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Method for detecting mutagenic substances |
The invention relates to a method and means for the rapid identification of mutations. The test strains used in the invention contain a reporter system and a selection marker. An antibiotic resistance protein which after mutation allows the selective growth of the revertant and the targeted induction of the inducible reporter system of the developing revertants acts as the selection marker. Proteins that are capable of directly or indirectly triggering a measuring signal are used as the reporters. The mutations are identified by means of the reporter signal of the developing revertants, in such a way that the evaluation can take place after only a few hours. |
1. Isolated prokaryotic test strain for the detection of mutagens, at least comprising a) a revertible mutated selection marker in the form of a resistance gene to bacteriotoxic or bacteriostatic substances or influences; b) a reporter system whose expression can be detected selectively in reverted bacteria. 2. Test strain according to claim 1, characterised in that the selection marker simultaneously also serves as reporter system. 3. Test strain according to claim 1, characterised in that the selection marker is an antibiotic resistance gene to an antibiotic which has a bacteriolytic action or inhibits protein biosynthesis. 4. Test strain according to claim 1 one or more of claims 1 to 3, characterised in that the selection marker is a tetracyclin resistance gene, an ampicillin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene or a streptomycin resistance gene. 5. Test strain according to claim 1, characterised in that the reporter system, on expression, leads directly or indirectly to a colour reaction, a luminescent signal or a fluorescent signal. 6. Test strain according to claim 1, characterised in that the reporter system used is the β-galactosidase, β-lactamase or luciferase gene or operon. 7. Test strain according to claim 1, additionally having one or more of the following properties: a) a cell wall which is permeable to large, lipophilic molecules; b) a defective excision repair; c) a functioning regulation unit of the SOS system; d) the mutator genes umuDC and/or mucAB. 8. Method for the detection of mutagens, essentially characterised by the following steps: a) provision of at least one test strain corresponding to claim 1; b) incubation of the test strain with the potential mutagen; c) selection of the revertants; d) if desired induction of the reporter system; e) detection of the gene product of the reporter system and/or detection of the growth of the reverted strains. 9. Method according to claim 8, characterised in that in step e), the detection of the gene product of the selection marker takes place as detection of the gene product of the reporter system. 10. Method according to claim 8 or 9, characterised in that the detection of the growth in step e) is carried out by means of a pH indicator in the medium. 11. Test kit for carrying out mutation tests, essentially containing one or more test strains corresponding to claim 1. 12. Use of at least one test strain according to claim 1 for HTP screening. |
Electrosynthesis of organic compounds |
Disclosed is a process for the electrochemical transformation of a compound to form a product, the process comprising (i) effecting the transformation in the presence of an electrolyte comprising at least one room temperature ionic liquid, wherein the ionic liquid is air-stable and moisture-stable, (ii) recovering the product, and optionally (iii) recovering the ionic liquid. The process can be used to effect the electrochemical transformation of a wide range of organic compounds. |
1. A process for the electrochemical transformation of a compound to form a product, said process comprising the steps of: (i) effecting said transformation in the presence of an electrolyte comprising at least one room temperature ionic liquid, said ionic liquid being air-stable and moisture-stable, and (ii) recovering the product. 2. A process according to claim 1 further comprising the recovery of said ionic liquid. 3. A process according to claim 1 wherein the electrochemical transformation is an oxidation, reduction, or a coupled pair of oxidation and reduction. 4. A process according to claim 1 wherein the compound includes at least one structural element selected from the following: a carbon-halogen bond, a C═C double bond, a C≡C triple bond, an ester group, an ether group, a carboxylic acid group, an amino group, an amido group, an imido group, —OH, —NO2, —C≡N, an aldehyde group and a keto group, and wherein said structural element is oxidized or reduced. 5. canceled 6. A process according to claim 1 wherein the compound includes at least one structural element selected from the following: NO2, an imido group, an aldehyde group and a carboxylic acid group. 7. A process according to claim 1 wherein the ionic liquid comprises an imidazolium, pyridinium, pyridazinium, pyrazinium, oxazolium, triazolium, pyrazolium, pyrrolidinium, piperidinium, tetraalkylammonium or tetraalkylphosphonium salt. 8. canceled 9. A process according to claim 1 wherein the ionic liquid is an air- and moisture stable room temperature ionic liquid selected from a compound of formula: wherein each Ra is independently selected from a C1 to C40 straight chain or branched alkyl or a C3 to C8 cycloalkyl group, wherein said alkyl or cycloalkyl group which may be substituted by one to three groups selected from: C1 to C6 alkoxy, C6 to C10 aryl, CN, OH, NO2, C1 to C30 aralkyl and C1 to C30 alkaryl; each Rb, Rc, Rd, Re, Rf, Rg and Rh can be the same or different and are each independently selected from H or any of the Ra groups as defined above; and [A]n− represents an anion having a charge n−; wherein n may be 1-3. 10-12. canceled 13. A process according to claim 1 wherein the ionic liquid is an air- and moisture stable room temperature ionic liquid selected from a compound of formula: wherein [A]n−, Ra, Rg and n are as defined in claim 7. 14. A process according to claim 7 wherein each Ra represents C1 to C40 straight chain or branched alkyl. 15. canceled 16. A process according to claim 7 wherein each Ra represents C1 to C8 straight chain or branched alkyl. 17. A process according to claim 7 wherein Rg and Rh represents C1 to C40 straight chain or branched alkyl. 18. canceled 19. A process according to claim 7 wherein Rg and Rh represents C1 to C8 straight chain or branched alkyl. 20. A process according to claim 7 wherein Rb, Rc, Rd, Re, Rf, Rg and Rh each represents hydrogen. 21. A process according to claim 7 wherein Ra, Rg and Rh each represents a C1-C20 alkyl group. 22. A process according to claim 7 wherein [A]n− represents a single species of anion having valency n. 23. A process according to claim 7 wherein n is 1. 24. A process according to claim 7 wherein [A]n− represents an anion selected from [C]l−, [Br]−, [I]−, boron or phosphorus fluorides, [NO3]−, [SO4]2−, [HSO4]−, [HCO3]−, [(CF3SO2)2N]−, [AsF6]−, [SbF6]−, aryl sulfonates, alkylsulfonates, mono- or difluorinated alkyl sulfonates including perfluorinated alkylsulfonates, carboxylic acid anions, fluorinated carboxylic acid anions and metal halides. 25. canceled 26. A process according to claim 7 wherein [A]n− represents an anion selected from [PF6]−, [BF4]−, [OSO2CF3]−, [OSO2(CF2)3CF3]−, [(CF3SO2)3C]−, [CH3CH2SO3]−, [OCO2CF3]−, [OCO2(CF2)3CF3]−, [OCO2CH3]−, nitrate, sulfate, hydrogensulfate, hydrogencarbonate, acetate, trifluoroacetate, lactate, [(CF3SO2)2N]−, [B(alkyl)4]− wherein each alkyl can be the same or different and can be any straight chain or branched C1 to C10 alkyl group, [SbF6]− and [AsF6]−. 27. A process according to claim 7 wherein [A]n− represents an anion selected from [PF6]−, [BF4]− and [(CF3SO2)2N]−. 28. A process according to claim 1 wherein the ionic liquid is 1-butyl-3-methylimidazolium hexafluorophosphate or N-butyl-N-methyl-pyrrolidinium bis(trifluoromethanesulfonyl)imide. 29. A process according to claim 7 wherein the ionic liquid has a melting point of up to 40° C. 30. canceled 31. A process according to claim 7 wherein the ionic liquid is substantially immiscible with water. 32. A process according to claim 1 wherein the electrochemical transformation is carried out in the presence of a proton source. 33. A process according to claim 32 wherein the proton source is selected from phenol, a mineral acid, an organic acid, a conjugate acid of the anion A, or water. 34. canceled 35. A process according to claim 32 wherein the proton source is phenol. 36. A process according to claim 1 wherein the electrochemical transformation is conducted at a temperature within the molten range of the electrolyte. 37. A process according to claim 1 wherein at least one product formed by the transformation is separated from the ionic liquid by solvent extraction, distillation, precipitation or as an immiscible liquid layer. 38. A process according to claim 1 wherein the ionic liquid is reusable after recovery of the product. 39. A process according to claim 1 wherein the ionic liquid is reusable directly after recovery of the product. 40. Use of an air- and moisture-stable room temperature ionic liquid electrolyte system as defined in claim 8 for carrying out an electrochemical transformation. 41. A process for the reduction of N-methylphthalimide wherein the reaction medium is 1-butyl-3-methylimidazolium hexafluorophosphate. |
Compositions comprising thylakoids useful in the modulation of inflammation process |
This invention relates to the use of a thylakoid extract that is preferably stabilized and activable for treating inflammation. Different types of cell or tissue targets and inflammatory stimuli have been used to evaluate the performance of the extract, which, in all cases successfully modulate inflammation through a balance of pro/anti-inflammatory cytokines. Compositions comprising the extract and other anti-inflammatory agents, namely glucocorticoids or NSAIDs are further disclosed and claimed. |
1. The use of a thylakoid extract in combination with an anti-inflammation agent in the making of an anti-inflammation medication for modulating the relative amounts of pro-inflammatory and anti-inflammatory cytokines induced during inflammation in a subject, said thylakoid extract comprising purified finctional photosynthetic pigments in their thylakoid membrane environment. 2. The use of claim 1, wherein said pro-inflammatory cytokines comprise TNF-α. 3. The use of claim 1, wherein said anti-inflammatory cytokines comprise interleukine-10. 4. The use of claim 1, wherein the relative amount of anti-inflammatory cytokines is higher. 5. The use of claim 1, wherein said anti-inflammatory agent is a glucocorticoid. 6. The use of claim 1, wherein said anti-inflammatory agent is a non-steroidal anti-inflammatory drug (NSAID). 7. The use of claim 1, wherein the extract is present in an amount achieving about 0.00005 to 500 mg per Kg of subject's body weight. 8. The use of claim 1, wherein the extract is present I an amount achieving about 0.05 to 5 mg per Kg of subject's body weight. 9. The use of claim 1, wherein the medication is topical. 10. The use of claim 9, wherein said medication comprises is about 0.1 μg to 1 mg of extract per cm2 of surface of subject's skin or mucosae. 11. The use of claim 9, wherein said medication comprises about 1 μg of extract per cm2 of surface of subject's skin or mucosae. 12. A composition to inhibit the expression of inflammation in a subject, comprising an effective amount of an anti-inflammatory agent and effective amount of a thylakoid extract comprising purified functional photosynthetic pigments in their thylakoid membrane environment. 13. The composition of claim 12, wherein said anti-inflammatory agent is a glucocorticoid. 14. The composition of claim 12, wherein said anti-inflammatory agent is a non-steroidal anti-inflammatory drug (NSAID). 15. The composition of claim 12, which is topical. 16. The composition of claim 15, wherein said effective amount of extract is about 0.1 μg to 1 mg per cm2 of surface of subject's skin or mucosae. 17. The composition of claim 15, wherein said effective amount of extract is about 1 μg to 200 μg per cm2 of subject's surface of skin or mucosae. 18. The composition of claim 12, wherein said effective amount of extract is about 0.00005 to 500 mg per Kg of subject's body weight. 19. The composition of claim 12, wherein said effective amount of extract is about 0.05 to 5 mg per Kg of subject's body weight. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Inflammation is a process well known for its implication in acute and chronic diseases and disorders in the biomedical field. Although inflammation is a natural process associated with cell and tissue defense and regeneration, disorganized inflammation can contribute to (or is implicated in) many processes that are harmful to cells and tissues. Inflammation is the body's reaction to infectious agents, antigen challenge or physical, chemical or traumatic injury (Stvrtinova et al., 1995). The main purpose of inflammation is to bring fluids, proteins, and cells from the blood into the damaged tissues. The main features of the inflammatory response are (i) vasodilation (widening of the blood vessels to increase blood flow); (ii) increased vascular permeability that allows diffusible components to enter the tissues; (iii) cellular infiltration by chemotaxic, or directed movement of inflammatory cells through the walls of blood vessels into the site of injury; (iv) changes in biosynthetic, metabolic, and catabolic profiles of the affected tissues; and (v) activation of cells of the immune system as well as enzymatic systems of the blood plasma. In general, the inflammation response is quite efficient in managing and repairing damages induced by injury or infectious agent. The degree to which these phenomena occur is normally proportional to the severity of the injury or the extent of the challenge. However, inflammation can become harmful to tissues when it develops in a disorganized, disproportionate or undesired manner and can lead to diseases and disorders. The acute inflammation response is short lasting and involves all of the previously mentioned features of inflammation. Acute inflammation, when it proceeds in a disorganized fashion, can cause many harmful effects such as the digestion/destruction of normal tissues, excessive swelling that may lead to obstruction of blood flow, resulting in ischemia damage, hypersensitive reaction to non threatening entities (e.g. allergens), etc. The chronic inflammation reaction may be seen as a long-lasting inflammation, where the inflammatory agent is continually present. In this context, chronic inflammation is essentially observed under conditions of delayed hypersensitivity. However, chronic inflammation may be seen is cases where the inflammatory agent is not continuously present, as is the case of in asthma, arthritis or inflammatory bowel disease, and it may also be related to neurological or genetic disorders. In this case, one or more inflammatory components contribute to the etiology and perpetuation of inflammation. The process of inflammation is driven and modulated by a complex interplay between products of the plasma enzyme systems, lipid mediators (arachidonic acid metabolites such as prostaglandins and leukotrienes), vasoactive mediators released from inflammatory cells, and, in particular, cytokines. Prostaglandins (derived from eicosanoic essential fatty acids) are produced during an inflammatory response by inflammation-related biochemical pathways and are responsible for mediating the clinical manifestations characteristic of inflammation. The major source for the production of inflammation-related prostaglandins is arachidonic acid. Arachidonic acid can be metabolized by one of two cyclo-oxygenases (COX-1 or COX-2) producing inflammatory metabolites. The increased production of pro-inflammatory metabolites in inflamed tissues is due to the specific up-regulation of COX-2 (Maier et al., 1990). The increased expression of COX-2 during an inflammatory response is believed to be induced (in part) by exposure to bacterial endotoxins and/or the release of pro-inflammatory cytokines (Isakson, 1995; Raz et al., 1989; O'Sullivan et al., 1992), although other materials may increase expression of COX-2 as well. In contrast, COX-1 is constitutively expressed in most tissues and has been proposed to be involved in the maintenance of physiological functions such as platelet aggregation, cytoprotection in the stomach, and in part, the regulation of normal kidney function (Prasit et al., 1995; Pinto et al., 1995; Whittle et al., 1980). In addition to the production of pro-inflammatory eicosanoid metabolites via the cyclo-oxygenase pathways, arachidonic acid also serves as the source for the production of another class of inflammation-related metabolites produced by a family of related enzymes called lipoxygenases (LOX). In particular, 5-LOX catalyzes the first step of a biochemical cascade that culminates in the biosynthesis of a class of molecules termed leukotrienes (Sirois, 1985). Leukotrienes have been implicated as important mediators of inflammatory responses, such as anaphylaxis, suggesting that potent inhibitors of 5-LOX would provide an approach to limit the deleterious effects of all the products of this pathway. Elevated 15-LOX activity has been associated with conditions such as asthma and hypereosinophilia. Selective inhibition of 5-, 12-, or 15-LOX may provide an agent with a definite therapeutic advantage. In addition to prostaglandins and leukotrienes, cytokines also play a critical role in the inflammatory response. They are produced at the onset of inflammation development and are responsible for the eventual outcome of the inflammation process as well as its resolution. When injury or challenge occurs, cytokines are released from inflammatory cells (mast cells, basophils, endothelial cells, macrophages and neutrophils). The release of many different cytokines is activated during this process including the pro-inflammatory interleukins IL-1, IL-6, IL-8, IL-12, and tumor necrosis factor (TNF-α). In order to counteract an exaggerated inflammation, anti-inflammatory cytokines such as IL4, IL-10, IL-13, and transforming growth factor (TGF-β) are also produced. Although many cytokines are involved in the inflammation process, some cytokines have a central role in the process and have recently been examined as possible targets for anti-inflammatory products. Acute and chronic inflammation is most often treated with compounds with anti-inflammatory activity. Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin are among the most frequently used drugs currently available. Originally, the medicinal utility of classical NSAIDs was suspected to be due to their ability to inhibit the activities of COX-1 (Mitchell et al., 1993; Meade et al., 1993). Today, it is recognized that NSAIDs also have anti-inflammatory activity due to inhibition of COX-2 as well. Other biochemical activities associated with NSAIDs include inhibition of inflammatory mediators other than those mentioned above (i.e. histamine, serotonin, kinins), inhibition of oxidative phosphorylation, displacement of anti-inflammatory peptides from serum albumin, or displacement of peptides that hyperpolarize neuronal membranes in inflamed tissue (Foye, 1989). The key roles played by arachidonic acid metabolites produced by COX-2 and 5-, 12-, 15-LOX in mediating inflammatory responses has prompted extensive research to identify compounds capable of specifically inhibiting the enzymatic activities of COX-2, 5-, 12-, 15-LOX, or more than one simultaneously (i.e., dual inhibitors). Compounds capable of inhibiting COX-2 (but not COX-1) and/or 5-LOX would be of great use as anti-inflammatory agents without the ensuing deleterious side effects common to most non-steroidal anti-inflammatory drugs. Alternatively, compounds inhibiting release of arachidonic acid or compounds antagonizing pro-inflammatory cytokines would be of potential therapeutic use, whether they are steroidal (SAID), non-steroidal. (NSAID), cytokine suppressive (CSAID) or other anti-inflammatory drugs. Such inhibitory compounds would have great clinical utility in the treatment of such conditions as pain, fever, asthma, allergic rhinitis, rheumatoid arthritis, osteoarthritis, gout, adult respiratory disease syndrome, inflammatory bowel disease, endotoxic shock, ischemia-induced myocardial injury, atherosclerosis, and brain damage caused by stroke. Such inhibitors could also be used topically for the treatment of acne, sunburn, psoriasis, eczema, and related conditions. Though anti-inflammatory drugs are widely used to effectively treat inflammation, side effects of anti-inflammatory drug use such as steroid resistance, high doses, osteoporosis, catabolism of proteins and lipids, redistribution of lipidic masse, etc. are a major concern in medical research and drug development. One approach to alleviate side effects is to develop anti-inflammatory drug that have specific biochemical targets such as the development of NSAIDs that inhibit COX-2 (but not COX-1). Although this strategy if current in terms of research and development of anti-inflammatory drugs, an alterative strategy would be to use current anti-inflammatory drugs in combination with a potentiation agent in order to heighten the efficacy of less effective anti-inflammatory drugs and, potentially, lower the dosage rate in order to alleviate some of the side effects. Cytokines play a critical role in the inflammatory response. They are produced at the onset of inflammation development and are responsible for the eventual outcome of the inflammation process as well as its resolution. When injury or challenge occurs, cytokines are released from inflammatory cells (mast cells, basophils, endothelial cells, macrophages and neutrophils). The release of many different cytokines is activated during this process including the pro-inflammatory interleukins IL-1, IL-6, IL-8, IL-12, and tumor necrosis factor (TNF-α). In order to counteract an exaggerated inflammation, anti-inflammatory cytokines such as IL-4, IL-10, IL-13, and transforming growth factor (TGF-β) are also produced. Pro- to anti-inflammatory cytokines will determine the eventual outcome of inflammation by their relative proportions, their affinities, and their interactions. More accurately, an appropriate balance and interaction of pro- to anti-inflammatory cytokines will modulate the inflammation process in order to deal with the injury or challenge in the most efficient manner. In order to limit or prevent the damaging effects of inflammation, the immune system is normally well equipped with methods to regulate the balance of pro- and anti-inflammatory cytokines. However, many diseases or disorders will occur when the injured tissue is unable to create this appropriate cytokine balance and interaction (Feghali and Wright, 1997). The onset of the inflammation process is, therefore, not attributable to a single cytokine. For example, an elevation in pro-inflammatory cytokines will not necessarily cause exaggerated inflammation if it is accompanied by an elevation in anti-inflammatory cytokine levels. Although many cytokines are involved in the inflammation process, some cytokines have a central role in the process and have recently been examined, as possible targets for anti-inflammatory products. For example, the pro-inflammatory cytokine TNF-α has been clearly established as playing a pivotal role in many chronic inflammatory diseases and has been targeted for such therapies as monoclonal antibodies, soluble TNF-α receptors, TNF-converting enzyme, and other anti-TNF-α therapies (Lewis and Manning, 1999). The anti-inflammatory cytokine IL-10 also plays a critical role in the inflammation process to down-regulate the acute inflammation response. Because of this property, IL-10 has been actively studied as a therapeutic means of controlling inflammation related diseases through gene therapy (Lewis and Manning, 1999; Sacca et al., 1997). A thylakoid extract that has anti-oxidant properties, as described in the patent publication WO01/49305 has been tested for its capacity as a modulator of cytokines, and in combination with other anti-inflammatory agents. This extract is provided in the form of specific formulations that ensure the integrity and stability thereof. To simplify terminology, the terms “thylakoids”, “thylakoid extract”, and “extract” are used hereinbelow and are meant to cover all the specific formulations comprising thylakoids. TNF-α and IL-10 have been selected as preferred examples of cytokines that are systematically involved in inflammation, notwithstanding the nature of the causative agent or the nature of the tissue or system. There is an increasing body of literature suggesting that these two cytokines are involved in the expression of inflammatory diseases and disorders exemplified but not limited to those affecting the following tissues: Skin: psoriasis (Reich et al., 2001), cutaneous inflammation (Berg et al., 1995), atopic dermatitis (Lee et al., 2000); Brain: encephalitis (Deckert et al., 2001); Gastrointestinal tract: inflammatory bowel disease (Gasche et al., 2000), Crohn's disease (Narula et al., 1998), colitis (Moriguchi et al., 1999); Eye: infected cornea (Yan et al., 2001); Lung: hypersensitivity pneumonitis (Gudmundsson et al., 1998), chronic lung inflammation (Jones et al., 1996); Multiorgan: ischemia-reperfusion injury (Daemen et al., 1999); Autoimmune disease: rheumatoid arthritis (Maini et al., 1997; van Roon et al., 1996); and Hyper-reactivity: asthma (Thomas, 2001). The state of the art and the availability of a performing and stabilized thylakoid extract prompted the present inventors to test the extract against IL-10 and/or TNF-α expression. Besides the capacity of affecting cytokines expression, the complementarity of the thylakoid extract with other anti-inflammatory agents has been investigated. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to the use of a thylakold extract, in nutraceutical, cosmeceutical, and pharmaceutical applications, in the modulation of the inflammatory process and, particularly, of the expression of cytokines involved in the inflammatory response, which can cause diseases or disorders stemming from disorganized, disproportionate or undesirable inflammatory response. This invention more specifically relates to the use of a thylakoid extract as an efficient and long-lasting modulator of both pro- and anti-inflammatory cytokines. More precisely, the invention relates to the use of a thylakoid extract in the regulation of pro-inflammatory cytokines such as TNF-α and anti-inflammatory cytokines such as IL-10, as well as the relative proportions (balance) between these two cytokines. Another object of the invention is to provide compositions comprising thylakoids and an anti-inflammatory agent. Preferred embodiments of anti-inflammatory agents are glucocorticoids or NSAIDs, examples of which are budesonide and mesalamine, respectively. Other objects, advantages and features of the present invention will become more apparent upon reading of the following non restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings. The contents of the documents cited in the present disclosure are incorporated by reference thereto. |
Broadcast system |
A broadcast system BS includes a broadcast apparatus 1 and a reception apparatus 2. The broadcast apparatus 1 first receives and stores therein a proxy request PR including at least an identifier assigned to the reception apparatus 2 and a command to be transmitted to the reception apparatus 2. Moreover, the broadcast apparatus 1 extracts the set of the identifier of the reception apparatus 2 and the command for the reception apparatus 2 from the proxy request PR stored therein, and assembles a command packet from the set of the identifier and the command. Then, the broadcast apparatus 1 generates a stream in which the assembled command packet is multiplexed, and sends it out to a broadcast channel. The reception apparatus 2 separates the command packet from the stream sent out to the broadcast channel, and restores the set of the identifier and the command of the reception apparatus 2. Then, the reception apparatus 2 determines whether or not the disassembled command is destined for the present reception apparatus based on the pre-stored identifier of the present reception apparatus and the disassembled identifier of the reception apparatus 2. If so, the reception apparatus 2 stores therein the command destined for the present reception apparatus. The reception apparatus 2 executes the command, which has bee stored as described above. |
1. A broadcast system, comprising a broadcast apparatus and a reception apparatus, the broadcast apparatus including: a request reception section for receiving a proxy request including at least an identifier assigned to the reception apparatus and a command to be transmitted to the reception apparatus; a proxy request storage device for storing the proxy request received by the request reception section; a command extraction section for extracting a set of the identifier of the reception apparatus and the command for the reception apparatus from the proxy request stored in the proxy request storage device; a command packet assembler for assembling a command packet from the set of the identifier and the command extracted by the command extraction section; a multiplexer for generating a stream in which the command packet assembled by the command packet assembler is multiplexed; and a transmitter for sending out the stream generated by the multiplexer to a broadcast channel, and the reception apparatus including: a command packet separation section for separating the command packet from the stream sent out by the transmitter to the broadcast channel; a command packet disassembling section for disassembling the command packet separated by the command packet separation section to restore the set of the identifier and the command of the reception apparatus; an apparatus identifier storage section for storing the identifier of the present reception apparatus; a command filtering section for determining whether or not the command disassembled by the command packet disassembling section is destined for the present reception apparatus based on the identifier of the reception apparatus disassembled by the command packet disassembling section and the identifier of the present reception apparatus stored in the apparatus identifier storage section; a command storage section for storing a command that is determined to be destined for the present reception apparatus by the command filtering section; and a command execution section for executing the command stored in the command storage section. 2. A broadcast apparatus for broadcasting a stream to the reception apparatus, comprising: a request reception section for receiving a proxy request including at least an identifier assigned to the reception apparatus and a command to be transmitted to the reception apparatus; a proxy request storage device for storing the proxy request received by the request reception section; a command extraction section for extracting a set of the identifier of the reception apparatus and the command for the reception apparatus from the proxy request stored in the proxy request storage device; a command packet assembler for assembling a command packet from the set of the identifier and the command extracted by the command extraction section; a multiplexer for generating a stream in which the command packet assembled by the command packet assembler is multiplexed; and a transmitter for sending out the stream generated by the multiplexer to a broadcast channel, wherein the command extraction section extracts the set of the identifier of the reception apparatus and the command for the reception apparatus from the same proxy request a plurality of times. 3. The broadcast apparatus according to claim 2, wherein, the proxy request received by the request reception section further includes an execution start time of the command to be transmitted to the reception apparatus; the broadcast apparatus further comprises a request determination section for determining whether or not to accept the proxy request received by the request reception section based on the execution start time included in the proxy request; and the proxy request storage section only stores the proxy request that is determined by the request determination section to be acceptable. 4. The broadcast apparatus according to claim 3, further comprising a timer for keeping a current time, wherein the request determination section determines whether or not to accept the proxy request received from the request reception section by further using the current time kept by the timer. 5. The broadcast apparatus according to claim 4, wherein, the command packet assembled by the command packet assembler has a predetermined data size, and the multiplexer multiplexes the assembled command packet with a predetermined bandwidth; the command extraction section extracts the set of the identifier of the reception apparatus and the command for the reception apparatus from the same proxy request a predetermined number of times at a predetermined time interval; and the request determination section: calculates a first amount of time required from when one command packet is sent out until the command packet arrives at the reception apparatus, based on the data size and the bandwidth; calculates a second amount of time required from when a first command packet is assembled until a last command packet, including the same set of the identifier and the command as the first packet, is assembled, based on the time interval and the number of times; calculates a reference time by which the proxy request received by the request reception section should arrive at the broadcast apparatus, based on the execution start time included in the proxy request and the calculated first and second amounts of time; and determines that the proxy request received by the request reception section is unacceptable if the current time kept by the timer is after the calculated reference time. 6. The broadcast apparatus according to claim 3, wherein the request reception section receives a proxy request transmitted by an external communication terminal apparatus, the broadcast apparatus further comprising: a request_accepted generation section for generating a request_accepted signal for notifying the communication terminal apparatus that the proxy request has been accepted by the request determination section; and a request_accepted transmission section for transmitting the request_accepted signal generated by the request_accepted generation section to the communication terminal apparatus. 7. The broadcast apparatus according to claim 3, wherein the request reception section receives a proxy request transmitted by an external communication terminal apparatus, the broadcast apparatus further comprising: a request determination section for discarding a proxy request that has been determined by the request determination section to be unacceptable; a request_rejected generation section for generating a request_rejected signal for notifying the communication terminal apparatus that the proxy request has been discarded by the request determination section; and a request_rejected transmission section for transmitting the request_rejected signal generated by the request_rejected generation section to the communication terminal apparatus. 8. The broadcast apparatus according to claim 2, further comprising: a broadcast program storage section for storing video data and audio data of a program to be broadcast from a predetermined broadcast start time to a predetermined broadcast end time; a video encoder for encoding the video data stored in the broadcast program storage section; a video packet assembler for assembling a video packet from the video data encoded by the video encoder; an audio encoder for encoding the audio data stored in the broadcast program storage section; and an audio packet assembler for assembling an audio packet from the audio data encoded by the audio encoder, wherein, the multiplexer generates a stream by multiplexing together the video packet assembled by the video packet assembler, the audio packet assembled by the audio packet assembler, and the command packet assembled by the command packet assembler; and the proxy request received by the request reception section includes a scheduled recording command to be transmitted to the reception apparatus, a broadcast start time of a broadcast program to be recorded by scheduled recording, and a broadcast end time of the program. 9. A reception apparatus for receiving a stream from a broadcast apparatus, wherein the broadcast apparatus broadcasts, over a pre-assigned broadcast channel, a stream in which a command packet is multiplexed, the command packet being assembled from an identifier assigned to the reception apparatus and a command to be transmitted to the reception apparatus, the reception apparatus comprising: a tuner section for receiving the stream sent over the broadcast channel; an apparatus identifier storage section for storing the identifier of the present reception apparatus; a command filtering section for determining whether or not the identifier multiplexed in the stream received by the tuner section matches the identifier of the present reception apparatus stored in the apparatus identifier storage section; a command storage section for storing the command multiplexed in the stream received by the tuner section if it is determined by the command filtering section that the identifiers match each other; and a command execution section for executing the command stored in the command storage section. 10. The reception apparatus according to claim 9, further comprising: a power supply that can be set to two states of ON and standby; a power supply monitoring section for monitoring the state of the power supply to send out a control signal to the tuner section after the power supply transitions from ON to standby; and a specified channel storage section for storing, as a specified broadcast channel, information indicating a broadcast channel assigned to the broadcast apparatus, wherein in response to the control signal from the power supply monitoring device, the tuner section sets a receiving frequency band thereof to the specified broadcast channel stored in the specified channel storage section. 11. The reception apparatus according to claim 9, wherein, the broadcast apparatus broadcasts, over a broadcast channel, a stream in which a command packet is multiplexed, the command packet being assembled from the identifier assigned to the reception apparatus, a password specified by a user, and a command to be transmitted to the reception apparatus; the reception apparatus further comprises a password storage section for storing the password specified by the user; the packet filtering section further determines whether or not the password multiplexed in the stream received by the tuner section matches the password stored in the password storage section; the command storage section further stores the command multiplexed in the stream received by the tuner section if it is determined by the command filtering section that the passwords match each other. 12. The reception apparatus according to claim 11, further comprising a determination/writing section for receiving the command from the packet filtering section if it is determined by the packet filtering section that the identifier and the password multiplexed in the stream received by the tuner section match the identifier and the password stored in the apparatus identifier storage section and the password storage section, respectively, wherein the determination/writing section determines whether or not to write the command received from the packet filtering section to the command storage section. 13. The reception apparatus according to claim 12, wherein, the broadcast apparatus broadcasts, over a broadcast channel, a stream in which a command packet is multiplexed, the command packet being assembled from the identifier assigned to the reception apparatus, the password specified by the user, the command to be transmitted to the reception apparatus, and an execution start time and an execution end time of the command; the packet filtering section passes the command, the execution start time and the execution end time multiplexed in the stream received by the tuner section to the determination/writing section if the identifier and the password multiplexed in the stream received by the tuner section match the identifier and the password stored in the apparatus identifier storage section and the password storage section, respectively; and the determination/writing section stores the received set of the command, the execution start time and the execution end time in the command storage section if there is no other set stored in the command storage section that has a temporal overlap with a time slot between the execution start time and the execution end time of the received command. 