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1.-20. (canceled) 21. An apparatus for monitoring an analyte comprising: (a) a urethral catheter; the urethral catheter including: (i) a positioning arrangement for positioning the catheter in the urethra, and (ii) at least one sensor oriented such that when the catheter is positioned within the urethra by the positioning arrangement, the at least one sensor is held in close proximity to a urethral wall. 22. An apparatus according to claim 21, wherein: (a) the catheter includes a distal end; and (b) the positioning arrangement comprises an expansible device arranged for holding the distal end of the catheter in the bladder in use; and the at least one sensor is located proximally of the expansible device so that the at least one sensor lies within the urethra. 23. An apparatus according to claim 21 wherein: (a) the positioning arrangement comprises an inflatable balloon. 24. An apparatus according to claim 21 wherein: (a) the at least one sensor is capable of measuring at least one analyte selected from pH, pO2 and pCO2. 25. An apparatus according to claim 21 wherein: (a) the at least one sensor is capable of measuring at least one analyte through optical detection. 26. An apparatus according to claim 24 wherein: (a) the at least one sensor includes at least two sensors, each of the at least two sensors being capable of measuring a respective different analyte; a respective sensor being present for measuring the respective different analyte. 27. An apparatus according to claim 21 wherein: (a) the at least one sensor is positioned within the catheter; and (b) a region of the catheter wall near the at least one sensor is capable of transmitting an analyte to be measured by the at least one sensor. 28. An apparatus according to claim 27 wherein: (a) the at least one sensor is a pH sensor positioned within the catheter; and (b) a region of the catheter wall is perforated or is porous to hydrogen ions. 29. An apparatus according to claim 27 wherein: (a) the at least one sensor is a sensor for dissolved carbon dioxide located within the catheter; and (b) at least a region of the catheter wall is permeable to carbon dioxide. 30. An apparatus according to claim 27 wherein: (a) the at least one sensor is a sensor for dissolved oxygen is located within the catheter; and (b) at least a region of the catheter wall is permeable to oxygen. 31. An apparatus according to claim 30 wherein: (a) the sensor is an optical device in which a fluorescent material is quenched by the analyte. 32. An apparatus according to claim 21 wherein: (a) the at least one sensor includes a common sensor device capable of measuring pO2 and pCO2 and pH. 33. An apparatus according to claim 21 further comprising: (a) a temperature sensing device for measuring temperature. 34. An apparatus according to claim 21 wherein: (a) the urethral catheter is a triple lumen catheter comprising: (i) a first lumen for inflating a positioning arrangement; (ii) a second lumen for draining urine; and (iii) a third lumen for housing the at least one sensor in close proximity to the urethral wall. 35. An apparatus for measuring at least one analyte selected from pH, oxygen and carbon dioxide in urethra epithelial tissue, the apparatus comprising: (a) an urethral catheter including: (i) an inflatable device to secure a distal end of the catheter within a bladder; and (ii) a sensor positioned with respect to the catheter to lie proximally of the inflatable device within the urethra and in a region of an urethral wall, when in use. 36. An apparatus according to claim 35 wherein: (a) the urethral catheter is a triple lumen catheter comprising: (i) a first lumen for inflating the inflatable device, (ii) a second lumen for draining urine; and (iii) a third lumen for housing the sensor in close proximity to the urethral wall; (A) the sensor including at least one or more sensors; each of the one or more sensors being housed by the third lumen. 37. A method of monitoring vital conditions of a patient, the method comprising: (a) positioning a sensor in a urethra of the patient for measuring a concentration of an analyte in urethral tissue. 38. A method according to claim 37 wherein: (a) the step of positioning includes introducing the sensor to the urethra with a catheter. 39. A method according to claim 38 further including: (a) draining urine from the patient through a lumen of the catheter; and (b) providing a closure distally of the sensor to prevent passage of urine into the urethra other than within the lumen. 40. A method of monitoring a patient; the method comprising: (a) introducing into a urethra of a patient a urethral catheter having a sensor for sensing an analyte and a positioning arrangement; and (b) positioning the catheter so that the sensor is held in close proximity to a urethral wall by using the positioning arrangement. 41. A method according to claim 40 wherein: (a) the step of introducing includes advancing a distal end of the catheter in to the patient's bladder; and (b) the step of positioning includes expanding an expansible device located at the distal end of the catheter to retain the distal end within the bladder, with the sensor being located proximally of the expansible device and within the urethra. 42. A method according to claim 40 wherein: (a) said step of positioning includes inflating an inflatable balloon. 43. A method according to claim 40 wherein: (a) said step of introducing includes introducing a urethral catheter having a sensor for sensing an analyte selected from pH, pO2 and pCO2. 44. A method according to claim 43 wherein: (a) said step of introducing includes introducing a urethral catheter having a sensor for sensing temperature. 45. A method according to claim 40 further including: (a) monitoring pH, pO2 and pCO2. 46. A method according to claim 40 further including: (a) monitoring at least one analyte with optical detection. 47. A method according to claim 46 further including: (a) allowing at least one analyte to diffuse into the sensor and interact with a chemical indicator. 48. A method according to claim 40 further including: (a) monitoring dissolved oxygen including permitting oxygen to permeate through a region of a wall of the catheter wall to gain access to an oxygen sensor located within the wall. |
Process for the preapartion 3-aryl-2-hydroxypropionic acid derivative |
A process for the preparation of a compound of formula I in which R represents H or an acid protecting group which comprises reacting a compound of formula II in which R is as previously defined with a compound of formula III wherein X is a suitable leaving group in the presence of a base and a phase transfer catalyst at a temperature in the range 50° C. to 150° C. |
1. A process for the preparation of a compound of formula I in which R represents H or an acid protecting group which comprises reacting a compound of formula II in which R is as previously defined with a compound of formula III wherein X is a suitable leaving group in the presence of a base and a phase transfer catalyst at a temperature in the range 50° C. to 150° C.: 2. A process according to claim 1, wherein the process is performed in the presence of an aqueous solution of a base and a phase transfer catalyst. 3. A process according to claim 1, wherein the process is performed in the presence of a base in solid form and a phase transfer catalyst. 4. A process according to claim 1, further comprising removing an acid protecting group to produce a compound of formula I in which R is H. 5. A process according to claim 4, in which the acid protecting group is removed by hydrolysis. 6. A process according to claim 3 or 4, in which the process is carried out as a melt. 7. A process according to claim 1, in which R is a (1-4C)alkyl group. 8. A process according to claim 1, in which X is halo, an optionally substituted phenylsulfonyloxy group or an alkylsulphonyloxy group. 9. A process according to claim 1, in which the base is selected from carbonates, hydrogen carbonates or hydroxides of alkali metals. 10. A process according to claim 1, in which the phase transfer catalyst is a crown ether, a polyethylene glycol or a quaternary ammonium salt. 11. A process according to claim 1 in which the process is carried out in the presence of a suitable solvent for the compounds of formulae II and III. 12. A process according to claim 11, in which the solvent is selected from 2-butanone, iso-butyl methyl ketone, acetone, dimethylsulfoxide, NN-dimethylformamide, or N-methylpyrrolidone. 13. A process according to claim 1, in which the compound of formula I is the S enantiomer. 14. A process for the preparation of a compound of formula I in which R represents H which comprises reacting a compound of formula II in which R represents an acid protecting group with a compound of formula III wherein X is a suitable leaving group in the presence of a base in solid form and a phase transfer catalyst at a temperature in the range 50° C. to 150° C. to give a compound of formula I in which R is an acid protecting group and then removing the protecting group to give a compound of formula I in which R is H. |
Valve device for musical instrument and metallic wind instrument comprising the same |
Since the casing newly developed for the entire valve block of a brasswind instrument is made of resin, the dimensions of all inner movable parts can be increased without increasing substantially the weight of the entire valve block. This additional degree of freedom in design allows for planning and manufacturing of both inner sound channels and switching channels of movable inner parts so that they have a perfectly circular profile and a smooth inner surface. Therefore, by providing an ideal sound channel without increasing the overall weight of the valve block, a considerable improvement in the sound quality of an instrument can be achieved. |
1. A valve block device for music instruments, comprising: a casing part that is entirely made of resin, contains a plurality of cylindrical inner holes, and has at least one pair of through holes in each side wall of the inner holes, the at least one pair of through holes being connected to the inner holes; a plurality of movable parts, each containing at least one channel, such parts being installed inside the cylindrical inner holes and capable of connecting through movement at least one of the pairs of through holes with each other; and at least one cover part being removably installed on at least one end of the cylindrical inner holes to cover all of the movable parts, wherein the casing part is formed integrally by molding. 2-3. (canceled) 4. A valve block device for music instruments according to claim 1, wherein the movable parts are the turning rotors, which rotate in the inner circular holes having circular profiles, and the at least one case cover part contains axle bearings supporting the rotors. 5. A valve block device for music instruments according to claim 1, wherein the movable parts are pistons which are located in the cylindrical holes and can displace the position along the direction of axis thereof. 6. A brasswind instrument, comprising a valve block device for music instruments, wherein the valve block device comprises: a casing part that is entirely made of resin, contains a plurality of cylindrical inner holes, and has at least one pair of through holes in each side wall of the inner holes, the at least one pair of through holes being connected to the inner holes; a plurality of movable parts, each containing at least one channel, such parts being installed inside the cylindrical inner holes and capable of connecting through movement at least one of the pairs of through holes with each other; and at least one cover part being removably installed on at least one end of the cylindrical inner holes to cover all of the movable parts, wherein the casing part is formed integrally by molding. 7. A valve block device for music instruments according to claim 1, wherein the casing part is made of either polyimide or a similar resin. 8. A valve block for music instruments according to claim 1, wherein the moveable parts are made of metal. 9. A brasswind instrument according to claim 6, wherein the movable parts are the turning rotors, which rotate in the inner circular holes having circular profiles, and the at least one case cover part contains axle bearings supporting the rotors 10. A brasswind instrument according to claim 6, wherein the movable parts are pistons which are located in the cylindrical holes and can displace the position along the direction of axis thereof. |
<SOH> BACKGROUND ART <EOH>Traditional instruments consist of a set of metal pipes, usually of brass, which are either connected to each other or connected to valves. In the case of a horn, the valve used to switch the bell pipe consists of a cylindrical enclosure, the valve casing and an internal rotor, the switcher, all forming an integrated unit. When several such valves are connected with each other by short pipes, a valve block device is formed. That in turn can be connected with short pipes, the valve slides, to the instrument and, one has a brasswind instrument with a valve block device. When such a valve block device is connected through valve slides to the sound tube, it contains, in addition, a link mechanism consisting of push and pull levers that permit adjustment of the pitch of sound by turning the switcher that is fixed on an axle inside the valve itself. It is a common belief, that, in order to minimize negative effects on both sound quality and tone, the cross-section of the air channel (sound channel) of the rotor, the switcher channel, should be identical to the cross-section of the pipes used as valve slides and connectors to the tuning slides. Therefore, manufacturers aim to achieve a perfectly circular switcher channel with a very smooth interior. However, perfectly circular switcher channels always result in an increase in valve dimensions, especially in those of the valve rotor. This in turn causes an unfortunate increase in the volume of materials used and in the overall weight of the valve block device. This contributes heavily to problems regarding the handling and maintenance of brasswind instruments. Moreover, it increases the manufacturing costs of such valves. For these reasons, the switcher channels of most valves used in today's brasswind instruments are elliptical. In this case, a portion of the outer side of the valve rotor sound channel is omitted. To minimize negative effects on tone, influencing the sound quality, great efforts are made to keep the diameter of the switcher, and thus the overall dimensions of the valve, small. This approach is believed to minimize disturbing factors which have a negative impact on true sound reproduction. It is also hoped, with this approach, to achieve a better balance regarding overall sound quality and handling of the instrument. Traditional brasswind instruments of today are actually the result of a compromise between reasonable price with easy handling and basic requirements regarding tone. However, for professionals striving to achieve the highest sound quality and perfect sound reproduction, the above mentioned problems have still not been resolved satisfactorily. State of the art technology does not permit high sound quality while keeping valves small and handy. Since it is still not possible to adhere to all requirements as mentioned above, we have decided to focus on achieving the best sound quality possible. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 2 shows an outline of a brasswind instrument with a valve block device installed in the form of the first embodiment. FIG. 2A shows an outline of the valve block device which can be installed in a brasswind instrument as shown, for example, in FIG. 1 . FIG. 2B shows a side view of the valve block device shown in FIG. 2A . FIG. 3 shows a cross-section of the valve block device shown in FIG. 2A , section AA. FIG. 4 shows a cross-section of the valve block device shown in FIG. 2B , section BB. FIG. 5 shows a cross-section of an individual valve in enlarged view. FIG. 6 shows a valve block device in the form of the second embodiment with a partially cut-away side wall. detailed-description description="Detailed Description" end="lead"? |
Method for removing oil fat and grease from water |
Free oil, fat and grease contaminate water and wastewater these contaminants can be removed by an efficient separator system and allow the contaminants to be recovered at their maximum concentration. |
1. A continuous flow separator for removing oil, fat, grease and the like from waste water comprising: a vertical cylindrical vessel having a centrally located waste water inlet at its base, a vertical riser pipe located centrally within the vessel and in fluid communication with the inlet and having an opening at its top, a first baffle located within the vessel and above the riser pipe, a free oil zone located between the top of the riser pipe and the oil outlet pipe, a first oil outlet pipe removing oil from the free oil zone, a first annulus chamber formed between the riser tube and a first sleeve surrounding the riser tube in fluid communication with the free oil zone, a second annulus chamber formed between a second sleeve surrounding the first sleeve and the top of which is in fluid communication with the top of the first annulus chamber, a third annulus chamber formed between the second sleeve and an outer wall of the cylindrical vessel and the bottom of which is in fluid communication with the second annulus chamber, a second baffle plate located above the first and second sleeves and separating the first and second annulus chambers from the free oil zone, at least one second oil outlet pipe for removing oil from the top of the second first and second annulus chambers, an air inlet connected to a sparge pipe located at the bottom of the first annulus chamber, and a waste water outlet located at the bottom of the first annulus chamber. 2. A continuous flow separator as defined in claim 1 further comprising a pump to supply waste water to the waste water inlet. 3. A continuous flow separator as defined in claim 2 wherein said pump is a non-emulsifying pump. 4. A continuous flow separator as defined in claim 1 wherein said first baffle plate has an inverted cone shape. 5. A continuous flow separator as defined in claim 1 wherein said outlet pipe can be adjusted in a vertical direction to control the level of liquid within the vessel. 6. A continuous flow separator as defined in claim 1 wherein the outlet chamber has a solid waste outlet to permit the removal of solids which may accumulate in the outlet chamber. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Most industries face significant problems with processing and disposal of liquid waste that is contaminated with free oil, fat and grease. This type of waste posses a major threat to the global environment and there is a need for simple and cost effective solution to remove these contaminants to allow clean water discharge to public sewer, waste treatment plants, inland waterways and seas or oceans. |
Method for detecting and identifying microorganism causative of infection |
Causative microorganisms of infectious diseases are detected and/or identified rapidly and high-sensitively by taking phagocytes from the clinical specimens containing active phagocytes, immobilizing the phagocytes so taken, treating the phagocytes to improve cell membrane permeabilities thereof, further treating the phagocytes to bare DNA in the causative microorganisms which might be existed in the phagocytes, and detecting the causative microorganisms with DNA probes which can hybridize with such DNA under stringent conditions. |
