Abstract: An apparatus for monitoring proteins and cells. The apparatus includes a plate having wells in which cells are disposed. The apparatus includes means for analyzing the effect or proteins and other biological and chemical moieties on the cells. A method for monitoring of proteins and cells. A method for analyzing a cells. An apparatus for aligning light in a well of a plate for holding cells. A method for lighting a well. A method for determining a condition of a cell. An apparatus for indicating a condition of a cell. A method for establishing a focus profile of a plate having wells for holding cells. A method for manipulating cells. An apparatus for manipulating cells.

Abstract: The present invention provides the enzyme and enzymatic procedures for cleaving the &bgr; secretase cleavage site of the APP protein and associated nucleic acids, peptides, vectors, cells and cell isolates and assays. The invention further provides a modified APP protein and associated nucleic acids, peptides, vectors, cells, and cell isolates, and assays that are particularly useful for identifying candidate therapeutics for treatment or prevention of Alzheimer's disease.

Abstract: Disclosed is a non-fluorescent cyanine dye that may be used as an acceptor in fluorescence energy transfer assays involving the detection of binding and/or cleavage events in reactions involving biological molecules, and assay methods utilising such dyes.

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: The present invention provides compounds useful for the detection of the enzyme tripeptidyl protease I (TPP-1). The invention also provides methods of making such compounds, methods of using such compounds, and kits and compositions containing such compounds. In one embodiment, Gly-L-Pro-L-Ser-1-anthraquinonylhydrazide, in combination with p-anisaldehyde, is used to detect TPP-1.

Abstract: The present invention relates to non-invasive methods for facilitating the diagnosis of a subject as having cancer. The described methods involve detecting the presence of a matrix metalloproteinase in urine samples obtained from a patient and correlating the presence or absence of the matrix metalloproteinase with the presence or absence of cancer. Methods for monitoring the prognosis of a patient by detection of matrix metalloproteinase are also provided.

Abstract: A drug delivery system compound comprising a carboxy(C1-4)alkyldextran polyalcohol modified with a saccharide compound and a residue of drug compound bound to the carboxy(C1-4)alkyldextran polyalcohol, and a method for measuring a drug delivery system compound in which a polymer carrier and a residue of drug compound are bound to each other by a spacer comprising 2 to 8 amino acids linked by peptide bond(s), which comprises treating the drug delivery system compound with a peptidase, and measuring the resulting hydrolysate.

Abstract: We describe a method for monitoring the activity of an enzyme, the method comprising the steps of: providing a binding domain which includes a site for enzymatic modification; providing a binding partner which binds to the binding domain in a manner which is dependent upon modification of the site. The binding domain is contacted with the enzyme; and binding of the binding domain to the binding partner is detected as an indication of the activity of the enzyme. One of the binding domain and binding partner comprises a polypeptide and the other of the binding domain and binding partner comprises a nucleic acid.

Abstract: The invention is directed in part to a purified form of stabilized activated thrombin-activatable fibrinolysis inhibitor (TAFIa). The invention is further directed to a method of producing stabilized TAFIa. The invention is also directed to methods for therapeutic use of stabilized TAFIa such as in the treatment, prevention or management of diseases via an anti-coagulant effect. The invention is also directed to methods for therapeutic use of inhibitors of TAFIa such as in the treatment, prevention or management of diseases via a procoagulant effect. The invention is also directed to methods of diagnostic use of stabilized TAFIa such as a standard in a chromogenic or a fluorometric carboxypeptidase activity assay. The present invention is also directed to kits comprising stabilized TAFIa useful in measuring carboxypepetidase activity.

Abstract: The present inventors have discovered that Threonine synthase is essential for fungal pathogenicity. Specifically, the inhibition of Threonine synthase gene expression in fungi results in no signs of successful infection or lesions. Thus, Threonine synthase can be used as a target for the identification of antibiotics, preferably antifungals. Accordingly, the present invention provides methods for the identification of compounds that inhibit Threonine synthase expression or activity. The methods of the invention are useful for the identification of antibiotics, preferably antifungals.

