Abstract: The mechanism by which pleiotrophin binds to the receptor protein tyrosine phosphatase ?/?(RPTP ?/?) is disclosed along with methods of modulating both pleiotrophin expression and signaling to treat, prevent and inhibit abnormal cell growth states. Specifically provided are methods of inhibiting tumor growth, promotion, metastasis, invasiveness and angiogenesis as well as methods of preventing or inhibiting cell adhesion.

Abstract: Methods, compositions and kits are disclosed for determining one or more target polypeptides in a sample where the target polypeptides have undergone phosphorylation. A mixture comprising the sample and a first reagent comprising a cleavage-inducing moiety and an IMAC resin for a binding site on a target polypeptide is subjected to conditions under which binding of respective binding moieties occurs. The binding site is the result of phosphorylation activity involving the target polypeptide. The method may be employed to determine the target polypeptide itself. In another embodiment the presence and/or amount of the target polypeptide is related to the presence and/or amount and/or activity of an agent such as an enzyme involved in phosphorylation of the target polypeptide.

Abstract: The ability to efficiently determine the state of enzyme expression in cells has long been desired as material to the diagnosis of disease. This invention relates to cytoenzymology, and more particularly to improved reagents for use in cell-based assays, especially those using fluorogenic substrates.

Abstract: The invention relates to assays for measuring ubiquitin ligase activity and for identifying modulators of ubiquitin ligase enzymes.

Abstract: The present invention relates to protein modulators, especially modulators of kinesin bioactivities. The present invention provides methods for screening protein modulators, e.g., kinesin modulators that bind to a region of a kinesin and/or that modulate kinesin bioactivities. The present invention also provide methods and compositions for treatment of disorders mediated by abnormal cellular proliferation activities.

Abstract: The invention discloses methods, compositions and kits for stabilizing a solubilized phenyl phosphate, preferably paranitrophenyl phosphate (PNPP), using charcoal. Also disclosed are methods, compositions and kits for recycling solubilized phenyl phosphate, preferably PNPP, that has an absorbance of less than 0.1 when measured at 405 nm due to non-enzymatic hydrolysis.

Abstract: The present invention provides modified Cdc14 phosphatase proteins, and novel constructs for expression thereof, in which the sequence C-terminal to the catalytic domain has been deleted. This modification results in the expression of a single form of the enzyme, enabling purification of a homogeneous preparation of active enzyme at a high yield.

Abstract: Provided are methods and compositions for assaying for ubiquitin agents that are enzymatic components of ubiquitin-mediated proteolysis and, more particularly, methods and compositions for assaying for agents that modulate the activity of such ubiquitin agents.

Abstract: A biphasic process for rapid screening of organic reactions comprising monitoring relative rates of parallel organic reactions. The screening process is suitable to determine the efficacy of different reactants, process conditions, and process enhancers such as catalysts or promoters. The biphasic process also allows multiple samples to be analyzed/monitored simultaneously. In addition because enzymes are used to monitor the reaction product in this invention, when that product is chiral and an enantio-discriminating enzyme is used to monitor the product, in addition to the relative rates, enantioselectivities of a set of parallel organic reactions can also be determined.

Abstract: The invention provides isolated nucleic acid and amino acid sequences of HsKif16a, antibodies to HsKif16a, methods of screening for HsKif16a modulators using biologically active HsKif16a, and kits for screening for HsKif16a modulators.

Abstract: The present invention provides an assay for an analyte in solution. The assay relies, in part, on associating two catalytic activities in close proximity to each other (e.g., by conjugating each enzyme to a ligand that binds the analyte) and providing substrates that produce a colored product only when both activities are bound to the same molecule in solution.

Abstract: Methods for delivering nucleic acid molecules into cells and methods for measuring nucleic acid delivery into cells and the expression of the nucleic acids are provided. The methods are designed for introduction of large nucleic acid molecules, including artificial chromosomes, into cells, and are practiced in vitro and in vivo.

Abstract: The invention relates to a combination of a protein phosphatase type 2C as an enzyme and a phospho-BAD as substrate. The protein BAD is dephosphorylated in position Ser155. Furthermore, the combination is used in an in vitro screening for ligands which modulate protein phosphatase type 2C, comprising the steps: incubating the protein phosphatase type 2C in combination with the substrate phospho-BAD and the ligand of the assay and detecting the decrease in phospho-BAD and/or the increase in phosphate and/or BAD. Therefore, new drugs for the treatment apoptosis may be found.

Abstract: Provided are methods and compositions for assaying for ubiquitin agents that are enzymatic components of ubiquitin-mediated proteolysis and, more particularly, methods and compositions for assaying for agents that modulate the activity of such ubiquitin agents.

