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1. A process of controlling a biochemical process (II) or biochemical cascade (III) of interest in a plant, said process comprising: (a) introducing into the nuclear genome of the plant one or more first heterologous DNA sequences; and (b) infecting the plant with at least one viral transfection vector containing in its genome one or more second heterologous DNA or RNA sequences, thus triggering a process of interaction (I) in the plant between (i) one or more first heterologous DNA sequences of the nuclear genome and/or expression products of the first heterologous DNA sequences, and (ii) one or more second heterologous DNA or RNA sequences of the transfection vector and/or expression products of the second heterologous DNA or RNA sequences, and (iii) optionally one or more externally added low molecular weight components, thus switching on the biochemical process (II) or biochemical cascade (II) of interest that was not operable prior to said interaction. 2. The process according to claim 1, wherein the process of interaction requires an expression product of a first heterologous DNA sequence stably integrated in the nuclear genome of the plant. 3. The process according to claim 1, wherein said interaction requires an expression product of a second heterologous DNA or RNA sequence of said transfer vector. 4. The process according to claim 1, wherein the infection of the plant in step (b) is achieved by an assembled virus particle or infectious viral nucleic acids, or by activating a transfection process by release of viral nucleic acids that were previously incorporated into the plant genome. 5. The process of claim 4, wherein said assembled virus particle or said infectious viral nucleic acid is or comprises RNA. 6. The process according to claim 1, wherein the infection of the plant in step (b) comprises Agrobacterium-mediated transfer of nucleic acid sequences into cells of said plant. 7. The process according to claim 1, wherein a further vector is introduced in step (b) and wherein a sequence and/or an expression product of said further vector is involved in said process of interaction. 8. The process according to claim 1, wherein the infection of the plant in step (b) is achieved by introducing one or more vectors into cells of said plant, whereby said vector(s) are adapted to undergo processing to generate said viral transfection vector in cells of said plant. 9. The process according to claim 1, wherein said process of interaction is a viral transfection vector-generated process. 10. The process according to claim 1, wherein the process of interaction involves DNA transposition. 11. The process according to claim 1, wherein the process of interaction involves DNA recombination. 12. The process according to claim 11, wherein the biochemical process or cascade of interest comprises expression of a first or second DNA or RNA sequence comprising a promoterless gene in anti-sense orientation which is placed into sense orientation towards a constitutive promoter in said process of interaction. 13. The process according to claim 1, wherein the process of interaction involves recognition of a heterologous promoter by a heterologous RNA polymerase. 14. The process according to claim 13, wherein said first and said second DNA or RNA sequence comprises a heterologous sequence to be expressed under the control of a heterologous promoter not recognized by a plant RNA polymerase, and transcription of said sequence to be expressed is switched on by interaction of said promoter with an RNA polymerase functional therewith and being encoded by said second or said first DNA sequence, respectively. 15. The process according to claim 14, wherein said RNA polymerase is a bacteriophage RNA polymerase and said heterologous promoter is a bacteriophage promoter. 16. The process according to claim 1, wherein the process of interaction involves a DNA reaction such as DNA replication, ligation, hybridisation, transcription, or DNA restriction. 17. The process according to claim 1, wherein the process of interaction involves an RNA reaction such as replication, processing, splicing, reverse transcription, hybridization or translation, or activiation, inhibition or modification thereof. 18. The process according to claim 1, wherein the process of interaction involves a protein reaction such as protein folding, assembly, activation, posttranslational modification, targeting, binding, enzymatic activity or signal transduction, or activation, inhibition or modification thereof. 19. The process according to claim 1, wherein (i) the biochemical process or cascade of interest comprises expression of a first or second DNA sequence separated from its promoter by a DNA insert capable of preventing transcription of the first or second DNA sequence, and (ii) the process of interaction triggered in step (b) results in the excision of the DNA insert whereby the first or second DNA sequence is expressed. 20. The process according to claim 19, wherein the DNA insert is a non-autonomous transposable element which is excised by a transposase (i) encoded by a second DNA sequence on the viral vector for an insert in the nuclear genome, or (ii) encoded by a first DNA sequence in the nuclear genome for an insert in the viral vector. 21. The process according to claim 19, wherein the DNA insert is flanked by unidirectional sites recognizable by a site-specific DNA recombinase (i) encoded by a second DNA sequence on the viral vector for an insert in the nuclear genome, or (ii) encoded by a first DNA sequence in the nuclear genome for an insert in the viral vector. 22. The process according to claim 1, wherein transcription of a first or a second DNA sequence is switched on by a heterologous or engineered transcription factor capable of recognizing a heterologous or engineered or chimaeric promoter operably linked to a heterologous gene of interest of said first or second DNA sequence, whereby said promoter is not recognizable by any natural plant transcription factor and said heterologous or engineered transcription factor is encoded by a second or a first DNA sequence, respectively. 23. The process according to claim 22, wherein the transcription factor is inducible by an externally applied low molecular weight component. 24. The process according claim 1, wherein said first heterologous DNA sequence of step (a) is not of plant viral origin. 25. A process of controlling a biochemical process (II) or biochemical cascade (III) of interest in a plant, said process comprising: (a) introducing into the nuclear genome of the plant one or more first heterologous nucleic acid sequences; and (b) infecting the plant with at least one vector containing in its genome one or more second heterologous nucleic acid sequences, thus triggering a process of interaction (I) in the plant between (i) one or more first heterologous nucleic acid sequences on the nuclear genome and/or expression products of the first heterologous nucleic acid sequences, and (ii) one or more second heterologous nucleic acid sequences of the trarsfection vector and/or expression products of the second heterologous nucleic acid sequences, and (iii) optionally one or more externally added low molecular weight components, whereby a viral transfection vector is generated in cells of said plant, thus switching on the biochemical process (II) or biochemical cascade (III) of interest that was not operable prior to said interaction. 26. The process of claim 5, wherein the process of interaction requires an expression product of a first heterologous DNA sequence stably integrated in the nuclear genome of the plant. 27. A process of producing a product in a transgenic plant comprising: (a) introducing into the nuclear genome of the plant one or more first heterologous DNA sequences: and (b) infecting the plant with at least one viral transfection vector containing in its genome one or more second heterologous DNA or RNA sequences, thus triggering a process of interaction (I) in the plant between (i) one or more first heterologous DNA sequences of the nuclear genome and/or expression products of the first heterologous DNA sequences, and (ii) one or more second heterologous DNA or RNA sequences of the transfection vector and/or expression products of the second heterologous DNA or RNA sequences, and (iii) optionally one or more externally added low molecular weight components, thus switching on the biochemical process (II) or biochemical cascade (III) of interest that was not operable prior to said interaction, thereby producing the product in the transgenic plant. 28. The process of claim 25 further comprising: (a) growing the transgenic plant to a desired stage; (b) infecting the plant with one or more viral transfection vectors, and optionally contacting the plant with one or more low molecular weight components, this switching on the biochemical process or cascade necessary to produce the product, said process or cascade not being operable prior to said interaction; and (c) producing the product in the plant. 29. Kit-of-parts for performing the process of claim 1, comprising (i) a transgenic plant or seeds thereof, and (ii) a vector, notably a viral transfection vector. 30. The vector for performing step (b) claim 1. 31. The plant used in the process of claim 27. 32. The plant used in the process of claim 28.

<SOH> BACKGROUND OF THE INVENTION <EOH>Controllable Transgene Expression Systems in Plants One of the major problems in plant biotechnology is the achievement of reliable control over transgene expression. Tight control over gene expression in plants is essential if a downstream product of transgene expression is growth inhibitory or toxic, like for example, biodegradable plastics (Nawrath, Poirier & Somerville, 1994, Proc. Natl. Acad. Sci, 91,12760-12764; John & Keller, 1996, Proc. Natl. Acad. Sci., 93, 12768-12773; U.S. Pat. No. 6,103,956; U.S. Pat. No. 5,650,555) or protein toxins (U.S. Pat. No. 6,140,075). Existing technologies for controlling gene expression in plants are usually based on tissue-specific and inducible promoters and practically all of them suffer from a basal expression activity even when uninduced, i.e. they are “leaky”. Tissue-specific promoters (U.S. Pat. No. 5,955,361;.WO09828431) present a powerful tool but their use is restricted to very specific areas of applications, e.g. for producing sterile plants (WO9839462) or expressing genes of interest in seeds (WO00068388; U.S. Pat. No. 5,608,152). Inducible promoters can be divided into two categories according to their induction conditions—those induced by abiotic factors (temperature, light, chemical substances) and those that can be induced by biotic factors, for example, pathogen or pest attack. Examples of the first category are heat-inducible (U.S. Pat. No. 5,187,287) and cold-inducible (U.S. Pat. No. 5,847,102) promoters, a copper-inducible system (Mett et al., 1993, Proc. Natl. Acad. Sci., 90, 4567-4571), steroid-inducible systems (Aoyama & Chua, 1997, Plant J., 11, 605-612; McNellis et al., 1998, Plant J., 14, 247-257; U.S. Pat. No. 6,063,985), an ethanol-inducible system (Caddick et al., 1997, Nature Biotech., 16, 177-180; WO09321334), and a tetracycline-inducible system (Weinmann et al., 1994, Plant J., 5, 559-569). One of the latest developments in the area of chemically inducible systems for plants is a chimaeric promoter that can be switched on by glucocorticoid dexamethasone and switched off by tetracycline (Bohner et al., 1999, Plant J., 19, 87-95). For a review on chemically inducible systems see: Zuo & Chua, (2000, Current Opin. Biotechnol., 11, 146-151). Other examples of inducible promoters are promoters which control the expression of patogenesis-related (PR) genes in plants. These promoters can be induced by treatment of the plant with salicylic acid, an important component of plant signaling pathways in response to pathogen attack, or other chemical compounds (benzo-1,2,3-thiadiazole or isonicotinic acid) which are capable of triggering PR gene expression (U.S. Pat. No. 5,942,662). There are reports of controllable transgene expression systems using viral RNA/RNA polymerase provided by viral infection (for example, see U.S. Pat. No. 6,093,554; U.S. Pat. No. 5,919,705). In these systems, a recombinant plant DNA sequence includes the nucleotide sequences from the viral genome recognized by viral RNA/RNA polymerase. The effectiveness of these systems is limited because of the low ability of viral polymerases to provide functions in trans, and their inability to control processes other than RNA amplification. The systems described above are of significant interest as opportunities of obtaining desired patterns of transgene expression, but they do not allow tight control over the expression patterns, as the inducing agents (copper) or their analogs (brassinosteroids in case of steroid-controllable system) can be present in plant tissues at levels sufficient to cause residual expression. Additionally, the use of antibiotics and steroids as chemical inducers is not desirable for the large-scale applications. When using promoters of PR genes or viral RNA/RNA polymerases as control means for transgenes the requirements of tight control over transgene expression are also not fulfilled, as casual pathogen infection or stress can cause expression. The tissue or organ-specific promoters are restricted to very narrow areas of applications, since they confine expression to a specific organ or stage of plant development, but do not allow the transgene to be switched on at will. Plant Viral Vectors and Their Use in the Field of Applied Plant Virology Presently, there are three distinct major fields in the area of applied plant virology: a) use of viruses as vectors for transgene overexpression; b) use of viruses as vectors for plant functional genomics, and c) use of viral components in the field of phytopathology for generating virus-resistant transgenic plants. Plant viruses can serve as efficient tools for high level expression of transgenes in host plant species. The use of transgenic plant virus in field does not seem to compromise any biosafety issues. For example, Animal and Plant Health Inspection Service, USDA, did not find any significant impact after field trials with genetically modified TMV (tobacco mosaic virus) and tobacco etch viruses containing heterologous genes of pharmaceutical interest. As a result, two permissions were issued in 1996 and 1998. Work has been conducted in the area of developing viral vectors for transferring foreign genetic material into plant hosts for the purposes of expression (U.S. Pat. No. 4,885,248; U.S. Pat. No. 5,173,410). There are several patents which describe the first viral vectors suitable for systemic expression of transgenic material in plants (U.S. Pat. No. 5,316,931; U.S. Pat. No. 5,589,367; U.S. Pat. No. 5,866,785). In general, these vectors can express foreign genes from an additional subgenomic promoter (U.S. Pat. No. 5,466,788; U.S. Pat. No. 5,670,353; U.S. Pat. No. 5,866,785), as translational fusions with viral proteins (U.S. Pat. No. 5,491,076; U.S. Pat. No. 5,977,438) or from polycistronic viral RNA using IRES elements for independent protein translation, also used herein, according to ANNEX A corresponding to German Patent Application No 10049587.7. Carrington et al., (U.S. Pat. No. 5,491,076) describe the use of an endogenous viral protease to cleave heterologous proteins from viral polyproteins. Another area of application for viral vectors is plant functional genomics. Della-Cioppa et al., (WO993651) describe the use of TMV-based viral vectors for expression of plant cDNA libraries with the purpose of silencing endogenous genes. Angell & Baulcombe (1997, EMBO J, 16, 3675-3684; WO9836083) describe a PVX-based system called “Amplicon™” designed for down-regulating the targeted genes in plants. The same system in combination with Hc-Pro that suppresses transgene silencing in plants (Pruss et al., 1997, Plant Cell, 9, 859-868; U.S. Pat. No. 5,939,541) is used for overexpression of transgenes. U.S. Pat. No. 5,939,541 describes an approach based on using the 5′proximal region (booster sequence, including the Hc-Pro gene) of the potyvirus to enhance expression of any gene in plants. This sequence can be stably integrated into the plant genome or delivered by a virus. It is worth mentioning that Hc-Pro has a pronounced pleiotropic effect and enhances the expression of both transgenes and endogenous plant genes. Thus, these systems provide at best a quantitative improvement of total protein expression over existing processes. They do so by influencing many components of the protein production machinery by an unknown mechanism and in a hardly controlled manner. There is an abundant literature including patent applications which describe the design of virus resistant plants by the expression of viral genes or mutated forms of viral RNA (e.g. U.S. Pat. No. 5,792,926; U.S. Pat. No. 6,040,496). It is also worth mentioning that an environmental risk is associated with the use of such plants due to the possibility of forming novel viruses by recombination between the challenging virus and transgenic viral RNA or DNA (Adair & Kearney, 2000, Arch. Virol, 145, 1867-1883). Therefore, it is an object of the present invention to provide an environmentally safe process of controlling a biochemical process or a biochemical cascade of interest in a plant whereby the process or cascade may be selectively switched on at any predetermined time. It is another object of this invention to provide a process for producing a product in a transgenic plant wherein the production of the product may be selectively switched on after the plant has grown to a desired stage, whereby the process is environmentally safe and does not lead to the release of potentially hazardous functional transgenes in the environment. Another object of this invention is to provide a kit of parts for performing such processes.

<SOH> BRIEF DESCRIPTION OF THE FIGURES <EOH>FIG. 1A is a schematic representation of a process according to the invention. FIG. 1B is a schematic representation of possible classes of processes of interaction in an infected plant cell. FIG. 2 depicts crTMV-based vectors pIC1111 and pIC1123 containing IRES cp,148 CR -Ac transposase and and IRES mp,75 CR -Ac transposase, respectively. Also shown is the T-DNA region of binary vector pSLJ744 containing p35S::Ds::GUS-3′ocs. FIG. 3 depicts crTMV-based vectors pIC2541 and pIC2531 containing IRES cp,148 CR -Cre recombinase and and IRES mp,75 CR -Cre recombinase, respectively. Also shown is the T-DNA region of the binary vector pIC2561 containing the GUS gene flanked by two loxP sites in direct orientation. FIG. 4 depicts crTMV-based vectors pIC2541 and pIC2531 (see also FIG. 3 ) in combination with the T-DNA region of the binary vector pIC1641 containing the GUS gene flanked by two inverted loxP sites. FIG. 5 depicts the T-DNA region of the binary vector pIC2691 carrying the GUS gene under control of T7 promoter and crTMV-based vector pIC2631 containing the T7 polymerase gene. FIG. 6 shows X-gluc stained leaves of transgenic Arabidopsis plants transformed with pSLJ744. Transcription of the GUS gene is prevented by the insertion of Ds element. A—leaves inoculated with the transcript from pIC1123. B—leaves inoculated with the transcript from pIC1111. C—leaves inoculated with water. FIG. 7 depicts the TMV-based viral provectors pICH4371 and pICH4461 end of provector (RdRp: RNA dependent RNA polymerase; MP: movement protein; sGFP: synthetic green fluorescent protein; 3′NTR: 3′non-translated region of TMV; sgp: subgenomic promoter). FIG. 8 depicts the T-DNA of binary vector pICH1754 providing a Cre recombinase expression cassette. FIG. 9 depicts a scheme of formation of viral vectors from provectors in the presence of Cre recombinase. detailed-description description="Detailed Description" end="lead"?

Orbital implant

An orbital implant includes a body of bioactive material having macropores of at least 400 μm, and a cap of bioactive material having substantially no pores or only micropores smaller than 50 μm. The cap covers a portion of the body.

1. An orbital implant which includes a body of bioactive material having macropores of at least 400 μm, and a cap of bioactive material having substantially no pores or only micropores smaller than 50 μm, with the cap covering a portion of the body. 2. An implant according to claim 1, which is substantially spherical. 3. An implant according to claim 2, which has a diameter of about 20 mm. 4. An implant according to claim 1, wherein the macropores in the body are substantially spherical so that they have diameters of at least 400 μm, and, optionally, wherein the diameters of the macropores do not exceed 1000 μm. 5. An implant according to claim 1, wherein some macropores are in communication with the outer surface of the body and wherein adjacent macropores in the body are interconnected by openings and/or passageways, so that open paths to the outer surface of the body are thereby provided in the body. 6. An implant according to claim 5, wherein substantially no isolated or closed macropores are present in the body. 7. An implant according to claim 5, wherein the openings and/or passageways which interconnect adjacent macorpores have diameters greater than 50 μm. 8. An implant according to claim 5, wherein the macropores in the body occupy from 40% to 85% by volume of the body. 9. An implant according to claim 5, wherein the body has micropores smaller than 50 μm. 10. An implant according to claim 9, wherein at least some of the micropores are of irregular shape, and have a maximum dimension smaller than 50 μm. 11. An implant according to claim 9, wherein at least some of the micropores are substantially spherical so that their diameters are thus smaller than 50 μm. 12. An implant according to claim 9, wherein adjacent micropores in the body are interconnected by openings and/or passageways and wherein some micropores are also interconnected to the macropores by openings and/or passageways so that the micropores, by means of those openings and/or passages, provide open paths to the macropores. 13. An implant according to claim 12, wherein substantially no isolated or closed micropores are present in the body. 14. An implant according to claim 9, wherein some of the micropores are of irregular shape and are in the form of interstitial spaces between incompletely sintered bioactive material particles, and wherein some of the micropores are of substantially spherical shape, with irregular micropores interconnecting adjacent spherical micropores, and also connecting spherical micropores to macropores. 15. An implant according to claim 14, wherein all the spherical micropores are of substantially the same size while all the irregular micropores are of substantially the same size, with the irregular micropores being smaller than the spherical micropores. 16. An implant according to claim 14, wherein substantially no isolated or closed micropores are present in the body. 17. An implant according to claim 9, wherein the micropores occupy from 3% to 70% by volume of the macropore-free bioactive material. 18. An implant according to claim 1, wherein the cap is in the form of a circular concave disc integrated with the body of bioactive material. 19. An implant according to claim 1, which is substantially spherical, and wherein the cap has a thickness which is no more than half the diameter of the implant. 20. An implant according to claim 1, wherein the bioactive material of the body and that of the cap are the same, and is hydroxyapatite.

Bumper airbag and system

An airbag (10) for mounting in the bumper (18) of a motor vehicle (12) is provided. The airbag can have an up-side-down “L” shape or a cylindrical shape. Further, multiple bags can be combined within one system. The airbag is configured to cover substantially the width of the vehicle upon deployment and also provide protection to the occupant of a struck vehicle (36) in the event the occupant is partially expelled from the struck vehicle in the direction of the bag. The airbag is combined with an inflation (23), collision sensor (34) and an electronic control unit (38) to form the airbag system.

1. A bumper airbag system comprising: a housing disposed in the bumper of a first vehicle and having a frangible door; a sensor for determining a pending collision between the first vehicle and another object and generating a signal thereof; at least one inflator for dispensing inflation fluid in response to the signal; and an airbag stored in the housing in a folded condition, the airbag member operatively connected to the inflator, whereby upon the firing of the inflator, the airbag inflates opening the frangible door and expanding out from and over the bumper to cover a substantial portion of the width of the vehicle. 2. The bumper airbag system of claim 1 further comprising an electronic control unit for processing the signal from the sensor and generating the signal to the inflator. 3. The bumper airbag system of claim 1 wherein upon inflation the bumper airbag has an up-side-down “L” shape defined a base portion extending away from the bumper and an arm portion extending downward from the base portion. 4. The bumper airbag system of claim 3 wherein the arm portion has a plurality of tubular members. 5. The bumper airbag system of claim 1 wherein the bumper airbag is fabricated from a woven polyester laminated material to provide a non-porous enclosure. 6. The bumper airbag system of claim 5 wherein the bumper airbag further comprises a nonabrasive, puncture resistant coating on its outer surface. 7. The bumper airbag system of claim 1 further comprising at least one pressure relief device. 8. The bumper airbag system of claim 7 wherein the pressure relief device is at least one hole in the airbag. 9. The bumper airbag system of claim 7 wherein the pressure relief device is a valve mounted in the housing. 10. The bumper airbag system of claim 1 further comprising a burst disk disposed between the inflator and the airbag. 11. The bumper airbag system of claim 1 wherein upon inflation the airbag takes on a cylindrical shape. 12. The bumper airbag system of claim 11 wherein the airbag further comprises a throat member having at least one hole for the inflating gas from the inflator to pass into the interior of the airbag. 13. The bumper airbag system of claim 12 wherein the throat member is attached at one end to the housing. 14. The bumper airbag system of claim 1 wherein the airbag upon inflation is comprised of a plurality of cylindrically shaped bags. 15. The bumper airbag system of claim 14 wherein each of the plurality of cylindrically shaped bags are secured together in such a manner that two of the bags cover the grille of the vehicle and another bag is disposed beyond the other two bags and away from the grille. 16. The bumper airbag system of claim 15 wherein the bags are secured by tethers. 17. The bumper airbag system of claim 14 wherein each of the bags abuts the others. 18. The bumper airbag system of claim 19 further comprising fluid communication ports between the bags. 19. A bumper airbag comprising: a first rectangular panel of fabric having four edges, the panel being rolled and two edges being attached to form a cylinder; a second rectangular panel of fabric substantially smaller than the first panel and sewn to the outer surface of the first panel near one of the edges; at least one hole extending through both panels; and a throat member attached to the first panel and encompassing the second panel. 20. The bumper airbag of claim 19 wherein the throat member has a flange at one end for attaching to the curved surface of the first panel and opening at its other end for receiving inflating gas. 21. The bumper airbag of claim 20 further comprising at least one support tether secured to the inside of the throat member. 22. The bumper airbag of claim 19 wherein having a plurality of holes extending through the two panels and a reinforcing member disposed between adjacent holes. 23. The bumper airbag of claim 19 further comprising a circular end cap attached at the open ends of the first panel. 24. The bumper airbag of claim 23 wherein each end cap has a vent hole.