14. The reception apparatus according to claim 13, further comprising: an error message generation section for generating an error message indicating that the set of the command, the execution start time and the execution end time received by the determination/writing section cannot be stored in the command storage section if there is a set that has a temporal overlap with the time slot between the execution start time and the execution end time of the received command; and a line connection section for sending out the error message generated by the error message generation section to a communication line. 15. The reception apparatus according to claim 14, wherein, in response to a request from a communication terminal apparatus operated by a user, the broadcast apparatus broadcasts, over a broadcast channel, a stream in which a command packet is multiplexed, the command packet being assembled from the identifier assigned to the reception apparatus, the password specified by the user, the command to be transmitted to the reception apparatus, and the execution start time and the execution end time of the command; and the line connection section transmits the error message generated by the error message generation section to the communication terminal apparatus via the communication line. 16. The reception apparatus according to claim 14, wherein the line connection section transmits the error message generated by the error message generation section to the broadcast apparatus via the communication line. 17. The reception apparatus according to claim 13, wherein, the reception apparatus further comprises a timer for keeping a current time; and the command execution section starts or ends an execution of the command if the current time received from the timer matches the execution start time or the execution end time, respectively, stored in the command storage section. 18. The reception apparatus according to claim 9, wherein, the broadcast apparatus broadcasts, over a broadcast channel, a stream obtained by multiplexing together a video packet, an audio packet and a command packet; a set of the video packet and the audio packet forms a program to be broadcast from a predetermined broadcast start time to a predetermined broadcast end time; the command packet is assembled from the identifier assigned to the reception apparatus, a password specified by a user, a scheduled recording command to be transmitted to the reception apparatus, a broadcast channel of a program to be recorded by scheduled recording, and an execution start time and an execution end time of the scheduled recording command; the command storage section stores a set of the identifier assigned to the reception apparatus, the password specified by the user, the scheduled recording command to be transmitted to the reception apparatus, the broadcast channel of the program to be recorded by scheduled recording, and the execution start time and the execution end time of the scheduled recording command; the reception apparatus further comprises: an AV packet separation section for separating the video packet and the audio packet multiplexed in the stream received by the tuner section; and a scheduled program storage section for storing the video packet and the audio packet separated by the AV packet separation section; and the command execution section: generates a control signal for setting a receiving frequency band of the tuner section to the broadcast channel stored in the command storage section and sends out the generated control signal to the tuner section at the execution start time of the scheduled recording command stored in the command storage section; and generates a recording instruction for instructing to send the video packet and the audio packet separated by the AV packet separation section to the scheduled program storage section and sends out the generated recording instruction to the AV packet separation section, after sending out the control signal to the tuner. 19. The reception apparatus according to claim 18, wherein, the reception apparatus further comprises a determination/writing section for receiving, from the packet filtering section, the scheduled recording command, the broadcast channel, the execution start time and the execution end time multiplexed in the stream received by the tuner section; and the determination/writing section writes a set of the scheduled recording command, the broadcast channel, the execution start time and the execution end time received from the packet filtering section to the command storage section if the scheduled program storage section has a free space sufficient for storing the program to be recorded by scheduled recording. 20. The reception apparatus according to claim 19, further comprising: an error message generation section for generating an error message indicating that the set of the scheduled recording command, the broadcast channel, the execution start time and the execution end time received from the packet filtering section cannot be written to the command storage section if it is determined by the determination/writing section that the scheduled program storage section does not have the free space; and a line connection section for sending out the error message generated by the error message generation section to a communication line. 21. The reception apparatus according to claim 9, wherein, the broadcast apparatus broadcasts, over a broadcast channel, a stream further including a video packet and an audio packet multiplexed therein; a set of the video packet and the audio packet forms a program to be broadcast from a predetermined broadcast start time to a predetermined broadcast end time; the tuner section includes: an AV packet tuner for setting a receiving frequency band of the tuner section to a broadcast channel specified by a user; and a command packet tuner for setting the receiving frequency band of the tuner section to a broadcast channel used for broadcasting a command packet destined for the present reception apparatus; and the reception apparatus further comprises: an AV packet separation section for separating the video packet and the audio packet from the stream received by the AV packet tuner; and a command packet separation section for separating the command packet from the stream received by the command packet tuner. 22. The reception apparatus according to claim 9, wherein, the broadcast apparatus broadcasts, over a broadcast channel, a stream obtained by multiplexing together a video packet, an audio packet and a command packet; a set of the video packet and the audio packet forms a program to be broadcast from a predetermined broadcast start time to a predetermined broadcast end time; the command packet is assembled from the identifier assigned to the reception apparatus, the password specified by a user, a scheduled recording command to be transmitted to the reception apparatus, a broadcast channel of a program to be recorded by scheduled recording, and an execution start time and an execution end time of the scheduled recording command; the command storage section stores a set of the identifier assigned to the reception apparatus, the password specified by the user, the scheduled recording command to be transmitted to the reception apparatus, the broadcast channel of the program to be recorded by scheduled recording, and the execution start time and the execution end time of the scheduled recording command; the reception apparatus is connected to an external recording device via a transmission path, the reception apparatus further comprising: an AV packet separation section for separating the video packet and the audio packet multiplexed in the stream received by the tuner section; and a bus control section for transmitting the video packet and the audio packet separated by the AV packet separation section to the external recording device via the bus; and the command execution section: generates a control signal for setting a receiving frequency band of the tuner section to the broadcast channel stored in the command storage section and sends out the generated control signal to the tuner section at the execution start time of the scheduled recording command stored in the command storage section; and generates a transfer instruction for instructing to transfer the video packet and the audio packet separated by the AV packet separation section to the external recording device and sends out the generated transfer instruction to the bus control section, after sending out the control signal to the tuner. 23. The reception apparatus according to claim 22, wherein the command execution section further converts the scheduled recording command stored in the command storage section into a format executable by the external recording device and then sends out the scheduled recording command to the bus control section. 24. A method for broadcasting a stream from a broadcast apparatus to a reception apparatus, wherein, the broadcast apparatus comprises: a request reception step of receiving a proxy request including at least an identifier assigned to the reception apparatus and a command to be transmitted to the reception apparatus; a proxy request storage step of storing the proxy request received in the request reception step; a command extraction step of extracting a set of the identifier of the reception apparatus and the command for the reception apparatus from the proxy request stored in the proxy request storage step; a command packet assembling step of assembling a command packet from the set of the identifier and the command extracted in the command extraction step; a multiplexing step of generating a stream in which the command packet assembled in the command packet assembling step is multiplexed; and a transmission step of sending out the stream generated in the multiplexing step to the broadcast channel; and the reception apparatus comprises: a command packet separation step of separating the command packet from the stream sent out to the broadcast channel in the transmission step; a command packet disassembling step of disassembling the command packet separated in the command packet separation step to restore set of the identifier and the command of the reception apparatus; a command filtering step of determining whether or not the command disassembled in the command packet disassembling step is destined for the present reception apparatus based on the identifier of the reception apparatus disassembled in the command packet disassembling step and an identifier of the present reception apparatus pre-stored in the reception apparatus; and a command execution step of executing the command that is determined in the command filtering step to be destined for the present reception apparatus. 25. A broadcast method for broadcasting a stream to the reception apparatus, comprising: a request reception step of receiving a proxy request including at least an identifier assigned to the reception apparatus and a command to be transmitted to the reception apparatus; a proxy request storage step of storing the proxy request received in the request reception step; a command extraction step of extracting the identifier of the reception apparatus and the command for the reception apparatus from the proxy request stored in the proxy request storage step; a command packet assembling step of assembling a command packet from the set of the identifier and the command extracted in the command extraction step; a multiplexing step of generating a stream in which the command packet assembled in the command packet assembling step is multiplexed; and a transmission step of sending out the stream generated in the multiplexing step to a broadcast channel, wherein the command extraction step extracts the set of the identifier of the reception apparatus and the command for the reception apparatus from the same proxy request a plurality of times. 26. A method for receiving a stream from a broadcast apparatus by a reception apparatus, wherein the broadcast apparatus broadcasts, over a pre-assigned broadcast channel, a stream in which a command packet is multiplexed, the command packet being assembled from an identifier assigned to the reception apparatus and a command to be transmitted to the reception apparatus, the reception method comprising: a reception step of receiving the stream sent over the broadcast channel; a command filtering step of determining whether or not the identifier multiplexed in the stream received in the reception step matches an identifier of the present reception apparatus pre-stored in the reception apparatus; a command storage step of storing the command multiplexed in the stream received in the reception step if it is determined in the command filtering step that the identifiers match each other; and a command execution step of executing the command stored in the command storage step. |
<SOH> BACKGROUND ART <EOH>FIG. 26 is a block diagram illustrating an overall configuration of a conventional broadcast system. The broadcast system of FIG. 26 is disclosed in Japanese Laid-Open Patent Publication No. 10-155131, and includes a communication terminal apparatus TA 1 , a server BSV as the broadcast apparatus, a communication terminal apparatus TA 2 as the reception apparatus, and a video tape recorder VTR as the unit to be controlled. The communication terminal apparatus TA 1 transmits information “recording scheduling request” to the server BSV, which is communicably connected to the communication terminal apparatus TA 1 via the Internet INT. In response to the recording scheduling request sent from the communication terminal apparatus TA 1 , the server BSV transmits personal recording schedule information, as an example of the command, to the communication terminal apparatus TA 2 over a text television channel CH. The communication terminal apparatus TA 2 , capable of receiving televised programs and text, receives a video signal sent over an ordinary broadcast channel, and supplies the received signal to the video tape recorder VTR, which is communicably connected to the communication terminal apparatus TA 2 . Furthermore, the communication terminal apparatus TA 2 sets a recording schedule requested by the communication terminal apparatus TA 1 in the video tape recorder VTR according to the personal scheduled recording information sent from the server BSV over the text television channel CH. Next, technical problems of the conventional broadcast system will be described. First, while the above publication discloses only one communication terminal apparatus TA 2 , an actual broadcast system accommodates a number of communication terminal apparatuses TA 2 . Nevertheless, the server BSV simply sends personal scheduled recording information that arrives at the server BSV while multiplexing it with other signals on the text television channel CH. Therefore, a communication terminal apparatus TA 2 may possibly receive personal scheduled recording information which was directed to another communication terminal apparatus TA 2 . Furthermore, there are a plurality of servers BSV, and they are assigned different text television channels CH. Moreover, the communication terminal apparatus TA 2 receives personal scheduled recording information sent over one of the plurality of text television channels CH that is specified by the user. Therefore, in order to reliably perform the scheduled recording, a communication terminal apparatus TA 2 needs to be set to a text television channel CH being used by a server BSV when the personal scheduled recording information from the server BSV arrives at the communication terminal apparatus TA 2 . Therefore, if the appropriate text television channel CH is not selected, the communication terminal apparatus TA 2 fails to receive the personal scheduled recording information from the server BSV, thus failing to perform the scheduled recording. As can be seen from the above, the first problem of the conventional broadcast system is that the reception apparatus is likely to fail to accurately receive only those commands that are destined for itself. Next, while the above publication discloses only one communication terminal apparatus TA 1 , as with the communication terminal apparatus TA 2 , personal scheduled recording information arrives at the server BSV from a plurality of communication terminal apparatuses TA 1 in an actual broadcast system. Due to the nature of scheduled recording, personal scheduled recording information needs to arrive at the communication terminal apparatus TA 2 before the start of the program requested by the communication terminal apparatus TA 1 . However, the conventional server BSV simply sends personal scheduled recording information that arrives at the server BSV. Therefore, the second problem is that the personal scheduled recording information may not arrive at the communication terminal apparatus TA 2 before the start of the program to be recorded by scheduled recording. Therefore, a first object of the present invention is to provide a broadcast system in which the reception apparatus can receive only those commands that are destined for itself with a higher reliability. Moreover, a second object of the present invention is to provide a broadcast system in which a command sent from the broadcast apparatus arrives at the reception apparatus by a predetermined time. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a schematic diagram illustrating an overall configuration of a broadcast system BS according to one embodiment of the present invention. FIG. 2 ( a ) and FIG. 2 ( b ) are schematic diagrams illustrating a data structure of a proxy request PR transmitted to a broadcast apparatus 1 by a communication terminal apparatus 3 of FIG. 1 . FIG. 3 is a block diagram illustrating a detailed configuration of the broadcast apparatus 1 of FIG. 1 . FIG. 4 ( a ) and FIG. 4 ( b ) are schematic diagrams illustrating a series including a header packet Phd and Nmux command packets Pct outputted from a command packet assembler 112 of FIG. 3 . FIG. 5 ( a ) and FIG. 5 ( b ) are schematic diagrams illustrating a transport stream TS sent out from a TS transmitter 119 of FIG. 3 . FIG. 6 is a block diagram illustrating a detailed configuration of a reception apparatus 2 of FIG. 1 . FIG. 7 is a schematic diagram illustrating an example of a command Cct that should not be stored in a command storage section 211 of FIG. 2 . FIG. 8 is a schematic diagram illustrating a data structure of an error message EM generated by an error message generation section 213 of FIG. 2 . FIG. 9 is a block diagram illustrating a detailed configuration of the communication terminal apparatus 3 of FIG. 1 . FIG. 10 ( a ) and FIG. 10 ( b ) are sequence charts illustrating a procedure for data communications performed in the broadcast system BS of FIG. 1 . FIG. 11 is a flow chart illustrating the procedure of a process in which an application execution section 32 of FIG. 9 generates and transmits the proxy request PR. FIG. 12 ( a ) and FIG. 12 ( b ) are schematic diagrams illustrating an example of an image to be displayed in step S 11 of FIG. 11 . FIG. 13 ( a ) is a schematic diagram illustrating an example of a data format of a command Cct that is stored in advance in the application execution section 32 of FIG. 3 , and FIG. 13 ( b ) is a schematic diagram illustrating an example of a generated command Cct. FIG. 14 is a flow chart illustrating the procedure of a process performed when the broadcast apparatus 1 of FIG. 1 receives the proxy request PR. FIG. 15 is a schematic diagram illustrating an example of a proxy request PRp stored in a request storage device 18 of FIG. 3 . FIG. 16 is a flow chart illustrating the procedure of a process that the broadcast apparatus 1 of FIG. 1 performs when transmitting the transport stream TS. FIG. 17 is a schematic diagram illustrating the command packet Pct of the same contents arriving at the reception apparatus 2 of FIG. 1 Nsc times at a time interval Tnv. FIG. 18 is a flow chart illustrating the procedure of a process that the reception apparatus 2 of FIG. 1 performs when receiving the transport stream TS. FIG. 19 is a flow chart illustrating the procedure of a process that the communication terminal apparatus 3 of FIG. 1 performs when receiving the error message EM. FIG. 20 is a schematic diagram illustrating an example of a command ACct that is written to the command storage section 211 by performing step S 416 of FIG. 18 . FIG. 21 is a flow chart illustrating the procedure of a process that a command execution section 216 of FIG. 6 performs when executing the command ACct stored in the command storage section 211 . FIG. 22 is a block diagram illustrating a detailed configuration of a first variant (reception apparatus 2 a ) of the reception apparatus 2 of FIG. 1 . FIG. 23 is a block diagram illustrating a detailed configuration of a dual tuner 41 of FIG. 22 . FIG. 24 is a block diagram illustrating a detailed configuration of a second variant (reception apparatus 2 b ) of the reception apparatus 2 of FIG. 1 . FIG. 25 is a flow chart illustrating the procedure of a process that a command execution section 51 of FIG. 24 performs when executing the command ACct stored in the command storage section 211 . FIG. 26 is a schematic diagram illustrating a configuration of a conventional broadcast system. detailed-description description="Detailed Description" end="lead"? |
Method of transporting physiological polymer using protein having rxp repeated sequence |
The present invention provides a method for efficiently transporting useful protein, peptide, gene, etc. to a target cell such as a diseased cell, which is characterized by that a complex in which protein having a structure of peptide unit repetition represented by the formula Arg-Xaa-Pro (in the formula, Xaa is a hydrophobic or acidic amino acid residue) at the C-terminal of the protein is bound to at least one of useful protein, peptide, gene, etc. is administered to a living body. |
1. A method for transporting at least one of (a) useful protein, (b) peptide and (c) gene in a living body, which is characterized by that at least one of (a) useful protein, (b) peptide and (c) gene is/are bound to protein having a structure of peptide unit repetition represented by the formula Arg-Xaa-Pro (in the formula, Xaa is a hydrophobic or acidic amino acid residue) at the C-terminal of the protein and the resulting complex is administered to a living body. 2. The method according to claim 1, wherein the gene is inserted into a plasmid. 3. The method according to claim 1, wherein transport in a living body is intercellular transport. 4. The method according to claim 3, wherein intercellular transport is transport to a target cell. 5. The method according to claim 4, wherein the target cell is a diseased cell. 6. The method according to claim 1, wherein the protein is a US11 protein of herpes simplex virus. 7. The method according to claim 1, wherein the useful protein is a cytokine or interferon-α, -β, -γ or -ω. 8. Use of a protein having a structure of peptide unit repetition represented by the formula Arg-Xaa-Pro (in the formula, Xaa is a hydrophobic or acidic amino acid residue) at the C-terminal of the protein for transporting at least one of (a) useful protein, (b) peptide and (c) gene in a living body. 9. A transport agent for transporting at least one of (a) useful protein, (b) peptide and (c) gene in a living body, which comprises a complex in which protein having a structure of peptide unit repetition represented by the formula Arg-Xaa-Pro (in the formula, Xaa is a hydrophobic or acidic amino acid residue) at the C-terminal of the protein is bound to at least one of (a) useful protein, (b) peptide and (c) gene. 10. A complex in which protein having a structure of peptide unit repetition represented by the formula Arg-Xaa-Pro (in the formula, Xaa is a hydrophobic or acidic amino acid residue) at the C-terminal of the protein is bound to at least one of (a) useful protein, (b) peptide and (c) gene. 11. The complex according to claim 10, wherein the gene is inserted into a plasmid. 12. The complex according to claim 10, wherein the protein is a US11 protein of herpes simplex virus. 13. A method for the treatment of human beings or animals, which is characterized by that a complex in which protein having a structure of peptide unit repetition represented by the formula Arg-Xaa-Pro (in the formula, Xaa is a hydrophobic or acidic amino acid residue) at the C-terminal of the protein is bound to at least one of (a) useful protein, (b) peptide and (c) gene is administered to a patient. |
<SOH> BACKGROUND ART <EOH>As a result of the progress in genetic engineering in recent years, genetic abnormality that causes many diseases such as hereditary disease has been explicated at the DNA level, whereupon there have been invented therapeutic methods in which the abnormal gene is returned to normal and expressed so that the disease is treated. In such therapeutic methods, one of the technical challenges is development of an art for transporting useful genes efficiently and safely to a target cell and expressing them. With regard to a method for introducing genes into a cell, microinjection, precipitation with calcium phosphate, cation liposome, virus vector, electroporation, etc. have been usually applied. However, there are disadvantages that those methods are troublesome and also have cytotoxicity. In addition, it takes as long as 12 to 80 hours to confirm the expression of the desired genes after introduction of genes. In order to overcome such disadvantages, there has been developed a method for introducing useful protein directly into a cell by using protein which has intercellular transportation ability. With regard to the protein having an intercellular transportation ability, there have been known HIV-1 TAT (Vives, E., Brodin, P. and Lebleu, B. (1997), A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus, J. Biol. Chem. 272, 16010-7), antennapedia (Derossi, D., Joliot, A/H/. Chassaing, G. and Prochiantz, A. (1994), The third helix of the Antennapedia homeodomain translocates through biological membrane, J. Biol. Chem. 269, 10444-50), etc. When those proteins are used, however, there is a problem that inactivation or denaturation of the useful protein to be introduced into a cell is resulted. |
Method and system for detecting inter-chromosomal imbalance by fluorescent in situ hybridization (fish) on interphase nuclei |
The invention concerns a system for detecting chromosomal imbalance by in situ hybridization of fluorescent probes on interphase nuclei, comprising the following phases: hybridizing in situ fluorescent probes on two separate chromosomes; exposing each probe with a different fluorochrome; measuring the intensity signals corresponding respectively to each probe thus exposed, on an assembly of nuclei, said measurement being carried out within a control cell population and within a cell population subjected to detection; calculating a ratio between the signals corresponding to each of the probes, said ratio calculation being carried out on said control cell population to provide a reference ratio and said population subjected to detection; comparing the fluorescence ratio corresponding to the population subjected to detection with the reference ratio; and processing the result of said comparison to detect an inter-chromosomal imbalance. |
1. A method for detecting interchromosomal imbalance by fluorescent in situ hybridization on interphase nuclei, comprising the following phases: in situ hybridizing of fluorescent probes on two separate chromosomes, visualizing each probe with a different fluorochrome, measuring intensity signals corresponding, respectively, to each probe thus visualized, on a set of nuclei, this measurement being carried out, firstly, in a control cell population and, secondly, in a cell population subjected to detection, calculating the ratio between said signals corresponding, respectively, to said probes, this ratio calculation being carried out, firstly, on said control cell population so as to provide a reference ratio and, secondly, on said cell population subjected to detection, comparing the ratio between signals corresponding to the cell population subjected to detection with the reference ratio, and processing the result of this comparison in order to detect an interchromosomal imbalance. 2. The process as claimed in claim 1, characterized in that the measurement of the two signals corresponding respectively to each probe is carried out by automated image cytometry. 3. The process as claimed in either of claims 1 and 2, characterized in that the two probes are visualized, respectively, with two different fluorochromes (for example, a green fluorochrome and a red fluorochrome). 4. The method as claimed in any one of the preceding claims, characterized in that the intensity signals from each probe are measured on a number of nuclei which can be defined by the operator, for example a few hundred. 5. The method as claimed in any one of the preceding claims, characterized in that the measuring phase comprises a first step consisting in acquiring, in a defined measuring plane, a given number of fields so as to obtain, for a given image magnification, at least a predetermined number of analyzable nuclei. 6. The method as claimed in claim 5, characterized in that the acquisition step also comprises acquisition, by means of successive optical filtering, of several images corresponding, for each field, respectively to a plurality of planes corresponding to the wavelengths of a plurality of fluorochromes (for example, blue for the counter-staining, green and red for the probe labeling), storage of said acquired images and superimposition of said acquired and stored images. 7. The method as claimed in either of claims 5 and 6, characterized in that the acquisition step is carried out under acquisition conditions which are substantially identical for a control cell population and a cell population subjected to detection. 8. The method as claimed in claim 7, characterized in that the acquisitions corresponding respectively to the control cell population and cell population subjected to detection are carried out on two fields included in the same measuring plane. 9. The method as claimed in one of claims 5 to 8, characterized in that the measuring phase also comprises a second step for detecting nuclei and quantifying fluorescence intensity signals. 10. The method as claimed in claim 9, characterized in that the detection and quantification step comprises segmentation of the nuclei in each field included in a measuring plane. 11. The method as claimed in claim 10, characterized in that the segmentation of the nuclei includes separation of nuclei in aggregates. 12. The method as claimed in either of claims 10 and 11, characterized in that the segmentation of the nuclei includes elimination of the artifacts by criteria of morphology and size. 13. The method as claimed in any one of claims 9 to 12, characterized in that the detection and quantification step comprises quantification of the integrated fluorescence signal intensity within each nucleus for each color corresponding to each probe. 14. The method as claimed in claim 13, characterized in that the detection and quantification step also comprises calculation of the background level for each color outside the nuclei in each of the fields. 15. The method as claimed in claim 14, characterized in that the detection and quantification step also comprises determination of the most common background level as noise reference level and correction with said reference level of the fluorescent signal intensity quantified within each nucleus. 16. A system for detecting chromosomal imbalances by fluorescent in situ hybridization on interphase nuclei, using the method as claimed in any one of the preceding claims, comprising: means for carrying out a fluorescent in situ hybridization on two separate chromosomes, means for visualizing each probe with a different fluorochrome, a device for measuring intensity signals corresponding respectively to each probe thus visualized, on a set of nuclei, firstly, within a control cell population and, secondly, within a cell population subjected to detection, means for calculating the ratio between said signals corresponding respectively to said probes, firstly, on said control cell population so as to provide a reference ratio and, secondly, on said cell population subjected to detection, means for comparing the ratio between signals corresponding to the cell population subjected to detection with the reference ratio, and means for processing the result of this comparison for the purpose of detecting an interchromosomal imbalance, even in the case of a mosaic. 17. The system of detection as claimed in claim 16, characterized in that it includes, as measuring means, an image cytometry device. 18. The system of detection as claimed in claim 17, characterized in that the image cytometry device comprises: multiple fluorescence microscopy means applied to cell populations subjected beforehand to a fluorescent in situ hybridization, means for acquiring images produced by the fluorescence microscopy means, means for storing said acquired images, and means for analyzing said acquired and stored images. 19. The system of detection as claimed in claim 18, characterized in that the fluorescence microscopy means and the image acquisition means cooperate so as to acquire, in a defined measuring plane, a given number of fields so as to obtain, for a given image magnification, at least a predetermined number of analyzable nuclei. 20. The system of detection as claimed in claim 19, characterized in that it also comprises filtering means which cooperate with the fluorescence microscopy means and the acquisition means in order to acquire several images corresponding, for each field, respectively to a plurality of planes corresponding to the wavelengths of a plurality of fluorochromes (for example: DAPI (blue) for the counter-staining, FITC (green) and Texas Red™ (red) for the probes). 21. The system of detection as claimed in one of claims 18 to 20, characterized in that the fluorescence microscopy means and the acquisition means cooperate so as to acquire, within the same plane of analysis, images corresponding to a control cell population and images corresponding to a cell population subjected to detection. 22. The system of detection as claimed in one of claims 17 to 21, characterized in that the image cytometry device also comprises means for detecting nuclei and quantifying fluorescence signals of multiple wavelengths. 23. The system of detection as claimed in claim 22, characterized in that the detection and quantification means are organized so as to segment nuclei on the basis of a morphometric and densitometric analysis, giving rise to the creation of a mask for all the fields of a measuring plane. 24. The system of detection as claimed in claim 22 or 23, characterized in that the detection and quantification means are organized so as to separate the nuclei in aggregates. 25. The system of detection as claimed in one of claims 22 to 24, characterized in that the detection and quantification means are organized so as to exclude artifacts by criteria of size and morphology. 26. The system of detection as claimed in one of claims 22 to 25, characterized in that the detection and quantification means are organized so as to quantify the integrated fluorescent signal intensity within each nucleus for each color. 27. The system of detection as claimed in one of claims 22 to 26, characterized in that the detection and quantification means are organized so as to calculate the background level for each color outside the nuclei. 28. The system of detection as claimed in one of claims 22 to 27, characterized in that the detection and quantification means are organized so as to determine the most common background level as background reference value and to subtract said reference value from the intensity quantified within each nucleus. 29. The application of the method for interchromosomal detection as claimed in any one of claims 1 to 15, to nuclei of fetal cells circulating in maternal blood. |
Dispensing indicator for an active substance dispenser |
The indicator is a non-electric indicator and is based on a thermochromic material which is applied on a diffuser or thermodiffuser device of volatile substances (1), so that when heating of the latter takes place to evaporate the volatile substance contained in a reservoir associated with said device (1), when a determined temperature is reached the indicator, constituted by a plastic insert (4) with thermochromic pigments, changes colour and indicates that the diffuser device (1) is in diffusion conditions. The colour change product can be constituted by the combination of some thermochromic and normal paints applied on the external surface of the device, so that when a determined temperature is reached the thermochromic paint changes colour and originates an indication of the device being in diffusion conditions. |
