diesel fuel contained in said reservoir into the discharge line. 28. The device of claim 27, wherein said chamber is supplied, on the one hand, with diesel fuel through a first line connecting it to said reservoir and, on the other hand, with compressed air through a second line connecting it to the engine, said chamber having an orifice through which the diesel fuel is injected into said filtration device. 29. The device of claim 23, further comprising an electroinc control module. 30. The device of claim 23, further comprising at least one temperature sensor, placed inside said enclosure and adapted to measure the temperature θm in its interior. 31. The device of claim 23, further comprising at least one pressure sensor, placed inside said enclosure and adapted to measure the pressure Pm in its interior. 32. The device of claim 30, wherein the electronic control module is connected to a temperature sensor and to a pressure sensor, and compares the respectively measured values of θm and optionally Pm with the reference values θr and optionally Pr, and initiates the injection of diesel fuel into the discharge line through said injection system when the measurements θm and optionally Pm are greater than or equal to the reference values θr and optionally Pr. 33. The device of claim 32, wherein the lines supplying the chamber of the injection system with diesel fuel and compressed air each have a solenoid valve controlled by the electronic control module, opening of the solenoid valves leading to the intake of diesel fuel and compressed air into the chamber and therefore to the injection of diesel fuel into the exhaust gas discharge line. 34. The device of claim 28, wherein the chamber of the injection system is provided with a nozzle in front of the orifice, making it possible to inject the diesel fuel in a nebulized form into the gas discharge line. 35. The device of claim 24, wherein the filtering units preferably consist of silicon carbide or any other equivalent material whose structure is of a honeycomb type. 36. The device of claim 23, wherein the combustion catalyst production means comprises at least one cartridge based on platinum or any equivalent material that catalyzes the conversion of the nitrogen monoxide (NO) contained in the exhaust gases into nitrogen dioxide (NO2), the NO2 which is produced catalyzing the combustion reaction of the particle clogging the filter. 37. The device of claim 23, wherein the capacity of the second reservoir is preferably equivalent to the maximum volume of diesel fuel injected during the post-injection. 38. The device of claim 25, wherein the particle filters are placed in parallel in the enclosure. |
Method of inducing the formation of neurofibrillary tangles in transgenic animals |
The present invention discloses a method and an in-vivo assay system useful for the identification and testing of modulating agents as well as for the validation of therapies of neurodegenerative diseases associated with the formation of neurofibrillary tangles, in particular Alzheimer's disease. The present invention is based on the surprising finding that injection of β-amyloid Aβ42 fibrils into brains of P301L mutant tau transgenic mice caused several-fold increases in the numbers and an accelerated production of neurofibrillary tangles in cell bodies predominantly within the amygdala. The induced neurofibrillary tangles occurred as early as 18 days after Aβ42 injections and displayed striking features of neurofibrillary tangles of several human neurodegenerative diseases, particularly Alzheimer's disease. |
1. A method of increasing the number of neurofibrillary tangles in a non-human transgenic animal which expresses a recombinant gene coding for tau protein wherein the increase of neurofibrillary tangles is induced through admission of APP, or a fragment, or derivative, or variant thereof, in particular β-amyloid. 2. A method of accelerating the production of neurofibrillary tangles in a non-human transgenic animal which expresses a recombinant gene coding for tau protein wherein the accelerated production of neurofibrillary tangles is induced through admission of APP, or a fragment, or derivative, or variant thereof, in particular β-amyloid. 3. The method according to claim 1 wherein said β-amyloid is Aβ42. 4. The method according to claim 3 wherein said Aβ42 is fibrillar, pre-aggregated, or aggregated. 5. The method according to claim 1 wherein said transgenic animal is a mouse. 6. The method according to claim 5 wherein said mouse expresses a recombinant gene coding for human tau. 7. The method according to claim 5 wherein said mouse expresses a recombinant gene coding for P301L mutant tau. 8. The method according to claim 1 wherein β-amyloid is admitted through injection into the brain of said animal, in particular through injection into the hippocampus or the cortex. 9. The method according to claim 1 wherein β-amyloid or a fragment, or derivative, or variant of APP is admitted through co-expressing a recombinant gene coding for APP, said APP being enzymatically processed to generate β-amyloid or said fragment, or derivative, or variant of APP. 10. The method according to claim 9 wherein said APP is a mutant APP. 11. The method according to claim 1 wherein the increase of neurofibrillary tangles is predominantly in the amygdala of the animal's brain, in particular the basolateral amygdala. 12. The method according to claim 1 wherein the increase of neurofibrillary tangles is several-fold, preferably at least two-fold, as compared to a control animal to which no APP or a fragment, or derivative, or variant thereof, in particular β-amyloid, has been administered. 13. The method according to claim 1 wherein the increase of neurofibrillary tangles is at least five-fold as compared to a control animal to which no APP or a fragment, or derivative, or variant thereof, in particular β-amyloid, has been administered. 14. The method according to claim 1 for inducing neuropil threads and/or degeneration of neurites. 15. A non-human transgenic animal which expresses a recombinant gene coding for tau protein wherein said animal is capable of producing neurofibrillary tangles. 16. A non-human transgenic animal which expresses a recombinant gene coding for tau protein wherein said animal is capable of producing an increased number of neurofibrillary tangles. 17. A non-human transgenic animal which expresses a recombinant gene coding for tau protein wherein said animal is capable of accelerated production of neurofibrillary tangles. 18. The animal according to claim 15 to 17 wherein neurofibrillary tangles are produced which are comparable to those commonly found in human neurodegenerative diseases. 19. The animal of claim 15 wherein said animal comprises APP or a fragment, or derivative, or variant thereof, in particular β-amyloid, in an amount sufficient to induce the production of neurofibrillary tangles. 20. The animal according to claim 16 wherein the increase of neurofibrillary tangles is induced through admission of APP, or a fragment, or derivative, or variant thereof, in particular β-amyloid. 21. The animal according to claim 15, wherein β-amyloid is admitted through injection into the brain of said animal, in particular through injection into the hippocampus or the cortex. 22. The animal according to claim 15 wherein said animal co-expresses a recombinant gene coding for APP, or a fragment, or derivative, or variant thereof, said APP being enzymatically processed to generate a cleavage product, said cleavage product being preferably β-amyloid. 23. The animal of claim 22 wherein said APP is a mutant APP. 24. Use of the animal according to claim 15 as an in-vivo assay to determine or validate the efficacy of modulating agents, or therapies, in particular amyloid-lowering therapies or NFT-reducing therapies. 25. The use according to claim 24 wherein said modulating agents, or therapies are for neurodegenerative diseases, in particular Alzheimer's disease, frontotemporal dementia (FTD), and other neurodegenerative diseases accompanied by neurofibrillary tangle formation. |
Process for the preparation of beta-ionylideneacetaldehyde |
The present invention relates to an industrially advantageous process for the preparation of beta-ionylidencacetaldehyde of structural Formula I, which is a key intermediate for the synthesis of vitamin A and related compounds such as tretinoin and isotretinoin. |
1. A process for the synthesis of β-ionylideneacetaldehyde of structural Formula I which comprises: (a) condensing β-ionone of structural Formula II with triethyl phosphonoacetate of structural Formula III to obtain ethyl β-ionylideneacetate of structural Formula IV, (b) reducing the ester of Formula IV with a reducing agent to β-ionylidene alcohol of Formula V in the presence of organic solvent, and (c) oxidizing the alcohol of Formula V in situ with manganese dioxide at 60-70° C. for about 2 to 4 hours to obtain β-ionylideneacetaldehyde of structural Formula I. 2. The process of claim I wherein the condensation of β-ionone with triethyl phosphonoacetate is carried out in the presence of sodium and toluene. 3. The process of claim 1 wherein the reducing agent is selected from the group consisting of lithium aluminium hydride, sodium bis (2-methoxyethoxy) aluminium hydride (Red-Al), and diisobutyl aluminium hydride (DIBAL). 4. The process of claim 3 wherein the reducing agent is lithium aluminium hydride (LAH). 5. The process of claim 1 wherein the organic solvent is selected from the group consisting of hexane, tetrahydrofuran, toluene, xylene, and mixture(s) thereof. 6. The process of claim 1 wherein the trans β-ionylideneacetaldehyde has less than 5% of the 9-cis isomer. 7. A process for the synthesis of ethyl β-ionylideneacetate of structural Formula IV which comprises the condensing β-ionone of structural Formula II with triethyl phosphonoacetate of structural Formula III in the presence of sodium amide. 8. A process for the preparation of β-ionylidene alcohol of Formula V which comprises reducing the ester of Formula IV with a reducing agent in the presence of organic solvent. 9. The process of claim 8 wherein the organic solvent is selected from the group consisting of hexane, tetrahydrofuran, toluene, xylene, and mixture(s) thereof. 10. The process of claim 8 wherein the reducing agent is selected from the group consisting of lithium aluminium hydride, sodium bis (2-methoxyethoxy) aluminium hydride (Red-Al), and diisobutyl aluminium hydride (DIBAL). 11. A process for the preparation of trans β-ionylideneacetaldehyde of Formula I which comprises oxidizing the alcohol of Formula V with manganese dioxide at 60-70° C. for about 2 to 4 hours. 12. The process according to claim 1 wherein the β-ionolideneacetaldehyde is converted into tretinoin. 13. The process according to claim 1 wherein the β-ionolideneacetaldehyde is converted into isotretinoin. 14. The process according to claim 1 wherein the β-ionolideneacetaldehyde is converted into Vitamin A. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The synthesis of β-ionylideneacetaldehyde utilizes β-ionone as the starting material. All the double bonds in β-ionylideneacetaldehyde have trans configuration and the major synthetic challenge has been to maintain the conjugated trans-polyene system in the molecule. The available synthetic approaches for β-ionylideneacetaldehyde are summarized below. J. Am. Chem. Soc., 1955; 77: 4111 discloses the synthesis of the cis and trans ethyl β-ionylideneacetates using Reformatsky reaction. This approach involves the condensation of ethyl bromoacetate with β-ionone in the presence of zinc to give β-ionylideneacetate as a mixture of cis and trans in the ratio of 7:3, respectively. This ester, upon saponification and selective crystallization, gives trans β-ionylideneacetic acid In very poor (˜20%) yield. The acid intermediate is esterified and reduced using lithium aluminium hydride to give trans β-ionylidene ethanol; oxidation of alcohol intermediate finally affords the desired β-ionylideneacetaldehyde. Although this approach maintains the trans geometry at the C-9 bond but it is not commercially viable as it involves several steps and extremely poor over-all yield; selectivity of the C-9 double bond formation at Reformatsky stage in ethyl β-ionylideneacetate lowers the yield of the desired trans isomer, rendering the process uneconomical. Bull. Chem. Japan, 1963, 1527 describes the synthesis of ethyl β-ionylideneacetate by means of a Wittig reaction using diethyl carboxymethylphosphonate prepared from triethyl phosphite and ethyl bromoacetate. β-ionylideneacetate is synthesized by condensing the β-ionone with diethylcarboxymethylphosphonate in the presence of sodium amide in tetrahydrofuran. This acetate is reduced with lithium aluminium hydride in ether to give β-ionylidene ethanol, followed by its oxidation with manganese dioxide to give the desired β-ionylideneacetaldehyde. The oxidation is performed in petroleum ether at room temperature for 24 hours. This process is unacceptable on a commercial scale because the process requires maintaining the temperature (30° C.) for 24 hours. More importantly, we found that this process was not stereoselective; the ester, alcohol and the desired aldehyde were not 100% trans, rather a mixture of 9-cis and 9-trans isomers were obtained. Gazz. Chem. 1973; 103: 117 discloses the synthesis of β-ionylideneacetaldehyde by the condensation of β-ionone with lithioacetonitrile (generated from n-butyl lithium and acetonitrile) to give β-ionylideneacetonitrile with almost 60%, trans selectively. After chromatographic purification, trans β-ionylideneacetonitrile is reduced with dilsobutylaluminium hydride (DIBAL) to afford β-ionylideneacetaldehyde which is further purified by chromatography. This process is not attractive for operation at commercial level, since it Involves column chromatography at the intermediate or penultimate stages of the preparation of β-ionylideneacetaldehyde. The trans selectivity of C-9 double bond in the preparation of β-ionylideneacetonitrile is poor and requires chromatographic purification before transformation to the aldehyde. The desired aldehyde also requires chromatographic purification, making this approach commercially difficult to implement. In Chem. Pharm. Bull. 1994; 42(3): 757 discloses an improved process by improving the trans-selectivity at the C-9 in the above approach. The process involves the preparation of a tricarbonyl iron complex of β-ionone by reacting β-ionone with triiron dodecacarbonyl in benzene which is condensed with lithium acetonitrile in tetrahydrofuran at −70° C. to afford the nitrile compound. The nitrile intermediate is subjected to the oxidative decomplexation with cupric chloride, followed by DIBAL reduction to afford the desired trans β-ionylideneacetaldehyde. This approach is also not suitable from commercial point of view as it involves a number of steps to generate the trans β-ionylidene-acetaldehyde, and also requires the use of expensive triiron dodecacarbonyl. In view of the above drawbacks in the prior art processes, there is a need for the development of a simpler and efficient process for the preparation of β-ionyledineacetaldehyde with desired ratio of trans-isomer. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention overcomes the problems associated with the prior art and provides a simpler way for obtaining β-ionylideneacetaldehyde in less time and in fewer steps. The invention also avoids the tedious and cumbersome purification process of column chromatography, usage of expensive chemicals, solvents and has obvious benefits with respect to economics and convenience to operate on a commercial scale. Thus, the present invention provides a more commercially viable process for the preparation of pharmaceutically important compounds such as isotretinoin, tretinoin, vitamin A, etc. Accordingly, the present invention provides a process for the synthesis of β-ionylideneacetaldehyde Formula I which comprises: (a) condensing β-ionone of structural Formula II with triethyl phosphonoacetate of structural Formula III in the presence of sodium amide and toluene to obtain ethyl β-ionylideneacetate of Formula IV, (b) reducing the ester of Formula IV to β-ionylidenealcohol of Formula V in the presence of organic solvent selected from hexane, tetrahydrofuran, toluene, xylene, and mixture(s) thereof, and (c) oxidizing the alcohol of Formula V In situ with manganese dioxide at 60-70° C. for about 2 to 4 hours to obtain trans β-ionylidene acetaldehyde of structural formula I. β-ionylideneacetaldehyde, so obtained may be converted into Vitamin A and related compounds such as tretinoin and isotretinoin by methods known in the art. The process of condensation in step (a) is achieved by the reaction of β-ionone of Formula II with triethyl phosphonoacetate of Formula III in the presence of sodium amide and an inert organic solvent such as toluene. After a suitable aqueous work up, the ethyl β-ionylideneacetate of Formula IV is obtained as a mixture of 9-cis and 9-trans isomers in the ratio of 1:7. The process of reduction in step (b) involves the reaction of ester of Formula IV with a reducing agent in organic solvent selected from hexane, tetrahydrofuran, toluene, xylene, and mixture (s) thereof at room temperature. The reducing agent used is selected from the group consisting of lithium aluminium hydride, sodium bis (2-methoxyethoxy) aluminium hydride (Red-Al) and diisobutyl aluminium hydride (DIBAL). The alcohol of Formula V obtained after aqueous acidic work up is oxidized in situ by reacting with manganese dioxide at 60-70° C. for 2 to 4 hours. After the reaction is completed, the desired trans β-ionylideneacetaldehyde is obtained in more than 90% yield having less than 5% of 9-cis isomer. Suitable aqueous work up involves the extraction with organic solvents. Any organic solvent may be used for extraction and such solvents are known to a person of ordinary skill In the art and include both water immisible and partially miscible solvent such as chloroform, methylene chloride, 1,2-dichloroethane, hexanes, cyclohexanes, toluene, methyl acetate, ethyl acetate, and the like. Methods known in the art may be used with the process of this invention to enhance any aspect of this process for example, the product obtained may be further purified by recrystallization from solvent(s). detailed-description description="Detailed Description" end="lead"? |
Compositions and methods relating to prostate specific genes and proteins |
