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<SOH> SUMMARY OF THE INVENTION <EOH>A novel gene product designated Antrum Mucosal Protein 18 (“AMP-18”) is a gastrokine. The protein was discovered in cells of the stomach antrum mucosa by analysis of cDNA clones obtained from humans, pigs, and mice. The protein is a member of a group of cellular growth factors or cytokines, more specifically gastrokines. The AMP-18 cDNA sequences predict a protein 185 amino acids in length for both pig and man. The nucleotide sequences also predict a 20-amino acid N-terminal signal sequence for secreted proteins. The cleavage of this N-terminal peptide from the precursor (preAMP-18) was confirmed for the pig protein; this cleavage yields a secreted protein 165 amino acids in length and ca. 18,000 Daltons (18 kD) in size. Human and mouse genomic DNA sequences were also obtained and sequenced. A human genomic DNA was isolated in 4 overlapping fragments of sizes 1.6 kb, 3 kb, 3.3 kb and 1.1 kb respectively. The mouse genomic DNA sequence was isolated in a single BAC clone. The gastrokine designated AMP-18 protein is expressed at high levels in cells of the gastric antrum. The protein is barely detectable in the rest of the stomach or duodenum, and was not found, or was found in low levels, in other body tissues tested. AMP-18 is synthesized in lumenal surface mucosal cells, and is secreted together with mucin granules. Compositions of AMP-18 isolated from mouse and pig antrum tissue stimulate growth of confluent stomach, intestinal, and kidney epithelial cells in culture; human, monkey, dog and rat cells are also shown to respond. This mitogenic (growth stimulating) effect is inhibited by specific antisera (antibodies) to AMP-18, supporting the conclusion that AMP-18, or its products, e.g. peptides derived from the protein by isolation of segments of the protein or synthesis, is a growth factor. Indeed, certain synthetic peptides whose amino acid sequences represent a central region of the AMP-18 protein also have growth-factor activity. The peptides also speed wound repair in tissue culture assays, indicating a stimulatory effect on cell migration, the process which mediates restitution of stomach mucosal injury. Thus, the protein and its active peptides are motogens. Unexpectedly, peptides derived from sub-domains of the parent molecule can inhibit the mitogenic effect of bioactive synthetic peptides and of the intact, natural protein present in stomach extracts. There are 3 activities of the gastrokine proteins and peptides of the present invention. The proteins are motogens because they stimulate cells to migrate. They are mitogens because they stimulate cell division. They function as cytoprotective agents because they maintain the integrity of the epithelium (as shown by the protection conferred on electrically resistant epithelial cell layers in tissue culture treated with damaging agents such as oxidants or non-steroidal anti-inflammatory drugs NSAIDs). The synthesis of AMP-18 is confined to lumenal mucosal lining epithelial cells of the gastric antrum of humans and other mammals. Inside cells the protein is co-localized with mucins in secretion granules, and appears to be secreted into the mucus overlying the apical plasma membrane. Recombinant human AMP-18 in E. coli exerts its mitogenic effect at a concentration an order of magnitude lower than growth-promoting peptides derived from the center of the mature protein. Peptide 77-97, the most potent mitogenic peptide, is amino acid sequence-specific AMP peptides appears to be cell-type specific as it does not stimulate growth of fibroblasts or HeLa cells. Mitogenesis by specific AMP peptides appears to be mediated by a cell surface receptor because certain peptides that are not active mitogens can competitively inhibit, in a concentration-dependent manner, the growth-stimulating effects of peptide 58-99 and antrum cell extracts. AMP-18 and its derived peptides exhibit diverse effects on stomach and intestinal epithelial cells which suggest they could play a critical role in repair after gastric mucosal injury. These include cytoprotection, mitogenesis, restitution, and maturation of barrier function after oxidant-and/or indomethacin-mediated injury. Possible mechanisms by which AMP-18 or its peptide derivatives mediate their pleiotropic effects include stimulation of protein tyrosine kinase activity, prolongation of heat shock protein expression after cell stress, and enhanced accumulation of the tight junction-associated protein ZO-1 and occludin. Certain of these physiological effects can occur at concentrations that are relatively low for rhAMP-18 (<50 nM) compared to the concentrations of other gastric peptide mediators such as trefoil peptides or the α-defensin, cryptdin 3 (>100 μM). Immunoreactive AMP-18 is apparently released by cells of the mouse antrum after indomethacin gavage, and by canine antrum cells in primary culture exposed to forskolin, suggest that the protein is subject to regulation. These results imply that AMP-18 could play a role in physiological and pathological processes such as wound healing in the gastric mucosal epithelium in vivo. The invention relates a group of isolated homologous cellular growth stimulating proteins designated gastrokines, that are produced by gastric epithelial cells and include the consensus amino acid sequence VKE(K/Q)KXXGKGPGG(P/A)PPK (SEQ ID NO: 10) wherein XX can be LQ or absent (which results in SEQ ID NOS: 25 and 26, respectively). An isolated protein of the group has an amino acid sequence as shown in FIG. 8 . The protein present in pig gastric epithelia in a processed form lacking the 20 amino acids which constitute a signal peptide sequence, has 165 amino acids and an estimated molecular weight of approximately 18 kD as measured by polyacrylamide gel electophoresis. Signal peptides are cleaved after passage through endoplasmic reticulum (ER). The protein is capable of being secreted. The amino acid sequence shown in FIG. 3 was deduced from a human cDNA sequence. An embodiment of the protein is shown with an amino acid sequence as in FIG. 6 , a sequence predicted from mouse RNA and DNA. A growth stimulating (bioactive) peptide may be derived from a protein of the gastrokine group. Bioactive peptides rather than proteins are preferred for use because they are smaller, consequently the cost of synthesizing them is lower than for an entire protein. In addition, a modified peptide may be produced by the following method: (a) eliminating major protease sites in an unmodified peptide amino acid sequence by amino acid substitution or deletion; and/or (b) introducing into the modified amino acid analogs of amino acids in the unmodified peptide. An isolated protein of the present invention include an amino acid sequence as in FIG. 8 , present in pig gastric epithelia in a processed form lacking the 20 amino acids which constitute a signal peptide sequence, having 165 amino acids and an estimated molecular weight of approximately 18 kD as measured by polyacrylamide gel electophoresis, said protein capable of being secreted. A protein of the present invention includes an amino acid sequence as in FIG. 3 , a sequence deduced from a human cDNA. A protein of the present invention includes an amino acid sequence as in FIG. 6 , a sequence predicted from mouse RNA and DNA. Embodiments of the present invention include a synthetic growth stimulating peptide, having a sequence of amino acids from positions 78 to 119 as shown in FIG. 3 ; having a sequence of amino acids from position 97 to position 117 as shown in FIG. 3 , or a sequence of amino acids from position 97 to position 121 as shown in FIG. 3 , or a sequence of amino acids from position 104 to position 117 as shown in FIG. 3 . An antibody to a protein of the present invention recognizies an epitope within a peptide of the protein that has an amino acid sequence from position 78 to position 119 as in FIG. 3 . An aspect of the invention also is an isolated genomic DNA molecule with the nucleotide sequence of a human as shown in FIG. 1 and an isolated cDNA molecule encoding a human protein with the amino acid sequence as shown in FIG. 3 . The invention includes a method to stimulate growth of epithelial cells in the gastrointestinal tract of mammals including the steps of: (a) contacting the epithelial cells with a composition comprising a protein of the present invention or a peptide derived from the protein; and (b) providing environmental conditions for stimulating growth of the epithelial cells. An embodiment of an isolated bioactive peptide has one of the following sequences: KKLQGKGPGGPPPK, (SEQ ID NO: 11) LDALVKEKKLQGKGPGGPPPK, (SEQ ID NO: 12) LDALVKEKKLQGKGPGGPPPKGLMY. (SEQ ID NO: 13) Embodiments of inhibitors are KKTCIVHKMKK (SEQ ID NO: 14) or KKEVMPSIQSLDALVKEKK. (SEQ ID NO: 15) (see also Table 1) Antibodies to the protein product AMP-18 encoded by the human cDNA expressed in bacteria were produced in rabbits; these antibodies reacted with 18 kD antrum antigens of all mammalian species tested (human, pig, goat, sheep, rat and mouse), providing a useful method to detect gastrokines. An antibody to a protein of the group recognizes an epitope within a peptide of the protein that includes an amino acid sequence from position 78 to position 119 as in FIG. 3 . The invention is also directed to an isolated genomic DNA molecule with the nucleotide sequence of a human as shown in FIG. 1 and an isolated cDNA molecule encoding a human protein, that the nucleotide sequence as shown in FIG. 2 . Another aspect of the invention is an isolated DNA molecule having the genomic sequence found in DNA derived from a mouse, as shown in FIG. 4 . Genomic DNA has value because it includes regulatory elements for gastric expression of genes, consequently, the regulatory elements can be isolated and used to express other gene sequences than gastrokines in gastric tissue. An aspect of the invention is a method to stimulate growth of epithelial cells in the gastrointestinal tract of mammals. The method includes the steps of: (a) contacting the epithelial cells with a composition comprising a gastrokine protein or a peptide derived from a protein of the group; and (b) providing environmental conditions for stimulating growth of the epithelial cells. A method to inhibit cellular growth stimulating activity of a protein of the group includes the steps of: (a) contacting the protein with an inhibitor; and (b) providing environmental conditions suitable for cellular growth stimulating activity of the protein. The inhibitor may be an antibody directed toward at least one epitope of the protein, e.g. an epitope with an amino acid sequence from position 78 to position 119 of the deduced amino acid sequence in FIG. 3 or an inhibitor peptide such as those in Table 1. A method of testing the effects of different levels of expression of a protein on mammalian gastrointestinal tract epithelia, includes the steps of: (a) obtaining a mouse with an inactive or absent gastrokine protein; (b) determining the effects of a lack of the protein in the mouse; (c) administering increasing levels of the protein to the mouse; and (d) correlating changes in the gastrointestinal tract epithelia with the levels of the protein in the epithelia. Kits are contemplated that will use antibodies to gastrokines to measure their levels by quantitative immunology. Levels may be correlated with disease states and treatment effects. A method to stimulate migration of epithelial cells after injury to the gastrointestinal tract of mammals, includes the steps of: (a) contacting the epithelial cells with a composition comprising a peptide derived from the protein; and (b) providing environmental conditions allowing migration of the epithelial cells. A method for cytoprotection of damaged epithelial cells in the gastrointestinal tract of mammals, includes the following steps: (a) contacting the damaged epithelial cells with a composition including a protein of the gastrokine group or a peptide derived from the protein; and (b) providing environmental conditions allowing repair of the epithelial cells. The damaged cells may form an ulcer.

Method and apparatus for enhanced particle collection efficiency
A method and apparatus for particle collection (30) that is characterized by co-aerosolizing fluids (62) into an air stream (34) containing the particles to be analyzed to significantly enhance their collection and identification efficiency is provided.
1. A method for particle collection comprising the steps of: guiding an air stream containing particles to be collected and analyzed toward an impaction surface; and introducing an aerosol containing aqueous droplets into the air stream upstream from the impaction surface to coagulate the particles with the aqueous droplets and to increase a size of the particles enhancing a collection efficiency of the coagulated particles on the impaction surface. 2. The method of claim 1, wherein the introduction of the aerosol includes directing a pressurized stream of carrier gas against a reservoir containing a liquid, thereby aspiring the liquid out of the reservoir while shearing liquid particulates apart to aerosolize the aspired liquid before co-aerosolizing the particles in the air stream. 3. The method of claim 1, wherein the introduction of the aerosol includes driving a selected liquid through a piezo-electric based element and vibrating the piezo-electric element at a desired frequency sufficient to shear apart liquid particulates to generate the aerosol as the liquid particulates traverse an outlet of the piezo-electric based element. 4. The method of claim 1, further comprising the step of wetting the impaction surface as the coagulated particles impinge against the impaction surface to form a pool of liquid on the impaction surface configured to minimize bouncing of the coagulated particle off the impaction surface to enhance the collection efficiency. 5. The method of claim 4, further comprising selecting additives and co-aerosolizing the selective additives to create an environment in the air stream or in the pool of liquid on the impaction surface capable of mechanically, chemically or biologically modifying the coagulated particles to enhance identification of the collected coagulated particles. 6. The method of claim 4, wherein the additives are so selected that the environment created in the air stream or in the pool of liquid allows accelerated growth of organisms to be cultured. 7. The method of claim 4, wherein the additives are so selected that the environment created in the pool of liquid is capable of reducing salts inhibiting further analysis of the collected coagulated particles. 8. The method of claim 4, further comprising removing an excess of liquid from the pool of liquid by either blowing the excess thereof off the impaction surface or by vacuuming the excess thereof from the pool of liquid. 9. The method of claim 1, further comprising identifying the collected coagulated particles in a mass spectrometer, PCR or a similar detection system. 10. The method of claim 1, wherein the air stream is guided through an impactor located upstream from the impaction surface, the impactor being selected from the group consisting of a virtual single stage impactor, a virtual multi-stage impactor, a real impactor and a combination thereof. 11. The method of claim 10, further comprising focusing the air stream entrained by the aerosol by guiding the air stream through a focusing mechanism located upstream from or downstream from the impactor. 12. The method of claim 11, wherein the air stream entrained by the aerosol is guided through a progressively narrowing guiding surface of the focusing mechanism directing the air stream towards a localized area on the impaction surface. 13. The method of claim 12, further comprising introducing the aerosol between the focusing mechanism and the impaction surface. 14. The method of claim 1, wherein the aerosolized aqueous droplets coagulate the particles having a size ranging from less than 1 micrometer to more than 1 micrometer. 15. An apparatus for collecting particles carried by an air stream, comprising: a guide configured to advance the air stream along a path; an impaction surface located downstream from the guide and configured to intercept the guided air stream and collect the particles; and an aerosolization generator configured to create an aerosol entraining the air stream upstream from the impaction surface to coagulate with the particles so as the coagulated particles increase an aerodynamic diameter thereof to enhance collection efficiency of the impaction surface upon impinging the coagulated particles thereagainst. 16. The apparatus of claim 15, wherein the coagulated particle wet the impaction surface to form a small pool of liquid, the apparatus further comprising a source of additives introducible upstream from impaction surface into the air stream to create an environment capable of mechanically, chemically or biologically modifying the collected coagulated particles to enhance identification thereof. 17. The apparatus of claim 16, wherein the aerosolization generator includes a standard nebulizer or a piezo-electric based nebulizer or an inkjet style aerosol generator in flow communication with the guide and located upstream from the impaction surface. 18. The apparatus of claim 17 further comprising a liquid reservoir in flow communication with the aerosolization generator, and a source of pressurized carrier gas aspiring the liquid out of the reservoir while shearing apart liquid particulates to create the aerosol. 19. The apparatus of claim 17 further comprising a positive displacement pump or a syringe pump in flow communication with the piezo-electric based nebulizer including a focused parabolic or conical piezoelectric ultrasonic transducer operative to vibrate at a desired frequency to create liquid droplets having desired aerodynamic characteristics to agglomerate the particles upon injection of the aerosol into the guide. 20. The apparatus of claim 19, wherein the guide includes an impactor selected from the group consisting of a virtual single-stage impactor, a virtual multi-stage impactor, and a real impactor, a focusing mechanism in flow communication with the impactor and located upstream or downstream therefrom along the path to guide the air stream towards a localized area on the impaction surface. 21. The apparatus of claim 20, wherein the focusing mechanism has an inner surface tapering towards the impaction surface and being either smooth or cascaded to direct the air stream entrained with the aerosol towards the localized area on the impaction surface. 22. The apparatus of claim 20, wherein the aerosolization generator is attached either to an input plenum of the impactor or between the impactor and the impaction surface or between the multiple stages of the multi-stage impactor. 23. The apparatus of claim 20, wherein the impaction surface is either a displaceable bare tape or a liquid. 24. The apparatus of claim 16, further comprising a device juxtaposed with the impaction surface and operative to remove excess of the liquid in the pool. 25. The apparatus of claim 15, further comprising a laser mass spectrometer or a PCR operative to identify the coagulated particles collected on the impaction surface. 26. A method for particle collection comprising the steps of: guiding an air stream containing particles to be collected and analyzed toward an impaction surface; and introducing an amount of liquid immediately upstream from the impaction surface to create a pool of small standing liquid droplets on the impaction surface to prevent the particles impinging upon the pool from bouncing off the impaction surface, thereby enhancing a collection efficiency of the particles.
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention generally relates to a sampling methodology. More particularly, the present invention is directed to a method and apparatus for improving capturing efficiency of airborne particulates and for providing immediate sample treatments which serve to enhance post-collection detection or identification systems. 2. Description of the Related Art Particulate materials, dispersed in air, pose major threats to the health and safety of the populace. For example, the EPA has strict regulations for respirable dust particles, which may bear allergens capable of triggering severe allergic reactions such as, for example, asthma, in susceptible individuals. Building engineers are plagued with particulate materials, which may include microorganisms, causing sick building syndrome. The FAA and the military have requirements to detect minute particles for identification of explosive materials that may pose a threat to personnel. Finally, the threat of biological warfare requires systems that can efficiently collect and analyze minute quantities of airborne toxins, bacteria, and viruses. All of these critical applications require efficient concentration and collection of airborne particulate materials over a broad particle size range. For optimum performance, the collection technology must rapidly provide the sample in a form that can be measured by the detector or sensor. For example, many biological sensors operate on a sample that is presented as a solution or suspension in water. For this type of detection device, the collection of sample must be gentle enough to minimize disruption of surface antigens present on the biological agents. On the other hand, sensors based on detecting chemical components of the particles generally require that the particles be disrupted in some manner to free the molecules of interest for detection. For these types of sensors, high-speed impaction onto solid phase surfaces, such as a metal plate coated with mineral oil, provides reasonable collection efficiency and the requisite disruption of the particulate into molecular components. Furthermore, for sensors that employ focused energy, such as a laser, in the detection scheme require that the sample be highly spatially localized on a surface. An example of this technology is laser ionization mass spectrometry. In this technique the particles in the air must be localized in a single sample spot of less than the diameter of the laser beam or ionization region to be effectively analyzed. In one embodiment, this area is required to be a circle with a diameter of the order of about one (1) millimeter. A variety of sampling devices configured to separate and deliver the material to be tested by a sensor are designed based on how the sample is originated: whether it comes from air, liquid, solid objects, surfaces, or from human tissue. There are several issues that make sampling for biological agents particularly challenging. Firstly, some forms of analysis require living organisms for detection and, therefore, the collection technology must not “harm” the sample. Secondly, the target microbe is generally only one component of a complex matrix of biological elements and chemical compounds that may affect the detection process, so the sample must often be purified to some extent. Finally, the sample must be highly concentrated for a rapid analysis. Among general types of sampling devices designed to accomplish one or more of these objectives that are of a particular interest within the context of the present invention are viable particle-size impactors and virtual impactors, cyclone samplers and bubblers. Each of these technologies is briefly described below. A viable particle-size impactors typically has multiple stages. Each stage contains a number of precision-drilled orifices that are appropriate for the size of the particles to be collected in that stage, and orifice sizes decrease with each succeeding impactor state. Particles in the air enter the instrument and are directed towards the collection surface by the jet orifices. Any particle not collected by that stage follows the stream of air around the edge of the collection surface to the next stage. The collection plate is typically a petri dish with agar or other suitable growth medium. A virtual impactor is similar to a viable particle-size impactor, but uses a collection probe instead of a flat plate as its impaction surface. Air flows through the collection probe and the collected particles are transported to other portions of the collector for additional concentration. By controlling the flow in the impactor, it is possible to adjust the cutoff size to the particles collected. By passing the collection probe airflow into successive virtual impactors, the particles can be concentrated many times the original air concentration before collection. A cyclone is an inertial device that is commonly used in industrial applications for removing particles from large airflows. A particle-laden air stream enters the cyclone body and forms an outer spiral moving downward towards the bottom of the cyclone. Larger particles are collected on the outer wall due to centrifugal force. Smaller particles follow the airstream that forms the inner spiral and leave the cyclone through the exit tube. A bubbler or impinger operates by drawing aerosols through a current inlet tube to create a jet. Usually the jet is submerged into the liquid contained in the sampler. As the air passes through the liquid, the aerosol particles are captured by the liquid surface at the base of the jet. In order to collect the smallest particles possible, the jet is typically made with a small critical orifice causing the flow to become sonic. Sampling small particles requires that due consideration be given to the variables that affect aerodynamic characteristics of the particulates, namely the size, number, randomness, and independence thereof. In particular, the size of the captured particulates can radically affect the collection efficiency of the cyclone and virtual impactor devices. The wetted-wall cyclone, and multi-stage virtual impactors feeding into true fluid impactors collect large volumes of air (on the order of about 1000 L/min), concentrate the respirable aerosol particles and impinge these into several milliliters of aqueous collection fluid. With proper tuning, these samplers provide high collection efficiency for particles greater than approximately one (1) micrometer in size. The collection efficiency drops precipitously for particles smaller than one micrometer. The central phenomenon exploited for operation of these devices is impaction of the particles, traveling at the proper velocity, onto a liquid collection surface. Upon the impact, the particles “splash down” into the fluid thereby minimizing particle bounce and reaerosolization into the exhaust airstream. While such samplers provide reasonably gentle capture, the relatively large volumes of collection fluid employed in this system lead to a dilution of the collected sample that makes sample cleanup and detection more difficult. Furthermore, the large volumes of collection fluid, when used for a long period of time, provides high logistics burden for fielded systems. An alternative strategy, directed to overcome the disadvantages of the above-discussed impactors, is based on collection of sample by impaction onto a polymer tape. Developed at the Applied Physics Laboratory of Johns Hopkins University (JHU/APL), a sampling system, equipped with a polymer tape, features a two-stage virtual impactor particulate concentrator. The latter typically collects air at a rate of 800 L/min and outputs air that is concentrated by a factor of 20×for particles greater than approximately 1 um, at a rate of 15 L/min. The output air enters a five (5) jet true impactor that directs the particles toward the polymer surface where a fraction of them collect on the surface. Generally, however, a large fraction of the particles strike the surface of the tape, bounce off, and are reaerosolized into the airstream. These particles either deposit on an undesirable surface in the sampler or are lost in the exhaust air. The collection efficiency and spot localization for this approach have been found to be low. To minimize particle bounce and therefore to enhance both the collection efficiency and spot localization, the sampling system designed by JHU/APL has been provided with a polymer tape coated with mineral oil, vacuum grease or an adhesive compound. However, this system requires an oily or sticky coating on the sample tape that tends to deposit on other mechanical motion components of the sampling system resulting in unacceptable performance. Furthermore, the collection efficiency for smaller particles such as viruses, certain types of bacteria and finely dispersed explosives still desires to be higher. Overall, the present technology may have the following deficiencies: (1) relatively low collection efficiency, particularly at small particle sizes; (2) harsh, dry environment in samplers that causes low viability for bacteria and viruses; and (3) poor focusing of deposited material. Thus, despite the intense activity in the recent past to advance and develop bioaerosol samplers, a need remains for a method and an associated aerosol collector that would enhance particle collection efficiency for current samplers and particularly, for collectors utilizing solid-surface sample tapes. It would also be desirable to provide a method and apparatus characterized by improved identification of the collected particles.
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention, which addresses the needs of the prior art and meets the objects of the present invention, provides an apparatus and a method for increasing the aerodynamic size of airborne particles. More specifically, the basic concept underlying the present invention provides for the introduction of an aqueous aerosol into an air stream to increase the agglomeration of the carried particles, particularly small ones. As a consequence, the aerodynamic diameter of the aerosol particles is increased leading to the enhanced collection efficiency of a tape impactor. Fluid droplets, contained in the aqueous aerosol and carried by the air stream towards the tape impactor, not only agglomerate with existing dry particulates making them larger and wet, thereby minimizing the bounce of the agglomerated particles, but also form small reservoirs of fluid on the impaction surface at the impaction location. Having such reservoirs is critical for the creation an environment beneficial for rapid growth or development of chemical or biological elements of interest immediately after the agglomerated particles have impinged upon the fluid contained in the reservoir. Thus, the formation of the fluid reservoir allows a variety of additives, introduced into the air stream and captured by the fluid, to chemically, biologically and/or mechanically provide beneficial conditions for the collected particles to be analyzed. Note, if not for the fluid reservoir, the introduced additives would rather bounce off the surface of the tape impactor and, thus, be ineffective for the intended purposes. Accordingly, results from reducing the inventive concept to practice indicate that the aqueous co-aerosolization protocol provides the following advantages: (1) enhanced collection efficiency of the airborne particulates; and (2) enhanced identification of the collected particles. In accordance with a further aspect of the present invention, an apparatus configured to implement the inventive process is provided comprising a focusing mechanism allowing the aerosolized particle to be collected in a localized area. To materialize this concept of the invention, the focusing mechanism includes a structure traversed by the aerosolized air stream and configured to have a cross-section gradually reducing towards a targeted area. As a consequence, a relatively narrow outlet port of the focusing mechanism is directly juxtaposed with the targeted area difficult to miss as the aerosolized particles leave the outlet port. This feature of the invention even further enhances the collection efficiency of the apparatus configured in accordance with the inventive concepts. It is, therefore, an object of the invention to provide a process and apparatus characterized by high collection efficiency for, among others, submicron particles. Still another object of the invention is to provide a process and apparatus for rapidly sampling and processing a sufficient volume of ambient air to ensure that a relatively large number of specific microorganisms of interest are collected to form a representative sample over a short time period. Still another object of the invention is to provide a process and apparatus for controllably generating an aerosol to increase the aerodynamic diameter of the particles of interest carried by an air stream. Yet a further object of the invention is to provide an apparatus carrying out the inventive concept in a simple and reliable manner.