1-18. (canceled) 19. A method for detecting and/or identifying microorganisms causative of infectious diseases, comprising: immobilizing phagocytes from a clinical specimen containing active phagocytes that may contain microorganisms causative of infectious diseases; treating the phagocytes to increase phagocyte cell membrane permeability; further treating the phagocytes to bare DNA in microorganisms that may exist in the phagocytes; in situ hybridizing the bared DNA with at least one DNA probe; and detecting and/or identifying the causative microorganisms by detecting hybridization of at least one probe to the bared DNA, wherein the method comprises at least one condition selected from the group consisting of: (1) the phagocytes in the immobilizing step have a cell density between 5×106 cells/ml and 1×108 cells/ml; (2) applying from about 1 Unit/ml to about 1,000 Units/ml of Lysostaphin to the phagocytes in the further treating step, to bare the DNA; (3) applying from about 1,000 Units/ml to about 1,000,000 Units/ml of Lysozyme to the phagocytes in the further treating step to bare the DNA; (4) applying from about 10 Units/ml to about 10,000 Units/ml of N-acetylmuramidase to the phagocytes in the further treating step to bare the DNA; (5) applying from about 50 Units/ml to about 500 Units/ml of Zymolyase to the phagocytes in the further treating step to bare the DNA; (6) applying surfactant in the step of in situ hybridization; (7) the at least one DNA probe is from about 350 to about 600 bases in length; and (8) the concentration of the at least one DNA probe is from about 0.1 ng/μl to about 2.2 ng/μl. 20. The method according to claim 19, wherein the step to bare the DNA employs at least one enzyme selected from the group consisting of: about 10 to about 100 Units/ml of Lysostaphin; about 10,000 to about 100,000 Units/ml of Lysozyme; about 100 to about 1,000 Units/ml of N-acetylmuramidase; and about 100 to about 500 Units/ml of Zymolyase. 21. The method according to claim 19, wherein the step to bare DNA employs at least one enzyme at a temperature of about 26° to about 59° C. for about 15 to about 120 minutes. 22. The method according to claim 19, wherein the step to bare the DNA further employs at least one substance to maintain phagocyte form. 23. The method according to claim 22, wherein the at least one substance comprises phenylmethylsulfonyl fluoride (PMSF). 24. The method according to claim 23, wherein the PMSF is employed at a concentration of about 10 μmol/l to about 10 mmol/l. 25. The method according to claim 22, wherein the at least one substance is dissolved into dimethylsulfoxide (DMSO). 26. The method according to claim 25, wherein the concentration of the DMSO in a solution to be employed in the step to bare DNA is less than about 5%. 27. The method according to claim 19, wherein the in situ hybridization is performed by hybridizing the DNA with the at least one DNA probe in the presence of at least one surfactant. 28. The method according to claim 27, wherein the at least one surfactant is an anionic surfactant. 29. The method according to claim 28, wherein the anionic surfactant is sodium dodecyl sulfate (SDS). 30. The method according to claim 19, wherein the in situ hybridization is performed at a temperature of about 25° to about 50° C. for about 30 to about 900 minutes. 31. The method according to claim 19, wherein the method comprises, prior to the immobilizing step, a step to mount the phagocytes onto a solid support. 32. The method according to claim 31, wherein the solid support is a slide coated with 3-aminopropyl triethoxysilane. 33. The method according to claim 19, wherein the detecting employs a pigment to distinguish between signals and cells. 34. The method according to claim 19, wherein the clinical specimen is blood. 35. A kit for detecting and/or identifying microorganisms causative of infectious diseases from phagocytes, comprising: (1) a composition comprising at least one enzyme selected from the group consisting of Lysostaphin, Lysozyme, N-acetylmuramidase and Zymolyase; and (2) at least one DNA probe for detecting DNA of a microorganism. 36. A method for monitoring a gene of exogenous microorganisms digested with the phagocytes in a clinical specimen containing active phagocytes comprising the step of detecting the gene with in situ hybridization method employed in the method according to claim 19, wherein the gene of exogenous microorganisms in the clinical specimens is monitored. 37. A method for diagnosing sepsis or bacteriemia comprising the step of identifying a gene of candidate causative microorganisms with in situ hybridization method employed in the method according to claim 19, wherein the causative microorganisms for sepsis or bacteriemia are determined based on the identification results. |
<SOH> BACKGROUND ARTS <EOH>Although the hemoculture methodologies have popularly been used conventionally as a mean to verify bacteria in the blood, since this methodology needs about from 3 to 14 days to culture and isolate the subjected bacteria and detection rates thereby are as low as about 10%, it was not well contributed in the diagnosis for treating serious diseases like sepsis. The present inventors had invented, to solve such problems, a method for detecting and identifying exogenous-microorganisms digested with phagocytes comprising a step of detecting genes from such exogenous-microorganisms in the phagocytes by in situ hybridization employing a probe which can specifically hybridize with the genes (Japanese Patent Publication No. 7-40). The method of Japanese Patent Publication No. 7-40 have been in the limelight in the field of infectious deseases because, in comparison with the conventional hemoculture methodology, the method allowed about four times rapidly detection of the subjected bacteria in bloods from patients who are under the suspicion about sepsis, and detection results were appeared within 24 hours. Objects of the present inventions is an improvement of detection effects and of detection sensitivity to be offered by the method according to Japanese Patent Publication No. 7-40 for detecting and/or identifying causative microorganisms of infectious diseases by taking phagocytes from the clinical specimens containing active phagocytes, immobilizing the phagocytes so taken, treating the phagocytes to improve cell membrane permeabilities thereof, further treating the phagocytes to bare DNA in the causative microorganisms which might be existed in the phagocytes, in situ hybridizing DNA so bared with detective DNA probe(s) which can hybridize with such bared DNA under stringent conditions, and detecting and/or identifying the causative microorganisms based on signals so detected. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 is a view of illustrating results of in situ hybridization performed under (a) the absence of surfactant (SDS) and (b) the presence of surfactant (SDS). FIG. 2 is a view of illustrating a manner of leukocytes immobilized with the various cell density. FIG. 3 is a view of illustrating a time-coursely change on lytic enzyme activity against (a) Staphylococcus aureus and Staphylococcus epidermidis , (b) Pseudomonas aeruginosa and Escherichia coli , and (c) Enterococcus faecalis. FIG. 4 is a view of illustrating concentration-dependency effects by addition of DMSO against lytic activity to be offered by (a) 300 Units/ml of N-acetylmuramidase, (b) 10,000 Units/ml of Lysozyme, and (c) 50 Units/ml of Lysostaphin. FIG. 5 is a view of illustrating results on addition of (a) protease 0.2 Units/ml only, (b) PMSF 1 μmol/ml, (c) PMSF 10 μmol/ml, (d) PMSF 0.1 mmol/ml, and (e) PMSF 1 mmol/ml to study effects of PMSF to be used to suppress the function of protease which changes a morphological form of leukocyte. FIG. 6 is a view of illustrating that, in the phagocytosis samples prepared according to the present invention, phagocytes digested bacteria and morphological forms of such digested bacteria were changed. FIG. 7 is a view of illustrating effects of enzyme-treatment in the phagocytosis samples and manner of the phagocytosis samples containing (a) Staphylococcus aureus prior to the treatment, (b) Enterococcus faecalis prior to the treatment, (c) Staphylococcus aureus of (a) with the treatment, and (d) Enterococcus faecalis of (b) with the treatment. FIG. 8 is a shematic view of the slide on which the phagocytosis samples were smeared to study optimum probe concentration under the in situ hybridization. FIG. 9 is a shematic view of the slide on which the phagocytosis samples were smeared to study optimum temperature under the in situ hybridization. FIG. 10 is a view of illustrating the signals appeared in the resluts of Southern Blot (the upper row) and Electrophoresis (the lower row) together with the length of the labelled detective probes prepared by putting digoxigenin-labels on (a) SA probes and (b) PA probes. FIG. 11 is a view of illustrating the signals detected by in situ hybridiozation of the digested Escherichia coli with detective probes of (a) EC-24, (b) EC-34, (c) EC-39 and (d) the mixed probes (MIX) of the foregoing probes (a)-(c). detailed-description description="Detailed Description" end="lead"? |
Thiochromenones used to combat painful conditions and neurodegenerative diseases |
The invention relates to novel thiochromenones and processes for their preparation, to their use for the treatment and/or prophylaxis of diseases, especially for the treatment and/or prophylaxis of states of pain and neurodegenerative disorders. |
1. A compound of the formula (I), in which the radical R1-A- is located at either of positions 2 or 3 of the thiochromenone ring, R1 is (C6-C10)-aryl or 5- to 10-membered heteroaryl, where aryl and heteroaryl are optionally substituted identically or differently by radicals selected from the group of halogen, formyl, carbamoyl, cyano, hydroxyl, trifluoromethoxy, nitro, —NR3R4, tetrazolyl, (C1-C6)-alkoxycarbonyl and optionally hydroxyl-, morpholinyl-, (C1-C6)-acyloxy- or halogen-substituted (C1-C6)-alkyl, (C1-C6)-alkoxy, (C1-C6)-acyl and (C1-C6)-alkylthio, in which R3 and R4 are, independently of one another, hydrogen, (C1-C6)-alkyl or (C1-C6)-acyl, is 3- to 12-membered carbocyclyl or 4- to 12-membered heterocyclyl, where carbocyclyl and heterocyclyl are optionally substituted identically or differently by radicals selected from the group of (C1-C6)-alkyl, (C1-C6)-alkoxy, (C1-C6)-acyl, (C1-C6)-alkoxycarbonyl or oxo, or is a group of the formula R5-E-, in which E is optionally unsaturated (C1-C10)-alkanediyl, and R5 is hydrogen, carbamoyl, halogen, hydroxyl, nitro, trifluoromethyl, amino, mono-(C1-C6)-alkylamino, di-(C1-C6)-alkylamino, (C1-C6)-alkoxy, (C6-C10)-aryl, 5- to 10-membered heteroaryl or 4- to 10-membered, optionally oxo- and/or (C1-C6)-alkyl-substituted, optionally benzo-fused heterocyclyl, where aryl, heteroaryl and benzo in turn may be substituted by radicals selected from the group of halogen, cyano, trifluoromethyl, trifluoromethoxy, nitro and (C1-C6)-alkyl, A is a bond or a group of the formula O, S, NR6, CO, SO, SO2, SO2—O, CO—NR7, SO2—NR8, O—SO2, NR9—CO, NR10—SO2, NR11—SO2—O, NR12—SO2—NR13 or NR14—CO—NR15, in which R6 R7, R8, R9, R10, R11, R12, R13, R14 and R15 are (C3-C8)-cycloalkyl or optionally unsaturated (C1-C6)-alkyl which is optionally substituted by hydroxyl, phenyl, (C1-C6)-alkoxy, (C1-C6)-alkoxycarbonyl or (C3-C8)-cycloalkyl, where phenyl in turn may be substituted by halogen or (C1-C4)-alkyl, or in which R6, R7, R9, R10, R11, R12, R14 and R15 are hydrogen, or the radical R1-A- is hydrogen or amino, R2 is hydrogen, halogen or (C1-C6)-alkyl or (C1-C6)-alkoxy, where alkyl and alkoxy are optionally substituted up to twice-identically or differently by radicals selected from the group of hydroxyl, (C1-C6)-alkoxy, mono- and di-(C1-C6)-alkylamino, and D is an optionally fluorine-substituted, divalent hydrocarbon radical having 3 to 10 carbon atoms, and the salts, hydrates and/or solvates thereof, with the exception of 2-chloro-6,7,8,9,10,10a-hexahydrocyclohepta[b]thio-chromen-11(5aH)-one. 2. A compound of the formula (I) as claimed in claim 1, where the radical R1-A- is located at position 3 of the thiochromenone ring, R1 is (C6-C10)-aryl or 5- to 10-membered heteroaryl, where aryl and heteroaryl are optionally substituted identically or differently up to twice by radicals selected from the group of halogen, formyl, cyano, hydroxyl, hydroxymethyl, (C1-C6)-alkyl, is 4- to 10-membered heterocyclyl, where heterocyclyl are optionally substituted identically or differently by radicals selected from the group of (C1-C6)-alkyl, (C1-C6)-alkoxy, (C1-C6)-alkoxycarbonyl or oxo, A is a bond or a group of the formula NR6, CO—NR7, SO2—NR8 or NR9—CO, in which R6, R7, R8 and R9 are optionally unsaturated (C1-C6)-alkyl which is optionally substituted up to twice, identically or differently, by hydroxyl or methoxy, or in which R6, R7 and R9 are hydrogen, R2 is hydrogen, and D is a group of the formula (CH2)m—CR16R17—(CH2)n, in which the total number of carbon atoms is 3 to 10, m and n are identical or different and are a natural number from the series 0 to 6, and R16 and R17 are identical or different and are hydrogen or (C1-C6)-alkyl which is optionally substituted identically or differently by (C3-C5)-cycloalkyl or halogen, or CR16R17 is (C3-C6)-cycloalkane-1,1-diyl, and the salts, hydrates and/or solvates thereof. 3. A compound of the formula (I) as claimed in claim 1, where the radical R1-A- is located at position 3 of the thiochromenone ring, R1 is phenyl or 5- to 6-membered heteroaryl, where phenyl and heteroaryl are optionally substituted identically or differently up to twice by radicals selected from the group of halogen, cyano, (C1-C3)-alkyl, is 5- to 7-membered heterocyclyl, where heterocyclyl are optionally substituted identically or differently by radicals selected from the group of (C1-C3)-alkyl or Oxo, A is a bond or a group of the formula NR6, SO2—NR8 or NR9—CO, in which R6, R8 and R9 are optionally unsaturated (C1-C3)-alkyl which is optionally substituted up to twice, identically or differently, by hydroxyl or methoxy, and in which R6, R8 and R9 are hydrogen, R2 is hydrogen, and D is a group of the formula (CH2)m—CR16R17—(CH2)n, in which the total number of carbon atoms is 3 to 6, m and n are identical or different and are a natural number from the series 0 to 2, and R16 and R17 are identical or different and are hydrogen or (C1-C3)-alkyl, or CR16R17 is (C3-C6)-cycloalkane-1,1-diyl, and the salts, hydrates and/or solvates thereof. 4. A compound of the formula (I) as claimed in claim 1 for the treatment and/or prophylaxis of diseases. 5. A medicament comprising at least one of the compounds of the formula (I) as claimed in any of claims 1 to 3 mixed with at least one pharmaceutically acceptable, essentially non-toxic carrier or excipient. 6. The use of compounds of the formula (I) as claimed in any of claims 1 to 3 for producing a medicament for the treatment and/or prophylaxis of states of pain and/or neurodegeinerative disorders. |
Process for treating an aqueous medium containing phosphate, cyclohexanone and cycloexanone oxime |
The invention relates to a process for treating an aqueous medium containing (i) phosphate and (ii) cyclohexanone and/or cyclohexanone oxime, said process comprising: feeding the aqueous medium to a stripping zone; passing steam through the aqueous medium in the stripping zone; and discharging a vapor stream from said stripping zone; wherein the joint content cyclohexanone and cyclohexanone oxime in the aqueous medium entering the stripping zone is less than 0.08 wt. %. |
1. Process for treating an aqueous medium containing (i) phosphate and (ii) cyclohexanone and/or cyclohexanone oxime, said process comprising: feeding the aqueous medium to a stripping zone; passing steam through the aqueous medium in the stripping zone; and discharging a vapor stream from said stripping zone; wherein the joint content of cyclohexanone and cyclohexanone oxime in the aqueous medium entering the stripping zone is less than 0.08 wt. %. 2. Process for preparing cyclohexanone oxime, said process comprising: passing an aqueous medium containing phosphate from a hydroxylammonium synthesis zone to a cyclohexanone oxime synthesis zone, from the cyclohexanone oxime synthesis zone to a stripping zone and from the stripping zone back to the hydroxylammonium synthesis zone; in said hydroxylammonium synthesis zone, preparing hydroxylammonium by catalytically reducing nitrate or nitrogen oxide with hydrogen; in said cyclohexanone oxime synthesis zone, preparing cyclohexanone oxime by reacting hydroxylammonium with cyclohexanone; passing steam through the aqueous medium in the stripping zone; and discharging a vapor stream from said stripping zone; wherein the joint content of cyclohexanone and cyclohexanone oxime in the aqueous medium entering the stripping zone is less than 0.08 wt. %. 3. Process according to claim 1 or claim 2, wherein the joint content of cyclohexanone and cyclohexanone oxime entering the stripping zone is less than 0.05 wt. %. 4. Process according to claim 1, wherein the process comprises, extracting cyclohexanone and cyclohexanone oxime from said aqueous medium, prior to feeding the aqueous medium to the stripping zone. 5. Process according to claim 4, wherein the process comprises, prior to feeding the aqueous medium to the stripping zone, extracting cyclohexanone and cyclohexanone oxime from said aqueous medium, such as to reduce the joint content of cyclohexanone and cyclohexanone oxime to a value below 0.08 wt. %. 6. Process according to claim 5, wherein the process comprises, prior to feeding the aqueous medium to the stripping zone, extracting cyclohexanone and cyclohexanone oxime from said aqueous medium, such as to reduce the joint content of cyclohexanone and cyclohexanone oxime to a value below 0.05 wt. %. 7. Process according to claim 6, wherein said extracting is effected in an extraction column. 8. Process according to claim 7, wherein said extraction column contains packing bodies. 9. Process according to claim 8, wherein said extraction column is a pulsed column. 10. Process according to claim 1, wherein the vapor stream comprises steam and cyclohexanone. 11. Process according to claim 1, wherein the process comprises: condensing the vapor stream to obtain a condensed aqueous fluid; and washing an organic product with the condensed aqueous fluid. 12. Process according to claim 11, wherein the organic product comprises cyclohexanone oxime. 13. Process according to claim 2, wherein the process comprises: condensing the vapor stream to obtain a condensed aqueous fluid; withdrawing an organic product comprising cyclohexanone oxime from the cyclohexanone oxime synthesis zone; and washing the organic product with the condensed aqueous fluid after said withdrawing. 14. Process according to claim 1, wherein the superficial gas velocity of the steam in the stripping zone is between 0.2 and 3 M/S. 15. Process according to claim 1, wherein the process comprises obtaining said steam by evaporating water from the aqueous medium. 16. Process according to claim 1, wherein the process comprises obtaining said steam by evaporating 20-400 kg water per m3 of aqueous medium. 17. Process according to claim 1, wherein said stripping zone is a column. 18. Process according to claim 17, wherein said column is a plate column or a packed column. 19. Process according to claim 1, wherein the aqueous medium is an acidic aqueous medium. 20. Process according to claim 19, wherein the aqueous medium entering the stripping zone has a pH of between 0 and 4. 21. Process according to claim 1, wherein the aqueous medium entering the stripping zone contains 2.0-8.0 mol phosphate, 0.5-8.0 mol ammonium and 0.1-5.0 mol nitrate per liter of aqueous medium. 22. Process for preparing cyclohexanone oxime, comprising: feeding an aqueous medium containing phosphate and hydroxylammonium to a cyclohexanone oxime synthesis zone; in said cyclohexanone oxime synthesis zone, preparing cyclohexanone oxime by contacting said aqueous medium with an organic stream comprising cyclohexanone and an organic solvent; withdrawing an organic product from said cyclohexanone oxime synthesis zone, said organic product comprising cyclohexanone oxime and organic solvent; washing said organic product with water. 23. Process according to claim 22, wherein the process comprises: evaporating part of the water from said aqueous medium; condensing at least part of the evaporated water; washing the organic product with said condensed water. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a schematic diagram of an embodiment of a stripping column. FIG. 2 is a schematic diagram of an embodiment of a process according to the present invention. detailed-description description="Detailed Description" end="lead"? |
Diagnostic methods for cardiovascular disease, low hdl-cholesterol levels, and high triglyceride levels |
A method for determining propensity toward developing a cardiovascular disease in a patient at risk thereof by determining the presence in an ABCA1 gene of said patient of a polymorphism in the DNA sequence of the gene wherein said polymorphism is present in a non-coding region of said gene is disclosed. Also described is a method of identifying a modulator of ABCA1 polynucleotide expression comprising by determining the ability of a test compound to modulate the activity of a polynucleotide comprising a polymorphism disclosed herein, or to mimic the effects of such polymorphism where such effects are beneficial. Treatment of cardiovascular disease, especially coronary artery disease, using agents identified by the disclosed methods is also described. |