The present invention relates to newly identified nucleic acid molecules and polypeptides present in normal and neoplastic prostate cells, including fragments, variants and derivatives of the nucleic acids and polypeptides. The present invention also relates to antibodies to the polypeptides of the invention, as well as agonists and antagonists of the polypeptides of the invention. The invention also relates to compositions containing the nucleic acid molecules, polypeptides, antibodies, agonists and antagonists of the invention and methods for the use of these compositions. These uses include identifying, diagnosing, monitoring, staging, imaging and treating prostate cancer and non-cancerous disease states in prostate, identifying prostate tissue, monitoring and identifying and/or designing agonists and antagonists of polypeptides of the invention. The uses also include gene therapy, production of transgenic animals and cells, and production of engineered prostate tissue for treatment and research. |
1. An isolated nucleic acid molecule comprising: (a) a nucleic acid molecule comprising a nucleic acid sequence that encodes an amino acid sequence of SEQ ID NO: 95, 96, 97, 99, 116, 119, 124, 125, 126, 127, 135 or 143; (b) a nucleic acid molecule comprising a nucleic acid sequence of SEQ ID NO: 22, 23, 24, 26, 47, 50, 55, 56, 57, 58, 66 or 75; (c) a nucleic acid molecule that selectively hybridizes to the nucleic acid molecule of (a) or (b); or (d) a nucleic acid molecule having at least 90% sequence identity to the nucleic acid molecule of (a) or (b). 2. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is a cDNA. 3. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is genomic DNA. 4. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is a mammalian nucleic acid molecule. 5. The nucleic acid molecule according to claim 4, wherein the nucleic acid molecule is a human nucleic acid molecule. 6. A method for determining the presence of a prostate specific nucleic acid (PSNA) in a sample, comprising the steps of: (a) contacting the sample with the nucleic acid molecule of SEQ ID NO: 21, 22, 23, 24, 26, 43, 47, 50, 55, 56, 57, 58, 66 or 75 under conditions in which the nucleic acid molecule will selectively hybridize to a prostate specific nucleic acid; and (b) detecting hybridization of the nucleic acid molecule to a PSNA in the sample, wherein the detection of the hybridization indicates the presence of a PSNA in the sample. 7. A vector comprising the nucleic acid molecule of claim 1. 8. A host cell comprising the vector according to claim 7. 9. A method for producing a polypeptide encoded by the nucleic acid molecule according to claim 1, comprising the steps of: (a) providing a host cell comprising the nucleic acid molecule operably linked to one or more expression control sequences, and (b) incubating the host cell under conditions in which the polypeptide is produced. 10. A polypeptide encoded by the nucleic acid molecule according to claim 1. 11. An isolated polypeptide selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence with at least 60% sequence identity to of SEQ ID NO: 95, 96, 97, 99, 116, 119, 124, 125, 126,127, 135 or 143 ; or (b) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having at least 90% sequence identity to a nucleic acid molecule comprising a nucleic acid sequence of SEQ ID NO: 22, 23, 24, 26, 47, 50, 55, 56, 57, 58, 66 or 75. 12. An antibody or fragment thereof that specifically binds to (a) a polypeptide comprising an amino acid sequence with at least 60% sequence identity to of SEQ ID NO: 94, 95, 96, 97, 99, 113, 116, 119, 124, 125, 126, 127, 135 or143; or (b) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having at least 90% sequence identity to a nucleic acid molecule comprising a nucleic acid sequence of SEQ ID NO: 21, 22, 23, 24, 26, 43, 47, 50, 55, 56, 57, 58, 66 or 75. 13. A method for determining the presence of a prostate specific protein in a sample, comprising the steps of: (a) contacting the sample with a suitable reagent under conditions in which the reagent will selectively interact with the prostate specific protein comprising an amino acid sequence with at least 60% sequence identity to of SEQ ID NO: 94, 95, 96, 97, 99, 113, 116, 119, 124, 125, 126, 127, 135 or 143; and (b) detecting the interaction of the reagent with a prostate specific protein in the sample, wherein the detection of binding indicates the presence of a prostate specific protein in the sample. 14. A method for diagnosing or monitoring the presence and metastases of prostate cancer in a patient, comprising the steps of: (a) determining an amount of the nucleic acid molecule of claim 1 or a polypeptide of claim 11 in a sample of a patient; and (b) comparing the amount of the determined nucleic acid molecule or the polypeptide in the sample of the patient to the amount of the prostate specific marker in a normal control; wherein a difference in the amount of the nucleic acid molecule or the polypeptide in the sample compared to the amount of the nucleic acid molecule or the polypeptide in the normal control is associated with the presence of prostate cancer. 15. A kit for detecting a risk of cancer or presence of cancer in a patient, said kit comprising a means for determining the presence the nucleic acid molecule of claim 1 or a polypeptide of claim 11 in a sample of a patient. 16. A method of treating a patient with prostate cancer, comprising the step of administering a composition according to claim 11 or 12 to a patient in need thereof, wherein said administration induces an immune response against the prostate cancer cell expressing the nucleic acid molecule or polypeptide. 17. A vaccine comprising the polypeptide or the nucleic acid encoding the polypeptide of claim 11. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Prostate cancer is the most prevalent cancer in men and is the second leading cause of death from cancer among males in the United States. AJCC Cancer Staging Handbook 203 (Irvin D. Fleming et al. eds., 5 th ed. 1998); Walter J. Burdette, Cancer: Etiology Diagnosis, and Treatment 147 (1998). In 1999, it was estimated that 37,000 men in the United States would die as result of prostate cancer. Elizabeth A. Platz et al., & Edward Giovannucci, Epidemiology of and Risk Factors for Prostate Cancer, in Management of Prostate Cancer 21 (Eric A Klein, ed. 2000). Cancer of the prostate typically occurs in older males, with a median age of 74 years for clinical diagnosis. Burdette, supra at 147. A man's risk of being diagnosed with invasive prostate cancer in his lifetime is one in six. Platz et al., supra at 21. Although our understanding of the etiology of prostate cancer is incomplete, the results of extensive research in this area point to a combination of age, genetic and environmental/dietary factors. Platz et al., supra at 19; Burdette, supra at 147; Steven K. Clinton, Diet and Nutrition in Prostate Cancer Prevention and Therapy, in Prostate Cancer: A Multidisciplinary Guide 246-269 (Philip W. Kantoff et al. eds. 1997). Broadly speaking, genetic risk factors predisposing one to prostate cancer include race and a family history of the disease. Platz et al., supra at 19, 28-29, 32-34. Aside from these generalities, a deeper understanding of the genetic basis of prostate cancer has remained elusive. Considerable research has been directed to studying the link between prostate cancer, androgens, and androgen regulation, as androgens play a crucial role in prostate growth and differentiation. Meena Augustus et al., Molecular Genetics and Markers of Progression, in Management of Prostate Cancer 59 (Eric A Klein ed. 2000). While a number of studies have concluded that prostate tumor development is linked to elevated levels of circulating androgen (e.g., testosterone and dihydrotestosterone), the genetic determinants of these levels remain unknown. Platz et al., supra at 29-30. Several studies have explored a possible link between prostate cancer and the androgen receptor (AR) gene, the gene product of which mediates the molecular and cellular effects of testosterone and dihydrotestosterone in tissues responsive to androgens. Id. at 30. Differences in the number of certain trinucleotide repeats in exon 1, the region involved in transactivational control, have been of particular interest. Augustus et al., supra at 60. For example, these studies have revealed that as the number of CAG repeats decreases the transactivation ability of the gene product increases, as does the risk of prostate cancer. Platz et al., supra at 30-31. Other research has focused on the α-reductase Type 2 gene, the gene which codes for the enzyme that converts testosterone into dihydrotestosterone. Id. at 30. Dihydrotestosterone has greater affinity for the AR than testosterone, resulting in increased transactivation of genes responsive to androgens. Id. While studies have reported differences among the races in the length of a TA dinucleotide repeat in the 3′ untranslated region, no link has been established between the length of that repeat and prostate cancer. Id. Interestingly, while ras gene mutations are implicated in numerous other cancers, such mutations appear not to play a significant role in prostate cancer, at least among Caucasian males. Augustus, supra at 52. Environmental/dietary risk factors which may increase the risk of prostate cancer include intake of saturated fat and calcium. Platz et al., supra at 19, 25-26. Conversely, intake of selenium, vitamin E and tomato products (which contain the carotenoid lycopene) apparently decrease that risk. Id. at 19, 26-28 The impact of physical activity, cigarette smoking, and alcohol consumption on prostate cancer is unclear. Platz et al., supra at 23-25. Periodic screening for prostate cancer is most effectively performed by digital rectal examination (DRE) of the prostate, in conjunction with determination of the serum level of prostate-specific antigen (PSA). Burdette, supra at 148. While the merits of such screening are the subject of considerable debate, Jerome P. Richie & Irving D. Kaplan, Screening for Prostate Cancer: The Horns of a Dilemma, in Prostate Cancer: A Multidisciplinary Guide 1-10 (Philip W. Kantoff et al. eds. 1997), the American Cancer Society and American Urological Association recommend that both of these tests be performed annually on men 50 years or older with a life expectancy of at least 10 years, and younger men at high risk for prostate cancer. Ian M. Thompson & John Foley, Screening for Prostate Cancer, in Management of Prostate Cancer 71 (Eric A Klein ed. 2000). If necessary, these screening methods may be followed by additional tests, including biopsy, ultrasonic imaging, computerized tomography, and magnetic resonance imaging. Christopher A. Haas & Martin I. Resnick, Trends in Diagnosis, Biopsy, and Imaging, in Management of Prostate Cancer 89-98 (Eric A Klein ed. 2000); Burdette, supra at 148. Once the diagnosis of prostate cancer has been made, treatment decisions for the individual are typically linked to the stage of prostate cancer present in that individual, as well as his age and overall health. Burdette, supra at 151. One preferred classification system for staging prostate cancer was developed by the American Urological Association (AUA). Id. at 148. The AUA classification system divides prostate tumors into four broad stages, A to D, which are in turn accompanied by a number of smaller substages. Burdette, supra at 152-153; Anthony V. D'Amico et al., The Staging of prostate Cancer, in Prostate Cancer: A Multidisciplinary Guide 41 (Philip W. Kantoff et al. eds. 1997). Stage A prostate cancer refers to the presence of microscopic cancer within the prostate gland. D'Amico, supra at 41. This stage is comprised of two substages: A1, which involves less than four well-differentiated cancer foci within the prostate, and A2, which involves greater than three well-differentiated cancer foci or alternatively, moderately to poorly differentiated foci within the prostate. Burdette, supra at 152; D'Amico, supra at 41. Treatment for stage Al preferentially involves following PSA levels and periodic DRE. Burdette, supra at 151. Should PSA levels rise, preferred treatments include radical prostatectomy in patients 70 years of age and younger, external beam radiotherapy for patients between 70 and 80 years of age, and hormone therapy for those over 80 years of age. Id. Stage B prostate cancer is characterized by the presence of a palpable lump within the prostate. Burdette, supra at 152-53; D'Amico, supra at 41. This stage is comprised of three substages: B1, in which the lump is less than 2 cm and is contained in one lobe of the prostate; B2, in which the lump is greater than 2 cm yet is still contained within one lobe; and B3, in which the lump has spread to both lobes. Burdette, supra, at 152-53. For stages B1 and B2, the treatment again involves radical prostatectomy in patients 70 years of age and younger, external beam radiotherapy for patients between 70 and 80 years of age, and hormone therapy for those over 80 years of age. Id. at 151. In stage B3, radical prostatectomy is employed if the cancer is well-differentiated and PSA levels are below 15 ng/mL; otherwise, external beam radiation is the chosen treatment option. Id. Stage C prostate cancer involves a substantial cancer mass accompanied by extraprostatic extension. Burdette, supra at 153; D'Amico, supra at 41. Like stage A prostate cancer, Stage C is comprised of two substages: substage C1, in which the tumor is relatively minimal, with minor prostatic extension, and substage C2, in which the tumor is large and bulky, with major prostatic extension. Id. The treatment of choice for both substages is external beam radiation. Burdette, supra at 151. The fourth and final stage of prostate cancer, Stage D, describes the extent to which the cancer has metastasized. Burdette, supra at 153; D'Amico, supra at 41. This stage is comprised of four substages: (1) D0, in which acid phophatase levels are persistently high, (2) D1, in which only the pelvic lymph nodes have been invaded, (3) D2, in which the lymph nodes above the aortic bifurcation have been invaded, with or without distant metastasis, and (4) D3, in which the metastasis progresses despite intense hormonal therapy. Id. Treatment at this stage may involve hormonal therapy, chemotherapy, and removal of one or both testes. Burdette, supra at 151. Despite the need for accurate staging of prostate cancer, current staging methodology is limited. The wide variety of biological behavior displayed by neoplasms of the prostate has resulted in considerable difficulty in predicting and assessing the course of prostate cancer. Augustus et al., supra at 47. Indeed, despite the fact that most prostate cancer patients have carcinomas that are of intermediate grade and stage, prognosis for these types of carcinomas is highly variable. Andrew A Renshaw & Christopher L. Corless, Prognostic Features in the Pathology of Prostate Cancer, in Prostate Cancer: A Multidisciplinary Guide 26 (Philip W. Kantoff et al. eds. 1997). Techniques such as transrectal ultrasound, abdominal and pelvic computerized tomography, and MRI have not been particularly useful in predicting local tumor extension. D'Amico, supra at 53 (editors' comment). While the use of serum PSA in combination with the Gleason score is currently the most effective method of staging prostate cancer, id., PSA is of limited predictive value, Augustus et al., supra at 47; Renshaw et al., supra at 26, and the Gleason score is prone to variability and error, King, C. R. & Long, J. P., Int'l. J. Cancer 90(6): 326-30 (2000). As such, the current focus of prostate cancer research has been to obtain biomarkers to help better assess the progression of the disease. Augustus et al., supra at 47; Renshaw et al., supra at 26; Pettaway, C. A., Tech. Urol. 4(1): 3542 (1998). Accordingly, there is a great need for more sensitive and accurate methods for predicting whether a person is likely to develop prostate cancer, for diagnosing prostate cancer, for monitoring the progression of the disease, for staging the prostate cancer, for determining whether the prostate cancer has metastasized and for imaging the prostate cancer. There is also a need for better treatment of prostate cancer. