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The present invention relates to process for the preparation of a 14-hydroxynormorphinone derivative of formula IV comprising reacting the compound of formula III, with a cobalt (II) oxidant in the presence of a mild base and air or oxygen as the cooxidant; wherein R1 is (1C-7C)alkyl optionally substituted with one or more chlorines, butenyl, vinyl, benzyl, phenyl or naphthyl; and R2 is benzyl or benzyl substituted with one or more (1C-6C)alkoxy group or benzyl substituted with one or more halogen. The process is very suitable in the production of noroxymorphone.
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1. A process for the preparation of a 14-hydroxynormorphinone derivative of formula IV comprising reacting the compound of formula III, with a cobalt (II) oxidant in the presence of a mild base and air or oxygen as the cooxidant; wherein R1 is (1C-7C)alkyl optionally substituted with one or more chlorines, butenyl, vinyl, benzyl, phenyl or naphthyl; and R2 is benzyl or benzyl substituted with one or more (1C-6C)alkoxy group or benzyl substituted with one or more halogen. 2. The process of claim 1, wherein the oxidant is Co(OAc)2. 3. The process of claim 1, wherein the cooxidant is oxygen. 4. The process of claim 1, wherein the mild base is sodium acetate, potassium acetate, sodium phosphate or potassium phosphate. 5. The process of claim 4, wherein the mild base is sodium acetate. 6. The process of claim 1, wherein R1 is (1-7C)alkyl. 7. The process of claim 6, wherein R1 is ethyl. 8. The process of claim 1, wherein R2 is benzyl. 9. A 14-hydroxynormorphinone derivative of the formula IV wherein R1 is (1C-7C)alkyl optionally substituted with one or more chlorines, butenyl, vinyl, benzyl, phenyl or naphthyl; and R2 is benzyl or benzyl substituted with one or more (1C-6C)alkoxy group or benzyl substituted with one or more halogen. 10. The 14-hydroxynormorphinone derivative of claim 9, wherein R1 is ethyl. 11. The 14-hydroxynormorphinone derivative of claim 9, wherein R2 is benzyl. 12. A morphinone derivative of the formula III wherein R1 is (1C-7C)alkyl optionally substituted with one or more chlorines, butenyl, vinyl, benzyl, phenyl or naphthyl; and R2 is benzyl or benzyl substituted with one or more (1C-6C)alkoxy group or benzyl substituted with one or more halogen. 13. The morphinone derivative of claim 12, wherein R1 is ethyl. 14. The morphinone derivative of claim 12, wherein R2 is benzyl. 15. A process for the preparation of a compound of formula III, comprising: reactively contacting a morphine derivative of formula II with an oxidizing agent effective for oxidizing allylic hydroxy groups to form keto groups, where the compound of formula III according to claim 12 is prepared. 16. The process of claim 15, wherein the oxidizing agent is sodium dichromate. 17. The process of claim 15, wherein R, is ethyl and R2 is benzyl. 18. A morphine derivative of formula II wherein R1 is (1C-7C)alkyl optionally substituted with one or more chlorines, butenyl, vinyl, benzyl, phenyl or naphthyl; and R2 is benzyl or benzyl substituted with one or more (1C-6C)alkoxy group or benzyl substituted with one or more halogen. 19. The morphine derivative of claim 18, wherein R1 is ethyl. 20. The morphine derivative of claim 17, wherein R2 is benzyl. 21. A process for the production of noroxymorphone, comprising: a reaction step wherein a morphinone derivative of formula III is oxidized into a 14-hydroxynormorphinone derivative of formula IV wherein R1 is (1C-7C)alkyl optionally substituted with one or more chlorines, butenyl, vinyl, benzyl, phenyl or naphthyl; and R2 is benzyl or benzyl substituted with one or more (1C-6C)alkoxy group or benzyl substituted with one or more halogen. 22. A process for the production of a compound of formula III, comprising: oxidizing a compound of formula II to form a morphinone derivative of formula III wherein R1 is (1C-7C)alkyl optionally substituted with one or more chlorines, butenyl, vinyl, benzyl, phenyl or naphthyl; and R2 is benzyl or benzyl substituted with one or more (1C-6C)alkoxy group or benzyl substituted with one or more halogen. 23. A process for the production of noroxymorphone, wherein morphine is converted into noroxymorphone, comprising: (a) converting morphine having the formula I by reaction with a haloformate ester of the formula X—C(═O)OR1, wherein R1 is (1C-7C)alkyl optionally substituted with one or more chlorines, butenyl, vinyl, benzyl, phenyl or naphthyl and X is a halogen, followed by a reaction with R2—X, wherein X is as previously defined and R2 is benzyl or benzyl substituted with one or more (1C-6C)alkoxy group or benzyl substituted with one or more halogen, to form a morphine derivative of formula II (b) oxidizing the morphine of formula II to form a morphinone derivative of formula III wherein the process is reactively contacting a morphine derivative of formula II with an oxidizing agent effective for oxidizing allylic hydroxy groups to form keto groups; (c) oxidizing the morphinone derivative of formula III to form a 14-hydroxynormorphinone derivative of formula IV according to process according to claim 1; (d) deprotecting the 3-position and reducing the double bond at the 7,8-position of the 14-hydroxynormorphinone derivative of formula IV to form a 3,14-hydroxynormorphinone derivative of formula V, (e) and hydrolyzing the 3,14-hydroxynormorphinone derivative of formula V into noroxymorphone of formula VI,
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Method and composition for treating immune complex associated disorders
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The present invention provides methods and compositions for treating immune complex associated diseases (ICAD), such as SLE, rheumatoid arthritis, and hepatitis-C related immune complex disease (e.g., cryoglobulinemia) in a subject having an ICAD or at risk for developing ICAD. The invention is based upon the surprising finding that chromatin-containing immune complexes activate autoreactive B cells and dendritic cells by a dual receptor engagement process which, in both cell types, involves a Toll-like receptor (TLR). The methods of treating ICAD comprise administering a compound to an individual in need thereof that either 1) inhibits formation of the immune complex either by preventing formation and/or binding to the TLR, or 2) interferes with binding of an autoantigen-containing immune complex (or the antigenic component thereof) to the TLR, or 3) inhibits signaling pathways initiated by dual engagement of BCR and TLR (in B cells) or FcR and TLR (in dendritic cells) via immune complexed or uncomplexed autoantigens.
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1. A method of treating a patient having or at risk of having an Immune Complex Associated Disease (ICAD) or a systemic autoimmune disease, comprising administering to a patient having or at risk of having an ICAD or a systemic autoimmune disease an effective amount of a compound that inhibits immune complexes or autoantigens from binding to or activating a Toll-like receptor (TLR), wherein the Toll-like receptor is chosen from Toll-like receptor-9 (TLR9) and Toll-like receptor-3 (TLR3) and wherein said immune complex comprises an autoantibody and an autoantigen bound to a cell receptor, to treat the ICAD or systemic autoimmune disease. 2. The method of claim 1, wherein the ICAD is systemic lupus erythematosus. 3. The method of claim 1, wherein the ICAD is rheumatoid arthritis. 4. The method of claim 1, wherein the ICAD is hepatitis-C related immune complex disease. 5. The method of claim 1 wherein the Toll-like receptor is TLR9. 6. The method of claim 1 wherein the Toll-like receptor is TLR3. 7. The method of claim 1, wherein the compound is selected from the group consisting of (a) compounds that bind components of the immune complex and prevent its formation or binding to the Toll-like receptor; (b) Toll-like receptor decoys; (c) compounds that inhibit the activity of MyD88 or other components of a TLR-initiated signaling cascade; (d) compounds that inhibit production of immune complex components; and (e) Toll-like receptor antagonists. 8. The method of claim 1, wherein the compound is an antibody that binds a Toll-like receptor. 9. The method of claim 8, wherein the antibody is a single chain antibody. 10. The method of claim 1, wherein the compound comprises a cocktail of compounds that inhibit binding to a combination of at least two of Toll-like receptor-2 (TLR2), TLR3, and TLR9. 11. The method of claim 7, wherein the Toll-like receptor antagonist is an inhibitory oligonucleotide. 12. The method of claim 7, wherein the Toll-like receptor antagonist is a dominant negative Toll-like receptor. 13. A method for screening for compounds that inhibit immune complex formation or binding to a Toll-like receptor, comprising contacting immune complex components with a compound being screened and with a TLR chosen from TLR3 and TLR9; measuring binding of an antigenic fragment of an immune complex to the TLR, wherein said immune complex comprises an autoantibody and an autoantigen; and identifying the compound being screened as an inhibitor of immune complex formation or of binding to a Toll-like receptor when the binding with the compound is reduced compared to binding without the compound. 14. A method for screening for compounds that inhibit immune complex formation or binding to a Toll-like receptor, comprising contacting immune complex components with a compound being screened and with a dendritic cell that expresses a TLR chosen from TLR3 and TLR9; measuring activation of the dendritic cell; and identifying the compound being screened as an inhibitor of immune complex formation or of binding to a Toll-like receptor when activation of the dendritic cell with the compound is reduced compared to activation of the dendritic cell without the compound. 15. (canceled) 16. (canceled) 17. The method of claim 13, wherein the TLR is TLR9. 18. The method of claim 13, wherein the binding with the compound is reduced at least 50 percent compared to binding without the compound. 19. The method of claim 14, wherein the TLR is TLR9. 20. The method of claim 14, wherein the measuring activation of the dendritic cell comprises measuring production of a cytokine chosen from TNF-α, interferon-α, and BAFF. 21. The method of claim 14, wherein the measuring activation of the dendritic cell comprises measuring upregulated expression on the dendritic cell of a costimulatory molecule chosen from CD80, CD86, and MHC class II. 22. The method of claim 14, wherein the activation of the dendritic cell with the compound is reduced at least 50 percent compared to activation of the dendritic cell without the compound. 23. The method of claim 14, wherein the immune complex components comprise an autoantibody and an autoantigen. 24. A method for screening for compounds that inhibit immune complex formation or binding to a Toll-like receptor, comprising contacting immune complex components with a compound being screened and with a B cell that expresses a TLR chosen from TLR3 and TLR9; measuring activation and/or proliferation of the B cell; and identifying the compound being screened as an inhibitor of immune complex formation or of binding to a Toll-like receptor when activation and/or proliferation of the B cell with the compound is reduced compared to activation and/or proliferation of the B cell without the compound. 25. The method of claim 24, wherein the TLR is TLR9. 26. The method of claim 24, wherein the measuring activation of the B cell comprises measuring upregulated expression on the B cell of a costimulatory molecule chosen from CD80, CD86, and MHC class II. 27. The method of claim 24, wherein the activation and/or proliferation of the B cell with the compound is reduced at least 50 percent compared to activation and/or proliferation of the B cell without the compound. 28. The method of claim 24, wherein the immune complex components comprise an autoantibody and an autoantigen.
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<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates to methods and compositions for treating immune complex associated diseases, preferably systemic lupus erythematosus (SLE), and other systemic autoimmune diseases associated with the subject having aberrant Toll-like receptor (TLR)/B cell receptor (BCR) dual engagement (in B cells) or TLR/Fc gamma receptor dual engagement (in dendritic cells and/or macrophages). 2. Background Autoimmune diseases are a fairly common but poorly understood group of diseases in which an individual's immune system either 1) begins recognizing self antigens as foreign and starts destroying tissues expressing such antigens thereby causing a disease, or 2) forms immune complexes with these antigens which then deposit in tissues and cause inflammatory pathology. Such autoimmune diseases include, for example, diabetes wherein the immune system turns against and destroys insulin producing pancreatic islet cells; multiple sclerosis, wherein the target antigen is the myelin sheath protecting neurons leading to destruction of function of motorneurons; psoriasis, where the target of the immune system is skin; rheumatoid arthritis, where the target organ is cartilage; and systemic lupus erythematosus (SLE), which presents itself as targeting a variety of tissues with no apparent specificity or selectivity although the target antigens themselves are extremely consistent and characteristic. Because the mechanisms leading to the development of autoimmune diseases in general are mostly unknown, their treatment is often directed to generally suppressing the immune system. Such general immunosuppressive therapies often cause a variety of undesirable side effects including cancer, infertility, and increased susceptibility to infections by viruses, fungi, yeast, and bacteria Therefore, it would be desirable to understand the mechanisms that cause the immune system to turn against self antigens to enable development of more specific therapies for the treatment of the autoimmune diseases. An example of a poorly understood autoimmune disease is systemic lupus erythematosus (SLE), commonly known as Lupus. SLE is characterized by dysregulation of the immune system resulting in the production of antinuclear antibodies, the generation of circulating immune complexes, and the activation of the complement system. The immune complexes build up in the tissues and joints causing inflammation, and degradation to both joints and tissues. While the word “systemic” correctly suggests that the disease affects the entire body and most organ systems, the disease most often involves inflammation and consequent injury to the joints, skin, kidney, brain, the membranes in body cavities, lung, heart, and gastrointestinal tract. An individual with SLE often experiences unpredictable acute episodes or “outbreaks” and equally unexpected remissions. The pathologic hallmark of the disease is recurrent, widespread, and diverse vascular lesions resembling a rash or changes on the surface of the skin. The prevalence of SLE in the United States is an issue of some debate. Estimates of prevalence range from 250,000 to 2,000,000 persons. Although reported in both the extremely old and the extremely young, the disease mainly affects women of childbearing age. Among children SLE is three times more common in females than in males. In the 60% of SLE patients who experience the onset of this disease between puberty and the fourth decade of life, the female to male ratio is 9:1. Thereafter, the female preponderance again falls to that observed in prepubescent children (i.e. 3:1). In addition, the disorder appears to be three times more common in persons of African and Asian descent than in persons of Caucasian descent. The etiology of SLE remains unknown. A genetic predisposition, the systemic proliferation of sex hormones, and various environmental triggers, such as viral infections have been suggested to play a role in triggering the aberrant immune responses that typify the disease. A role for genetics is suggested by the increased percentage of two histocompatibility antigens, HLA-DR2 and HLA-DR3, in patients with SLE. In addition, there is an increased frequency of the extended haplotypes HLA-A1, B8, and DR3 in affected individuals. The role of heredity is further supported by the concordance for this illness among monozygotic twins. The polygenic nature, however, of this genetic predisposition as well as the contribution of environmental factors is suggested by the concordance rate, which is only moderate and reported to be between 25% and 60%. The precise initiating etiology of SLE is unknown. However, it is generally accepted that most of the clinical manifestations of the disease are caused either directly or indirectly by autoantibody production and the subsequent formation of pathogenic immune complexes. These autoantibodies, which are produced by dysregulated B lymphocytes, have distinct specificities recognizing discrete nuclear autoantigens including, among others, DNA, nucleosomes and subnucleosomes. Certain RNA/protein complexes including the Sm antigen and small nuclear ribonucleoproteins (snRNP) are additional characteristic autoantigenic specificities. The pathogenic immune complexes are formed by binding of the autoantibodies to their respective nuclear autoantigens. Autoantibodies in SLE often circulate as immune complexes (IC) bound with their respective autoantigens. Chromatin or chromatin fragments such as DNA, nucleosomes or subnucleosome particles are especially common autoantigenic specificities in both mice and humans (Tan, E. Adv. Immunol. 44, 93-151 (1989); Monestier and Novick, Mol. Immunol. 33: 89-99., 1996) The central goals in the treatment of SLE, therefore, are either to attempt to suppress the dysfunctional B lymphocytes thereby decreasing the production of autoantibody or, to attempt to diminish the pathogenicity of the immune complexes once they have formed. At present these goals can only be achieved, and often incompletely so, by the use of intensive systemic immunosuppressive drug therapy using drugs such as cortisone, azathioprine, hydroxychloroquine and cyclophosphamide. These therapies are associated with many serious and undesirable side-effects including infections, infertility, retinopathy and cancer. Therefore, new treatments for SLE, and other autoimmune diseases, would be desirable.
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<SOH> SUMMARY OF THE INVENTION <EOH>It is therefore the purpose of the present invention to provide methods and compositions for treating immune complex associated diseases (ICAD), such as SLE, rheumatoid arthritis, and hepatitis-C related immune complex disease (e.g., cryoglobulinemia) in a subject having an ICAD or at risk for developing ICAD. We have discovered that chromatin-containing immune complexes activate autoreactive B cells and dendritic cells by a dual receptor engagement process. In both cell types a Toll-like receptor (TLR) is involved. TLR9, located in a cytoplasmic compartment, is the essential second receptor required for cell activation. In the case of the B cell, the B cell antigen receptor located on the cell surface is the essential first receptor required for cell activation. In the case of the dendritic cell, a stimulatory Fc gamma receptor located on the cell surface is the essential first receptor required for cell activation. We have found a method of treating ICAD by administering a compound to an individual in need thereof that either 1) inhibits formation of the immune complex (i.e., autoantibody and nuclear autoantigen) either by preventing formation and/or binding to the Toll-like receptor (TLR), or 2) interferes with binding of an autoantigen-containing immune complex (or the antigenic component thereof) to the TLR, or 3) inhibits signaling pathways initiated by dual engagement of BCR and TLR (in B cells) or FcR and TLR (in dendritic cells) via immune complexed or uncomplexed autoantigens. The compound is administered in a pharmaceutically acceptable carrier. Preferably, the ICAD is SLE, rheumatoid arthritis or hepatitis-C related immune complex disease (e.g., cryoglobulinemia). In an other embodiment, the ICAD is related to an immune reaction in a host after organ transplantation. Although not working to be bound by theory, we believe that immune complexes (IC) containing an autoantigen, such as chromatin, but not IC containing a foreign antigen, are able to activate autoreactive B cells and that this activation is absolutely dependent on the ability of the autoantigen-containing IC to sequentially engage either the B cell receptor (BCR) in B cells or FcγR in dendritic cells, and a second receptor, a Toll-like receptor. This finding establishes a novel link between the innate and adaptive immune systems and suggests a general mechanism whereby autoreactive B cells or dendritic cells specific for protein/nucleic acid autoantigens are activated. According to one aspect of the invention a method is provided for treating a patient having an ICAD or at risk for an ICAD by identifying an individual with ICAD and administering an effective amount of a compound capable of inhibiting the autoantigen or autoantigen/immune complex from forming and/or from activating B cells or dendritic cells. A person at risk for ICAD or systemic autoimmune disease includes individuals having at least a parent, grandparent or sibling who has an ICAD or systemic autoimmune disease. The compound is selected from a group consisting of compounds that bind components of the immune complex and either prevent its formation or prevent the autoantigen from activating a Toll-like receptor (TLR). Such compounds include Toll-like receptor decoys, compounds that inhibit the activity of MyD88, compounds that inhibit production of immune complex components (e.g., antisense nucleotides), dominant-negative TLR, a Toll-like receptor antagonist, and compounds that inhibit signaling pathways activated by the interaction or binding of the immune complex or autoantigen to the TLR. Preferably, the compound binds and/or inhibits function of Toll-like receptors TLR2, TLR3, and TLR9 or functional domains thereof. More preferably, the TLR is TLR3 or TLR9 or a functional fragment thereof. Compounds that bind components of the immune complex and prevent its formation or prevent binding of the complex to the Toll-like receptor and compounds that inhibit MyD88 signaling include polyclonal and monoclonal antibodies, dominant negative proteins that can block wildtype TLR activity or block components of the TLR-mediated signaling cascade, inhibitory oligodeoxynucleotides (ODN), such as S-ODN 2088 (Lenart, et al., Antisense Nucleic Acid Drug Dev. 4, 247-256 (2001)), antisense nucleotides including RNA and modified nucleotides, or other pathway specific kinase inhibitors. The compound is a compound other than chloroquine. In one embodiment the invention provides methods for screening compounds or agents that inhibit immune complex formation or binding to the Toll-like receptor and/or inhibit B cell/dendritic cell activation. The methods comprise contacting immune complex components with a test agent and measuring B cell or dendritic cell activation and/or proliferation and/or binding of the complex to the Toll-like receptor. In an other embodiment, a method of diagnosing an ICAD is provided. The method comprises taking a biological sample comprising IgG of an individual suspected of having ICAD, incubating the biological sample together with RF+ B cells or dendritic cells, and measuring the activation of the RF+ B cells or dendritic cells, wherein a change in activity in the RF+ B cell or dendritic cell cultures exposed to the biological sample from the individual suspected of having ICAD relative to the RF+ B cells or dendritic cells exposed to a biological sample comprising IgG from a control individual is indicative of ICAD. Preferably, the change is an increase in activity. Activation of the B cells can be measured, for example by measuring proliferation or upregulation of co-stimulatory molecules such as CD80 and CD86 as well as upregulation of MHC class II molecules. Activation of dendritic cells can be assessed by measuring the expression of co-stimulatory molecules, production of cytokines, e.g., TNF-α, or changes in the dendritic cell phenotype. In yet another embodiment, the invention provides an in vivo model system for evaluating a compound or an agent for its efficacy in treating ICAD. The model system comprises administering a test agent to an ICAD model animal including mouse and rat models, and measuring B cell or dendritic cell activation and/or proliferation and/or binding of the complex to the Toll-like receptor in such animal, wherein decreased activation is indicative of an agent which is capable of treating ICAD. Alternatively, one can use cell lines expressing Toll-like receptors and screen for compounds that modulate, preferably inhibit or block, such receptors. Other aspects of the invention are disclosed infra.