<SOH> BACKGROUND OF THE INVENTION <EOH>For years, the automotive industry has tried various methods and products to reduce the damage to passengers and vehicles in collisions. Of prime importance are the various systems of vehicle airbags that are deployed upon the sensing of an actual collision. These airbags are located in and about the passenger compartment of the motor vehicle and are inflated to surround and protect the occupants from serious injury. Other methods of reducing, to some extent, the forces created in a collision from injuring the occupants are various attempts to provide “crush zones” at the front and the rear of the vehicle to absorb some of the collision forces. Still other methods also deal with design of the vehicle frame, engine mounts and other structural members to absorb the forces by means of controlled structural collapsing. Airbags have been fabricated to the front end of the vehicle that just prior to the instance of a crash, inflate and form a fluid-filled structure between the striking object or vehicle and the struck object or vehicle. PCT application number WO98/50254 “Collision Protection System for Vehicles” teaches airbags mounted to the front of a vehicle. U.S. Pat. No. 3,656,791 “Vehicle Impact-Cushioning Device” teaches an airbag mounted to deploy from the front end of a vehicle. U.S. Pat. No. 3,708,194 “Vehicle Safety Apparatus” teaches a front-end mounted airbag that includes a fire extinguishing material. Several prior art patents deal with bumper improvements. U.S. Pat. No. 4,518,183 “Extendible Safety Impact Bags for Vehicles” teaches mechanisms for extending bumpers outwardly of the vehicle upon the sensing of a potential crash. Air is supplied to airbags to form a somewhat rigid member supporting the bumpers for the duration of the crash and then the airbags are deflated and the bumpers return to their normal position. U.S. Pat. No. 4,930,823 “Vehicle Bumper” teaches front and rear bumpers having airbags that are inflated upon contact of the bumper with an object. U.S. Pat. No. 5,106,137 “Vehicle Bumper with Combination Foam and Airbag Energy Absorber” teaches a bumper having an internal cavity surrounded by compressible energy absorbing plastic. Inside the cavity is an airbag that is inflated upon the onset of a crash to provide more protection to the front or rear end of the vehicle. U.S. Pat. No. 5,651,569 “Inflatable Bumper System” teaches a bumper having an enclosed airbag that is permanently inflated to provide a permanent cushion bumper. U.S. Pat. No. 5,725,265 “Airbag System For Vehicle Bumpers” teaches an airbag concealed inside a bumper that is inflated and extends outwardly of the bumper to reduce the effects of the crash. The bumper has an expellable panel on the outer surface of the bumper that is removed by the inflation of the airbag. U.S. Pat. No. 6,056,336 “Airbag with Internal Shock Absorber” teaches a bumper airbag having an internal shock absorber. The airbag is deployed in a circular shape. U.S. Pat. No. 6,126,214 “Air Bumper” teaches an air inflatable bumper that responds to a crash to provide an air-supported member to protect the car from damages due to collision. Several prior art patents show a system for the detection of a crash and the deployment of airbags. U.S. Pat. No. 3,822,076 “Fluid Shock Absorbing Buffer, teaches a front or rear mounted airbags that are inflated when a telescopic rod extending from the vehicle touches a barrier. U.S. Pat. No. 4,176,858 “Energy Absorbing Bumper System” teaches a combination of a pneumatic bumper system supporting an airbag system that deploys in response to increased pressure in the pneumatic system as a result of an impact with an object. U.S. Pat. No. 5,431,463 “Air Cell Bumper Device” teaches a plurality of air cells containing grouped around a much larger air cell that stores inflation fluid. Upon impact, the material of cells is such that the larger cell ruptures and the fluid therein flows to the smaller cells buffering the impact. The invention is particularly useful on the sides of a vehicle. U.S. Pat. No. 5,646,613 “System for Minimizing Automobile Collision Damage” teaches the storage and deployment of various airbags around the vehicle as a result of proximity sensing. The different sides of the vehicle are uniquely controlled. U.S. Pat. No. 5,732,785 “Proactive Exterior Airbag System and Its Deployment Method for a Motor Vehicle” teaches a system having a detection unit, a control unit, and a deployment unit together will deploy airbags mounted on the vehicle. This system deploys the airbags before the crash and describes the method used to determine distance and speed between the striking and struck vehicles or objects. European Patent Application EP 1,024,063 “Vehicle Bumper and Hood Airbag System” teaches a bumper and hood bag that is inflated prior to the collision of a pedestrian and the vehicle. The airbag is inflated to absorb the collision forces between the areas from the waist down of a pedestrian and the vehicle. JP 6,144,154 “Shock Relaxing Device” teaches an airbag deployed in front of a bumper to reduce the shock of a pedestrian or bike collision with a car. The increased popularity of sports utility vehicles, passenger trucks and other retail motor vehicles that stand higher than a standard motor vehicle, such as a sedan or sports car, has created new problems in the area of vehicle collisions. Specifically, when one of these higher standing vehicles broadsides a standard vehicle, because of the difference in height between the two vehicles, the bumper of the high vehicle will contact the side window portion of the struck vehicle instead of the door portion. If the collision happens at high speeds, the head of the occupant sitting adjacent the window portion may move outward past the window and into contact with the bumper of the higher vehicle. Accordingly, there is a need for an airbag that can reduce the severity of such collisions.

<SOH> SUMMARY OF THE INVENTION <EOH>An advantage of the present invention is that it can reduce the severity of a collision between high standing vehicle and a low standing vehicle. Another advantage of the present invention is that it offers protection to the occupant of the struck vehicle in the event the occupant is partially expelled in the direction of the invention. These and other advantages will be found in the present invention that is directed to an airbag assembly mounted behind a vehicle's bumper coupled with sensors, electronic control units, and inflators to inflate by means of a fluid pressure to expand and provide an interface between a striking and struck motor vehicles. The airbag can have an up-side-down “L” shape or a cylindrical shape. Further, multiple bags can be combined within one system. The airbag is configured to cover substantially the width of the vehicle upon deployment and also provide protection to the occupant of a struck vehicle in the event the occupant is partially expelled from the struck vehicle in the direction of the bag.

5-azido-laevulinic acid, method for the production thereof and its use

The present invention relates to 5-azido levulinic acid, a process for its preparation, its use. Using 5-azido levulinic acid as starting material for the synthesis of 5-amino levulinic acid hydrochloride it is possible to obtain the latter in good yield an in pharmaceutical acceptable quality. 5-Azido levuliniv acid is synthesized in that methyl 5-bromo levulinate and/or methyl 5-chloro levulinate is converted with aqueous hydrochloric acid and as a result of an incomplete bromine/chlorine exchange at the C-5-postion a mixture of 5-chloro levulinic acid and 5-bromo levulinic acid is obtained, and the obtained 5-chloro levulinic acid, a mixture of 5-chloro levulinic acid and 5-bromo levulinic acid and the pure 5-bromo levulinic acid is transferred into 5-azido levulinic acid by conversion with a nucleophilic azide.

1. 5-azido levulinic acid 2. Process for the preparation of 5-azidolevuliniv acid, wherein (a) methyl 5-bromo levulinate and/or methyl 5-chloro levulinate is converted with aqueous hydrochloric acid and as a result of an incomplete bromine/chlorine exchange at the C-5-postion a mixture of 5-chloro levulinic acid and 5-bromo levulinic acid is obtained, and (b) the obtained 5-chloro levulinic acid, a mixture of 5-chloro levulinic acid and 5-bromo levulinic acid and the pure 5-bromo levulinic acid is transferred into 5-azido levulinic acid by conversion with a nucleophilic azide. 3. Process according to claim 2, wherein in stage (b) the conversion with an azide is carried out in a polar solvent, especially acetone, a C1-C4-alkanol or water as reaction medium. 4. Process according to claim 2, wherein in stage (b) an alkali metal azide, in particular sodium azide is used as nucleophilic azide. 5. Use of 5-azido levulinic acid for the preparation of 5-amino levulinic acid hydrochloride, whereas 5-azido levulinic acid is converted into 5-amino levulinic acid hydrochloride by catalytic hydrogenation. 6. Use according to claim 5, wherein (a) the catalytic hydrogenation ist carried out in aqueous hydrochloric acid, (b) the solvent and excess hydrochloric acid is seperated and (c) 5-amino levulinic acid hydrochloride is obtained by cristallisation from 2-propanol or t-butyl methyl ether/methanol. 7. Use of 5-azido levulinic acid as explosive and/or priming fuse. 8. Use of 5-azido levulinic acid as explosive and/or priming fuse in airbags in the motor vehicle technique.

Medical metal implants that can be decomposed by corrosion

An in vivo, decomposable medical implant is provided and comprises a metal material that contains, as a main alloying constituent, tungsten or a metal from the group rhenium, osmium, and molybdenum. The method for decomposing the implant, via corrosion in a bio system, includes the step of changing the pH level of the bio system, at least at the site of the implant, from a corrosion-inhibiting level to a corrosion-promoting level.

1-11. (cancelled) 12. An in vivo, decomposable medical implant from the group including: stents (coronary stents, peripHeral stents, tracheal stents, bile duct stents, esopHagus stents), surgical clips, osteosynthesis material, biological matrix (foam), metal wiring, metal threads, active substance depots, comprising a metal material, wherein said material contains, as a main alloying constituent, tungsten or a metal selected from the group consisting of rhenium, osmium and molybdenum. 13. A medical implant according to claim 12, wherein said material contains, as a secondary constituent, at least one element selected from the group consisting of lanthanides, actinides, iron, osmium, tantalum, platinum, gold, rhenium, gadolinium, yttrium and scandium. 14. A medical implant according to claim 13, wherein said lanthanide is cerium. 15. A medical implant according to claim 12, wherein said main alloying constituent represents more than 75% of said material, with any remainder, to form 100%, being formed by at least one secondary constituent. 16. A medical implant according to claim 15, wherein said main alloying constituent represents 95 to 99.5% of said material. 17. A medical implant according to claim 12, wherein said material has a crystalline structure having a particle size of 0.5 to 30 μm. 18. A medical implant according to claim 17, wherein said particle size is 0.5 to 5 μm. 19. A medical implant according to claim 12, wherein said implant, with the exception of said material, contains metal or non-metal inclusions that comprise an essentially pure alkali or alkaline earth metal, a drug, mRNA or a vector. 20. A medical implant according to claim 12, wherein said implant has an essentially tubular base. 21. A method for decomposition of the implant of claim 12 via corrosion in a bio system, including the step of: changing the pH level of the bio system, at least at a site of the implant, from a corrosion-inhibiting level to a corrosion-promoting level. 22. A method according to claim 21, wherein within the vicinity of a cardiovascular system, the pH level of said bio system is changed to a level of at least 7.4. 23. A method according to claim 22, wherein the pH level of said bio system is changed to a level of at least 7.5. 24. A method according to claim 22, wherein the pH level of said bio system is changed to a level of at least 7.6. 25. A method according to claim 21, wherein within the vicinity of a urine or bio system, the pH level of said bio system is changed from a lower pH level to a higher pH level. 26. A method according to claim 21, wherein the pH level of said bio system is changed by supplying or stopping alkalizing or acidifying substances. 27. A method according to claim 26, wherein said alkalizing or acidifying substances are at least one of the group consisting of ascorbic acid, sodium bicarbonate, citrates, and diuretics. 28. A method according to claim 21, wherein the pH level of said bio system is changed by supplying or stopping drugs that alkalize said bio system. 29. A method according to claim 28, wherein said drugs are loop diuretics.

Piezoelectric actuator

An apparatus for actuating a brake comprising force transmission means for transmitting force to a friction pad of a brake, at least one piezo-electric device operable when energised to change shape and/or size so as to apply a force to the force transmission means in a direction for actuating the brake, and retaining means operable to resist movement of the force transmission means in an opposite direction

1. An apparatus for actuating a brake comprising: a force transmission means that transmits force to a friction pad of a brake; at least one piezo-electric device operable when energized to change at least one of shape and size so as to apply a force to the force transmission means in a first direction for actuating the brake; and a retaining means operable to resist movement of the force transmission means in a second direction opposite the first direction, wherein the retaining means is a ratchet device. 2. An apparatus as defined in claim 1, wherein, wherein the ratchet device comprises a mechanical device. 3. An apparatus as defined in claim 2, wherein the mechanical device comprises a ratchet member and a biasing means that biases the ratchet member into engagement with the force transmission means for resisting movement thereof. 4. An apparatus as defined in claim 3, wherein the ratchet member has a sharp edge for engaging the force transmission means. 5. An apparatus as defined in claim 3, wherein expansion of the piezo-electric device causes the ratchet member to move out of engagement with the force transmission means, and wherein contraction of the piezo-electric device permits the biasing means to move the ratchet member into engagement with the force transmission means. 6. An apparatus as defined in claim 1, wherein the apparatus comprises a support, wherein a portion of the force transmission means passes through an opening in the support, the piezo-electric device being interposed between said portion and the support to act against the support when applying force to the force transmission means. 7. An apparatus as defined in claim 1, wherein the apparatus further comprises at least one oppositely-acting piezo-electric device operable to apply force to move the force transmission means in the second direction. 8. An apparatus as defined in claim 7, wherein the ratchet device comprises a ratchet member and a biasing means that applies a biasing force to bias the ratchet member into engagement with the force transmission means so as to resist movement thereof, wherein the at least one oppositely-acting piezo-electric device is also operable to overcome the biasing force. 9. An apparatus for actuating a brake comprising force transmission means for transmitting force to a friction pad of a brake, a structural element, a shaft of the force transmission means passing through an opening in the structural element, and a plurality of piezo-electric devices being interposed between the shaft and the structural element, such that when energized, the piezo-electric devices change shape and/or size so as to act against the structural element and cause a force to be applied to the force transmission means in a direction for actuating the brake, and in a tangential and/or radial direction with respect to the shaft, the piezo-electric devices further being operable to resist movement of the force transmission means in an opposite direction. 10. An apparatus for actuating a brake as defined in claim 9, wherein the piezo-electric devices are operable to apply a force to move the force transmission means in the opposite direction. 11. An apparatus for actuating a brake as defined in claim 9, wherein sub-sets of the piezo-electric devices are arranged to change size and/or shape when energized so as to act against a radially inner surface of the structural element, the sub-sets of piezo-electric devices capable of being repeatedly energized in sequence so as to apply a large angle of rotation or sustained application of torque to the shaft. 12. An apparatus for actuating a brake as defined in claim 9, wherein some of the plurality of piezo-electric devices are arranged to provide axial force to the shaft, and others arranged to provide torque to the shaft, selective expansion of the piezo-electric devices enabling a combined axial and rotational force to be applied to the shaft. 13. An apparatus for actuating a brake as defined in claim 9, wherein a first group of the plurality of piezo-electric devices are arranged to provide a lateral force in a first radial direction, and a second group of the plurality of piezo-electric devices are arranged to provide a lateral force in a second radial direction perpendicular to the first radial direction. 14. An apparatus for actuating a brake, comprising: a plurality of piezo-electric devices, each device being associated with a respective one of a plurality of device groups, wherein the devices in each group are energizable together; and an oscillating power supply connected to the plurality of piezo-electric devices, wherein the power supply has a phase difference between power supplied to associated groups, to change size and/or shape in groups; sequentially; to apply force directly to a brake pad so as to actuate a brake. 15. An apparatus for actuating a brake as defined in claim 14, wherein each group of piezo-electric devices is arranged to apply a force to an associated area of the brake pad so as to vary the force across the brake pad.

Autonomous bird predation reduction device

A self-guided apparatus for repelling birds through various means of deterrence. The device comprises a chassis, a floatation assembly, a propulsion system, and a guidance and control system. The device may operate in either of at least two modes: passive and active. In the passive mode, the device traverses a predefined area, scaring birds away. In the active mode, the device surveys a designated area and, upon detection of birds, propels itself towards the birds and drives them away.

1. A device for traversing a water body and for reducing the number of birds in the vicinity of the water body, said device comprising: (a) one or more floats having sufficient buoyancy to maintain a portion of said device above the surface of the water body; (b) an electrically-powered propulsion system adapted to transport said device across the surface of the water body, without the need for continuous monitoring or input from a human; (c) an electrically-powered collision prevention system to detect potential collisions before collisions occur, and to cause said propulsion system to alter the direction of transport of said device to avoid at least some detected potential collisions before collisions occur; all without the need for continuous monitoring or input from a human; (d) an electrically-powered navigation system adapted to cause said propulsion system to periodically transport the device within the characteristic distance of at least 75% of the surface of the water body at least once every two hours; and (e) a power source to supply electrical power, directly or indirectly, to said propulsion system, to said collision prevention system, and to said guidance system; whereby: (f) the transport of the device on the water body causes a reduction in the number of birds in the vicinity of the water body as compared to the number of birds that would be present in the absence of the transport of the device. 2. A device as recited in claim 1, wherein said device is adapted to operate in an active and a passive mode; wherein in the active mode, said device autonomously traverses a surveyed area of the water body either continuously or upon detection of at least one bird; and wherein in the passive mode, said device traverses the water body without regard to the location of any birds. 3. A device as recited in claim 1, wherein when said collision prevention system detects a potential collision said device is adapted to stop, back up, rotate between 0° and 360°, and then proceed forward, while operating either in a passive or active mode. 4. A device as recited in claim 1, wherein said propulsion system comprises a drive source and a motive source; wherein said drive source comprises at least two paddle wheels; and wherein said motive source comprises at least one electric motor to activate each paddle wheel. 5. A device as recited in claim 4, wherein said motive source operate said paddle wheels independently to propel and steer said device. 6. A device as recited in claim 1, wherein said collision prevention system comprises proximity feelers pivotally mounted on said device and attached to a triggering mechanism. 7. A device as recited in claim 6, wherein said triggering mechanism comprises a magnetic switch. 8. A device as recited in claim 1, wherein said navigation system is capable of identifying birds and guiding said device in the direction of said birds. 9. A device as recited in claim 1, wherein said power source comprises solar panels. 10. A device as recited in claim 9, wherein said power source additionally comprises one or more batteries adapted to store energy from said solar panels. 11. A device as recited in claim 1, wherein said electrically-powered navigation system is adapted to cause the propulsion system to periodically transport the device within the characteristic distance of 90% of the surface of the water body at least once every thirty minutes. 12. A collection comprising a plurality of devices for traversing a water body and for reducing the number of birds in the vicinity of the water body, said device comprising: (a) one or more floats having sufficient buoyancy to maintain a portion of said devices above the surface of the water body; (b) an electrically-powered propulsion system adapted to transport said devices across the surface of the water body, without the need for continuous monitoring or input from a human; (c) an electrically-powered collision prevention system to detect potential collisions before collisions occur, and to cause said propulsion system to alter the direction of transport of said devices to avoid at least some detected potential collisions before collisions occur; all without the need for continuous monitoring or input from a human; (d) an electrically powered navigation system adapted to cause the propulsion system to transport said devices across the water body, wherein at least one of said devices in said collection is periodically transported within the characteristic distance of at least 75% of the surface of the water body at least once every two hours; and (e) a power source to supply electrical power, directly or indirectly, to said propulsion system, to said collision prevention system, and to said guidance system; whereby: (f) the transport of said devices on the water body causes a reduction in the number of birds in the vicinity of the water body as compared to the number of birds that would be present in the absence of the transport of said devices. 13. A collection as recited in claim 12, wherein said devices are adapted to operate in an active and a passive mode; wherein in the active mode, said devices autonomously traverse a surveyed area of the water body either continuously or upon detection of at least one bird; and wherein in the passive mode, said devices traverse the water body without regard to the location of any birds. 14. A collection as recited in claim 12, wherein when said collision prevention system detects a potential collision said devices are adapted to stop, back up, rotate between 0° and 360°, and then proceed forward, while operating either in a passive or active mode. 15. A collection as recited in claim 12, wherein said propulsion system comprises a drive source and a motive source; wherein said drive source comprises at least two paddle wheels; and wherein said motive source comprises at least one electric motor to activate each paddle wheel. 16. A collection as recited in claim 15, wherein said motive source operate said paddle wheels independently to propel and steer said devices. 17. A collection as recited in claim 12, wherein said collision prevention system comprises proximity feelers pivotally mounted on said devices and attached to a triggering mechanism. 18. A collection as recited in claim 17, wherein said triggering mechanism comprises a magnetic switch. 19. A collection as recited in claim 12, wherein said navigation system is capable of identifying birds and guiding said devices in the direction of said birds. 20. A collection as recited in claim 12, wherein said power source comprises solar panels. 21. A collection as recited in claim 20, wherein said power source additionally comprises one or more batteries adapted to store energy from said solar panels. 22. A collection as recited in claim 12, wherein said electrically-powered navigation system is adapted to cause the propulsion system to periodically transport said devices within the characteristic distance of 90% of the surface of the water body at least once every thirty minutes. 23. A device for traversing a surveyed area and for reducing the number of birds in the vicinity of the surveyed area, said device comprising: (a) one or more floats having sufficient buoyancy to maintain a portion of said device above the surface of a water body when operating the device in water; (b) an electrically-powered propulsion system adapted to transport said device across the surface of the surveyed area, without the need for continuous monitoring or input from a human; (c) an electrically-powered collision prevention system to detect potential collisions before collisions occur, and to cause said propulsion system to alter the direction of transport of said device to avoid at least some detected potential collisions before collisions occur; all without the need for continuous monitoring or input from a human; (d) an electrically-powered navigation system adapted to cause the propulsion system to periodically transport the device within the characteristic distance of at least 75% of the surface of the surveyed area at least once every two hours; and (e) a power source to supply electrical power, directly or indirectly, to said propulsion system, to said collision prevention system, and to said guidance system; whereby: (f) the transport of the device on the surface of the surveyed area causes a reduction in the number of birds in the vicinity of the surveyed area as compared to the number of birds that would be present in the absence of the transport of the device. 24. A device as recited in claim 23, wherein said device is adapted to operate in an active and a passive mode; wherein in the active mode, said device autonomously traverses a surveyed area either continuously or upon detection of at least one bird; and wherein in the passive mode, said device traverses the surveyed area without regard to the location of any birds. 25. A device as recited in claim 23, wherein when said collision prevention system detects a potential collision said device is adapted to stop, back up, rotate between 0° and 360°, and then proceed forward, while operating either a passive or active mode. 26. A device as recited in claim 23, wherein said propulsion system comprises a drive source and a motive source; wherein said drive source comprises at least two paddle wheels; and wherein said motive source comprises at least one electric motor to activate each paddle wheel. 27. A device as recited in claim 26, wherein said motive source operate said paddle wheels independently to propel and steer said device. 28. A device as recited in claim 23, wherein said collision prevention system comprises proximity feelers pivotally mounted on said device and attached to a triggering mechanism. 29. A device as recited in claim 28, wherein said triggering mechanism comprises a magnetic switch. 30. A device as recited in claim 23, wherein said navigation system is capable of identifying birds and guiding said device in the direction of said birds. 31. A device as recited in claim 23, wherein said power source comprises solar panels. 32. A device as recited in claim 23, wherein said power source is a docking station adapted to recharge said device. 33. A device as recited in claim 31, wherein said power source additionally comprises one or more batteries adapted to store energy. 34. A device as recited in claim 32, wherein said power source additionally comprises one or more batteries adapted to store energy. 35. A device as recited in claim 23, wherein said electrically-powered navigation system is adapted to cause the propulsion system to periodically transport the device within the characteristic distance of 90% of the surface of the surveyed area at least once every thirty minutes.