Abstract: The present invention provides methods whereby the positions of peptide amide groups that are labeled with a heavy hydrogen in a polypeptide or protein can be localized at high resolution. The methods are useful for determining which peptide amide groups in a polypeptide or protein are accessible to solvent, mapping the binding site and/or binding surface of a binding protein, and/or studying allosteric or other conformational changes in a polypeptide or protein which alter the rates at which certain peptide amide hydrogens exchange with solvent.

Abstract: A search method in a biological sample containing an HIV 2 viral strain for possible resistance of said strain to treatment by an anti-protease agent, and nucleotide probes for the implementation thereof. According to methods known per se, the presence of at least one mutation at certain, specified, particular positions of the proteinic sequence of the protease of said viral strain from a biological sample taken from a patient contaminated by HIV 2 is searched. If said mutation is observed, the existence of a resistance to said anti-protease agent is assumed in the patient.

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: This invention relates to a soluble form of PHEX, PHEX being a type II integral membrane glycoprotein. This enzyme is the gene product of a phosphate-regulating gene with homologies to endopeptidases on the X chromosome. To produce a soluble form of PHEX, the transmembrane anchor domain has been modified to encode a signal peptidase coding sequence. The soluble PHEX therefore comprises the active ectodomain. An inactive mutant of PHEX is also an object of this invention. Both soluble and inactive mutant forms of PHEX can be used to screen ligands to PHEX. These ligands can also be used as substrates or inhibitors of PHEX. PHEX being phosphaturic, an inhibitor thereof will be used to treat phosphaturia and/or hypophosphatemia. On the opposite, a substrate for PHEX or PHEX itself can be used to treat hyperphosphatemia.

Abstract: This invention describes methods and kits for detecting and quantifying viable cells in a sample using fluorescent dyes that can be internalized predominantly by viable cells and have fluorescent properties measurably altered when bound to target components. These methods and kits provide a rapid and cost-effective means of detecting potential biological threats in the field.

Abstract: The present invention provides a method of identifying a membrane dipeptidase (MDP)-binding homing molecule that selectively homes to lung endothelium. The method includes the steps of contacting MDP with one or more molecules; and determining specific binding of a molecule to the MDP, where the presence of specific binding identifies the molecule as a MDP-binding homing molecule that selectively homes to lung endothelium. Such MDP-binding homing molecules can be linked to a moiety and, when administered to a subject as a conjugate, can selectively direct the moiety to lung endothelium in the subject.

Abstract: This invention relates to positively charged non-natural amino acids, methods of making thereof, and utilization thereof in peptides. In one embodiment, the invention relates to non-natural amino acids that closely replicate the natural amino acids lysine and arginine.

Abstract: The present invention provides methods relating to chemotherapeutic treatment of a cell proliferative disorder. In particular, a method is provided for predicting the clinical response to certain types of chemotherapeutic agents. Alkylating agents, used for the treatment of certain types of tumors including tumors of the nervous system and lymph system, are efficacious agents when the damage they do to tumor cell DNA is not repaired by cellular DNA repair mechanisms. The present invention provides a method for determining the activity of a gene encoding a DNA repair enzyme, thus providing a prediction of the clinical response to alkylating agents.

Abstract: The present invention relates to novel fluorescent dyes, novel fluorogenic and fluorescent reporter molecules and new enzyme assay processes that can be used to detect the activity of caspases and other enzymes involved in apoptosis in whole cells, cell lines and tissue samples derived from any living organism or organ. The reporter molecules and assay processes can be used in drug screening procedures to identify compounds which act as inhibitors or inducers of the caspase cascade in whole cells or tissues. The reagents and assays described herein are also useful for determining the chemosensitivity of human cancer cells to treatment with chemotherapeutic drugs. The present invention also relates to novel fluorogenic and fluorescent reporter molecules and new enzyme assay processes that can be used to detect the activity of type 2 methionine aminopeptidase, dipeptidyl peptidase IV, calpain, aminopeptidase, HIV protease, adenovirus protease, HSV-1 protease, HCMV protease and HCV protease.