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Digital time stamping system
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A digital time-stamping system for issuing stamps on messages by means of a cryptographic digital signature includes a front end unit (10), a load balancer (50) and one or more public key signature units (60). Incoming messages are rapidly and temporarily time-stamped by means of a MAC generator (30) in the front end unit, and the message is then passed on to the load balancer. The load balancer queues the messages, and passes them on at an appropriate speed to the public key signature units (60), which verifies the MAC and resigns the message with a public key signature. The front end unit (10) and the public key signature units (60) are preferably individual secure modules with the load balancer (50) being outside the security boundaries. The system enables large numbers of incoming messages to be accurately time-stamped in a secure manner.
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1. A digital time-stamping system comprising: (a) a first signature generator for receiving an incoming message, combining the message with time of arrival information, and digitally signing it to create a temporary signed message; (b) a signature verifier for verifying the signature of the temporary signed message; and (c) a second signature generator for creating, where authorised to do so by the signature verifier, an output time-stamped message by digitally signing a combination of the message and the time of arrival information. 2. A digital time-stamping system as claimed in claim 1 in which the first signature generator digitally signs using a cryptographic Message Authentication Code. 3. A digital time-stamping system as claimed in claim 1 in which the second signature generator digitally signs using a cryptographic public key system. 4. A digital time-stamping system as claimed in claim 1 including a load balancer for receiving and holding the temporary signed message and for passing it on to the signature verifier. 5. A digital time-stamping system as claimed in claim 4 in which the load balancer includes a temporary message queue. 6. A digital time-stamping system as claimed in claim 4 including a plurality of signature verifiers and corresponding second signature generators, the load balancer controlling message transmission to the respective signature verifiers. 7. A digital time-stamping system as claimed in claim 1 in which the first signature generator uses a first key to create the temporary signed message and the signature verifier uses a copy of the first key to verify the signature. 8. A digital time-stamping system as claimed in claim 7 in which the first key and the copy of the first key are both securely stored. 9. A digital time-stamping system as claimed in claim 1 in which the first signature generator is contained within a first secure area. 10. A digital time-stamping system as claimed in claim 9 in which the first secure area is a secure first hardware module. 11. A digital time-stamping system as claimed in claim 10 in which the first secure area contains a first key store which holds a first key used by the first signature generator to create the temporary signed message. 12. A digital time-stamping system as claimed in claim 11 in which the first secure area includes a time source for generating time of arrival information. 13. A digital time-stamping system as claimed in claim 10 in which the signature verifier is contained within a second secure area. 14. A digital time-stamping system as claimed in claim 13 in which the second secure area is a second secure hardware module. 15. A digital time-stamping system as claimed in claim 14 in which the second secure area includes a second key storage which holds a copy of the first key for use by the signature verifier to verify the signature of the temporary signed message. 16. A digital time-stamping system as claimed in claim 13 in which the second signature generator is contained within the second secure area. 17. A digital time-stamping system as claimed in claim 16 in which the second secure area includes a second key store which holds a second key for use by the second signature generator to create the output time stamped message. 18. A digital time-stamping system as claimed in claim 1 including a first secure area containing the first signature generator and a second secure area containing the signature verifier. 19. A digital time-stamping system as claimed in claim 18 in which the first and second secure areas comprise respective first and second secure hardware modules. 20. A digital time-stamping system as claimed in claim 18 in which the first signature generator uses a first key stored within the first secure area and the signature verifier uses a copy of the first key stored in the second secure area. 21. A digital time-stamping system as claimed in claim 18 including a load balancer for receiving and holding the temporary signed message and for passing it on to the second secure area, the load balancer being outside both the first and second secure areas. 22. A digital time-stamping system as claimed in claim 21 including a plurality of second secure areas each containing a respective signature verifier, the load balancer controlling message transmission to the second secure areas. 23. A digital time-stamping system as claimed in claim 18 in which the temporary signed message passes out of the first secure area on its way to the second secure area. 24. A digital time-stamping system as claimed in claim 1 including a time source for generating the time of arrival information. 25. A method of digital time-stamping comprising: (a) receiving an incoming message, combining the message with time of arrival information, and digitally signing it to create a temporary signed message; (b) verifying the signature of the temporary signed message; and (c) where the verification is successful, creating an output time-stamped message by digitally signing a combination of the message and the time of arrival information. 26. A computer program for carrying out the method as claimed in claim 25. 27. A computer-readable media carrying the computer program as claimed in claim 26.
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Nucleic acid compositions conferring altered metabolic characteristics
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This invention encompasses the identification and isolation genes and gene fragments that confer altered metabolic characteristics in Nicotiana benthamiana plants, when expressed using GENEWARE™ viral vectors. These genes are derived from a variety of sources. Expression of these genes resulted in alterations of the levels of at least one of the following metabolites: acids, fatty acids, amino acids and related compounds, branched fatty acids, carbohydrates, hydrocarbons, alkaloids and other bases, esters, glycerides, phenols and related compounds, alcohols, alkenes and alkynes, sterols, oxygenated terpenes, and other isoprenoids, and ketones and quinones.
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1. An isolated nucleic acid selected from the group consisting of SEQ ID NOs: 1-7554 and nucleic acid sequences that hybridize to any thereof under conditions of low stringency, wherein expression of said isolated nucleic acid in a plant results in an altered metabolic characteristic. 2. An isolated nucleic acid of claim 1 selected from SEQ ID NOs: 162, 212, 3781, 3970, 3990, 492, 3796, 3975, and 4028, wherein expression of said nucleic acid in a plant results in altered acid metabolism. 3. An isolated nucleic acid of claim 1 selected from SEQ ID NOs: 4049, 210, 4045, 229, 3825, 4015, 3835, 4039, 1048 and 1106, wherein expression of said nucleic acid in a plant results in altered alcohol metabolism. 4. An isolated nucleic acid of claim 1 selected from SEQ ID NOs: 7548, 283, 3957, 3734, 3739, 3797, 7516, 3762, 4020 and 1062, wherein expression of said nucleic acid in a plant results in altered fatty acid metabolism. 5. An isolated nucleic acid of claim 1 selected from SEQ ID NOs: 1148, 4147, 273, 281, 299, 3920, 450, 7463 and 4074, wherein expression of said nucleic acid in a plant results in altered branched fatty acid metabolism. 6. An isolated nucleic acid of claim 1 selected from SEQ ID NOs: 258, 456, 3859, 3817, 4018, 3848, 3862, 4008 and 1000, wherein expression of said nucleic acid in a plant results in altered alkaloid or other base metabolism. 7. An isolated nucleic acid of claim 1 selected from SEQ ID NOs: 372, 3714, 3717, 3963, 3775, 3757, 7462, 3743, 3744 and 7480, wherein expression of said nucleic acid in a plant results in altered amino acid metabolism. 8. An isolated nucleic acid of claim 1 selected from SEQ ID NOs: 7404, 180, 181, 225, 231, 366, 3983, 3833, 1121 and 1062, wherein expression of said nucleic acid in a plant results in altered ester metabolism. 9. An isolated nucleic acid of claim 1 selected from SEQ ID NOs: 3773, 583, 3821, 7403, 988, 1002, 1007 and 1129, wherein expression of said nucleic acid in a plant results in altered glyceride metabolism. 10. An isolated nucleic acid of claim 1 selected from SEQ ID NOs: 150, 7410, 175, 7553, 619, 1078, 1122 and 1124, wherein expression of said nucleic acid in a plant results in altered phenolic compound metabolism. 11. An isolated nucleic acid of claim 1 selected from SEQ ID NOs: 3891, 7545, 7551, 4121, 157, 159, 7411, 3792, 3799 and 3997, wherein expression of said nucleic acid in a plant results in altered carbohydrate metabolism. 12. An isolated nucleic acid of claim 1 selected from SEQ ID NOs: 7405, 7406, 173, 183, 220, 227, 3778, 3803, 3847 and 1005, wherein expression of said nucleic acid in a plant results in altered sterol, oxygenated terpene, or isoprenoid metabolism. 13. An isolated nucleic acid of claim 1 selected from SEQ ID NOs: 7408, 351, 378, 3864, 4103, 996, 1006 and 1098, wherein expression of said nucleic acid in a plant results in altered alkene or alkyne metabolism. 14. An isolated nucleic acid of claim 1 selected from SEQ ID NOs: 177, 7442, 4038, 3836, 3855, 1012, 1015, 1119 and 1024, wherein expression of said nucleic acid in a plant results in altered hydrocarbon metabolism. 15. An isolated nucleic acid of claim 1 selected from SEQ ID NOs: 360, 4001, 3703, 7399, 645, 3849 and 7552, wherein expression of said nucleic acid in a plant results in altered ketone or quinone metabolism. 16. A vector comprising the isolated nucleic acid of claim 1. 17. The vector of claim 16, wherein said isolated nucleic acid is operably linked to a plant promoter. 18. A vector according to claims 16, wherein said isolated nucleic acid is in sense orientation. 19. A vector according to claims 16, wherein said isolated nucleic acid is in antisense orientation. 20. A plant transfected with an isolated nucleic acid or vector according to claim 1. 21. A seed from the plant of claim 20. 22. A leaf, root or stem from the plant of claim 21. 23. An isolated nucleic acid according to claim 1, for use in conferring an altered metabolic characteristic in a plant. 24. A process for making a transgenic plant comprising: a. providing a vector according to claims 16 and a plant, b. and transfecting said plant with said vector. 25. A process for providing disease resistance in a plant comprising: a. providing a vector according to claim 16 and a plant, b. and transfecting said plant with said vector under conditions such that an altered metabolic characteristic is conferred by expression of said isolated nucleic acid from said vector. 26. An isolated nucleic acid selected from the group consisting of SEQ ID NOs: 1-7554 and nucleic acid sequences that hybridize to any thereof under conditions of low stringency for use in producing plants with an altered metabolic characteristic. 27. canceled. 28. A method for identifying altered metabolic characteristics in a biological system comprising isolation of metabolites, comparing for altered metabolic characteristics relative to a control or reference using a bioinformatics system and suitable analytical methodology.
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<SOH> BACKGROUND OF THE INVENTION <EOH>Plants are photosynthetic organisms able to fix inorganic carbon (CO 2 ) in organic matter via energy from light and minerals contained in water. All carbons fixed primarily via the pentose/triose phosphate cycle are converted in numerous anabolic pathways necessary to sustain life (primary metabolism). To survive plants must adapt to their environment and synthesize an extremely wide range of organic compounds required to interact with the elements of their microenvironment (secondary metabolism). To capture the biochemical diversity of this particular kingdom both primary and secondary metabolism have to be taken into account. The primary metabolism is represented by the biosynthesis of building blocks of macromolecules such as amino acids, fatty acids, carbohydrates, and sterols. Each of these groups of compounds is of economic importance. Fatty acids can also be used as a raw material for industrial applications in a variety of products, including soaps, lubricants, paints, detergents, adhesives, and plasticizers. Furthermore, fatty acids are the major components of edible oils. For example, fatty acid compounds are involved in building blocks for protection (cell membrane, epicuticular polymers), storage of energy in the plant seeds and as secondary messengers in the plant cell. As another example, carbohydrates are intermediates in the biosynthesis of energy reserves (starch, cellulose) and building blocks of the cell wall giving the plant shape and structure. The carbohydrates are the carbon skeletons of many biosynthetic reactions. As such, the ability to alter carbohydrate metabolism could lead to many improvements in plants, including increased transport and accumulation of starch by accumulation of hexose phosphate that could improve starch yield in the seed and the plant; alterations in the cell wall for better resistance to pest and drought; better digestibility for forage plants; and better processivity for pulp production in paper industry (e.g., less lignin and hemicellulose). The advent of modern biology, particularly molecular biology and genetics, has opened up new avenues for altering the production of compounds of economic importance by plants. Scientists have focused on utilizing recombinant DNA (rDNA) methods, that allow new varieties of plants to be produced much faster than by conventional breeding. rDNA techniques allow the introduction of genes from distantly related species or even from different biological kingdoms into crop plants, conferring traits that provide significant agronomic advantages. Furthermore, detailed knowledge of the traits being introduced, such as cellular function and localization, can lead to less variability in offspring, and fine-tuning of secondary effects (e.g., permitting variation from what is customarily observed). After a trait has been introduced into a plant by transgenic methods, conventional breeding can be used to hybridize the transgenic line with useful varieties and elite germplasms, resulting in crops containing numerous advantageous properties. Most efforts to engineer plants with specific traits thus far have been based on the rational design paradigm of transforming a plant with a gene of known function with the intent of introducing a known trait. As agricultural biotechnology hurtles into the genomics and post-genomics era, the massive amounts of genetic and functional data being generated are being used to direct the search for genes that can be utilized with recombinant methods. However, if the use of this information is limited to the rational design paradigm, the identification of genes with truly profound effects on the production of desired compounds by plants could be extremely time-consuming and slow. Accordingly, what is needed in the art are methods for rapidly screening and identifying gene sequences and polypeptide sequences of previously unknown function whose expression causes altered metabolic characteristics in biological systems, including, but not limited to, plants.
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<SOH> SUMMARY OF THE INVENTION <EOH>This invention relates to deoxyribonucleic acid (DNA) and amino acid sequences that confer altered metabolic characteristics in plants. In some embodiments, the present invention provides polynucleotides and polypeptides that confer altered metabolic characteristics when expressed in plants. The present invention is not limited to the alteration of amounts or levels of any particular metabolite. Indeed, the alteration of the levels or amounts of a variety of metabolites is contemplated, including, but not limited to acids, fatty acids, amino acids, hydroxy fatty acids, branched fatty acids, carbohydrates, hydrocarbons, glycerides, phenols, strerols, oxygenated terpenes, and other isoprenoids, alcohols, alkenes and alkynes. The present invention is not limited to any particular polypeptide or polynucleotide sequences that confer altered metabolic characteristics. Indeed, a variety of such sequences are contemplated. Accordingly, in some embodiments the present invention provides an isolated nucleic acid selected from the group consisting of SEQ ID NOs: 1-7554 and nucleic acid sequences that hybridize to any thereof under conditions of low stringency, wherein expression of the isolated nucleic acid in a plant results in a altered metabolic characteristic. In some embodiments, the present invention provides an isolated nucleic acid selected from SEQ ID NOs: 162, 212, 3781, 3970, 3990, 492, 3796, 3975, and 4028, wherein expression of the nucleic acid in a plant results in altered acid metabolism. In other embodiments, the present invention provides an isolated nucleic acid selected from SEQ ID NOs: 4049, 210, 4045, 229, 3825, 4015, 3835, 4039, 1048 and 1106, wherein expression the nucleic acid in a plant results in altered alcohol metabolism. In still other embodiments, the present invention provides an isolated nucleic acid selected from SEQ ID NOs: 7548, 283, 3957, 3734, 3739, 3797, 7516, 3762, 4020 and 1062, wherein expression of the nucleic acid in a plant results in altered fatty acid metabolism. In further embodiments, the present invention provides an isolated nucleic acid of selected from SEQ ID NOs: 1148, 4147, 273, 281, 299, 3920, 450, 7463 and 4074, wherein expression of the nucleic acid in a plant results in altered branched fatty acid metabolism. In still further embodiments, the present invention provides an isolated nucleic acid selected from SEQ ID NOs: 258, 456, 3859, 3817, 4018, 3848, 3862, 4008 and 1000, wherein expression of the nucleic acid in a plant results in altered alkaloid or other base metabolism. In some embodiments, the present invention provides an isolated nucleic acid selected from SEQ ID NOs: 372, 3714, 3717, 3963, 3775, 3757, 7462, 3743, 3744 and 7480, wherein expression of the nucleic acid in a plant results in altered amino acid metabolism. In some other embodiments, the present invention provides an isolated nucleic acid selected from SEQ ID NOs: 7404, 180, 181, 225, 231, 366, 3983, 3833, 1121 and 1062, wherein expression of the nucleic acid in a plant results in altered ester metabolism. In some further embodiments, the present invention provides an isolated nucleic acid selected from SEQ ID NOs: 3773, 583, 3821, 7403, 988, 1002, 1007 and 1129, wherein expression of the nucleic acid in a plant results in altered glyceride metabolism. In still other embodiments, the present invention provides an isolated nucleic acid selected from SEQ ID NOs: 150, 7410, 175, 7553, 619, 1078, 1122 and 1124, wherein expression of the nucleic acid in a plant results in altered phenolic compound metabolism. In further embodiments, the present invention provides an isolated nucleic acid selected from SEQ ID NOs: 3891, 7545, 7551, 4121, 157, 159, 7411, 3792, 3799 and 3997, wherein expression of the nucleic acid in a plant results in altered carbohydrate metabolism. In other embodiments, the present invention provides an isolated nucleic acid selected from SEQ ID NOs: 7405, 7406, 173, 183, 220, 227, 3778, 3803, 3847 and 1005, wherein expression of the nucleic acid in a plant results in altered sterol, oxygenated terpene, or isoprenoid metabolism. In still other embodiments, the present invention provides an isolated nucleic acid selected from SEQ ID NOs: 7408, 351, 378, 3864, 4103, 996, 1006 and 1098, wherein expression of the nucleic acid in a plant results in altered alkene or alkyne metabolism. In further embodiments, the present invention provides an isolated nucleic acid selected from SEQ ID NOs: 177, 7442, 4038, 3836, 3855, 1012, 1015, 1119 and 1024, wherein expression of the nucleic acid in a plant results in altered hydrocarbon metabolism. In still further embodiments, the present invention provides an isolated nucleic acid selected from SEQ ID NOs: 360, 4001, 3703, 7399, 645, 3849 and 7552, wherein expression of the nucleic acid in a plant results in altered ketone or quinone metabolism. In further preferred embodiments, the present invention provides vectors comprising the foregoing polynucleotide sequences. In still further embodiments, the foregoing sequences are operably linked to an exogenous promoter, most preferably a plant promoter. However, the present invention is not limited to the use of any particular promoter. Indeed, the use of a variety of promoters is contemplated, including, but not limited to, 35S, 19S, heat shock, and Rubisco promoters, subgenomic promoters such as the CaMV promoter and TMV coat protein promoter, and dual promoters systems such as DHSPES (see U.S. Pat. No. 6,303,848, incorporated herein by reference). In some embodiments, the nucleic acid sequences of the present invention are arranged in sense orientation, while in other embodiments, the nucleic acid sequences are arranged in the vector in antisense orientation. In still further embodiments, the present invention provides a plant comprising one of the foregoing nucleic acid sequences or vectors, as well as seeds, leaves, roots, stems and fruit from the plant. In some particularly preferred embodiments, the present invention provides at least one of the foregoing sequences for use in conferring altered metabolism in a plant. In still other embodiments, the present invention provides processes for making a transgenic plant comprising providing a vector as described above and a plant, and transfecting the plant with the vector. In other preferred embodiments, the present invention provides processes for providing an altered metabolic characteristic in a plant or population of plants comprising providing a vector as described above and a plant, and transfecting the plant with the vector under conditions such that an altered metabolic characteristic is conferred by expression of the isolated nucleic acid from the vector. In still further embodiments, the present invention provides an isolated nucleic acid selected from the group consisting of SEQ ID NOs: 1-7554 and nucleic acid sequences that hybridize to any thereof under conditions of low stringency for use in producing a plant with altered metabolism. In other embodiments, the present invention provides an isolated nucleic acid, composition or vector substantially as described herein in any of the examples or claims.