1. Active substance diffuser which incorporates a diffusion indicator, which being based on the heating of the active substance contained in the actual diffuser, for its use as air freshener, insecticide and the like, and being of the type of those which include a plug for connection to an electricity mains supply, in connection with a heater element to produce the heating of the active substance and its corresponding evaporation, is characterised in that the corresponding diffusion indicator is constituted by a thermochromic element or mounted on a visible part of the body of the diffuser. 2. Active substance diffuser, according to claim 1, characterised in that the thermochromic product is constituted by a plastic insert provided with one or more pigments of which at least one has thermochromic properties, such insert forming an integral part of the diffuser device, this insert having a function in the design of the diffuser besides that whereby it confers upon it its thermochromic properties. 3. Active substance diffuser, according to claim 1, characterised in that the thermochromic product is constituted by a plastic insert fitted with one or more pigments of which at least one has thermochromic properties, such insert not forming an integral part of the diffuser, whereby this insert has no functional role in the device apart from that which its thermochromic properties confer upon it, whereby this insert can be considered optional from the point of view of the release of the active substance by the diffuser. 4. Active substance diffuser, according to claim 12, characterised in that the pigment of thermochromic properties is incorporated in the plastic insert by means of extrusion. 5. Active substance diffuser, according to claim 12, characterised in that the insert is obtained by means of moulding. 6. Active substance diffuser, according to claim 12, characterised in that the insert is obtained by means of extrusion. 7. Active substance diffuser, according to claim 12, characterised in that the insert is obtained by means of thermoforming moulding. 8. Active substance diffuser, according to claim 12 characterised in that the insert includes a part of higher thermal conductivity, mounted next to the corresponding heater element of the diffuser device, for transmission of the heat to the external part or surface of the actual insert. 9. Active substance diffuser, according to claim 1, characterised in that the product is constituted by a thermochromic paint blended or not with a non-thermochromic paint, located on the external surface of the diffuser device, with the particularity that the colour of said paints is the same at low temperature, whilst heating up to a determined temperature causes the thermochromic pigment of the paint to lose its colour allowing a message or indication of operation of the diffuser device to appear. 10. Active substance diffuser, according to claim 9, characterised in that the paints and are applied by printing by means of tampography or serigraphy or flexography or printing by photolithography or thermotransfer or lithography on the external surface of the diffuser device, thereafter a fixing agent can be applied to fix the paint by drying or chemical reticulation. 11. Active substance diffuser, according to claim 9, characterised in that the paints and are located on a label fixed by means of adhesive to the surface of the diffuser device. 12. Active substance diffuser, according to claim 1, characterized in the thermochromic product is constructed by a plastic insert provided with one or more pigments of which at least one has thermochromic properties. 13. Active substance diffuser, according to claim 2, characterised in that the product is constituted by a thermochromic paint blended or not with a non-thermochromic paint, located on the external surface of the diffuser device, with the particularity that the colour of said paints is the same at low temperature, whilst heating up to a determined temperature causes the thermochromic pigment of the paint to lose its colour allowing a message or indication of operation of the diffuser device to appear. 14. Active substance diffuser, according to claim 2, characterised in that the paints and are applied by printing by means of tampography or serigraphy or flexography or printing by photolithography or thermotransfer or lithography on the external surface of the diffuser device, thereafter a fixing agent can be applied to fix the paint by drying or chemical reticulation. 15. Active substance diffuser, according to claim 10, characterised in that the paints and are located on a label fixed by means of adhesive to the surface of the diffuser device. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Determined diffuser devices of active substances, used as air fresheners, insecticides and the like, are 4 based on the heating of the active substance contained in the actual diffuser device, so that the evaporation of said substance permeates the surroundings in which the device in question is located. This type of device can come with a plug forming an integral part thereof so that in this particular case it is applied directly in an electric plug socket. In general, heat is released by means of an exothermic process based on a heater element included in the diffuser device. With this the heating takes place of the volatile substance contained in the diffuser device, which gives rise to the evaporation of said substance. For this reason, it is convenient to refer in a general manner to that kind of active substance diffuser device as “thermodiffuser”. Such active substance thermodiffusers are marketed in some cases with no indicator whatever as to whether they are releasing heat or not, whereby it is not known whether the device is fulfilling its diffusion function or not, unless it is touched and it is observed by touch whether or not it is hot, assuming that if it is hot the device is diffusing and if it is cold the device is not diffusing. In the particular case in which these active substance thermodiffusers have a plug for the supply of electric power to a heater, they can moreover be marketed with a luminous indicator, generally a light-emitting diode (hereinafter a LED), so that if said LED is on, it indicates that the thermodiffuser is being fed electrically, but not with certainty that the heating being produced is characteristic of its correct operation. There is a third type of active substance thermodiffuser whose operation is determined either by the position of an electric switch which can be light-emitting or not, or by the relative position of one or more than one moving piece which forms part of the design of the device and acts as an electric switch. In all cases, and specifically in those in which there is a light-emitting indicator or indicator of another type to signal the supply of power, it is known if the device is being fed electrically, but it is not known if evaporation of the active substance is taking place by means of heating. On the other hand, thermochromic elements are known applicable as informative means or as indicators of the temperature of certain devices. In this sense mention can be made of the European Patent with Publication Number 0294136, in which a heating unit is described with thermochromic indicator of the temperature, wherein the indicator includes an electrically resistant thick film indicator track, with a defined configuration printed on a substrate of a layer of thermochromic material applied to the substrate to cover and surround the defined configuration. For its part, the heating unit includes elements to respond to the current flowing through the heating element, generating a current in the indicator track. Consequently, when the element heats up, so too the indicator track, an area of colour change being visible which shows the defined configuration. Likewise, mention can be made of the European Patent with Publication Number 0287336 with respect to one a heating unit with thermochromic region which comprises a warmable surface with a region of that surface coated or provided with a layer which has a thermochromic material in sufficient quantity to be affected by the received heat, the thermochromic material having a limiting wavelength of absorption which changes reversibly with temperature in the range from 20 to 400° C., at least. The absorption wavelength limit is at least 540 nm at 20° C. and increases progressively up to 700 nm at 400° C. This heating unit can provide a visible indication of whether the warmable surface is or is not yet hot when the electric power has been disconnected. Likewise, mention can be made of the Spanish Patent with Publication Number 2147513, with respect to a temperature indicator system which comprises a support which is printed with one or more thermochromic inks and which is coupled to a temperature indicator scale, which is calibrated according to the characteristic temperature of the inks mentioned. The system is based on printing the aforementioned support with a band of ink, thermochromic or not thermochromic, on which are printed in turn windows of whatever form, placed successively with a thermochromic ink of characteristic temperature different to that of the band. This indicator system is applicable in the production of devices which can be adapted to containers of products the temperature of which should be controlled in a continuous manner. Mention could also be made of the Spanish Patent with Publication Number 2137129 with respect to a procedure to obtain the chromatic variation of objects, in the presence of external stimuli and product obtained thereby. In any case, no non-electric indicators of operation of an active substance thermodiffuser device are known, based on the thermochromic property of certain products. |
Silicon compounds |
The present invention relates to a novel silicon compound characterized by having a polymerization-initiating ability to polymerizable monomers and a polymer obtained using the same. Provided is a so-called organic-inorganic composite material of cage type silsesquioxane in which an organic substituent is bonded to a silicon atom and an organic polymer. A preparing method thereof is mixing with an organic polymer or copolymerization making use of a polymerizable functional group of cage type silsesquioxane. However, it is considered to be very difficult to evenly disperse an inorganic component in an organic polymer in either method and maintain characteristics thereof. The present invention provides a silicon compound represented by Formula (1): in Formula (1), A1 is a group having a polymerization-initiating ability to a monomer; R1 is alkyl having 2 to 10 carbon atoms; in these alkyls, at least one hydrogen may be replaced by halogen; one —CH2— may be replaced by —O—; one hydrogen may be replaced by an aromatic group or an alicyclic group; this aromatic group or alicyclic group may have a substituent; R2 and R3 are independently alkyl having 1 to 8 carbon atoms, phenyl or cyclohexyl; n is an integer of 2 to 30; e is 0 or 1; a is an integer of 1 to 2n; b is an integer of 0 to (2n−1); c is an integer of 0 to (2n−1); and the total of a, b and c is 2n. The preferred examples of A1 are a group having halogenated alkylphenyl, a group having an MgBr group and a group having a dithiocarbamate group. If initiating the polymerization of, for example, an acrylic monomer with this silsesquioxane derivative acting as a starting point, a very homogeneous organic-inorganic composite material is obtained without causing coagulation of silsesquioxane, and therefore problems in conventional methods are solved. |
1. A silicon compound represented by Formula (1): wherein symbols in Formula (1) are defined as follows; A1 is a group having a polymerization-initiating ability to a monomer; R1 is alkyl having 2 to 10 carbon atoms; in these alkyls, at least one hydrogen may be replaced by halogen, one —CH2— may be replaced by —O—, and one hydrogen may be replaced by an aromatic group or an alicyclic group; and this aromatic group or alicyclic group may have a substituent; R2 and R3 are independently alkyl having 1 to 8 carbon atoms, phenyl or cyclohexyl; n is an integer of 2 to 30; e is 0or 1; a is an integer of 1 to 2n, b is an integer of 0 to (2n−1), c is an integer of 0 to (2n−1); and the total of a, b and c is 2n. 2. The silicon compound according to claim 1, wherein A1 in Formula (1) is a group selected from a group having halogenated alkylphenyl, a group having an MgBr group and a group having a dithiocarbamate group. 3. The silicon compound according to claim 1, wherein A1 in Formula (1) is the group having halogenated alkylphenyl. 4-8. (Cancelled) 9. The silicon compound according to claim 1, wherein A1 in Formula (1) is the group having a dithiocarbamate group. 10-17. (Cancelled) 18. A silicon compound obtained by polymerizing a vinyl type monomer with the compound represented by Formula (1) as described in claim 1 being used as an initiator. 19-24. (Cancelled) 25. The silicon compound according to claim 1, wherein A1 is a group represented by Formula (2): wherein symbols and the bonding positions of the substituents in Formula (2) are defined as follows; X is halogen; Z1 is alkylene having 1 to 3 carbon atoms; Z2 is alkylene having 2 to 10 carbon atoms; in these alkylenes, one —CH2— may be replaced by —O—; and a bonding position of Z1 to the benzene ring is a meta position or a para position to a bonding position of Z2; R6 is alkyl having 1 to 3 carbon atoms; and a bonding position of R6 to the benzene ring is an optional position excluding the respective bonding positions of Z1 and Z2; and d is an integer of 0 to 2. 26. The silicon compound according to claim 25, wherein Z2 in Formula (2) is Z3—C2H4—; Z3 is a single bond or alkylene having 1 to 8 carbon atoms; and in this alkylene, one —CH2— may be replaced by —O—. 27. The silicon compound according to claim 1, wherein in Formula (1), both of R2 and R3 are methyl, and n is an integer of 3 to 5. 28. The silicon compound according to claim 1, wherein in Formula (1), A1 is a group represented by Formula (2); both of R2 and R3 are methyl; a is 8; and n is 4: wherein symbols and the bonding positions of the substituents in Formula (2) are defined as follows; X is halogen; Z1 is alkylene having 1 to 3 carbon atoms; Z2 is alkylene having 2 to 10 carbon atoms; in these alkylenes, one —CH2— may be replaced by —O—; and a bonding position of Z1 to the benzene ring is a meta position or a para position to a bonding position of Z2; R6 is alkyl having 1 to 3 carbon atoms; and a bonding position of R6 to the benzene ring is an optional position excluding the respective bonding positions of Z1 and Z2; and d is 0. 29. The silicon compound according to claim 28, wherein in Formula (2), Z1 is —CH2— and Z2 is —CH2CH2—. 30. The silicon compound according to claim 1, wherein A1 is a group represented by Formula (3): wherein symbols and the bonding positions of the substituents in Formula (3) are defined as follows; R4 and R5 are independently hydrogen, alkyl having 1 to 12 carbon atoms, an alicyclic group having 5 to 10 carbon atoms or an aromatic group having 6 to 10 carbon atoms; R4 and R5 may be combined with each other to form a ring together with N; Z1 is alkylene having 1 to 3 carbon atoms; Z2 is alkylene having 2 to 10 carbon atoms; in these alkylenes, one —CH2— may be replaced by —O—; and a bonding position of Z1 to the benzene ring is a meta position or a para position to a bonding position of Z2; R6 is alkyl having 1 to 3 carbon atoms; and a bonding position of R6 to the benzene ring is an optional position excluding the respective bonding positions of Z1 and Z2; and d is an integer of 0 to 2. 31. The silicon compound according to claim 30, wherein Z2 in Formula (3) is a group represented by Z3—C2H4—; Z3 is a single bond or alkylene having 1 to 8 carbon atoms; and in this alkylene, one —CH2— may be replaced by —O—. 32. The silicon compound according to claim 1, wherein in Formula (1), A1 is a group represented by Formula (3); both of R2 and R3 are methyl, and n is an integer of 3 to 5: wherein symbols and the bonding positions of the substituents in Formula (3) are defined as follows; R4 and R5 are independently hydrogen, alkyl having 1 to 12 carbon atoms, an alicyclic group having 5 to 10 carbon atoms or an aromatic group having 6 to 10 carbon atoms; R4 and R5 may be combined with each other to form a ring together with N; Z1 is alkylene having 1 to 3 carbon atoms; Z2 is alkylene having 2 to 10 carbon atoms; in these alkylenes, one —CH2— may be replaced by —O—; and a bonding position of Z1 to the benzene ring is a meta position or a para position to a bonding position of Z2 ; R6 is alkyl having 1 to 3 carbon atoms; and a bonding position of R6 to the benzene ring is an optional position excluding the respective bonding positions of Z1 and Z2; and d is an integer of 0 to 2. 33. The silicon compound according to claim 1, wherein in Formula (1), A1 is a group represented by Formula (3); both of R2 and R3 are methyl; a is 8; and n is 4: wherein symbols and the bonding positions of the substituents in Formula (3) are defined as follows; R4 and R5 are ethyl; Z1 is alkylene having 1 to 3 carbon atoms; Z2 is alkylene having 2 to 10 carbon atoms; in these alkylenes, one —CH2— may be replaced by —O—; and a bonding position of Z1 to the benzene ring is a meta position or a para position to a bonding position of Z2; and d is 0. 34. The silicon compound according to claim 33, wherein in Formula (3), Z1 is —CH2— and Z2 is —CH2CH2—. 35. A production process for a compound represented by Formula (1-1), characterized by carrying out, in the presence of a transition metal base catalyst, any of a process in which a compound represented by Formula (5) is reacted with a compound represented by Formula (6), a process in which the compound represented by Formula (5) is reacted with the compound represented by Formula (6) and a compound represented by Formula (7) at the same time, a process in which the compound represented by Formula (5) is reacted with the compound represented by Formula (6) and then reacted with the compound represented by Formula (7), and a process in which the compound represented by Formula (5) is reacted with the compound represented by Formula (7) and then reacted with the compound represented by Formula (6): wherein symbols in Formula (1-1) are defined as follows; R2 and R3 are independently alkyl having 1 to 8 carbon atoms, phenyl or cyclohexyl; n is an integer of 2 to 30; e is 0 or 1; a is an integer of 1 to 2n, b is an integer of 0 to (2n−1), c is an integer of 0 to (2n−1), and the total of a, b and c is 2n; R7is hydrogen, halogen or alkyl having 1 to 8 carbon atoms; in these alkyls, at least one hydrogen may be replaced by halogen, one —CH2— may be replaced by —O— and one hydrogen may be replaced by an aromatic group or an alicyclic group; and this aromatic group or alicyclic group may have a substituent; B1 is a group represented by the following Formula (4): wherein symbols and the bonding positions of the substituents in Formula (4) are defined as follows; X is halogen; Z1 is alkylene having 1 to 3 carbon atoms; Z2 is alkylene having 2 to 10 carbon atoms; in these alkylenes, one —CH2— may be replaced by —O—; and a bonding position of Z1 to the benzene ring is a meta position or a para position to a bonding position of Z2; R 6is alkyl having 1 to 3 carbon atoms; and a bonding position of R6 to the benzene ring is an optional position excluding the respective bonding positions of Z1 and Z2 ; and d is an integer of 0 to 2; Z3 is a single bond or alkylene having 1 to 8 carbon atoms; in this alkylene, one —CH2— may be replaced by —O—; a bonding position of Z1 to the benzene ring is a meta position or a para position to a bonding position of Z3 ; and a bonding position of R6 to the benzene ring is an optional position excluding the respective bonding positions of Z1 and Z3; wherein R2, R3, e and n in Formula (5) each are the same as these symbols in Formula (1-1); wherein symbols and the bonding positions of the substituents in Formula (6) each are the same as the symbols and the bonding positions of the substituents in Formula (4); R7—CH═CH2 (7) wherein R7 in Formula (7) is the same as R7 in Formula (1-1). 36. A production process for a silicon compound represented by Formula (1-2), characterized by reacting the compound represented by Formula (1-1) as described in claim 35 with a compound represented by Formula (9): wherein symbols in Formula (1-2) are defined as follows; R2, R3, R7, a, b, c, e and n each are the same as these symbols in Formula (1-1) as described in claim 35; A2 is a group represented by the following Formula (8): wherein symbols and the bonding positions of the substituents in Formula (8) are defined as follows; R4 and R5 are independently hydrogen, alkyl having 1 to 12 carbon atoms, an alicyclic group having 5 to 10 carbon atoms or an aromatic group having 6 to 10 carbon atoms; R4 and R5 may be combined with each other to form a ring together with N; Z1 is alkylene having 1 to 3 carbon atoms; R6 is alkyl having 1 to 3 carbon atoms; d is an integer of 0 to 2; Z3 is a single bond or alkylene having 1 to 8 carbon atoms; in this alkylene, one —CH2— may be replaced by —O—; a bonding position of Z1 to the benzene ring is a meta position or a para position to a bonding position of Z3; and a bonding position of R6 to the benzene ring is an optional position excluding the respective bonding positions of Z1 and Z3; wherein symbols in Formula (9) are defined as follows; R4 and R5 each are the same as these symbols in Formula (8); M is a metal element of the first group or the second group in the periodic table; and p is the same value as an atomic value of M. 37. A production process for the compound represented by Formula (1-2), characterized by carrying out the following step (a) and then (b): wherein symbols in Formula (1-2) are defined as follows; R2 and R3 are independently alkyl having 1 to 8 carbon atoms, phenyl or cyclohexyl; n is an integer of 2 to 30; e is 0 or 1; a is an integer of 1 to 2n, b is an integer of 0to (2n−1), c is an integer of 0 to (2n−1), and the total of a, b and c is 2n; R7 is hydrogen, halogen or alkyl having 1 to 8 carbon atoms; in these alkyls, at least one hydrogen may be replaced by halogen, one —CH2— may be replaced by —O— and one hydrogen may be replaced by an aromatic group or an alicyclic group; and this aromatic group or alicyclic group may have a substituent; A is a group represented by the following Formula (8): wherein symbols and the bonding positions of the substituents in Formula (8) are defined as follows; R4 and R5 are independently hydrogen, alkyl having 1 to 12 carbon atoms, an alicyclic group having 5 to 10 carbon atoms or an aromatic group having 6 to 10 carbon atoms; R4 and R5 may be combined with each other to form a ring together with N; Z1 is alkylene having 1 to 3 carbon atoms; R6 is alkyl having 1 to 3 carbon atoms; d is an integer of 0 to 2; Z3 is a single bond or alkylene having 1 to 8 carbon atoms; in this alkylene, one —CH2— may be replaced by —O—; a bonding position of Z1 to the benzene ring is a meta position or a para position to a bonding position of Z3; and a bonding position of R6 to the benzene ring is an optional position excluding the respective bonding positions of Z1 and Z3; (a) a step in which a compound represented by Formula (6) is reacted with a compound represented by Formula (9) to obtain a compound represented by Formula (10): wherein symbols and the bonding positions of the substituents in Formula (6) are defined as follows; X is halogen; Z1, Z3, R6, d and the bonding positions of the substituents each are the same as these symbols and the bonding positions of the substituents in Formula (8); wherein symbols in Formula (9) are defined as follows; R4 and R5 are the same as these symbols in Formula (8); M is a metal element of the first group or the second group in the periodic table; and p is the same value as an atomic value of M; wherein symbols and the bonding positions of the substituents in Formula (10) each are the same as the symbols and the bonding positions of the substituents in Formula (8); (b) a step for obtaining the compound represented by Formula (1-2) by carrying out, in the presence of a transition metal base catalyst, any of a process in which a compound represented by Formula (5) is reacted with the compound represented by Formula (10), a process in which the compound represented by Formula (5) is reacted with the compound represented by Formula (10) and a compound represented by Formula (7) at the same time, a process in which the compound represented by Formula (5) is reacted with the compound represented by Formula (10) and then reacted with the compound represented by Formula (7), and a process in which the compound represented by Formula (5) is reacted with the compound represented by Formula (7) and then reacted with the compound represented by Formula (10): wherein R2, R3, e and n in Formula (5) each are the same as these symbols in Formula (1-2); R7—CH═CH2 (7) wherein R7 in Formula (7) is the same as R7 in Formula (1-2). 38. A silicon compound represented by Formula (11) obtained by polymerizing a vinyl type monomer with the compound represented by Formula (1-1) as described in claim 35 being used as an initiator and a transition metal complex being used as a catalyst: wherein symbols in Formula (11) are defined as follows; R2, R3, R7, a, b, c, e and n each are the same as these symbols in Formula (1-1) as described in claim 35; B2is represented by the following Formula (4-1): wherein symbols and the bonding positions of the substituents in Formula (4-1) are defined as follows; X, Z1 , R6, d, Z3 and the bonding positions of the substituents each are the same as these symbols and the bonding positions in Formula (4) as described in claim 35; and P is a polymeric chain derived from a vinyl type monomer. 39. A silicon compound represented by Formula (12) obtained by photopolymerizing a vinyl type monomer with the compound represented by Formula (1-2) as described in claim 36 being used as an initiator: wherein symbols and the bonding positions of the substituents in Formula (12) are defined as follows; R2, R3, R7, a, b, c, e and n each are the same as these symbols in Formula (1-2) as described in claim 36; A3is represented by the following Formula (8-1): wherein symbols and the bonding positions of the substituents in Formula (8-1) are defined as follows; R4 to R6, Z1, d, Z3 and the bonding positions of the substituents each are the same as these symbols and the bonding positions of the substituents in Formula (8) as described in claim 36; and P is a polymeric chain derived from a vinyl type monomer. 40. The silicon compound according to claim 38, wherein the vinyl type monomer is at least one compound selected from the group consisting of styrene derivatives and (meth)acrylic acid derivatives. 41. The silicon compound according to claim 39, wherein the vinyl type monomer is at least one compound selected from the group consisting of styrene derivatives and (meth)acrylic acid derivatives. 42. The silicon compound according to claim 38, wherein the vinyl type monomer is at least one compound selected from the group consisting of the (meth)acrylic acid derivatives. 43. The silicon compound according to claim 39, wherein the vinyl type monomer is at least one compound selected from the group consisting of the (meth)acrylic acid derivatives. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Si—H functional cage type silsesquioxane is a compound which can be synthesized by hydrolytic condensation of trialkoxysilane or trichlorosilane. A well known example is cage type silsesquioxane having 8 silicon atoms. In addition thereto, known are various cage type silsesquioxanes including cage type silsesquioxane having 10 silicon atoms and cage type silsesquioxane having 12 silicon atoms. A cage type silsesquioxane derivative having a dimethylsiloxy group is known as well. A production process for this derivative is disclosed in J. Organometallic Cem., 441 (1992), 373-, J. Chem. Soc., Chem. Comm., 4 (1989), 208-, J. Sol-Gel Science and Technology, 1 (1) (1993), 57- and Sol-Gel Science and Technology, 2 (1/2/3) (1994), 127-. Both of Si—H functional cage type silsesquioxane and a cage type silsesquioxane derivative having a dimethylsiloxy group have a structure similar to the structural units of silica and zeolite, and therefore they are expected to be applied to inorganic materials of the next generation such as precursors of organic/inorganic hybrid materials, low dielectric materials, optical crystals and liquid crystal materials. Cage type silsesquioxane in which an organic substituent such as methyl and phenyl is bonded to a silicon atom is well known as well. Those having a functional group such as 3-chloroammoniumpropyl, epoxy, vinyl ether, methacrylate and phenylpropyl as an organic substituent are known as well. In recent years, so-called organic-inorganic composite materials of organic polymers with silsesquioxane are prepared by making use of these functional groups. An example making use of, for example, an intermolecular hydrogen bond includes an example in which cage type silsesquioxane having a 3-chloroammoniumpropyl group is added to polyvinylpyrrolidone, polydimethylacrylamide, polyvinyl alcohol or polyethylene glycol. This example is reported in “Polymer Preprints, Japan”, vol. 48, No. 14, 4253 (1999). An example making use of π-π electron interaction includes an example in which phenylprpopyl group-containing cage type silsesquioxane is added to polystyrene. This example is reported in “Polymer Preprints, Japan”, vol. 48, No. 14, 4255 (1999). An organic-inorganic composite material is obtained by polymerizing cage type silsesquioxane having a polymerizable functional group such as γ-methacryloyloxypropyl. An example in which this cage type silsesquioxane is radically polymerized with azobisisobutyronitrile being used as a catalyst is described in Macromolecules, 28 (1995), 8435-. An example in which this cage type silsesquioxane is subjected to atom transfer radical copolymerization in the coexistence of other acrylic monomers is described in Macromolecules, 33 (2000), 217-. In addition thereto, it is tried to introduce polyethylene oxide by coupling reaction of Si—H functional cage type silsesquioxane with a polymer having a double bond at a terminal (hereinafter the coupling reaction of Si—H functional cage type silsesquioxane with the polymer shall be referred to as a polymer coupling method). This example is described in Langmuir, 15 (1999), 4752-. In the case of a cage type silsesquioxane derivative having a dimethylsiloxy group, reported are derivatives in which bonded to a silicon atom in the dimethylsiloxy group are hydrogen and functional groups such as a hydroxyl group (J. Am. Chem. Soc., 122 (2000), 6979-), an epoxy-containing group (Chemistry of Materials, 8 (1996), 1592-) and methacryloyloxy (Macromolecules, 29 (1996), 2327-). Organic-inorganic composite materials of organic polymers with silsesquioxane are prepared by making use of these functional groups. In the case of cage type silsesquioxane derivative having methacryloyloxy, organic-inorganic composite materials can be prepared as well by radically polymerizing this compound alone or in the presence of other acrylic monomers. Organic-inorganic composite materials making use of intermolecular interaction are obtained by mechanically blending the silsesquioxanes described above with organic polymers, and therefore aggregation of silsesquioxane is not avoided. That is, it is considered that it is very difficult in this material to evenly disperse silsesquioxane as an inorganic component. This composite material makes use of weak intermolecular interaction and is considered to have problems on a water resistance and a chemical resistance. In the case of cage type silsesquioxane having a polymerizable functional group, it is not liable to be radically polymerized in terms of a structure thereof, and therefore it is considered to be very difficult that all functional groups thereof participate in polymer formation in either case of homopolymerization and copolymerization. Accordingly, a lot of unreacted matters remain in the product, and it is presumed that aggregation of silsesquioxane which does not contribute to polymerization reaction takes place. Accordingly, it is considered to be very difficult in this method to evenly disperse an inorganic component in an organic polymer and maintain the stable characteristics. In a polymer coupling method, a polymer itself has a structure of a very low motility, and a concentration of the terminal group which contributes to the reaction is low, so that it is difficult that the whole of the polymer takes part in coupling reaction with cage type silsesquioxane. As a result thereof, it is considered that an amount of the polymer chain introduced into the inorganic component is restricted. In the foregoing conventional technique for preparing an organic-inorganic composite material, it is considered to be difficult in any case to evenly disperse the inorganic component in the organic polymer. detailed-description description="Detailed Description" end="lead"? |
Composite compositions |
There is described a composite composition comprising a layered mineral and at least one polymer precursor which is substantially miscible therewith; characterised in that: (i) the composition and/or polymer precursor are polymerisible by radiation to form a polymeric composition; and/or (ii) the polymer precursor comprises a polymer obtained and/or obtainable by polymerisation with radiation. Further described are composite compositions comprising a layered mineral and intercalated therein at least one polymer which is further polymerisable by radiation to form a polymeric network, such as a polymeric coating. Preferred minerals are nanoclays such a montmorillonite in which the counter cation comprises a radiation curable organo functional group covalently bonded thereto (e.g. an acrylate functional onium ion). Processes for making the composites are also described. |
1. A composite composition comprising a layered mineral and at least one polymer precursor which is substantially miscible therewith; characterised in that: (i) the polymer precursor is polymerisible by radiation to form a polymeric composition; and/or (ii) the polymer precursor comprises a polymer obtained and/or obtainable by polymerisation with radiation. 2. A composite composition comprising a layered mineral such as nanoclay intercalated therein at least one polymer which is further polymerisable by radiation to form a polymeric network, such as a polymeric coating. 3. A composition as claimed in claim 1, in which the layered mineral is a natural or synthetic mineral selected from: kaolins, serpentines, talcs, pyrophyllites, ferropyrophyllites; smectites montmorillonites; illites micas glauconites calcdonia; chlorites vermiculites; palygorskites; sepiolites; mixed layer minerals which may comprise two three or more mineral components in random and/or regular order; amorphous clays, high alumina clays and/or any suitable mixtures and combinations thereof. 4. A composition as claimed in claim 1, in which the layered mineral selected from: smectite, montmorillonite, bentonite; beidellite, nontronite, hectrorite, fluorohectorite, saponite, sauconite; volkhonskoite, medmontite, pimelite, stevensite, stephanite; vermiculite, and/or halloysite. 5. A composition as claimed in claim 1, in which the polymer or polymer precursor is formed from oligomers having free radical polymerisable double bonds. 6. A composition as claimed in claim 1, in which the polymer or polymer precursor comprises (meth)acrylated oligomers. 7. A composition as claimed in claim 1, in which the layered mineral comprises nano-layers. 8. A composition as claimed in claim 1, which the layered mineral is exfolliated exfoliated. 9. A composition as claimed in claim 1, in which the layered mineral comprise from about 1% to about 15% by weight of the total composition. 10. A composition as claimed in claim 1, in which the counter cation comprises an activated unsaturated moiety represented by Formula 1′ which may be univalent if a substituent on a cation (such as an onium ion) and/or may bear a positive charge if comprising the whole cation, where Formula 1′ comprises: In which n′ is 0 or 1, X′1 is oxy or, thio X′2 is oxy, thio or NR′5 (where R′5 represents H or optionally substituted organo), R′1, R′2, R′3 and R′4 each independently represent H, optional substituents and/or optionally substituted organo groups; and all suitable isomers thereof, combinations thereof on the same species and/or mixtures thereof. 11. A composition as claimed in claim 1, in which the counter cation is an optionally substituted onium ion. 12. A composition as claimed in claim 1, in which the counter cation is those represented by the formulae +(MR1R2R3R4); and/or +(PyR4); where M is nitrogen or phosphorus; R1, R2, R3, and R4 independently denote hydrogen or an optionally substituted hydrocarbo group, preferably H or hydrocarbyl optionally substituted by one or more hydroxy or carboxy group(s), more preferably H or alkyl, aryl or allyl group, most preferably at least one of which is other than hydrogen and is substituted by a hydroxy or carboxy group; and Py denotes a pyridinium or an alkyl substituted pyridinium group. 13. A composition as claimed in claim 1, in which the counter cation is represented by one or more of the following formulae in which R, R′ and R″ independently represent H or any suitable optionally substituted organo group such as optionally substituted C1-30hydrocarbyl, for example C1-20alkyl; and R′″ independently represents any suitable optionally substituted divalent organo linking group such as optionally substituted C1-30hydrocarbylene, for example C1-20alkylene, and where optionally the OH or COOH in these formulae may be replaced by any one of the moieties described in claim 15. 14. A composition as claimed in claim 1, in which the counter cation is selected from the group consisting of: where HT denotes a hydrogenated tallow residue comprising the following mixture of linear alkyl groups in the proportions about 65% C18alkyl, about 30% C16alkyl and about 5% C14alkyl. 15. A composition as claimed in claim 1, in which the counter cation comprises at least one moiety, which may comprises an optional substituent on the cation and/or replace any hydroxy and/or carboxy thereon, the moiety selected from group consisting of: where L independently denotes any suitable divalent organic linking group and R independently denotes in each instance within each moiety any suitable optionally substituted hydrocarbo group. 16. A composition as claimed in claim 1, in which the counter cation comprises at least one moiety, which may comprises an optional substituent on the cation and/or replace any hydroxy and/or carboxy thereon, the moiety selected from group consisting of: the arrow denotes a covalent bond with the remaining counter cation, a dashed double line indicates a carbon to carbon bond which may be a single or double bond; and/or any of these moieties may be further optionally substituted. 17. A composition as claimed in claim 1, in which the counter cation can be further modified to be polymerisable by radiation. 18. A process for preparing a nanoclay as claimed in claim 1 comprising the step of: (a) replacing cations in a nanoclay with an onium ion functionalised with at least one hydroxy group, (b) reacting the onium ion with an (optionally substituted) reagent selected from: (i) maleic or succinic anhydride followed by an (poly)oxirane e.g. glycidyl methacrylate (ii) (poly)isocyanate (such as those comprising at least one R—NCO); optionally in the absence of additional catalyst for urethanisation (e.g. omit dibutyltindilaurate (DBTL)); (iii) methacrylate isocyanate optionally in the absence of additional catalyst for urethanisation (e.g. omit dibutyltindilaurate (DBTL)); (iv) (meth)acrylate where it is preferred that R′ is H or alkyl; (v) a (poly)oxirane (vi) a polycarboxylic acid 19. A process for preparing a nanoclay as claimed in claim 18 comprising the step of (a) replacing cations in a nanoclay with an onium ion functionalised with at least one carboxy group (b) reacting the onium ion with an optionally substituted cyclo(alkoxy) (meth)acrylate (c) reacting the onium salt with an (poly)oxirane (d) reacting the onium salt with a polyol 20. A process as claimed in claim 19 in which the cyclo(alkoxy) (meth)acrylate is of formulae where L independently denotes any suitable divalent organic linking group and R independently denotes in each instance any suitable optionally substituted hydrocarbo group. 21. A process as claimed in claim 20 in which the cyclo(alkoxy)(meth) acrylate is an oxirane (alkyl)(meth)acrylate. 22. A process as claimed in claim 21 in which the oxirane (alkyl)(meth)acrylate is an epoxy (meth)acrylate such as either glycidyl methacrylate 23. A process for preparing a nanoclay as claimed in claim 18 comprising the steps of (a) protonating and/or alkylating an amino functional radiation polymerisable oligomer by radiation to form a cation thereof (b) ion exchanging the oligomer cation with a cation from a nanoclay to form a clay intercalated with cationic oligomer as counter ion. 24. A process as claimed in claim 23, where the oligomer is an amino (meth)acrylate. 25. A process as claimed in claim 18 which comprises further step of exfoliating the clay. 26. A process as claimed in claim 18 in which the steps are carried out in a single container without intermediate isolation or purification steps. 27. A process as claimed in claim 23 which comprises further step of exfoliating the clay. 28. A nanoclay obtained and/or obtainable by a process as claimed in claim 18 in which the mineral layers are intercalated with polymer. 29. A nanoclay as claimed in claim 28 in which the mineral layers are substantially completely exfoliated. 30. A nanoclay, mineral, coating and/or composition as claimed in claim 28 which is substantially free of tin catalyst. 31. A substrate coated with a film comprising a composition as claimed claim 1. |
Preparation of polymer particles |
A process for the preparation of polymer magnetic particles, which comprises: providing polymer particles having a porous interior, and contacting the polymer particles with a magnetic fluid comprising a homogeneous dispersion of magnetic particles, whereby the magnetic particles are incorporated into the porous interior to produce polymer magnetic particles. |
1. A process for the preparation of polymer magnetic particles, which comprises: (a) providing polymer particles having a porous interior; and (b) contacting the polymer particles with a magnetic fluid comprising a homogeneous dispersion of magnetic particles, whereby the magnetic particles are incorporated into the porous interior to produce polymer magnetic particles. 2. A process for the preparation of polymer magnetic particles, which comprises: (a) providing polymer particles having a porous interior; and (b) contacting the polymer particles with a magnetic fluid comprising a homogeneous dispersion of magnetic particles, whereby the magnetic particles are incorporated into the porous interior to produce polymer magnetic particles, and wherein the polymer particles are dry when contacted with the magnetic fluid. 3. A process according to claim 2, wherein the magnetic fluid enters the porous interior by capillary action. 4. A process according to claim 1, wherein the polymer particles are contacted with the magnetic fluid at sub-atmospheric pressure. 5. A process according to claim 1, wherein the polymer particles are dispersible in an aqueous medium. 6. A process according to claim 5, wherein the porous interior of the polymer particles bears hydrophilic groups. 7. A process according to claim 6, wherein the hydrophilic groups are selected from one or more of esters, amines, alcohols, carboxylic acid, amides, halides, aldehydes, keto compounds, imines, nitro compounds, thiols, thioethers, nitrites, acid anhydrides and sulfonic compounds. 8. A process according to claim 1, wherein the porous interior of the polymer particles bears charged groups and the magnetic particles bear a charge opposite to that of the charged groups of the porous interior. 9. A process according to claim 8, wherein the magnetic particles are positively charged. 10. A process according to claim 9, wherein the charged groups of the porous interior comprise sulphonate groups, phosphonate groups, carboxylate groups, nitrate groups, or inorganic bound species of carbonates or chlorates. 11. A process according to claim 9, wherein the magnetic particles are negatively charged. 12. A process according to claim 11, wherein the charged groups of the porous interior comprise imine groups, ammonia groups, ammonium groups, phosphonium groups, or sulphonium groups. 13. A process according to claim 1, wherein the magnetic fluid comprises a ferrofluid. 14. A process according to claim 1, wherein the magnetic particles have a maximum dimension in the range of 1 to 50 nm. 15. A process according to claim 14, wherein the maximum dimension is less than 10 nm. 16. A process according to claim 1, wherein the polymer particles have a pore volume in the range 30 to 90%. 17. A process according to claim 16, wherein the pore volume is no greater than 75%. 18. A process according to claim 1, wherein the porous interior of the polymer particles comprises small pores with a radius in the range 1 to 10 nm. 19. A process according to claim 1, wherein the porous interior of the polymer particles comprises large pores with a radius in the range 100 to 500 nm. 20. Polymer magnetic particles obtained by the process according to claim 1 or 2, in which the magnetic particles are substantially uniformly distributed. 21. A process for the preparation of polymer magnetic particles, which comprises: (a) providing polymer particles having a porous interior; and (b) contacting the polymer particles with a magnetic fluid comprising a homogeneous dispersion of magnetic particles, whereby the magnetic particles are incorporated into the porous interior to produce polymer magnetic particles, wherein the magnetic fluid comprises a ferrofluid. 22. A method of separating a target substance from a sample using the polymer magnetic particles according to claim 20. |