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention solves many needs in the art by providing nucleic acid molecules, polypeptides and antibodies thereto, variants and derivatives of the nucleic acids and polypeptides, agonists and antagonists that may be used to identify, diagnose, monitor, stage, image and treat prostate cancer and non-cancerous disease states in prostate; identify and monitor prostate tissue; and identify and design agonists and antagonists of polypeptides of the invention. The invention also provides gene therapy, methods for producing transgenic animals and cells, and methods for producing engineered prostate tissue for treatment and research. One aspect of the present invention relates to nucleic acid molecules that are specific to prostate cells, prostate tissue and/or the prostate organ. These prostate specific nucleic acids (PSNAs) may be a naturally-occurring cDNA, genomic DNA, RNA, or a fragment of one of these nucleic acids, or may be a non-naturally-occurring nucleic acid molecule. If the PSNA is genomic DNA, then the PSNA is a prostate specific gene (PSG). In a preferred embodiment, the nucleic acid molecule encodes a polypeptide that is specific to prostate. More preferred is a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 79-146. In another preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence of SEQ ID NO: 1-78. For the sequences listed herein, DEX0236 — 1 corresponds to SEQ ID NO: 1, DEX0236 — 79 corresponds to SEQ ID NO: 79, etc. This aspect of the present invention also relates to nucleic acid molecules that selectively hybridize or exhibit substantial sequence similarity to nucleic acid molecules encoding a Prostate Specific Protein (PSP), or that selectively hybridize or exhibit substantial sequence similarity to a PSNA. In one embodiment of the present invention the nucleic acid molecule comprises an allelic variant of a nucleic acid molecule encoding a PSP, or an allelic variant of a PSNA. In another embodiment, the nucleic acid molecule comprises a part of a nucleic acid sequence that encodes a PSP or a part of a nucleic acid sequence of a PSNA. In addition, this aspect of the present invention relates to a nucleic acid molecule further comprising one or more expression control sequences controlling the transcription and/or translation of all or a part of a PSNA or the the transcription and/or translation of a nucleic acid molecule that encodes all or a fragment of a PSP. Another aspect of the present invention relates to vectors and/or host cells comprising a nucleic acid molecule of this invention. In a preferred embodiment, the nucleic acid molecule of the vector and/or host cell encodes all or a fragment of a PSP. In another preferred embodiment, the nucleic acid molecule of the vector and/or host cell comprises all or a part of a PSNA. Vectors and host cells of the present invention are useful in the recombinant production of polypeptides, particularly PSPs of the present invention. Another aspect of the present invention relates to polypeptides encoded by a nucleic acid molecule of this invention. The polypeptide may comprise either a fragment or a full-length protein. In a preferred embodiment, the polypeptide is a PSP. However, this aspect of the present invention also relates to mutant proteins (muteins) of PSPs, fusion proteins of which a portion is a PSP, and proteins and polypeptides encoded by allelic variants of a PSNA as provided herein. Another aspect of the present invention relates to antibodies and other binders that specifically binds to a polypeptide of the instant invention. Accordingly antibodies or binders of the present specifically bind to PSPs, muteins, fusion proteins, and/or homologous proteins or a polypeptides encoded by allelic variants of an PSNA as provided herein. Another aspect of the present invention relates to agonists and antagonists of the nucleic acid molecules and polypeptides of this invention. The agonists and antagonists of the instant invention may be used to treat prostate cancer and non-cancerous disease states in prostate and to produce engineered prostate tissue. Another aspect of the present invention relates to methods for using the nucleic acid molecules to detect or amplify nucleic acid molecules that have similar or identical nucleic acid sequences compared to the nucleic acid molecules described herein. Such methods are useful in identifying, diagnosing, monitoring, staging, imaging and treating prostate cancer and non-cancerous disease states in prostate. Such methods are also useful in identifying and/or monitoring prostate tissue. In addition, measurement of levels of the nucleic acid molecules of this invention may be useful for diagnostics as part of panel in combination with other markers. Another aspect of the present invention relates to use of the nucleic acid molecules of this invention in gene therapy, for producing transgenic animals and cells, and for producing engineered prostate tissue for treatment and research. Another aspect of the present invention relates to methods for detecting polypeptides this invention, preferably using antibodies thereto. Such methods are useful to identify, diagnose, monitor, stage, image and treat prostate cancer and non-cancerous disease states in prostate. In addition, measurement of levels of the polypeptides of this invention may be useful to identify, diagnose, monitor, stage, image prostate cancer in combination with other prostate cancer markers. The polypeptides of the present invention can also be used to identify and/or monitor prostate tissue, and to produce engineered prostate tissue. Yet another aspect of the present invention relates to a computer readable means of storing the nucleic acid and amino acid sequences of the invention. The records of the computer readable means can be accessed for reading and displaying of sequences for comparison, alignment and ordering of the sequences of the invention to other sequences. In addition, the computer records regarding the nucleic acid and/or amino acid sequences and/or measurements of their levels may be used alone or in combination with other markers to diagnose prostate related diseases. detailed-description description="Detailed Description" end="lead"? |
Compositions and methods relating to ovary specific genes and proteins |
The present invention relates to newly identified nucleic acid molecules and polypeptides present in normal and neoplastic ovarian cells, including fragments, variants and derivatives of the nucleic acids and polypeptides. The present invention also relates to antibodies to the polypeptides of the invention, as well as agonists and antagonists of the polypeptides of the invention. The invention also relates to compositions containing the nucleic acid molecules, polypeptides, antibodies, agonists and antagonists of the invention and methods for the use of these compositions. These uses include identifying, diagnosing, monitoring, staging, imaging and treating ovarian cancer and non-cancerous disease states in ovarian, identifying ovarian tissue, monitoring and identifying and/or designing agonists and antagonists of polypeptides of the invention. The uses also include gene therapy, production of transgenic animals and cells, and production of engineered ovarian tissue for treatment and research. |
1. An isolated nucleic acid molecule comprising: (a) a nucleic acid molecule comprising a nucleic acid sequence that encodes an amino acid sequence of SEQ ID NO: 68, 70, 73, 75, 80, 93, 96, 97 or 110; (b) a nucleic acid molecule comprising a nucleic acid sequence of SEQ ID NO: 7, 9, 13, 16, 21, 34, 36, 40, 41 or 54; (c) a nucleic acid molecule that selectively hybridizes to the nucleic acid molecule of (a) or (b); or (d) a nucleic acid molecule having at least 90% sequence identity to the nucleic acid molecule of (a) or (b). 2. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is a cDNA. 3. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is genomic DNA. 4. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is a mammalian nucleic acid molecule. 5. The nucleic acid molecule according to claim 4, wherein the nucleic acid molecule is a human nucleic acid molecule. 6. A method for determining the presence of an ovarian specific nucleic acid (OSNA) in a sample, comprising the steps of: (a) contacting the sample with the nucleic acid molecule of SEQ ID NO: 7, 9, 13, 16, 18, 21, 34, 36, 40, 41 or 54 under conditions in which the nucleic acid molecule will selectively hybridize to an ovarian specific nucleic acid; and (b) detecting hybridization of the nucleic acid molecule to an OSNA in the sample, wherein the detection of the hybridization indicates the presence of an OSNA in the sample. 7. A vector comprising the nucleic acid molecule of claim 1. 8. A host cell comprising the vector according to claim 7. 9. A method for producing a polypeptide encoded by the nucleic acid molecule according to claim 1, comprising the steps of: (a) providing a host cell comprising the nucleic acid molecule operably linked to one or more expression control sequences, and (b) incubating the host cell under conditions in which the polypeptide is produced. 10. A polypeptide encoded by the nucleic acid molecule according to claim 1. 11. An isolated polypeptide selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence with at least 60% sequence identity to of SEQ ID NO: 68, 70, 73, 75, 80, 93, 96, 97 or 110 ; or (b) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having at least 90% sequence identity to a nucleic acid molecule comprising a nucleic acid sequence of SEQ ID NO: 7, 9, 13, 16, 21, 34, 36, 40, 41 or 54. 12. An antibody or fragment thereof that specifically binds to (a) a polypeptide comprising an amino acid sequence with at least 60% sequence identity to of SEQ ID NO: 68, 70, 73, 75, 77, 80, 93, 96, 97 or 110; or (b) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having at least 90% sequence identity to a nucleic acid molecule comprising a nucleic acid sequence of SEQ ID NO: 7, 9, 13, 16, 18, 21, 34, 36, 40, 41 or 54. 13. A method for determining the presence of an ovarian specific protein in a sample, comprising the steps of: (a) contacting the sample with a suitable reagent under conditions in which the reagent will selectively interact with the ovarian specific protein comprising an amino acid sequence with at least 60% sequence identity to of SEQ ID NO: 68, 70, 73, 75, 77, 80, 93, 96, 97 or 110; and (b) detecting the interaction of the reagent with an ovarian specific protein in the sample, wherein the detection of binding indicates the presence of an ovarian specific protein in the sample. 14. A method for diagnosing or monitoring the presence and metastases of ovarian cancer in a patient, comprising the steps of: (a) determining an amount of the nucleic acid molecule of claim 1 or a polypeptide of claim 11 in a sample of a patient; and (b) comparing the amount of the determined nucleic acid molecule or the polypeptide in the sample of the patient to the amount of the ovarian specific marker in a normal control; wherein a difference in the amount of the nucleic acid molecule or the polypeptide in the sample compared to the amount of the nucleic acid molecule or the polypeptide in the normal control is associated with the presence of ovarian cancer. 15. A kit for detecting a risk of cancer or presence of cancer in a patient, said kit comprising a means for determining the presence the nucleic acid molecule of claim 1 or a polypeptide of claim 11 in a sample of a patient. 16. A method of treating a patient with ovarian cancer, comprising the step of administering a composition according to claim 11 or 12 to a patient in need thereof, wherein said administration induces an immune response against the ovarian cancer cell expressing the nucleic acid molecule or polypeptide. 17. A vaccine comprising the polypeptide or the nucleic acid encoding the polypeptide of claim 11. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Cancer of the ovaries is the fourth-most cause of cancer death in women in the United States, with more than 23,000 new cases and roughly 14,000 deaths predicted for the year 2001. Shridhar, V. et al., Cancer Res. 61(15): 5895-904 (2001); Memarzadeh, S. & Berek, J. S., J. Reprod. Med. 46(7): 621-29 (2001). The incidence of ovarian cancer is of serious concern worldwide, with an estimated 191,000 new cases predicted annually. Runnebaum, I. B. & Stickeler, E., J. Cancer Res. Clin. Oncol. 127(2): 73-79 (2001). Unfortunately, women with ovarian cancer are typically asymptomatic until the disease has metastasized. Because effective screening for ovarian cancer is not available, roughly 70% of women diagnosed have an advanced stage of the cancer with a five-year survival rate of ˜25-30%. Memarzadeh, S. & Berek, J. S., supra; Nunns, D. et al., Obstet. Gynecol. Surv. 55(12): 746-51. Conversely, women diagnosed with early stage ovarian cancer enjoy considerably higher survival rates. Wemess, B. A. & Eltabbakh, G. H., Int'l. J. Gynecol. Pathol. 20(1): 48-63 (2001). Although our understanding of the etiology of ovarian cancer is incomplete, the results of extensive research in this area point to a combination of age, genetics, reproductive, and dietary/environmental factors. Age is a key risk factor in the development of ovarian cancer: while the risk for developing ovarian cancer before the age of 30 is slim, the incidence of ovarian cancer rises linearly between ages 30 to 50, increasing at a slower rate thereafter, with the highest incidence being among septagenarian women. Jeanne M. Schilder et al., Heriditary Ovarian Cancer: Clinical Syndromes and Management , in Ovarian Cancer 182 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001). With respect to genetic factors, a family history of ovarian cancer is the most significant risk factor in the development of the disease, with that risk depending on the number of affected family members, the degree of their relationship to the woman, and which particular first degree relatives are affected by the disease. Id. Mutations in several genes have been associated with ovarian cancer, including BRCA1 and BRCA2, both of which play a key role in the development of breast cancer, as well as hMSH2 and hMLH1, both of which are associated with heriditary non-polyposis colon cancer. Katherine Y. Look, Epidemiology, Etiology, and Screening of Ovarian Cancer , in Ovarian Cancer 169, 171-73 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001). BRCA1, located on chromosome 17, and BRCA2, located on chromosome 13, are tumor supressor genes implicated in DNA repair; mutations in these genes are linked to roughly 10% of ovarian cancers. Id. at 171-72; Schilder et al., supra at 185-86. hMSH2 and hMLH1 are associated with DNA mismatch repair, and are located on chromsomes 2 and 3, respectively; it has been reported that roughly 3% of heriditary ovarian carcinomas are due to mutations in these genes. Look, supra at 173; Schilder et al., supra at 184, 188-89. Reproductive factors have also been associated with an increased or reduced risk of ovarian cancer. Late menopause, nulliparity, and early age at menarche have all been linked with an elevated risk of ovarian cancer. Schilder et al., supra at 182. One theory hypothesizes that these factors increase the number of ovulatory cycles over the course of a woman's life, leading to “incessant ovulation,” which is thought to be the primary cause of mutations to the ovarian epithelium. Id.; Laura J. Havrilesky & Andrew Berchuck, Molecular Alterations in Sporadic Ovarian Cancer , in Ovarian Cancer 25 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001). The mutations may be explained by the fact that ovulation results in the destruction and repair of that epithelium, necessitating increased cell division, thereby increasing the possibility that an undetected mutation will occur. Id. Support for this theory may be found in the fact pregnancy, lactation, and the use of oral contraceptives, all of which suppress ovulation, confer a protective effect with respect to developing ovarian cancer. Id. Among dietary/environmental factors, there would appear to be an association between high intake of animal fat or red meat and ovarian cancer, while the antioxidant Vitamin A, which prevents free radical formation and also assists in maintaining normal cellular differentiation, may offer a protective effect. Look, supra at 169. Reports have also associated asbestos and hydrous magnesium trisilicate (talc), the latter of which may be present in diaphragms and sanitary napkins. Id. at 169-70. Current screening procedures for ovarian cancer, while of some utility, are quite limited in their diagnostic ability, a problem that is particularly acute at early stages of cancer progression when the disease is typically asymptomatic yet is most readily treated. Walter J. Burdette, Cancer: Etiology, Diagnosis, and Treatment 166 (1998); Memarzadeh & Berek, supra; Runnebaum & Stickeler, supra; Wemess & Eltabbakh, supra. Commonly used screening tests include biannual rectovaginal pelvic examination, radioimmunoassay to detect the CA-125 serum tumor marker, and transvaginal ultrasonography. Burdette, supra at 166. Pelvic examination has failed to yield adequate numbers of early diagnoses, and the other methods are not sufficiently accurate. Id. One study reported that only 15% of patients who suffered from ovarian cancer were diagnosed with the disease at the time of their pelvic examination. Look, supra at 174. Moreover, the CA-125 test is prone to giving false positives in pre-menopausal women and has been reported to be of low predictive value in post-menopausal women. Id. at 174-75. Although transvaginal ultrasonography is now the preferred procedure for screening for ovarian cancer, it is unable to distinguish reliably between benign and malignant tumors, and also cannot locate primary peritoneal malignancies or ovarian cancer if the ovary size is normal. Schilder et al., supra at 194-95. While genetic testing for mutations of the BRCA1, BRCA2, hMSH2, and hMLH1 genes is now available, these tests may be too costly for some patients and may also yield false negative or indeterminate results. Schilder et al., supra at 191-94. The staging of ovarian cancer, which is accomplished through surgical exploration, is crucial in determining the course of treatment and management of the disease. AJCC Cancer Staging Handbook 187 (Irvin D. Fleming et al. eds., 5th ed. 1998); Burdette, supra at 170; Memarzadeh & Berek, supra; Shridhar et al., supra. Staging is performed by reference to the classification system developed by the International Federation of Gynecology and Obstetrics. David H. Moore, Primary Surgical Management of Early Epithelial Ovarian Carcinoma , in Ovarian Cancer 203 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001); Fleming et al. eds., supra at 188. Stage I ovarian cancer is characterized by tumor growth that is limited to the ovaries and is comprised of three substages. Id. In substage IA, tumor growth is limited to one ovary, there is no tumor on the external surface of the ovary, the ovarian capsule is intact, and no malignant cells are present in ascites or peritoneal washings. Id. Substage IB is identical to A1, except that tumor growth is limited to both ovaries. Id, Substage IC refers to the presence of tumor growth limited to one or both ovaries, and also includes one or more of the following characteristics: capsule rupture, tumor growth on the surface of one or both ovaries, and malignant cells present in ascites or peritoneal washings. Id. Stage II ovarian cancer refers to tumor growth involving one or both ovaries, along with pelvic extension. Id. Substage IIA involves extension and/or implants on the uterus and/or fallopian tubes, with no malignant cells in the ascites or peritoneal washings, while substage IIB involves extension into other pelvic organs and tissues, again with no malignant cells in the ascites or peritoneal washings. Id. Substage IIC involves pelvic extension as in IIA or IIIB, but with malignant cells in the ascites or peritoneal washings. Id. Stage III ovarian cancer involves tumor growth in one or both ovaries, with peritoneal metastasis beyond the pelvis confirmed by microscope and/or metastasis in the regional lymph nodes. Id. Substage IIIA is characterized by microscopic peritoneal metastasis outside the pelvis, with substage IIIB involving macroscopic peritoneal metastasis outside the pelvis 2 cm or less in greatest dimension. Id. Substage IIIc is identical to IIIB, except that the metastasis is greater than 2 cm in greatest dimension and may include regional lymph node metastasis. Id. Lastly, Stage IV refers to the presence distant metastasis, excluding peritoneal metastasis. Id. While surgical staging is currently the benchmark for assessing the management and treatment of ovarian cancer, it suffers from considerable drawbacks, including the invasiveness of the procedure, the potential for complications, as well as the potential for inaccuracy. Moore, supra at 206-208, 213. In view of these limitations, attention has turned to developing alternative staging methodologies through understanding differential gene expression in various stages of ovarian cancer and by obtaining various biomarkers to help better assess the progression of the disease. Vartiainen, J. et al., Int'l J. Cancer, 95(5): 313-16 (2001); Shridhar et al. supra; Baekelandt, M. et al., J. Clin. Oncol. 