Abstract: The invention provides chemiluminescent assays that incorporate a film including at least one chemiluminescent precursor immobilized therewith which produces a triggerable chemiluminescent compound, the film being free of compounds which generate singlet oxygen and being adapted for use with a sensitizer-labeled agent or agent probative of the analyte.

Abstract: A method for measuring protein kinase activity comprising: (a) providing a first solution comprising ATP and a protein kinase to be tested, and a second solution comprising ATP in the absence of said kinase to be tested; (b) adding a substrate capable of being phosphorylated by the protein kinase to be tested to the first and second solutions of step (a); (c) measuring the concentration of ATP and/or ADP, or the rate of change thereof with respect to time, in each of the reaction mixtures formed in step (b) using a bioluminescence reaction; and (d) using the information about the concentration of ATP and/or ADP to determine the activity of the protein kinase to be tested.

Abstract: Reagents which regulate human inositol polyphosphate 5-phosphatase and reagents which bind to human inositol polyphosphate 5-phosphatase gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, COPD, asthma, diabetes, and cancer.

Abstract: A conjugate of a tissue non-specific alkaline phosphatase (tns-AP) and dextran which can be obtained by reacting unglycosylated tns-AP with activated dextran by incubation in aqueous solution, stopping the reaction and isolating the conjugate from the solution. The conjugate obtained in this manner is suitable as a standard for the determination of alkaline phosphatase.

Abstract: Disclosed is a method and corresponding kit for assaying the presence, activity, or both, of an enzyme classified within an enzyme classification selected from the group consisting of EC 2.7.1, EC 3.1.3, and EC 3.1.4. The method generally includes the steps of reacting an enzyme with a substrate for a time sufficient to yield phosphorylated or dephosphorylated product; contacting the product with a binding matrix, whereby product is adhered to the matrix; and then analyzing the matrix for presence of, amount of, or both the presence and the amount of the product fixed to the matrix, whereby the presence, the activity, or both the presence and activity of the enzyme can be determined.

Abstract: The present invention provides compositions and methods for modulating B-lymphocyte activation. Nucleic acids encoding proteins and proteins so encoded which are capable of modulating B-lymphocyte activation are provided. Compositions and methods for the treatment of disorders related to dysfunction or dysregulation of B-lymphocyte activation are also provided. Prophylactics and methods for the prevention of such disorders are also provided. Also provided are compositions and methods for diagnostic and prognostic determination of such disorders. Further provided are assays for the identification of bioactive agents capable of modulating B-lymphocyte activation.

Abstract: A method of identifying a compound which modulates the interaction between a PP1c and a regulatory subunit thereof, the method comprising determining whether a compound enhances or disrupts the interaction between (a) a PP1c or a fragment, variant, derivative or fusion thereof or a fusion of a fragment, variant or derivative and (b) a regulatory subunit which is able to bind to PP1c or a PP1c-binding fragment, variant, derivative or fusion of the subunit or a fusion of the fragment, variant or derivative. A method of affecting cellular metabolism or function, the method comprising administering to a cell (a) a compound which modulates the interaction between a PP1c and a regulatory subunit thereof or (b) a compound which mimics the effect of a regulatory subunit of PP1c or (c) a peptide capable of binding a PP1c and which affects the ability of PP1c to bind to a particular target and/or affects the regulation of PP1c activity, or a functional equivalent thereof.

Abstract: The invention provides novel polypeptides which are associated with the transcription complex NF-AT, polynucleotides encoding such polypeptides, antibodies which are reactive with such polypeptides, polynucleotide hybridization probes and PCR amplification probes for detecting polynucleotides which encode such polypeptides, transgenes which encode such polypeptides, homologous targeting constructs that encode such polypeptides and/or homologously integrate in or near endogenous genes encoding such polypeptides, nonhuman transgenic animals which comprise functionally disrupted endogenous genes that normally encode such polypeptides, and transgenic nonhuman animals which comprise transgenes encoding such polypeptides.

Abstract: A method for screening of modulators of calcineurin is provided, which uses the interaction between calcineurin and superoxide dismutase. Modulators of calcineurin are potential candidates for drugs, e.g. for immunosuppressive drugs. The forming of a complex comprising calcineurin and superoxide dismutase is monitorable in the presence of potential activators or inhibitors of calcineurin. Complex formation is performed within the cell by the use of appropriate expression vectors or in vitro using isolated proteins. Preferably, complex formation is monitored by fluorescence detection, especially by laser fluctuation correlation spectroscopy.