<SOH> BACKGROUND ART <EOH>Bird depredation of fish, crawfish, and shrimp in aquaculture ponds poses major problems. For example, pelicans can consume 1 to 3 lb (0.45 to 1.36 kg) of fish per day, and may arrive with hundreds per flock. Cormorants, anhingas, herons, and egrets may also do significant damage to aquaculture ponds. It is estimated that one egret can eat ⅓ lb (0.15 kg) of fish per day, while a great heron can eat ⅔ to ¾ lb (0.30 to 0.34 kg) per day. See G. A. Littauer etal., “Control of Bird Predation at Aquaculture Facilities: Strategies and Cost Estimates,” Southern Regional Aquaculture Center, Publ. No. 402 (1997). This problem can be especially troublesome in ponds that have just been stocked with young fish. M. D. Hoy et al., Eastern Wildlife Damage Control Conference, vol. 4, pp. 109-112 (1989), estimated that wading birds could cause profound losses during fall migration, up to $10,000 per week on bait fish farms. The Louisiana State University Ben Hur Aquaculture Facility in Baton Rouge, La. recently experienced this problem with the white pelican during December 2000, when many fish were eaten and several ponds were completely de-stocked of fish. A. R. Stickley et al., Eastern Wildlife Damage Control Conference, vol. 4, pp. 105-108 (1989), estimated that in 1988 catfish losses due to double-crested cormorants amounted to $3.3 million. Currently, several different methods are employed to attempt to scare birds from aquaculture ponds. One of the most common methods is the use of sonic cannons. Sonic cannons emit loud bursts of noise. However, birds eventually become accustomed to the noise emitted by sonic cannons. Also, the loud “boom” produced by the sonic cannon can be disturbing to surrounding communities, and typically causes birds to migrate to other parts of the farm where the noise is more tolerable. See M. Bomford et al., “Sonic Deterrents in Animal Damage Control: A Review of Device Test and Effectiveness,” Wildlife Society Bulletin, vol. 18, pp. 411-422 (1990). Poisons, scarecrows, and nets have also been used. However, these methods have several faults. For example, poisons are usually fatal to birds and may cause casualties in non-target species. Scarecrows are effective for short-term periods only, because birds typically adapt and become accustomed to them. Nets typically have a high initial cost and are not practical for large ponds (>5 acres/˜2 hectares). An unfilled need exists for a cost-effective device and method for effectively reducing bird predation of aquatic organisms over a relatively long period of time. The device should be environmentally friendly, harmless to birds, and capable of withstanding expected environmental elements (e.g., water, wind, sun, and rain) and animal attacks. The device should also be able to endure biological challenges (e.g., wind, weeds, and slime), and should have some level of intelligence to adapt to the evolving conduct of birds.

<SOH> BRIEF DESCRIPTION OF THE FIGURES <EOH>FIG. 1 illustrates a front plan view of one embodiment of the predation reduction device. FIG. 2 illustrates a perspective view of one embodiment of the predation reduction device with the solar panels removed. FIG. 3 is an illustrative trajectory diagram depicting typical paths traversed by one embodiment of the predation reduction device. detailed-description description="Detailed Description" end="lead"?

Method of designing addressable array for detection of nucleic acid sequence differences using ligase detection reaction

The present invention is directed to a method of designing a plurality of capture oligonucleotide probes for use on a support to which complementary oligonucleotide probes will hybridize with little mismatch, where the plural capture oligonucleotide probes have melting temperatures within a narrow range. The first step of the method involves providing a first set of a plurality of tetramers of four nucleotides linked together, where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, (3) no tetramers within a set are palindromic or dinucleotide repeats, and (4) no tetramer within a set has one or less or three or more G or C nucleotides. Groups of 2 to 4 of the tetramers from the first set are linked together to form a collection of multimer units. From the collection of multimer units, all multimer units formed from the same tetramer and all multimer units having a melting temperature in ° C. of less than 4 times the number of tetramers forming a multimer unit are removed to form a modified collection of multimer units. The modified collection of multimer units is arranged in a list in order of melting temperature. The order of the modified collection of multimer units is randomized in 2° C. increments of melting temperature.

1. A method of designing a plurality of capture oligonucleotide probes for use on a support to which complementary oligonucleotide probes will hybridize with little mismatch, wherein the plural capture oligonucleotide probes have melting temperatures within a narrow range, said method comprising: providing a first set of a plurality of tetramers of four nucleotides linked together, wherein (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, (3) no tetramers within a set are palindromic or dinucleotide repeats, and (4) no tetramer within a set has one or less or three or more G or C nucleotides; linking groups of 2 to 4 of the tetramers from the first set together to form a collection of multimer units; removing from the collection of multimer units all multimer units formed from the same tetramer and all multimer units having a melting temperature in ° C. of less than the 4 times the number of tetramers forming a multimer unit, to form a modified collection of multimer units; arranging the modified collection of multimer units in a list in order of melting temperature; randomizing, in 2° C. increments of melting temperature, the order of the modified collection of multimer units; dividing alternating multimer units in the list into first and second subcollections, each arranged in order of melting temperature; inverting the order of the second subcollection; linking in order the first collection of multimer units to the inverted second collection of multimer units in order to form a collection of double multimer units; and removing from the collection of double multimer units those units (1) having a melting temperature in ° C. of less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, to form a modified collection of double multimer units. 2. A method according to claim 1 further comprising: placing the modified collection of double multimer units at positions on a support so that complementary oligonucleotides to be immobilized on the support can be captured at the positions. 3. A method according to claim 2, wherein the collection of double multimer units is shown in FIG. 26. 4. A method according to claim 2, wherein the collection of double multimer units is shown in FIG. 27. 5. A method according to claim 2, wherein the collection of double multimer units removed has a melting temperature in ° C. of less than 12.5 times the number of tetramers and more than 14 times the number of tetramers. 6. A method according to claim 1, wherein the multimer units have 12 mers, the double multimer units have 24 mers, and the melting point of the double multimer units is 75-84° C. 7. A method according to claim 1 further comprising: reclaiming double multimer units having a melting temperature in ° C. of less than 11 times the number of tetramers and more than 15 times the number of tetramers; unlinking the reclaimed double multimers units to each form a pair of multimer units; selecting multimer units with a melting temperature in ° C. of more than 11 times the number of tetramers and less than 17 times the number of tetramers; and reintegrating the selected multimer units into said method. 8. A method according to claim 7, wherein the the method of claim 7 is repeated. 9. A method according to claim 1 further comprising: making additional different sets of a plurality of tetramers by producing, base by base, circular permutations of the tetramers within the first set and complements thereof. 10. A method according to claim 1, wherein the set of tetramers is shown in Table 1 and complements thereof. 11. A method according to claim 10, wherein the set of tetramers are one base circular permutations of the tetramers shown in Table 1 and complements thereof. 12. A method according to claim 10, wherein the set of tetramers are two base circular permutations of the tetramers shown in Table 1 and complements thereof. 13. A method according to claim 10, wherein the set of tetramers are three base circular permutations of the tetramers shown in Table 1 and complements thereof. 14. A method according to claim 1, wherein the collection of double multimer units is shown in FIG. 26. 15. A method according to claim 1, wherein the modified collection of double multimer units is shown in FIG. 27. 16. A method according to claim 1, wherein the collection of double multimer units have a melting temperature in ° C. of less than 12.5 times the number of tetramers and more than 14 times the number of tetramers. 17. An oligonucleotide array comprising: a support and a collection of double multimer unit oligonucleotides at different positions on the support so that complementary oligonucleotides to be immobilized on the support can be captured at the different positions, wherein the complementary oligonucleotides will hybridize, within a narrow temperature range of greater than 24° C. with little mismatch, to members of the collection of double multimer unit oligonucleotides, the double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats, and the collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. less than 12.5 times the number of tetramers and more than 14 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) multimer units with the same 4 tetramers linked together with or without interruption. 18. An oligonucleotide array according to claim 17, wherein the collection of double multimer units is shown in FIG. 26. 19. An oligonucleotide array according to claim 17, wherein the collection of double multimer units is shown in FIG. 27. 20. An oligonucleotide array according to claim 17, wherein the collection of double multimer units has a melting temperature in ° C. less than 12.5 times the number of tetramers and more than 14 times the number of tetramers. 21. An oligonucleotide array according to claim 17, wherein the double multimer units have 24 mers and the melting temperature of the double multimer units is 75-84° C. 22. An oligonucleotide array according to claim 17, wherein the set of tetramers is shown in Table 6 and complements thereof. 23. An oligonucleotide array according to claim 17, wherein the set of tetramers are one base circular permutations of the tetramers shown in Table 6 and complements thereof. 24. A oligonucleotide array according to claim 17, wherein the set of tetramers are two base circular permutations of the tetramers shown in Table 6 and complements thereof. 25. An oligonucleotide array according to claim 17, wherein the set of tetramers are three base circular permutations of the tetramers shown in Table 6 and complements thereof. 26. A method for identifying one or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences comprising: providing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences; providing a plurality of oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a target-specific portion and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target-specific portion and a detectable reporter label, wherein the oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence present in the sample; providing a ligase, blending the sample, the plurality of oligonucleotide probe sets, and the ligase to form a mixture; subjecting the mixture to one or more ligase detection reaction cycles comprising a denaturation treatment, wherein any hybridized oligonucleotides are separated from the target nucleotide sequences, and a hybridization treatment, wherein the oligonucleotide probe sets hybridize at adjacent positions in a base-specific manner to their respective target nucleotide sequences, if present in the sample, and ligate to one another to form a ligated product sequence containing (a) the addressable array-specific portion, (b) the target-specific portions connected together, and (c) the detectable reporter label, and, wherein the oligonucleotide probe sets may hybridize to nucleotide sequences in the sample other than their respective target nucleotide sequences but do not ligate together due to a presence of one or more mismatches and individually separate during the denaturation treatment; providing a support with different capture oligonucleotides immobilized at different positions, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions and are formed from a collection of double multimer unit oligonucleotides, wherein oligonucleotide with addressable array-specific portions will hybridize, within a narrow temperature range of more than 4 times the number of tetramers in the multimer unit with little mismatch, to the capture oligonuncleotides, the double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats, and the collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. of 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, to form a modified collection of double multimer units; contacting the mixture, after said subjecting, with the support under conditions effective to hybridize the addressable array-specific portions to the capture oligonucleotides in a base-specific manner, thereby capturing the addressable array-specific portions on the support at the site with the complementary capture oligonucleotide; and detecting the reporter labels of ligated product sequences captured on the support at particular sites, thereby indicating the presence of one or more target nucleotide sequences in the sample. 27. A method according to claim 26, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, have a mismatch at a base at the ligation junction which interferes with such ligation. 28. A method according to claim 27, wherein the mismatch is at the 3′ base at the ligation junction. 29. A method according to claim 26, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, there is a mismatch at a base adjacent to a base at the ligation junction which interferes with such ligation. 30. A method according to claim 29, wherein the mismatch is at the base adjacent to the 3′ base at the ligation junction. 31. A method according to claim 26, wherein the sample potentially contains unknown amounts of one or more of a plurality of target sequences with a plurality of sequence differences, said method further comprising: quantifying, after said detecting, the amount of the target nucleotide sequences in the sample by comparing the amount of captured ligated product sequences generated from the sample with a calibration curve of captured ligated product sequences generated from samples with known amounts of the target nucleotide sequence. 32. A method according to claim 26, wherein the sample potentially contains unknown amounts of one or more of a plurality of target nucleotide sequences with a plurality of sequence differences, said method further comprising: providing a known amount of one or more marker target nucleotide sequence; providing a plurality of marker-specific oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a target-specific portion complementary to the marker target nucleotide sequence and an addressable array-specific portion complementary to capture oligonucleotides on the support, and (b) a second oligonucleotide probe, having a target-specific portion complementary to the marker target nucleotide sequence and a detectable reporter label, wherein the oligonucleotide probes in a particular marker-specific oligonucleotide set are suitable for ligation together when hybridized adjacent to one another on a corresponding marker target nucleotide sequence, but, when hybridized to any other nucleotide sequence present in the sample or added marker sequences, there is a mismatch which interferes with such ligation, wherein said blending comprises blending the sample, the marker target nucleotide sequences, the plurality of oligonucleotide probe sets, the plurality of marker-specific oligonucleotide probe sets, and the ligase to form a mixture; detecting the reporter labels of the ligated marker-specific oligonucleotide sets captured on the support at particular sites, thereby indicating the presence of one or more marker target nucleotide sequences in the sample; and quantifying the amount of target nucleotide sequences in the sample by comparing the amount of captured ligated product generated from the known amount of marker target nucleotide sequences with the amount of captured other ligated product. 33. A method according to claim 31, wherein the one or more marker target nucleotide sequences differ from the target nucleotide sequences in the sample at one or more single nucleotide positions. 34. A method according to claim 33, wherein the oligonucleotide probe sets and the marker-specific oligonucleotide probe sets form a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probe sets of each group, the first oligonucleotide probes have a common target-specific portion, and the second oligonucleotide probes have a differing target-specific portion which hybridize to a given allele or a marker nucleotide sequence in a base-specific manner. 35. A method according to claim 33, wherein the oligonucleotide probe sets and the marker-specific oligonucleotide probe sets form a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probe sets of each group, the second oligonucleotide probes have a common target-specific portion and the first oligonucleotide probes have differing target-specific portions, which hybridize to a given allele or a marker nucleotide sequence in a base-specific manner. 36. A method according to claim 26, wherein the sample potentially contains unknown amounts of two or more of a plurality of target nucleotide sequences with a plurality of sequence differences, said method further comprising: quantifying, after said detecting, the relative amount of each of the plurality of target nucleotide sequences in the sample by comparing the relative amount of captured ligated product sequences generated by each of the plurality of target sequences within the sample, thereby providing a quantitative measure of the relative level of two or more target nucleotide sequences in the sample. 37. A method according to claim 26, wherein the target-specific portions of the oligonucleotide probe sets have substantially the same melting temperature so that they hybridize to target nucleotide sequences under similar hybridization conditions. 38. A method according to claim 26, wherein multiple allele differences at one or more nucleotide position in a single target nucleotide sequence or multiple allele differences at one or more positions in multiple target nucleotide sequences are distinguished, the oligonucleotide probe sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probes of each group, the second oligonucleotide probes have a common target-specific portion and the first oligonucleotide probes have differing target-specific portions which hybridize to a given allele in a base-specific manner, wherein, in said detecting, the labels of ligated product sequences of each group, captured on the support at different sites, are detected, thereby indicating a presence, in the sample of one or more allele at one or more nucleotide position in one or more target nucleotide sequences. 39. A method according to claim 38, wherein the oligonucleotide probes in a given set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when hybridized to any other nucleotide sequence present in the sample, the first oligonucleotide probe has a mismatch at a base at the ligation junction which interferes with such ligation. 40. A method according to claim 38, wherein multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence or multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences are distinguished with oligonucleotide probe groups having oligonucleotide probes with target-specific portions which overlap. 41. A method according to claim 40, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, there is a mismatch at a base at the ligation junction which interferes with such ligation. 42. A method according to claim 26, wherein multiple allele differences consisting of insertions, deletions, microsatellite repeats, translocations, or other DNA rearrangements at one or more nucleotide positions which require overlapping oligonucleotide probe sets in a single target nucleotide sequence or multiple allele differences consisting of insertions, deletions, microsatellite repeats, translocations, or other DNA rearrangements at one or more nucleotide positions which require overlapping oligonucleotide probe sets in multiple target nucleotide sequences are distinguished, the oligonucleotide probe sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences selected from the group consisting of insertions, deletions, microsatellite repeats, translocations, and other DNA rearrangements at one or more nucleotide positions which require overlapping oligonucleotide probe sets, wherein, in the oligonucleotide probe sets of each group, the second oligonucleotide probes have a common target-specific portion and the first oligonucleotide probes have differing target-specific portions which hybridize to a given allele in a base-specific manner, wherein, in said detecting, the labels of ligated product sequences of each group, captured on the support at different sites, are detected, thereby indicating a presence, in the sample, of one or more allele differences selected from the group consisting of insertions, deletions, microsatellite repeats, translocations, and other DNA rearrangements in one or more target nucleotide sequences. 43. A method according to claim 42, wherein the oligonucleotide probe sets are designed for distinguishing multiple allele differences selected from the group consisting of insertions, deletions, and microsatellite repeats, at one or more nucleotide positions which require overlapping oligonucleotide probe sets, wherein, in the oligonucleotide probe sets of each group, the second oligonucleotide probes have a common target-specific portion, and the first oligonucleotide probes have differing target-specific portions which contain repetitive sequences of different lengths to hybridize to a given allele in a base-specific manner. 44. A method according to claim 26, wherein a low abundance of multiple allele differences at multiple adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence, in the presence of an excess of normal sequence, or a low abundance of multiple allele differences at multiple nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences, in the presence of an excess of normal sequence, are distinguished, the oligonucleotide probe sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein one or more sets within a group share common second oligonucleotide probes and the first oligonucleotide probes have differing target-specific portions which hybridize to a given allele excluding the normal allele in a base-specific manner, wherein, in said detecting, the labels of ligated product sequences of each group captured on the support at different sites, are detected, thereby indicating a presence, in the sample, of one or more low abundance alleles at one or more nucleotide positions in one or more target nucleotide sequences. 45. A method according to claim 44, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, the first oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation. 46. A method according to claim 44, wherein a low abundance of multiple allele differences at multiple adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence, in the presence of an excess of normal sequence, or a low abundance of multiple allele differences at multiple nucleotide positions which require overlapping oligonucleotide probe sets in multiple target nucleotide sequences, in the presence of an excess of normal sequence, are quantified in a sample, said method further comprising: providing a known amount of one or more marker target nucleotide sequences; providing a plurality of marker-specific oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe having a target-specific portion complementary to the marker target nucleotide sequence and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target-specific portion complementary to the marker target nucleotide sequence and a detectable reporter label, wherein the oligonucleotide probes in a particular marker-specific oligonucleotide set are suitable for ligation together when hybridized adjacent to one another on a corresponding marker target nucleotide sequence, but, when hybridized to any other nucleotide sequence present in the sample or added marker sequences, have a mismatch which interferes with such ligation; providing a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets or marker-specific oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, including marker nucleotide sequences, wherein one or more sets within a group share a common second oligonucleotide probe and the first oligonucleotide probes have different target-specific probe portions which hybridize to a given allele or a marker nucleotide sequence excluding the normal allele, in a base-specific manner, wherein said blending comprises blending the sample, the marker target nucleotide sequences, the plurality of oligonucleotide probe sets, the plurality of marker-specific oligonucleotide probe sets, and the ligase to form a mixture; detecting the reporter labels of the ligated marker-specific oligonucleotide sets captured on the support at particular sites, thereby indicating the presence of one or more marker target nucleotide sequences in the sample; and quantifying the amount of target nucleotide sequences in the sample by comparing the amount of captured ligated products generated from the known amount of marker target nucleotide sequences with the amount of other captured ligated product generated from the low abundance unknown sample. 47. A method according to claim 46, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence under selected conditions due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, the first oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation. 48. A method according to claim 26, wherein multiple allele differences at one or more nucleotide position in a single target nucleotide sequence or multiple allele differences at one or more positions in multiple target nucleotide sequences are distinguished, the oligonucleotide sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probes of each group, the first oligonucleotide probes have a common target-specific portion and the second oligonucleotide probes have differing target-specific portions which hybridize to a given allele in a base-specific manner, wherein, in said detecting, different reporter labels of ligated product sequences of each group captured on the support at particular sites are detected, thereby indicating a presence, in the sample, of one or more alleles at one or more nucleotide positions in one or more target nucleotide sequences. 49. A method according to claim 48, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, the second oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation. 50. A method according to claim 48, wherein multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence, or multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences are distinguished, the oligonucleotide probe groups containing oligonucleotide probes with target-specific portions which overlap. 51. A method according to claim 50, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, the second oligonucleotide probe has a mismatch at a base at the ligation junction which interferes with such ligation. 52. A method according to claim 26, wherein multiple allele differences at one or more nucleotide position in a single target nucleotide sequence or multiple allele differences at one or more positions in multiple target nucleotide sequences are distinguished, the oligonucleotide sets forming a plurality of probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probes of different groups, the second oligonucleotide probes have a common target-specific portion or the first oligonucleotide probes have a common target-specific portion, wherein, in said detecting, the one of a plurality of labeled ligated product sequences of each group captured on the support at particular sites are detected, thereby indicating a presence of one or more allele at one or more nucleotide positions in one or more target nucleotide sequences in the sample. 53. A method according to claim 52, wherein the oligonucleotide probes in a given set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction but, when the oligonucleotides in the set are hybridized to any other nucleotide sequence present in the sample, the first or second oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation. 54. A method according to claim 52, wherein multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a target nucleotide sequence or multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences, are distinguished, the oligonucleotide probe groups containing probes with target-specific portions which overlap. 55. A method according to claim 54, wherein oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotides in the set are hybridized to any other nucleotide sequence present in the sample, the first or second oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation. 56. A method according to claim 53, wherein all possible single-base mutations for a single codon in a single target nucleotide sequence, all possible single-base mutations for multiple codons in a single target nucleotide sequence, and all possible single-base mutations for multiple codons in multiple target nucleotide sequences are distinguished, the oligonucleotide sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing all possible single-base mutations for a single codon, wherein, in the oligonucleotide probes of each group, the second oligonucleotide probes differ only in their 5′ bases at their ligation junction and contain different reporter labels, the first oligonucleotide probes differ only in their 3′ bases at their ligation junction and contain different addressable array-specific portions, or the first oligonucleotide probes differ only in their 3′ bases adjacent to the base at the ligation junction and contain different addressable array-specific portions. 57. A method according to claim 53, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotides in the set are hybridized to any other nucleotide sequence present in the sample, the first oligonucleotide probes have a mismatch at the 3′ base at the ligation junction or the 3′ base adjacent the base at the ligation junction or the second oligonucleotide probes have a mismatch at the 5′ base at the ligation junction which interferes with such ligation. 58. A method according to claim 57, wherein all possible single-base mutations for a single codon in a single target nucleotide sequence, or all possible single-base mutations for two or more adjacent codons, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences are distinguished, the oligonucleotide probe groups containing oligonucleotide probes with target-specific portions which overlap. 59. A method according to claim 26, wherein the denaturation treatment is at a temperature of about 80°-105° C. 60. A method according to claim 26, wherein each cycle, comprising a denaturation treatment and a hybridization treatment, is from about 30 seconds to about five minutes long. 61. A method according to claim 26, wherein said subjecting is repeated for 2 to 50 cycles. 62. A method according to claim 26, wherein total time for said subjecting is 1 to 250 minutes. 63. A method according to claim 26, wherein the ligase is selected from the group consisting of Thermus aquaticus ligase, Thermus thermophilus ligase, E. coli ligase, T4 ligase, and Pyrococcus ligase. 64. A method according to claim 26, wherein the detectable reporter label is selected from the group consisting of chromophores, fluorescent moieties, enzymes, antigens, heavy metals, magnetic probes, dyes, phosphorescent groups, radioactive materials, chemiluminescent moieties, and electrochemical detecting moieties. 65. A method according to claim 26, wherein the target-specific portions of the oligonucleotide probes each have a hybridization temperature of 20-85° C. 66. A method according to claim 26, wherein the target-specific portions of the oligonucleotide probes are 20 to 28 nucleotides long. 67. A method according to claim 26, wherein the mixture further includes a carrier DNA. 68. A method according to claim 26 further comprising: amplifying the target nucleotide sequences in the sample prior to said blending. 69. A method according to claim 68, wherein said amplifying is carried out by subjecting the sample to a polymerase-based amplifying procedure. 70. A method according to claim 68, wherein said polymerase-based amplifying procedure is carried out with DNA polymerase. 71. A method according to claim 68, when rein said polymerase-based amplifying procedure is carried out with reverse transcriptase. 72. A method according to claim 68, wherein said polymerase-based amplifying procedure is carried out with RNA polymerase. 73. A method according to claim 68, wherein said amplifying is carried out by subjecting the target nucleotide sequences in the sample to a ligase chain reaction process. 74. A method according to claim 26, wherein the oligonucleotide probe sets are selected from the group consisting of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, peptide nucleic acids, modified peptide nucleic acids, modified phosphate-sugar backbone oligonucleotides, nucleotide analogues, and mixtures thereof. 75. A method according to claim 26, wherein said method is used to detect infectious diseases caused by bacterial, viral, parasitic, and fungal infectious agents. 76. A method according to claim 75, wherein the infectious disease is caused by a bacteria selected from the group consisting of Escherichia coli Salmonella, Shigella, Klebsiella, Pseudomonas, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium-intracellulare, Yersinia, Francisella, Pasteurella, Brucella, Clostridia, Bordetella pertussis, Bacteroides, Staphylococcus aureus, Streptococcus pneumonia, B-Hemolytic strep., Corynebacteria, Legionella, Mycoplasma, Ureaplasma, Chlamydia, Neisseria gonorrhea, Neisseria meningitides, Hemophilus influenza, Enterococcus faecalis, Proteus vulgaris, Proteus mirabilis, Helicobacter pylori, Treponema palladium, Borrelia burgdorferi, Borrelia recurrentis, Rickettsial pathogens, Nocardia, and Acitnomycetes. 77. A method according to claim 75, wherein the infectious disease is caused by a fungal infectious agent selected from the group consisting of Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Paracoccicioides brasiliensis, Candida albicans, Aspergillus fumigautus, Phycomycetes (Rhizopus), Sporothrix schenckii, Chromomycosis, and Maduromycosis. 78. A method according to claim 75, wherein the infectious disease is caused by a viral infectious agent selected from the group consisting of human immunodeficiency virus, human T-cell lymphocytotrophic virus, hepatitis viruses (e.g., Hepatitis B Virus and Hepatitis C Virus), Epstein-Barr Virus, cytomegalovirus, human papillomaviruses, orthomyxo viruses, paramyxo viruses, adenoviruses, corona viruses, rhabdo viruses, polio viruses, toga viruses, bunya viruses, arena viruses, rubella viruses, and reo viruses. 79. A method according to claim 75, wherein the infectious disease is caused by a parasitic infectious agent selected from the group consisting of Plasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodium ovale, Onchoverva volvulus, Leishmania, Trypanosoma spp., Schistosoma spp., Entamoeba histolytica, Cryptosporidum, Giardia spp., Trichimonas spp., Balatidium coli, Wuchereria bancrofti, Toxoplasma spp., Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Dracunculus medinesis, trematodes, Diphyllobothrium latum, Taenia spp., Pneumocystis carinii, and Necator americanis. 80. A method according to claim 26, wherein said method is used to detect genetic diseases. 81. A method according to claim 80, wherein the genetic disease is selected from the group consisting of 21 hydroxylase deficiency, cystic fibrosis, Fragile X Syndrome, Turner Syndrome, Duchenne Muscular Dystrophy, Down Syndrome, heart disease, single gene diseases, HLA typing, phenylketonuria, sickle cell anemia, Tay-Sachs Syndrome, thalassemia, Klinefelter's Syndrome, Huntington's Disease, autoimmune diseases, lipidosis, obesity defects, hemophilia, inborn errors in metabolism, and diabetes. 82. A method according to claim 26, wherein said method is used to detect cancer involving oncogenes, tumor suppressor genes, or genes involved in DNA amplification, replication, recombination, or repair. 83. A method according to claim 82, wherein the cancer is associated with a gene selected from the group consisting of BRCA1 gene, p53 gene, Familial polyposis coli, Her2/Neu amplification, Bcr/Ab1, K-ras gene, human papillomavirus Types 16 and 18, leukemia, colon cancer, breast cancer, lung cancer, prostate cancer, brain tumors, central nervous system tumors, bladder tumors, melanomas, liver cancer, osteosarcoma and other bone cancers, testicular and ovarian carcinomas, ENT tumors, and loss of heterozygosity. 84. A method according to claim 26, wherein said method is used for environmental monitoring, forensics, and food and feed industry monitoring. 85. A method according to claim 26, wherein said detecting comprises: scanning the support at the particular sites and identifying if ligation of the oligonucleotide probe sets occurred and correlating identified ligation to a presence or absence of the target nucleotide sequences. 86. A method according to claim 85, wherein said scanning is carried out by scanning electron microscopy, electron microscopy, confocal microscopy, charge-coupled device, scanning tunneling electron microscopy, infrared microscopy, atomic force microscopy, electrical conductance, and fluorescent or phosphor imaging. 87. A method according to claim 85, wherein said correlating is carried out with a computer. 88. A method according to claim 26, wherein said contacting the mixture with the support is at a temperature of 45-90° C. and for a time period of up to 60 minutes. 89. A method according to claim 26, wherein some of the plurality of capture oligonucleotides have identical nucleotide sequences and different labels are used for some different target nucleotide sequence. 90. A method according to claim 26, wherein the plurality of capture oligonucleotides each have different nucleotide sequences. 91. A method according to claim 89, wherein each capture oligonucleotide has adjacent capture oligonucleotides separated from adjacent capture oligonucleotides by barrier oligonucleotides to which ligated oligonucleotide probe sets will not hybridize during said contacting. 92. A method according to claim 26, wherein the oligonucleotide probe sets hybridize to the target nucleotide sequences at temperatures which are less than that at which the capture oligonucleotides hybridize to the addressable array-specific portion of oligonucleotide probe sets. 93. A method according to claim 26 further comprising: treating the mixture chemically or enzymatically, after said subjecting the mixture to a series of ligase detection reaction cycles, to destroy unligated oligonucleotide probes. 94. A method according to claim 93, wherein said treating is carried out with an exonuclease. 95. A method according to claim 26 further comprising: removing oligonucleotides bound to the capture oligonucleotides to permit reuse of the support with immobilized capture oligonucleotides. 96. A method according to claim 26, wherein the support includes different capture oligonucleotides immobilized at different sites with different capture oligonucleotides being complementary to different addressable array-specific portions, whereby different oligonucleotide probe sets are captured and detected at different sites on the support. 97. A method according to claim 26, wherein the support includes identical capture oligonucleotides immobilized on the support with the capture oligonucleotides being complementary to all the addressable array-specific portions and the labels attached to the oligonucleotide probe sets being different, whereby the different oligonucleotide probe sets are detected and distinguished by the different labels. 98. A method according to claim 26, wherein the collection of double multimer units is shown in FIG. 26. 99. A method according to claim 26, wherein the collection of double multimer units is shown in FIG. 27. 100. A method according to claim 26, wherein the collection of double multimer units removed has a melting temperature in ° C. of less than 12.5 times the number of tetramers and more than 14 times the number of tetramers. 101. A method according to claim 26, wherein the double multimer units have 24 mers and the melting point of the double multimer units is 75-84° C. 102. A method according to claim 26, wherein the set of tetramers is shown in Table 1 and complements thereof. 103. A method according to claim 26, wherein the set of tetramers are one base circular permutations of the tetramers shown in Table 1 and complements thereof. 104. A method according to claim 26, wherein the set of tetramers are two base circular permutations of the tetramers shown in Table 1 and complements thereof. 105. A method according to claim 26, wherein the set of tetramers are three base circular permutations of the tetramers shown in Table 1 and complements thereof. 106. A kit for identifying one or more of a plurality of sequences differing by single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences comprising: a ligase; a plurality oligonucleotide probe sets, each characterized by (a) a first oligonucleotide probe, having a target sequence-specific portion and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target sequence-specific portion and detectable reporter label, wherein the oligonucleotide probes in a particular set are suitable for ligation together when hybrided adjacent to one another on a respective target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence, present in the sample; and a support with different capture oligonucleotides immobilized at different positions, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions and are formed from a collection of double multimer unit oligonucleotides, wherein oligonucleotide with addressable array-specific portions will hybridize, within a narrow temperature range of greater than 4 times the number of tetramers in the multimer unit with little mismatch, to members of the capture oligonuncleotides, the double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats, and the collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. of less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions. 107. A kit according to claim 106, wherein the mismatch of oligonucleotide probe sets to nucleotide sequences other than their respective target nucleotide sequences is at a base at a ligation junction at which the oligonucleotide probe of each set ligate together when hybridized to their respective target nucleotide sequences. 108. A kit according to claim 106, wherein the mismatch is on the oligonucleotide probe of the oligonucleotide probe sets which have 3′ ends at the ligation junction. 109. A kit according to claim 106, wherein the mismatch of oligonucleotide probe sets to nucleotide sequences other than their respective target nucleotide sequence is at a base adjacent to a ligation junction at which the oligonucleotide probes of each set ligate together when hybridized to their respective target nucleotide sequences. 110. A kit according to claim 109, wherein the mismatch is on the oligonucleotide probe of the oligonucleotide probe sets which have 3′ ends at the ligation junction. 111. A kit according to claim 106, wherein the ligase is selected from the group consisting of Thermus aquaticus ligase, Thermus thermophilus ligase, E. coli ligase, T4 ligase, and Pyrococcus ligase. 112. A kit according to claim 106 further comprising: amplification primers suitable for preliminary amplification of the target nucleotide sequences and a polymerase. 113. A kit according to claim 106, wherein the support includes different capture oligonucleotides immobilized at different particular sites with different capture oligonucleotides being complementary to different addressable array-specific portions, whereby different oligonucleotide probe sets are hybridized and detected at different sites on the support. 114. A kit according to claim 106, wherein the support includes identical capture oligonucleotides immobilized on the support with the capture oligonucleotides complementary to all the addressable array-specific portions and the labels attached to the oligonucleotide probe sets being different, whereby the oligonucleotide probe sets are detected and distinguished by the different labels. 115. A kit according to claim 106, wherein the oligonucleotide probe sets and the capture oligonucleotides are configured so that the oligonucleotide probe sets hybridize, respectively, to the target nucleotide sequences at temperatures which are less than that at which the capture oligonucleotides hybridize to the addressable array-specific portions of the oligonucleotide probes sets. 116. A kit according to claim 106, wherein the collection of double multimer units is shown in FIG. 26. 117. A kit according to claim 106, wherein the collection of double multimer units is shown in FIG. 27. 118. A kit according to claim 106, wherein the collection of double multimer units removed has a melting temperature in ° C. of less than 12.5 times the number of tetramers and more than 14 times the number of tetramers. 119. A kit according to claim 106, wherein the double multimer units have 24 mers and the melting point of the double multimer units is 75-84° C. 120. A kit according to claim 106, wherein the set of tetramers is shown in Table 1 or complements thereof. 121. A kit according to claim 106, wherein the set of tetramers are one base circular permutations of the tetramers shown in Table 1 and complements thereof. 122. A kit according to claim 106, wherein the set of tetramers are two base circular permutations of the tetramers shown in Table 1 and complements thereof. 123. A kit according to claim 106, wherein the set of tetramers are three base circular permutations of the tetramers shown in Table 1 and complements thereof. 124. A method to avoid synthesizing ligase detection reaction oligonucleotides that will inappropriately cross-hybridize to capture oligonucleotides on a solid support comprising comparing the ligase detection reaction oligonucleotides with the capture oligonucleotides and identifying any capture oligonucleotides likely to cross-hybridize to the ligase detection reaction oligonucleotides.