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Nucleic acid compositions conferring disease resistance
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This invention encompasses the identification and isolation of genes that confer disease control properties in plants, as well as plants comprising such genes. These genes are derived from the following sources: Nicotiana benthamiana, Oryzae sativa (var. Indica IR7), Papaver rhoeas, Saccharomyces cerevisiae and Trichoderma harzianum (Rifai 1295-22). The control conferred is against the one or more of the following phytopathogens: Aspergillus flavus, Cercospora zeae-maydis, Fusarium monilforme, Fusarium graminearum, Helminthosporium maydis, Phoma lingam, Phomopsis helianthi, Phytopthera infestans, Pyricularia oryzae, Pythium ultimum, Rhizoctonia solani, Sclerotinia sclerotiorum, Ustilago maydis, and Verticillium dahliae. Further, this invention encompasses other homologous and heterologous sequences with a high degree of functional similarity.
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1. An isolated nucleic acid selected from the group consisting of SEQ ID NOs: 1-2318 and nucleic acid sequences that hybridize to any thereof under conditions of low stringency, wherein expression of said isolated nucleic acid in a plant results in a disease resistance phenotype. 2. A vector comprising the isolated nucleic acid of claim 1. 3. The vector of claim 2, wherein said isolated nucleic acid is operably linked to a plant promoter. 4. A vector according to claim 2, wherein said isolated nucleic acid is in sense orientation. 5. A vector according to claim 2, wherein said isolated nucleic acid is in antisense orientation. 6. A plant transfected with an isolated nucleic acid or vector according to claims 1. 7. A seed from the plant of claim 6. 8. A leaf from the plant of claim 6. 9. An isolated nucleic acid according to claim 1, for use in conferring disease resistance. 10. A method for providing disease resistance in a plant comprising: a. providing a vector according to claim 2 and a plant, b. and transfecting said plant with said vector under conditions such that a disease resistance phenotype is conferred by expression of said isolated nucleic acid from said vector. 11. Cancelled. 12. An isolated nucleic acid selected from the group consisting of SEQ ID NOs: 1-2318 and nucleic acid sequences that hybridize to any thereof under conditions of low stringency.
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<SOH> BACKGROUND OF THE INVENTION <EOH>Ever since the advent of agriculture thousands of years ago, farmers have been engaged in an ongoing battle to minimize the impact of crop pests. Plant diseases caused by bacteria and fungi are currently a major factor in limiting crop production worldwide. For example, it was estimated that head blight, caused by the fungal pathogens Fusarium graminearum and F. poae caused $3 billion in losses to wheat and barley production in the US between 1991-1996. The impact in less developed countries, where food production is usually at or below sustenance levels, is much more severe. Diseases not only adversely affect overall yield, but have a significant impact on the quality of foods produced. This destruction of crops and decrease in food quality has profound socioeconomic effects, as exemplified by the widespread starvation and subsequent emigration caused by the Irish potato famine of the 1800's. Furthermore, some plant disease agents pose human health hazards, such as mycotoxins produced by fungal phytopathogens. Fungi are a highly diverse and versatile group of organisms that successfully occupy most natural habitats. Less than 10% of the ˜100,000 known fungal species can colonize plants, and of these, a small fraction are responsible for plant diseases. However, virtually all flowering plants are attacked by and susceptible to some form of phytopathogenic fungus, and the specificity of these interactions is determined by host range limitations of both plant and microbe. In general, fungal phytopathogens may be categorized into three classes: 1) opportunistic parasites, that usually have a broad host range but relatively low virulence, 2) facultative pathogens that rely on living plants to grow but can survive as free-living under some circumstances, and 3) obligate pathogens, for which a living host is an absolute requirement for survival. Many of the most serious and virulent plant disease agents fall into the second class of phytopathogen, the facultative parasites, and it is this group of organisms that is of primary interest to agricultural researchers. Agronomically important diseases caused by fungal phytopathogens include: glume and leaf blotch, late blight, stalk/head rot, rice blast, leaf blight and spot, corn smut, wilt, sheath blight, stem canker, root rot, blackleg and kernel rot. Plant Defense Over the course of evolution and natural selection, plants have developed several mechanisms of defense against phytopathogens. This process, often referred to as the “evolutionary arms-race,” continues due to the rapid ability of most pathogens to overcome plant defenses. Plants have several defensive modes-of-action, that often act in concert to mount responses in both generalized and specific manners. Defense mechanisms such as bark, trichomes and waxy cuticles form physical barriers protecting the plant from contact with disease organisms. Additionally, some plants secrete compounds, such as resins or gums, that not only provide a barrier to pathogen contact, but in some cases may act as a repellent. In addition to physical barriers, plants have developed the ability to mount defense responses when challenged by pathogens. These induced responses require that a plant recognize a pathogen, activate and elaborate a defense pathway, and localize the infection, preventing invasion/spread of the pathogen and full-blown disease. This type of resistant plant-microbe interaction is described as incompatible, since the pathogen is not able to successfully parasitize and infect the host plant. Incompatible interactions involve a complex set of distinct and networked signal transduction pathways, the study of which has been facilitated by molecular analyses of both plant and microbe genes identified in various mutant screens. Generally, the defense pathways induced during incompatible interactions fall into two categories: the hypersensitive response (HR), and systemic acquired resistance (SAR). However, it is clear that there are many intersecting and overlapping branch points in these pathways. The HR consists of localized, induced cell death in a host plant at the site of pathogen invasion. HR is frequently associated with the appearance of necrotic flecks containing dead plant cells within a few hours of pathogen contact. This plant cell death deprives the pathogen of access to further nutrients, causing pathogen arrest and protecting the rest of the plant from the disease agent. The mechanisms of HR include both the activation of programmed cell death (apoptosis) by the plant and/or a switch in plant cell metabolism, activating biochemical pathways that produce compounds toxic to both pathogen and plant. The triggering of HR is associated with the presence of reactive oxygen species such as superoxide anions and H 2 O 2 , that can act as signal molecules in addition to being converted to highly reactive and damaging oxygen radicals. HR is also associated with the induction of benzoic acid (BA), salicylic acid (SA), and their respective glucoside conjugates, which also play signaling roles and may be directly antimicrobial, as well as several classes of PR (pathogen response) proteins. SAR is a broad-spectrum, inducible plant immunity that is activated after the formation of a necrotic lesion, either as a part of HR or as a symptom of disease. Therefore, it is not limited to incompatible interactions, but may be induced by compatible interactions with disease-causing microbes. This immunity or resistance spreads systemically and develops in distal, unchallenged parts of the plant. SAR acts nonspecifically throughout the plant and reduces the severity of disease symptoms caused by all classes of pathogens, including highly virulent ones. It can be induced by the elicitor SA in a dose-dependent manner, and involves a complex set of signal transduction molecules and downstream elicitors. The SAR response is characterized by the coordinate induction in uninfected leaves of several gene families, including chitinases, β-1,3 glucanases, PR-1 proteins and many others. The exact mechanisms of SAR and HR are still being elucidated, and are also targets for bioengineering of plant disease resistance. Traditional Agricultural Approaches to Plant Disease Control Over several centuries of agricultural development, farmers have devised methods for controlling plant disease. Husbandry techniques such as crop rotation, controlled irrigation, manure application, and tilling date back to the Roman Empire. Alone, these methods are limited in their efficacy to control diseases (by modern standards). However, they are still considered standard practice, and contribute significantly to any comprehensive pest management program. In addition to husbandry, breeding methods have been employed to develop disease-resistant cultivars. The ability to select and propagate cultivars of crops containing desirable traits has enabled plant breeders to take advantage of natural genetic variation and/or induced mutations. There are numerous genetic methods and techniques available to breeders, including crossing and hybridization, embryo rescue, cell fusion and mutagenesis. The programs breeders implement depend on both the type of cultivar they want to improve (e.g., hybrid vs. inbred) and the reproductive biology of the particular species (self-pollinated vs. out-crossed). One example of a successful breeding program is that of blight-resistant potatoes that are a result of introducing traits from a Mexican species into >50% of all cultivars. Conventional breeding methods will undoubtedly continue to play a significant role in the improvement of agricultural crops, however, the time-scale and labor requirements of breeding programs may not be adequate to meet increasing demands for many agronomic traits, including disease resistance. Furthermore, the ability of pathogens to rapidly overcome resistance bred into new races of plants limits the utility and useful lifetime of these crops. Within the last several decades, agricultural techniques have expanded to include widespread and intensive use of chemicals. A recent study of US farm-sector sales of pesticides estimated that for 11 major crops, a total of approximately $8.83 billion was spent in 1997 alone. This represents a significant portion of the US agriculture economy. In addition to chemical control, bio-control methods have gained a smaller, but constantly growing, following among farmers. As concern for the global environment and human health increases, it is imperative that new agricultural practices be developed and implemented. AgBiotech Approaches to Plant Disease Control The advent of modern biology, particularly molecular biology and genetics, has opened up new avenues for disease control research and practice. Scientists have focused on utilizing recombinant DNA (rDNA) methods, which allow new varieties of plants to be produced much faster than by conventional breeding. rDNA techniques allow the introduction of genes from distantly related species or even from different biological kingdoms into crop plants, conferring traits that provide significant agronomic advantages. Furthermore, detailed knowledge of the traits being introduced, such as cellular function and localization, can lead to less variability in offspring, and fine-tuning of secondary effects. After a trait has been introduced into a plant by transgenic methods, conventional breeding can be used to hybridize the transgenic line with useful varieties and elite germplasms, resulting in crops containing numerous advantageous properties. Agricultural biotechnology (AgBiotech) approaches to disease resistance are typically three-fold. First, specific crops that undergo compatible (disease causing) interactions with specific pathogens are analyzed to determine the endogenous factors that enable this interaction, in an effort to prevent the particular disease(s) via bio-engineering. Second, researchers look for exogenous factors (compounds and proteins) from other species/sources that, when produced in crop plants, provide protection from phytopathogens. Finally, efforts are being made to hyper-activate the plant's own defense responses, in order to provide crops with broad-spectrum immunity against several disease agents simultaneously. Each of these approaches has it's advantages and disadvantages, and has met with some limited success to date. However, intensive research and testing continues; between 1987 and May 1999, there were 61 publicly-sponsored and 272 privately-sponsored field trials testing genes for fungal disease resistance in transgenic crops. A recent example of a successfull disease-resistance bioengineered product was described by the Monsanto Company, which demonstrated that a potato engineered to express an alfalfa antifungal peptide (defensin) showed robust resistance to the fungal pathogen Verticillium dahliae (Verticillium wilt) under both greenhouse and field conditions. As AgBiotech hurtles into the genomics and post-genomics era, the massive amounts of genetic and functional data being generated are being used to direct the search for genes that can be utilized with recombinant methods. Additionally, transgenic technology itself is overcoming some of it's rate-limiting obstacles, allowing expression and modulation of several genes simultaneously in transgenic crops. These advances in both the informational and technological tools available to agricultural biotechnologists has and will continue to increase the pace of discovery and product development with regards to disease resistance. As the regulatory and commercial framework is developed, many of these AgBiotech products will be entering the marketplace. It is therefore reasonable to expect that in the very near future, bioengineered crops will be part of a comprehensive, integrated disease management program throughout the agricultural enterprise. Accordingly, what is needed in the art are gene sequences and polypeptide sequences whose expression in plants, plant seeds, plant tissues and/or plant cells causes resistance to plant pathogens.
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<SOH> SUMMARY OF THE INVENTION <EOH>This invention relates to deoxyribonucleic acid (DNA) and amino acid sequences that confer disease resistance phenotypes in plants, as well as disease resistant plants, plant seeds, plant tissues and plant cells comprising such sequences. In some embodiments, the present invention provides polynucleotides and polypeptides that confer disease resistance phenotypes when expressed in plants. The present invention is not limited to any particular polypeptide or polynucleotide sequences that confer disease resistance phenotypes. Indeed, a variety of such sequences are contemplated. Accordingly, in some embodiments the present invention provides an isolated nucleic acid selected from the group consisting of SEQ ID NOs:1-2318 and nucleic acid sequences that hybridize to any thereof under conditions of low stringency, wherein expression of the isolated nucleic acid in a plant results in a disease resistance phenotype. In further preferred embodiments, the present invention provides vectors comprising the foregoing polynucleotide sequences. In still further embodiments, the foregoing sequences are operably linked to an exogenous promoter, most preferably a plant promoter. However, the present invention is not limited to the use of any particular promoter. Indeed, the use of a variety of promoters is contemplated, including, but not limited to, 35S and 19S of Cauliflower Mosaic Virus, Cassava Vein Mosaic Virus, ubiquitin, heat shock and rubisco promoters. In some embodiments, the nucleic acid sequences of the present invention are arranged in sense orientation, while in other embodiments, the nucleic acid sequences are arranged in the vector in antisense orientation. In still further embodiments, the present invention provides a plant comprising one of the foregoing nucleic acid sequences or vectors, as well as seeds, leaves, and fruit from the plant. In some particularly preferred embodiments, the present invention provides at least one of the foregoing sequences for use in conferring pathogen or disease resistance in a plant. In still other embodiments, the present invention provides processes for making a transgenic plant comprising providing a vector as described above and a plant, and transfecting the plant with the vector. In other preferred embodiments, the present invention provides processes for providing a disease or pathogen resistance phenotype in a plant or population of plants comprising providing a vector as described above and a plant, and transfecting the plant with the vector under conditions such that a disease resistance phenotype is conferred by expression of the isolated nucleic acid from the vector. In still further embodiments, the present invention provides an isolated nucleic acid selected from the group consisting of SEQ ID NOs:1-2318 and nucleic acid sequences that hybridize to any thereof under conditions of low stringency for use in producing a disease or pathogen resistant plant. In other embodiments, the present invention provides an isolated nucleic acid, composition or vector substantially as described herein in any of the examples or claims.
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Manhole structure, flexible water shut off joint for manhole structure and method for installing manhole structure
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There is provided a manhole structure 1 formed by joining a cast-in-place concrete manhole wall 2 to a pipe 3. In the manhole structure 1, the pipe 3 is driven and laid in a vertical shaft 4, and a flexible cut-off joint 5 for manhole is disposed on an outer periphery of the pipe 3. The flexible cut-off joint 5 for manhole comprises a rigid cylindrical body 6 and a tubular flexible body 7 located at an inside of the cylindrical body 6 in a space between the cylindrical body and the pipe, and the tubular flexible body 7 is made of an elastic body. An end 7a of the tubular flexible body 7 is press-fixed to an inner surface of the cylindrical body 6 with an expansion band 8, and the other end 7b of the tubular flexible body 7 is press-fixed to an outer periphery of the pipe 3 by tightening with a fastening band 9. The manhole wall 2 is formed by pouring a concrete on the outer periphery of the cylindrical body 6. The tubular flexible body 7 in the manhole structure 1 absorbs an expansion-contraction displacement, a bending displacement, a shearing displacement and the like between the manhole wall 2 and the pipe 3 without applying a load to the pipe 3.
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1. A manhole structure comprising a manhole and a pipe joined thereto, in which the pipe is driven and laid in a vertical shaft, and a flexible cut-off joint for manhole structure is disposed on an outer periphery of the pipe, and the flexible cut-off joint for manhole structure comprises a rigid cylindrical body and a tubular flexible body located in a space between the cylindrical body and the pipe and at an inside of the cylindrical body, and the tubular flexible body is made of an elastic body absorbing a displacement between the cylindrical body and the pipe, and at least a part of the tubular flexible body is fixed to the cylindrical body and the pipe, respectively, and the outer periphery of the cylindrical body is fixed with a filler for manhole wall. 2. A manhole structure according to claim 1, wherein said manhole is a cast-in-place concrete manhole, and a wall of said manhole is formed by pouring said filler for manhole wall. 3. A manhole structure according to claim 1, wherein said manhole is a ready-made build-up manhole, and a drilled hole surface is provided on a wall of said manhole, and said filler for manhole wall is poured between an outer periphery of said cylindrical body and said drilled hole surface. 4. A manhole structure according to claim 1, wherein said cylindrical body has a strip-shaped protrusion portion protruding toward a wall side of said manhole, and an end of said filler for manhole wall pored into the outer periphery of said cylindrical body is positioned by said protrusion portion. 5. A manhole structure according to claim 1, wherein a flange is provided on an outer periphery of said cylindrical body. 6. A manhole structure according to claim 5, wherein a non-curing butyl rubber adhering to said filler for manhole wall is attached to a face of said flange contacting with said filler for manhole wall. 7. A manhole structure according to claim 1, wherein an end of said tubular flexible body is press-fixed to an inner surface of said cylindrical body with an expansion band, and the other end of said tubular flexible body is press-fixed to an outer periphery of said pipe by tightening with a fastening band. 8. A flexible cut-off joint for manhole structure for joining a manhole to a pipe, in which said flexible cut-off joint for manhole comprises a rigid cylindrical body and a tubular flexible body located at an inside of said cylindrical body in a space between said cylindrical body and said pipe, and said tubular flexible body is made of an elastic body absorbing a displacement between said cylindrical body and said pipe, and at least a part of said tubular flexible body is fixed to said cylindrical body and said pipe, respectively, and when the manhole structure is formed, said pipe is driven and laid in a vertical shaft and said flexible cut-off joint for manhole is mounted on the outer periphery of said pipe and the outer periphery of said cylindrical body is fixed with a filler for manhole wall. 9. A flexible cut-off joint for manhole structure according to claim 8, wherein an end of said tubular flexible body is press-fixed to an inner surface of said cylindrical body with an expansion band and the other end of said tubular flexible body is press-fixed to an outer periphery of said pipe by tightening with a fastening band. 10. A method for the construction of a manhole structure by joining a manhole and a pipe, which comprises driving and laying said pipe in a vertical shaft, mounting a flexible cut-off joint for manhole structure on an outer periphery of said pipe in which said flexible cut-off joint for manhole structure comprises a rigid cylindrical body and a tubular flexible body located at an inside of said cylindrical body in a space between said cylindrical body and said pipe and said tubular flexible body is made of an elastic body absorbing a displacement between said cylindrical body and said pipe and at least a part of said tubular flexible body is fixed to said tubular body and said pipe, and pouring a filler for manhole wall into the outer periphery of said cylindrical body. 11. A manhole structure according to claim 2, wherein said cylindrical body has a strip-shaped protrusion portion protruding toward a wall side of said manhole, and an end of said filler for manhole wall pored into the outer periphery of said cylindrical body is positioned by said protrusion portion. 12. A manhole structure according to claim 3, wherein said cylindrical body has a strip-shaped protrusion portion protruding toward a wall side of said manhole, and an end of said filler for manhole wall pored into the outer periphery of said cylindrical body is positioned by said protrusion portion. 13. A manhole structure according to claim 2, wherein a flange is provided on an outer periphery of said cylindrical body. 14. A manhole structure according to claim 3, wherein a flange is provided on an outer periphery of said cylindrical body. 15. A manhole structure according to claim 4, wherein a flange is provided on an outer periphery of said cylindrical body. 16. A manhole structure according to claim 2, wherein an end of said tubular flexible body is press-fixed to an inner surface of said cylindrical body with an expansion band, and the other end of said tubular flexible body is press-fixed to an outer periphery of said pipe by tightening with a fastening band. 17. A manhole structure according to claim 3, wherein an end of said tubular flexible body is press-fixed to an inner surface of said cylindrical body with an expansion band, and the other end of said tubular flexible body is press-fixed to an outer periphery of said pipe by tightening with a fastening band. 18. A manhole structure according to claim 4, wherein an end of said tubular flexible body is press-fixed to an inner surface of said cylindrical body with an expansion band, and the other end of said tubular flexible body is press-fixed to an outer periphery of said pipe by tightening with a fastening band. 19. A manhole structure according to claim 5, wherein an end of said tubular flexible body is press-fixed to an inner surface of said cylindrical body with an expansion band, and the other end of said tubular flexible body is press-fixed to an outer periphery of said pipe by tightening with a fastening band. 20. A manhole structure according to claim 6, wherein an end of said tubular flexible body is press-fixed to an inner surface of said cylindrical body with an expansion band, and the other end of said tubular flexible body is press-fixed to an outer periphery of said pipe by tightening with a fastening band.