Abstract: A dual enzyme chemiluminescent substrate formulation useful for the independent or simultaneous analysis of the enzymes is disclosed, the formulation includes, as one substrate component, dihydrophthalazinedione, such as luminol or isoluminol, and a peroxide source and, as the other substrate component, a 1,2-dioxetane. The formulation further includes a polymeric quaternary onium salt as an isolating agent to defeat adverse interactions between the substrate pairs.

Abstract: Chemiluminescent 1,2-dioxetane compounds capable of producing light energy when decomposed, substantially stable at room temperature, represented by the formulas I or II: wherein T is:

Abstract: The invention relates to a novel lipid kinase which is part of the PI3 Kinase family. PI3 Kinases catalyze the addition of phosphate to inositol generating inositol mono, di and triphosphate. Inositol phosphates have been implicated in regulating intracellular signaling cascades resulting in alternations in gene expression which, amongst other effects, can result in cytoskeletal remodeling and modulation of cellular motility. More particularly the invention relates to a novel human PI3 Kinase, p110? which interacts with p85, has a broad phosphinositide specificity and is sensitive to the same kinase inhibitors as PI3 Kinase p110A. However, in contrast to previously identified PI3 Kinases which show a ubiquitous pattern of expression, p110? is selectively expressed in leucocytes. Importantly, p110? shows enhanced expression in most melanomas tested and therefore may play a crucial role in regulating the metastatic property exhibited by melanomas.

Abstract: The present invention relates to rapid, quantitative, specific, high through-put methods for screening test substances for their ability to inhibit activity of an ouabain-resistant Na+-K+-ATPase involved in a variety of biological processes such as regulation of osmotic balance and cell volume, maintenance of the resting membrane potential, establishment of the ionic composition of cerebrospinal fluid and aqueous humor, electrical activity of muscle and nerve, and receptor-mediated endocytosis, cardiac muscle contractility, neurotransmitter metabolism and vascular muscle cell contraction. These methods can be employed to identify compounds for use in therapeutic applications for disease processes in which dysfunction of the Na+-K+-ATPase contributes to a pathological process. The present invention also includes kits which are used in the methods provided herein.

Abstract: The present invention provides filter-based assays for measuring binding of compounds to phosphodiesterases (PDEs). The assay permits stoichiometric binding of compounds to PDEs, thereby providing a highly sensitive measure of a PDE binding and inhibition.

Abstract: Methods for quantifying tartrate-resistant acid phosphotase (TRAP) activity, or determining the distribution of TRAP in a tissue are provided. More specifically, the fluorescent assay of the present invention is useful for identifying or quantifying TRAP in isolated osteoclast cells or osteoclasts differentiated from RAW264.7 monocyte-macrophage cells.

Abstract: Method for measuring the presence or absence of chemical groups, in particular phosphate groups, attached to biological molecules in a sample in which these molecules are tagged with fluorescent markers and these fluorescent markers are activated by means of irradiating the sample with light. The method is characterized by the following steps: a) Use of a fluorescent marker, the fluorescence lifetime of which assumes a different value depending upon the presence or absence of phosphate groups attached to the biomolecule; b) Measurement of the fluorescence lifetime of the fluorescent marker attached to a biomolecule and selected in accordance with Step a); c) Classification of the biomolecules in accordance with the presence or absence of phosphate groups attached to these, based on the different lifetime of each.

Abstract: Arrays modified with chemiluminescent enhancing materials and methods of detecting chemiluminescent emissions on solid supports in the presence of chemiluminescent enhancing materials are described. The arrays include a solid support having a surface layer, a plurality of probes disposed on the surface layer in discrete regions and a chemiluminescent enhancing material. The array may be a high density array. At least some of the probes can be bound either directly or indirectly to an enzyme conjugate comprising an enzyme capable of activating a chemiluminescent substrate. The surface layer can be contacted with a composition comprising the chemiluminescent substrate in the presence of the chemiluminescent enhancing material and the resulting chemiluminescent emissions can be detected. The probes can be polynucleotide or polypeptide probes.

Abstract: Novel RGS polypeptides, proteins, and nucleic acid molecules are disclosed. In addition to isolated, full-length RGS proteins, the invention further provides isolated RGS fusion proteins, antigenic peptides, and anti-RGS antibodies. The invention also provides RGS nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which an RGS gene has been introduced or disrupted. Diagnostic, screening, and therapeutic methods utilizing compositions of the invention are also provided.

Abstract: The present invention relates to novel chemical compounds, methods for their discovery, and their therapeutic use. In particular, the present invention provides benzodiazepine derivatives and methods of using benzodiazepine derivatives as therapeutic agents to treat a number of conditions associated with the faulty regulation of the processes of programmed cell death, autoimmunity, inflammation, and hyperproliferation, and the like.