Use of herpesviruses, herpesvirus proteins and nucleic acids encoding the proteins to inhibit ccr5-tropic hiv-1 infection and replication
It has been discovered that herpesviruses can trigger an increase in the production of HIV-suppressive chemokines, and that these chemokines block the CCR5 receptor, which is used as a co-receptor with CD4 in the CCR5-tropic forms of HIV-1 that predominate in early stage HIV-1 infection. Use of live, attenuated or killed herpesviruses, or of herpesvirus proteins which trigger an increase in production of HIV-suppressive chemokines, or of nucleic acids encoding those proteins, can likewise be used to prevent establishment of HIV-1 infection or to inhibit HIV-1 replication. The invention provides uses, methods and compositions related to these discoveries.
1. A use of an attenuated herpesvirus which increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, for the manufacture of a medicament to inhibit infection by or replication of CCR5-tropic HIV-1. 2. A use of claim 1, wherein said herpesvirus is selected from the group consisting of human herpesvirus (“HHV”)-6A, HHV-6B, and HHV-7. 3. A use of claim 2, wherein said HHV-6 is HHV-6A. 4. A use of claim 1, wherein the chemokine is RANTES. 5. A use of a isolated protein from a herpesvirus, which protein alone or in combination with other herpesvirus proteins, increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, for the manufacture of a medicament to inhibit infection by or replication of CCR5-tropic HIV-1. 6. A use of claim 5, wherein said herpesvirus is selected from the group consisting of human herpesvirus (“HHV”)-6A, HHV-6B, and HHV-7. 7. A use of claim 6, wherein said HHV-6 is HHV-6A. 8. A use of claim 7, wherein the chemokine is RANTES. 9. A use of an isolated nucleic acid encoding a protein from a herpesvirus, which protein increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, for the manufacture of a medicament to inhibit infection by or replication of CCR5-tropic HIV-1. 10. A use of claim 9, wherein said herpesvirus is selected from the group consisting of human herpesvirus (“HHV”)-6A, HHV-6B, and HHV-7. 11. A use of claim 10, wherein said HHV-6 is HHV-6A. 12. A use of claim 9, wherein the chemokine is RANTES. 13. A use of a lymphoid tissue-tropic herpesvirus which increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, for the manufacture of a medicament to inhibit infection by or replication of CCR5-tropic HIV-1. 14. A use of claim 13, wherein said herpesvirus is selected from the group consisting of human herpesvirus (“HHV”)-6A, HHV-6B, and HHV-7. 15. A use of claim 14, wherein said HHV-6 is HHV-6A. 16. A use of a killed lymphoid tissue-tropic herpesvirus, which killed herpesvirus increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, for the manufacture of a medicament to inhibit infection by or replication of CCRS-tropic HIV-1. 17. A use of claim 16, wherein said herpesvirus is selected from the group consisting of human herpesvirus (“HHV”)-6A, HHV-6B, and HHV-7. 18. A use of claim 17, wherein said HHV-6 is HHV-6A. 19. A method of inhibiting growth or replication of CCR5-tropic HIV-1, said method comprising administering an agent selected from the group consisting of: an attenuated herpesvirus which increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, a protein from a herpesvirus, which protein increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, a nucleic acid encoding a protein from a herpesvirus, which protein increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, and a killed or living herpesvirus which killed or living virus increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β. 20. A method of claim 19, in which the agent is an attenuated lymphoid tissue-tropic herpesvirus which increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β. 21. A method of claim 19, wherein said herpesvirus is selected from the group consisting of human herpesvirus (“HHV”)-6A, HHV-6B, and HHV-7. 22. A method of claim 21, wherein said HHV-6 is HHV-6A. 23. A method of claim 19, wherein the chemokine is RANTES. 24. A composition comprising an agent selected from the group consisting of: an attenuated herpesvirus which increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, a protein from a herpesvirus, which protein increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, a nucleic acid encoding a protein from a herpesvirus, which protein increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, and a lymphoid tissue-tropic herpesvirus which increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, in a pharmaceutically acceptable carrier. 25. A composition of claim 23, in which the agent is an attenuated lymphoid tissue-tropic herpesvirus which increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β. 26. A composition of claim 23, wherein said herpesvirus is selected from the group consisting of human herpesvirus (=HHV”)-6A, HHV-6B, and HHV-7. 27. A composition of claim 26, wherein said HHV-6 is HHV-6A. 28. A composition of claim 24, wherein said agent comprises one or more proteins from a herpesvirus, which agent increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β. 29. A composition of claim 24, wherein said agent is a nucleic acid encoding one or more proteins from a herpesvirus, which protein increases or proteins increase production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β. 30. A composition of claim 24, wherein said agent is a killed or living lymphoid tissue-tropic herpesvirus, which killed or living herpesvirus increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β.
<SOH> BACKGROUND OF THE INVENTION <EOH>HIV-1 infects target cells via a receptor complex formed by CD4 and a chemokine receptor, primarily CCR5 or CXCR4 (Berger, E. et al., Ann. Rev. Immunol. 17:657-700 (1999)). Commonly, HIV-1 transmission is mediated by CCR5-tropic variants, also designated slow/low, non-syncytia-inducer, or macrophage-tropic, which dominate the early stages of HIV-1 infection and frequently persist during the entire course of the disease (Fenyo, E. M. et al. J. Virol. 62:4414-9 (1988); Tersmette, M. et al. Lancet 1:983-5 (1989); Schuitemaker, H. et al. J. Virol. 66:1354-60 (1992); Fenyo, E. M. AIDS 7:1673-4 (1993); Koot, M. et al. Ann. Intern. Med. 118:681-688 (1993); Bjorndal, A. et al. J. Virol. 71:7478-87 (1997); Scarlatti, G. et al. Nature Med. 3:1259-65 (1997); Connor, R. I. et al., J. Exp. Med. 185:621-8 (1997)). In contrast, HIV-1 variants that use CXCR4 are typically detected at the later stages, and are associated with a rapid decline in CD4 T cells and progression to AIDS (Fenyo, E. M. et al., 1988, supra; Bjorndal, A. et al. J. Virol. 71:7478-87 (1997); Scarlatti, G. et al., 1997, supra; Connor, R. I. et al., 1997, supra; Fouchier, R. A. et al., Virology 219:87-95 (1996); Koot, M. et al. J. Infect. Dis. 179:254-8 (1999)). Disease progression is also associated with the emergence of concurrent infections that may affect the course of HIV disease by mechanisms that remain unknown. Human herpesvirus 6 (HHV-6) is a double-stranded DNA virus originally isolated from immunocompromised patients with different lymphoproliferative disorders and described as a B-lymphotropic virus (Salahuddin, S. Z. et al., Science 234:596-601 (1986)). Shortly thereafter, however, a primary tropism for CD4+ T lymphocytes was documented both in vitro (Lusso, P. et al., J. Exp. Med. 167:1659-1670 (1988)) and in vivo (Takahashi, K. et al., J. Virol. 63:3161-3163 (1989)). Two major viral subgroups, designated A and B, have been defined, which form two segregated clusters with unique genetic, biologic and immunologic characteristics (Lusso, P. Antiviral Research, 31:1-21 (1996)). While HHV-6 B is highly prevalent in the human population in all geographic areas (Lusso, P. Antiviral Research, 31:1-21 (1996)), the epidemiology of HHV-6 A is still largely undefined. Primary infection with HHV-6 B, which occurs almost universally during early childhood, has been linked to the etiology of exanthema subitum, a usually benign febrile disease (Yamanishi, K. et al., Lancet i, 1065 (1988)). Conversely, little is known about the time and pathologic consequences of primary HHV-6 A infection. In the adult, HHV-6 infection and/or reactivation have been associated with a wide variety of diseases, although most of these associations have not been substantiated by rigorous epidemiological and virological studies. Considerable interest was raised by recent data on a possible link between HHV-6 and multiple sclerosis (Challoner, P. B. et al., Proc. Natl. Acad. Sci. USA 92:7440-4 (1995)), but the evidence hitherto accumulated is controversial (Soldan, S. S. et al., Nat. Med. 3:1394-7 (1997); Martin C et al. Acta Neurol Scand; 95:280-283 (1997)). In immunocompromised people, HHV-6 acts as an opportunistic agent that may cause life-threatening infections of the respiratory tract and the central nervous system, as well as bone marrow and organ graft failure (Lusso, P. Antiviral Research , supra). Several lines of evidence have led to speculation that HHV-6 may act as an immunosuppressive agent in vivo. Both HHV-6 subgroups induce severe thymocyte depletion in heterochimeric SCID hu thy/liv mice, showing a predominant tropism and cytopathicity for immature thymic precursor cells (Gobbi, A. et al., Journal of Experimental Medicine, 189:1953-1960 (1999)). Disseminated HHV-6 A and B coinfection has been linked with a progressive and ultimately fatal immunodeficiency syndrome in an HIV-negative child (Knox, K. K. et al., Clin. Infect. Dis. 20:406-13 (1995)). Moreover, active HHV-6 infection, as revealed by the detection of plasma viremia, was associated with lymphocytopenia and with defective lymphocyte proliferation to recall antigens in patients who received allogeneic stem cell transplantation. Consistent with these observations, it has been suggested that HHV-6, particularly subgroup A, may act as a cofactor in the progression of human immunodeficiency virus (HIV) disease, which might contribute, either directly or indirectly, to the depletion of CD4 + T cells and the other dysfunctions of the immune system associated with full-blown AIDS (Lusso, P. et al., Immunology Today 16:67-71 (1995)). It has also been suggested that HHV-6 down-regulates the CXCR4 receptor, making CD4+ lymphocytes resistant to T lymphocyte-tropic HIV-1 strains. (Yasukawa et al., J. Immunol. 162:5417-5422 (1999). Progress in this area of investigation has been hampered by the lack of reliable and easily accessible study models.
<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>The invention provides uses, methods and compositions useful to prevent establishment of HIV-1 infection or, in an individual already infected with HIV-1, to delay or to inhibit progression of CCR5-tropic HIV infection to CXCR4-tropic infection, which is associated with rapid declines in T-cell counts and progression to AIDS. Specifically, the invention provides the use of attenuated herpesviruses which increase production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, for the manufacture of a medicament to inhibit infection by or replication of CCR5-tropic HIV-1. In preferred forms, the herpesvirus is selected from the group consisting of human herpesvirus (“HHV”)-6A, HHV-6B, and HHV-7. In particularly preferred embodiments, the HHV-6 is HHV-6A. The chemokine can be RANTES, MIP-1α, or MIP-1β, or a combination of these. In preferred forms, the chemokine is RANTES or MIP-1β. In another group of embodiments, the invention provides for the use of a isolated protein from a herpesvirus, which protein alone or in combination with other herpesvirus proteins, increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, for the manufacture of a medicament to inhibit growth or replication of CCR5-tropic HIV-1. In preferred forms, the herpesvirus is selected from the group consisting of human herpesvirus (“HHV”)-6A, HHV-6B, and HHV-7. In particularly preferred embodiments, the HHV-6 is HHV-6A. The chemokine can be ANTES, MIP-1α, or MIP-1β, or a combination of these. In preferred forms, the chemokine is RANTES or MIP-1β. The invention further provides the use of an isolated nucleic acid encoding a protein from a herpesvirus, which protein increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, for the manufacture of a medicament to inhibit growth or replication of CCR5-tropic HIV-1. In preferred forms, the herpesvirus is selected from the group consisting of human herpesvirus (“HHV”)-6A, HHV-6B, and HHV-7. In particularly preferred embodiments, the HHV-6 is HHV-6A. The chemokine can be RANTES, MIP-1α, or MIP-1β, or a combination of these. In preferred forms, the chemokine is RANTES or MIP-1β. In yet another set of embodiments, the invention provides the use of a lymphoid tissue-tropic herpesvirus which increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, for the manufacture of a medicament to inhibit growth or replication of CCR5-tropic HIV-1. In preferred forms, the herpesvirus is selected from the group consisting of human herpesvirus (“HHV”)-6A, HHV-6B, and HHV-7. In particularly preferred embodiments, the HHV-6 is HHV-6A. The chemokine can be RANTES, MIP-1α, or MIP-1β, or a combination of these. In preferred forms, the chemokine is RANTES or MIP-1β. Further, the invention provides the use of a killed herpesvirus, which killed herpesvirus increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, for the manufacture of a medicament to inhibit growth or replication of CCR5-tropic HIV-1. 17. In preferred forms, the herpesvirus is lymphoid tissue-tropic. In preferred forms, the herpesvirus is selected from the group consisting of human herpesvirus (“HHV”)-6A, HHV-6B, and HHV-7. In particularly preferred embodiments, the HHV-6 is HHV-6A. The chemokine can be RANTES, MIP-1α, or MIP-1β, or a combination of these. In preferred forms, the chemokine is RANTES or MIP-1β. In another important group of embodiments, the invention provides methods of inhibiting growth or replication of CCR5-tropic HIV-1. The methods comprise administering an agent selected from the group consisting of: an attenuated herpesvirus which increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, a protein from a lymphoid tissue-tropic virus, which protein increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, a nucleic acid encoding a protein from a lymphotropic virus, which protein increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, and a killed or living lymphotropic herpesvirus which killed or living virus increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β. 20. In preferred forms, the agent is an attenuated lymphoid tissue-tropic herpesvirus which increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β. In especially preferred forms, the herpesvirus is selected from the group consisting of human herpesvirus (“HHV”)-6A, HHV-6B, and HHV-7 and in the most preferred embodiment is HHV-6A. In yet another important group of embodiments, the invention provides compositions comprising an agent selected from the group consisting of: an attenuated herpesvirus which increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, a protein from a herpesvirus, which protein increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, a nucleic acid encoding a protein from a herpesvirus, which protein increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, and a lymphoid tissue-tropic herpesvirus which increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β, in a pharmaceutically acceptable carrier. In one set of preferred embodiments, the agent in the composition is an attenuated lymphoid tissue-tropic herpesvirus which increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β. In particularly preferred embodiments, the herpesvirus is selected from the group consisting of human herpesvirus (“HHV”)-6A, HHV-6B, and HHV-7, with HHV-6A being the most preferred. In some preferred embodiments, the agent comprises one or more proteins from a herpesvirus, which agent increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β. In some other preferred embodiments, the agent in the composition is a nucleic acid encoding one or more proteins from a herpesvirus, which protein increases or proteins increase production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β. Finally, in some preferred embodiments, the agent in the composition is a killed or living lymphoid tissue-tropic herpesvirus, which killed or living herpesvirus increases production in lymphoid tissue of one or more chemokines selected from the group consisting of RANTES, MIP-1α, or MIP-1β.

Control of growth and repair of gastro-intestinal tissues by gastrokines and inhibitors
A novel group of gastrokines called Gastric Antrum Mucosal Protein is characterized. A member of the group is designated AMP-18. AMP-18 genomic DNA, cDNA and the AMP-18 protein are sequenced for human, mouse and pig. The AMP-18 protein and active peptides derived from it are cellular growth factors. Surprisingly, peptides capable of inhibiting the effects of the complete protein, are also derived from the AMP-18 protein. Cytoprotection and control of mammalian gastro-intestinal tissue growth and repair (restitution) is facilitated by the use of the proteins, making the proteins candidates for therapies in inflammatory bowel disease and gastric ulcers.
1. An inhibitor of a protein, said protein selected from a group of isolated homologous cellular growth stimulating proteins designated gastrokines, said protein produced by gastric epithelial cells and comprising a consensus amino acid sequence selected from the group consisting of VKE(K/Q)KXXGKGPGG(P/A)PPK (SEQ ID NO: 10), VKE(K/Q)KLQGKGPGG(P/A)PPK (SEQ ID NO: 25) and VKE(K/Q)KGKGPGG(P/A)PPK (SEQ ID NO: 26), said inhibitor selected from the group of peptides having an amino acid sequence consisting of KKTCIVHKMKK (SEQ ID NO: 14) and KKEVMPSIQSLDALVKEKK (SEQ ID NO: 15). 2. A pharmaceutical composition used for the treatment of a gastrointestinal disorder, said composition comprising a growth stimulating peptide derived from a gastrokine protein. 3. The pharmaceutical composition of claim 2 wherein the gastrokine protein comprises a consensus amino acid sequence selected from the group consisting of VKE(K/Q)KXXGKGPGG(P/A)PPK (SEQ ID NO: 10), VKE(K/Q)KLQGKGPGG(P/A)PPK (SEQ ID NO: 25) and VKE(K/Q)KGKGPGG(P/A)PPK (SEQ ID NO: 26). 4. The pharmaceutical composition of claim 2 comprising a growth stimulating peptide selected from the group consisting of amino acid sequences KKLQGKGPGGPPPK (SEQ ID NO: 11), LDALVEKKLQGKGPGGPPPK (SEQ ID NO: 12), and LDALVEKKLQGKGPGGPPPKGLMY (SEQ ID NO: 13). 5. A pharmaceutical composition for the treatment of diseases associated with overgrowth of gastric epithelia, said compositions comprising an inhibitor according to claim 1. 6. The pharmaceutical composition of claim 5, wherein diseases are diseases of the colon and small intestine, said diseases selected from the group consisting of ulcerative colitis and Crohn's Disease. 7. An isolated cDNA molecule encoding a human protein, said protein having the amino acid sequence as shown in FIG. 2. 8. An isolated DNA molecule comprising the genomic sequence found in DNA derived from a mouse, said nucleotide sequence shown in FIG. 4. 9. A mouse with a targeted deletion in a nucleotide sequence in the mouse genome that when expressed without the deletion encodes a protein of the group of gastrokine proteins comprising a consensus amino acid sequence selected from the group consisting of VKE(K/Q)KXxGKGPGG(P/A)PPK (SEQ ID NO: 10), VKE(K/Q)KLQGKGPGG(P/A)PPK (SEQ ID NO: 25) and VKE(K/Q)KGKGPGG(P/A)PPK (SEQ ID NO: 26). 10. A method of making a peptide or protein derived from a gastrokine protein, the gastrokine protein comprising a consensus amino acid sequence selected from the group consisting of VKE(K/Q)KXXGKGPGG(P/A)PPK (SEQ ID NO: 10), VKE(K/Q)KLQGKGPGG(P/A)PPK (SEQ ID NO: 25) and VKE(K/Q)KGKGPGG(P/A)PPK (SEQ ID NO: 26), said method comprising: (a) obtaining an isolated cDNA molecule comprising a sequence encoding the protein or peptide; (b) placing the molecule in a recombinant DNA expression vector; (c) transecting a host cell with the recombinant DNA expression vector (d) providing environmental conditions allowing the transfected host cell to produce a protein encoded by the cDNA molecule; and (e) purifying the protein from the host cell. 11. A method to inhibit cellular growth stimulating activity of a protein derived from a gastrokine protein comprising a consensus amino acid sequence selected from the group consisting of VKE(K/Q)KXXGKGPGG(P/A)PPK (SEQ ID NO: 10), VKE(K/Q)KLQGKGPGG(P/A)PPK (SEQ ID NO: 25) and VKE(K/Q)KGKGPGG(P/A)PPK (SEQ ID NO: 26), said method comprising: (a) contacting the protein with an inhibitor; and (b) providing environmental conditions suitable for cellular growth stimulating activity of the protein. 12. The method of claim 11, wherein the inhibitor is an antibody directed toward at least one epitope of the protein, said epitope comprising an amino acid sequence from position 78 to position 119 of the deduced amino acid sequence in FIG. 3. 13. The method of claim 11, wherein the inhibitor is selected from the group of inhibitor peptides consisting of KKTCIVHKMKK (SEQ ID NO: 14) and KKEVMPSIQSLDALVKEKK (SEQ ID NO: 15). 14. A method of testing the effects of different levels of expression of a gastrokine protein, on mammalian gastrointestinal tract epithelia, said method comprising: (a) obtaining a mouse in accord with claim 9; (b) determining the effects of a lack of the protein in the mouse; (c) administering increasing levels of the protein to the mouse; and (d) correlating changes in the gastrointestinal tract epithelia with the levels of the protein in the epithelia. 15. A method to stimulate migration of epithelial cells after injury to the gastrointestinal tract of mammals, said method comprising: (a) contacting the epithelial cells with a composition comprising a protein derived from a gastrokine protein, or a peptide derived from the protein; and (b) providing environmental conditions allowing migration of the epithelial cells. 16. A method for cytoprotection of damaged epithelial cells in the gastrointestinal tract of mammals, said method comprising: (a) contacting the damaged epithelial cells with a composition comprising a gastrokine protein comprising a consensus amino acid sequence selected from the group consisting of VKE(K/Q)KXXGKGPGG(P/A)PPK (SEQ ID NO: 10), VKE(K/Q)KLQGKGPGG(P/A)PPK (SEQ ID NO: 25) and VKE(K/Q)KGKGPGG(P/A)PPK (SEQ ID NO: 26); and (b) providing environmental conditions allowing repair of the epithelial cells. 17. The method of claim 16, wherein the damaged cells are an ulcer.
<SOH> BACKGROUND <EOH>A novel group of Gastric Antrum Mucosal Proteins that are gastrokines, is characterized. A member of the gastrokine group is designated AMP-18. AMP-18 genomic DNA, and cDNA molecules are sequenced for human and mouse, and the protein sequences are predicted from the nucleotide sequences. The cDNA molecule for pig AMP-18 is sequenced and confirmed by partial sequencing of the natural protein. The AMP-18 protein and active peptides derived from its sequence are cellular growth factors. Surprisingly, peptides capable of inhibiting the effects of the complete protein, are also derived from the AMP-18 protein sequence. Control of mammalian gastro-intestinal tissues growth and repair is facilitated by the use of the protein or peptides, making the protein and the derived peptides candidates for therapies. Searches for factors affecting the mammalian gastro-intestinal (GI) tract are motivated by need for diagnostic and therapeutic agents. A protein may remain part of the mucin layer, providing mechanical (e.g., lubricant or gel stabilizer) and chemical (e.g. against stomach acid, perhaps helping to maintain the mucus pH gradient and/or hydrophobic barrier) protection for the underlying tissues. The trefoil peptide family has been suggested to have such general cytoprotectant roles (see Sands and Podolsky, 1996). Alternatively, a cytokine-like activity could help restore damaged epithelia. A suggestion that the trefoil peptides may act in concert with other factors to maintain and repair the epithelium, further underlines the complexity of interactions that take place in the gastrointestinal tract (Podolsky, 1997). The maintenance of the integrity of the GI epithelium is essential to the continued well-being of a mammal, and wound closing after damage normally occurs very rapidly (Lacy, 1988), followed by proliferation and differentiation soon thereafter to reestablish epithelial integrity (Nursat et al., 1992). Thus protection and restitution are two critical features of the healthy gastrointestinal tract, and may be important in the relatively harsh extracellular environment of the stomach. Searches for GI proteins have met with some success. Complementary DNA (cDNA) sequences to messenger RNAs (mRNA) isolated from human and porcine stomach cells were described in the University of Chicago Ph.D. thesis “Characterization of a novel messenger RNA and immunochemical detection of its protein from porcine gastric mucosa,” December 1987, by one of the present inventors working with the other inventors. However, there were several cDNA sequencing errors that led to significant amino acid changes from the AMP-18 protein disclosed herein. The protein itself was isolated and purified only as an aspect of the present invention, and functional analyses were performed to determine utility. Nucleic acid sequences were sought.
<SOH> SUMMARY OF THE INVENTION <EOH>A novel gene product designated Antrum Mucosal Protein 18 (“AMP-18”) is a gastrokine. The protein was discovered in cells of the stomach antrum mucosa by analysis of cDNA clones obtained from humans, pigs, and mice. The protein is a member of a group of cellular growth factors or cytokines, more specifically gastrokines. The AMP-18 cDNA sequences predict a protein 185 amino acids in length for both pig and man. The nucleotide sequences also predict a 20-amino acid N-terminal signal sequence for secreted proteins. The cleavage of this N-terminal peptide from the precursor (preAMP-18) was confirmed for the pig protein; this cleavage yields a secreted protein 165 amino acids in length and ca. 18,000 Daltons (18 kD) in size. Human and mouse genomic DNA sequences were also obtained and sequenced. A human genomic DNA was isolated in 4 overlapping fragments of sizes 1.6 kb, 3 kb, 3.3 kb and 10.1 kb respectively. The mouse genomic DNA sequence was isolated in a single BAC clone. The gastrokine designated AMP-18 protein is expressed at high levels in cells of the gastric antrum. The protein is barely detectable in the rest of the stomach or duodenum, and was not found, or was found in low levels, in other body tissues tested. AMP-18 is synthesized in lumenal surface mucosal cells, and is secreted together with mucin granules. Studies in humans confirm the location and expression of the AMP-18 peptide in human gastric mucosa. Compositions of AMP-18 isolated from mouse and pig antrum tissue stimulate growth of confluent stomach, intestinal, and kidney epithelial cells in culture; human, monkey, dog and rat cells are also shown to respond. This mitogenic (growth stimulating) effect is inhibited by specific antisera (antibodies) to AMP-18, supporting the conclusion that AMP-18, or its products, e.g. peptides derived from the protein by isolation of segments of the protein or synthesis, is a growth factor. Indeed, certain synthetic peptides whose amino acid sequences represent a central region of the AMP-18 protein also have growth-factor activity. The peptides also speed wound repair in tissue culture assays, indicating a stimulatory effect on cell migration, the process which mediates restitution of stomach mucosal injury. Thus, the protein and its active peptides are motogens. Unexpectedly, peptides derived from sub-domains of the parent molecule can inhibit the mitogenic effect of bioactive synthetic peptides and of the intact, natural protein present in stomach extracts. There are 3 activities of the gastrokine proteins and peptides of the present invention. The proteins are motogens because they stimulate cells to migrate. They are mitogens because they stimulate cell division. They function as cytoprotective agents because they maintain the integrity of the epithelium (as shown by the protection conferred on electrically resistant epithelial cell layers in tissue culture treated with damaging agents such as oxidants or non-steroidal anti-inflammatory drugs NSAIDs). The synthesis of AMP-18 is confined to lumenal mucosal lining epithelial cells of the gastric antrum of humans and other mammals. Inside cells the protein is co-localized with mucins in secretion granules, and appears to be secreted into the mucus overlying the apical plasma membrane. Recombinant human AMP-18 in E. coli exerts its mitogenic effect at a concentration an order of magnitude lower than growth-promoting peptides derived from the center of the mature protein. Peptide 77-97, the most potent mitogenic peptide, is amino acid sequence-specific AMP peptides appears to be cell-type specific as it does not stimulate growth of fibroblasts or HeLa cells. Mitogenesis by specific AMP peptides appears to be mediated by a cell surface receptor because certain peptides that are not active mitogens can competitively inhibit, in a concentration-dependent manner, the growth-stimulating effects of peptide 58-99 and antrum cell extracts. AMP-18 and its derived peptides exhibit diverse effects on stomach and intestinal epithelial cells which suggest they could play a critical role in repair after gastric mucosal injury. These include cytoprotection, mitogenesis, restitution, and maturation of barrier function after oxidant-and/or indomethacin-mediated injury. Possible mechanisms by which AMP-18 or its peptide derivatives mediate their pleiotropic effects include stimulation of protein tyrosine kinase activity, prolongation of heat shock protein expression after cell stress, and enhanced accumulation of the tight junction-associated protein ZO-1 and occludin. Certain of these physiological effects can occur at concentrations that are relatively low for rhAMP-18 (<50 nM) compared to the concentrations of other gastric peptide mediators such as trefoil peptides or the α-defensin, cryptdin 3 (>100 μM). Immunoreactive AMP-18 is apparently released by cells of the mouse antrum after indomethacin gavage, and by canine antrum cells in primary culture exposed to forskolin, suggest that the protein is subject to regulation. These results imply that AMP-18 could play a role in physiological and pathological processes such as wound healing in the gastric mucosal epithelium in vivo. The invention relates a group of isolated homologous cellular growth stimulating proteins designated gastrokines, that are produced by gastric epithelial cells and include the consensus amino acid sequences VKE(K/Q)KXXGKGPGG(P/A)PPK (SEQ ID NO: 10) wherein XX can be LQ or absent (which results in SEQ ID NOS 25 and 26, respectively). An isolated protein of the group has an amino acid sequence as shown in FIG. 7 . The protein present in pig gastric epithelia in a processed form lacking the 20 amino acids which constitute a signal peptide sequence, has 165 amino acids and an estimated molecular weight of approximately 18 kD as measured by polyacrylamide gel electophoresis. Signal peptides are cleaved after passage through endoplasmic reticulum (ER). The protein is capable of being secreted. The amino acid sequence shown in FIG. 3 was deduced from a human cDNA sequence. An embodiment of the protein is shown with an amino acid sequence as in FIG. 6 , a sequence predicted from mouse RNA and DNA. A growth stimulating (bioactive) peptide may be derived from a protein of the gastrokine group. Bioactive peptides rather than proteins are preferred for use because they are smaller, consequently the cost of synthesizing them is lower than for an entire protein. In addition, a modified peptide may be produced by the following method: (a) eliminating major protease sites in an unmodified peptide amino acid sequence by amino acid substitution or deletion; and/or (b) introducing into the modified amino acid analogs of amino acids in the unmodified peptide. An aspect of the invention is a synthetic growth stimulating peptide, having a sequence of amino acids from positions 78 to 119 as shown in FIG. 3 . Another peptide has a sequence of amino acids from position 97 to position 117 as shown in FIG. 3 . Another peptide has a sequence of amino acids from position 97 to position 121 as shown in FIG. 3 . Another peptide has a sequence of amino acids from position 104 to position 117 as shown in FIG. 3 . An embodiment of an isolated bioactive peptide has one of the following sequences: KKLQGKGPGGPPPK (SEQ ID NO: 11), LDALVKEKKLQGKGPGGPPPK (SEQ ID NO: 12), or LDALVKEKKLQGKGPGGPPPKGLMY (SEQ ID NO: 13). An embodiment of an inhibitor of a protein of the gastrokine group has the amino acid sequence KKTCIVHKMKK (SEQ ID NO: 14) or KKEVMPSIQSLDALVKEKK. (SEQ ID NO: 15) (see also Table 1) The invention also relates a pharmaceutical composition including at least a growth stimulating peptide. A pharmaceutical composition for the treatment of diseases associated with overgrowth of gastric epithelia, includes an inhibitor of a protein of the group of gastrokines or of a growth stimulating peptide derived from the gastrokine proteins. A pharmaceutical composition for the treatment of diseases of the colon and small intestine includes at least a growth stimulating peptide of the present invention. Examples of such diseases include ulcerative colitis and Crohn's Disease. Antibodies to the protein product AMP-18 encoded by the human cDNA expressed in bacteria were produced in rabbits; these antibodies reacted with 18 kD antrum antigens of all mammalian species tested (human, pig, goat, sheep, rat and mouse), providing a useful method to detect gastrokines. An antibody to a protein of the group recognizes an epitope within a peptide of the protein that includes an amino acid sequence from position 78 to position 119 as in FIG. 3 . The invention is also directed to an isolated genomic DNA molecule with the nucleotide sequence of a human as shown in FIG. 1 and an isolated cDNA molecule encoding a human protein, that the nucleotide sequence as shown in FIG. 2 . Another aspect of the invention is an isolated DNA molecule having the genomic sequence found in DNA derived from a mouse, as shown in FIG. 4 . Genomic DNA has value because it includes regulatory elements for gastric expression of genes, consequently, the regulatory elements can be isolated and used to express other gene sequences than gastrokines in gastric tissue. An aspect of the invention is a mouse with a targeted deletion in a nucleotide sequence in the mouse genome that, when expressed without the deletion, encodes a protein of the group of gastrokines of the present invention. An aspect of the invention is a method of making a gastrokine protein or a peptide derived from a gastrokine protein. The method includes: a) obtaining an isolated cDNA molecule with a sequence such as that shown in FIG. 2 ; (b) placing the molecule in a recombinant DNA expression vector; (c) transfecting a host cell with the recombinant DNA expression vector; (d) providing environmental conditions allowing the transfected host cell to produce a protein encoded by the cDNA molecule; and (e) purifying the protein from the host cell. Host cells in which expression has been successful include baculovirus, which allows large amounts of gastrokines to be provided for commercial and research uses. For example, human AMP-18 protein without the signal peptide was produced. A recombinant human protein AMP-18 expressed in E. coli has the sequence in FIG. 14 , left panel. An aspect of the invention is a method to stimulate growth of epithelial cells in the gastrointestinal tract of mammals. The method includes the steps of: (a) contacting the epithelial cells with a composition comprising a gastrokine protein or a peptide derived from a protein of the group; and (b) providing environmental conditions for stimulating growth of the epithelial cells. A method to inhibit cellular growth stimulating activity of a protein of the group includes the steps of: (a) contacting the protein with an inhibitor; and (b) providing environmental conditions suitable for cellular growth stimulating activity of the protein. The inhibitor may be an antibody directed toward at least one epitope of the protein, e.g. an epitope with an amino acid sequence from position 78 to position 119 of the deduced amino acid sequence in FIG. 3 or an inhibitor peptide such as those in Table 1. A method of testing the effects of different levels of expression of a protein on mammalian gastrointestinal tract epithelia, includes the steps of: (a) obtaining a mouse with an inactive or absent gastrokine protein; (b) determining the effects of a lack of the protein in the mouse; (c) administering increasing levels of the protein to the mouse; and (d) correlating changes in the gastrointestinal tract epithelia with the levels of the protein in the epithelia. Kits are contemplated that will use antibodies to gastrokines to measure their levels by quantitative immunology. Levels may be correlated with disease states and treatment effects. A method to stimulate migration of epithelial cells after injury to the gastrointestinal tract of mammals, includes the steps of: (a) contacting the epithelial cells with a composition comprising a peptide derived from the protein; and (b) providing environmental conditions allowing migration of the epithelial cells. A method for cytoprotection of damaged epithelial cells in the gastrointestinal tract of mammals, includes the following steps: (a) contacting the damaged epithelial cells with a composition including a protein of the gastrokine group or a peptide derived from the protein; and (b) providing environmental conditions allowing repair of the epithelial cells. The damaged cells may form an ulcer.