<SOH> BACKGROUND OF THE INVENTION <EOH>Detection of Sequence Differences Large-scale multiplex analysis of highly polymorphic loci is needed for practical identification of individuals, e.g., for paternity testing and in forensic science (Reynolds et al., Anal. Chem., 63: 2-15 (1991)), for organ-transplant donor-recipient matching (Buyse et al., Tissue Antigens, 41: 1-14 (1993) and Gyllensten et al., PCR Meth. Appl, 1: 91-98 (1991)), for genetic disease diagnosis, prognosis, and pre-natal counseling (Chamberlain et al., Nucleic Acids Res., 16: 11141-11156 (1988) and L. C. Tsui, Human Mutat., 1: 197-203 (1992)), and the study of oncogenic mutations (Hollstein et al., Science, 253: 49-53 (1991)). In addition, the cost-effectiveness of infectious disease diagnosis by nucleic acid analysis varies directly with the multiplex scale in panel testing. Many of these applications depend on the discrimination of single-base differences at a multiplicity of sometimes closely space loci. A variety of DNA hybridization techniques are available for detecting the presence of one or more selected polynucleotide sequences in a sample containing a large number of sequence regions. In a simple method, which relies on fragment capture and labeling, a fragment containing a selected sequence is captured by hybridization to an immobilized probe. The captured fragment can be labeled by hybridization to a second probe which contains a detectable reporter moiety. Another widely used method is Southern blotting. In this method, a mixture of DNA fragments in a sample are fractionated by gel electrophoresis, then fixed on a nitrocellulose filter. By reacting the filter with one or more labeled probes under hybridization conditions, the presence of bands containing the probe sequence can be identified. The method is especially useful for identifying fragments in a restriction-enzyme DNA digest which contain a given probe sequence, and for analyzing restriction-fragment length polymorphisms (“RFLPs”). Another approach to detecting the presence of a given sequence or sequences in a polynucleotide sample involves selective amplification of the sequence(s) by polymerase chain reaction. U.S. Pat. No. 4,683,202 to Mullis, et al. and R. K. Saiki, et al., Science 230: 1350 (1985). In this method, primers complementary to opposite end portions of the selected sequence(s) are used to promote, in conjunction with thermal cycling, successive rounds of primer-initiated replication. The amplified sequence may be readily identified by a variety of techniques. This approach is particularly useful for detecting the presence of low-copy sequences in a polynucleotide-containing sample, e.g., for detecting pathogen sequences in a body-fluid sample. More recently, methods of identifying known target sequences by probe ligation methods have been reported. U.S. Pat. No. 4,883,750 to N. M. Whiteley, et al., D. Y. Wu, et al., Genomics 4: 560 (1989), U. Landegren, et al., Science 241: 1077 (1988), and E. Winn-Deen, et al., Clin. Chem. 37: 1522 (1991). In one approach, known as oligonucleotide ligation assay (“OLA”), two probes or probe elements which span a target region of interest are hybridized with the target region. Where the probe elements match (basepair with) adjacent target bases at the confronting ends of the probe elements, the two elements can be joined by ligation, e.g., by treatment with ligase. The ligated probe element is then assayed, evidencing the presence of the target sequence. In a modification of this approach, the ligated probe elements act as a template for a pair of complementary probe elements. With continued cycles of denaturation, hybridization, and ligation in the presence of the two complementary pairs of probe elements, the target sequence is amplified exponentially, i.e., exponentially allowing very small amounts of target sequence to be detected and/or amplified. This approach is referred to as ligase chain reaction (“LCR”). F. Barany, “Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase,” Proc. Nat'l Acad. Sci. USA, 88: 189-93 (1991) and F. Barany, “The Ligase Chain Reaction (LCR) in a PCR World,” PCR Methods and Applications, 1: 5-16 (1991). Another scheme for multiplex detection of nucleic acid sequence differences is disclosed in U.S. Pat. No. 5,470,705 to Grossman et. al. where sequence-specific probes, having a detectable label and a distinctive ratio of charge/translational frictional drag, can be hybridized to a target and ligated together. This technique was used in Grossman, et. al., “High-density Multiplex Detection of Nucleic Acid Sequences: Oligonucleotide Ligation Assay and Sequence-coded Separation,” Nucl. Acids Res. 22(21): 4527-34 (1994) for the large scale multiplex analysis of the cystic fibrosis transmembrane regulator gene. Jou, et. al., “Deletion Detection in Dystrophin Gene by Multiplex Gap Ligase Chain Reaction and Immunochromatographic Strip Technology,” Human Mutation 5: 86-93 (1995) relates to the use of a so called “gap ligase chain reaction” process to amplify simultaneously selected regions of multiple exons with the amplified products being read on an immunochromatographic strip having antibodies specific to the different haptens on the probes for each exon. There is a growing need, e.g., in the field of genetic screening, for methods useful in detecting the presence or absence of each of a large number of sequences in a target polynucleotide. For example, as many as 400 different mutations have been associated with cystic fibrosis. In screening for genetic predisposition to this disease, it is optimal to test all of the possible different gene sequence mutations in the subject's genomic DNA, in order to make a positive identification of “cystic fibrosis”. It would be ideal to test for the presence or absence of all of the possible mutation sites in a single assay. However, the prior-art methods described above are not readily adaptable for use in detecting multiple selected sequences in a convenient, automated single-assay format. Solid-phase hybridization assays require multiple liquid-handling steps, and some incubation and wash temperatures must be carefully controlled to keep the stringency needed for single-nucleotide mismatch discrimination. Multiplexing of this approach has proven difficult as optimal hybridization conditions vary greatly among probe sequences. Allele-specific PCR products generally have the same size, and a given amplification tube is scored by the presence or absence of the product band in the gel lane associated with each reaction tube. Gibbs et al., Nucleic Acids Res., 17: 2437-2448 (1989). This approach requires splitting the test sample among multiple reaction tubes with different primer combinations, multiplying assay cost. PCR has also discriminated alleles by attaching different fluorescent dyes to competing allelic primers in a single reaction tube (F. F. Chehab, et al., Proc. Natl. Acad. Sci. USA, 86: 9178-9182 (1989)), but this route to multiplex analysis is limited in scale by the relatively few dyes which can be spectrally resolved in an economical manner with existing instrumentation and dye chemistry. The incorporation of bases modified with bulky side chains can be used to differentiate allelic PCR products by their electrophoretic mobility, but this method is limited by the successful incorporation of these modified bases by polymerase, and by the ability of electrophoresis to resolve relatively large PCR products which differ in size by only one of these groups. Livak et al., Nucleic Acids Res., 20: 4831-4837 (1989). Each PCR product is used to look for only a single mutation, making multiplexing difficult. Ligation of allele-specific probes generally has used solid-phase capture (U. Landegren et al., Science, 241: 1077-1080 (1988); Nickerson et al., Proc. Natl. Acad. Sci. USA, 87: 8923-8927 (1990)) or size-dependent separation (D. Y. Wu, et al., Genomics, 4: 560-569 (1989) and F. Barany, Proc. Natl. Acad. Sci., 88: 189-193 (1991)) to resolve the allelic signals, the latter method being limited in multiplex scale by the narrow size range of ligation probes. The gap ligase chain reaction process requires an additional step—polymerase extension. The use of probes with distinctive ratios of charge/translational frictional drag technique to a more complex multiplex will either require longer electrophoresis times or the use of an alternate form of detection. The need thus remains for a rapid single assay format to detect the presence or absence of multiple selected sequences in a polynucleotide sample. Use of Oligonucleotide Arrays for Nucleic Acid Analysis Ordered arrays of oligonucleotides immobilized on a solid support have been proposed for sequencing, sorting, isolating, and manipulating DNA. It has been recognized that hybridization of a cloned single-stranded DNA molecule to all possible oligonucleotide probes of a given length can theoretically identify the corresponding complementary DNA segments present in the molecule. In such an array, each oligonucleotide probe is immobilized on a solid support at a different predetermined position. All the oligonucleotide segments in a DNA molecule can be surveyed with such an array. One example of a procedure for sequencing DNA molecules using arrays of oligonucleotides is disclosed in U.S. Pat. No. 5,202,231 to Drmanac, et. al. This involves application of target DNA to a solid support to which a plurality of oligonucleotides are attached. Sequences are read by hybridization of segments of the target DNA to the oligonucleotides and assembly of overlapping segments of hybridized oligonucleotides. The array utilizes all possible oligonucleotides of a certain length between 11 and 20 nucleotides, but there is little information about how this array is constructed. See also A. B. Chetverin, et. al., “Sequencing of Pools of Nucleic Acids on Oligonucleotide Arrays,” BioSystems 30: 215-31 (1993); WO 92/16655 to Khrapko et. al.; Kuznetsova, et. al., “DNA Sequencing by Hybridization with Oligonucleotides Immobilized in Gel. Chemical Ligation as a Method of Expanding the Prospects for the Method,” Mol. Biol. 28(20): 290-99(1994); M. A. Livits, et. al., “Dissociation of Duplexes Formed by Hybridization of DNA with Gel-Immobilized Oligonucleotides,” J. Biomolec. Struct . & Dynam. 11(4): 783-812 (1994). WO 89/10977 to Southern discloses the use of a support carrying an array of oligonucleotides capable of undergoing a hybridization reaction for use in analyzing a nucleic acid sample for known point mutations, genomic fingerprinting, linkage analysis, and sequence determination. The matrix is formed by laying nucleotide bases in a selected pattern on the support. This reference indicates that a hydroxyl linker group can be applied to the support with the oligonucleotides being assembled by a pen plotter or by masking. WO 94/11530 to Cantor also relates to the use of an oligonucleotide array to carry out a process of sequencing by hybridization. The oligonucleotides are duplexes having overhanging ends to which target nucleic acids bind and are then ligated to the non-overhanging portion of the duplex. The array is constructed by using streptavidin-coated filter paper which captures biotinylated oligonucleotides assembled before attachment. WO 93/17126 to Chetverin uses sectioned, binary oligonucleotide arrays to sort and survey nucleic acids. These arrays have a constant nucleotide sequence attached to an adjacent variable nucleotide sequence, both bound to a solid support by a covalent linking moiety. The constant nucleotide sequence has a priming region to permit amplification by PCR of hybridized stands. Sorting is then carried out by hybridization to the variable region. Sequencing, isolating, sorting, and manipulating fragmented nucleic acids on these binary arrays are also disclosed. In one embodiment with enhanced sensitivity, the immobilized oligonucleotide has a shorter complementary region hybridized to it, leaving part of the oligonucleotide uncovered. The array is then subjected to hybridization conditions so that a complementary nucleic acid anneals to the immobilized oligonucleotide. DNA ligase is then used to join the shorter complementary region and the complementary nucleic acid on the array. There is little disclosure of how to prepare the arrays of oligonucleotides. WO 92/10588 to Fodor et. al., discloses a process for sequencing, fingerprinting, and mapping nucleic acids by hybridization to an array of oligonucleotides. The array of oligonucleotides is prepared by a very large scale immobilized polymer synthesis which permits the synthesis of large, different oligonucleotides. In this procedure, the substrate surface is functionalized and provided with a linker group by which oligonucleotides are assembled on the substrate. The regions where oligonucleotides are attached have protective groups (on the substrate or individual nucleotide subunits) which are selectively activated. Generally, this involves imaging the array with light using a mask of varying configuration so that areas exposed are deprotected. Areas which have been deprotected undergo a chemical reaction with a protected nucleotide to extend the oligonucleotide sequence where imaged. A binary masking strategy can be used to build two or more arrays at a given time. Detection involves positional localization of the region where hybridization has taken place. See also U.S. Pat. Nos. 5,324,633 and 5,424,186 to Fodor et. al., U.S. Pat. Nos. 5,143,854 and 5,405,783 to Pirrung, et. al., WO 90/15070 to Pirrung, et. al., A. C. Pease, et. al., “Light-generated Oligonucleotide Arrays for Rapid DNA Sequence Analysis”, Proc. Natl. Acad. Sci USA 91: 5022-26 (1994). K. L. Beattie, et. al., “Advances in Genosensor Research,” Clin. Chem. 41(5): 700-09 (1995) discloses attachment of previously assembled oligonucleotide probes to a solid support. There are many drawbacks to the procedures for sequencing by hybridization to such arrays. Firstly, a very large number of oligonucleotides must be synthesized. Secondly, there is poor discrimination between correctly hybridized, properly matched duplexes and those which are mismatched. Finally, certain oligonucleotides will be difficult to hybridize to under standard conditions, with such oligonucleotides being capable of identification only through extensive hybridization studies. The present invention is directed toward overcoming these deficiencies in the art.