1. A method for determining propensity toward developing a cardiovascular disease in a patient at risk of developing said disease comprising determining the presence in an ABCA1 gene of said patient of a polymorphism in the DNA sequence of said gene wherein said polymorphism is present in a non-coding region of said gene. 2. The method of claim 1 wherein said polymorphism is present in the promoter region of said gene. 3. The method of claim 1 wherein said polymorphism is present in an intronic region of said gene. 4. The method of claim 1 wherein said disease is coronary artery disease. 5. The method of claim 1 wherein said disease involves increased triglyceride levels in the plasma of said patient. 6. The method of claim 1 wherein said disease involves decreased high density lipoprotein (HDL-C) levels in the plasma of said patient. 7. The method of claim 1 wherein said disease involves elevated cholesterol levels in the plasma of said patient. 8. The method of claim 1 wherein said disease involves decreased lipid transport in the cells of said patient. 9. The method of claim 1 wherein said polymorphism is a single nucleotide polymorphism. 10. The method of claim 9 wherein said polymorphism is a polymorphism shown in Table 1. 11. A method for identifying a modulator of ABCA1 polynucleotide expression comprising: (a) contacting a compound with a polynucleotide that encodes ABCA1 polypeptide, which polynucleotide comprises a polymorphism in a non-coding region of said polynucleotide, under conditions promoting said contacting and promoting expression of ABCA1 polypeptide by said polynucleotide; (b) determining the activity of said polynucleotide in expressing said ABCA1 polypeptide after said contacting wherein a difference in the expression of said polynucleotide relative to when said compound and said polynucleotide are not contacted indicates polynucleotide modulating activity, thereby identifying a modulator of ABCA1 polynucleotide expression. 12. The method of claim 11 wherein said ABCA1 polynucleotide is present in a cell. 13. The method of claim 11 wherein said difference in expression in step (b) is an increase in expression. 14. The method of claim 11 wherein said polymorphism is present in an intronic region of said polynucleotide. 15. The method of claim 11 wherein said polymorphism occurs in a promoter region of said polynucleotide. 16. The method of claim 11 wherein said polymorphism is a single nucleotide polymorphism (SNP). 17 The method of claim 14 wherein said SNP is a member selected from the SNPs shown in Table 1. 18. The method of claim 11 wherein said polymorphism has the effect of decreasing the activity of said polynucleotide. 19. A method of identifying an agent that modulates plasma lipid levels comprising administering to an animal an effective amount of a compound first identified as an ABCA1 modulator using the method of claim 11. 20. The method of claim 19 wherein said compound has the effect of reducing plasma triglyceride levels. 21. The method of claim 19 wherein said compound has the effect of reducing plasma cholesterol levels. 22. The method of claim 19 wherein said compound has the effect of increasing plasma HDL-C levels. 23. A method of treating a patient for cardiovascular disease comprising administering to a patient afflicted therewith of an effective amount of a compound first identified as an ABCA1 modulator using the method of claim 11 or 19. 24. The method of claim 23 wherein said disease is coronary artery disease. 25. The method of claim 23 wherein said disease is atherosclerosis. 26. A method of protecting a patient against developing cardiovascular disease comprising administering to a patient at risk thereof of an effective amount of a compound first identified as an ABCA1 modulator using the method of claim 11 or 19. 27. The method of claim 26 wherein said disease is coronary artery disease. 28. The method of claim 26 wherein said disease is atherosclerosis. 29. A method for identifying a therapeutic agent for administration to a patient in need thereof, comprising comparing a nucleotide sequence of a non-coding region of an ABCA1 gene of said patient to a database that correlates nucleic acid sequences of ABCA1 genes with the effectiveness of therapeutic agents in beneficially regulating lipid levels in a patient, thereby identifying a therapeutic agent for administration to said patient. 30. The method of claim 29 wherein said database comprises ABCA1 nucleotide sequences comprising the polymorphic sequences disclosed in Table 1. 31. A method for identifying a candidate for enrolment in a program of clinical trials of a potential therapeutic agent, comprising comparing a nucleotide sequence of a non-coding region of an ABCA1 gene of said candidate to a database that correlates nucleic acid sequences of ABCA1 genes with the effectiveness of therapeutic agents in beneficially regulating lipid levels in a patient, thereby identifying a candidate for enrolment in a program of clinical trials. 32. The method of claim 31 wherein said database comprises ABCA1 nucleotide sequences comprising the polymorphic sequences disclosed in Table 1. 33. A method for producing a product comprising identifying an agent according to the process of claim 11 or 19 wherein said product is the data collected with respect to said agent as a result of said process and wherein said data is sufficient to convey the chemical structure and/or properties of said agent. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Atherosclerotic cardiovascular disease is the leading cause of death worldwide 1 . Altered lipoprotein levels are pivotal risk factors for atherosclerosis 2,3 . In particular, low HDL cholesterol (HDL-C) levels are a major independent risk factor for the development of premature coronary disease 4-6 . The anti-atherogenic function of HDL is generally attributed to its role in reverse cholesterol transport (RCT), whereby excess cholesterol is transported from peripheral cells to HDL particles for subsequent delivery to the liver 7,8 . The protein crucial for the initial step of RCT, namely ABC1, was recently identified 9-12 . Complete ABC1 deficiency is the underlying cause of Tangier disease (TD) 9,11,12 , a rare disorder associated with a near absence of HDL-C and apolipoprotein AI and with remarkably decreased cholesterol efflux from cells 13 . Clinically, TD is associated with hepatosplenomegaly, neuropathy and cholesterol ester accumulation in specific cells 13 . Individuals heterozygous for ABC1 mutations are characterized by low HDL-C levels, increased triglycerides (TG), depressed levels of cholesterol efflux and an increased risk of coronary artery disease (CAD), but have no obvious clinical manifestations of cholesterol ester accumulation 9,10,14 . Cholesterol efflux levels are highly correlated with HDL-C levels in these individuals 14 . The frequency of individuals with severe mutations in the ABC1 gene is low, but common variants having minor functional effects could be of great clinical relevance for the general population. We have previously shown that individuals heterozygous for mutations in the ABC1 gene (also called ABCA1) have decreased HDL cholesterol (HDL-C), increased triglycerides (TG) and a greater than threefold increased frequency of coronary artery disease (CAD) and that single nucleotide polymorphisms in the coding region (cSNPs) of the ABC1 gene may significantly impact plasma lipid levels and the severity of CAD in the general population. We have now identified several SNPs in non-coding regions of ABC1 that may be important for the appropriate regulation of ABC1 expression (i.e. in the promoter, intron 1 and the 5′ untranslated region (UTR)), and have examined the phenotypic effects of these SNPs in the REGRESS population. Of 12 SNPs, 4 were associated with a clinical outcome. A 3-fold increase in coronary events and an increased family history of CAD was evident for the G-191C variant. Similarly, the C69T SNP was also associated with a 2-fold increase in events. In contrast, the C-17G was associated with decreased coronary events, and the InsG319 SNP was associated with less focal and diffuse atherosclerosis. For all these SNPs, the changes in atherosclerosis and CAD occurred independent of changes in plasma lipid levels, findings which were replicated in a second cohort. These data suggest that common variation in non-coding regions of ABC1 may significantly alter the severity of atherosclerosis, without necessarily influencing plasma lipid levels. We have previously presented a complete analysis of 10 single nucleotide polymorphisms in the coding region of the ABC1 gene (cSNPs) 15 . We have shown that cSNPs of the ABC1 gene influence plasma lipid levels and the severity of CAD. Interestingly, the R219K cSNP is associated with decreased TG, increased HDL-C and a decreased severity of CAD, compatible with a gain of function, while other cSNPs were associated with more moderate effects 15 . Here, we describe 12 non-coding SNPs in potential regulatory regions and have examined the functional effects of these SNPs in the promoter, the 5′ untranslated region (UTR) and first intron. Several studies have shown that SNPs in these regions from other genes indeed have functional consequences 16-18 . We have also recently shown that sequences within the first intron of ABC1 constitute an alternate promoter with three alternate transcription start sites, and thus may have direct effects on the regulation of ABC1 (Singaraja et al, manuscript submitted). An alternate transcription start site within intron 1 has also recently been reported by another group 19 . We have now examined the phenotypic effects of these 12 non-coding SNPs in a large ethnically uniform cohort (REGRESS) and show that they indeed are associated with altered risk and severity of CAD, without associated changes in lipid and lipoprotein levels. This provides evidence that sequences in these regions are important for the proper regulation of ABC1 and suggest that changes in ABC1 regulation can alter risk for CAD presumably through influencing RCT without necessarily having an effect on lipid levels. |
<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>In one aspect, the present invention relates to a method for determining propensity toward developing a cardiovascular disease in a patient at risk of developing said disease comprising determining the presence in an ABCA1 gene of said patient of a polymorphism in the DNA sequence of said gene wherein said polymorphism is present in a non-coding region of said gene. In preferred embodiments, the polymorphism is present in the promoter region of said gene or in an intronic region. In preferred embodiments, the disease is coronary artery disease or atherosclerosis, or a disease that involves increased triglyceride or cholesterol levels, or decreased HDL-C levels, in a patient, especially in the plasma of said patient. In preferred embodiments, the disease involves decreased lipid transport in the cells of the patient, especially decreased HDL-C transport. In additional preferred embodiments, the polymorphism is a single nucleotide polymorphism, most preferably any of the polymorphisms depicted in Table 1 (SEQ ID NOS: 1-24). In another aspect, the present invention relates to method for identifying a modulator of ABCA1 polynucleotide expression comprising: (a) contacting a compound with a polynucleotide that encodes ABCA1 polypeptide, which polynucleotide comprises a polymorphism in a non-coding region of said polynucleotide, under conditions promoting said contacting and promoting expression of ABCA1 polypeptide by said polynucleotide; (b) determining the activity of said polynucleotide in expressing said ABCA1 polypeptide after said contacting wherein a difference in the expression of said polynucleotide relative to when said compound and said polynucleotide are not contacted indicates polynucleotide modulating activity, thereby identifying a modulator of ABCA1 polynucleotide expression. In a preferred embodiment, the ABCA1 polynucleotide is present in a cell, which cell then expresses the ABCA1 polypeptide and such expression is readily measured, such as by measuring lipid transport across the membrane of the cell whereby an increase in transport shows increased expression of the polypeptide. Thus, in a preferred embodiment, the difference in expression in step (b) is an increase in expression. Preferably, the polymorphism is present in an intronic region or promoter region, or some other non-coding region, such as an enhancer region, of the polynucleotide. In a preferred embodiment, the polymorphism is a single nucleotide polymorphism (SNP), most preferably one of the SNPs shown in Table 1 (SEQ ID NOS: 1-24). Such polymorphisms may also have the effect of decreasing the activity of said polynucleotide. In a further aspect, the present invention relates to a method for identifying an agent that modulates plasma lipid levels comprising administering to an animal an effective amount of a compound first identified as an ABCA1 modulator using a screening method as disclosed herein. In preferred embodiments thereof, the compound has the effect of reducing plasma triglyceride levels, reducing plasma cholesterol levels, or increasing plasma HDL-C levels. In an additional aspect, the present invention relates to a method of treating a patient for cardiovascular disease comprising administering to a patient afflicted therewith of an effective amount of a compound first identified as an ABCA1 modulator using a screening method as disclosed herein. In preferred embodiments, the disease is coronary artery disease or atherosclerosis. In yet a further aspect, the present invention relates to a method of protecting a patient against developing cardiovascular disease comprising administering to a patient at risk thereof of an effective amount of a compound first identified as an ABCA1 modulator using the method as disclosed herein. In preferred embodiments thereof, the disease is coronary artery disease or atherosclerosis. |
Method for the diagnosis and therapy of renal cell carcinoma |
The invention relates to a novel approach for the diagnosis and therapy of renal cell carcinoma, and other renal tumors, for example Wilms' tumor or other tumors not originating from the kidney or non-malignant kidney pathologies. The areas of application of the invention include the field of medicine and the pharmaceutical industry but also basic biology. The aim of the invention is to provide novel forms of treatment which are urgently required regarding the present state-of-the-art in treatment of renal carcinoma and other cancers. It was discovered that the Nbk protein which is highly expressed in normal renal tissue, is either not expressed at all or only weakly expressed in the tumor tissue. The protein expression and the loss of Nbk was examined in renal cell carcinoma samples by the use of immunohistochemistry, PCR strategies, mutation and deletion analyses. The inventive method for diagnosis is characterized by that the Nbk protein concentration or the Nbk RNA quantity or Nbk mutation or deletion or Nbk gene modification, preferentially methylation, in tissue-derived material is determined. The inventive agent for the therapy of the renal carcinoma and other cells with low Nbk protein or RNA expression is that Nbk expression is increased and directly initiates the therapeutic effect in these cells. The implementation of a novel agent in non-tumorous kidney diseases is based on the inhibition of Nbk RNA or protein expression or Nbk activity. |
1. Method for the diagnosis of renal carcinoma and other kidney tumors including the wilms' tumor and other tumors, characterized by a measurement of the Nbk concentration in the tissue at the protein or RNA level. 2. Method according to claim 1, characterized by an analysis for Nbk gene alterations such as mutations and polymorphisms, preferentially by DNA sequence analysis and measurement of the Nbk concentration of the protein or RNA level. 3. Agent for the treatment of renal carcinoma and other renal tumors, for example the wilms' tumor, and other tumors not originating from the kidney characterized by causing an increase in the Nbk protein concentration in renal or other tissues and thereby mediating a therapeutic effect. 4. Agent according to claim 3, characterized by that it increases Nbk protein expression in consequence of a transfer of Nbk cDNA or the entire Nbk gene or parts of it. 5. Agent according to claims 3 and 4, characterized by that it increases the Nbk protein expression by use of viral or non-viral expression vectors. 6. Agent according to claims 3 to 5, characterized by that the Nbk protein expression is increased by use of a conditional adenoviral expression vector, preferentially by use of a vector as depicted FIG. 6, constructed by cloning the expression cassette in the plasmid pAD3 as shown in FIG. 3. 7. Agent according to claim 3, characterized by tissue specific expression of Nbk by preferential use of tissue specific promoters or other, e.g. hormonal or other pharmacological regulators. 8. Agent according to claim 3, characterized by tissue specific expression of Nbk that is achieved by the use of chimeric molecules consisting of Nbk and components of transcription factors or signal peptides at the level of the Nbk gene, the RNA or the protein. 9. Agent according to claim 3, characterized by the property to induce re-expression of downregulated endogenous Nbk, e.g. by use of pharmacological stimulation of Nbk protein expression by activating the gene expression and by activating regulation of the Nbk promotor. 10. Agent according to claim 3, characterized by increasing the Nbk protein expression by stabilisation of the Nbk protein expression, e.g. by interference with Nbk degradation. 11. Agent according to claim 3, characterized by that the Nbk activity is increased or inhibited by activation or inhibition of regulatory parts of the Nbk protein. 12. Agent for the therapy of hereditary or somatic renal diseases such as degenerative kidney disease, infectious and non-infectious inflammable or toxic renal damage in which cells die by apoptotic cell death, characterizied by that inhibitors of the expression or activity of Nbk protein or Nbk RNA are brought into renal cells. 13. Agent according to claim 12, characterized by that the activity of the Nbk protein or the Nbk RNA is decreased by activation or inhibition of regulatory parts of the Nbk gene. 14. Agent according to claim 12, characterized by that the activity of the Nbk protein or Nbk RNA is decreased by activation or inhibition of downstream signaling pathways. |
Method for patterning of three-dimensional surfaces |
In a method for patterning of three-dimensional surfaces, the paterns is first formed on a medium, from which it is transferred to the surface of an object to be patterned. The method comprises the following steps: a) providing a medium (1) having a release layer (1b), b) treating the release layer (1b) on the surface of the medium (1) with an adhesion promoter, c) forming a pattern (2) onto the surface of the treated medium (1), on top of the adhesion promoter, d) separating the pattern (2) from the medium (1) at the release layer (1b) in such a way that the adhesion promoter remains attached to the pattern (2), and e) placing the pattern (2) onto the three-dimensional surface of an object (4), with the adhesion promoter against the surface of the object (4). |
1. A method for patterning of three-dimensional surfaces, in which the image is first formed on a medium, from which it is transferred to the surface of the object to be patterned so that the image is visible on the surface of the object, the method comprising: a) providing a medium having a release layer or release surface, b) treating the release layer or release surface on top of the medium with a material which forms a film, c) forming a pattern onto the surface of the medium, on top of the film, d) separating the pattern from the medium at the release layer or release surface in such a way that the film remains attached to the pattern, and e) placing the pattern on the three-dimensional surface of an object by means of an elastic-plastic layer formed by an adhesion promoter, which comes against the surface of the object. 2. A The method according to claim 1, wherein the release layer or release surface on the surface of the medium is treated with an adhesion promoter which forms a film. 3. The method according to claim 1, wherein the film, onto which the pattern is formed, is of an elastic-plastic material. 4. The method according to claim 3, wherein the adhesion promoter is of an elastic-plastic hot melt adhesive, hot seal coating, or (pressure sensitive adhesive). 5. The method according to claim 3, wherein the pattern is formed of an elastic-plastic ink. 6. The method according to claim 1, wherein before the separation of the pattern from the medium, a protective film is applied on the pattern to facilitate the mechanical treatment of the pattern. 7. The method according to claim 1, wherein the release layer of the medium consists of a water-soluble material, such as a water-soluble polymer. 8. The method according to claim 1, wherein the release layer or release surface of the medium consists of a material, from whose surface the film can be mechanically detached, such as silicone or a corresponding material forming a low-energy surface. 9. The method according to claim 1, wherein the fixing of the layer formed by the adhesion promoter to the surface of the object is promoted by means of heat. 10. The method according to claim 1, wherein the surface of the object to be patterned is curved in two sectional planes perpendicular to each other and the surface. 11. The method according to claim 10, wherein the object to be patterned is the cover of an electronic device, such as the cover of a mobile phone. 12. The method according to claim 1, wherein the method also comprises the forming of an image file corresponding to the pattern by a data processing technique before the image is formed on the surface of the medium. |
Polymorphisms in the human gene for cytochrome p450 polypeptide 2c8 and their use in diagnostic and therapeutic applications |
The present invention relates to a polymorphic CYP2C8-polynucleotide. Moreover, the invention relates to genes or vectors comprising the polynucleotides of the invention and to a host cell genetically engineered with the polynucleotide or gene of the invention. Further, the invention relates to methods for producing molecular variant polypeptides or fragments thereof, methods for producing cells capable of expressing a molecular variant polypeptide and to a polypeptide or fragment thereof encoded by the polynucleotide or the gene of the invention or which is obtainable by the method or from the cells produced by the method of the invention. Furthermore, the invention relates to an antibody which binds specifically the polypeptide of the invention. Moreover, the invention relates to a transgenic non-human animal. The invention also relates to a solid support comprising one or a plurality of the above mentioned polynucleotides, genes, vectors, polypeptides, antibodies or host cells. Furthermore, methods of identifying a polymorphism, identifying and obtaining a prodrug or drug or an inhibitor are also encompassed by the present invention. In addition, the invention relates to methods for producing of a pharmaceutical composition and to methods of diagnosing a disease. Further, the invention relates to a method of detection of the polynucleotide of the invention. Furthermore, comprised by the present invention are a diagnostic and a pharmaceutical composition. Even more, the invention relates to uses of the polynucleotides, genes, vectors, polypeptides or antibodies of the invention. Finally, the invention relates to a diagnostic kit. |