Separate control line for maintenance of repeaters of a digital subscriber line
Digital subscriber line (DSL) system comprising a digital subscriber line access multiplexer (DSLAM, 20) having a line trunk unit (LTU, 13), a repeater container (23) containing a repeater (16) and a customer premises equipment (CPE, 11) having a network terminating unit (NTU, 15) in which the repeater (16) connects a line trunk unit (13) to a network terminating unit (15) through respective lengths (18, 19) of a digital subscriber line and in which both the digital subscriber line access multiplexer (20) and the repeater container (23) comprise an operation and maintenance unit (OMU, 21, 24) which are connected to each other by a line (25) which is separate from the digital subscriber line for controlling the operation and maintenance of the repeater (16) or repeaters (16) of the repeater container (23) from the digital subscriber line access multiplexer (20).
1. Digital subscriber line system (DSL) comprising a digital subscriber line access multiplexer (DSLAM, 20) having a line trunk unit (LTU, 13), a repeater container (23), which contains a repeater (16) and a customer premises equipment (CPE, 11) having a network terminating unit (NTU, 15), in which lengths (18, 19) of a subscriber line connect the repeater to the line trunk unit (LTU, 13) and to the network terminating unit (NTU, 15) respectively, characterized in that the digital line access multiplexer (DSLAM, 20) comprises a first operations and maintenance unit (OMU, 21) and the repeater container (23) comprises a second operations and maintenance unit (OMU, 24) which is connected to the repeater (16), the first and second operations and maintenance units are connected by a control line (25) which is apart from the digital subscriber line, and the operations and maintenance units being suitable to operate and maintain the repeater in co-operation through control line (25). 2. Digital subscriber line system according to claim 1, characterized in that the control line (25) is of a type of a length of subscriber line. 3. Digital subscriber line system according to claim 1 or 2, characterized in that the line trunk unit (13) and the network terminating unit (15) are connected by a series of line lengths and repeaters (16) of different repeater containers (23) in between, and the second operations and maintenance units (24) of the different repeater containers are connected to at least one first operations and maintenance unit (21) of the digital subscriber line access multiplexer (20). 4. Digital subscriber line system according to claim 3, characterized in that the second operations and maintenance units (24) are connected to the at least one first operations and maintenance unit (21) by individual control lines (25). 5. Digital subscriber line system according to claim 3, characterized in that the second operations and maintenance units (24) are connected in series and to one first operations and maintenance unit (21) by control lines (25) in between. 6. Method for operating and managing a repeater (16) for repeating a digital subscriber line signal which is supplied to it and which is communicated through it between a digital subscriber line access multiplexer (20) and a customer premises equipment (CPE, 11), in which control signals to operate and manage the repeater (16) are communicated between the repeater (16) and a processing unit (14) of the multiplexer (20), characterized in that control signals from and to the multiplexer (20) are communicated over a communication path which is distinct from a communication path for the digital subscriber line signal. 7. Method according to claim 6, characterized in that the processing unit (14) delegates generating, transmitting, receiving and processing of control signals to a first operations and managing unit (21) of the multiplexer (20). 8. Method according to claim 6 or 7, characterized in that the repeater (16) delegates generating, transmitting, receiving and processing of control signals to a second operations and managing unit (24). 9. Method according to one of the claims 6-8, characterized in that control signals from and to the multiplexer (20) and a plurality of the repeaters (16) are communicated over different communication paths between the multiplexer (20) and each repeater (16) respectively. 10. Method according to claim 9, characterized in that the control signals to and from different ones of the repeaters are communicated to and from the multiplexer (20) over a common communication path.



Lipstick mechanism or the like with frictional braking
This lipstick mechanism comprising an inner tubular sleeve (1), an outer tubular sleeve (3), a stick-holder cup (2) arranged in the inner tubular sleeve (1). The inner sleeve comprises a cylindrical base (12) in the wall (21) in which are formed friction pads designed to be pressed elastically on a cylindrical bearing surface (7) linked to the outer sleeve (3), a thinned wall (23) completely surrounding the periphery of each pad (22).
1. Lipstick mechanism or the like of the type comprising an inner tubular sleeve (1) free to rotate inside an outer tubular sleeve (3), the sleeves being provided with two longitudinal slots (10, 11) and two helical slots (5) to guide the studs (6) fitted on a stick-holder cup (2) located in the inner tubular sleeve (1) in translation, the inner sleeve being provided with a cylindrical base (12) in the wall (21) of which is formed elastic friction relief designed to be pressed elastically on a cylindrical bearing surface (7) linked to the outer sleeve (3), the relief being formed of pads (22) placed on a thinned part (23) of the wall (21) of the cylindrical base, characterized in that the thinned wall (23) completely surrounds the periphery of each pad. 2. Mechanism according to claim 1, characterized in that each pad (22) has a small overthickness (e) with respect to the wall (21) of the cylindrical base. 3. Mechanism according to claim 2, characterized in that the overthickness (e) of the pad (22) is of the order of a few hundredths of a millimetre. 4. Mechanism according to claims 1, characterized in that the pads (22) are grouped in pairs. 5. Mechanism according to claim 4, characterized in that there are two opposite pairs of pads (22). 6. Mechanism according to claims 1, characterized in that it is made from an injected plastic material.



Method of treatment
This invention relates to a method of maintaining glycemic control in diabetic hypertensive patients which comprises administering carvedilol to a subject in need thereof.
1. A method of maintaining glycemic control in diabetic hypertensive patients which comprises administering carvedilol to a subject in need thereof. 2. The method according to claim 1 which comprises administering carvedilol in a dosage range from about 6.25 to about 25 mg given twice daily. 3. The method according to claim 1 which comprises administering carvedilol in a maintenance dose of about 25 mg given twice daily. 4-6. (Cancelled)
<SOH> BACKGROUND OF THE INVENTION <EOH>Hypertension is common in diabetic patients, and the coexistence of diabetes and hypertension confers an increased risk for the development of cardiovascular and renal disease. As many as 40% of hospitalized non-insulin-dependent diabetics are hypertensive (Pacy et al., Prevalence of hypertension in white, black, and Asian diabetics in a district hospital diabetic clinic. Diabetic Medicine 1982;5:125-130). Medical management of these patients is complex as either condition and/or its pharmacological treatment has the potential to exacerbate the pathophysiology of the other. For example, the adverse renal effects of diabetes can produce hypertension while thiazide diuretics prescribed for hypertension may induce insulin resistance and consequent compensatory hyperinsulinemia (Furman, Impairment of glucose tolerance produced by diuretics and other drugs. Pharmacol Ther. 1981; 12:613-649; Lewis et al., Deterioration of glucose tolerance in hypertensive patients on prolonged diuretic treatment. Lancet 1976;1:564-566′). Selective β 1 -blockers also reportedly increase insulin resistance and worsen glycemic control (Giugliano et al., Metabolic and cardiovascular effects of carvedilol, and atenolol in non-insulin-dependent diabetes mellitus and hypertension. Ann Int Med 1997; 126:955-959; Jacob et al., Differential effect of chronic treatment with two beta-blocking agents on insulin sensitivity: the carvedilol-metoprolol study. J Hypertension 1996; 14:489494; Pollare et al., Sensitivity to insulin during treatment with atenolol and metoprolol: a randomised, double-blind study of the effects on carbohydrate and lipoprotein metabolism in hypertensive patients. Br Med J 1989;298:1152-7; UK Prospective Diabetes Study Group, Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes. Br Med J 317:703-713, 1998; Wright et al., Beta-adrenoceptor blocking drugs and blood sugar control in diabetes mellitus. Br Med J 1:159-161, 1979). Consequently, some physicians are reluctant to prescribe β-blockers to hypertensive diabetic patients. Hypertension and diabetes are also recognized risk factors for development of congestive heart failure, and approximately 15-20% of patients with congestive heart failure have concurrent diabetes mellitus. Thus, despite the recent acceptance of beta-blockers as part of standard poly-therapy in heart failure, studies that report that beta-blocking agents such as metoprolol, atenolol, and propranolol significantly reduce glycemic control in hypertensive patients (Groop L, et al., Influence of beta-blocking drugs on glucose metabolism in patients with non-insulin-dependent diabetes mellitus. Acta Med Scand 1982; 211:7-12; Pollare et al., Sensitivity to insulin during treatment with atenolol and metoprolol: a randomised, double-blind study of the effects on carbohydrate and lipoprotein metabolism in hypertensive patients. Br Med J 1989;298:1152-7) potentially elicit concerns regarding the use of beta-blockers in diabetic heart failure patients. Carvedilol is a novel, multiple action drug approved for treatment of mild-to-moderate hypertension and mild-to-moderate heart failure (Ruffolo et al., Recent observations with beta-adrenoceptor blockade—beneficial effects in hypertension and heart failure. Am J Hypertension 11(1 Part 2 Suppl S):9S-14S, 1998). Carvedilol is a non-selective β-blocker and selective α 1 -receptor blocker with coincident anti-oxidant properties. In extensive clinical trials, carvedilol has been shown to be as effective as other β-blockers, angiotensin converting enzyme inhibitors or calcium channel blockers in lowering blood pressure. Importantly, carvedilol increases insulin sensitivity in both nondiabetic (Jacob et al., Differential effect of chronic treatment with two beta-blocking agents on insulin sensitivity: the carvedilol-metoprolol study. J Hypertension 1996; 14:489-494), and diabetic (Giugliano et al., Metabolic and cardiovascular effects of carvedilol, and atenolol in non-insulin-dependent diabetes mellitus and hypertension. Ann Int Med 1997; 126:955-959) hypertensive individuals. In a randomized, double-blind, controlled trial involving 45 hypertensive diabetic patients carvedilol increased insulin sensitivity and glucose disposal while it decreased HbA1c, fasting glucose, fasting insulin, and lipid peroxidation (Giugliano et al., 1997). In contrast, despite equivalent antihypertensive effect, atenolol decreased insulin sensitivity and glucose disposal and increased HbA1c, fasting glucose, and fasting insulin. It had no effect lipid peroxidation (Giugliano et al., 1997). According to the instant invention, carvedilol represents an antihypertensive therapeutic option for treatment of hypertensive type II diabetic patients with a unique ability, relative to selective β 1 -blockers, to reduce blood pressure without compromising glycemic control. The purpose of this study is to compare the effects of carvedilol to the widely-prescribed β 1 -selective antagonist metoprolol on a measure of glycemic control, glycosylated hemoglobin (HbA1c), in hypertensive patients with non-insulin-dependent diabetes mellitus.
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a method of maintaining glycemic control in diabetic hypertensive patients which comprises administering carvedilol to a subject in need thereof. detailed-description description="Detailed Description" end="lead"?

G protein-coupled receptor structural model and a method of designing ligand binding to g protein-coupled receptor by using the structural model
The present invention provides a method for constructing a structural model of a complex that a G protein-coupled protein receptor forms with a ligand capable of binding the G protein-coupled receptor and a three-dimensional structural model of an activated intermediate in the structural model of the complex. The present invention also provides a method for identifying, screening for, searching for, evaluating, or designing a ligand capable of binding a GPCR by using the three-dimensional model. In one specific method by the present invention, a three-dimensional structural model of a photoactivated intermediate of rhodopsin is constructed by using a molecule modeling software and by using the three-dimensional structural coordinate of the crystal structure of rhodopsin in such a manner that amino acid residues highly conserved among GPCRs are taken into consideration. The three-dimensional stractural model of the photoactivated intermediate of rhodopsin is subsequently used to construct structural models of activated intermediates of other GPCRs. The present invention further provides a method for identifying, screening for, searching for, evaluating, or designing a ligand that binds a GPCR to act as an agonist or an antagonist. This method employs the three-dimensional structural model constructed by the above-described method.
1. A method for constructing a three-dimensional structural model of an activated intermediate of a G protein-coupled receptor for use in identifying, screening for, searching for, evaluating, or designing a ligand that binds the G protein-coupled receptor to act as an agonist or an antagonist. 2. The method according to claim 1, wherein the activated intermediate of the G protein-coupled receptor is an intermediate of activated rhodopsin. 3. The method according to claim 1 or 2, wherein the structural model of the intermediate of the activated rhodopsin is a structural model of metarhodopsin II. 4. The method according to claim 1 or 2, wherein the structural model of the intermediate of the activated rhodopsin is a structural model of metarhodopsin I. 5. The method according to claim 1 or 2, wherein the structural model of the intermediate of the activated rhodopsin is a structural model of metarhodopsin Ib. 6. The method according to claim 1 or 2, wherein the structural model of the intermediate of the activated rhodopsin is a structural mode of metarhodopsin I380. 7. The method according to claim 1, characterized in that the three-dimensional structural model of the activated intermediate of the G protein-coupled receptor is constructed based on a structural model of metarhodopsin II. 8. The method according to claim 1, characterized in that the three-dimensional structural model of the activated intermediate of the G protein-coupled receptor is constructed based on a structural model of metarhodopsin I. 9. The method according to claim 1, characterized in that the three-dimensional structural model of the activated intermediate of the G protein-coupled receptor is constructed based on a structural model of metarhodopsin Ib. 10. The method according to claim 1, characteristics in that the three-dimensional structural model of the activated intermediate of the G protein-coupled receptor is constructed based on a structural model of metarhodopsin I380. 11. A three-dimensional structural model of the activated intermediate of the G protein-coupled receptor obtained by the method according to any one of claims 1 to 10, or a three-dimensional coordinate for determining the structural model. 12. A three-dimensional coordinate shown in Table 1 or Table 2. 13. A computer storage medium that stores all or part of the three-dimensional coordinate according to claim 11 or 12 for use in identifying, screening for, searching for, evaluating, or designing a ligand that binds the G protein-coupled receptor to act as an agonist or an antagonist. 14. A method for identifying, screening for, searching for, evaluating, or designing a ligand that binds a G protein-coupled receptor to act as an agonist, the method comprises the step of using the three-dimensional strut model according to claim 11 or the three-dimensional coordinate for determining the structural model according to claim 11 or 12, or the computer storage medium according to claim 13. 15. The method according to claim 14, wherein the agonist is a full agonist of the G protein-coupled receptor. 16. The method for identifying, screening for, searching for, evaluating, or designing the full agonist of the G protein-coupled receptor of claim 15, characterized in that, of the three-dimensional structural models according to claim 11 or the three-dimensional coordinates for determining the structural models according to claim 11 or 12, the metarhodopsin II structural model or the three-dimensional coordinate for determining the structural model, or the structural model constructed based on the metarhodopsin II structural model or the three-dimensional coordinate for determining the structural model is used. 17. The method according to claim 14, herein the agonist is a partial agonist of the G protein-coupled receptor. 18. The method for identifying, screening for, searching for, evaluating, or designing the partial agonist of the G protein-coupled receptor of claim 17, characterized in that, of the three-dimensional structural models according to claim 11 or the three-dimensional coordinates for determining the structural medals according to claim 11 or 12, the metarhodopsin I380 structural model or the three-dimensional coordinate for determining the structural model, or the structural model constructed based on the metarhodopsin I380 structural model or the three-dimensional coordinate for determining the structural model is used. 19. A method for identifying, screening for, searching for, evaluating, or designing a ligand capable of binding a G protein-coupled protein to act as an antagonist, the method comprises the step of using the three-dimensional structural model according to claim 11 or the three-dimensional coordinate for determining the structural model according to claim 11 or 12, or the computer storage medium according to claim 13. 20. The method according to claim 19, wherein the antagonist is an inverse agonist of the G protein-coupled receptor. 21. The method for identifying, screening for, searching for, evaluating, or designing the inverse agonist of the G protein-coupled protein of claim 20, characterized in that, of the three-dimensional structural modes according to claim 11 or the three-dimensional coordinates for determining the structural models according to claim 11 or 12, the metarhodopsin I structural model or the three-dimensional coordinate for determining the structural model, or the structural model constructed based on the metarhodopsin I structural model or the three-dimensional coordinate for determining the structural model is used. 22. The method for identifying, screening for, searching for, evaluating, or designing the antagonist of the G protein-coupled protein of claim 19, characterized in that, of the three-dimensional structural models according to claim 11 or the three-dimensional coordinates for determining the structural models according to claim 11 or 12, the metarhodopsin Ib structural model or the three-dimensional coordinate for determining the structural model, or the structural model constructed based on the metarhodopsin Ib structural model or the three-dimensional coordinate for determining the structural model is used. 23. A method for identifying, screening for, seeing for, evaluating, or designing a mutant of a G protein-coupled receptor, the method comprises the step of using the three-dimensional structural model according to claim 11 or the three-dimensional coordinate for determining the structural model according to claim 11 or 12, or the computer storage medium according to claim 13. 24. The method according to claim 22, wherein the mutant of the G protein-coupled receptor is a constitutively active mutant. 25. The method according to any one of claims 14 to 24, wherein the G protein-coupled receptor is selected from the group consisting of rhodopsin, adrenaline receptor, muscarinergic acetylcholine receptor, histamine H2 receptor, serotonin receptor, and amine receptor. 26. The method according to claims 1 to 6, characterized in that the structural model of the intermediate is generated by using coordinates of existing amino acid sequences and existing crystal structures of amino acids and by using an ordinary molecule modeling software in such a manner that amino acid residues highly conserved among the transmembrane helices of the G protein-coupled receptor are taken into consideration, and structural optimization is performed at 300 K according to molecular kinetics and molecular dynamics in such a manner with C α carbons of the amino acids fixed as firmly as possible. 27. The method according to any one of claims 7 to 10, corresponding the steps of: introducing amino acid substitution and insertion or deletion of amino acid residues on the loop regions by means of a three-dimensional structural model of a rhodopsin/ligand complex or a three-dimensional model of rhodopsin in the structural model of the complex based on the homology between the amino acid sequence of rhodopsin and the amino acid sequence of a G protein-coupled receptor for which to construct a model; generating a structure using a molecule modeling software; and performing structural optimization with C α carbons of the amino acids fixed as firmly as possible.
<SOH> BACKGROUND ART <EOH>Transmission of extracellular information into the cell in most cases requires mediation by membrane proteins that have transmembrane domains. G protein-coupled receptors (GPCRs) are signal-transmitting membrane proteins that have seven transmembrane domains and make up a receptor family that can bind various physiological peptide ligands, including biological amines such as dopamine and serotonin, lipid derivatives such as prostaglandin, nucleic acids such as adenosine, amino acids such as GABA, angiotensin II, bradykinin, and cholecystokinin. Serving also as receptors for extracellular transmitters responsible for the senses of vision, taste and smell, GPCRs are important membrane proteins that play a key role in signal transduction. The recent progress in completing the human genome sequence is expected to lead to discovery of many orphan receptors that are suspected of being GPCR. If successfully identified, the ligands for these GPCRs will allow for more effective development of pharmaceutical products. Thus, devising a structural model for G protein-coupled receptor/ligand complexes and devising a three-dimensional structural model for G protein-coupled receptors in the structural model of the Complexes will provide an important approach to the future development of pharmaceutical products, as will the identification, screening, searching, evaluation, and designing methods of ligands that take advantage of these structural models. In fact, a number of patent applications entitled “novel G protein-coupled receptor protein and its DNA” have recently been filed, including Japanese Laid-Open Patent Publications No. 2001-29083, No. 2001-29084, No. 2001-54388, No. 2001-54389, No. 2000-23676, No. 2000-23677, No. 2000-50875, No. 2000-152792, No. 2000-166576, No. 2000-175690, No. 2000-175691, and No. 2000-295995, to name a few. Some applications, such as Japanese Patent Laid-Open Publication No. 2000-354500, disclose methods for screening for ligands that bind to G protein-coupled receptors while other applications concern methods for cloning expression of G protein-coupled receptors. Ligands that bind to a particular G are generally classified into agonists and antagonists. According to the latest pharmacological classification standards, the former is further divided into full agonists and partial agonists and the latter into inverse agonists and antagonists. These ligands are classified not by their affinity for the receptor, but by the degree to which the ligand activates the receptor. For example, assuming the activity elicited by binding of a full agonist to be 100%, a partial agonist elicits a 50 to 70% activity. In comparison, binding of an antagonist suppresses the activity to 5 to 10% of what is elicited by the binding of a full agonist, and binding of an inverse agonist completely eliminates the activity (0% activity). Even when unbound to ligands, many GPCRs exhibit 5 to 10% of the activity led by the binding of a full agonist. Thus, it is believed that antagonists bind to physiologically inactive receptor conformations. This suggests that binding of other types of ligands brings about conformational change of GPCR. Thus, the binding of ligands and subsequent conformational change of receptors are believed to play an important role in information transmission mediated by GPCR. G protein-coupled receptors (GPCR), which share seven transmembrane domains, are classified into different families based on the homology of their amino acid sequences. In one such GPCR family, each member has high homology to rhodopsin, a photoreceptor membrane protein. The GPC of this family share highly conserved amino acid residues in their transmembrane domains. These amino acid residues are believed to play an important role in the functioning of GPCRs. Structural and functional studies of GPCR have been conducted by analyzing three-dimensional structure of rhodopsin through two-dimensional cryoelectron diffraction crystallography and X-ray crystallography (Palczewski, K. et al., Science 289, 739-745. (2000)). Also, structures of the receptor proteins and the chromophores to serve as ligands, as well as the receptors' conformational changes, have been studied using FT-IR and Raman spectroscopy (Sakmar, T. P., Prog. Nucleic Acid Res. 59, 1-34 (1998)). Based on the results of two-dimensional, low-resolution, cryoelectron diffraction crystallography, a three-dimensional structural model of rhodopsin was first constructed. More recently, more detailed three-dimensional structure of rhodopsin was revealed by X-ray crystallography. This structure was consistent with the structural characteristics previously expected from the results of FT-IR and Raman spectroscopy and made it possible to formulate assumptions about the roles of some parts of the highly conserved amino acid residues of GPCRs. For example, of the highly conserved amino acid residues of rhodopsin, the Glu134-Arg135-Tyr136 triplet (ERY triplet, which corresponds to Asp-Arg-Tyr, or DRY triplet, in other GPCRs) of the third transmembrane helix (TM3) (hereinafter, each of the seven transmembrane helixes may be denoted by abbreviation followed by respective consecutive numbers: n th helix is denoted as TMn (e.g., TM3)) located on the inside of the cell plays a significant role in the activation of G protein. It has been shown that the protonation of ionized Glu134 in metarhodopsin II (described later), an activated conformation of rhodopsin, triggers activation of G-protein (Arnis, S. & Hofmann, K. P., Proc. Natl. Acad. Sci. USA, 90, 7849-7853, 1993). Also, a significant involvement of Glu and Arg in the activation of G is suggested. On the other hand, it is suggested that a highly conserved Pro residue found in TM6 and TM7 (Pro 267 in TM6) is responsible for the kink structure characteristic of these two helices. However, the role of the kink in the functioning of GPCRs still remains unclear. Hydrophilic amino acid residues Asn55, Asp83, Asn302 found in TM1, TM2, and TM7, respectively, are linked to one another via hydrogen bonds. Also, Tyr306 residue conserved among TM7s is linked, through hydrophobic interaction, to a residue of C-terminal helix located on the inside of the cell. These interactions are believed to contribute to stabilizing the structure. Rhodopsin is also one of the G closely studied for its conformational change and functions. Rhodopsin consists of 11-cis-retinal, a chromophore, and rhodopsin, a protein component with the seven transmembrane domains. 11-cis-retinal is covalently bonded to Lys296 to form a Schiff base. This Schiff base is protonated and is thus responsible for the shift of the maximum UV absorbance (λmax) of the chromophore to a long-wavelength range of 498 nm. When illuminated, rhodopsin is converted to highly unstable bathorhodopsin (which may be referred to simply as ‘Batho,’ hereafter), which has the UV absorbance shifted to an even longer wavelength range. Upon this, 11-cis-retinal is converted to 11-trans-retinal, an all-trans chromophore. The unstable, high-energy Batho is then sequentially converted to different intermediates in the order of lumirhodopsin (‘Lumi,’ hereinafter), metarhodopsin I (‘Meta I,’ hereinafter), metarhodopsin Ib (‘Meta I,’ hereinafter), and metarhodopsin II (‘Meta II,’ hereafter) as the chromophore and opsin thermally undergo conformational changes (Tachibanaki, S. et al., Biochemistry 36, 14173-14180 (1997)) (the photoreaction process is shown in FIG. 1 ). Under physiological conditions, Lumi is converted to Meta II via an intermediate known as metarhodopsin I 380 (‘Meta I 380 ,’ hereinafter) (T. E. et al., Biochemistry 32, 13861-13872 (1993)) ( FIG. 1 ). Because the activation of G protein (transducin) takes place at Meta II stage, 11-cis-retinal attached to rhodopsin is regarded as an inverse agonist while all-trans retinal attached to Meta II can be regarded as a full agonist. Since the same chromophore of rhodopsin changes from an inverse agonist to a full agonist upon illumination of light, its conformational changes can be studied by observing changes in absorption spectrum. The conversion of rhodopsin to Batho is a rapid process that takes place within 200 fs. Each conformational change leading to Meta II takes about a few milliseconds, which is long enough to allow a protein to undergo a significant conformational change involving spatial displacement of the secondary structures of the protein. It has been shown that the conformational change of opsin causes the beta-ionon moiety of the retinal chromophore to change its direction from the 6th helix (TM6) to the 4th helix (TM4) (Bean, B. et al., Science, 288, 2209-2212 (2000)). This implies that the arrangement of helices has been altered as a result of photoisomerization. Also, Khorana and Hubbell in their experiment illuminated light onto a mutant rhodopsin, which has been spin-labeled in a site-directed manner by taking advantage of SH groups in the mutant site-specifically substituted with cysteine, and demonstrated that the conformational changes of rhodopsin to Meta II are accompanied by conformational changes of the intracellular loops and helices. They proposed a model in which the entire TM6 helix undergoes significant rotation. The model implies considerable conformational changes of membrane proteins (Farms, D. L. et al., Science 274, 768-770 (1996)). Light energy absorbed by the chromophore is harnessed to cause initial conformational change of opsin. Transition to the final active form, the Meta II conformation, begins with proton transfer from the protonated Schiff base to its counterion, Glu 134 in TM3, to form neutral Schiff base. The neutralization of the Schiff base allows movement of the helix and, ultimately, the rotation of TM6, causing the shift to the Meta II conformation. Of the different photoactivated intermediates of rhodopsin, the final Meta II conformation has proven to be the only form that has been fully activated (Khorana, H. G. J. Biol. Chem., 267, 1-4 (1992)). Ha, However, opsin without the chromophore is known to exhibit approximately 5% activity, and mutant opsin in which Glu134, which serves as a counterion of the protonated Schiff base, has been substituted with Gln exhibits approximately 50% activity even in the absence of the chromophore. This mutant opsin is known to be deactivated when 11-cis-retinal is added and irrational with light converts it to all-trans-retinal, which in turn is converted to fully activated Meta II conformation. Thus, it has been shown that opsin has several active forms (Kim, J.-M. et al., Proc. Natl. Acad. Sci. USA, 94, 14273-14278 (1997)). It is also known that G-protein (transducin) does not bind opsin when rhodopsin in is in its Meta I state while it binds opsin without activating it when rhodopsin is in its Meta Ib state (Tachibanki, S. et al., Biochemistry 36, 14173-14180 (1997)). As described, a series of events, including conformational changes of opsin and its interaction with G-protein, and suit activation of G-protein, take place over the curse of the process from Lumi to Meta II. During this process, the rotation of TM6, essential for the activation of rhodopsin, provides the G protein-coupled receptor with the structural specificity required for ligand recognition. Specifically, it has been shown that the amino acid residues in the ligand binding site involved with TM6 before the rotation of TM6 are different than the ones involved with TM6 after the rotation of TM6, and amino acid residues that serve to recognize full agonists are different than those that serve to recognize antagonists. In fact, mutants are often found in which alteration of some of the amino acid residues in TM6 affects the binding of full agonists but not the binding of antagonists. Such phenomenon will be explained by taking into account the conformational changes of the receptors. Studies on conformational changes of rhodopsin suggested that the arrangement of TMs is significantly different between the receptors that bind antagonists and the receptors that bind agonists. For this reason, the crystal structure of rhodopsin does not solely provide a structural model for every receptor/ligand complex. A comparison between the crystal structure of rhodopsin and a structural model for Meta II in accordance with the present invention is shown in FIG. 2 . The significant displacement of highly conserved Trp265 in TM6 suggests that different amino acid residues are involved in recognizing agonists and antagonists. As described above, several experiments demonstrated that photoactivation of rhodopsin brings about conformational changes of opsin (See, for example, Farrens, D. L. et al., Science 274, 768-770 (1996), Kim, J.-M. et al., Proc. Natl. Acad. Sci. USA, 94, 14273-14278, (1997)). Nonetheless, the nature of specific conformational change has yet to be understood. Accordingly, it is an objective of the present invention to simulate three-dimensional structures of these photoactivated intermediates of rhodopsin by means of computer graphics and scientific calculation and to thereby construct structural models for their complexes formed with ligands (chromophores) that can bind rhodopsin as well as three-dimensional structural models for the activated rhodopsin intermediates in the structural models of the complexes. It is another objective of the present invention to provide a method for identifying, screening for, searching for, or evaluating whether a given ligand is a fell agonist, a partial agonist, an antagonist, or an inverse agonist by constructing three-dimensional models for general G protein-coupled receptors (GPCRs) other than rhodopsin from the three-dimensional structural models for the activated intermediates of rhodopsin and, for each of the three-dimensional constructing structural models for their complexes formed with ligands and analyzing the interaction of GPCRs with corresponding ligands. It is still another objective of the present invention to provide a method for designing a novel ligand molecule that acts as an agonist or an antagonist of a GPCR.
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a diagram showing the photoreaction process of rhodopsin. FIG. 2 is a diagram showing a comparison between crystal structural model of rhodopsin and a structural of Meta II in a accordance with the present invention. FIG. 3 is a structural model for a Meta II-ligand (chromophore) complex in accordance with the present invention. FIG. 4 is a structural model for a Meta I-ligand (chromophore) complex in accordance with the present invention. FIG. 5 is a structural model for a Meta Ib-ligand (chromophore) complex in accordance with the present invention. FIG. 6 is a structural model for a Meta I 380 -ligand (chromophore) complex in accordance with the present invention. FIG. 7 is a structural model in one embodiment of the present invention showing a complex that an adrenergic beta-2 receptor to serve as a Meta I-like structure of the present invention forms with an inverse agonist propranolol. FIG. 8 is a structural model in another embodiment of the present invention showing a complex that an adrenergic beta-2 receptor to serve as a Meta II-like structure of the present invention forms with a full agonist (S)-isoproterenol. FIG. 9 is a structural model in another embodiment of the present invention showing a complex that a muscarinic acetylcholine receptor to serve as a Meta II-like structure of the present invention forms with a full agonist acetylcholine. FIG. 10 is a structural model in another embodiment of the present invention showing a complex that a muscarinic acetylcholine receptor to serve as a Meta Ib-like structure of the present invention forms with an antagonist N-methylscopolamine. FIG. 11 is a structural model in another embodiment of the present invention showing a complex that a histamine H2 receptor to serve as a Meta II-like structure of the present invention forms with a full agonist histamine. FIG. 12 is a structural model in another embodiment of the present invention showing a complex that a histamine H2 receptor to serve as a Meta Ib-like structure of the present invention forms with an antagonist tiotidine. FIG. 13 is a structural model in another embodiment of the present invention showing a complex that a serotonin receptor to serve as a Meta II-like structure of the present invention forms with a full agonist serotonin. FIG. 14 is a structural model in another embodiment of the present invention showing a complex that a serotonin receptor to serve as a Meta I 380 -like structure of the present invention forms with a partial agonist lysergic acid diethylamide (LSD). FIG. 15 is a structural model in another embodiment of the present invention showing a complex that a serotonin receptor to serve as a Meta Ib-like structure of the present invention forms with an antagonist ketanserine. FIG. 16 is a structural model in another embodiment of the present invention showing a complex that a dopamine receptor to serve as a Meta II-like structure of the present invention forms with a full agonist dopamine. FIG. 17 is a structural model in another embodiment of the present invention showing a complex that a dopamine receptor to serve as a Meta Ib-like structure of the present invention forms with an antagonist sulpiride. FIG. 18 is a diagram showing a homology in amino acid sequences of the seven transmembrane domains among rhodopsin and other GPCRs. FIG. 19 is a structural model of a human adrenergic alpha-1A receptor bound to an antagonist. FIG. 20 is a structural model of a human adrenergic alpha-1B receptor bound to an antagonist. FIG. 21 is a structural model of a human adrenergic alpha-1D receptor bound to an antagonist. FIG. 22 is a structural model of a human adrenergic alpha-2A receptor bound to an antagonist. FIG. 23 is a structural model of a human adrenergic alpha-2B receptor bound to an antagonist. FIG. 24 is a structural model of a human adrenergic alpha-2C-1 receptor bound to an antagonist. FIG. 25 is a structural model of a human adrenergic alpha-2C-2 receptor bound to an antagonist. FIG. 26 is a structural model of a human adrenergic beta-1 receptor bound to an antagonist. FIG. 27 is a structural model of a human adrenergic beta-2 receptor bound to an antagonist. FIG. 28 is a structural model for a human adrenergic alpha-1A receptor isoform 4 bound to an antagonist. FIG. 29 is a structural model of a human adrenergic alpha-1C receptor isoform 2 bound to an antagonist. FIG. 30 is a structural model of a human adrenergic alpha-1C receptor isoform 3 bound to an antagonist. FIG. 31 is a structural model of a human adrenergic alpha-1C-AR receptor bound to an antagonist. detailed-description description="Detailed Description" end="lead"?