<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is directed to a method of designing a plurality of capture oligonucleotide probes for use on a support to which complementary oligonucleotide probes will hybridize with little mismatch, where the plural capture oligonucleotide probes have melting temperatures within a narrow range. The first step of the method involves providing a first set of a plurality of tetramers of four nucleotides linked together, where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, (3) no tetramers within a set are palindromic or dinucleotide repeats, and (4) no tetramer within a set has one or less or three or more G or C nucleotides. Groups of 2 to 4 of the tetramers from the first set are linked together to form a collection of multimer units. From the collection of multimer units, all multimer units formed from the same tetramer and all multimer units having a melting temperature in ° C. of less than 4 times the number of tetramers forming a multimer unit are removed to form a modified collection of multimer units. The modified collection of multimer units is arranged in a list in order of melting temperature. The order of the modified collection of multimer units is randomized in 2° C. increments of melting temperature. Alternating multimer units in the list are then divided into first and second subcollections, each arranged in order of melting temperature. After the order of the second subcollection is inverted, the first collection is linked in order to the inverted second collection to form a collection of double multimer units. From the collection of double multimer units those units (1) having a melting temperature in ° C. less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption are removed, to form a modified collection of double multimer units. Another aspect of the present invention relates to an oligonucleotide array which includes a support and a collection of double multimer unit oligonucleotides at different positions on the support so that complementary oligonucleotides to be immobilized on the solid support can be captured at the different positions. The complementary oligonucleotides will hybridize, within a narrow temperature range of greater than 24° C. with little mismatch, to members of the collection of double multimer unit oligonucleotides, the double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats, and the collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. less than 12.5 times the number of tetramers and more than 14 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) multimer units with the same 4 tetramers linked together with or without interruption. Yet another aspect of the present invention relates to a method for identifying one or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences. This method involves providing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences. A plurality of oligonucleotide probe sets are also provided with each set characterized by (a) a first oligonucleotide probe, having a target-specific portion and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target-specific portion and a detectable reporter label. The oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence present in the sample. A ligase is also provided with the sample, the plurality of oligonucleotide probe sets, and the ligase being blended to form a mixture. The mixture is subjected to one or more ligase detection reaction cycles comprising a denaturation treatment, where any hybridized oligonucleotides are separated from the target nucleotide sequences, and a hybridization treatment, where the oligonucleotide probe sets hybridize at adjacent positions in a base-specific manner to their respective target nucleotide sequences, if present in the sample, and ligate to one another to form a ligated product sequence containing (a) the addressable array-specific portion, (b) the target-specific portions connected together, and (c) the detectable reporter label. The oligonucleotide probe sets may hybridize to nucleotide sequences in the sample other than their respective target nucleotide sequences but do not ligate together due to a presence of one or more mismatches and individually separate during the denaturation treatment. A support is provided with different capture oligonucleotides immobilized at different positions, where the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions and are formed from a collection of double multimer unit oligonucleotides. The oligonucleotide with addressable array-specific portions will hybridize, within a narrow temperature range of more than 4 times the number of tetramers in the multimer unit with little mismatch, to members of the capture oligonuncleotides. The double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats. The collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. of less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, to form a modified collection of double multimer units. After subjecting the mixture to one or more ligase detection reaction cycles, the mixture is contacted with the support under conditions effective to hybridize the addressable array-specific portions to the capture oligonucleotides in a base-specific manner, thereby capturing the addressable array-specific portions on the support at the site with the complementary capture oligonucleotide. The reporter labels of ligated product sequences captured on the support at particular sites are detected, indicating the presence of one or more target nucleotide sequences in the sample. Another aspect of the present invention is directed to a kit for identifying one or more of a plurality of sequences differing by single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences. In addition, to a ligase, the kit includes a plurality oligonucleotide probe sets, each characterized by (a) a first oligonucleotide probe, having a target sequence-specific portion and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target sequence-specific portion and detectable reporter label, wherein the oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a respective target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence, present in the sample. Also found in the kit is a support with different capture oligonucleotides immobilized at different positions, where the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions and are formed from a collection of double multimer unit oligonucleotides. The oligonucleotide with addressable array-specific portions will hybridize, within a narrow temperature range of greater than 4 times the number of tetramers in the multimer unit with little mismatch, to members of the capture oligonuncleotides. The double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats. The collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. of less than 11 times the number of tetramers and more than 15 times then number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, where the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions. Another aspect of the present invention relates to a method to avoid synthesizing ligase detection reaction oligonucleotides that will inappropriately cross-hybridize to capture oligonucleotides on a solid support. This method includes comparing the ligase detection reaction oligonucleotides with the capture oligonucleotides and identifying any capture oligonucleotides likely to cross-hybridize to the ligase detection reaction oligonucleotides.

Lactic acid bacteria overproducing exopolysaccharides

The invention concerns lactic acid bacteria overproducing exopolysaccharides following mutation in the gene coding for α-phosphoglucomutase. Said mutants are useful, in particular for preparing fermented products or for producing exopolysaccharides.

1. A lactic acid bacteria mutant overproducing exopolysaccharides, in which the pgm gene of alpha-phosphoglucomutase is totally or partially inactivated. 2. The mutant as claimed in claim 1, in which the galU gene is overexpressed. 3. The mutant as claimed in either one of claims 1 and 2, characterized in that said lactic acid bacterium is Streptococcus thermophilus. 4. The mutant as claimed in claim 3, characterized in that it is obtained from a Streptococcus thermophilus strain capable of using galactose. 5. The use of a lactic acid bacterium mutant as claimed in any one of claims 1 to 4, for producing a fermented product. 6. The use of a lactic acid bacterium mutant as claimed in any one of claims 1 to 4, for producing an exopolysaccharide. 7. A nucleic acid encoding an α-phosphoglucomutase the amino acid sequence of which exhibits at least 70% identity or at least 85% similarity with the α-phosphoglucomutase represented by the sequence SEQ ID NO: 2.

Method for measuring cylinder specific parameters in a combustion engine

The invention concerns a method to measure parameters in the combustion chamber of a piston engine that comprises an ignition system. An alternating voltage is applied across the secondary winding of the ignition coil and the value of the current that arises in a measurement circuit that co-operates with the secondary winding is measured. The value of the current depends on the resistance (R1) of the measurement circuit, on the inductance (L1) and resistance of the secondary winding, and on the impedance of the combustion chamber, i.e. on its capacitance (C1) and its resistance. For example, top dead centre, the pressure in the cylinder, analysis of the ionic current and change of the burning time can be determined by means of the method.

1. A method to measure and determine cylinder-specific parameters such as pressure, piston position and impedance in a combustion chamber of a piston engine having an ignition system with an ignition coil and spark plugs, characterised in that measurement occurs with the aid of the ignition system through the application of an alternating voltage, that can be changed by a control program, across a secondary winding of the ignition coil, after which the value of the current that arises is detected as a function of time in a measurement circuit that co-operates with the secondary winding, whereby the value of current detected depends on a measurement resistance of the measurement circuit, on an inductance and resistance of the secondary winding and on a characteristic impedance of the combustion chamber, which comprises a capacitance and a resistance, and whereby in addition, the pressure in a cylinder and/or a position of a piston can be determined by means of the value of the current. 2. The method according to claim 1, characterised in that the measurement is carried out during revolutions of the piston engine in which combustion does not take place. 3. The method according to claim 2, characterised in that the measurement is carried out while the piston engine is mechanically motored. 4. The method according to claim 2 or 3, characterised in that the maximum value of the current that arises in the measurement circuit that co-operates with the secondary winding is measured, whereby the highest value of the capacitance of the combustion chamber and this maximum value of the secondary current, that is, the top dead centre, is determined. 5. The method according to claim 2 or 3, characterised in that the frequency of the alternating voltage is varied, whereby the value of the resistance of the combustion chamber is determined, which value of the resistance is equivalent to the insulation resistance in the high-tension section of the ignition system. 6. The method according to claim 1, characterised in that the measurement is carried out during revolutions of the engine during which combustion takes place. 7. The method according to claim 6, characterised in that the measurement is carried out during that part of the engine revolution during which combustion does not take place, whereby the frequency of the alternating voltage is varied and the value of the resistance of the combustion chamber is determined, which value of the resistance is equivalent to the insulation resistance of the high-tension section of the ignition system. 8. The method according to claim 7, characterised in that the burning time of the ignition spark can be changed by means of applying a changed voltage level across the measurement circuit. 9. The method according to claim 7, characterised in that an ionic current from the combustion process is analysed by means of the measurement circuit. 10. The method according to claim 7, characterised in that both analysis of an ionic current and increased burning time of the spark are generated by the measurement circuit. 11. The method according to any one of the preceding claims, characterised in that the alternating voltage that can be changed is applied through a primary winding of an existing ignition coil. 12. The method according to claim 11, characterised in that the ignition coil comprises more than one primary winding. 13. The method according to any one of claims 1-10, characterised in that the alternating voltage that can be changed is applied through a primary winding of a separate transformer, the secondary winding of which is placed in series with the secondary winding of the ignition coil. 14. The method according to any one of claims 1-10, characterised in that the alternating voltage that can be changed is applied via a primary winding of a separate transformer, the secondary winding of which is connected in series with the secondary windings of several ignition coils, which coils are connected in parallel. 15. The method according to any one of claims 1-14, characterised in that the ignition voltage of the ignition system is generated in an inductive ignition system. 16. The method according to any one of claims 1-14, characterised in that an ignition voltage of the ignition system is generated in an inductive ignition system.

<SOH> TECHNICAL FIELD <EOH>The present invention concerns a method for measuring and determining cylinder-specific parameters in a combustion chamber in a piston engine that comprises an ignition system.

<SOH> SUMMARY OF THE INVENTION <EOH>The method of measuring and determining cylinder-specific parameters in a combustion chamber of a piston engine that comprises an ignition system is characterised in that the measurement occurs with the aid of the ignition system in that an alternating voltage is applied across the secondary winding of the ignition coil, after which the value of the current that arises in a co-operating measurement circuit on the secondary side is detected as a function of time. The absolute value of the current depends on the measurement resistance of the measurement circuit, on the inductance and the resistance of the secondary winding and on the characteristic impedance of the combustion chamber, which comprises a capacitance and a resistance. The capacitance of the combustion chamber changes when the position of the piston changes and it has its highest value when the piston is situated at the top dead centre. The secondary current has its highest value when the capacitance reaches its maximum. The measurement according to the invention can be carried out during revolutions of the engine when no combustion takes place. The measurement can even be carried out by “motoring” the engine before it is started. The measurement can also be carried out during revolutions of the engine when combustion does take place. In this case, by applying a suitable voltage level across the measurement circuit, the ionic current from the combustion process can also be analysed and, in the same way, the burning time of the spark can be increased, or multisparks can be supplied through the application of a voltage across the measurement circuit that is suitable for this purpose. Other characteristics of the invention are specified in the following claims.

Method and device for the mechanical-thermal separation of different materials

The invention relates to a method and system for the mechanical-thermal separation of different materials such as textile flat structures, threads, and plastic films by heating and simultaneously compacting the material. According to the invention, the material is fed through a gap which is formed between a tool (2) and an opposing surface (3) that touches the tool while forming a point. An adjustable electrical current is conducted over the contact point (5) in such a manner that a temperature profile (14), which is adapted to the density of the material, in the area of the contact point (5) is set such that the temperature of the tool (2) and opposing surface (3), independent of their shape and cross-section up to the contact point, is, in the advance direction of the material (1), increased to the same maximum value, and the contact point is heated in a defined manner.

1. Method for the mechanical/thermal severing of different materials, such as textile fabrics, yarns and plastic films by heating and simultaneously compacting the material, the material being passed in the transporting direction through a gap, which is closed at one end and formed between a tool and a counter surface, contacting the tool at one point, a controllable electric current being passed through this contact point so that a temperature profile, adapted to the thickness of the material, develops in the area of the contact point, the temperatures of the tool and of the counter surface, independently of their shape and cross-section, increasing in the direction of the contact point, that is, in the transporting direction of the material, to the same maximum value. 2. The method of claim 1, characterized in that the gap is wedge-shaped and that the tool and/or the counter surface can be constructed to be stationary and/or rotating and/or oscillating. 3. The method of claim 1, characterized in that a directly contacting or contactless temperature sensor is used at the contact point for shortening the adjustment times. 4. The method of claim 1, characterized in that the tool and/or the counter surface are preheated to a basic temperature, which is related to maximum value of the temperature, which is to be attained at the contact point. 5. The method of claim 1, characterized in that the current flowing through the contact point is used as an error signal. 6. Arrangement for mechanically/thermally severing certain materials such textile fabrics, yarns or plastic sheets, by means of a tool (2) and a counter surface (3), the tool (2) being disposed with respect to the counter surface (3), so that a wedge-shaped gap (4) is formed, which is closed at one end by a contact point (5) and through which the material (1) is passed, the tool (2) and the counter surface (3) being connected with a controllable circuit (6), so that a controllable current flows through the contact point (5). 7. The arrangement of claim 6, characterized in that, for obtaining am error signal and/or for controlling the temperature of the contact site (5), a current detector (8) is disposed in the lead from or to the contact site (5) and a temperature sensor (7) is disposed at the contact site (5). 8. The arrangement of claim 6, characterized in that the geometry and material properties as well as the surface treatment of the tool (2) and of the counter surface (3) are matched to one another, in order to obtain a desired temperature profile in the area of the contact point (5).

Heterocyclic cyclopentyl tetrahydroisoquinoline and tetrahydropyridopyridine modulators of chemokine receptor activity

The present invention is directed to compounds of the formula I: Wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, X, n and the broken lines are as defined herein which are useful as modulators of chemokine receptor activity. In particular, these compounds are useful as modulators of the chemokine receptor CCR-2.

1. A compound of formula I: wherein: X is selected from the group consisting of: C, N, O, S and SO2; Y is N or C; R1 is selected from the group consisting of: hydrogen, —C1-6alkyl, —C0-6alkyl-O—C1-6alkyl, —C0-6alkyl-S—C1-6alkyl, —(C0-6alkyl)-(C3-7cycloalkyl)-(C0-6alkyl), hydroxy, heterocycle, —CN, —NR12R12, —NR12COR13, —NR12SO2R14, —COR11, —CONR12R12, and phenyl, where R11 is independently selected from the group consisting of: hydroxy, hydrogen, C1-6 alkyl, —O—C1-6alkyl, benzyl, phenyl and C3-6 cycloalkyl where the alkyl, phenyl, benzyl, and cycloalkyl groups can be unsubstituted or substituted with 1-3 substituents where the substituents are independently selected from the group consisting of: halo, hydroxy, C1-3alkyl, C1-3alkoxy, —CO2H, —CO2—C1-6 alkyl, and trifluoromethyl, and where R12 is selected from the group consisting of: hydrogen, C1-6 alkyl, benzyl, phenyl and C3-6 cycloalkyl where the alkyl, phenyl, benzyl and cycloalkyl groups can be unsubstituted or substituted with 1-3 substituents where the substituents are independently selected from the group consisting of: halo, hydroxy, C1-3alkyl, C1-3alkoxy, —CO2H, —CO2—C1-6 alkyl, and trifluoromethyl, and where R13 is selected from the group consisting of: hydrogen, C1-6 alkyl, —O—C1-6alkyl, benzyl, phenyl and C3-6 cycloalkyl where the alkyl, phenyl, benzyl, and cycloalkyl groups can be unsubstituted or substituted with 1-3 substituents where the substituents are independently selected from the group consisting of: halo, hydroxy, C1-3alkyl, C1-3alkoxy, —CO2H, —CO2—C1-6 alkyl, and trifluoromethyl, and where R14 is selected from the group consisting of: hydroxy, C1-6 alkyl, —O—C1-6alkyl, benzyl, phenyl and C3-6 cycloalkyl where the alkyl, phenyl, benzyl, and cycloalkyl groups can be unsubstituted or substituted with 1-3 substituents where said substituents are independently selected from the group consisting of: halo, hydroxy, C1-3alkyl, C1-3alkoxy, —CO2H, —CO2—C1-6 alkyl, and trifluoromethyl, and where said alkyl and said cycloalkyl are unsubstituted or substituted with 1-7 substituents where said substituents are independently selected from the group consisting of: (a) halo, (b) hydroxy, (c) —O—C1-13alkyl, (d) trifluoromethyl, (f) C1-3alkyl, (g) —O—C1-3alkyl, (h) —COR11, (i) —SO2R14, (j) —NHCOCH3, (k) —NHSO2CH3, (l) -heterocycle, (m) ═O and (n) —CN, and where said phenyl and heterocycle are unsubstituted or substituted with 1-3 substituents where said substituents are independently selected from the group consisting of: halo, hydroxy, C1-3alkyl, C1-3alkoxy and trifluoromethyl; R2 is selected from the group consisting of: (a) hydrogen, (b) hydroxy, (c) halo, (d) C1-3alkyl, where the alkyl is unsubstituted or substituted with 1-6 substituents independently selected from fluoro and hydroxy, (e) —NR12R12, (f) —COR11, (g) —CONR12R12, (h) —NR12COR13, (i) —OCONR12R12, (j) —NR12CONR12R12, (k) -heterocycle, (l) —CN, (m) —NR12—SO2—NR12R12, (n) —NR12—SO2—R14, (p) —SO2—NR12R12, and (p) ═O, where R2 is connected to the ring via a double bond; R3 is oxygen or is absent when Y is N; R3 is selected from when Y is C: (a) hydrogen, (b) hydroxy, (c) halo, (d) C1-3alkyl, where said alkyl is unsubstituted or substituted with 1-6 substituents independently selected from: fluoro, hydroxy, and —COR11, (e) —NR12R12, (f) —COR11, (g) —CONR12R12, (h) —NR12COR13, (i) —OCONR12R12, (j) —NR12CONR12R12, (k) -heterocycle, (l) —CN, (m) —NR12—SO2—NR12R12, (n) —NR12—SO2—R14, (o)—SO2—NR12R12 and (p) nitro; R4 is selected from the group consisting of: (a) hydrogen, (b) C1-6alkyl, (c) trifluoromethyl, (d) trifluoromethoxy, (e) chloro, (f) fluoro, (g) bromo, and (h) phenyl; R5 is selected from the group consisting of: (a) C1-6alkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro and optionally substituted with hydroxyl, (b) —O—C1-6alkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro, (c) —CO—C1-6alkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro, (d) —S—C1-6alkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro, (e)-pyridyl, which may be unsubstituted or substituted with one or more substituents selected from the group consisting of: halo, trifluoromethyl, C1-4alkyl, and COR11, (f) fluoro, (g) chloro, (h) bromo, (i) —C4-6cycloalkyl, (j) —O—C4-6cycloalkyl, (k) phenyl, which may be unsubstituted or substituted with one or more substituents selected from the group consisting of: halo, trifluoromethyl, C1-4alkyl, and COR11, (l) —O-phenyl, which may be unsubstituted or substituted with one or more substituents selected from the group consisting of: halo, trifluoromethyl, C1-4alkyl, and COR11, (m) —C3-6cycloalkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro, (n) —O—C3-6cycloalkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro, (o) -heterocycle, (p) —CN, and (q) —COR11; R6 is selected from: (a) hydrogen, (b) C1-6alkyl, (c) trifluoromethyl, (d) fluoro, (e) chloro, and (f) bromo; R7 is selected from: hydrogen, (C0-6alkyl)-phenyl, (C0-6alkyl)-heterocycle, (C0-6alkyl)-C3-7cycloalkyl, (C0-6alkyl)-COR11, (C0-6alkyl)-(alkene)-COR11, (C0-6alkyl)-SO3H, (C0-6alkyl)—W—C0-4alkyl, (C0-6alkyl)-CONR12-phenyl, (C0-6alkyl)-CONR15—V—COR11, and nothing (when X is O, S, or SO2), where V is C1-6alkyl or phenyl, where W is selected from the group consisting of: a single bond, —O—, —S—, —SO—, —SO2—, —CO—, —CO2—, —CONR12— and —NR12—, where the R15 can be hydrogen, C1-4alkyl, or where R15 is joined via a 1-5 carbon tether to one of the carbons of V to form a ring, where the C0-6alkyl is unsubstituted or substituted with 1-5 substituents, where said substituents are independently selected from: (a) halo, (b) hydroxy, (c) —C0-6alkyl (d) —O—C1-3alkyl, (e) trifluoromethyl, and (f) —C0-2alkyl-phenyl, where said phenyl, heterocycle, cycloalkyl, and C0-4alkyl is unsubstituted or substituted with 1-5 substituents where said substituents are independently selected from the group consisting of: (a) halo, (b) trifluoromethyl, (c) hydroxy, (d) C1-3alkyl, (e) —O—C1-3alkyl, (f) —C0-3—COR11, (g) —CN, (h) —NR12R12, (i) —CONR12R12, and (j) —C0-3-heterocycle, or where the phenyl and heterocycle may be fused to another heterocycle, which itself may be unsubstituted or substituted with 1-2 substituents independently selected from hydroxy, halo, —COR11, and —C1-3alkyl, and where alkene is unsubstituted or substituted with 1-3 substituents which are independently selected from the group consisting of: (a) halo, (b) trifluoromethyl, (c) C1-3alkyl, (d) phenyl, and (e) heterocycle; R8 is selected from the group consisting of: (a) hydrogen, (b) nothing when X is either O, S, SO2 or N or when a double bond joins the carbons to which R7 and R10 are attached, (c) hydroxy, (d) C1-6alkyl, (e) C1-6alkyl-hydroxy, (f) —O—C1-3alkyl, (g) —COR11, (h) —CONR12R12, and (i) —CN; or where R7 and R8 are joined together to form a ring which is selected from the group consisting of: (a) 1H-indene, (b) 2,3-dihydro-1H-indene, (c) 2,3-dihydro-benzofuran, (d) 1,3-dihydro-isobenzofuran, (e) 2,3-dihydro-benzothiofuran, (f) 1,3-dihydro-isobenzothiofuran, (g) 6H-cyclopenta[d]isoxazol-3-ol (h) cyclopentane, and (i) cyclohexane, where said ring formed may be unsubstituted or substituted with 1-5 substituents independently selected from the group consisting of: (a) halo, (b) trifluoromethyl, (c) hydroxy, (d) C1-3alkyl, (e) —O—C1-3alkyl, (f) —C0-3—COR11, (g) —CN, (h) —NR12R12, (i) —CONR12R12, and (j) —C0-3-heterocycle, or where R7 and R9 or R8 and R10 may be joined together to form a ring which is phenyl or heterocycle, wherein said ring is unsubstituted or substituted with 1-7 substituents where said substituents are independently selected from the group consisting of: (a) halo, (b) trifluoromethyl, (c) hydroxy, (d) C1-3alkyl, (e) —O—C1-13alkyl, (f) —COR11 (g) —CN, (h) —NR12R12, and (i) —CONR12R12; R9 and R10 are independently selected from the group consisting of: (a) hydrogen, (b) hydroxy, (c) C1-6alkyl, (d) C1-6alkyl-COR11, (e) C1-6alkyl-hydroxy, (f) —O—C1-3alkyl, (g)═O, when R9 or R10 is connected to the ring via a double bond (h) halo; n is selected from 0, 1 and 2; the dashed line represents a single or a double bond; and pharmaceutically acceptable salts thereof and individual diastereomers thereof. 2. The compound of claim 1 having formula Ia: wherein R16 and R17 are independently selected from the group consisting of: (a) hydrogen, (b) halo, (c) trifluoromethyl, (d) hydroxy, (e) C1-3alkyl, (f) —O—C1-3alkyl, (g) —C0-3—CO2H, (h) —C0-3—CO2C1-3alkyl, (i) —CN, and (j) —C0-3-heterocycle, or where the R16 and R17 are joined together to form a heterocycle which is fused to the phenyl ring, and which itself may be unsubstituted or substituted with 1-2 substituents independently selected from hydroxy, halo, —COR11, and —C1-3alkyl; and pharmaceutically acceptable salts and individual diastereomers thereof. 3. The compound of claim 1 having the formula Ib: and pharmaceutically acceptable salts and individual diastereomers thereof. 4. The compound of claim 1 having formula: and pharmaceutically acceptable salts and individual diastereomers thereof. 5. The compound of claim 1 having formula Id: wherein said C1-4 carbon chain may be unsubstituted, or substituted with 1-4 substituents which are independently selected from the group consisting of: (a) halo, (b) hydroxy, (c) —C0-6alkyl (d) —O—C1-3alkyl, (e) trifluoromethyl, and (f) —C0-2alkyl-phenyl, where said C1-4 carbon chain optionally is included within a C3-7cycloalkyl ring, and pharmaceutically acceptable salts and individual diastereomers thereof. 6. The compound of claim 1 having formula Ie: wherein the dotted lines represent a single or double bond, o is 1 or 2, and A, B, and D are independently selected from C, N, O, or S, and pharmaceutically acceptable salts and individual diastereomers thereof. 7. The compound of claim 1 having formula If: wherein X is N or O, and pharmaceutically acceptable salts and individual diastereomers thereof. 8. The compound of claim 1 having formula Ig: wherein R16 and R17 optionally join to form a heterocycle which is fused to the phenyl ring, and wherein said ring is optionally substituted with 1-2 substituents independently selected from hydroxy, halo, —COR11, and —C1-3alkyl; and pharmaceutically acceptable salts and individual diastereomers thereof. 9. The compound of claim 1 having formula Ih: and pharmaceutically acceptable salts and individual diastereomers thereof. 10. The compound of claim 1 having formula Ii: wherein Het is a heterocycle, and pharmaceutically acceptable salts and individual diastereomers thereof. 11. The compound of claim 1 having formula Ij: wherein said C1-4 carbon chain is optionally substituted with 1-4 substituents independently selected from the group consisting of: (a) halo, (b) hydroxy, (c) —C0-6alkyl (d) —O—C1-3alkyl, (e) trifluoromethyl, and (f) —C0-2alkyl-phenyl, and pharmaceutically acceptable salts and individual stereoisomers thereof. 12. The compound of claim 1 having formula Ik: and pharmaceutically acceptable salts and individual diastereomers thereof. 13. The compound of claim 1 wherein R1 is selected from the group consisting of: —C1-6alkyl, —C0-6alkyl-O—C1-6alkyl, and —(C0-6alkyl)-(C3-7cycloalkyl)-(C0-6alkyl), where the alkyl and the cycloalkyl are unsubstituted or substituted with 1-7 substituents where the substituents are independently selected from: (a) halo, (b) hydroxy, (c) —O—C1-3alkyl, (d) trifluoromethyl, (f) C1-3alkyl, (g) —O—C1-3alkyl, (h) —COR11, (i) —CN, (j) —NR12R12, and (k) —CONR12R12, and pharmaceutically acceptable salts and individual diastereomers thereof. 14. The compound of claim 1 wherein R1 is selected from the group consisting of: (1) —C1-6alkyl, which is unsubstituted or substituted with 1-6 substituents where the substituents are independently selected from the group consisting of: (a) halo, (b) hydroxy, (c) —O—C1-3alkyl, (d) trifluoromethyl, and (e) —COR11, (2) —C0-6alkyl-O—C1-6alkyl-, which is unsubstituted or substituted with 1-6 substituents where the substituents are independently selected from the group consisting of: (a) halo, (b) trifluoromethyl, and (c) —COR11, (3) and —(C3-5cycloalkyl)-(C0-6alkyl), which is unsubstituted or substituted with 1-7 substituents where the substituents are independently selected from the group consisting of: (a) halo, (b) hydroxy, (c) —O—C1-3alkyl, (d) trifluoromethyl, and (e) —COR11. 15. The compound of claim 14 wherein R1 is selected from the group consisting of: (a) C1-6alkyl, (b) C1-6alkyl substituted with hydroxy and (c) C1-6alkyl substituted with 1-6 fluoro. 16. The compound of claim 15 wherein R1 is selected from the group consisting of: (a) —CH(CH3)2 (b) —CH(OH)CH3, and (c) —CH2CF3. 17. A compound selected from: 18. A pharmaceutical composition which comprises an inert carrier and a compound of claim 1. 19. A method for modulation of chemokine receptor activity in a mammal which comprises the administration of an effective amount of the compound of claim 1. 20. A method for treating, ameliorating, controlling or reducing the risk of an inflammatory and immunoregulatory disorder or disease which comprises the administration to a patient of an effective amount of the compound of claim 1. 21. A method for treating, ameliorating, controlling or reducing the risk of rheumatoid arthritis which comprises the administration to a patient of an effective amount of the compound of claim 1. 22. A method for treating, ameliorating, controlling or reducing the risk of stroke which comprises the administration to a patient of an effective amount of the compound of claim 1. 23. A method for treating, ameliorating, controlling or reducing the risk of cancer which comprises the administration to a patient of an effective amount of the compound of claim 1.