18(22): 3775-81. The treatment of ovarian cancer typically involves a multiprong attack, with surgical intervention serving as the foundation of treatment. Dennis S. Chi & William J. Hoskins, Primary Surgical Management of Advanced Epithelial Ovarian Cancer , in Ovarian Cancer 241 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001). For example, in the case of epithelial ovarian cancer, which accounts for ˜90% of cases of ovarian cancer, treatment typically consists of: (1) cytoreductive surgery, including total abdominal hysterectomy, bilateral salpingo-oophorectomy, omentectomy, and lymphadenectomy, followed by (2) adjuvant chemotherapy with paclitaxel and either cisplatin or carboplatin. Eltabbakh, G. H. & Awtrey, C. S., Expert Op. Pharnacother. 2(10): 109-24. Despite a clinical response rate of 80% to the adjuvant therapy, most patients experience tumor recurrence within three years of treatment. Id. Certain patients may undergo a second cytoreductive surgery and/or second-line chemotherapy. Memarzadeh & Berek, supra. From the foregoing, it is clear that procedures used for detecting, diagnosing, monitoring, staging, prognosticating, and preventing the recurrence of ovarian cancer are of critical importance to the outcome of the patient. Moreover, current procedures, while helpful in each of these analyses, are limited by their specificity, sensitivity, invasiveness, and/or their cost As such, highly specific and sensitive procedures that would operate by way of detecting novel markers in cells, tissues, or bodily fluids, with minimal invasiveness and at a reasonable cost, would be highly desirable. Accordingly, there is a great need for more sensitive and accurate methods for predicting whether a person is likely to develop ovarian cancer, for diagnosing ovarian cancer, for monitoring the progression of the disease, for staging the ovarian cancer, for determining whether the ovarian cancer has metastasized, and for imaging the ovarian cancer. There is also a need for better treatment of ovarian cancer. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention solves many needs in the art by providing nucleic acid molecules, polypeptides and antibodies thereto, variants and derivatives of the nucleic acids and polypeptides, agonists and antagonists that may be used to identify, diagnose, monitor, stage, image and treat ovarian cancer and non-cancerous disease states in ovarian; identify and monitor ovarian tissue; and identify and design agonists and antagonists of polypeptides of the invention. The invention also provides gene therapy, methods for producing transgenic animals and cells, and methods for producing engineered ovarian tissue for treatment and research. One aspect of the present invention relates to nucleic acid molecules that are specific to ovarian cells, ovarian tissue and/or the ovarian organ. These ovarian specific nucleic acids (OSNAs) may be a naturally-occurring cDNA, genomic DNA, RNA, or a fragment of one of these nucleic acids, or may be a non-naturally-occurring nucleic acid molecule. If the OSNA is genomic DNA, then the OSNA is an ovarian specific gene (OSG). In a preferred embodiment, the nucleic acid molecule encodes a polypeptide that is specific to ovarian. More preferred is a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 62-116. In another preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence of SEQ ID NO: 1-61. For the sequences listed herein, DEX0237 — 1 corresponds to SEQ ID NO: 1, DEX0237 — 62 corresponds to SEQ ID NO: 62, etc. This aspect of the present invention also relates to nucleic acid molecules that selectively hybridize or exhibit substantial sequence similarity to nucleic acid molecules encoding a Ovarian Specific Protein (OSP), or that selectively hybridize or exhibit substantial sequence similarity to an OSNA. In one embodiment of the present invention the nucleic acid molecule comprises an allelic variant of a nucleic acid molecule encoding an OSP, or an allelic variant of an OSNA. In another embodiment, the nucleic acid molecule comprises a part of a nucleic acid sequence that encodes an OSP or a part of a nucleic acid sequence of an OSNA. In addition, this aspect of the present invention relates to a nucleic acid molecule further comprising one or more expression control sequences controlling the transcription and/or translation of all or a part of an OSNA or the transcription and/or translation of a nucleic acid molecule that encodes all or a fragment of an OSP. Another aspect of the present invention relates to vectors and/or host cells comprising a nucleic acid molecule of this invention. In a preferred embodiment, the nucleic acid molecule of the vector and/or host cell encodes all or a fragment of an OSP. In another preferred embodiment, the nucleic acid molecule of the vector and/or host cell comprises all or a part of an OSNA. Vectors and host cells of the present invention are useful in the recombinant production of polypeptides, particularly OSPs of the present invention. Another aspect of the present invention relates to polypeptides encoded by a nucleic acid molecule of this invention. The polypeptide may comprise either a fragment or a full-length protein. In a preferred embodiment, the polypeptide is an OSP. However, this aspect of the present invention also relates to mutant proteins (muteins) of OSPs, fusion proteins of which a portion is an OSP, and proteins and polypeptides encoded by allelic variants of an OSNA as provided herein. Another aspect of the present invention relates to antibodies and other binders that specifically binds to a polypeptide of the instant invention. Accordingly antibodies or binders of the present specifically bind to OSPs, muteins, fusion proteins, and/or homologous proteins or a polypeptides encoded by allelic variants of an OSNA as provided herein. Another aspect of the present invention relates to agonists and antagonists of the nucleic acid molecules and polypeptides of this invention. The agonists and antagonists of the instant invention may be used to treat ovarian cancer and non-cancerous disease states in the ovary and to produce engineered ovarian tissue. Another aspect of the present invention relates to methods for using the nucleic acid molecules to detect or amplify nucleic acid molecules that have similar or identical nucleic acid sequences compared to the nucleic acid molecules described herein. Such methods are useful in identifying, diagnosing, monitoring, staging, imaging and treating ovarian cancer and non-cancerous disease states in the ovary. Such methods are also useful in identifying and/or monitoring ovarian tissue. In addition, measurement of levels of the nucleic acid molecules of this invention may be useful for diagnostics as part of panel in combination with other markers. Another aspect of the present invention relates to use of the nucleic acid molecules of this invention in gene therapy, for producing transgenic animals and cells, and for producing engineered ovarian tissue for treatment and research. Another aspect of the present invention relates to methods for detecting polypeptides this invention, preferably using antibodies thereto. Such methods are useful to identify, diagnose, monitor, stage, image and treat ovarian cancer and non-cancerous disease states in the ovary. In addition, measurement of levels of the polypeptides of this invention may be useful to identify, diagnose, monitor, stage, image ovarian cancer in combination with other ovarian cancer markers. The polypeptides of the present invention can also be used to identify and/or monitor ovarian tissue, and to produce engineered ovarian tissue. Yet another aspect of the present invention relates to a computer readable means of storing the nucleic acid and amino acid sequences of the invention. The records of the computer readable means can be accessed for reading and displaying of sequences for comparison, alignment and ordering of the sequences of the invention to other sequences. In addition, the computer records regarding the nucleic acid and/or amino acid sequences and/or measurements of their levels may be used alone or in combination with other markers to diagnose ovarian related diseases. detailed-description description="Detailed Description" end="lead"? |
Organic luminous diode, method for the production thefeof and uses thereof |
The invention relates to an organic light emitting diode (OLED), also referred to as a light emitting diode, which comprises at least one substrate, one anode, one perforated transport layer, one emitter layer, one cathode and one encapsulation. It is proposed that an energy carrier, which supplies the voltage that causes the OLED to emit light, be integrated into the OLED. The energy carrier can be a battery (energy storage device) or an energy converter (photovoltaic element). |
1-12. (canceled) 13. A light emitting device, comprising: one or more substrates; an organic light emitting diode supported by the one or more substrates, wherein the organic light emitting diode comprises: a first electrode; an emitter layer disposed on the first electrode; and a second electrode disposed on the emitter layer; and an energy source to apply a voltage to the organic light emitting diode, wherein the energy source is supported by the one or more substrates. 14. The device according to claim 13, further comprising: an encapsulation that encapsulates the energy source. 15. The device according to claim 14, wherein: the encapsulation includes a sealing layer. 16. The device according to claim 13, further comprising: an encapsulation that encapsulates the organic light emitting diode. 17. The device according to claim 16, wherein: the encapsulation includes a sealing layer. 18. The device according to claim 13, wherein: the energy source is an energy converter. 19. The device according to claim 18, wherein: the energy converter comprises a photovoltaic element. 20. The device according to claims 18, wherein: the one or more substrates includes a semitransparent insulation layer and the semitransparent insulation layer is between the organic light emitting diode and the energy converter. 21. The device according to claims 20, wherein: the semitransparent insulation layer comprises at least one of a planarizing layer, a thin substrate, a flexible layer and a getter layer. 22. The device according to claim 13, wherein: the energy source is an energy storage device. 23. The device according to claim 22, wherein: the one or more substrates include a semitransparent insulation layer and the semitransparent insulation layer is between the organic light emitting diode and the energy storage device. 24. The device according to claim 23, wherein: the semitransparent insulation layer comprises at least one of a planarizing layer, a thin substrate, a flexible layer and a getter layer. 25. The device according to claim 13, wherein: the energy source is at least semitransparent. 26. The device according to claim 13, wherein: the organic light emitting diode is supported by a first substrate of the one or more substrates; the energy source is supported by a second substrate of the one or more substrates; and the first and second substrates encapsulate the organic light emitting diode and the energy source. 27. The device according to claim 13, wherein: the organic light emitting diode is supported by a first side of a substrate of the one or more substrates; and the energy source is supported by a second side of the substrate of the one or more substrates. 28. The device according to claim 13, wherein: the organic light emitting diode contacts the energy source. 29. The device according to claim 13, wherein: the energy source includes the first electrode of the organic light emitting diode. 30. The device according to claim 29, wherein: the first electrode is an anode. 31. A method for producing a light emitting device, comprising a substrate and an organic light emitting diode having an anode, an emitter layer and a cathode, wherein an energy source is integrated into the light emitting device to provide a voltage to the organic light emitting diode, comprising: providing one or more substrates; forming an energy source on the one or more substrates; forming an organic light emitting diode on the one or more substrates; encapsulating the energy source. 32. The method of claim 31, wherein: forming the energy source includes forming a solar cell. 33. The method of claim 31, wherein: encapsulating the energy source includes encapsulating the organic light emitting diode. 34. A method for producing a light emitting device, comprising a substrate and an organic light emitting diode having an anode, an emitter layer and a cathode, wherein an energy source is integrated into the light emitting device to provide a voltage to the organic light emitting diode, comprising: constructing an energy source on a substrate; encapsulating the energy source; constructing an organic light emitting diode on the encapsulated energy source to form a combined energy source and organic light emitting diode; and encapsulating the combined energy source and organic light emitting diode. 35. The method of claim 34, wherein: constructing an energy source on a substrate includes constructing a solar cell. 36. A method for producing a light emitting device, comprising a substrate and an organic light emitting diode having an anode, an emitter layer and a cathode, wherein an energy source is integrated into the light emitting device to provide a voltage to the organic light emitting diode, comprising: forming a first cathode on a substrate; forming a first photoactive layer on the first cathode; forming an anode on the first photoactive layer; forming a second photoactive layer on the anode; forming a second cathode on the second photoactive layer; and forming an encapsulation to encapsulate the first and second cathodes. 37. The method of claim 36 wherein: the first photoactive layer forms a layer of an energy carrier; and the second photoactive layer forms a layer of an organic light emitting diode. 38. The method of claim 36 wherein: the first photoactive layer forms a layer of an organic light emitting diode; and the second photoactive layer forms a layer of an energy carrier. 39. A method of operating a light emitting device that comprises a light emitting diode having a first electrode, an emitter layer and a second electrode, wherein the emitter layer is between the first and second electrodes and at least one energy source is integrated into the organic light emitting device such that the first and second electrodes, emitter layer and energy source are encapsulated, the method comprising: supplying a voltage from the energy source to the organic light emitting diode; and applying the supplied voltage across the first electrode and the second electrode; and emitting light when the voltage is applied across the first and second electrodes. 40. The method of claim 39, further comprising: detecting an environmental influence selected from the group consisting of radiation, heat, pressure and sound, wherein detecting the environmental influence initiates the step of supplying a voltage. 41. The method of claim 39, wherein: emitting light includes emitting light through the energy source. 42. An electronic paper, comprising: one or more substrates; an organic light emitting diode supported by the one or more substrates, wherein the organic light emitting diode comprises: a first electrode; an emitter layer disposed on the first electrode; and a second electrode disposed on the emitter layer; and an energy source to apply a voltage to the organic light emitting diode wherein the energy source is supported by the one or more substrates. |
Nitroreductase enzymes |
Improved nitroreductase enzymes, particularly for use as prodrug converting enzymes are provided. In particular, single and double mutants of the E. coli NFSB nitroreductase, having improved properties for the activation of the prodrug CB 1954 for use in gene therapy are disclosed. |