Immunoglobulin construct containing tumor- specific p53bp2 sequenes for eliciting an anti-tumor response
The present invention provides immunoglobulins specific for p53BP2 ligand polypeptides. In a preferred embodiment, the present invention provides a variant of an immunoglobulin variable domain, wherein the immunoglobulin variable domain contains (A) at least one CDR region and (B) framework regions flanking the CDR, and the variant includes: (a) the CDR region having added or substituted therein at least one binding sequence, and (b) the flaking framework regions, wherein the binding sequence is heterologous to the CDR and the binding sequence is derived from a human ligand having immunogenic properties relevant to human lung cancer. In a preferred embodiment, the binding sequence is an antigenic sequence. In a further preferred embodiment, the variant contains a variable domain lacking an intrachain disulfide bond.
1. A variant of an immunoglobulin variable domain, said immunoglobulin variable domain comprising (A) at least one CDR region and (B) framework regions flanking said CDR, said variant comprising: (a) said CDR region having added or substituted therein at least one binding sequence and (b) said flanking framework regions, wherein said binding sequence is heterologous to said CDR and is an antigenic sequence from p53 binding protein having immunogenic properties relevant to lung human cancer. 2. The variant as define in claim 1, wherein the variable domain lacks an intrachain disulfide bond. 3. A variant as defined in claim 1, wherein (i) one or more amino acid residues in one or more of said flanking framework regions has been substituted or deleted, (ii) one or more amino acid residues has been added in one or more of said flanking framework regions, or (iii) a combination of (i) and (ii). 4. A variant as defined in claim 1, wherein (i) one or more amino acid residues in one or more framework regions other than said framework regions flanking said CDR has been substituted or deleted, (ii) one or more amino acid residues has been added in one or more framework regions other than said framework regions flanking said CDR, or (iii) a combination of (i) and (ii). 5. A variant as defined in claim 1, wherein (i) one or more amino acid residues in one or more of said flanking framework regions has been substituted or deleted, (ii) one or more amino acid residues has been added in one or more of said flanking framework regions, or (iii) a combination of (i) and (ii); and wherein (iv) one or more amino acid residues in one or more framework regions other than said framework regions flanking said CDR has been substituted or deleted, (v) one or more amino acid residues has been added in one or more framework regions other than said framework regions flanking said CDR, or (vi) a combination of (iv) and (v). 6. A variant of an immunoglobulin variable domain, said immunoglobulin variable domain comprising (A) at least one CDR region and (B) framework regions flanking said CDR, said variant comprising: (a) said CDR region having added or substituted therein at least one amino acid sequence which is heterologous to said CDR and (b) said flanking framework regions, wherein said heterologous sequence is an antigenic sequence from a p53 binding protein having immunogenic properties relevant to lung human cancer. 7. A variant as defined in claim 6, wherein the variable domain lacks an intrachain disulfide bond. 8. A variant as defined in claim 6, wherein (i) one or more amino acid residues in one or more of said flanking framework regions has been substituted or deleted, (ii) one or more amino acid residues has been added in on or more of said flanking framework regions, or (iii) a combination of (i) and (ii). 9. A variant as defined in claim 6, wherein (i) one or more amino acid residues in one or more framework regions other than said framework regions flanking said CDR has been substituted or deleted, (ii) one or more amino acid residues has been added in one or more framework regions other than said framework regions flanking said CDR, or (iii) a combination of (i) and (ii). 10. A variant as defined in claim 6, wherein (i) one or more amino acid residues in one or more of said flanking framework regions has been substituted or deleted, (ii) one or more amino acid residues has been added in one or more of said flanking framework regions, (iii) a combination of (i) and (ii); and wherein (iv) one or more amino acid residues in one or more framework regions other than said framework regions flanking said CDR has been substituted or deleted, (v) one or more amino acid residues has been added in one or more framework regions other than said framework regions flanking said CDR, or (vi) a combination of (iv) and (v). 11. A variant as defined in claim 6, wherein said CDR is more than one CDR. 12. A variant as defined in claim 6, wherein said heterologous sequence is a CDR of a heavy chain variable region. 13. A variant as defined in claim 6, wherein said heterologous sequence is a CDR of a light chain variable region. 14. A variant as defined in claim 6, wherein said antigenic sequence is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, amino acids 669 to 677 of SEQ ID NO: 2, and SEQ ID NO: 21. 15. A variant as defined in claim 6, which is an antibody. 16. A molecule comprising a variant as defined in claim 6. 17. A molecule comprising a variant as defined in claim 7. 18. A molecule comprising a variant as defined in claim 8. 19. A molecule comprising a variant as defined in claim 9. 20. A molecule comprising a variant as defined in claim 10. 21. A molecule comprising a variant as defined in claim 14. 22. A molecule as defined in claim 16, further comprising one or more constant domains from an immunoglobulin. 23. A molecule as defined in claim 16, further comprising a second variable domain linked to said variant. 24. A molecule as defined in claim 16, further comprising a second variable domain linked to said variant, and one or more constant domains from an immunoglobulin. 25. A molecule as defined in claim 16, wherein said CDR region is CDR 1. 26. A molecule as defined in claim 16, wherein said CDR region is CDR 2. 27. A molecule as defined in claim 16, wherein said CDR region is CDR 3. 28. A molecule as defined in claim 16, which is an antibody. 29. A molecule as defined in claim 16, which is derived from a human antibody. 30. A molecule as defined in claim 16, which is derived from a chimeric or a humanized antibody. 31. An immunoglobulin comprising a heavy chain and a light chain, wherein said heavy chain comprises a variant as defined in claim 6 and three constant domains from an immunoglobulin heavy chain, and said light chain comprises a second variable domain associated with said variant and a constant domain from an immunoglobulin light chain. 32. An immunoglobulin comprising a heavy chain and a light chain, wherein said light chain comprises a variant as defined in claim 6 and a constant domain from an immunoglobulin light chain, and said heavy chain comprises a second variable domain associated with said variant and three constant domains from an immunoglobulin heavy chain. 33. An isolated nucleic acid encoding a variant as defined in claim 1. 34. An isolated nucleic acid encoding a variant as defined in claim 6. 35. An isolated nucleic acid encoding a molecule as defined in claim 16. 36. An isolated nucleic acid encoding an immunoglobulin as defined in claim 29. 37. An isolated nucleic acid encoding an immunoglobulin as defined in claim 30. 38. A cell containing nucleic acid as defined in claim 31. 39. A cell containing nucleic acid as defined in claim 32. 40. A cell containing nucleic acid as defined in claim 33. 41. A cell containing nucleic acid as defined in claim 34. 42. A cell containing nucleic acid as defined in claim 35. 43. A recombinant non-human host containing nucleic acid as defined in claim 31. 44. A recombinant non-human host containing nucleic acid as defined in claim 32. 45. A recombinant non-human host containing nucleic acid as defined in claim 33. 46. A recombinant non-human host containing nucleic acid as defined in claim 34. 47. A recombinant non-human host containing nucleic acid as defined in claim 35. 48. A vaccine composition comprising a therapeutically or prophylactically effective amount of a variant as defined in claim 1, and an adjuvant. 49. A vaccine composition comprising a therapeutically or prophylactically effective amount of a variant as defined in claim 6, and an adjuvant. 50. A vaccine composition comprising a therapeutically or prophylactically effective amount of a variant as defined in claim 16, and an adjuvant. 51. A vaccine composition comprising a therapeutically or prophylactically effective amount of an immunoglobulin as defined in claim 29, and an adjuvant. 52. A vaccine composition comprising a therapeutically or prophylactically effective amount of an immunoglobulin as defined in claim 30, and an adjuvant. 53. A method of treating or preventing a lung cancer tumor in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of a variant as defined in claim 1. 54. A method of treating or preventing a lung cancer tumor in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of a variant as defined in claim 6. 55. A method of treating or preventing a lung cancer tumor in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of a molecule as defined in claim 16. 56. A method of treating or preventing a lung cancer tumor in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of an immunoglobulin as defined in claim 29. 57. A method of treating or preventing an adenocarcinoma tumor in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of an immunoglobulin as defined in claim 30. 58. A method of treating or preventing a lung cancer tumor in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of a nucleic acid as defined in claim 31. 59. A method of treating or preventing a lung cancer tumor in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of a vaccine composition as defined in claim 46. 60. A method of treating or preventing a lung cancer tumor in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of a vaccine as defined in claim 48. 61. A variant of an immunoglobulin variable domain, said immunoglobulin variable domain comprising at least one CDR region, said variant comprising said CDR region having added or substituted therein at least one antigenic sequence from a p53 binding protein having immunogenic properties relevant to human lung cancer, said at least one sequence being selected from the group consisting of (a) a binding sequence heterologous to said CDR; (b) a CTL-epitope sequence; (c) a T-helper cell sequence; (d) a B-helper cell sequence; and (e) combinations thereof, wherein said at least one sequence is heterologous to said CDR and the variable domain lacks an intrachain disulfide bond. 62. A variant as claimed in claim 59 wherein said variable region comprises (a) a CDR1 region having said CTL epitope sequence substituted or added therein; (b) a CDR2 region having said T-helper cell substituted or added therein; and (c) a CDR3 region having said binding sequence of B-helper cell sequence substituted or added therein. 63. A variant as claimed in claim 59 wherein said CTL sequence is selected from the group consisting of HLA-A1, HLA-A2, and HLA-A3. 64. A variant as claimed in claim 59 wherein said T-helper cell sequence is DRB1 0101, and A 0201. 65. A variant as claimed in claim 59 wherein said binding sequence is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, amino acids 669 to 677 of SEQ ID NO: 2, and SEQ ID NO: 21. 66. A variant as claimed in claim 59 which is an antibody. 67. A molecule comprising a variant as claimed in claim 59. 68. A molecule as claimed in claim 67 further comprising one or more constant domains from an immunoglobulin. 69. A molecule as claimed in claim 67 further comprising a second variable domain linked to said variant. 70. A molecule as claimed in claim 67 further comprising a second variable domain linked to said variant and one or more constant domains from an immunoglobulin. 71. A molecule as claimed in claim 67 which is an antibody. 72. A molecule as claimed in claim 67 which is derived from a human antibody. 73. A molecule as claimed in claim 67 which is derived from a chimeric or humanized antibody. 74. An immunoglobulin comprising a heavy chain and a light chain, wherein said heavy chain comprises a variant as claimed in claim 59 and three constant domains from an immunoglobulin heavy chain, and said light chain comprises a second variable domain associated with said variant and a constant domain from an immunoglobulin light chain. 75. An immunoglobulin comprising a heavy chain and a light chain, wherein said light chain comprises a variant as claimed in claim 59 and a constant domain from an immunoglobulin light chain, and said heavy chain comprises a second variable domain associated with said variant and three constant domains from an immunoglobulin heavy chain. 76. An isolated nucleic acid encoding a variant as claimed in claim 59. 77. An isolated nucleic acid encoding a molecule as claimed in claim 67. 78. An isolated nucleic acid encoding an immunoglobulin as claimed in claim 73. 79. An isolated nucleic acid encoding an immunoglobulin as claimed in claim 74. 80. A cell containing nucleic acid as claimed in claim 75. 81. A cell containing nucleic acid as claimed in claim 76. 82. A cell containing nucleic acid as claimed in claim 77. 83. A cell containing nucleic acid as claimed in claim 78. 84. A recombinant non-human host containing nucleic acid as claimed in claim 75. 85. A recombinant non-human host containing nucleic acid as claimed in claim 76. 86. A recombinant non-human host containing nucleic acid as claimed in claim 77. 87. A recombinant non-human host containing nucleic acid as claimed in claim 78. 88. A vaccine composition comprising a therapeutically or prophylactically effective amount of a variant as claimed in claim 59 and an adjuvant. 89. A vaccine composition comprising a therapeutically or prophylactically effective amount of a molecule as claimed in claim 67 and an adjuvant. 90. A vaccine composition comprising a therapeutically or prophylactically effective amount of an immunoglobulin as claimed in claim 73 and an adjuvant. 91. A vaccine composition comprising a therapeutically or prophylactically effective amount of an immunoglobulin as claimed in claim 74 and an adjuvant. 92. A method of treating or preventing cancer in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of a variant as claimed in claim 59 and an adjuvant. 93. A method of treating or preventing cancer in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of a molecule as claimed in claim 67 and an adjuvant. 94. A method of treating or preventing cancer in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of an immunoglobulin as claimed in claim 73 and an adjuvant. 95. A method of treating or preventing cancer in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of an immunoglobulin as claimed in claim 74 and an adjuvant. 96. A method of treating or preventing cancer as claimed in claim 91, wherein said cancer is selected from the group consisting of gastrointestinal cancer, breast cancer, small cell lung cancer, and medullary carcinoma of the thyroid. 97. A method of treating or preventing cancer as claimed in claim 92, wherein said cancer is selected from the group consisting of gastrointestinal cancer, breast cancer, small cell lung cancer, and medullary carcinoma of the thyroid. 98. A method of treating or preventing cancer as claimed in claim 93, wherein said cancer is selected from the group consisting of gastrointestinal cancer, breast cancer, small cell lung cancer, and medullary carcinoma of the thyroid. 99. A method of treating or preventing cancer as claimed in claim 94, wherein said cancer is selected from the group consisting of gastrointestinal cancer, breast cancer, small cell lung cancer, and medullary carcinoma of the thyroid. 100. A method of treating or preventing tumor metastasis in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of a variant as claimed in claim 59 and an adjuvant. 101. A method of treating or preventing tumor metastasis in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of a molecule as claimed in claim 67 and an adjuvant. 102. A method of treating or preventing tumor metastasis in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of an immunoglobulin as claimed in claim 73 and an adjuvant. 103. A method of treating or preventing tumor metastasis in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of an immunoglobulin as claimed in claim 74 and an adjuvant. 104. A method of eliciting an anti-idiotypic response to a tumor antigen in a subject in need of treatment or prevention of a disease condition associated with said tumor antigen, said method comprising administering to said subject a disease treating or preventing effective amount of a variant as claimed in claim 59 and an adjuvant. 105. A method of eliciting an anti-idiotypic response to a tumor antigen in a subject in need of treatment or prevention of a disease condition associated with said tumor antigen, said method comprising administering to said subject a disease treating or preventing effective amount of a molecule as claimed in claim 67 and an adjuvant. 106. A method of eliciting an anti-idiotypic response to a tumor antigen in a subject in need of treatment or prevention of a disease condition associated with said tumor antigen, said method comprising administering to said subject a disease treating or preventing effective amount of an immunoglobulin as claimed in claim 73 and an adjuvant. 107. A method of eliciting an anti-idiotypic response to a tumor antigen in a subject in need of treatment or prevention of a disease condition associated with said tumor antigen, said method comprising administering to said subject a disease treating or preventing effective amount of an immunoglobulin as claimed in claim 74 and an adjuvant.
<SOH> BACKGROUND OF THE INVENTION <EOH>Cancer remains the second leading cause of death in the United States. There were an estimated 563,100 cancer deaths in 1999. Each year, about 1,222,000 new cancer cases are diagnosed Non-surgical therapy for breast, lung, colon, and ovarian cancers, as well as many other solid tumors, is presently poor. While initial therapies for breast and ovarian cancer with taxanes result in some response by most patients, nearly all patients with ovarian cancer and some patients with breast cancer relapse. There is substantial evidence indicating that the immune system plays a critical role in the prevention of cancer and the control of tumor growth. This includes the occasional observation of spontaneous tumor regression, the correlation of spontaneous regressions with the presence of tumor-infiltrating lymphocytes (TILs) and the identification of TILs that are specific for tumor antigens. However, as evidenced by the incidence rates of cancer, the immune response is often not sufficient to successfully combat the tumor. During the past 40 years, an increased understanding of the immune mechanism has led to the development of approaches to enhance the immune response against the tumor. These have included the expression in tumor cells of genes encoding immunostimulatory molecules and several approaches to enhance tumor antigen presentation. Unfortunately, only limited indications of beneficial effects have been seen and attempts to utilize these new approaches to immunotherapy have been disappointing.
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides immunoglobulins specific for p53BP2 ligand polypeptides. In a preferred embodiment, the present invention provides a variant of an immunoglobulin variable domain, wherein the immunoglobulin variable domain contains (A) at least one CDR region and (B) framework regions flanking the CDR, and the variant includes: (a) the CDR region having added or substituted therein at least one binding sequence, and (b) the flanking framework regions, wherein the binding sequence is heterologous to the CDR and the binding sequence is derived from a human ligand having immunogenic properties relevant to human lung cancer. In a preferred embodiment, the binding sequence is an antigenic sequence. In a further preferred embodiment, the variant contains a variable domain lacking an intrachain disulfide bond. The variant described above may also include one or more of the following: (i) one or more amino acid residues in one or more of said flanking framework regions has been substituted or deleted, (ii) one or more amino acid residues has been added in one or more of said flanking framework regions, or (iii) a combination of (i) and (ii). Alternatively, the variant described above may also include one or more of the following additional features: (i) one or more amino acid residues in one or more framework regions other than said framework regions flanking said CDR has been substituted or deleted, (ii) one or more amino acid residues has been added in one or more framework regions other than said framework regions flanking said CDR, or (iii) a combination of (i) and (ii). The invention also provides a variant as defined above wherein (i) one or more amino acid residues in one or more of said flanking framework regions has been substituted or deleted, (ii) one or more amino acid residues has been added in one or more of said flanking framework regions, or (iii) a combination of (i) and (ii); and wherein (iv) one or more amino acid residues in one or more framework regions other than said framework regions flanking said CDR has been substituted or deleted, (v) one or more amino acid residues has been added in one or more framework regions other than said framework regions flanking said CDR, or (vi) a combination of (iv) and (v). In a further embodiment, the invention provides a variant of an immunoglobulin variable domain, wherein the immunoglobulin variable domain includes (A) at least one CDR region and (B) framework regions flanking the CDR, the variant containing: (a) the CDR region having added or substituted therein at least one amino acid sequence which is heterologous to the CDR, and (b) the flanking framework regions, wherein the heterologous sequence is an antigenic sequence from a human ligand having immunogenic properties relevant to human lung cancer. In a preferred embodiment, the variable domain lacks an intrachain disulfide bond. The variant may also include one or more of the following: (i) one or more amino acid residues in one or more of said flanking framework regions has been substituted or deleted, (ii) one or more amino acid residues has been added in on or more of said flanking framework regions, or (iii) a combination of (i) and (ii). Alternatively, the variant may include: (i) one or more amino acid residues in one or more framework regions other than said framework regions flanking said CDR has been substituted or deleted, (ii) one or more amino acid residues has been added in one or more framework regions other than said framework regions flanking said CDR, or (iii) a combiantion of (i) and (ii). The variant may also contain (i) one or more amino acid residues in one or more of said flanking framework regions has been substituted or deleted, (ii) one or more amino acid residues has been added in one or more of said flanking framework regions, (iii) a combination of (i) and (ii); and wherein (iv) one or more amino acid residues in one or more framework regions other than said framework regions flanking said CDR has been substituted or deleted, (v) one or more amino acid residues has been added in one or more framework regions other than said framework regions flanking said CDR, or (vi) a combination of (iv) and (v). The variant described above may contain more than one CDR. The variant of the invention may also contain a heterologous sequence that is a CDR of a heavy chain variable region or a light chain variable region. The variant of the invention may contain antigenic sequence having the amino acid sequences selected from the group consisting of FLLWAEFTV (SEQ ID NO:3), FIRMEVMV (SEQ ID NO:5), FLVETEKEV (SEQ ID NO:7), FLILGEFEV (SEQ ID NO: 9), FLNEWIVMV (SEQ ID NO:11), FLLDQPQFV (SEQ ID NO:13), FLVWDETQV (SEQ ID NO:15), FLVDFEHMV (SEQ ID NO:17), FLIYGEAEV (SEQ ID NO:19), and LHVEPEKEV (amino acids 669 to 677 of SEQ ID NO:2, also SEQ ID NO: 21). The invention also provides a variant as defined above which is an antibody. In addition, the invention provides a molecule containing the variant(s) described above. The molecule may be derived from a human antibody, a chimeric or a humanized antibody. The invention also provides an immunuglobulin containing a heavy chain and a light chain, wherein said heavy chain comprises a variant as described above and three constant domains from an immunoglobulin heavy chain, and said light chain comprises a second variable domain associated with said variant and a constant domain from an immunoglobulin light chain. The invention further provides an immunoglobulin containing a heavy chain and a light chain, wherein said light chain comprises a variant as defined above and a constant domain from an immunoglobulin light chain, and said heavy chain comprises a second variable domain associated with said variant and three constant domains from an immunoglobulin heavy chain. Moreover, the invention provides a variant of an immunoglobulin variable domain, said immunoglobulin variable domain comprising at least one CDR region, said variant comprising said CDR region having added or substituted therein at least one antigenic sequence from a human ligand having immunogenic properties relevant to human lung cancer, said at least one sequence being selected from the group consisting of (a) a binding sequence heterologous to said CDR; (b) a CTL-epitope sequence; (c) a T-helper cell sequence; (d) a B-helper cell sequence; and (e) combinations thereof, wherein said at least one sequence is heterologous to said CDR and the variable domain lacks an intrachain disulfide bond. In a preferred embodiment, the variable region comprises (a) a CDR1 region having said CTL epitope sequence substituted or added therein; (b) a CDR2 region having said T-helper cell substituted or added therein; and (c) a CDR3 region having said binding sequence of B-helper cell sequence substituted or added therein. This variant may be an antibody, and the invention also contemplates incorporating such a variant in a molecule, which may further comprise one or more constant domains from an immunoglobulin, a second variable domain linked to said variant, and/or a second variable domain linked to said variant and one or more constant domains from an immunoglobulin. The molecule may be an antibody, and it may also be derived from a human antibody or from a chimeric or humanized antibody. Also provided is an immunoglobulin comprising a heavy chain and a light chain, wherein said heavy chain comprises a variant described above and three constant domains from an immunoglobulin heavy chain, and said light chain comprises a second variable domain associated with said variant and a constant domain from an immunoglobulin light chain; alternatively, the invention provides an immunoglobulin comprising a heavy chain and a light chain, wherein said light chain comprises a variant as described above and a constant domain from an immunoglobulin light chain, and said heavy chain comprises a second variable domain associated with said variant and three constant domains from an immunoglobulin heavy chain. In a further embodiment, the invention provides an isolated nucleic acid encoding encoding a variant or immunoglobulin as described above. Also provided is a cell and/or recombinant non-human host containing such a nucleic acid. The invention also provides a vaccine composition containing a therapeutically or prophylactically effective amount of a variant, molecule, immunoglobulin, nucleic acid, or cell as defined above and an adjuvant. The constructs of the invention may be used in a method of treating or preventing a lung cancer tumor in a subject in need of such treatment or prevention, said method comprising administering to said subject a disease treating or preventing effective amount of a variant, molecule, immunoglobulin, nucleic acid, cell, or vaccine, as described above. Finally, the invention provides a method of eliciting an anti-idiotypic response to a tumor antigen in a subject in need of treatment or prevention of a disease condition associated with said tumor antigen, said method comprising administering to said subject a disease treating or preventing effective amount of a variant, molecule, immunogloboulin, nucleic acid, cell, or vaccine, as described above.

