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1. Versatile hydraulic shock absorber for a vehicle, consisting of: a shock absorber body (2) divided by a piston (6) and a large-cross-section chamber (G) and a small-cross-section chamber (P), an oleopneumatic tank (R) whose hydraulic chamber (H) connects to the large-cross-section chamber (G) of the shock absorber body, a mobile assembly comprised of a piston (6) with its tubular rod (5) and having an annular channel (42) connecting with the small-cross-section chamber (P) and ending with the seat (22), fitting into an axial check valve (29), closed during the compression phase, with the piston (6) being traversed by longitudinal channels (27) that are closed in the expansion phase by means of a lamellar check valve (50), an axial rod (33) arranged in the mobile assembly and whose end (34), protruding into the large-cross-section chamber (G), bears the calibration means (31, 40) of the axial valve (29), whereas the other end protrudes out of the tubular rod (5) in order to fit into the means for adjusting the calibration of the axial valve (29), a tank head (61) equipped with compression adjustment means, at least for the lower speeds, these means consisting of an adjusting screw (70) whose rod, inside a channel (69a) connecting to the large-cross-section chamber (G), bears, at its end leading into the hydraulic chamber (H) of the tank (R), an adjustable-calibration valve (72) that fits into a seat, and a helicoidal spring (15) arranged around the body (2) of the shock absorber and resting on mounts (13, 14) that are connected, respectively, to the body (2) and to the end of the piston rod (5), wherein all of the adjustment means (70, 71) that determine the shock absorption conditions in the compression phase are located in the head (61) of the tank (R) and are accessible from the outside without disassembly, and the ones (71) that regulate low-speed compression, include the tubular body (75) for adjusting the preload that screws into the head (61) of the tank and protrudes out of the latter by a lift nipple (75a) and, inside the tank, by an active end shaped like a seat (82), with this tubular body (75): a) connecting to the large-cross-section chamber (G) of the shock absorber, b) being axially traversed by an adjusting screw (70), which screws into its upper part and bears at its end inside the tank (R), the valve (72) with its spring-equipped calibration means, c) and including, in its part shaped like a seat (82) for the valve (72), at least one radial groove (81) forming an outlet determining the preload, with the flow area of this channel determined between a zero value and a maximum value by the distance (S) between, on the one hand, the end face (82) of the tubular body (75) and, on the other hand, the inner face (61a) of the tank head (61). 2. Versatile hydraulic shock absorber according to claim 1, wherein the lamellar valve (50), which fits into the longitudinal channels (27) in the piston (6), is constituted by a sole lock washer (50) whose inner edge is clamped inside a recess (6e) of the piston (6) by the end face of a shouldered ring (52), which is itself screwed onto an extension (17) of the piston (6). 3. Versatile hydraulic shock absorber according to claim 2, wherein the face (6a) of the piston (6) against which the lamellar valve (50) presses when it is at rest or in the compression phase, includes, in the support zone of the valve (50), at least one circular groove (53) connecting the outlets of the channels (27) traversing the piston, reducing the surface area between the valve (27) and the face (6a) of the piston where pressing and sticking might occur, and forming a cushion of shock absorbing fluid. 4. Versatile hydraulic shock absorber according to claim 1, wherein the high-speed compression adjustment means, inside the head (61) of the tank with the very-high-speed adjustment means, are constituted by a tubular body (85) traversing the head (61) and whose end outside this head is screwed into this head, whereas its inner end, protruding from a channel (69b) in the head bears a valve (88) fits into a seat formed at the end of the channel (69b), with the conditions for opening this valve (88) depending, at high speed, on the calibration of lock washers (89) arranged around the inner end of the tubular body (75) and between the valve (88) and the nut (90) screwed onto this end and, at very high speeds, on the calibration of lock washers (93) arranged around the end below the tank (R) of an adjustment rod (86) that traverses the tubular body (75) and screws into it, with these washers (93) being located between, on the one hand, a nut (94) screwed onto this rod (86) and, on the other hand, the diametric wall (92b) of a bell (92), resting on the end of the tubular body (75) and whose skirt (92a), surrounding the high-speed calibration means, extends out toward the valve (88) while providing, between its edge and this valve, play (E) corresponding to its opening stroke during high-speed operation. 5. Versatile hydraulic shock absorber according to claim 1, wherein the piston (6) is monolithic with a tail piston rod (17) and a tubular funnel (16) protruding into the large-cross-section chamber (G) and whose internal cylinder bore (20) ensures the translatory guiding of the axial check valve head (29) that opens during expansion, with the wall of this funnel (16) being traversed, near the piston (6) and the seat for the axial valve, by several radial channels (44) for the fluid to pass through, whereas the valve seat (29) is formed by a tapered axle end (22) made in the piston (6) and able to accommodate the tapered valve head. This axle end (22) is extended into the piston by a cylinder bore (19) that fits into the cylindrical valve rod (30) of the valve (29) to form an annular channel (42) for stabilizing the oil flow that passes through during expansion. This channel (42) is supplied by the radial holes (43) made in the corresponding tubular part of the tail piston rod (17) of the piston (6). 6. Versatile hydraulic shock absorber according to claim 5, wherein the funnel (16) of the piston fits into a blind cylinder bore (7) made in the head (3) of the shock absorber and connecting to the tank (R) by a connecting channel (8) to form a hydraulic stop that gradually stops the piston (6) at the end of the compression stroke. 7. Versatile hydraulic shock absorber according to claim 6, wherein the blind cylinder bore (7) of the shock absorber head (3) has a diameter that is equal, except for the oil clearance, to the outer diameter of the funnel (16) such that, at the end of the compression phase, the funnel (16), entering into this blind cylinder bore (7), gradually blocks the connection to the tank (R). 8. Versatile hydraulic shock absorber according to claim 6, wherein a blind cylinder bore (6) of the cylinder bore (7) of the head (3) of the shock absorber contains a bushing (120) that is recalled by a spring (123) against a stop (125) such that its rear edge frees the connecting channel (8) with tank (R), this bushing (120) being equipped on its front edge with a flange (122) arranged in the displacement path of the funnel (16) of the piston, in such a way that at the end of the compression phase, the funnel (16) displaces the bushing (120) into the blind cylinder bore (7) by gradually blocking, by its rear edge, the connecting channel (8) with the tank (R). 9. Versatile hydraulic shock absorber according to claim 6, wherein the funnel (16) of the piston (6) is capped by a mount (100) whose outer diameter is equal, except for oil clearance, to the diameter of the blind cylinder bore (7) of the head (3) of the shock absorber, this mount (100) being adjustable by displacement in longitudinal translation in relation to the funnel (16), by means of an axial control rod (102), mounted so that it slides into the rod (33, 38) for adjusting the check valve (29) and connected, by its end that protrudes beyond the end of this screw adjustment rod, to adjustment means (104, 405, 108) that make it possible to change the position at which this mount (100) starts to block the channel (8) connected to the tank (R). 10. Versatile hydraulic shock absorber according to claim 1, wherein the sum of the flow areas of the longitudinal channels (27) traversing the piston (6) is at least 50% greater than the cross section of the outlet channel (8) leading to the tank (R). |
Stereoselective preparation of cyclic l-amino acids |
The invention concerns a method for producing a cyclic L-amino acid of formula (I), characterised in that it consists in reacting a L-diamino acid of formula (II) or an enantiomeric mixture comprising such a L-diamino acid and a corresponding D-diamino acid in variable proportions, in the presence of an ornithine cyclodeaminase or a polypeptide homologous to the ornithine cyclodeaminase. |
1: Method of production of a cyclic L-amino acid of formula (I) or of one of the salts or derivatives thereof: R1 is selected from amongst the hydrogen atom, a linear or branched alkyl radical having from 1 to 6 carbon atoms and a linear or branched acyl radical having from 1 to 6 carbon atoms; and X represents a saturated, or partially or totally unsaturated, linear or branched C1-C9, preferably C2-C4, hydrocarbon chain, optionally comprising in the chain and/or at the end of the chain one or several heteroatoms or heterogroups selected from amongst O, S, P, NR2, R2 representing H or a C1-C4 alkyl or acyl group, the said chain optionally being substituted by one or several identical or different radicals chosen from amongst —R, —OR, —SR, ═O, —C(O)OR, —C(S)OR, —C(O)N′RR″, —C(S)NR′RR″, —CN, —NO2, —X, —MgX, —NR′R″, —NR′C(O)R, —SiR and —SiOR,R′, and R″, identical or different, representing hydrogen or a linear or branched, saturated, or totally or partially unsaturated, hydrocarbon radical and having from 2 to 20 carbon atoms, it being understood that R′ and R″ can form a ring with the atom carrying them, wherein: a) a L-diamino acid of formula (II): in which X and R1 are as defined above; or a salt or derivative thereof, or an enantiomeric mixture comprising a L-diamino acid of formula (II) and a corresponding D-diamino acid, the salts or derivatives thereof in variable proportions, is made to react in the presence of an ornithine cyclodeaminase, or a polypeptide homologous to ornithine cyclodeaminase, the enzyme or the homologous polypeptide being obtained from a recombinant expression vector expressing the said enzyme or the said homologous polypeptide, b) the cyclic L-amino acid of formula (I) or a salt or derivative thereof is recovered in an enantiomeric excess of at least 80%. 2: Method as claimed in claim 1, wherein the compound of formula (I) comprises a ring with six bonds, X representing a hydrocarbon chain with four bonds. 3: Method as claimed in claim 1, wherein the hydrocarbon chain X is a linear or branched alkylene chain. 4: Method as claimed in claim 1, wherein the compound of formula (II) or the enantiomeric mixture comprising an L-diamino acid of formula (II) and a corresponding D-amino acid in variable proportions is placed in the presence of an enzyme in the purified state. 5: Method as claimed in claim 1, wherein the compound of general formula (I) is in the form of an ammonium salt in aqueous solution. 6: Method as claimed in claim 1, wherein L-piperidine-2-carboxylic acid or one of its salts is prepared from L-lysine. 7: Method as claimed in claim 1, wherein L-piperazine-2-carboxylic acid or one of its salts is prepared from L-azalysine. 8: Method as claimed in claim 1, wherein L-thiomorpholine-2-carboxylic acid or one of its salts is prepared from L-thialysine. 9: Method as claimed in claim 1, wherein the enzyme is an ornithine cyclodeaminase of Agrobacterium or a peptide having an activity homologous to that of the said ornithine cyclodeaminase. 10: Method as claimed in claim 1, wherein the ornitbine cyclodeaminase is that of the strain of Agrobacterium C 58. 11: Method as claimed in claim 1, wherein the enzyme is recombinant enzyme expressed by the ocd gene of the strain of Agrobacterium tumefaciens C58 or pipA of Streptomyces pristinaespiralis or rapL of Sfreptomyces hygroscopicus or a homologous gene in a host microorganism. 12: Method as claimed in claim 11, wherein the recombinant enzyme is expressed in E. coli. 13: Method as claimed in claim 12, wherein the recombinant enzyme is obtained by expression of a modified gene of a native ornithine cyclodeaminase, in which some bases G or C are replaced by bases A or T, in such a way as to obtain modified codons coding for the same amino acid as the native codon. 14: Method as claimed in claim 13, wherein the modified genes have less than 65%, advantageously less than 55% of bases G+C. 15: Method as claimed in claim 12, wherein the modified gene is the gene pipA* of sequence SEQ ID No. 1 or the gene rapL* of the sequence SEQ ID No. 2, or the gene rapL** of the sequence SEQ ID No. 5. 16: Method as claimed in claim 1, wherein no exogenous NAD is added to the reaction medium. 17: Polynucleotide comprising the sequence SEQ ID No. 1. 18: Polynucleotide comprising the sequence SEQ ID No. 2. 19: Polynucleotide comprising the sequence SEQ ID No. 5. 20: Compound of formula (I) or salt or derivative of the compound of formula (I): in which: R1 is selected from amongst the hydrogen atom, a linear or branched alkyl radical having from 1 to 6 carbon atoms and a linear or branched acyl radical having from 1 to 6 carbon atoms; and X represents a saturated, or partially or totally unsaturated, linear or branched C1-C9, preferably C2-C4, hydrocarbon chain, optionally comprising in the chain and/or at the end of the chain one or several heteroatoms or heterogroups chosen from amongst O, S, P, NR2, R2 representing H or a C1-C4 alkyl or acyl group, the said chain also being optionally substituted by one or several identical or different radicals chosen from amongst —R, —OR, —SR, ═O, —C(O)OR, —C(S)OR, —C(O)N′RR″, —C(S)NR′RR″, —CN, —NO2, —X, —MgX, —NR′R″, —NR′C(O)R, —SiR and —SiOR,R′, and R″, identical or different, representing hydrogen or a linear or branched, saturated, or totally or partially unsaturated, hydrocarbon radical having from 2 to 20 carbon atoms, it being understood that R′ and R″ can form a ring with the atom carrying them, substantially obtained by the method as claimed in claim 1. |
Organic electroluminescene device and luminance material |
An organic EL device is provided which is capable of providing high luminance and high luminous efficiency. In an organic EL device, a hole injecting electrode lies on a glass substrate, and a hole injecting layer, a hole transporting layer, and a light emitting layer are sequentially formed thereon. An electron injecting electrode lies on the light emitting layer. The light emitting layer contains a host material, a light emitting dopant, and a first light-emission assisting dopant. The first light-emission assisting dopant is composed of a rubrene derivative. |
1. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer containing a rubrene derivative having a molecular structure represented by Formula (1): wherein R11-R15, R21-R25, R31-R35 and R41-R45 are the same or different and each represent a hydrogen atom or a substituent, where the structure in which all are hydrogen atoms is excluded, and adjacent two of R11-R15, adjacent two of R21-R25, adjacent two of R31-R35, and adjacent two of R41-R45 may be bonded together to form rings, and adjacent three of R11-R15, adjacent three of R21-R25, adjacent three of R31-R35, and adjacent three of R41-R45 may be bonded together to form rings. 2. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer containing a rubrene derivative having a molecular structure represented by Formula (2): wherein R 11-R15 and R21-R25 are the same or different and each represent a hydrogen atom or a substituent, adjacent two of R 11-R15 and adjacent two of R21-R25 may be bonded together to form rings, and adjacent three of R11-R15 and adjacent three of R21-R25 may be bonded together to form rings. 3. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer containing a rubrene derivative having a molecular structure represented by Formula (3): wherein Ar1-Ar6 are the same or different and each represent a hydrogen atom or a substituent, R1 and R2 are the same or different and each represent a hydrogen atom or a substituent, and adjacent R1 and R2 may be bonded together to form a ring. 4. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer containing a rubrene derivative having a molecular structure represented by Formula (4): wherein Ar1-Ar10 are the same or different and each represent a hydrogen atom or a substituent. 5. The organic electroluminescent device according to claim 1, wherein said rubrene derivative is 5,6,11,12-tetrakis(naphth-2-yl)-naphthacene having a molecular structure represented by Formula (A1). 6. The organic electroluminescent device according to claim 1, wherein said rubrene derivative is 5,12-bis(4-(6-methylbenzothiazole-2-yl)phenyl)-6,11-diphenylnaphthacene represented by Formula (A2). 7. The organic electroluminescent device according to claim 1, wherein said rubrene derivative is 5,6,11,12-tetrakis(4-tert-butylphenyl)-naphthacene represented by 8. The organic electroluminescent device according to claim 2, wherein said rubrene derivative is 5,12-bis(4-tert-butylphenyl)-naphthacene represented by Formula (A4). 9. The organic electroluminescent device according to claim 2, wherein said rubrene derivative is 5,12-diphenylnaphthacene represented by Formula (A5). 10. The organic electroluminescent device according to claim 2, wherein said rubrene derivative is 5,12-bis(naphth-2-yl)-naphthacene represented by Formula (A6). 11. The organic electroluminescent device according to claim 2, wherein said rubrene derivative is 5,12-bis(pyrene-1-yl)-naphthacene represented by Formula (A7). 12. The organic electroluminescent device according to claim 3, wherein said rubrene derivative is 5,6,13,14-6-tetrakisphenyl-pentacene represented by Formula (A8). 13. The organic electroluminescent device according to claim 3, wherein said rubrene derivative is 6,13-bis(4-(6-methylbenzothiazole-2-yl)phenyl)-pentacene represented by Formula (A9). 14. The organic electroluminescent device according to claim 4, wherein said rubrene derivative is 5,6,11,12-tetrakisphenyl-1,2-benzo-(3,4-benzo-)naphthacene represented by Formula (A10). 15. The organic electroluminescent device according to claim 1, wherein said light emitting layer contains a host material, a light emitting dopant, and a first light-emission assisting dopant, and said first light-emission assisting dopant is composed of said rubrene derivative. 16. The organic electroluminescent device according to claim 15, wherein said light emitting layer further contains a second light-emission assisting dopant. 17. The organic electroluminescent device according to claim 1, wherein said light emitting layer contains a host material and a light emitting dopant, and said light emitting dopant is composed of said rubrene derivative. 18. The organic electroluminescent device according to claim 15, wherein the content of said light emitting dopant is not less than 0.1 percent by weight nor more than 50 percent by weight, with respect to said host material. 19. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C1), and a second light emitting layer that contains a luminescent material that emits blue light. 20. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C2), and a second light emitting layer that contains a luminescent material that emits blue light. 21. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C3), and a second light emitting layer that contains a luminescent material that emits blue light. 22. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C4), and a second light emitting layer that contains a luminescent material that emits blue light. 23. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C5), and a second light emitting layer that contains a luminescent material that emits blue light. 24. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C6), and a second light emitting layer that contains a luminescent material that emits blue light. 25. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C7), and a second light emitting layer that contains a luminescent material that emits blue light. 26. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C8), and a second light emitting layer that contains a luminescent material that emits blue light. 27. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C9), and a second light emitting layer that contains a luminescent material that emits blue light. 28. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C10), and a second light emitting layer that contains a luminescent material that emits blue light. 29. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C11), and a second light emitting layer that contains a luminescent material that emits blue light. 30. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C12), and a second light emitting layer that contains a luminescent material that emits blue light. 31. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C13), and a second light emitting layer that contains a luminescent material that emits blue light. 32. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C14), and a second light emitting layer that contains a luminescent material that emits blue light. 33. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C15), and a second light emitting layer that contains a luminescent material that emits blue light. 34. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C16), and a second light emitting layer that contains a luminescent material that emits blue light. 35. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C17), and a second light emitting layer that contains a luminescent material that emits blue light. 36. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C18), and a second light emitting layer that contains a luminescent material that emits blue light. 37. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C19), and a second light emitting layer that contains a luminescent material that emits blue light. 38. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising, a first light emitting layer that contains a compound having a molecular structure represented by Formula (C20), and a second light emitting layer that contains a luminescent material that emits blue light. 39. An organic electroluminescent device comprising: a hole injecting electrode; an electron injecting electrode; and a light emitting layer provided between said hole injecting electrode and said electron injecting electrode, said light emitting layer comprising a first light emitting layer that contains at least one compound selected from the group consisting of compounds having molecular structures represented by Formulas (C1)-(C20) and at least one compound selected from the group consisting of compounds represented by Formulas (A4)-(A7), (A10), and (C21)-(C27), and a second light emitting layer that contains a luminescent material that emits blue light. 40. A luminescent material having a molecular structure represented by Formula (1): wherein R11-R15, R21-R25, R31-R35 and R41-R45 are the same or different and each represent a hydrogen atom or a substituent, where the structure in which all are hydrogen atoms is excluded, and adjacent two of R11-R15, adjacent two of R21-R25, adjacent two of R31-R35, and adjacent two of R41-R45 may be bonded together to form rings, and adjacent three of R11-R15, adjacent three of R21-R25, adjacent three of R31-R35, and adjacent three of R41-R45 may be bonded together to form rings. 41. A luminescent material having a molecular structure represented by Formula (2): wherein R11-R15 and R21-R25 are the same or different and each represent a hydrogen atom or a substituent, adjacent two of R11-R15 and adjacent two of R21-R25 may be bonded together to form rings, and adjacent three of R11-R15 and adjacent three of R21-R25 may be bonded together to form rings. 42. A luminescent material having a molecular structure represented by Formula (3): wherein Ar1-Ar6 are the same or different and each represent a hydrogen atom or a substituent, and R1 and R2 are the same or different and each represent a hydrogen atom or a substituent, and adjacent R1 and R2 may be bonded together to form a ring. 43. A luminescent material having a molecular structure represented by Formula (4): wherein Ar1-Ar10 are the same or different and each represent a hydrogen atom or a substituent. |
<SOH> BACKGROUND ART <EOH>Organic electroluminescent devices (hereinafter referred to as organic EL devices) are new self-emitting devices of great prospects. The organic EL devices have a stacked structure in which a carrier transporting layer (an electron or hole transporting layer) and a light emitting layer are sandwiched between a hole injecting electrode and an electron injecting electrode. An electrode material having a large work function, such as gold or ITO (indium-tin oxide), is used to form the hole injecting electrode, and an electrode material having a small work function, such as Mg (magnesium) or Li (lithium), is used to form the electron injecting electrode. The hole transporting layer, light emitting layer, and electron transporting layer are formed of organic materials. A material having p-type semiconductor properties is used to form the hole transporting layer and a material having n-type semiconductor properties is used to form the electron transporting layer. The light emitting layer is formed of a fluorescent or phosphorescent organic material which, too, has a carrier transporting property, as an electron or hole transporting property. The hole injecting electrode, hole transporting layer, light emitting layer, electron transporting layer, and electron injecting electrode are stacked in this order to form an organic EL device. The functional layers, i.e. the hole transporting layer, electron transporting layer, and light emitting layer, may each be composed of a plurality of layers, or may be omitted, depending on the organic materials used. For example, Chihaya Adachi et al., Appl. Phys. Lett., Vol. 55, pp. 1489-1491 (1989) discloses a device structure that has only two organic layers, light emitting and electron transporting layers, between the hole injecting electrode and electron injecting electrode. In this device, the light emitting layer made of a luminescent material called NSD has a good hole transporting property, so that the light emitting layer can act also as a hole transporting layer. C. W. Tang et al., Appl. Phys. Lett., Vol. 51, pp. 913-915 (1987) discloses a device structure that has two organic layers: hole transporting and light emitting layers. In this case, tris(8-hydroxyquinolinato)aluminum (hereinafter referred to as Alq) in the light emitting layer performs two functions: light emitting and electron transporting functions. S. A. VanSlyke et al., Appl. Phys. Lett., Vol. 69, pp. 2160-2162 (1996) discloses a device structure that has three organic layers: a hole injecting layer, hole transporting layer, and light emitting layer. In this case, the hole injecting layer, made of copper phthalocyanine, serves like a hole transporting layer; thus the entire device includes two hole transporting layers. In this way, the stacked structure of the electron transporting layer, hole transporting layer and light emitting layer can be freely designed, depending on the organic materials used. Organic EL devices can be used to obtain visible light from blue to red, by properly selecting organic material of the light emitting layer. Therefore a full-color display can be implemented by using monochromatic organic EL devices individually emitting red, green or blue light, i.e. the three primary colors of light (RGB). Among red, green and blue lights obtained with organic EL devices, green light and blue light are stable. On the other hand, it is difficult to obtain light with high luminance and high luminous efficiency in the range from red to orange. Therefore developing full-color displays requires red-emitting organic EL devices with good color purity, high luminous efficiency, and high luminance. JP2000-164362, A suggests a method in which rubrene having the molecular structure represented by Formula (15) below is used as a light-emission assisting dopant. While this method offers improved red color purity, it fails to provide sufficient luminous efficiency and sufficient luminance. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a schematic diagram showing the structure of an organic EL device according to an embodiment of the invention; and FIG. 2 is a schematic diagram showing the structure of an organic EL device according to another embodiment of the invention. detailed-description description="Detailed Description" end="lead"? |
Bleaching device using electro-optical and chemical means namely in the medical and dental field |
A complete bleaching unit is made up of an electrochemical system, finding its application in particular in the dental and medical fields, allowing, thanks to the creation of an electrophoretic field, a strong, fast penetration and directing the bleaching agents, their removal under the same conditions together with the molecules responsible for coloration and an effective and controlled penetration of the stabilizing agents for the apatite crystal, basic component of the tooth, such as fluorine. Also thanks to its light-based optical system, the invention provides that the activation of the photosensitive bleaching products by decreasing the heat effect while increasing the photonic effect thanks to a selection of wavelength in the range of 400-500 nm and, finally, thanks to an electro-optical unit, the control of the evolution of coloration by objective spectrocolorimetry-based methods. |