Abstract: Amounts of components in a specimen can be analyzed with excellent quantitativity. The analysis includes: measuring an amount of a component to be analyzed in a specimen; measuring an amount of a standard component present originally and homeostatically in the specimen other than the component to be analyzed; determining the amount of the specimen from the amount of the standard component thus measured and a known concentration of the standard component in the specimen; and determining a concentration of the component to be analyzed in the specimen from the amount of the specimen thus determined and the amount of the component to be analyzed thus measured. The quantitative analysis of the present invention allows a component to be analyzed to be measured with high quantitativity as shown in FIG. 1.

Abstract: This invention describes a method of removing N-terminal alanine residues from polypeptides, preferably recombinant proteins, using an aminopeptidase derived from the marine bacterium Aeromonas proteolytica. Accordingly, Aeromonas aminopeptidase (AAP; E.C. 3.4.11.10) can be used to remove N-terminal alanyl residues from derivatives of human somatotropin (hST, human growth hormone, or hGH), porcine somatotropin (pST), and bovine somtotropin (bST), for example, to yield proteins having their native amino acid sequences. The enzyme reactions can be carried out in free solution, or the AAP can be immobilized on a solid support, for reactions carried out in vitro. An efficient method for converting Ala-hGH to hGH, for example, comprises expression of Ala-hGH in E. coli, recovery of inclusion bodies, solubilization and refolding in detergent, detergent removal by ultrafiltration, selective precipitation, enzyme cleavage, followed by two column chromatography steps.

Abstract: Described herein are methods which identify candidate agents as binding to a protein or as a modulator of the binding characteristics or biological activity of a protein. Generally, the methods involve the use of ADP or phosphate. The assays can be used in a high throughput system to obviate the cumbersome steps of using gels or radioactive materials.

Abstract: A process is provided for the assessment of an active proteolytic enzyme in a blood or another biological fluid sample possibly comprising a complex of said proteolytic enzyme and &agr;2-macroglobulin, wherein said sample is contacted with a substrate comprising a molecule of sufficient size coupled to a signal-substrate, said signal-substrate comprising a detectable leaving group, wherein said substrate is hydrolysed by said proteolytic enzyme but not by said complex. The proteolytic enzyme is preferably selected from the group consisting of thrombin, activated clotting factor, activated fibrinolytic factor, and activated component of the complement system. Of these, thrombin is most preferred. The molecules of sufficient size are preferably water soluble and selected from the group consisting of inert protein, preferably ovalbumin, polysaccharide, and synthetic polymer. The size of these molecules is such that they will not fit into the cavity of the &agr;2M molecules.

Abstract: The present invention provides a method for distinguishing bacterial from non-bacterial exacerbations of chronic lung disease. The method comprises detecting the presence of elastase in patient sputum containing secretions of the lower respiratory tract.

Abstract: The method for recognizing damage and determining the extent of the damage to a keratin-containing material, such as hair or wool, includes subjecting a sample of it in aqueous solution to an enzymatic and/or chemical treatment for proteolytic or hydrolytic degradation of the sample, and subsequently measuring the turbidity of the resulting liquid sample to determine the extent of the damage, either by visual observation with the naked eye or by physical measurement methods. An apparatus suitable for performing the method and enzymes, such as proteases and proteinases, and chemical agents for carrying out the method are also described.

Abstract: The present invention provides the enzyme and enzymatic procedures for cleaving the &bgr; secretase cleavage site of the APP protein and associated nucleic acids, peptides, vectors, cells and cell isolates and assays. The invention further provides a modified APP protein and associated nucleic acids, peptides, vectors, cells, and cell isolates, and assays that are particularly useful for identifying candidate therapeutics for treatment or prevention of Alzheimer's disease.