Abstract: Methods are provided for diagnosing and/or characterizing chronic immune disease activity in a subject. In the subject methods, a sample is obtained from a subject suspected of having or known to have a chronic immune disease. The sample is then assayed for the presence of low molecular actin fragments. The assay results are used to diagnose the presence of chronic immune disease activity and/or characterize chronic immune disease activity in the subject, e.g. to confirm an initial chronic immune disease diagnosis, to determine the stage of the disease, to monitor disease progression, to predict disease attacks, and the like. Also provided by the subject invention are kits for practicing the methods.

Abstract: Methods of using a heme-deficient mutant sGC with a substituted His105 residue, which has a high basal specific activity and displays properties similar to NO-stimulated wild type sGC, are disclosed. Preferred embodiments aid in the prevention and treatment of cyclic GMP-dependent pathophysiologies, and are useful in the development of drugs that inhibit or activate sGC. Certain embodiments provide a method of treating angina and other chronic heart diseases comprising delivery of a constitutively active &agr;&bgr;Cys105 mutant gene or enzyme to an in vivo cell are described.

Abstract: The invention relates to a method for identifying fungicides, to the use of farnesyl-pyrophosphate synthase for identifying fungicides, and to the use of farnesyl-pyrophosphate synthase inhibitors as fungicides.

Abstract: The present invention provides a set of methods and compositions for homogeneous coupled luminescent assays of cytotoxicity and/or proliferation of cells, as well as for enzymatic activity. In both cases the activities of the enzymes of interest are coupled to production of a high-energy molecule, which serves as a substrate for the production of light by a luciferase, typically in a single reagent mixture, with a useful readout available in 1—5 minutes. Individual cytotoxicity and proliferation signals can be measured from a single sample in 6 minutes or less. The invention also provides a 3-minute, one-step phosphatase assay. The ability to couple the activity of interest to light production in a one-step procedure gives rise to extremely rapid and flexible methods for measurement of cytotoxic effects and enzymatic activities. The assay methods are highly suitable for use in high-throughput screening procedures.

Abstract: A homogeneous assay for determining the deoxynivalenol (DON) content in grains uses the technique of fluorescence polarization. A grain extract is prepared by shaking a crushed grain sample with water. A mixture is prepared by combining the grain extract with a tracer and with monoclonal antibodies specific to DON. The tracer is able to bind to the monoclonal antibodies to produce a detectable change in fluorescence polarization. The tracer is prepared by conjugating DON to a suitable fluorophore. The fluorescence polarization of the mixture is measured. The DON concentration of the mixture may be calculated using a standard curve obtained by measuring the fluorescence polarization of a series of DON solutions of known concentration.

Abstract: The present invention relates to a package of screening methods for developing drugs against pathogenic microbes having two-component system of DevR-DevS and/or DevR-Rv2027c and its homologues, said method comprising steps of over-expressing DevR, DevS, and Rv2027c and their single domain derivatives including mutant variant proteins, autophosphorylating DevS, and Rv2027c proteins and thereafter, phosphotransfering to DevR and its derivatives in SDS-PAGE or High-throughput format in the presence of a test compound, and determining the drug-potential of the test compound, wherein the potential of the drug is inversely proportional to (i) the degree of autophosphorylation of DevS and Rv2027c, (ii). the degree of phosphotransfer-based dephosphorylation of DevR and/its single domain derivative, and (iii). the degree of dephosphorylation of phosphorylated species of DevS and Rv2027c and/their single domain derivatives, and a method of treatment, and a composition thereof.

Abstract: A method for determining the level of tyrosine kinase activity in a biological sample is disclosed. The method employs an anti-phosphotyrosine antibody as both the capture agent and the detecting agent. The detecting antibody is labeled with a fluorescent label, for instance, Cy5, Cy5.5 or Cy7 or a lanthanide ion, such as europium, as the signal generating entity. The method is particularly well suited to high throughput screening, for example, for compounds which modulate tyrosine kinase activity.

Abstract: A lateral flow chromatographic assay format for the performance of rapid enzyme-driven assays is described. A combination of components necessary to elicit a specific enzyme reaction, which are either absent from the intended sample or insufficiently present therein to permit completion of the desired reaction, are predeposited as substrate in dry form together with ingredients necessary to produce a desired color upon occurrence of the desired reaction. The strip is equipped with a sample pad placed ahead of the substrate deposit in the flowstream, to which liquid sample is applied. The sample flows from the sample pad into the substrate zone where it immediately reconstitutes the dried ingredients while also intimately mixing with them and reacting with them at the fluid front. The fluid front moves rapidly into the final “read zone” wherein the color developed is read against predetermined color standards for the desired reaction. Pretreatment pads for the sample, as needed, (e.g.