Methods and apparatus for treating diseased tissue
Methods and devices for treating diseased tissue using ultrasound waves are provided. The ultrasound waves are not focused, and advantageously are administered in conjunction with radiation therapy and/or chemotherapy. The methods and devices provide controlled delivery of ultrasound, and are particularly effective in treating cancers of non-bony tissue such as the breast.
1. A method for treating diseased tissue in a patient, comprising inducing in said tissue regional hyperthermia by applying unfocused ultrasound waves to said tissue. 2. A method of claim 1, further comprising applying to said tissue high-energy radiation. 3. A method of claim 2, wherein said ultrasound waves and said high-energy radiation are applied simultaneously. 4. A method of claim 2, wherein said ultrasound waves and said high-energy radiation are applied sequentially. 5. A method of claim 4, wherein said diffuse ultrasound waves and radiation are applied according to a treatment schedule in which radiation is administered for from about 1 minute to about two minutes, and after a delay of at least about 30 minutes, diffuse ultrasound is applied for at least about ten minutes. 6. A method of claim 5, wherein said radiation is administered for 1.5 minute, said delay is about 30 minutes, and said diffuse ultrasound is applied such that the temperature of the diseased tissue is maintained at 42° C. or higher for at least about 10 minutes. 7. A method of claim 6 wherein said temperature of the diseased tissue is maintained within the range of about 42° C. to about 46° C. for at least about 10 minutes. 8. A method of claim 5 wherein at least one of said administration of said radiation for 1.5 minute and said application of diffuse ultrasound such that the temperature of the diseased tissue is maintained at 42° C. or higher is repeated. 9. A method of claim 5 wherein said treatment schedule is repeated within not less than 3 days. 10. A method of claim 9 wherein said treatment schedule is repeated within not less than one week. 11. A method of claim 1, further comprising administering to said patient one or more pharmacological agents. 12. A method of claim 5, wherein said pharmacological agent is selected from the group consisting of TAXOL®, Herceptin, cytoxan, methotrexate, 5-fluorouracil, adriamycin, Ellence®, and Xeloda. 13. A method of claim 1, wherein said ultrasound waves have a frequency from about 0.6 MHz to about 8 MHz. 14. A method of claim 1 wherein said patient is a human. 15. A method of claim 1 wherein said diseased tissue comprises one or more microtumors. 16. A method of claim 1 wherein said diseased tissue comprises breast cancer. 17. A method of claim 1, wherein said application of ultrasound waves is computer controlled. 18. A method of claim 17, wherein said computer controlling comprises obtaining data from said tissue, processing said data, and controlling at least one of the duration and frequency of ultrasound waves applied to said tissue. 19. A device for providing ultrasound-generated hyperthermic treatment to a patient having diseased tissue in need of treatment, comprising: a means for generating unfocused ultrasound waves; a means for obtaining data from said tissue in situ; a central processing unit; at least one of an exposure tank and an isolated chamber; and a means for directing said ultrasound waves to said tissue.
<SOH> BACKGROUND OF THE INVENTION <EOH>Ultrasonic waves are effective in providing therapeutic heating of tissue, which can be a useful technique in treating diseased tissue such as cancerous tissue. The use of therapeutic heating for treating diseased tissue relies on the effects of hyperthermia on tissue, which effects can be enhanced by the simultaneous application of radiation, among other ways. Hyperthermia may be achieved by the application of energy in the form of, for example, microwaves, ultrasound waves, or radio-frequency waves. Hyperthermic toxicity, the direct killing of cells by overheating, is described in Overgaard, “The Current and Potential Role of Hyperthermia in Radiotherapy”, Int. J. Radiation Oncology Biol. Phys., Vol. 16, pp. 535-549 (1989). When hyperthermia and radiation are applied to tissue, an effect known as “hyperthermic radiosensitization” occurs. Radiation and hyperthermic treatment may be applied either simultaneously or sequentially. A need continues for new and/or improved methods and devices for using hyperthermia, and particularly hyperthermia generated by ultrasonic waves, in medical treatment applications. Methods that allow increased control of the level and duration of local heating are desired, as well as methods that provide effective treatment, while minimizing unnecessary exposure of patients to radiation and excessive damage to healthy cells, are also desired. The present invention is directed to these and other important ends.
<SOH> SUMMARY OF THE INVENTION <EOH>One aspect of the invention is a method for treating diseased tissue in a mammal. The method includes inducing regional hyperthermia in the tissue by applying unfocused ultrasound waves to the tissue so as to achieve a diffuse area of heating. The heating can be regulated and can be applied as a therapeutic regimen. In preferred embodiments the method is used to treat a human patient. In one embodiment of the invention, the method further includes applying to the tissue high-energy radiation, such as ionizing radiation, preferably concentrated on a treatment zone. In some preferred embodiments, the unfocused ultrasound waves are applied in conjunction with the high-energy radiation. In another preferred embodiment of the invention, the method further includes administering to the mammal one or more chemotherapeutic agents in conjunction with the other steps, either simultaneously or sequentially. An inventive aspect is use of a particular device for providing ultrasound-generated hyperthermic treatment to a patient having diseased tissue in need of treatment. The device includes a means for generating unfocused ultrasound waves; a means for obtaining data from the tissue in situ; a central processing unit; an exposure tank; and a means for directing said ultrasound waves to said tissue. Optionally, the device can include an isolated chamber. The optional isolated chamber allows for coupling of acoustic energy to targeted tissue. These and other aspects of the present invention will be apparent to one skilled in the art in view of the following disclosure and the appended claims.

Carburized and quenched member and method for production thereof
A carburizing and hardening method enhances strength while sufficiently reducing hardening strain, without increasing the production cost, and a carburized and hardened member produced thereby. The raw material is an alloy steel which contains Fe as a main component, 0.10 to 0.50 wt. % C and 0.50 to 1.50 wt. % Si and having a hardenability J, based on an end quenching test, in a range of 35 to 50 (at 12.5 mm). After the raw material is formed into the desired shape, a carburized layer is formed by carburizing in an oxidation inhibiting atmosphere. After the carburizing, quenching is performed with cooling, uninterrupted by temperature rise, from a pearlite transformation point (A1 point) to a martensite transformation start point (Ms point), and with a severity of quenching H in a range of 0.01 to 0.08 (cm−1).
1-15. (canceled) 16. A steel carburizing and hardening method comprising: providing an alloy steel, as a raw material, which contains Fe as a main component, 0.10 to 0.50 wt. % C and 0.50 to 1.50 wt. % Si, said alloy steel having a hardenability J, based on an end quenching test, in a range of 35 to 50 at 12.5 mm; forming the alloy steel into a desired shape; carburizing the shaped alloy steel in an oxidation inhibiting atmosphere; and quenching the carburized alloy steel by cooling from a pearlite transformation point (A1 point) to a martensite transformation start point (Ms point), with severity of quenching H in a range of 0.01 to 0.08 (cm−1), and without interruption by any rise in temperature. 17. A method according to claim 16 wherein said carburizing is performed in an atmosphere having a reduced pressure of 1 to 30 hPa. 18. A method according to claim 16 wherein said carburizing is performed in an atmosphere containing an inert gas as a main component. 19. A method according to claim 16 wherein said carburizing produces 0.6 to 1.5 wt. % carbon in a carburized layer. 20. A method according to claim 16 wherein intergranular oxidation progresses from a surface of the raw material to a depth which is at most 3 μm. 21. A method according to claim 16 wherein the raw material has a surface compression residual stress of 300 to 800 Mpa. 22. A method according to claim 16 wherein said quenching is performed with the severity of quenching H being in said range during transition from a temperature in an austenite region to 300° C. 23. A method according to claim 16 wherein said quenching is accomplished by gas cooling. 24. A method according to claim 23 wherein said quenching accomplished by the gas cooling uses an inert gas. 25. A method according to claim 24 wherein the inert gas is nitrogen. 26. A carburized and hardened steel alloy member produced by the method of claim 16 having a carburized layer with a surface hardness in a range of 700 to 900 Hv, and an internal non-carburized portion, located inward of the carburized layer, having a hardness in a range of 250 to 450 Hv. 27. The carburized and hardened steel member according to claim 26 wherein the area of retained austenite in section of the carburized layer is at most 25%. 28. The carburized and hardened steel alloy member according to claim 26 wherein the area of troostite structure in section at the surface of the carburized layer is at most 10%. 29. The carburized and hardened steel alloy member according to claim 26 having an internal structure of bainite. 30. The carburized and hardened member according to claim 26 in the form of a toothed gear.
<SOH> BACKGROUND ART <EOH>For example, for power transmission component parts of an automatic transmission, for example, gears and the like, carburized and hardened members subjected to a carburizing and quenching process are often used in order to increase the surface hardness and the toughness. Conventional carburized and hardened members are normally produced by forming a case hardening steel (JIS: SCM420H, SCR420H, SNCM220) or the like into a desired shape, and then gas-carburizing the steel in a carburizing atmosphere, and then quenching it in an oil or the like. As for the carburized and hardened members, cost cut and performance improvement are demanded more strongly than ever. In order to achieve both a cost cut and a performance improvement, it is necessary to remove each of problems of the conventional carburized and hardened members produced from a conventional case hardening steel by an ordinary carburizing and quenching method. One of goals regarding the carburized and hardened members is to further improve the post-carburizing and quenching process strength and, at the same time, improve the dimensional accuracy by reducing or suppressing the hardening strain. However, improved hardenability normally leads to increased hardening strain, as well known. There is a possibility that the strength prior to the carburizing and quenching process may increase resulting in degraded processability and therefore increased cost of processing. The present invention has been accomplished in view of the aforementioned problems of the conventional art. It is an object of the present invention to provide a carburized and hardened member that allows strength enhancement while sufficiently reducing the hardening strain, and a production method for the carburized and hardened member.
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is an illustration of a rotating bending fatigue test piece. FIG. 2 a is a plan view of a toothed gear for evaluation. FIG. 2 b is a sectional view the toothed gear for evaluation. detailed-description description="Detailed Description" end="lead"?

Oral absorbed drugs
Orally nonabsorbed or poorly absorbed drugs may be converted to orally absorbed prodrug derivatives by derivatization of free functional groups selected from amino, hydroxyl, mercapto, phosphate and/or carboxyl with groups sensitive to mild basic conditions such as 9-fluorenylmethoxycarbonyl (Fmoc), 2-sulfo-9-fluorenylmethoxycarbonyl (Fms), and fluorenylmethyl (Fm).
1-34. (canceled) 35: A compound of the formula: X—Y wherein Y is a moiety of an orally nonabsorbed or poorly absorbed drug bearing at least one functional group selected from free amino, hydroxyl, mercapto, phosphate and/or carboxyl, and X is at least one radical selected from the group consisting of radicals of the formulas (i) to (iv): wherein R1 and R2, the same or different, are each hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkaryl, aralkyl, halogen, nitro, sulfo (SO3H), amino, ammonium, carboxyl, PO3H2, or OPO3H2; R3 and R4, the same or different, are each hydrogen, alkyl or aryl; and A is a covalent bond when the radical is linked to a carboxyl, phosphate or mercapto group of the drug Y, or A is OCO— when the radical is linked to an amino or hydroxyl group of the drug Y; and pharmaceutically acceptable salts thereof. 36: A compound according to claim 35, wherein X is at least one radical (i) wherein R1 is hydrogen or sulfo and R2, R3 and R4 are hydrogen. 37: A compound according to claim 36, wherein free amino and/or carboxyl groups, and optionally free hydroxyl groups, of the drug moiety Y, are substituted by at least one said radical (i). 38: A compound according to claim 37, wherein one or more amino and/or hydroxyl groups of the moiety Y are substituted by the radical (i) in which R1 to R4 are hydrogen and A is OCO— (9-fluorenylmethoxycarbonyl, hereinafter “Fmoc”). 39: A compound according to claim 37, wherein one or more amino and/or hydroxyl groups of the moiety Y are substituted by the radical (i) in which R1 is sulfo at position 2, R2, R3 and R4 are hydrogen, and A is OCO— (2-sulfo-9-fluorenylmethoxycarbonyl, hereinafter “Fms”). 40: A compound according to claim 37, wherein one or more carboxyl, mercapto or phosphate groups are substituted by the radical (i) in which R1 to R4 are hydrogen and A is a covalent bond (9-fluorenylmethyl, hereinafter “Fm”). 41: A compound according to claim 35, wherein one or more amino groups are substituted by the Fmoc or Fms radical and one or more carboxyl groups are substituted by the Fm radical. 42: A compound according to claim 35, wherein one or more hydroxyl groups are substituted by the Fmoc or Fms radical and one or more carboxyl groups are substituted by the Fm radical. 43: A compound according to claim 35, wherein Y is a moiety of an orally nonabsorbed or poorly absorbed drug containing at least one amino-sugar moiety. 44: A compound according to claim 43, wherein said drug containing an amino-sugar moiety is selected from the group consisting of an anthracycline antibiotic, an antibacterial aminoglycoside antibiotic or an antifungal polyene antibiotic. 45: A compound according to claim 44, wherein said anthracycline antibiotic is daunorubicin or doxorubicin. 46: A compound according to claim 45, selected from the group consisting of Fmoc-daunorubicin, Fms-daunorubicin, Fmoc-doxorubicin and Fms-doxorubicin. 47: A compound according to claim 43, wherein said antibacterial aminoglycoside antibiotic is selected from the group consisting of streptomycin, tobramycin and gentamicin. 48: A compound according to claim 47 consisting of (Fmoc)3-gentamicin or (Fms)3-gentamicin. 49: A compound according to claim 44, wherein said antifungal polyene antibiotic is amphotericin B. 50: A compound according to claim 49, consisting of Fmoc-amphotericin B or Fms-amphotericin B. 51: A compound according to claim 35, wherein Y is a moiety of an orally nonabsorbed or poorly absorbed beta-lactam antibiotic in which the carboxyl group is substituted by a radical Fm. 52: A compound according to claim 51, wherein said beta-lactam antibiotic is ceftazimide or meropenem and said compound is Fm-ceftazimide or Fm-meropenem. 53: A compound according to claim 35, wherein Y is a moiety of an orally nonabsorbed or poorly absorbed peptide. 54: A compound according to claim 53, wherein said peptide belongs to the endorphin class and is Met5-enkephalin (SEQ ID NO: 1), or Leu5-enkephalin (SEQ ID NO: 2). 55: A compound according to claim 53, wherein said compound is selected from the group consisting of Fmoc-Met5-enkephalin (SEQ ID NO: 3), Fms-Met5-enkephalin (SEQ ID NO: 3), Fmoc-Leu5-enkephalin (SEQ ID NO: 4), and Fms-Leu5-enkephalin (SEQ ID NO: 4). 56: A compound according to claim 53, wherein said peptide is a peptide hormone selected from the group consisting of gonadotropin releasing hormone (GnRH) (SEQ ID NO: 5), an analogue thereof, and octreotide (SEQ ID NO: 15). 57: A compound according to claim 56, wherein said GnRH analogue is selected from the group consisting of leuprolide (SEQ ID NO: 6), nafarelin (SEQ ID NO: 7), goserelin (SEQ ID NO: 8), histrelin (SEQ ID NO: 9) and D-Lys6-GnRH D-Lys-GnRH (SEQ ID NO: 10). 58: A compound according to claim 57, of the sequence denoted as SEQ ID NO: 11: R5-5-oxo-Pro-His-Trp-Ser-Tyr-R6-Leu-Arg-Pro-R7 wherein R5 is a Fmoc or Fms substitution at a free amino or hydroxyl group of an amino acid residue; R6 is Gly or a D-amino acid residue of a natural or nonnatural amino acid, and R7 is Gly-NH2, NHCH2CH3 or NHNHCONH2. 59: A compound according to claim 58 wherein R6 is a D-Leu or D-Lys, or a residue of a non-natural amino acid selected from the group consisting of D-Nal [D-3(2-naphthyl)-alanine], D-Ser(t-Bu) and D-His(Nτ-PhCH2). 60: A compound according to claim 58, consisting of Fms-leuprolide, in which the free hydroxyl group of the tyrosine at position 5 is substituted by Fms, of the SEQ ID NO: 12: 5-oxo-Pro-His-Trp-Ser-(Fms)Tyr-D-Leu- SEQ ID NO: 12 Leu-Arg-Pro-NHCH2CH3 61: A compound according to claim 58 consisting of: 5-oxo-Pro-His Trp-Ser-Tyr-(Fmoc)D- SEQ ID NO: 13 Lys-Leu-Arg-Pro-Gly-NH2 or 5-oxo-Pro-His-Trp-Ser-Tyr-(Fms)D-Lys- SEQ ID NO: 14 Leu-Arg-Pro-Gly-NH2. 62: A compound according to claim 56, wherein said peptide hormone is octreotide (SEQ ID NO: 15) and said compound is Fmoc-octreotide or Fms-octreotide, wherein the Lys residue at position 5 is substituted by Fmoc or Fms (SEQ ID NO: 16). 63: A compound according to claim 53, wherein said orally nonabsorbed or poorly absorbed peptide is eptifibatide and said compound is selected from the group consisting of Fmoc-eptifibatide and Fms-eptifibatide, wherein the free amino group of the Lys residue is substituted by Fmoc or Fms. 64: A pharmaceutical composition for oral administration comprising a compound according to claim 35 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 65: A method for oral treatment of a disease or disorder that can be treated with an orally nonabsorbed or poorly absorbed drug Y, which comprises administering to an individual in need thereof a suitable amount of a compound X—Y according to claim 35. 66: A method for converting an orally nonabsorbed or poorly absorbed drug Y to an orally absorbed drug suitable for oral delivery, which comprises attaching to at least one free amino, hydroxy, mercapto, phosphate and/or carboxyl group of said orally nonabsorbed or poorly absorbed drug at least one radical selected from the group consisting of the radicals of the formulas (i) to (iv): wherein R1 and R2, the same or different, are each hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkaryl, aralkyl, halogen, nitro, sulfo (SO3H), amino, ammonium, carboxyl, PO3H2, or OPO3H2; R3 and R4, the same or different, are each hydrogen, alkyl or aryl; and A is a covalent bond when the radical is linked to a carboxyl, phosphate or mercapto group of the drug Y, or A is OCO— when the radical is linked to an amino or hydroxyl group of the drug Y. 67: A Fmoc- or Fms-derivative of a compound selected from the group consisting of Met-enkephalin, Leu-enkephalin, doxorubicin, gentamicin, amphotericin B, leuprolide and D-Lys6-GnRH. 68: A Fmoc- or Fms-derivative according to claim 67 selected from the group consisting of Fmoc-Met-enkephalin, Fms-Met-enkephalin, Fmoc-Leu-enkephalin, Fms-Leu-enkephalin, Fms-amphotericin B, Fmoc-amphotericin B, (Fms)3-gentamicin, Fms-doxorubicin, Fms-leuprolide, Fms-D-Lys6-GnRH and Fmoc-D-Lys6-GnRH.
<SOH> BACKGROUND OF THE INVENTION <EOH>Certain therapeutical drugs used in human therapy and/or in veterinary are not absorbed or poorly absorbed orally and must be administered by other routes e.g. by injection, in order to reach the blood circulation. Oral absorption of drugs is a highly desirable goal in the treatment of human diseases, particularly in prolonged therapeutical treatments. Major efforts are being made to convert orally non-absorbed or poorly absorbed drugs into orally absorbed drugs by encapsulation or by chemical modification. Structural alteration of drugs may result in an increase of the oral absorption, and, eventually, in biostability of the drugs. International PCT Publication No. WO 98/05361 of the present applicants describes a new conceptual approach for prolonging the half-life of drugs, particularly proteins such as insulin, in vivo, and suggests that this approach may represent alternative possibilities for drug administration, e.g. oral and transdermal, and for penetration of the drug through physiological barriers. According to WO 98/05361, a drug containing a group selected from free amino, carboxyl, hydroxyl and/or mercapto is derivatized with a hydrophobic group such as 9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl (Fms), chemical modifications which are reversible under physiological conditions. Moreover, such covalent modification renders the drugs, particularly proteins such as insulin, more stable towards enzymatic degradation, and increases the hydrophobicity index of the drug. However, although Fmoc- and Fms-drug derivatives having increased resistance to proteolysis and increased lipophilicity may be candidates for use in orally active delivery systems, it is not obvious that polar hydrophlic molecules such as peptides, amino-sugars, amino acids and the like, that are not orally absorbed or are only poorly absorbed orally, will turn into orally absorbed species following enhancement of hydrophobicity. It has to be stressed that Fmoc- and Fms-modified drugs consist schematically of two domains: a polar domain and a hydrophobic domain, and the effect of each of these domains on oral delivery of the drug cannot be predicted, especially when the molecule has a high molecular weight.
<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to provide orally deliverable prodrugs derived from orally nonabsorbed or poorly absorbed drugs. The present invention thus relates to an orally absorbed prodrug of the formula: in-line-formulae description="In-line Formulae" end="lead"? X—Y in-line-formulae description="In-line Formulae" end="tail"? wherein Y is a moiety of an orally nonabsorbed or poorly absorbed drug bearing at least one functional group selected from free amino, hydroxyl, mercapto, phosphate and/or carboxyl, and X is a radical selected from radicals of the formulas (i) to (iv). wherein R 1 and R 2 , the same or different, are each hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkaryl, aralkyl, halogen, nitro, sulfo (SO 3 H), amino, ammonium, carboxyl, PO 3 H 2 , or OPO 3 H 2 , R 3 and R 4 , the same or different, are each hydrogen, alkyl or aryl; and A is a covalent bond when the radical is linked to a carboxyl, phosphate or mercapto group of the drug Y, or A is OCO— when the radical is linked to an amino or hydroxyl group of the drug Y, and pharmaceutically acceptable salts thereof. In preferred embodiments of the invention, at least one functional group of the drug molecule Y is attached to at least one radical X wherein said radical X is the radical (i), wherein either R 1 , R 2 , R 3 and R 4 are hydrogen and A is OCO—, i.e. the 9-fluorenylmethoxycarbonyl radical (herein designated “Fmoc”) or R 1 is sulfo at position 2, R 2 , R 3 and R 4 are hydrogen and A is OCO—, i.e. the 2-sulfo-9-fluorenylmethoxycarbonyl or 2-sulfo-Fmoc radical (herein designated “Fms”). Several types of orally nonabsorbed or poorly absorbed drugs can be derivatized according to the invention thus obtaining orally absorbed prodrugs that are administered orally and are hydrolyzed to the original active drug molecule under physiological conditions. The invention also relates to a method for converting an orally nonabsorbed or poorly absorbed drug to an orally absorbed drug suitable for oral delivery, which comprises attaching to at least one free amino, hydroxy, mercapto, phosphate and/or carboxyl group of said orally nonabsorbed or poorly absorbed drug at least one radical selected from the group consisting of the radicals of the formulas (i) to (iv) as described above. The invention further relates to pharmaceutical compositions for oral administration comprising a prodrug X—Y of the invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In another aspect, the invention provides a method for oral treatment of a disease or disorder that can be treated with an orally nonabsorbed or poorly absorbed drug Y, which comprises administering to an individual in need thereof a suitable amount of a prodrug X—Y of the invention or a pharmaceutically acceptable salt thereof.

Industrial robot
A communication system for safe wireless control of an manoeuvrable object (3), comprising a control unit (1) having means for operating the object and receiving means for wireless information, and a portable operating unit (2) having sending means for wireless information, where said information is divided in time slots (ts), each containing a data package (21). The receiving means comprises detecting means for detecting in a time part (tp) of a time slot the presence of said data package and calculating means for calculating upon a reception failure the number of time parts having no presence of said data package following a time slot with presence of said data package. The control unit exercises a control command when a data package with a correct information has been received within a number of time parts just exceeding a full time slot.
1. A communication system for safe wireless control of an maneuverable object, comprising a control unit having means for operating the object and receiving means for wireless information, and a portable operating unit having sending means for wireless information, where said information is divided in time slots, each containing a data package, characterized in that the receiving means comprises detecting means for detecting in a time part of a time slot the presence of said data package and calculating means for calculating upon a reception failure the number of time parts having no presence of said data package following a time slot with presence of said data package whereupon the control unit exercises a control command when a data package with correct information has been received within a watch-time comprising a number of time parts just exceeding a full time slot. 2. The communication system according to claim 1, wherein the control unit further comprises means for putting the object in a safety stop condition after a reception failure during a first predetermined number of time parts. 3. The communication system according to claim 2, wherein the first predetermined number of time parts exceeds a full time slot. 4. The communication system according to claim 2, wherein the first predetermined number of time parts exceeds two full time slots. 5. The communication system according to claim 1, wherein the control unit further comprises means for exercising an emergency stop after a reception failure during a second predetermined number of time parts. 6. The communication system according to claim 1, wherein the time part is equal to half a time slot. 7. The communication system according to claim 1, wherein the sending means and the receiving means are parts of a wireless local area network. 8. The communication system according to claim 7, wherein the wireless local area network is a Bluetooth network. 9. A communication system according to claim 1, wherein the data package contains an address part, an instruction part, a data part and an identifying part. 10. The communication system according to claim 9, wherein the data part is encrypted. 11. The communication system according to claim 9, wherein the data part upon operating failure contains an error detection code. 12. The communication system according to claim 9, wherein the data part contains an operating command that enables the object to move during more than one time slot. 13. The communication system according to claim 9, wherein the data part contains an operating command that enables the object to move during more than two time slots. 14. An industrial robot comprising a manipulator having a plurality of moveable members, a control unit having means for automatically operating the manipulator and receiving means for wireless information, and a portable operating unit having sending means for wireless information, where said information is divided into time slots, each containing a data package, wherein the receiving means comprises detecting means for detecting in a time part (tp) of a time slot the presence of said data package and calculating means for calculating the number of time parts having no presence of said data package following a time slot with presence of said data package, whereupon the control unit on presence of an enabling signal in the data package exercises a control command when a data package has been received within watch-time (tw) comprising a number of time parts just exceeding a full time slot. 15. The industrial robot according to claim 14, wherein the control unit further comprises means for putting the manipulator in a safety stop condition after a reception failure during a number of time parts exceeding a full time slot. 16. The industrial robot according to claim 14, wherein the control unit further comprises means for exercising an emergency stop after a reception failure during a predetermined number of time parts. 17. The industrial robot according to claim 14, wherein the portable operating unit comprises display means, jogging means, and an emergency stop unit. 18. The industrial robot according to claim 14, characterized in that the time part is equal to half a time slot. 19. The industrial robot according to claim 14 wherein the data package contains an address part an instruction part, a data part and an identifying part. 20. The industrial robot according to claim 14, wherein the data package contains control command which enables the manipulator to move for a predefined time exceeding one time slot. 21. The industrial robot according to claim 14, wherein the data part is encrypted. 22. An industrial robot according to claim 14, wherein the data upon operating failure contains an error detection code. 23. A method for safe wireless control of an industrial robot comprising a manipulator, a control unit having means for automatically operating the manipulator and a portable operating unit, comprising the operation unit sending information to the control unit, where said information is divided in time slots, each containing a data package, comprising: dividing each time slot into time parts, determining the presence of a data package received by the control unit within each of said time part, calculating upon reception failure the number of time parts with no presence of said data package following a time slot with presence of said data package, and exercising the control of the manipulator upon reception of said data package within a watch-time comprising a number of time parts exceeding a full time slot. 24. The method according to claim 23, wherein the exercising step further comprises putting the robot in a safety stop condition upon a reception failure during a time period corresponding to a number of time parts exceeding a full time slot. 25. The method according to claim 23, wherein the exercising step further comprises exercising an emergency stop command upon reception failure during a time period corresponding to a predefined number of time parts exceeding a full time slot. 26. The method according to any of claims 23, wherein the time part is equal to half a time slot. 27. The method according to any of claims 23, wherein the data package is brought to contain information on addressee, enabling, position of emergency switch, and movement instructions.
<SOH> BACKGROUND OF THE INVENTION <EOH>In order to accomplish a safe control of a moveable object such as an industrial robot it is essential to have absolute confidence in the control of the object. As an object in this context should be understood any stationary or mobile moveable object. The operator must be fully confident that the commanded order is effected. Any other person or machine must not be able to interfere with the control of the object on purpose or by accident. In a system where an operating unit is connected to a control unit via a cable this is easy. Such a cable is preferably shielded and no other communication system can interfere with the two connected units. The cable is however heavy and stiff thus making the operation of the operating unit cumbersome. Another problem with a cable connection is that the cable often is getting entangled in loops thus making it difficult to stretch it out. When a plurality of robots are placed together in a cell the cables often are entangled themselves thus making it difficult to find out which operating unit belongs to which robot. Finally there is the risk of the cable being damaged especially when a vehicle is running over it. If the cable is damaged it is involving much work to have it repaired or exchanged. It is therefore a long lasting desire in the industry of industrial robots to carry out robot control without a cable. The obvious solution is a wireless control system. The need described above then will be very obviously solved. Controlling an object with a wireless system where the air could contain a plurality of simultaneous command messages involves, however, other problems. The receiving part of a specific control system must find out and recognize a control message addressed to that specific control unit and effectuate that command. It is in such an environment obvious that sometimes different signals will interfere with each other. It is also obvious as the operating unit is moved around that sometimes the aerial will be located in a “radio shadow” thus no signal from the unit will be received by the control unit. In such cases there must be a control system which accounts for time slots where no signal is received and for a correct action accordingly. From “Gebrauchsmuster” DE 297 10 026 U1 a wireless control of an industrial robot is previously known. A mobile operating unit has a wheel knob that sends pulses via a Digital European Cordless Telephone (DECT) module to a numeric control device. The safety aspects of this wireless control is not mentioned in the document. There is thus nothing mentioned on how to proceed when a signal is not received by the receiving part. Another wireless control system is previously known from JP 11073201. The problem solved in this document is the communication of an emergency stop control carried out by wireless means in a manner equivalent to a cable communication. Under normal conditions the controller periodically transmits data to the operating unit. Upon receiving this data the operating unit responds by transmitting a response signal back to the controller. Thus the control unit asks the operating unit if this is in function. The reason for making the communication intermittent is that data, if transmitted and received at every time, increases the electric power consumption of the battery-operated teaching unit. The length of a time period however is not mentioned. The controller judges from said response signal that the operating unit is in normal condition. If the emergency switch has been activated the response signal carries that information and the controller executes the emergency stop. Under abnormal situations, that means when the operating unit is not in a correct function mood, the operating unit transmits no response signal. Based on the absence of a response from the operating unit the controller outputs the conclusion that there is an “emergency stop condition”. From JP 9117888 another control system is known the object of which is to provide a remote control with an emergency stop function. The system is said to ensure safety while cutting costs by making possible to carry out the remote control of a robot etc by transmitters for wireless remote controllers or by serial wired communication means. According to the system an operator is actuating a control permit switch by means of a key operation. A base signal is then continuously transmitted from the transmitter of the operating unit. When driving control switches are activated a driving control signal is transmitted in addition to the base signal. A control part of the system is provided in the object to be controlled. When a continuous base signal is received and driving control signals are received in addition to this the driving means is then controlled. When, however, the base signal is interrupted the driving is stopped irrespective of the existence of other signals. Thus an emergency stop function is achieved by reliably stopping the driving means when the actuation of said control permit switch is released. The signal is sent in time slots, which has a typical length of 108 ms, and contain a leader code (A), a control permit code (C) also known as base signal and a switch code (B). In the absence of the base signal the power source of the driving control means is cut of and the motor is stopped irrespective of the other signals. Thus when the need for an emergency stop arises the motor is reliably stopped when the operator stops operating the key of the servo control permit switch. Also when the signal between the transmitter and the robot is cut off or when the transmission is poor the power source is cut of from the driving control means thus effecting an emergency stop function. Both of these Japanese documents describe the execution of an emergency stop in the absence of a base signal sent from an operating device. However, an emergency stop is not wanted when a signal from the operating unit is just lacking for a short moment. There will always be moments when such a base signal is not received for one or more time slots. This, however, does not call for an emergency stop execution. When an emergency stop is executed the whole robot cell or even several cells will be cut of from electric power. To reset an emergency stop a plurality of checks has to be made and the cell will be out of work for a considerable time. This is therefore a not wanted situation.
<SOH> SUMMARY OF THE INVENTION <EOH>A primary object of the present invention is to devise ways to find a solution to a safe wireless control of a moveable object. Such a control system shall control the object even under conditions when communication is poor and when a data signal is missing for short periods of time. The control system shall also execute an emergency stop on command or when the wireless communication is broken. These objects are achieved according to the invention in a first aspect with a communication system according to the features in the characterizing part of the independent claim 1 , in a second aspect according to an industrial robot according to the characterizing features of the independent claim 13 , and in a third aspect according to a method according to the characterizing features of the independent claim 21 . Preferred embodiments are described in the dependent claims. According to the first aspect of the invention a communication system for safe wireless control of a maneuverable object is disclosed where a receiving means comprises detecting means for detecting in a time part of a time slot the presence of a data package and calculating means for calculating upon a reception failure the number of time parts having no presence of said data package following a time slot with presence of said data package whereupon the control unit exercises a control command when a data package has been received within a number of time parts just exceeding a full time slot. The principle of a wireless safety loop is based on transmitting a continues stream of data packages, each within a time slot of a specified maximum gap, from the operating unit to the control unit. This stream of data packages will be sent even when the operating unit does not actively control the robot. On the controller side a watchdog device with a specified timeout is implemented. The control unit thus comprises an electronic hardware means with a processor, which in the absence of a software product running the processor will execute a safety stop of the object. The watchdog device looks for the necessary software pieces in the data packages received from the operating unit. If there is no such software piece in the data package or there is no data package at all the watchdog executes a safety stop. If during the following time slots there is no software piece or no data packages for a predefined time interval the watchdog executes an emergency stop. A typical time slot would be 100 ms. In a wireless link there is no guarantee for a continues connection. There will be short breaks caused by multipath fading etc. There could also be longer breaks caused by radio path obstructions and such. Finally there could be long-lasting breaks caused by battery failure. There are thus three different time windows that calls for different action by the watchdog. For short breaks, that is in the area of 1.5 time slots the control of the object should not be affected. The optimum watch-time for the watchdog when there is no time mismatch would be a time slot plus the length of a data package. For longer breaks, that is, up to about 500 ms the watchdog should execute a safety stop. That means that when the watchdog detects a new data package within that time the control of the object should continue. But when the break last for longer than 500 ms an emergency stop should be executed. In a further embodiment of this aspect of the invention the sending means and the receiving means are parts of a wireless local area network. In yet another embodiment, this local area network is a Bluetooth network. In a further embodiment of the invention the data package contains an address part, an instruction part, a data part and an identifying part. In one embodiment the data is encrypted. The data part is carrying data for controlling the object. The data is also carrying enabling information, emergency stop execution and software pieces for the watchdog means. Preferably the data comprises operating commands that contain movement instructions that allow the object to move for at least one time slot in advance. Preferably it will last for one and a half time slot. If thus these operating commands arrive in each time slot, the commands are overlapping each other thus giving the object a smooth movement. The overlapping will also ensure a smooth operation if there is a clock mismatch between the operating unit and the control unit. In one embodiment, the command contains a movement allowance for just over two time slots. This will assure a smooth operation if there is a missing data package in one time slot only. On such reception failure in just one time slot the next command will still overlap the command under execution, thus giving the object a smooth movement. According to the second aspect of the invention an industrial robot is disclosed where a receiving means comprises detecting means for detecting in a time part of a time slot the presence of said data package and calculating means for calculating the number of time parts having no presence of said data package following a time slot with presence of said data package, whereupon the control unit on presence of an enabling signal in the data package exercises a control command when a data package has been received within a number of time parts just exceeding a full time slot. In a further embodiment of the invention, the control unit further comprises means for putting the manipulator in a safety stop condition after a reception failure during a number of time parts exceeding a full time slot. An emergency stop will be exercised after a reception failure during a predetermined number of time parts. In a further embodiment of this aspect of the invention, the portable operating unit comprises display means, jogging means, and an emergency stop unit. Yet in another embodiment the data package contains control command which enables the manipulator to move for a predefined time exceeding one time slot. According to the third aspect of the invention, a method is disclosed which involves the steps of: dividing each time slot into time parts, determining the presence of a data package received by the control unit within each of said time part, calculating upon reception failure the number of time parts with no presence of said data package following a time slot with presence of said data package, and exercising the control of the manipulator upon reception of said data package within a number of time parts exceeding a full time slot. In a further embodiment of this aspect of the invention the exercising step of the method further comprises putting the robot in a safety stop condition upon reception failure during a time period corresponding to a number of time parts exceeding a full time slot. An emergency stop is exercised upon reception failure during a time period corresponding to a predefined number of time parts exceeding a full time slot.