1. Bleaching device using electro-optical and chemical means, in particular in the medical and dental field, for bleaching parts of the body such as the teeth, the hairs or the nails, said bleaching device comprising: an electric, electrophoretic or electromagnetic wave-current generator/controller with a varying profile and/or modulation, means for transmitting current to said bleaching parts to be bleached, means for creating a non-traumatizing electric, electrophoretic or electromagnetic field through the treated area, comprising electrodes and a conductive gel, means for containing said gel in contact with the poles and body, allowing the continuity of this field, active products used for bleaching, sensitive to the electrophoretic currents, means for activating said products either by means of light or by means of heat. 2. Device according to claim 1, further comprising: a central unit for control, storage and direct monitoring by an operator or indirect monitoring by memory or over the Internet of the parameters of operation of the electric, electrophoretic or electromagnetic source, in order to allow to control and simplify the penetration of all or part of the molecules sensitive to the electric, electrophoretic or electromagnetic field, active for the bleaching and the protection of the part target of the treatment and the exit of all or part of the molecules sensitive to the electric, electrophoretic or electromagnetic field and which are undesirable or have reacted in this bleaching process and can be removed from the body, but also means for direct control by feedback at the central unit, or manual control by the operator of the parameters allowing to know the evolution and the results of the bleaching action carried out, as are the thermal or electric variations and the modification in coloration induced by the treatment. 3. Device according to claim 1, further comprising: an electrophoretic current generator/controller, a conduction circuit and a resistor creating a pole in the means for containing the gel, the second pole being in contact with a portion of the body. 4. Device according to claim 1, wherein said means for containing the gel are modeled on the portion of the patient's body. 5. Device according to claim 1, wherein said second pole located in contact with the body is adapted to be placed in a tube containing an electrophoretic bleaching gel as the root of a devitalized tooth. 6. Device according to claim 1, further comprising: a photonic activator capable of activating the products used for bleaching and present in the gel in a less active form. 7. Device according to claim 1, wherein said means for containing the gel are associated with a source of light proceeding from halogen, arc light or from LEDs, transmitted by means of a fiber. 8. Device according to claim 1, wherein said means for containing the gel incorporate light-emitting diodes (LEDs) generating a source of light. 9. Device according to claim 1, further comprising: inside the means for containing the gel, means for controlling the electric or thermal evolution of the bleaching treatment, in the form of sensors. 10. Device according to claim 9, wherein said means for controlling are comprised of a spectrocolorimeter or a colorimeter comprised of a light generator and a differential analyzer capable of carrying out a comparative study between the value of the source and the value being reflected onto the object. 11. Device according to claim 1, wherein electrical, electromagnetic or electrophoretic currents used for the displacement of the molecules have a varying profile and modulation in tension, frequency and amplitude. 12. Device according to claim 1, wherein said basic electrophoretic gel allowing the passing through of the electrical, electromagnetic or electrophoretic current contains electrically charged molecules responsible for the bleaching action, so that they move through the gel towards the body to be treated under the action of the current used. 13. Device according to claim 1, wherein said basic electrophoretic gel allowing the passing through of the electrical, electromagnetic or electrophoretic current contains electrically charged molecules responsible for the action of reinforcement of the body to be treated, so that they move through the gel towards the body to be treated under the action of the current used and that they compensate for the possible degradations caused by the bleaching action. 14. Device according to claim 1, wherein said basic electrophoretic gel allowing the passing through of the electrical, electromagnetic or electrophoretic current is electrically charged, thus allowing, by its own movement, to drag the molecules responsible for the bleaching or reinforcing action which would not be electrically charged, so that they move passively towards the body under the action of the displacement of the gel. 15. Device according to claim 1, further comprising: a button for reversing the polarity of the electrodes responsible for creating the electric, electrophoretic and electromagnetic fields used for the displacement of the gel or of the molecules responsible in the bleaching process. 16. Device according to that claim 1, wherein said thermal means for the activation of the molecules used in the process of bleaching and moved by the electrophoretic, electromagnetic or electric current are comprised of an electric resistor, a photonic emission or a circulating fluid. 17. Device according to claim 1, wherein said gel is designed capable of changing color according to the evolution of the bleaching reaction, the degree of activation of the molecules responsible for the bleaching or the percentage of molecules responsible for coloration removed from the body and present in said gel. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Bleaching a tooth, or providing it its original natural, specific to the patient, has been an action commonly carried out in dentist's surgeries for more than 100 years and is very significant for the psychological balance of the patients. A very good explanation for this aesthetic action was given, and constitutes a reference in this matter: the special addendum to JADA (Journal American Dental Assoc.) of April, 1997, No. 128 (addendum) pp. S1-S64 entitled “non-restorative treatment of discolored teeth, reports for an international symposium” and summarizing the congress of Chapel Hill, North Carolina of Sep. 25 and 26, 1996. An update can be found in CRA (Clinical Research Associates newsletter) Vol. 24, Apr. 4, 2000 issue or, more recently, in “Incidence of tooth sensitivity after home whitening treatment” by Jorgensen and coll. JADA, August, 2002, Vol. 133 pp. 1076-1082. From this work can be seen that there are nowadays three important methods of bleaching used in the dentist's and medical surgeries: a mechanical method consisting in associating to the plaque control of teeth by mechanical means (manual and ultrasonic) abrasive polishing pastes. a chemical method, in general following the previous one, and consisting in applying to the tooth a product capable of removing the surface deposits as those due to tea or coffee. These products are very low-concentration carboxide- or peroxide-based products, and can be used by the patient himself at home. a more invasive chemical method having higher concentrations of peroxide-based product, often requiring the dental surgeon's intervention, taking into consideration the risks incurred by the patient if it does not follow its therapeutics according to the rules of the dental and medical field, and allowing to reduce the coloration of the teeth inside the dental body itself. Recently this method was modified and reduced in action in order to be usable by the patient himself at home (home kit) under cover of a periodic medical control. Unfortunately and very quickly, both the practitioners and their patients realized that: the application time was very long and required the immobilization of the patient during more than 5 minutes per tooth, or 20 minutes per half arch. the cost of the intervention was therefore painful and prohibitive! For this reason products were developed reacting more quickly while being activated by the light or heat. This method allows to reduce by four the time spent for the medical bleaching action. Based on these promising results a number of products known as photosensitive were put on the market and used abundantly and successfully, associating perborate and hydrogen peroxide or peroxide carbamide activated by camphoroquinone, itself photosensitive between 400 and 500 nm. These products result from the techniques developed and introduced originally by CORCORAN and ZILLICH (1974) and by RENNEBOOG (1989). These studies highlight the role of heat and radiation brought by the halogen lamps in the activation of the bleaching products. Thus, nowadays there exist on the market many bleaching products that are directly usable by the patient at home or that can be applied at higher concentration by the dentists. These products act directly or after activation by light or heat. They use in great majority as basic formula hydrogen peroxide at approximately 35% as described for first time by HALON in 1884. To activate even more the reaction and to further divide by two the already considerably reduced time, it was asked to develop even more powerful lamps and it is for this purpose that the xenon-arc plasma lamp “Apollo 95 E ”, patents FR 2,773,986 and FR 2,782,000, was invented and developed, which included a “bleaching” function and which supposed an action of about 30 seconds on the product placed in contact with the tooth. This product is often in the form of a gel maintained in a transparent gutter. A typical example has been sold for a long time under the name of “Apollo secrete whitening kit” (DMDS Corp. Los Angeles, USA). Admittedly the results obtained were spectacular and many manufacturers followed this technology. However this process, even if it reduced the time considerably, had many limits. It was indeed shown that: with some products the action of the lamp, therefore its effectiveness, was not only due to the photonic, but also to the thermal emissions and it is under these two effects that these products were activated. since the cost of these lamps is very high, the treatment remained relatively expensive, since these methods require high peroxide concentrations, they impeded their use by the patient at home, the thermal rise observed in the tooth was disproportionate compared to the activation of the product and could even be dangerous when the action was too long, it was impossible to properly control the thermal value at the very level of the tooth, to the risk of causing significant disorders in the health of dental pulp itself. Moreover, the movement even of the hand could result into changes in localization of the point of luminous or thermal impact. by replacing the light emitted by the lamps with high thermal emission, such as the xenon-arc or halogen lamps, by lamps known as cold lamps, such as those described in FR 2,805,148 and FR 2,318,892, the heating effect was removed, which allowed the operator to increase without any risk his time of action to activate the photosensitive products, but in parallel obliged him again to a long exposure because of the elimination of the source of heat. if a dispersed light is used over the whole arcade (for example in the form of luminous gutter), the time is again reduced, but not more than by using the high powers. the doses implemented for treatment in the dentist's surgery (30% peroxide) as well as at the patient's home (8-10% peroxide) are extremely high compared to the aim searched for and explains the side effects observed (dental pains with the cold). there are many repetitions because the molecules responsible for coloration are modified, even cut, but never actually removed from the site. The fact that they remain allows the recombination of the initial chemical bonds explaining the need for a re-treatment at increasingly closer intervals. finally, the aggressive action of peroxide in contact with the gum always obliges the practitioner as well as the patient to many precautions to avoid the bums during and after the treatment. Moreover, at no time has been solved a fundamental problem which justified the putting under monitoring of the bleaching products and in particular the peroxide by all the safety committees in the field of health of the EC and the FDA, irrespective of the degree of degradation of the tooth itself under the effect of these products, whether they are massively applied in high doses, or slowly at home by the patient himself. Do the post-operational pains result from a degradation of the tooth such as many authors affirmed? How to correct some significant decalcifications observed among patients after erroneous applied treatments? Finally, though it is possible through known techniques to allow a faster action of the bleaching products, there is no method allowing to control, at low cost, the activation of the chemical bleaching components and to correct the degradation of the tooth following their effects. Even more serious is what will happen with the molecules responsible for coloration. Indeed, after a treatment primarily based on the division of large colored molecules into smaller ones, the residue of this reaction remains, with the active agent, inside the dental body, which cunningly continues its action and significantly limits the penetration of fluorine or calcium. In addition to the consequences of this steric space occupation, we see well that the arrival on the site of intra-dental action of the bleaching molecule occurs by pure passive permeability, undoubtedly explaining its weak penetration, the time necessary to allow its action, the absence of a possible control of the reaction by the operator, but also a total lack of knowledge of its actual concentration in the area of action, i.e. on the molecules responsible for coloration in dental tissue. Finally, the absence of objective and inexpensive reference to the beginning and the follow-up of the evolution of bleaching of the tooth makes its estimate perfectly subjective. |
<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>The purpose of the present invention is to solve the above-mentioned drawbacks by providing a complete bleaching unit made up of an electrochemical system, finding its application in particular in the dental and medical fields, allowing, thanks to the creation of an electrophoretic field, a strong, fast penetration and directing the bleaching agents, their removal under the same conditions together with the molecules responsible for coloration and an effective and controlled penetration of the stabilizing agents for the apatite crystal, basic component of the tooth, such as fluorine, but also, thanks to its light-based optical system, the activation of the photosensitive bleaching products by decreasing the heat effect while increasing the photonic effect thanks to a selection of wavelength in the range of 400-500 nm and, finally, thanks to an electro-optical unit, the control of the evolution of coloration by objective spectrocolorimetry-based methods. The purpose of the present invention is to solve these problems by providing a flexible and cheap solution usable both in a dentist's surgery and, in a simplified form, at the patient's home. In particular it solves the many problems mentioned above, because: by the means implemented, the device provides a natural source of energy for activating the components responsible for the bleaching, which is the ohm effect due to the passage of the electrophoretic current in a gel, the supplement brought by electric heat sources or also the addition of a photonic emission (LEDs), the cost of such a system, which allows avoiding the use of the lamps, is extremely low because an electrophoretic source is only an electric power supply. This cost becomes very low if the same power supply is used for an electric tooth-brush and the device of this invention. the means for controlling the electrophoretic energy, provided by a simple power supply similar to a power supply for an electric tooth-brush, allows its use at the patient's home. The communication by modem allows the dentist to follow up the evolution of the treatment over the Internet. the thermal rise caused by the device allows to activate the bleaching molecules before moving them towards the center of the tooth. the thermal value can be fully controllable thanks to a feedback device independent from the operator. if LEDs are used, it is not necessary to increase the time, compared to lamps with high thermal emission, because to the photonic effect is added the natural heating effect accompanying any electrophoretic current. since LEDs are tiny, they can be placed against the operation site, avoiding the use of a lamp with fibers, which reduces the cost of the device considerably. the doses implemented for the treatment at the dentist's as well as at home are very low for an identical effect and much higher effect at equal doses, because the active molecules are brought on the colored site and the molecules responsible for this coloration are evacuated by the electrophoretic currents. the evacuation of the colored molecules from the tooth reduces the repetitions. the reduction in the doses for a similar effect allows to reduce the side or undesirable effects such as the caustic action on the gum. To this end, the present invention relates to a device for activating reactive molecules responsible for bleaching and for selectively amplifying their movements towards the inside of the tooth, of the opposite movement of the products resulting from their internal action and their replacement, by a movement also captured by the device, of the molecules stabilizing and reinforcing the structure of the tooth, the whole under the control of a system for measuring their effectiveness. Thus the device according to the invention comprises: a central unit allowing to control the parameters defining thermal dynamics (such as the intensity, the variation, the speed or the acceleration), photonic dynamics (such as the power and the wavelength), electrophoretic dynamics (such as the intensity, the power, the frequency, the profile and the modulation, but also a system for controlling through feedback of the action in progress or obtained), the time, the storage, the reading and the transmission of the data on various carriers a primarily electro-opto-numerical extrabuccal unit, whether included or not in the central unit and containing: an electronic system creating a polarization field enabling us to obtain an electric, electromagnetic or electrophoretic field between the gel deposited on the tooth and the interior of the tooth and the function of which is to activate and direct a direct ion flow. By direct action, we understand that the electric field that we have created acts directly on the loads present (and total loads) of the molecules which are of interest to us in bleaching. We will thus have an electric or magnetic field acting directly on the positive or negative loads of the active molecules, such as the peroxide responsible for bleaching, carrying them towards the interior of the tooth, so that they can penetrate into it quickly and strongly. We will also have an opposite movement, whether simultaneous or not, of the whole or degraded coloring molecules, by the bleaching agents, so that they do not remain in the enamel and that they are not likely to recombine. We will finally have a last movement, whether combined or not with the bleaching agents, of the reconstituting ions of the tooth towards the interior of the enamel (for example, the negative ion fluorine at doses of 1.1 ppm). This action can be indirect when the molecules to be moved are not electrically active. In this case, it is no longer the acting molecules (peroxide, dye and fluorine . . . ) which are pulled by the electric field, but a complementary product sensitive to the electric field and capable, because of its electric characteristics, of being pulled by this field, and, because of its chemical characteristics, of fixing itself on the acting molecules or simply of pulling them without chemical bond, but by passive carrying away, like any moving fluid is capable of carrying particles. It should be specified that the incorporation of ions stabilizing and reinforcing the tooth fills the possible gaps resulting from the bleaching action. According to an additional feature of the device according to the invention, it includes a device activating the photosensitive products thanks to a LED, halogen or arc light system associated with a complementary thermal system in the event cold light (LED) is used and/or when the active products are heat-sensitive. The transport of the necessary energy to the area to be bleached occurs by means of fiber when light energy is involved, by a fluid when thermal energy is involved and by wires when electric power is involved. According to another additional feature of the device according to the invention, it includes a spectro or colorimetric sensor capable of storing and indicating, according to the comparative principle, the specific adjustments and the evolution of the coloration of the teeth. This spectrocolorimeter of elementary and inexpensive design, allows to measure, before and after treatment, the progressive change in coloration of the gel being loaded with products degraded and/or proceeding from the treated tooth released from them. The device according to the invention also comprises an endobuccal unit containing: a generally transparent gutter capable of containing the bleaching products such as the peroxide solutions, the dyes degraded by peroxide, and the fluorinated gel (for example). These gutters can be standard or individual for each patient and/or each type of product, in particular, in the case of indirect action. eventually a gutter included in the first one or adaptable to it, carrying the source of lights (LEDs for example) or guiding the (halogen or plasma) light to all or part of the arch, an electrophoretic connection ensuring, according to its polarization, the fast evacuation of the components degraded under the action of the light or of the activated peroxide, and inversely, the penetration of the bleaching products or the gel loaded with reconstituent components of the tooth, eventually a thermal source, such as for example electric filaments, allowing a controlled rise in the temperature at the level of the teeth to be treated. a gel containing the bleaching products and/or the reconstituent ions used for conducting current through the gutter from outside towards the interior of the tooth, and, eventually, another type of gel of polarization reversed or identical to the previous one, but the polarization of which has been reversed and aimed at receiving, and eventually at trapping, the coloring molecules moved out of the tooth. This gel constitutes the first pole of polarization. The device according to the invention also comprises a polar element, a kind of handle held in the hand or in the tooth or a flexible conductive surface applied on the patient's back and allowing to bring the reversed polarity in the human body with respect to that applied in the gel of the endobuccal gutter. It is now known that this polarity will be transmitted into the dental pulp thanks to the liquids present in the human body. The human body is thus the second pole creating the electrophoretic field with the gel of the gutter. This invention also consists in associating with a coherent aggregate of known bleaching processes the electrophoretic movements described here and in controlling their results obtained thanks to a simple monitoring system such as for example by colorimetry. The invention thus consists in more quickly bleaching the teeth, to reduce the cost of the treatment by transporting in an active way the bleaching products very close to the molecules responsible for their coloration, in ensuring a new, unique and actual evacuation of the degraded components, and in reinforcing the apatite structure of the enamel, thanks to the active bringing in of ions such as calcium or fluorine onto the site. This new and unique design is implemented thanks to the use of electrophoretic currents combined with a supporting system of thermal or photonic activation which can result from the electrophoresis. It is also known that a electrophoresis current causes the environmental temperature to increase during the passage of the current without another external intervention. Advantageously, the present invention thus consists in selecting a gel carrying the active bleaching ingredients capable of optimizing the well-known ohm effect accompanying the passage of the current and activating at the same time the active peroxide molecules, while avoiding the use of external heat or photonic sources. Advantageously and according to an additional feature of the device according to the invention, if the normal rise in the temperature is not sufficient, it comprises a gutter containing a unique filament serving both as electrophoretic pole and as electric resistor allowing a thermal rise during the passage of the current, thus creating the heat source. It is known, indeed, that any passage of current is accompanied by an ohm effect. It is thus proposed here to use a polarizing wire having, in addition, a fast and consequent thermal rise during the passage of current, even if a low current, and thus allowing to reinforce the chemical and electrophoretic effect, already described, of a thermal effect activating the molecules by increasing the energy vibration, making them more reactive. Advantageously and according to another additional feature of the device according to the invention, the electric filament will be of different size or made of different materials, according to the desired effect and, for example, and without this being restrictive, it will be of a larger size in the case of use under the control of a professional, such as a dental surgeon, in order to achieve a higher and faster thermal rise. It will be less powerful in the case of a private use at home, in order to avoid any risk. According to another additional feature of the device according to the invention, the means for adjusting the parameters of the operation of the heat source consist of means for selecting in a memory connected to said central unit and the selection of a determined profile among several pre-stored profiles in the memory. Indeed, it is known that an optimal temperature must be obtained in order to have the best possible reaction of activation of the active ingredients of peroxide. Below or above this temperature the molecule is less active. The purpose of the memory is to optimize the time of action and the temperature obtained by natural ohm effect or by the electric resistor according to the desired degree of action. These parameters resulting from tests in laboratory and well-known to the specialists allow one to preset a priori the value of the difference in potential between the two terminals of the electrophoresis, therefore, the power and the duration of application according to the desired degree of action. According to another additional feature of the device according to the invention, the means for adjusting the parameters of operation of the electrophoresis and the heat source are pre-stored in the memory of the central unit according to determined parameters, such as the time and the power and are adjustable by the practitioner. Advantageously, in another case, only one parameter, such as the time, can be changed, thus avoiding any false handling and this in particular within the framework of a private use. Advantageously, the memory connected to said central unit can be of a programmable type for recording the thermal profiles and/or the data relating to one or several adjustable parameters such as the voltage or the intensity of the current, capable of being selected. Preferably, the device includes data-entering means, such as a keyboard with keys and/or a touch screen and/or any other data-entering means, namely remote ones, for storing in the memory the energy profile and/or data corresponding to an adjustable parameter of the memory. In the latter case, the practitioner or the patient will be able to follow up the evolution of the treatment on a screen, over the Internet or a confidential modem or any other data-transmission means. Among the means of remote entering are the parameters for adjusting the profiles, the control of the operation of the apparatus and the detection of a possible breakdown thus allowing its remote monitoring, remote diagnosis or remote maintenance without physical intervention on the site of use. According to an additional device of the invention, the gutter contains sensors for measuring the current or thermal or optical sensors allowing to know the evolution of the treatment before removing the gutter. Indeed we now know that it is impossible to follow up and to know the actual state of the evolution of the bleaching treatment before the removal of the gutter. According to an additional device of this invention, this consists in providing the expert with means for knowing this evolution, thus for removing the gutter or for acting in feedback on the parameters such as the difference in potential, the intensity or the time by following the behavior of the current, because we know that the more an electrophoresis evolves, the more the characteristics of the current also evolve. A priori the variation of the electrical current can be tested on an in vitro bench and reported in vivo in order to follow up a priori the bleaching without being obliged to remove the gutter. According to the invention, there is an alternative to the thermal action. It is a photonic activation that can be included in the gutter and comprising a source of light and a routing of the light to the site that must receive it. This mode of action can be a compulsory alternative, because we know that some peroxides such as those present in the “ultra light” marketed by “SHOFU” are more photosensitive than heat-sensitive products. There is thus a source of light which can be halogen light, with LEDs or plasma light, such as Apollo 95 E , or any association between them and light of which is guided by a fiber to inside the gutter. This radiation ensures, on the one hand, a photonic activation corresponding to the wavelength selected according to the absorption spectrum of the peroxide molecule and, on the other hand, a thermal activation due to the release of heat accompanying any halogen or plasma radiation. Advantageously, this source of light is comprised of one or more light-emitting diodes (LEDs) capable of emitting in the photosensitive area of the activators of the bleaching reaction for the teeth ranging between 380 and 900 nanometers. These diodes, uniformly distributed over the gutter, for example opposite each tooth, allow the light to act at once on the complete arch. Advantageously, these diodes use 2 sources of wavelength, one activating camphoroquinone or PPDA (470 and 430 nanometers) and/or another one producing a thermal radiation thanks to LEDs emitting in the red and the infra-red (above 650 nanometers). Since the LEDs are small, they can be uniformly deposited on a die-carrier and alternately, in order to properly distribute the wavelengths over the arch. No light-conducting fiber is required, because LEDs are sufficiently small to be inserted into the patient's mouth. Only a current-supply cord connects the LEDs to the source of energy. This card will preferentially be a DC-DC supply. such as the one described in Patent FR 2,818,092. Indeed the supply mode can be the same, whether an activation reaction (peroxide) or condensation reaction (polymerization) is concerned. Advantageously, these sources of light have a profile of the programmable type varying in time, power or wavelength, drawn or not, allowing to optimize this activation reaction. Advantageously and according to this mode of action, there will be a uncoupling between the activation of the peroxide molecule, via the camphoroquinone, and the release of the fluorine ion. Indeed it is known that the fluorine ion can be an inhibitor in determined reactions. It is thus desirable for it to be released specifically in the electrophoretic action after the action of peroxide, i.e. after the degradation of the molecules responsible for coloration. To this end, the photonic activation of the camphoroquinone molecule and the release of fluorine will occur, according to an alternative embodiment of the invention, by two different wavelengths, one emitted by the LEDs at 470 nm for the camphoroquinone, and the other one by of LEDs the radiation of which will be different and will release the fluorine molecule at the end of the clinical bleaching operation. Indeed, and according to the body of the invention, there is a device allowing to fill the degradations of the dental structure during the evacuation of the coloring molecules. We realized that the coloring molecules penetrating most deeply into the tooth, locate themselves either in spaces between crystals or in the crystal itself when the latter is not completely structured following food deficits or drug treatments during its formation (tetracycline-based antibiotics therapy, NATHOO 1997). This situation, on the one hand, makes difficult its evacuation, which justifies high concentrations of peroxide, and, on the other hand, and this is dangerous for the tooth, a degradation of the crystal itself, which justifies the restriction of the use of bleaching products or the limitation in percentage of the active agents such as hydrogen peroxide by the international health authorities. It is known for a long time that the extraction of an ion, hydroxyl, calcium or its absence, makes the hydroxyapatite crystal unstable and vulnerable. This is often leads to dental carie. That is why we recommended fluoridations of the tooth, in order to fill these ionic lacks of fluorine or calcium. The present invention thus proposes to use the electric resistor or a complementary resistor as polarization field, allowing the active evacuation of the coloring molecules used for bleaching (peroxide), but also the creation of a reversed field, simultaneously or a posteriori, allowing a repairing ionic action as is the fluoridation of the dental body. Indeed, it is known that fluorine is one of the determining components in allowing a restructuring of the crystal of the tooth, enamel or dentine. For this reason this ion is administered to children and adults in the event of decalcification. This ion is indeed aimed at making up the calcium deficits in the apatite crystal, allowing a stabilization of the molecular structure. This is also the reason why in the bleaching kits is included, for example, “Opalescence F1” which contains 0.11% (that is 1.1 ppm) for 5% of carbamide peroxide, but which it must nowadays diffuse in a passive, hence random way. Advantageously, the present invention proposes to inject this ion, or a similar ion as to its action on protecting the tooth, by creating an electric field reversed with respect to the one used for the evacuation of the molecules responsible for dental coloration or of the same polarity (positive to attract fluorine, negative inside the tooth) but during evacuation and after the action of peroxide. The action can occur at the same time as the action of peroxide, since the ion used is no inhibitor. The present invention proposes to sequence the polarities according to the desired effect: action of peroxide, evacuation of the dyestuffs and reinforcement of the tooth according to the possible interrelationships of the molecules with each other. Using an electrophoretic mode not only allows to move these molecules in an active way, but also guarantees the separation of the chemical actions in order to avoid any interactive action that could be negative between the various reactions. It is the only method that allows to affirm that there is no chemical interrelationship in phases that must be separated. To this end, it can be summarized the present invention as being an electrophoretic and chemical, incidentally thermal, bleaching device, comprising a source of energy supplying a electrophoretic current varying in polarity and the conductors of which are connected, on the one hand, to the patient's body (first pole) and, on the other hand (second pole), to a gutter containing the chemicals responsible for a bleaching treatment. These electric conductors present in the gutter allow both thermal rise by Joule effect (which can be replaced by a photonic source), but also, because of their varying electrophoretic polarization, the displacement of the molecules towards the interior of the tooth so that they are quickly and deeply active (such as the peroxide molecules or the fluorine ions, reconstituent molecules of the tooth after bleaching), and the opposite displacement towards the outside of the tooth ensuring an active evacuation of the molecules responsible for coloration. The electrophoretic field is located at the level of the tooth. One of the poles is held in the hands or on the body of the patient, this polarity is transmitted via the blood and lymphatic ways to the dental pulp, and the other pole is transmitted by the electric resistor present in the gutter. |