Abstract: The present invention is related to an inhibitor of the inositol polyphosphate 5-phosphatase SHIP2 protein or its encoding nucleotide sequence identified by SEQ ID NO. 1 or of SHIP2 mRNA expression.

Abstract: The present inventors have discovered that Lipid Transfer Protein (LTP) is essential for plant growth. Specifically, the inhibition of LTP gene expression in plant seedlings results in reduced growth and chlorosis. Thus, LTP is useful as a target for the identification of herbicides. Accordingly, the present invention provides methods for the identification of herbicides by measuring the activity of an LTP in the presence and absence of a compound, wherein an alteration of LTP activity in the presence of the compound indicates the compound as a candidate for a herbicide.

Abstract: A competitive assay to determine the presence and concentration of an intracellular analyte (e.g., cAMP) in a sample is provided. All of the steps of the assay can be performed on the same assay plate, thereby eliminating the need to transfer the cells from a tissue culture plate on which the cells are grown, induced and lysed to a separate assay plate. The assay procedure includes combining, in a reaction chamber provided with a capture antibody, an antibody for the analyte, the sample to be assayed, and a conjugate of the analyte and an enzyme such as alkaline phosphatase. The mixture is incubated and washed and an enzyme labile substrate (e.g., a chemiluminescent, fluorescent or calorimetric substrate) is added. The assay can also be performed with a tagged analyte (e.g., an analyte having a radioactive or fluorescent tag) instead of an enzyme conjugate.

Abstract: The present invention provides cell-based assays for inositol phosphates involving the preferential binding of radio-labeled inositol phosphates to a solid phase containing a scintillant within. The assay allows one to screen for inhibitors of inositol phosphate phosphatases or GPCRs which are coupled to phosphoinositide hydrolysis. The assays are fast, convenient, and avoid the column chromatography steps that prior art methods employed.

Abstract: Disclosed is a process for obtaining an enzyme having a specified enzyme activity derived from a heterogeneous DNA population by screening, for the specified enzyme activity, a library of clones containing DNA from the heterogeneous DNA population which have been exposed to directed mutagenesis towards production of the specified enzyme activity. Also disclosed is a process for obtaining an enzyme having a specified enzyme activity by screening, for the specified enzyme activity, a library of clones containing DNA from a pool of DNA populations which have been exposed to directed mutagenesis in an attempt to produce in the library of clones DNA encoding an enzyme having one or more desired characteristics which can be the same or different from the specified enzyme activity.

Abstract: The present invention relates, first, to methods for the modulation of acid sphingomyelinase (ASM)-related processes, including apoptosis. Such apoptosis can include, but is not limited to, environmental stress-induced apoptosis such as, for example, ionizing radiation and/or chemotherapeutic agent-induced apoptosis. Apoptosis can be characterized by a cellular morphology comprising cellular condensation, nuclear condensation or zeiosis. The present invention further relates to methods for the identification of compounds which modulate (i.e., either increase or decrease) sensitivity to ASM-related processes, including apoptosis.

Abstract: Two novel phosphorylation sites of myosin light chain 1 (MLC1) are described. Methods of monitoring phosphorylation of MLC1 to identify new cardiac and skeletal muscle protective agents, monitor the extent of preconditioning of cardiac and skeletal muscles, and monitoring the status of a subject with cardiac or skeletal muscle damage are provided. Also provided are methods and compositions for altering MLC1 to change contractility of cardiac and skeletal muscles and to protecting cardiac and skeletal muscles from damage caused by conditions and/or agents including, but not limited to, cardiomyopathies, hypertension, free radicals ischemia, hypoxia, and ischemia/hypoxia with reperfusion.

Abstract: The present invention provides polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins. In particular, the polypeptides and polynucleotides of the invention comprise amino acid and nucleic acid sequences of novel CD39-like gene and gene products.

Abstract: Provided are novel complexes containing a crosslinked avidin, an analyzing method and analyzing reagents and kits whereby a compound to be analyzed can be quickly, conveniently and accurately analyzed while taking advantage of the avidin-biotin reaction. The complexes contain at least two homogeneous or heterogeneous biotin-introduced products and one crosslinked avidin sandwiched therebetween. In the analyzing method, the homogeneous or heterogeneous biotin-introduced products and the crosslinked avidin are used. The analyzing reagent contains the crosslinked avidin. The analyzing kit contains the crosslinked avidin and a biotinylating agent.