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<SOH> BACKGROUND ART <EOH>There is known a manhole structure constructed by the pipe jacking method as shown in FIG. 13 . In FIG. 13 is shown a vertical cross-sectional view of the conventional manhole structure constructed by the pipe jacking method. In the conventional manhole structure, as shown in FIG. 13 , vertical shafts 42 are first dug in both of start and arrive by the pipe jacking method, and then a pipe 43 such as a sewerage pipe or the like is compressed into the vertical shaft 42 by a jack while with digging with a transverse boring machine. In the conventional pipe jacking method, as shown in FIG. 13 , a mold is subsequently built in the vertical shaft 42 laying the pipe 43 such as sewerage pipe or the like therein, and concrete is poured into the mold to form a manhole wall 44 , whereby the manhole structure is constructed. In case of digging the vertical shaft, a steel pipe is usually used as an earth retaining wall for rendering the working area into a minimum range. In the formation of the manhole wall 44 , therefore, a casing steel pipe 45 forms an outer mold. The previously laid pipe 43 is embedded in the concrete of the manhole wall 44 . 8 Problems to be Solved in the Invention] In such a manhole structure 41 , however, the joining portion between the manhole wall 44 and the pipe 43 such as sewerage pipe or the like is rigid joining, so that the joining portion between the manhole wall 44 and the pipe 43 is broken due to uneven settlement of ground in earthquakes or the like. It is an of the invention to prevent the breakage of the joining portion between the manhole wall and the pipe in the earthquake or the like by providing a manhole structure in which the joining between a cast-in-place concrete manhole wall and a pipe is rendered into flexible joining by a flexible cut-off joint for manhole. It is another object of the invention to prevent the breakage of the joining portion between the manhole wall and the pipe in the earthquake or the like by providing a manhole structure in which the joining between the manhole and the pipe is rendered into flexible joining by a flexible cut-off joint for manhole structure. Means for Solving Problems] The invention concerns a manhole structure comprising a manhole and a pipe joined thereto, in which the pipe is driven and laid in a vertical shaft, and a flexible cut-off joint for manhole structure is disposed on an outer periphery of the pipe, and the flexible cut-off joint for manhole structure comprises a rigid cylindrical body and a tubular flexible body located in a space between the cylindrical body and the pipe and at an inside of the cylindrical body, and the tubular flexible body is made of an elastic body absorbing a displacement between the cylindrical body and the pipe, and at least a part of the tubular flexible body is fixed to the cylindrical body and the pipe, respectively, and the outer periphery of the cylindrical body is fixed with a filler for manhole wall. Also, the invention concerns a flexible cut-off joint for use in such a manhole structure and a method for construction of such a manhole structure. The inventor has prepared and examined various manhole structures for rendering the joining portion between a cast-in-place concrete manhole wall and a pipe such as sewerage pipe or the like into flexible joining. As a result, the inventor has found that by using a given flexible cut-off joint for manhole is obtained a manhole structure wherein the joining portion between a pipe laid by the pipe jacking method and the cast-in-place concrete manhole wall is rendered into the flexible joining, and the invention has been accomplished. Moreover, the inventor has prepared and examined various manhole structures for rendering a joining portion between a ready-made build-up manhole and a pipe such as sewerage pipe or the like into a flexible joining. Consequently, the inventor has found that by using a given flexible cut-off joint for manhole is obtained a manhole structure wherein the joining portion between a laid by the pipe jacking method and a manhole, and the invention has been accomplished. The flexible cut-off joint for manhole according to the invention comprises a rigid cylindrical body and a tubular flexible body located at the inside of the cylindrical body, in which the tubular flexible body is made of an elastic body. The tubular flexible body according to the invention connects between the rigid cylindrical body and the pipe and acts to absorb a displacement therebetween. The tubular flexible body is formed so as to expand radially in a direction from the inside of manhole to the outside thereof, whereby a fixing portion can be fixed to an outer periphery of the pipe from the inside of the manhole. The joint according to the invention is a flexible cut-off joint for manhole applied on the joining portion between the pipe and the manhole wall of the manhole such as a cast-in-place concrete manhole, a build-up manhole or the like. The cylindrical body according to the invention serves as a dam to ensue a space between the cylindrical body and the pipe when the manhole wall is formed by pouring a filler for manhole wall such as a concrete or the like on the outer periphery of the cylindrical body. Also, the cylindrical body according to the invention serves as a dam on a filler for manhole wall between a drilled surface of the manhole wall and a flexible cut-off joint for manhole structure to ensue a space between the cylindrical body and the pipe when the filler for the manhole wall such as mortar concrete or the like is poured on the outer periphery of the cylindrical body. In the invention, the tubular flexible body made of an elastic body is arranged in the space between the cylindrical body and the pipe. Such a tubular flexible body serves to absorb a displacement between the cylindrical body and the pipe. According to the manhole structure of the invention, the manhole wall is formed on the outer periphery of the cylindrical body and the cylindrical body and the pipe are connected through the tubular flexible body made of the elastic body, so that even if different load is applied between the manhole and the pipe or a relatively different displacement is produced there between to cause a position displacement by large-scale diastrophism such as earthquake or the like, the tubular flexible body can absorb such load and displacement to prevent the breakage of the joining portion between the manhole wall and the pipe. Moreover, according to the manhole structure of the invention, the outer periphery of the cylindrical body is fixed to the manhole wall through the filler for manhole wall embedding a space between the manhole wall and the pipe, and the cylindrical body and the pipe are connected through the tubular flexible body made of the elastic body, so that even if different load is applied between the manhole and the pipe or a relatively different displacement is produced there between to cause a position displacement by large-scale diastrophism such as earthquake or the like, the tubular flexible body can absorb such load and displacement to prevent the breakage of the joining portion between the manhole and the pipe.
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<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a vertical cross-sectional view of an embodiment of the manhole structure according to the invention. FIG. 2 is a vertical cross-sectional view of another embodiment of the manhole structure according to the invention. FIG. 3 is a transverse sectional view of the manhole structure of FIG. 2 . FIG. 4 is a vertical cross-sectional view of the other embodiment of the manhole structure according to the invention. FIG. 5 is a perspective view of a flexible cut-off joint for manhole used in the manhole structure of FIG. 4 . FIG. 6 is a perspective view of another embodiment of the flexible cut-off joint for manhole structure according to the invention. FIG. 7 is a vertical cross-sectional view of an embodiment of the method for constructing the manhole structure of FIG. 4 . FIG. 8 is a vertical cross-sectional view illustrating a step of an embodiment of constructing the manhole structure according to the invention. FIG. 9 is a vertical cross-sectional view illustrating another step of the embodiment of constructing the manhole structure according to the invention. FIG. 10 is a vertical cross-sectional view illustrating the other step of the embodiment of constructing the manhole structure according to the invention. FIG. 11 is a vertical cross-sectional view illustrating a further step of the embodiment of constructing the manhole structure according to the invention. FIG. 12 is a vertical cross-sectional view of further embodiment of the manhole structure according to the invention. FIG. 13 is a vertical cross-sectional view of the conventional manhole structure constructed by the pipe jacking method. FIG. 14 is a front view of the conventional manhole structure as viewed from the inside of vertical shaft. FIG. 15 is a vertical cross-sectional view of the conventional embodiment using a flexible cut-off joint. FIG. 16 is a partially enlarged view of FIG. 15 . detailed-description description="Detailed Description" end="lead"?
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Catheter assembly
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A catheter assembly is provided comprising a catheter (3;530;630;730) including a catheter tube (32) and a connector (31;531) arranged in one end of the catheter tube. The catheter is preferably a urinary, hydrophilic catheter. Further, the assembly comprises a catheter receptacle (2;520;620;720) for accommodation of at least the catheter tube, wherein the receptacle is provided with an opening (22;622;722), said opening being connected to and closed by the connector (31;531) of the catheter. A method for producing such a catheter assembly is also provided.
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1. A catheter assembly comprising: a catheter (3;530;630;730) having on at least part of its surface a hydrophilic surface layer intended to produce a low-friction surface character of the catheter by treatment with a wetting fluid prior to use of the catheter; a catheter receptacle (2;520;620;720) forming a cavity for accommodation of at least part of the catheter; and a compartment accommodating said wetting fluid, wherein said compartment forms an integrated part of the receptacle, but being separated from said cavity, characterised by an additional outer layer attached to the receptacle, said layer being arranged to at least partly cover the part of the receptacle forming said wetting fluid compartment. 2. The catheter assembly of claim 1, wherein the receptacle is formed of a flexible plastics material. 3. The catheter assembly of claim 1 or 2, wherein the receptacle is formed of a transparent material. 4. The catheter assembly of any one of claim 1, wherein the catheter is a hydrophilic urinary catheter. 5. The catheter assembly of claim 1, wherein the wetting fluid compartment is arranged in a part of the receptacle extending rearwardly from the catheter. 6. The catheter assembly of claim 5, wherein the rearward part of the receptacle accommodating the wetting fluid compartment is in fluid communication with the forward part housing the catheter. 7. The catheter assembly of claim 1, wherein the separation between the wetting fluid compartment and the cavity accommodating the catheter provides a sealed closure, said closure being openable for enabling of a fluid communication between the compartment and the cavity and thereby an activation of the catheter. 8. The catheter assembly of claim 7, wherein the openable closure is formed by a separable joint between the compartments. 9. The catheter assembly of claim 1, wherein the additional cover material is gas-impermeable. 10. The catheter assembly of claim 1, wherein the additional cover material provides a protection for the wetting fluid against a sterilizing agent used on the assembly. 11. The catheter assembly of claim 10, wherein the cover material provides protection against ethylene oxide. 12. The catheter assembly of claim 1, wherein the additional outer layer is arranged to cover essentially the whole part of the receptacle forming said wetting fluid compartment. 13. The catheter assembly of claim 1, wherein the additional outer layer comprises at least one of aluminium foil laminate, poly(vinylidene chloride) or a laminate comprising metallised film such as metallised poly(ethylene terepthalate), or a silicon oxide coated film, or a laminate comprising aluminum oxide. 14. The catheter assembly of claim 1, wherein the additional outer layer is attached to the receptacle by means of an adhesive. 15. The catheter assembly of claim 1, wherein the additional outer layer is attached to the receptacle by means of welding. 16. The catheter assembly according to claim 1, wherein the receptacle comprises opening means for opening of the receptacle, said opening means preferably being arranged in an end of the receptacle being opposite to the connection between the receptacle and the catheter. 17. The catheter assembly according to claim 16, wherein the opening means comprises a peel-off joint. 18. The catheter assembly according to claim 16, wherein the opening means comprises a tear line. 19. The catheter assembly of claim 1, wherein the whole catheter is arranged within the bounds of the receptacle. 20. The catheter assembly of claim 1, wherein the catheter has a part of which forms an insertable length to be inserted through a body opening, and wherein the receptacle is connected to the catheter, thereby enclosing the insertable length of the catheter, but leaving at least part of the catheter outside the receptacle. 21. A method for producing a catheter assembly, comprising: providing a receptacle; providing a hydrophilic catheter; arranging at least part of the catheter tube in a cavity of the receptacle; arranging a wetting fluid in a compartment forming an integrated part of the receptacle, but being separated from said cavity; and attaching an additional outer layer to the receptacle to at least partly cover the part of the receptacle forming said wetting fluid compartment. 22. The method of claim 21, comprising the subsequent step of sterilizing the assembly, whereby the additional cover serves as a protection for the wetting fluid against the sterilizing agent. 23. A catheter assembly comprising: a catheter (3;530;630;730) having on at least part of its surface a hydrophilic surface layer intended to produce a low-friction surface character of the catheter by treatment with a wetting fluid prior to use of the catheter; a catheter receptacle (2;520;620;720) forming a cavity for accommodation of at least part of the catheter; and a compartment accommodating said wetting fluid, wherein said compartment forms an integrated part of the receptacle, but being separated from said cavity, wherein the separation between the wetting fluid compartment and the cavity accommodating the catheter provides a rupturable sealed closure, characterised in that said closure is provided with at least one point of weakness, in order for an induced rupture to occur in a predetermined position, thereby enabling fluid communication between the compartment and the cavity housing the catheter. 24. The catheter assembly of claim 23, wherein the closure is formed by a rupturable joint between the compartments. 25. The catheter assembly of claim 24, wherein the joint is a welded joint with a welding width variation, thus providing the at least one point of weakness. 26. The catheter assembly of claim 24, wherein the joint is a welded joint with a welding strength variation, thus providing the at least one point of weakness. 27. The catheter assembly of claim 24, wherein the joint is arranged in a non-linear arrangement, thus providing the at least one point of weakness. 28. The catheter assembly of claim 27, wherein the joint is arranged with at least one knee directed towards the wetting fluid compartment, thus providing the at least one point of weakness. 29. The catheter assembly of claim 28, wherein the knee is provided has an angled peak portion directed towards the wetting fluid compartment. 30. The catheter assembly of claim 29, wherein the angled peak portion forms an obtuse angle. 31. The catheter assembly of claim 29, wherein the angled peak portion forms an acute angle. 32. The catheter assembly of claim 28, wherein the knee is has an curved peak portion directed towards the wetting fluid compartment. 33. The catheter assembly of claim 23, wherein the receptacle is formed of a flexible plastics material. 34. The catheter assembly of claim 23, wherein the receptacle is formed of a transparent material. 35. The catheter assembly of claim 23, wherein the catheter is a hydrophilic urinary catheter. 36. The catheter assembly of claim 23, wherein the wetting fluid compartment is arranged in a part of the receptacle extending rearwardly from the catheter. 37. The catheter assembly of claim 36, wherein the rearward part of the receptacle accommodating the wetting fluid compartment is in fluid communication with the forward part housing the catheter. 38. The catheter assembly of claim 23, wherein an additional outer layer is attached to the receptacle, said layer being arranged to at least partly cover the part of the receptacle forming said wetting fluid compartment. 39. The catheter assembly of claim 38, wherein the additional cover material is gas-impermeable. 40. The catheter assembly of claim 38, wherein the additional cover material provides a protection for the wetting fluid against a sterilizing agent used on the assembly. 41. The catheter assembly of claim 40, wherein the cover material provides protection against ethylene oxide. 42. The catheter assembly of claim 38, wherein the additional outer layer is arranged to cover essentially the whole part of the receptacle forming said wetting fluid compartment. 43. The catheter assembly of claim 38, wherein the additional outer layer comprises at least one of aluminium foil laminate, poly(vinylidene chloride) or a laminate comprising metallised film such as metallised poly(ethylene terepthalate), or a silicon oxide coated film, or a laminate comprising aluminum oxide. 44. The catheter assembly of claim 38, wherein the additional outer layer is attached to the receptacle by means of an adhesive. 45. The catheter assembly of claim 38, wherein the additional outer layer is attached to the receptacle by means of welding. 46. The catheter assembly according to claim 23, wherein the receptacle comprises opening means for opening of the receptacle, said opening means preferably being arranged in an end of the receptacle being opposite to the connection between the receptacle and the catheter. 47. The catheter assembly according to claim 46, wherein the opening means comprises a peel-off joint. 48. The catheter assembly according to claim 46, wherein the opening means comprises a tear line. 49. The catheter assembly of claim 23, wherein the whole catheter is arranged within the bounds of the receptacle. 50. The catheter assembly of claim 23, wherein the catheter has a part of which forms an insertable length to be inserted through a body opening, and wherein the receptacle is connected to the catheter, thereby enclosing the insertable length of the catheter, but leaving at least part of the catheter outside the receptacle. 51. The catheter assembly of claim 23, additionally comprising the features claim 1. 52. A method for producing a catheter assembly, comprising: providing a receptacle; providing a hydrophilic catheter; arranging at least part of the catheter tube in a cavity of the receptacle; arranging a wetting fluid in a compartment forming an integrated part of the receptacle, but being separated from said cavity; and wherein the separation between the wetting fluid compartment and the cavity accommodating the catheter is formed as a rupturable sealed closure, wherein said closure is provided with at least one point of weakness, in order for an induced rupture to occur in a predetermined position, thereby enabling fluid communication between the compartment and the cavity housing the catheter. 53. The method of claim 52, additionally comprising the features of claim 21. 54. A catheter assembly comprising: a catheter (3;530;630;730), part of which forms an insertable length to be inserted through a body opening; and a catheter receptacle (2;520;620;720) for accommodation of at least part of the catheter, characterised in that the receptacle is connected to the catheter, thereby enclosing the insertable length of the catheter, but leaving at least part of the catheter outside the receptacle. 55. The catheter assembly of claim 54, wherein the compartment formed by the receptacle and the catheter forms a microbial barrier towards the environment. 56. The catheter assembly of claim 54 or 55, wherein the catheter is a hydrophilic urinary catheter, and wherein the assembly further comprises a wetting fluid. 57. The catheter assembly of claim 56, wherein the wetting fluid is arranged in a compartment of the receptacle. 58. The catheter assembly of claim 57, wherein the wetting fluid compartment is formed by a compartment of the receptacle housing the catheter, for preservation of the hydrophilic surface layer in a wetted state during accommodation in said receptacle and provision of a ready-to-use catheter assembly. 59. The catheter assembly of claim 56, wherein the wetting fluid compartment is arranged to keep the wetting fluid separated from at least an insertable part of the catheter during storage, the compartment container being openable for activation of the catheter. 60. The catheter assembly of claim 56, wherein the receptacle is gas impermeable. 61. The catheter assembly of claim 56, wherein the wetting fluid container forms a compartment being separated from the receptacle. 62. A method for producing a catheter assembly, comprising: providing a receptacle (2;520;620;720) having an opening; providing a catheter (3;530;630;730) including a catheter tube and a connector arranged on one end thereof; arranging the catheter tube in the receptacle; and connecting the receptacle to the connector, thereby closing said opening. 63. The method of claim 62, wherein the receptacle is connected to the connector thereby sealingly closing the receptacle.
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<SOH> BACKGROUND OF THE INVENTION <EOH>Catheters find their use in many different medical applications, such as urinary catheters for bladder drainage. Catheters are normally pre-packed in a receptacle by the manufacturer, in order to maintain the catheter in a clean and preferably sterile condition. However, a problem with such catheter assemblies are that they are bulky, making them difficult and expensive to store, transport and handle. Further, a lot of packaging and wrapping material is required, making the assemblies expensive and harmful to the environment. Urinary catheters in general need to have a lubricant applied to the outer surfaces thereof to facilitate insertion into the urethra. Especially, for lubrication purposes hydrophilic urinary catheters may have a hydrophilic outer surface coating which should be wetted by a fluid such as water or saline for a certain time period prior to insertion thereof into the urethra of a patient. In order to facilitate the use and to improve cleanliness of the catheter, the assemblies have in recent years developed to comprise a rupturable wetting fluid pouch or container as well. This is e.g. known from WO 98/19729. However, unfortunately the inclusion of such a wetting fluid container makes the above-described problem with bulkiness, etc, even more severe. Further, there has recently been a trend towards so-called “ready-to-use” catheters, where the catheter is arranged in the receptacle together with a wetting fluid, so that the catheter is maintained in a wetted, activated condition. Such a catheter assembly is e.g. known from WO 00/47494. However, in such a catheter, a relatively large amount of wetting fluid is required to fill the receptacle to a certain degree and to ensure that an adequate wetting of the catheter is maintained. accordingly, even this type of catheter assembly suffers from the bulkiness discussed above, and is further relatively heavy. Further, a problem with this type of catheter assemblies are that the catheter becomes wet and slippery, which makes it uncomfortable and difficult to handle. There is further a problem with known catheter assemblies that the catheter must normally be removed from the receptacle or package before it could be connected to other devices, such as drainage tubes, urine bags, etc. Hereby, the handling of the catheter becomes more difficult, since more work has to be done by the person responsible for the catheterization, and especially during the stressed situation when the catheter is exposed to the environment, instead of in advance, or even as a step during the production process. Further, the time period when the catheter is exposed, and accordingly vulnerable to contamination and the like, is prolonged, which increases the risk for the patient. Accordingly, there is a need for a leaner and less bulky catheter assembly and/or a catheter assembly which is easier and more convenient to handle and/or a catheter assembly which is less expensive to produce, and especially for hydrophilic urinary catheters. The present invention therefore proposes to address this need. This object is achieved with the catheter assembly and the method according to the appended claims.