<SOH> BACKGROUND OF THE INVENTION <EOH>The chemokines are a family of small (70-120 amino acids), proinflammatory cytokines, with potent chemotactic activities. Chemokines are chemotactic cytokines that are released by a wide variety of cells to attract various cells, such as monocytes, macrophages, T cells, eosinophils, basophils and neutrophils to sites of inflammation (reviewed in Schall, Cytokine, 3, 165-183 (1991) and Murphy, Rev. Immun., 12, 593-633 (1994)). These molecules were originally defined by four conserved cysteines and divided into two subfamilies based on the arrangement of the first cysteine pair. In the CXC-chemokine family, which includes IL-8, GROα, NAP-2 and IP-10, these two cysteines are separated by a single amino acid, while in the CC-chemokine family, which includes RANTES, MCP-1, MCP-2, MCP-3, MIP-1α, MIP-1β and eotaxin, these two residues are adjacent. The α-chemokines, such as interleukin-8 (IL-8), neutrophil-activating protein-2 (NAP-2) and melanoma growth stimulatory activity protein (MGSA) are chemotactic primarily for neutrophils, whereas β-chemokines, such as RANTES, MIP-1α, MIP-1β, monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3 and eotaxin are chemotactic for macrophages, monocytes, T-cells, eosinophils and basophils (Deng, et al., Nature, 381, 661-666 (1996)). The chemokines are secreted by a wide variety of cell types and bind to specific G-protein coupled receptors (GPCRs) (reviewed in Horuk, Trends Pharm. Sci., 15, 159-165 (1994)) present on leukocytes and other cells. These chemokine receptors form a sub-family of GPCRs, which, at present, consists of fifteen characterized members and a number of orphans. Unlike receptors for promiscuous chemoattractants such as C5a, fMLP, PAF, and LTB4, chemokine receptors are more selectively expressed on subsets of leukocytes. Thus, generation of specific chemokines provides a mechanism for recruitment of particular leukocyte subsets. On binding their cognate ligands, chemokine receptors transduce an intracellular signal though the associated trimeric G protein, resulting in a rapid increase in intracellular calcium concentration. There are at least seven human chemokine receptors that bind or respond to β-chemokines with the following characteristic pattern: CCR-1 (or “CKR-1” or “CC-CKR-1”) [MIP-1α, MIP-1β, MCP-3, RANTES] (Ben-Barruch, et al., J. Biol. Chem., 270, 22123-22128 (1995); Beote, et al, Cell, 72, 415-425 (1993)); CCR-2A and CCR-2B (or “CKR-2A”/“CKR-2A” or “CC-CKR-2A”/“CC-CKR-2A”) [MCP-1, MCP-2, MCP-3, MCP-4]; CCR-3 (or “CKR-3” or “CC-CKR-3”) [Eotaxin, Eotaxin 2, RANTES, MCP-2, MCP-3] (Rollins, et al., Blood, 90, 908-928 (1997)); CCR-4 (or “CKR-4” or “CC-CKR-4”) [MIP-1α, RANTES, MCP-1] (Rollins, et al., Blood, 90, 908-928 (1997)); CCR-5 (or “CKR-5” or “CC-CKR-5”) [MIP-1α, RANTES, MIP-1β] (Sanson, et al., Biochemistry, 35, 3362-3367 (1996)); and the Duffy blood-group antigen [RANTES, MCP-1] (Chaudhun, et al., J. Biol. Chem., 269, 7835-7838 (1994)). The β-chemokines include eotaxin, MIP (“macrophage inflammatory protein”), MCP (“monocyte chemoattractant protein”) and RANTES (“regulation-upon-activation, normal T expressed and secreted”) among other chemokines. Chemokine receptors, such as CCR-1, CCR-2, CCR-2A, CCR-2B, CCR-3, CCR-4, CCR-5, CXCR-3, CXCR-4, have been implicated as being important mediators of inflammatory and immunoregulatory disorders and diseases, including asthma, rhinitis and allergic diseases, as well as autoimmune pathologies such as rheumatoid arthritis and atherosclerosis. Humans who are homozygous for the 32-basepair deletion in the CCR-5 gene appear to have less susceptibility to rheumatoid arthritis (Gomez, et al., Arthritis & Rheumatism, 42, 989-992 (1999)). A review of the role of eosinophils in allergic inflammation is provided by Kita, H., et al., J. Exp. Med. 183, 2421-2426 (1996). A general review of the role of chemokines in allergic inflammation is provided by Lustger, A. D., New England J. Med., 338(7), 426-445 (1998). A subset of chemokines are potent chemoattractants for monocytes and macrophages. The best characterized of these is MCP-1 (monocyte chemoattractant protein-1), whose primary receptor is CCR2. MCP-1 is produced in a variety of cell types in response to inflammatory stimuli in various species, including rodents and humans, and stimulates chemotaxis in monocytes and a subset of lymphocytes. In particular, MCP-1 production correlates with monocyte and macrophage infiltration at inflammatory sites. Deletion of either MCP-1 or CCR2 by homologous recombination in mice results in marked attenuation of monocyte recruitment in response to thioglycollate injection and Listeria monocytogenes infection (Lu et al., J. Exp. Med., 187, 601-608 (1998); Kurihara et al. J. Exp. Med., 186, 1757-1762 (1997); Boring et al. J. Clin. Invest., 100, 2552-2561 (1997); Kuziel et al. Proc. Natl. Acad. Sci., 94, 12053-12058 (1997)). Furthermore, these animals show reduced monocyte infiltration into granulomatous lesions induced by the injection of schistosomal or mycobacterial antigens (Boring et al. J. Clin. Invest., 100, 2552-2561 (1997); Warmington et al. Am J. Path., 154, 1407-1416 (1999)). These data suggest that MCP-1-induced CCR2 activation plays a major role in monocyte recruitment to inflammatory sites, and that antagonism of this activity will produce a sufficient suppression of the immune response to produce therapeutic benefits in immunoinflammatory and autoimmune diseases. Accordingly, agents which modulate chemokine receptors such as the CCR-2 receptor would be useful in such disorders and diseases. In addition, the recruitment of monocytes to inflammatory lesions in the vascular wall is a major component of the pathogenesis of atherogenic plaque formation. MCP-1 is produced and secreted by endothelial cells and intimal smooth muscle cells after injury to the vascular wall in hypercholesterolemic conditions. Monocytes recruited to the site of injury infiltrate the vascular wall and differentiate to foam cells in response to the released MCP-1. Several groups have now demonstrated that aortic lesion size, macrophage content and necrosis are attenuated in MCP-1−/− or CCR24−/− mice backcrossed to APO-E −/−, LDL-R −/− or Apo B transgenic mice maintained on high fat diets (Boring et al. Nature, 394, 894-897 (1998); Gosling et al. J. Clin. Invest., 103, 773-778 (1999)). Thus, CCR2 antagonists may inhibit atherosclerotic lesion formation and pathological progression by impairing monocyte recruitment and differentiation in the arterial wall.

<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is further directed to compounds which are modulators of chemokine receptor activity and are useful in the prevention or treatment of certain inflammatory and immunoregulatory disorders and diseases, allergic diseases, atopic conditions including allergic rhinitis, dermatitis, conjunctivitis, and asthma, as well as autoimmune pathologies such as rheumatoid arthritis and atherosclerosis. The invention is also directed to pharmaceutical compositions comprising these compounds and the use of these compounds and compositions in the prevention or treatment of such diseases in which chemokine receptors are involved. detailed-description description="Detailed Description" end="lead"?

Production of high grade and high concentration of free fatty acids from residual residual oils, fats and greases

The present invention relates to a process for the production of unsaturated and saturated free fatty acids. The process comprises the steps of: (a) selecting a starting material from the group consisting of trap oils, trap greases, yellow greases and brown greases, (b) pre-treating the oils and/or greases selected in step (a) in order to separate the oils and/or greases, from residual solids and water and obtain a mixture consisting principally of saturated and unsaturated free fatty acids, (c) bleaching the mixture of free fatty acids obtained in step (b) in order to obtain a suitable coloration thereof, (d) fractionating the bleached free fatty acids obtained in step (c) in two fractions: saturated and unsaturated fatty acids, (e) purifying the unsaturated fatty acids obtained from step (d), and purifying the saturated fatty acids obtained from step (d).

1. A process for producing unsaturated and saturated free fatty acids, the process being characterized in that it comprises the steps of: a) selecting a starting material from the group consisting of trap oils, trap greases, yellow greases and brown greases, b) pre-treating the oils and/or greases selected in step a) in order to separate the oils and/or greases from water and obtain a mixture consisting principally of saturated and unsaturated free fatty acids, c) bleaching the mixture of free fatty acids obtained in step b) in order to obtain a suitable coloration thereof, d) fractionating the bleached free fatty acids obtained in step c) in two fractions: saturated and unsaturated, e) purifying the unsaturated fatty acids obtained from step d), and f) purifying the saturated fatty acids obtained from step d). 2. A process according to claim 1, characterized in that it further comprises a fat splitting step of the mixture of saturated and unsaturated free fatty acids obtained in step b), prior to the bleaching step c). 3. A process according to claim 2, characterized in that the fat splitting step is carried out by hydrolysis or saponification of the oil and/or greases obtained in step b), 4. A process according to claim 3, characterized in that the fat splitting is carried out by hydrolysis at a temperature varying between 150° C. to 260° C. and at a pressure varying between 76 psi and 500 psi. 5. A process according to claim 3, characterized in that the fat splitting is carried out by saponification at a temperature varying between 100° C. and 150° C. and at a pressure varying between 20 psi and 50 psi. 6. A process according to any one of claims 1 to 5, characterized in that in step b), the pretreatment comprises hot filtration and decantation of oils or/and greases selected in step a). 7. A process according to any one of claims 2 to 6, characterized in that in step c), the bleaching is carried out by adsorption with an adsorbent selected from the group consisting of silica gel, crystalline silica, bentonite, Fuller's earth and a mixture thereof. 8. A process according to claim 7, characterized in that the bleaching of the free fatty acids is carried out in a batch or continuous mode by percolation in different columns. 9. A process according to claim 8, characterized in that the bleaching is carried out at a temperature ranging from 100° C. to 150° C. for a period ranging from 15 minutes to 1 hour. 10. A process according to claim 8 or 9, characterized in that the bleaching is carried in a batch mode under vacuum. 11. A process according to claim 8 or 9, characterized in that the bleaching is carried out in continuous mode under nitrogen atmosphere. 12. A process according to any one of claims 2 to 6, characterized in that the bleaching step c) is carried by a treatment with a sufficient amount of hydrogen peroxide at a temperature varying from 60° C. to 90° C. for a period varying from 20 minutes to 3 hours. 13. A process according to claim 12, characterized in that the concentration of the hydrogen peroxide ranges from 10 to 30% by weight 14. A process according to claim 12 or 13, characterized in that the bleaching step is carried out at a temperature of 80° C. for a period of 1 hour. 15. A process according to any one of claims 2 to 6, characterized in that the bleaching step c) is carried out by molecular distillation by a vacuum thin-film distillation step at a temperature varying from 150° C. to 200° C. and at a pressure varying from 0.1 to 5 mm Hg. 16. A process according to claim 15, characterized in that the bleaching step is carried out at a temperature varying from 165° C. to 185° C. and at a pressure varying from 0.2 to 0.5 mm Hg. 17. A process according to claim 1, characterized in that the bleaching step is carried out by molecular distillation by a vacuum thin-film distillation step.

<SOH> BACKGROUND OF THE INVENTION <EOH>Most commercial unsaturated acids (i.e. oleic acid) are derived from animal tallow (by-product of the meat industry), tall oil (by-product of paper mills) or natural vegetable oils. Fat splitting processes are well known in the art. The most common methods are: 1) Twichell process; 2) Batch autoclave process; 3) Continuous process; and 4) Enzymatic process. In Twichell process, the fat is hydrolyzed at a temperature of 100° C. to 105° C. and at atmospheric pressure for 12 to 48 hours. Alkyl-aryl acid or cycloaliphatic sulfonic acid with sulfuric acid (0.75-1.25% w/w) are used as catalysts. Yields of 85%-95% are obtained. The main inconvenients of this process are the catalyst handling, long reaction time, tendency to form dark-colored acid and high labor cost. In the batch autoclave operations, the fat is hydrolyzed in the presence or absence of a catalyst. Live steam is injected continuously at the bottom while venting a small amount to maintain the desired agitation and operating pressure. After settling and formation of an aqueous and a fatty acids phase, the fatty acids phase is treated with mineral acid to separate the soap formed. The fatty acids phase is further washed with water to remove traces of the mineral acid. Under catalytic conditions (i.e. zinc, calcium or magnesium oxides) the fatty acids phase is reacted for a period of 5 to 10 hours at 150° C. -175° C. A high yield of about 85%-95% is obtained. Without catalyst the fatty acids phase is reacted for a period of 2 to 4 hours at a high temperature (240° C.) to give similar yields. The principal inconvenient of this process is the catalyst handling, and high labor cost. In continuous operations also known as the Colgate—Emery process, a single-stage countercurrent high pressure splitting is carried out for fat hydrolysis. The fat is introduced by means of a sparge ring from the bottom of the splitting tower while water is introduced by the top. The crude fat passes as a coherent phase from the bottom to the top, while heavier splitting water travels downward as a dispersed phase through the mixture of fat and fatty acids. The high temperatures involved (250° C. -260° C.) associated to high pressures (725 psi) assures degrees of splitting up to 98% in only 2 to 3 hours. The principal inconvenient of this process is the high cost associated with the equipment and the restriction to relative clean starting materials. In enzymatic operations, the lipase from Candida rugosa, Aspergillus niger ; and Rhizopus arrhizus had been studied at temperatures of 26° C. to 46° C. for periods of 48 to 72 hours. Even though 98% of splitting is claimed there is no commercial process available until now. The principal inconvenient of this process is that because enzymes work very well over a specific substrate under specific conditions, when the starting material is composed of more than one product, the reaction is less selective. Long reaction times and great volumes required to satisfy the optimal concentration are also current problems involved in this kind of procedure. Fractionation of free fatty acids is commonly performed by distillation of tall oil. Tall oil is recovered in most paper mills by acidulation of the soap skimming from black liquor. Crude tall oil (CTO) consists of a mixture of fatty acids (40%-45%), resin acid (40%-45%) and various neutral components (i.e. hydrocarbons, wax alcohols, sterols, esters and residues). About 40% to 50% of the fatty acids contained in tall oil are oleic acid, while another 35% to 45% are linoleic acid. Higher quality of tall oil fatty acids, TOFA, (less than 2% of resins acid) can be obtained by distillation through two columns: a rosin column and a fatty acids column. Oleic acid is probably the most important unsaturated fatty acids (UFA) because many applications have been developed for its use in different fields (i.e. cosmetics, chemicals, lubricants, textiles, etc.). Separation of oleic acid form tall oil distillates requires additional refining steps. Best known-process for fractionation of fatty acids by crystallization from solvent is the “Emersol” process, developed by Emery Industries Inc. in 1934. Different American patents used different solvents (methanol: U.S. Pat. No. 2,421,157; acetone: U.S. Pat. No. 2,450,235 and methyl formate: U.S. Pat. No. 3,755,389) to separate saturated fatty acids from unsaturated fatty acids. The process was optimized by addition of crystallizing promoters (neutral fats, tallow, and glycerol tri-stearate). One more refined promoter is described in Australian patent AU-28434/92. It is the reaction product of: 1) a polyhydric alcohol (i.e. glycerol, pentaerythritol, trimethylol pentane, etc.), 2) a dicarboxylic acid (i.e. adipic, oxalic, succinic, azelaic, glutaric and tartaric) and 3) a fatty acids. All these process require explosion proof installations and low temperature refrigeration systems. Other methods for producing oleic acid involve separation over molecular sieves (U.S. Pat. No. 4,529,551 and U.S. Pat. No. 4,529,551); lithium soap separation (U.S. Pat. No. 4,097,507), urea complexation (U.S. Pat. No. 2,838,480 and U.S. Pat. No. 4,601,856) and complexation with dienophiles (U.S. Pat. No. 5,194,640). All these process have the inconvenient of a high cost operation associated to the use of chemicals required. Dry fractionation technology was originally developed for treatment of animal fat (i.e. beef tallow) in the 60's. Since this time, many improvements were performed in response to the ever-increasing demand of the industry for new products with very specific requirements. Two main sources are now the target of this technology: 1) vegetable oils such as palm oil, soybean oil, sunflower oil, rapeseed oil, groundnuts oil, cottonseed oil and palm kernel oil and 2) animal fats such as beef tallow, milk fat, lard and fish oil. These fats and oils are mainly composed of triglycerides, diglycerides and monoglycerides (i.e. a broad range of melting points) constituting a large number of intersoluble glycerides that are very difficult to separate by dry fractionation (i.e. solvent free crystallization). The separation of a liquid fraction (i.e. olein, used in food oil) and a solid fraction (i.e. stearin, used in shortening and margarine) can be achieved through dry fractionation. In the present invention, dry fractionation was used to separate purified free fatty acid obtained by splitting the residual oils and greases recuperated from industrial and commercial operations (i.e. trap greases, yellow greases and brown greases). The free fatty acids obtained from these starting materials are mainly constituted by unsaturated fatty acids, such as mainly oleic acid, linoleic acid, linolenic acid and saturated fatty acids such as palmitic acid and stearic acid. The range of melting points for these limited number of products, in comparison with all the possible combinations presented by glycerides, was shown to be wide enough to perform a highly selective separation.

<SOH> SUMMARY OF THE INVENTION <EOH>An object of the present invention is to provide a process for the production of free fatty acids that uses residual oils, fats and greases as the starting material. Another object of the present invention is to provide an inexpensive and simple way to produce high grade and high concentration of free fatty acids. A further object of the invention is to overcome most of the drawbacks mentioned hereinabove. More precisely, the objects of the present invention are provided by a process for producing unsaturated and saturated free fatty acids, the process comprising the steps of: a) selecting a starting material from the group consisting of trap oils and greases, yellow greases and brown greases, b) pre-treating the oils and/or greases selected in step a) in order to separate the oils and/or greases, from residual solids and water and obtain a mixture consisting principally of saturated and unsaturated free fatty acids, c) bleaching the mixture of free fatty acids obtained in step b) in order to obtain a suitable coloration thereof, d) fractionating the bleached mixture of free fatty acids obtained in step c) in two fractions: saturated and unsaturated fatty acids, e) purifying the unsaturated fatty acids obtained from step d), and f) purifying the saturated fatty acids obtained from step d). The process of the present invention has the, advantage of using inexpensive starting material thereby reducing the cost all the while allowing the recycling of the starting material that is normally eliminated through costly treatments thereof. The process of the present invention also has the advantage of giving the option of eliminating a hydrolysis step in the production of the free fatty acids, thereby simplifying the process for the production of fatty acids and reducing the production cost of same.