1. A polynucleotide comprising a polynucleotide selected from the group consisting of: (a) a polynucleotide having the nucleic acid sequence of SEQ ID NO: 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 183, 189, 192, 195, 198, 201, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 306, 309, 318, 321, 324, 327, 330, 333, 342, 345, 348, 351, 354, 357, 360, 363, 366, 369, 384, 387, 390, 393, 396 or 399; (b) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 6, 8, 10, 12, 18, 377, 379 or 381; (c) a polynucleotide capable of hybridizing to a CYP2C8 gene, wherein said polynucleotide is having at a position corresponding to position 411, 560, 713, 817, 824, 831, 879, 886, 1058, 1627, 1668, 1767, 1887, 1905 or 1952 (GenBank accession No: AF136830.1), at a position corresponding to position 171 or 258 (GenBank accession No: AF136832.1), at a position corresponding to position 122, 150, 182, 334, 339 or 378 (GenBank accession No: AF136833.1), at a position corresponding to position 162, 163, 243 (GenBank accession No: AF136834.2) or at position 583 (GenBank accession No: NM—000770.1), at a position corresponding to position 13 or 180 (GenBank accession No: AF136835.1), at a position corresponding to position 116, 132, 172 or 189 (GenBank accession No: AF136836.1), at a position corresponding to position 42 or 101 (GenBank accession No: AF136837.1), at a position corresponding to position 309 (GenBank accession No: AF136838.1), at a position corresponding to position 1135 (GenBank accession No: NM—000770.1), at a position corresponding to position 232 (GenBank accession No: AF136840.1), at a position corresponding to position 206 (GenBank accession No: AF136842.1), at a position corresponding to position 30, 87, 167, 197, 212, 221, 255 or 271 (GenBank accession No: AF136843.1), at a position corresponding to position 118 (GenBank accession No: AF136844.1), at a position corresponding to position 44 (GenBank accession No: AF136845.1) of the cytochrome 2C8 gene (GenBank accession No: GI: 13787189) a nucleotide substitution, at a position corresponding to position 306 to 307, 1271 to 1273 or 1397 to 1398 of the CYP2C8 gene (GenBank accession No: AF136830.1), at a position corresponding to position 329 of the CYP2C8 gene (GenBank accession No: AF136833.1), at a position corresponding to position 87 of the CYP2C8 gene (GenBank accession No: AF136834.2) a deletion of one or more nucleotides or at a position corresponding to position 1785/1786 of the CYP2C8 gene (GenBank accession No: AF136830.1) or at a position corresponding to positionl80/181 of the CYP2C8 gene (GenBank accession No: AF136833.1) an insertion of one or more nucleotides; (d) a polynucleotide capable of hybridizing to a CYP2C8 gene, wherein said polynucleotide is having at a position corresponding to position 411, 817, 824, 831, 879, 1058, 1767 or 1887 of the CYP2C8 gene (GenBank accession No: AF136830.1) an A, at a position corresponding to position 560 or 1668 of the CYP2C8 gene (GenBank accession No: AF136830.1) a G, at a position corresponding to position 713 or 886 of the CYP2C8 gene (GenBank accession No: AF1.36830.1) a T, at a position corresponding to position 1627, 1905 or 1952 of the CYP2C8 gene (GenBank accession No: AF136830.1) a C, at a position corresponding to position 258 of the CYP2C8 gene (GenBank accession No: AF136832.1) a T, at a position corresponding to position 171 of the CYP2C8 gene (GenBank accession No: AF136832.1) a C, at a position corresponding to position 122, 150 or 334 of the CYP2C8 gene (GenBank accession No: AF136833.1) an A, at a position corresponding to position 182 or 378 of the CYP2C8 gene (GenBank accession No: AF136833.1) a C, at a position corresponding to position 162, 163, 243 [identical to position corresponding to position 583 of the CYP2C8 gene (GenBank accession No: NM—000770.1) of the CYP2C8 gene (GenBank accession No: AF136834.2) an A, at a position corresponding to position 180 of the CYP2C8 gene (GenBank accession No: AF136835.1) an A, at a position corresponding to position 13 of the CYP2C8 gene (GenBank accession No: AF136835.1) a G, at a position corresponding to position 116 or 132 of the CYP2C8 gene (GenBank accession No: AF136836.1) a G, at a position corresponding to position 172 of the CYP2C8 gene (GenBank accession No: AF136836.1) a G, at a position corresponding to position 189 of the CYP2C8 gene (GenBank accession No: AF136836.1) a C, at a position corresponding to position 42 or 101 of the CYP2C8 gene (GenBank accession No: AF136837.1) a G, at a position corresponding to position 1135 of the CYP2C8 gene (GenBank accession No: GI: 13787189) an A, at a position corresponding to position 309 of the CYP2C8 gene (GenBank accession No: AF136838.1) a T, at a position corresponding to position 232 (GenBank accession No: 136840.1) a T, at a position corresponding to position 30 or 212 of the CYP2C8 gene (GenBank accession No: AF136843.1) a T, at a position corresponding to position 87 of the CYP2C8 gene (GenBank accession No: AF136843.1) a G, at a position corresponding to position 167 or 197 of the CYP2C8 gene (GenBank accession No: AF136843.1) an A, at a position corresponding to position 221, 255 or 271 of the CYP2C8 gene (GenBank accession No: AF136843.1) a C, at a position corresponding to position 118 of the CYP2C8 gene (GenBank accession No: AF136844.1) an A, at a position corresponding to position 44 of the CYP2C8 gene (GenBank accession No: AF136845.1) a T; (e) a polynucleotide encoding a molecular CYP2C8 variant polypeptide or fragment thereof, wherein said polypeptide comprises an amino acid substitution at a position corresponding to any one of position 159, 181, 209, 244, 263, 274, 343 or 365 of the CYP2C8 polypeptide (GI: 13787189); and (f) a polynucleotide encoding a molecular CYP2C8 variant polypeptide or fragment thereof, wherein said polypeptide comprises an amino acid substitution of T to P at position corresponding to position 159 (frameshift), V to I at a position corresponding to position 181, N to S at a position corresponding to position 209, I to V at a position corresponding to position 244, F to L at a position corresponding to position 263, E to Stop at a position corresponding to position 274, G to S at a position corresponding to position 365 or S to I at a position corresponding to position 343 of the CYP2C8 polypeptide (GenBank accession No: GI: 13787189). 2. A polynucleotide of claim 1, wherein said polynucleotide is associated with an incompatibility or disease related to arachidonic acid metabolism, cancer, or cardiovascular diseases. 3. A polynucleotide of claim 1 which is DNA or RNA. 4. An isolated gene comprising the polynucleotide of claim 1. 5. The gene of claim 4, wherein a nucleotide deletion, addition and/or substitution results in altered expression of the variant gene compared to the corresponding wild type gene. 6. A vector comprising a polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene. 7. The vector of claim 6, wherein the polynucleotide is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells or isolated fractions thereof. 8. A host cell genetically engineered with the polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, or a vector comprising said polynucleotide, said isolated gene or said variant gene. 9. A method for producing a molecular variant CYP2C8 polypeptide or fragment thereof comprising (a) culturing the host cell of claim 8; and (b) recovering said protein or fragment thereof from the culture. 10. A method for producing cells capable of expressing a molecular variant CYP2C8 polypeptide comprising genetically engineering cells with the polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, or a vector comprising said polynucleotide, said isolated gene or said variant gene gene. 11. A polypeptide or fragment thereof encoded by the polynucleotide of claim 1 an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene. 12. An antibody which binds specifically to the polypeptide of claim 11. 13. An antibody which specifically recognizes an epitope containing one or more amino acid substitution(s) resulting from a nucleotide exchange as defined in claim 1. 14. The antibody of claim 12 which is monoclonal or polyclonal antibody. 15. A transgenic non-human animal comprising at least one polynucleotide of claim 1 an isolated gene comprising said polynucleotide, said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, or a vector comprising said polynucleotide, said isolated gene or said variant gene. 16. The transgenic non-human animal of claim 15 which is a mouse, a rat or a zebrafish. 17. A solid support comprising one or a plurality of the polynucleotide of claim 1, an isolated gene comprising said polynucleotide, said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, a vector comprising said polynucleotide, said isolated gene or said variant gene, a polypeptide or fragment thereof encoded by the polynucleotide, an antibody which specifically binds to the polypeptide or fragment thereof, and a host cell genetically engineered with the polynucleotide in immobilized form. 18. The solid support of claim 17, wherein said solid support is a membrane, a glass-or polypropylene- or silicon-chip, oligonucleotide-conjugated beads or a bead array, which is assembled on an optical filter substrate. 19. An in vitro method for identifying a single nucleotide polymorphism said method comprising: (a) isolating a polynucleotide of claim 1, an isolated gene comprising said polynucleotide, said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene from a plurality of subgroups of individuals, wherein one subgroup has no prevalence for a CYP2C8 associated disease and at least one or more further subgroup(s) do have prevalence for a CYP2C8 associated disease; and (b) identifying a single nucleotide polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having no prevalence for a CYP2C8 associated disease with said at least one or more further subgroup(s) having a prevalence for a CYP2C8 associated disease. 20. A method for identifying and obtaining a pro-drug or a drug capable of modulating the activity of a molecular variant of a CYP2C8 polypeptide comprising: (a) contacting a polypeptide encoded by the polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, a solid support comprising one or a plurality of the polynucleotide of claim 1, an isolated gene comprising said polynucleotide, said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, a vector comprising said polynucleotide, said isolated gene or said variant gene, a polypeptide or fragment thereof encoded by the polynucleotide, an antibody which specifically binds to the polypeptide or fragment thereof, and a host cell genetically engineered with the polynucleotide in immobilized form, a cell expressing a molecular variant gene comprising a polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, or a vector comprising said polynucleotide, said isolated gene or said variant gene, in the presence of components capable of providing a detectable signal in response to drug activity with a compound to be screened for pro-drug or drug activity; and (b) detecting the presence or absence of a signal or increase or decrease of a signal generated from the pro-drug or the drug activity, wherein the absence, presence, increase or decrease of the signal is indicative for a putative pro-drug or drug. 21. A method for identifying and obtaining an inhibitor of the activity of a molecular variant of a CYP2C8 polypeptide comprising: (a) contacting a polypeptide encoded by the polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, a solid support comprising one or a plurality of the polynucleotide of claim 1, an isolated gene comprising said polynucleotide, said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, a vector comprising said polynucleotide, said isolated gene or said variant gene, a polypeptide or fragment thereof encoded by the polynucleotide, an antibody which specifically binds to the polypeptide or fragment thereof, and a host cell genetically engineered with the polynucleotide in immobilized form, a cell expressing a molecular variant gene comprising a polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, or a vector comprising said polynucleotide, said isolated gene or said variant gene, in the presence of components capable of providing a detectable signal in response to drug activity with a compound to be screened for inhibiting activity; and (b) detecting the presence or absence of a signal or increase or decrease of a signal generated from the inhibiting activity, wherein the absence or decrease of the signal is indicative for a putative inhibitor. 22. The method of claim 20, wherein said cell is a host cell genetically engineered with the polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, or a vector comprising said polynucleotide, said isolated gene or said variant gene, or obtained by a transgenic non-human animal comprising the polynucleotide. 23. A method of identifying and obtaining a pro-drug or drug capable of modulating the activity of a molecular variant of a CYP2C8 polypeptide comprising the steps of: (a) contacting a host cell, genetically engineered with the polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, or a vector comprising said polynucleotide, said isolated gene or said variant gene, the polypeptide encoded by the polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, or the solid support comprising one or a plurality of the polynucleotide of claim 1, an isolated gene comprising said polynucleotide, said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, a vector comprising said polynucleotide, said isolated gene or said variant gene, a polypeptide or fragment thereof encoded by the polynucleotide, an antibody which specifically binds to the polypeptide or fragment thereof, and a host cell genetically engineered with the polynucleotide in immobilized form with the first molecule known to be bound by a CYP2C8 polypeptide to form a first complex of said polypeptide and said first molecule; (b) contacting said first complex with a compound to be screened, and (c) measuring whether said compound displaces said first molecule from said first complex. 24. A method of identifying and obtaining an inhibitor capable of modulating the activity of a molecular variant of a CYP2C8 polypeptide or its gene product comprising the steps of: (a) contacting a host cell, genetically engineered with the polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, or a vector comprising said polynucleotide, said isolated gene or said variant gene, the polypeptide encoded by the polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene or the solid support comprising one or a plurality of the polynucleotide of claim 1, an isolated gene comprising said polynucleotide, said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, a vector comprising said polynucleotide, said isolated gene or said variant gene, a polypeptide or fragment thereof encoded by the polynucleotide, an antibody which specifically binds to the polypeptide or fragment thereof, and a host cell genetically engineered with the polynucleotide in immobilized form with the first molecule known to be bound by a CYP2C8 polypeptide to form a first complex of said polypeptide and said first molecule; (b) contacting said first complex with a compound to be screened, and (c) measuring whether said compound displaces said first molecule from said first complex. 25. The method of claim 23, wherein said measuring step comprises measuring the formation of a second complex of said polypeptide and said compound. 26. The method of claim 23, wherein said measuring step comprises measuring the amount of said first molecule that is not bound to said polypeptide. 27. The method of claim 23, wherein said first molecule is labeled. 28. A method for the production of a pharmaceutical composition comprising the steps of the method of claim 23; and the further step of formulating the compound identified and obtained or a derivative thereof in a pharmaceutically acceptable form. 29. A method of diagnosing a disorder related to the presence of a molecular variant of a CYP2C8 gene or susceptibility to such a disorder comprising determining the presence of a polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene in a sample from a subject. 30. The method of claim 29 further comprising determining the presence of a polypeptide encoded by the polynucleotide, the isolated gene or variant gene or the antibody which binds specifically to the polypeptide. 31. A method of diagnosing a disorder related to the presence of a molecular variant of a CYP2C8 gene or susceptibility to such a disorder comprising determining the presence of a polypeptide of claim 11 or the antibody which binds specifically to the polypeptide. 32. The method of claim 29, wherein said disorder is a incompatibility or a disease related to arachidonic acid metabolism, cancer or cardiovascular diseases. 33. The method of claim 29 comprising PCR, ligase chain reaction, restriction digestion, direct sequencing, nucleic acid amplification techniques, hybridization techniques or immunoassays. 34. A method of detection of a polynucleotide in a sample comprising the steps of (a) contacting the solid support comprising one or a plurality of the polynucleotide of claim 1, an isolated gene comprising said polynucleotide, said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, a vector comprising said polynucleotide, said isolated gene or said variant gene, a polypeptide or fragment thereof encoded by the polynucleotide, an antibody which specifically binds to the polypeptide or fragment thereof, and a host cell genetically engineered with the polynucleotide in immobilized form with the sample under conditions allowing interaction of the polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene with the immobilized targets on a solid support and (b) determining the binding of said polynucleotide or said gene to said immobilized targets on a solid support. 35. An in vitro method for diagnosing a disease comprising the steps of the method of claim 34, wherein binding of said polynucleotide or gene to said immobilized targets on said solid support is indicative for the presence or the absence of said disease or a prevalence for said disease. 36. A diagnostic composition comprising the polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, or a vector comprising said polynucleotide, said isolated gene or said variant gene, or a vector comprising said polynucleotide, said isolated gene or said variant gene, a polypeptide or the antibody of any one of the claims 12 to 14. 37. A pharmaceutical composition comprising the polynucleotide encoded by the polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, or a vector comprising said polynucleotide, said isolated gene or said variant gene, the polypeptide encoded by the polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, or a vector comprising said polynucleotide, said isolated gene or said variant gene or the antibody which binds specifically to the polypeptide. 38.-40. (canceled) 41. A diagnostic kit for detection of a single nucleotide polymorphism comprising the polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, or a vector comprising said polynucleotide, said isolated gene or said variant gene, the polypeptide encoded by the polynucleotide of claim 1, an isolated gene comprising said polynucleotide or said isolated gene further comprising a nucleotide deletion, addition and/or substitution resulting in altered expression of the variant gene compared to the corresponding wild type gene, or a vector comprising said polynucleotide, said isolated gene or said variant gene or the antibody which binds specifically to said polypeptide. |
Method for determining an analyte |
The present invention relates to a method for determining an analyte in a sample. According to said method, a probe is transferred from a stamp member onto an analyte that is bound to a support member. The invention is particularly suitable for microarrays. The invention also relates to an apparatus for performing said method. |