<SOH> BACKGROUND TO THE INVENTION <EOH>Various types of separation media are available for use in isolating target substances in chemical or biological samples. In molecular biology, various materials are used in fractionation procedures, including agarose and polyacrylamide in electrophoretic fractionation, and gel permeation, ion exchange and affinity materials for chromatography. Among such materials, both organic polymer and silicon-based particles find use in separations of targets such as nucleic acids. In each type of separation there is a need at some point physically to remove a liquid phase from the particles in the solid phase. This may be achieved by immobilising the particles, for example in the form of a column or on a gel plate, and eluting the liquid phase or by aggregating the particles by applying a force under centrifugation or using magnetism. The use of magnetism to separate particles from other sample components requires the particles to respond to a magnetic field. Because organic or silicon-based materials are not themselves magnetically responsive, there is a need to devise methods of production of magnetic particles which incorporate both a magnetic component and a polymer component. WO83/03920 describes preparation of polymer magnetic particles in which iron is introduced into porous polymer particles in solution. Non-magnetic iron salts are mixed with the porous particles and the iron is subsequently precipitated in the pores as magnetite deposits. In order to achieve this, there is a requirement that an additional step comprising an oxidation reaction be carried out in situ to convert the non-magnetic iron into magnetite. More recently, U.S. Pat. No. 5,648,124 describes a process for preparing magnetically responsive micro particles which are presently available commercially from Seradyn Inc. According to this process, a non-porous core particle is provided onto which is deposited magnetite from a ferrofluid in the presence of a heterocoagulant. A coating of magnetite is deposited on the surface of the core. In order to achieve a sufficiently high concentration of magnetite, there is a need in this process to use the heterocoagulant, which acts as a binding agent to enable further magnetite to be deposited on the coated core. The particles obtained by this process have a further disadvantage that the magnetic component therein is not uniformly distributed. The present invention aims to overcome the drawbacks of the prior art by providing a simplified process for the production of useful polymer magnetic particles. |
<SOH> SUMMARY OF THE INVENTION <EOH>Accordingly, in a first aspect, the present invention provides a process for the preparation of polymer magnetic particles, which comprises: (a) providing polymer particles having a porous interior; and (b) contacting the polymer particles with a magnetic fluid comprising a homogeneous dispersion of magnetic particles, whereby the magnetic particles are incorporated into the porous interior to produce polymer magnetic particles. It has surprisingly been found that magnetic particles can be incorporated into the porous interior of polymer particles in a stable manner without the need for additional chemical reactions and produce polymer magnetic particles containing sufficiently high concentrations of magnetic particles for use in separating the target substance from a sample. Advantageously, the polymer particles are contacted with the magnetic fluid at sub-atmospheric pressure. Use of the reduced pressure is found to promote entry of the magnetic fluid into the porous interior of the polymer particles. It is preferred that the polymer particles are dry when contacted with the magnetic fluid particularly so that the magnetic fluid enters the porous interior by capillary action and may do so in a relatively short time. Entry of the magnetic fluid into the porous interior by capillary action is promoted where the porous interior of the polymer particles is wettable by the liquid phase in which the particles are suspended. When the polymer particles are wet table in this way, they are dispersible in an aqueous medium. This may be achieved if the porous interior of the polymer particles bears hydrophilic groups. Hydrophilic groups may be borne by polymer particles containing both hydrocarbon and heteroatomic structures. Among those hydrophilic groups useful according to the present invention may be included esters, amines, alcohols, carboxylic acid, amides, halides, aldehydes, keto componds, imines, nitro componds, thiols, thioethers, nitriles, acid anhydrides and sulfonic compounds. It is preferred that the polymer particles have a porous interior of which more than 60% bears hydrophilic groups which may be charged or uncharged. In one embodiment, it is preferred that the porous interior of the polymer particles bears charge groups and the magnetic particles bear a charge opposite to that of the charge groups in the porous interior. Charged hydrophilic groups are particularly useful. The magnetic particles bear a charge opposite to that of the charge borne by the porous interior so that ionic interactions between the two charges enable the magnetic particles to be incorporated in a stable manner. In one aspect, the magnetic particles are positively charged and this is conveniently achieved by preparing the magnetic fluid to have an acidic pH, generally in the range 1 to 6, preferably in the range 1 to 3. According to this aspect, the charged groups of the porous interior are negatively charged. Among negatively charged functional groups may be mentioned sulphonates, phosphonates, carboxylates, nitrates, and inorganic bound species of carbonates and chlorates. In a further aspect, the magnetic particles are negatively charged and this is conveniently achieved by preparing the magnetic fluid to have a basic pH, generally in the range 8 to 14. According to this aspect, the charged groups of the porous interior are positively charged. Among positively charged groups or chemical groups with the potential to be so, may be mentioned imines, ammonia, ammonium, phosphonium and sulphonium. The groups of the porous interior may comprise any suitable functional groups which are readily incorporated into the polymer structure by derivatisation or as monomers or co monomers or via the adsorption of monomers, co monomers or polymers consisting of these and other chemical groups into a pre-formulated porous interior. The magnetic fluid of the invention comprises a homogeneous dispersion of magnetic particles which are not in solution and are therefore not required to be subjected to further chemical reaction to render them magnetic or to retain them in the porous interior of the polymer particles. The magnetic particles are magnetic in the sense that they are capable of being magnetised in the presence of a magnetic field but are not themselves magnetic in the absence of such a field at the operational temperature of the polymer magnetic particles. Such magnetic materials include paramagnetic and superparamagnetic materials. They also include ferromagnetic and ferrimagnetic materials, provided that they have a sufficiently low Curie temperature that, in operation, the polymer magnetic particles are not magnetic in the absence of an applied magnetic field. Typical materials include magnetic metal oxides especially the iron oxides. Useful magnetic metal oxides include iron oxides in which, optionally, all or a part of the ferrous iron thereof is substituted by a divalent transition metal such as cadmium, chromium, cobalt, copper, magnesium, manganese, nickel, vanadium, and/or zinc. It is preferred that the magnetic fluid is provided as a ferrofluid. The magnetic fluids useful in the present invention particularly include those which have magnetic particles of maximum dimension typically up to about 50 nm, advantageously up to about 25 nm and preferably in the range of 1 to less than 10 nm. Particles of this size are able to be dispersed in any of the small pores of the polymer particles. This leads to a very high iron loading capacity for the particles and a uniform distribution of magnetic particles throughout the polymer particles. The polymer particles may be organic polymer particles or inorganic polymer particles. Typically, the polymer particles have a pore volume in the range 30 to 90%, preferably in the range 30 to 75%. The polymer particles may be prepared by known methods, including those described in WO83/03920. This includes the use of vinyl monomers and polyvinyl monomers and mixtures thereof. Vinyl monomers include styrene, styrene derivatives, maleic anhydride, acrylates, methacrylates and vinyl esters. Where high ligand binding to the polymer particles is required, particles must be produced with a high specific surface area. This can be achieved using prior art methodology only if a relatively small particle size is used. A disadvantage in use of such small particles is that they separate only very slowly in an applied magnetic field. If larger particles are used instead, a much bigger pore size is required. This is not readily achievable with prior art methodology because the deposition of magnetite crystals in large pores gives rise to residual magnetisation or remanence in use. In contrast, the present invention provides a controllable method capable of application to both large and small polymer particles. It is possible to make particles with controlled pore size distribution. Relatively small pores mainly in the range 1 to 20 nm, preferably in the range 1 to 15 nm and especially in the range 1 to 10 nm can be made in addition to or alternatively to relatively large pores of radius 100 to 500 nm. Using a magnetic fluid according to the present invention, magnetic particles would tend to enter the small pores by capillary action in preference to the large pores. In this way there is no problem with remanence and relatively large polymer particles can contain high concentrations of magnetic particles evenly distributed throughout whilst leaving the large pores for ligand binding. The polymer magnetic particles obtainable by the present process in which the magnetic component is substantially uniformly distributed are thought to be superior to those polymer magnetic particles presently available. Using light microscopy it is found that magnetic polymer particles currently available incorporate magnetite or other metal oxides only in an aggregated non-homogenous form as visible metal cores coated with layers of silica. In contrast, the present invention provides a magnetic component which is homogeneously and uniformly distributed throughout the particle matrix. This has an important advantage over the prior art magnetic particles in providing a uniform and potentially rapid response to a magnetic field when in use. This makes possible aggregation of the particles at a uniform speed onto the surface by application of a magnetic field; this is an essential property when using such particles, for example, in an automated separation system. detailed-description description="Detailed Description" end="lead"? |
Method and nucleic acids for the analysis of colorectal cell proliferative disorders |
The invention provides methods and nucleic acids for detecting, differentiating or distinguishing between colon cell proliferative disorders as well as therapy thereof by analysis of the gene EYA4 and its promoter and regulatory sequences. The invention further provides novel nucleic acid sequences useful for the cell proliferative disorder specific analysis of said gene as well as methods, assays and kits thereof. |
1. A method of diagnosing a colon cell proliferative disorder in a subject, comprising the steps of: (a) obtaining one or more test samples from colon tissue or serum or both of said subject; and (b) detecting a decrease in the amount or expression of a polypeptide expressed from the EYA4 gene 2. The method of claim 1, wherein said colon cell proliferative disorders are taken from the group comprising adenocarcinomas, squamous cell cancers, carcinoid tumours, sarcomas, and lymphomas. 3. The method of claim 1 or 2, wherein said detection is by immunoassay, in particular by an ELISA. 4. The method of claim 3, wherein said immunoassay is a radioimmunoassay. 5. A method of diagnosing a colon cell proliferative disorder in a subject, comprising the steps of: a) obtaining one or more test samples from colon tissue or serum or both of said subject; and b) contacting said sample with an antibody immunoreactive with the EYA4 polypeptide to form an immunocomplex; c) detecting said immunocomplex; d) comparing the quantity of said immunocomplex to the quantity of immunocomplex formed under identical conditions with the same antibody and a control sample from one or more subjects known not to have colon cancer; wherein a decrease in quantity of said immunocomplex in the sample from said subject relative to said control sample is indicative of colon cancer. 6. The method of claim 5, wherein said immunocomplex is detected in a Western blot assay. 7. The method of claim 5, wherein said immunocomplex is detected in an ELISA. 8. The method of claim 1, wherein said detection is by expression analysis. 9. The method of claim 8, comprising detecting the presence or absence of mRNA encoding a EYA4 polypeptide in a sample from a patient, wherein a decreased concentration of said mRNA above the concentration for a healthy individual indicates the presence of colon cell proliferative disorder cells. 10. The method of claim 8, comprising the steps of: (a) providing a polynucleotide probe which specifically hybridises or is identical to a polynucleotide consisting of SEQ ID NO: 1, (b) incubating said sample with said polynucleotide probe under high stringency conditions to form a specific hybridisation complex between an mRNA and said probe; (c) detecting said hybridisation complex. 11. The method according to claim 10 wherein the detecting step further comprises the steps of: a) producing a cDNA from mRNA in the sample; b) providing two oligonucleotides which specifically hybridise to regions flanking a segment of the cDNA; c) performing a polymerase chain reaction on the cDNA of step a) using the oligonucleotides of step b) as primers to amplify the cDNA segment; and d) detecting the amplified cDNA segment. 12. A method for repressing transformation in a colon cell, the method comprising contacting said cell with a EYA4 polypeptide in an amount effective to inhibit a transformed phenotype. 13. The method of claim 12, wherein said EYA4 polypeptide is introduced into said cell by the direct introduction of said EYA4 polypeptide. 14. The method of claim 12, wherein said EYA4 polypeptide is introduced into the cell through the introduction of a EYA4-encoding polynucleotide. 15. The method of claim 14, wherein said EYA4-encoding polynucleotide further comprises control sequences operatively linked to said EYA4 encoding polynucleotide. 16. The method of claim 14, wherein said EYA4-encoding polynucleotide is present in a vector. 17. Use of the vector according to claim 16 for therapy of colon cell proliferative disorders. 18. A method for preventing or treating a colon cell proliferative disorder in a subject which comprises administering to the subject a therapeutically effective amount of a compound that agonises EYA4. 19. Use of a polypeptide expressed from the EYA4 gene for detecting, differentiating or distinguishing between colon cell proliferative disorders. 20. Use of a polypeptide expressed from the EYA4 gene for therapy of colon cell proliferative disorders. 21. The method of claim 8, wherein said detection comprises methylation analysis of the gene EYA 4, its promoter and/or regulatory elements, in particular through the methylation analysis of a genomic DNA sequence according to SEQ ID NO: 1. 22. A nucleic acid comprising a sequence at least 18 bases in length of a segment of the chemically pretreated genomic DNA according to one of the sequences taken from the group comprising SEQ ID NO: 2 to SEQ ID NO: 5 and sequences complementary thereto. 23. An oligomer, in particular an oligonucleotide or peptide nucleic acid (PNA)-oligomer, said oligomer comprising in each case at least one base sequence having a length of at least 9 nucleotides which is complementary to, or hybridises under moderately stringent or stringent conditions to a pretreated genomic DNA according to one of the SEQ ID NO: 2 to SEQ ID NO: 5 and sequences complementary thereto. 24. The oligomer as recited in claim 23; wherein the base sequence includes at least one CpG, TpG or CpA dinucleotide. 25. The oligomer as recited in claim 24; characterised in that the cytosine of the CpG dinucleotide is located approximately in the middle third of the oligomer. 26. A set of oligomers, comprising at least two oligomers according to any of claims 23 to 25. 27. A set of at least two oligonucleotides as recited in claims 23 to 26, which can be used as primer oligonucleotides for the amplification of DNA sequences of one of SEQ ID NO: 2 to SEQ ID NO: 5 and sequences complementary thereto. 28. A set of oligonucleotides as recited in claim 23, characterised in that at least one oligonucleotide is bound to a solid phase. 29. Use of a set of oligomer probes comprising at least ten of the oligomers according to any of claims 24 to 28 for detecting the cytosine methylation state and/or single nucleotide polymorphisms (SNPs) within one of the sequences according to SEQ ID NO: 1, and sequences complementary thereto. 30. A method for manufacturing an arrangement of different oligomers (array) fixed to a carrier material for analysing diseases associated with the methylation state of the CpG dinucleotides of one of SEQ ID NO: 1, and sequences complementary thereto wherein at least one oligomer according to any of the claims 23 through 26 and 28 is coupled to a solid phase. 31. An arrangement of different oligomers (array) obtainable according to claim 30. 32. An array of different oligonucleotide- and/or PNA-oligomer sequences as recited in claim 31, characterised in that these are arranged on a plane solid phase in the form of a rectangular or hexagonal lattice. 33. The array as recited in any of the claims 31 or 32, characterised in that the solid phase surface is composed of silicon, glass, polystyrene, aluminium, steel, iron, copper, nickel, silver, or gold. 34. A composition of matter comprising the following: A nucleic acid comprising a sequence at least 18 bases in length of a segment of the chemically pretreated genomic DNA according to one of the sequences taken from the group comprising SEQ ID NO: 1 to SEQ ID NO: 5 and sequences complementary thereto, and A buffer comprising at least one of the following substances: 1 to 5 mM Magnesium Chloride, 100-500 μM dNTP, 0.5-5 units of taq polymerase, an oligomer, in particular an oligonucleotide or peptide nucleic acid (PNA)-oligomer, said oligomer comprising in each case at least one base sequence having a length of at least 9 nucleotides which is complementary to, or hybridises under moderately stringent or stringent conditions to a pretreated genomic DNA according to one of the SEQ ID NO: 2 to SEQ ID NO: 5 and sequences complementary thereto. 35. Use of the gene EYA 4, its promoter and/or regulatory elements for detecting, differentiating or distinguishing between colon cell proliferative disorders. 36. A method for detecting, differentiating or distinguishing between colon cell proliferative disorders according to claim 21, comprising: a) obtaining, from a subject, a biological sample having subject genomic DNA; b) treating the genomic DNA, or a fragment thereof, with one or more reagents to convert 5-position unmethylated cytosine bases to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridisation properties; c) contacting the treated genomic DNA, or the treated fragment thereof, with an amplification enzyme and at least two primers comprising, in each case a contiguous sequence at least 18 nucleotides in length that is complementary to, or hybridises under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NOS: 2 to 5, and complements thereof, wherein the treated DNA or a fragment thereof is either amplified to produce one or more amplificates, or is not amplified; and d) determining, based on the presence or absence of, or on a property of said amplificate, the methylation state of at least one CpG dinucleotide sequence of SEQ ID NO: 1, or an average, or a value reflecting an average methylation state of a plurality of CpG dinucleotide sequences of SEQ ID NO: 1. 37. A method for detecting, differentiating or distinguishing between colon cell proliferative disorders according to claim 21, comprising the following steps of a) obtaining, from a subject, a biological sample having subject genomic DNA; b) treating the genomic DNA, or a fragment thereof, with one or more reagents to convert 5-position unmethylated cytosine bases to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridisation properties; c) amplifying one or more fragments of the treated DNA such that only DNA originating from colon or colon cell proliferative disorder cells are amplified d) detecting the amplificates or characteristics thereof and thereby deducing on the presence or absence of a colon cell proliferative disorder. 38. The method of one of claims 36 or 37, wherein in step a) the biological sample obtained from the subject is selected from the group consisting of histological slides, biopsies, paraffin-embedded tissue, bodily fluids, serum, plasma, stool, urine, blood, and combinations thereof. 39. The method of one of claims 36 to 38, wherein in step b) treating the genomic DNA, or the fragment thereof, comprises use of a solution selected from the group consisting of bisulfite, hydrogen sulfite, disulfite, and combinations thereof. 40. The method of one of claims 36 to 39, wherein treating in b) is subsequent to embedding the DNA in agarose. 41. The method of one of claims 36 to 40, where treating in b) comprises treating in the presence of at least one of a DNA denaturing reagent or a radical trap reagent. 42. The method of one of claims 36 to 41, wherein contacting or amplifying in step c) comprises use of at least one method selected from the group consisting of: use of a heat-resistant DNA polymerase as the amplification enzyme; use of a polymerase lacking 5′-3′ exonuclease activity; use of a polymerase chain reaction (PCR); generation of a amplificate nucleic acid molecule carrying a detectable labels; and combinations thereof. 43. The method of claim 42, wherein the detectable amplificate label is selected from the label group consisting of: fluorescent labels; radionuclides or radiolabels; amplificate mass labels detectable in a mass spectrometer; detachable amplificate fragment mass labels detectable in a mass spectrometer; amplificate, and detachable amplificate fragment mass labels having a single-positive or single-negative net charge detectable in a mass spectrometer; and combinations thereof. 44. The method of claim 43, comprising in step d) use of mass spectrometry for detecting the amplificate, or detachable amplificate fragment mass labels. 45. The method of claim 44, wherein the mass spectrometry technique is selected from the group consisting of matrix assisted laser desorption/ionisation mass spectrometry (MALDI), electron spray mass spectrometry (ESI), and combinations thereof. 46. The method according to one of claims 36 to 45 wherein in step c) of the method 6 or more different fragments are amplified. 47. The method according to one of claims 36 to 46 wherein said amplificates are between 100 and 200 base pairs in length. 48. The method according to one of claims 36 to 47 wherein said amplificates are between 100 and 350 base pairs in length. 49. The method according to one of claims 36 to 48 wherein one or more of said primers comprise sequences taken from the group according to SEQ ID NO: 11 to SEQ ID NO: 15. 50. The method according to one of claims 36 to 48 wherein one or more of said primers comprise one or more CpG, TpG or CpA dinucleotides. 51. The method of claim 50 wherein said primers comprise between two to four CpG, TpG or CpA dinucleotides. 52. The method according to one of claims 50 or 51 wherein said one or more CpG, TpG or CpA dinucleotides are located within the 3′ half of the primer. 53. The method according to one of claims 50 to 52 wherein said primers comprise one or more bases which hybridise to positions that were converted in the treatment of step b). 54. The method of claim 53 wherein said bases are located within the 3′ half of the primer. 55. The method according to one of claims 50 to 54 wherein said primers do not comprise more than 2 cytosine or guanine bases within the first 5 bases at the 3′ end. 56. The method according to one of claims 36 to 55 wherein said amplificates obtained in step d) comprise at least one 20 base pair sequence that comprises 3 or more CpG, TpG or CpA dinucleotides. 57. The method according to one of claims 36 to 56, further comprising in step c) the use of at least one nucleic acid molecule or peptide nucleic acid molecule at least 18 base pairs in length comprising one or more CpG, TpG or CpA dinucleotides and wherein the sequence of said molecule is complementary or identical to a sequence selected from the group consisting of SEQ ID NOS: 2 to 5, and complements thereof, and wherein said nucleic acid molecule or peptide nucleic acid molecule suppresses amplification of the nucleic acid to which it is hybridised. 58. The method according to claim 57 wherein the sequence of said nucleic acid(s) or peptide nucleic acid(s) is selected from the group consisting SEQ ID NO: 6 to SEQ ID NO: 10, and sequences complementary thereto. 59. The method of claim 57, wherein amplification of DNA that was unmethylated prior to treatment of step b) is suppressed. 60. The method of one of claims 57 to 59, wherein said nucleic acid molecule or peptide nucleic acid molecule is in each case modified at the 5′-end thereof to preclude degradation by an enzyme having a 5′-3′ exonuclease activity. 61. The method of one of claims 57 to 60, wherein said nucleic acid molecule or peptide nucleic acid molecule in each case lack a 3′ hydroxyl group. 62. The method of one of claims 57 to 61, wherein the amplification enzyme is a polymerase lacking 5′-3′ exonuclease activity. 63. The method of one of claims 57 to 62, wherein the binding site of the oligonucleotide or PNA oligomer is identical to, or overlaps with that of the primer and thereby hinders hybridisation of the primer to its binding site. 64. The method of one of claims 57 to 63, wherein the binding sites of at least two of the oligonucleotides or PNA oligomers are identical to, or overlap with those of at least two of the primers, and thereby hinder hybridisation of the primers to their binding site. 65. The method of claim 64, wherein hybridisation of at least one of the oligonucleotides or peptide nucleic acid oligomers hinders hybridisation of a forward primer, and the hybridisation of at least one of the oligonucleotides or peptide nucleic acid oligomers hinders the hybridisation of a reverse primer that binds to the elongation product of said forward primer. 66. The method of one of claims 57 to 64, wherein said oligonucleotide or peptide nucleic acid oligomer hybridises between the binding sites of the forward and reverse primers. 67. The method of one of claims 36 or 37, wherein determining in step d), comprises hybridisation of at least one nucleic acid molecule or peptide nucleic acid (PNA) molecule in each case comprising a contiguous sequence at least 9 nucleotides in length comprising one or more CpG, TpG or CpA dinucleotides and wherein the sequence of said molecule that is complementary or identical to a sequence selected from the group consisting of SEQ ID NOS: 2 to 5, and complements thereof. 68. The method of claim 67, wherein at least one such hybridising nucleic acid molecule or peptide nucleic acid molecule is bound to a solid phase. 69. The method of claim 68, wherein a plurality of such hybridising nucleic acid molecules or peptide nucleic acid molecules are bound to a solid phase in the form of a nucleic acid or peptide nucleic acid array selected from the array group consisting of linear, hexagonal, rectangular, and combinations thereof. 70. The method of one of claims 36 or 37, wherein determining in step d), comprises sequencing of the amplificate. 71. The method of one of claims 36 or 37, wherein determining in step d), comprises: hybridising at least one nucleic acid molecule comprising a contiguous sequence at least 9 nucleotides in length that is complementary to, or hybridises under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NOS: 2 to 5, and complements thereof; and extending at least one such hybridised nucleic acid molecule by at least one nucleotide base. 72. The method according to claim 71 wherein the sequence of said nucleic acid(s) or peptide nucleic acid(s) is selected from the group consisting SEQ ID NO: 11 to SEQ ID NO: 15, and sequences complementary thereto. 73. The method according to claim 67 wherein said oligonucleotides or PNA-oligomers are fluorescently labelled, and wherein detection thereof is by either an increase or a decrease in fluorescence or fluorescence polarisation. 74. The method according to claim 73 wherein the hybridisation of the oligonucleotides or PNA oligomers is detectable by fluorescence resonance energy transfer, and wherein the detection is by either an increase or a decrease in fluorescence. 75. The method of one of claims 36 or 37, wherein the background DNA concentration is at between 100 to 1000 fold excess of the concentration of the DNA to be investigated. 76. A method for detecting a colon cell proliferative disorder according to claim 21, comprising: a) obtaining, from a subject, a biological sample having subject genomic DNA; b) extracting the genomic DNA; c) contacting the genomic DNA, or a fragment thereof, comprising SEQ ID NO:1 or a sequence that hybridises under stringent conditions to SEQ ID NO:1, with one or more methylation-sensitive restriction enzymes, wherein the genomic DNA is either digested thereby to produce digestion fragments, or is not digested thereby; and d) determining, based on a presence or absence of, or on property of at least one such fragment, the methylation state of at least one CpG dinucleotide sequence of SEQ ID NO: 1, or an average, or a value reflecting an average methylation state of a plurality of CpG dinucleotide sequences of SEQ ID NO: 1, whereby at least one of detecting the prostate cell proliferative disorder, or distinguishing between a transitional and a peripheral zone of origin of the prostate cell proliferative disorder is, at least in part, afforded. 77. The method of claim 76, further comprising, prior to determining in step d), amplifying of the digested or undigested genomic DNA. 78. The method of claim 77, wherein amplifying comprises use of at least one method selected from the group consisting of: use of a heat resistant DNA polymerase as an amplification enzyme; generation of a amplificate nucleic acid carrying a detectable label; and combinations thereof. 79. The method of claim 78, wherein the detectable amplificate label is selected from the label group consisting of: fluorescent labels; radionuclides or radiolabels; amplificate mass labels detectable in a mass spectrometer; detachable amplificate fragment mass labels detectable in a mass spectrometer; amplificate, and detachable amplificate fragment mass labels having a single-positive or single-negative net charge detectable in a mass spectrometer; and combinations thereof. 80. The method of claim 79, comprising use of mass spectrometry for detecting amplificate, or detachable amplificate fragment mass labels. 81. The method of claim 80, wherein the mass spectrometry is selected from the group consisting of matrix assisted laser desorption/ionisation mass spectrometry (MALDI), electron spray mass spectrometry (ESI), and combinations thereof. 82. The method of claim 76, wherein the biological sample obtained from the subject is selected from the group consisting of histological slides, biopsies, paraffin-embedded tissue, bodily fluids, urine, serum, plasma, stool, blood, and combinations thereof. 83. A kit useful for detecting, differentiating or distinguishing between colon cell proliferative disorders, comprising: a) a bisulfite reagent; and b) at least one nucleic acid molecule or peptide nucleic acid molecule comprising, in each case a contiguous sequence at least 9 nucleotides in length that is complementary to, or hybridises under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NOS: 1 to 5, and complements thereof. 84. The kit of claim 83, further comprising standard reagents for performing a methylation assay selected from the group consisting of MS-SNuPE, MSP, MethylLight™, HeavyMethyl™, COBRA, nucleic acid sequencing, and combinations thereof. 85. The use of a nucleic acid according to claim 22, of an oligonucleotide or PNA-oligomer according to one of the claims 23 to 28, of a kit according to claims 83 or 84, of an array according to one of the claims 32 to 33, of a set of oligonucleotides according to one of claims 26 to 28, or a method according to claims 36 to 82, for the classification, differentiation and/or diagnosis of colon cell proliferative disorders or the predisposition to colon cell proliferative disorders. 86. The use of a nucleic acid according to claim 22, of an oligonucleotide or PNA-oligomer according to one of the claims 23 to 28, of a kit according to claims 83 or 84, of an array according to one of the claims 32 to 33, of a set of oligonucleotides according to one of claims 26 to 28, or a method according to claims 36 to 82, for the therapy of colon cell proliferative disorders. |