Abstract: The present invention is based on the findings that BACE 2, a homolog of &bgr;-secretase BACE, is able to stimulate processing of APP in a non-amyloidogenic pathway, thereby suppressing the level of A&bgr;. Accordingly, the present invention provides methods and means for the identification and use of modulators of this unique activity of BACE 2 to suppress A&bgr; production. The compounds identified using the methods and means provided herein may be used as potential candidates for the treatment of Alzheimer's disease and other neurological diseases.

Abstract: The invention provides isolated nucleic acid and amino acid sequences of HsKifC2, antibodies to HsKifC2, methods of screening for HsKifC2 modulators using biologically active HsKifC2, and kits for screening for HsKifC2 modulators.

Abstract: Methods, compositions and articles of manufacture for assaying a sample for a GHB source are provided. A sample suspected of containing a GHB source is contacted with a first oxidoreductase selective for GHB and an oxidized cofactor. In the presence of GHB in the sample, the first oxidoreductase oxidizes GHB to succinic semialdehyde and reduces the cofactor. The reduced cofactor thus produced can be detected directly, or a hydride abstractor can be used that abstracts a hydride from the reduced cofactor and produces a detectable change. The hydride abstractor can be a second oxidoreductase that oxidizes the reduced cofactor and produces a detectable change in a chromogen or dye. Preferably a visual change is produced, allowing performance of the assay outside of a laboratory setting. Fusion proteins comprising the first oxidoreductase, polynucleotides encoding such proteins, host cells expressing such proteins, and vectors comprising such polynucleotides are also provided.

Abstract: The invention describes catabolism of A&bgr; by endothelin converting enzymes (ECEs). Methods of identifying compounds that upregulate ECEs are provided by the invention. Further provided by the invention are methods of regulating A&bgr; catabolism in a cell and methods of decreasing the amount of A&bgr; in a cell. The invention discloses methods of diagnosing an individual with AD and methods of treating such an individual. The invention further discloses methods of identifying compounds that have anti-hypertension activity but do not cause an increase in the level of A&bgr;. Further, the invention provides mutant ECE nucleic acids and mutant ECE polypeptides.

Abstract: The present invention provides a method and a kit for detecting or quantitatively determining homocysteine rapidly and simply and with high sensitivity by oxidizing the residual homocysteine cosubstrate, the produced homocysteine-converting enzyme product or an enzyme reaction product thereof in the presence of an SH reagent to produce hydrogen peroxide and determining the produced hydrogen peroxide by color development using an oxidative color-developing agent. By using the method and kit of the present invention, homocysteine in biological samples, in particular, in body fluids such as blood and urine can be detected and quantitatively determined rapidly and simply and with high sensitivity.

Abstract: A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of enzymes, such as proteases. The substrates contain a fluorogenic-leaving group, such as 7-amino-4-carbamoylmethyl-coumarin (ACC). Substrates incorporating the ACC leaving group show comparable kinetic profiles as those with the traditionally used 7-amino-4-methyl-coumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries using solid-phase synthesis techniques. The approximately 3-fold increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library.

Abstract: The instant methods and compositions represent an advance in controlling drug resistance in microbes. AcrAB-like efflux pumps have been found to control resistance to drugs, even in highly resistant microbes. Accordingly, methods of treating infection, methods of screening for inhibitors of AcrAB-like efflux pumps, and methods of enhancing antimicrobial activity of drugs are provided. Pharmaceutical composition comprising an inhibitor of an AcrAB-like efflux pump and an antimicrobial agent are also provided.

Abstract: A sensor for detecting an analyte enzyme includes at least one substrate compound and at least one indicator compound selected to produce a measurable change of state as a result of the interaction of the substrate and at least one target or analyte enzyme. Each of the indicator(s) and substrate(s) are incorporated within a single polymer.