1. Method for determining an analyte in a sample, characterized in that a sample that might contain the analyte being determined is brought into contact with a first surface (i.e. support member) so that the analyte possibly contained in the sample is bound to the support member; a second surface (i.e. stamp member) to which a probe that can bind the analyte is bound is brought closer to the support member so that the probe and the analyte can interact, the binding of the probe to the analyte and the binding of the analyte to the support member being more stable under an externally applied tensile force than the binding of the probe to the stamp member; the stamp member and support member are separated from each other; and it is determined whether the probe is bound to the support member and/or how much probe is bound to the support member. 2. Method according to claim 1, characterized in that molecules that can bind the analyte (i.e. receptors) are immobilized on the support member, the binding of the analytes to the receptor being more stable under an externally applied tensile force than the binding of the probe to the stamp member. 3. Method according to claim 2, characterized in that the receptors and/or probes are selected from the group comprising polyclonal or monoclonal antibodies, antibody fragments or antibody derivatives that can recognize and bind the analyte(s). 4. Method according to claim 3, characterized in that the receptors and/or probes used are antibody fragments or derivatives selected from Fv-, Fab-, Fab′- or F(ab′)2 fragments, “single-chain antibody fragments”, bispecific antibodies, chimeric antibodies, humanized antibodies and fragments containing CDRs (complementarity determining regions) that recognize an epitope of the analyte. 5. Method according to claim 1, characterized in that the receptors and/or probes used are nucleic acids such as DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), synthetic nucleic acids such as LNA (locked nucleic acid) and PNA (peptide nucleic acid), and three-dimensional structures made of nucleic acids, preferably aptamers. 6. Method according to claim 1, characterized in that the analyte is selected from the group comprising proteins, polypeptides, peptides, nucleic acids, modified nucleic acids, molecules similar to nucleic acids such as peptide nucleic acid (PNA) or locked nucleic acid (LNA), three-dimensional nucleic acid structures, aptamers, carbohydrates and modified variants thereof. 7. Method according to claim 6, characterized in that the analyte is a protein, polypeptide or a peptide. 8. Method according to claim 1, characterized in that the probe binds specifically to the analyte. 9. Method according to claim 1, characterized in that the probe is selected from the group comprising antibodies, antibody fragments and antibody derivatives. 10. Method according to claim 1, characterized in that the probe carries a marker. 11. Method according to claim 1, characterized in that the probe is immobilized at the stamp member by a sensor complex. 12. Method according to claim 11, characterized in that the sensor complex comprises a first binding partner that is bound to the stamp member and a second binding partner that is bound fixedly to the probe, wherein the first binding partner and the second binding partner can bind non-covalently to each other. 13. Method according to claim 11, characterized in that the first binding partner and the second binding partner are nucleic acids. 14. Method according to claim 13, characterized in that the first binding partner and the second binding partner are selected from the group comprising single-stranded DNA and/or single-stranded RNA. 15. Method according to claim 14, characterized in that the 5′ end of the first binding partner is bound to the stamp member and the 3˜ end of the second binding partner is bound to the probe, or that the 3′ end of the first binding partner is bound to the stamp member and the 5′ end of the second binding partner is bound to the probe. 16. Method according to claim 1, characterized in that the stamp member comprises a plurality of areas separated from each other, in which probes with different specificity for different analytes are bound. 17. Method according to claim 2, characterized in that the support member comprises a plurality of areas separated from each other, in which receptors with different specificity for different analytes are bound. 18. Method according to claim 16, characterized in that the support member has a plurality of areas separated from each other, in which receptors with different specificity for different analytes are bound in such a way that they are disposed symmetrically to the areas on the stamp member, so that when the stamp member and the support member approach each other, the areas on the probes and receptors that have specificity for the same analyte become aligned. 19. Method according to claim 16, characterized in that both the support member and the stamp member have areas in which receptors are immobilized, as well as areas in which probes are immobilized, the respective areas being so arranged that when the stamp member and the support member are brought into contact, areas with receptors for an analyte and areas with probes for the same analyte are positioned facing each other. 20. Method according to claim 16, characterized in that the stamp member and/or support member have sub-areas on which a plurality of spots are arranged. 21. Method according to claim 20, characterized in that the stamp member and/or support member have sub-areas with a plurality of spots, the spots on a sub-area each having different receptors or probes that can bind to an analyte. 22. Method according to claim 20, characterized in that the stamp member and/or the support member have at least one spot where receptors capable of binding directly with the probe are immobilized, said spots being used to monitor coupling. 23. Apparatus for determining an analyte in a sample, comprising two surfaces each having sub-areas to which receptors and probes capable of binding with different analytes are bound, said receptors and probes being respectively so arranged that when the two surfaces come into contact, the receptors and/or probes capable of binding with the same analyte face each other, wherein the probes are each immobilized by a sensor complex and bear a marker. 24. Apparatus according to claim 23, characterized in that the apparatus comprises a stamp member and a support member, and that a first binding partner is bound fixedly to the stamp member and a second binding partner is bound fixedly to the probe, wherein the first binding partner and the second binding partner can bind non-covalently to each other. 25. Apparatus according to claim 24, characterized in that the first binding partner and the second binding partner are nucleic acids. 26. Apparatus according to claim 25, characterized in that the first binding partner and the second binding partner are selected from the group comprising single3o stranded DNA and/or single-stranded RNA. 27. Apparatus according to claim 26, characterized in that the 5, end of the first binding partner is bound to the stamp member and the 3′ end of the second binding partner is bound to the probe, or that the 3˜ end of the first binding partner is bound to the stamp member and the 5′ end of the second binding partner is bound to the probe. 28. Apparatus according to claim 23, characterized in that the apparatus also comprises a device for bringing close together and separating the two surfaces. 29. Apparatus according to claim 23, characterized in that the apparatus also comprises a device for detecting whether and/or in what quantity the probe is bound to the support member. 30. Apparatus according to claim 29, characterized in that the apparatus has a device for detecting the marker. 31. Method according to claim 23, characterized in that the stamp member comprises a plurality of areas separated from each other, in which probes with different specificity for different analytes are bound. 32. Apparatus according to claim 23, characterized in that the support member comprises a plurality of areas separated from each other, in which receptors with different specificity for different analytes are bound. 33. Apparatus according to claim 31, characterized in that the support member has a plurality of areas separated from each other, in which receptors with different specificity for different samples are bound in such a way that they are disposed symmetrically to the areas on the stamp member, so that when the stamp member and the support member approach each other, the areas on the probes and receptors that have specificity for the same sample become aligned. 34. Apparatus according to claim 23, characterized in that the areas each have a plurality of spots on each of which different receptors and probes capable of binding with an analyte are arranged. |
Method and apparatus for magnetic field measurement |
The invention provides for measurement of an actual magnitude of an applied magnetic field, rather than providing a value of magnetic field which is relative to an unknown quiescent value. In particular, by providing a SQUID (100) having an effective area which varies in response to applied flux, an absolute value of magnetic field can be determined due to the change in effective area of the SQUID (100). |
1. A method of measurement of absolute magnitude of a magnetic field, the method comprising the steps of: providing a superconducting quantum interference device (SQUID) having an effective flux-collection area which varies with applied flux; and determining an absolute magnitude of an applied magnetic field based on variations in said effective area. 2. The method of claim 1 wherein said step of determining comprises monitoring a periodicity of an output voltage waveform of the SQUID in order to determine when a variation in the effective flux-collection area has occurred. 3. The method of claim 1 wherein said step of determining comprises the steps of recording a magnetic field value at which the effective flux-collection area alters; and determining a change of the magnetic field from said magnetic field value. 4. The method of claim 1 wherein said step of providing comprises providing a flux dam in a pick up loop of the SQUID. 5. The method of claim 4 wherein said step of determining comprises: calculating a critical value of applied magnetic field at which a current in the pick up loop is equal to a critical current of the flux-dam; and determining that an applied magnetic field is equal to the calculated critical value when a periodicity of an output voltage of the SQUID changes. 6. The method of claim 4 wherein said flux dam is provided by forming a grain boundary in the material of the pick up loop, the grain boundary being formed over a step edge. 7. The method of claim 4, wherein said step of providing the flux dam comprises controlling formation of the flux dam such that a critical current of the flux dam arises when an applied magnetic field is in a range of interest. 8. The method of claim 7, wherein said flux dam is provided by forming a grain boundary in the material of the pick up loop, the grain boundary being formed over a step edge, and wherein formation of the flux dam is controlled by controlling a step height and a step angle of the step edge. 9. A superconducting quantum interference device for measurement of absolute magnitude of a magnetic field, the device having an effective flux-collection area which varies with applied flux. 10. The SQUID of claim 9, wherein the effective flux-collection area comprises a pick-up loop, and wherein a flux dam is provided in the pick up loop such that the effective area of the SQUID changes when a current in the pick up loop exceeds the critical current of the flux dam. 11. The SQUID of claim 10 wherein the critical current of the flux dam arises when an applied magnetic field is in a range of interest for an intended application of the SQUID. 12. The SQUID of claim 9, wherein the SQUID comprises a superconducting ring of HTS material interrupted by a Josephson Junction. 13. The SQUID of claim 12 wherein the Josephson Junction is implemented by formation of a grain boundary in the HTS material. 14. The SQUID of claim 13 wherein the Josephson Junction is formed over a step-edge in a substrate. 15. The SQUID of claim 13 wherein the Josephson Junction is formed by one of a microbridge, an ion-irradiated link, a superconductor-insulator-superconductor (SIS) junction, and a superconductor-normal metal-superconductor (SNS) junction. 16. The SQUID of claim 10 wherein the flux-dam is implemented by forming a grain boundary at a step edge in a substrate. 17. The SQUID of claim 10 wherein the flux dam is implemented by use of a microbridge. 18. The SQUID of claim 9 wherein the SQUID is an rf-SQUID. 19. The SQUID of claim 9 wherein the SQUID is a dc-SQUID. 20. A method of measurement of absolute value of a magnetic field, the method comprising the steps of: providing a pick-up loop for a SQUID, the pick-up loop having a flux dam having a critical current, the critical current occurring in the pick-up loop when a critical magnetic field is applied to the SQUID; and determining an absolute value of an applied magnetic field by comparison to said critical magnetic field. 21. The method of claim 20 further comprising the step of fabricating the flux-dam such that the critical magnetic field is in a magnetic field range of interest. 22. The method of claim 21, wherein the flux dam is fabricated by forming a grain boundary in the material of the pick-up loop, the grain boundary being formed over a step edge in a substrate. 23. The method of claim 21 wherein the flux dam is fabricated by forming by a microbridge. 24. A pick-up loop for a SQUID for measurement of absolute value of a magnetic field, the pick-up loop having a flux dam having a critical current, the critical current arising when a critical magnetic field is applied to the SQUID, and the flux dam being formed such that the critical magnetic field is in a magnetic field range of interest. 25. The pick-up loop of claim 24, wherein the flux dam comprises a grain boundary formed over a step edge in a substrate. 26. The pick up loop of claim 25, wherein an angle and height of the step edge serve to control the critical current of the flux dam to be in the magnetic field range of interest. 27. The pick-up loop of claim 24 wherein the flux dam comprises a microbridge. |
<SOH> BACKGROUND ART <EOH>Superconducting Quantum Interference Devices (SQUIDs) are often used as highly sensitive magnetic field sensors. Such SQUID sensors are becoming increasingly popular due to the capabilities of high sensitivity sensing in areas such as geophysical mineral prospecting and biological magnetic field detection, such as magnetic field emanations from the human brain. With the advent of high critical temperature superconducting (HTS) materials such as YBa 2 Cu 3 O x (YBCO), HTS-SQUIDs can be cooled by relatively inexpensive liquid nitrogen, and can be made in a compact form. The HTS radio frequency (rf) SQUID is essentially a superconducting ring made of YBCO or the like, the ring being interrupted by a Josephson Junction or weak link. When the superconducting ring is energised by an inductively coupled resonant rf-oscillator, tunnelling of electrons takes place at the junction and a periodic signal, being a function of flux through the ring, can be detected across the junction. The periodic signal is substantially a triangular waveform, usually having a period (ΔB) in the order of a nanotesla. Therefore, in order to yield a sensitivity in the femtotesla range, the SQUID is operated in a nulling bridge mode, or flux locked loop (FLL) mode. In this mode, magnetic flux is fed back to the SQUID so as to cause the output voltage to remain relatively constant. The feedback voltage, being proportional to the difference between the applied flux and the quiescent flux level, gives a highly accurate measurement of relative magnetic flux. The feedback voltage V can therefore be written as in-line-formulae description="In-line Formulae" end="lead"? V=M ( A eff B+u ) (1) in-line-formulae description="In-line Formulae" end="tail"? where M is a constant in a specific SQUID system; A eff is the effective area of the SQUID; B is the applied magnetic field; and u is the quiescent flux. However, since the quiescent flux u is unknown, SQUIDs provide only relative measurements of magnetic field, and do not provide a measurement of an absolute magnitude of magnetic field. Further, when the applied flux changes too quickly, at a rate which is greater than the “slew rate” of the SQUID, the loop loses lock, and a discontinuous output results. Due to the periodic nature of the SQUID response, it is not possible to determine from the output whether the SQUID has regained lock at a same position in the periodic waveform, and thus such interrupted results are of limited use. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Throughout this specification, the terms ‘superconducting material’, ‘superconducting device’ and the like are used to refer to a material or device which, in a certain state and at a certain temperature, is capable of exhibiting superconductivity. The use of such terms does not imply that the material or device exhibits superconductivity in all states or at all temperatures. |
<SOH> SUMMARY OF THE INVENTION <EOH>According to a first aspect the present invention resides in a method of measurement of absolute magnitude of a magnetic field, the method comprising the steps of: providing a superconducting quantum interference device having an effective flux-collection area which varies with applied flux; and determining an absolute magnitude of an applied magnetic field based on variations in said effective area. According to a second aspect, the present invention provides a superconducting quantum interference device for measurement of absolute magnitude of a magnetic field, the device having an effective flux-collection area which varies with applied flux. It has been realised that periodicity of the output voltage function of a SQUID relies on the effective area of the SQUID. Accordingly, providing a SQUID with an effective area which alters or varies at one or more known absolute values of flux density, enables the SQUID to detect when the one or more known flux densities are applied, due to the changing periodicity of the output voltage of the SQUID at those flux densities. Hence, absolute magnetic field values may be measured by the SQUID. Further, the absolute value of an applied flux which is different to the one or more known absolute values of flux may be determined with reference to the one or more known flux densities. Accordingly, the method and device of the present invention allow measurement of the absolute value of an applied field to be measured, at least when the strength of that field is in the vicinity of the one or more known flux values to allow comparison to the one or more known flux values. It has further been realised that Provision of a flux-dam in the pick-up loop of a SQUID is an effective manner in which to provide a SQUID having an effective area which varies with applied flux. In such embodiments, the flux-dam ‘opens’ and ‘closes’, depending on whether the circulating current in the pick-up loop is greater than or less than the critical current of the flux-dam. That is, the flux-dam becomes resistive when the circulating current in the pick-up loop exceeds the critical current of the flux-dam. As the circulating current is caused by applied flux, there exists a critical (and calculable) value of applied magnetic field at which the flux-dam becomes resistive. At that point, the flux-dam becomes resistive, causing the effective area of the SQUID to change, and so the periodicity of the output voltage of the SQUID changes, enabling the absolute value of the applied magnetic field to be measured. The absolute value of an applied magnetic field of different magnitude to the critical magnetic field may be determined by reference to the critical magnetic field. Accordingly, in a third aspect the present invention resides in a method of measurement of absolute value of a magnetic field, the method comprising the steps of: providing a pick-up loop for a SQUID, the pick-up loop having a flux dam having a critical current, the critical current occurring in the pick-up loop when a critical magnetic field is applied to the SQUID; and determining an absolute value of an applied magnetic field by comparison to said critical magnetic field. The method of the third aspect of the present invention may further comprise the step of fabricating the flux-dam such that the critical magnetic field is in a magnetic field range of interest. According to a fourth aspect, the present invention resides in a pick-up loop for a SQUID for measurement of absolute value of a magnetic field, the pick-up loop having a flux dam having a critical current, the critical current arising when a critical magnetic field is applied to the SQUID, and the flux dam being formed such that the critical magnetic field is in a magnetic field range of interest. The SQUID may comprise a superconducting ring of HTS material, such as YBCO, interrupted by a Josephson Junction. The Josephson Junction may be implemented by formation of a grain boundary in the HTS material, for example by forming the junction over a step-edge in a substrate. The step edge could, for example, be formed in accordance with the teachings of International Patent Publication No. WO 00/16414, the contents of which are incorporated herein by reference. Of course, the Josephson Junction may be formed in a different manner, for example by use of a microbridge, an ion-irradiated link, a superconductor-insulator-superconductor (SIS) junction, a superconductor-normal metal-superconductor (SNS) junction or the like. Similarly, where a flux-dam is used to provide an effective area dependent on flux, the flux dam may be implemented by forming a grain boundary at a step edge in a substrate, or by use of a microbridge, or the like. Further, it will be appreciated that the present invention is applicable to both rf-SQUIDs and dc-SQUIDs. |
Uniform broad ion beam deposition |
Apparatus for the generation of a plurality of ion beams for use in vacuum sputtering methods is disclosed comprising: a discharge chamber, defined by a plasma confinement vessel, for generation of a plasma therein; and a plurality of facets located on the discharge chamber, each facet comprising acceleration and extraction means for extracting ions from the plasma in the discharge chamber in an ion beam. |