Controller and method of controlling an apparatus |
In the field of electronic or computerised control apparatuses, known methods of compensating for disturbance signals rely on less than ideal analytical or empirical models. There is provided a controller (200) or method of controlling which observes and learns the correlation between various measured signals and automatically learns how to control the apparatus. It has a primary input (xR) for the measured state of the apparatus, and signal processing means (604, 606) responsive to the input signal for generating a control signal (V) to maintain the apparatus in a desired state, the controller (200) having further input(s) (X1, Xn) for additional measurements of the apparatus or its environment, the signal processing means using a weighted average (606) of results of different fixed impulse responses (611-615) to each input to modify the control signal, and means (630, 800, 802) for conditioning the response automatically in response to temporal cross-correlation observed between the measurement signal(s) and the control signal. The controller can be pre-taught with an appropriate response, or it can learn it during initial operation. |
1. A controller for controlling a physical apparatus, the controller comprising first input means for receiving a primary input signal representing a measured state of the apparatus, and signal processing means responsive to said primary input signal for generating a control signal for influencing the state of the apparatus so as to maintain a desired state, the controller having at least one further input for a signal representing additional measurements of the apparatus or its environment, said signal processing means including correcting means for including corrections in said control signal in response to said additional measurement signal, and means for conditioning the response of said correcting means automatically in response to temporal cross-correlation observed between said additional measurement signal and said control signal observed during operation of the controller and apparatus together. 2. A controller as claimed in claim 1, wherein the conditioning means comprises means for adjusting a gain and frequency response of a signal path in the correcting means. 3. A controller as claimed in claim 1, wherein the correcting means comprises means for filtering the additional signal via a plurality of filters having different fixed impulse responses and means for forming a weighted sum of the differently filtered signals to derive the correction to be applied, the conditioning means comprising means for adjusting the weightings of the different signal paths in response to their respective observed correlation with the control signal. 4. A controller as claimed in claim 1, wherein the conditioning means is arranged to observe said correlation by multiplying the signal of each path with a derivative of the control signal and to integrate the product of said signals over time to derive said weighting. 5. A controller as claimed in claim 4, wherein said integration is capable of being held at a fixed value such that said conditioning is maintained in the absence of stimulation. 6. A controller as claimed in claim 1 having a plurality of additional inputs, each said input having associated correcting means and conditioning means within the signal processing means. 7. A controller as claimed in claim 6, wherein different quantities of filters are used by each said correcting means. 8. A controller as claimed in claim 6, wherein different filter responses are used by each said correcting means. 9. A controller as claimed in claim 1 arranged to generate plural control signals based on at least some of the same input signals, the signal processing means including correcting means and conditioning means for generation of each control signal. 10. A controller as claimed in claim 9, wherein a first control signal is connected to serve as an additional measurement input to a correcting means for generating a second control signal. 11. A controller as claimed in claim 10, wherein a derivative or other transformation is applied to at least one of said measurement inputs as a preliminary processing step. 12. A controller as claimed in claim 1, wherein plural correcting means share filter components. 13. A controller substantially as claimed in claim 1, wherein the correcting means is replaced by one having a fixed response, which has been transferred from another controller which has learned said fixed response in operation. 14. A controller as claimed in claim 1, wherein a non-zero response has been set in the correcting means as a starting condition, said controller then being capable of adjusting the response of the correcting means during its continuing operation. 15. A method of controlling a physical apparatus, the method comprising on a continuous basis: receiving a primary input signal representing a measured state of the apparatus; generating a control signal for influencing the state of the apparatus in response to said primary input signal so as to maintain a desired state; receiving at least one further input for a signal representing additional measurements of the apparatus or its environment; including corrections in said control signal in response to said additional measurement signal; and conditioning the response of said correcting means automatically in response to temporal cross-correlation observed between said additional measurement signal and said control signal observed during operation of the controller and apparatus together. 16. A method as claimed in claim 15, wherein said correcting step comprises filtering the additional signal via a plurality of filter functions having different fixed impulse responses, and forming a weighted sum of the differently filtered signals to derive the correction to be applied, the conditioning step comprising adjusting the weightings of the different signal paths in response to their respective observed correlation with the control signal. 17. A method as claimed in claim 15, wherein the conditioning step begins from a zero condition. 18. A method as claimed in claim 15, wherein the conditioning step begins with a non-zero set of conditions. 19. A method as claimed in claim 18, wherein the conditioning step begins with a non-zero set of conditions learned in another apparatus. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which: FIG. 1 is a generalised block diagram of a conventional chemical processing plant, showing a typical closed loop control system; FIG. 2 shows schematically the values of certain signals over time in a typical closed loop control system responding in a reactive manner to an external disturbance; FIG. 3 shows the chemical processing plant of FIG. 1 modified with a CMP filter-based controller performing closed loop control of the steam entering the plant based on both the input and the output parameters of the plant; FIG. 4 shows the signals corresponding to those in FIG. 2 , in a closed loop control system using a cross-modal predictive filter responding directly to the same external disturbance after a learning phase; FIG. 5 is a schematic block diagram of a generalised process control system incorporating a CMP-filter based controller according to a generalised embodiment of the present invention; FIG. 6 shows in more detail a multi-stage CMP filter within the control system of FIG. 5 ; FIG. 7 shows response curves of a set of resonators in a CMP filter of the type shown in FIG. 6 , when presented with a square pulse stimulus; FIG. 8 shows in more detail a gain control circuit (GCC) associated with each resonator in the CMP filter of FIG. 6 ; FIG. 9 shows relationship between a single resonator output signal u i , a control signal v driving the process and its derivative v′, with respect to time; FIG. 10 is a schematic block diagram of an alternative form of CMP filter in which each resonator has a variable frequency response; FIG. 11 is a schematic block circuit diagram for a robot control system forming a more specific embodiment of the present invention; FIG. 12 is a simulation of the path of the robot under control of the circuit of FIG. 11 as it encounters obstacles in an environment over a period of time; and FIG. 13 illustrates the learning of optimum resonator weights for one disturbance signal within the robot circuit during the time period represented in FIG. 12 . detailed-description description="Detailed Description" end="lead"? |
Detection of single nucleotide polymorphisms |
A method determining the presence or absence of a single nucleotide polymorphism at a SNP site in a nucleic acid target. Capture probes are designed, each of which has a different SNP base and a sequence of probe bases on each side of the SNP base. The probe bases are complementary to the corresponding target sequence adjacent to the SNP site. Each capture probe is immobilized on a different electrode having a non-conductive outer layer on a conductive working surface of a substrate. The extent of hybridization between each capture probe and the nucleic acid target is detected by detecting the oxidation-reduction reaction at each electrode, utilizing a transition metal complex. These differences in the oxidation rates at the different electrodes are used to determine whether the selected nucleic acid target has a single nucleotide polymorphism at the selected SNP site. |
1. A method of determining the presence or absence of a single nucleotide polymorphism at a selected SNP site in a target nucleic acid, comprising: a) obtaining information about base sequences adjacent to the selected SNP site; b) providing a plurality of SNP capture probes, each of said SNP capture probes comprising a SNP base having a sequence of probe bases on at least one side of said SNP base, each said sequence of probe bases being complementary to a corresponding base sequence adjacent the selected SNP site, each of said SNP capture probes having a different SNP base, wherein the length of each of the SNP capture probes is sufficient so that the SNP capture probe having the SNP base that is complementary to the base at the SNP site in the target nucleic acid binds to the SNP target nucleic acid; c) providing an electrode for each SNP capture probe, each of said electrodes having a non-conductive immobilization layer on a conductive working surface of a substrate; d) immobilizing each SNP capture probe on its immobilization layer on its electrode; e) contacting each electrode with the target nucleic acid; f) contacting the target nucleic acid with a transition metal complex that oxidizes guanine in an oxidation-reduction reaction under conditions that cause an oxidation-reduction reaction between the transition metal complex and guanine, wherein there is electron transfer from guanine to the transition metal complex, resulting in regeneration of the reduced form of the transition metal complex as part of a catalytic cycle; g) detecting whether there is hybridization between each SNP capture probe and the target nucleic acid, by detecting the oxidation-reduction reaction at each electrode; and h) using the hybridization between each of the plurality of SNP capture probes and the target nucleic acid to determine whether the target nucleic acid has a single nucleotide polymorphism at the selected SNP site. 2. The method according to claim 1, wherein the nonconductive immobilization layer comprises a self-assembled monolayer. 3. The method according to claim 1, wherein the nonconductive immobilization layer comprises a polymer. 4. The method according to claim 1, wherein each of the capture probes comprises a neutral backbone. 5. The method according to claim 4, wherein the neutral backbone is selected from the group consisting of peptide backbones, p-ethoxy backbones and morpholine backbones. 6. The method according to claim 1, wherein each of the capture probes has a sugar-phosphate backbone. 7. The method according to claim 6, wherein the capture probe is an oligonucleotide. 8. The method according to claim 1, wherein there are four SNP capture probes. 9. The method according to claim 1, wherein three different bases are possible at the SNP site and there are three SNP capture probes. 10. The method according to claim 1, wherein two different bases are possible at the SNP site and there are two SNP capture probes. 11. The method according to claim 1, wherein each of the SNP capture probes has a sequence of probe bases on each side of said SNP base. 12. The method according to claim 11, wherein the sequence of probe bases on at least one side of the SNP base comprises at least four probe bases. 13. The method according to claim 1, wherein the sample is selected from the group consisting of: synthetic or natural oligonucleotides, surgical specimens, specimens used for medical diagnostics, specimens used for genetic testing, environmental specimens, food specimens, dental specimens and veterinary specimens. 14. The method according to claim 1, wherein each capture probe is immobilized on an electrode through a covalent bond to a silane molecule. 15. The method according to claim 1, wherein each capture probe is immobilized on an electrode through a covalent bond to a phosphonate molecule. 16. The method according to claim 1, wherein the immobilization layer comprises the immobilized capture probe. 17. The method according to claim 1, wherein the capture probe comprises 9-31 bases. 18. The method according to claim 1, wherein each SNP capture probe is on a soluble probe sequence that also comprises a second sequence that is complementary to a second capture probe immobilized on the electrode. 19. The method according to claim 18, wherein each of the SNP capture probes has a sequence of probe bases on each side of said SNP base. 20. The method according to claim 18, wherein the sequence of probe bases on at least one side of the SNP base comprises at least four probe bases. 21. The method according to claim 18, wherein the capture probe comprises 9-31 bases. 22. A method of determining the presence or absence of a single nucleotide polymorphism at a selected SNP site in a target nucleic acid, comprising: a) providing a plurality of SNP capture probes, each of said SNP capture probes comprising a SNP base having a sequence of probe bases on at least one side of said SNP base, each said sequence of probe bases being complementary to a corresponding base sequence adjacent the selected SNP site, each of said SNP capture probes having a different SNP base, wherein the length of each of the SNP capture probes is sufficient so that the SNP capture probe having the SNP base that is complementary to the base at the SNP site in the target nucleic acid binds to the SNP target nucleic acid; b) contacting each electrode with the target nucleic acid; c) contacting the target nucleic acid with a transition metal complex that oxidizes guanine in an oxidation-reduction reaction under conditions that cause an oxidation-reduction reaction between the transition metal complex and guanine, wherein there is electron transfer from guanine to the transition metal complex, resulting in regeneration of the reduced form of the transition metal complex as part of a catalytic cycle; d) detecting whether there is hybridization between each SNP capture probe and the target nucleic acid, by detecting the oxidation-reduction reaction at each electrode; and e) using the hybridization between each of the plurality of SNP capture probes and the target nucleic acid to determine whether the target nucleic acid has a single nucleotide polymorphism at the selected SNP site. 23. The method according to claim 22, wherein the nonconductive immobilization layer comprises a self-assembled monolayer. 24. The method according to claim 22, wherein the nonconductive immobilization layer comprises a polymer. 25. The method according to claim 22, wherein each of the capture probes comprises a neutral backbone. 26. The method according to claim 25, wherein the neutral backbone is selected from the group consisting of peptide backbones, p-ethoxy backbones and morpholine backbones. 27. The method according to claim 22, wherein each of the capture probes has a sugar-phosphate backbone. 28. The method according to claim 27, wherein the capture probe is an oligonucleotide. 29. The method according to claim 22, wherein there are four SNP capture probes. 30. The method according to claim 22, wherein three different bases are possible at the SNP site and there are three SNP capture probes. 31. The method according to claim 22, wherein two different bases are possible at the SNP site and there are two SNP capture probes. 32. The method according to claim 22, wherein each of the SNP capture probes has a sequence of probe bases on each side of said SNP base. 33. The method according to claim 32, wherein the sequence of probe bases on at least one side of the SNP base comprises at least four probe bases. 34. The method according to claim 22, wherein the sample is selected from the group consisting of: synthetic or natural oligonucleotides, surgical specimens, specimens used for medical diagnostics, specimens used for genetic testing, environmental specimens, food specimens, dental specimens and veterinary specimens. 35. The method according to claim 22, wherein each capture probe is immobilized on an electrode through a covalent bond to a silane molecule. 36. The method according to claim 22, wherein each capture probe is immobilized on an electrode through a covalent bond to a phosphonate molecule. 37. The method according to claim 22, wherein the immobilization layer comprises the immobilized capture probe. 38. The method according to claim 22, wherein the capture probe comprises 9-31 bases. 39. The method according to claim 22, wherein each SNP capture probe is on a soluble probe sequence that also comprises a second sequence that is complementary to a second capture probe immobilized on the electrode. 40. The method according to claim 39, wherein each of the SNP capture probes has a sequence of probe bases on each side of said SNP base. 41. The method according to claim 39, wherein the sequence of probe bases on at least one side of the SNP base comprises at least four probe bases. 42. The method according to claim 39, wherein the capture probe comprises 9-31 bases. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention This invention relates to a method of determining single nucleotide polymorphisms. 2. Description of the Related Art Single Nucleotide Polymorphism Studies. A single nucleotide polymorphism (SNP) is a single base change or point mutation resulting in genetic variation between individuals. SNPs occur in the human genome approximately once for every kilobase of the genome, and can occur in coding or non-coding regions of the genome. A SNP in the coding region may or may not change the amino acid sequence of a protein product. A SNP in a non-coding region can alter promoters or processing sites and affect gene transcription and processing. Knowledge of whether an individual has a particular SNP may provide sufficient information to develop diagnostic, preventative and therapeutic applications for a variety of diseases. In particular, such information allows prediction of drug responses, selection of clinical trial subjects, and identification of genetic subgroups, such as those susceptible to drug side-effects. A number of databases have been constructed of known SNPs and the effect of a SNP at a particular site. Examples of genetic conditions that are related to SNPs include sickle-cell anemia and long QT syndrome for sudden death from ventricular tachyarrhythmias. Other general information on SNPs is found in Wang et al. (1998, Science 280 , 1077 - 1082 ); and Collins et al. (1997, Science 278 , 1580 - 1581 ). Rutter et al. (1998, Cancer Research 58 , 5321 - 5325 ) discusses the impact a particular SNP has in a defined biochemical system. The disclosure of all patents and publications referred to herein is incorporated herein by reference. A wide variety of techniques have been devised to determine whether a particular genome or gene has a particular SNP present. Generally, unlike the instant invention, these prior techniques require fluorescent or enzymatic labeling. Some of these techniques require that the target material be amplified using a method such as the polymerase chain reaction (PCR). For example, the amplification of gene sequences has enabled sequencing of particular PCR products, as in the products of PE Biosystems (Foster City, Calif.). The specific hybridization of PCR primers to either wild-type or mutant alleles in PCR products enables accumulation of evidence on the genotype being investigated (AndCare, Inc., Durham, N.C., and Thetagen, Inc., Bothell, Wash.). Another technique for determining SNPs includes use of the mass spectrometer to measure probes that hybridize to the SNP. This technique varies in how rapidly it can be performed, from a few samples per day to a high throughput of 40,000 SNPs per day, using mass code tags. Companies using such techniques include Rapigene Inc. (Bothell, Wash.), Perseptive Biosystems (Foster City, Calif.) and Orchid Biocomputer (Princeton, N.J.). See Ross et al., 1997 , Anal. Chem. 69, 4197-4202. SNPs can also be determined by ligation-bit analysis. This analysis requires two primers that hybridize to a target with a one nucleotide gap between the primers. Each of the four nucleotides is added to a separate reaction mixture containing DNA polymerase, ligase, target DNA and the primers. The polymerase adds a nucleotide to the 3′end of the first primer that is complementary to the SNP, and the ligase then ligates the two adjacent primers together. Upon heating of the sample, if ligation has occurred, the now larger primer will remain hybridized and a signal, for example, fluorescence, can be detected. A further discussion of these methods can be found in U.S. Pat. Nos. 5,919,626; 5,945,283; 5,242,794; and 5,952,174. The techniques of Affymetrix (Santa Clara, Calif.) and Nanogen Inc. (San Diego, Calif.) utilize the fact that DNA duplexes containing single base mismatches are much less stable than duplexes that are perfectly base-paired. The presence of a matched duplex is detected by fluorescence. Another fluorescent technique for SNP analysis involves allowing a primer to hybridize to the DNA sequence adjacent to the SNP site on the test sample under investigation. The primer is extended by one nucleotide using all four differentially tagged fluorescent dideoxynucleotides (A,C,G, or T), and a DNA polymerase. Only one of the four nucleotides (homozygous case) or two of the four nucleotides (heterozygous case) is incorporated. The base that is incorporated is complementary to the nucleotide at the SNP position. This technique is used by Packard Instrument Company (Meriden, Conn.), PE Biosystems (Foster City, Calif.) and Orchid Biocomputer Inc. (Princeton, N.J.). The technique of Lynx Therapeutics (Hayward, Calif.) using MEGATYPE™ technology can genotype very large numbers of SNPs simultaneously from small or large pools of genomic material. This technology uses fluorescently labeled probes and compares the collected genomes of two populations, enabling detection and recovery of DNA fragments spanning SNPs that distinguish the two populations, without requiring prior SNP mapping or knowledge. Finally, other techniques rely on conformational differences between molecules. PCR products amplified with the same primers but containing different SNPs will have a different conformation after denaturing and annealing. This change in conformation results in a mobility shift in non-denaturing acrylamide gels that can be used to differentiate SNPs, relying on single-stranded conformational polymorphism (SSCP). The need to amplify and label the sample, and the difficulty of performing large numbers of analyses in the prior methods for SNP determination mean that these techniques are generally more time-intensive or labor-intensive than is desirable. Electrochemical Detection of Nucleic Acid Hybridization. The invention herein utilizes the prior method of electrochemical detection of nucleic acid hybridization of Thorp et al. (U.S. Pat. No. 5,871,918), the disclosure of which patent is incorporated herein by reference. Briefly, this patent discloses a new method of sequencing, and methods of qualitatively and quantitatively detecting a nucleic acid, such as DNA or RNA, that contains at least one preselected base, for example, adenine, guanine, 6-mercaptoguanine, 8-oxo-guanine, 8-oxo-adenine, or other base that undergoes oxidation upon reaction with a selected oxidizing agent. The method of Thorp et al. comprises: a) reacting the nucleic acid with a transition metal complex (mediator) capable of oxidizing the preselected base in an oxidation-reduction reaction; b) detecting the oxidation-reduction reaction; and c) determining the presence or absence of the nucleic acid from the detected oxidation-reduction reaction at the preselected base. Depending on the particular embodiment of the method of Thorp et al. that is employed, the method of Thorp et al. may optionally include the step of contacting the nucleic acid with a complementary nucleic acid probe to form a hybridized nucleic acid, generally as an initial step. The probe used in the method of Thorp et al. may be from about 4-6 bases to about 100 bases or more, and preferably is about 12-30 bases. The nucleic acid being analyzed may optionally be amplified using methods known in the art prior to contacting with the nucleic acid probe. A preferred transition metal complex for use as an oxidizing agent in the electrochemical detection of Thorp et al., which is reactive with the preselected base at a unique oxidation potential, is ruthenium 2+ (2,2′-bipyridine) 3 (Ru(bpy) 3 2+ ). Other suitable transition metal complexes are disclosed in U.S. Pat. No. 5,871,918, the disclosure of which is incorporated herein. The detection of the oxidation-reduction reaction of Thorp et al. typically utilizes a detection electrode that is sensitive to the transfer of electrons between the oxidizing agent (the transition metal complex) and the hybridized nucleic acid. Such an electrode is placed in contact with the solution containing the reacted hybridized nucleic acid and the oxidizing agent, along with a reference electrode and an auxiliary electrode. Suitable electrodes are known in the art, with a preferred electrode being an indium tin oxide electrode. The step of determining the presence or absence of hybridized nucleic acid typically includes (i) measuring the reaction rate of the oxidation-reduction reaction; (ii) comparing the measured reaction rate to the oxidation-reduction reaction rate of the transition metal complex with a single-stranded nucleic acid; and (iii) determining whether the measured reaction rate is essentially the same as the oxidation-reduction rate of the transition metal complex with the single-stranded nucleic acid. The oxidation-reduction rate may be determined by comparing the current as a function of scan rate, probe concentration, target concentration, transition metal complex, buffer, temperature, and/or electrochemical method. Typically, the oxidation-reduction reaction rate in the Thorp method is measured by measuring the electronic signal associated with the occurrence of the oxidation-reduction reaction, for example, by providing a suitable apparatus (e.g., a potentiostat) in electronic communication with the detection electrode, using methods such as cyclic voltammetry, normal pulse voltammetry, chronoamperometry, and square-wave voltammetry. Cyclic voltammetry and chronoamperometry are the preferred methods. The patent of Thorp et al. also teaches various types of apparatus, electrode structures and microelectronic devices for carrying out the nucleic acid detection. It is therefore an object of the invention to provide a method of SNP determination, utilizing a method of Thorp et al., that is both simple and sensitive, and that can rapidly be performed on many samples, because it does not require amplification of genomic material, and can be performed using a small volume of sample. In addition, the method of the invention does not generally require purification of the target. It is also an object of the invention to provide a method of SNP determination using a straightforward interrogation technique that provides a simple yes or no qualitative result that is universal in its application. Other objects and advantages will be more fully apparent from the following disclosure and appended claims. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention herein is a method of SNP determination comprising a method of determining the presence or absence of a single nucleotide polymorphism at a selected site (the “SNP site”) in a selected nucleic acid target. Using information about the normal base composition of the target at the selected SNP site and adjacent to the selected SNP site, capture probes are designed, each of which capture probes has a SNP base and a sequence of probe bases on each side of the SNP base. Preferably, there are about 9-31 nucleotides in the capture probe. The probe bases on each side of the SNP base are complementary to the corresponding target sequence adjacent to the selected SNP site. The SNP base is different in each of the capture probes that are being used to determine whether there is a single nucleotide polymorphism at the SNP site in the target nucleic acid. Each capture probe is immobilized on a different electrode having a non-conductive outer layer on a conductive working surface of a substrate. The extent of hybridization between each capture probe and the nucleic acid target is detected by detecting the oxidation-reduction reaction at each electrode, utilizing a transition metal complex. There will be different oxidation-reduction rates at the different electrodes depending on whether the nucleic acid target has hybridized to the capture probe. These differences in the oxidation rates at the different electrodes are used to determine whether the selected nucleic acid target has a single nucleotide polymorphism at the selected SNP site. Other objects and features of the inventions will be more fully apparent from the following disclosure and appended claims. |
Structural and cytoskeleton-associated protein |
Various embodiments of the invention provide human structural and cytoskeleton-associated proteins (SCAP) and polynucleotides which identify and encode SCAP. Embodiments of te invention also provide expression vectors, host cells, anti-bodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing discorders associated with aberrant expression of SCAP. |