Isolated human ras-like proteins acid molecules encoding these human ras-like proteins, and uses thereof
The present invention provides amino acid sequences of polypeptides that are encoded by genes within the human genome, the Ras-like protein polypeptides of the present invention. The present invention specifically provides isolated polypeptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the Ras-like protein polypeptides, and methods of identifying modulators of the Ras-like protein polypeptides.
1. An isolated polypeptide consisting of an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids. 2. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids. 3. An isolated antibody that selectively binds to a polypeptide of claim 2. 4. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d). 5. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d). 6. A gene chip comprising a nucleic acid molecule of claim 5. 7. A transgenic non-human animal comprising a nucleic acid molecule of claim 5. 8. A nucleic acid vector comprising a nucleic acid molecule of claim 5. 9. A host cell containing the vector of claim 8. 10. A method for producing any of the polypeptides of claim 1 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the polypeptides are expressed from the nucleotide sequence. 11. A method for producing any of the polypeptides of claim 2 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the polypeptides are expressed from the nucleotide sequence. 12. A method for detecting the presence of any of the polypeptides of claim 2 in a sample, said method comprising contacting said sample with a detection agent that specifically allows detection of the presence of the polypeptide in the sample and then detecting the presence of the polypeptide. 13. A method for detecting the presence of a nucleic acid molecule of claim 5 in a sample, said method comprising contacting the sample with an oligonucleotide that hybridizes to said nucleic acid molecule under stringent conditions and determining whether the oligonucleotide binds to said nucleic acid molecule in the sample. 14. A method for identifying a modulator of a polypeptide of claim 2, said method comprising contacting said polypeptide with an agent and determining if said agent has modulated the function or activity of said polypeptide. 15. The method of claim 14, wherein said agent is administered to a host cell comprising an expression vector that expresses said polypeptide. 16. A method for identifying an agent that binds to any of the polypeptides of claim 2, said method comprising contacting the polypeptide with an agent and assaying the contacted mixture to determine whether a complex is formed with the agent bound to the polypeptide. 17. A pharmaceutical composition comprising an agent identified by the method of claim 16 and a pharmaceutically acceptable carrier therefor. 18. A method for treating a disease or condition mediated by a human Ras-like protein, said method comprising administering to a patient a pharmaceutically effective amount of an agent identified by the method of claim 16. 19. A method for identifying a modulator of the expression of a polypeptide of claim 2, said method comprising contacting a cell expressing said polypeptide with an agent, and determining if said agent has modulated the expression of said polypeptide. 20. An isolated human Ras-like protein polypeptide having an amino acid sequence that shares at least 70% homology with an amino acid sequence shown in SEQ ID NO:2. 21. A polypeptide according to claim 20 that shares at least 90 percent homology with an amino acid sequence shown in SEQ ID NO:2. 22. An isolated nucleic acid molecule encoding a human Ras-like protein polypeptide, said nucleic acid molecule sharing at least 80 percent homology with a nucleic acid molecule shown in SEQ ID NOS:1 or 3. 23. A nucleic acid molecule according to claim 22 that shares at least 90 percent homology with a nucleic acid molecule shown in SEQ ID NOS:1 or 3.
<SOH> BACKGROUND OF THE INVENTION <EOH>Ras-like proteins, particularly members of the Ras-like GTPase subfamilies, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of Ras-like proteins. The present invention advances the state of the art by providing a previously unidentified human Ras-like proteins that have homology to members of the Ras-like GTPase subfamilies. Ras Protein Ras proteins are small regulatory GTP-binding proteins, or small G proteins, which belong to the Ras protein superfamily. They are monomeric GTPases, but their GTPase activity is very slow (less than one GTP molecule per minute). Ras proteins are key relays in the signal-transducing cascade induced by the binding of a ligand to specific receptors such as receptor tyrosine kinases (RTKs), since they trigger the MAP kinase cascade. The ligand can be a growth factor (epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin, an interleukin (IL), granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF). Ras proteins contain sequences highly conserved during evolution. Their tertiary structure includes ten loops connecting six strands of beta-sheet and five alpha helices. In mammalians, there are four Ras proteins, which are encoded by Ha-ras, N-ras, Ki-rasA and Ki-rasB genes. They are composed of about 170 residues and have a relative molecular mass of 21 kD. Ras proteins contain covalently attached modified lipids allowing these proteins to bind to the plasma membrane. Ha-Ras has a C-terminal farnesyl group, a C-terminal palmitoyl group and a N-terminal myristoyl group. In Ki-Ras(B), a C-terminal polylysine domain replaces the palmitoyl group. Ras proteins alternate between an inactive form bound to GDP and an active form bound to GTP. Their activation results from reactions induced by a guanine nucleotide-exchange factor (GEF). Their inactivation results from reactions catalyzed by a GTPase-activating protein (GAP). When a Ras protein is activated by a GEF such as a Sos protein, the N-terminal region of a serine/threonine kinase, called “Raf protein”, can bind to Ras protein. The C-terminal region of the activated Raf thus formed binds to another protein, MEK, and phosphorylates it on both specific tyrosine and serine residues. Active MEK phosphorylates and activates, in turn, a MAP kinase (ERK1 or ERK2), which is also a serine/threonine kinase. This phosphorylation occurs on both specific tyrosine and threonine residues of MAP kinase. MAP kinase phosphorylates many different proteins, especially nuclear transcription factors (TFs) that regulate expression of many genes during cell proliferation and differentiation. Recent researches suggest that, in mammalians, phosphatidyl inositol 3′-kinase (PI3-kinase) might be a target of Ras protein, instead of Raf protein. In certain mutations, the translation of ras genes may produce oncogenic Ras proteins. Ras-Like Protein Guanine nucleotide-binding proteins (GTP-binding proteins, or G proteins) participate in a wide range of regulatory functions including metabolism, growth, differentiation, signal transduction, cytoskeletal organization, and intracellular vesicle transport and secretion. These proteins control diverse sets of regulatory pathways in response to hormones, growth factors, neuromodulators, or other signaling molecules. When these molecules bind to transmembrane receptors, signals are propagated to effector molecules by intracellular signal transducing proteins. Many of these signal-transducing proteins are members of the Ras superfamily. The Ras superfamily is a class of low molecular weight (LMW) GTP-binding proteins that consist of 21-30 kDa polypeptides. These proteins regulate cell growth, cell cycle control, protein secretion, and intracellular vesicle interaction. In particular, the LMW GTP-binding proteins activate cellular proteins by transducing mitogenic signals involved in various cell functions in response to extracellular signals from receptors (Tavitian, A. (1995) C. R. Seances Soc. Biol. Fil. 189:7-12). During this process, the hydrolysis of GTP acts as an energy source as well as an on-off switch for the GTPase activity of the LMW GTP-binding proteins. The Ras superfamily is comprised of five subfamilies: Ras, Rho, Ran, Rab, and ADP-ribosylation factor (ARF). Specifically, Ras genes are essential in the control of cell proliferation. Mutations in Ras genes have been associated with cancer. Rho proteins control signal transduction in the process of linking receptors of growth factors to actin polymerization that is necessary for cell division. Rab proteins control the translocation of vesicles to and from membranes for protein localization, protein processing, and secretion. Ran proteins are localized to the cell nucleus and play a key role in nuclear protein import, control of DNA synthesis, and cell-cycle progression. ARF and ARF-like proteins participate in a wide variety of cellular functions including vesicle trafficking, exocrine secretion, regulation of phospholipase activity, and endocytosis. Despite their sequence variations, all five subfamilies of the Ras superfamily share conserved structural features. Four conserved sequence regions (motifs I-IV) have been studied in the LMW GTP-binding proteins. Motif I is the most variable but has the conserved sequence, GXXXXGK. The lysine residue is essential in interacting with the beta.- and .gamma.-phosphates of GTP. Motif II, III, and IV contain highly conserved sequences of DTAGQ, NKXD, and EXSAX, respectively. Specifically, Motif II regulates the binding of gamma-phosphate of GTP; Motif III regulates the binding of GTP; and Motif IV regulates the guanine base of GTP. Most of the membrane-bound LMW GTP-binding proteins generally require a carboxy terminal isoprenyl group for membrane association and biological activity. The isoprenyl group is added posttranslationally through recognition of a terminal cysteine residue alone or a terminal cysteine-aliphatic amino acid-aliphatic amino acid-any amino acid (CAAX) motif. Additional membrane-binding energy is often provided by either internal palmitoylation or a carboxy terminal cluster of basic amino acids. The LMW GTP-binding proteins also have a variable effector region, located between motifs I and II, which is characterized as the interaction site for guanine nucleotide exchange factors (GEFs) or GTPase-activating proteins (GAPs). GEFs induce the release of GDP from the active form of the G protein, whereas GAPs interact with the inactive form by stimulating the GTPase activity of the G protein. The ARF subfamily has at least 15 distinct members encompassing both ARF and ARF-like proteins. ARF proteins identified to date exhibit high structural similarity and ADP-ribosylation enhancing activity. In contrast, several ARF-like proteins lack ADP-ribosylation enhancing activity and bind GTP differently. An example of ARF-like proteins is a rat protein, ARL184. ARL184 has been shown to have a molecular weight of 22 kDa and four functional GTP-binding sites (Icard-Liepkalns, C. et al., (1997) Eur. J. Biochem. 246: 388-393). ARL184 is active in both the cytosol and the Golgi apparatus and is closely associated with acetylcholine release, suggesting that ARL184 is a potential regulatory protein associated with Ca.sup.2+-dependent release of acetylcholine. A number of Rho GTP-binding proteins have been identified in plasma membrane and cytoplasm. These include RhoA, B and C, and D, rhoG, rac 1 and 2, G25K-A and B, and TC10 (Hall, A. et al. (1993) Philos. Trans. R. Soc. Lond. (Biol.) 340:267-271). All Rho proteins have a CAAX motif that binds a prenyl group and either a palmitoylation site or a basic amino acid-rich region, suggesting their role in membrane-associated functions. In particular, RhoD is a protein that functions in early endosome motility and distribution by inducing rearrangement of actin cytoskeleton and cell surface (Murphy, C. et al. (1996) Nature 384:427-432). During cell adhesion, the Rho proteins are essential for triggering focal complex assembly and integrin-dependent signal transduction (Hotchin, N. A. and Hall, A. (1995) J. Cell Biol. 131:1857-1865). The Ras subfamily proteins already indicated supra are essential in transducing signals from receptor tyrosine kinases (RTKs) to a series of serine/threonine kinases which control cell growth and differentiation. Mutant Ras proteins, which bind but cannot hydrolyze GTP, are permanently activated and cause continuous cell proliferation or cancer. TC21, a Ras-like protein, is found to be highly expressed in a human teratocarcinoma cell line (Drivas, G. T. et al. (1990) Mol. Cell. Biol. 10: 1793-1798). Rin and Rit are characterized as membrane-binding, Ras-like proteins without the lipid-binding CAAX motif and carboxy terminal cysteine (Lee, C.-H. J. et al. (1996) J. Neurosci. 16: 6784-6794). Further, Rin is shown to localize in neurons and have calcium-dependant calmodulin-binding activity. Ras-Like GTPase Proteins The novel human protein, and encoding gene, provided by the present invention is related to the family of Ras-like GTPase proteins (also referred to as Ras-like GTP-binding proteins), which includes Rab proteins. The protein of the present invention is similar to Rab8 and low molecular weight (LMW) GTP-binding proteins isolated from an electrode lobe library of the marine ray Discopyge ommata (Ngsee et al., J. Biol. Chem. 266 (4), 2675-2680 (1991)), some of which were determined to be homologs of the rab1, ra1, Krev, and rho LMW GTP-binding proteins. These proteins were localized to cholinergic synaptic vesicles and at least two of these proteins, oral and o-rho, were localized to the pre-synaptic terminals (Ngsee et al., J. Biol. Chem. 266 (4), 2675-2680 (1991)). Rab proteins are important for regulating the targeting and fusion of membranous vesicles during organelle assembly and transport. Rab proteins undergo controlled exchange of GTP for GDP, and they hydrolyze GTP in a reaction that may regulate the timing and unidirectional nature of these assemblies. Generally, known Rab proteins terminate in sequences such as cys-X-cys (e.g., RAB3A), cys-cys (e.g., RAB1A), or a similar sequence, and generally all are geranylgeranylated. Rab GTP-binding proteins are similar to YPT1/SEC4 in Saccharomyces cerevisiae , which are critical for transport along the exocytic route (Chavrier et al., Mol Cell Biol 1990 December;10(12):6578-85). Different Rab proteins are presumed to control different steps in membrane traffic, leading to a high level of diversity and complexity within the Rab family (Chavrier et al., Mol Cell Biol 1990 December;10(12):6578-85). The Rab1 gene maps in close viscinity to the ‘wobbler’ spinal muscular atrophy gene. Rab1 and Rab2 from the snail Lymnaea stagnalis share a very high degree (95-97%) of sequence identity with mammalian Rab1 and Rab2 over the first 178-191 N-terminal amino acids; however, the C-terminal region is almost completely divergent, except for the final 24 amino acids at the extreme ends. Rab1 was found to be highly expressed in the albumin gland of Lymnaea stagnalis , suggesting an important role in that gland (Agterberg et al., Eur. J. Biochem. 217 (1), 241-246 (1993)). The tethering factor p115 is a RAB1 effector that binds directly to activated RAB1. It is thought that RAB1-regulated assembly of functional effector-SNARE complexes serves as a conserved molecular mechanism for regulating recognition between different subcellular compartments such as endoplasmic reticulum and Golgi apparatus (Allan et al., Science 289: 444-448, 2000). GTPases play important roles in a wide variety of cell functions such as signal transduction, cytoskeletal organization, and membrane trafficking. Rab GTPases are particularly important for regulating cellular membrane dynamics by modulating the activity of effector proteins that then regulate vesicle trafficking. The Rab8 GTPase plays important roles in Golgi to plasma membrane vesicle trafficking. Studies have suggested that Rab37 plays an important role in mast cell degranulation. Thus, novel human Rab GTPases may be valuable as potential therapeutic targets for the development of allergy treatments (Masuda et al., FEBS Lett 2000 Mar. 17;470). Rab5 may act, together with Rab3A, to regulate synaptic vesicle membrane flow within nerve terminals, thereby regulating neurotransmitter release. Rab15 and Rab3A are low molecular weight GTP-binding proteins. Rab proteins are generally comprised of four conserved structural domains necessary for GTP binding, as well as additional domains for membrane localization and effector protein interactions. Rab15 is expressed primarily in neural tissues such as the brain and is localized to synaptic vesicles (Elferink et al., J. Biol. Chem. 267 (9), 5768-5775 (1992)). For a further review of Rab proteins, see Wedemeyer et al., Genomics 32: 447-454, 1996 and Zahraoui et al., J. Biol. Chem. 264: 12394-12401, 1989. Due to their importance in human physiology, particularly in regulating membrane trafficking, novel human Ras-like GTPase proteins/genes, such as provided by the present invention, are valuable as potential targets for the development of therapeutics to treat a wide variety of diseases/disorders caused or influenced by defects in membrane trafficking. Furthermore, SNPs in Ras-like GTPase genes are valuable markers for the diagnosis, prognosis, prevention, and/or treatment of such diseases/disorders. Using the information provided by the present invention, reagents such as probes/primers for detecting the SNPs or the expression of the protein/gene provided herein may be readily developed and, if desired, incorporated into kit formats such as nucleic acid arrays, primer extension reactions coupled with mass spec detection (for SNP detection), or TaqMan PCR assays (Applied Biosystems, Foster City, Calif.). The discovery of new human Ras-like proteins and the polynucleotides that encode them satisfies a need in the art by providing new compositions that are useful in the diagnosis, prevention, and treatment of inflammation and disorders associated with cell proliferation and apoptosis.
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is based in part on the identification of amino acid sequences of human Ras-like protein polypeptides and proteins that are related to the Ras-like GTPase protein subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate Ras-like protein activity in cells and tissues that express the Ras-like protein. Experimental data as provided in FIG. 1 indicates expression in bone marrow, stem cells, and leukocytes.