1. A recombinant mutant nitroreductase encoded by a mutated equivalent of the E.coli NFSB gene, characterised in that it has an increased nitroreductase activity for CB1954 compared to the wild-type enzyme. 2. A nitroreductase according to claim 1, characterised in that said nitroreductase comprises a substitution of serine 40 with an amino acid selected from a group consisting of alanine, glycine and threonine. 3. A nitroreductase according to claim 1, characterised in that said nitroreductase corresponds to the wild-type sequence of FIG. 9 (SEQ ID NO: 1), wherein serine 40 is substituted by an amino acid selected from the group consisting of alanine, glycine and threonine, and optionally also having substitutions, insertions or deletions at residues other than serine 40. 4. A nitroreductase according to claim 1, characterised in that said nitroreductase comprises a substitution of threonine 41 with an amino acid selected from a group consisting of asparagine, glycine, isoleucine, leucine and serine. 5. A nitroreductase according to claim 1, characterised in that said nitroreductase corresponds to the wild-type sequence of FIG. 9 (SEQ ID NO: 1), wherein threonine 41 is substituted by an amino acid selected from the group consisting of asparagine, glycine, isoleucine, leucine and serine, and optionally also having substitutions, insertions or deletions at residues other than threonine 41. 6. A nitroreductase according to claim 1, characterised in that said nitroreductase comprises a substitution of tyrosine 68 with an amino acid selected from a group consisting of alanine, asparagine, aspartate, cysteine, glutamine, glycine, histidine, serine and tryptophan. 7. A nitroreductase according to claim 1, characterised in that said nitroreductase corresponds to the wild-type sequence of FIG. 9 (SEQ ID NO:1), wherein tyrosine 68 is substituted by an amino acid selected from the group consisting of alanine, asparagine, aspartate, cysteine, glutamine, glycine, histidine, serine and tryptophan, and optionally also having substitutions, insertions or deletions at residues other than tyrosine 68. 8. The nitroreductase of claim 7, characterised in that said nitroreductase is a double mutant comprising a first substitution of tyrosine 68 to glycine (Y68G) and a second substitution of phenylalanine 124 to tryptophan (F124W). 9. A nitroreductase according to claim 1, characterised in that said nitroreductase comprises a substitution of phenylalanine 70 with an amino acid selected from a group consisting of alanine, cysteine, glutamine, glutamate, glycine, isoleucine, leucine, proline, serine, threonine and valine. 10. A nitroreductase according to claim 1, characterised in that said nitroreductase corresponds to the wild-type sequence of FIG. 9 (SEQ ID NO:1), wherein phenylalanine 70 is substituted by an amino acid selected from the group consisting of alanine, cysteine, glutamine, glutamate, glycine, isoleucine, leucine, proline, serine, threonine and valine, and optionally also having substitutions, insertions or deletions at residues other than phenylalanine 70. 11. A nitroreductase according to claim 1, characterised in that said nitroreductase comprises a substitution of asparagine 71 with an amino acid selected from a group consisting of aspartate, glutamine and serine. 12. A nitroreductase according to claim 1, characterised in that said nitroreductase corresponds to the wild-type sequence of FIG. 9 (SEQ ID NO: 1), wherein asparagine 71 is substituted by an amino acid selected from the group consisting of aspartate, glutamine and serine, and optionally also having substitutions, insertions or deletions at residues other than asparagine 71. 13. The nitroreductase of claim 12 characterised in that said nitroreductase is a double mutant comprising a first substitution of asparagine 71 to serine (N71S) and a second substitution of phenylalanine 124 to lysine (F124K). 14. A nitroreductase according to claim 1 characterised in that said nitroreductase comprises a substitution of glycine 120 with an amino acid selected from a group consisting of alanine, serine and threonine. 15. A nitroreductase according to claim 1, characterised in that said nitroreductase corresponds to the wild-type sequence of FIG. 9 (SEQ ID NO:1), wherein glycine 120 is substituted with an amino acid selected from a group consisting of alanine, serine and threonine. 16. A nitroreductase according to claim 1, characterised in that said nitroreductase comprises a substitution of phenylalanine 124 with an amino acid selected from a group consisting of asparagines, cysteine, glycine, lysine, methionine, tryptophan and tyrosine. 17. A nitroreductase according to claim 1, characterised in that said nitroreductase corresponds to the wild-type sequence of FIG. 9 (SEQ ID NO:1), wherein phenylalanine 124 is substituted by an amino acid selected from the group consisting of asparagine, cysteine, glycine, lysine, methionine, tryptophan and tyrosine, and optionally also having substitutions, insertions or deletions at residues other than phenylalanine 124. 18. An isolated polynucleotide encoding a nitroreductase according to claim 1. 19-24. (canceled) 25. A recombinant mutant nitroreductase encoded by a mutated E.coli NfsB gene, characterised in that it has an increased nitroreductase activity compared to the wild-type enzyme and comprises the substitution of phenylalanine 124 with an amino acid selected from the group consisting of alanine, glutamine, histidine, isoleucine, leucine, serine, threonine or valine. 26. A recombinant E.coli NfsB nitroreductase mutant corresponding to the wild-type sequence of FIG. 9 (SEQ ID NO: 1), characterised in that phenylalanine 124 is substituted by an amino acid selected from the group consisting of alanine, glutamine, histidine, isoleucine, leucine, serine, threonine or valine, having nitroreductase activity greater than that of the wild-type protein, and optionally also having substitutions, insertions or deletions at residues other than phenylalanine 124. 27. An isolated polynucleotide encoding a nitroreductase according to claim 25. 28-34. (canceled) 35. A vector comprising an isolated polynucleotide according to claim 18. 36. A vector according to claim 35 characterised in that said vector provides tissue-specific expression of the encoded nitroreductase. 37. A vector according to claim 36 characterised in that said vector comprises a TCF-responsive element operably linked to said polynucleotide. 38. A vector according to any of claims 35 to 37 characterised in that said vector is a virus. 39. A vector according to claim 38 characterised in that said viral vector is an adenovirus. 40. (canceled) 41. A host cell comprising an isolated polynucleotide according to of either of claims 18 or 27 or a vector according to any one of claims 35 to 39. 42-44. (canceled) 45. A method of treating cancer in a mammalian subject, comprising administering the isolated polynucleotide of either of claims 18 or 27, or the vector according to any one of claims 35 to 39, allowing a suitable time for expression of the encoded nitroreductase to occur, and administering a prodrug capable of being activated by said expressed nitroreductase. 46. A recombinant mutant nitroreductase with increased nitroreductase activity as compared to the wild-type enzyme, characterised in that said nitroreductase is encoded by a mutated Salmonella NFSB gene. 47. A recombinant mutant nitroreductase with increased nitroreductase activity as compared to the wild-type enzyme, characterised in that said nitroreductase is encoded by a mutated Enterobacter NFSB gene. 48. A nitroreductase according to either of claims 46 or 47 characterised in that it has an increased nitroreductase activity for prodrugs. 49. A nitroreductase according to claim 48, characterised in that it has an increased nitroreductase activity for nitrobenzamide prodrugs. 50. A nitroreductase according to claim 49, characterised in that it has an increased nitroreductase activity for CB1954. 51. A method for treating cancer comprising administering a nitroreductase according to any one of claims 1-17 to a mammalian subject in the presence of a prodrug capable of being activated by said nitroreductase. 52. A method for converting a prodrug into a cytotoxic agent comprising administering a nitroreductase according to any one of claims 1-17 or an isolated polynucleotide according to claim 18 in the presence of said prodrug. 53. A method for converting a nitrobenzamide prodrug into a cytotoxic agent comprising administering a nitroreductase according to any one of claims 1-17 or an isolated polynucleotide according to claim 18 in the presence of said prodrug. 54. A method for converting CB1954 into a cytotoxic agent comprising administering a nitroreductase according to any one of claims 1-17 or an isolated polynucleotide according to claim 18 in the presence of CB1954. 55. A method for treating cancer by converting a prodrug into a cytotoxic agent comprising administering a nitroreductase according to any one of claims 1-17 or an isolated polynucleotide according to claim 18 in the presence of said prodrug. 56. A method for treating cancer comprising administering a nitroreductase according to claims 25 or 26 to a mammalian subject in the presence of a prodrug capable of being activated by said nitroreductase. 57. A method for converting a prodrug into a cytotoxic agent comprising administering a nitroreductase according to claims 25 or 26 or an isolated polynucleotide according to claim 27 in the presence of said prodrug. 58. A method for converting a nitrobenzamide prodrug into a cytotoxic agent comprising administering a nitroreductase according to claims 25 or 26 or an isolated polynucleotide according to claim 27 in the presence of said prodrug. 59. A method for converting CB1954 into a cytotoxic agent comprising administering a nitroreductase according to claims 25 or 26 or an isolated polynucleotide according to claim 27 in the presence of CB1954. 60. A method for treating cancer by converting a prodrug into a cytotoxic agent comprising administering a nitroreductase according to claims 25 or 26 or an isolated polynucleotide according to claim 27 in the presence of said prodrug. 61. A pharmaceutical composition comprising the nitroreductase according to any one of claims 1, 25, or 26 or of an isolated polynucleotide according to either of claims 18 or 27, in a pharmaceutically acceptable diluent or excipient. 62. A pharmaceutical composition comprising the vector of any one of claims 35 to 37 in a pharmaceutically acceptable diluent or excipient. 63. A pharmaceutical composition comprising the host cell of claim 41 in a pharmaceutically acceptable diluent or excipient. 64. A method for the design of, or screening for, improved prodrugs comprising contacting a test prodrug with the nitroreductase according to any one of claims 1 to 17, 25, or 26, and screening for cytotoxic activity. |
<SOH> BACKGROUND TO THE INVENTION <EOH>The present invention relates to mutated nitroreductase enzymes and the DNA encoding them, and their use in the conversion of prodrugs for the treatment of cancer. One approach to treating cancer is to introduce a gene into the tumour cells that encodes an enzyme capable of converting a prodrug of relatively low toxicity into a potent cytotoxic drug. Systemic administration of the prodrug is then tolerated since it is only converted into the toxic derivative locally, in the tumour, by cells expressing the prodrug-converting enzyme. This approach is known as gene-directed enzyme prodrug therapy (GDEPT), or when the gene is delivered by means of a recombinant viral vector, virus-directed prodrug therapy (VDEPT) (McNeish et al, 1997). An example of an enzyme/prodrug system is nitroreductase and the aziridinyl prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) (Knox et al 1988). Following the observation that the Walker rat carcinoma cell line was particularly sensitive to CB1954, it was shown that this was due to the expression of the rat nitroreductase DT diaphorase. However, since CB 1954 is a poor substrate for the human form of this enzyme, human tumour cells are far less sensitive to CB1954. GDEPT was conceived as a way of introducing a suitable nitroreductase, preferably with greater activity against CB1954, in order to sensitise targeted cells. The Escherichia coli nitroreductase (EC1.6.99.7, alternatively known as the oxygen-insensitive NAD(P)H nitroreductase or dihydropteridine reductase, and often abbreviated to NTR) encoded by the NFSB gene (alternatively known as NFNB, NFSI, or DPRA) has been widely used for this purpose (Reviewed in Grove et al, 1999). The NFSB-encoded nitroreductase (NTR) is a homodimer that binds two flavin mononucleotide (FMN) cofactor molecules. Using NADH or NADPH as an electron donor, and bound FMN as a reduced intermediate, NTR reduces one or other of the two nitro-groups of CB 1954 to give either the highly toxic 4-hydroxylamine derivative or the relatively non-toxic 2-hydroxylamine. Within cells, 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, probably via a further toxic metabolite, becomes very genotoxic (Knox et al, 1991). The exact nature of the lesion caused is unclear, but is unlike that caused by other agents. A particularly high rate of inter-strand cross-linking occurs and the lesions seem to be poorly repaired, with the result that CB 1954 is an exceptionally affective anti-tumour agent (Friedlos et al, 1992). The structure of the NFSB NTR has been analysed by X-ray crystallography (Parkinson et al 2000, Lovering et al, 2001). Each monomer consists of 217 amino acids forming a four-stranded beta sheet (a fifth parallel strand is contributed by the other subunit) and ten α helices (A-K) and comprises a large hydrophobic core (residues 2-91 and 131-217), a two helix domain (E and F, residues 92-130) that protrudes from the core region, and an extensive dimer interface formed by parts of helices A, B, G, J and K. (NB: the domain assignments are from Lovering et al, and differ slightly from the earlier structure solved by Parkinson et al). Residues in what Parkinson et al designated as Helix G (residues 113-131) have been identified as being in or near the active site and are important in determining substrate specificity. Lovering et al assigns residues 110-131 to helix F and 135-157 to helix G. However, both papers agree that residues in this region form part of the opening to the substrate- and cofactor-binding pocket and that phenylalanine 124 is particularly important. The NFSB NTR has sequence homology to a number of other enzymes, in particular FRase I, a flavin reductase enzyme from Vibrio fischeri (Zenno et al 1996). By random mutagenesis, Zenno et al generated a number of nfsb mutants that had greatly increased flavin reductase activity. These mutants all had substitutions of phenylalanine 124 (F124), a crucial position in the αG helix. F124 mutants having substitutions with serine, alanine, threonine, leucine, valine, isoleucine, aspartate, glutamine, arginine and histidine were generated, all of which had substantially increased flavin reductase activity. However, with one exception, the nitroreductase activity of these mutants was either broadly similar or substantially reduced, as judged with nitrofurazone and nitrofurantoin as substrates. The histidine mutant (F124H) had approximately double the wild-type activity for these substrates. However, firstly, these disclosures give no information as to what the effects on other substrates, such as CB1954, might be. Secondly, such data as are disclosed suggest that mutations of the F124 position have, at best, an unpredictable effect on nitroreductase activity and, in general, a deleterious effect. International patent application WO 00/47725 (Minton et al) discloses bacterial nitroreductases that are structurally unrelated to the E.coli NFSB-encoded enzyme and that are derived from Bacillus species. The aim of GDEPT is to obtain efficient conversion of a prodrug such as CB1954 in target cells in order to kill not only NTR-expressing cells but also bystander tumour cells that may not have been successfully transfected or transduced. It is therefore desirable to have efficient delivery of the NTR-encoding DNA, prodrugs with as high a therapeutic index as possible, and a nitroreductase enzyme that is as efficient as possible in the conversion of CB1954 and other nitro-based prodrugs to toxic DNA cross-linking products. To address the latter, it is desirable to develop modified nitroreductase enzymes, since these would allow more efficient therapy and/or lower systemic doses of the prodrug. Although prodrugs are of relatively low toxicity in comparison with their activated derivatives, it is nevertheless desirable to reduce the chances of adverse effects by minimising the required dose. |
Air-bag |
An air-bag (1) has a primary gas flow duct (11) leading from a gas generator to one or more zones within the air-bag which are to be inflated. The air-bag has a separate inner gas flow duct (12) mounted within the outer gas flow duct. The inner gas flow duct (12) and the outer gas flow duct (11) have areas (22 and 23) thereof secured together in the region that is to be connected to the gas generator. |
1. An air-bag for use in connection with a gas generator comprsing an outer gas flow duct to lead from the gas generator to one or more zones within the air-bag to be inflated, and a separate inner gas flow duct mounted within the outer gas flow duct, the inner gas flow duct, and the outer gas flow duct, having areas thereof secured together in a region to be connected to a gas generator. 2. An air-bag according to claim 1 wherein first and second areas of the inner gas flow duct are secured to two respective first and second areas of the outer gas flow duct. 3. An air-bag according to claim 2 wherein the outer gas flow duct is formed from a first set of two superimposed layers of fabric including an upper layer and a lower layer, and the inner gas flow duct is formed from a second set of two superimposed layers of fabric including an upper layer and a lower layer, the upper layer of fabric forming the outer gas flow duct being secured to an the first area of the upper layer of fabric forming the inner gas flow duct, and the lower layer of fabric of the outer gas flow duct being secured to the second area of the lower layer of fabric of the inner gas flow duct. 4. An air-bag according to claim 3 wherein the first and second areas are off-set and not superimposed. 5. An air-bag according to claim 1 wherein the inner gas flow duct is provided with projecting ears which extend beyond an end of the outer gas flow duct, and the ears being folded back about a fold-line to lie adjacent the end of the outer gas flow duct, the ears being secured to the fabric forming the outer gas flow duct. 6. An air-bag according to claim 5 wherein at least one of the ears has a dimension which is no greater than the diameter, when flat, of the inner gas flow duct, at least one of the ears being folded back to lie adjacent a respective layer of fabric forming the outer gas flow duct, at least one of the ears being secured to the layer of fabric that it lies adjacent. 7. An air-bag according to claim 5 wherein at least one of the ears has a dimension greater than the diameter of the inner gas flow duct when flat, at least one of the ears being folded back to lie adjacent the exterior of the outer gas flow duct, with parts of the ears not lying over the inner gas flow duct, the parts of the ears not lying over the inner gas flow duct being secured together, and being secured to the fabric forming the outer gas flow duct. 8. An air-bag according to any one of claim 5 wherein the fold-line of a projecting ear is provided with a slot and the air-bag is provided with a tab located to pass through the slot. 9. An air-bag according to any one of claim 5 wherein at least one ear is provided with an ear aperture, and the outer gas flow duct and the inner gas flow duct are also provided with duct apertures so that the ear apertures is aligned with the duct apertures. 10. An air-bag according to claim 9 wherein the inner gas flow duct is provided with at least one further duct aperture at a point spaced from the projecting ears. 11. An air-bag according to claim 2 wherein the a areas are on axially extending tabs which project axially beyond the ends of the inner and outer gas flow ducts. |
Method for surface treating a semiconductor |
The invention relates to a method for the thermal treatment of a surface layer (4) on a semiconductor substrate (5). Laser pulses (2) generated by a laser (1) are emitted onto the surface layer (4). This method can be used to produce, in particular, ohmic contacts to III-V compound semiconductors. |