Abstract: The invention concerns the structure of the sample support plates for mass spectrometric analysis of organic samples ionized by matrix-assisted laser desorption. The invention consists of a highly flat plate, electrically conductive at least on its surface, rigidly bonded to a base structure in such a way that together they form a body having the external dimensions of a microtitre plate, but such that thermal distortions of the surface cannot occur. The base structure may have both depressions for frictional gripping by a robot as well as a machine-readable identifier.

Abstract: Novel compositions comprising one or more of an acid protease and an acidic buffer, the acidic buffer comprising an acid and a pharmaceutically or cosmetically acceptable carrier, vehicle or excipient, useful for treating or preventing abnormal biological conditions, diseases or disorders, and/or for improving the texture or appearance of the skin, and/or for enhancing epidermal exfoliation and/or for enhancing epidermal cell renewal and to methods for the use of the compositions. The acid protease comprises one or more proteolytic enzymes which exhibit proteolytic activity at pH values below that of the surface of the skin, i.e., approximately pH 5.5. The acidic buffer comprises at least one acidic buffering component that can reversibly disassociate hydrogen ions and has buffering capacity at pH values below that of the surface of the skin, i.e., approximately pH 5.5. or mixtures thereof with a pharmaceutically or cosmetically acceptable carrier, vehicle or excipient.

Abstract: Compounds of formula X—NH—R, in which X represents an optionally substituted indol-3-yl group and R represents the acyl residue of an amino acid or of a peptide, enabling the detection of peptidase activity in a micro-organism culture medium, including a gelled medium, by forming a stain or fluorescence in the medium.

Abstract: The present inventors have discovered that homocitrate synthase is essential for fungal pathogenicity. Specifically, the inhibition of homocitrate synthase gene expression in fungi results in no signs of successful infection or lesions. Thus, homocitrate synthase can be used as a target for the identification of antibiotics, preferably antifungals. Accordingly, the present invention provides methods for the identification of compounds that inhibit homocitrate synthase expression or activity. The methods of the invention are useful for the identification of antibiotics, preferably antifungals.

Abstract: Enzyme assays are performed in microfluidic devices including, e.g., in-line labeling, separation, and detection of assay products. In-line labeling allows assays, e.g., protease assays, to be performed in a continuous flow microfluidic format. Also included are microfluidic devices and integrated systems for performing in-line labeling in continuous flow enzyme assays.

Abstract: Provided is a method of screening gene libraries derived from a mixed population of organisms for a bioactivity or biomolecule of interest. The mixed population of organisms can be a cultured population or an uncultured population from, for example, the environment. Also provided are methods of screening isolates or enriched populations of organisms, which isolates include a population that is spatially, temporally, or hierarchical, for example, of a particular species, genus, family, or class of organisms. Identified clones containing a biomolecule or bioactivity of interest can be further variegated or the DNA contained in the clone can be variegated to create novel biomolecules or bioactivities of interest.

Abstract: Enzymatic digests of whey protein isolates were prepared using animal, bacterial and fungal proteases, and evaluated for antihypertensive activities. The antihypertension activity was obtained with a hydrolysate of whey protein isolate prepared with a porcine trypsin. The recovered hydrolysate is used to treat hypertension in mammals such as humans and domestic pets such as dogs and cats.

Abstract: This invention provides a method of determining the proteolytic activity of the in vivo secretases, particularly the &bgr;-secretease and &ggr;-secretease that produce the A&bgr; peptides found in the plaques of Alzheimer Dementia (AD) patients. The invention also provides methods of isolating such secretases and methods of selecting agents that affect the activity of such secretases for developing drugs to treat or prevent dementia.

Abstract: Described is a method for the preparation of a mixture of peptides having a cysteine content between 7-20 w/w % from a protein source, comprising cysteine containing proteins, comprising the steps of: a) cleaving the proteins of the protein source into peptides; b) digesting the peptides obtained in step a) by an exopeptidase, the action of which is at least attenuated at the position of a cysteine in the peptide, therewith forming digested peptides having a terminal cysteine; c) purifying the digested peptides, and the use of the preparation as active component in a medicament, especially for the treatment of conditions mediated by oxidative damage and for the elevation of cellular glutathion levels in the human or animal body.

