Abstract: A microfluidic sensor device includes a substrate having patterned thereon at least one Ag/AgCl electrode (working electrode) and an inner chamber overlying the at least one Ag/AgCl electrode. The device includes an ion selective permeable membrane permeable to TPP+ disposed on one side of the first chamber and a sensing chamber overlying the ion selective permeable membrane. A separate reference electrode is inserted into the sensing chamber. The working electrode and reference electrode are coupled to a voltmeter to measure voltage. This voltage can then be translated into a TPP+ concentration which is used to determine the mitochondrial membrane potential (??m).

Abstract: This invention relates to methods of isolating cell nuclei from the other cell components in cell samples, e.g., cell samples from cell cultures or tissue samples. The method does not comprise ultracentrifugation or super-centrifugation rather the method comprises centrifuging cell samples in a table-top conventional centrifuge or microfuge. The method also comprises the use of buffers that are substantially devoid of protease inhibitor or enzyme treatments. The methods facilitate separation of nuclei from nuclear outer membranes leaving the cellular structures and inner membranes of nuclei intact. The method also provides for rapid and consistent results.

Abstract: The present invention concerns a method for modulating the enzymatic activity of an enzyme, wherein said enzyme interacts with BMP, said method comprising the step of administering or inducing Hsp70, or a functional fragment or variant thereof, in a form suitable for allowing interaction between BMP and Hsp70, or said functional fragment or variant thereof, and thereby modulating the enzymatic activity of an enzyme interacting with BMP.

Abstract: An apparatus for disrupting cells or viruses comprises a container having a chamber for holding the cells or viruses. The container includes at least one flexible wall defining the chamber. The apparatus also includes a transducer for impacting an external surface of the flexible wall to generate pressure waves in the chamber. The apparatus also includes a pressure source for increasing the pressure in the chamber. The pressurization of the chamber ensures effective coupling between the transducer and the flexible wall. The apparatus may also include beads in the chamber for rupturing the cells or viruses.

Abstract: The present invention provides protein tyrosine phosphatases (PTP) in which the invariant aspartate residue and the invariant glutamine residue are each replaced with a replacement amino acid residue, wherein the replacement residues together cause a reduction in catalytic rate (kcat) of the enzyme and an increase in substrate-binding affinity (Kd) of the enzyme. The present invention further provides methods for identifying a substrate of a PTP. Also provided are kits for identifying a substrate of a PTP. Additionally, the present invention provides methods for identifying an agent that alters interaction between a PTP and a substrate of the PTP. The invention also provides methods for reducing the activity of a substrate of a PTP.

Abstract: A method to recover receptors without removing microenvironmental components associated with them is described. The membranes containing the receptors are first associated with ligand-coupled solid supports to achieve association of the membranes through a receptor/ligand complex which includes the microenvironment to the solid support and then removing the membrane from the receptor and its microenvironment by shear forces. The thus isolated receptors and their microenvironments can then be analyzed for their role in cellular differentiation, apoptosis, transformation and the like.

Abstract: A method and device for performing lysing on a cell-containing fluid, in which the fluid flows through a vibrating micromachined tube to physically rupture the cell walls (mechanical lysis), and/or to mix, agitate or homogenize the fluid during chemical lysis, and/or to mix, agitate or homogenize the lysate for analysis or other processing after lysing. The tube includes a freestanding portion spaced apart from a surface of a substrate on which the tube is formed. The device further includes means for vibrating the freestanding portion of the tube at a level sufficient to rupture the walls of cells in a fluid flowing through the freestanding portion (for mechanical lysing) or to mix the fluid and a chemical lysing additive within the freestanding portion (for chemical lysing).

Abstract: A method for separating and purifying HA-positive progenitor toxin(s) (LL and/or L toxins) and HA-negative progenitor toxin (M toxin) from a Clostridium botulinum strain is provided. The method comprises applying a liquid containing both the HA-positive progenitor toxin(s) and the HA-negative progenitor toxin to a lactose column. Also provided is a method for separating and purifying neurotoxin (7S toxin) from HA-positive progenitor toxins, which comprises treating HA-positive progenitor toxins with an alkaline buffer and then applying the resulting liquid containing dissociated neurotoxin and non-toxic components to a lactose column. Activated pure HA-positive toxins (L and LL toxins) and neurotoxin are obtained by simple procedures.

Abstract: The present invention relates to methods of preparing biological material, for use in various experimental, diagnostic or therapeutic uses, including immunotherapy treatment or prophylaxy of tumors. More particularly, the present invention relates to methods of preparing membrane vesicles (in particular exosomes) released by various types of mammalian cells, comprising diafiltration and/or density cushion centrifugation. The invention also provides novel methods for characterizing and analyzing exosome preparations, which can be used in quality control assay for the purpose of pharmaceutical product production. The invention is suitable to produce pharmaceutical grade preparations of such membrane vesicles and to fully characterize said preparations, for use in human beings.

Abstract: Poly(ethylene glycol) carbamate derivatives useful as water-soluble pro-drugs are disclosed. These degradable poly(ethylene glycol) carbamate derivatives also have potential applications in controlled hydrolytic degradation of hydrogels. In such degradable hydrogels, drugs may be either trapped in the gel and released by diffusion as the gel degrades, or they may be covalently bound through hydrolyzable carbamate linkages. Hydrolysis of these carbamate linkages releases the amine drug at a controllable rate as the gel degrades.

Abstract: The present invention concerns new electrochemical biosensors for the determination of analytes concentration in sample solutions or suspensions, based on composite transducers containing an electro-conducting material, in the form of powder or grains, a chemical mediator, optionally a substance capable of sorption of said chemical mediator, and a solid binding maker, which is a compound in solid state at room temperature; said biosensors are prepared by incorporating a biocatalyst into the bulk of said composite transducers or by applying a biocatalytic layer onto their surface.