1. Apparatus for the generation of a plurality of ion beams, comprising: a discharge chamber, defined by a plasma confinement vessel, for generation of a plasma therein; and a plurality of facets located on the discharge chamber, each facet comprising a respective acceleration and extraction means for extracting ions from the plasma in the discharge chamber in an ion beam. 2. Apparatus according to claim 1, wherein the accelerator has two facets to produce two ion beams. 3. Apparatus according to claim 1, wherein the accelerator has a least three facets to produce at least three ion beams. 4. Apparatus according to claim 1, wherein each facet includes an array of apertures such that each ion beam is made up of a plurality of beamlets. 5. Apparatus according to claim 1, wherein each facet is pivotably mounted on the discharge chamber. 6. Apparatus according to claim 1, including means by which each ion beam may be steered to illuminate a discrete area of the target. 7. Apparatus according to claim 1, including means by which each ion beam may be steered to illuminate an area of the target which abuts at least one area of the target illuminated by a further ion beam. 8. Apparatus according to claim 1, including means by which the ion beams are steered to illuminate overlapping areas of the target. 9. Apparatus according to claim 1, additionally including r.f. plasma generation means. 10. Vacuum sputtering apparatus capable of depositing multi-layer materials on a substrate comprising: a vacuum chamber; target support means within the vacuum chamber for supporting at least one deposition target; a substrate table within the vacuum chamber for supporting a substrate oriented such that material sputtered from the at least one deposition target is deposited on the substrate when supported by the substrate table; and apparatus for the generation of a plurality of ion beams in accordance with claim 1, arranged so as to project the plurality of ion beams into the vacuum chamber so as to illuminate a respective area of the deposition target. 11. Apparatus according to claim 10 wherein the substrate table is rotatable and the apparatus includes means for rotating the substrate table. 12. Apparatus according to claim 10, additionally including a neutraliser. 13. A method of depositing multi-layer materials on a substrate by vacuum sputtering comprising: a) providing a substrate supported by a rotatable substrate table and a deposition target within a vacuum chamber, the substrate table, the substrate and the deposition target being oriented relative to each other such that material sputtered from the deposition target is deposited on the substrate; b) illuminating the target with a plurality of deposition target illuminating ion beams, from ion beam generating apparatus according to claim 1 so as to cause material to be sputtered from the deposition target; and c) rotating the substrate while the deposition target is illuminated by the deposition target illuminating ion beams. 14. A method according to claim 13, wherein the thin film deposited on the substrate has a level of non-uniformity of at most ±0.01%. 15. Apparatus according to claim 2 wherein each facet includes an array of apertures such that each ion beam is made up of a plurality of beamlets. 16. Apparatus according to claim 3, wherein each facet includes an array of apertures such that each ion beam is made up of a plurality of beamlets. 17. Apparatus according to claim 2, wherein each facet is pivotably mounted on the discharge chamber. 18. Apparatus according to any one of claim 3, wherein each facet is pivotably mounted on the discharge chamber. 19. Apparatus according to claim 4, wherein each facet is pivotably mounted on the discharge chamber. 20. Apparatus according to claim 2, including means by which each ion beam may be steered to illuminate a discrete area of the target. 21. Apparatus according to claim 3, including means by which each ion beam may be steered to illuminate a discrete area of the target. 22. Apparatus according to claim 4, including means by which each ion beam may be steered to illuminate a discrete area of the target. 23. Apparatus according to claim 5, including means by which each ion beam may be steered to illuminate a discrete area of the target. 24. Apparatus according to claim 2, including means by which each ion beam may be steered to illuminate an area of the target which abuts at least one area of the target illuminated by a further ion beam. 25. Apparatus according to claim 3, including means by which each ion beam may be steered to illuminate an area of the target which abuts at least one area of the target illuminated by a further ion beam. 26. Apparatus according to claim 4, including means by which each ion beam may be steered to illuminate an area of the target which abuts at least one area of the target illuminated by a further ion beam. 27. Apparatus according to claim 5, including means by which each ion beam may be steered to illuminate an area of the target which abuts at least one area of the target illuminated by a further ion beam. 28. Apparatus according to claim 2, including means by which the ion beams are steered to illuminate overlapping areas of the target. 29. Apparatus according to claim 3, including means by which the ion beams are steered to illuminate overlapping areas of the target. 30. Apparatus according to claim 4, including means by which the ion beams are steered to illuminate overlapping areas of the target. 31. Apparatus according to claim 5, including means by which the ion beams are steered to illuminate overlapping areas of the target. 32. Apparatus according to claim 2, additionally including r.f. plasma generation means. 33. Apparatus according to claim 3, additionally including r.f. plasma generation means. 34. Apparatus according to claim 4, additionally including r.f. plasma generation means. 35. Apparatus according to claim 5, additionally including r.f. plasma generation means. 36. Apparatus according to claim 6, additionally including r.f. plasma generation means. 37. Apparatus according to claim 7, additionally including r.f. plasma generation means. 38. Apparatus according to claim 8, additionally including r.f. plasma generation means. 39. Vacuum sputtering apparatus capable of depositing multi-layer materials on a substrate comprising: a vacuum chamber; target support means within the vacuum chamber for supporting at least one deposition target; a substrate table within the vacuum chamber for supporting a substrate oriented such that material sputtered from the at least one deposition target is deposited on the substrate when supported by the substrate table; and apparatus for the generation of a plurality of ion beams in accordance with claim 2, arranged so as to project the plurality of ion beams into the vacuum chamber so as to illuminate a respective area of the deposition target. 40. Vacuum sputtering apparatus capable of depositing multi-layer materials on a substrate comprising: a vacuum chamber; target support means within the vacuum chamber for supporting at least one deposition target; a substrate table within the vacuum chamber for supporting a substrate oriented such that material sputtered from the at least one deposition target is deposited on the substrate when supported by the substrate table; and apparatus for the generation of a plurality of ion beams in accordance with claim 3, arranged so as to project the plurality of ion beams into the vacuum chamber so as to illuminate a respective area of the deposition target. 41. Vacuum sputtering apparatus capable of depositing multi-layer materials on a substrate comprising: a vacuum chamber; target support means within the vacuum chamber for supporting at least one deposition target; a substrate table within the vacuum chamber for supporting a substrate oriented such that material sputtered from the at least one deposition target is deposited on the substrate when supported by the substrate table; and apparatus for the generation of a plurality of ion beams in accordance with claim 4, arranged so as to project the plurality of ion beams into the vacuum chamber so as to illuminate a respective area of the deposition target. 42. Vacuum sputtering apparatus capable of depositing multi-layer materials on a substrate comprising: a vacuum chamber; target support means within the vacuum chamber for supporting at least one deposition target; a substrate table within the vacuum chamber for supporting a substrate oriented such that material sputtered from the at least one deposition target is deposited on the substrate when supported by the substrate table; and apparatus for the generation of a plurality of ion beams in accordance with claim 5, arranged so as to project the plurality of ion beams into the vacuum chamber so as to illuminate a respective area of the deposition target. 43. Vacuum sputtering apparatus capable of depositing multi-layer materials on a substrate comprising: a vacuum chamber; target support means within the vacuum chamber for supporting at least one deposition target; a substrate table within the vacuum chamber for supporting a substrate oriented such that material sputtered from the at least one deposition target is deposited on the substrate when supported by the substrate table; and apparatus for the generation of a plurality of ion beams in accordance with claim 6, arranged so as to project the plurality of ion beams into the vacuum chamber so as to illuminate a respective area of the deposition target. 44. Vacuum sputtering apparatus capable of depositing multi-layer materials on a substrate comprising: a vacuum chamber; target support means within the vacuum chamber for supporting at least one deposition target; a substrate table within the vacuum chamber for supporting a substrate oriented such that material sputtered from the at least one deposition target is deposited on the substrate when supported by the substrate table; and apparatus for the generation of a plurality of ion beams in accordance with claim 7, arranged so as to project the plurality of ion beams into the vacuum chamber so as to illuminate a respective area of the deposition target. 45. Vacuum sputtering apparatus capable of depositing multi-layer materials on a substrate comprising: a vacuum chamber; target support means within the vacuum chamber for supporting at least one deposition target; a substrate table within the vacuum chamber for supporting a substrate oriented such that material sputtered from the at least one deposition target is deposited on the substrate when supported by the substrate table; and apparatus for the generation of a plurality of ion beams in accordance with claim 8, arranged so as to project the plurality of ion beams into the vacuum chamber so as to illuminate a respective area of the deposition target. 46. Vacuum sputtering apparatus capable of depositing multi-layer materials on a substrate comprising: a vacuum chamber; target support means within the vacuum chamber for supporting at least one deposition target; a substrate table within the vacuum chamber for supporting a substrate oriented such that material sputtered from the at least one deposition target is deposited on the substrate when supported by the substrate table; and apparatus for the generation of a plurality of ion beams in accordance with claim 9, arranged so as to project the plurality of ion beams into the vacuum chamber so as to illuminate a respective area of the deposition target. 47. Apparatus according to claim 11, additionally including a neutraliser. 48. A method of depositing multi-layer materials on a substrate by vacuum sputtering comprising: a) providing a substrate supported by a rotatable substrate table and a deposition target within a vacuum chamber, the substrate table, the substrate and the deposition target being oriented relative to each other such that material sputtered from the deposition target is deposited on the substrate; b) illuminating the target with a plurality of deposition target illuminating ion beams, from ion beam generating apparatus according to claim 2, so as to cause material to be sputtered from the deposition target; and c) rotating the substrate while the deposition target is illuminated by the deposition target illuminating ion beams. 49. A method of depositing multi-layer materials on a substrate by vacuum sputtering comprising: a) providing a substrate supported by a rotatable substrate table and a deposition target within a vacuum chamber, the substrate table, the substrate and the deposition target being oriented relative to each other such that material sputtered from the deposition target is deposited on the substrate; b) illuminating the target with a plurality of deposition target illuminating ion beams, from ion beam generating apparatus according to claim 3, so as to cause material to be sputtered from the deposition target; and c) rotating the substrate while the deposition target is illuminated by the deposition target illuminating ion beams. 50. A method of depositing multi-layer materials on a substrate by vacuum sputtering comprising: a) providing a substrate supported by a rotatable substrate table and a deposition target within a vacuum chamber, the substrate table, the substrate and the deposition target being oriented relative to each other such that material sputtered from the deposition target is deposited on the substrate; b) illuminating the target with a plurality of deposition target illuminating ion beams, from ion beam generating apparatus according to claim 4, so as to cause material to be sputtered from the deposition target; and c) rotating the substrate while the deposition target is illuminated by the deposition target illuminating ion beams. 51. A method of depositing multi-layer materials on a substrate by vacuum sputtering comprising: a) providing a substrate supported by a rotatable substrate table and a deposition target within a vacuum chamber, the substrate table, the substrate and the deposition target being oriented relative to each other such that material sputtered from the deposition target is deposited on the substrate; b) illuminating the target with a plurality of deposition target illuminating ion beams, from ion beam generating apparatus according to claim 5, so as to cause material to be sputtered from the deposition target; and c) rotating the substrate while the deposition target is illuminated by the deposition target illuminating ion beams. 52. A method of depositing multi-layer materials on a substrate by vacuum sputtering comprising: a) providing a substrate supported by a rotatable substrate table and a deposition target within a vacuum chamber, the substrate table, the substrate and the deposition target being oriented relative to each other such that material sputtered from the deposition target is deposited on the substrate; b) illuminating the target with a plurality of deposition target illuminating ion beams, from ion beam generating apparatus according to claim 6, so as to cause material to be sputtered from the deposition target; and c) rotating the substrate while the deposition target is illuminated by the deposition target illuminating ion beams. 53. A method of depositing multi-layer materials on a substrate by vacuum sputtering comprising: a) providing a substrate supported by a rotatable substrate table and a deposition target within a vacuum chamber, the substrate table, the substrate and the deposition target being oriented relative to each other such that material sputtered from the deposition target is deposited on the substrate; b) illuminating the target with a plurality of deposition target illuminating ion beams, from ion beam generating apparatus according to claim 7, so as to cause material to be sputtered from the deposition target; and c) rotating the substrate while the deposition target is illuminated by the deposition target illuminating ion beams. 54. A method of depositing multi-layer materials on a substrate by vacuum sputtering comprising: a) providing a substrate supported by a rotatable substrate table and a deposition target within a vacuum chamber, the substrate table, the substrate and the deposition target being oriented relative to each other such that material sputtered from the deposition target is deposited on the substrate; b) illuminating the target with a plurality of deposition target illuminating ion beams, from ion beam generating apparatus according to claim 8, so as to cause material to be sputtered from the deposition target; and c) rotating the substrate while the deposition target is illuminated by the deposition target illuminating ion beams. 55. A method of depositing multi-layer materials on a substrate by vacuum sputtering comprising: a) providing a substrate supported by a rotatable substrate table and a deposition target within a vacuum chamber, the substrate table, the substrate and the deposition target being oriented relative to each other such that material sputtered from the deposition target is deposited on the substrate; b) illuminating the target with a plurality of deposition target illuminating ion beams, from ion beam generating apparatus according to claim 9, so as to cause material to be sputtered from the deposition target; and c) rotating the substrate while the deposition target is illuminated by the deposition target illuminating ion beams. |
Protein expression profile database |
This invention describes the use of peptide profiling to identify, characterize, and classify biological samples. In complex samples, many thousands of different peptides will be present at varying concentrations. The invention uses liquid chromatography and similar methods to separate peptides, which are then identified and quantified using mass spectrometry. By identification it is meant that the correct sequence of the peptide is established through comparisons with genome sequence databases, since the majority of peptides and proteins are unannotated and have no ascribed name or function. Quantification means an estimate of the absolute or relative abundance of the peptide species using mass spectrometry and related techniques including, but not limited to, pre- or post-experimental stable or unstable isotope incorporation, molecular mass tagging, differential mass tagging, and amino acid analysis. |
1. A method for identifying the constituent proteins for a cell type, tissue or pathological sample using a database comprising peptide profile libraries wherein the libraries have multiple peptide sequences, comprising: a) deriving a plurality of peptides from the cell type, tissue or pathological sample; b) identifying the peptide species by liquid phase tandem mass spectroscopy sequencing; c) compiling a data set or peptide profile containing the collection of peptide sequences obtained thereby; and d) cross-tabulating with a collection of peptide sequences in the database. 2. The method of claim 1, wherein the step of deriving a plurality of peptides from the cell type, tissue or pathological sample further comprises the step of: a) obtaining a peptide-containing extract of the cell type, tissue or pathological sample; b) digesting the extract producing peptides with an enzyme, the enzyme capable of localizing mobile protons to the N-terminal amine and the side chains of the carboxy-terminal arginine or lysine residues; c) separating the peptides by high pressure liquid chromatography apparatus; 3. The method of claim 2, wherein the enzyme comprises one selected from the group consisting of trypsin and endoproteinase LysC. 4. The method of claim 2, wherein the step of digesting the extract producing peptides further comprises the steps of: a) dividing the extract into two equal portions; b) derivatizing one of the two equal portions with a reagent, the reagent comprising one selected from the group consisting of o-methylisourea, homoarginine, canavanine, hydrazine, phenylhydrazine, and butyric acid derivatives. c) combining the two portions. 5-7. Canceled. 8. A method for quantitating the relative abundance of proteins in two samples of a cell type, tissue or pathological sample using a database comprising peptide profile libraries wherein the libraries have multiple peptide sequences, comprising: a) deriving a plurality of peptides from each sample of the cell type, tissue or pathological sample; b) identifying the peptide species by tandem mass spectroscopy sequencing; compiling a data set or peptide profile containing the collection of peptide sequences obtained thereby; c) cross-tabulating with a collection of peptide sequences in the database of peptide sequences; and d) determining the relative abundance of the peptides and/or proteins. 9. Canceled. 10. The method of claim 8, wherein the step of deriving a plurality of peptides in two samples further comprises the step of: a) obtaining a peptide-containing extract of each sample; b) digesting separately the extracts producing peptides with an enzyme, the enzyme capable of localizing mobile protons to the N-terminal amine and the side chains of the carboxy-terminal arginine or lysine residues; c) combining the two extracts; and d) separating the peptides by high pressure liquid chromatography. 11. The method of claim 10, wherein the enzyme comprises one selected from the group consisting of trypsin and endoproteinase LysC. 12. The method of claim 8, wherein the step of digesting the extracts further comprises the step of derivatizing completely one of the two extracts with a reagent, the reagent comprising one selected from the group consisting of o-methylisourea, homoarginine, canavanine, hydrazine, phenylhydrazine, and butyric acid derivatives. 13-19. Canceled. 20. A method of comparing quantitative peptide profiles using a database of a plurality of peptide profile libraries, the method comprising: a) receiving a selection of two or more of the peptide profile libraries; b) determining the peptide profiles common to the selected peptide profile libraries and identifying profiles unique to each of selected peptide profile library; and c) displaying the results of the determination. 21. The method of claim 20, wherein the correlation of a peptide profile against selected peptide profile libraries is determined by Px,y=[1n(j=1 to n)Σ(Xj−μx)(Yj−μy)]/[∂x·∂y] where peptides common to two profiles score ‘1’ and peptides not shared between profiles score ‘0’. 