Mobile sleeve structure for maintaining spatial relationship between vertebrae
An arrangement and method of use for supporting a plurality of vertebrae in a cervical column in predetermined spatial relation and for facilitating a spinal fusion procedure. A plurality of templates (12, 14) couple to respectively associated vertebrae at anterior lateral surfaces thereof. Such coupling is effected with fasterners, such as bone screws (19). The templates are slidingly coupled to each other by a coupler assembly (22) whereby they are displaceable along a path over a limited distance that is substantially parallel to the longitudinal axis of the cervical column. The sliding displacement between the templates is limited by a pair of protruding tongues (27) that extend through elongated apertures (29). The elongated dimension of the apertures determines the extent of sliding travel, which extent is established to accommodate subsidence of the bone fusion and to prevent separation of the sliding elements of the coupler assembly. The coupler assembly is coupled to each template by engagement between posts (18) and post holes (15). Additionally, locking plates and fasteners, such as threaded fasteners, are used to ensure the security of the engagement between the templates and the coupler assembly. Sequential replication of the arrangement enables three or more vertebra to be supported in the determined spatial relation.
1. An implantable mobile sleeve for stabilizing the anterior side of a vertebral column, comprising: a lowermost template having respective anterior and posterior surfaces, and a through-hole penetrating through the anterior and posterior surfaces, wherein the through-hole is configured to receive a fastener, said lowermost template having a width dimension that is greater than a height dimension; an uppermost template having respective anterior and posterior surfaces, and a through-hole penetrating through the anterior and posterior surfaces, wherein the through-hole is configured to receive a fastener, said uppermost template having a width dimension that is greater than a height dimension; a coupling assembly for coupling said lowermost and uppermost templates adjustably to one another, said coupling assembly extending substantially vertically and orthogonal with respect to the width dimensions of said lowermost and uppermost templates, said coupling assembly having; a bottom sleeve portion having a first end for coupling to said lowermost template and a second end extending upward toward said uppermost template, and a top sleeve portion having a first end with at least one post aperture for coupling to said uppermost template, and a second end that extends toward said lowermost template and configured to terminate at an opening that forms a hollow longitudinal channel that extends between the first and second ends of the top sleeve portion, the second end of the top sleeve portion being configured to engage slidably with the second end of the bottom sleeve portion, and a locking plate for securing a selectable one of the top and bottom sleeve portions to its corresponding template. 2. The implantable mobile sleeve structure of claim 1 wherein the bottom sleeve of said coupling assembly has a greater thickness at the second end thereof than at its first end. 3. The implantable mobile sleeve structure of claim 1 wherein the lowermost template is arranged for coupling to the anterior region of a first cervical vertebra and the uppermost template is arranged for coupling to the anterior region of a second cervical vertebra. 4. The implantable mobile sleeve structure of claim 3 wherein the uppermost and lowermost templates are each formed in response to the curvatures of corresponding surfaces of the first and second vertebrae. 5. The implantable mobile sleeve structure of claim 1, wherein said top sleeve portion is configured to have a channel for accommodating slidingly the second end of said bottom sleeve portion. 6. The implantable mobile sleeve structure of claim 5 further comprising: a tongue extending orthogonally from the second end of said bottom sleeve portion, and an elongated aperture disposed in the second portion of said top sleeve portion, said elongated aperture accommodating therein said tongue, whereby a displacement limit is imposed on the slidable engagement between the bottom and top sleeve portions. 7. The implantable mobile sleeve structure of claim 6, wherein the elongated aperture is disposed in a base portion of the channel. 8. The implantable mobile sleeve structure of claim 1, wherein said uppermost and lowermost templates communicate exclusively on an anterior substantially vertical portion of their corresponding vertebrae. 9. An implantable mobile sleeve for stabilizing the anterior side of a vertebral column, comprising: a lowermost template having a generally rectangular configuration having an elongated width dimension perpendicular to the axis of a vertebral column, said lowermost template having an anterior surface, a posterior surface for communicating with exclusively with a lateral anterior surface of a vertebra, and an outer edge extending between the anterior surface and the posterior surface, there being further provided at least two pre-drilled through-holes penetrating through the lowermost template between the anterior surface to the posterior surface; an uppermost template having a generally rectangular configuration having an elongated width dimension perpendicular to the axis of a vertebral column, the uppermost template having an anterior surface, a posterior surface that will rest against a second vertebra and, an outer edge between extending between the anterior surface and the posterior surface, there being further provided two or more pre-drilled through-holes penetrating through the uppermost template between the anterior surface to the posterior surface; at least two posts attached to the anterior surface of the uppermost template and the lowermost template, and arranged to extend orthogonally outward from the anterior surface; a plurality of fasteners disposed through the at least two pre-drilled holes of the uppermost template and the lowermost template, corresponding ones of said plurality of fasteners attaching the lowermost template to the first vertebra and the uppermost template to the second vertebra, wherein the lengthwise side of the lowermost template and the lengthwise side of the uppermost template are parallel when the lowermost template is attached to the first vertebra and the uppermost template is attached to the second vertebra, and a generally rectangular shaped adjustable sleeve having a longitudinal dimension thereof extending parallel to a longitudinal axis of the vertebral column, the adjustable sleeve having a bottom sleeve portion having a first end with at least two pre-drilled post holes for coupling the bottom sleeve portion to the posts located on the lowermost template and a second end that extends upward toward a top sleeve portion of the adjustable sleeve having a first end with two or more pre-drilled post holes that couple the top sleeve portion to the posts located on the uppermost template, wherein the top sleeve portion extends downward toward the lowermost template and terminates at a second end that forms an opening to a hollow longitudinal channel that extends between the first end and second end of the top sleeve portion, the second end and the hollow sleeve being configured to receive slidably the second end of the bottom sleeve portion; a sliding displacement limiter arrangement located on the anterior surface of the top sleeve portion of the adjustable sleeve and having a tongue portion attached to the anterior surface of the bottom sleeve portion that is positioned in the hollow channel of the top sleeve portion, wherein the tongue extends through and slidably connects to an elongated aperture disposed through the anterior surface of the top sleeve portion, and first and second locking plates each associated with one of said uppermost and lowermost templates, said first and second locking plates each having at least two pre-drilled post holes for engaging with the posts of the lowermost template and the uppermost template and thereby securing the adjustable sleeve to the uppermost template and the lowermost template. 10. The implantable mobile sleeve structure of claim 9 further comprising: a second bottom sleeve portion of a second adjustable sleeve placed onto the posts of the uppermost template over the top sleeve portion and under said first locking plate; a second uppermost template having a generally rectangular shape with the lengthwise side of the rectangle extending perpendicular to the axis of a vertebral column, the second uppermost template having an anterior surface, a posterior surface that communicates with a third vertebra, and an outer edge between extending between the anterior surface and the posterior surface, and at least two pre-drilled through-holes penetrating through the second uppermost template between the anterior surface to the posterior surface, wherein the pre-drilled holes are configured to receive two or more fasteners; two posts attached to the anterior surface of the second uppermost template and arranged to extend orthogonally outward therefrom; two fasteners respectively disposed through the two pre-drilled holes of the second uppermost template, each fastener attaching the second uppermost template to the third vertebra, wherein the lengthwise side of the second lowermost template and the lengthwise side of the second uppermost template are parallel when the lowermost template is attached the uppermost template and the second uppermost template is attached to the third vertebra, and a second top sleeve portion of a second adjustable sleeve installed on the posts. 11. The implantable mobile sleeve structure of claim 10 wherein the vertebra are part of an anterior cervical vertebral column. 12. A method of attaching a mobile sleeve between two adjacent vertebra, the method comprising the steps of: attaching a lowermost template to the anterior vertebral body of a first vertebra of a vertebral column using fasteners disposed through pre-drilled holes in the lowermost template; attaching an uppermost template to the anterior vertebral body of a second vertebra of the vertebral column located above and adjacent to the first vertebra using fasteners disposed through pre-drilled holes in the uppermost template; bending a bottom sleeve portion of an adjustable sleeve to conform to the curvature of the vertebral column; placing a first end of the bottom sleeve portion of the adjustable sleeve using pre-drilled post holes located at the first end of the bottom sleeve portion onto a set of posts that extend outward from the anterior surface of the lowermost template; placing a first end of a top sleeve portion of the adjustable sleeve using pre-drilled post holes located at the first end of the top sleeve portion onto posts that extend out of the anterior surface of the uppermost template, and attaching a locking plate onto the posts of the lowermost template and the uppermost template over the adjustable sleeve and the lowermost template and the uppermost template. 13. The method of claim 12 further comprising the further steps of: removing the locking plate located on top of the uppermost template; attaching a second uppermost template of a second mobile sleeve to the anterior vertebral body of a third vertebra located above and adjacent to the second vertebra using fasteners disposed through pre-drilled holes in the second uppermost template; bending a second bottom sleeve portion of a second adjustable sleeve to conform to the curvature of the vertebral column; placing a first end of the second bottom sleeve portion of the second adjustable sleeve using pre-drilled post holes located at the first end the second bottom sleeve portion onto a set of posts that extend outward from the anterior surface of the top plate of the first mobile sleeve; placing a first end of a second top sleeve portion of the second adjustable sleeve using pre-drilled post holes located at the first end the second top sleeve portion onto posts that extend out of the anterior surface of the second uppermost template; attaching a locking plate onto the posts of the uppermost template over the adjustable sleeve and the second adjustable sleeve, and attaching a locking plate onto the posts of the second uppermost template over the adjustable sleeve and the second uppermost template. 14. The method of claim 13 wherein the vertebral column is the an anterior cervical column.
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention This invention relates generally to spinal implant systems, and more particularly, to a mobile sleeve structure that maintains a determinable spatial relationship between adjacent vertebrae. 2. Description of the Related Art There is a need in the field of neurosurgery for spinal implant systems that enable surgeons to maintain a determinable spatial relationship between adjacent vertebrae during vertebral fusion procedures. A typical vertebral fusion procedure involves implanting a bone graft or cage element into the intervertebral region known that normally would be occupied by a disc. Patients that suffer from degenerative disc disorders may be required to undergo a surgical procedure that involves the fusing together ofthe adjacent vertebrae that are located immediately above and below the disc region. During a vertebral fusion procedure, bone grafts are inserted into the disc region to facilitate fusion of the vertebrae by promoting growth of bone in the disc. The result is that the vertebrae will eventually fuse together. Such patients may additionally require a series of such bone grafts that will result in the fusing together of several vertebrae in the cervical vertebral column. A problem with vertebral fusion procedures is that once the bone grafts are implanted they are prone to becoming misplace due to movement of the adjacent vertebrae. In response to this problem several systems have been developed that will immobilize the adjacent vertebrae by attaching a plate between the adjacent vertebrae. The attached plate will maintain the spatial relationship between the vertebrae and promote fusion by preventing slippage of the bone grafts during the fusion process. One known system for maintaining spinal elements in a desired spatial relationship incorporates a series of plates having predetermined heights and shapes. Each such plate has a plurality of apertures disposed at lengthwise distal ends of the plate. Bone screws are threaded through the apertures to attach the plate to a corresponding one of a pair of anterior cervical vertebrae. The plates so attached maintain a spatial relationship between the two adjacent vertebrae that are located immediately above and below a bone graft implant. In the known system, the plates are attached to extend longitudinally with respect to the axis of an anterior cervical vertebral column. It is a deficiency of the above system that plates of various heights must be prefabricated and made available during the procedure because the distance between respective pairs of vertebrae will vary along the length of the anterior cervical column. Thus, upon the conclusion of the procedure there will remain several unused plates that will have to be disposed of or sterilized. Another problem with the known system is that the plates are only used between two vertebrae and cannot be used to maintain a desired spatial relationship between three or more vertebrae. Three or more vertebrae may need to be secured in predetermined relation to one another when the patient is diagnosed to require more than one bone graft implant. Some systems have been developed that use a one-piece rod that can be attached across several vertebrae. A problem with the known rod system is that it does not accommodate the natural lordotic curvature of an individual patient's spinal column as the lordotic curvature varies from patient to patient.
<SOH> SUMMARY OF THE INVENTION <EOH>The foregoing and other objects are achieved by this invention which provides an implantable mobile sleeve for stabilizing the anterior side of a vertebral column. The implantable mobile sleeve has a group ofgenerally rectangular components that generally have an “T” shape. The mobile sleeve has a lowermost template with a generally rectangular shape, in that the lowermost template has two distal length dimensions that are longer than the dimensions of two opposing sides that constitute the width of the plate. When mounted to a vertebra the lengthwise sides of the rectangular shaped lowermost template will extend in a direction substantially perpendicular to the axis of the vertebral column. The lowermost template has a posterior surface that rests against the surface of a vertebra, and an opposing anterior surface that faces outward from the surface of the vertebra. The lowermost template has two or more pre-drilled apertures that penetrate the lowermost template from the posterior surface to the anterior surface. The pre-drilled apertures are configured to receive a fastener to attach the lowermost template to the vertebra. Two posts extend substantially orthogonally outward from the anterior surface. The posts facilitate the fastening of other components of the mobile sleeve to the lowermost template. In some embodiments, the posts are threaded so that a fastener can be used to fix components that are installed on the posts. In one embodiment of the invention, the mobile sleeve is provided with an uppermost template having a generally rectangular shape. The uppermost template is substantially identical to the lowermost template, except that the uppermost template is mounted to a second vertebra located above the first vertebra where the bottom plate is mounted. During attachment, the mobile sleeve is attached between adjacent vertebrae on the vertebral column. The lowermost template will be coupled to a first vertebra and the uppermost template will be coupled to a second vertebra disposed above the first vertebra. In a preferred embodiment, the templates are secured to the anterior region of a cervical vertebral column. However, the mobile sleeve structure described herein may additionally be attached to other regions of the cervical vertebral column, or to other areas of the spine. Both the uppermost template and the lowermost templates are each affixed to vertebrae using two or more fasteners disposed through the two or more pre-drilled through holes located on the top and lowermost templates. The fasteners, in this embodiment, are be inserted into the vertebrae at respective angles in order to prevent the templates from being pulled away from the vertebrae. The fasteners attach the lowermost template to the first vertebra and the uppermost template to the second vertebra. Such fasteners can, in certain embodiments of the invention, be threaded bone screws, pins, or any other suitable fastener for attaching the templates to the vertebrae. While only two bone screws are described herein as being used in the practice of the illustrative embodiment of the invention, persons of skill in the art can, in light of the teaching herein, employ a different number of fasteners depending on conditions encountered during the procedure, such as vertebral size or bone density, spatial positioning of the vertebrae, and the strength of attachment required by the physician. In other embodiments, one or both of the top and lowermost templates are affixed to the vertebrae using adhesives that are applied over the posterior surface of the templates. Once the bottom plate and the uppermost template are attached to the respective first and second vertebrae, it is preferred that the lengthwise sides of the bottom and uppermost templates be parallel. In a further embodiment of the invention, a generally rectangular adjustable sleeve slidably connects the uppermost template and the lowermost template by means of the aforementioned posts and/or apertures through, the templates. It is an advantage of the invention that perfect placement of the adjustable sleeve is achieved because the posts extend generally perpendicular with respect to the midportion of the anterior vertebral body. The adjustable sleeve has a bottom sleeve portion that perpendicularly couples at a first end to the middle region of the lowermost template. A second end of the bottom sleeve portion extends upward toward the uppermost template. With respect to the distance between the anterior and posterior surfaces, the bottom sleeve portion will have a thickness at the first end that differs from that of its second end. In some embodiments of the invention, the thickness at the first end is about 1 mm and will increase to about 2 mm in the middle region and at second end of the bottom sleeve portion. The reason for the varying thickness is so that several adjustable sleeves can be stacked onto the posts of the lowermost template without any large protrusions in the lowermost template area. The thickness of the lowermost template is not limited to 1 mm and 2 mm since sleeves of various thicknesses will be needed depending on the needs of a particular surgical procedure. In a further embodiment, the adjustable sleeve has a top sleeve portion with a first end that couples to post holes in the middle region of the uppermost template. The top sleeve portion also extends downward toward the bottom sleeve portion and terminates at a second end. With regard to thickness (i.e., the distance between the anterior and posterior surfaces), the top sleeve portion will have a different thickness at the first end than at the second end of the top sleeve portion. Illustratively, the thickness at the first end can be about 1 mm and will increase to about 2 mm as for the middle and second end of the top sleeve portion. The reason for the varying thickness is so that several adjustable sleeves can be stacked onto the posts of the uppermost template without any large protrusions in the uppermost template area. The thickness of the uppermost template is not limited to 1 mm and 2 mm since sleeves of various thicknesses will be needed depending on the needs of a particular surgical procedure. The second end of the top sleeve portion also has an opening to a hollow channel that extends upward toward the first end of the top sleeve portion. The second end of the bottom sleeve portion enters the bottom end of the top sleeve portion and is slidably disposed through the hollow channel. The hollow channel affords mobility to the top third of the adjustable sleeve and allows the height of the mobile sleeve to adjust for post-operative subsidence of the vertebrae. The movement of the adjustable sleeve during subsidence enables the resulting load to be shared equally by all of the components of the mobile sleeve. Such distribution of the load increases the integrity of the mobile sleeve by preventing the damage to the templates, adjustable sleeve, or the vertebrae that may occur when the load is not equally distributed. In order to control sliding movement between the top and lowermost templates, a tongue and groove mechanism can, in certain embodiments, be incorporated along the anterior surface of the adjustable sleeve. One or more tongues protrude from the anterior surface of the bottom sleeve portion that is disposed through the hollow channel, extending orthogonally outward from the anterior surface. One or more grooves are located on the anterior surface of the top sleeve portion and are arranged to extend completely through the anterior side of the top sleeve portion, whereby the tongues will extend into and slide freely within the grooves. In the practice of this embodiment, the grooves are of sufficient length to allow a top sleeve portion and a bottom sleeve portion to slide relative to each other and thereby accommodate subsidence of the vertebrae. However, the grooves will not be so long as to permit the bottom sleeve portion to become separated from the top sleeve portion. The lowermost two thirds portion of the adjustable sleeve (i.e., the lower portion of the bottom sleeve portion) is formed of a solid material that can be bent with a plate bender so that the adjustable sleeve will correspond substantially to the curvature of the vertebral column. Although the adjustable sleeve is described herein as having a top third portion that can be adjusted for length and a bottom two thirds that are solid, different ratios are achievable depending upon the region to which the mobile sleeve is to be attached, or the particular needs of a patient. In a further embodiment, a locking plate holds the adjustable sleeve and template together to allow the distribution of stress to all the parts of the mobile sleeve. Controlling the distribution of stress contributes to advantageous distribution of the load by ensuring that no one particular portion of the mobile sleeve is burdened excessively by the stress that is caused by the subsidence of the vertebrae. The locking plate in this embodiment has two or more post holes that are aligned and placed onto the two or more posts of the bottom or uppermost templates. In order to hold the locking plate in place, the post holes of the locking plate are configured to be secured onto the posts as the locking plate moves into place. In other embodiments, the posts of the uppermost template and the lowermost template are threaded so that a fastener, such as a nut, can be used to lock the locking plate in place. In a yet further embodiment of the invention, additional mobile sleeve structures are linked to form additional levels of structure to the spinal support system. The post holes can be used as a way of linking together mobile sleeve structures when a patient needs more than one implant. A second mobile sleeve is linked to a first mobile sleeve by removing the locking plate of either the uppermost template or the lowermost template, depending on whether the site of the next bone graft implant is above or below the uppermost template or the lowermost template of the first mobile sleeve. A second adjustable sleeve is then placed onto the post holes over the first adjustable sleeve and the locking plate is then placed back on to the holes. A second uppermost template or lowermost template (i. e., depending on whether the second sleeve is being place above or below the first mobile sleeve) is then attached to a vertebra so that the second end of the second adjustable sleeve is placed onto the posts of the second uppermost template or the second lowermost template. A locking plate then is placed over either the second uppermost template or the second lowermost template and the second end of the adjustable sleeve. While the second mobile sleeve has been described as attaching so that the mobile sleeves are attached across a series of three adjacent vertebra, the mobile sleeve structure may be attached to three vertebrae that span across more than three vertebrae of the vertebral column. In addition, more levels may be attached to the mobile sleeve structure to create a device that has any multiple number (i.e., two, three, four, etc.) of mobile sleeves in the structure. In accordance with a further aspect of the present invention, there is provided a method of attaching a mobile sleeve between two adjacent cervical vertebrae. The method includes the steps of: attaching a lowermost template to the anterior vertebral body of a first vertebra using fasteners disposed through pre-drilled holes in the lowermost template; attaching an uppermost template to the anterior vertebral body of a second vertebra located above and adjacent to the first vertebra; and installing fasteners through pre-drilled holes in the uppermost template. In a further embodiment of this method aspect of the invention, the first and second vertebrae are separated by one or more vertebrae, and therefore are not adjacent to one another. For instance, the uppermost template and the lowermost template can be attached to respective vertebra in a manner wherein the mobile sleeve spans across any number of vertebrae. In a further embodiment, there is provided the step of bending the bottom sleeve portion of an adjustable sleeve in response to the curvature of the vertebral column. Additionally, there are provided the steps of installing the first end of the bottom sleeve portion of the adjustable sleeve onto posts that extend outward of the anterior surface of the lowermost template, and further installing the first end of a top sleeve portion of the adjustable sleeve is placed onto posts that extend out of the anterior surface of the uppermost template. As previously described, each adjustable sleeve fits onto the posts using pre-drilled post holes located at the first ends of the bottom and top sleeve portions, respectively. In a further embodiment, there is provided the step of coupling the adjustable sleeve to the top and lowermost templates at a generally perpendicular angle. In a further embodiment of this method aspect of the invention, there is provided the step of installing a locking plate onto the posts of the uppermost template and the lowermost template over the adjustable sleeve and the uppermost template and lowermost template. Additionally, there is provided the step of locking the locking plate with a fastener. Illustratively, when a second level (i.e., a second mobile sleeve) is added to a vertebra located above the second vertebra, there is provided a step of removing the locking plate located on the uppermost template. Then, there is performed a step of attaching the second uppermost template of the second mobile sleeve to the anterior vertebral body of a third vertebra located above and adjacent to the second vertebra using fasteners disposed through pre-drilled holes in the uppermost template. In a highly advantageous embodiment of the method aspect of the invention, there is provided the step of bending the bottom sleeve portion of a second adjustable sleeve in response to the curvature of the vertebral column. A first end of the bottom sleeve portion of the second adjustable sleeve is placed onto posts that extend out of the anterior surface of the uppermost template of the first mobile sleeve. The first end of a top sleeve portion of the adjustable sleeve is placed onto posts that extend out of the anterior surface of the second uppermost template attached to the third vertebra. The second adjustable sleeve is configured to fit onto the posts using, pre-drilled post holes located at the first ends of the bottom sleeve portion and top sleeve portion. The adjustable sleeve couples the top and lowermost templates at a generally perpendicular angle. There is additionally provided the step of placing locking plates onto the posts of the uppermost template of the first mobile sleeve and the second uppermost template. In this embodiment, the locking plates are placed over the ends of the second adjustable sleeve and uppermost template of the first mobile sleeve and the second uppermost template. Then, in this embodiment, there is provided the step of affixing the locking plate with a fastener. It is to be understood that once the two mobile sleeve devices have been connected, additional levels can be added to this structure by simply repeating the steps recited for attaching the second mobile sleeve. The mobile sleeves may be coupled in a manner that permits the adjustable sleeve portion to span across more than one vertebrae, as described above. This provides the significant advantage that the interconnection of several levels of mobile sleeves will constitute a support structure that will be custom fitted to the lordotic curvature of the anterior cervical column.

Automatic optimization of a splice loss estimator for optical fiber splicers
A technique is provided for automatic optimization of a splice loss estimator of a fiber splicer (1), where the splice loss estimator is adapted, in a splice loss estimation procedure, to estimate the splice losses (Lti) of splices (i) of optical fibers as produced by the fiber splicer from images taken of the optical fibers at the splicing thereof, and the splice loss estimation procedure includes the use of splice loss estimation parameters (Pj). The estimator estimates splice losses based on information (Cij) obtained from the images and the estimation parameters. Further, the splice losses are measured by means of a measurement instrument (3). The estimated (Lti) and measured (LMi) splice losses, and the information obtained from the images are uploaded (71) into an off-line computer (5) and the key estimation parameters are automatically optimized by the selection of any solution within the Bellcore accuracy criteria (75), whereafter the optimized estimation parameters are downloaded (81) to the splicer.
1. An apparatus for automatic optimization of a splice loss estimator of an optical fiber splicer apparatus, said splice loss estimator being adapted, in a splice loss estimation procedure, to estimate the splice losses (LTi) of splices (i) of end portions of optical fibers as produced by said fiber splicer apparatus from at least one respective image taken of the respective end portions of the optical fibers prior to, during, or subsequent to the splicing thereof, said splice loss estimation procedure including the use of a set of splice loss estimation parameters (Pj), comprising a first input line for receiving, from said splice loss estimator, data of a plurality of splices of end portions of optical fibers as produced by said fiber splicer apparatus, wherein said data for each splice (i) include information (Cij) derived from the at least one image taken of the end portions of the optical fibers constituting that splice, and the estimated splice loss (LTi) of that splice; a second input line for receiving a splice loss value (LMi) for each of said plurality of splices of end portions of optical fibers as measured by a measurement equipment; means for determining a new set of splice loss estimation parameters from said data and said estimated and measured splice loss values such that said splice loss estimation procedure, using the new set of splice loss estimation parameters, estimates the splice loss of each of said plurality of splices of end portions of optical fibers with a given accuracy; and an output line for outputting said determined new set of splice loss estimation parameters to said splice loss estimator to replace the set of splice loss estimation parameters used in the splice loss estimation procedure of the splice loss estimator for the estimation of the splice losses of any further splices of end portions of optical fibers to be produced by said fiber splicer apparatus. 2. The apparatus as claimed in claim 1 wherein said data for each splice (i) include information (Cij) derived from hot images taken of the end portions of the optical fibers constituting that splice. 3. The apparatus as claimed in claim 2 wherein said data for each splice include information regarding mismatch of mode field diameter (MFD), including micro bending loss (LD), or fiber core/cladding deformation including cladding-to-core eccentricities, non-circularity of fibers, cleave angle of fiber ends, and/or cladding/core offsets. 4. The apparatus as claimed in claim 2 wherein said data for each splice include information regarding mismatch of hot core index (LI), including hot core index offset (V), fiber dopant concentrations, and/or fiber core diameters. 5. The apparatus as claimed in claim 1 wherein said data for each splice (i) include information (Cij) derived from cold images taken of the end portions of the optical fibers constituting that splice. 6. The apparatus as claimed in claim 5 wherein said data for each splice include information regarding macro bending, including view angle offset, fiber alignment, and/or presence of microdust on fiber surfaces. 7. The apparatus as claimed in claim 1 wherein said apparatus is a computer, preferably an off-line computer and/or a PC-platform based splicer, provided with suitable software and information as regards the splice loss estimation procedure used in the splice loss estimator. 8. The apparatus as claimed in claim 1 wherein said set of splice loss estimation parameters include any of an M factor, an F factor, a loss factor, a macro factor, a mode field diameter left (MFDL) factor, a mode field diameter right (MFDR) factor, and a splice loss shift (LS) factor, where the M and F factors are employed in the estimation of a core deformation induced splice loss (LM), the loss factor is employed in the estimation of a hot core index mismatch induced splice loss (LI) the macro factor, the mode field diameter left (MFDL) and right (MFDR) factors are employed in the estimation of a macro bending induced splice loss (LA) and the splice loss shift (LS) factor is employed in the estimation of a splice loss correction term, which corrects for any systematic errors in the estimation procedure. 9. The apparatus as claimed in claim 1 wherein said apparatus is adapted to receive, on said first input line, the set of estimation parameters as used by said splicer apparatus. 10. The apparatus as claimed in claim 1 wherein said apparatus is adapted to receive, on said first input line, a set of image processing parameters, wherein said set of image processing parameters are used in said splice loss estimation procedure by said splice loss estimator of the splicer apparatus; said means for determining is adapted to determine a new set of image processing parameters from said data and said estimated and measured splice loss values such that said splice loss estimation procedure, using the new set of image processing parameters, estimates the splice loss of each of said plurality of splices of end portions of optical fibers with a given accuracy; and said apparatus for automatic optimization is adapted to output, via said output line, said determined new set of image processing parameters to said splice estimator to replace the set of image processing parameters used in the splice loss estimation procedure of the splice estimator for the estimation of the splice losses of any further splices of end portions of optical fibers to be produced by said fiber splicer apparatus. 11. The apparatus as claimed in claim 1 wherein said plurality of splices are at least 50, more preferably at least 100, and most preferably between 100 and 500. 12. The apparatus as claimed in claim 1 wherein said means for determining is adapted to determine said new set of splice loss estimation parameters such that said new set of splice loss estimation parameters fulfils the Bellcore splice loss estimator criteria, preferably the 90% Bellcore criteria, and most preferably the 100% Bellcore criteria. 13. The apparatus as claimed in claim 1 wherein said means for determining is adapted to determine said new set of splice loss estimation parameters by using linear regression criteria to increase the probability that the estimated splice loss values using said new set of estimation parameters represent the measured splice losses. 14. (canceled) 15. A method for automatic optimization of a splice loss estimator of an optical fiber splicer apparatus, where said splice loss estimator is adapted, in a splice loss estimation procedure, to estimate the splice losses (LTi) of splices (i) of end portions of optical fibers as produced by said fiber splicer apparatus from at least one respective image taken of the respective end portions of the optical fibers prior to, during, or subsequent to the splicing thereof, said splice loss estimation procedure including the use of a set of splice loss estimation parameters (Pj), comprising the steps of: receiving, from said splice loss estimator, data of a plurality of splices of end portions of optical fibers as produced by said fiber splicer apparatus, wherein said data for each splice (i) include information (Cij) derived from the at least one image taken of the end portions of the optical fibers constituting that splice, and the estimated splice loss (LTi) of that splice; receiving a splice loss value (LMi) for each of said plurality of splices of end portions of optical fibers as measured by a measurement equipment; determining a new set of splice loss estimation parameters from said data and said estimated and measured splice loss values such that said splice loss estimation procedure, using the new set of splice loss estimation parameters, estimates the splice loss of each of said plurality of splices of end portions of optical fibers with a given accuracy; and outputting said determined new set of splice loss estimation parameters to said splice loss estimator to replace the set of splice loss estimation parameters used in the splice loss estimation procedure of the splice loss estimator for the estimation of the splice losses of any further splices of end portions of optical fibers to be produced by said fiber splicer apparatus. 16. The method as claimed in claim 15 wherein said data for each splice (i) include information (Cij) derived from hot images taken of the end portions of the optical fibers constituting that splice. 17. The method as claimed in claim 15 wherein said data for each splice (i) include information (Cij) derived from cold images taken of the end portions of the optical fibers constituting that splice. 18. The method as claimed in claim 15 wherein said set of splice loss estimation parameters include any of an M factor, an F factor, a loss factor, a macro factor, a mode field diameter left (MFDL) factor, a mode field diameter right (MFDR) factor, and a splice loss shift (LS) factor, where the M and F factors are employed in the estimation of a core deformation induced splice loss (LM), the loss factor is employed in the estimation of a hot core index mismatch induced splice loss (LI) the macro factor, the mode field diameter left (MFDL) and right (MFDR) factors are employed in the estimation of a macro bending induced splice loss (LA) and the splice loss shift (LS) factor is employed in the estimation of a splice loss correction term, which corrects for any systematic errors in the estimation procedure. 19. The method as claimed in claim 15 further comprising the steps of: receiving, from said splice estimator, a set of image processing parameters, wherein said set of image processing parameters are used in said splice loss estimation procedure by said splice loss estimator of the splicer apparatus; determining a new set of image processing parameters from said data and said estimated and measured splice loss values such that said splice loss estimation procedure, using the new set of image processing parameters, estimates the splice loss of each of said plurality of splices of end portions of optical fibers with a given accuracy; and outputting said determined new set of image processing parameters to said splice estimator to replace the set of image processing parameters used in the splice loss estimation procedure of the splice estimator for the estimation of the splice losses of any further splices of end portions of optical fibers to be produced by said fiber splicer apparatus. 20. The method as claimed in claim 15 wherein said new set of splice loss estimation parameters is determined such that said new set of splice loss estimation parameters fulfils the Bellcore splice loss estimator criteria, preferably the 90% Bellcore criteria, and most preferably the 100% Bellcore criteria. 21. The method as claimed in claim 15 wherein said new set of splice loss estimation parameters is determined by using linear regression criteria to increase the probability that the estimated splice loss values using said new set of estimation parameters represent the measured splice losses. 22. (canceled) 23. A method for automatic optimization of a splice loss estimator of an optical fiber splicer apparatus, where said splice loss estimator is adapted, in a splice loss estimation procedure, to estimate the splice losses (LTi) Of splices of end portions of optical fibers as produced by said fiber splicer apparatus from at least one respective image taken of the respective end portions of the optical fibers prior to, during, or subsequent to the splicing thereof, said splice loss estimation procedure including the use of a set of splice loss estimation parameters (Pj), comprising the steps of: producing a plurality of splices of end portions of optical fibers by means of said fiber splicer apparatus; deducing information from the at least one image taken for each splice produced; estimating the splice loss based on the information deduced from the at least one image for each splice produced; measuring the splice loss of each splice produced by means of a measurement instrument,; uploading the deduced information, the estimated splice losses and the measure splice losses to a processing device; determining a new set of splice loss estimation parameters from the deduced information and the estimated and measured splice loss values such that said splice loss estimation procedure, using the new set of splice loss estimation parameters, estimates the splice loss of each of said plurality of splices of end portions of optical fibers with a given accuracy; and downloading said determined new set of splice loss estimation parameters to said splice loss estimator of the fiber splicer apparatus to replace the set of splice loss estimation parameters used in the splice loss estimation procedure of the splice loss estimator for the estimation of the splice losses of any further splices of end portions of optical fibers to be produced by said fiber splicer apparatus.
<SOH> DESCRIPTION OF RELATED ART AND BACKGROUND OF THE INVENTION <EOH>The technology for forming low-loss optical fibers has advanced to a point where there is widespread commercial manufacturing of optical fibers. Most processing includes drawing an optical fiber from a previously manufactured glass boule, sometimes referred to as a fiber preform. Extremely long lengths of fiber can be obtained by splicing a plurality of lengths, which are obtained using current fusion splicer techniques. Additionally, it has become increasingly more common to splice optical fibers, which have broken, either accidentally, or during appropriate proof testing. For these and other applications, splicing in which the coating material is removed from end portions of two fibers, which are then fused together end to end, provides a suitable means for joining the ends of two glass fibers with an acceptably low splice loss. Fusion splice losses depend mainly on the mismatch of mode-field diameter (MFD) and misalignment of the cladding/core of two fibers due to either the fiber refractive index design or the inappropriate fusion processes being used. With the help of image techniques, information on the MFD mismatch can be obtained and analyzed by monitoring the deformation/misalignment of the cladding/core of the fibers during the fusion process, see e.g. EP 1 014 070. Such a monitoring system includes typically a charged-coupled device (CCD) camera equipped with an image processor. By use of suitable theoretical models and the information obtained from the images, an estimation method for evaluation of splice losses can be established. The estimation method as a passive technique for evaluation of splice losses is widely used in most automated fusion splicers of today. Different models for evaluation of splice loss have been explored and developed during the past two decades. Well-known methods for splice loss estimation include the butt-joint approximation and the mode coupling theory. More advanced methods, e.g. hot-image techniques for real-time analysis of cladding and core deformation have also been developed. In order to achieve the best performance and maximize the flexibility of the estimation method, the models used for splice loss estimation include usually a number of free parameters, called estimation parameters. The optimization of these estimation parameters in order to achieve the best performance of the estimation method has nowadays become one of essential features in the development of splicing techniques. In practice, the estimation parameters are manually optimized according to different types of on-test fibers and the fusion processes being used. Due to primarily technical reasons and rather complicated nature of the involved splicing processes, the optimization of the estimation parameters is a quite difficult and time consuming job that may only be performed by very experienced engineers. The time typically needed for manually optimizing the estimation parameters on a given fiber combination is as long as a few days which is hardly to be accepted by splicer users, especially, when frequently changes of fusion processes and fiber combinations are involved, e.g. in the manufacture of erbium doped fiber amplifiers (EDFA). This is hardly acceptable for the splicer user, and such optimization has thus typically to be performed by the fiber splicer manufacturer.
<SOH> SUMMARY OF THE INVENTION <EOH>The present inventors have realized that the splicer user does not need to re-optimize the image processing parameters, but only the estimation parameters. For a fiber splicer user, only a limited number of the image processing parameters can be optimized, and most of these parameters are already designed to be linear to the splice loss by an embedded algorithm in the splicer. In the present invention a new technique for fast automatic optimization of the performance of the estimator is proposed. The technique leaves the image processing of the cold and hot images taken during the fusion processes unchanged. The built-in estimator in the splicer analyzes the causes of splice losses based on the information obtained from the images, and based on the fusion process employed and the kind of spliced fibers. Further, splice losses are measured by means of a measurement instrument, e.g. a power meter. The results of the analysis and the measured splice losses are uploaded into a spreadsheet-based worksheet in an off-line computer and/or a PC-platform based splicer to form a splice database. Then, the manual procedures for optimization of estimation parameters are simulated. The key estimation parameters are automatically optimized by the selection of any solution within the Bellcore accuracy criteria, or the best solution within the Bellcore accuracy criteria by assistance of regression analysis criteria, whereafter the optimized estimation parameters are downloaded to splicer. The time needed for optimizing a splice database with hundred splices is significantly reduced down to a few seconds only. Thus, an enhanced performance of the estimator is automatically achieved. The tedious and complicated work for manually optimizing the estimator is automated. It is a main object of the present invention to provide an apparatus for automatic optimization of a splice loss estimator of an optical fiber splicer, wherein the splice loss estimator is adapted, in a splice loss estimation procedure, to estimate the splice losses of splices of end portions of optical fibers as produced by said fiber splicer apparatus from at least one respective image taken of the respective end portions of the optical fibers prior to, during, or subsequent to the splicing thereof, and wherein the splice loss estimation procedure includes the use of a set of splice loss estimation parameters, which apparatus for automatic optimization is fast, and which is particularly suited to be used by a fiber splicer user. It is in this respect a particular object of the invention to provide such apparatus, which is simple, easy to use, accurate, precise and of low cost. These objects, among others, are according to a first aspect of the present invention attained by an apparatus comprising a first input line; a second input line; means for determining (preferably a personal computer provided with suitable software); and an output line. The first input line is provided for receiving, from the splice loss estimator, data of a plurality of splices of end portions of optical fibers as produced by the fiber splicer apparatus, wherein the data for each splice include information derived from the image taken of the end portions of the optical fibers constituting that splice, and the estimated splice loss of that splice; and the second input line is provided for receiving a splice loss value for each of the plurality of splices of end portions of optical fibers as measured by a measurement equipment. The means for determining is adapted to determine or calculate a new set of splice loss estimation parameters from the data and the measured splice loss values such that the splice loss estimation procedure, using the new set of splice loss estimation parameters, estimates the splice loss of each of the plurality of splices of end portions of optical fibers with a given accuracy. Finally, the output line is provided for outputting the determined or calculated new set of splice loss estimation parameters to the splice loss estimator of the fiber splicer apparatus to replace the set of splice loss estimation parameters used in the splice loss estimation procedure of the splice loss estimator for the estimation of the splice losses of any further splices of end portions of optical fibers to be produced by the fiber splicer apparatus. Further, the above-mentioned objects, among others, are according to a second aspect of the present invention attained by an optical fiber splicer system comprising such an apparatus for automatic optimization. It is a further object of the present invention to provide a method for automatic optimization of the above-depicted splice loss estimator, which is fast and which is thus suitable to be used by the fiber splicer user. This object is according to a third aspect of the present invention attained by a method comprising the following steps: (i) receiving, from said splice loss estimator, data of a plurality of splices of end portions of optical fibers as produced by said fiber splicer apparatus, wherein said data for each splice include information derived from the at least one image taken of the end portions of the optical fibers constituting that splice, and the estimated splice loss of that splice; (ii) receiving a splice loss value for each of said plurality of splices of end portions of optical fibers as measured by a measurement equipment; (iii) determining a new set of splice loss estimation parameters from said data and said measured splice loss values such that said splice loss estimation procedure, using the new set of splice loss estimation parameters, estimates the splice loss of each of said plurality of splices of end portions of optical fibers with a given accuracy; and (iv) outputting said determined new set of splice loss estimation parameters to said splice loss estimator to replace the set of splice loss estimation parameters used in the splice loss estimation procedure of the splice loss estimator for the estimation of the splice losses of any further splices of end portions of optical fibers to be produced by said fiber splicer apparatus. Finally, according to a fourth aspect of the present invention there is provided a computer program product loadable into the internal memory of a computer, which comprises software code portions for performing the above-described method when the computer program product is run on the computer. Further characteristics of the invention and advantages thereof will be evident from the detailed description of preferred embodiments of the invention given hereinbelow and the accompanying drawings, which are given by way of illustration only, and thus are not limitative of the present invention.