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<SOH> SUMMARY OF THE INVENTION <EOH>According to one aspect of the invention, it relates to a catheter assembly comprising a catheter including a catheter tube and a connector arranged in one end of the catheter tube; and a catheter receptacle for accommodation of at least the catheter tube. Further, the receptacle is provided with an opening, said opening being connected to and closed by the connector of the catheter. The term “connector” is, in the context of this application, to be understood in a broad sense, meaning any part of the catheter functioning as a means for connecting the catheter tube to external means, such as external tubing, or simply functioning as an outlet for drainage through the catheter tube. Further, the connector need not be a separate part of the catheter, but could be integrated with the catheter tube. Further, “end of the catheter”, is in the context of this application, also to be understood in a broad sense, meaning the end section of any part projecting or protruding from the rest of the catheter. Consequently, a catheter may have more than two ends, and an end must not be positioned farthest away from the main body of the catheter in any direction. With the catheter assembly according to the first aspect of the invention, a very advantageous catheter assembly is provided. Generally, the invention relates to a catheter assembly, comprising a catheter receiving receptacle and a catheter having a connector at one end thereof, wherein the receptacle is sealed by said connector. Since the receptacle is closed by the connector of the catheter, only part of the catheter is enclosed in the receptacle. Hereby, the assembly could be made leaner and less bulky since a receptacle of smaller volume could be used, and at the same time less material is required making the assembly easier and less costly to manufacture. This also makes the product more environment friendly, since less material is required. Still, all parts of the catheter intended to be inserted into the patient, i.e. all insertable parts of the catheter, could nonetheless be kept in a sterile and medically safe condition, making this catheter assembly at least as reliable as previously known catheter assemblies where the whole catheter is arranged inside the receptacle. Further, by the arrangement with the receptacle being closed by the connector of the catheter, whereby at least part of the connector can be accessed from the outside of the receptacle, the catheter could be connected to other devices, such as drainage tubes, urine bags, etc. before removal of the catheter from the receptacle or package. Hereby, the handling of the catheter becomes both simpler and safer, since the connection work need not be done by the person responsible for the catheterization, and especially not during the stressed situation when the catheter is removed from the receptacle and, thus, exposed to the environment. Hereby, the catheterization becomes easier and more efficient, and the time period when the catheter is exposed, and accordingly vulnerable to contamination and the like, is also shortened significantly, which decreases the risk for the patient. The connection of the catheter to other parts could, with the inventive concept, even be made already during production. Hereby, the production could comprise a module system, where the catheter assembly could be produced as a standard component, which is connectable during production with other parts, such as drainage tubes, urine bags and the like, into different end products. Hereby, the production could be made simpler and more cost efficient, requiring less production machinery, less storages, etc. Hereby, the production also becomes more environment friendly. Consequently, the invention relates to a catheter assembly comprising a catheter, part of which forms an insertable length to be inserted through a body opening; and a catheter receptacle for accommodation of at least part of the catheter. Further, the receptacle is connected to the catheter, thereby enclosing the insertable length of the catheter, but leaving at least part of the catheter outside the receptacle. Further, by the arrangement of at least part of the connector outside the receptacle, the catheter could be connected to other devices, such as drainage tubes, urine bags, etc. before removal of the catheter from the receptacle or package. Some preferred embodiments of the above-discussed aspect of the invention will now be discussed briefly. Preferably, the receptacle is sealingly connected to the catheter, and/or the compartment formed by the receptacle and the catheter is sealed. Hereby, at least an insertable part of the catheter could be maintained in a clean, and preferably sterile, condition during storage. Alternatively or additionally, the compartment formed by the receptacle and the catheter could form a microbial barrier against the environment. The catheter is preferably a hydrophilic catheter. A hydrophilic catheter is a catheter in which the catheter has, on at least a part of its surface, a hydrophilic surface layer intended to be wetted with a wetting fluid prior to use in order to provide a low-friction surface. In case a hydrophilic urinary catheter is used in the catheter assembly, the assembly could further comprise a wetting fluid. Hereby, no additional wetting fluid is needed for activation of the catheter, which entails many advantages. For example, activation of the catheter could easily be accomplished in places where it is normally difficult to find an appropriate wetting fluid for this specific use. Further, it could be ensured that only a sufficiently clean and sterile fluid is used, thereby decreasing the risk for unwanted contamination of the catheter. Still further, the wetting of the catheter may be accomplished in a simpler and more convenient manner. In one line of embodiments, the catheter assembly comprises a container containing the wetting fluid, said container being arranged to keep the wetting fluid separated from at least the insertable part of the catheter during storage and the container being openable for activation of the catheter. In such an embodiment, the wetting fluid may be kept separated from the insertable part of the catheter, i.e. the part of the catheter to be inserted through a body opening of the patient, until the time when the catheter is intended to be used. Then, the wetting fluid container may be opened, e.g. by application of a pressure, a pulling force or the like to the container, whereby the wetting fluid is allowed to discharge into the compartment housing the catheter. The container may be a compartment integrated with the receptacle, or a separate container. In case a separate container is used, said container may be arranged completely inside the receptacle, partly inside the receptacle or outside the receptacle but preferably with some sort of fluid communication with the receptacle. In another line of embodiments, the catheter assembly comprises a container containing the wetting fluid, said container being formed by a compartment of the receptacle housing the catheter, for preservation of the hydrophilic surface layer in a wetted state during accommodation in said receptacle and provision of a so called “ready-to-use” catheter assembly. In such an embodiment, the catheter is continuously maintained in an activated, ready-to-use condition. In all embodiments where the catheter assembly comprises a wetting fluid container, the amount of wetting fluid provided is preferably sufficient for filling the receptacle to a certain degree and to ensure that an adequate wetting of the catheter is maintained. In case the container is a compartment formed by the receptacle and the catheter, said compartment is preferably gas sealed, wherein the longevity of the product is increased. For the same reason, the receptacle is preferably gas impermeable. A suitable wetting fluid is sterile water or a saline solution. It is further preferred that the receptacle of the catheter assembly forms an elongate pocket. The receptacle could be connected to the catheter by means of a welding joint, said joint preferably being arranged between the catheter and the receptacle. This is a simple and cost effective way of providing a tight and sufficiently strong connection. Alternatively, the receptacle could be connected to the catheter by means of a shrink fit, which is also a simple and cost effective way of providing a tight and sufficiently strong connection. The receptacle is preferably connected to the connector of the catheter. The receptacle of the catheter assembly preferably comprises opening means for opening of the receptacle, said opening means preferably being arranged in an end of the receptacle being opposite to the connection between the receptacle and the catheter. Hereby, unpacking of the catheter before use becomes very easy. The opening means could comprise a peel-off joint, a tear line or the like. A typical sterilizing agent which could be used for sterilizing the catheter assembly of the invention is ethylene oxide. Moreover, the fluid in the fluid container would normally already be sterile when packed. For these reasons, the wetting fluid container is preferably made of a material which is impermeable or substantially impermeable to ethylene oxide as well as the fluid contained therein. Non-limiting examples of materials satisfying this condition when the fluid is water or saline are aluminium foil laminate, poly(vinylidene chloride) or a laminate comprising metallised film such as metallised poly(ethylene terepthalate), or a silicon oxide coated film, or a laminate comprising aluminum oxide. Other sterilization processes could of course be used instead, for example by irradiation. Steam treatment may also be used for sterilization. According to another aspect of the invention, it relates to a method for producing a catheter assembly, comprising: providing a receptacle having an opening; providing a catheter including a catheter tube and a connector arranged on one end thereof; arranging the catheter tube in the receptacle; and connecting the receptacle to the connector, thereby closing said opening. With a production method according to this aspect of the invention, similar advantages are achieved as discussed above in relation to the first and second aspect of the invention. Preferably, the catheter is first assembled by connecting the catheter tube and the connector, where after the catheter tube is arranged in the receptacle and the receptacle is connected to the catheter connector. However, the manufacturing may also be in a reversed order. E.g. the connector may be connected to the receptacle before assembly of the connector and the catheter tube. In this case, the catheter tube may e.g. be introduced from another opening of the receptacle, which is subsequently closed. According to another aspect of the invention, it relates to a catheter assembly comprising: a catheter having on at least part of its surface a hydrophilic surface layer intended to produce a low-friction surface character of the catheter by treatment with a wetting fluid prior to use of the catheter; a catheter receptacle forming a cavity for accommodation of at least part of the catheter; and a compartment accommodating said wetting fluid, wherein said compartment forms an integrated part of the receptacle, but being separated from said cavity. Further, an additional outer layer is attached to the receptacle, said layer being arranged to at least partly cover the part of the receptacle forming said wetting fluid compartment. In accordance with this aspect of the invention, an additional cover is provided in order to achieve a stronger and preferably gas impermeable wetting fluid compartment. Due to the use of this additional cover, the requirements on the material of the receptacle could be lowered, and the material need e.g. not be gas-impermeable. Hereby, the additional cover could provide the impermeability of the compartment wall necessary to alleviate evaporation and maintain the wetting fluid in the compartment during storage. At the same time, only a limited amount of cover material is needed, making the product cost effective to produce. The additional cover may also be used as a protection for the wetting fluid against a sterilizing agent used for the sterilization of the catheter and the rest of the catheter assembly. A typical sterilizing agent which could be used for sterilizing the wetting apparatus of the invention is ethylene oxide. Moreover, the fluid in the fluid container would normally already be sterile when packed, and need not be further sterilized. Additionally, the sterilizing agent may leave unwanted residual products in the wetting fluid if exposed to the same. For these reasons, it is preferred that the additional cover of the wetting fluid container is made of a material which is impermeable or substantially impermeable to ethylene oxide as well as the fluid contained therein. Non-limiting examples of materials satisfying this condition when the fluid is water or saline are aluminium foil laminate, poly(vinylidene chloride) or a laminate comprising metallised film such as metallised poly(ethylene terepthalate), or a silicon oxide coated film, or a laminate comprising aluminum oxide. Other sterilization processes could of course be used instead, for example by irradiation in which case the fluid in the container could be sterilized in situ at the same time as the rest of the components of the assembly. Steam treatment may also be used for sterilization. The additional cover may be attached to the compartment by means of an adhesive, welding or any other suitable connection means. According to a corresponding aspect, the invention further relates to a method for producing a catheter assembly, comprising: providing a receptacle; providing a hydrophilic catheter; arranging at least part of the catheter tube in a cavity of the receptacle; arranging a wetting fluid in a compartment forming an integrated part of the receptacle, but being separated from said cavity; and attaching an additional outer layer to the receptacle to at least partly cover the part of the receptacle forming said wetting fluid compartment. According to this method, similar advantages as discussed above are achieved. According to another aspect of the invention, it relates to a catheter assembly comprising: a catheter having on at least part of its surface a hydrophilic surface layer intended to produce a low-friction surface character of the catheter by treatment with a wetting fluid prior to use of the catheter; a catheter receptacle forming a cavity for accommodation of at least part of the catheter; and a compartment accommodating said wetting fluid, wherein said compartment forms an integrated part of the receptacle, but being separated from said cavity, wherein the separation between the wetting fluid compartment and the cavity accommodating the catheter provides a rupturable sealed closure. Further, said closure is provided with at least one point of weakness, in order for an induced rupture to occur in a predetermined position, thereby enabling fluid communication between the compartment and the cavity housing the catheter. In accordance with this embodiment of the invention, a rupture of the fluid compartment could be effectively controlled, in order for it to occur in a predetermined position. Hereby, it could be avoided that the compartment, upon application of pressure or the like, is ruptured in a random position, which could lead to leakage of the fluid, insufficient wetting of the hydrophilic surface, etc. Instead, the rupture will always occur in the most effective position, leading to an effective wetting of the catheter surface. Preferably, the closure is formed by a rupturable joint between the compartments, and most preferably the joint is a welded joint with a welding width variation, or a welding strength variation, thus providing the at least one point of weakness. Alternatively, the joint could be arranged in a non-linear arrangement, thus providing the at least one point of weakness. In this case, the joint could be arranged with at least one knee directed towards the wetting fluid compartment. The knee could e.g. have an angled peak portion directed towards the wetting fluid compartment, with an obtuse or acute angle. However, the knee may alternatively have an curved peak portion directed towards the wetting fluid compartment. Hereby, effective rupture control may be achieved, and at the same time a very cost effective and easily producible joint is provided. According to a corresponding aspect of the invention, a method is provided for producing a catheter assembly, comprising: providing a receptacle; providing a hydrophilic catheter; arranging at least part of the catheter tube in a cavity of the receptacle; arranging a wetting fluid in a compartment forming an integrated part of the receptacle, but being separated from said cavity; and attaching an additional outer layer to the receptacle to at least partly cover the part of the receptacle forming said wetting fluid compartment. All the various detailed embodiments and different features discussed above in relation to different aspects of the invention discussed above are also usable together with the other aspects of the invention, and thus combinable in other ways than those specifically disclosed, if nothing else is explicitly stated. Further scope of the applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
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Machine-detectable adhesive tape
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The invention relates to an adhesive tape for a flying reel change of a flat material wound up over a reel, provided with at least one detachable adhesive system. The invention is characterized in that the main support is made of a material to which a machine-detectable admixture has been added.
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1-5. (Cancel) 6. An adhesive tape for the flying reel change of flat web material wound up on reels, comprising at least one main carrier, at least one cleavable adhesive system and a material comprising at least one machine-detectable additive. 7. The adhesive tape according to claim 6, comprising a layer of a self-adhesive compound on a top side thereof and at least one cleavable adhesive system on a bottom side thereof. 8. The adhesive tape as claimed in claim 6, wherein said machine-detectable additive is comprised of a metal powder or a metal granulate or a combination thereof. 9. A method for splicing during a flying reel change of flat web material wound up on reels, said method comprising; a) providing the adhesive tape of claim 6; b) adhering an uppermost paper web of a new reel to the web lying underneath with said adhesive tape; c) exposing part of the self-adhesive compound which is needed for the splicing process; d) placing the new reel beside an old reel which is unwinding and is to be replaced; e) accelerating the new reel to substantially the same rotational speed as the old reel; f) pressing the new reel against the old reel, wherein the exposed self-adhesive compound of the adhesive tape bonds adhesively to the uppermost paper web of the old reel, while at the same time cleaving the cleavable adhesive system wherein, following the cleaving, no adhesive regions are exposed; and g) determining the time of the splicing operation by mechanically detecting at least one machine-detectable additive. 10. A method according to claim 9, wherein said machine-detectable additive is comprised of a metal powder, metal granulate or a combination thereof.
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Surface for the immobilisation of nucleic acids
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The invention relates to a surface for the immobilization of one or several first nucleic acids as recognition elements (“immobilization surface”), for the production of a recognition surface for the detection of one or several second nucleic acids in one or more samples which are brought into contact with the recognition surface, the first nucleic acids being applied to a layer of the graft copolymer poly(L-lysine)-g-poly(ethyleneglycol) (PLL-g-PEG) as surface for immobilization, characterized in that the grafting ratio g, in other words the ratio between the number of lysine units and the number of polyethylene glycol side chains (“PEG” side chains) has an average value between 7 and 13. The invention also relates to a method for the qualitative and/or quantitative detection of one or more second nucleic acids in one or more samples, characterized in that said samples and optionally further reagents are brought into contact with an immobilization surface according to the invention, upon which one or several first nucleic acids are immobilized as recognition elements for specific binding/hybridization with said second nucleic acids and changes in optical or electronic signals resulting from the binding/hybridization of said second nucleic acid, or further, resulting from applied tracer substances applied for analyte detection, are measured.