1. A method for the thermal treatment of a surface layer (4) on a semiconductor substrate (5), characterized in that the surface layer (4) is thermally treated with the aid of a laser pulse having a duration of <0.1 μsec and an irradiation energy density of between 10 and 1 000 mJ/cm2. 2. The method as claimed in claim 1, in which the semiconductor substrate (5) comprises a III-V compound semiconductor material with a band gap of >2.5 eV and the surface layer (4) has, in particular, a thickness of between 1 and 150 nm. 3. The method as claimed in claim 1 or 2, in which the surface layer (4) comprises donors or acceptors. 4. The method as claimed in claim 1 or 2, in which the surface layer (4) is produced from a metal. 5. The method as claimed in claim 4, in which the surface layer (4) is produced from a material with at least one element from the group Pt, Mg, Zn with in each case a proportion of >0.01% by weight. 6. The method as claimed in one of claims 1 to 5, in which the semiconductor substrate (5) is produced at least partly from a III-V compound semiconductor. 7. The method as claimed in claim 6, in which the semiconductor substrate (5) is produced at least partly from AlxInyGa1−x−yN where 0≦x≦1, 0≦y≦1 and x+y≦1. 8. The method as claimed in one of claims 1 to 7, in which a laser pulse having a duration of <1 nsec is used. 9. The method as claimed in one of claims 1 to 8, in which laser radiation having a wavelength of <450 nm is used for the laser pulse. 10. The method as claimed in one of claims 1 to 9, in which the surface layer (4) is melted by the laser pulse. 11. The method as claimed in one of claims 1 to 10, in which a sequence of laser pulses is emitted onto the surface layer (4). 12. The method as claimed in claim 11, in which the laser pulses are emitted at a time interval which is greater than ten thousand times the pulse duration of the laser pulses. 13. The method as claimed in one of claims 1 to 12, in which laser pulses are applied to the semiconductor substrate (5) in a predetermined pattern with the aid of a mask. 14. The method as claimed in one of claims 1 to 13, in which the semiconductor substrate (5) is spatially displaced between two laser pulses. 15. The method as claimed in one of claims 1 to 14, in which laser pulses are applied to the edges of the areas provided for contacts on the surface layer (4). 16. The method as claimed in one of claims 1 to 14, in which laser pulses are applied to the areas of the surface layer (4) which are provided for contacts. 17. The method as claimed in one of claims 1 to 16, in which laser pulses are applied to the surface layer (4) after a measurement of components formed in the semiconductor substrate (5) for the purpose of influencing the measured parameters. 18. The method as claimed in one of claims 1 to 17, in which a further reinforcement layer is applied to the surface layer (4). 19. The method as claimed in claim 18, in which the reinforcement layer contains at least one element from the group Zn and Mg. 20. The method as claimed in one of claims 1 to 19, in which a passivation layer made of Al2O3, or SiOxNy where 0<x≦2, 0≦y≦1, is subsequently arranged on a side of the surface layer facing away from the substrate. 21. The method as claimed in one of claims 1 to 20, in which the surface of the semiconductor substrate (5) is irradiated with laser pulses before the application of the surface layer (4) on the semiconductor substrate (5). |
Reformate stream cooler with a catalytic coating for use in a gas generation system |
A gas generation system comprises a reformer (1) to generate a hydrogen-containing reformate stream (4), a reformate stream cooler (2), and a shift stage (3) down-stream of the reformate stream cooler to purify the reformate stream. The surfaces of the cooler that come into contact with the reformate stream are coated with a material that contains at least one catalytically active constituent. The coating is selected such that it also protects against corrosion and sooting in the presence of oxidizing, reducing, and carbon-containing gases. By directly utilizing the coated reformate stream cooler as a catalytically active reactor unit, a water-gas shift reaction to reduce the carbon monoxide concentration takes place to some extent in the cooler prior to the reformate stream entering the actual shift stage. This enables the size of subsequent shift stage(s) to be reduced. |
1. A gas generation system comprising: a reformer to generate a reformate stream, a cooler downstream of the reformer to cool the reformate stream, and a shift stage downstream of the cooler to reduce the carbon monoxide concentration in the reformate stream, wherein surfaces of the cooler that come into contact with the reformate stream are coated with a cooler coating that is soot-inhibiting and is catalytically active with respect to the water-gas shift reaction. 2. The system of claim 1, wherein the composition of the cooler coating varies along the reformate stream flow path. 3. The system of claim 2, wherein the cooler coating comprises at least two areas of different composition, which differ with respect to soot-inhibiting activity, or catalytic activity with respect to the water-gas shift reaction, or both. 4. The system of any one of claims 1 to 3, wherein the system further comprises a cooler feed line connecting the reformer to the cooler and a cooler discharge line connecting the cooler to the shift stage, and wherein the cooler feed line and the cooler discharge line are coated with a line coating that is soot-inhibiting material and is catalytically active with respect to the water-gas shift reaction. 5. The system of claim 4 wherein the line coating exhibits greater soot-inhibiting activity and lesser activity with respect to the water-gas shift reaction than the cooler coating. 6. The system of claim 4 wherein the cooler coating comprises a first material of the same composition as the line coating, and a second material that exhibits greater catalytic activity with respect to the water-gas shift reaction and a lower soot-inhibiting activity than the material. 7. The system of claim 1, wherein the surfaces of the cooler that come into contact with the reformate stream are coated with at least one layer of a base material, wherein the base material is disposed between the surfaces and the cooler coating, and wherein the base material comprises a metal-containing substance, selected from the group consisting of chromium, silicon, aluminum, magnesium, manganese, titanium, rare earths, compounds of chromium, silicon, aluminum, magnesium, manganese, titanium and rare earths, and alloys of chromium, silicon, aluminum, magnesium, manganese, titanium and rare earths. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The invention concerns a reformate stream cooler in which the surfaces of the cooler that come into contact with the reformate stream are coated with a catalytically active material. 2. Description of the Related Art Gas generation systems comprising a reformer to generate a reformate stream, a reformate stream cooler, and a downstream shift stage to purify the reformate stream are used in fuel-cell-powered motor vehicles to provide the hydrogen that is needed to operate the fuel cells. In addition to hydrogen, the reforming of carbon-containing fuels produces by-products such as carbon monoxide, which must only be present in very small quantities in proton-exchange membrane (PEM) fuel cells. Accordingly gas purification upstream of the fuel cell is required. Present technology for the reduction of the carbon monoxide concentration in hydrogen-rich streams include the water-gas shift reaction and the selective oxidation of carbon monoxide in fixed-bed reactors with suitable selective oxidation catalysts. However, at the necessarily low operating temperatures, the water-gas shift reaction Is comparatively slow, so that higher amounts of catalyst are required, leading to larger shift stages and/or increased costs due to the higher noble metal content. The selective oxidation units (for selective oxidation of carbon monoxide) are not able to handle high carbon monoxide concentrations (>2%). Moreover, as for example described in U.S. Pat. No. 5,873,951, the generation of hydrogen from carbon-containing fuels brings with it the problem of sooting or coking (i.e. the formation of carbon black or carburization). This problem, also known as metal dusting corrosion, is encountered where hot carbon monoxide-containing gas cools on a metal surface and the carbon monoxide breaks down into carbon and carbon dioxide in the air-carbon reaction. In this manner carbides are formed in the metal structure, which leads to the degradation of the material structure. Metal dusting corrosion not only affects steel, but also nickel-based materials, for example. The intensity of the corrosion increases with carbon monoxide partial pressure and the molar carbon monoxide/carbon dioxide ratio at the metal surface. Metal dusting corrosion can be prevented by carrying out the desired process outside of the critical temperature range for metal dusting corrosion, or by “bypassing” the critical temperature range as rapidly as possible. A common method of bypassing the critical temperature range is quenching the reformate stream by introducing water into the reformate stream between the reformer and the shift stage. The disadvantage of this method is that the thermal energy contained in the reformate stream can not then be utilized elsewhere, which leads to a significantly lower efficiency. To provide a solution to the problem of efficiently reducing carbon monoxide in a compact system, EP 0 974 393 A2 provides a gas generation system comprising a reformer, a carbon monoxide shift reactor, and a catalytic burner. The publication describes that the fuel gas is conducted in counter-current flow, which allows efficient cooling of the carbon monoxide shift stage (which shifts the shift gas balance in the reformate stream towards a lower carbon monoxide concentration), in a more compact design. However, there remains a need for an improved gas generation system for real-life applications that offers consistent high performance and reliability over the entire lifetime of the system, as well as a more compact design. |
<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>A gas generation system comprises a reformer to generate a hydrogen-containing reformate stream, a reformate stream cooler (or heat exchanger), and a shift stage downstream of the reformate cooler to purify the reformate stream. The surfaces of the cooler that come into contact with the reformate stream are coated with a material that contains at least one catalytically active constituent. The coating is selected such that it also protects against corrosion and sooting in the presence of oxidizing, reducing, and carbon-containing gases. By directly utilizing the coated reformate stream cooler as a catalytically active reactor unit, a water-gas shift reaction to reduce the carbon monoxide concentration takes place to some extent in the cooler prior to the reformate stream entering the actual shift stage. This enables the size of the subsequent shift stage(s) to be reduced, resulting in a more lightweight and compact gas generation system. These and other aspects will be evident upon reference to the attached Figures and following detailed description. |
Organic electroluminescent element and lumiscent device or display including the same |
The present invention provides an organic electroluminescent element which is superior in luminance, reliability, and thermal stability and is capable of selectively emitting light with comparative long wavelengths such as red and good color purity and a light-emitting device or display device incorporated therewith. The organic electroluminescent element consists of a glass substrate (1), an anode (2), a hole transporting layer (10), an emitting layer (11), an electron transporting layer (12), and a cathode (3), which are sequentially laminated on top of the other. The emitting layer (11) is formed from a mixture composed of at least one species of the styryl compound represented by the general formula [I] below and a material with charge transporting capability. Y—CH═CH—X General formula [I] (where X denotes an aryl group (such as phenyl group) which has a substituent group (such as cyano group and methyl group), and Y denotes a group having a skeleton of aminophenyl group or the like.) |
1. An organic electroluminescent element in which organic layers having a light emitting region are formed between the anode and the cathode, characterized in that at least one of the organic layers is formed from a mixture composed of at least one species of the styryl compounds represented by the general formula [I] given below and a material with charge transporting capability: Y—CH═CH—X General formula [I] [where, in the general formula [I], X denotes any group represented by the general formulas (1) to (13) shown below; (where, in the general formula (1), R1 to R5 may be identical or different, and at least one of them is a group selected from halogen atom, nitro group, cyano group, trifluoromethyl group, alkyl group (optionally substituted), and alkoxyl group (optionally substituted); and in the general formulas (2) to (13), each of R6 to R109, which may be identical or different, denotes a group selected from hydrogen atom, halogen atom, nitro group, cyano group, trifluoromethyl group, alkyl group (optionally substituted), aryl group (optionally substituted), and alkoxyl group (optionally substituted); also, in the general formula [I], Y denotes any group represented by the general formulas (14) to (16) shown below. (where, in the general formula (14) above, each of Z1 and Z2, which may be identical or different, is a group selected from hydrogen atom, alkyl group (optionally substituted), and aryl group (optionally substituted); and in the general formulas (15) and (16) above, each of R110 to R126, which may be identical or different, denotes a group selected from hydrogen atom, alkyl group (optionally substituted), aryl group (optionally substituted), alkoxyl group (optionally substituted), halogen atom, nitro group, cyano group, and trifluoromethyl group.)] 2. An organic electroluminescent element as defined in claim 1, wherein the organic layer is a laminate layer composed of a hole transforming layer and an electron transforming layer, and at least the electron transporting layer of the organic layers of laminate structure is formed from a mixture containing at least one species of the styryl compounds represented by the general formula [I] given above. 3. An organic electroluminescent element as defined in claim 1, wherein the organic layer is a laminate layer composed of a hole transforming layer and an electron transforming layer, and at least the hole transporting layer of the organic layers of laminate structure is formed from a mixture containing at least one species of the styryl compounds represented by the general formula [I] given above. 4. An organic electroluminescent element as defined in claim 1, wherein the organic layer is a laminate layer composed of a hole transforming layer and an electron transforming layer, and the hole transporting layer is formed from the mixture containing at least one species of the styryl compounds represented by the general formula [I] given above and the electron transporting layer is formed from the mixture containing at least one species of the styryl compounds represented by the general formula [I] given above. 5. An organic electroluminescent element as defined in claim 1, wherein the organic layer is a laminate layer composed of a hole transforming layer, en emitting layer, and an electron transforming layer, and at least the emitting layer of the organic layers of laminate structure is formed from a mixture containing at least one species of the styryl compounds represented by the general formula [I] given above. 6. An organic electroluminescent element as defined in claim 1, wherein at least one species of the styryl compounds represented by the general formula [I] given above is mixed in concentrations ranging from 5 to 90 wt % with the material having the charge transporting capability in the mixture layer. 7. An organic electroluminescent element as defined in claim 1, wherein the mixture contains at least one species of the styryl compounds represented by the general formula [I] given above and a red or orange emitting dye which has the emission maximum in the range of 600 nm to 700 nm. 8. A light-emitting device or display device incorporated with the organic electroluminescent element defined in any of claims 1 to 7. 9. A light-emitting device or display device as defined in claim 8, which has pixels constructed at least partially of the organic electroluminescent element. 10. An organic electroluminescent element in which organic layers having a light emitting region are formed between the anode and the cathode, characterized in that at least one of the organic layers is formed from a mixture composed of at least one species of the styryl compounds represented by the structural formulas (17)-1 to (17)-86 given below and a material with charge transporting capability. 11. An organic electroluminescent element as defined in claim 10, wherein the organic layer is a laminate layer composed of a hole transforming layer and an electron transforming layer, and at least the electron transporting layer of the organic layers of laminate structure is formed from a mixture containing at least one species of the styryl compounds represented by the structural formulas (17)-1 to (17)-86 given above. 12. An organic electroluminescent element as defined in claim 10, wherein the organic layer is a laminate layer composed of a hole transforming layer and an electron transforming layer, and at least the hole transporting layer of the organic layers of laminate structure is formed from a mixture containing at least one species of the styryl compounds represented by the structural formulas (17)-1 to (17)-86 given above. 13. An organic electroluminescent element as defined in claim 10, wherein the organic layer is a laminate layer composed of a hole transforming layer and an electron transforming layer, and the hole transporting layer is formed from the mixture containing at least one species of the styryl compounds represented by the structural formulas (17)-1 to (17)-86 given above and the electron transporting layer is formed from the mixture containing at least one species of the styryl compounds represented by the structural formulas (17)-1 to (17)-86 given above. 14. An organic electroluminescent element as defined in claim 10, wherein the organic layer is a laminate layer composed of a hole transforming layer, en emitting layer, and an electron transforming layer, and at least the emitting layer of the organic layers of laminate structure is formed from a mixture containing at least one species of the styryl compounds represented by the structural formulas (17)-1 to (17)-86 given above. 15. An organic electroluminescent element as defined in claim 10, wherein at least one species of the styryl compounds represented by the structural formulas (17)-1 to (17)-86 given above is mixed in concentrations ranging from 5 to 90 wt % with the material having the charge transporting capability in the mixture layer. 16. An organic electroluminescent element as defined in claim 10, wherein the mixture contains at least one species of the styryl compounds represented by the structural formulas (17)-1 to (17)-86 given above and a red or orange emitting dye which has the emission maximum in the range of 600 nm to 700 nm. 17. A light-emitting device or display device incorporated with the organic electroluminescent element defined in any of claims 10 to 16. 18. A light-emitting device or display device as defined in claim 17, which has pixels constructed at least partially of the organic electroluminescent element. |