Micro-arrayed organization of transcription factor target genes

The following invention outlines methodologies for the construction and utilization of transcription factor direct target gene microarrays of both DNA and corresponding protein/peptide target origin. The technology entails the array/microarray annotation and organization of transcription factor direct loci and corresponding protein products identified through modified and improved versions of chromosomal immunoprecipitation (CHIP) and molecular cloning procedures. It allows for the formulation of physiologically directed arrays which result in a thorough, focused characterization of the genetic and biochemical regulation occurring within a give population of cells or a given tissue. Arrays and microarrays of direct targets for any given transcription factor created utilizing this technology are substantially more clinically relevant for purposes of medical diagnostics and patient prognostics than conventional microarrays due to the physiologically focused nature and the transcription factor targets. In addition, the characterization and array organization of transcription factor target protein products and the assessment of their interactions with other proteins and/or small molecules is of critical importance for the purposes of understanding cellular and ultimately the design of therapeutics for human anomalies.

1. A method according to the present invention which utilizes modified sequential chromosomal immunoprecipitation and cloning procedures for the discovery of transcription factor target genes from cells whereby said genes are organized into an array format. 2. A method according to claim 1 comprising the process of: a) cross-linking protein/DNA complexes in cells or tissues; b) immunoprecipitating said protein/DNA complexes with antibodies which recognize transcription factors; c) purifying DNA present within immunoprecipitated protein/DNA samples; d) organizing said purified DNA sequences into an array format. 3. A method according to claim 2 in which said purification of DNA present within immunoprecipitated protein/DNA samples includes amplification via inverse polymerase chain reaction (I-PCR) utilizing oligonucleotides corresponding to transcription factor binding sites to determine flanking nucleotide sequences present within discovered DNA fragments. 4. A method according to claim 2 in which said arrays consist of DNA templates bound to solic supports, for purposes of assessing the expression patterns or levels of transcription factor target genes. 5. A method according to claim 4 in which said transcription factor target genes consist of transcribed sequences, including coding sequences which correspond to amino acid composition. 6. A method according to claim 2 in which purified DNA fragments are utilized to cross hybridize against libraries of DNA sequences for the purposes of creating transcription factor target 7. An antibody according to claim 2 whereby said antibody allows for the purification of protein/protein and/or protein/DNA complexes from cells, for purposes of creating arrays and/or microarrays of transcription factor target genes. 8. A protein/DNA complex isolated from cells according to claim 2 whereby said protein DNA/complex results in the identification of transcription factor target genes, for purposes of constructing arrays and/or microarrays of said target genes. 9. DNA fragments isolated from protein/DNA complexes according to claim 8 whereby said DNA fragments encode transcription factor target genes, for purposes of constructing arrays and/or microarrays of said target genes. 10. Nucleotide sequences present in DNA fragments isolated according to methods described in claim 2 wherein said sequences represent transcription factor target genes and are utilized for purposes of constructing arrays of said sequences. 11. Arrays of transcription factor target gene sequences, for purposes of monitoring the expression patterns of transcription factor targets in given samples. 12. A method according to claim 2 which further comprises the process of translating isolated transcription factor target gene sequences for the purposes of constructing target protein arrays. 13. A method according to claim 12 in which said arrays are of a chemical/“nonliving” nature or biological/“living” nature. 14. Arrays of transcription factor target proteins as described in claim 13. 15. A transcription factor target protein/protein interaction complex identified by arrays described in claim 14 in which said protein/protein complex represents the interaction between transcription factor target protein sequences and other protein sequences, for the purposes of characterizing transcription factor target protein interacting molecules. 16. A transcription factor target protein/small molecule complex identified by arrays described it claim 14 in which said protein/small molecule complex represents the interaction between transcription factor target protein sequences and small molecules, for the purposes of characterizing transcription factor target protein interacting molecules. 17. A transcription factor target protein/metal complex identified by arrays described in claim 14 in which said protein/metal complex represents the interaction between transcription factor target protein sequences and charged or uncharged metals, for the purposes of characterizing transcription factor target protein interacting molecules. 18. A transcription factor target protein/nucleotide sequence complex identified by arrays described in claim 14 in which said protein/nucleotide sequence complex represents the interaction between transcription factor target protein sequences and nucleotide sequences of DNA or RNA origin, for the purposes of characterizing transcription factor target protein interacting molecules. 19. Proteins which are discovered as specifically interacting with transcription factor target protein sequences through the use of arrays according to claim 14. 20. Metals which are discovered as specifically interacting with transcription factor target proteii sequences through the use of arrays described by claim 14. 21. Nucleotide sequences which are discovered as specifically interacting with transcription factor target protein sequences through the use of arrays described in claim 14. 22. Simple sugars and oligosaccharides which are discovered as specifically interacting with transcription factor target protein sequences through the use of arrays described by claim 14. 23. Therapies designed as a result of the knowledge obtained from the discovery of interactions between transcription factor target protein sequences and proteins, amino acid or peptide sequences, nucleotide sequences, small molecules, metals, simple sugars and oligosaccharides through the use of arrays according to claim 14.

<SOH> 2.0 BACKGROUND OF THE INVENTION <EOH>Genetic activity, i.e. the activation or repression of gene transcription, has long been directly correlated with gene function. Transcriptional regulation is the first and perhaps most crucial mechanism by which cells regulate the functions of genes. By providing or denying mRNA templates for translation it is possible to tightly control the intricate cellular mechanisms of determination, division, survival etc. ( FIG. 1 and for review see Moroy et al., 2000 , Cellular and Molecular Life Sciences, 57(6): 957-75). Recently, a number of methods have been developed which allow for the rapid assessment of gene expression in a given sample and thus give insight as genetic profiles for various aspects of physiology and disease. These include, but are not limited to, two dimensional arrays and microarrays of either cDNAs or oligonucleotides representing corresponding mRNAs on solid supports. The arrayed aspect of the technology provides an organized, unbiased method for determining the quantitative and qualitative aspects of gene expression in a given sample population in a massive high-throughput format (a representative set of examples includes U.S. Pat. Nos. 6,136,592, 6,100,030, 6,040,138 herein incorporated by reference Debouck et al., 1999 , Nature Genetics Supplement, 21: 48-50). It is this macromolecular ability to monitor the expression patterns and levels of genes involved in physiology and disease which allows for many basic science as well as clinical applications such as the assessment of predisposition to particular disorders as well as the possibility of disease prevention or early treatment. It is clear that array technology enables researchers to efficiently ascertain expression patterns and levels of a multitude of loci within a particular sample. In addition, some effort has been directed towards the construction of microarrays which contain templates organized by physiology or functional entity such as cell cycle control or tissue specificity, yet these “focused arrays” are considerably lacking in gene content and limited in number. In addition, it still remains that the majority of genetic microarrays consist of random sequences, the identity and composition of which are often even unknown. Thus, for the most part, arrayed templates of either a nucleotide or peptide origin have yet to be developed such that the array of genes itself depicts something about physiology. It is therefore imperative that more focused, biologically relevant arrays and microarrays of genes be created. For example, arrays of genes known or hypothesized to be involved in a particular disease such as cancer, for example, would be of much more relevance clinically than arrayed organization of random gene sequences. By clustering arrays and microarrays in the context of specific physiologic and disease categories, these arrays can then be more readily subjected to the appropriate sample populations for analysis. This prevents the endless costly analysis of expression data which very well may not be relevant to the sample being studied. Therefore, an initial establishment of clusters and “families” of genes predicted to play particular roles in physiology or disease, and subsequent organization of these clusters in an array and microarray format will allow for a new level of discrete and focused genetic profiling for basic science and medical diagnostics. One method for clustering genes into particular physiologic and disease categories relies upon the exploitation of either the direct or indirect interaction between transcriptional regulators and terminal target genes (for review see Tjian and Maniatis, 1994 , Cell, 77: 5-8). Many transcription factors have been extensively demonstrated to play specific roles in very “focused” areas of physiology and disease, primarily through the regulation of target genes. It is possible to exploit this knowledge for the creation and production of functionally relevant arrays. By establishing arrays and microarrays of transcription factor target loci it is possible to narrow the purpose of said arrays for the characterization of expression profiles for specific aspects of physiology. In addition to transcription factor target genetic expression pattern profiling, it is clear that characterization of the biochemical interaction properties of transcription factor targets will enhance therapeutic discovery and development. The ability to characterize protein/protein, chemical/protein, small molecule/protein and enzymatic reaction interactions in a high-throughput and saturable format is of unparalleled value for the eventual design of therapeutic intervention strategies for the treatment of disease. In order to efficiently search for and analyze these types of interactions in a high-throughput yet sensitive format it is necessary to implement variations of array and microarray technology. A number of groups have begun to focus upon the organization of proteins and/or peptide and amino acid sequences in array and microarray formats similar to that for nucleotides sequences. Such an organization has been successfully implemented for the efficient identification of specific interactions between arrayed protein samples and other entities which include, but are not limited to, other proteins, enzymes, metals, sugars, oligosaccharides, chemical compounds, DNA and RNA molecules (a representative set of examples includes U.S. Pat. Nos. 5,591,646, 6,156,511, 5,834,318 herein incorporated by reference; MacBeath et al., 2000 , Science, 289: 1760-1763 and for review see Emili et al., 2000 , Nature Biotechnology, 18: 393-397). These arrays allow for the high-throughput sensitive and specific characterization of interactions between arrayed proteins and other molecules. Yet in order to fully take advantage of protein array technology it is necessary to focus its application to discrete realms of physiology and disease. By concentrating the identities of protein arrays on particular facets of biology a great deal of irrelevant biochemical screening and the costs associated with it can be eliminated. It is the modification and narrowing of protein array and microarray technology in the context of transcription factor target proteins which is described in the present invention. The creation and utilization of transcription factor target protein microarrays will allow for the high-throughput identification of small molecules, enzymes and other proteins which interact specifically with these targets. Such characterizations will reveal novel enzymatic modification of protein targets as well as protein/protein, protein/DNA, protein/RNA and protein/small molecule interactions. The resulting transcription factor target protein biochemical interaction data will enable researchers to more efficiently focus their efforts on specific aspects of human physiology and disease in order to optimize the design of novel therapeutic intervention strategies for particular human anomalies. In order to create arrays and microarrays of transcription factor target genes and the corresponding target protein sequences, it is necessary to discover and isolate the target genes in a complete and saturable fashion, as the more target genes present in a defined array the more thorough and complete the assessment of the genetic profile for the sample being analyzed. The chromosomal immunoprecipitation (ChIP) assay has been developed previously as a method for the analysis and characterization of transcription factor and/or regulatory protein interactions with known target sequences (Solomon et al., 1988 , Cell, 53: 937-947). Recent advances in this technology now make it possible to identify and establish both direct and indirect relationships between transcriptional regulatory proteins and known as well as unknown target loci. Optimized in a high-throughput format, it is now possible to manipulate regulatory protein/DNA interactions in order to “scan the genome” in search of genes involved in discrete, focused aspects of physiology and disease (PCT patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference). By combining both modified chromosomal immunoprecipitation/target gene cloning methodologies and array/microarray technology, the presently described invention allows for creation of gene expression and protein interaction analysis tools such as expression and function-restricted arrays of particular focused physiologic relevance. FIG. 2 illustrates the construction of transcription factor target nucleotide microarrays through an application of modified chromosomal immunoprecipitation procedures in combination with molecular cloning methodologies. FIG. 4 diagrams methodology for the construction and implementation of transcription factor target protein “nonliving” arrays. These arrays and microarrays eliminate random nucleotide and peptide sequence characterization and enhance the detailed analysis of physiologically directed expression and biochemical profiling. Originally, in order to take advantage of the inherent ability of transcription factors to dictate the regulation of specific downstream target genes for purposes of target gene identification, technologies such as CHIP were developed to extract transcription factor/known target gene interactions from living cells and tissues (Solomon et al., 1988 , Cell, 53: 937-947). This technology, however, was limited to the identification of only known transcription factor targets. More recently, the ChIP methodology has been significantly improved upon and implemented for the efficient high-throughput identification and characterization of actively transcribed transcription factor target genes of both known and unknown origin (PCT patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference). Yet in order to fully take advantage of the knowledge of transcription factor target sequences for the purposes of therapeutic development it is apparent that efficient methodologies must be developed and employed which will reveal the genetic activity and biochemical nature of these target loci. The herein described technology accomplishes these goals and further extends the value of transcription factor target gene identification at the biochemical level for purposes of therapeutic development.

<SOH> 3.0 SUMMARY OF THE INVENTION <EOH>The application of array and microarray technologies for purposes of assessing genetic as well as biochemical interaction profiles of sample populations has been considerably limited by the construction of both nucleotide and peptide or protein arrays which do not represent discrete aspects of physiology and disease. This lack of focus impairs the analysis of expression patterns by including a great deal of loci which are often not relevant to the particular sample being studied, thereby resulting in an unnecessary allocation of resources to nonrelevant gene expression and biochemical interaction analysis. In addition, significant costs are associated with large-scale microarrays as well as misdirected analysis of valuable limited sample sources. The presently described invention, based upon transcription factor function, circumvents these hindrances by allowing for the construction of physiologic and disease oriented arrays and microarrays. By focusing the creation and implementation of arrays and microarrays on transcription factor target genes and the corresponding proteins, the presently described invention achieves significantly concentrated and discrete genetic and biochemical profiling. Furthermore, the employment of protein arrays and microarrays for purposes of identifying protein/protein, protein/small molecule and enzymatic interactions is becoming increasing valuable for the high-throughput efficient analysis and characterization of potential avenues for therapeutic intervention. It is the discrete organization and annotation of protein amino acid sequences in a format which allows for rapid assessment of interacting partners which drives the rapid accumulation of biochemical information. Yet this organization is of limited value if the microarrayed proteins themselves are of limited utility with respect to the long-term goal of identifying therapeutics for the treatment of human anomalies. The presently described invention lends significant improvement to protein array and microarray technologies by narrowing the arrayed material in a physiological context. By arraying and microarraying proteins which are of known function and value due to their classification as specific transcription factor targets, it will be possible to considerably eliminate the analysis and characterization of irrelevant biochemical interactions. Such narrowing of focus streamlines the drug discovery process, resulting in the requirement of fewer resources and a significant increase in the inherent value of the interaction data obtained. Transcription factors such as p53, for example, are strategically chosen which have been previously demonstrated to play critical roles in certain aspects of disease and physiology ( FIG. 1 ). In vivo cross-linkage of protein/DNA complexes is performed in cell lines expressing the factor of interest and immunoprecipitation of protein/chromosomal complexes is subsequently employed through the utilization of antibodies specific for the transcription factor being studied (Solomon et al., 1988 , Cell, 53: 937-947). Cross-linkage is reversed and purified DNA fragments representing target genes for the factor of interest are subjected to gene sequence or corresponding protein microarray construction. The transcribed downstream target sequences represent the functionality of the transcription factor in question as they directly carry out its function with respect to physiology. The protein and peptide outputs for transcription factor target genes represent downstream biochemical effectors for transcription factor function and potentially encode therapeutic targets. The aforementioned nucleotide and peptide or protein sequences are arrayed on solid supports such as nylon membrane, plastic or glass chips or even in vivo (see “living” arrays described below) and utilized to monitor the expression and interaction profiles of samples in question. In order to successfully generate complex, saturable arrays and microarrays for particular aspects of physiology, the chromosomal immunoprecipitation assay has been modified and optimized for the high-throughput identification of both known and unknown transcription factor target loci ( FIG. 2 , FIG. 4 and PCT Patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference). Improvements include preimmunoprecipitation-immunoprecipitation (“preIP-IP”) utilizing antibodies specific for basal transcriptional machinery, which results in preisolation of only actively transcribed genes thus significantly reducing the acquisition of background random sequences. Subsequent immunoprecipitation is conducted on isolated complexes with antibodies which recognize particular transcription factors involved in discrete aspects of physiology and disease. In addition, sequences are isolated proximal to the transcriptional initiation site which often include 5′ untranslated and coding regions. The ability to direct immunoprecipitation of protein/DNA complexes to only actively transcribed regions of the genome is accomplished in the present invention through the use of antibodies specific for the large subunit of RNA polymerase II, the central component of the basal transcriptional machinery (Chang et al., 1998 , Clinical Immunology and Immunopathology, 89(1): 71-8). In addition, the use of antibodies conjugated to solid supports such as magnetic beads results in significant increases in yield and sensitivity, thus making high-throughput capability feasible (Dynal Corporation Technical Handbook, 1998, Biomagnetic Applications in Cellular Immunology). These solid supports aid in the retrieval of protein/DNA complexes during initial and subsequent immunoprecipitation procedures by providing a matrix for retrieval of complexed material. It is also stated that sequential immunoprecipitation may be performed in any order with the end result being decreased background random sequences and increased yield obtained. Additionally, a further elimination of background random sequences is obtained through the employment of inverse polymerase chain reaction (1-PCR) utilizing oligonucleotides specific for the transcription factor binding site (Ochlnan et al., 1988 , Genetics, 120(3): 621-623; PCT Patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference). Acquisition of PCR products obtained by this methodology strongly infers direct target identity as products will only be obtained upon successful PCR extension from the inherent transcription factor binding sites present within immunoprecipitated fragments. The combination of these novel technologies along with standard cloning procedures and the creation of arrays and microarrays of target sequences obtained allows for the discrete assessment of expression profiling for virtually any aspect of physiology or disease. The proposed strategy would be indispensable for correct diagnostic tracing of disease progression and ultimately therapeutic intervention. One embodiment of the present invention includes arrays and/or microarrays of transcription factor target genes, for the purposes of focusing genetic expression profiling experiments to particular specific entities of physiology and disease. An additional embodiment of the present invention includes the methodology utilized to create the physiology, cellular morphology and disease oriented nucleotide arrays and microarrays. Said methodology, described herein, includes chromosomal immunoprecipitation, double immunoprecipitation utilizing antibodies to the basal transcriptional machinery, solid phase separation technologies and inverse-PCR combined with standard molecular cloning methods. Another embodiment of the present invention is the antibodies utilized to immunoprecipitate crosslinked protein/DNA complexes from intact cells and/or tissues for purposes of creating arrays of transcription factor target genes and ultimately transcription factor target proteins. Yet another embodiment of the present invention includes antibodies conjugated to solid phase supports, such as but not limited to magnetic beads, for purposes of increasing the yield of DNA template obtained and/or reducing the background of nonspecific random sequences obtained, for the further purposes of creating arrays and microarrays of transcription factor target genes. Another embodiment of the present invention includes protein/DNA complexes isolated by modified ChIP methodologies described herein, for purposes of creating arrays and microarrays of transcription factor target genes. Still another embodiment of the present invention includes DNA fragments isolated by the methodology described herein, for the purposes of creating arrays and microarrays of transcription factor target genes. An additional embodiment of the present invention includes the nucleotide sequences corresponding to the transcription factor target genes identified by the methodology described herein, for purposes of creating physiologically and disease focused arrays and microarrays of transcription factor target genes. Still another embodiment of the present invention includes the genetic profile information gleaned from application of transcription factor target nucleotide arrays and microarrays. It is this information which provides valuable insight with respect to particular realms of physiology and disease. Yet another embodiment of the present invention is the application of transcription factor target gene sequence arrays and microarrays for purposes of medical diagnostics and patient prognostics. Another embodiment of the present invention entails the peptide and amino acid sequences of the transcription factor target proteins which are organized and annotated in a microarrayed fashion. It is these sequences which are analyzed for interactions with other proteins, nucleotide sequences and chemical small molecule entities. Yet another embodiment of the present invention includes the methodology for constructing transcription factor target protein arrays. It is the combination of modified chromosomal immunoprecipitation and molecular cloning and protein translation methods with biochemical array technology which results in the creation of valuable array reagents for therapeutic discovery. An additional embodiment of the present invention includes “living”/biological arrays of transcription factor target proteins, for example, in the context of yeast colonies grown in a multiwel format which express the transcription factor target protein of interest. Living arrays allow for the characterization of interactions with the protein of interest in a biological context in which other components or factors may be required and thus provided by the yeast machinery to catalyze interactions with arrayed transcription factor target proteins. Yet another embodiment of the present invention includes “nonliving”/chemical arrays and microarrays of transcription factor target proteins, for example, in the context of amino acid sequences bound either covalently or noncovalently to membranes or glass microchips. An additional embodiment of the present invention includes the proteins, metals, small molecules and nucleotide sequences which are tested for interaction specificities with transcription factor target protein arrays and microarrays. Yet another embodiment of the present invention includes the knowledge obtained from protein microarray studies revealing specific interaction data on transcription factor target proteins and their interactions with other proteins, enzymes or small molecule chemicals. It is the rapid accumulation of transcription factor target protein/protein and protein small molecule interaction data that will result in significant improvements in the efficiency and success of therapeutic development. Still another embodiment of the present invention includes therapies developed as a result of knowledge obtained from the construction and implementation of transcription factor target protein arrays and microarrays.

Eeg feedback controlled sound therapy for tinnitus

An automated method for treating tinnitus by habituation through use of neurological feedback, comprising the steps of connecting a subject through a set of attached headphones to an electronic sound player that is connected to a PC workstation presenting sound examples by software to the subject who can refine them by manipulating a series of controllers on the player, making an electronic recording of the sound in a digital music format, storing the recording in the computer, transferring a copy of the electronic sound file to the subject's electronic music player, generating an EEC signature of the subject's brain activity in response to the presented sound, sound using the customized sound to stimulate the auditory system while the brain activity is recorded, wherein the computer continuously monitors for the feedback signatures and drives the sound stimuli appropriately.