<SOH> FIELD OF THE INVENTION <EOH>Colorectal cancer is the fourth leading cause of cancer mortality in men and women. The 5-year survival rate is 61% over all stages with early detection being a prerequisite for curative therapy of the disease. Up to 95% of all colorectal cancers are adenocarcinomas of varying differentiation grades. Sporadic colon cancer develops in a multistep process starting with the pathological transformation of normal colonic epithelium to an adenoma which consecutively progresses to invasive cancer. The progression rate of colonic adenomas is currently predicted based on their histological appearance, location, degree of spread and extent of bowel involvement. For example, tubular-type benign adenomas rarely progress to malignant tumours, whereas villous benign adenomas, particularly if larger than 2 cm in diameter, have a significant malignant potential. During progression from benign proliferative lesions to malignant neoplasms several genetic and epigenetic alterations are known to occur. Somatic mutation of the APC gene seems to be one of the earliest events in 75 to 80% of colorectal adenomas and carcinomas. Activation of K-RAS is thought to be a critical step in the progression towards a malignant phenotype. Consecutively, mutations in other oncogenes as well as alterations leading to inactivation of tumour suppressor genes accumulate. Aberrant DNA methylation within CpG islands is among the earliest and most common alterations in human cancers leading to abrogation or overexpression of a broad spectrum of genes. In addition, abnormal methylation has been shown to occur in CpG rich regulatory elements in intronic and coding parts of genes for certain tumours. In contrast to the specific hypermethylation of tumour suppressor genes, an overall hypomethylation of DNA can be observed in tumour cells. This decrease in global methylation can be detected early, far before the development of frank tumour formation. Also, correlation between hypomethylation and increased gene expression was reported for many oncogenes. In colon cancer, aberrant DNA methylation constitutes one of the most prominent alterations and inactivates many tumour suppressor genes such as p14ARF, p161NK4a, THBS1, MINT2, and MINT31 and DNA mismatch repair genes such as hMLH1. In the molecular evolution of colorectal cancer, DNA methylation errors have been suggested to play two distinct roles. In normal colonic mucosal cells, methylation errors accumulate as a function of age or as time-dependent events predisposing these cells to neoplastic transformation. For example, hypermethylation of several loci could be shown to be already present in adenomas, particularly in the tubulovillous and villous subtype. At later stages, increased DNA methylation of CpG islands plays an important role in a subset of tumours affected by the so called CpG island methylator phenotype (CIMP). Most CIMP+ tumours, which constitute about 15% of all sporadic colorectal cancers, are characterised by microsatellite instability (MIN) due to hypermethylation of the hMLHI promoter and other DNA mismatch repair genes. By contrast, CIMP— colon cancers evolve along a more classic genetic instability pathway (CIN), with a high rate of p53 mutations and chromosomal changes. However, the molecular subtypes do not only show varying frequencies regarding molecular alterations. According to the presence of either micro satellite instability or chromosomal aberrations, colon cancer can be subclassified into two classes, which also exhibit significant clinical differences. Almost all MIN tumours originate in the proximal colon (ascending and transversum), whereas 70% of CIN tumours are located in the distal colon and rectum. This has been attributed to the varying prevalence of different carcinogens in different sections of the colon. Methylating carcinogens, which constitute the prevailing carcinogen in the proximal colon have been suggested to play a role in the pathogenesis of MIN cancers, whereas CIN tumours are thought to be more frequently caused by adduct-forming carcinogens, which occur more frequently in distal parts of the colon and rectum. Moreover, MIN tumours have a better prognosis than do tumours with a CIN phenotype and respond better to adjuvant chemotherapy. The identification of markers for the differentiation of colon carcinoma as well as for early detection are main goals of current research. EYA4 is the most recently identified member of the vertebrate Eya (eyes-absent) gene family, a group of four transcriptional activators that interact with other proteins in a conserved regulatory hierarchy to ensure normal embryologic development. The EYA4 gene is mapped to 6q22.3 and encodes a 640 amino acid protein. The structure of EYA4 conforms to the basic pattern established by EYA1-3, and includes a highly conserved 271 amino acid C-terminus called the eya-homologous region (eyaHR; alternatively referred to as the eya domain or eya homology domain 1) and a more divergent proline-serine-threonine (PST)-rich (34-41%) transactivation domain at the N-terminus (Borsani G, et al., EYA4, a novel vertebrate gene related to Drosophila eyes absent. Hum Mol Genet 1999 January;8(1):11-23). EYA proteins interact with members of the SIX and DACH protein families during early embryonic development. Mutations in the EYA4 gene are responsible for postlingual, progressive, autosomal dominant hearing loss at the DFNA10 locus (Wayne S, Robertson NG, DeClau F, Chen N, Verhoeven K, Prasad S, Tranebjarg L, Morton CC, Ryan A F, Van Camp G, Smith R J: Mutations in the transcriptional activator EYA4 cause late-onset deafness at the DFNA10 locus. Hum Mol Genet 2001 Feb. 1;10(3):195-200 with further references). A link between the Methylation of Cytosine positions in the EYA 4 gene and cancer has not yet been established. 5-methylcytosine is the most frequent covalent base modification in the DNA of eukaryotic cells. It plays a role, for example, in the regulation of the transcription, in genetic imprinting, and in tumorigenesis. Therefore, the identification of 5-methylcytosine as a component of genetic information is of considerable interest. However, 5-methylcytosine positions cannot be identified by sequencing since 5-methylcytosine has the same base pairing behaviour as cytosine. Moreover, the epigenetic information carried by 5-methylcytosine is completely lost during PCR amplification. A relatively new and currently the most frequently used method for analysing DNA for 5-methylcytosine is based upon the specific reaction of bisulfite with cytosine which, upon subsequent alkaline hydrolysis, is converted to uracil which corresponds to thymidine in its base pairing behaviour. However, 5-methylcytosine remains unmodified under these conditions. Consequently, the original DNA is converted in such a manner that methylcytosine, which originally could not be distinguished from cytosine by its hybridisation behaviour, can now be detected as the only remaining cytosine using “normal” molecular biological techniques, for example, by amplification and hybridisation or sequencing. All of these techniques are based on base pairing which can now be fully exploited. In terms of sensitivity, the prior art is defined by a method which encloses the DNA to be analysed in an agarose matrix, thus preventing the diffusion and renaturation of the DNA (bisulfite only reacts with single-stranded DNA), and which replaces all precipitation and purification steps with fast dialysis (Olek A, Oswald J, Walter J. A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res. 1996 Dec. 15;24(24):5064-6). Using this method, it is possible to analyse individual cells, which illustrates the potential of the method. However, currently only individual regions of a length of up to approximately 3000 base pairs are analysed, a global analysis of cells for thousands of possible methylation events is not possible. However, this method cannot reliably analyse very small fragments from small sample quantities either. These are lost through the matrix in spite of the diffusion protection. An overview of the further known methods of detecting 5-methylcytosine may be gathered from the following review article: Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 1998, 26, 2255. To date, barring few exceptions (e.g., Zeschnigk M, Lich C, Buiting K, Doerfler W, Horsthemke B. A single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur J Hum Genet. 1997 March-April;5(2):94-8) the bisulfite technique is only used in research. Always, however, short, specific fragments of a known gene are amplified subsequent to a bisulfite treatment and either completely sequenced (Olek A, Walter J. The pre-implantation ontogeny of the H19 methylation imprint. Nat Genet. 1997 November;17(3):275-6) or individual cytosine positions are detected by a primer extension reaction (Gonzalgo ML, Jones P A. Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 1997 Jun. 15;25(12):2529-31, WO 95/00669) or by enzymatic digestion (Xiong Z, Laird P W. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 1997 Jun. 15;25(12):2532-4). In addition, detection by hybridisation has also been described (Olek et al., WO 99/28498). Further publications dealing with the use of the bisulfite technique for methylation detection in individual genes are: Grigg G, Clark S. Sequencing 5-methylcytosine residues in genomic DNA. Bioessays. 1994 June;16(6):431-6, 431; Zeschnigk M, Schmitz B, Dittrich B, Buiting K, Horsthemke B, Doerfler W. Imprinted segments in the human genome: different DNA methylation patterns in the Prader-Willi/Angelman syndrome region as determined by the genomic sequencing method. Hum Mol Genet. 1997 March;6(3):387-95; Feil R, Charlton J, Bird A P, Walter J, Reik W. Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing. Nucleic Acids Res. 1994 Feb. 25;22(4):695-6; Martin V, Ribieras S, Song-Wang X, Rio M C, Dante R. Genomic sequencing indicates a correlation between DNA hypomethylation in the 5′ region of the pS2 gene and its expression in human breast cancer cell lines. Gene. 1995 May 19;157(1-2):261-4; WO 97/46705 and WO 95/15373. An overview of the Prior Art in oligomer array manufacturing can be gathered from a special edition of Nature Genetics (Nature Genetics Supplement, Volume 21, January 1999), published in January 1999, and from the literature cited therein. Fluorescently labelled probes are often used for the scanning of immobilised DNA arrays. The simple attachment of Cy3 and Cy5 dyes to the 5′-OH of the specific probe are particularly suitable for fluorescence labels. The detection of the fluorescence of the hybridised probes may be carried out, for example via a confocal microscope. Cy3 and Cy5 dyes, besides many others, are commercially available. Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-TOF) is a very efficient development for the analysis of biomolecules (Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988 Oct. 15;60(20):2299-301). An analyte is embedded in a light-absorbing matrix. The matrix is evaporated by a short laser pulse thus transporting the analyte molecule into the vapour phase in an unfragmented manner. The analyte is ionised by collisions with matrix molecules. An applied voltage accelerates the ions into a field-free flight tube. Due to their different masses, the ions are accelerated at different rates. Smaller ions reach the detector sooner than bigger ones. MALDI-TOF spectrometry is excellently suited to the analysis of peptides and proteins. The analysis of nucleic acids is somewhat more difficult (Gut I G, Beck S. DNA and Matrix Assisted Laser Desorption Ionization Mass Spectrometry. Current Innovations and Future Trends. 1995, 1; 147-57). The sensitivity to nucleic acids is approximately 100 times worse than to peptides and decreases disproportionally with increasing fragment size. For nucleic acids having a multiply negatively charged backbone, the ionisation process via the matrix is considerably less efficient. In MALDI-TOF spectrometry, the selection of the matrix plays an eminently important role. For the desorption of peptides, several very efficient matrixes have been found which produce a very fine crystallisation. There are now several responsive matrixes for DNA, however, the difference in sensitivity has not been reduced. The difference in sensitivity can be reduced by chemically modifying the DNA in such a manner that it becomes more similar to a peptide. Phosphorothioate nucleic acids in which the usual phosphates of the backbone are substituted with thiophosphates can be converted into a charge-neutral DNA using simple alkylation chemistry (Gut IG, Beck S. A procedure for selective DNA alkylation and detection by mass spectrometry. Nucleic Acids Res. 1995 Apr. 25;23(8):1367-73). The coupling of a charge tag to this modified DNA results in an increase in sensitivity to the same level as that found for peptides. A further advantage of charge tagging is the increased stability of the analysis against impurities which make the detection of unmodified substrates considerably more difficult. Genomic DNA is obtained from DNA of cell, tissue or other test samples using standard methods. This standard methodology is found in references such as Sambrook, Fritsch and Maniatis eds., Molecular Cloning: A Laboratory Manual, 1989. |
Category based, extensible and interactive system for document retrieval |
In information retrieval (IR) systems with high-speed access, especially to search engines applied to the Internet and/or corporate intranet domains for retrieving accessible documents automatic text categorization techniques are used to support the presentation of search query results within high-speed network environments. An integrated, automatic and open information retrieval system (100) comprises an hybrid method based on linguistic and mathematical approaches for an automatic text categorization. It solves the problems of conventional systems by combining an automatic content recognition technique with a self-learning hierarchical scheme of indexed categories. In response to a word submitted by a requester, said system (100) retrieves documents containing that word, analyzes the documents to determine their word-pair patterns, matches the document patterns to database patterns that are related to topics, and thereby assigns topics to each document. If the retrieved documents are assigned to more than one topic, a list of the document topics is presented to the requester, and the requester designates the relevant topics. The requester is then granted access only to documents assigned to relevant topics. A knowledge database (1408) linking search terms to documents and documents to topics is established and maintained to speed future searches. Additionally, new strategies are presented to deal with different update frequencies of changed Web sites. |
1. An interactive document retrieval system (100) designed to search for documents after receiving a search query from a requestor, said system comprising: a knowledge database (200) containing at least one data structure (202, 208, 210, 212, 214, 216 and/or 218) that relates text patterns to topics, and a query processor (400) that, in response to the receipt of a search query from a requester, performs the following steps: searching for and trying to capture documents containing at least one term related to the search query, if any documents are captured, analyzing the captured documents to determine their text patterns, categorizing the captured documents by comparing each document's text pattern to the text patterns in the knowledge database (200), and if a document's text pattern is similar to a text pattern in the knowledge database (200), assigning to that document the similar word pattern's related topic, presenting at least one list of the topics assigned to the categorized documents to the requester, and asking the requester to designate at least one topic from the list as a topic that is relevant to the requestor's search, and granting the requestor access to the subset of captured and categorized documents to which topics designated by the requestor have been assigned, wherein the word patterns determined by analysis are pairings of words, each pairing comprising two searchable words with one word occurring frequently within the document and the other word occurring near the one word frequently within the document. 2. An interactive document retrieved system according to claim 1, characterized in that, the query processor performs the step of analyzing using an hybrid method based on linguistic and mathematical approaches for an automatic text categorization. 3. An interactive document retrieval system (100) in accordance with claim 1, wherein the knowledge base (200) is initially constructed by analyzing indexed documents to which topics have previously been assigned, thereby determining the indexed document's word patterns, and then storing in the knowledge database (200) these word patterns for the indexed documents and the topics assigned to these documents, and then relating the word pattern of an indexed document to the topics assigned to that same indexed document. 4. An interactive document retrieval system (100) in accordance with claim 1, wherein the search query contains a phrase, and the term searched for is that phrase. 5. An interactive document retrieval system (100) in accordance with claim 1, wherein the search query contains at least one word, and the term searched for is at least one searchable word taken from the search query. 6. An interactive document retrieval system (100) in accordance with claim 1, wherein the search query contains several words, the term searched for is a searchable word taken from the search query, and several words in the search query are searched for in separate searches. 7. An interactive document retrieval system (100) in accordance with claim 1, wherein the search query contains at least one operator and at least one word, and the presentation of documents to the requester scope is limited by the search query. 8. An interactive document retrieval system (100) in accordance with claim 1, wherein the knowledge database (200) retains a record of words previously searched for, the documents captured by such previous searches, and the index terms assigned to the captured documents, and the knowledge database (200) also retains linkages between the words previously searched for and the documents captured by such previously-conducted searches, such that the search, analysis, and categorizing steps may be bypassed when a word previously searched for is encountered in a later search query. 9. An interactive document retrieval system (100) in accordance with claim 8, wherein the knowledge database (200) is initially constructed by analyzing indexed documents to which topics have previously been assigned, thereby determining the indexed document's word patterns, and then storing in the knowledge database (200) these word patterns for the indexed documents and the topics assigned to these documents, and then relating the word pattern of an indexed document to the topics assigned to that same indexed document. 10. An interactive document retrieval system (100) in accordance with claim 8, wherein the knowledge database (200) is maintained by periodically checking to see if documents entered into the knowledge database (200) have changed or been deleted from the searchable universe of documents, and if they have, then deleting all reference to such documents, as well as the words searched for that caused their capture, from the knowledge database (200), thereby forcing all searches for such words likely to capture such documents to be repeated anew if encountered in a later search query. 11. An interactive document retrieval system (100) in accordance with claim 8, wherein the knowledge database (200) is maintained by periodically checking to see if documents entered into the knowledge database (200) have been changed, and if so, reanalyzing and re-categorizing such documents and also removing from the knowledge database (200) linkages between such documents and words that they no longer contain. 12. An interactive document retrieval system (100) in accordance with claim 1, wherein the knowledge database (200) is updated by periodically checking for new documents at some locations within the searchable universe of documents, and analyzing and categorizing such documents prior to those documents being captured by a search. 13. An interactive document retrieval system (100) in accordance with claim 1, wherein said knowledge database (200) includes a topic combination table (212) containing replacement topics for certain combinations of other topics that may appear within a captured document and that are assigned to such a document as a replacement for said other topics to improve categorization. 14. An interactive document retrieval system (100) in accordance with claim 1, wherein plural topics are assigned to at least some documents during categorization and are arranged hierarchically and linked to the at least some documents in the knowledge database (200), and wherein as many lists of topics as there are hierarchical topics associated with the categorized documents are presented to the requestor in sequence, such that the requestor designates multiple topics and subtopics, and such that search precision is improved by eliminating documents irrelevant to the requestor's designated topics from those to which the requestor is granted access. 15. An interactive document retrieval system (100) in accordance with claim 14, wherein the presentation of topics to the requester at any given hierarchical level is suppressed when all the documents are associated with the same topic at that level. 16. An interactive document retrieval system (100) in accordance with claim 1, wherein analysis includes the following steps: reduce the document data to a list of words; address inflection and synonym problems; eliminate non-searchable words; select the most frequently occurring words; and select frequently occurring pairings of those words with adjacent words in the document. 17. An interactive document retrieval system (100) in accordance with claim 16, wherein up to a predefined number of the most frequently occurring words are selected. 18. An interactive document retrieval system (100) in accordance with claim 16, wherein a word occurs frequently if the number of times it appears within a document divided by the total word content of the document exceeds a predetermined value. 19. An interactive document retrieval system (100) in accordance with claim 1, wherein a pairing occurs frequently if the number of occurrences of a given pairing within a given document, divided by the number of occurrences of the frequently-occurring adjacent word of the pairing within the document, is greater than a predetermined value. 20. An interactive document retrieval system (100) in accordance with claim 1, wherein: the query processor (400) is installed in at least one Web server connecting to the Internet or to an intranet; the knowledge database (200) is installed on a database engine (1124) accessible to the Web server; the requestor communicates with the Web server (1114, 1116, 1118 or 1120) using a computer (1102) having a browser (1104) also connecting to the Internet or to the same intranet; and searches are performed by a search engine (1128) accessible to the Web server (1114, 1116, 1118 or 1120) and conducting searches on the Internet or on the same intranet. 21. An interactive document retrieval system (100) in accordance with claim 20, wherein the predetermined value is in the neighborhood of 0.0001. 22. An interactive document retrieval system (100) in accordance with claim 20, wherein multiple Web servers (1114, 1116, 1118 or 1120) are employed, interconnected to the Internet or to an intranet by a router (1112) and a firewall (1110); and the status of any given search procedure is maintained on the requestor's computer (1102) and is resubmitted to one of the Web servers (1114, 1116, 1118 or 1120) each time a search query or designation is submitted by the requestor. 23. An interactive document retrieval system (100) in accordance with claim 1, wherein the knowledge database (200) contains a word table (202), a dictionary (204) and synonyms (206), a topic table (208), a word combination table (210), a topic combination table (212), a query word table (214), a query linkage table (216), and an URL table (218). 24. An interactive method of searching for and retrieving documents after receiving a search query from a requestor, said method comprising the steps of: providing a knowledge database (200) containing at least one data structure (202, 208, 210, 212, 214, 216 and/or 218) that relates text patterns to topics, in response to the receipt of a search query from a requester, searching for and attempting to capture documents containing at least one term related to the search query, if any documents are captured, analyzing the captured documents to determine their text patterns, categorizing the captured documents by comparing each document's text pattern to the text patterns in the knowledge database (200), and when a document's word pattern is similar to a text pattern in the knowledge database (200), assigning to that document the similar text pattern's related topic, presenting at least one list of the topics assigned to the categorized documents to the requester, and asking the requester to designate at least one topic from the list as a topic that is relevant to the requestor's search, and granting the requestor access to the subset of captured and categorized documents to which topics designated by the requester have been assigned, wherein the word patterns determined by analysis are pairings of words, each pairing comprising two searchable words with one word occurring frequently within the document and the other word occurring near the one word frequently within the document. 25. An interactive method according to claim 24, wherein the step of analyzing is carried out using an hybrid method based on linguistic and mathematical approaches for an automatic text categorization. 26. An interactive method of searching in accordance with claim 24, which further includes constructing the knowledge database (200) by analyzing indexed documents to which topics have previously been assigned, thereby determining the indexed document's word patterns, and then storing in the knowledge database (200) these word patterns for the indexed documents and the topics assigned to these documents, and then relating the word pattern of an indexed document to the topics assigned to that same indexed document. 27. An interactive method of searching in accordance with claim 24, which accepts at search queries that contain a phrase and that search for the phrase. 28. An interactive method of searching in accordance with claim 24, which accepts search queries that contain at least one word and that search for the word. 29. An interactive method of searching in accordance with claim 24, which accepts search queries that contain several words and search for each word in separate searches. 30. An interactive method of searching in accordance with claim 24, which accept at least some search queries that contain at least one operator and at least one word and that search for the word and later use the operator to limit the scope of the documents presented to the requestor. 31. An interactive method of searching in accordance with claim 24, which further includes retaining in the knowledge database (200) a record of words previously searched for, the documents captured by such previous searches, and the index terms assigned to the captured documents, and retaining within the knowledge database (200) linkages between the words previously searched for and the documents captured by such previously-conducted searches, such that the search, analysis, and categorizing steps may be bypassed when a word previously searched for is encountered in a later search query. 32. An interactive method of searching in accordance with claim 31, which further includes initially constructing the knowledge database (200) by analyzing indexed documents to which topics have previously been assigned, thereby determining the indexed document's word patterns, and then storing in the knowledge database (200) these word patterns for the indexed documents and the topics assigned to these documents, and then relating the word pattern of an indexed document to the topics assigned to that same indexed document. 33. An interactive method of searching in accordance with claim 31, which further includes maintaining the knowledge database (200) by periodically checking to see if documents entered into the knowledge database (200) have changed or been deleted from the searchable universe of documents; and if they have, then deleting all reference to such documents, as well as the words searched for that caused their capture, from the knowledge database (200), thereby forcing all searches for such words likely to capture such documents to be repeated anew if encountered in a later search query. 34. An interactive method of searching in accordance with claim 31, which further includes maintaining the knowledge database (200) by periodically checking to see if documents entered into the knowledge database (200) have been changed, and if so, reanalyzing and re categorizing such documents and also removing from the knowledge database (200) linkages between such documents and words that they no longer contain. 35. An interactive method of searching in accordance with claim 24, which further includes updating the knowledge database (200) by periodically checking for new documents at some locations within the searchable universe of documents, and analyzing and categorizing such documents prior to those documents being captured by a search. 36. An interactive method of searching in accordance with claim 24, which further includes including in said knowledge database (200) a topic combination table (212) containing replacement topics for certain combinations of other topics that may appear within a captured document, and assigning a replacement topic to such a document as a replacement for said other topics to improve categorization. 37. An interactive method of. searching in accordance with claim 24, which further includes assigning plural topics to at least some documents during categorization, arranging them hierarchically, and linking them to the at least some documents in the knowledge database (200), and presenting to the requester in hierarchical sequence as many lists of topics as there are hierarchical topics associated with the categorized documents, such that the requestor designates multiple topics and subtopics, and such that search precision is improved by eliminating documents irrelevant to the requestor's designated topics from those to which the requester is granted access. 38. An interactive method of searching in accordance with claim 37, which further includes suppressing the presentation of topics to the requester at any given hierarchical level when all the documents are associated with the same topic at that level. 39. An interactive method of searching in accordance with claim 24, which further includes reducing the document data to a list of words; addressing inflection and synonym problems; eliminating non-searchable words; selecting the most frequently occurring words; and selecting frequently-occurring pairings of those words with adjacent words in the document. 40. An interactive method of searching in accordance with claim 39, which further includes selecting up to a predefined number of the most frequently occurring words. 41. An interactive method of searching in accordance with claim 39, which further includes determining whether a word occurs frequently by determining if the number of times the word appears within a document divided by the total word content of the document exceeds a predetermined value. 42. An interactive method of searching in accordance with claim 39, which further includes determining whether a pairing occurs frequently by determining whether the number of occurrences of a given pairing within a given document, divided by the number of occurrences of the adjacent word of the pairing within the document, is greater than a predetermined value. 43. An interactive method of searching in accordance with claim 24, which further includes an arranging for communication with the requestor using the Internet protocol. 44. An interactive method of searching in accordance with claim 43, which further includes maintaining the status of any given search procedure with the requestor. 45. An interactive method of searching in accordance with claim 24, which further includes building into the knowledge database (200) a word table (202), a dictionary (204) and synonyms (206), a topic table (208), a word combination table (210), a topic combination table (212), a query word table (214), a query linkage table (216), and an URL table (218). 46. Computer software program implementing a method according to claim 24 when run on a computing device. 47. An interactive document retrieval system (100) in accordance with claim 1, characterized by a specially designed user interface (1402) presenting the user an uniform access to all accessible documents, thereby enabling a search in heterogeneous environments, regardless whether they are retrieved from the domain of any corporate networks or from the Internet, and irrespective of their file format. 48. An interactive document retrieval system (100) in accordance with claim 1, characterized by, a specially developed updating function (1312) is employed for visiting Web sites dependent on their individual modification cycles and providing them for a further analysis. 49. An interactive document retrieval system (100) in accordance with claim 1, comprising means for recognizing existing security structures used in the domain of individual companies for securing electronically stored data which enable an integration of said interactive document retrieval system (100) into said security structures without changing them. 50. An interactive document retrieval system (100) in accordance with claim 1, wherein a portability of said interactive document retrieval system (100) into different operating system environments is supported. 51. An interactive document retrieval system (100) in accordance with claim 1, wherein the user is provided with a set of data spaces, each comprising a set of thematically connected documents. 52. An interactive document retrieval system (100) in accordance with claim 1, wherein a specially designed user interface (1402) comprising presentation programs for generating appropriately formatted texts suitable for the presentation of documents retrieved from the Internet is applied. 53. An interactive document retrieval system (100) in accordance with claim 1, wherein agent programs are applied which continuously process entered search queries in the background. 54. An interactive document retrieval system (100) in accordance with claim 1, wherein each document of a selected category is classified according to its origin, such as public places, media and/or encyclopedias, enterprises or other sources. 55. An interactive document retrieval system (100) in accordance with claim 1, wherein an universally applicable thesaurus with different categories and associated start documents is applied. 56. An interactive document retrieval system (100) in accordance with claim 1, wherein a user interface is applied comprising means for to entering search queries by means of voice commands being automatically recognized and interpreted with the aid of an underlying automatic voice recognition application. 57. An interactive document retrieval system (100) in accordance with claim 1, wherein search results are presented by means of a voice data output. 58. An interactive document retrieval system (100) in accordance with claim 1, wherein a multilingual operation of said interactive document retrieval system (100) is enabled. 59. An interactive method of searching in accordance with claim 24, wherein the user is provided with an uniform access to all accessible documents, thereby enabling a search in heterogeneous environments, regardless whether they are retrieved from the domain of any corporate networks or from the Internet, and irrespective of their file format. 60. An interactive method of searching in accordance with claim 24, wherein predefined exemplary archives are employed comprising the category information for a set of pre-categorized documents in order to save implementation costs which would arise if a new archive structure had to be installed. 61. An interactive method of searching in accordance with claim 24, wherein a specially developed updating function (1312) is employed for visiting Web sites dependent on their individual modification cycles and providing them for a further analysis, thereby guaranteeing a maximum topicality of the employed Internet archive structure. 62. An interactive method of searching in accordance with claim 24, comprising means for recognizing existing security structures used in the domain of individual companies for securing electronically stored data which enable an integration of said interactive document retrieval system (100) into said security structures without changing them. 63. An interactive method of searching in accordance with claim 24, wherein a portability of said interactive document retrieval system (100) into different operating system environments is supported. 64. An interactive method of searching in accordance with claim 24, wherein the user is provided with a set of data spaces, each comprising a set of thematically connected documents. 65. An interactive method of searching in accordance with claim 24, wherein a specially designed user interface (1402) comprising presentation programs for generating appropriately formatted texts suitable for the presentation of documents retrieved from the Internet is applied. 66. An interactive method of searching in accordance with claim 24, wherein agent programs are applied which continuously process entered search queries in the background. 67. An interactive method of searching in accordance with claim 24, wherein each document of a selected category is classified according to its origin, such as public places, media and/or encyclopedias, enterprises or other sources. 68. An interactive method of searching in accordance with claim 24, wherein an universally applicable thesaurus with different categories and associated start documents is applied. 69. An interactive method of searching in accordance with claim 24, wherein a user interface is applied comprising means for to entering search queries by means of voice commands being automatically recognized and interpreted with the aid of an underlying automatic voice recognition application. 70. An interactive method of searching in accordance with claim 24, wherein search results are presented by means of a voice data output. 71. An interactive method of searching in accordance with claim 24, wherein a multilingual operation of said interactive document retrieval system (100) is enabled. 72. A mobile computing and/or telecommunications device, comprising a graphical user interface capable of applying the WAP standard for accessing documents from the Internet and/or any corporate network, characterized by an interactive document retrieval system (100) in accordance with claim 1. 73. An interactive document retrieval system, comprising a knowledge database (1408) for relating identifications of analyzed documents to topics, a user interface (1402) for inputting a search query, a search engine (1406) for searching a resource for documents essentially matching an input search query and for outputting identifications of documents as a search result, a finding machine (1404) being supplied with the search result of the search engine (1406), for accessing the knowledge database (1408) to check whether a document identified in the search result has already been analyzed before in relation with other search terms than the present search term, forwarding the identification of a document along with its related topic as retrieved from the knowledge database (1408) to the user interface (1402) in case the document has already been analyzed before and its identification been stored together with its related topic in the knowledge database (1408), and analyzing the identified document in case the document has not yet been analyzed before to relate a topic to the identification of the document and forwarding the identification of the document along with its related topic to the user interface (1402). 74. An interactive document retrieval method, the method comprising the steps of relating (1408) identifications of analyzed documents to topics in a database, inputting (1402) a search term by means of an user interface, searching (1406) a resource for documents essentially matching an input search query and outputting identifications of documents as a search result, accessing the database (1408) to check whether a document identified in the search result has already been analyzed before in relation with other search terms than the present search term, forwarding the identification of a document along with its related topic as retrieved from the knowledge database (1408) to the user interface (1402) in case the document has already been analyzed before and its identification been stored together with its related topic in the knowledge database (1408), and analyzing the identified document in case the document has not yet been analyzed before to relate a topic to the identification of the document and forwarding the identification of the document along with its related topic to the user interface (1402). |
<SOH> FIELD AND BACKGROUND OF THE INVENTION <EOH>The invention generally relates to the field of information retrieval (IR) systems with high-speed access, especially to search engines applied to the Internet and/or corporate intranet domains for retrieving accessible documents using automatic text categorization techniques to support the presentation of search query results within high-speed network environments. As the volume of published information which can be accessed with the aid of a plurality of corporate networks and particularly via the Internet continues to increase, there is growing interest in helping people better find, filter, and manage these resources. Since said networks represent a young, dynamic and still not much standardized market, they comprise an enormous volume of non-structured documents and text material. Particularly the Internet as an open medium being freely accessible to everyone represents a gigantic knowledge base that is still unused to a great extend, since there are no syntactic rules at all for the retrieval of the stored information. The insufficient information structure of the Internet (and other networks) is often criticized. Moreover, search engines often fail in coverage or present broken links to publications. What the user would actually like to find can not be found, or the user is strained by a large number of unsuitable matches when receiving the results of an entered search query. Although the desired information possibly is available within these networks, it can not easily be obtained. Simultaneously, the demands for the availability of qualified information rapidly increase both in the commercial and in the private area. Efficient indexing, retrieval and management of digital media is therefore becoming more and more important due to the vast volume of digital information available within the Internet and a plurality of intranet domains. Manual Indexing of Text Documents Librarians and other trained professionals have worked for years on manually indexing new items using controlled vocabularies such as in the scope of Medical Subject Headings (MeSH), Dewey Decimal, Yahoo! or CyberPatrol. For instance, Yahoo! currently uses human experts to manually categorize its documents. Likewise, at legal publishing houses such as West Group, legal documents are manually indexed by human experts. This process is very time-consuming and costly, thus limiting its applicability. Consequently, there is an increased interest in developing techniques for automatic text categorization. Rule-based approaches similar to those used in expert systems are common (cf. Hayes and Weinstein's CONSTRUE system for classifying news stories, 1990), but they generally require manual construction of the rules, make rigid binary decisions about category membership, and are typically difficult to modify. Automatic Text Categorization The increasing amount of information available in different areas of knowledge creates the need to automate part of the process described above. Automatic indexing algorithms based on statistical patterns of natural language appeared during the 1960's, and 1970's. During the 1980's several systems were created for computer-aided indexing. During the late 1980's several expert systems were applied to create knowledge-based indexing systems, for instance MedIndeEx System at the National Library of Medicine (Humphrey, 1988). The 1990's can be characterized by the advent of the World Wide Web (WWW) which has made available a vast amount of information that is potentially useful. The information overload created by the WWW has stimulated the creation of reliable automatic indexing methods that could help users filter large amounts of documents. Today several researchers around the world are trying to solve the automatic text categorization problem by using two major approaches: firstly, to capture the rules used in human communications and apply them to a system, and secondly, to employ methods for automatically training categorization rules from a training set of already categorized text material. Previous similar works were mainly related to speech recognition, e.g. in the scope of automatic telephone services. For this purpose several topics are predefined, and the recognition system tries to detect the topics from input texts. Once a topic is detected, a statistical model for the text is applied to assist the process of speech recognition. In general, automatic classification schemes can essentially facilitate the process of categorization. The process of automatic text categorization—the algorithmic analysis and automatic assignment of electronically accessible natural language text documents to a set of prespecified topics (categories or index terms) that concisely describe the content of said documents—is an important component in a plurality of information organization and management tasks. Its most widespread application up to now has been the support of text retrieval, routing and filtering for assigning subject categories to input documents. Automatic text categorization can play an important role in a wide variety of more flexible, dynamic and personalized information management tasks as well. These tasks comprise: real-time sorting of emails or other text files into predefined folder hierarchies, thematic identification to support topic-specific processing operations, structuring of search and/or browsing techniques, and finding documents that refer to static, long-term interests or more dynamic, task-based interests. In any case, classification techniques should be able to support category structures that are very general, commonly accepted, and relatively static like Dewey Decimal or Library of Congress classification systems, Medical Subject Headings (MeSH), or Yahoo!'s topic hierarchy, as well as those that are more dynamic and customized to individual interests or tasks. |