<SOH> BACKGROUND ART <EOH>Enormous efforts are being directed to research and development of light-weight high-efficiency flat-panel displays for use as computer screens or television screens. One reason for this is that the conventional CRT now in general use as the display device is bulky, heavy, and power-consumptive despite its high luminance and good color reproduction. Among the light-weight high-efficiency flat-panel displays which have recently been commercialized is, for example, a liquid-crystal display of active matrix drive type. Unfortunately, it has a narrow viewing angle and needs back light (which consumes a large amount of electric power) for use in a dark place because it does not emit light by itself. Moreover, it cannot quickly respond to high-definition high-speed video signals to be put to practical use in near future. Another problem is high production cost and technical difficulties involved in production of large-sized display. A possible substitute for liquid-crystal display is a display with light-emitting diodes. However, it also has a problem with high production cost and technical difficulties in forming light-emitting diodes in matrix structure on a single substrate. It is not an inexpensive display to replace CTRs in near future. There has recently appeared a flat panel display which is expected to solve the above-mentioned problems. It is an organic electroluminescent element (organic EL element) made with an organic luminescent material. It is expected that a specific organic compound as a luminescent material will help realize a flat display panel which emits light by itself, responds quickly, and ensures good visibility free from viewing angles. The organic EL element is composed of a transparent anode and a metal cathode and an organic thin film interposed between the anode and the cathode which contains a luminescent material which emits light upon injection of electric current. That of single hetero structure, which was developed by C. W. Tang and S. A. VanSlyke, is reported in Applied Physics Letters, vol. 51, No. 12, pp. 913 to 915 (1987). It has an organic thin film of double-layer structure, which is composed of a thin film of hole transporting material and a thin film of electron transporting material, so that luminescence takes place upon recombination of holes and electrons injected into the organic thin film from each electrode. The organic EL element of this structure permits either the hole transfer material or the electron transfer material to function as a luminescent material. Luminescence takes place in the wavelength region corresponding to the energy gap between the ground state and the excited state which are assumed by the luminescent material. The double-layer structure brings about a considerable reduction in driving voltage and a great improvement in luminescence efficiency. At a later time, an organic EL element of double hetero structure was reported by C. Adachi, S. Tokita, T. Tsutsui, and S. Saito in Japanese Journal of Applied Physics, vol. 27, No. 2, pp. L269 to L271 (1988). The structure is composed of three layers of hole transporting material, luminescent material, and electron transporting material. Further, another organic EL element was reported by C. W. Tang, S. A. VanSlyke, and C. H. Chen in Journal of Applied Physics, vol. 65, No. 9, pp. 3610 to 3616 (1989). The structure has a luminescent material contained in the electron transporting material. These researches have proved the feasibility of intensive luminescence at a low voltage. Active research and development works in this field are going on. The fact that there exists a large variety of organic compounds used as luminescent materials suggests that it would be possible, at least theoretically, to produce light of any desired color if their molecular structure is properly modified. In other words, properly designed organic compounds would give three pure colors (red, green, and blue) necessary for full color display more easily than inorganic compounds used for thin-film EL elements. It is reported in non-patent literature (1) listed below that the emission of red color is possible with an electron transporting material which is tris(8-quinolyl)aluminum (Alq 3 for short hereinafter) doped with 4-dicyanomethylene-6-(p-dimethylaminostyryl)-2-methyl-4H-pyran (DCM for short hereinafter). It is also reported in non-patent literature (2) listed below that BSB-BCN gives a luminescence of 1000 cd/m 2 or above. It is proposed in patent literature (1) listed below that a specific styryl compound be used as the electroluminescent material. Non-patent literature (1): Chem. Funct. Dyes, Proc. Int. Symp., 2nd. p.536 (1993) Non-patent literature (2): T. Tsutsui, D. U. Kim, Inorganic and Organic Electroluminescence Conference (1996, Berlin) Patent literature (1): Japanese Patent Laid-open No. Hei 7-188649 (Claim, from p. 5, right column, line 8, to p. 22, right column, line 5, and FIGS. 1 to 3) Despite these prior art technologies, the actual organic electroluminescent element still have problems to be solved. Difficulties are involved in development of an element capable of stably emitting red light with a high luminance. The electron transporting material (DCM-doped Alq 3 ) reported in the non-patent literature (1) above does not exhibit satisfactory luminance and reliability required of the display material. The BSB-BCN reported in the non-patent literature (2) above gives a luminance of 1000 cd/m 2 or above but does not give a complete red chromaticity necessary for full color display. There is a need for development of an electroluminescent element capable of stably emitting red light with a high purity and a high luminance. The specific styryl compound, which proposed for use as the organic electroluminescent material in the patent literature (1) above, is intended only for blue light emission. It is not intended for emission of light with wavelengths of red or other colors. It is an object of the present invention to provide an organic electroluminescent element and a light-emitting device or display device incorporated therewith. The organic electroluminescent element is formed from a compound which has a high fluorescence yield and a good thermal stability. Moreover, it stably and selectively emits light of red and other colors (in the region of comparatively long wavelengths) with a high color purity and a high luminance. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 is a schematic sectional view showing important parts of the organic electroluminescent element in one example according to the present invention. FIG. 2 is a schematic sectional view showing important parts of the organic electroluminescent element in another example according to the present invention. FIG. 3 is a schematic sectional view showing important parts of the organic electroluminescent element in further another example according to the present invention. FIG. 4 is a schematic sectional view showing important parts of the organic electroluminescent element in further another example according to the present invention. FIG. 5 is a schematic sectional view showing important parts of the organic electroluminescent element in further another example according to the present invention. FIG. 6 is a schematic sectional view showing important parts of the organic electroluminescent element in further another example according to the present invention. FIG. 7 is a diagram showing the structure of the full-color flat panel display incorporated with the organic electroluminescent element. detailed-description description="Detailed Description" end="lead"? |
Novel human bmcc1 gene |
Human BMCC1 protein having an amino acid sequence set forth in SEQ ID NO:1 in the Sequence Listing and its variant protein, as well as human BMCC1 gene having a base sequence set forth in SEQ ID NO:2 in the Sequence Listing and its variant gene. |
1. A protein comprising an amino acid sequence set forth in SEQ ID NO:1 in the Sequence Listing or a salt thereof. 2. A protein comprising a deletion, a substitution, an insertion or an addition of one or more amino acids in an amino acid sequence set forth in SEQ ID NO:1 in the Sequence Listing or a salt thereof. 3. The protein or a salt thereof according to claim 2, wherein the protein has the amino acid sequence and is provided with apoptosis-inducing activity. 4. A nucleic acid encoding the protein according to claim 1 or a partial peptide thereof. 5. A nucleic acid comprising a base sequence set forth in SEQ ID NO:2. 6. An isolated nucleic acid capable of hybridizing to the nucleic acid according to claim 4 or a complementary nucleic acid thereof under stringent conditions. 7. The nucleic acid according to claim 6, wherein the protein encoded by the nucleic acid is provided with apoptosis-inducing activity. 8. A nucleic acid comprising a portion of a base sequence set forth in SEQ ID NO:2. 9. The nucleic acid according to any of claims 4-8, wherein the nucleic acid has enhancement in expression in neuroblastomas with favorable prognosis based on comparison between neuroblastomas with favorable prognosis and neuroblastomas with unfavorable prognosis. |
<SOH> BACKGROUND ART <EOH>(Tumorgenesis and Genes) Individual tumors exhibit distinct characteristic natures, and their biological properties are not necessarily identical even though the basic principle of oncogenesis is the same. Rapid advances in the understanding of cancer from a molecular biological and molecular genetic perspective in recent years have opened the way to an explanation of oncogenesis and tumor cell biology on the genetic level. (Neuroblastomas) Neuroblastoma is a pediatric cancer occurring in sympathetic gangliocytes and adrenal medullary cells which originate from cells of the peripheral sympathetic nervous system. Of these sympathetic nervous system cells, neural crest cells in the initial stage of development migrate to the abdomen, differentiating and maturing at sites where sympathetic ganglia are formed. Some of these cells migrate further to the adrenal bodies, penetrating through the adrenal cortex which is already in the process of formation, and reaching the medulla and forming medullary substance there. The neural crest cells also serve as a source of other peripheral nerve cells, differentiating into dorsal root ganglia (sensory nerves), skin pigment cells, thyroid C cells, some pulmonary cells, intestinal gangliocytes, and the like. (Prognosis for Neuroblastoma) Neuroblastoma is characterized by a varied clinical profile (Nakagawara, Shinkeigashu no Hassei to Sono Bunshi Kiko [Neuroblastoma Development and Molecular Mechanism], Shoni Naika 30, 143, 1998). For example, neuroblastoma occurring at less than one year of age has very favorable prognosis, with the majority undergoing differentiation and cell death, and spontaneous regression. Currently, most neuroblastomas discovered by a positive result in the commonly performed mass screening of 6-month-old infant urine are of the type which tend to undergo this spontaneous regression. On the other hand, neuroblastoma occurring at age 1 or higher is highly malignant and leads to death of the infant in the majority of cases. It is also hypothesized that a somatic mutation occurs in highly malignant neuroblastomas in infants older than one year of age, which are of monoclonal nature, whereas in naturally regressing neuroblastomas, the genetic mutation remains at only a germline mutation. See Knudson A G, et al.: Regression of neuroblastoma IV-S: A genetic hypothesis, N. Engl. J. Med. 302, 1254 (1980)). (Tumor Markers which Allow the Diagnosis of Prognosis for Neuroblastoma) With recent advances in molecular biology research, it has become clear that expression of the high affinity nerve growth factor (NGF) receptor TrkA is closely connected with control of differentiation and cell death. See Nakagawara A., The NGF story and neuroblastoma, Med. Pediatr. Oncol., 31, 113 (1998). Trk is a membrane-spanning receptor, existing as the three main-types, Trk-A, -B and -C. These Trk family receptors play an important role in specific nerve cell differentiation and survival in the central nervous and peripheral nervous systems. See Nakagawara, et al., Shinkeigasaiboushu ni Okeru Neurotrophin Juyoutai no Hatsugen to Yogo [Expression of Neurotrophin Receptors and Prognosis in Neuroblastoma], Shoni Geka (Pediatric Surgery), 29: 425-432, 1997. The survival and differentiation of tumor cells is controlled by signals from Trk tyrosine kinase and Ret tyrosine kinase. In particular, the role of TrkA receptor is most significant, with TrkA expression being notably high in neuroblastomas with favorable prognosis, and its signals exerting a powerful control over survival and differentiation of tumor cells, and cell death (apoptosis). In neuroblastomas with unfavorable prognosis, on the other hand, TrkA expression is significantly suppressed, while tumor development is aided by a mechanism in which survival is promoted by signals from TrkB and Ret. It has become clear that amplification of the neural oncogene N-myc has become clearly associated with the prognosis of neuroblastoma. See Nakagawara, Nou-shinkeishuyo no Tadankai Hatsugan [Multistage Oncogenesis of Cerebral and Neural Tumors], Molecular Medicine, 364, 366 (1999). This gene, first cloned in neuroblastoma, is ordinarily only present in a single copy per haploid set in normal cells and neuroblastomas with favorable prognosis, whereas it has been found to be amplified several dozen times in neuroblastomas with unfavorable prognosis. Up till the present time, however, no oncogene other than N-myc is known to be expressed in neuroblastomas, and absolutely no genetic information other than that of N-myc has been known in relation to favorable or unfavorable prognosis. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a figure corresponding to an electropherogram showing the results of the expression of the BMCC1 gene in a clinical sample of neuroblastoma with favorable prognosis as confirmed by Northern hybridization. FIG. 2 is a figure corresponding to an electropherogram showing the results of determination of the expression levels of the BMCC1 gene in clinical samples of neuroblastomas with favorable prognosis and with unfavorable prognosis by semi-quantitative PCR. FIG. 3 is a figure corresponding to an electropherogram showing the results of determination of the expression levels of the BMCC1 gene in normal human tissues by semi-quantitative PCR. FIG. 4 is a figure corresponding to an electropherogram showing the results of determination of the expression levels of the BMCC1 gene in human cancer cell lines by semi-quantitative PCR. FIG. 5 is a figure corresponding to an electropherogram showing the results of determination of the expression levels of the BMCC1 gene in differentiated states of HeLa cell by semi-quantitative PCR. FIG. 6 is a figure corresponding to an electropherogram showing the results of determination of the expression levels of the BMCC1 gene in different cell cycle phases of HeLa cell by semi-quantitative PCR. FIG. 7 is a schematic representation of the predicted domain structures of BMCC1, BNIP2 and Cdc42GAP-BCH by PSORT2. detailed-description description="Detailed Description" end="lead"? |
Optical disc medium, data recording method, and data recording apparatus |
An optical disc medium 10 having a wobbled track groove 12 includes three wobble patterns including a flat wobble pattern, a positive pulse wobble pattern, and a negative pulse wobble pattern. In the flat wobble pattern, the amplitude of the wobble from the lengthwise direction of the track groove is zero such that the track groove is flat. In the positive pulse wobble pattern, the amplitude of the wobble from the length-wise direction of the track groove is positive such that the track groove is wobbled in a first direction perpendicular to the length-wise direction. Further, in the negative pulse wobble pattern, the amplitude of the wobble from the lengthwise direction of the track groove is negative such that the track groove is wobbled in a second direction opposite to the first direction. Then, three data values can be recorded using three distinctive wobble patterns. |