1. An automated method for treating tinnitus by habituation to customized sound through use of neurological feedback, comprising the steps of: connecting a subject through headphones to an electronic sound player that is connected to a PC workstation; creating a customized sound profile for the subject's particular tinnitus by presenting a plurality of audible sound examples from tinnitus sound library software to the subject; allowing the subject to choose and refine the presented sounds that most closely resemble the tinnitus sound; recording the refined sound in a digital music format to create a custom sound profile; storing the custom sound profile recording in a computer; generating an EEG signature of the subject's brain activity in response to the presented sound by downloading a copy of the electronic sound file to the subject's electronic music player, presenting custom sound most closely matching the tinnitus to stimulate the auditory system, recording the subject's response to sounds adjacent to but not specifically corresponding to his tinnitus signature, recording the subject's brain activity during absence of sound stimuli uploading the EEG responses to sound stimuli and to the absence of sound into the computer to create the EEG signature; and exposing the subject to the custom sound for as long as practical each day wherein the EEG is actively monitored by the computer, which generates sound in response and periodically tests the signatures for tinnitus and silence and determines if the tinnitus is decreasing and the silence signal is strengthening, and wherein if these changes are not present, the computer slightly alters the sound stimuli and again checks for feedback, and wherein the computer continuously monitors for the feedback signatures and drives the sound stimuli appropriately to habituate the subject to his tinnitus. 2. A method for customized habituation treatment of tinnitus without masking sound or using subthreshold sound, comprising: matching narrowband sound frequency to a patient's perceived tinnitus; presenting the matched sound frequency to the patient wherein the presented matched sound activates the same population of neurons affected by tinnitus, and wherein habituation occurs when the tinnitus and the habituating stimulus sound are as much alike as possible; and periodically updating frequency changes as required for maintaining maximum habituation. 3. An objective method for diagnosing tinnitus by detecting changes in the dynamic response characteristics of the auditory cortex induced by tinnitus, comprising: characterizing a subject's tinnitus perception by matching the pitch of his or her tinnitus to the frequericy of a pure sine tone generated by a function generator a programmable logarithmic amplifier that controlled in real time by a stimulus presentation and data collection program set the intensity of each stimulus; recording an auditory evoked potential to a variety of tone pitches, including the tinnitus pitch wherein the increased activation of the auditory cortex manifests itself as an increased slope of the AR; and calculating the slope of the AR response for tinnitus frequency tones wherein the observed increase in the slope of the AR in tinnitus is due to tinnitus-related activity present in the auditory cortex. 4. A method for treating tinnitus by habituation to its sound frequency, comprising the steps of: determining the “matching frequency” (pitch) of a subject's tinnitus; determining the hearing threshold for the tinnitus frequency; determining the “matching intensity” of the subject's tinnitus; determining the hearing threshold for the “off” frequencies; stimulating the auditory system in two series of tonal stimulation first at the tinnitus frequency, and second at the “off” frequency; collecting EEG data; and giving the subjects an electronic music player having the habituation stimulus downloaded wherein the subjects are asked to listen to it for as long as it is comfortable each day. 5. The method of claim 4, wherein the “matching frequency” is determined by 25 presenting the subjects with a continuous, audible tone varying in pitch, and asking them to indicate the frequency most closely matching the frequency of their tinnitus. 6. The method of claim 4, wherein to determine the threshold, subjects are presented with a continuous tone that gradually increases in volume, and are asked to indicate when they begin to hear the tone. 7. The method of claim 6, wherein the intensity at which the subjects begin to. hear the tone is considered the hearing threshold. 8. The method of claim 4, wherein to determine the “matching intensity” subjects are presented with a continuous tone that increases or decreases in volume and are asked to indicate the moment when they perceive the tone as being of the same loudness as their tinnitus. 9. An EEG marker of tinnitus suitable for diagnosing the presence of tinnitus, comprising: a replica of a subject's sound experience; and a measure of the subject's EEG response to increasing intensity of the sound wherein the replica of sound is constructed from a patients subjective determination of sound most closely related to the annoying sound experienced by the patient, and the patient's EEG response is measured to determine peak amplitude of the N100 component.

<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention generally concerns the diagnosis and treatment of tinnitus. The invention more specifically relates to the use of custom designed sound and feedback to determine the precise treatment sound matching the tinnitus, its neurophysiological effect, and monitoring the treatment effect by feedback from the brain. 2. Description of Related Art Commonly perceived as a “ringing in the ear,” tinnitus is a very frequent disorder of the auditory system, affecting about 17% of the general population and up to 33% in the elderly. About a quarter of these people are sufficiently bothered by their tinnitus that they seek professional help [Jastreboff et al, 1996]. Tinnitus is a phantom perception and thus not associated with any auditory stimulus. Until very recently, there were no objective measurements that could be related to tinnitus [Jastreboff et al, 1994], and diagnosis of tinnitus had to rely on various questionnaires, e.g. [Wilson et al, 1991]. The fact that tinnitus is perceived as a sound, however, indicates that it is associated with aberrant neural activity in the auditory pathways. Furthermore, the fact that tinnitus is associated with perception leads to the conclusion that central auditory structures such as the thalamus and auditory cortex must be involved. Neural correlates of tinnitus have indeed been found in central auditory structures [Norena et al, 1 999; Mühlnickel et al, 1 998; Wallhäusser-Franke et al, 1996]. Previously, tinnitus had been viewed as being caused in the auditory periphery [Eggermont, 1990; Tonndorf, 1981; Salvi and Ahroon, 1983], and even though neuronal activity related to tinnitus has been found in the central auditory system rather than in the periphery, it remains possible that the chain of events that leads to the development of tinnitus may be set off by events taking place in the periphery.

<SOH> SUMMARY OF THE INVENTION <EOH>To address the beforementioned problem and the above solution the inventors disclose their invention as follows. The invention contemplates an automated method for treating tinnitus by habituation to customized sound through use of neurological feedback. The methodology comprises the following steps. The tinnitus-suffering subject is connected through headphones to an electronic sound player that in turn is connected to a PC workstation. A customized sound profile is created for the subject's particular tinnitus by presenting a plurality of audible sound examples from a tinnitus sound library in special software. The subject is allowed to choose and refine the presented sounds that most closely resemble his or her tinnitus sound. The refined sound is recorded in a digital music format to create a custom sound profile for that particular subject, and the custom sound profile recording is stored in a computer. An EEG signature of the subject's brain activity in response to the presented sound is generated by downloading a copy of the electronic sound file to the subject's electronic music player, presenting custom sound most closely matching the tinnitus to stimulate the auditory system, and recording the subject's neurological response to sounds adjacent to but not specifically corresponding to his tinnitus signature. The subject's brain activity during absence of sound stimuli is also recorded. The EEG profile is uploaded into the computer to create the EEG signature. When undergoing treatment, the EEG response is actively monitored by the computer, which generates sound in response. The computer periodically tests the signatures for tinnitus and silence and determines if the tinnitus is decreasing and the silence signal is strengthening, and when if these desirable changes are not present, the computer slightly alters the sound stimuli and again checks for feedback. Thus, the computer continuously monitors for the feedback signatures and drives the sound stimuli appropriately to habituate the subject to his tinnitus. A method is also contemplated by this invention for customized habituation treatment of tinnitus without masking sound or using subthreshold sound. This method involves matching narrowband sound frequency to a patient's perceived tinnitus and presenting the matched sound frequency to the subject, wherein the presented matched sound activates the same population of neurons affected by tinnitus, and wherein habituation occurs when the tinnitus and the habituating stimulus sound are as much alike as possible. Periodically, frequency changes are updated as required for maintaining maximum habituation. An objective method for diagnosing tinnitus by detecting changes in the dynamic response characteristics of the auditory cortex induced by tinnitus is further contemplated. This method comprises characterizing a subject's tinnitus perception by matching the pitch of his or her tinnitus to the frequency of a pure sine tone generated by a function generator, having a programmable logarithmic amplifier that is controlled in real time by a stimulus presentation and data collection program software to set the intensity of each stimulus. An auditory evoked potential to a variety of tone pitches, including the tinnitus pitch is recorded, wherein the increased activation of the auditory cortex manifests itself as an increased slope of the AR. The slope of the AR response for tinnitus frequency tones is calculated, wherein an observed increase in the slope of the AR in tinnitus indicates tinnitus-related activity present in the auditory cortex. Another preferred method for treating tinnitus by habituation to its sound frequency comprises the steps of determining the “matching frequency” (pitch) of a subject's tinnitus, determining the hearing threshold for the tinnitus frequency; determining the “matching intensity” of the subject's tinnitus, and determining the hearing threshold for the “off” frequencies. This is followed by stimulating the auditory system in two series of tonal stimulation; first at the tinnitus frequency, and second at the “off” frequency. EEG data is collected and the subject is given an electronic music player having the habituation stimulus downloaded into it. The subject is asked to listen to the player for as long as it is comfortable each day. The “matching frequency” is determined by presenting the subjects with a continuous, audible tone varying in pitch, and asking them to indicate the frequency most closely matching the frequency of their tinnitus. To determine the threshold, subjects are presented with a continuous tone that gradually increases in volume, and are asked to indicate when they begin to hear the tone. The intensity at which the subjects begin to hear the tone is considered the hearing threshold. “Matching intensity” is determined when subjects are presented with a continuous tone that increases or decreases in volume and are asked to indicate the moment when they perceive the tone as being of the same loudness as their tinnitus. An EEG marker of tinnitus suitable for diagnosing the presence of tinnitus is also contemplated by this invention. This marker comprises a replica of a subject's tinnitus sound experience and a measure of the subject's EEG response to increasing intensity of the sound. The replica of sound is constructed from a subject's subjective determination of sound most closely related to the annoying sound experienced by him, and the patients EEG response is measured to determine peak amplitude of the N100 component. These and other aspects and attributes of the present invention will become increasingly clear upon reference to the following drawings and accompanying specification.

Method for applying a layer containing at least polymeric material

In a method for applying a layer containing at least polymer material to a substrate, there is applied to the substrate a film of polymer particles dispersed in a non-reactive liquid. By subjecting these at least polymer material-containing particles to an energy flow, which is locally at least substantially converted into heat, the particles will fuse with each other as a result of such a heat treatment. In particular, the heat treatment is done with the aid of a laser device operative in the UV region, or a laser device operative outside this region, though then in combination with a heat transferring agent in the film.

1. A method for applying a layer containing at least polymer material to a substrate, wherein the layer is obtained by applying, to the substrate, a film including at least polymer material-containing particles dispersed in a non-reactive liquid, and subjecting the particles to an energy flow which is locally substantially converted into heat, during which the particles fuse with each other. 2. A method according to claim 1, wherein the energy flow is provided with the aid of an Atomic Force Microscope thermal analyzer after evaporating the liquid in the film. 3. A method according to claim 1 wherein the energy flow is provided with the aid of a laser device. 4. A method according to claim 3 wherein the heat generated by the laser device is utilized first to remove the liquid from the film and then to fuse the polymer material-containing particles with each other. 5. A method according to claim 3 wherein, after the liquid has at least substantially disappeared from the film, the polymer material-containing particles fuse with each other with the aid of the laser device. 6. A method according to claim 1, wherein the film is formed by polymer material-containing particles dispersed in water. 7. A method according to claim 1, wherein the size of the dispersed particles is between about 5 and 30,000 nm. 8. A method according to claim 1, wherein polymer material-containing particles of different composition and/or size are present. 9. A method according to claim 1, wherein polymer material-containing particles have a multicomponent structure. 10. A method according to claim 1, wherein the film contains one or more further non-reactive components, from the group including: colors, pigments, and flowing property improving agents. 11. A method according to claim 1, wherein the film contains one or more reactive components, comprising crosslinking agents to be activated in a post-treatment. 12. A method according to claim 1, wherein a heat transferring agent is dispersed in the film. 13. A method according to claim 1, characterized in that the solids content in the wet film is less than 70 vol. %. 14. A method according to claim 3 wherein the laser device is operative in the UV region is used. 15. A method according to claim 14, wherein the wavelength at which the laser device is operative is between 190 and 360 nm. 16. A method according to claim 14 wherein the laser devise is an excimer laser device. 17. A method according to claim 14 wherein the laser devise is an Nd:YAG laser device. 18. A method according to claim 14 wherein the laser devise is a solid state diode pumped laser. 19. A method according to claim 3 wherein an Nd:YAG laser device is used which is operative in the infrared region, and carbon black has been added to the film as heat transferring agent. 20. A method according to claim 14, wherein the laser device transmits a pulsed activation signal. 21. A method according to claim 14, wherein the laser device transmits a continuous activation signal. 23. A method according to claim 14, wherein the laser device, viewed over the width of the beam, has a “tophat” distribution intensity profile. 24. A method for manufacturing an object by building it up layer-by layer, wherein each of the layers to be successively applied onto each other is obtained by the use of the method according to claim 1. 25. An apparatus for forming one or more layers containing at least polymer material to a substrate, wherein the apparatus is provided with a laser device and with a processing space having a substrate on which a layer, containing at least a polymer material, can be formed by the method defined in claim 1. 26. An apparatus according to claim 25, wherein the laser device is provided with means for setting the intensity profile over the beam cross section. 27. An apparatus according to claim 25 wherein a mask is arranged in the optical path. 28. A coating obtained by the use of the method according to claim 1. 30. A method according to claim 1, characterized in that the size of the dispersed particles is between about 100 and 1,000 nm. 31. A method according to claim 1, characterized in that the solids content in the wet film is less than 60 vol. %. 32. A method according to claim 12 wherein the heat transferring agent is carbon black. 33. An apparatus according to claim 25 wherein a mask is arranged directly above the substrate.

Optical recording medium and its manufacturing method

A multi-layered film made of sequentially stacked first dielectric layer, recording layer, record-assist layer, second dielectric layer and first reflection layer on a disc substrate having a concavo-convex structure at least on one surface thereof. An ultraviolet-setting resin layer is formed to cover the multi-layered film. The record-assist layer is controlled in thickness to satisfy the relation d/4<a<3d, or more preferably the relation d/2≦a≦2d, between the thickness d of the recording layer and the thickness a of the record-assist layer. The stacking order of the multi-layered film may be opposite. In this case, a light-transmitting sheet is provided to cover the multi-layered film via a bonding layer.

1. An optical recording medium comprising: a disc substrate having formed a concavo-convex structure formed at least on one surface thereof; and a multi-layered film made by stacking at least a recording layer capable of recording information signals by irradiation of a laser beam and a record-assist layer adjacent to the recording layer on the surface of the disc substrate having the concavo-convex structure, wherein d/4<a<3d is satisfied where d is the thickness of the recording layer and a is the thickness of the auxiliary recording layer. 2. An optical recording medium according to claim 1 wherein said recording layer can record the information signals by reversible changes at least between two different states. 3. An optical recording medium according to claim 1 wherein said recording layer is made of a phase-changeable material capable of recording the information signals by phase changes between a crystal phase and an amorphous phase. 4. An optical recording medium according to claim 1 wherein the laser beam used for recording and/or reproducing information signals is irradiated to said recording layer from one side of the multi-layered film where the disc substrate exists. 5. An optical recording medium according to claim 4 wherein said multi-layered film includes a first dielectric layer, said recording layer, said record-assist layer, a second dielectric layer and a first reflection layer that are stacked sequentially from near the surface of the disc substrate having formed the concavo-convex structure. 6. An optical recording medium according to claim 4 wherein a layer of a synthetic resin is formed to cover the multi-layered film on the disc substrate. 7. An optical recording medium according to claim 1 wherein the laser beam used for recording and/or reproducing information signals is irradiated to the recording layer from one side of the disc substrate where the multi-layered film exists. 8. An optical recording medium according to claim 7 wherein said multi-layered film is made of a second reflection layer, third dielectric layer, said record-assist layer, said recording layer and a fourth dielectric layer that are stacked sequentially from near one surface of the disc substrate having said concavo-convex structure. 9. An optical recording medium according to claim 7 wherein a light-transmitting layer permitting the laser beam to pass through is formed to cover the multi-layered film on the disc substrate. 10. An optical recording medium according to claim 1 wherein, in a portion of the disc substrate having the concavo-convex structure, width Dg of a level difference of a portion nearer to the injection side of the laser beam and width Dl of a level difference of a portion remoter from the injection side of the laser beam satisfy the ratio of 0.5≦Dl/Dg≦2.0. 11. An optical recording medium according to claim 1 wherein, in a portion of the disc substrate having the concavo-convex structure, width Dg of a level difference of a portion nearer to the injection side of the laser beam and width Dl of a level difference of a portion remoter from the injection side of the laser beam satisfy the ratio of 0.8≦Dl/Dg≦1.2. 12. An optical recording medium according to claim 1 wherein an objective lens used for recording and/or reproducing the information signals has a numerical aperture not smaller than 0.45 and not larger than 0.60. 13. An optical recording medium according to claim 1 wherein thickness of the recording layer is in the range not smaller than 5 nm and not larger than 50 nm. 14. An optical recording medium according to claim 1 wherein thickness of said recording layer is in the range not smaller than 10 nm and not larger than 40 nm. 15. An optical recording medium according to claim 1 wherein thickness of said record-assist layer is in the range not smaller than 3 nm and not larger than 100 nm. 16. An optical recording medium according to claim 1 wherein thickness of said record-assist layer is in the range not smaller than 5 nm and not larger than 60 nm. 17. An optical recording medium according to claim 1 wherein said recording layer is capable of recording the information signals in a write only mode. 18. An optical recording medium according to claim 1 wherein said recording layer is made of GeTe alloy or GeSbTe alloy. 19. An optical recording medium according to claim 1 wherein said record-assist layer is made of a material containing as its major component at least one compound selected from the group consisting of SnTe, SiN, SiC, SnSe, GeN, PbSe, PbTe, Bi2Te3 and Sb2Te3. 20. An optical recording medium according to claim 1 wherein record marks can be recorded on both portions of the recording layer on top surfaces of ridges and portions of the recording layer on bottoms of the furrows of the concavo-convex structure of the disc substrate by said laser beam. 21. An optical recording medium according to claim 1 wherein said recording layer is located nearer to the irradiation side of the laser beam. 22. A method of manufacturing an optical recording medium, which forms a multi-layered film made by stacking at least a recording layer capable of recording information signals by irradiation of a laser beam and a record-assist layer adjacent to the recording layer on the surface of the disc substrate having the concavo-convex structure, comprising: adjusting the thickness of the record-assist layer to satisfy d/4<a<3d where d is the thickness of the recording layer and a is the thickness of the record-assist layer. 23. A method of manufacturing an optical recording medium according to claim 22 wherein said recording layer is made of a material capable of recording the information signals by reversible changes at least between two different states 24. A method of manufacturing an optical recording medium according to claim 22 wherein said recording layer is made of a phase-changeable material capable of recording the information signals by phase changes between a crystal phase and an amorphous phase. 25. A method of manufacturing an optical recording medium according to claim 22 wherein the optical recording medium is configured to record the information signals in the recording layer by irradiation of the laser beam to the recording layer from one side of the multi-layered film where the disc substrate exists. 26. A method of manufacturing an optical recording medium according to claim 25 wherein said multi-layered film is made by stacking a first dielectric layer, said recording layer, said record-assist layer, a second dielectric layer and a first reflection layer that are stacked sequentially from near the surface of the disc substrate having formed the concavo-convex structure. 27. A method of manufacturing an optical recording medium according to claim 25 wherein a layer of a synthetic resin is formed to cover the multi-layered film after the multi-layered film is formed on the disc substrate. 28. A method of manufacturing an optical recording medium according to claim 22 wherein the optical recording medium is configured to record the information signals in the recording layer by irradiation of the laser beam to the recording layer from one side of the disc substrate where the multi-layered film exists. 29. A method of manufacturing an optical recording medium according to claim 28 wherein said multi-layered film is made by stacking a second reflection layer, third dielectric layer, said record-assist layer, said recording layer and a fourth dielectric layer sequentially from near one surface of the disc substrate having said concavo-convex structure. 30. A method of manufacturing an optical recording medium according to claim 28 wherein a light-transmitting layer permitting the laser beam to pass through is formed to cover the multi-layered film after the multi-layered film is formed on the disc substrate. 31. A method of manufacturing an optical recording medium according to claim 22 wherein, in a portion of the disc substrate having the concavo-convex structure, width Dg of a level difference of a portion nearer to the injection side of the laser beam and width Dl of a level difference of a portion remoter from the injection side of the laser beam satisfy the ratio of 0.5≦Dl/Dg≦2.0. 32. A method of manufacturing an optical recording medium according to claim 22 wherein, in a portion of the disc substrate having the concavo-convex structure, width Dg of a level difference of a portion nearer to the injection side of the laser beam and width Dl of a level difference of a portion remoter from the injection side of the laser beam satisfy the ratio of 0.8≦Dl/Dg≦1.2. 33. A method of manufacturing an optical recording medium according to claim 22 wherein an objective lens used for recording and/or reproducing the information signals has a numerical aperture not smaller than 0.45 and not larger than 0.60. 34. A method of manufacturing an optical recording medium according to claim 22 wherein the recording layer is formed to have a thickness in the range not smaller than 5 nm and not larger than 50 nm. 35. A method of manufacturing an optical recording medium according to claim 22 wherein the recording layer is formed to have a thickness in the range not smaller than 10 nm and not larger than 40 nm. 36. A method of manufacturing an optical recording medium according to claim 22 wherein said record-assist layer is formed to have a thickness in the range not smaller than 3 nm and not larger than 100 nm. 37. A method of manufacturing an optical recording medium according to claim 22 wherein said record-assist layer is formed to have a thickness in the range not smaller than 5 nm and not larger than 60 nm. 38. A method of manufacturing an optical recording medium according to claim 22 wherein said recording layer is formed to be capable of recording the information signals in a write only mode. 39. A method of manufacturing an optical recording medium according to claim 22 wherein said recording layer is made of GeTe alloy or GeSbTe alloy. 40. A method of manufacturing an optical recording medium according to claim 22 wherein said record-assist layer is made of a material containing as its major component at least one compound selected from the group consisting of SiC, SiN, SnTe, SnSe, GeN, PbSe, PbTe, Bi2Te3 and Sb2Te3. 41. A method of manufacturing an optical recording medium according to claim 22 wherein the optical recording medium is formed to be capable of recording record marks in top portions of ridges and bottom portions of furrows of the concavo-convex structure of the disc substrate.

<SOH> BACKGROUND ART <EOH>Recently, along with enhancement of recording density of optical recording mediums such as optical discs, development of the technique of effectively using the area on the recording surface of an optical disc has been pushed forward to enhance the recording density. The recording surface of any disc substrate has concavo-convex called lands and grooves respectively. In conventional optical discs, only lands or only grooves were used to record recording marks. However, with the need of enhancing the recording density of optical discs, a technique for recording record marks on both lands and grooves, i.e. a land/groove recording technique, has been brought into use as a format of optical discs. An example of optical discs combined with the land/groove recording as their format is DVD-RAM (Digital Versatile Disc-Random Access Memory). However, such optical discs employing the land/groove recording suffer a difference of the thermal property exerted to the recording layer because of the difference in physical configuration between lands and grooves when recording laser beams are irradiated to lands and grooves respectively. In addition, lands and grooves form a periodical structure close to the wavelength of the laser beams. Therefore, vector diffraction of light causes uneven distribution of irradiated laser beams between lands and grooves, and it has been difficult to record or reproduce record marks from both lands and grooves with a high C/N ratio (carrier-to-noise ratio). To match lands and grooves in property, it is contemplated, for example, to change widths of lands and grooves and thereby change the duty ratio. However, only change of the duty ratio cannot bring about ample freedom. Under the circumstances, an optical disc was proposed, in which the recording layer on grooves is formed thicker than the recording layer on lands by selecting the sputtering condition, for example (Japanese Patent Laid-Open Publication No. 2000-215511, referred to as Literature 1). However, in case of the optical disk disclosed in Literature 1, the condition for deposition of the recording layer is changed to form the recording layer on grooves thicker than the recording layer on lands. This condition digresses the optimum condition for deposition of the recording layer and invites deterioration of signal characteristics of the recording layer. Therefore, it has been demanded to develop a method capable of independently controlling the recording characteristics of lands and grooves. It is therefore an object of the invention to provide an optical recording medium having a multi-layered film of a recording layer and a record-assist layer stacked adjacently on one surface of a disc substrate having formed a concavo-convex structure, and its manufacturing method, which enable freer designing while ensuring good signal characteristics in the recording layer on the concavo-convex structure and ensuring high reliability.

<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 is a cross-sectional view that shows an optical disc according to the first embodiment of the invention FIG. 2 is a graph that shows dependency or a carrier upon the thickness of a record-assist layer in the optical disc according to the first embodiment of the invention; FIG. 3 is a graph that shows dependency of the signal amplitude upon the thickness of the record-assist layer in the optical disc according to the first embodiment of the invention; FIG. 4 is a graph that shows dependency of a cross talk signal upon the thickness of the record-assist layer in the optical disc according to the first embodiment of the invention; FIG. 5 is a cross-sectional view that shows an optical disc according to the second embodiment of the invention; FIG. 6 is a triangular graph that shows suitable composition of a phase-changed recording layer in an optical recording medium according to the invention; FIG. 7 is a triangular graph that shows suitable composition of a phase-changed recording layer in an optical recording medium according to the invention; and FIG. 8 is a triangular graph that shows suitable composition of a phase-changed recording layer in an optical recording medium according to the invention. detailed-description description="Detailed Description" end="lead"?