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1. A surface for the immobilization of one or several first nucleic acids as recognition elements (“immobilization surface”), for the production of a recognition surface for the detection of one or several second nucleic acids in one or more samples which are brought into contact with the recognition surface, the first nucleic acids being applied to a layer of PLL-g-PEG (graft copolymer poly(L-lysine)-g-poly(ethyleneglycol)) as a surface for immobilization, characterized in that the grafting ratio g, in other words the ratio between the number of lysine units and the number of polyethylene glycol side chains (“PEG” side chains) has an average value between 7 and 13. 2. A surface for the immobilization of one or several first nucleic acids according to claim 1, wherein the grafting ratio g has a medium value between 8 and 12. 3. A surface for the immobilization of one or several first nucleic acids according to claim 1, wherein the molecular weight of the polyetheyleneglycol side chains (“PEG” side chains) is between 500 Da and 7000 Da. 4. A surface for the immobilization of one or several first nucleic acids according to claim 1, wherein the molecular weight of the polyetheyleneglycol side chains (“PEG” side chains) is between 1500 Da and 5000 Da. 5. A surface for the immobilization of one or several first nucleic acids according to claim 1, wherein said surface is deposited on a solid carrier. 6. A surface for the immobilization of one or several first nucleic acids according to claim 5, wherein said solid carrier is an essentially optically transparent carrier. 7. A surface for the immobilization of one or several first nucleic acids according to claim 6, wherein the essentially optically transparent carrier comprises a material from the group comprising moldable, sprayable or millable plastics, metals, metal oxides, silicates, such as glass, quartz or ceramics. 8. A surface for the immobilization of one or several first nucleic acids according to claim 1, wherein said surface is essentially optically transparent. 9. A surface for the immobilization of one or several first nucleic acids according to claim 1, wherein said surface (as a PLL-g-PEG layer) has a thickness of less than 200 nm, preferably of less than 20 nm. 10. An immobilization surface according to claim 6, wherein said surface for immobilization is deposited on a solid carrier, in the surface of which are structured recesses for generation of sample compartments. 11. An immobilization surface according to claim 10, wherein said recesses in the surface of the carrier have a depth of 20 μm to 500 μm, especially preferably 50 μm to 300 μm. 12. An immobilization surface according to claim 6, wherein the essentially optically transparent carrier comprises a continuous optical waveguide or an optical waveguide divided into individual waveguiding areas. 13. An immobilization surface according to claim 12, wherein the optical waveguide is an optical film waveguide with a first essentially optically transparent layer (a) facing the immobilization surface on a second essentially optically transparent layer (b) with a refractive index lower than that of layer (a). 14. An immobilization surface according to claim 13, wherein said optical film waveguide is essentially planar. 15. An immobilization surface according to claim 13, wherein, for the in-coupling of excitation light into the optically transparent layer (a), this layer is in optical contact with one or more optical in-coupling elements from the group comprising prism couplers, evanescent couplers with combined optical waveguides with overlapping evanescent fields, butt-end couplers with focusing lenses, preferably cylinder lenses, arranged in front of one face of the waveguiding layer, and grating couplers. 16. An immobilization surface according to claim 15, wherein the excitation light is in-coupled into the optically transparent layer (a) using one or more grating structures (c) which are featured in the optically transparent layer (a). 17. An immobilization surface according to claim 15, wherein light guided in the optically transparent layer (a) is out-coupled using one or more grating structures (c′) which are featured in the optically transparent layer (a) and have the same or different period and grating depth as grating structures (c). 18. An immobilization surface according to claim 1, wherein the nucleic acids immobilized thereon as recognition elements are arranged in discrete (laterally separated) measurement areas. 19. An immobilization surface according to claim 18, wherein up to 1,000,000 measurement areas are provided in a 2-dimensional arrangement and a single measurement area covers an area of 10−4 mm2-10 mm2. 20. An immobilization surface according to claim 18, wherein the measurement areas are arranged in a density of more than 10, preferably more than 100, especially preferably more than 1000 measurement areas per square centimeter. 21. An immobilization surface according to claim 18, wherein discrete (laterally separated) measurement areas are generated on said immobilization surface by the laterally selective application of nucleic acids as recognition elements, preferably using one or more methods from the group of methods comprising ink-jet spotting, mechanical spotting by means of pin, pen or capillary, micro-contact printing, fluidic contact of the measurement areas with the biological or biochemical or synthetic recognition elements through their application in parallel or intersecting microchannels, upon exposure to pressure differences or to electric or electromagnetic potentials, and photochemical or photolithographic immobilization methods. 22. A method for the simultaneous or sequential, qualitative and/or quantitative detection of one or more second nucleic acids in one or more samples, wherein said samples and if necessary further reagents are brought into contact with an immobilization surface according to claim 1, on which surface one or several first nucleic acids are immobilized as recognition elements for the specific binding/hybridization with said second nucleic acids, and changes in optical or electronic signals resulting from the binding/hybridization with these second nucleic acids or of further tracer substances used for analyte detection are measured. 23. A method according to claim 22, wherein the one or more samples are pre-incubated with a mixture of the various tracer reagents for determining the second nucleic acids to be detected in said samples, and these mixtures are then brought into contact with the first nucleic acids immobilized on said immobilization surface in a single addition step. 24. A method according to claim 22, wherein the detection of the one or more second nucleic acids is based on the determination of the change in one or more luminescences. 25. A method according to claim 22, wherein the excitation light from one or more light sources for the excitation of one or more luminescences is delivered in an epi-illumination configuration. 26. A method according to claim 22, wherein the excitation light from one or more light sources for the excitation of one or more luminescences is delivered in a transillumination configuration. 27. A method according to one of claims 22-24 claim 22, wherein the immobilization surface is arranged on an optical waveguide which is preferably essentially planar, wherein one or more samples with second nucleic acids to be detected therein and, if necessary further tracer reagents, are brought sequentially or in a single addition step after mixture with said tracer reagents, into contact with said first nucleic acids immobilized as recognition elements on said immobilization surface, and wherein the excitation light from one or more light sources is in-coupled into the optical waveguide using one or more optical coupling elements from the group comprising prism couplers, evanescent couplers with combined optical waveguides with overlapping evanescent fields, butt-end couplers with focusing lenses, preferably cylinder lenses, arranged in front of one face of the waveguiding layer, and grating couplers. 28. A method according to claim 27, wherein the detection of one or more second nucleic acids is performed on a grating structure (c) or (c′) formed in the layer (a) of an optical film waveguide, based on changes in the resonance conditions for the in-coupling of excitation light into layer (a) of a carrier formed as film waveguide or for out-coupling of light guided in layer (a), these changes resulting from binding/hybridization of said second nucleic acids or further tracer reagents to the first nucleic acids immobilized as recognition elements in the region of said grating structure on said immobilization surface. 29. A method according to claim 27, wherein said optical waveguide is designed as an optical film waveguide with a first optically transparent layer (a) on a second optically transparent layer (b) with lower refractive index than layer (a), wherein excitation light is further in-coupled into the optically transparent layer (a) with the aid of one or more grating structures, which are featured in the optically transparent layer (a), and delivered as a guided wave to measurement areas (d) located, and wherein the luminescence of molecules capable of luminescence, generated in the evanescent field of said guided wave, is further determined using one or more detectors, and the concentration of one or more nucleic acids to be detected is determined from the intensity of these luminescence signals. 30. A method according to claim 29, wherein (1) the isotropically emitted luminescence or (2) luminescence in-coupled into the optically transparent layer (a) and out-coupled via grating structure (c) or (c′) or luminescences of both (1) and (2) are measured simultaneously. 31. A method according to claim 29, wherein, for the generation of luminescence, a luminescence dye or luminescent nanoparticle is used as a luminescence label, which can be excited and emits at a wavelength between 300 nm and 1100 nm. 32. A method according to claim 31, wherein the luminescence label is bound to the second nucleic acids themselves to be detected as analytes or, in a competitive assay, to nucleic acids with the same sequence as said second nucleic acids to be detected and added to the sample as competitors at a known concentration, or, in a multistep assay, to one of the binding partners of the first nucleic acids immobilized as recognition elements, or to said immobilized first nucleic acids. 33. A method according to claim 31, wherein a second luminescence label or further luminescence labels are used with excitation wavelengths either the same as or different from that of the first luminescence label and the same or different emission wavelength. 34. A method according to claim 33, wherein the second or further luminescence labels can be excited at the same wavelength as the first luminescence label, but emit at different wavelengths. 35. A method according to claim 33, wherein the excitation spectra and emission spectra of the luminescence dyes used overlap only little or not at all. 36. A method according to claim 33, wherein charge or optical energy transfer from a first luminescence label serving as donor to a second luminescence label serving as acceptor is used for the purpose of detecting the second nucleic acids as analytes. 37. A method according to claim 29, wherein changes in the effective refractive index on the measurement areas are determined in addition to the determination of one or more luminescences. 38. A method according to claim 29, wherein the one or more luminescences and/or determinations of light signals at the excitation wavelength are carried out using a polarization-selective procedure. 39. A method according to claim 29, wherein the one or more luminescences are measured at a polarization different from that of the excitation light. 40. A method according to claim 22, wherein the samples to be analyzed are aqueous solutions, especially buffer solutions, or naturally occurring body fluids such as blood, serum, plasma, urine or tissue fluids. 41. A method according to claim 22, wherein the sample to be analyzed is an optically turbid fluid, surface water, a soil or plant extract, a biological or synthetic process broth. 42. A method according to claim 22, wherein the samples to be analyzed are prepared from biological tissue parts or cells. 43. Use of an immobilization surface according to claim 1 for quantitative or qualitative analyses in screening methods in pharmaceutical research, clinical and pre-clinical development, for real-time binding studies and the determination of kinetic parameters in affinity screening and in research, for qualitative and quantitative analyte determinations, especially for DNA and RNA analytics and for the determination of genomic or proteomic differences in the genome, such as single nucleotide polymorphisms, for the measurement of protein-DNA interactions, for the determination of control mechanisms for mRNA expression and for protein (bio)synthesis, for the generation of toxicity studies and the determination of expression profiles, especially for the determination of biological and chemical marker compounds, such as mRNA, pathogens or bacteria in pharmaceutical product research and development, human and veterinary diagnostics, agrochemical product research and development, for symptomatic and pre-symptomatic plant diagnostics, for patient stratification in pharmaceutical product development and for therapeutic drug selection, for the determination of pathogens, nocuous agents and germs, especially of salmonella, prions, viruses and bacteria, especially in food and environmental analytics. 44. An immobilization surface according to claim 8, wherein said surface for immobilization is deposited on a solid carrier, in the surface of which are structured recesses for generation of sample compartments. 45. An immobilization surface according to claim 9, wherein said surface for immobilization is deposited on a solid carrier, in the surface of which are structured recesses for generation of sample compartments. 46. An immobilization surface according to claim 8, wherein the essentially optically transparent carrier comprises a continuous optical waveguide or an optical waveguide divided into individual waveguiding areas. 47. An immobilization surface according to claim 46, wherein the optical waveguide is an optical film waveguide with a first essentially optically transparent layer (a) facing the immobilization surface on a second essentially optically transparent layer (b) with a refractive index lower than that of layer (a). 48. An immobilization surface according to claim 9, wherein the essentially optically transparent carrier comprises a continuous optical waveguide or an optical waveguide divided into individual waveguiding areas. 49. An immobilization surface according to claim 48, wherein the optical waveguide is an optical film waveguide with a first essentially optically transparent layer (a) facing the immobilization surface on a second essentially optically transparent layer (b) with a refractive index lower than that of layer (a). 50. A method for the simultaneous or sequential, qualitative and/or quantitative detection of one or more second nucleic acids in one or more samples, wherein said samples and if necessary further reagents are brought into contact with an immobilization surface according to claim 6, on which surface one or several first nucleic acids are immobilized as recognition elements for the specific binding/hybridization with said second nucleic acids, and changes in optical or electronic signals resulting from the binding/hybridization with these second nucleic acids or of further tracer substances used for analyte detection are measured. 51. A method according to claim 50, wherein the immobilization surface is arranged on an optical waveguide which is preferably essentially planar, wherein one or more samples with second nucleic acids to be detected therein and, if necessary further tracer reagents, are brought sequentially or in a single addition step after mixture with said tracer reagents, into contact with said first nucleic acids immobilized as recognition elements on said immobilization surface, and wherein the excitation light from one or more light sources is in-coupled into the optical waveguide using one or more optical coupling elements from the group comprising prism couplers, evanescent couplers with combined optical waveguides with overlapping evanescent fields, butt-end couplers with focusing lenses, preferably cylinder lenses, arranged in front of one face of the waveguiding layer, and grating couplers. 52. A method for the simultaneous or sequential, qualitative and/or quantitative detection of one or more second nucleic acids in one or more samples, wherein said samples and if necessary further reagents are brought into contact with an immobilization surface according to claim 8, on which surface one or several first nucleic acids are immobilized as recognition elements for the specific binding/hybridization with said second nucleic acids, and changes in optical or electronic signals resulting from the binding/hybridization with these second nucleic acids or of further tracer substances used for analyte detection are measured. 53. A method according to claim 52, wherein the immobilization surface is arranged on an optical waveguide which is preferably essentially planar, wherein one or more samples with second nucleic acids to be detected therein and, if necessary further tracer reagents, are brought sequentially or in a single addition step after mixture with said tracer reagents, into contact with said first nucleic acids immobilized as recognition elements on said immobilization surface, and wherein the excitation light from one or more light sources is in-coupled into the optical waveguide using one or more optical coupling elements from the group comprising prism couplers, evanescent couplers with combined optical waveguides with overlapping evanescent fields, butt-end couplers with focusing lenses, preferably cylinder lenses, arranged in front of one face of the waveguiding layer, and grating couplers. 54. A method for the simultaneous or sequential, qualitative and/or quantitative detection of one or more second nucleic acids in one or more samples, wherein said samples and if necessary further reagents are brought into contact with an immobilization surface according to claim 9, on which surface one or several first nucleic acids are immobilized as recognition elements for the specific binding/hybridization with said second nucleic acids, and changes in optical or electronic signals resulting from the binding/hybridization with these second nucleic acids or of further tracer substances used for analyte detection are measured. 55. A method according to claim 54, wherein the immobilization surface is arranged on an optical waveguide which is preferably essentially planar, wherein one or more samples with second nucleic acids to be detected therein and, if necessary further tracer reagents, are brought sequentially or in a single addition step after mixture with said tracer reagents, into contact with said first nucleic acids immobilized as recognition elements on said immobilization surface, and wherein the excitation light from one or more light sources is in-coupled into the optical waveguide using one or more optical coupling elements from the group comprising prism couplers, evanescent couplers with combined optical waveguides with overlapping evanescent fields, butt-end couplers with focusing lenses, preferably cylinder lenses, arranged in front of one face of the waveguiding layer, and grating couplers. 56. Use of a method according to claim 22 for quantitative or qualitative analyses in screening methods in pharmaceutical research, clinical and pre-clinical development, for real-time binding studies and the determination of kinetic parameters in affinity screening and in research, for qualitative and quantitative analyte determinations, especially for DNA and RNA analytics and for the determination of genomic or proteomic differences in the genome, such as single nucleotide polymorphisms, for the measurement of protein-DNA interactions, for the determination of control mechanisms for mRNA expression and for protein (bio)synthesis, for the generation of toxicity studies and the determination of expression profiles, especially for the determination of biological and chemical marker compounds, such as mRNA, pathogens or bacteria in pharmaceutical product research and development, human and veterinary diagnostics, agrochemical product research and development, for symptomatic and pre-symptomatic plant diagnostics, for patient stratification in pharmaceutical product development and for therapeutic drug selection, for the determination of pathogens, nocuous agents and germs, especially of salmonella, prions, viruses and bacteria, especially in food and environmental analytics.
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Apparatus, composition and method for proteome profiling
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The present invention is directed to a high throughput method for producing a large number of different antibodies, more specifically organized antibody microarrays. These antibodies and antibody microarrays can be used to rapidly assay protein abundance and identify types of proteins that are expressed in cells and tissues under a variety of conditions, or to compare protein expression profiles of different cells.
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1. A method for a fast high-throughput determination of proteins differentially expressed by a cell or a plurality of cells comprising: (a) subjecting a first biological sample to a microarray containing at least 100 different antibodies; (b) comparing the protein profiles of said first biological sample with a second biological sample; and (c) identifying proteins that are differentially expressed in at least part of the cells of said biological samples. 2. The method of claim 1, wherein the first and second biological sample are from a similar tissue but differ from each other by developmental stage. 3. The method of claim 1, wherein the first and second biological sample are from a similar tissue but differ from each other by hormone expression. 4. The method of claim 1, wherein the first biological sample is from a normal or non-diseased tissue and the second biological sample is from a diseased-tissue or tissue suspected of being diseased. 5. The method of claim 1, wherein the first biological sample is from a normal or non-malignant tissue and the second biological sample is from a malignant tissue or a tissue suspected of being malignant. 6. The method of claim 1, wherein the first biological sample is from a normal or non-infected tissue and the second biological sample is from an infected tissue or from a tissue suspected of being infected. 7. The method of claim 1, wherein said microarray contains at least 1,000 antibodies. 8. The method of claim 1, wherein said microarray contains at least 10,000 antibodies. 9. The method of claim 1, wherein said microarray contains at least 100,000 antibodies. 10. A method of high-throughput synthesis of a plurality of antibodies comprising: (a) synthesizing a peptide microarray on a suitable substrate using virtual masking; (b) forming a random antibody library; (c) screening the random antibody library with the peptide microarray and selecting antibodies that bind with a suitable affinity and specificity to said peptide microarray; (d) purifying the selected antibodies; (e) amplifying the purified antibodies; and (d) binding the antibodies to a substrate thereby forming a microarray of antibodies suitable for high-throughput analysis. 11. The method of claim 10, wherein the substrate is glass or nylon. 12. The method of claim 10, wherein the microarray of antibodies contains at least 1,000 antibodies. 13. The method of claim 10, wherein the microarray of antibodies contains at least 10,000 antibodies. 14. The method of claim 10, wherein the microarray of antibodies contains at least 100,000 antibodies. 15. The method of claim 10, wherein the random antibody library is formed using a phage-display library.
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<SOH> BACKGROUND OF THE INVENTION <EOH>Significant attention in recent years has been directed to understanding and categorizing the genome of various organisms including humans. That field has been referred to as genomics. Attention has also been focused on understanding and identifying the various proteins an organism expresses. This field is referred to as proteomics. Comparisons of genes expressed by various organisms show greater similarity than might be expected by the physical differences between the species. Thus, understanding the proteins that are expressed, when they are expressed, and in what cells they are expressed takes on increasing importance. This is also important with respect to diseases, malignancies, etc. Consequently, ascertaining the set of proteins expressed by a particular cell type at various times and states such as resting vs. developing, normal (wild type), malignant, diseased, etc. has been an important challenge. Any method that could even partially meet this challenge, for example by determining a fraction of the protein profile rapidly and cost effectively, would be extremely desirable. The typical approach used in assessing the number and identity of expressed proteins is 2D gel electrophoresis and its extensions. The method, which was introduced 25 years ago, separates proteins on the basis of size and charge, and typically resolves several thousand proteins (1). More recently, mass spectrometry (MS) has been used in conjunction with the 2D gels after proteolytic cleavage to quantitatively ascertain the mass associated with each spot and to help identify the protein. However, these methods have various drawbacks. Among the problems associated with the use of gels and MS are preparation and purification of proteins, resolution and throughput. Although MS solves some of the problem of spot identification, its application to large numbers of spots (100 or more) is slow. Other problems are limitations in dynamic range of abundance and mass, For example, proteins expressed in low amounts are frequently missed. Further, the use of denaturants can prevent related functional studies. Ciphergen Biosystems Inc., has reported a chip technology that it claims should allow researchers to capture, separate and quantitatively analyze proteins directly on the chip. Their system is said to integrate mass spectrometry (particularly, surface enhanced laser desorption/ionization (SELDI)) and biochip technology on a single chip. They claim that their ProteinChip™ uses various molecular substrates, including antibodies and receptors, having affinities for proteins of interest. The chips are stated to be made of aluminum, about three inches long and one centimeter wide, containing eight sites and a group of 12 is alleged to be processed as the equivalent of a 96-well format. This system is intended to measure the mass of the captured proteins rather than their activity. The system is also limited in the number of kinds of proteins that can be identified. Therefore, it is not broadly applicable. Zyomyx Inc. and CombiMatrix Corp., both California companies, have stated that they are working on creating large-scale standardized methods for producing protein biochips. Zyomyx Inc., has claimed to develop a biochip, covered with a multi-component organic thin film to reduce non-specific protein binding and a protein capture agent such as an antibody or a peptide to fish for specific proteins of interest. The binding of proteins to capture agents is said to be detected by fluorescence among other methods. However, Zyomyx's technology is concerned with immobilizing a correctly oriented protein on a solid surface which is a complex and expensive process. CombiMatrix Corp., has reported it is developing a method, utilizing electrochemistry and semiconductor technology, to synthesize peptides (one amino acid at a time), antibodies, and proteins directly on the chip. The chip is said to consist of a large number of virtual flasks (up to one million per square centimeter) arranged in a grid pattern on the surface of a semiconductor wafer. This, too, is a very complex and expensive process. MacBeath et al. of Harvard University have described a method of immobilizing proteins by covalently attaching them to glass surfaces that is stated as using standard laboratory equipment. MacBeath et al. reports that they were able to create protein microarrays (with about 10,800 spots per standard microscope slide). These microarrays were alleged to be effective in detecting interactions between one protein and another that are known to interact with a small molecule (for which specific protein receptors are available) and a protein, and an enzyme and its substrate by identifying phosphorylation by means of phosographic emulsion and a light microscope. Genomic Solutions has stated it is developing robots to prepare samples (protein digestion) and to excise spots for MS. However, such a method is expensive and technologically complex. Accordingly, a need exists for a method of determining proteins expressed by a particular cell that is relatively simple. It would be desirable if this method was fast. It would be more desirable if the method was simple.
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<SOH> SUMMARY OF THE INVENTION <EOH>We have here discovered a high throughput method for producing a large number of different antibodies. These antibodies can be used to rapidly assay protein abundance in cells under a variety of conditions or to compare protein expression profiles of different cells. Additionally, we have discovered a method for the determination of proteins expressed by a specific cell or tissue. In one embodiment, the present invention permits targets of such proteins to be obtained. Still another embodiment of the present invention is directed to a method of making a microarray that can be used in such a method. The method of making a microarray utilizes microarrays of peptides, wherein one or more of the peptides are from a coding region of a genome of interest. Preferably, the peptides cover at least a part of the coding region of the genes that are of interest. For example, peptides can be selected from a family of proteins such as chemokine receptors, G-coupled protein receptors, a family of related proteins such as tumor associated antigens, oncogene products, etc. or combinations thereof. Preferably, the peptides chosen contain an antigenic epitope. More preferably, the peptide has an epitope that approximates the wild type conformation of the protein. The arrays are used to screen an antibody library such as a large, combinatorially generated library of antibodies that specifically bind to the peptides. Preferably, the antibodies bind to the peptides in a conformation that approximates their native state (i.e. when they are part of the protein). In this way a large library of antibodies that will bind specific native proteins is obtained. These antibodies can be for any species whose coding genome is known for any desired group of proteins. The antibodies can then be expressed by known means such as simple bacterial amplification. The antibodies are arrayed on a substrate such as on a chip or sphere. Any type of substrate will be a suitable “chip” as long as the antibodies can be substantially immobilized and used as bait to fish for expressed proteins in a sample, such as a cell of interest. Such antibody arrays can be used to screen a biological sample of interest. The proteins in the sample that bind to the array can readily be determined. These arrays can be used for a wide range of purposes. For example, to determine proteins that are differentially expressed in different cells. For instance, malignant cells versus non-malignant cells, diseased cells versus normal, cells in a pregnant woman versus non-pregnant, menopausal versus non-menopausal, stem cells versus nerve cells, etc. The antibody array of the present invention can be used, for example, in the diagnosis and treatment of a cancer, and immunopathology, a neuropathology, and the like. In another aspect, the present invention provides an expression profile that can reflect the expression levels of a plurality of proteins in a sample. The expression profile comprises an antibody array and a plurality of detectable proteins. The profiles can be collected, for example, to a database which can consequently be used for diagnostic and prognostic purposes, and for “pharmacoproteomic” applications. Such diagnostic and prognostic purposes include, for example, classification of different types of cancers according to their protein expression profile. Pharmacoproteomic applications include, for example, classification of individuals according to their responsiveness to pharmaceuticals or propensity to harmful side effects according to their protein expression profiles.
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Embossing tools and tape printers
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A tool (10) for providing an image on a tape medium (14), said tool comprising: means for inputting an image; means for providing said image on said tape medium; means for obtaining input image information from said input means; and sound generation means for using said information to provide an audible output in dependence on said input image.