1. An optical disc medium having a wobbled track groove comprising: a flat wobble pattern in which the amplitude of the wobble from the lengthwise direction of the track groove is zero such that the track groove is flat; a positive pulse wobble pattern in which the amplitude of the wobble from the lengthwise direction of the track groove is positive such that the track groove is wobbled in a first direction perpendicular to the lengthwise direction; and a negative pulse wobble pattern in which the amplitude of the wobble from the lengthwise direction of the track groove is negative such that the track groove is wobbled in a second direction opposite to the first direction; whereby three data values can be recorded using three distinctive wobble patterns. 2. An optical disc medium according to claim 1, wherein the pulse width of the positive pulse wobble pattern and the negative pulse wobble pattern is greater than or equal to the track groove width and is less than or equal to ¼ period of a reference signal recorded to the track groove wobble pattern. 3. An optical disc medium according to claim 1, wherein the phase difference of the wobble patterns of adjacent first and second track grooves is greater than or equal to the pulse width and is less than or equal to ¼ period of the reference signal recorded to the wobble pattern. 4. An optical disc medium comprising: a first wobble pattern forming a wobble of a first frequency in a unit period to record first data; and a second wobble pattern forming a wobble of a second frequency in a unit period to record second data. 5. An optical disc medium having a wobbled track groove, comprising: a first wobble pattern with a first pulse projecting in the outside circumference direction and a second pulse projecting in the inside circumference direction in a single period for recording first data; and a second wobble pattern with a second pulse waveform of consecutive pulse projecting in either the outside circumference direction or the inside circumference direction in a single period for recording second data. 6. An optical disc medium according to claim 1, wherein the phase difference between wobble patterns in adjacent first and second track grooves is effectively 90 degrees. 7. An optical disc medium according to claim 1 having a Constant Angular Velocity format. 8. A data recording method for recording information to an optical disc medium, the method comprising: signal generating a wobble signal having at least one frequency from a data signal; displacing an optical axis of an optical head relative to the optical disc medium according to the wobble signal; and emitting light to the optical disc medium by means of the optical head to form a wobble pattern according to the wobble signal in a track groove on the optical disc medium. 9. A data recording method according to claim 8, wherein the wobble signal generating generates: a positive pulse with a positive amplitude and specific pulse width at a rising edge of the data signal; a negative pulse with a negative amplitude and a specific pulse width at a falling edge of the data signal; and a reference signal with 0 amplitude when there is no change in the data signal. 10. A data recording method according to claim 9, wherein the pulse width is greater than or equal to the track groove width and is less than or equal to ¼ period of a reference signal recorded to the track groove wobble pattern. 11. A data recording method according to claim 9, wherein the phase difference of the wobble patterns of adjacent first and second track grooves is greater than or equal to the pulse width and is less than or equal to ¼ period of the reference signal recorded to the wobble pattern. 12. A data recording method according to claim 8, wherein the wobble signal generating generates: a first wobble signal of a first frequency for a first data signal; and a second wobble signal of a second frequency for a second data signal. 13. A data recording method according to claim 8, wherein the wobble signal generating generates: a first wobble signal for a first data signal, the first wobble signal having a first pulse projecting upward or downward from a reference value, and a second pulse projecting downward or upward opposite the direction of the first pulse; and a second wobble signal for a second data signal, the second wobble signal having two consecutive pulses projecting in one direction either upward or downward from the reference value. 14. A data recording method according to claim 11, wherein using a single frequency reference signal the wobble signal generating generates: a first wobble signal for the first data signal based on the waveform of one period of the reference signal; and a second wobble signal for the second data signal based on two consecutive same-sign waveforms inverting the sign of different-sign waveform parts in one period of the reference signal. 15. A data recording method according to claim 8, wherein the phase difference of wobble signals forming wobble patterns in adjacent first and second track grooves is effectively 90 degrees. 16. A data recording method according to claim 8, further comprising: generating a rotational synchronization signal synchronized to rotation of the spindle motor rotating the optical disc medium; and generating a reference signal of which the frequency and phase have a specific relationship to the rotational synchronization signal; wherein the wobble signal generating generates a two-valued or greater wobble signal synchronized to the reference signal. 17. A data recording method according to claim 16, wherein for circumferential length R of a track groove on the optical disc medium and one reference period length T of the reference signals in one circumference of the track groove the following equation is true for integer n. R = ( n ± 1 4 ) × T 18. A data recording method according to claim 8, wherein the wobble signal generating comprises: frequency modulating the data signal and generating a FM signal; and generating a two-valued or greater wobble signal from the FM signal synchronized to the reference signal. 19. A data recording apparatus for recording information to an optical disc medium by forming a wobble pattern according to a wobble signal in track grooves of the optical disc medium, comprising: a wobble signal generator which generates a wobble signal having at least one frequency from a data signal; an optical head which emits light to the optical disc medium to form a wobble pattern according to the wobble signal in a track groove; an optical head displacing arrangement which displaces an optical axis of the optical head relative to the optical disc medium according to the wobble signal; and a controller which controls the wobble signal generator, the optical head displacing arrangement, and the optical head. 20. A data recording apparatus according to claim 19, further comprising: a spindle motor for rotating the optical disc medium; a rotational synchronization signal generator which generates a rotational synchronization signal synchronized to rotation of the spindle motor; and a reference signal generator which generates a reference signal of which the frequency and phase have a specific relationship to the rotational synchronization signal; wherein the wobble signal generator generates a wobble signal of at least a first frequency synchronized to the reference signal from the data signal. 21. A data recording apparatus according to claim 19, further comprising: a frequency modulator which frequency modulates the data signal and generating a FM signal; the wobble signal generator which generates a wobble signal of at least a first frequency from the FM signal. 22. An optical disc medium according to claim 4, wherein the phase difference between wobble patterns in adjacent first and second track grooves is effectively 90 degrees. 23. An optical disc medium according to claim 5, wherein the phase difference between wobble patterns in adjacent first and second track grooves is effectively 90 degrees. 24. An optical disc medium according to claim 4 having a Constant Angular Velocity format. 25. An optical disc medium according to claim 5 having a Constant Angular Velocity format. |
<SOH> BACKGROUND ART <EOH>An optical disc medium 50 normally has a track groove 52 travelling in a spiral from the inside circumference side to the outside circumference side of the disc while the radius of the spiral increases as shown in FIG. 14 . The disc also normally has a management area with prerecorded data at the inside circumference side, and a data recording area at the outside circumference side. Optical disc media with a high recording density for handling video data, for example, are also in demand. As the recording density of optical disc media has increased, the management data recorded to the management area at the inside circumference side has also increased dramatically, and high recording density is also needed in the management area. The area at the inside circumference side is limited, however, and high density recording of the management data using prepits as is done conventionally when the management data is voluminous leads to the problem of crosstalk between adjacent tracks. |
<SOH> BRIEF DESCRIPTION OF THE INVENTION <EOH>The present invention will become readily understood from the following description of preferred embodiments thereof made with reference to the accompanying drawings, in which like parts are designated by like reference numeral and in which: FIG. 1 is an enlarged view of the wobble pattern in three adjacent track grooves; FIG. 2A shows the data in the data signal; FIG. 2B shows the FM signal obtained by frequency modulation of the data signal; FIG. 2C shows the tristate wobble signal obtained by conversion from the FM signal, and shows the wobble pattern of the track groove in an optical disc medium according to a first embodiment of the present invention; FIG. 2D shows wobble pattern in adjacent tracks; FIG. 3 is an enlarged view showing the wobble pattern in three adjacent track grooves; FIG. 4A is an enlarged view of adjacent track grooves in an optical disc medium according to a first embodiment of the present invention; FIG. 4B is an enlarged view for reference of a wobble pattern formed to the track groove using the FM signal as shown in FIG. 2B ; FIG. 4C is a comparison of the amplitude of positive and negative pulses obtained using the tristate wobble signal and the amplitude when using the FM signal; FIG. 5 is a block diagram showing the configuration of a data recording apparatus for recording data to an optical disc medium according to a first embodiment of the present invention; FIG. 6 is a flow chart of a method for recording a data signal to an optical disc medium; FIG. 7 is a flow chart showing the specific phase difference in adjacent track grooves during conversion to the tristate wobble signal; FIG. 8 is a flow chart showing a method first frequency modulating the data signal to obtain the FM signal; FIG. 9A is a photograph of the playback waveform obtained from the wobble pattern of the track groove in an optical disc medium according to the first embodiment of the present invention; FIG. 9B is the waveform of the reference clock signal; FIG. 10A shows the data of the data signal; FIG. 10B shows the FM signal obtained by frequency modulation of the data signal; FIG. 10C shows the tristate wobble signal obtained by conversion from the FM signal, and shows the wobble pattern of the track groove in an optical disc medium according to a variation of the first embodiment of the present invention; FIG. 10D shows wobble pattern in adjacent tracks; FIG. 11A shows the data of the data signal; FIG. 11B shows the FM signal obtained by frequency modulation of the data signal; FIG. 11C shows a two-value wobble signal obtained by conversion from the FM signal, and shows the wobble pattern of the track groove in an optical disc medium according to a second embodiment of the present invention; FIG. 11D shows wobble pattern in adjacent tracks; FIG. 12 is an enlarged view of the track groove in FIG. 11C and the wobble pattern of three mutually adjacent track grooves; FIG. 13A is schematic view of the playback wave obtained from the wobble pattern of the track groove in an optical disc medium according to a second embodiment of the present invention; FIG. 13B is a waveform of the reference clock signal; and FIG. 14 is a schematic view showing the management area and data recording area of an optical disc medium. detailed-description description="Detailed Description" end="lead"? |
Device and method for fine synchronization on the sampling of spread-coded received signals |
In order to determine the optimal sampling point for a series of spread-coded data, received by a radio, a correlation data series is used that is produced by convoluting the channel impulse response of the transmission channel with the spread data series, known at the receiving end. The channel impulse response is determined using a channel estimator according to a training sequence. An early-late correlator correlates the correlation data series with the received data in order to generate a correction signal for the optimal sampling point. |
1. An apparatus for tracking the optimum sampling time for a series of received, spread-coded data in a radio receiver, where the received data have been spread-coded at a transmitter end using a spreading sequence which is known at a receiver end, comprising: means for channel estimation which use a training sequence known at the receiver end to ascertain a channel impulse response for the transmission channel, means for convoluting the channel impulse response with the spreading sequence known at the receiver end, means for early/late correlation which correlate the training sequence data known at the receiver end at an early time and at a late time with the correlation sequence obtained by convolution, and also, means for generating a tracking signal which take the correlation ascertained at the early time and the correlation ascertained at the late time as a basis for generating a tracking signal for tracking an optimum sampling time. 2. The apparatus as claimed in claim 1, wherein the series of received, spread-coded data comprises a series of baseband signal values. 3. The apparatus as claimed in claim 2, wherein the baseband signal values are complex baseband signal values which respectively comprise an inphase signal value and a quadrature signal value. 4. The apparatus as claims in claim 1, wherein the means for channel estimation deliver the channel impulse response for the transmission channel as a set of channel coefficients (h0, h1, . . . hL). 5. The apparatus as claimed in claim 1, wherein the tracking signal tracks the optimum sampling time such that the correlation ascertained at the early time and the correlation ascertained at the late time match. 6. The apparatus as claimed in claim 1, wherein the means for generating the tracking signal generate the tracking signal by dividing the correlation ascertained at the early time and the correlation ascertained at the late time. 7. The apparatus as claimed in claim 6, wherein the optimum sampling time is shifted in a first direction if the result of the division is greater than one, and the optimum sampling time is shifted in the direction opposite to the first direction if the result of the division is less than one. 8. The apparatus as claimed in claim 1, wherein the means for generating the tracking signal generate the tracking signal by subtracting the correlation ascertained at the early time and the correlation ascertained at the late time. 9. The apparatus as claimed in claim 8, wherein the optimum sampling time is shifted in a first direction if the result of the subtraction is greater than zero, and the optimum sampling time is shifted in the direction opposite to the first direction if the result of the subtraction is less than zero. 10. The apparatus as claimed in claim 1, wherein the early time is half a chip period ( Tc 2 ) in front of the current sampling time, and the late time is half a chip period ( Tc 2 ) behind the current sampling time. 11. The apparatus as claimed in claim 1, wherein the sampling time at which the received, spread-coded data are sampled is a movable sampling time which is tracked in line with the tracking signal in order to set optimum sampling conditions. 12. The apparatus as claimed in claim 1, wherein the apparatus for tracking the optimum sampling time comprises a unit for linearly interpolating a series of oversampled data values in the received, spread-coded data which uses linear interpolation between consecutive data values in the series of oversampled data values to generate a new series of data values sampled at the tracked optimum sampling time. 13. The apparatus as claimed in claim 12, wherein the unit for linearly interpolating the received, spread-coded data generates the new series of data values sampled at the tracked optimum sampling time in line with the tracking signal, with the tracking signal stipulating the weightings which are used for linearly combining the received spread-coded data to form the data values in the new series. 14. A multiuser detector which comprises an apparatus as claimed in claim 1. 15. A mobile radio station, which comprises an apparatus as claimed in claim 1. 16. The mobile radio station as claimed in claim 15, wherein a standard used for the data transmission is the UMTS standard. 17. A method for tracking an optimum sampling time for a series of received, spread-coded data in a radio receiver, where the received data have been spread-coded at a transmitter end using a spreading sequence which is known at a receiver end, comprising a) performing channel estimation to ascertain a channel impulse response for the transmission channel using a training sequence known at the receiver end; b) convoluting the channel impulse response ascertained in step a) with the spreading sequence known at the receiver end to generate a correlation sequence; c) correlating the training sequence known at the receiver end with the correlation sequence generated in step b) at an early time and at a late time; d) generating a tracking signal for tracking an optimum sampling time on the basis of the correlation ascertained at the early time and the correlation ascertained at the late time. 18. The method as claimed in claim 17, wherein performing the channel estimation comprises ascertaining the channel impulse response for the transmission channel as a set of channel coefficients (h0, h1, . . . hL). 19. The method as claimed in claim 17, wherein generating the tracking signal for tracking the optimum sampling time comprises dividing the correlation ascertained at the early time and the correlation ascertained at the late time. 20. The method as claimed in claim 17, wherein the tracking signal for tracking the optimum sampling time comprises subtracting the correlation ascertained at the early time and the correlation ascertained at the late time. 21. The method as claimed in claim 17, wherein the early time is half a chip period ( Tc 2 ) in front of a current sampling time, and the late time is half a chip period ( Tc 2 ) behind the current sampling time. 22. The method as claimed in claim 17, further comprising shifting the sampling time at which the received spread-coded data are sampled in line with the tracking signal in order to set optimum sampling conditions. 23. The method as claimed in claim 17, further comprising linearly interpolating between consecutive data values in the series of received, spread-coded data is used to generate a new series of data values sampled at the tracked optimum sampling time. 24. The method as claimed in claim 23, wherein the linear interpolation is based on the generated tracking signal. |
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