<SOH> SUMMARY OF THE INVENTION <EOH>The information retrieval system according to the underlying invention is basically dedicated to the idea of an automatic document and/or text categorization technique, concerning the question how an arbitrary text (the content of a document in electronic form) can automatically be recognized and assigned to a predefined category. This basic technology can be applied to a plurality of products and within a plurality of different environments. In any case, the idea to facilitate the frequently occurring task of selectively searching for documents that can be accessed via the Internet, which is a very time-consuming procedure due to the plurality of the herein contained documents, and to automatically perform this task in the background is the same—irrespective of the underlying application and its environment. The proposed solution according to the underlying invention thereby involves the creation of a framework to define services for retrieving, filtering and categorizing documents from the Internet and/or corporate network domains organized in a common category scheme. To achieve this, specialized information retrieval and text classification tools are needed. Briefly summarized, the present invention is an interactive document retrieval system that is designed to search for documents after receiving a search query from a requestor. It contains a knowledge database that contains at least one data structure which assigns document word patterns to topics. This knowledge database can be derived from an indexed collection of documents. The underlying invention utilizes a query processor that, in response to the receipt of a search query from a requester, searches for and tries to capture documents containing at least one term that is related to the search query. If any documents are captured, the processor analyzes the captured documents to determine their word patterns, and it then categorizes the captured documents by comparing each document's word pattern to the word patterns in the database. When a word pattern of a document is similar to a word pattern in the database, the processor assigns the similar word pattern's related topic to that document. In this manner, each document is assigned to one or several topics. Next, a list of the topics assigned to the categorized documents is presented to the requester, and the requestor is asked to designate at least one topic from the list as a topic that is relevant to the requestor's search. Finally, the requester is granted access to the subset of the captured and categorized documents to which topics designated by the requestor have been assigned. The system may rely on a server connected to the Internet or to an intranet, and the requester may access the system from a personal computer equipped with a Web browser. To save time, queries once processed are saved along with the list of documents retrieved by those queries and the topics to which they are assigned. Periodic update and maintenance searches are performed to keep the system up-to-date, and analysis and categorization performed during update and maintenance is saved to speed the performance of searches later on. The system may be set up initially and trained by having it analyze a set of documents that have been manually indexed, saving a record of the word patterns of these documents in a word combination table within the knowledge database and relating these word patterns to the topics assigned to each document. These word patterns may be adjacent pairs of searchable words (not including non-searchable words such as articles, prepositions, conjunctions, etc.), wherein at least one of the words in each such pairing frequently occurs within the document. The main idea of the concept according to the underlying invention is to process the documents of the Internet and the information contained therein by means of a classical, natural language based archive structure. The requester shall no longer be strained by a large number of unsuitable results. Instead, he should interactively be lead towards a suitable set of results with the aid of universally applicable or individually defined archive structures. In the foreground stands an easy and fast operability with a minimum of technical expenditure. This object can only be achieved by employing two essential functions: 1. The content of the documents must automatically be analyzed, categorized and inserted into the archive structure. 2. The user must intuitively be lead towards the set of the results by means of an interactive query system performed by a novel user surface. The proposed solution according to the underlying invention represents an integrated, automatic and open information retrieval system, comprising an hybrid method based on linguistic and mathematical approaches for an automatic text categorization. On the one hand it is possible to meet the requirements of all Internet users by means of the novel Internet archive according to the preferred embodiment of the underlying invention providing desired information in a quick, simple and accurate manner. On the other hand significant advantages arise for the data management within individual companies. Newly developed analysis tools and categorization techniques form the basis of the system architecture consisting of a framework of substantiated linguistic rules. Thereby, arbitrary data supplies of any size can automatically be analyzed, structured and managed. The proposed system solves the problems of conventional systems by combining an automatic content recognition technique with a self-learning hierarchical scheme of indexed categories. Nevertheless, it still works fast. Instead of performing a crude semantic full-text research, the system can be used for thematically analyzing all available documents in a context-sensitive and sensible manner. An hierarchically structured topical search—which could only be performed in the domain of corporate networks so far for reasons of capacity—can now be extended to the Internet domain. In this way, different intranets and the Internet can grow together towards a conjoint data space with a homogeneous structure. The information retrieval system according to the preferred embodiment of the underlying invention can flexibly be adapted to the archive structure and the data management of individual companies. Available information supplies can be read in by incorporating already available hierarchical structures, thereby being associated with new information. Vertically organized information chains are thus rebuilt by an horizontally organized archive structure that permits a permanent and decentralized access on needed data supplies and documents. Thus, a virtual archive of the information and knowledge supplies of an individual enterprise is given which can completely be updated at any time since the information retrieval system according to the preferred embodiment of the underlying invention also serves as an interface between corporate network domains and the Internet. The intern archive structure of an individual company can be applied to all documents stored within the Internet without needing additional expenditure. The system thereby enables an unification of searches in both domains. |
Mutations in ion channels |
A method of identifying a subject predisposed to a disorder associated with ion channel dysfunction, comprising ascertaining whether at least one of the genes encoding ion channel subunits in said subject has undergone a mutation event such that a cDNA derived from said subject has the sequence set forth in one of SEQ ID NOS: 1-134. |
1. A method of identifying a subject predisposed to a disorder associated with ion channel dysfunction, comprising ascertaining whether at least one of the genes encoding ion channel subunits in said subject has undergone a mutation event such that a cDNA derived from said subject has the sequence set forth in one of SEQ ID NOS: 1-134. 2. A method as claimed in claim 1, wherein said mutation event disrupts the functioning of an assembled ion channel so as to produce an epilepsy phenotype in said subject. 3. A method as claimed in claim 1, wherein said mutation event disrupts the functioning of an assembled ion channel so as to produce one or more disorders associated with ion channel dysfunction, including but not restricted to, hyper- or hypo-kalemic periodic paralysis, myotonias, malignant hyperthermia, myasthenia, cardiac arrhythmias, episodic ataxia, migraine, Alzheimer's disease, Parkinson's disease, schizophrenia, hyperekplexia, anxiety, depression, phobic obsessive symptoms, neuropathic pain, inflammatory pain, chronic/acute pain, Bartter's syndrome, polycystic kidney disease, Dent's disease, hyperinsulinemic hypoglycemia of infancy, cystic fibrosis, congenital stationary night blindness and total color-blindness in said subject. 4. A method as claimed in claim 1, wherein said mutation event disrupts the functioning of an assembled ion channel so as to produce an epilepsy phenotype when expressed in combination with one or more additional mutations or variations in said ion channel subunit genes. 5. A method as claimed in claim 1, wherein said mutation event disrupts the functioning of an assembled ion channel so as to produce one or more disorders associated with ion channel dysfunction, including but not restricted to, hyper- or hypo-kalemic periodic paralysis, myotonias, malignant hyperthermia, myasthenia, cardiac arrhythmias, episodic ataxia, migraine, Alzheimer's disease, Parkinson's disease, schizophrenia, hyperekplexia, anxiety, depression, phobic obsessive symptoms, neuropathic pain, inflammatory pain, chronic/acute pain, Bartter's syndrome, polycystic kidney disease, Dent's disease, hyperinsulinemic hypoglycemia of infancy, cystic fibrosis, congenital stationary night blindness and total color-blindness, when expressed in combination with one or more additional mutations or variations in said ion channel subunit genes. 6. An isolated nucleic acid molecule encoding a mutant or variant ion channel subunit wherein a mutation event has occurred such that a cDNA derived therefrom has the sequence set forth in one of SEQ ID NOS: 1-134. 7. An isolated nucleic acid molecule encoding a mutant or variant ion channel subunit as claimed in claim 6, wherein said mutation event disrupts the functioning of an assembled ion channel so as to produce an epilepsy phenotype. 8. An isolated nucleic acid molecule encoding a mutant or variant ion channel subunit as claimed in claim 6, wherein said mutation event disrupts the functioning of an assembled ion channel so as to produce one or more disorders associated with ion channel dysfunction, including but not restricted to, hyper- or hypo-kalemic periodic paralysis, myotonias, malignant hyperthermia, myasthenia, cardiac arrhythmias, episodic ataxia, migraine, Alzheimer's disease, Parkinson's disease, schizophrenia, hyperekplexia, anxiety, depression, phobic obsessive symptoms, neuropathic pain, inflammatory pain, chronic/acute pain, Bartter's syndrome, polycystic kidney disease, Dent's disease, hyperinsulinemic hypoglycemia of infancy, cystic fibrosis, congenital stationary night blindness and total color-blindness. 9. An isolated nucleic acid molecule encoding a mutant or variant ion channel subunit as claimed in claim 6, wherein said mutation event disrupts the functioning of an assembled ion channel so as to produce an epilepsy phenotype when expressed in combination with one or more additional mutations or variations in said ion channel subunit genes. 10. An isolated nucleic acid molecule encoding a mutant or variant ion channel subunit as claimed in claim 6, wherein said mutation event disrupts the functioning of an assembled ion channel so as to produce one or more disorders associated with ion channel dysfunction, including but not restricted to, hyper- or hypo-kalemic periodic paralysis, myotonias, malignant hyperthermia, myasthenia, cardiac arrhythmias, episodic ataxia, migraine, Alzheimer's disease, Parkinson's disease, schizophrenia, hyperekplexia, anxiety, depression, phobic obsessive symptoms, neuropathic pain, inflammatory pain, chronic/acute pain, Bartter's syndrome, polycystic kidney disease, Dent's disease, hyperinsulinemic hypoglycemia of infancy, cystic fibrosis, congenital stationary night blindness and total color-blindness, when expressed in combination with one or more additional mutations or variations in said ion channel subunit genes. 11. An isolated nucleic acid molecule comprising any one of the nucleotide sequences set forth in SEQ ID NOS: 1-134. 12. An isolated nucleic acid molecule consisting of any one of the nucleotide sequences set forth in SEQ ID NOS: 1-134. 13. Canceled. 14. Canceled. 15. Canceled. 16. Canceled. 17. Canceled. 18. Canceled. 19. Canceled. 20. Canceled. 21. An expression vector comprising a nucleic acid molecule as claimed in claim 6 or claim 77. 22. A cell comprising one or more nucleic acid molecules claimed in claim 6 or claim 77. 23. Canceled. 24. A cell comprising at least one ion channel type, wherein the or each ion channel type incorporates at least one mutant polypeptide encoded by a nucleic acid molecule as claimed in claim 6 or claim 77. 25. Canceled. 26. Canceled. 27. A method of preparing a polypeptide, comprising the steps of: (1) culturing cells as claimed in either one of claims 22 or claim 24 under conditions effective for polypeptide production; and (2) harvesting the polypeptide. 28. A polypeptide prepared by the method of claim 27. 29. Canceled. 30. Canceled. 31. Canceled. 32. Canceled. 33. Canceled. 34. Canceled. 35. Canceled. 36. Canceled. 37. Canceled. 38. Canceled. 39. Canceled. 40. Canceled. 41. Canceled. 42. Canceled. 43. Canceled. 44. Canceled. 45. Canceled. 46. Canceled. 47. Use of a nucleic acid molecule as claimed in claim 6 or claim 77 for the screening of candidate pharmaceutical agents. 48. Use as claimed in claim 47 for the screening of candidate pharmaceutical agents useful for the treatment of epilepsy or a disorder associated with ion channel dysfunction, including but not restricted to, hyper- or hypo-kalemic periodic paralysis, myotonias, malignant hyperthermia, myasthenia, cardiac arrhythmias, episodic ataxia, migraine, Alzheimer's disease, Parkinson's disease, schizophrenia, hyperekplexia, anxiety, depression, phobic obsessive symptoms, neuropathic pain, inflammatory pain, chronic/acute pain, Bartter's syndrome, polycystic kidney disease, Dent's disease, hyperinsulinemic hypoglycemia of infancy, cystic fibrosis, congenital stationary night blindness or total color-blindness. 49. Canceled. 50. Use of a polypeptide as claimed in either claim 28 or claim 96 for the screening of candidate pharmaceutical agents. 51. Use as claimed in claim 50 for the screening of candidate pharmaceutical agents useful for the treatment of epilepsy or a disorder associated with ion channel dysfunction, including but not restricted to, hyper- or hypo-kalemic periodic paralysis, myotonias, malignant hyperthermia, myasthenia, cardiac arrhythmias, episodic ataxia, migraine, Alzheimer's disease, Parkinson's disease, schizophrenia, hyperekplexia, anxiety, depression, phobic obsessive symptoms, neuropathic pain, inflammatory pain, chronic/acute pain, Bartter's syndrome, polycystic kidney disease, Dent's disease, hyperinsulinemic hypoglycemia of infancy, cystic fibrosis, congenital stationary night blindness or total color-blindness. 52. Canceled. 53. Use of a cell as claimed in claims 22 or 24 for the screening of candidate pharmaceutical agents. 54. Use as claimed in claim 53 for the screening of candidate pharmaceutical agents useful for the treatment of epilepsy or a disorder associated with ion channel dysfunction, including but not restricted to, hyper- or hypo-kalemic periodic paralysis, myotonias, malignant hyperthermia, myasthenia, cardiac arrhythmias, episodic ataxia, migraine, Alzheimer's disease, Parkinson's disease, schizophrenia, hyperekplexia, anxiety, depression, phobic obsessive symptoms, neuropathic pain, inflammatory pain, chronic/acute pain, Bartter's syndrome, polycystic kidney disease, Dent's disease, hyperinsulinemic hypoglycemia of infancy, cystic fibrosis, congenital stationary night blindness or total color-blindness. 55. Canceled. 56. Canceled. 57. Canceled. 58. A genetically modified non-human animal comprising one or more nucleic acid molecules as claimed in claim 6 or claim 77. 59. Canceled. 60. Canceled. 61. Canceled. 62. Canceled. 63. Use of a genetically modified non-human animal as claimed in claim 58 in the screening of candidate pharmaceutical compounds. 64. Use as claimed in claim 63 in the screening of candidate pharmaceutical compounds useful in the treatment of epilepsy or a disorder associated with ion channel dysfunction, including but not restricted to, hyper- or hypo-kalemic periodic paralysis, myotonias, malignant hyperthermia, myasthenia, cardiac arrhythmias, episodic ataxia, migraine, Alzheimer's disease, Parkinson's disease, schizophrenia, hyperekplexia, anxiety, depression, phobic obsessive symptoms, neuropathic pain, inflammatory pain, chronic/acute pain, Bartter's syndrome, polycystic kidney disease, Dent's disease, hyperinsulinemic hypoglycemia of infancy, cystic fibrosis, congenital stationary night blindness or total color-blindness. 65. Canceled. 66. The use of a nucleic acid molecule as claimed in claim 6 or claim 77 for the diagnosis of epilepsy or a disorder associated with ion channel dysfunction, including but not restricted to, hyper- or hypo-kalemic periodic paralysis, myotonias, malignant hyperthermia, myasthenia, cardiac arrhythmias, episodic ataxia, migraine, Alzheimer's disease, Parkinson's disease, schizophrenia, hyperekplexia, anxiety, depression, phobic obsessive symptoms, neuropathic pain, inflammatory pain, chronic/acute pain, Bartter's syndrome, polycystic kidney disease, Dent's disease, hyperinsulinemic hypoglycemia of infancy, cystic fibrosis, congenital stationary night blindness or total colour-blindness. 67. Canceled. 68. The use of a polypeptide as claimed in claim 28 or claim 96 in the diagnosis of epilepsy or a disorder associated with ion channel dysfunction, including but not restricted to, hyper- or hypo-kalemic periodic paralysis, myotonias, malignant hyperthermia, myasthenia, cardiac arrhythmias, episodic ataxia, migraine, Alzheimer's disease, Parkinson's disease, schizophrenia, hyperekplexia, anxiety, depression, phobic obsessive symptoms, neuropathic pain, inflammatory pain, chronic/acute pain, Bartter's syndrome, polycystic kidney disease, Dent's disease, hyperinsulinemic hypoglycemia of infancy, cystic fibrosis, congenital stationary night blindness or total color-blindness. 69. Canceled. 70. Canceled. 71. Canceled. 72. A method for the diagnosis of epilepsy comprising the steps of: (1) obtaining DNA from a subject; and (2) comparing the DNA of one or more subunits of ion channels from said subject to the DNA of the corresponding native subunits; wherein identification of one or more DNA molecules as claimed in claim 6 or claim 77 is an indication of epilepsy, or a predisposition thereto. 73. A method for the diagnosis of a disorder associated with ion channel dysfunction, including but not restricted to, hyper- or hypo-kalemic periodic paralysis, myotonias, malignant hyperthermia, myasthenia, cardiac arrhythmias, episodic ataxia, migraine, Alzheimer's disease, Parkinson's disease, schizophrenia, hyperekplexia, anxiety, depression, phobic obsessive symptoms, neuropathic pain, inflammatory pain, chronic/acute pain, Bartter's syndrome, polycystic kidney disease, Dent's disease, hyperinsulinemic hypoglycemia of infancy, cystic fibrosis, congenital stationary night blindness or total color-blindness, comprising the steps of: (1) obtaining DNA from a subject; and (2) comparing the DNA of one or more subunits of ion channels from said subject to the DNA of the corresponding native subunits; wherein identification of one or more DNA molecules as claimed in claim 6 or claim 77 is an indication of the disorder, or a predisposition thereto. 74. Canceled. 75. Canceled. 76. Canceled. 77. An isolated nucleic acid molecule encoding a mutant subunit of a mammalian nicotinic acetylcholine receptor (nAChR), wherein a mutation event selected from the group consisting of point mutations, deletions, insertions and rearrangements has occurred in the nucleotides outside of the M2 domain of the subunit of said mammalian nicotinic acetylcholine receptor, so as to produce an epilepsy phenotype or so as to produce a disorder associated with ion channel dysfunction, including but not restricted to, hyper- or hypo-kalemic Periodic paralysis, myotonias, malignant hyperthermia, myasthenia, cardiac arrhythmias, episodic ataxia, migraine, Alzheimer's disease, Parkinson's disease, schizophrenia, hyperekplexia, anxiety, depression, phobic obsessive symptoms, neuropathic pain, inflammatory pain, chronic/acute pain, Bartter's syndrome, polycystic kidney disease, Dent's disease, hyperinsulinemic hypoglycemia of infancy, cystic fibrosis, congenital stationary night blindness or total colour-blindness. 78. Canceled. 79. An isolated nucleic acid molecule as claimed in claim 77 wherein said mutant subunit is the CHRNA4 subunit. 80. An isolated nucleic acid molecule as claimed in claim 78 wherein said mutation event takes place in exon 5. 81. An isolated nucleic acid molecule as claimed in claim 79 wherein said mutation event is selected from the group consisting of a C to T nucleotide substitution at position 1006, a G to A nucleotide substitution at position 1106 and a C to G nucleotide substitution at position 1421. 82. An isolated nucleic acid molecule as claimed in claim 77 wherein said mutant subunit is the CHRNB2 subunit. 83. An isolated nucleic acid molecule as claimed in claim 81 wherein said mutation event takes place in exon 2 or exon 5. 84. An isolated nucleic acid molecule as claimed in claim 82 wherein said mutation event is selected from the group consisting of a C to T nucleotide substitution at position 77, a C to G nucleotide substitution at position 901, a T to C nucleotide substitution at position 923 and a G to A nucleotide substitution at position 1235. 85. Canceled. 86. Canceled. 87. Canceled. 88. Canceled. 89. Canceled. 90. Canceled. 91. Canceled. 92. Canceled. 93. Canceled. 94. Canceled. 95. Canceled. 96. Canceled. 97. A polypeptide encoded by a nucleic acid molecule as claimed in claim 6 or 77. |
<SOH> BACKGROUND ART <EOH>Epilepsies constitute a diverse collection of brain disorders that affect about 3% of the population at some time in their lives (Annegers, 1996). An epileptic seizure can be defined as an episodic change in behaviour caused by the disordered firing of populations of neurons in the central nervous system. This results in varying degrees of involuntary muscle contraction and often a loss of consciousness. Epilepsy syndromes have been classified into more than 40 distinct types based upon characteristic symptoms, types of seizure, cause, age of onset and EEG patterns (Commission on Classification and Terminology of the International League Against Epilepsy, 1989). However the single feature that is common to all syndromes is the persistent increase in neuronal excitability that is both occasionally and unpredictably expressed as a seizure. A genetic contribution to the aetiology of epilepsy has been estimated to be present in approximately 40% of affected individuals (Gardiner, 2000). As epileptic seizures may be the end-point of a number of molecular aberrations that ultimately disturb neuronal synchrony, the genetic basis for epilepsy is likely to be heterogeneous. There are over 200 Mendelian diseases which include epilepsy as part of the phenotype. In these diseases, seizures are symptomatic of underlying neurological involvement such as disturbances in brain structure or function. In contrast, there are also a number of “pure” epilepsy syndromes in which epilepsy is the sole manifestation in the affected individuals. These are termed idiopathic and account for over 60% of all epilepsy cases. Idiopathic epilepsies have been further divided into partial and generalized sub-types. Partial (focal or local) epileptic fits arise from localized cortical discharges, so that only certain groups of muscles are involved and consciousness may be retained (Sutton, 1990). However, in generalized epilepsy, EEG discharge shows no focus such that all subcortical regions of the brain are involved. Although the observation that generalized epilepsies are frequently inherited is understandable, the mechanism by which genetic defects, presumably expressed constitutively in the brain, give rise to partial seizures is less clear. The molecular genetic era has resulted in spectacular advances in classification, diagnosis and biological understanding of numerous inherited neurological disorders including muscular dystrophies, familial neuropathies and spinocerebellar degenerations. These disorders are all uncommon or rare and have simple Mendelian inheritance. In contrast, common neurological diseases like epilepsy, have complex inheritance where they are determined by multiple genes sometimes interacting with environmental influences. Molecular genetic advances in disorders with complex inheritance have been far more modest to date (Todd, 1999). Most of the molecular genetic advances have occurred by a sequential three stage process. First a clinically homogeneous disorder is identified and its mode of inheritance determined. Second, linkage analysis is performed on carefully characterized clinical populations with the disorder. Linkage analysis is a process where the chromosomal localization of a particular disorder is narrowed down to approximately 0.5% of the total genome. Knowledge of linkage imparts no intrinsic biological insights other than the important clue as to where to look in the genome for the abnormal gene. Third, strategies such as positional cloning or the positional candidate approach are used to identify the aberrant gene and its specific mutations within the linked region (Collins, 1995). Linkage studies in disorders with complex inheritance have been bedevilled by negative results and by failure to replicate positive findings. A sense of frustration permeates current literature in the genetics of complex disorders. Carefully performed, large scale studies involving hundreds of sibpairs in disorders including multiple sclerosis and diabetes have been essentially negative (Bell and Lathrop, 1996; Lernmark and Ott, 1998). An emerging view is that such disorders are due to the summation of many genes of small effect and that identification of these genes may only be possible with very large-scale association studies. Such studies on a genome-wide basis are currently impossible due to incomplete marker sets and computational limitations. The idiopathic generalized epilepsies (IGE) are the most common group of inherited human epilepsy and do not have simple inheritance. Like other complex disorders, linkage studies in IGE have generated controversial and conflicting claims. Previous authors have suggested the possibility of multifactorial, polygenic, oligogenic or two locus models for the disease (Andermann, 1982; Doose and Baier, 1989; Greenberg et al., 1988a; 1992; Janz et al., 1992). Two broad groups of IGE are now known—the classical idiopathic generalized epilepsies (Commission on Classification and Terminology of the International League Against Epilepsy, 1989) and the newly recognized genetic syndrome of generalized epilepsy with febrile seizures plus (GEFS + ) (Scheffer and Berkovic, 1997; Singh et al., 1999). The classical IGEs are divided into a number of clinically recognizable but overlapping sub-syndromes including childhood absence epilepsy, juvenile absence epilepsy, juvenile myoclonic epilepsy etc (Commission on Classification and Terminology of the International League Against Epilepsy, 1989; Roger et al., 1992). The sub-syndromes are identified by age of onset and the pattern of seizure types (absence, myoclonus and tonic-clonic). Some patients, particularly those with tonic-clonic seizures alone do not fit a specifically recognized sub-syndrome. Arguments for regarding these as separate syndromes, yet recognizing that they are part of a neurobiological continuum, have been presented previously (Berkovic et al. 1987; 1994; Reutens and Berkovic, 1995). GEFS + was originally recognized through large multi-generation families and comprises a variety of sub-syndromes. Febrile seizures plus (FS + ) is a sub-syndrome where children have febrile seizures occurring outside the age range of 3 months to 6 years, or have associated febrile tonic-clonic seizures. Many family members have a phenotype indistinguishable from the classical febrile convulsion syndrome and some have FS + with additional absence, myoclonic, atonic, or complex partial seizures. The severe end of the GEFS + spectrum includes myoclonic-astatic epilepsy. The cumulative incidence for epilepsy by age 30 years (proportion suffering from epilepsy at some time) is 1.5% (Hauser et al., 1993). Accurate estimates for the cumulative incidence of the IGEs are unavailable. In epidemiological studies where attempts are made to subclassify epilepsies, rather few cases of IGE are diagnosed, and many cases are unclassified. This is probably because cases are rarely directly examined by epileptologists. In clinic- or office-based series seen by experts, most cases are classifiable and IGEs account for about 25% of cases. This suggests that about 0.3% of the population suffer from IGE at some time. In outbred populations many patients with classical IGE appear to be sporadic as siblings and parents are usually unaffected. Systematic EEG studies on clinically unaffected family members show an increase in age-dependent occurrence of generalized epileptiform discharges compared to controls. In addition, to the approximate 0.3% of the population with clinical IGE, systematic EEG studies suggest that about 1% of healthy children have generalized epileptiform discharges while awake (Cavazutti et al., 1980; Okubo et al., 1994). Approximately 5-10% of first degree relatives of classical IGE probands have seizures with affected relatives usually having IGE phenotypes or febrile seizures. While nuclear families with 2-4 affected individuals are well recognized and 3 generation families or grandparent-grandchild pairs are occasionally observed (Italian League Against Epilepsy Genetic Collaborative Group, 1993), families with multiple affected individuals extending over 4 or more generations are exceptionally rare. For GEFS + , however, a number of large multi-generation families showing autosomal dominant inheritance with incomplete penetrance are known. Similar to classical IGE, analysis of sporadic cases and small families with GEFS + phenotypes does not suggest simple Mendelian inheritance. Indeed, bilineal inheritance, where there is a history of epilepsy on maternal and paternal sides, is observed in both GEFS + and classical IGE families (Singh et al., 1999; Italian League Against Epilepsy Genetic Collaborative Group, 1993). Within single families with classical IGE or GEFS + , affected individuals often have different sub-syndromes. The closer an affected relative is to the proband, the more similar are their sub-syndromes, and siblings often have similar sub-syndromes (Italian League Against Epilepsy Genetic Collaborative Group, 1993). Less commonly, families are observed where most, or all, known affected individuals have one classical IGE sub-syndrome such as childhood absence epilepsy or juvenile myoclonic epilepsy (Italian League Against Epilepsy Genetic Collaborative Group, 1993). Importantly, sub-syndromes are identical in affected monozygous twins with IGE. In contrast, affected dizygous twins, may have the same or different sub-syndromes. Classical IGE and GEFS + sub-syndromes tend to segregate separately (Singh et al., 1999). In some inbred communities, pedigree analysis strongly suggests recessive inheritance for juvenile myoclonic epilepsy and other forms of IGE (Panayiotopoulos and Obeid, 1989; Berkovic et al., 2000). In such families, sub-syndromes are much more similar in affected siblings than in affected sib-pairs from outbred families. Recently, a family with an infantile form of IGE with autosomal recessive inheritance, confirmed by linkage analysis, was described in Italy (Zara et al., 2000). Most work on the molecular genetics of classical IGEs has been done on the sub-syndrome of juvenile myoclonic epilepsy where a locus in proximity or within the HLA region on chromosome 6p was first reported in 1988 (Greenberg et al., 1988b). This finding was supported by two collaborating laboratories, in separate patient samples, and subsequently three groups provided further evidence for a 6p locus for juvenile myoclonic epilepsy in some, but not all, of their families. However, genetic defects have not been found and the exact locus of the gene or genes, in relationship to the HLA region, remains controversial. Strong evidence for linkage to chromosome 6 also comes from a study of a single large family with juvenile myoclonic epilepsy, but in this pedigree the locus is well outside the HLA region. A locus on chromosome 15q has also been suggested for juvenile myoclonic epilepsy, but was not confirmed by two other studies. In general, the results of studies of the putative chromosomal 6p locus in the HLA region in patients with absence epilepsies or other forms of idiopathic generalized epilepsies have been negative. The major exception is that study of probands with tonic-clonic seizures on awakening, a sub-syndrome closely related to juvenile myoclonic epilepsy, suggests linkage to 6p. Linkage for classical remitting childhood absence epilepsy remains elusive, but in a family with persisting absence evolving into a juvenile myoclonic epilepsy phenotype, linkage to chromosome 1p has been claimed. An Indian pedigree with persisting absence and tonic-clonic seizures may link to 8q24. Linkage to this region was also suggested by a non-parametric analysis in IGE, irrespective of subsyndrome, but was not confirmed in another study. Other loci for IGEs that have been reported in single studies include 3 p14, 8p, 18 and possibly 5p. The unusual example of recessively inherited infantile onset IGE described in Italy maps to 16p in a single family. Thus, like most disorders with complex inheritance, the literature on genetics of classical IGEs is confusing and contradictory. Some, and perhaps much, of this confusion is due to heterogeneity, with the likelihood of a number of loci for IGEs. The studies reviewed above were principally performed on multiple small families, so heterogeneity within and between samples is probable. Whether all, some, or none of the linkages reported above will be found to harbour relevant genes for IGE remains to be determined. Most of the studies reviewed above used analysis methods assuming Mendelian inheritance, an assumption that is not correct for outbred communities. Some studies used multiple models (autosomal recessive, autosomal dominant). Although parametric linkage analysis may be reliable in some circumstance of analyzing complex disease, it can lead to spurious findings as highlighted by the literature on linkage in major psychoses (Risch and Botstein, 1996). In so far as GEFS + is concerned, linkage analysis on rare multi-generation large families with clinical evidence of a major autosomal dominant gene have demonstrated loci on chromosomes 19q and 2q. Both the 19q and 2q GEFS + loci have been confirmed in independently ascertained large families, and genetic defects have been identified. Families linked to 19q are known and a mutation in the gene for the β1 subunit of the neuronal sodium channel (SCN1B) has been identified (Wallace et al., 1998). This mutation results in the loss of a critical disulphide bridge of this regulatory subunit and causes a loss of function in vitro. Families linked to 2q are also known and mutations in the pore-forming α subunit of the neuronal sodium channel (SCN1A) have been identified (Australian provisional patent PR2203; Wallace et al., 2001b; Escayg et al., 2000). Studies on the more common small families with GEFS + have not revealed these or other mutations to date. In addition to the SCN1B and SCN1A mutations in GEFS + , four other gene defects have been discovered for human idiopathic epilepsies through the study of large families. Mutations in the alpha-4 subunit of the neuronal nicotinic acetylcholine receptor (CHRNA4) occur in the focal epilepsy syndrome of autosomal dominant nocturnal frontal lobe epilepsy (Australian patent AU-B-56247/96; Steinlein et al., 1995). Mutations in the gamma-2 subunit of the GABA A receptor (GABRG2) have been identified in childhood absence epilepsy, febrile seizures (including febrile seizures plus) and myoclonic epilepsy (PCT/AU01/00729; Wallace et al., 2001a). Finally, mutations in two potassium channel genes (KCNQ2 and KCNQ3) were identified in benign familial neonatal convulsions (Singh et al., 1998; Biervert et al., 1998; Charlier et al., 1998). Although initially regarded as a special form of IGE, this unusual syndrome is probably a form of inherited focal epilepsy. Further to these studies, mutations in other genes have been identified to be causative of epilepsy. These include mutations in the beta-2 subunit (CHRNB2) of the neuronal nicotinic acetylcholine receptor (PCT/AU01/00541; Phillips et al., 2001) and the delta subunit (GABRD) of the GABA A receptor (PCT/AU01/00729). A number of mouse models approximating human IGE are known. These mice mutants have ataxia in addition to generalized spike-and-wave discharges with absences or tonic-clonic seizures. Recessive mutations in calcium channel subunit genes have been found in lethargic (CACNB4), tottering/leaner (CACNA1A), and stargazer (CACNG2) mutants. The slow-wave epilepsy mouse mutant has a mutation in the sodium/hydrogen exchanger gene, which may have important downstream effects on pH-sensitive ion channels. The human and mouse literature is now suggesting that the idiopathic epilepsies comprise a family of channelopathies with mutations in ion channel subunits of voltage-gated (eg SCN1A, SCN1B, KCNQ2, KCNQ3) or ligand-gated (eg CHRNA4, CHRNB2, GABRG2, GABRD) types. These channels are typically comprised of a number of subunits, specified by genes on different chromosomes. The stoichiometry and conformation of ion channel subunits are not yet well understood, but many have multiple subunits in a variety of combinations. The involvement of ion channels in other neuro/physiological disorders has also been observed (reviewed in Dworakowska and Dolowy, 2000). Mutations in voltage-gated sodium, potassium, calcium and chloride channels as well as ligand-gated channels such as the acetylcholine and GABA receptors may lead to physiological disorders such as hyper- and hypo-kalemic periodic paralysis, myotonias, malignant hyperthermia, myasthenia and cardiac arrhythmias. Neurological disorders other than epilepsy that are associated with ion channel mutations include episodic ataxia, migraine, Alzheimer's disease, Parkinson's disease, schizophrenia, hyperekplexia, anxiety, depression, phobic obsessive symptoms, as well as neuropathic pain, inflammatory pain and chronic/acute pain. Some kidney disorders such as Bartter's syndrome, polycystic kidney disease and Dent's disease, secretion disorders such as hyperinsulinemic hypoglycemia of infancy and cystic fibrosis, and vision disorders such as congenital stationary night blindness and total colour-blindness may also be linked to mutations in ion channels. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>Preferred forms of the invention will now be described, by way of example only, with reference to the following examples and the accompanying drawings, in which: FIG. 1 provides an example of ion channel subunit stoichiometry and the effect of multiple versus single ion channel subunit mutations. FIG. 1A : A typical channel may have five subunits of three different types. FIG. 1B : In outbred populations complex diseases such as idiopathic generalized epilepsies may be due to mutations in two (or more) different subunit genes. Because only one allele of each subunit gene is abnormal, half the expressed subunits will have the mutation. FIG. 1C : In inbred populations, both alleles of a single subunit gene will be affected, so all expressed subunits will be mutated. FIG. 1D : Autosomal dominant disorders can be attributed to single ion channel subunit mutations that give rise to severe functional consequences; FIG. 2 represents the location of mutations identified in the ion channel subunits constituting the sodium channel. These examples include both novel and previously identified mutations; FIG. 3 provides examples of epilepsy pedigrees where mutation profiles of ion channel subunits for individuals constituting the pedigree have begun to be determined. These examples have been used to illustrate how the identification of novel ion channel subunit mutations and variations in IGE individuals can combine to give rise to the disorder. detailed-description description="Detailed Description" end="lead"? |