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1. A tool for providing a an image on a tape medium, said tool comprising: means for inputting an image; means for providing said image on said tape medium; means for obtaining input image information from said input means; and sound generation means for using said information to provide an audible output in dependence on said input image. 2. A tool as claimed in claim 1, wherein said sound generation means comprises a speaker. 3. The tool of claim 1, wherein said sound generation means comprises a voice generator. 4. The tool of claim 1, wherein said sound generation means is arranged to provide an output when said means for providing said image on said tape is activated. 5. The tool of claim 1, wherein said sound generation means is arranged to provide an output as the image is input. 6. The tool of claim 1, wherein said sound generation means is arranged to provide an output only when all of said image has been input. 7. The tool of claim 1, wherein said tool comprises means for determining if the tape medium is present, said sound generation means is arranged to provide an audible output only if said tape medium is present. 8. The tool of claim 1, wherein said image comprises one or more characters, numerals, symbols, or combinations thereof. 9. A tool as claimed in claim 8, wherein said sound generation means provides voice output for said input characters, numerals and/or symbols. 10. The tool of claim 1, wherein said tool is an embosser. 11. A tool as claimed in claim 10, wherein said tool has a handle portion, said handle portion accommodating one or more batteries. 12. The tool of claim 1, wherein said tool is a tape printer. 13. The tool of claim 1, wherein said input means comprises a keyboard. 14. The tool of claim 10, wherein said input means comprises a rotatable wheel. 15. A tape embosser adapted to emboss an image on a tape comprising: an image input adapted to allow a user to select an image to be embossed; an actuator; an embossing die set comprising a plurality of selectable die and a presser adapted to press tape into the selected die, wherein the image input selects the die corresponding to the selected image; a sound generator adapted to receive information about the selected image and to provide audible information about the selected image; and a detector which detects the selected die and conveys information about the image to the sound generator. 16. The tape embosser of claim 15 wherein a plurality of dies in the die set are each individually associated with a series of conductive connectors adapted to interact with the detector to convey information about the die to the detector. 17. The tape embosser of claim 15 wherein the sound generator comprises a voice generator. 18. The tape embosser of claim 15 further comprising a handle having the actuator attached thereto and a supply of tape contained therein. 19. The tape embosser of claim 15 further comprising a detector to detect whether tape is present, wherein the sound generator is adapted to provide a sound only if tape is present. 20. The tape embosser of claim 16 wherein the voice generator is adapted to provide a sound only after the actuator is activated. 21. A tape printer adapted to print an image on a tape comprising: a keyboard adapted to allow entry of data about the image to be printed; a visual display providing information about the a printing head adapted to print the image onto a tape; a detector to detect the presence of tape; an actuator; and a sound generator adapted to provide an audible output in dependence on said input image. 22. The tape printer of claim 20 wherein the sound generator comprises a voice generator adapted to provide an audible output about characteristics of the image, wherein if the image contains a word, the audible output includes that pronounced word.
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Use of pregnane-diones or diols as neuropathic analgesic agents
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The present invention relates to the use of pregnanes in inducing analgesia, preferably without overt sedation, in a mammal in response to neuropathic pain, and compositions and kits therefore.
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1. A method of inducing analgesia in response to neuropathic pain in a mammal which comprises administering to the mammal an effective amount of a compound of formula I wherein R1 is H, OH, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl or —OR; R2 is H, OH, OR or ═O; R3 is H, OH or C1-C4 alkyl; R4 is H, OH, ═O, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl or —OR; R5 is H, OH, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl or —OR; R6 is H, OH, ═CH2 or C1-C4 alkyl; R7 is H, OH, halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl, SH, SR or —OR; and R is C1-C4 alkyl, C2-C2 alkenyl or C2-C4 alkanoyl; or a pharmaceutically acceptable derivative thereof. 2. A method according to claim 1 wherein R7 is OH, SH, OR or SR. 3. The method according to claim 1 wherein R1 is H, OH or methyl; R2 is OH; R3 is H or methyl; R4 is H, OH or ═O; R5 is H, OH or methyl; R6 is H or methyl; R7 is OH, OCOCH3, SH, SCOCH3, Cl, Br or F. 4. The method according to claim 2 wherein R1 is H, R2 is OH in alpha conformation, R3 is methyl and R7 is OH or OR. 5. The method according to claim 3 wherein R3 is methyl in alpha conformation. 6. The method according to claim 1 wherein R2 or R4 is ═O. 7. The method according to claim 1 wherein R2 and R7 are independently selected from OH and OR. 8. The method according to claim 1 wherein the compound according to formula I is alphadolone acetate. 9. The method according to claim 1 wherein the compound according to formula I is administered orally. 10. The method according to claim 1 wherein the compound according to formula I is administered intravenously, intramuscularly, intraperitoneally, intragastrically, intestinally, transdermally or intrathecally. 11. The method according to claim 1 wherein the neuropathic pain is selected from the group consisting of monoradiculopathies, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, complex regional pain syndromes, neuropathic pain associated with AIDS or infection with the human immunodeficiency virus and drug-induced and diabetic neuropathy. 12. The method according to claim 1 wherein the compound according to formula I is administered up to a maximum dose of about 2 grams/70 kg every 6 hours. 13. The method according to claim 1 wherein the mammal is a human. 14. A method of inducing analgesia, without overt sedation, in response to neuropathic pain in a mammal which comprises administering to the mammal an effective amount of a compound of formula I wherein R1 is H, OH, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl or —OR; R2 is H, OH, OR or ═O; R3 is H, OH or C1-C4 alkyl; R4 is H, OH, ═O, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl or —OR; R5 is H, OH, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl or —OR; R6 is H, OH, ═CH2 or C1-C4 alkyl; R7 is H, OH, halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl, SH, SR or —OR; and R is C1-C4 alkyl, C2-C2 alkenyl or C2-C4 alkanoyl; or a pharmaceutically acceptable derivative thereof. 15. A method according to claim 14 wherein R7 is OH, SH, OR or SR. 16. The method according to claim 14 wherein R1 is H, OH or methyl; R2 is OH; R3 is H or methyl; R4 is H, OH or ═O; R5 is H, OH or methyl; R6 is H or methyl; R7 is OH, OCOCH3, SH, SCOCH3, Cl, Br or F. 17. The method according to claim 16 wherein R1 is H, R2 is OH in alpha conformation, R3 is methyl and R7 is OH or OR. 18. The method according to claim 16 wherein R3 is methyl in alpha or beta conformation. 19. The method according to claim 14 wherein R2 or R4 is ═O. 20. The method according to claim 14 wherein R2 and R7 are independently selected from OH and OR. 21. The method according to claim 14 wherein the compound according to formula I is alphadolone acetate. 22. The method according to claim 14 wherein the compound according to formula I is administered orally. 23. The method according to claim 14 wherein the compound according to formula I is administered intravenously, intramuscularly, intraperitoneally, intragastrically, intestinally, transdermally or intrathecally. 24. The method according to claim 14 wherein the neuropathic pain is selected from the group consisting of monoradiculopathies, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, complex regional pain syndromes, neuropathic pain associated with AIDS or infection with the human immunodeficiency virus and drug-induced and diabetic neuropathy. 25. The method according to claim 14 wherein the compound according to formula I is administered up to a maximum dose of 2 grams/70 kg every 6 hours. 27. The method according to claim 14 wherein the mammal is a human. 27. A method of inducing analgesia in response to neuropathic pain in a mammal which comprises concurrently or sequentially administering to the mammal effective amounts of an analgesic compound and a compound of formula I or a pharmaceutically acceptable derivative thereof. wherein R1 is H, OH, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl or —OR; R2 is H, OH, OR or ═O; R3 is H, OH or C1-C4 alkyl; R4 is H, OH, ═O, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl or —OR; R5 is H, OH, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl or —OR; R6 is H, OH, ═CH2 or C1-C4 alkyl; R7 is H, OH, halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl, SH, SR or —OR; and R is C1-C4 alkyl, C2-C2 alkenyl or C2-C4 alkanoyl; or pharmaceutically acceptable derivatives thereof. 28. A method according to claim 27 wherein R7 is OH, SH, OR or SR. 29. The method according to claim 27 wherein R1 is H, OH or methyl; R2 is OH; R3 is H or methyl; R4 is H, OH or; R5 is H, OH or methyl; R6 is H or methyl; R7 is OH, OCOCH3, SH, SCOCH3, Cl, Br or F. 30. The method according to claim 29 wherein R1 is H, R2 is OH in alpha conformation, R3 is methyl and R7 is OH or OR. 31. The method according to claim 29 wherein R3 is methyl in alpha or beta conformation. 32. The method according to claim 27 wherein R2 or R4 is ═O. 33. The method according to claim 27 wherein R2 and R7 are independently selected from OH and OR. 34. The method according to claim 27 wherein the compound according to formula I is alphadolone acetate. 35. The method according to claim 27 wherein the analgesic compound is an opioid. 36. The method according to claim 35 wherein the opioid is selected from one or more of fentanyl, oxycodone, codeine, dihydrocodeine, dihydrocodeinone enol acetate, morphine, desomorphine, apomorphine, pethidine, methadone, dextropropoxyphene, pentazocine, dextromoramide, oxymorphone, hydromorphone, dihydromorphine, noscapine, papaverine, papaveretum, alfentanil, buprenorphine and tramadol pharmaceutically acceptable derivatives, salts, pro-drugs and/or tautomers thereof. 37. The method according to claim 36 wherein the opioid is morphine or a pharmaceutically acceptable salt thereof. 38. The method according to claim 36 wherein the opioid is oxycodone or a pharmaceutically acceptable salt thereof. 39. The method according to claim 36 wherein the opioid is fentanyl or a pharmaceutically acceptable salt thereof. 40. The method according to claim 27 wherein at least one of the compounds are administered orally. 41. The method according to claim 27 wherein at least one of the compounds are administered intravenously, intramuscularly, intraperitoneally, intragastrically, intestinally, transdermally or intrathecally. 42. The method according to claim 27 wherein the neuropathic pain is selected from the group consisting of monoradiculopathies, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, complex regional pain syndromes, neuropathic pain associated with AIDS or infection with the human immunodeficiency virus and drug-induced and diabetic neuropathy. 43. The method according to claim 27 wherein the mammal is a human. 44. The method according to claim 27 wherein the compound according to formula I or pharmaceutically acceptable derivative thereof is administered at a maximum dose of 2 grams/70 kg every six hours. 45. The method according to claim 27 which does not result in overt sedation. 46. The method according to claim 27 wherein the compound of formula I, or pharmaceutically acceptable derivative, and the opioid are administered in a synergistically effective amount. 47. A kit for inducing analgesia in response to neuropathic pain in a mammal which comprises an analgesic compound and a compound of formula I wherein R1 is H, OH, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl or —OR; R2 is H, OH, OR or ═O; R3 is H, OH or C1-C4 alkyl; R4 is H, OH, ═O, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl or —OR; R5 is H, OH, C1-C4 alky, C2-C4 alkenyl, C2-C4 alkanoyl or —OR; R6 is H, OH, ═CH2 or C1-C4 alkyl; R7 is H, OH, halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl, SH, SR or —OR; and R is C1-C4 alkyl, C2-C2 alkenyl or C2-C4 alkanoyl; or a pharmaceutically acceptable derivative thereof. 48. A composition for inducing analgesia, without overt sedation, in response to neuropathic pain in a mammal comprising a compound of formula I wherein R1 is H, OH, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl or —OR; R2 is H, OH, OR or ═O; R3 is H, OH or C1-C4 alkyl; R4 is H, OH, ═O, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl or —OR; R5 is H, OH, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl or —OR; R6 is H, OH, ═CH2 or C1-C4 alkyl; R7 is H, OH, halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkanoyl, SH, SR or —OR; and R is C1-C4 alkyl, C2-C2 alkenyl or C2-C4 alkanoyl; or a pharmaceutically acceptable derivative thereof, together with at least one pharmaceutically acceptable additive.
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<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention relates generally to the induction of analgesia in response to neuropathic pain. In considering the approaches to treatment of pain it is important to understand the distinction between acute and chronic pain. Acute pain occurs as a result of tissue injury or inflammation and is mediated by chemical, mechanical or thermal stimulation of pain receptors. In contrast, chronic pain in itself constitutes a disease which serves no protective biological function. Chronic pain is unrelenting and can persist for years after an initial injury. Chronic, non-malignant pain predominantly constitutes neuropathic pain which can be defined as pain initiated or caused by a primary lesion or dysfunction within the nervous system 1 . Neuropathic pain is associated with a variety of disease states and presents in the clinic with a range of symptoms 2 . Neuropathic pain is often reported as having a lancinating or continuous burning character and is often associated with the appearance of abnormal sensory signs such as allodynia and hyperalgesia Allodynia is defined as pain resulting from a stimulus which does not normally elicit a painful response and hyperalgesia is characterised by an increased pain response to a stimulus which is normally painful. Some disorders characterised by neuropathic pain include monoradiculopathies, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, complex regional pain syndromes and the various peripheral neuropathies. Whereas there are numerous effective therapies for acute pains caused by inflammatory processes or acute injury, especially including treatment with opioid and non-steroidal anti-inflammatory drugs (NSAIDs), neuropathic pain is an area of largely unmet therapeutic need. Due to the distinct pathophysiological mechanisms associated with neuropathic pain relative to inflammatory pains, agents useful in treatment of inflammatory and other pains have reduced effectiveness in neuropathic pain treatment. In particular, the effectiveness of opioids in treatment of neuropathic pain is diminished relative to inflammatory pain treatment and the dose response curve of opioids in neuropathic pain is shifted to the right of that for inflammatory pain 5 . The conventional pharmacological mainstays of clinical management of neuropathic pain are the tricyclic anti-depressants and certain anti-convulsants 3,4 , but even these achieve clinically significant pain relief (that is greater than 50% pain relief) in less than 50% of patients. These agents are also associated with significant side effect profiles. There is therefore a pressing need for improved treatment regimes for addressing the problem of neuropathic pain and it is in this context that the present invention has been conceived. Other objects of the present invention will become apparent from the following detailed description thereof
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<SOH> SUMMARY OF THE INVENTION <EOH>According to one embodiment of the present invention there is provided a method of inducing analgesia in response to neuropathic pain in a mammal which comprises administering to the mammal an effective amount of a compound of formula I wherein R 1 is H, OH, C 1 -C 4 alkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkanoyl or —OR; R 2 is H, OH, OR or ═O; R 3 is H, OH or C 1 -C 4 alkyl; R 4 is H, OH, ═O, C 1 -C 4 alkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkanoyl or —OR; R 5 is H, OH, C 1 -C 4 alkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkanoyl or —OR; R 6 is H, OH, ═CH 2 or C 1 -C 4 alkyl; R 7 is H, OH, halogen, C 1 -C 4 alkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkanoyl, SH, SR or —OR; and R is C 1 -C 4 alkyl, C 2 -C 2 alkenyl or C 2 -C 4 alkanoyl; or a pharmaceutically acceptable derivative thereof. According to another embodiment of the present invention there is provided a method of inducing analgesia, without overt sedation, in response to neuropathic pain in a mammal which comprises administering to the mammal an effective amount of a compound of formula I or a pharmaceutically acceptable derivative thereof. Another embodiment of the invention provides a composition for inducing analgesia, without overt sedation, in response to neuropathic pain in a mammal comprising a compound of formula I, or a pharmaceutically acceptable derivative thereof, together with at least one pharmaceutically acceptable additive. In a still further embodiment of the present invention there is provided a method of inducing analgesia in response to neuropathic pain in a mammal which comprises concurrently or sequentially administering to the mammal effective amounts of an analgesic compound, such as an opioid, and a compound of formula I or a pharmaceutically acceptable derivative thereof. Preferably the analgesic compound and compound of formula I, or pharmaceutically acceptable derivative thereof are administered in synergistically effective amounts. Preferably the method does not result in overt sedation. The invention also relates to the use of a compound of formula I, or a pharmaceutically acceptable derivative thereof, in the manufacture of a medicament for inducing analgesia, preferably without overt sedation, in response to neuropathic pain. In a still further embodiment of the present invention there is provided a kit for inducing analgesia in response to neuropathic pain in a mammal which comprises an analgesic compound, such as an opioid, and a compound of formula I or a pharmaceutically acceptable derivative thereof. For example, the analgesic compound may be an opioid selected from one or more of fentanyl, oxycodone, codeine, dihydrocodeine, dihydrocodeinone enol acetate, morphine, desomorphine, apomorphine, pethidine, methadone, dextropropoxyphene, pentazocine, dextromoramide, oxymorphone, hydromorphone, dihydromorphine, noscapine, papaverine, papaveretum, alfentanil, buprenorphine and tramadol and pharmaceutically acceptable derivatives thereof. In a preferred embodiment, R 7 is OH, OR, SH, SR or halogen, more preferably OH, OR, SH or SR. In another preferred embodiment R 2 is OH or OR, more preferably in the α-conformation. Preferred compounds of formula I are those wherein R 1 is H, OH or methyl; R 2 is OH; R 3 is H or methyl; R 4 is H, OH or ═O; R 5 is H, OH or methyl; R 6 is H or methyl; R 7 is OH, OC 2-4 alkanoyl (such as OCOCH 3 ), SH, SCOCH 3 , Cl, Br or F. In another preferred embodiment R 1 is H, R 2 is OH in alpha conformation, R 3 is methyl (in alpha or beta conformation) and R 7 is OH or OR. In another preferred embodiment, the compound of formula I is a pregnane-dione, ie R 2 or R 4 is ═O. In a particularly preferred embodiment of the invention, the compound according to formula I is alphadolone acetate. The compounds according to the invention may be administered, inter alia, orally, intravenously, intramuscularly, intraperitoneally, intragastrically, intrathecally. transdermally or intestinally. In a particularly preferred form, the compounds are administered orally. Preferably the compound according to formula I is administered up to a maximum dose of about 2 grams/70 kg every 6 hours. In a particularly preferred embodiment of the invention the mammal is a human.
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Method and apparatus for generating hydrogen gas
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A method of generating hydrogen gas is disclosed. The method includes a first step of contacting an aqueous solution of a chemical hydride and a catalyst and producing hydrogen gas and a heated hydrogen-depleted solution. The hydrogen gas is recovered and used as required, for example in a fuel cell. The heated solution is brought into direct or indirect heat exchange relationship with a metal hydride, thereby heating the metal hydride and causing desorption of hydrogen from the metal hydride and producing hydrogen gas and cooling the heated solution and producing a cooled solution. The hydrogen gas is recovered and used as required.
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1. A method of generating hydrogen gas which includes the steps of: (a) contacting an aqueous solution of a chemical hydride and a catalyst and producing hydrogen gas and a heated hydrogen-depleted solution; (b) recovering hydrogen gas produced in step (a); (c) bringing the heated solution produced in step (a) into direct or indirect heat exchange relationship with a metal hydride and heating the metal hydride and causing desorption of hydrogen from the metal hydride and producing hydrogen gas and cooling the heated solution and producing a cooled solution; and (d) recovering hydrogen gas produced in step (c). 2. The method defined in claim 1 further including contacting the cooled solution produced in step (c) with a metal and producing hydrogen gas and recovering the hydrogen gas in situations in which the cooled solution is alkaline. 3. The method defined in claim 2 wherein the metal is aluminium. 4. The method defined in claim 1 further including treating the cooled solution produced in step (c) to regenerate the chemical hydride. 5. The method defined in claim 1 further including treating the cooled solution produced in step (c) to regenerate the chemical hydride by electrolysis of the cooled solution in an electrolytic cell that contains an ionic liquid as an electrolyte. 6. The method defined in claim 5 wherein electrolysis is indirect electrolysis with the cooled solution and the electrolyte being in separate compartments of the electrolytic cell and being separated by a barrier that is selectively permeable to ions that can form the chemical hydride, whereby the ions migrate from the compartment containing the cooled solution into the compartment containing the ionic liquid in response to an applied potential. 7. The method defined in claim 6 wherein the chemical hydride or a precursor of the chemical hydride form as a gas or as an insoluble compound in the ionic liquid. 8. The method defined in claim 7 further includes extracting the chemical hydride or precursor from the ionic liquid. 9. The method defined in claim 1 wherein step (c) includes heating the metal hydride by at least 30° C. 10. The method defined in claim 9 wherein step (c) includes heating the metal hydride by at least 40° C. 11. The method defined in claim 10 wherein step (c) includes heating the metal hydride by at least 50° C. 12. The method defined in claim 1 wherein the chemical hydride includes any one or more of lithium hydride, lithium aluminium hydride, sodium borohydride, and dimethyl borane. 13. The method defined in claim 1 wherein the chemical hydride is sodium borohydride. 14. The method defined in claim 1 wherein the aqueous solution of the chemical hydride supplied to step (a) is in the form of a slurry that includes a suspension of chemical hydride particles in water that contains the chemical hydride in solution. 15. The method defined in claim 1 wherein the metal hydride includes any one or more of iron titanium hydride and lanthanum nickel hydride. 16. A hydrogen gas generator comprising: (a) a chemical hydride reactor for allowing contact between an aqueous solution of a chemical hydride and a catalyst and producing hydrogen gas and a heated hydrogen-depleted solution; (b) a metal hydride reactor for generating hydrogen gas by heating metal hydride by direct or indirect heat exchange with heated hydrogen-depleted solution from the chemical hydride reactor and causing desorption of hydrogen from the metal hydride and generating hydrogen gas and producing a cooled solution; and (c) a means for transferring heated solution from the chemical hydride reactor to the metal hydride reactor. 17. The generator defined in claim 16 further comprising means for regenerating the chemical hydride from the cooled solution produced in the metal hydride reactor. 18. The generator defined in claim 17 wherein the means for regenerating the chemical hydride includes an electrolytic cell. 19. The generator defined in claim 18 wherein the electrolytic cell includes two compartments separated by a barrier that is selectively permeable to ions that can form the chemical hydride. 20. A system for generating electricity comprising a fuel cell and a hydrogen gas generator comprising: (a) a chemical hydride reactor for allowing contact between an aqueous solution of a chemical hydride and a catalyst and producing hydrogen gas and a heated hydrogen-depleted solution; (b) a metal hydride reactor for generating hydrogen gas by heating metal hydride by direct or indirect heat exchange with heated hydrogen-depleted solution from the chemical hydride reactor and causing desorption of hydrogen from the metal hydride and generating hydrogen gas and producing a cooled solution; and (c) a means for transferring heated solution from the chemical hydride reactor to the metal hydride reactor. 21. An electric-powered motor vehicle which comprising a system for generating electricity, said electricity generating system comprising a fuel cell and a hydrogen gas generator, wherein said hydrogen gas generator comprises: (a) a chemical hydride reactor for allowing contact between an aqueous solution of a chemical hydride and a catalyst and producing hydrogen gas and a heated hydrogen-depleted solution; (b) a metal hydride reactor for generating hydrogen gas by heating metal hydride by direct or indirect heat exchange with heated hydrogen-depleted solution from the chemical hydride reactor and causing desorption of hydrogen from the metal hydride and generating hydrogen gas and producing a cooled solution; and (c) a means for transferring heated solution from the chemical hydride reactor to the metal hydride reactor and a means for controlling the relative amounts of hydrogen gas generated by the chemical hydride reactor and the metal hydride reactor of said system.