1. An isolated polypeptide selected from the group consisting of: a) a poypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-5, SEQ ID NO:7-13, SEQ ID NO:15-16, SEQ ID NO:18-19, and SEQ ID NO:21-25, c) a polypeptide comprising a naturally occurring amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO:6, d) a polypeptide comprising a naturally occurring amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:14, e) a polypeptide comprising a naturally occurring amino acid sequence at least 94% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:17 and SEQ ID NO:20, f) a biological active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and g) an immonogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. 2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. 3. An isolated polynucleotide encoding a polypeptide of claim 1. 4. An isolated polynucleotide encoding a polypeptide of claim 2. 5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50. 6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3. 7. A cell transformed with a recombinant polynucleotide of claim 6. 8. A transgenic organism comprising a recombinant polynucleotide of claim 6. 9. A method of producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed. 10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. 11. An isolated antibody which specifically binds to a polypeptide of claim 1. 12. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-41 and SEQ ID NO:43-50, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO:42, d) a polynucleotide complementary to a polynucleotide of a), e) a polynucleotide complementary to a polynucleotide of b), f) a polynucleotide complementary to a polynucleotide of c), and g) an RNA equivalent of a)-f). 13. (CANCELED). 14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof. 15. (CANCELED). 16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof. 17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient. 18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. 19. (CANCELED). 20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample. 21. (CANCELED). 22. (CANCELED). 23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample. 24. (CANCELED). 25. (CANCELED). 26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1. 27. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1. 28. (CANCELED). 29. A method of assessing toxicity of a test compound, the method comprising: a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. 30-105. (CANCELED). |
<SOH> BACKGROUND OF THE INVENTION <EOH>The cytoskeleton is a cytoplasmic network of protein fibers that mediate cell shape, structure, and movement. The cytoskeleton supports the cell membrane and forms tracks along which organelles and other elements move in the cytosol. The cytoskeleton is a dynamic structure, that allows cells to adopt various shapes and to carry out directed movements. Major cytoskeletal fibers include the microtubules, the microflaments, and the intermediate filaments. Motor proteins, including myosin, dynein, and kinesin, drive movement of or along the fibers. The motor protein dynamin drives the formation of membrane vesicles. Accessory or associated proteins modify the structure or activity of the fibers while cytoskeletal membrane anchors connect the fibers to the cell membrane. Microtubules and Associated Proteins Tubulins Microtubules, cytoskeletal fibers with a diameter of about 24 nm, have multiple roles in the cell. Bundles of microtubules form cilia and flagella, which are whip-like extensions of the cell membrane that are necessary for sweeping materials across an epithelium and for swimming of sperm, respectively. Marginal bands of microtubules in red blood cells and platelets are important for these cells' pliability. Organelles, membrane vesicles, and proteins are transported in the cell along tracks of microtubules. For example, microtubules run through nerve cell axons, allowing bi-directional transport of materials and membrane vesicles between the cell body and the nerve terminal. Failure to supply the nerve terminal with these vesicles blocks the transmission of neural signals. Microtubules are also critical to chromosomal movement during cell division. Both stable and short-lived populations of microtubules exist in the cell Microtubules are polymers of GTP-binding tubulin protein subunits. Bach subunit is a heterodimer of α- and β-tubulin, multiple isoforms of which exist The hydrolysis of GTP is linked to the addition of tubulin subunits at the end of a microtubule. The subunits interact head to tail to form protofilaments; the protofilaments interact side to side to form a microtubule. A microtubule is polarized, one end ringed with α-tubulin and the other with β-tubulin, and the two ends differ in their rates of assembly. Generally, each microtubule is composed of 13 protofilaments although 11 or 15 protofilament-microtubules are sometimes found. Cilia and flagella contain doublet microtubules. Microtubules grow from specialized structures known as centrosomes or microtubule-organizing centers (MTOCs). MTOCs may contain one or two centrioles, which are pinwheel arrays of triplet microtubules. The basal body, the organizing center located at the base of a cilium or flagellum, contains one centriole. Gamma tubulin present in the MTOC is important for nucleating the polymerization of α- and β-tubulin heterodimers but does not polymerize into microtubules. The protein pericentrin is found in the MTOC and has a role in microtubule assembly. Microtubule-Associated Proteins Microtubule-associated proteins (MAPs) have roles in the assembly and stabilization of microtubules. One major family of MAPs, assembly MAPs, can be identified in neurons as well as non-neuronal cells. Assembly MAPs are responsible for cross-linking microtubules in the cytosol. These MAPs are organized into two domains: a basic microtubule-binding domain and an acidic projection domain. The projection domain is the binding site for-membranes, intermediate-filaments, or other microtubules. Based on sequence analysis, assembly MAPs can be further grouped into two types: Type I and Type II Type I MAPs, which include MAP1A and MAP1B, are large, filamentous molecules that co-purify with microtubules and are abundantly expressed in brain and testes. Type I MAPs contain several repeats of a positively-charged amino acid sequence motif that binds and neutralizes negatively charged tubulin, leading to stabilization of microtubules. MAP1A and MAP1B are each derived from a single precursor polypeptide that is subsequently proteolytically processed to generate one heavy chain and one light chain. Another light chain, LC3, is a 16.4 kDa molecule that binds MAP1A, MAP1B, and microtubules. It is suggested that LC3 is synthesized from a source other than the MAP1A or MAP1B transcripts, and that the expression of LC3 maybe important in regulating the microtubule binding activity of MAP1A and MAP1B during cell proliferation (Mann, S. S. et al. (1994) J. Biol. Chem. 269:11492-11497). Type II MAPs, which include MAP2a, MAP2b, MAP2c, MAP4, and Tau, are characterized by three to four copies of an 18-residue sequence in the microtubule-binding domain. MAP2a, MAP2b, and MAP2c are found only in dendrites, MAP4 is found in non-neuronal cells, and Tau is found in axons and dendrites of nerve cells. Alternative splicing of the Tau mRNA leads to the existence of multiple forms of Tau protein. Tau phosphorylation is altered in neurodegenerative disorders such as Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia and Parkinsonism linked to chromosome 17. The altered Tau phosphorylation leads to a collapse of the microtubule network and the formation of intraneuronal Tau aggregates (Spillantini, M. G. and M. Goedert (1998) Trends Neurosci. 21:428-433). Another microtubule associated protein, STOP (stable tubule only polypeptide), is a calmodulin-regulated protein that regulates stability (Denarier, E. et al. (1998) Biochem. Biophys. Res. Commun. 24:791-796). In order for neurons to maintain conductive connections over great distances, they rely upon axodendritic extensions, which in turn are supported by microtubules. STOP proteins function to stabilize the microtubular network. STOP proteins are associated with axonal microtubules, and are also abundant in neurons (Guillaud, L. et at (1998) J. Cell Biol. 142:167-179). STOP proteins are necessary for normal neurite formation, and have been observed to stabilize microtubules, in vitro, against cold-, calcium-, or drug-induced dissassembly (Margolis, R. L. et al. (1990) EMBO 9:4095-502). Microfilaments and Associated Proteins Actins Microfilaments, cytoskeletal filaments with a diameter,of about 7-9 nm, are vital to cell locomotion, cell shape, cell adhesion, cell division, and muscle contraction. Assembly and disassembly of the microfilaments allow cells to change their morphology. Microfilaments are the polymerized form of actin, the most abundant intracellular protein in the eukaryotic cell. Human cells contain six isoforms of actin. The three α-actins are found in different kinds of muscle, nonmuscle β-actin and nonmuscle γ-actin are found in nonmuscle cells, and another γ-actin is found in intestinal smooth muscle cells. G-actin, the monomeric form of actin, polymerizes into polarized, helical F-actin filaments, accompanied by the hydrolysis of ATP to ADP. Actin filaments associate to form bundles and networks, providing a framework to support the plasma membrane and determine cell shape. These bundles and networks are connected to the cell membrane. In muscle cells, thin filaments containing actin slide past thick filaments containing the motor protein myosin during contraction. A family of actin-related proteins exist that are not part of the actin cytoskeleton, but rather associate with microtubules and dynein. Actin-Associated Proteins Actin-associated proteins have roles in cross-linking, severing, and stabilization of actin filaments and in sequestering actin monomers. Several of the actin-associated proteins have multiple functions. Bundles and networks of actin filaments are held together by actin cross-linking proteins. These proteins have two actin-binding sites, one for each filament. Short cross-liking proteins promote bundle formation while longer, more flexible cross-linking proteins promote network formation. Actin-interacting proteins (AIPs) participate in the regulation of actin filament organization. Other actin-associated proteins such as TARA, a novel F-actin binding protein, function in a similar capacity by regulating actin cytoskeletal organization. Calmodulin-like calcium-binding domains in actin cross-linking proteins allow calcium regulation of cross-linking. Group I cross-linking proteins have unique actin-binding domains and include the 30 kD protein, EF-1a, fascin, and scruin. Group II cross-linking proteins have a 7,000-MW actin-binding domain and include villin and dematin. Group III cross-linking proteins have pairs of a 26,000-MW actin-binding domain and include fimbrin, spectrin, dystrophin, ABP 120, and filamin. Severing proteins regulate the length of actin filaments by breaking them into short pieces or by blocking their ends. Severing proteins include gCAP39, severin (fragmin), gelsolin, and villin. Capping proteins can cap the ends of actin filaments, but cannot break filaments. Capping proteins include CapZ and tropomodulin. The proteins thymosin and profilin sequester actin monomers in the cytosol, allowing a pool of unpolymerized actin to exist. The actin-associated proteins tropomyosin, troponin, and caldesmon regulate muscle contraction in response to calcium. Microtubule and actin filament networks cooperate in-processes such as vesicle and organelle transport, cleavage furrow placement, directed cell migration, spindle rotation, and nuclear migration. Microtubules and actin may coordinate to transport vesicles, organelles, and cell fate determinants, or transport may involve targeting and capture of microtubule ends at cortical actin sites. These cytoskeletal systems may be bridged by myosin-kinesin complexes, myosin-CLIP170 complexes, formin-homology (FH) proteins, dynein, the dynactin complex, Kar9p, coronin, ERM proteins, and kelch repeat-containing proteins (for a review, see Goode, B. L. et al. (2000) Curr. Opin. Cell Biol. 12:63-71). The kelch repeat is a motif originally observed in the kelch protein, which is involved in formation of cytoplasmic bridges called ring canals. A variety of mammalian and other kelch family proteins have been identified. The kelch repeat domain is believed to mediate interaction with actin (Robinson, D. N. and L. Cooley (1997) J. Cell Biol. 138:799-810). ADF/cofilins are a family of conserved 15-18 kDa actin-binding proteins that play a role in cytokinesis, endocytosis, and in development of embryonic tissues, as well as in tissue regeneration and in pathologies such as ischemia, oxidative or osmotic stress. LIM kinase 1 downregulates ADF (Carlier, M. F. et al. (1999) J. Biol. Chem. 274:33827-33830). LIM is an acronym of three transcription factors, Lin-11, Isl-1, and Mec-3, in which the motif was first identified. The LIM domain is a double zinc-finger motif that mediates the protein-protein interactions of transcription factors, signaling, and cytoskeleton-associated proteins (Roof, D. J. et al. (1997) J. Cell Biol. 138:575-588). These proteins are distributed in the nucleus, cytoplasm, or both (Brown, S. et al. (1999) J. Biol. Chem. 274:27083-27091). Recently, ALP (actinin-associated LIM protein) has been shown to bind alpha-actinin-2 (Bouju, S. et al. (1999) Neuromuscul. Disord. 9:3-10). The Frabin protein is another example of an actin-filament binding protein (Obaishi, H. et al. (1998) J. Biol. Chem. 273:18697-18700). Frabin (FGD1-related F-actin-binding protein) possesses one actin-filament binding (FAB) domain, one Dbl homology (DH) domain, two pleckstrin homology (PH) domains, and a single cysteine-rich FYVE (Fab1p, XOTB, Yac1p, and EEA1 (early endosomal antigen 1)) domain. Frabin has shown GDP/GTP exchange activity for Cdc42 small G protein (Cdc42), and indirectly induces activation of Rac small G protein (Rac) in intact cells. Through the activation of Cdc42 and Rac, Frabin is able to induce formation of both filopodia- and lamellipodia-like processes (Ono, Y. et al. (2000) Oncogene 19:3050-3058). The Rho family of small GTP-binding proteins are important regulators of actin-dependent cell functions including cell shape change, adhesion, and motility. The Rho family consists of three major subfamilies: Cdc42, Rac, and Rho. Rho family members cycle between GDP-bound inactive and GTP-bound active forms by means of a GDP/GTP exchange factor (GEF) (Umikawa, M. et al. (1999) J. Biol. Chem. 274:25197-25200). The Rho GEF family is crucial for microfilament organization. Intermediate Filaments and Associated Proteins Intermediate filaments (IFs) are cytoskeletal fibers with a diameter of about 10 nm, intermediate between that of microfilaments and microtubules. IFs serve structural roles in the cell, reinforcing cells and organizing cells into tissues. IFs are particularly abundant in epidermal cells and in neurons. IFs are extremely stable, and, in contrast to microfilaments and microtubules, do not function in cell motility. Five types of IF proteins are known in mammals. Type I and Type II proteins are the acidic and basic keratins, respectively. Heterodimers of the acidic and basic keratins are the building blocks of keratin IFs. Keratins are abundant in soft epithelia such as skin and cornea, hard epithelia such as nails and hair, and in epithelia that line internal body cavities. Mutations in keratin genes lead to epithelial diseases including epidermolysis bullosa simplex, bullous congenital ichthyosiform erythroderma (epidermolytic hyperkeratosis), non-epidermolytic and epidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens, pachyonychia congenita, and white sponge nevus. Some of these diseases result in severe skin blistering. (See, e.g., Wawersik, M. et al. (1997) J. Biol. Chem. 272:32557-32565; and Corden L. D. and W. H. McLean (1996) Exp. Dermatol. 5:297-307.) Type III IF proteins include desmin, glial fibrillary acidic protein, vimentin, and peripherin. Desmin filaments in muscle cells link myofibrils into bundles and stabilize sarcomeres in contracting muscle. Glial fibrillary acidic protein filaments are found in the glial cells that surround neurons and astrocytes. Vimentin filaments are found in blood vessel endothelial cells, some epithelial cells, and mesenchymal cells such as fibroblasts, and are commonly associated with microtubules. Vimentin filaments may have roles in keeping the nucleus and other organelles in place in the cell. Type IV IFs include the neurofilaments and nestin. Neurofilaments, composed of three polypeptides, NF-L, NF-M, and NF-H, are frequently associated with microtubules in axons. Neurofilaments are responsible for the radial growth and diameter of an axon, and ultimately for the speed of nerve impulse transmission. Changes in phosphorylation and metabolism of neurofilaments are observed in neurodegenerative diseases including amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease (Julien, J. P. and W. E. Mushynski (1998) Prog. Nucleic Acid Res. Mol. Biol. 61:1-23). Type V IFs, the lamins, are found in the nucleus where they support the nuclear membrane. IFs have a central α-helical rod region interrupted by short nonhelical linker segments. The rod region is bracketed, in most cases, by non-helical head and tail domains. The rod regions of intermediate filament proteins associate to form a coiled-coil dimer. A highly ordered assembly process leads from the dimers to the IFs. Neither ATP nor GTP is needed for IP assembly, unlike that of microfilaments and microtubules. IF associated proteins (IFAPs) mediate the interactions of IFs with one another and with other cell structures. IFAPs cross-link IFs into a bundle, into a network, or to the plasma membrane, and may cross-link IFs to the microfilament and microtubule cytoskeleton. Microtubules and IFs are, particularly closely associated. IFAPs include BPAG1, plakoglobin, desmoplakin I, desmoplakin II, plectin, filaggrin, and lamin B receptor. Cytoskeletal-Membrane Anchors Cytoskeletal fibers are attached to the plasma membrane by specific proteins. These attachments are important for maintaining cell shape and for muscle contraction. In erytbrocytes, the spectrin-actin cytoskeleton is attached to the cell membrane by three proteins, band 4.1, ankyrin, and adducin. Defects in this attachment result in abnormally shaped cells which are more rapidly degraded by the spleen, leading to anemia. In platelets, the spectrin-actin cytoskeleton is also linked to the membrane by a ankyrin; a second actin network is anchored to the membrane by filamin. In muscle cells the protein dystrophin links actin filaments to the plasma membrane; mutations in the dystrophin gene lead to Duchenne muscular dystrophy. Focal Adhesions Focal adhesions are specialized structures in the plasma membrane involved in the adhesion of a cell to a substrate, such as the extracellular matrix (ECM). Focal adhesions form the connection between an extracellular substrate and the cytoskeleton, and affect such functions as cell shape, cell motility and cell proliferation. Transmembrane integrin molecules form the basis of focal adhesions. Upon ligand binding, integrins cluster in the plane of the plasma membrane. Cytoskeletal linker proteins such as the actin binding proteins α-actinin, talin, tensin, vinculin, paxillin, and filamin are recruited to the clustering site. Key regulatory proteins, such as Rho and Ras family proteins, focal adhesion kinase, and Src family members are also recruited. These events lead to the reorganization of actin filaments and the formation of stress fibers. These intracellular rearrangements promote further integrin-ECM interactions and integrin clustering. Thus, integrins mediate aggregation of protein complexes on both the cytosolic and extracellular faces of the plasma membrane, leading to the assembly of the focal adhesion. Many signal transduction responses are mediated via various adhesion complex proteins, including Src, FAY, paxillin, and tensin. For a review, see Yamada, K. M. and B. Geiger, (1997) Curr. Opin. Cell Biol. 9.76-85.) IFs are also attached to membranes by cytoskeletal-membrane anchors. The nuclear lamina is attached to the inner surface of the nuclear membrane by the lamin B receptor. Vimentin IFs are attached to the plasma membrane by ankyrin and plectin. Desmosome and hemidesmosome membrane junctions hold together epithelial cells of organs and skin. These membrane junctions allow shear forces to be distributed across the entire epithelial cell layer, thus providing strength and rigidity to the epithelium. IFs in epithelial cells are attached to the desmosome by plakoglobin and desmoplakins. The proteins that link IFs to hemidesmosomes are not known. Desmin IFs surround the sarcomere in muscle and are linked to the plasma membrane by paranemin, synemin, and ankyrin. Motor Proteins Myosin-Related Motor Proteins Myosins are actin-activated ATPases; found in eukaryotic cells, that couple hydrolysis of ATP with motion. Myosin provides the motor function for muscle contraction and intracellular movements such as phagocytosis and rearrangement of cell contents during mitotic cell division (cytokinesis). The contractile unit of skeletal muscle, termed the sarcomere, consists of highly ordered arrays of thin actin-containing filaments and thick myosin-containing filaments. Crossbridges form between the thick and thin filaments, and the ATP-dependent movement of myosin heads within the thick filaments pulls the thin filaments, shortening the sarcomere and thus the muscle fiber. Myosins are composed of one or two heavy chains and associated light chains. Myosin heavy chains contain an amino-terminal motor or head domain, a neck that is the site of light-chain binding, and a carboxy-terminal tail domain. The tail domains may associate to form an α-helical coiled coil. Conventional myosins, such as those found in muscle tissue, are composed of two myosin heavy-chain subunits, each associated with two light-chain subunits that bind at the neck region and play a regulatory role. Unconventional myosins, believed to function in intracellular motion, may contain either one or two heavy chains and associated light chains. There is evidence for about 25 myosin heavy chain genes in vertebrates, more than half of them unconventional. Dynein-Related Motor Proteins Dyneins are (−) end-directed motor proteins which act on microtubules. Two classes of dyneins, cytosolic and axonemal, have been identified. Cytosolic dyneins are responsible for translocation of materials along cytoplasmic microtubules, for example, transport from the nerve terminal to the cell body and transport of endocytic vesicles to lysosomes. As well, viruses often take advantage of cytoplasmic dyneins to be transported to the nucleus and establish a successful infection (Sodeik, B. et al. (1997) J. Cell Biol. 136:1007-1021). Virion proteins of herpes simplex virus 1, for example, interact with the cytoplasmic dynein intermediate chain (Ye, G. J. et al. (2000) J. Virol. 74:1355-1363). Cytoplasmic dyneins are also reported to play a role in mitosis. Axonemal dyneins are responsible for the beating of flagella and cilia. Dynein on one microtubule doublet walks along the adjacent microtubule doublet. This sliding force produces bending that causes the flagellum or cilium to beat. Dyneins have a native mass between 1000 and 2000 kDa and contain either two or three force-producing heads driven by the hydrolysis of ATP. The heads are linked via stalks to a basal domain which is composed of a highly variable number of accessory intermediate and light chains. Cytoplasmic dynein is the largest and most complex of the motor proteins. Nebulin-Related Proteins Nebulin is a large sarcomeric protein that interacts with actin filaments in skeletal muscle (Wang, K. et al. (1996) J. Biol. Chem. 271:4304-4314). Nebulin contains 185 or more copies of a 35-residue module that has a consensus sequence and a predicted α-helical structure. The 35-residue module comprises an actin-binding domain. In the central region of nebulin, the 35-residue modules exhibit a seven module super-repeat pattern. This super-repeat pattern is not present in the C-terminal 100 kDa region of nebulin. The N-terminal region of nebulin contains 8 linker modules and an 8 kDa acidic domain. The C-terminal region is distinct and contains an SH3 domain. Within the sarcomere, nebulin is oriented with its C-terminus located at the Z-line, and its N-terminus at the pointed slow-growing end of thin filaments in the acto-myosin overlap region. Nebulin exists as different isoforms which range in size from 600-900 kDa (Kruger, M. et al. (1991) J. Cell. Biol. 115:97-107). The size of nebulin is tissue- and species-specific and is developmentally regulated. Based on the observation that isoform size correlates with the length of thin filaments in skeletal muscle, nebulin is proposed to play a role as a molecular ruler that regulates the length of thin filaments. Each nebulin 35-residue module may associate with one actin monomer; thus, isoforms with different numbers of modules could determine the length of thin filaments. The N-terminal region of nebulin interacts with tropomodulin, which may assist in this function (McElhinny, A. S. et al. (2001) J. Biol. Chem. 276:583-592). Tropomodulin caps actin at the pointed end of thin filaments and maintains filament length by preventing actin monomer dissociation or addition. Nebulin is absent from cardiac muscle, but related proteins with nebulin-like modules may provide similar functions. Nebulette, for example, is specifically expressed in heart and has a C-terminal region containing twenty-three 35-residue nebulin-like modules (Moncman, C. L. and Wang, K. (2000) J Muscle Res. Cell Motil. 21:153-169; Millevoi, S. et al. (1998) J. Mol. Biol. 282:111-123). The domain structure of nebulette is similar to nebulin, though it is a smaller protein of only 107 kDa. It has an acidic N-terminal domain, a repeat domain containing nebulin-like modules, a linker domain, and an SH3 domain. The repeat domain of nebulette is about one-tenth the size of that of nebulin. The 35-residue modules of nebulette have a consensus motif, and a subfamily of modules 15-22 share a conserved motif. Unlike nebulin, nebulette modules do not display a super-repeat pattern. Nebulette binds to actin as well as other sarcomeric proteins including myosin, calmodulin, tropomyosin, troponin, and α-actinin (Moncman, C. L. and Wang, K. (1999) Cell Motil. Cytoskeleton 44:1-22). The orientation of nebulette in the sarcomere is analogous to that of nebulin with its C-terminus at the Z-line and its N-terminus in the I-band. Nebulin-related anchoring protein (N-RAP) is expressed in cardiac and skeletal muscle (Luo, G. et al. (1997) Cell Motil. Cytoskeleton 38:75-90). It is a 133 kDa protein found at the ends of myofibrils at muscle myotendon junctions and intercalated disks. The C-terminal region of N-RAP has 27 copies of the 35-residue nebulin-like modules. Seventeen of the modules are organized in a super-repeat pattern. The N-terminal region contains a cysteine-rich LIM domain. LIM domains bind two zinc ions in two adjacent zinc finger-like structures and are known to mediate protein-protein interactions. N-RAP may mediate interactions between actin filaments of myofibrils and other sarcomeric proteins. N-RAP binds to actin, talin, and vinculin (Luo, G. et al. (1999) Biochemistry 38:6135-6143). It interacts with actin and vinculin through its super-repeat region and with talin through its LIM domain. Talin and vinculin are also located at myotendon junctions and together with N-RAP may provide a link between actin filaments of the myofibril and the sarcolemma and transmit tension from the myofibril to the extracellular matrix Mutations of sarcomeric proteins are associated with muscle weakness and disease (Laing, N. G. (1999) Curr. Opin. Neurol. 12:513-518). Autosomal recessive nemaline myopathy in some cases is caused by nebulin deficiency (Pelin, K. et al. (1999) Proc. Natl. Acad. Sci. 96:2305-2310). The disease is characterized by the presence of nemaline bodies in muscle fibers. The nemaline bodies contain proteins normally associated with the Z disc and thin filament. Defects in nebulin apparently perturb interactions among sarcomeric proteins and result in the pathological aggregation of proteins in nemaline bodies. Kinesin-Related Motor Proteins Kinesins are (+) end-directed motor proteins which act on microtubules. The prototypical kinesin molecule is involved in the transport of membrane-bound vesicles and organelles. This function is particularly important for axonal transport in neurons. Kinesin is also important in all cell types for the transport of vesicles from the Golgi complex to the endoplasmic reticulum. This role is critical for maintaining the identity and functionality of these secretory organelles. Kinesins define a ubiquitous, conserved family of over 50 proteins that can be classified into at least 8 subfamilies based on primary amino acid sequence, domain structure, velocity of movement, and cellular function. (Reviewed in Moore, J. D. and S. A. Endow (1996) Bioessays 18:207-219; and Hoyt, A. M. (1994) Curr. Opin. Cell Biol. 6:63-68.) The prototypical kinesin molecule is a heterotetramer comprised of two heavy polypeptide chains (KHCs) and two light polypeptide chains (KLCs). The KHC subunits are typically referred to as “kinesin.” KHC is about 1000 amino acids in length, and KLC is about 550 amino acids in length. Two KHCs dimerize to form a rod-shaped molecule with three distinct regions of secondary structure. At one end of the molecule is a globular motor domain that functions in ATP hydrolysis and microtubule binding. Kinesin motor domains are highly conserved and share over 70% identity. Beyond the motor domain is an α-helical coiled-coil region which mediates dimerization. At the other end of the molecule is a fan-shaped tail that associates with molecular cargo. The tail is formed by the interaction of the KHC C-termini with the two KLCs. Members of the more divergent subfamilies of kinesins are called kinesin-related proteins (KRPs), many of which function during mitosis in eukaryotes (Hoyt, supra). Some KRPs are required for assembly of the mitotic spindle. In vivo and in vitro analyses suggest that these KRPs exert force on microtubules that comprise the mitotic spindle, resulting in the separation of spindle poles. Phosphorylation of KRP is required for this activity. Failure to assemble the mitotic spindle results in abortive mitosis and chromosomal aneuploidy, the latter condition being characteristic of cancer cells. In addition, a unique KRP, centromere protein E, localizes to the kinetochore of human mitotic chromosomes and may play a role in their segregation to opposite spindle poles. Dynamin-Related Motor Proteins Dynamin is a large GTPase motor protein that functions as a “molecular pinchase,” generating a mechanochemical force used to sever membranes. This activity is important in forming clathrin-coated vesicles from coated pits in endocytosis and in the biogenesis of synaptic vesicles in neurons. Binding of dynamin to a membrane leads to dynamin's self-assembly into spirals that may act to constrict a flat membrane surface into a tubule. GTP hydrolysis induces a change in conformation of the dynamin polymer that pinches the membrane tubule, leading to severing of the membrane tubule and formation of a membrane vesicle. Release of GDP and inorganic phosphate leads to dynamin disassembly. Following disassembly the dynamin may either dissociate from the membrane or remain associated to the vesicle and be transported to another region of the cell. Three homologous dynamin genes have been discovered, in addition to several dynamin-related proteins. Conserved dynamin regions are the N-terminal GTP-binding domain, a central pleckstrin homology domain that binds membranes, a central coiled-coil region that may activate dynamin's GTPase activity, and a C-terminal proline-rich domain that contains several motifs that bind SH3 domains on other proteins. Some dynamin-related proteins do not contain the pleckstrin homology domain or the proline-rich domain (See McNiven, M. A. (1998) Cell 94:151-154; Scaife, R. M. and R. L. Margolis (1997) Cell. Signal. 9:395401.) The cytoskeleton is reviewed in Lodish, H. et al. (1995) Molecular Cell Biology , Scientific American Books, New York N.Y. Cyclic Nucleotide Signaling Cyclic nucleotides (cAMP and cGMP) function as intracellular second messengers to transduce a variety of extracellular signals including hormones, light, and neurotransmitters. In particular, cyclic-AMP dependent protein kinases (PKA) are thought to account for all of the effects of cAMP in most mammalian cells, including various hormone-induced cellular responses. Visual excitation and the phototransmission of light signals in the eye is controlled by cyclic-GMP regulated, Ca 2+ -specific channels. Because of the importance of cellular levels of cyclic nucleotides in mediating these various responses, regulating the synthesis and breakdown of cyclic nucleotides is an important matter. Thus adenylyl cyclase, which synthesizes cAMP from AMP, is activated to increase cAMP levels in muscle by binding of adrenaline to β-adrenergic receptors, while activation of guanylate cyclase and increased cGMP levels in photoreceptors leads to reopening of the Ca 2+ -specific channels and recovery of the dark state in the eye. There are nine known transmembrane isoforms of mammalian adenylyl cyclase, as well as a soluble form preferentially expressed in testis. Soluble adenylyl cyclase contains a P-loop, or nucleotide binding domain, and may be involved in male fertility (Buck, J. et al. (1999) Proc. Natl. Acad. Sci. USA 96:79-84). In contrast, hydrolysis of cyclic nucleotides by cAMP and cGMP-specific phosphodiesterases (PDEs) produces the opposite of these and other effects mediated by increased cyclic nucleotide levels. PDEs appear to be particularly important in the regulation of cyclic nucleotides, considering the diversity found in this family of proteins. At least seven families of mammalian PDEs (PDE1-7) have been identified based on substrate specificity and affinity, sensitivity to cofactors, and sensitivity to inhibitory drugs (Beavo, J. A. (1995) Physiol. Rev. 75:725-748). PDE inhibitors have been found to be particularly useful in treating various clinical disorders. Rolipram, a specific inhibitor of PDE4, has been used in the treatment of depression, and similar inhibitors are undergoing evaluation as anti-inflammatory agents. Theophylline is a nonspecific PDE inhibitor used in the treatment of bronchial asthma and other respiratory diseases (Banner, K. H. and C. P. Page (1995) Eur. Respir. J. 8:996-1000). Expression Profiling Microarrays are analytical tools used in bioanalysis. A microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry. One area in particular in which microarrays find use is in gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder. |