Alphanubeta3 integrin-binding polypeptide monobodies and their use
The present invention relates to a polypeptide monobody which includes a modified acid sequence and renders the polypeptide monobody able to bind selectively to αvβ3 integrin. Fusion proteins and conjugates which include the polypeptide monobody, as well as compositions containing the polypeptide monobody, fusion proteins, or conjugates are also disclosed. Uses thereof include: treating or preventing an αvβ3 integrin-mediated disease or disorder, inhibiting αvβ3 integrin activity, treating a cancerous or precancerous condition, imaging tissues using positron emission tomography or magnetic resonance imaging, assessing the metastatic characteristics of a tumor, and delivering DNA to a cell.
1. A polypeptide monobody comprising: at least two Fn3 β-strand domain sequences with a loop region sequence linked between adjacent β-strand domain sequences; and optionally, an N-terminal tail of at least about 2 amino acids, a C-terminal tail of at least about 2 amino acids, or both; wherein at least one loop region sequence, the N-terminal tail, or the C-terminal tail comprises a modified amino acid sequence which varies by deletion, insertion, or replacement of at least two amino acids from a corresponding loop region, N-terminal tail, or C-terminal tail in a wild-type Fn3 domain of fibronectin, and wherein the polypeptide monobody binds selectively to αvβ3 integrin. 2. The polypeptide monobody according to claim 1, wherein the modified amino acid sequence comprises XRGDWXEX where X is any amino acid (SEQ ID No: 26). 3. The polypeptide monobody according to claim 2, wherein the modified amino acid sequence comprises LRGDWSED (SEQ ID No: 5), VRGDWYEY (SEQ ID No: 6), GRGDWTEH (SEQ ID No: 10), ARGDWVEG (SEQ ID No: 11), PRGDWTEG (SEQ ID No: 12), PRGDWIEF (SEQ ID No: 15), PRGDWNEG (SEQ ID No: 23), or FRGDWIEL (SEQ ID No: 24). 4. The polypeptide monobody according to claim 1, wherein said at least one loop region sequence is selected from the group consisting of a BC loop region sequence, a DE loop region sequence, an FG loop region sequence, and combinations thereof. 5. The polypeptide monobody according to claim 4, wherein said at least one loop region sequence is an FG loop region sequence. 6. The polypeptide monobody according to claim 5, wherein the FG loop region sequence comprises, as the modified amino acid sequence, XRGDWXEX where X is any amino acid (SEQ ID No: 26). 7. The polypeptide monobody according to claim 6, wherein the modified amino acid sequence comprises LRGDWSED (SEQ ID No: 5), VRGDWYEY (SEQ ID No: 6), GRGDWTEH (SEQ ID No: 10), ARGDWVEG (SEQ ID No: 11), PRGDWTEG (SEQ ID No: 12), PRGDWIEF (SEQ ID No: 15), PRGDWNEG (SEQ ID No: 23), or FRGDWIEL (SEQ ID No: 24). 8. A nucleic acid molecule encoding the polypeptide monobody of claim 1. 9. The nucleic acid molecule according to claim 8, wherein the nucleic acid is DNA. 10. A DNA construct comprising: the DNA molecule of claim 9; a promoter-effective DNA molecule operably coupled 5′ of the DNA molecule; and a transcription termination DNA molecule operably coupled 3′ of the DNA molecule. 11. An expression vector into which is inserted a DNA construct according to claim 9. 12. A host cell transformed with a DNA construct according to claim 10. 13. The host cell according to claim 12, wherein the host cell is a prokaryote. 14. The host cell according to claim 12, wherein the host cell is a eukaryote. 15. A fusion protein comprising: a polypeptide monobody according to claim 1 and a second polypeptide linked by peptide bond to the polypeptide monobody, the second polypeptide being (i) an epitope tag polypeptide, (ii) a detectable marker polypeptide, (iii) a metal ion-complexing polypeptide, or (iv) a DNA-binding polypeptide. 16. The fusion protein according to claim 15 wherein the modified amino acid sequence of the polypeptide monobody comprises XRGDWXEX (SEQ ID No: 26) where X is any amino acid. 17. The fusion protein according to claim 15 wherein the second polypeptide is an epitope tag polypeptide. 18. The fusion protein according to claim 17 wherein the epitope tag comprises a polyhistidine amino acid sequence. 19. The fusion protein according to claim 15 wherein the second polypeptide is a detectable marker polypeptide. 20. The fusion protein according to claim 19 wherein the detectable marker polypeptide is an alkaline phosphatase. 21. The fusion protein according to claim 15 wherein the second polypeptide is a metal ion-complexing polypeptide. 22. The fusion protein according to claim 21 wherein the metal-ion complexing polypeptide is a polyhistidine amino acid sequence. 23. The fusion protein according to claim 15 wherein the second polypeptide is a DNA-binding polypeptide. 24. The fusion protein according to claim 23 wherein the DNA-binding polypeptide comprises a polylysine amino acid sequence. 25. A fusion protein-metal ion complex comprising a fusion protein according to claim 21 which is complexed with a metal ion. 26. The fusion protein-metal ion complex according to claim 25 wherein the metal ion is a positron emitting metal ion, a paramagnetic metal ion, a radioactive metal ion, or a non-radioactive metal ion. 27. The fusion protein-metal ion complex according to claim 25 wherein the modified amino acid sequence of the polypeptide monobody comprises XRGDWXEX where X is any amino acid (SEQ ID No: 26). 28. A fusion protein-DNA complex comprising a fusion protein according to claim 23 which is complexed with a DNA molecule via the DNA-binding polypeptide. 29. The fusion protein-DNA complex according to claim 28 wherein the DNA-binding polypeptide comprises a polylysine amino acid sequence. 30. The fusion protein-DNA complex according to claim 28 wherein the modified amino acid sequence of the polypeptide monobody comprises XRGDWXEX where X is any amino acid (SEQ ID No: 26). 31. A nucleic acid molecule encoding the fusion protein of claim 15. 32. The nucleic acid molecule according to claim 31, wherein the nucleic acid is DNA. 33. A DNA construct comprising: the DNA molecule of claim 32; a promoter-effective DNA molecule operably coupled 5′ of the DNA molecule; and a transcription termination DNA molecule operably coupled 3′ of the DNA molecule. 34. An expression vector into which is inserted a DNA construct according to claim 33. 35. A host cell transformed with a DNA construct according to claim 33. 36. The host cell according to claim 35, wherein the host cell is a prokaryote. 37. The host cell according to claim 35, wherein the host cell is a eukaryote. 38. A conjugate comprising a polypeptide monobody according to claim 1 conjugated to (i) a chemotherapeutic agent, (ii) a contrasting agent, or (iii) an organic chelating agent. 39. The conjugate according to claim 38 wherein the modified amino acid sequence of the polypeptide monobody comprises XRGDWXEX where X is any amino acid (SEQ ID No: 26). 40. The conjugate according to claim 38 wherein the polypeptide monobody is conjugated to a chemotherapeutic agent. 41. The conjugate according to claim 38 wherein the polypeptide monobody is conjugated to a contrasting agent or an organic chelating agent. 42. The conjugate according to claim 41, wherein the contrasting agent is a dextran-coated superparamagnetic iron oxide particle, a lipid vesicle with Gd3+ attached to a surface thereof, or poly-lysine with Gd3+ attached to an amino group thereof 43. The conjugate according to claim 41, wherein the organic chelating agent is diethylenetriamine pentaacetic acid. 44. A composition comprising a pharmaceutically acceptable carrier and a polypeptide monobody according to claim 1. 45. A composition comprising a pharmaceutically acceptable carrier and the fusion protein according to claim 15. 46. A composition comprising a pharmaceutically acceptable carrier and the conjugate according to claim 38. 47. A method of treating or preventing an αvβ3 integrin-mediated disease or disorder comprising: administering to a patient in need thereof, an effective amount of a polypeptide or polypeptide monobody according to claim 1 which binds to the αvβ3 integrin to inhibit activity thereof, thereby treating or preventing the αvβ3 integrin-mediated disease or disorder. 48. The method according to claim 47 wherein said administering is carried out under conditions which are effective to contact one or more cells expressing αvβ3 integrin with a sufficient amount of the polypeptide monobody to inhibit the activity of αvβ3 integrin on such one or more cells. 49. The method according to claim 47 wherein the αvβ3 integrin-mediated disease or disorder is tumor metastasis, solid tumor growth, osteoporosis, Paget's disease, humoral hypercalcemia of malignancy, angiogenesis, retinopathy, arthritis, periodontal disease, psoriasis, smooth muscle cell migration, viral infection, fungal infection, and bacterial infection. 50. The method according to claim 47 wherein the αvβ3 integrin-mediated disease or disorder is tumor metastasis or solid tumor growth, the polypeptide monobody being present in the form of a conjugate comprising the polypeptide monobody conjugated to a chemotherapeutic agent. 51. The method according to claim 47 wherein the modified amino acid sequence of the polypeptide monobody comprises XRGDWXEX where X is any amino acid (SEQ ID No: 26). 52. A method of treating a cancerous or precancerous condition comprising: administering to a patient in need thereof an effective amount of a polypeptide monobody according to claim 1, wherein the polypeptide monobody binds to cancerous or precancerous cells expressing αvβ3 integrin to inhibit αvβ3 integrin-induced activity of the cancerous or precancerous cells. 53. The method according to claim 52 wherein the modified amino acid sequence of the polypeptide monobody comprises XRGDWXEX where X is any amino acid (SEQ ID No: 26). 54. A method of treating a cancerous or precancerous condition comprising: administering to a patient in need thereof an effective amount of a conjugate according to claim 40, wherein the polypeptide monobody binds to cancerous or precancerous cells expressing αvβ3 integrin to inhibit αvβ3 integrin-induced activity of the cancerous or precancerous cells and delivers the chemotherapeutic agent to the cancerous or precancerous cells. 55. The method according to claim 54 wherein the modified amino acid sequence of the polypeptide monobody comprises XRGDWXEX where X is any amino acid (SEQ ID No: 26). 56. A method of imaging tissues using positron emission tomography or magnetic resonance imaging, the method comprising: administering a fusion protein-metal ion complex according to claim 25 to a patient and detecting, by positron emission tomography or magnetic resonance imaging, whether the conjugate is localized within any tissues of the patient. 57. The method according to claim 56 wherein the modified amino acid sequence of the polypeptide monobody comprises XRGDWXEX where X is any amino acid (SEQ ID No: 26). 58. A method of imaging tissues using positron emission tomography or magnetic resonance imaging, the method comprising: administering a conjugate according to claim 41 to a patient and detecting, by positron emission tomography or magnetic resonance imaging, whether the conjugate is localized within any tissues of the patient. 59. The method according to claim 58 wherein the modified amino acid sequence of the polypeptide monobody comprises XRGDWXEX where X is any amino acid (SEQ ID No: 26). 60. A method of assessing the metastatic characteristics of a tumor, the method comprising: administering a fusion protein-metal ion complex according to claim 25 to a patient and detecting, by positron emission tomography or magnetic resonance imaging, whether the conjugate is localized within any tissues of the patient, wherein localization of the fusion protein-metal ion complex indicates an angiogenic site containing a potentially metastatic tumor. 61. The method according to claim 60 wherein the modified amino acid sequence of the polypeptide monobody comprises XRGDWXEX where X is any amino acid (SEQ ID No: 26). 62. A method of assessing the metastatic characteristics of a tumor, the method comprising: administering a conjugate according to claim 41 to a patient and detecting, by positron emission tomography or magnetic resonance imaging, whether the conjugate is localized within any tissues of the patient, wherein localization of the conjugate indicates an angiogenic site containing a potentially metastatic tumor. 63. The method according to claim 62 wherein the modified amino acid sequence of the polypeptide monobody comprises XRGDWXEX where X is any amino acid (SEQ ID No: 26). 64. A method of inhibiting αvβ3 integrin activity comprising: contacting αvβ3 integrin with a polypeptide monobody according to claim 1 under conditions effective to inhibit the activity of the αvβ3 integrin. 65. The method according to claim 64 wherein the modified amino acid sequence of the polypeptide monobody comprises XRGDWXEX where X is any amino acid (SEQ ID No: 26). 66. A method of delivering DNA to a cell comprising: providing a fusion protein-DNA complex according to claim 28 and contacting a cell expressing an αvβ3 integrin with the fusion protein-DNA complex under conditions effective to cause uptake of the fusion protein-DNA complex, thereby delivering the DNA into the cell. 67. The method according to claim 66 wherein the modified amino acid sequence of the polypeptide monobody comprises XRGDWXEX where X is any amino acid (SEQ ID No: 26). 68. An isolated polypeptide which binds to an αvβ3 integrin and comprises the amino acid sequence XRGDWXEX where X is any amino acid (SEQ ID No: 26). 69. The polypeptide according to claim 68, wherein the polypeptide comprises the amino acid sequence of LRGDWSED (SEQ ID No: 5), VRGDWYEY (SEQ ID No: 6), GRGDWTEH (SEQ ID No: 10), ARGDWVEG (SEQ ID No: 11), PRGDWTEG (SEQ ID No: 12), PRGDWIEF (SEQ ID No: 15), PRGDWNEG (SEQ ID No: 23), or FRGDWIEL (SEQ ID No: 24).
<SOH> BACKGROUND OF THE INVENTION <EOH>αvβ3 (CD51/CD61) is a member of the integrin family of cell surface adhesion receptors. Over 20 different αβ integrin heterodimers exist, each with different tissue and ligand specificities. Normal tissue distribution of αvβ3 is generally limited to high levels of expression on osteoclasts, with lower levels observed on platelets, megakaryocytes, kidney, vascular smooth muscle, placenta, dendritic cells (Weiss et al., 2001), and in varying amounts on normal endothelium (reviewed in Horton, 1997). In contrast to α5β1, which binds to only fibronectin, αvβ3 binds to a wide range of RGD-containing integrin ligands, including but not limited to fibronectin, vitronectin, osteopontin, von Willebrandt factor, and fibrinogen (Horton, 1997). αvβ3 integrin is a multifunctional cell surface receptor that has pleiotropic roles in normal cell growth and survival. αvβ3 integrin can also contribute to oncogenesis. Consistent with this, upregulation of αvβ3 expression has been observed on the endothelial cells of angiogenic vessels, and binding of αvβ3 to the basement membrane is a critical step in the angiogenesis induced by basic fibroblast growth factor and tumor necrosis factor-α (Friedlander et al., 1995). Expression of αvβ3 has also been implicated in tumor invasion, and it has been shown that αvβ3 binds matrix metalloproteinase-2 (MM-2) and presents MMP-2 on the surface of invasive carcinomas and on invasive angiogenic endothelial cells (Brooks et al., 1996; Silletti et al., 2001). αvβ3 also regulates cell growth and survival, since ligation of this receptor can, under some circumstances, induce apoptosis in tumor cells (Kozlova et al., 2001). Furthermore, disruption of cell adhesion with anti-αvβ3 antibodies, RGD peptides, and other integrin antagonists has been shown to slow tumor growth (Chatterjee et al., 2000; Chatterjee et al., 2001; and Brooks et al., 1996). Finally, the selective upregulation of αvβ3 expression on tumor blood vessels is also being explored as the basis for imaging of neoplastic lesion, and the αvβ3-specific antibody LM609 has been successfully used for this purpose in vivo (Sipkins et al., 1998). Novel molecules capable of binding with high specificity to αvβ3 integrin have potential utility in several applications, and as a consequence, αvβ3 has been a frequent target for drug discovery and selection of new binding ligands. Phage display technology, in which combinatorial peptide libraries are expressed on the surface of bacteriophages, has been used to select for peptide ligands capable of binding to αvβ3—yielding a wide range of RGD-containing peptide sequences capable of interacting at moderate affinity with αvβ3 and other integrins (Koivunen et al., 1994; Healy et al., 1995). Phage-displayed random peptide libraries have also been constructed and screened using framework proteins such as the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). This resulted in the identification of phage clones which could be used to stain human umbilical vein endothelial cells in a flow cytometric assay. However, the ability of the purified recombinant CTLA-4 protein to stain cells, and its cross-reactivity with other integrins was not reported (Hufton et al., 2000). Fibronectin is a natural ligand of integrins, and it contains repeats of three types of domains. The tenth fibronectin type III domain (FNfn10) includes the RGD sequence in the loop connecting the F and G β-strands (FG loop) (Main et al., 1992). FNfn10 was developed as a scaffold for phage display of peptides because of its small size (94 residues), monomeric assembly, and ability to retain its global fold while exposed loops were randomized (Koide et al., 1998). In addition, FNfn10 lacks cysteine residues and requires no post-translational modification, allowing for large-scale bacterial expression. It has been shown that residues in the FG loop including the RGD sequence are highly flexible (Main et al., 1992; Carr et al., 1997) and this flexibility of the FG loop has been implicated as the origin of the ability of FNfn10 to interact with multiple integrins (Main et al., 1992). While the stability of monobodies makes them well suited for intracellular studies, there has been no prior use of monobodies to probe for modified FG loop or other loop sequences with enhanced binding affinity for and selectivity between integrins, particularly αvβ3 integrin. The present invention overcomes these and other deficiencies in the art.
<SOH> SUMMARY OF THE INVENTION <EOH>A first aspect of the present invention relates to a polypeptide monobody which includes: at least two Fn3 β-strand domain sequences with a loop region sequence linked between adjacent β-strand domain sequences; and optionally, an N-terminal tail of at least about 2 amino acids, a C-terminal tail of at least about 2 amino acids, or both; wherein at least one loop region sequence, the N-terminal tail, or the C-terminal tail comprises a modified amino acid sequence which varies by deletion, insertion, or replacement of at least two amino acids from a corresponding loop region, N-terminal tail, or C-terminal tail in a wild-type Fn3 domain of fibronectin, and wherein the polypeptide monobody binds selectively to αvβ3 integrin. Compositions containing the polypeptide monobody are also disclosed. A second aspect of the present invention relates to a nucleic acid molecule encoding a polypeptide monobody of the present invention. A third aspect of the present invention relates to a DNA construct which includes a DNA molecule encoding a polypeptide monobody of the present invention; a promoter-effective DNA molecule operably coupled 5′ of the DNA molecule; and a transcription termination DNA molecule operably coupled 3′ of the DNA molecule. Expression vectors and host cells which include the DNA construct are also disclosed. A fourth aspect of the present invention relates to a fusion protein which includes: a polypeptide monobody of the present invention and a second polypeptide linked by peptide bond to the polypeptide monobody, the second polypeptide being (i) an epitope tag polypeptide, (ii) a detectable marker polypeptide, (iii) a metal ion-complexing polypeptide, or (iv) a DNA-binding polypeptide. Compositions containing the fusion protein are also disclosed. A fifth aspect of the present invention relates to a fusion protein-metal ion complex which includes a fusion protein of the present invention including a metal ion-complexing polypeptide as the second polypeptide, where the metal ion-complexing polypeptide is complexed with a metal ion. A sixth aspect of the present invention relates to a fusion protein-DNA complex which includes a fusion protein of the present invention including a DNA-binding polypeptide as the second polypeptide, where the DNA-binding polypeptide is complexed with a DNA molecule via the DNA-binding polypeptide. A seventh aspect of the present invention relates to a nucleic acid molecule encoding a fusion protein of the present invention. An eighth aspect of the present invention relates to a DNA construct which includes a DNA molecule encoding a fusion protein of the present invention; a promoter-effective DNA molecule operably coupled 5′ of the DNA molecule; and a transcription termination DNA molecule operably coupled 3′ of the DNA molecule. Expression vectors and host cells which include the DNA construct as also disclosed. A ninth aspect of the present invention relates to a conjugate comprising a polypeptide monobody of the present invention conjugated to (i) a chemotherapeutic agent, (ii) a contrasting agent, or (iii) an organic chelating agent. Compositions containing the conjugate are also disclosed. A tenth aspect of the present invention relates to a method of treating or preventing an αvβ3 integrin-mediated disease or disorder which includes: administering to a patient in need thereof an effective amount of a polypeptide monobody of the present invention which binds to the αvβ3 integrin to inhibit activity thereof, thereby treating or preventing the αvβ3 integrin-mediated disease or disorder. An eleventh aspect of the present invention relates to a method of treating a cancerous or precancerous condition which includes: administering to a patient in need thereof an effective amount of a polypeptide monobody of the present invention, wherein the polypeptide monobody binds to cancerous or precancerous cells expressing αvβ3 integrin to inhibit αvβ3 integrin-induced activity of the cancerous or precancerous cells. A twelfth aspect of the present invention relates to a method of treating a cancerous or precancerous condition which includes: administering to a patient in need thereof an effective amount of a conjugate of the present invention, wherein the polypeptide monobody binds to cancerous or precancerous cells expressing αvβ3 integrin to inhibit αvβ3 integrin-induced activity of the cancerous or precancerous cells and delivers the chemotherapeutic agent to the cancerous or precancerous cells. A thirteenth aspect of the present invention relates to a method of imaging tissues using positron emission tomography or magnetic resonance imaging, the method including: administering a fusion protein-metal ion complex of the present invention to a patient and detecting, by positron emission tomography or magnetic resonance imaging, whether the complex is localized within any tissues of the patient. A fourteenth aspect of the present invention relates to a method of imaging tissues using positron emission tomography or magnetic resonance imaging, the method including: administering to a patient a conjugate of the present invention which includes a contrasting agent and detecting, by positron emission tomography or magnetic resonance imaging, whether the conjugate is localized within any tissues of the patient. A fifteenth aspect of the present invention relates to a method of assessing the metastatic characteristics of a tumor, the method including: administering a fusion protein-metal ion complex of the present invention to a patient and detecting, by positron emission tomography or magnetic resonance imaging, whether the conjugate is localized within any tissues of the patient, wherein localization of the fusion protein-metal ion complex indicates an angiogenic site containing a potentially metastatic tumor. A sixteenth aspect of the present invention relates to a method of assessing the metastatic characteristics of a tumor, the method including: administering to a patient a conjugate of the present invention which includes a contrasting agent and detecting, by positron emission tomography or magnetic resonance imaging, whether the conjugate is localized within any tissues of the patient, wherein localization of the fusion protein-metal ion complex indicates an angiogenic site containing a potentially metastatic tumor. A seventeenth aspect of the present invention relates to a method of inhibiting αvβ3 integrin activity which includes: contacting αvβ3 integrin with a polypeptide monobody of the present invention under conditions effective to inhibit the activity of the αvβ3 integrin. An eighteenth aspect of the present invention relates to a method of delivering DNA to a cell which includes: providing a fusion protein-DNA complex of the present invention and contacting a cell expressing an αvβ3 integrin with the fusion protein-DNA complex under conditions effective to cause uptake of the fusion protein-DNA complex, thereby delivering the DNA into the cell. A nineteenth aspect of the present invention relates to an isolated polypeptide which binds to an αvβ3 integrin and includes the amino acid sequence XRGDWXEX where X is any amino acid (SEQ ID No: 26). The structure of the polypeptide monobodies of the present invention are advantageous for multiple applications because they (1) lack disulfide bonds, rendering them resistant against reducing agents; (2) are very stable even at high temperature; and (3) can be produced in a bacterial expression system with yields of up to 50-100 mg per liter of culture. Moreover, because they are derived from the endogenous human protein fibronectin, which is non-immunogenic, it is believed that the polypeptide monobodies will not elicit an immune response. As a small, single-chain molecule, DNA encoding the polypeptide monobodies can be incorporated into gene delivery vectors (e.g. viral vectors, liposomes) for cell or tissue-specific gene expression. As a result of these properties, the polypeptide monobodies of the present invention can be used to treat or prevent a number of αvβ3 integrin-associated diseases or disorders as well as for diagnostic imaging of αvβ3 integrin-expressing tissues.

Controller of internal combustion engine
Disclosed is an internal combustion engine controller, which is used with a fuel-based torque-on-demand control type, multi-cylinder, direct-injection internal combustion engine to provide an air flow rate control means that excels in response and convergence. This internal combustion engine controller comprises means for computing a target throttle opening from operating conditions. The means for computing a target throttle opening includes: a first computing means for determining a target throttle opening by exercising feedback control in accordance with operating conditions including an intake air flow rate; a second computing means for determining a target throttle opening by exercising feed-forward control in accordance with operating conditions; and a third computing means for determining a target throttle opening in accordance with a target throttle opening value determined by the first computing means and a target throttle opening value determined by the second computing means.

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