Abstract: The invention relates to methods for measuring proteasome activity in biological samples. More particularly, then invention relates to methods for monitoring drug action following in vivo administration of a proteasome inhibitor. The invention provides methods and kits for monitoring pharmacodynamic drug action and for determining dose regimen for a proteasome inhibitor. The invention also provides methods for determining baseline proteasome activity in a mammal.

Abstract: The present invention relates to growing and testing microorganisms in a multitest format which utilizes a gel forming matrix for the rapid screening of clinical and environmental cultures. The present invention is suited for the characterization of commonly encountered microorganisms (e.g., E. coli, S. aureus, etc.), as well as commercially and industrially important organisms from various and diverse environments (e.g., the present invention is particularly suited for the growth and characterization of the actinomycetes and fungi). The present invention is also particularly suited for comparative analysis of phenotypic differences between cell types, including strains of microorganisms that have been designated as the same genus and species, as well as other cell types (e.g., mammalian, insect, and plant cells).

Abstract: A system for the rapid characterization of multi-analyte fluids, in one embodiment, includes a light source, a sensor array, and a detector. The sensor array is formed from a supporting member into which a plurality of cavities may be formed. A series of chemically sensitive particles are, in one embodiment positioned within the cavities. The particles may be configured to produce a signal when a receptor coupled to the particle interacts with the analyte. Using pattern recognition techniques, the analytes within a multi-analyte fluid may be characterized.

Abstract: Provided herein are methods for measuring protein biosynthesis by using 2H2O or radioactive 3H2O and applicable uses thereof.

Abstract: Deficiencies in certain physiological pathways are linked with ACE or vasopeptidase inhibitor associated angioedema. Additionally, detection and/or measurement of dipeptidyl peptidase IV (DPP IV) enzyme activity and aminopeptidase P (APP) enzyme activity is a predictor of this risk. The present invention provides biological markers, diagnostic tests, and pharmaceutical indications that are useful in the diagnosis and treatment of angioedema and in the marketing and safety of certain medications. This ability can be important for the treatment of a subject that is in need of or are taking an angiotensin-converting enzyme (ACE) inhibitor and/or a vasopeptidase inhibitor (combined ACE and neutral endopeptidase (NEP) inhibitor), which are commonly used in the treatment of hypertension (high blood pressure), diabetes, and cardiac and renal diseases.

Abstract: The present invention provides a method for identifying a test compound that binds to a target species. The method includes: incubating at least one test mixture under isothermal denaturing conditions, each test mixture comprising at least one test compound, and at least one target species, wherein the isothermal denaturing conditions are effective to cause at least a portion of the target species to denature to a measurable extent; detecting a denaturation signal of each target species in the presence of the at least one test compound by a change in the diffusion properties of the target molecule using fluorescence correlation spectroscopy; and comparing the denaturation signal of each target species in the presence of at least one test compound with a denaturation signal of the same target species in the absence of the at least one test compound under the same isothermal denaturing conditions.

Abstract: A method for the direct analysis of analyte in keratinized structures, e.g., hair, fingernails and toenails, which comprises preparing a mixture containing a low redox potential compound such as dithiothreitol or dithioerythritol, an enzyme suitable for the degradation of the keratin structure and a sample of the keratin structure; permitting the enzyme to at least substantially degrade the sample of keratin structure, filtering the digest solution to remove substances which may interfere with ligand based analytical methods and subjecting the filtered digest solution to analysis to determine the identity and amount of analyte in the keratin substance sample. To accelerate the method, cupric sulfate may be added to the mixture after degradation of the keratin sample. The enzyme may be a peptidase, endopeptidase or proteinase, with papain, chymopapain, and proteinase K being preferred for use in the invention.