<SOH> SUMMARY OF THE INVENTION <EOH>Various embodiments of the invention provide purified polypeptides, structural and cytoskeleton-associated proteins, referred to collectively as “SCAP” and individually as “SCAP-1,” “SCAP-2,” “SCAP-3,” “SCAP-4,” “SCAP-5,” “SCAP-6,” “SCAP-7, ” “SCAP-8,” “SCAP-9,” “SCAP-10,” “SCAP-11,” “SCAP-12,” “SCAP-13,” “SCAP-14,” “SCAP-15,” “SCAP-16, “SCAP-17,” “SCAP-18,” “SCAP-19,” “SCAP-20,” “SCAP-21,” “SCAP-22,” “SCAP-23,” “SCAP-24,” and “SCAP-25,” and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions. Embodiments also provide methods for utilizing the purified structural and cytoskeleton-associated proteins and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology. Related embodiments provide methods for utilizing the purified structural and cytoskeleton-associated proteins and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions. An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:1-25. Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-25. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:26-50. Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from tie group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide. Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed. Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. Still yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). In other embodiments, the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides. Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex. In a related embodiment, the method can include detecting the amount of the hybridization complex. In still other embodiments, the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides. Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof. In a related embodiment, the method can include detecting the amount of the amplified target polynucleotide or fragment thereof. Another embodiment provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and a pharmaceutically acceptable excipient. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional SCAP, comprising administering to a patient in need of such treatment the composition. Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional SCAP, comprising administering to a patient in need of such treatment the composition. Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional SCAP, comprising administering to a patient in need of such treatment the composition. Another embodiment provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide. Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide. Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. |
Methods for preventing and treating diseases and conditions associated with cellular stress |
This invention relates to methods of preventing and treating diseases and conditions associated with cellular stress. The methods involve administration of an effective amount of a compound of the formula. |
1. A method of ameliorating one or more symptoms of an alcohol-induced or alcohol-exacerbated injury to, or condition of, the gastrointestinal tract in a person, said method comprising administering to said person an effective amount of a compound of the formula: 2. The method of claim 1, wherein said injury or condition is selected from the group consisting of non-steroidal anti-inflammatory drug-induced gastroduodenal injury, gastritis, dyspepsia, nausea, gastrointestinal distress, abdominal pain, esophagitis, gastroesophageal reflux disease, colitis, irritable bowel syndrome, inflammatory bowel disease, diverticulosis, diverticulitis, celiac disease, Zollinger-Ellison disease, Helicobacter pylori infection, heartburn, hiatal hernia, and Barrett's esophagus. 3. The method of claim 1, wherein said injury or condition is hangover. 4. The method of claim 1, wherein administration is carried out either: (i) within two hours prior to drinking an alcoholic beverage; (ii) during the period of drinking alcoholic beverages; (iii) within one hour after drinking the last in a series of alcoholic beverages; (iv) within fourteen hours after drinking the last in a series of alcoholic beverages; or (v) two or more of (i)-(iv). 5. The method of claim 1, wherein the total amount of said compound administered over a fourteen-hour period is 10-1,000 mg. 6. The method of claim 5, wherein the total amount of said compound administered in said period is 50-500 mg. 7. The method of claim 6, wherein the total amount of said compound administered is 100-300 mg. 8. The method of claim 2, wherein said inflammatory bowel disease is Crohn's disease. 9. The method of claim 2, wherein said inflammatory bowel disease is ulcerative colitis. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The body responds to cellular stress by a highly conserved response that includes the induction of a family of proteins known as stress proteins, which are also referred to as heat shock proteins and chaperones. These proteins play essential roles in the normal functioning of cells, and also have been implicated in several diseases and conditions, including heart disease, diabetes, autoimmune disease, cancer, and cellular stress caused by, for example, exposure to high temperatures, low oxygen levels (i.e., ischemia), inflammation, infection, heavy metal exposure, and alcohol. Consumption of ethanol-containing drinks, such as beer, wine, and liquor, can cause cellular stress in, and thereby induce or exacerbate a number of conditions of, the gastrointestinal tract including, for example, gastritis, abdominal pain, gastrointestinal distress, dyspepsia, and ulcers. In addition, excessive alcohol consumption can cause the physical discomforts often felt by persons the morning after such consumption, which are collectively known as hangover. The severity of a hangover depends on several factors, including the ability of the person to process alcohol, the non-alcoholic components of the drinks (called congeners), the amount of alcohol consumed over a particular time period, and the severity of the dehydration caused by the excessive alcohol consumption. There are many supposed treatments for hangover, including black coffee and further consumption in the morning of small amounts of alcohol. None of these measures adequately treats the symptoms of hangover, which can include abdominal discomfort, nausea, fatigue, and headache. |
<SOH> SUMMARY OF THE INVENTION <EOH>This invention provides methods of ameliorating one or more symptoms of a disease or condition associated with cellular stress. For example, the methods of the invention can be used in the prevention or treatment of an alcohol-induced or alcohol-exacerbated injury to, or condition of, the gastrointestinal tract. As a specific example, the methods of the invention can be used in the prevention and treatment of hangover. Additional examples of diseases and conditions that can be prevented or treated using the methods of the invention are provided below. The methods of the invention involve use of a drug known as Selbex®, which has the formula: Selbex® is a drug that was first marketed by Eisai Co., Ltd. in Japan in 1994 for the treatment of peptic ulcers. Selbex®, its synthesis, and its formulations are described in U.S. Pat. No. 4,169,157, which is incorporated herein by reference. According to the invention, administration of Selbex® is carried out to ameliorate one or more symptoms of gastrointestinal injuries or conditions such as, for example, hangover, non-steroidal anti-inflammatory drug (NSAID)-induced gastroduodenal injury (caused by, e.g., aspirin, acetaminophen, or ibuprophen), gastritis, dyspepsia, nausea, gastrointestinal distress, abdominal pain, esophagitis, gastroesophageal reflux disease (GERD), colitis, irritable bowel syndrome, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), diverticulosis, diverticulitis, celiac disease, Zollinger-Ellison disease, Helicobacter pylori infection, heartburn, hiatal hernia, and Barrett's esophagus. Additional examples of diseases and conditions that can be prevented or treated using the methods of the invention are provided below. When used to prevent or to treat hangover, the administration can take place, for example, either: (i) within two hours prior to drinking an alcoholic beverage; (ii) during the drinking of alcoholic beverages; (iii) within one hour after drinking the last in a series of alcoholic beverages; (iv) within fourteen hours after drinking the last in a series of alcoholic beverages; or (v) at two or more of the time points specified in (i)-(iv). The total amount of Selbex® administered over a fourteen hour period can be, for example, 10-1,000 mg, e.g., 50-500 mg, e.g., 100-300 mg, as determined to be appropriate by those of skill in this art. The amount of Selbex® administered can vary, depending on the body weight of the patient, the alcohol tolerance of the patient,,and the amount of alcohol consumed. The invention also includes the use of Selbex® in the preparation of medicaments for preventing and treating the diseases and conditions described herein. The methods of the invention are effective and produce little in the way of side effects. Other features and advantages of the invention will be apparent from the following detailed description and from the claims. detailed-description description="Detailed Description" end="lead"? |
Mechanic skill control system |
A mechanic skill managing system includes a first database storing an average operation time for every kind of check/repair, the average operation time being a time required for an average mechanic with an average skill to complete the check/repair, and a second database storing a skill value indicative of skill of a mechanic, both of which are connected to a processor. The processor calculates the skill value of the mechanic based on a difference or a ratio between the average operation time stored in the first database and an actual operation time spent by the mechanic to actually complete the check/repair, and registers the skill value in the second database. Thus, the skill of the mechanic can be grasped accurately and can be applied to various operations. |
1. A mechanic skill managing system comprising: a first database storing an average operation time for every kind of check/repair, said average operation time being a time required for an average mechanic having an average skill to complete said check/repair; a second database storing a skill value indicative of skill of a mechanic; and a processor calculating said skill value of said mechanic based on a difference or a ratio between said average operation time stored in said first database and an actual operation time spent by said mechanic to actually complete said check/repair, and registering said skill value in said second database. 2. The mechanic skill managing system according to claim 1, wherein said average operation time stored in said first database is calculated by averaging a plurality of times spent by a plurality of mechanics to actually complete said check/repair, except a peculiar time. 3. The mechanic skill managing system according to claim 1, wherein said second database further comprises information indicative of history of training attendances and years of experience, and said skill value of said mechanic is calculated in said processor based on said difference or said ratio, and said information indicative of said history of training attendances and said years of experience stored in said second database. 4. The mechanic skill managing system according to claim 2, wherein said second database further comprises information indicative of history of training attendances and years of experience, and said skill value of said mechanic is calculated in said processor based on said difference or said ratio, and said information indicative of said history of training attendances and said years of experience stored in said second database. 5. The mechanic skill managing system according to claim 1, wherein said processor further determines whether a requested check/repair of a vehicle is acceptable or not, based on said skill value of said mechanic registered in said second database. 6. The mechanic skill managing system according to claim 2, wherein said processor further determines whether a requested check/repair of a vehicle is acceptable or not, based on said skill value of said mechanic registered in said second database. 7. The mechanic skill managing system according to claim 3, wherein said processor further determines whether a requested check/repair of a vehicle is acceptable or not, based on said skill value of said mechanic registered in said second database. 8. The mechanic skill managing system according to claim 4, wherein said processor further determines whether a requested check/repair of a vehicle is acceptable or not, based on said skill value of said mechanic registered in said second database. 9. The mechanic skill managing system according to claim 1, wherein said processor further determines compensation for work of said mechanic, based on said skill value of said mechanic registered in said second database. 10. The mechanic skill managing system according to claim 2, wherein said processor further determines compensation for work of said mechanic, based on said skill value of said mechanic registered in said second database. 11. The mechanic skill managing system according to claim 3, wherein said processor further determines compensation for work of said mechanic, based on said skill value of said mechanic registered in said second database. 12. The mechanic skill managing system according to claim 4, wherein said processor further determines compensation for work of said mechanic, based on said skill value of said mechanic registered in said second database. 13. The mechanic skill managing system according to claim 5, wherein said processor further determines compensation for work of said mechanic, based on said skill value of said mechanic registered in said second database. 14. The mechanic skill managing system according to claim 6, wherein said processor further determines compensation for work of said mechanic, based on said skill value of said mechanic registered in said second database. 15. The mechanic skill managing system according to claim 7, wherein said processor further determines compensation for work of said mechanic, based on said skill value of said mechanic registered in said second database. 16. The mechanic skill managing system according to claim 8, wherein said processor further determines compensation for work of said mechanic, based on said skill value of said mechanic registered in said second database. |
<SOH> BACKGROUND ART <EOH>Conventionally, a check of a vehicle has been carried out in a maintenance shop by request of a user. In this case, in the maintenance shop, a total man-hour is calculated by adding up respective man-hours in a plurality of check items defined for each kind of the check. Here, the man-hour means a work amount carried out by one worker in a unit time when a work load is represented by using time as a unit. Then, an operation is carried out by referring to a maintenance schedule controlled in the maintenance shop, assigning a day and an hour for the requested check on which the total man-hour can be accomplished, and carrying out the maintenance. As such a technique, for example, Japanese Laid Open Patent Application (JP-P2000-20581A) discloses a “System for Managing Vehicle Storage for Maintenance”. In this system, a client enters desired maintenance contents and desired storage terms by using a terminal. In response to this procedure, a host computer calculates a period necessary for the maintenance on the basis of the desired maintenance contents, and then searches a storing/retrieving management table stored in a memory apparatus of the host computer for an available period for the maintenance on the basis of the desired storage terms of the client. Then, an optimal date in this period for the storage is automatically determined and displayed on the terminal. In response to a confirmation of the client through the terminal, the optimal date of storage and a date of retrieval determined from the date of storage are automatically registered in the storing/retrieving management table, and so a storage reservation is made. Thus, clients can register the storage by themselves. When the check of the vehicle is requested as mentioned above, the man-hour is calculated on the basis of a standard man-hour. This standard man-hour is pre-determined by averaging experience and performance of a plurality of mechanics. Therefore, a time necessary for the check varies widely from a mechanic with a high skill and a mechanic without that high skill. Incidentally, in recent years, the request for the check of the vehicle is made in various ways. For example, there are many clients in recent years who want the check to be completed in a short time such as 0.5 hours or one hour on their way to offices. However, conventionally, the time necessary for the requested check has been calculated on the basis of the standard man-hour, and whether the requested check can be accepted or not is determined on the basis of the calculated time. Hence, the requested check is sometimes determined to be unacceptable even when a mechanic with the high skill can complete the requested check within the time to meet the client's requirement. It is therefore desired to develop a system in which the skill of the mechanics can be grasped accurately and a proper mechanic can be assigned to the requested check so that the requested check is completed within the appointed date according to the user's requirement. The present invention is proposed in order to cope with the above-mentioned request. The object is to provide a mechanic skill managing system, in which skill of a mechanic can be grasped accurately and can be applied to various operations. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 is a block diagram showing a configuration of a mechanic skill managing system according to a best mode for carrying out the present invention. FIG. 2 is a flowchart describing an operation of the mechanic skill managing system according to a best mode for carrying out the present invention. detailed-description description="Detailed Description" end="lead"? |
Printer or other media processor with on-demand selective media converter and variable peeler |
On demand apparatus for use in a printer, printer module, stand-alone media converter, or other media processor comprises a print device and a converting system. The print device receives a series of labels, tickets, tags, cards or other media samples and responds to a set of form and content print instructions which direct the print device regarding what and where to print on selected media samples. The converting system includes an applicator which receives the series of media samples from the print device and a series of value-adding elements. The applicator responds to a set of application instructions which direct the applicator to apply a value-adding element to selected media samples. A corresponding method is disclosed. |
1. A method of selectively peeling or not peeling a succession of peelable web-carried transponders, comprising: a. providing an adjustable peel member having a first position wherein it is effective to peel a transponder from the web and a second position wherein it is ineffective to peel a transponder from the web, when the web is moved relative to the peel member; b. providing a web carrying a spaced series of peelable transponders which are responsive to electromagnetic wave energy; c. moving said peel member from said first position to said second position in a first pattern of movement when it is desired not to peel a predetermined transponder from the web; and d. while said peel member is being moved to said second position, moving said web across said peel member in a second pattern of movement different from said first pattern of movement. 2. The method of claim 1 wherein said first pattern of movement is substantially linear, and second pattern of movement is nonlinear. 3. The method of claim 2 wherein said first pattern of movement is unidirectional and said second pattern of movement is bidirectional. 4. The method of claim 3 wherein when peel member is moved unidirectionally from said first position to said second position, said web is moved initially in the opposite direction and is then moved in the same direction as said peel member surface is moved, so as to fix the relative positions of said web and peel member during said adjustment of said peel member position. 5. The method of claim 1 wherein said peel member has a peel edge and a convex body surface, and wherein said peel member is rotated between said first position in which said peel edge is effective to peel a transponder from the web and said second position wherein said convex body surface is ineffective to peel a transponder from the web. 6. The method of claim 2 wherein said peel member has a peel edge and a convex body surface, and wherein said peel member is rotated between said first position in which said peel edge is effective to peel a transponder from the web and said second position wherein said convex body surface is ineffective to peel a transponder from the web. 7. The method of claim 1 wherein said peeler is moved to the second position and stopped, after which the web is moved to draw a length of web over the peeler. 8. The method of claim 7 wherein said length of web is equal to an element length plus an inter-element spacing. 9. The method of claim 7 wherein said length of web is such as to place an element at a position adjacent the peeler where it can be peeled by the peeler. 10. The method of claim 1 wherein as said peeler is moved to the second position and before reaching the second position, the web is accelerated to draw a length of web over the peeler effective to place an element at a position adjacent the peeler where it can be peeled by the peeler. 11. A method of selectively peeling or not peeling a succession of peelable web-carried elements, comprising: a. providing an adjustable peel member having a first position wherein it is effective to peel an element from the web and a second position wherein it is ineffective to peel an element from the web when the web is moved relative to the peel member; b. providing a web carrying a spaced series of peelable elements; c. unidirectionally moving said peel member from said first position to said second position when it is desired not to peel a predetermined element from the web; and d. while said peel member is being moved unidirectionally to said second position, moving said web bidirectionally across said peel member such that said the position of said element relative to said peel member remains substantially fixed and said element is not peeled. 12. The method of claim 11 wherein said elements comprise transponders responsive to electromagnetic wave energy. 13. The method of claim 12 wherein said peel member has a peel edge and a convex body surface, and wherein said peel member is rotated between said first position in which said peel edge is effective to peel a transponder from the web and said second position wherein said convex body surface is ineffective to peel a transponder from the web. 14. The method of claim 13 wherein when peel member is moved unidirectionally from said first position to said second position, said web is moved initially in the opposite direction and is then moved in the same direction as said peel member surface is moved so as to fix the relative positions of said web and peel member during said adjustment of said peel member position. 15. A method of selectively peeling or not peeling a succession of peelable web-carried transponders, comprising: a. providing an adjustable peel member having a first position wherein it is effective to peel a transponder from the web and a second position wherein it is ineffective to peel a transponder from the web when the web is moved relative to the peel member; b. providing a web carrying a spaced series of peelable transponders which are responsive to electromagnetic wave energy; c. unidirectionally moving said peel member from said first position to said second position when it is desired not to peel a predetermined transponder from the web; d. while said peel member is being moved unidirectionally to said second position, moving said web bidirectionally across said peel member such that said the position of said transponder relative to said peel member remains substantially fixed and said transponder is not peeled; e. with the peel member in said second position, moving the web a distance substantially equal to the length of a transponder plus the inter-transponder spacing on the web; and f. returning the peel member to said first position. 16. In an RF transponder applicator for applying RF transponders to objects or to labels, tickets, tags, cards or other media samples, a method comprising: providing a web having a series of RF transponders; inspecting a transponder; if the transponder passes inspection, at a predetermined peel station and in response to program-controlled, on-demand variable application instructions, selectively peeling the transponder and applying it to a media sample or object; and if the transponder fails inspection, moving the transponder having the failed transponder past the peel station and storing it. 17. The method of claim 16 including: providing an adjustable peel member having a first position wherein it is effective to peel a transponder from the web and a second position wherein it is ineffective to peel a transponder from the web, when the web is moved relative to the peel member; adjusting the position of said peel member to said first position to accomplish said selective peeling of the transponder having the transponder which passed inspection. 18. The method of claim 17 including adjusting the position of said peel member to said second position to prevent the transponder having the failed transponder to be peeled as it is moved past the peel station. 19. The method of claim 18 further including: a. moving said peel member from said first position to said second position in a first pattern of movement when it is desired not to peel a predetermined transponder from the web; and b. while said peel member is being moved to said second position, moving said web across said peel member in a second pattern of movement different from said first pattern of movement. 20. The method of claim 19 wherein said first pattern of movement is substantially linear, and second pattern of movement is nonlinear. 21. The method of claim 20 wherein said first pattern of movement is unidirectional and said second pattern of movement is bidirectional. 22. The method of claim 18 including unidirectionally moving said peel member from said first position to said second position and while said peel member is being moved unidirectionally, moving said web bidirectionally across said peel member such that said the position of said element relative to said peel member remains substantially fixed. 23. The method of claim 17 wherein said peel member has a peel edge and a convex body surface, and wherein said peel member is rotatable between said first position in which said peel edge is effective to peel a transponder from the web and said second position wherein said convex body surface is ineffective to peel a transponder from the web. 24. A method of selectively peeling or not peeling a succession of peelable web-carried elements, comprising: a. providing an adjustable peel member having a first position wherein it is effective to peel an element from the web and a second position wherein it is ineffective to peel an element from the web when the web is moved relative to the peel member; b. providing a web carrying a spaced series of peelable elements which are responsive to electromagnetic wave energy and which have a memory for storing information; c. verifying that information stored in an element is correct and that the element is not defective; d. unidirectionally moving said peel member from said first position to said second position if said verifying step indicates that the stored information is not correct or that the element is defective; and e. while said peel member is being moved unidirectionally, moving said web bidirectionally across said peel member such that the position of said element relative to said peel member remains substantially fixed and said element is not peeled. 25. The method of claim 24 wherein said peel member has a peel edge and a convex body surface, and wherein said peel member is rotated between said first position in which said peel edge is effective to peel an element from the web and said second position wherein said convex body surface is ineffective to peel an element from the web. 26. The method of claim 25 wherein when peel member is moved unidirectionally from said first position to said second position, said web is moved initially in the opposite direction and then moved in the same direction as said peel member is rotated so as to fix the relative positions of said web and peel member during said adjustment of said peel member position. 27. The method of claim 24 wherein said element comprises a transponder including an RFID transponder. 28. An improved selective peeling system for use with a web carrying a series of spaced peelable elements, comprising: a. an adjustable peel member having a first position wherein it is effective to peel an element from the web and a second position wherein it is ineffective to peel an element from the web; b. a web drive responsive to commands from a control system and configured to move the web bidirectionally; c. a peel member drive responsive to commands from the control system and configured to adjust said peel member between said first and second positions; and d. said control system being coupled to said web drive and to said peel member drive and configured to cause a selected element to not be peeled by issuing coordinated drive commands to said web drive and said peel member drive effective to cause said peel member to move linearly from said first position to said second position while said web is caused to move nonlinearly as said peel member moves from said first position to said second position. 29. The system of claim 28 wherein said control system commands cause said web drive to move said web backward and forward as necessary to maintain said web in a substantially fixed position on said peel member as said peel member is moved from said first position to said second position. 