22. The method of claim 21, wherein the peptides profiles are of cell fractions, the cell fractions comprising high molecular weight proteins, soluble proteins, membrane proteins, modified proteins, phosphoproteins, peptides terminating in lysine or arginine or the specific products of proteolytic enzymes or chemical derivatives of those products, peptides containing rare amino acids, and proteins isolated by binding to disease-specific affinity reagents. 23-24. Canceled. 25. The method of claim 22, wherein the rare amino acids comprise tryptophan and cysteine and amino acids comprising 5% or less of the amino acid representation. 26. The method of claim 22, wherein the disease-specific affinity reagents comprise polyclonal antibodies, toxin or drugs. 27. The method of claim 20, wherein the peptide profiles are of peptide sequences, the peptide sequences comprising mammalian peptide sequences. 28. The method of claim 20, wherein the peptide profiles are of peptide sequences, the peptide sequences comprising microbial peptide sequences. 29-30. Canceled. 31. The method of claim 20, wherein the step of receiving a selection of two or more of the peptide profile libraries for comparison comprises receiving an electronically transmitted file containing sequence and quantitative data. 32. The method of claim 20, wherein the results of the determination comprise a unique identifier for related peptide profiles. 33-34. Canceled. 35. The method of claim 20, further comprising the step of displaying the peptide profiles common to the selected peptide profile libraries. 36. The method of claim 20, further comprising the step of displaying the peptide profiles unique to the selected peptide profile libraries. 37. Canceled. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Modem biochemistry and molecular medicine is entering the post-genomic era. While genome sequencing has generated a large amount of genetic data, the focus in the biological sciences is now changing to the full characterization of proteins. Protein post-translational modifications, protein localization, protein-protein interactions, and analysis of protein structure and folding have become subjects of major importance. Proteomics is the study of patterns of protein expression by complex biological systems. It involves, in principle, the determination of the relative abundance, post-translational modification, and/or stability of large numbers of cellular proteins at specific time-points within the life cycle of an organism. There is growing recognition that qualitative and quantitative analysis of protein expression profiles on a genome-wide scale will accelerate the development of powerful new diagnostic tools and therapeutics, including novel biomarkers and drug targets, as well as lead to a better understanding of the basic molecular logic that governs cell biology. This is because most, if not all, complex biological processes are ultimately regulated by means of protein turnover and not simply through the control of gene expression. The study of protein expression will bring researchers closer to the actual biological function of genes than studies of gene sequence or gene expression alone. This is because molecular regulation of proteins, and not simply their corresponding genes, holds the key to the function of most, if not all, complex biological processes. In contrast to genomics, which captures DNA information that is largely stable throughout the lifetime of an organism, proteomics efforts seek to summarize the protein-expression patterns of dynamic biological systems at different times; While there are a finite number of genes in a given genome, a cell's proteome is constantly fluctuating in response to environment and cellular perturbations. Hence, understanding how proteins work together requires systematic data on the entire spectrum of protein status in a cell at any given time. Biology Enters the Post-Genomic Era By the late 1990's the DNA sequences of numerous bacterial and eukaryotic organisms had been published and in 2000 the nearly complete DNA sequence of Homo sapiens was completed. The availability of large-scale genomic sequencing efforts now offers investigators a unique opportunity to perform comparative analysis from an evolutionary perspective which can both help to annotate and validate completed genome sequences and also help identify conserved protein function, regulation, or pathways based on protein sequence homology. Today several disciplines, in particular bioinformatics, functional genomics, and proteomics, are converging in efforts to exploit this newly-available genome sequence information. The long-term objective of these efforts is to understand the function and interrelationships of the many thousands of genes and proteins present in human cells, with the implicit expectation that this understanding will lead to dramatic progress in the clinical sciences. In the last few years, laboratories have begun to investigate the functions of the protein products of genes and their respective regulatory pathways in a systematic global manner. Several approaches are now commonly used. First, systematic two-hybrid experiments can be used to define interactions among large sets of proteins (Flores et al, 1999), including whole yeast proteome (Ito et al., 2000; Uetz et al, 2000). Second, comprehensive screening of mutant genetic loci as a means for dissecting networks of interacting gene products has recently been adapted to automated high-throughput formats. Finally, powerful experimental tools for identifying the components of protein samples, including large complexes such as the ribosome (Link et al., 1999) and nuclear pore (Rout et al., 2000), and most recently whole organelles and whole cells have been described. Tandem Mass Spectrometry Because the amino acid sequence of a protein is encoded in DNA, and because the rules for determining the primary amino acid sequence of a protein are known, vast numbers of hypothetical proteins with no known function await classification and characterization. Clearly, many of these genes and proteins play a role in human disease and other phenomena of biological or commercial interest. The emerging field of proteomics research relies on enabling technologies that can accurately and rapidly characterize the numerous diverse proteins typically found in biological samples This requires scalable, robust, and automated methods for protein analysis. To reveal biochemical pathways and regulatory networks, and help define new targets for structure-function analysis, proteomics studies require high-resolution, high-sensitivity techniques for separation, detection, and quantitation of proteins as well as methods for linking proteins to their corresponding cognate gene sequences. Mass spectrometry (MS) is currently the method of choice for identifying proteins present in biological mixtures. The primary advantages of MS are its high-sensitivity, accuracy and capacity. Mass spectrometry is the study of gas phase ions as a means to characterize the structures, and hence identities, of molecules. Proteomics began with the commercialization of soft ionization techniques in the 1990s, in particular electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI), which permitted analysis of proteins for the first time. Commercial MS instruments are designed as high performance instruments for structural characterization of ions produced by these soft ionization techniques and have largely replaced traditional Edman chemical sequencing for the analysis of proteins. MS has proven to be very successful at identifying limited numbers of proteins, such as single polypeptide bands cut from polyacrylamide gels, and it is currently possible to identify proteins at picomolar to sub picomolar levels. Recent advances in mass spectrometry and data analysis described below are providing the necessary tools for implementation of high-throughput protein identification and characterization. As the scope of protein analysis has shifted from a molecule-by-molecule approach to a genomic scale, the ability of both academia and industry to generate new MS data has dramatically outstripped the ability to validate, manage, and interrogate the data. For these studies, routine access to state-of-the-art mass spectrometry instrumentation with an adequate infrastructure is essential. Two new ionization techniques, MALDI and ESI, have revolutionized the analysis of proteins. The MALDI and ESI techniques can be coupled with various types of mass analyzers, such as quadrupoles (Quad, Q), time-of-flight (TOF), ion-trap, Fourier transform ion cyclotron resonance (ICR) and hybrid instruments with two different mass analyzers (Q-TOF). Each kind of instrument has advantages and disadvantages and, in practice, the achievement of high throughput in conjunction with reliable protein identification requires access to both MALDI and ESI instruments. Mass spectrometry is the most powerful physical technique in its ability to resolve and identify rapidly the thousands of proteins expressed by a genome. Mass spectrometric techniques are particularly effective when coupled with classical biochemical techniques such as proteolytic digestion, immunoprecipitation and separation techniques such as affinity chromatography, HPLC or capillary electrophoresis. Tandem mass spectrometry (MS/MS) provides a means for fragmenting a mass-selected ion and measuring the mass-to-charge ratio (m/z) of the product ions that are produced during the fragmentation process. The MS/MS process used most often is based on collision-induced dissociation (CID), in which a mass-selected ion is transmitted to a high-pressure region of the instrument where it undergoes low energy collisions with inert gas molecules. As a molecular ion collides, a portion of its kinetic energy is converted into excess internal energy rendering the ion unstable, and driving unimolecular fragmentation reactions prior to leaving the collision cell. Detailed structural information is generated as a result of fragmentation. The mass selectivity of many commercial MS systems permit the isolation of single precursor peptide ions from mixtures, thereby removing the contribution of any other peptide or contaminant from the sequence analysis step. The product ion spectra can subsequently be interpreted to deduce the amino acid sequence of a protein. A protein to be identified by MS is first digested enzymatically with a site-specific protease such as trypsin (which cleaves after lysine and arginine residues) in order to produce peptides with structures suitable for MS. Tryptic peptides are particularly amenable to MS/MS analysis since mobile protons localize to the N-terminal amine and the side chains of the carboxy-terminal arginine or lysine residues at which proteolysis occurs. These protons cause peptides to fragment in a somewhat predictable manner following activation in a tandem MS, leading to production of two broad classes of fragment ions—the so-called amino-terminal b-type ions and carboxy-terminal y-type ions. Recognition of the members of these series is a fundamental process of MS-based protein sequence interpretation. Tandem mass spectrometry is a uniquely powerful technology for identifying the components of low abundance protein complexes (Andersen et al., 1996). Using this technique, the molecular weight of individual ionized peptides resulting from trypsin digestion of protein sample is initially determined by the mass spectrometer. The peptides are then isolated based on their mass/charge properties, fragmented using low energy collision with inert gas (or with resonance excitation), and the fragments are analyzed using a second round of mass spectrometry. The relative abundance of daughter product ions in peptide tandem mass spectra varies considerably, and some are not observed. This variation reflects subtle differences between favored and disfavored fragmentation sites, the nature of the amino acid side chains, and their position on the peptide backbone. CID of protonated peptides also leads to other fragmentation reaction products that can complicate spectral interpretation. Molecular losses of water or ammonia for instance, are commonly observed in the product ion scans of tryptic peptide ions. Spectra often also contain non-peptide noise peaks. Because of this, de novo interpretation of spectra is extremely difficult to automate and most MS-based identification techniques rely on reducing the computational scale of the problem by searching protein sequence databases using a relatively simple correlation algorithm. The fragmentation patterns of the peptides can be used to obtain amino acid sequence information by comparison with predicted patterns obtained from translated protein databases. In addition, advances in tandem mass spectrometry mean that polypeptides can now be identified at a low picomolar to femtomolar level in a rapid, sensitive, and versatile manner. By revealing the composition of biologically relevant, low abundance protein complexes, the technology can provide fundamental insight into the circuitry of interacting proteins. Tryptic peptides are particularly amenable to MS/MS analysis since mobile protons localize to the N-terminal amine and the side chains of the carboxy-terminal arginine or lysine residues at which proteolysis occurs. These protons cause peptides to fragment in a somewhat predictable manner following activation in a tandem MS, leading to production of two broad classes of fragment ions—the so-called amino-terminal b-type ions and carboxy-terminal y-type ions (a typical MS/MS peptide spectra showing prominent b- and y-ions is shown below). The fragmentation pattern reflects the dissociation of the peptides along the peptide bond backbone, and therefore correlates with the sequence of amino acids for those peptides. Recognition of the members of the b- and y-ion series is a fundamental process of MS-based protein sequence interpretation. Since de novo interpretation of spectra is difficult to automate, most MS-based identification techniques rely on reducing the computational scale of the problem by searching protein sequence databases using a relatively simple correlation algorithm. The SEQUEST program (U.S. Pat. No. 5,538.897), for instance, uses uninterpreted product ion spectra to search databases of theoretical spectra derived from protein and translated gene sequence databases. Recent developments in tandem mass spectrometry (MS/MS) now allow for the identification of hundreds of proteins per sample in a single run using available technology. This represents a major breakthrough compared to traditional methods, for example, 2D gel electrophoresis, and permits, for the first time, protein analysis on a truly proteomic scale. Accurate mass measurement of peptides derived from proteins provides information not available from DNA sequence, such as post-translational modifications and correction to errors in the DNA databank. Database searching with masses of peptides obtained from proteolytic digests is a well-established technique in many laboratories around the world. The searching of databases with partial sequence information obtained from MS/MS sequencing experiments is even more reliable because it imposes statistical constraints on the identification. The ability of mass spectrometry techniques to quantify the levels of individual peptides in a sample has been limiting. Recent approaches, such as ICAT (isotope-coded affinity tags; Gygi et al, 2000), have begun to address this issue. Using ICAT and similar strategies, the proteins of two samples are differentially modified with a reagent that quantitatively adds a molecular tag of defined molecular mass to one of the protein samples. By combining the samples after this treatment, the relative abundance of different protein species in each sample can be estimated by comparing the signal intensities of the corresponding peptides in the mass spectrometer. Another quantitative approach, limited to culturable organisms, is to label growth media with stable isotopes such as N15. The isotope becomes incorporated into the peptide or protein and the isotope-treated peptide is offset in the mass spectrum by multiples of 1 amu (the difference in mass between the naturally abundant isotope N14 and the heavy isotope derivative N15) depending on the number of N atoms in the peptide. These spectra can be deconvoluted to determine the relative abundance of the labeled and unlabeled peptide species. Alternatively, non-isotopic mass tags, whereby the ‘labeled’ or tagged species is offset by the mass of the tag, can be used. Thus methods suitable for high-throughput and efficient identification and quantitation of large numbers of proteins from complex mixtures are now available. HPLC High-resolution separation techniques are required to separate the peptide components of complex biological mixtures prior to mass spectrometry. A particularly powerful approach to identifying the components of complex protein mixtures is direct analysis of the protease-digested proteins using high-performance, high-resolution multi-dimensional liquid separation techniques coupled online to mass spectrometry/database searching (HPLC-MS/MS)(Link et al., 1999). This strategy enables the separation of very complex peptide mixtures, such as the whole cell extracts or nuclear extracts (Washburn, 2000). One aspect of the method separates complex peptide mixtures by strong cation exchange in the first dimension and by reverse phase in the second. However, many combinations of separation media and more than two dimensions could be used. One advantage of the strategy is that it eliminates the need to separate proteins on gels or to identify them using antibody- or affinity-based techniques that are both time-consuming and difficult to standardize. Therefore this technique circumvents the technical and analytical limitations associated with traditional proteomics technologies. Bioinformatics The interpretation of peptide mass spectra for the purposes of generating protein identifications can be carried out manually but requires experience and skill and is prohibitively time-consuming. For this reason, computer algorithms have been developed that, while not capable of interpreting all spectra they encounter, can easily outperform human identifications for even minimally complex peptide mixtures. Any of several generally available algorithms may be used for this purpose. For instance, the SEQUEST program (Eng et al., 1994) uses uninterpreted product ion spectra to search databases of theoretical spectra derived from protein and translated gene sequence databases. SEQUEST first generates a list of theoretical peptide masses for each entry in the database that match the experimentally determined peptide mass, producing a list of candidate peptides. The program then calculates the fragment ion masses expected for each of the candidate peptides, generating a predicted MS/MS spectrum. Finally, the experimentally determined MS/MS spectrum is compared with the predicted spectra using a correlation function. Each comparison receives a score, and the highest-scoring peptide(s) are reported. When high scoring matches are detected, one effectively jumps from spectral data directly to a peptide identity, which in turn can be linked to the entire amino acid and DNA sequence of the corresponding gene. Ideally, a protein is positively identified when the spectra of one or more peptides in a tryptic digest can be matched unambiguously. Mass spectral reference libraries representing stored tandem mass spectra, or validated chemical signatures, are routinely used for the identification of small chemical compounds by MS (eg. Wiley Registry, NIST database). Unknown compounds can then be both identified by searching experimental spectra against a comprehensive database of these reference mass spectra, which are in turn derived from pure compounds, so that only hits of strong similarity or identity are produced. A similar reference spectral database approach would likewise facilitate MS-based identification of proteins. Compared to mRNA expression analysis the development of corresponding ‘proteomics’ technologies has lagged, with only a few laboratories addressing complex phenotypes on a global scale. Nonetheless, protein expression profiling holds great promise for rapid genome functional analysis. It is plausible that the protein expression profile could serve as a universal and rich cellular phenotype: provided that the cellular response to disruption of different steps of a given biochemical process or pathway is similar, and that there are sufficiently unique cellular responses to the perturbation of most cellular pathways, systematic characterization of novel genetic mutants could be carried out with a single genome-wide protein expression measurement. To date the only studies focusing on peptides or proteins that includes a quantitative component has been the separation of bacterial and yeast cell lysates on 2-dimensional electrophoretic gels (refs). These approaches do not directly identify the resolved proteins, are relatively insensitive, and are unlikely to scale up to the study of larger proteomes (e.g. that of vertebrates). Furthermore, no attempt was made to use the data to identify or characterize unknown samples. |