Solid oxide fuel cell stack configuration |
A fuel cell stack (2) comprises a stack (3) of alternating solid oxide fuel cell and gas separator plates within a housing (4). Each fuel cell plate has apertures therethough aligned with corresponding apertures through adjacent separator plates. A first aligned series of apertures in the fuel cell and separator plates opens to the anode side of each fuel cell to form a first manifold (5) for incoming fuel gas. A second aligned series of apertures in the fuel cell and separator plates opens from the anode side of each fuel cell to form a second manifold (6) for exhaust fuel gas. A third manifold (7) for in coming air is formed between the stack (3) and housing (4) and opens to the cathode side of each fuel cell. A fourth manifold (8) for exhaust air is formed between the stack (3) and housing (4) and opens from the cathode side of each fuel cell. In a preferred embodiment a third aligned series of apertures in the plates opens from the anode side of each fuel cell to form a second exhaust fuel gas manifold (6) and a second exhaust air manifold (8) is formed between the stack (3) and housing (4). Sliding fibrous seals (9) are provided are provided between the stack (3) and housing (4) to separate the air manifolds (7) and (8). |
1. A fuel cell stack comprising alternating solid oxide fuel cell plates and gas separator plates stacked face to face within a housing, each of said fuel cell plates having an electrolyte layer with an anode layer on one side of the electrolyte layer and a cathode layer on an opposite side of the electrolyte layer, wherein each of said fuel cell plates has apertures therethough aligned with corresponding apertures through adjacent gas separator plates, a first aligned series of said apertures in the fuel cell plates and the gas separator plates opening to the respective anode side of each of the fuel cell plates to form a first manifold through which incoming fuel gas is distributed and a second aligned series of said apertures in the fuel cell plates and the gas separator plates opening from the respective anode side of each of the fuel cell plates to form a second manifold through which exhaust fuel gas is discharged from the stack, and wherein a third manifold is formed between the plates and the housing and opens to the respective cathode side of each of the fuel cell plates for distributing oxygen-containing gas to the fuel cell plates and a fourth manifold is formed between the plates and the housing and opens from the respective cathode side of each of the fuel cell plates for discharging exhaust oxygen-containing gas from the stack. 2. A fuel cell stack according to claim 1 wherein a third aligned series of said apertures in the fuel cell plates and the gas separator plates opens from the respective anode side of each of the fuel cell plates to form a further exhaust fuel gas manifold. 3. A fuel cell stack according to claim 2 wherein the first and second manifolds and the further exhaust fuel gas manifold are angularly spaced about the fuel cell plates and the gas separator plates. 4. A fuel cell stack according to claim 3 wherein the fuel cell plates and the gas separator plates are each generally circular with three lobes extending therefrom through which the apertures of the first, second and third aligned series of apertures respectively extend. 5. A fuel cell stack according to any one of claims 2 to 4 wherein each of the apertures of the first series of apertures has a greater cross-sectional area than each of the apertures of the second and third series of apertures. 6. A fuel cell stack according to any of claims 1 to 5 wherein a gas-tight seal extends around each of the apertures of the series of apertures, between said opposite side of each fuel cell plate and the adjacent gas separator plate. 7. A fuel cell stack according to claim 6 wherein each of said gas tight seals comprises a groove having a glass sealant in the bottom thereof in an upwardly facing surface of one of said fuel cell plate and said adjacent gas separator plate and a rib on the oppositely facing surface of the other of said fuel cell plate and said adjacent gas separator plate that closes the open top of the groove to retain the sealant in the groove. 8. A fuel cell stack according to claim 7 wherein the groove is formed between a pair of spaced ribs extending around the respective aperture in said upwardly facing surface. 9. A fuel cell stack according to any one of claims 1 to 8 wherein a respective gas-tight seal between said one side of each fuel cell plate and the adjacent gas separator plate extends around said plates outwardly of the apertures of said series of apertures through said plates. 10. A fuel cell stack according to claim 9 wherein said gas-tight seal comprises a groove having a glass sealant in the bottom thereof in an upwardly facing surface of one of said fuel cell plate and said adjacent gas separator plate and a rib on the oppositely facing surface of the other of said fuel cell plate and said adjacent gas separator plate that closes the open top of the groove to retain the sealant in the groove. 11. A fuel cell stack according to claim 10 wherein the groove is formed between a pair or spaced ribs on said upwardly facing surface. 12. A fuel cell stack according to any one of claims 1 to 11 wherein a further exhaust oxygen-containing gas manifold opening from the respective cathode side of each of the fuel cell plates is formed between the plates and the housing. 13. A fuel cell stack according to any one of claims 1 to 12 wherein the manifolds formed between the plates and the housing are separated by seals extending along the stack between the plates and the housing. 14. A fuel cell stack according to claim 13 wherein the seals are fibrous seals. 15. A fuel cell stack according to any one of claims 1 to 14 wherein the fuel gas flow across the anode layer of each of the fuel cell plates is in counter-flow to the oxygen containing gas flow across the cathode side of said fuel cell plate. 16. A fuel cell stack according to any one of claims 1 to 15 wherein the housing is cylindrical. 17. A fuel cell stack according to any one of claims 1 to 16 wherein the housing is constructed from sheet material formed of heat resistant steel. 18. A fuel cell stack according to any one of claims 1 to 17 wherein a respective current collector is provided between each adjacent pair of said fuel cell plates and said gas separator plates. 19. A fuel cell stack according to any one of claims 1 to IS wherein gas flow control formations are provided on the plates between each adjacent pair of said fuel cell plates and said gas separator plates. 20. A fuel cell stack according to claim 19 wherein the gas flow control formations act as current collectors. 21. A fuel cell stack according to any one of claims 1 to 20 wherein the solid oxide electrolyte is an yttria-stabilized zirconia and wherein each of said gas separator plates is formed at least substantially of zirconia. 22. A fuel cell stack according to claim 21 wherein the zirconia of the gas separator plates includes up to about 20 wt. % alumina. 23. A fuel cell stack according to claim 21 or claim 22 wherein each of said gas separator plates has electrically conductive paths therethrough from the anode-facing side to the cathode-facing side. 24. A fuel cell stack according to claim 23 wherein the material of the electrically conductive paths comprises silver. |
<SOH> BACKGROUND OF THE INVENTION <EOH>While planar solid oxide fuel cells are a proven technology in terms of individual cells, the problem of combining the individual cells into stacks, having collectively useful power at reasonable cost and with acceptable durability, has proven to be an elusive goal to those skilled in the art. Specific difficulties include providing satisfactory fuel and oxygen-containing gas inlet and exhaust manifolding systems as well as reliable sealing of the system at a commercially acceptable cost while at the same time providing a structure that is robust to thermal cyling. There have been many different patent proposals for manifolding fuel cell systems comprising alternating fuel cell members and gas separator members, both for SOFCs and other fuel cell technologies such as molten carbonate fuel cells. Many such proposals are for fully internally manifolding the fuel gas and oxygen-containing gas supply and exhaust, that is all of the manifolds pass through apertures in the fuel cell members and the gas separator members, requiring reliable and complex sealing arrangements around each aperture to ensure the fuel gas and oxygen-containing gas remain separated from each other at all times during operation and thermal cycling of the fuel cell stack. By way of example only, the following patent specifications are all directed to internally manifolded fuel cell systems: U.S. Pat. Nos. 6,103,415, 6,040,076, 5,945,232 and 4,963,442, EP 959511 and 459940, WO 95/16287 and 92/09116 and JP 2001-202984, 06-275304 and 04-149966. Other internally manifolded fuel cell systems are proposed in U.S. Pat. Nos. 5,288,569 and 5,230,966, EP 425939 and JP 08-190921. In contrast, an externally manifolded proton exchange membrane fuel cell is proposed in WO 01/99219. U.S. Pat. No. 5,688,610 is also directed to external manifolding, for planar SOFC stacks. In this proposal fuel gas and exhaust fuel gas are directed to and from the stacks by respective manifolds on the sides of the stack and exhaust air gas is directed from plural stacks by a common manifold, but incoming air is freely directed to the stacks from within a housing enclosing all of the stacks. In the prior art discussion of U.S. Pat. No. 5,688,610, similar proposals to that in the patent are said to be disclosed in DE 4,324,907 and EP 450336. Although U.S. Pat. No. 6,040,076 referenced above is directed to an internally manifolded gas separator plate, it also mentions internal manifolding being provided for fuel gas or oxidant communication to one side of the gas separator plate while the other of fuel gas or oxidant gas is provided to the opposite side of the gas separator plate through an external manifold. However, there is no suggestion of how this is achieved. It is an aim of the present invention to simplify the manifolding of an SOFC stack, and to thereby simplify the sealing of the stack. |
<SOH> SUMMARY OF THE INVENTION <EOH>According to the invention there is provided a fuel cell stack comprising alternating solid oxide fuel cell plates and gas separator plates stacked face to face within a housing, each of said fuel cell plates having an electrolyte layer with an anode layer on one side of the electrolyte layer and a cathode layer on an opposite side of the electrolyte layer, wherein each of said fuel cell plates has apertures therethough aligned with corresponding apertures through adjacent gas separator plates, a first aligned series of said apertures in the fuel cell plates and the gas separator plates opening to the respective anode side of each of the fuel cell plates to form a first manifold through which incoming fuel gas is distributed and a second aligned series of said apertures in the fuel cell plates and the gas separator plates opening from the respective anode side of each of the fuel cell plates to form a second manifold through which exhaust fuel gas is discharged from the stack, and wherein a third manifold is formed between the plates and the housing and opens to the respective cathode side of each of the fuel cell plates for distributing oxygen-containing gas to the fuel cell plates and a fourth manifold is formed between the plates and the housing and opens from the respective cathode side of each of the fuel cell plates for discharging exhaust oxygen-containing gas from the stack. Provision of fuel inlet and exhaust manifolds internally of the plates and oxygen-containing gas (usually air) inlet and exhaust manifolds externally of the plates can optimise the structure of the plates from both economic and power producing perspectives. If the manifolds were fully internalised, the construction of the plates would be more complex and a significant portion of the plates would need to be dedicated to the formation of the respective manifolds, i.e. each plate would have an increased aperture area compared to the plates in the stack of the invention. Relatively increasing the functional area of the plates allows for maximised generation of electric current from the stack. Externalising the air manifolds simplifies the inter-plate sealing since there are no air apertures through the plates around which individual seals must be provided, and providing the air manifolds between the plates and the housing can allow for simple seals between the air manifolds. However, internalising the fuel manifolds also means the overall structure may be robust since external connections which may otherwise be subject to fatigue or leakage are minimised. There may be more than one incoming fuel gas manifold and/or more than one exhaust fuel gas manifold to enhance fuel gas flow through the stack. In a preferred embodiment, a third aligned series of said apertures in the fuel cell plates and the gas separator plates opens from the respective anode side of each of the fuel cell plates to form a further exhaust fuel gas manifold. Preferably, the first and second manifolds and the further exhaust fuel gas manifold are angularly spaced about the fuel cell plates and the gas separator plates. In this arrangement, the fuel cell plates and the gas separator plates may each be generally circular with three lobes extending therefrom through which the apertures of the first, second and third aligned series of apertures respectively extend. Advantageously, each of the apertures of the first series of apertures has a greater cross-sectional area than each of the apertures of the second and third series of apertures. The incoming and exhaust fuel gas manifolds must be sealed to prevent leakage of the fuel gas into the cathode sides of the fuel cell plates, and advantageously a gas-tight seal extends around each of the apertures of the series of apertures, between said opposite side of each fuel cell plate and the adjacent gas separator plate. The gas-tight seals may take any suitable form, including gasket seals and opposed formations ill the plates. In one embodiment, each of said gas-tight seals comprises a groove having a glass sealant in the bottom thereof in an upwardly facing surface of one of said fuel cell plate and said adjacent gas separator plate and a rib on the oppositely facing surface of the other of said fuel cell plate and said adjacent gas separator plate and said rib projects into contact with the glass in the groove. Conveniently, the rib, or the plate from which the rib protrudes closes the open top of the groove to retain the sealant in the groove. Conveniently, the groove is formed between a pair of spaced ribs extending around the respective aperture in said upwardly facing surface. It is important that the incoming and exhaust air is prevented from leaking into the anode sides of the fuel cell plates, and there is advantageously provided a respective gas-tight seal between said one of each fuel cell plate and the adjacent gas separator plate around said plates outwardly of the apertures of said series of apertures through said plates. This gas-tight seal may extend wholly around the periphery of the one side of the respective fuel cell plate and adjacent gas separator plate, but conveniently it is also used to direct the fuel gas flow across the anode. The same options for the gas-tight seal between the one side of each fuel cell plate and the adjacent gas separator plate apply as for the gas-tight seal that extends around the apertures between the opposite side of each fuel cell plate and the adjacent gas separator plate described above. The fuel cell and gas separator plates of the stack are preferably physically in a columnar series and enclosed within a cylindrical housing, with the walls of the housing at least partially defiling the inlet air and exhaust air manifolds. The air manifolds, being external to the fuel cell plates and the separator plates, are also defined by peripheral portions of the fuel cell plates and the gas separator plates and seals between the plates. The inlet air and exhaust air manifolds formed between the plates and the housing are preferably separated by seals, most preferably simple fibre seals, extending along the stack between the plates and the housing. There may be a small degree of gas leakage between the inlet air and exhaust manifolds past the seals, but this need not significantly affect the performance of the stack. There may be more than one inlet air manifold and/or more than one exhaust air manifold to enhance the flow of the oxygen-containing gas through the stack. In a preferred embodiment, a further exhaust air manifold opening from the respective cathode side of each of the fuel cell plates is formed between the plates and the housing. In the preferred embodiment described above, the three air manifolds are partly defined by the portions of the plate peripheries between the three lobes through which the apertures of the first, second and third aligned series of apertures respectively extend. Advantageously, the fuel gas flow across the anode layer of each of the fuel cell plates is in counter-flow to the air flow across the cathode side of the fuel cell plates, in which case the inlet air manifold is conveniently defined between the lobes through which the apertures of the second and third series of apertures extend, while the two exhaust air manifolds are defined between the lobes through which the apertures of the first series of apertures extend and the apertures of the second and third series of apertures, respectively. Advantageously, the inlet air manifold had a greater angular extent than each of the two exhaust air manifolds. The stack may be arranged so that the air and fuel gases pass through the manifolds in co-flow or counter-flow. The current may be passed along the stack from, for example, an anode of one of the cell plates, via a fuel side current collector between the plates either through or around an adjacent separator plate, to an air side current collector at a cathode of an adjoining fuel cell plate. If the current passes around the separator plate, it may do so by means of conductive wires or foils or some other means. If the current passes through the separator plate, it may do so either by use of the bulk material of that plate, or by specific conductive elements in the plate. Gas flow control formations may be provided on the plates between each adjacent pair of said fuel cell plates and said gas separator plates, and such gas flow control formations may also act as current collectors. They may also act as spacers to control the spacing of the respective separator plate from an adjacent fuel cell plate. The fuel cell plates and the gas separator plates are preferably both constructed of ceramic material such as zirconia, but the gas separator plates may be formed of a suitable metal such as self-aluminising stainless steel. |
Fuel cell gas separator plate |
A fuel cell gas separator (212) for use between two solid oxide fuel cells (210) and having a separator body with an anode-facing side and a cathode-facing side and with paths (234) of electrically conductive material therethrough in an electrode-contacting zone (236). In a first aspect, the electrically conductive material comprises a silver-glass composite, preferably containing 15 to 30 wt % glass. In this aspect the material of the separator body is preferably zirconia and the silver is commercially pure, a silver mixture or a silver alloy. In another aspect, the material of the separator body is zirconia, the electrically conductive material comprises silver or a silver-based material, a coating of nickel is formed on the electrode-contacting zone (236) on the anode-facing side preferably with an undercoating of Ag, and a coating of Ag—Sn alloy is formed on the electrode-contacting zone (236) on the cathode side. |
1. A fuel cell gas separator for use between two solid oxide fuel cells, the gas separator having a separator body with an anode-facing side and a cathode-facing side and with paths of electrically conductive material therethrough from the anode-facing side to the cathode-facing side in an electrode-contacting zone, wherein the electrically conductive material forming at least part of the length of each path is a silver-glass composite. 2. A gas separator according to claim 1 wherein the material of the separator body is zirconia. 3. A gas separator according to claim 2 wherein the zirconia contains up to about 20 wt % alumina. 4. A gas separator according to any one of claims 1 to 3 wherein the silver-glass composite contains from about 10 to about 40 wt % glass. 5. A gas separator according to claim 4 wherein the silver-glass composite contains from about 15 to about 30 wt % glass. 6. A gas separator according to any one of claims 1 to 5 wherein the silver in the silver-glass composite is commercially pure silver. 7. A gas separator according to any one of claims 1 to 5 wherein the silver in the silver-glass composite is a silver alloy or mixture. 8. A gas separator according to claim 7 wherein the silver is alloyed or mixed with any one or more of gold, palladium, platinum and stainless steel. 9. A gas separator according to any one of claims 1 to 8 wherein the glass in the silver-glass composite is stable against crystallisation. 10. A gas separator according to any one of claims 1 to 9 wherein the glass in the silver-glass composite is a high silica glass. 11. A gas separator according to claim 10 wherein the composition of the glass is 0-5.5 wt % Na2O, 8-14 wt % K2O, 0-2.2 wt % MgO, 1-3 wt % CaO, 0-6 wt % SrO, 0-8 wt % BaO, 6-20 wt % B2O3, 3-7 wt % Al2O3, 58-76 wt % SiO2 and 0-10 wt % ZrO2. 12. A gas separator according to claim 11 wherein the composition of the glass is 0-2.0 wt % Na2O, 8-13.5 wt % 120, 0-0.05 wt % MgO, 1-1.6 wt % CaO, 0.5-1 wt % SrO, 0-4.4 wt % BaO, 6-20 wt % B2O3, 3-6.0 wt % Al2O3, 60-75 wt % SiO2 and 0-5.0 wt % ZrO2. 13. A gas separator according to any one of claims 1 to 12 wherein a respective electrically conductive coating is provided on the silver-glass composite at the anode-facing side and at the cathode-facing side of the separator body. 14. A gas separator according to claim 13 wherein each of said coatings extends over the respective electrode-contacting zone. 15. A gas separator according to claim 13 or claim 14 wherein the coating on the cathode-facing side is of Ag or Ag alloy. 16. A fuel cell gas separator for use between two solid oxide fuel cells, the gas separator having a zirconia-based body with an anode-facing side and a cathode-facing side and with paths of electrically conductive material therethrough from the anode-facing side to the cathode-facing side in an electrode-contacting zone of the separator body, wherein the electrically conductive material forming at least part of the length of each path is silver or a silver-based material and wherein a coating of nickel on the electrode-contacting zone on the anode-facing side overlies said silver or silver-based material in the paths of electrically conductive material and a coating of Ag or Ag—Sn alloy on the electrode contacting zone on the cathode-facing side overlies said silver or silver-based material in the paths of electrically conductive material. 17. A gas separator according to claim 16 wherein the zirconia of the separator body is yttria-stabilised. 18. A gas separator according to claim 16 wherein the zirconia of the separator body contains up to about 20 wt % alumina. 19. A gas separator according to any one of claims 16 to 18 wherein the silver or silver-based material is metallic silver. 20. A gas separator according to any one of claims 16 to 18 wherein the silver or silver-based material is a silver alloy or mixture. 21. A gas separator according to claim 20 wherein the silver is alloyed or mixed with any one or more of gold, palladium, platinum and stainless steel. 22. A gas separator according to any one of claims 16 to 18 wherein the silver or silver-based material is a silver-glass composite. 23. A gas separator according to any one of claims 16 to 22 wherein the coating on the anode-facing side is of commercially pure nickel. 24. A gas separator according to any one of claims 16 to 23 wherein the layer of nickel on the anode-facing side has a thickness in the range of 10 to 1000 μm. 25. A gas separator according to any one of claims 16 to 24 wherein a layer of silver is disposed on the electrode-contacting zone between the coating of nickel and the anode-facing side of the gas separator body. 26. A gas separator according to claim 25 wherein the layer of silver comprises commercially pure silver. 27. A gas separator according to claim 25 or claim 26 wherein the layer of silver has a thickness in the range of 10 to 1000 μm. 28. A gas separator according to any one of claims 15 to 27 wherein the coating on the cathode-facing side is Ag—Sn alloy that contains from about 4 to about 20 wt % Sn. 29. A gas separator according to any one of claims 15 to 28 wherein the coating on the cathode-facing side is Ag—Sn alloy that includes up to 10 wt % of dopants to improve the electrical conductivity of said coating. 30. A gas separator according to any one of claims 15 to 29 wherein the coating on the cathode-facing side is Ag—Sn alloy and has a thickness in the range of 10 to 1000 μm. 31. A gas separator according to any one of claims 15 to 30 wherein the coating on the cathode-facing side is Ag—Sn alloy having a surface layer of SnO2. 32. A gas separator according to any one of claims 15 to 27 wherein the coating on the cathode-facing side is of commercially pure silver and has a thickness in the range of 50 to 250 μm. 33. A gas separator according to any one of claims 1 to 32 wherein the paths of electrically conductive material are formed in perforations through the separator body. 34. A gas separator according to claim 33 wherein the perforations extend perpendicularly through the thickness of the separator body. 35. A gas separator according to claim 34 wherein each path of electrically conductive material at the anode side of the separator body is offset relative to a connected path of electrically conductive material at the cathode side. 36. A gas separator according to any one of claims 1 to 35 wherein each path of electrically conductive material has an average cross-sectional dimension in the range of 50 to 1000 μm. 37. A gas separator according to any one of claims 1 to 36 wherein the total area of the paths of electrically conductive material through the separator body is in the range of 0.1 to 20 mm2 per 1000 mm2 surface area (measured on one side-only) of the electrode-contacting zone. 38. A gas separator according to any one of claims 1 to 37 wherein the separator body is in the form of a plate. 39. A gas separator according to any one of claims 1 to 38 wherein surface formations defining gas flow passages therebetween are provided on each of the anode-facing side and cathode-facing side in the electrode-contacting zone, said surface formations being electrically conductive and overlying the paths of electrically conductive material. 40. A gas separator according to claim 39 wherein the surface formations on the anode side are formed of solid oxide fuel cell anode material and the surface formations on the cathode side are formed of solid oxide fuel cell cathode material, said surface formations being bonded to the separator body or to any coating in the electrode-contacting zone. 41. A gas separator according to claim 39 or claim 40 wherein a respective electrically conductive coating is provided over the surface formations on the anode-facing side and on the cathode-facing side. 42. A gas separator according to claim 41 wherein the coating on the surface formations on the cathode-facing side is of metallic silver. 43. A gas separator according to claim 41 or claim 42 wherein the coating on the surface formations on the anode-facing side is of nickel. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The purpose of a gas separator in a fuel cell assembly is to keep the oxygen containing gas supplied to the cathode side of one fuel cell separate from the fuel gas supplied to the anode side of an adjacent fuel cell, and to conduct heat generated in the fuel cells away from the fuel cells. The gas separator may also conduct electricity generated in the fuel cells between or away from the fuel cells. Although it has been proposed that this function may alternatively be performed by a separate member between each fuel cell and the gas separator, much development work has been carried out on electrically conductive gas separators. Sophisticated ceramics for use in gas separators for solid oxide fuel cells have been developed which are electrically conductive, but these suffer from a relatively high fragility, low thermal conductivity and high cost. Special metallic alloys have also been developed, but it has proved difficult to avoid the various materials of the fuel cell assembly and the interfaces between them degrading or changing substantially through the life of the fuel cell, particularly insofar as their electrical conductivity is concerned, because of the tendency of different materials to chemically interact at the high temperatures that are required for efficient operation of a solid oxide fuel cell. For example, most metallic gas separators contain substantial quantities of the element chromium, which is used to impart oxidation resistance to the metal as well as other properties. It has been found that where chromium is present in more than minute quantities it may combine with oxygen or oxygen plus moisture to form highly volatile oxide or oxyhydroxide gases under conditions that are typical of those experienced in operating solid oxide fuel cells. These volatile gases are attracted to the cathode-electrolyte interface where they may react to form compounds that are deleterious to the efficiency of the fuel cell. If these chromium reactions are not eliminated or substantially inhibited, the performance of the fuel cell deteriorates with time to the point where the fuel cell is no longer effective. Several of these metallic alloys and one proposal for alleviating this problem are described in our patent application WO96/28855, in which a chromium-containing gas separator is provided with an oxide surface layer that reacts with the chromium to form a spinel layer between the substrate and the oxide surface layer and thereby tie in the chromium. However, these specialist alloys remain expensive for substantial use in fuel cell assemblies and it would be preferable to have a lower cost alternative. Special stainless steels have also been developed that are stable at high temperature in the atmospheres concerned, but they generally contain substantial amounts of chromium to provide the desired oxidation resistance, and special coatings or treatments are required to prevent the chromium-based gases escaping from a gas separator formed of these steels. Another approach to a heat resistant steel gas separator is described in our patent application WO 99/25890. However, all of these heat resistant steels are specialist materials and their cost will remain high unless substantial amounts can be produced. Furthermore, the thermal and electrical conductivities of heat resistant steels are low relative to many other metals and alloys, for example 22-24 W/m.K compared to 40-50 W/m.K for the Siemens-Plansee alloy described in WO96/28855. To compensate for this, the thickness of the steel gas separator has to be increased, increasing the mass and cost of a fuel cell stack. In yet another proposal, disclosed in our patent application WO 00/76015, we have found that copper-based gas separators may be successfully utilised in solid oxide fuel cell assemblies without poisoning the anode. Such a gas separator member comprises a layer of copper or copper-based alloy having a layer of oxidation-resistant material on the cathode side. One of the major difficulties with developing a satisfactory gas separator is ensuring that its coefficient of thermal expansion (“CTE”) is at least substantially matched to that of the other components of the fuel cell assembly. For example, solid oxide fuel cells comprising an oxide electrolyte with a cathode and an anode on opposed surfaces operate at temperatures in excess of about 700° C., and the alternating gas separators and fuel cells are generally bonded or otherwise sealed to each other. Thus, any substantial mismatch in the CTE between the two components can lead to cracking of one or both of them, with resultant leakage of the fuel gas and oxygen-containing gas across the component or components, and eventually to failure of the fuel cell stack. A particular difficulty with developing a suitable fuel cell gas separator is providing a material that provides all four functions of separating the fuel gas on one side from the oxygen-containing gas on the other side, being thermally conductive, having a CTE substantially matched to that of the other fuel cell components, and being electrically conductive. In order to meet these requirements, it has been proposed to provide a gas separator formed principally of a material that may not be electrically conductive, or not adequately electrically conductive, but that meets the other requirements, and to provide electrically conductive feedthroughs through the thickness of the separator. One such proposal is made in Kendall et al. in Solid Oxide Fuel Cells IV, 1995, pp.229-235, in which the gas separator plate is formed of a zirconia material and lanthanum chromite rivets extend through holes in the plate. Another proposal for electrically conductive feedthroughs through the thickness of the separator is made in EP 0993059. In this proposal, a ceramic gas separator plate, preferably stabilized zirconia, has passages therethrough that in the preferred embodiment are filled with cathode material from the cathode side and with anode material from the anode side. Alternatively, they may be filled with a single material composition such as doped chromite, silver-palladium or Plansee alloy. Thus, the feedthrough material is different to that of the principal separator material and will generally have a higher electrical conductivity. However, as the gas separator is subjected to thermal cycling, this can lead to the disadvantage of the feedthrough material becoming loose in the plate material, due to their different CTEs, and to the leakage of gas through the passages in which the feedthroughs are formed. Additionally in EP 0993059, individual contacts for the feethroughs of, for example, Ni, Plansee metal or Ag—Pd on the anode side and Ag—Pd or lanthanum strontium manganite on the cathode side, are bonded to the respective electrode by means of a bond layer that overlies the entire electrode surface. Such a bond layer will tend to inhibit free gas flow through the electrode and the individual contacts must be located very accurately on the electrodes to overlie the respective feedthrough when the fuel cell plates carrying the electrodes and the individual contacts are assembled with the gas separator plates An alternative proposal published in US Patent Application 20020068677 on 6 Jun. 2002 includes a gas separator plate in which the principal plate material is a high silica glass matrix having a metal conductor incorporated therein formed of a material such as silver, Ag—Pd alloy, gold and ferritic stainless steel. An aim of each aspect the present invention is to provide a fuel cell gas separator that alleviates at least some of the abovementioned disadvantages. |