30. The system of claim 28 wherein said peel member has a peel edge and a convex body surface, wherein said peel member drive rotates said peel member between said first position in which said peel edge is effective to peel an element from the web and said second position wherein said convex body surface is ineffective to peel an element from the web. 31. The system of claim 30 wherein when said peel member drive moves the peel member continuously and unidirectionally from said first position to said second position, said web drive initially moves the web in the opposite direction and then moves the web in the same direction as said peel member is moved so as to fix the relative positions of said web and peel member during said adjustment of said peel member position. 32. The system of claim 30 wherein said element comprises a transponder including an RFID transponder. 33. An improved selective peeling system for use with a web carrying a series of spaced peelable RFID transponders, comprising: a. an adjustable peel member having a first position wherein it is effective to peel a transponder from the web and a second position wherein it is ineffective to peel a transponder from the web; b. a web drive responsive to commands from a control system and configured to move the web bidirectionally; c. a peel member drive responsive to commands from the control system and configured to adjust said peel member between said first and second positions; and d. said control system being coupled to said web drive and to said peel member drive and configured to cause a selected transponder to not be peeled by issuing coordinated drive commands to said web drive and said peel member drive effective to cause said peel member to move linearly from said first position to said second position while said web is caused to move nonlinearly as said peel member moves from said first position to said second position. 34. The system of claim 33 wherein said control system commands cause said web drive to move said web backward and forward as necessary to maintain said web in a substantially fixed position on said peel member as said peel member is moved from said first position to said second position. 35. The system of claim 33 wherein said peel member has a peel edge and a convex body surface and wherein said peel member drive rotates said peel member between said first position in which said peel edge is effective to peel a transponder from the web and said second position wherein said convex body surface is ineffective to peel a transponder from the web. 36. The system of claim 35 wherein when said peel member drive moves the peel member continuously and unidirectionally from said first position to said second position, said web drive initially moves the web in the opposite direction and then moves the web in the same direction as said peel member is moved so as to substantially fix the relative positions of said web and peel member during said adjustment of said peel member position. 37. For use in a system which processes a series of RFID transponders carried on a web, a peeler receiving the series of transponders and being configured to selectively delaminate transponders from the web, said peeler being adjustable between a first state wherein it is effective to delaminate transponders from said web and a second state wherein it is ineffective to delaminate media samples from said web. 38. The peeler of claim 37 comprising a peeler member mounted for movement between first and second positions which create said first and second states. 39. The peeler of claim 38 wherein said peeler member is mounted for rotation and is configured to present a rounded surface to said web when in said first position and to present a peeling edge to said web when rotated to said second position. 40. The peeler of claim 38 including a means for controlling the movement of said peeler member between said first and second states. 41. The peeler of claim 39 including a motor for rotating said peeler member between said first and second positions. 42. The peeler of claim 41 located in an on-demand printer, said motor being configured to receive computer-generated rotation instructions. 43. The peeler of claim 37 in combination with an RFID transceiver positioned and configured to verify the transponder before it is processed by the peeler. 44. The peeler of claim 42 in combination with an RFID transceiver positioned and configured to verify the transponder before it is processed by the peeler, the peeler motor receiving instructions to rotate the peeler to said second position when a transponder which failed verification is processed by the peeler. 45. A method comprising: providing on a web a series of peelable RF transponders of like or unlike type; selecting a particular RF transponder to be peeled; providing a peeler adjustable between a peel and a no-peel state; adjusting the peeler to the peel state; moving the web to bring the selected particular transponder in a position to be peeled by said peeler; peeling said selected transponder; and applying the peeled transponder to an object or to a ticket, tag, label, card or other media sample. 46. The method of claim 45 wherein the transponder is applied to a media sample, the method including the step of printing on said media sample before the applying step. 47. The method of claim 45 wherein said peeler has a convex no-peel surface presented to the web when the peeler is in its no-peel state, and a peeling edge presented to said web when said peeler is in its peel state. 48. The method of claim 45 wherein said transponder is applied to a media sample which is then applied to an object. 49. The method of claim 48 including printing on said media sample before it is applied to an object. 50. The method of claim 45 including a step of testing the functionality of the selected transponder. 51. The method of claim 50 wherein if the tested transponder fails the testing step, the peeler is adjusted to its no-peel state and the transponder is maintained on the web and not peeled. 52. The method of claim 45 wherein the series of transponders on the web include different types of transducers which are sequentially selected, peeled and applied. 53. The method of claim 45 wherein selecting is performed by one of: identifying a particular transponder by interrogating it with an RF signal, sensing an electrical or physical attribute of the transponder, or employing a computer to catalog the location and identity of transponders on the web. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention concerns, in one aspect, a method and apparatus by which, both selectively and on-demand, individual labels, tickets, tags, cards, and the like (hereinafter collectively and in individual units referred to as “media,” or individually as “media samples”) having selected characteristics may be custom configured by causing one or more value-adding elements that have chosen characteristics to be associated with said media. More particularly, the invention is directed to method and apparatus for selectively incorporating one or more value-adding elements such as, for example, radio frequency identification (hereinafter called RFID) transponders with selected individual media samples on an on-demand basis. Other types of value-adding elements that could be incorporated into media samples include, for example, shipping documents; parts to be inventoried, stored or shipped; promotional devices such as coupons, tokens, currency or other objects having a value to the recipient; integrated circuits on labels with leads to be connected to printed antennas; and attached or embedded objects that have associated information on the printed media relating to their identification or use. The process of coupling or associating an RFID transponder (typically in the form of an inlay) with a label, ticket, tag, card or other media is commonly termed “converting,” and the device used to accomplish converting is termed a “converter”. As used herein this terminology will be extended to cover associating or coupling any value-adding element with a media sample. A particularly suitable environment for the converting apparatus and method of this invention is a printer of the type commonly used to print bar codes, text and graphics. Such printers typically are offered as tabletop or portable devices or as part of label print and apply systems, and are used in factories, warehouses, shipping centers and a wide variety of other applications. Another favored environment is in card printers of the type used to create identification or security badges and the like. The global installed base of such media printers is immense. These media printers are typically networked and print on demand from a central computer under software program control. Because of the flexibility of systems containing such printers, each media sample is capable of being unique in the text, graphics or codes imprinted on the media samples, as well as the attributes and number of value-adding elements. With the burgeoning adoption of RFID technology, many users of media printers, for example, would like to have the capability of generating media samples with associated RFID transponders, herein termed “smart media,” “smart labels,” or the like. However, typically today such users may have only a part-time or occasional need to generate a smart label or other smart media. Currently, to acquire the capability of generating an occasional smart media prior to this invention, it is necessary for users to acquire one or more additional multi-function printers having the capability of encoding and testing smart media. Such a printer(s) is loaded with smart media and stands ready for occasional use. If such a printer is the only equipment available, and the user wishes to generate a conventional (non-smart) bar code label, for example, he must take the printer off line, uninstall the smart labels, install standard (non-smart) labels, set up the printer and print the standard label. To then generate a smart label, the process must be reversed. An operation that called for mixed smart and standard labels or other media would obviously be difficult to execute in a single printer, or require duplication of equipments and supplies to support the use of both standard (non-smart) labels and smart labels in the same environment. If different types of transponder formats are called for, the smart media printer must be taken down and re-setup for the alternate transponder format. Floor or table space for the duplicative equipment is often not available, and the extra equipment and inventory of rolls of smart media in various needed transponder formats increases operating costs. Today, smart media printers necessarily print on media in which the transponders are already embedded. The printing process inevitably breaks transponder leads, creating expensive media rejects which must be removed or labeled as rejects. Ideally, separation of the printing process from the embedding of the transponder would lead to fewer smart labels with damaged transponders. It has been estimated that more than half the cost of a smart label is in the fabrication. Less than half the cost is in the materials (transponder, transponder carrier, media, media carrier, etc.). Much of the cost is incurred by the multiple processing steps to conventionally fabricate a smart label. The transponder (including antenna and typically, but not always, an integrated circuit) is mounted on a carrier to create an “inlay”. Next a series of such inlays are mounted on a liner and wound on a roll for storage. Media, such as label stock, is mounted on a liner. To create smart labels, rolls of the label stock are brought together with rolls of transponder inlays. The label stock and liner is separated, the transponder inlays are inserted serially and the label stock and liner are rejoined to capture the inlays. The smart labels are then die cut and otherwise finished. The multiple processing steps, (such as inlay insertion into the labelstock and liner), including the high scrap rate in certain of such processing steps, are among the chief reasons for the high cost of smart labels today. Although ink jet and various other printer technologies are employed in printers of the type discussed, a thermal transfer printer is commonly used to print individual media samples and will be described to frame the ensuing discussion of the present invention. Referring to FIG. 1 , a side view of a standard thermal transfer printer mechanism 10 is illustrated. A label carrier 12 (also generally referred to as a release liner) carries adhesive-backed, (typically unprinted) diecut labels 14 through the mechanism. Typically, the top surface of each label is printed with a pattern of ink dots from a thermal transfer ribbon 16 melted onto the label surface as the ribbon and label pass under a computer-controlled thermal printhead 18 . An elastomer-coated platen roller 20 typically is driven by a stepping motor (not shown) to provide both the movement force for the ribbon and label by means of a friction drive action on the label carrier 12 , as well as acting as the receiver for the required pressure of the printhead on the ribbon-label sandwich. This pressure assists in transferring the molten ink dots under printhead 18 from the thermal transfer ribbon 16 onto the diecut label 14 surface. The thermal transfer ribbon 16 is unwound from a printer ribbon supply 22 , and is guided under the thermal printhead 18 by idler rollers 24 . After the ink is melted from the ribbon 16 onto the printed diecut label 26 , the spent ribbon is wound on a printer ribbon take-up spindle 28 . Typically, a media exit 30 is located immediately after the printhead 18 . The now-printed diecut label 26 is often dispensed on its label carrier 12 . If a user desires that the printed diecut labels be automatically stripped from label carrier, then an optional peeler bar 32 is utilized. As the label carrier 12 passes over the sharp radius of peeler bar 32 , the adhesive bond is broken, thereby releasing the printed diecut label 26 from its label carrier 12 . The peeled, printed diecut label 26 is dispensed at media exit 30 . The excess label carrier 12 is both tensioned for peeling and rewound using optional label carrier take-up mechanism 34 . As will be described in detail hereinafter, an exemplary embodiment of the present invention involves selectively and on demand associating, in the environment of a thermal or thermal transfer or other type of printer, an RFID transponder with a label, e.g., to create a “smart” label. Although much of the following discussion will be in the context of media in the form of labels, it should be understood that application of the invention is not limited to labels, and is equally applicable to tickets, tags, cards and other media. Although “chipless” RFID transponders exist and may be utilized as one example of a value-added element with certain aspects of this invention, the most common form of an RFID transponder used in smart labels comprises an antenna and an RFID integrated circuit. Such RFID transponders include both DC powered active transponders and batteryless passive transponders, and are available in a variety of form factors. Commonly used passive inlay transponders 36 shown in FIG. 2 have a substantially thin, flat shape. For automatic insertion into labels, the inlay transponders 36 are typically, but not always prepared with a pressure-sensitive adhesive backing, and are delivered individually diecut and mounted with a uniform spacing on an inlay carrier. Inlay transponders have been used as layers of identification tags and labels to carry encoded data, stored in a non-volatile memory area data, that may be read wirelessly at a distance. For example, a camera having a radio-frequency identification transponder that can be accessed for writing and reading at a distance is disclosed in U.S. Pat. No. 6,173,119. The antenna 38 for an inlay transponder 36 is in the form of a conductive trace deposited on a non-conductive support 40 , and has the shape of a flat coil or the like. Antenna leads 42 are also deposited, with non-conductive layers interposed as necessary. The RFID integrated circuit 44 of the inlay transponder 36 includes a non-volatile memory, such as an EEPROM (Electrically Erasable Programmable Read Only Memory); a subsystem for power generation from the RF field generated by the reader; RF communications capability; and internal control functions. The RFID integrated circuit 44 is mounted on the non-conductive support 40 and operatively connected through the antenna leads 42 . The inlays are typically packaged singulated or on a Z-form or roll inlay carrier 46 as shown in FIG. 2 . It is known how to utilize on-press equipment for insertion of transponders into media to form “smart labels,” and then to print information on a surface of the smart labels. See, for example, an application white paper entitled “RFID Technology & Smart Labels,” dated Sep. 14, 1999, P/N 11315 L Rev. 1 of Zebra Technologies Corporation. See also, for example, a document entitled “A White Paper On The Development Of AIM Industry Standards For 13.56 MHz RFID Smart Labels And RFID Printer/Encoders” by Clive P. Hohberger, PhD, dated May 24, 2000. Both of these documents are incorporated by reference into this application as if fully set forth herein. It also is known how to utilize label applicator equipment to attach pressure-sensitive labels to business forms. Such equipment has been commercially available on the U.S. market from several companies for more than one year prior to the filing of this application. Zebra Technologies Corporation is a leading manufacture of a number of printer related products, including a number of on-demand thermal transfer printers that incorporate a number of the aspects of the technology that is disclosed in the two above-referenced white papers. An example of such a “smart label” printer commercially available for more than a year prior to the filing of this application includes Zebra model number R-140. Such products are satisfactory for their intended uses. However, the need for a smart printer or other media processor with on-demand selective converting capability has become urgent, but unmet prior to this invention. Certain features and advantages of the invention will become apparent from the description that follows. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>The objects and advantages of the present invention will become more readily apparent to those of ordinary skill in the relevant art after reviewing the following detailed description and accompanying drawings, wherein: FIG. 1 is a side, schematic view of a standard thermal transfer label printer mechanism; FIG. 2 is a schematic view of a plurality of passive inlay-type RFID transponders as delivered with an adhesive backing on an inlay carrier; FIG. 3 is a side, schematic view of a thermal transfer printer that incorporates a number of aspects of an exemplary embodiment of the present invention disclosed in this application; FIG. 4 is a front, sectional view of a portion of the thermal transfer printer shown in FIG. 3 detailing a tamping applicator mechanism; FIG. 5 is a front, sectional, schematic view of the thermal transfer printer shown in FIG. 3 , wherein a transponder dispensing mechanism is disposed in a fully retracted initial position; FIG. 6 is a schematic, block diagram of some of the key electronic subsystems and components of the thermal transfer printer shown in FIG. 3 ; FIG. 7 is a program flow-chart that illustrates certain key program steps that are executed by the processor unit shown in FIG. 6 for each print job that is performed by the thermal transfer label printer shown in FIGS. 3-6 ; FIG. 8 is a front, sectional, schematic view of the thermal transfer printer shown in FIG. 3 , wherein the transponder dispensing mechanism shown in FIG. 5 is disposed in an extended position so that an RFID transponder is positioned in a desired position and orientation with respect to a delaminated diecut label printed by the thermal transfer printer; FIG. 9 is a front, sectional, schematic view of the thermal transfer printer shown in FIG. 5 , wherein the tamping applicator mechanism detailed in FIG. 4 is utilized to permanently affix a programmed RFID transponder to a media sample that is to be printed by the thermal transfer printer mechanism and wherein a linear actuator is used to retract the dispensing mechanism to peel the inlay carrier from the back of the programmed transponder thereby exposing its adhesive layer; FIG. 10 is a side, sectional, schematic view of the thermal transfer printer shown in FIG. 3 , wherein a diecut label/programmed transponder sandwich is formed and relaminated to the diecut label carrier; FIG. 11 is a side schematic view of a thermal transfer printer mechanism, similar to that disclosed in FIG. 3 , that incorporates a number of aspects of a further exemplary embodiment of the present invention disclosed in this application, and that allows adhesive-backed value-adding devices such as RFID transponders to be affixed to stiff media that does not include its own adhesive layer; FIG. 12 is a side schematic view of the thermal transfer printer shown in FIG. 11 , wherein an adhesive-backed, programmed RFID transponder is disposed in a dispensing position with respect to the value-adding mechanism; FIG. 13 is a side schematic view of the thermal transfer printer shown in FIG. 11 , wherein an adhesive-backed, programmed RFID transponder is affixed to a stiff media; FIG. 14 is a side schematic view of the thermal transfer printer shown in FIG. 11 , wherein the stiff media, upon which an adhesive-backed, programmed RFID transponder is affixed, is advanced to a dispensing position; FIG. 15 is a flow-chart that illustrates certain key program steps that are executed by the processor unit shown in FIG. 6 for each print job that is performed by the thermal transfer printer shown in FIGS. 11-14 ; FIGS. 16A though 16 D are schematic views of two types of RFID integrated circuit labels and their attachment to two corresponding types of printed antennae in order to form actual RFID transponders in a process using an exemplary variation of the thermal transfer printer shown in FIGS. 11-15 ; FIGS. 17A and 17B are schematic views of the front and reverse sides postcard set media that is on-demand printed and to which various value-added elements are added in a production process according to an exemplary embodiment of the present invention; FIG. 18 is a representation of the four value-added elements which are added in certain combinations to the postcard set media of FIG. 17 by the exemplary production process that is shown in FIG. 19 ; FIG. 19 is an overhead schematic view of an exemplary production process incorporating forms of two exemplary embodiments invention embodiments that is used for selectively and on-demand configuring the postcard media of FIG. 17 by addition of one or more value-added elements of FIG. 18 ; FIGS. 20-23 are side, schematic views of a thermal transfer printer mechanism that incorporates a number of aspects of the present invention disclosed in this application, and that an RFID transponder to be selectively and on demand, under program control, RFID transponder encoded, and attached to an adhesive backed previously printed diecut label; FIG. 24 is a side, schematic view of a thermal transfer printer mechanism, similar to FIGS. 20-23 , that allows an RFID transponder to be selectively and on demand, under program control, RFID transponder encoded, and attached to a linerless media; FIG. 25 is a schematic illustration of an additional embodiment of the present invention; FIG. 26 is a schematic illustration of an additional embodiment of the present invention; FIG. 26A is a schematic illustration of an additional embodiment of the present invention; FIG. 27 is a schematic illustration of an additional embodiment of the present invention; FIG. 27A is a schematic illustration of an additional embodiment of the present invention; FIG. 27B is a schematic illustration of an additional embodiment of the present invention; FIG. 28 is a detailed schematic illustration of a specific embodiment of the present invention; FIGS. 29A-29F is a pictorial sequential view of operation of the apparatus of FIG. 28 ; FIG. 30A is a pictorial illustration showing a length of the web when the peeler is in four sequential positions; FIG. 30B is a graph showing the number of steps taken by the peeler motor and the take-up reel motor, respectively, during the sequential operation of the peeler shown in FIGS. 29A-20B ; FIG. 31 is a schematic illustration of an additional embodiment of the present invention; and FIG. 32 is a schematic illustration of an additional embodiment of the present invention. detailed-description description="Detailed Description" end="lead"? |
Method and device for tank leakage diagnosis at elevated fuel degassing |
A method for operating a tank leakage diagnosis device, especially of a motor vehicle, volatile fuel (to be degassed) being temporarily stored using an adsorption filter of known absorption capacity or absorption characteristics, and the adsorption filter being regenerated from time to time by purging, using fresh air, to avoid faulty measurements in the tank leakage diagnosis, in particular at elevated fuel degassing. It is provided that the adsorption filter be purged, and, in this context, the volatile fuel removed from the adsorption filter over a predefined time span be integrated, and from that, the loading of the adsorption filter with the volatile fuel, changing during the time span, is ascertained, from the absorption capacity and the absorption characteristics of the adsorption filter, as well as the integrated fuel quantity or the changing loading. The quantity of fuel to be degassed supplied to the adsorption filter from the fuel container in the time span is calculated, and, as a function of the calculated quantity of fuel supplied to the adsorption filter, an intervention is undertaken at the tank leakage diagnosis device. |
1-16. (Canceled). 17. A method for operating a tank leakage diagnosis device in a motor vehicle, for testing a fuel container, which is connected to an internal combustion engine, for tightness, the method comprising: regenerating an adsorption filter from time to time by purging by fresh air drawn in by the internal combustion engine, wherein degassing fuel is temporarily stored with an adsorption filter having at least one of an adsorption capacity and adsorption characteristics, the adsorption filter being connected to the fuel container; providing a loading factor specifying the loading of the adsorption filter with the degassing fuel, wherein the adsorption filter is purged and, in this context, the degassing fuel removed from the adsorption filter over a predefined time span is recorded; determining, from at least one of the absorption capacity, the absorption characteristics of the adsorption filter, the loading factor, a recorded, removed fuel quantity and a changing at the loading, a quantity of the degassing fuel supplied to the adsorption filter from the fuel container over the predefined time span; and performing, as a function of a determined quantity of the degassing fuel supplied to the adsorption filter, at least one of: (i) one of an interrupting and a blocking of at least one tank leakage diagnosis function, and (ii)a correcting of the data as to an actual tank degassing ascertained with the at least one tank leakage diagnosis function. 18. The method of claim 17, wherein one of the following is satisfied:(i) the at least one tank leakage diagnosis function is one of interrupted and blocked only when a determined quantity of the degassing fuel supplied to the adsorption filter exceeds a predefinable threshold value; and (ii) the data with respect to the actual tank degassing, ascertained with the at least one tank leakage diagnosis function, is corrected only when the determined quantity of the degassing fuel supplied to the adsorption filter exceeds a predefinable threshold value. 19. The method of claim 17, wherein the degassing fuel removed from the adsorption filter over the predefined time span is recorded in an integrating manner. 20. The method of claim 17, wherein a balance equation of the form AKF_HC-loading=the integral(tank_HC-loading)−the integral(AKF_HC-venting) is used to determine the loading factor. 21. The method of claim 17, wherein an exceeding of a defined threshold is assumed to be satisfied until it is possible to determine the quantity of the degassing fuel within a predefined tolerance during operation of the internal combustion engine. 22. The method of claim 17, wherein a quantity of temporarily stored degassing fuel removed from the adsorption filter by purging is ascertained from at least one of: (i) a fuel quantity required for combustion is determined in a load sensing of the internal combustion engine, and (ii) by taking, as a basis, a mixture correction factor resulting from a lambda regulation. 23. The method of claim 17, wherein the quantity of the degassing fuel is recorded only at sufficiently constant operating conditions of the internal combustion engine. 24. The method of claim 17, wherein a value of the loading factor of the adsorption filter is low-pass filtered. 25. A control apparatus for operating a tank leakage diagnosis device in a motor vehicle, for testing a fuel container, which is connected to an internal combustion engine, for tightness, degassing fuel being temporarily stored with an adsorption filter that has at least one of an adsorption capacity and adsorption characteristics, connected to the fuel container, comprising: a regenerating arrangement to regenerate the adsorption filter from time to time by purging fresh air drawn in by the internal combustion engine; a loading factor arrangement to provide a loading factor specifying a loading of the adsorption filter with the degassing fuel; a recording arrangement to record engine characteristics data and the loading of the adsorption filter with the degassing fuel, and for determining the degassing fuel supplied to the adsorption filter from the degassing fuel removed from the adsorption filter and the loading; and an arrangement for one of: (i) one of blocking and interrupting at least one tank leakage diagnosis function; and (ii) for correcting data as to an actual tank degassing, ascertained with at least one tank leakage diagnosis function, as a function of a determined quantity of degassing fuel supplied to the adsorption filter. 26. The control apparatus of claim 25, wherein the blocking arrangement includes a comparing arrangement to compare a determined amount of emitted gas, supplied to the adsorption filter, to a predefined threshold value, and when the predefined threshold value is exceeded, performing the one of (i) the blocking and interrupting and (ii) the correcting. 27. The control apparatus of claim 25, further comprising: a timer; an integrator for integrating calculated values of the degassing fuel supplied to the adsorption filter; and a starting arrangement to actively start a purging of the adsorption filter. 28. The control apparatus of claim 25, further comprising: a testing arrangement to test whether a calculation of the emitted gas removed from the adsorption filter is possible, based on recorded operating variables of the internal combustion engine. 29. The control apparatus of claim 25, further comprising: a filtering arrangement to low-pass filter the recorded loading of the adsorption filter. 30. A tank leakage diagnosis apparatus comprising: a tank leakage diagnosis device for a motor vehicle, for testing a fuel container, which is connected to an internal combustion engine, for tightness, the device performing the following: regenerating an adsorption filter from time to time by purging by fresh air drawn in by the internal combustion engine, wherein degassing fuel is temporarily stored with an adsorption filter having at least one of an adsorption capacity and adsorption characteristics, the adsorption filter being connected to the fuel container; providing a loading factor specifying the loading of the adsorption filter with the degassing fuel, wherein the adsorption filter is purged and, in this context, the degassing fuel removed from the adsorption filter over a predefined time span is recorded; determining, from at least one of the absorption capacity, the absorption characteristics of the adsorption filter, the loading factor, a recorded, removed fuel quantity and a changing at the loading, a quantity of the degassing fuel supplied to the adsorption filter from the fuel container over the predefined time span; and performing, as a function of a determined quantity of the degassing fuel supplied to the adsorption filter, at least one of: (i) one of an interrupting and a blocking of at least one tank leakage diagnosis function, and (ii)a correcting of the data as to an actual tank degassing ascertained with the at least one tank leakage diagnosis function. 31. A tank leakage diagnosis apparatus comprising: a control apparatus to control a diagnosing of a tank leakage in a motor vehicle, for testing a fuel container, which is connected to an internal combustion engine, for tightness, degassing fuel being temporarily stored with an adsorption filter that has at least one of an adsorption capacity and adsorption characteristics, the adsorption filter being connected to the fuel container, the control apparatus including: a regenerating arrangement to regenerate the adsorption filter from time to time by purging fresh air drawn in by the internal combustion engine; a loading factor arrangement to provide a loading factor specifying a loading of the adsorption filter with the degassing fuel; a recording arrangement to record engine characteristics data and the loading of the adsorption filter with the degassing fuel, and for determining the degassing fuel supplied to the adsorption filter from the degassing fuel removed from the adsorption filter and the loading; and an arrangement for one of: (i) one of blocking and interrupting at least one tank leakage diagnosis function; and (ii) for correcting data as to an actual tank degassing, ascertained with at least one tank leakage diagnosis function, as a function of a determined quantity of degassing fuel supplied to the adsorption filter. |