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Valve seal
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A valve gasket for sealing a fluid dispenser device comprising a valve, in particular a metering valve, mounted on a fluid reservoir, said gasket being characterized in that it comprises an alloy of vulcanized ethylene vinyl acetate (EVA) and/or of butyl (IIR) or of vulcanized halobutyl (CIIR or BIIR) with one or more materials selected from the group constituted by polyoctene ethylene (POE), ethylene propylene (EP), ethylene propylene diene monomer (EPDM) and chloroprene rubber (CR), all of these materials being vulcanized.
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1. A valve gasket for sealing a fluid dispenser device comprising a valve, in particular a metering valve, mounted on a fluid reservoir, said gasket being characterized in that it comprises an alloy of vulcanized ethylene vinyl acetate (EVA) and/or of butyl (IIR) or of vulcanized halobutyl (CIIR or BIIR) with one or more materials selected from the group constituted by polyoctene ethylene (POE), ethylene propylene (EP) ethyl propylene diene monomer (EPDM) and chloroprene rubber (CR), all of these materials being vulcanized. 2. A gasket according to claim 1, in which said gasket comprises ethylene propylene (EP) or ethylene propylene diene monomer (EPDM). 3. A gasket according to claim 1, in which said gasket comprises polyoctene ethylene (POE). 4. A gasket according to claim 1, in which said gasket comprises one or more other ingredients such as inorganic fillers and/or carbon black fillers and/or vulcanization agents and/or dyes and/or processing agents and/or plasticizers. 5. A fluid dispenser device comprising a valve, in particular a metering valve, provided with a moving valve member, said valve being mounted on a reservoir containing the fluid and a propellant, the device including at least one neck gasket between the valve and the reservoir, and at least one dynamic gasket in which said valve member slides, said dispenser device being characterized in that at least one of said gaskets is made in accordance with claim. 6. A fluid dispenser according to claim 5, in which the propellant comprises an HFC-134a or HFC-227 gas, with or without alcohol.
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Nonaqueous electrolyte
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Disclosed is a nonaqueous electrolyte for a lithium-ion secondary battery that contains an addictive for preventing destruction of a solid electrolyte interface (SEI) layer and hence decomposition of the electrolyte that may cause generation of a gas, thereby preventing swelling of the battery caused by the gas generated from the inside of the battery at a high temperature. More specifically, the present invention provides an electrolyte that includes, as an additive, 2-sulfobenzoic acid cyclic anhydride represented by the formula 1: The electrolyte contains the additive in an amount of 0.1 to 10.0 wt. %.
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1. A nonaqueous electrolyte for a lithium-ion secondary battery which uses at least one of LiCoO2, LiMnO2, LiMn2O4, LiNiO2, or a composite compound, LiM1xM2yO2 as an anodic active material, and crystalline or amorphous carbon or lithium as a cathodic active material, wherein M1 and M2 are a metal element; and x and y are a rational number from 0 to 2, the electrolyte comprising: a mixed solvent comprising at least one carbonate solvent, as a solvent; at least one lithium salt selected from LiPF6, LiBF4, LiClO4, LiN(SO2CF3)2 and LiN(SO2CF2CF3)2, as an electrolytic salt; and 2-sulfobenzoic acid cyclic anhydride as an additive, represented by the formula 1: 2. The electrolyte as claimed in claim 1, wherein the 2-sulfobenzoic acid cyclic anhydride as the additive is used in an amount of 0.1 to 10.0 wt. %. 3. The electrolyte as claimed in claim 1, wherein the lithium salt as an electrolytic salt has a concentration of 0.5 to 2.0 M.
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<SOH> BACKGROUND ART <EOH>The battery refers to a device for converting chemical energy generated from an electrochemical oxidation-reduction reaction of chemicals, which are contained in the battery, to electrical energy. According to its characteristic in use, the battery is classified into a primary battery that has to be disposed when the energy in the battery is used up, and a secondary battery that is rechargeable. With a rapid progress of electronic, communication and computer industries, the related equipment has recently been miniaturized with lightweight and high performance and portable electronic appliances such as camcorder, mobile phone, notebook personal computer, etc. have been in popular use. There is thus a need for high performance lightweight and small-sized batteries that have a long use life with high reliability. In regard to this requirement, a lithium-ion battery is the very promising secondary battery. Lithium (Li) is the most lightweight metal existing in the earth that has the highest electrical capacity per unit mass and a high thermodynamic oxidation potential, and can be used as a material for a high-voltage battery. Lithium is a positive electrode material most preferred for a battery that has to generate the maximum energy from a limited amount of chemicals, especially, for a secondary battery. The lithium-ion secondary battery is composed of a lithium-mixed oxide as an anodic active material capable of deintercalation and intercalation of lithium ions, a carbon material or metal lithium as a cathode, and an electrolyte containing an adequate amount of a lithium electrolytic salt dissolved in a mixed organic solvent. Such a lithium-ion secondary battery has a high energy density, which is about 200% of a nickel-cadmium (Ni—Cd) battery and about 160% of a nickel-hydrogen (Ni—H) battery per weight, and about 170% of the Ni—Cd battery and about 105% of the Ni—H battery per density, and a low self-discharge rate of less than 5% per month at 20° C., which is no more than about ⅓ of the Ni—Cd or Ni—H battery. The lithium-ion secondary battery is also an environment-friendly battery that is free from heavy metals such as cadmium (Cd) or mercury (Hg) causing environmental contamination and rechargeable at least 500 times in a normal state so as to have a long use life. Also, the lithium-ion secondary battery has an average discharge voltage of 3.6 to 3.7 V, which is advantageous to provide a very high power relative to another alkali batteries or Ni—MH or Ni—Cd batteries. To generate such a high driving voltage, the lithium-ion secondary battery needs an electrolyte that is electrochemically stable in the charge/discharge voltage range of the lithium-ion battery, i.e., 2.75 to 4.2 V. Examples of such an electrolyte include a nonaqueous mixed solvent composed of mixed carbonates such as ethylene carbonate (EC), propylene carbonate (PC), dimethyl carbonate (DMC), ethylmethyl carbonate (EMC) or diethyl carbonate (DEC). The electrolyte of such a composition is disadvantageous to have a high charge/discharge rate because of its strikingly low ion conductance relative to aqueous electrolytes used in Ni—MH or Ni—Cd battery. For example, EC has a melting point of 36° C. with a disadvantage in low-temperature characteristic but is necessary for an electrical performance of the battery; PC is susceptible to decomposition during a charge/discharge of the battery in an artificial graphite most preferred as a negative electrode material; DMC that has a melting point of 3° C. and a boiling point of 90° C. is inferior in low-temperature characteristic as EC and high-temperature storage characteristic; DEC that has a melting point of less than −40° C. and a boiling point of about 126° C. is superior in performance but has a poor mixability with other solvents; EMC that has a melting point of less than 30° C. and a boiling point of about 107° C. is a most popular solvent but still unsatisfactory in temperature characteristic or the like. These solvents have their own advantages and disadvantages and a combination of the solvents in an actual use makes a great difference in the performance of the battery. So, many attempts have been made to find out an optimized combination of the solvents to have a high performance and now EC/DMC/EMC, EC/EMC/DEC and EC/DMC/EMCIPC are widely used in the battery industry. The high-temperature characteristic of the lithium-ion secondary battery is dependent on the type of an electrolytic salt. LiPF 6 , LiBF 4 , LiCIO 4 , LiN(SO 2 CF 3 ) 2 and LiN(SO 2 CF 2 CF 3 ) 2 , which are generally used as a solute of the electrolyte in the lithium-ion secondary battery, act as a source of lithium ions in the battery cell to enable the basic operation of the lithium-ion secondary battery. Among these electrolytic salts, LiBF 4 is known to be superior in thermal stability at high temperature. The electrolyte for lithium-ion secondary battery that is composed of a solvent and an electrolytic salt reacts with carbon constituting the cathode to form a thin layer called SEI (Solid Electrolyte Interface) on the surface of the cathode. The SEI layer is an important factor that affects the migration of ions and charges to cause a change in the performance of the battery, and its properties are greatly dependent on the type of the solvents used in the electrolyte and the properties of the additives (See. Shoichiro Mori, Chemical properties of various organic electrolytes for lithium rechargeable batteries, J. Power Source 68 (1997)). Now, a description will be given in further detail to the SEI layer formed on the surface of the cathode due to a reaction of the electrolyte and carbon that constitute the cathode. During the initial charge of the lithium-ion secondary battery, lithium ions generated from the lithium oxide used as the positive electrode material migrate to carbon (crystalline or amorphous) electrode used as the negative electrode material and intercalated into the carbon electrode of the negative electrode material. At this time the ion, since it has high reactivity, reacts with the carbon to produce Li 2 CO 3 , Li 2 O and LiOH, which form a so-called SEI layer on the surface of the negative electrode material. Once formed during the initial charge of the battery, the SEI layer prevents a reaction of lithium ions and the carbon negative electrode or another material during a repeated charge/discharge of the battery and acts as an ion tunnel that lets only the lithium ions pass between the electrolyte and the negative electrode. Such an ion tunnel effect of the SEI blocks the migration of organic solvents, having a large molecular weight in which is dissolved the Lithium ion and flown together in the electrolyte, such as EC, DMC or DEC to the carbon negative electrode and the cointercalation of the organic solvents together with the lithium ions into the carbon negative electrode, thereby preventing the destruction of the carbon negative electrode. Thus the SEI layer reversibly maintains the amount of lithium ions during the subsequent charge/discharge of the battery by preventing a side reaction of the lithium ions and the carbon negative electrode or another material. More specifically, the carbonaceous material of the negative electrode reacts with the electrolyte during the initial charge of the battery to form a passivation layer on the surface of the negative electrode and maintains a stable charge/discharge without further decomposition of the electrolyte (See. J. Power Sources, 51 (1994) 79-104). The quantity of electric charge used in formation of the passivation layer on the surface of the negative electrode is an irreversible volume that hardly involves in reversible reaction during the discharge of the battery. As such, the lithium-ion battery can maintain a stable life cycle without any irreversible reaction after the initial charge. When the lithium-ion battery is stored at a high temperature in a fully charged state (for example, at 85° C. for 4 days after a 100% charge with 4.2 V), the electrochemical and heat energies increased with an elapse of time gradually destroy the SEI layer. In this case, the carbonate solvent in the electrolyte reacts with the surface of the negative electrode exposed due to the destruction of the SEI layer, causing a continuous side reaction. Such a side reaction continuously generates a gas, such as CO, CO 2 , CH 4 or C 2 H 6 , which depends on the types of the carbonate and the active material (See. J. Power Sources, 72 (1998) 66-70). The continuous generation of the gas irrespective of the type causes an increase in the internal pressure of the lithium-ion battery at a high temperature and swells the battery. The formation of the SEI layer is dependent on the characteristic of an additive as well as the type of the electrolyte. When using no additive or an additive poor in characteristic, a non-uniform SEI layer is formed to cause the above-mentioned decomposition of the electrolyte, increasing the irreversible quantity of the active material of the battery and swelling the battery with generation of a gas at a high temperature. This disables set installation of the battery in equipment or apparatuses to reduce the capacity and use life of the battery and make realization of a lightweight battery impossible. In regard to the function of the SEI layer, many studies have been made to enhance the physicochemical characteristics of the electrolyte by using a high-performance additive to the electrolyte and changing the reaction related to the formation of the SEI layer. For example, some documents disclose an addition of CO 2 to the electrolyte (Japanese Patent Laid-open No. 95-176323A), or an addition of a sulfide compound to the electrolyte (Japanese Patent Laid-open No. 95-320779A) to prevent decomposition of the electrolyte. However, these studies cannot completely solve the problem related to destruction of the SEI layer at a high temperature in the lithium-ion battery. There is thus a need for an additive for forming a high-performance uniform SEI layer and reducing destruction of the SEI layer at a high temperature.
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Method and device for operating a fluorescent tube in an energy saving manner
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The invention relates to an energy-saving operating method and an apparatus for energy-saving operation of a fluorescent tube, especially a T5 fluorescent tube. In a first operating mode, heat current is applied to an incandescent filament at one end of the fluorescent tube. Moreover, in the first operating mode, another heat current is applied to another incandescent filament at an end of the fluorescent tube opposite to said one end. The other incandescent filament is connected to energy-saving circuitry. In a second operating mode, the application of the heat current to the incandescent filament and of the other heat current and the other incandescent filament is interrupted. Monitoring means are provided, which are comprised by electronic circuitry, to monitor an operating parameter of the other incandescent filament in the first and second operating modes. The electronic circuitry controls a time period of application of the other heat current to the other incandescent filament in dependence on a time period of application of the heat current to the incandescent filament, in response to the operating parameter being monitored.
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1. An energy-saving operating method for a fluorescent tube, the method comprising the following steps: applying heat current to an incandescent filament at one end of the fluorescent tube, in a first operating mode, the incandescent filament being connected to electronic energy-saving circuitry; applying another heat current to another incandescent filament at an end opposite to said one end of the fluorescent tube, the other incandescent filament being connected to electronic circuitry which is separate from the electronic energy-saving circuitry; and interrupting the application of the heat current and the other heat current to the incandescent filament and the other incandescent filament, respectively, in a second operating mode; wherein an operating parameter of the other incandescent filament is monitored, in the first and second operating modes, by monitoring means which are comprised by the electronic circuitry so as to control a time period of application of the other heat current to the other incandescent filament in dependence on a time period of application of the heat current to the incandescent filament by means of the electronic circuitry in response to the operating parameter being monitored. 2. The method as claimed in claim 1, wherein the operating parameter being monitored of the other incandescent filament is a maintaining voltage dependent on frequency at the other incandescent filament. 3. The method as claimed in claim 2, wherein a voltage dependent on frequency and induced in a resonant circuit is utilized for monitoring the frequency of the maintaining voltage. 4. The method as claimed in any claim 1, wherein in the first operating mode, the fluorescent tube is operated in a dimming mode. 5. An apparatus for energy-saving operation of a fluorescent tube, especially a T5 fluorescent tube, comprising: electronic energy-saving circuitry adapted to be coupled to an incandescent filament at one end of the fluorescent tube to control application of heat current to the incandescent filament; and electronic circuitry, separate from the electronic energy-saving circuitry, adapted to be coupled to another incandescent filament at an end opposite to said one end of the fluorescent tube to control application of another heat current to said other incandescent filament; the electronic circuitry comprising monitoring means to monitor an operating parameter of said other incandescent filament so that switch-on/switch-off of the application of the other heat current to the other incandescent filament can be controlled in dependence on the switch-off/switch-on of the application of the heat current to the incandescent filament by means of the electronic circuitry in response to the operating parameter being monitored. 6. The apparatus as claimed in clam 5, wherein the monitoring means comprise means for monitoring a frequency of a maintaining voltage applied to the other incandescent filament. 7. The apparatus as claimed in claim 6, wherein the means for monitoring the frequency of the maintaining voltage applied to the other incandescent filament comprise a resonant circuit. 8. An apparatus for coupling to an incandescent filament of a fluorescent tube, comprising electronic circuitry for controlling the application of heat current to the incandescent filament in dependence on the operating mode, the electronic circuitry comprising monitoring means to monitor a frequency of a maintaining voltage at the incandescent filament so that the application of heat current to the incandescent filament can be switched on in a first mode of operation and interrupted in a second mode of operation by means of the electronic circuitry in response to the frequency being monitored. 9. (Canceled)
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Process for the isolation of sterols and/or wax alcohols from tall oil products
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The invention relates to a process for recovering sterols and/or wax alcohols from a crude tall oil based source material comprising sterols and/or wax alcohols in esterified form and fatty and/or rosin acids and optionally sterols and/or wax alcohols in free form, said method comprising the steps of: a) converting free acids in the source material to corresponding salts, b) removing water if present, c) transesterifying the esterified sterols and/or wax alcohols present in the dry material obtained in step a or step b to liberate sterols and/or wax alcohols, d) evaporative fractionating the transesterified material, and e) isolating sterols and/or wax alcohols from the obtained fraction(s) and/or the residue.
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1. A process for recovering sterols and/or wax alcohols from a crude tall oil based source material comprising sterols and/or wax alcohols in esterified from and fatty and/or rosin acids and optionally sterols and/or wax alcohols in free form, said method comprising the steps of: a) converting free acids in the source material to corresponding salts, b) removing water is present, c) transesterifying the esterified sterols and/or wax alcohols present in the dry material obtained in step a or step b to liberate sterols and/or wax alcohols, d) evaporative fractioning the transesterified material, and e) isolating sterols and/or wax alcohols from the obtained fraction(s) and/or the residue. 2. The process according to claim 1, wherein the conversion step is performed using an oxide, hydroxide or alkoxide of an alkali metal or alkaline earth metal. 3. The process according to claim 1, wherein the transesterification step c is performed using as catalyst an alkoxide or hydroxide of an alkali metal. 4. The process according to claim 1, wherein the transesterification step c is performed using a lower alcohol as alcohol reagent. 5. The process according to claim 1 wherein the transesterification step c is performed using a high boiling polyhydric alcohol such as ethylene glycol, propylene glycol, glycerol of diethylene glycol as alcohol reagent. 6. The process according to claim 1, wherein the fractionation step d includes the steps of d1) evaporation of excess of lower alcohol reagent partly or completely, and d2) evaporation fractionation of the transesterifled material. 7. The process according to claim 1, wherein after transesterification step c the organic salts formed during conversion step a are completely or partially acidulated to corresponding acids before performing fractionation step d. 8. The process according to claim 1, wherein free acids present in the source material are partially esterified before performing conversion step a. 9. The process according to claim 8 wherein the esterification is performed using a lower alcohol or a high boiling polyhydric alcohol such as ethylene glycol, propylene glycol, glycerol or diethylene glycol as alcohol reagent. 10. The process according to claim 1 wherein step a and step c are performed in the same stage under anhydrous conditions using an alkoxide of an alkali metal or alkaline earth metal such as sodium methoxide as reagent in the conversion step a and as catalyst in the transesterification step c.
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