<SOH> SUMMARY OF THE INVENTION <EOH>The protein profiling approach proposed has both a qualitative and a quantitative component such that each profile generated can be directly compared to other profiles present in a reference database. This invention describes the use of peptide profiling to identify, characterize, and classify biological samples. In complex samples, many thousands of different peptides will be present at varying concentrations. The invention uses liquid chromatography and similar methods to separate peptides, which are then identified and quantified using mass spectrometry. By identification it is meant that the correct sequence of the peptide is established through comparisons with genome sequence databases, since the majority of peptides and proteins are unannotated and have no ascribed name or function. Quantification means an estimate of the absolute or relative abundance of the peptide species using mass spectrometry and related techniques including, but not limited to, pre or post-experimental stable or unstable isotope incorporation, molecular mass tagging, differential mass tagging, and amino acid analysis. The principle experimental strategy of the present invention is centered on rapid high-throughput protein identification using coupled tandem mass spectrometry (MS/MS) and sequence database searching. Quantitation is based on either metabolic labeling with stable isotopes or with chemical derivation. Below, an example of a non-isotopic tag based on the lysine-specific guanidylation reagent O-methylisourea is described in detail. Significant patterns of peptide expression are identified with software and data mining algorithms. Below, a method is described for identifying, classifying and characterizing functions of known and unknown gene products, peptides and proteins, for characterizing metabolic and other functional pathways in cells, and for identifying the proteins and pathways targeted by drugs and other reagents. The method is based on the comparison of protein profiles obtained following global proteomics or other comprehensive protein studies from cells, cell fractions, tissues, organisms or other defined sources. The invention further contemplates the use of high-throughput robotic screening of diverse chemical compound libraries to systematically identify small molecules that perturb cellular pathways associated with disease. The protein targets of the lead compounds will be isolated and identified by the tandem mass spectrometry profiling techniques described herein. Protein profiling acts as an optimal assay since the profile of a healthy cell or tissue is the goal. The invention relates to a method for identifying the constituent proteins for a cell type, tissue or pathological sample using a database comprising peptide profile libraries wherein the libraries have multiple peptide sequences, comprising; 1. deriving a plurality of peptides from the cell type, tissue or pathological sample; 2. identifying the peptide species by liquid phase tandem mass spectroscopy sequencing; 3. compiling a data set or peptide profile containing the collection of peptide sequences obtained thereby; and 4. cross-tabulating with a collection of peptide sequences in the database. The step of deriving a plurality of peptides from the cell type, tissue or pathological sample preferably further comprises the step of: a) obtaining a peptide-containing extract of the cell type, tissue or pathological sample; b) digesting the extract producing peptides with an enzyme, the enzyme capable of localizing mobile protons to the N-terminal amine and the side chains of the carboxy-terminal arginine or lysine residues; c) separating the peptides by high pressure liquid chromatography apparatus, The enzyme preferably comprises one selected from the group consisting of trypsin and endoproteinase LysC. The step of digesting the extract producing peptides preferably further comprises the steps of: a) dividing the extract into two equal portions; b) derivatizing completely one of the two equal portions with a reagent, the reagent comprising one selected from the group consisting of o-methylisourea, homoarginine, canavanine, hydrazine, phenylhydrazine, and butyric acid derivatives. c) combining the two portions. The methods of the invention may be used in toxicology analysis. The methods optionally comprise administering a candidate compound to a cell. As described above, samples suitable for MS anaylsis are generated and a peptide profile is produced. Relative abundance of peptides in samples is also preferably determined. This candidate compound peptide profile is compared to peptide profiles in a database or library (for example, profiles showing the cell in a normal state and in varied states of toxicity). If the candidate compound sample profile is highly similar to (for example, greater than 90%, 95%, or 99% similarity), or identical to a profile in the database or library, then that similarity shows the amount of toxicity of the candidate compound to the cell. If the candidate compound sample profile is highly similar to a normal cell profile, than the candidate compound is less likely to be toxic than if the candidate compound sample profile is similar to the peptide profile of the cell in state of toxicity. The relative abundance of the test sample peptides is also preferably compared to other profiles to determine the amount of toxicity of a candidate compound. In a similar manner, candidate drugs compounds may be screened against cells, such as diseased cells. If the candidate drug shifts the profile from a disease profile and relative abundance towards a normal, healthy profile and relative abundance with substantial similarity (eg. Over 90%, 95%, 95% similarity), or identical to the healthy profile and relative abundance, the drug compound is likely to be useful as a therapeutic. Another embodiment relates to a method for identifying a peptide sequence for a cell type, tissue or pathological sample using a database comprising peptide profile libraries wherein the libraries have multiple peptide sequences, comprising; a) obtaining a peptide-containing extract of the cell type, tissue or pathological sample; b) digesting the extract producing peptides with an enzyme capable of localizing mobile protons to the N-terminal amine and the side chains of the carboxy-terminal arginine or lysine residues; c) separating the peptides by high pressure liquid chromatography apparatus; d) identifying the peptide species by tandem mass spectroscopy sequencing; and e) compiling a data set or peptide profile containing the collection of peptide sequences obtained thereby. The enzyme is preferably selected from the group consisting of trypsin and endoproteinase LysC. The step of digesting the extract producing peptides preferably further comprises the steps of: a) dividing the extract into two equal portions; b) derivatizing completely one of the two equal portions with a reagent, the reagent comprising one selected from the group consisting of o-methylisourea, homoarginine, canavanine, hydrazine, phenylhydrazine, and butyric acid derivatives. c) combining the two portions. Another aspect of the invention includes a method for quantitating the relative abundance of proteins in two samples of a cell type, tissue or pathological sample using a database comprising peptide profile libraries wherein the libraries have multiple peptide sequences, comprising: a) deriving a plurality of peptides from each sample of the cell type, tissue or pathological sample, b) identifying the peptide species by tandem mass spectroscopy sequencing; c) compiling a data set or peptide profile containing the collection of peptide sequences obtained thereby; d) cross-tabulating with a collection of peptide sequences in the database of peptide sequences; and e) determining the relative abundance of the proteins. In the methods of the invention, a pathological sample may have been contacted with a candidate drug compound and the peptide profile and/or relative abundance of the peptides and/or proteins is compared to a database comprising peptide profile libraries of the cell in varied states of toxicity (ie. exposed to known toxic compounds which injure and/or kill the cell). The toxicity of the candidate drug compound may be determined by comparison of the profile and relative abundance for the cell type, tissue or pathological sample exposed to the candidate drug compound with the profile and relative abundance for the cell type, tissue or pathological sample in varied states of toxicity and a normal state. A similar method may be used to determine whether a compound is likely to be useful as a therapeutic, for example by comparison of the profile and relative abundance for a pathological (diseased) cell type, tissue or sample exposed to the candidate drug compound with the profile and relative abundance for the cell type, tissue or sample in a normal, healthy state. The invention includes a method for quantitating the relative abundance of proteins in two samples of a cell type, tissue or pathological sample using a database comprising peptide profile libraries wherein the libraries have multiple peptide sequences, comprising: a) deriving a plurality of peptides from each sample of the cell type, tissue or pathological sample; b) identifying the peptide species by tandem mass spectroscopy sequencing: c) compiling a data set or peptide profile containing the collection of peptide sequences obtained thereby; d) determining the degree of relatedness of a collection of peptide sequences in the database of peptide sequences using clustering and related statistical methods The step of deriving a plurality of peptides in two samples preferably further comprises the step of: a) obtaining a peptide-containing extract of each sample; b) digesting separately the extracts producing peptides with an enzyme, the enzyme capable of localizing mobile protons to the N-terminal amine and the side chains of the carboxy-terminal arginine or lysine residues; c) combining the two extracts, and d) separating the peptides by high pressure liquid chromatography. The enzyme preferably comprises one selected from the group consisting of trypsin and endoproteinase LysC. The step of digesting the extracts preferably further comprises the step of derivatizing completely one of the two extracts with a reagent, the reagent comprising one selected from the group consisting of o-methylisourea, homoarginine, canavanine, hydrazine, phenylhydrazine, and butyric acid derivatives. The invention also includes a method for identifying a peptide sequence for a cell type, tissue or pathological sample, comprising: a) obtaining a peptide-containing extract of a cell type, tissue or pathological sample; b) digesting the extract producing peptides with an enzyme capable of localizing mobile protons to the N-terminal amine and the side chains of the carboxy-terminal arginine or lysine residues; c) separating the peptides by high pressure liquid chromatography apparatus: d) identifying the peptide species by tandem mass spectroscopy sequencing; and e) compiling a data set or peptide profile containing the collection of peptide sequences obtained thereby. The enzyme preferably comprises one selected from the group consisting of trypsin and endoproteinase LysC. The step of digesting the extract producing peptides preferably further comprises the steps of: a) dividing the extract into two equal portions; b) derivatizing completely one of the two equal portions with a reagent, the reagent comprising one selected from the group consisting of o-methylisourea, homoarginine, canavanine, hydrazine, phenylhydrazine, and butyric acid derivatives. c) combining the two portions. Another embodiment of the invention is a computer system for identifying quantitative peptide profiles, comprising: (a) a database including peptide profile libraries for a plurality of types of organisms wherein the libraries have multiple peptide profiles each profile comprising an array of at least 50 peptide species each having a unique identifier cross-tabulated with quantitative data indicating relative and/or absolute abundance of each peptide species in a sample; and (b) a user interface capable of receiving a selection of one or more queries to the database for use in determining a rank-ordered similarity of peptide profiles in the database. The invention includes a method of producing a computer database comprising a computer and software for storing in computer-retrievable form a collection of peptide profiles for cross-tabulating with data specifying the source of the peptide-containing sample from which each peptide profile was obtained. Optionally, at least one of the sources is from a sample known to be free of pathological disorders. Optionally, at least one of the sources is a known pathological specimen. The invention also includes a method of comparing quantitative peptide profiles using a database of a plurality of peptide profile libraries, the method comprising: a) receiving a selection of two or more of the peptide profile libraries; b) determining the peptide profiles common to the selected peptide profile libraries and identifying profiles unique to each of selected peptide profile library; and c) displaying the results of the determination. The correlation of a peptide profile against selected peptide profile libraries may be determined by in-line-formulae description="In-line Formulae" end="lead"? P x,y =[1 n (j=1 to n) Σ( X j −μ x )( Y j −μ y )]/[∂ x ·∂ y ] in-line-formulae description="In-line Formulae" end="tail"? where peptides common to two profiles score ‘1’ and peptides not shared between profiles score ‘0’. The peptides profiles are preferably of cell fractions, the cell fractions comprising high molecular weight proteins, soluble proteins, membrane proteins, modified proteins, phosphoproteins, peptides terminating in lysine or arginine or the specific products of proteolytic enzymes or chemical derivatives of those products, peptides containing rare amino acids, and proteins isolated by binding to disease-specific affinity reagents. The specific products of proteolytic enzymes may be comprise chemical derivatives of these products wherein de novo sequencing or relative abundance measurements of the peptides is facilitated. The chemical derivatives may be obtained by guanidinylation and related modifications. The rare amino acids may comprise tryptophan and cysteine and amino acids comprising 5% or less of the amino acid representation. The disease-specific affinity reagents may comprise polyclonal antibodies, toxin or drugs. The peptide profiles may be of peptide sequences, the peptide sequences comprising mammalian peptide sequences. Thee peptide profiles may be of peptide sequences, the peptide sequences comprising microbial peptide sequences. The step of receiving a selection of two or more of the peptide profile libraries for comparison may include receiving a user selection from two or more pull-down menus using a graphical user interface. The step of receiving a selection of two or more of the peptide profile libraries for comparison may comprise command line entry using a computer. The step of receiving a selection of two or more of the peptide profile libraries for comparison may comprise receiving an electronically transmitted file containing sequence and quantitative data. The results of the determination may comprise a unique identifier for related peptide profiles. The results of the determination may comprise annotated information relating to the related peptide profiles obtained from a public database. The results of the determination may comprise quantitative or relative abundance information relating to the related peptide profiles obtained from a public. database. The method may further comprise the step of displaying the peptide profiles common to the selected peptide profile libraries. The method may further comprise the step of displaying the peptide profiles unique to the selected peptide profile libraries. The invention also includes a method of identifying peptide profiles common to a set of environments, organisms, organs, tissues, cells, cellular fractions or isolated molecular complexes using a database comprising peptide profile libraries for a plurality of types of organisms wherein the libraries have multiple peptide sequences, the method comprising: (a) displaying at least one list of peptide profile libraries; (b) receiving a selection of one or more peptide profile libraries from at least one list of peptide profile libraries; (c) determining peptide profiles common to the selected peptide profile libraries; and (d) displaying the results of said determination. |
Head mounted display device |
A head mounted display device comprises a display which is imaged on the eye at an adjustable angle of incidence. On the basis of information about a position of the head of the wearer, the angle of incidence is controlled such that, upon movement of the head, the angle of incidence of the image moves along with that of the outside world. |
1. A head mounted display device. 2. A head mounted display device according to claim 1. 3. A head mounted display device according to claim 1. 4. A head mounted display device according to claim 1. 5. A head mounted display device according to claim 1. 6. A head mounted display device according to claim 1. |
Single-step or muti-step piston compressor |
The invention relates to a novel piston compressor whose return valves have a very good closure force and are extremely tight. The inlet return valve (15) is provided with a first sealing membrane (17) and the outlet return valve (30) is provided with a third sealing membrane. The two sealing membranes (17,32) are produced from an elastic polymer which has high disruptive strength, high temperature compatibility and memory properties. |
1. Multistage piston compressor comprising a valve casing (4) and a shiftable valve piston (9) formed as a single piece and driven linearly oscillating by a drive motor (2), wherein the multistage piston compressor is furnished with at least one volume changeable low-pressure chamber (5) with an intake check valve (15) and with at least one volume changeable high-pressure chamber (6) with a discharge check valve (30), wherein the valve piston (9) includes a low-pressure piston (10) and the high-pressure piston (11) and wherein the low-pressure chamber (5) and the high-pressure chamber (6) are connected to each other through an overflow duct (19), wherein an overflow check valve (20) opening in the direction toward the high-pressure chamber (6) is inserted in the overflow duct (19), characterized in that the intake check valve (15) is equipped with an intake sealing membrane (17) and wherein the discharge check valve (30) is equipped with a discharge sealing membrane (32) and wherein the two sealing membranes (17,32) comprise an elastic polymer with a high rupture strength, with a high compatibility to temperature and with memory properties. 2. Multistage piston compressor according to claim 1, wherein the intake check valve (15) is equipped with several intake openings (16) disposed on a partial circle, characterized in that the intake sealing membrane (17) of the intake check valve (15) is fitted into a sunk bore hole of the valve case floor (7) with a ball shaped or angular bore hole base and wherein the first sealing membrane (17) is fixed under tension by a centrally placed and mushroom shaped attachment element (18), wherein the attachment element (18) immerses only to such an extent into the sunk bore hole that the attachment element (18) closes flush with the inner face of the valve case floor (7) and wherein the intake sealing membrane (17) is only pretensioned by the attachment element (18) to such an extent that the sealing membrane (17) still remains rotatable. 3. Multistage piston compressor according to claim 1 wherein the discharge check valve (30) is furnished with several outlet openings (33) disposed on a common part circle, characterized in that the discharge openings (33) are entered into a valve plate (31), wherein the valve plate (31) is tensioned between the valve casing (4) and a valve casing cover (8) and wherein the discharge sealing membrane (32) is formed as a ring and is held with the outer circumference of the discharge sealing membrane (32) without tension between the valve plate (31) and the valve casing cover (8), wherein the third sealing membrane (32) with its inner circumference covers over the outlet openings (33). 4. Multistage piston compressor according to claim 3, wherein the overflow check valve (20) is equipped with a sealing disk, characterized in that the overflow duct (19) joins at least two passage bore holes (26, 27) and wherein the sealing disk of the overflow check valve (20) is formed as a loosely guided and stroke limited overflow sealing membrane (28), wherein the passage bore holes (26, 27) of the overflow duct (19) exhibit different diameters and wherein the passage bore holes (26, 27) are disposed on a common part circle with a radial distance to the axis of the overflow sealing membrane (28) and wherein the passage bore holes (26, 27) are completely covered by the overflow sealing membrane (28). 5. Multistage piston compressor according to claim 4, characterized in that the overflow sealing membrane (28) comprises an elastic polymer with a high rupture strength, with a high compatibility to temperature and with memory properties. 6. Multi-stage piston compressor according to claim 5, characterized in that the overflow sealing membrane (28) is fitted into a recess (24) of a valve support (23) and wherein the sealing membrane (28) is covered by a stop grid (29). 7. Multistage piston compressor according to claim 6, characterized in that the two passage bore holes (26, 27) are entered with the different diameters into the cylindrical recess (24) of the valve support (23) and have connection to the overflow duct (19), wherein the overflow duct (19) is formed as a kidney shaped chamber (21) in the joining region. |
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