<SOH> BACKGROUND INFORMATION <EOH>In a fuel storage tank of a motor vehicle that contains fuel, volatile hydrocarbons are continuously escaping. This effect increases with temperature and the agitation or sloshing of the fuel. In motor vehicles driven by internal combustion engines, for a flawless fuel supply, venting of the fuel storage tank is absolutely essential. For, as fuel is used up, air has to be able to flow in behind it, since otherwise a vacuum would form in the tank, and the fuel flow would come to a stop. However, the tank also has to be vented so as to give the tank's contents sufficient opportunity to expand as it warms up. Also, when the tank is filled up, sufficient air has to be able to exit the tank so that the fuel being filled up does not bubble out of the filler pipe again. Therefore, in such vehicles, increasingly tank venting systems are used in which the evaporating and excess fuel vapor is guided not into the open air but, via a venting line, into an active charcoal filter (AKF). This fuel vapor is stored temporarily in the AKF, and, during the operation of the motor vehicle, is guided via a clocked activatable electromagnetic tank venting valve (TEV) to the intake manifold of the internal combustion engine, and thus to combustion. This prevents emission of the environmentally harmful fuel vapors from the tank into the environment to the greatest extent, and at the same time the vapors supplied to the internal combustion engine are themselves used as fuel, whereby fuel usage is considerably reduced. Based on the limited absorption volume of the active charcoal used in the AKF, one should intermittently regenerate the AKF. In order to do this, while the internal combustion engine is running, fresh air is drawn in via the AKF, and the fuel vapor removed in the process is supplied to the internal combustion engine as a mixture for combustion. The respective flushing quantity is controlled by the TEV via a performance characteristics adaptation using the parameters load and rotary speed, so that the running properties of the internal combustion engine are not impaired. A lambda control additionally monitors and regulates the regeneration. The lambda deviation resulting from this can then be drawn upon as a measurement of the loading state of the AKF. In this connection, intensified legal regulations on the operation of internal combustion engines will apply in the future in some countries, such as the USA. Thereafter, it will be required for motor vehicles, in which volatile fuels like gasoline are used, that a possibly existing leakage in the entire fuel tank system be tracked down using an on-board arrangement. Corresponding methods and devices for tank leakage diagnosis in a tank venting system of a motor vehicle are referred to, for example, in the U.S. Pat. No. 5,349,935, DE 196 36 431.0 A1, DE 198 09 384.5 A1 and DE 196 25 702 A1. In these, an overpressure is applied to the tank venting system, and a conclusion as to the presence of a leak is drawn from the subsequent course of the pressure. In the system of DE 196 36 431.0 A1, one may form a ram pressure between a pump and a reference leak, whereby the pump's rotary speed is lowered and the pump's current consumption increases. If the tank is leakproof, a higher pressure develops than when against the reference leak. The current consumption is consequently higher. It may be observed that the tank leak diagnosis, instead of by the use of overpressure, may also be performed with the aid of underpressure. A relatively high fuel degassing leads to erroneous measurements in tank leakage diagnosis. Therefore, as a measure of increased fuel degassing, a filtered loading factor of the AKF is used as a basis. The loading factor is calculated during travel, and filtered via a time constant. To do this, with the engine running, the TEV is controlled to open, and the deviation coming about in the lambda regulator, in this context, is recorded. Using the recorded deviation, together with the volume stream through the TEV that is also present in the engine control, the hydrocarbon (HC) concentration of the drawn in flushing volume stream is calculated. The HC concentration of the air drawn in through the AKF thus ascertained is valid as the measure of the magnitude of the AKF's loading. If the loading value exceeds a predefined threshold, the leakage diagnosis is interrupted or temporarily blocked. Since the loading is a function not only of the magnitude of the fuel degassing, using the loading value alone no accurate statement can be made concerning the actual magnitude of the instantaneous degassing. Thus, even at a very great fuel degassing under certain travel conditions, the loading factor may be artificially kept low using a high purging rate. In such a case, the leakage diagnosis would be enabled because of the low loading factor and the low degassing supposed from this. In actual fact, however, because of the actually present high degassing, this would lead to erroneous results in the leakage diagnosis. In the case of the overpressure diagnosis method discussed above, the leakage quantities specified by law in the USA would not be met. In the underpressure methods also named, such an erroneous detection may lead to the mistaken diagnosis of a non-leakproof tank system. |
<SOH> SUMMARY OF THE INVENTION <EOH>Therefore, the exemplary method of the present invention is intended to avoid erroneous measurements in tank leakage diagnosis, particularly at elevated fuel degassing. The exemplary embodiment and/or exemplary method of the present invention is based on ascertaining the actual instantaneously present fuel degassing, and, as a function of the ascertained degassing value, of suppressing affected diagnosis functions in order thereby to avoid false diagnoses. According to one variant, a substantial improvement in the quality of the diagnosis may be brought about, depending on the diagnosis function affected, by compensation of the disturbance measured by the degassing present during the tank leakage diagnosis. For this, the exemplary method according to the present invention provides that the adsorption filter be flushed, and in this context, the volatile fuel removed over a predefined time span from the adsorption filter be integrated, and from that there be ascertained loading of the adsorption filter with the volatile fuel that changes within the time span, that from the adsorption capacity or adsorption characteristics of the adsorption filter, the loading factor made available as well as the integrated fuel quantity or the changing loading, the quantity of degassing fuel supplied to the adsorption filter from the fuel container in the time span be calculated, and, as a function of the calculated quantity of the fuel supplied to the adsorption filter, an intervention is undertaken at the tank leakage diagnosis unit. According to this, a balance calculation is carried out or performed from which a conclusion is drawn on the fuel mass supplied to the adsorption filter from the fuel mass removed during the purging of the adsorption filter. In this context, the fuel mass supplied to the adsorption filter is assumed to be the actual degassing mass. In a first variant it is provided that at least one leakage diagnosis function is interrupted or blocked as a function of the calculated quantity of degassing fuel. According to a second variant, also as a function of the calculated quantity of degassing fuel, there takes place an immediate, or possibly time-delayed, compensation of the disturbance in the tank leakage diagnosis conditioned upon the calculated quantity of degassing fuel. The interventions, at the tank leakage diagnosis device mentioned, take place either in response to each present calculated value, in the way of a compensation, or in each case only when the calculated quantity of degassing fuel exceeds a predefinable threshold value. |
Method of encapsulating an active substance |
The invention provides a method of encapsulating an active substance in an interpolymer complex, to make an encapsulated product in particulate form. The method comprises forming a mixture of a supercritical fluid, an interpolymer complex and an active substance and then causing or allowing the interpolymer complex to encapsulate the active substance. The encapsulated product is then separated from the supercritical fluid and, if necessary, the product is subjected to size reduction to ontain particles in which the active substance is encapsulated by the interpolymer complex. |
1. A method of encapsulating an active substance in a polymeric encapsulating material to make an encapsulated product in particulate form by forming a mixture of a supercritical fluid, a polymeric encapsulating material and an active substance, causing or allowing the encapsulating material to encapsulate the active substance to form an encapsulated product, separating the encapsulated product from the supercritical fluid and, if necessary, subjecting the encapsulated product to size reduction to obtain encapsulated product particles in which the active substance is encapsulated by the encapsulating material, the method being characterised in that, the forming of the mixture is of the supercritical fluid, the active substance and a polymeric encapsulating material in the form of an interpolymer complex, so that the encapsulated product comprises particles of the active substance encapsulated by the interpolymer complex. 2. A method as claimed in claim 1, characterised in that the forming of the mixture comprises the step of dissolving a pre-prepared interpolymer complex in the supercritical fluid so that the mixture comprises a solution of the interpolymer complex as solute in the supercritical fluid as solvent. 3. A method as claimed in claim 1, characterised in that the forming of the mixture comprises the steps of dissolving in the supercritical fluid each of at least two complementary polymers capable of interacting together in solution in a supercritical fluid to form an interpolymer complex, to form a solution in which they are solutes and the supercritical solution is a solvent, and causing or allowing the complementary polymers to interact together to form the interpolymer complex in the supercritical fluid. 4. A method as claimed in claim 3, characterised in that the complementary polymers interact together to form an interpolymer complex which is soluble in the supercritical fluid, the forming of the mixture comprising the step of dissolving each of the complementary polymers in the supercritical fluid to form a solution in which the complementary polymers respectively form solutes in the supercritical fluid as solvent, the causing or allowing of the complementary polymers to Interact together acting to form the interpolymer complex as solute dissolved in the supercritical fluid as solvent. 5. A method as claimed in claim 3, characterised in that the complementary polymers interact together to form an interpolymer complex which is insoluble in the supercritical fluid, the forming of the mixture comprising the steps of separately dissolving each of the complementary polymers in the supercritical fluid to form separate solutions in which the complementary polymers respectively form solutes in the supercritical fluid as solvent, and mixing the separate solutions together to cause or allow the complementary polymers to interact together to form the interpolymer complex, the forming of the interpolymer complex resulting in precipitation thereof from the supercritical-fluid. 6. A method as claimed in any one of claims 2-5 inclusive, characterised in that the forming of the mixture includes the step of admixing a solubilizing agent into the mixture, the solubilizing agent acting to facilitate dissolving in the supercritical fluid of at least one member of the group consisting of the complementary polymers and the interpolymer complex. 7. A method as claimed in any one of the preceding claims, characterised in that the forming of the mixture comprises dispersing the active substance as a suspension of particles in the supercritical fluid, the causing or allowing of the interpolymer complex to encapsulate the particles of the active substance being by causing or allowing the interpolymer complex to precipitate from the supercritical fluid on to the surfaces of the particles. 8. A method as claimed in any one of claims 1-6 inclusive, characterised in that the forming of the mixture comprises the step of dissolving the active substance as solute in the supercritical fluid as solvent to form a solution of the active substance in the supercritical fluid, the causing or allowing of the interpolymer complex to encapsulate the active substance comprising atomizing the mixture in an atmosphere having a temperature and pressure such that the super critical fluid solvent evaporates to leave a residue comprising particles in which the active substance is encapsulated by the interpolymer complex. 9. A method as claimed in claim 1, characterised in that the forming of the mixture comprises the step of dissolving the supercritical fluid in the interpolymer complex to liquefy or plasticise the interpolymer complex. 10. A method as claimed in claim 9, characterised in that the forming of the mixture comprises the steps of blending at least two complementary polymers, capable of interacting together when blended and in liquefied or plasticised form, to obtain a blend comprising the polymers, dissolving the supercritical fluid in the polymers, and causing or allowing the polymers to interact together in blended liquefied or plasticised form to form the interpolymer complex. 11. A method as claimed in claim 10, characterised in that the blending of the polymers is to form a particle blend comprising polymer particles having a particle size of at most 1000 μm, after which the supercritical fluid is dissolved in the polymer particles. 12. A method as claimed in claim 10, characterised in that the supercritical fluid is separately dissolved in the complementary polymers in particle form comprising particles having a particle size of at most 1000 μm, after which the polymers in liquefied or plasticised form are blended to form the blend. 13. A method as claimed in any one of claims 9-12 inclusive, characterised in that the causing or allowing of the interpolymer complex to encapsulate the active substance comprises atomizing the mixture in an atmosphere having a temperature and pressure such that the supercritical fluid evaporates to leave a residue comprising particles in which the active substance is encapsulated by the interpolymer complex. 14. A method as claimed in claim 13, characterised in that the dissolving of the supercritical fluid in the interpolymer complex to liquefy or plasticise the interpolymer complex includes the step of dispersing a viscosity-reducing agent in the interpolymer complex to reduce the viscosity of the interpolymer complex to facilitate the atomizing. 15. A method as claimed in any one of claims 8-12 inclusive, characterised IR that the causing or allowing of the interpolymer complex to encapsulate the active substance comprises allowing the supercritical fluid to evaporate to leave a solid residue comprising the active substance dispersed In the interpolymer complex, and subjecting the residue to size reduction to obtain particles in which the active substance is encapsulated by the interpolymer complex. 16. A method as claimed in any one of the preceding claims, characterised in that the forming of the mixture includes the step of admixing a polymeric surfactant into the mixture. 17. A method as claimed in any one of the preceding claims, characterised in that it comprises selecting the polymers which form the interpolymer complex by interpolymer complexation from complementary members of the group consisting of hydrophilic polymers, hydrophobic polymers, hydrophobically modified hydrophilic polymers and hydrophilically modified hydrophobic polymers, namely alginates, alkyd- and hydroxyalkylcelluloses, carboxymethyl cellulose and its salts, carrageenan, cellulose and its derivatives, gelatin, gellan, guar gum, gum arabic, maleic acid copolymers, methacrylic acid copolymers, methyl vinyl ether/maleic anhydride copolymers, pectins, polyacrylamide, poly(acrylic acid) and its salts, polyethylene glycol), poly(ethylene imine), polyethylene oxide), poly(propylene oxide), poly(methacrylic acid), polystyrene and sulphonated polystyrene, poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl amine), poly(vinyl pyrrlidone), poly(vinyl sulphonic acid), starches and their derivatives, styrene maleic anhydride copolymers, crotonic acid copolymers, xanthan gum and copolymers of the aforegoing. |
Cement binder based plate |
The invention concerns a plate whereof the body comprises a binder containing Portland cement, a sulphoaluminous clinker and a calcium sulphate source: the binder comprises, by weight 30 to 80% of Portland cement, 20 to 70% of sulphoaluminous binder, 5 to 20% of a calcium sulphate source, and 0.4 to 7% of a water-reducing plasticizer or highly water-reducing superplasticizer admixture; it contains light aggregates in an amount such that the final density is close to 1, of the order of 0.8 to 1.5. |
1-30. (cancelled) 31. A board wherein the board body comprises a binder containing Portland cement, a sulfoaluminous clinker and a source of calcium sulfate, characterized in that the binder includes, by weight: 30 to 80% of Portland cement, 20 to 70% of sulfoaluminous clinker, 5 to 20% of a source of calcium sulfate and 0.4 to 7% of at least one water reducer plasticizer additive or high water reducer superplasticizer additive, and in that said board body includes light aggregates. 32. A board according to claim 31, characterized in that the light aggregates are in proportions such that the final specific gravity of the board body is close to 1. 33. A board according to claim 31, characterized in that the final specific gravity of the board body is of the order of 0.8 to 1.5. 34. A board according to claim 31, characterized in that the sulfoaluminous clinker has a C4A3—{overscore (S)} content greater than approximately 30%. 35. A board according to claim 31, characterized in that the additive includes a polymelamine sulfonate. 36. A board according to claim 31, characterized in that the additive includes a poly(meth)acrylate. 37. A board according to claim 31, characterized in that it includes a second additive which is a retarder. 38. A board according to claim 37 characterized in that it contains up to approximately 2% of the second additive. 39. A board according to claim 37, characterized in that the second additive contains poly(meth)acrylate, citric acid or a gluconate. 40. A board according to claim 31, characterized in that it further includes an alkaline carbonate additive. 41. A board according to claim 40, characterized in that the alkaline carbonate is Li2CO3. 42. A board according to claim 31, characterized in that the source of calcium sulfate is plaster, gypsum or anhydrite. 43. A board according to claim 31, characterized in that the contribution of sulfate by the source of calcium sulfate is such that the mass ratio r is from 2 to 2.5, where the mass ratio r is defined by the following equation: r=[SO3)a+(SO3)b]/(SO3)c in which: (SO3)a is the content of sulfate coming from the source of calcium sulfate, (SO3)b is the content of free sulfate coming from the sulfoaluminous clinker, and (SO3)c is the content of sulfate coming from the calcium sulfoaluminate contained in the sulfoaluminous clinker. 44. A board according to claim 31, characterized in that the Portland cement content is from 50 to 70% by weight of the binder. 45. A board according to claim 31, characterized in that the approximate Blaine specific surface area of the Portland cement is from 2500 to 6000 cm2/g and that of the sulfoaluminous clinker is from 2500 to 7000 cm2/g. 46. A board according to claim 31, characterized in that water is added to it in an approximate water/binder ratio by weight from 0.2 to 0.5 and preferably from 0.25 to 0.4. 47. A board according to claim 31, characterized in that it comprises 60 g of Portland cement, 30 g of sulfoaluminous clinker, 10 g of gypsum, 2 to 4 g of polymelamine sulfonate and 30 g of water. 48. A board according to claim 31, characterized in that it comprises 60 g of Portland cement, 30 g of sulfoaluminous clinker containing 56% C4A3{overscore (S)}, 10 g of plaster, 2 g of polymelamine sulfonate and 30 g of water. 49. A board according to claim 31, characterized in that it comprises 50 g of Portland cement, 43 g of sulfoaluminous clinker containing 35% of C4A3{overscore (S)}, 7 g of plaster, 2 g of polymelamine sulfonate and 30 g of water. 50. A board according to claim 31, characterized in that it comprises 36 to 76 g of Portland cement, 54 to 14 g of sulfoaluminous clinker, 10 g of plaster, 2 g of polymelamine sulfonate and water in a water/binder mass ratio of 0.3. 51. A board according to claim 31, characterized in that it comprises 60 g of Portland cement, 30 g of sulfoaluminous clinker, 10 g of plaster, 2 g of polymelamine sulfonate, 30 g of water and 2 g of expanded polystyrene balls. 52. A board according to claim 31, characterized in that it comprises 47 to 68 g of Portland cement, 43 to 22 g of sulfoaluminous clinker, 10 g of plaster, 2 g of polymelamine sulfonate, 30 g of water and 2 g of expanded polystyrene balls. 53. A board according to claim 31 characterized in that it comprises 60 g of Portland cement with a Blaine specific surface area from 3720 to 5040 cm2/g, 30 g of sulfoaluminous clinker with a Blaine specific surface area equal to 4500 cm2/g, 10 g of plaster, 2 g of polymelamine sulfonate, 30 g of water and 2 g of expanded polystyrene balls. 54. A board according to claim 31, characterized in that it comprises 60 g of Portland cement with a Blaine specific surface area equal to 3720 cm2/g, 30 g of sulfoaluminous clinker having a Blaine specific surface area from 3800 to 5000 cm2/g, 10 g of plaster, 2 g of polymelamine sulfonate, 30 g of water and 2 g of expanded polystyrene balls. 55. A board according to claim 31, characterized in that it comprises 60 g of Portland cement, 30 g of sulfoaluminous clinker, 10 g of gypsum, 2 g of polymelamine sulfonate, 0.3 to 1 g of poly(meth)acrylate and 30 g of water. 56. A board according to claim 31, characterized in that it includes 60 to 69 g of Portland cement, 30 to 21 g of sulfoaluminous clinker, 10 g of plaster, 2 g of polymelamine sulfonate, 1 g of poly(meth)acrylate, 30 g of water and 2 g of expanded polystyrene balls with a size ≦1 mm. 57. A board according to claim 31, characterized in that it comprises 40 g of Portland cement, 45 g of sulfoaluminous clinker, 15 g of plaster, 1.5 g of polymelamine sulfonate, 0.3 g of poly(meth)acrylate, 30 g of water and 1.5 g of expanded polystyrene balls with a size ≦1 mm. 58. A board according to claim 31, characterized in that it comprises 40 g of Portland cement, 45 g of sulfoaluminous clinker, 15 g of plaster, 1.5 g of polymelamine sulfonate, 0.3 g of poly(meth)acrylate, 0.5 g of alkaline carbonate (Li2CO3), 30 g of water and 1.5 g of expanded polystyrene balls with a size ≦1 mm. 59. A board according to claim 31, characterized in that it comprises 60 g of Portland cement, 30 g of sulfoaluminous clinker, 10 g of gypsum, 2 g of polymelamine sulfonate, 0.4 to 1.5 g of citric acid and 30 g of water. 60. A board according to claim 31, characterized in that it comprises 60 g of Portland cement, 30 g of sulfoaluminous clinker, 10 g of gypsum, 2 g of polymelamine sulfonate, 0.25 to 1.5 g of gluconate and 30 g of water. 61. A board according to claim 31, characterized in that it comprises 60 g of Portland cement, 30 g of sulfoaluminous clinker, 10 g of gypsum, 0.6 to 2 g of poly(meth)acrylate and 30 g of water. 62. Use of a board according to claim 31 to form or cover walls, partition walls, floors or roofs, inside or outside buildings, such as industrial kitchens, agriculture-foodstuffs laboratories, showers, bathrooms, pools or swimming pools, and/or rooms frequently washed with a water jet, such as rooms of agricultural buildings and industrial slaughterhouses. |
Process for producing and storing an unvulcanized rubber product |
A process for producing and storing a semi-finished product includes preparing an elastomeric composition, forming the composition to obtain a semi-finished product in strip form, and winding up the semi-finished product on a storage reel. At least one polyalkenamer is added during preparing the composition. A tyre for a vehicle wheel includes a belt structure, a tread, and a reinforcing layer disposed between the belt structure and tread. The reinforcing layer includes reinforcing cords coated and held together with a crosslinked elastomeric composition including at least one polyalkenamer. A process for producing the tyre includes manufacturing an unvulcanized tyre including at least one element made of crosslinkable elastomeric material, moulding the unvulcanized tyre in a moulding cavity defined in a vulcanizing mould, and crosslinking the elastomeric material by heating the tyre to a given temperature for a given time. The at least one element includes at least one polyalkenamer. |
1-23. (canceled) 24. A process for producing and storing a semi-finished product, comprising: preparing an elastomeric composition; forming the elastomeric composition to obtain a semi-finished product in strip form; and winding up the semi-finished product in strip form on a storage reel; wherein at least one polyalkenamer is added during preparing the elastomeric composition. 25. The process of claim 24, wherein the semi-finished product is a tape consisting of a crosslinkable elastomeric composition comprising the at least one polyalkenamer. 26. The process of claim 24, wherein the semi-finished product is a band comprising filiform reinforcing elements at least partially embedded in a crosslinkable elastomeric composition comprising the at least one polyalkenamer. 27. The process of claim 24, wherein the at least one polyalkenamer comprises a polyoctenamer. 28. The process of claim 24, wherein the at least one polyalkenamer is a polyoctenamer. 29. The process of claim 27 or 28, wherein the polyoctenamer comprises a percentage of double bonds in trans configuration of at least 60 mol %. 30. The process of claim 27 or 28, wherein the polyoctenamer comprises a Mooney viscosity ML (1+4) at 125° C. greater than 2 and less than 20. 31. The process of claim 27 or 28, wherein the polyoctenamer comprises a melting point greater than 25° C. and less than 80° C. 32. The process of claim 27 or 28, wherein the polyoctenamer comprises a glass transition temperature (Tg) greater than −90° C. and less than −50° C. 33. The process of claim 27 or 28, wherein the polyoctenamer comprises at least 25% by weight of macrocycles with a molecular weight up to 100,000. 34. The process of claim 24, wherein the at least one polyalkenamer is present in an amount greater than 3 phr and less than 40 phr. 35. The process of claim 24, wherein the at least one polyalkenamer is present in an amount greater than 5 phr and less than 30 phr. 36. The process of claim 24, wherein the at least one polyalkenamer is present in an amount greater than 8 phr and less than 25 phr. 37. The process of claim 24, wherein the elastomeric composition comprises at least one elastomeric polymer, and wherein the at least one elastomeric polymer comprises one or more of: natural rubber; homopolymers and copolymers of butadiene, isoprene, or 2-chlorobutadiene, such as polybutadiene (BR), polyisoprene (IR), styrene-butadiene (SBR), nitrile-butadiene (NBR), and polychloroprene (CR); ethylene/propylene copolymers (EPM); and ethylene/propylene/non-conjugated diene terpolymers (EPDM). 38. The process of claim 24, wherein the elastomeric composition comprises at least one elastomeric polymer, and wherein the at least one elastomeric polymer comprises one or more of: natural rubber; styrene-butadiene (SBR); nitrile-butadiene (NBR); polychloroprene (CR); ethylene/propylene copolymers (EPM); and ethylene/propylene/non-conjugated diene terpolymers (EPDM). 39. A process for producing a tyre for a vehicle wheel, comprising: manufacturing an unvulcanized tyre comprising at least one element made of crosslinkable elastomeric material; moulding the unvulcanized tyre in a moulding cavity defined in a vulcanizing mould; and crosslinking the elastomeric material by heating the tyre to a given temperature for a given time; wherein the at least one element is obtained by unwinding a semi-finished product in strip form from a storage reel and coiling the semi-finished product onto a building drum, and wherein the at least one element comprises at least one polyalkenamer. 40. The process of claim 39, wherein the unwinding is carried out at a speed greater than 50 m/min and less than 400 m/min. 41. The process of claim 39, wherein the unwinding is carried out at a speed greater than 250 m/min and less than 350 m/min. 42. The process of claim 39, wherein the at least one polyalkenamer comprises a polyoctenamer. 43. The process of claim 39, wherein the at least one polyalkenamer is a polyoctenamer. 44. The process of claim 42 or 43, wherein the polyoctenamer comprises a percentage of double bonds in trans configuration of at least 60 mol %. 45. The process of claim 42 or 43, wherein the polyoctenamer comprises a Mooney viscosity ML (1+4) at 125° C. greater than 2 and less than 20. 46. The process of claim 42 or 43, wherein the polyoctenamer comprises a melting point greater than 25° C. and less than 80° C. 47. The process of claim 42 or 43, wherein the polyoctenamer comprises a glass transition temperature (Tg) greater than −90° C. and less than −50° C. 48. The process of claim 42 or 43, wherein the polyoctenamer comprises at least 25% by weight of macrocycles with a molecular weight up to 100,000. 49. The process of claim 39, wherein the at least one polyalkenamer is present in an amount greater than 3 phr and less than 40 phr. 50. The process of claim 39, wherein the crosslinkable elastomeric material comprises at least one elastomeric polymer, and wherein the at least one elastomeric polymer comprises one or more of: natural rubber; homopolymers and copolymers of butadiene, isoprene, or 2-chlorobutadiene, such as polybutadiene (BR), polyisoprene (IR), styrene-butadiene (SBR), nitrile-butadiene (NBR), and polychloroprene (CR); ethylene/propylene copolymers (EPM); and ethylene/propylene/non-conjugated diene terpolymers (EPDM). 51. The process of claim 39, wherein the crosslinkable elastomeric material comprises at least one elastomeric polymer, and wherein the at least one elastomeric polymer comprises one or more of: natural rubber; styrene-butadiene (SBR); nitrile-butadiene (NBR); polychloroprene (CR); ethylene/propylene copolymers (EPM); and ethylene/propylene/non-conjugated diene terpolymers (EPDM). 52. A tyre for a vehicle wheel, comprising: at least one rubberized carcass ply; a belt structure applied along a circumference of the at least one carcass ply; a tread applied along a circumference of the belt structure; a reinforcing layer disposed between the belt structure and the tread; and a pair of sidewalls applied externally to the at least one carcass ply; wherein the at least one carcass ply is shaped in a substantially toroidal configuration, wherein opposite lateral edges of the at least one carcass ply are associated with respective bead wires, wherein each bead wire is enclosed in a respective bead, wherein the belt structure comprises at least one belt strip, wherein the reinforcing layer comprises a plurality of reinforcing cords coated and held together with a crosslinked elastomeric composition comprising at least one polyalkenamer, and wherein the sidewalls extend in an axially external position from a respective bead to a respective edge of the belt structure. 53. The tyre of claim 52, wherein the reinforcing cords consist of textile fibers. 54. The tyre of claim 52, wherein the at least one polyalkenamer comprises a polyoctenamer. 55. The tyre of claim 52, wherein the at least one polyalkenamer is a polyoctenamer. 56. The tyre of claim 54 or 55, wherein the polyoctenamer comprises a percentage of double bonds in trans configuration of at least 60 mol %. 57. The tyre of claim 54 or 55, wherein the polyoctenamer comprises a Mooney viscosity ML (1+4) at 125° C. greater than 2 and less than 20. 58. The tyre of claim 54 or 55, wherein the polyoctenamer comprises a melting point greater than 25° C. and less than 80° C. 59. The tyre of claim 54 or 55, wherein the polyoctenamer comprises a glass transition temperature (Tg) greater than −90° C. and less than −50° C. 60. The tyre of claim 54 or 55, wherein the polyoctenamer comprises at least 25% by weight of macrocycles with a molecular weight up to 100,000. 61. The tyre of claim 52, wherein the at least one polyalkenamer is present in an amount greater than 3 phr and less than 40 phr. 62. The tyre of claim 52, wherein the crosslinked elastomeric composition comprises at least one elastomeric polymer, and wherein the at least one elastomeric polymer comprises one or more of: natural rubber; homopolymers and copolymers of butadiene, isoprene, or 2-chlorobutadiene, such as polybutadiene (BR), polyisoprene (IR), styrene-butadiene (SBR), nitrile-butadiene (NBR), and polychloroprene (CR); ethylene/propylene copolymers (EPM); and ethylene/propylene/non-conjugated diene terpolymers (EPDM). 63. The tyre of claim 52, wherein the crosslinked elastomeric composition comprises at least one elastomeric polymer, and wherein the at least one elastomeric polymer comprises one or more of: natural rubber; styrene-butadiene (SBR); nitrile-butadiene (NBR); polychloroprene (CR); ethylene/propylene copolymers (EPM); and ethylene/propylene/non-conjugated diene terpolymers (EPDM). |