<SOH> SUMMARY OF THE INVENTION <EOH>According to a first aspect of the present invention there is provided a fuel cell gas separator for use between two solid oxide fuel cells, the gas separator having a separator body with an anode-facing side and a cathode-facing side and with paths of electrically conductive material therethrough from the anode-facing side to the cathode-facing side in an electrode-contacting zone, wherein the electrically conductive material forming at least part of the length of each path is a silver-glass composite. By this aspect of the present invention, the advantage of separating the desired level of electrical conductivity of the gas separator from the material of the separator body is achieved by the use of silver in the electrically conductive paths through the separator body, and the risk of leakage of gases through the gas separator is alleviated by the use of glass in the electrically conductive paths. The glass may soften at the operating temperature of the fuel cell and, if necessary, can flow with expansion and contraction of the separator body as the separator is subjected to thermal cycling. The ductility of the silver facilitates this. The silver-glass composite may effectively be in the form of pure silver or a silver-based material in a glass matrix. The material of the separator body is preferably selected with a CTE that substantially matches that of other ftiel cell components, but any suitable material may be selected, including electrically conductive materials such as metals and alloys. In a solid oxide fuel cell assembly, in which the electrolyte material is preferably a zirconia and may be the principal layer that supports the electrode layers, the material of the separator body is advantageously zirconia, as described hereinafter. The silver-glass composite preferably comprises from about 10 to about 40 wt % glass, more preferably from 15 to 30 wt % glass. About 10 wt % glass is believed to be the lower limit to provide adequate sealing advantages in the separator body, while at a level above about 40 wt % glass there may be insufficient silver in the composite to provide the desired level of electrical conductivity. Potentially, the proportions of silver and glass in the composite may be varied to best suit the CTE of the separator body but the major advantages of the composite lie in the ability of the material to deform with expansion and contraction of the separator body and to conduct electricity. The silver-glass composite may be formed by a variety of suitable processes, including mixing glass and silver powders, mixing glass powder with silver salts, and mixing sol-gel glass precursors and silver powder or silver salts. Alternatively, for example, the silver or silver salt may be introduced to the glass matrix after the glass particles have been provided in the principal material of the gas separator, as described hereinafter. The material is then fired. One suitable silver salt is silver nitrate. In a preferred embodiment the glass powder has a particle size of less than 100 μm, most preferably with an average particle size in the range of 13 to 16 μm, and the silver powder has a particle size less than 45 μm. A suitable binder is for example an organic screen printing medium or ink. After mixing, the composition is introduced to passages through the principal separator material and fired. The silver may be commercially pure, a material mixture in which Ag is the major component or, for example, a silver alloy. Silver may advantageously be used alone in the glass matrix provided the operating temperature of the fuel cell is not above about 900° C., for example in the range 800 to 900° C. There may be some ion exchange of the silver at the interface with the glass that may strengthen the Ag-glass bond and may spread interface stresses. Particularly if the fuel cell operating temperature will be higher than about 900° C., above the melting point of the silver, for example up to 1100° C., the silver may be alloyed with any suitable ductile metal or metals having a sufficiently high melting point, for example one or more noble metals such as gold, palladium and platinum. Preferably, there will be no less than 50 wt % Ag present in the alloy. If the high melting temperature alloying metal or metals excessively reduces the ability of the silver alloy to bond with the glass by ion exchange at the interface, a lower melting temperature metal such as copper may be also included. An alternative and cheaper material to combine with the Ag is stainless steel. The Ag and stainless steel may be mixed to powders prior to being combined with the glass. A variety of different glass compositions can be used with the selected principal separator material. The glass composition should be stable against crystallisation (for example, less than 40% by volume crystallisation) at the temperatures and cool-down rates at which the, fuel cell gas separator will be used. Advantageously, the glass composition has a small viscosity change over the intended fuel cell operating range of, for example, 700 to 1100° C., preferably 800 to 900° C. At the maximum intended operating temperature, the viscosity of the glass should not have decreased to the extent that the glass is capable of flowing out of the separator body under its own weight. Preferably, the glass is low (for example, less than 10 wt %) in or free of fuming components, for example no lead oxide, no cadmium oxide, no zinc oxide, and no or low sodium oxide and boron oxide. The type of glasses that exhibit a small viscosity change over at least the 100° C. temperature range at the preferred fuel cell operating range of 800° C. to 900° C. are typically high silica glasses, for example in the range 55 to 80 wt % SiO 2 . Such glasses generally have a relatively low CTE. Preferred and more preferred compositions of such a high silica glass, particularly for use with a zirconia gas separator body, are set out in Table 1. TABLE 1 Preferred Range More Preferred Range Oxide wt % wt % Na 2 O 0-5.5 0-2.0 K 2 O 8-14 8-13.5 MgO 0-2.2 0-0.05 CaO 1-3 1-1.6 SrO 0-6 0.5-1 BaO 0-8 0-4.4 B 2 O 3 6-20 6-20 Al 2 O 3 3-7 3-6.0 SiO 2 58-76 60-75 ZrO 2 0-10 0-5.0 The electrically conductive material may be introduced to the paths by any suitable means. For example, after the glass powder or particles have been introduced to the paths or perforations, a solution of a silver salt or very fine suspension of the silver material, for example as a liquid coating applied to one or both surfaces of the separator body, may be permitted or caused to be drawn through the glass particles in the paths or perforations, such as by capillary action. Alternatively, the solution or suspension could be injected in. More preferably, a mixture of the glass and silver material powders in a binder is printed, for example by screen or stencil printing, onto one or both surfaces of the separator body to at least partly fill the paths in the body. The mixture is then heated to melt the glass and sinter the silver. The molten glass-silver composite then flows in the paths to seal them. A suitable heating/firing temperature is dependent upon the glass composition and the silver material but is preferably in the range 650 to 950° C. for pure silver in a high silica glass matrix for optimum melting of the glass without undue evaporation of the silver. In order to ensure that the fuel cell gas separator does transmit electricity between the surfaces defined by the anode-facing and cathode-facing sides of the separator body, the silver-glass composite in the paths may extend to the outer surfaces of the separator body. Alternatively, the silver-glass composite may have an electrically conductive coating on it in the paths which extends to the respective surface and which may protect the silver-glass composite and/or the interface between the gas separator and the adjacent electrode. For example, in accordance with the second aspect of the invention, a Ni protective coating may be provided at the anode side, optionally with an undercoating of Ag, and a Ag or Ag alloy such as Ag—Sn protective coating may be provided at the cathode side to alleviate loss of the silver-glass composite through evaporation or “wiclcing” to other nearby components. In particular, the coating may alleviate loss of the glass in the silver-glass composite to the adjacent fuel cell electrode or other porous component by capillary action at the fuel cell operating temperature. The coatings also enhance electrical contacts and provide a degree of compliance. To enhance electrical current flow between the adjacent fuel cell and gas separator, the aforementioned protective coatings advantageously extend across the electrode-contacting zones of the separator body, for example with a thickness in the range of 10 to 1000 μm, preferably 60 to 150 μm. Alternatively or in addition, a respective mesh or other current collector may be interposed between the gas separator and the electrodes of the adjacent fuel cells. The mesh or other current collector may define, or partly define, gas passages through which the air or other oxygen-containing gas on the cathode side of the gas separator and the fuel gas on the anode side of the gas separator is passed over the adjacent fuel cell electrode. According to a second aspect of the invention, there is provided a fuel cell gas separator for use between two solid oxide fuel cells, the gas separator having a zirconia-based body with an anode-facing side and a cathode-facing side and with paths of electrically conductive material therethrough from the anode-facing side to the cathode-facing side in an electrode-contacting zone of the separator body, wherein the electrically conductive material forming at least part of the length of each path is silver or a silver-based material and wherein a coating of nickel on the electrode-contacting zone on the anode-facing side overlies said silver or silver-based material in the paths of electrically conductive material and a coating of Ag or of Ag—Sn alloy on the electrode contacting zone on the cathode-facing side overlies said silver or silver-based material in the paths of electrically conductive material. By this aspect of the invention, the electrically conductive paths are protected by the opposed surface coatings over the electrode contacting zones of the separator body, electrical contact with an integral of separate device or devices between the gas separator and the adjacent electrode for current collection and/or gas flow control may be enhanced, and the coatings may give a degree of compliance by distributing uneven loads due to components of the fuel cell stack having slightly different heights. By the term “electrode-contacting zone” as used throughout this specification is meant the portion of the gas separator body that is opposed to and aligned with the respective electrodes of the adjacent fuel cell plates. Any contact of the electrode-contacting zone with the adjacent electrodes may be indirect, through interposed current collection and/or gas flow control devices. It will be understood therefore that the use of the term “electrode-contacting zone” does not require that zone of the gas separator body to directly contact the adjacent electrodes. The silver or silver-based electrically conductive material may be the silver-glass composite used and described with reference to the first aspect of the invention, but could alternatively be metallic silver (commercially pure), a metallic mixture in which Ag is the major component, or a silver alloy. Particularly if the fuel cell operating temperature will be higher than about 900° C., above the melting point of the silver, for example up to 1100° C., the silver may be alloyed with any suitable ductile metal or metals having a sufficiently high melting point. Examples of such metals are one or more noble metals such as gold, palladium and platinum. Preferably, there will be no less than 50 wt % Ag present in the alloy. An alternative and cheaper material to combine with the Ag is stainless steel. The Ag and stainless steel may be mixed as powders and sintered together by firing in the paths through the separator body. The metallic silver, silver mixture or silver alloy electrically conductive material may be introduced to the pores by any suitable method, including screen or stencil printing a slurry of the metal, mixture or alloy in an organic binder into the paths, or coating the surfaces of the electrode-contacting zone by, for example, printing, vapour deposition or plating and causing the coated metal, mixture or alloy to enter the paths. The layer of nickel, preferably commercially pure nickel, on the anode-facing side may have a thickness in the range of about 10 to 1000 μm, preferably 60 to 100 μm. To ensure continued contact of the Ni layer with the separator body during extended thermal cycling of the fuel cell stack particularly where the separator body is zirconia, a layer of silver, preferably commercially pure Ag, may be disposed on the electrode-contacting zone between the coating of nickel and the anode-facing side of the gas separator body. Such a layer of silver may have a thickness in the range of about 10 to 1000 μm, preferably 20 to 200 μm, and conveniently provides enhanced compliance of the overall coating on the anode side due to its ductility. Ag—Sn alloy on the catlhode-facing side of the separator body preferably contains from about 4 to about 20 wt % Sn, and may have a thickness in the range of about 10 to 1000 μm, preferably 100 to 150 μm. An Ag—Sn alloy coating may have a surface layer of SnO 2 , formed for example in the oxidising atmosphere on the cathode-facing side of the gas separator. The SnO 2 surface layer alleviates loss of Ag by “evaporation” at the elevated temperatures of use of the gas separator. To improve the electrical conductivity of the coating on the cathode-facing side, the coating must include up to about 10 wt % of dopants such as Pd and La. Each of the coating layers may be applied by any suitable means, including screen printing, spin coating, a vapour deposition process such as magnetron sputtering, slurry coating and tape casting. As an alternative to the Ag—Sn coating, a layer of silver may be formed on the cathode-facing side of the separator body. The silver coating on the cathode side may have a thickness of 10 to 1000 μm, preferably 50 to 250 μm. The following description applies to both aspects of the invention, as does the above discussion of surface coatings if they are provided on the gas separator of the first aspect of the invention. The zirconia of the gas separator may be yttria-stabilised, for example 3 to 10 wt % Y. Alternatively, the zirconia may include other materials while retaining a zirconia-based structure. For example, the zirconia may be a zirconia alumina having up to 15 wt %, or even up to about 20 wt %, alumina. For convenience, all such materials are hereinafter referred to as zirconia. The thickness of the separator body is preferably no more than 500 μm, more preferably substantially less than this in order to minimize the overall thickness or height and mass of a full cell stack utilizing the gas separator or separators, for example in the range 50 to 250 μm. While a lesser thickness could be used, the gas separator becomes difficult to manufacture and it becomes more difficult to ensure that the material of the separator body is dense, that is that it is gas tight to the gases in the fuel cell assembly. Greater thicknesses may be used but are unnecessary, and more preferably the thickness is no more than 200 μm. The separator body may be formed by any suitable means, depending particularly upon the material and the shape of the separator. A gas separator for use with a planar fuel cell will generally be in the form of a plate, and a zirconia plate, for example, may be formed by tape casting the green material and sintering. Suitable manufacturing methods may be readily identified and do not form part of the present invention. The separator body may be formed in two or more layers, for example of zirconia, that may be separated by a layer of electrically conductive material in contact with the paths of electrically conductive material through the layers of the separator body. Preferably the electrically conductive material in the paths and the separating layer is the same. As noted already, the gas separator must be gas tight to the gases used in the fuel cell assembly, and most preferably the material of the separator body is dense. However, the material could be porous, with the electrically conductive material plugging the pores through the thickness of the material. Preferably, however, the paths of electrically conductive material are defined by perforations through the separator body. For convenience, such perforations preferably extend substantially perpendicularly through the thickness of the separator body. However, this is not essential and it may be advantageous for the paths of electrically conductive material to be inclined to the perpendicular. Each path at the anode-side of the separator body may be offset relative to a connected path at the cathode-side to further alleviate the risk of leakage of gases through the separator, and/or the separator body may be formed as two or more layers separated by a layer or layers of electrically conductive material that may be the same as or different to the electrically conductive material in the paths through the separator body, as described above. Each path of electrically conductive material through the separator body preferably has a diameter or average cross-sectional dimension in the range of 50 to 1000 μm. If the paths are defined by perforations, the perforations may be formed during manufacture of the separator body or subsequently, for example by laser cutting. The minimum size of the perforations is a function of the difficulty of forming them and plugging them with the electrically conductive material. More preferably, the average cross-sectional dimension is in the range 200 to 400 μm, for example about 300 μm. The minimum number of perforations is a function of their size, the electrical conductivity of the plug material and the electrical current to be passed through the gas separator. If the perforations have an average cross-sectional dimension towards the upper end of the preferred range, they may be fewer in number and more widely spaced. Preferably, the total area of the paths of electrically conductive material through the separator body is in the range of 0.1 mm 2 to 20 mm 2 per 1000 mm 2 surface area (measured on one side only) of the electrode-contacting zone of the separator body, more preferably in the range 0.2 mm 2 to 5 mm 2 per 1000 mm 2 . In a currently preferred embodiment, there are 19 paths of electrically conductive material having an average diameter of about 300 μm through a gas separator plate having an electrode-contacting zone or functional gas separating area of about 5400 mm 2 . Advantageously, the paths of electrically conductive material also provide thermally conductive paths for transmission of heat away from the fuel cells on opposite sides of the gas separator. Surface formations may be provided in the electrode-contacting zone of the gas separator to define gas flow passages, optionally in conjunction with a separate current collector. The surface formations may be in the form of parallel ridges which may be integrally formed with the separator body, or may be affixed to the surfaces of the separator body. The surface formations may have any suitable height to provide for the necessary gas flow, for example up to about 750 μm, preferably about 500 μm high. Advantageously, in one embodiment, the electrically conductive paths in the separator body are covered by an array of parallel ridges on both sides, which extend parallel to the desired direction of the gas flow. The ridges on opposed sides of the gas separator may extend parallel to each other or perpendicularly to each other, depending upon whether the fuel gas and oxygen-containing gas are to be in co- or counter-flow, or in transverse- or cross-flow. The ridges may be formed of any suitable material that is electrically conductive and structurally and chemically stable in the fuel cell operating environment, and at least in the second aspect of the invention are conveniently bonded to the Ni and the silver or Ag—Sn coatings on the separator body, or possibly through the Ni coating to the Ag undercoating if it is present. In one embodiment, the ridges on each side of the gas separator are made of the same material as the respective electrode that they contact. Thus, on the cathode side the ridges may be formed of a conductive perovskite such as lanthanum strontium manganate, preferably coated with a metallic silver coating up to about 100 μm, preferably about 50 μm, thick. On the anode side, the ridges may be formed of a nickel-zirconia cermet, preferably with a metallic nickel coating up to about 100 μm, preferably about 50 μm, thick. |
Small intestine and colon genes |
The present invention relates to all facets of novel polynucleotides, the polypeptides they encode, antibodies and specific binding partners thereto, and their applications to research, diagnosis, drug discovery, therapy, clinical medicine, forensic science, pathology, and medicine, etc. The polynucleotides are expressed in small intestine or colon and are therefore useful in variety of ways, including, but not limited to, as molecular markers, as drug targets, and for detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, determining predisposition to, etc., diseases and conditions relating to small intestine or colon. |
1. An isolated polynucleotide comprising, a polynucleotide sequence set forth in SEQ ID NOS 1-14, 19-24, or 25, or a complement thereto. 2. An isolated polynucleotide of claim 1 which codes without interruption for an amino acid sequence set forth in SEQ ID NOS 1-14, 19-24, or 25, or a complement thereto. 3. An isolated polynucleotide comprising, polynucleotide sequence having 95% or more sequence identity to the polynucleotide sequence set forth in SEQ ID NOS 1-14, 19-24, or 25 and which codes without interruption for SEQ ID NOS 1-14, 19-24, or 25, or a complement thereto. 4. An isolated polynucleotide of claim 3 which is selective for small intestine or colon. 5. An isolated polynucleotide of claim 1 consisting of: SEQ ID NOS 1-14, 19-24, or 25, or a complement thereof. 6. An isolated polynucleotide consisting of: a polynucleotide fragment of SEQ ID NO 1-14, 19-24, or 25 which is specific for SEQ ID NO 1-14, 19-24, or 25, or a complement thereof. 7. An isolated polynucleotide of claim 6, wherein said fragment is effective in a polymerase chain reaction. 8. An isolated polypeptide coded for by a polynucleotide of claim 1. 9. An isolated polypeptide, selected from the amino acid sequence of SEQ ID NOS 1-14, 19-24, or 25 of claim 8 which is specific-for a polypeptide selected from SEQ ID NOS 1-14, 19-24, or 25. 10. An isolated polypeptide comprising an amino acid sequence having 95% or more sequence identity to the polypeptide of claim 8. 11. A method of diagnosing a small intestine or colon disease associated with abnormal SEQ ID NO 1-14, 19-24, or 25, comprising: assessing the expression of SEQ ID NO 1-14, 19-24, or 25 of claim 1 in a tissue sample comprising small intestine or colon, or cells derived from small intestine or colon. 12. A method of claim 1, wherein assessing is: measuring expression levels of said gene, determining the genomic structure of said gene, determining the mRNA structure of transcripts from said gene, or measuring the expression levels of polypeptide coded for by said gene. 13. A method of claim 12, further comprising: comparing said expression to the expression of said gene of a known normal tissue. 14. A method of claim 1, wherein said assessing detecting is performed by: Northern blot analysis, polymerase chain reaction (PCR), reverse transcriptase PCR, RACE PCR, or in situ hybridization, and using a polynucleotide probe having a sequence selected from SEQ ID NO 1-14, 19-24, or 25, a polynucleotide having 95% sequence identity or more to a sequence set forth in SEQ ID NO 1-14, 19-24, or 25, effective specific fragments thereof, or complements thereto. 15. A method of assessing a therapeutic or preventative intervention in a subject having a small intestine or colon disease, comprising, determining the expression levels of SEQ ID NO 1-14, 19-24, or 25 of claim 1 in a tissue sample comprising small intestine or colon cells, or cells derived from small intestine or colon. 16 (cancelled). 17. A method of detecting a nucleic acid coding for a polynucleotide of claim 1, comprising, contacting a sample comprising nucleic acid with a polynucleotide probe specific for a polynucleotide of claim 1 under conditions effective for said probe to hybridize specifically with said gene, and detecting hybridization between said probe and said nucleic acid. 18. An antibody which is specific-for a polypeptide of claim 8. 19 (cancelled). 20 (cancelled). 21 (cancelled). |
Method and computer system for graphical assignments in hierarchies |
Computer-implemented method, computer system and computer program product for creating graphical assignments between objects. An object hierarchy (110) and an object type selector (130) are displayed on an output device. The object type selector (130) provides a plurality of predefined object types (131-134). A rule database stores for each object type at least one predefined object type assignment. A first object (111) is inserted (438) into the object hierarchy (110) in accordance with one of the object type assignments, wherein the first object (111) has a first object type (131) and is graphically assigned to a second object (112) having a second object type (132). An object type can result from a combination of a predefined object type (134) with an object instance of any predefined object type. Graphical assignments between objects of at least two object hierarchies can also be created. |
1. A computer-implemented method (400) for creating graphical assignments between objects, the method (400) comprising the following steps: displaying (410) a predefined object hierarchy (110) and an object type selector (130), wherein the object type selector (130) provides a plurality of predefined object types (131-134); providing (420) a rule database (190), the rule database storing for each object type (131-134) at least one predefined object type assignment (191-194) of a corresponding predefined subset (130-1, 130-2, 130-3, 130-4) of object types to the object type (131-134); and inserting (430) a first object (111) into the object hierarchy (110) in accordance with one of the object type assignments (191, 192), wherein the first object (111) has a first object type (131) and is assigned to a second object (112) having a second object type (132). 2. The method of claim 1, wherein a first subset (130-1) of object types comprises parent object types assigned to the first object type (131) and the inserting step (430) comprises the following steps: selecting (431) the first object type (131) from the plurality of object types (131-134) in the object type selector (130); indicating (432) in the object hierarchy (110) a subset (110-1) of objects, wherein the subset (110-1) of objects comprises objects having an object type which is included in the first subset (130-1) of object types; selecting (433) the second object (112) from the subset (110-1) of objects as parent node for the first object (111); and adding (434) the first object (111) as child of the second object (112) to the object hierarchy (110). 3. The method of claim 1, wherein a second subset (130-2) of object types comprises child object types assigned to the second object type (132) and the inserting step (430) comprises the following steps: selecting (435) the second object (112) within the object hierarchy (110) as parent node for the first object (131); indicating (436) in the object type selector (130) the second subset (130-2) of object types; selecting (437) the first object type (131) from the subset (130-2); and adding (438) the first object (111) as child of the second object (112) to the object hierarchy (110). 4. The method of claim 1 comprising the further steps: displaying (440) a further predefined object hierarchy (120) having a third object (121) that has a third object type (133), wherein the further object hierarchy (120) is functionally equivalent to the object hierarchy (110); and creating (450) a graphical assignment (150) from the first object (111) to the third object (121) in accordance with one of the object type assignments (193, 194), wherein the graphical assignment (150) is specified by an assignment category (151) and an assignment value (152). 5. The method of claim 4, wherein a third subset (130-3) comprises receiver object types assigned to the first object type (133) and the creating step (450) comprises the following steps: selecting (451) the first object (111) as sender object of the graphical assignment (150); indicating (452) in the further object hierarchy (120) a receiver subset (120-1) of objects, wherein the receiver subset (120-1) of objects comprises objects having a receiver object type which is included in the third subset (130-3) of object types; selecting (453) from the receiver subset (120-1) of objects the third object (121) as receiver object of the graphical assignment (150); creating (454) the graphical assignment (150); and receiving (455) the assignment category (151) and the assignment value (152). 6. The method of claim 4, wherein a forth subset (130-4) comprises sender object types assigned to the third object type (133) and the creating step (450) comprises the following steps: selecting (456) the third object (121) as receiver object of the graphical assignment (150); indicating (457) in the object hierarchy (110) a sender subset (110-2) of objects, wherein the sender subset (110-2) of objects comprises objects having a sender object type which is included in the forth subset (130-4) of object types; selecting (458) from the sender subset (110-2) of objects the first object (111) as sender object of the graphical assignment (150); creating (459) the graphical assignment (150); and receiving (460) the assignment category (151) and the assignment value (152). 7. The method of claim 1, wherein in the providing step (420) each object type assignment (191-194) has an assignment type (P, C, R, S) and is a N:1 assignment of N object types (131-134) to a corresponding object type (131-134). 8. The method of claim 1, wherein in the providing step (420), a further predefined object type (135) results from a combination of a predefined object type (134) with an object instance (119) of any predefined object type (133). 9. The method of claim 1, wherein in the providing step (420), a further predefined object type (136) results from a combination of a predefined object type (134) with at least one other predefined object type (133). 10. The method of claim 4, comprising the further steps: displaying (470) a still further predefined object hierarchy (120-10) that is functionally equivalent to the object hierarchy (110); and creating (480) a further graphical assignment (150-1) from an object of one other object hierarchy (110, 120) to an object of the still further object hierarchy (120-10) in accordance with one of the object type assignments, wherein the further graphical assignment (150-1) is specified by a further assignment category (151-1) and a further assignment value (152-1). 11. A computer system (999) for creating graphical assignments between objects, the computer system (999) comprising: a first means for displaying (410) a predefined object hierarchy (110) and an object type selector (130), wherein the object type selector (130) provides a plurality of predefined object types (131-134); a second means for providing (420) a rule database (190), the rule database storing for each object type (131-134) at least one predefined object type assignment (191-194) of a corresponding predefined subset (130-1, 130-2, 130-3, 130-4) of object types to the object type (131-134); and a third means for inserting (430) a first object (111) into the object hierarchy (110) in accordance with one of the object type assignments (191, 192), wherein the first object (111) has a first object type (131) and is assigned to a second object (112) having a second object type (132). 12. The computer system (999) of claim 11, further comprising: a forth means for displaying (440) a further predefined object hierarchy (120) having a third object (121) that has a third object type (133), wherein the further object hierarchy (120) is functionally equivalent to the object hierarchy (110); and a fifth means for creating (450) a graphical assignment (150) from the first object (111) to the third object (121) in accordance with one of the object type assignments (193, 194), wherein the graphical assignment (150) is specified by an assignment category (151) and an assignment value (152). 13. The computer system (999) of claim 12, further comprising: a sixth means for displaying (470) a still further predefined object hierarchy (120-10) that is functionally equivalent to the object hierarchy (110); and a seventh means for creating (480) a further graphical assignment (150-1) from an object of one other object hierarchy (110, 120) to an object of the still further object hierarchy (120-10) in accordance with one of the object type assignments, wherein the further graphical assignment (150-1) is specified by a further assignment category (151-1) and a further assignment value (152-1). 14. The computer system (999) of claim 11, wherein a further predefined object type (135) results from a combination of a predefined object type (134) with an object instance (119) of any predefined object type (133). 15. The computer system (999) of claim 11, wherein a further predefined object type (136) results from a combination of a predefined object type (134) with at least one other predefined object type (133). 16. A computer program product (100/101) causing at least one processor (910) to process a plurality of program instructions; the processor (910) performing the steps of: displaying (410) a predefined object hierarchy (110) and an object type selector (130), wherein the object type selector (130) provides a plurality of predefined object types (131-134); providing (420) a rule database (190), the rule database storing for each object type (131-134) at least one predefined object type assignment (191-194) of a corresponding predefined subset (130-1, 130-2, 130-3, 130-4) of object types to the object type (131-134); and inserting (430) a first object (111) into the object hierarchy (110) in accordance with one of the object type assignments (191, 192), wherein the first object (111) has a first object type (131) and is assigned to a second object (112) having a second object type (132). 17. The computer program product (100/101) of claim 16, causing at least one processor (910) to process a plurality of program instructions; the processor (910) performing the further steps of: displaying (440) a further predefined object hierarchy (120) having a third object (121) that has a third object type (133), wherein the further object hierarchy (120) is functionally equivalent to the object hierarchy (110); and creating (450) a graphical assignment (150) from the first object (111) to the third object (121) in accordance with one of the object type assignments (193, 194), wherein the graphical assignment (150) is specified by an assignment category (151) and an assignment value (152). 18. The computer program product (100/101) of claim 17, causing at least one processor (910) to process a plurality of program instructions; the processor (910) performing the still further steps of: displaying (470) a still further predefined object hierarchy (120-10) that is functionally equivalent to the object hierarchy (110); and creating (480) a further graphical assignment (150-1) from an object of one other object hierarchy (110, 120) to an object of the still further object hierarchy (120-10) in accordance with one of the object type assignments, wherein the further graphical assignment (150-1) is specified by a further assignment category (151-1) and a further assignment value (152-1). 19. The computer program product (100/101) of claim 16, causing at least one processor (910) to provide a further predefined object type (135) resulting from a combination of a predefined object type (134) with an object instance (119) of any predefined object type (133). 20. The computer program product (100/101) of claim 16, causing at least one processor (910) to provide a further predefined object type (136) resulting from a combination of a predefined object type (134) with at least one other predefined object type (133). 21. A computer readable medium (970), embodying program instructions causing at least one processor (910) to execute the steps of: displaying (410) a predefined object hierarchy (110) and an object type selector (130), wherein the object type selector (130) provides a plurality of predefined object types (131-134); providing (420) a rule database (190), the rule database storing for each object type (131-134) at least one predefined object type assignment (191-194) of a corresponding predefined subset (130-1, 130-2, 130-3, 130-4) of object types to the object type (131-134); and inserting (430) a first object (111) into the object hierarchy (110) in accordance with one of the object type assignments (191, 192), wherein the first object (111) has a first object type (131) and is assigned to a second object (112) having a second object type (132). 22. The computer readable medium (970) of claim 21, embodying program instructions causing at least one processor (910) to execute the further steps of: displaying (440) a further predefined object hierarchy (120) having a third object (121) that has a third object type (133), wherein the further object hierarchy (120) is functionally equivalent to the object hierarchy (110); and creating (450) a graphical assignment (150) from the first object (111) to the third object (121) in accordance with one of the object type assignments (193, 194), wherein the graphical assignment (150) is specified by an assignment category (151) and an assignment value (152). 23. The computer readable medium (970) of claim 22, embodying program instructions causing at least one processor (910) to execute the still further steps of: displaying (470) a still further predefined object hierarchy (120-10) that is functionally equivalent to the object hierarchy (110); and creating (480) a further graphical assignment (150-1) from an object of one other object hierarchy (110, 120) to an object of the still further object hierarchy (120-10) in accordance with one of the object type assignments, wherein the further graphical assignment (150-1) is specified by a further assignment category (151-1) and a further assignment value (152-1). 24. The computer readable medium of claim 21, embodying program instructions causing at least one processor (910) to provide a further predefined object type (135) resulting from a combination of a predefined object type (134) with an object instance (119) of any predefined object type (133). 25. The computer readable medium of claim 21, embodying program instructions causing at least one processor (910) to provide a further predefined object type (135) resulting from a combination of a predefined object type (134) with at least one other predefined object type (133). |
<SOH> BACKGROUND OF THE INVENTION <EOH>Typically, hierarchies that help to structure assignments between objects are administrated through graphical user interfaces, such as the Microsoft Windows Explorer or the cost assignment view of ABC Technologies OROS program. For example, in the Microsoft Windows Explorer, a folder structure is built in the form of a hierarchy. Objects of three different object types (folders, files and shortcuts) can be graphically assigned to a folder. The computer user, in the following called user, maintains the hierarchy through a) a combination of menu entries in the drop down menus of the windows explorer, b) menu entries in a context menu launched with the right mouse button and c) “drag & drop” functions launched with the left or right mouse buttons. Also shortcuts via the keyboard can be used. For example, new folders or shortcuts can be created by selecting an appropriate menu entry from the “File” menu or from the context menu; for instance, folders or files can be moved or copied by using the right mouse button for “drag & drop”. Files, folders or shortcuts can be deleted by using the context menu with the right mouse button. For each object type only specific activities are allowed. For instance, a folder cannot be graphically assigned to a file or shortcut. A shortcut cannot be graphically assigned to a file but is assigned to the file in a logical relationship. The OROS program allows the user to graphically define assignments between objects of two different hierarchies. Both hierarchies work similar to the Windows Explorer logic for adding, moving or deleting objects. Both hierarchies support three different object types: center type, account type and cost element type. For example, a first hierarchy shows the resource view of an enterprise. A center can define a group of resources. Centers can be assigned to other centers. An account can define a specific resource, such as a machine or a building. Accounts can be assigned to centers. A cost element defines a specific type of cost, such as salary, rent etc. Cost elements can be assigned to accounts. A second hierarchy, for example, shows the activity view of an enterprise, where centers define groups of activities, accounts define activities and cost elements, again, define specific types of cost. Similar to the first hierarchy, cost elements are assigned to accounts and accounts are assigned to centers. When the user creates an assignment between objects of the first hierarchy and the second one, only accounts of the first hierarchy can be assigned to accounts of the second hierarchy and vice versa. The graphical user interface supports this assignment, for example, by displaying the first hierarchy (sender objects) within a first frame and only the accounts of the second hierarchy (receiver objects) as a flat object list within a second frame. When the user selects an account of the first hierarchy in the first frame, accounts in the second frame become a possible target for the assignment. This is indicated by little arrows icons next to each of the accounts in the second frame. To create the assignment the user selects one account in the second frame by clicking on the corresponding arrow icon and finally enters an assignment category and an assignment value to specify the assignment. These examples work very well with applications that only use a limited number of object types (three in the examples above) with a limited number of possible relationships between these objects. Therefore, they provide an easy-to-use solution for graphical maintenance of hierarchical assignments in application systems that do not show a high degree of complexity as far as the number of object types and their possible dependencies are concerned. However, more complex application systems, such as Enterprise Resource Planning (ERP) systems (e.g. SAP R/3) usually support a much higher number of object types. Also the number of possible relations between the various object types is very high. When applying the prior art user interface models to complex application systems, such as ERP systems, the user encounters some inconveniences when creating assignments within or across hierarchies. This becomes obvious, when looking at a typical organizational structure of an enterprise in an ERP system from a cost management point of view. For example, in the SAP R/3 system, a controlling area defines an area that is relevant for an enterprise from a cost management point of view. In each controlling area a hierarchy of cost center groups is defined. Multiple cost centers are assigned to a cost center group. For each cost center multiple cost elements are assigned either directly to the cost center or to activities of the cost center. Cost elements can be grouped into cost element groups. In a further hierarchy internal orders can be defined. Cost centers with cost element groups or activities can be assigned to internal orders. On the other hand internal orders can be assigned back to cost centers. The same is true for projects and project elements. Cost center activities can also be assigned to production or sales orders. For convenience of explanation, further object types and object type relations are not listed here. Numerous further object types and their possible relations to other object types within the same or across hierarchies can be taken from “CO-I Overhead Cost Controlling”, published in September 1999 by SAP AG. The user desires a clear visualization of all actual and possible dependencies between object types. For assignments across hierarchies a flat list of receiver objects, as in the prior art example, would contain a large amount of objects of different object types. This leaves the task to identify the right receiver object for an assignment completely with the user (e.g., by applying the right mouse button to every single object). To identify the receiver object is difficult because the information about the location of the receiver object within the hierarchy is hidden. |
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