Method and device for shaping a lengthwise corrugated web |
In a method and device for continuous shaping of a plane web to a lengthwise corrugated web, wherein the web in the shaping area is continuously spatially guided and shaped, in that any imaginary line positioned within the center plane of the web and extending in the transport direction during shaping in the shaping area travels approximately the same travel distance, with increasing shaping only those web lines not positioned at the edge are subjected to a deflection perpendicularly to the plane of the web. The maximum value of the deflection is the greater the farther the web line is removed from the edge of the web, respectively. |
1. A method for continuous shaping of a plane web to a lengthwise corrugated web, wherein the web in the shaping area is continuously spatially guided and shaped, in that any imaginary line positioned within the center plane of the web and extending in the transport direction during shaping in the shaping area travels approximately the same travel distance, wherein with increasing shaping only those web lines not positioned at the edge are subjected to a deflection perpendicularly to the plane of the web whose maximum value is the greater the farther the web line is removed from the edge of the web, respectively. 2. The method according to claim 1, wherein the deflection of the web lines that is perpendicular to the web plane is realized in the same direction at any location. 3. The method according to claim 1, wherein the deflection of the web lines that is perpendicular to the web plane is subjected to a directional reversal approximately at the center of the shaping area. 4. The method according to claim 1, wherein the web is pre-shaped from its planar state first into a curved state and is then shaped into a web with corrugated cross-section. 5. The method according to claim 4, wherein a shaping shoulder is used for shaping the web curvature. 6. A device for continuous shaping of a plane web to a lengthwise corrugated web, wherein one or several shaping elements are arranged in the device for guiding the web to be shaped for deflecting the web over portions thereof perpendicularly to the web plane, wherein the shaping travel of all web lines defined by the shaping elements is approximately identical in length. 7. The device according to claim 6, wherein the shaping elements are formed as solid bodies on which the desired shaping contour is impressed on the side facing the web, respectively, such that they provide in the mounted state a forced guiding action for the web to be shaped, wherein the shaping elements in the shaping path of the device according to the invention are arranged such that those two sides are facing one another in which the shaping contour is impressed, wherein between the facing sides of the shaping elements a gap is located that is so large that the web can pass it without impairment. 8. The device according to claim 6, wherein glide elements circulating together with the web are embedded within the contour of the shaping elements facing the web, for example, balls or belts circulating together with the web. 9. The device according to claim 6, wherein the shaping contour itself is formed by balls or by belts circulating with the web. 10. The device according to claim 6, wherein a shaping shoulder is arranged in front of the shaping elements in the travel direction of the web. 11. A shaping element for continuous shaping of a plane web to a web that is corrugated in the transport direction, comprising at least one doubly curved functional surface that is surrounded by four boundary lines that intercept one another in pairs at a point, respectively, wherein the functional surface, starting from a straight or curved intake cross-section line to the curved exit cross-section line has an increasing shaping, wherein every imaginary web line positioned within the functional center plane and extending from the intake cross-section line to the exit cross-section line has substantially the same length in the shaping area and wherein, with increasing x coordinate, the web lines not positioned on the edge are subjected to a deflection perpendicularly to the plane of the functional surface whose maximum value is the greater the farther the respective web line is removed from the edge of the web. |
Mutants of igf binding proteins and methods of production of antagonists thereof |
The present invention provides a crystal suitable for X-ray diffraction, comprising a complex of insulin-like growth factor I or II (IGF) and a polypeptide consisting of the amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, the amino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or a fragment thereof consisting at least of the 9th to 12th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7th to 10th cysteine of IGFBP-6; methods for the determination of the atomic coordinates of such a crystal; IGFBP mutants with enhanced binding affinity for IGF-I and/or IGF-II, and methods to identify and optimize small molecules which displace IGFs from their binding proteins. |
1. A crystal suitable for X-ray diffraction, comprising a complex of insulin-like growth factor I or II (IGF) and a polypeptide consisting of the amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, the amino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or a fragment thereof consisting at least of the 9th to 12th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7th to 10th cysteine of IGFBP-6, to form a complex which exhibits restricted conformation mobility. 2. A crystal of claim 1, which effectively diffracts X-ray for the determination of the atomic coordinates of the complex to a resolution of 1.5 to 3.5 Å. 3. (Canceled) 4. A method for producing a crystal suitable for X-ray diffraction, comprising (a) contacting IGF with a polypeptide consisting of the amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, the amino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or a fragment thereof consisting at least of the 9th to 12th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7th to 10th cysteine of IGFBP-6, to form a complex which exhibits restricted conformation mobility, and (b) obtaining a crystal from the complex so formed suitable for X-ray diffraction. 5. A method for the determination of the atomic coordinates of a crystal suitable for X-ray diffraction obtained by (a) contacting IGF with a polypeptide consisting of the amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, the amino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or a fragment thereof consisting at least of the 9th to 12th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7th to 10th cysteine of IGFBP-6, to form a complex which exhibits restricted conformation mobility; and (b) obtaining a crystal from the complex so formed suitable for X-ray diffraction; (c) determining the atomic coordinates of said crystal. 6. A method for identifying a mutant of IGFBP (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5 or IGFBP-6 or a mutant of a fragment thereof consisting at least of the 9th to 12th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7th to 10th cysteine of IGFBP-6) having an enhanced binding affinity for IGF, comprising (a) constructing a three-dimensional structure of the complex of IGF and a polypeptide consisting of the amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP4, the amino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 consisting at least of the 9th to 12th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7th to 10th cysteine of IGFBP-6, based on the atomic coordinates of a crystal consisting of IGFI and said IGFBP or a fragment thereof; (b) employing said three-dimensional structure and modeling methods to identify said mutant of an IGFBP in which a residue within a distance of 5 Å to a hydrophobic amino acid residue of IGF is modified in that the hydrophobic interaction between IGF and said mutant of IGFBP is enhanced; (c) producing said mutant; (d) assaying said mutant to determine said enhanced binding affinity for IGF. 7. A method for identifying a mutant of IGFBP-5 with enhanced binding affinity for IGF-1, said method comprising (a) constructing a three-dimensional structure of the complex of IGF and IGFBP-5 defined by the atomic coordinates shown in FIGS. 5 and 6; (b) employing said three-dimensional structure and modeling methods to identify an amino acid residue in IGFBP-5 within a distance of 5 Å or shorter to an amino acid residue of IGFI, wherein said residue of IGFBP-5 can be modified hydrophobically in that the hydrophobic interaction between IGF and IGFBP-5 is enhanced; (c) producing said mutant; (d) assaying said mutant to determine said enhanced binding affinity for IGF. 8. A mutant of IGFBP containing one or more of the mutations as depicted in Tables 1 to 6. 9. A mutant of IGFBP containing one or more mutations of amino acid residues 49, 70 and/or 73 corresponding to IGFBP-5 sequence alignment according to Tables 1 to 6. 10. A method for identifying a non-proteinaceous compound capable of binding to IGFBP, comprising (a) constructing a three-dimensional structure of a complex of insulin-like growth factor 1 or 11 and a polypeptide consisting of the amino acids 40-92 of insulin-like growth factor binding protein 5, amino acids 39-91 of IGFBP-1, amino acids 55-107 of IGFBP-2, amino acids 47-99 of IGFBP-3, amino acids 39-91 of IGFBP-4, amino acids 40-92 of IGFBP-5, amino acids 40-92 of IGFBP-6 or a fragment thereof consisting at least of the 9th to 12th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7th to 10th cysteine of IGFBP-6, based on the atomic coordinates of a crystal consisting of IGF-I and said IGFBP; (b) employing said three-dimensional structure and modeling methods to identify a non-proteinaceous compound forming a complex with said IGFBP by hydrophobic binding with amino acids 49, 50, 70, 71 and 74 in the case of IGFBP-5 and in the case of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4 and IGFBP-6 with the corresponding amino acids according to Table 7; (c) producing said compound; (d) determining the binding between the compound and said IGFBP. 11. A crystal of claim 1, wherein the crystal is arranged in the cubic space group P2,3 having unit cell dimensions of 74.385 Å.×74.385 Å×74.385 Å. 12. A crystal of claim 2, wherein the crystal is arranged in the cubic space group P2,3 having unit cell dimensions of 74.385 Å.×74.385 Å×74.385 Å. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides a crystal suitable for X-ray diffraction, comprising a complex of insulin-like growth factor I or II and a polypeptide consisting of the amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, the amino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or a fragment thereof consisting at least of the 9 th to 12 th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7 th to 10 th cysteine of IGFBP-6 (such polypeptides and fragments are hereinafter also referred to as “mini-IGFBPs). Such a crystal is suitable for determining the atomic coordinates of the binding sites of IGF-I, IGF-II, and IGFBPs, and therefore allows the optimization of these molecules to identify and improve stabilizing interactions between IGF-I or IGF-II and IGFBPs. Preferably, the crystal effectively diffracts X-ray for the determination of the atomic coordinates of said complex to a resolution of 1.5 to 3.5 Å. The crystal is arranged in the cubic space group P2,3 having unit cell dimensions of 74.385 Å×74.385 Å×74.385 Å. The invention further provides a method for producing a crystal suitable for X-ray diffraction, comprising (a) contacting IGF-I or IGF-II with a polypeptide consisting of the amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, the amino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or a fragment thereof consisting at least of the 9 th to 12 th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7 th to 10 th cysteine of IGFBP-6, to form a complex which exhibits restricted conformation mobility, and (b) obtaining a crystal from the complex so formed suitable for X-ray diffraction. Using this crystal, the atomic coordinates of the complex can be determined. The invention further comprises a method for identifying a mutant of IGFBP or a mutant of a fragment thereof consisting at least of the 9 th to 12 th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7 th to 10 th cysteine of IGFBP-6, and having enhanced binding affinity for IGF-I and/or IGF-II comprising (a) constructing a three-dimensional structure of the complex of IGF-I or IGF-II and a polypeptide consisting of the amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, the amino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or a fragment thereof consisting at least of the 9 th to 12 th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7 th to 10 th cysteine of IGFBP-6, based on the atomic coordinates of a crystal consisting of IGF-I or IGF-II and said polypeptide; (b) employing said three-dimensional structure and modeling methods to identify said mutant in which an amino acid residue within a distance of 5 Å to a hydrophobic amino acid residue of IGF-I or IGF-II is modified in that the hydrophobic interaction between IGF-I or IGF-II and said mutant of IGFBP is enhanced; (c) producing said mutant; (d) assaying said mutant to determine said enhanced binding affinity for IGF. The invention further comprises a method for identifying a mutant of IGFBP-5 with enhanced binding affinity for IGF-I, said method comprising (a) constructing a three-dimensional structure of the complex of IGF-1 and IGFBP-5 defined by the atomic coordinates shown in FIGS. 5 and 6 ; (b) employing said three-dimensional structure and modeling methods to identify an amino acid residue in IGFBP-5 within a distance of 5 Å or shorter to an amino acid residue of IGF-I, wherein said residue of IGFBP-5 can be modified hydrophobically in that the hydrophobic interaction between IGF and IGFBP-5 is enhanced; (c) producing said mutant; (d) assaying said mutant to determine said enhanced binding affinity for IGF. The amino acid residue(s) in which IGFBP(s) is/are modified is/are preferably selected from the amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, the amino acids 49-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6. Especially preferred IGFBP mutants are modified at amino acid residues 49, 70 and/or 73 corresponding to IGFBP-5 sequence alignment and according to Table 7. The invention therefore provides mutant IGFBPs (“IGFBPs” as used herein means IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5 and/or IGFBP-6) with enhanced affinity (preferably about 3-fold to 10-fold increased affinity to the corresponding wild-type IGFBP) for IGF (“IGF” as used herein means IGF-I and/or IGF-II), improved inhibitory potency for the activity of IGF in vitro and in vivo and therefore improved therapeutic effectiveness. The invention further provides a method for identifying a compound capable of binding to IGFBP, comprising (a) constructing a three-dimensional structure of a complex of IGF-I or IGF-II and a polypeptide consisting of the amino acids 39-91 of IGFBP-1, amino acids 55-107 of IGFBP-2, amino acids 47-99 of IGFBP-3, amino acids 39-91 of IGFBP-4, amino acids 40-92 of IGFBP-5, amino acids 40-92 of IGFBP-6 or a fragment thereof consisting at least of the 9 th to 12 th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7 th to 10 th cysteine of IGFBP-6, based on the atomic coordinates of a crystal consisting of IGF-I and said IGFBP; (b) employing said three-dimensional structure and modeling methods to identify a compound forming a complex with said IGFBP by hydrophobic binding with amino acids 49, 50, 70, 71 and 74 in the case of IGFBP-5 and in the case of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4 and IGFBP-6 with corresponding amino acids according to Table 7; (c) producing said compound; (d) determining the binding between the compound and IGFBP. The invention further provides a method of inhibiting the binding of IGF to the IGFBP in a subject, preferably a human subject, comprising administering an effective amount of an above-described mutant of IGFBP to the subject. |
Bcl-2 dnazymes |
The present invention provides DNAzymes which specifically cleaves mRNA transcribed from a member of the bcl-2 gene family selected from the group consisting of bcl-2, bcl-xl, bcl-w, bfl-1, brag-1, Mcl-1 and A1. The DNAzymes comprise (a) a catalytic domain that has the nucleotide sequence GGCTAGCTACAACGA (SEQ ID NO.1) and cleaves mRNA at any purine: pyrimidine cleavage site at which it is directed, (b) a binding domain contiguous with the 5′ end of the catalytic domain, and (c) another binding domain contiguous with the 3′ end of the catalytic domain. The binding domains are complementary to, and therefore hybridise with, the two regions immediately flanking the purine residue of the cleavage site within the bcl-2 gene family mRNA, at which DNAzyme-catalysed cleavage is desired. Each binding domain is at least six nucleotides in length, and both binding domains have a combined total length of at least 14 nucleotides. |
1. A DNAzyme which specifically cleaves mRNA transcribed from a member of the bcl-2 gene family selected from the group consisting of bcl-2, bcl-xl, bcl-w, bfl-1, brag-1, Mcl-1 and A1, the DNAzyme comprising (a) a catalytic domain that has the nucleotide sequence GGCTAGCTACAACGA and cleaves mRNA at any purine:pyrimidine cleavage site at which it is directed, (b) a binding domain contiguous with the 5′ end of the catalytic domain, and (c) another binding domain contiguous with the 3′ end of the catalytic domain, wherein the binding domains are complementary to, and therefore hybridise with, the two regions immediately flanking the purine residue of the cleavage site within the bcl-2 gene family mRNA, at which DNAzyme-catalysed cleavage is desired, and wherein each binding domain is at least six nucleotides in length, and both binding domains have a combined total length of at least 14 nucleotides. 2. A DNAzyme according to claim 1 wherein the DNAzyme is 29 to 39 nucleotides in length. 3. A DNAzyme according to claim 1 wherein the bcl-2 gene family member is bcl-2 or bcl-xl. 4. A DNAzyme according to claim 1 selected from the group consisting of those listed in SEQ ID NOS. 7 to 61. 5. A DNAzyme according to claim 1 wherein the DNAzyme cleaves bcl-2 mRNA at position 455, 729, 1432, 1806 or 2093. 6. A DNAzyme according to claim 1 wherein the sequence of the DNAzyme is set out in SEQ ID NOS 24, 45, 53, 55 or 57. 7. A DNAzyme according to claim 1 selected from the group consisting of those listed in SEQ ID NOS. 62 to 87. 8. A DNAzyme according to claim 1 wherein the DNAzyme cleaves bcl-xl mRNA at position 126, 129 or 135. 9. A DNAzyme according to claim 1 wherein the sequence of the DNAzyme sequence is set out in SEQ ID NOS 82, 83 or 84. 10. A DNAzyme according to claim 1 wherein 1 to 6 phosphorothioate linkages are introduced into each of the 5′ and 3′ ends of the DNAzymes. 11. A DNAzyme according to claim 1 wherein the DNAzyme comprises at least one modification selected from the group consisting of 3′-3′ inversion, N3′-P5′ phosphoramidate linkages, peptide-nucleic acid linkages, and 2′-O-methyl. 12. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one DNAzyme according to claim 1. 13. A pharmaceutical composition according to claim 13 wherein the composition further comprises at least one chemotherapeutic agent selected from the group consisting of taxol, daunorubicin, dacitinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-flurouracil, floxuridine, methotrexate, colchicine, vincristine, vinlastin, etoposide and cisplatin. 14. A method of treating tumours in a subject, the method comprising administering to the subject a composition according to claim 13. 15. A method of enhancing the sensitivity of malignant or virus infected cells to therapy, the method comprising modulating expression level of a member of the bcl-2 gene family selected from the group consisting of bcl-2, bcl-xl, bcl-w, bfl-1, brag-1, Mcl-1 and A1 using a DNAzyme according to claim 1. 16. A method of treating tumours in a subject, the method comprising administering to the subject a first composition comprising at least one DNAzyme according to claim 1 and a second composition comprising an anticancer agent. 17. A method as claimed in claim 16 in which the anticancer agent is selected from the group consisting of tazol, daunorubicin, dacitinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-flurouracil, floxuridine, methotrexate, colchicine, vincristine, vinlastin, etoposide and cisplatin. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Apoptosis and Bcl-2 Gene Family Apoptosis is a complex process resulting in the regulated destruction of a cell, which plays a major role in normal development, cellular response to injury and carcinogenesis(Ellis et al., 1991). It has been suggested that an apoptotic component either contributes to, or accounts for, many human disease pathologies including cancer, viral infection and some neurological disorders (Ashkenazi and Dixit, 1998; Vocero-Akbani et al., 1999; Yakovlev et al., 1997). The Bcl-2 family of proteins are among the most studied molecules in the apoptotic pathway. Bcl-2 gene was first identified in B-cell lymphomas where the causal genetic lesion has been characterised as a chromosomal translocation (t(14:18)) which places the Bcl-2 gene under the control of the immunoglobulin promoter. The resulting overexpression of Bcl-2 retards the normal course of apoptotic cell death that otherwise maintains B-cell homeostasis, resulting in B-cell accumulation and follicular lymphoma (Adams and Cory, 1998). This observation showed that cancers do not strictly arise from unrestrained cell proliferation, but could also be due to insufficient apoptotic turnover. In addition to follicular lymphomas, Bcl-2 levels are elevated in a broad range of other human cancers, indicating that this molecule may have a role in raising the apoptotic threshold in a broad spectrum of cancerous disorders. The Bcl-2 gene family has at least 16 members involved in the apoptosis pathway. Some genes in this family are apoptosis inducers, including, bax, bak, bcl-Xs, bad, bid, bik and hrk, and others, such as bcl-2, bcl-XL, bcl-w, bfl-1, brag-1, Mcl-1 and A1 are apoptosis suppressors (Reed, 1998). Bcl-2 family members have been suggested to act through many different mechanisms, including pore formation in the outer mitochondrial membrane, through which cytochrome c(Cyt c) and other intermembrane proteins can escape; and heterodimerization between pro- and anti-apoptotic family members (Reed, 2000). It has been suggested that a decrease in Bcl-2 levels or the inhibition of Bcl-2 activity might provoke apoptosis or at least sensitise cells to apoptotic death. In the absence of a clearly defined biochemical mechanism of action or activity for this family of cell-death regulatory proteins (for which conventional inhibitors could therefore be developed), gene therapy and antisense approaches have become a reasonable alternative. For example, an 18-mer all-phosphorothioate Bcl-2 antisense oligodeoxynucleotide (ODN), G-3139 that targets the first six codons of the human Bcl-2 open reading frame, has shown very promising results in both preclinical and clinical studies (Jansen et al., 1998; Waters et al., 2000). This antisense molecule binds to the Bcl-2 mRNA blocking translation of the mRNA into Bcl-2 protein and targeting the message for RNAse H-mediated degradation. The resultant decrease in bcl-2 levels in the treated cells alters the balance between pro-apoptotic and anti-apoptotic family members in favour of pro-apoptotic members resulting in apoptosis. Using a similar strategy, antisense oligonucleotides to another member of the bcl-2 gene family bcl-xL has also been shown to be active in down-regulation of the bcl-xL expression, leading to an increased chemosensitivity in a range of cancer cells (Zangemeister-Wittke et al., 2000). Catalytic DNA (DNAzyme) In human gene therapy, antisense nucleic acid technology has been one of the major tools of choice to inactivate genes whose expression causes disease and is thus undesirable. The anti-sense approach employs a nucleic acid molecule that is complementary to, and thereby hybridizes with, a mRNA molecule encoding an undesirable gene. Such hybridization leads to the inhibition of gene expression. Anti-sense technology suffers from certain drawbacks. Anti-sense hybridization results in the formation of a DNA/target mRNA heteroduplex. This heteroduplex serves as a substrate for RNAse H-mediated degradation of the target mRNA component. Here, the DNA anti-sense molecule serves in a passive manner, in that it merely facilitates the required cleavage by endogenous RNAse H enzyme. This dependence on RNAse H confers limitations on the design of anti-sense molecules regarding their chemistry and ability to form stable heteroduplexes with their target mRNA's. Anti-sense DNA molecules also suffer from problems associated with non-specific activity and, at higher concentrations, even toxicity. As an alternative to anti-sense molecules, catalytic nucleic acid molecules have shown promise as therapeutic agents for suppressing gene expression, and are widely discussed in the literature (Haseloff and Gerlach 1988; Breaker 1994; Koizumi et al 1993; Kashani-Sabet et al 1992; Raillard et al 1996; and Carmi et al 1998) Thus, unlike a conventional anti-sense molecule, a catalytic nucleic acid molecule functions by actually cleaving its target mRNA molecule instead of merely binding to it. Catalytic nucleic acid molecules can only cleave a target nucleic acid sequence if that target sequence meets certain minimum requirements. The target sequence must be complementary to the hybridizing regions of the catalytic nucleic acid, and the target must contain a specific sequence at the site of cleavage. Catalytic RNA molecules (“ribozymes”) are well documented (Haseloff and Gerlach 1988; Symonds 1994; and Sun et al 1997), and have been shown to be capable of cleaving both RNA (Haseloff and Gerlach 1988) and DNA (Raillard et al 1996) molecules. Indeed, the development of in vitro selection and evolution techniques has made it possible to obtain novel ribozymes against a known substrate, using either random variants of a known ribozyme or random-sequence RNA as a starting point (Pan 1997; Tsang and Joyce 1996; and Breaker 1994). Ribozymes, however, are highly susceptible to enzymatic hydrolysis within the cells where they are intended to perform their function. This in turn limits their pharmaceutical applications. Recently, a new class of catalytic molecules called “DNAzymes” was created (Breaker and Joyce 1995; Santoro and Joyce 1997). DNAzymes are single-stranded, and cleave both RNA (Breaker (1994; Santoro and Joyce 1997) and DNA (Carmi et al 1998). A general model for the DNAzyme has been proposed, and is known as the “10-23” model. DNAzymes following the “10-23” model, also referred to simply as “10-23 DNAzymes”, have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. In vitro analyses show that this type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions under physiological conditions (Santoro and Joyce 1998). Several groups have examined the activity of DNAzymes in biological systems. DNAzyme molecules targeting c-myc were found to suppress SMC proliferation after serum stimulation (Sun et al 1997). Two studies have explored the activity and specificity of DNAzymes targeting the bcr-abI fusion in Philadelphia chromosome positive leukemia cells; Wu et al., 1999). The activity of these DNAzymes compared favourably with previous work with hammerhead ribozymes and antisense oligonucleotides (Gewirtz et al., 1998). More recently a 10-23 DNAzyme targeting the transcription factor Egr-1 has been shown to inhibit smooth muscle cell proliferation in cell culture and neointima formation in the rat carotid artery damaged by ligation injury or balloon angioplasty. (Santiago et al., 1999). Suppression of Egr-1 was also monitored at the RNA and protein level in treated smooth muscle cells by northern and western blot analysis respectively. This was the first evidence of DNAzyme efficacy in vivo, and furthermore the activity displayed by this anti-Egr-1 molecule could potentially find application in various forms of cardiovascular disease such as restenosis. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present inventors have determined that the level of expression of bcl-2 gene family members can be inhibited by DNAzymes. Accordingly in a first aspect the present invention consists in a DNAzyme which specifically cleaves mRNA transcribed from a member of the bcl-2 gene family, the DNAzyme comprising (a) a catalytic domain that has the nucleotide sequence GGCTAGCTACAACGA (SEQ ID No.1) and cleaves mRNA at any purine:pyrimidine cleavage site at which it is directed, (b) a binding domain contiguous with the 5′ end of the catalytic domain, and (c) another binding domain contiguous with the 3′ end of the catalytic domain, wherein the binding domains are complementary to, and therefore hybridise with, the two regions immediately flanking the purine residue of the cleavage site within the bcl-2 gene family mRNA, at which DNAzyme-catalysed cleavage is desired, and wherein each binding domain is at least six nucleotides in length, and both binding domains have a combined total length of at least 14 nucleotides. This invention also provides a method to enhance the sensitivity of malignant or virus infected cells to therapy by modulating expression level of a member of the bcl-2 gene family using catalytic DNA. It is preferred that the bcl-2 gene family member is selected from the group consisting of bcl-2, bcl-xl, bcl-w, bfl-1, brag-1, Mcl-1 and A1. It is particularly preferred that the bcl-2 gene family member is bcl-2 or bcl-xl. |
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