Abstract: This invention provides an apparatus for extracting nucleic acids from nucleic acid-containing samples. The nucleic acid extraction apparatus of the invention comprises (1) a group of extraction vessels each comprising a reactor tube in which a sample, a reagent solution, and a magnetic carrier are admixed and reacted, a drain cup for pooling an unwanted component solution, and a nucleic acid recovery tube all as secured to a support, (2) a distribution means for introducing a solution into each of the extraction vessels, (3) a stirring means for mixing the solution and magnetic carrier in the reactor tube, (4) a holding means for holding the magnetic carrier stationary within the vessel, (5) a discharging means for discharging the solution from the reactor tube while the magnetic carrier is held stationary, (6) a heating means for heating the solution and magnetic carrier in the reactor tube, and (7) a transfer means for serially transferring the vessels to the given positions.

Abstract: The present invention provides a coloring agent comprising glucan-containing yeast cell ghosts which comprise a proportion of substantially intact yeast cell walls and at least one color source. The color source is selected to give the desired color in the coloring agent and in any product in which the coloring agent is used and may be any dye or pigment. For uses such as in foodstuffs, pharmaceuticals and cosmetics, the coloring agents must be acceptable for food and/or pharmaceutical use.

Abstract: The present invention relates to a novel bacterial respiratory poultry disease and the identification of the causative agent. A vaccine derived from this agent was effective in preventing the disease in chickens challenged with the virulent field strains.

Abstract: This invention relates to a method for removing fumarase activity from a microorganism or processed product thereof having ethylenediamine-N,N'-disuccinic acid ethylenediamine lyase activity, which includes treating the microorganism or processed product thereof with an aqueous alkaline solution at a pH of 8.0 to 10.5 in the presence of at least one salt with a concentration of 5 mM to 1000 mM. The salt is preferably selected from the group consisting of sodium, potassium, ammonium and C.sub.2-6 alkanediamine salts of boric acid, phosphoric acid, hydrochloric acid, sulfuric acid, acetic acid, oxalic acid, fumaric acid, maleic acid and ethylenediamine-N,N'-disuccinic acid, and mixtures thereof.

Abstract: A micro-sonicator for spore lysis. Using micromachining technology, the micro-sonicator uses ultrasonic excitation of spores to perform spore and cell lysis. The micro-sonicator comprises a container with a cavity therein for retaining the sample in an ultrasonic transmission medium, the cavity being closed by a silicon membrane to which an electrode and piezoelectric material are attached, with the electrode and piezoelectric material being electrically connected to an AC signal generator which causes the membrane to flex and vibrate at the frequency of the applied voltage.

Abstract: Method and apparatus are described for purifying and separating biopolymers from a homogenized biological milieu using a pipette tip. The biopolymers are complexed with first magnetic particles. A magnet is applied to the pipette tip to attract the biopolymer/magnetic particle complex to the inner surface of the pipette tip adjacent to the magnet for separation from the biological milieu. The magnet is removed and the magnetic particles dissociated and removed from the biopolymers. Second magnetic particles are bound to the biopolymers. The magnet is replaced against the exterior surface of the pipette tip, thereby attracting the bound biopolymers to the inner surface of the pipette tip adjacent to the magnet to allow separation from impurities. The biopolymers are then labeled for detection, which may include detecting the concentration and identity of the biopolymer.

Abstract: The present invention is directed towards isolated lactobacillus biosurfactants and the process for producing same. The present invention is also directed to methods for preventing urogenital infection in mammals using the isolated lactobacillus biosurfactant. The present invention is further directed to methods of inhibiting microbial biofilm formation using the isolated lactobacillus biosurfactant to prevent the formation of bacterial biofilms, and to displace adherent biofilm-forming bacteria from surfaces.

Abstract: A method is provided for in vitro protein synthesis in a bacterial extract wherein the reaction mixture comprises a reducing agent and dissolved oxygen (DO.sub.2) wherein the DO.sub.2 concentration is regulated such that complete oxidation of the reducing agent concentration does not occur for a period of at least about 30 minutes following initiation of protein synthesis in the reaction mixture. Also provided is a method for in vitro protein synthesis in bacterial extracts wherein the reaction mixture comprises an initial methionine concentration of at least about 1.0 mM, or wherein the reaction mixture comprises both labeled and unlabeled methionine, and the initial concentration of unlabeled methionine is at least about 0.1 mM.

Abstract: This invention provides a useful protein-bound magnetic particle which includes a magnetic particle produced in the cell of a magnetic bacterium, and a hybrid protein bound to an organic membrane covering the magnetic particle, and of which the hybrid protein comprises a membrane protein which is originally produced in a state of being bound to the organic membrane, and one or more useful proteins bound biologically through fusion or other binding means to the membrane protein. The protein biologically immobilized does not suffer reduced activity. It is possible to obtain a useful protein such as an enzyme, antibody, etc. immobilized on a magnetic particle, only by cultivating a transformed bacterium, and separating a magnetic particle produced in the cell of the bacterium. When a functional protein is immobilized in this way, it is possible to guide the functional protein magnetically, and to move it to a desired location effectively.

Abstract: A method of preparing a recipient oocyte from a target zona-intact or zona-free oocyte by centrifuging the oocyte against a density gradient material to force the chromosomal material to pass through the cytoplasmic membrane.

Abstract: Methods of isolating and purifying caveolae, microdomains of GPI-anchored proteins, and membranes consisting essentially of caveolae associated with microdomains of GPI-anchored proteins from endothelial cell membranes are disclosed. The methods comprise coating a luminal surface of an endothelial cell membrane with an adherent first ionic material by perfusion from a luminal cavity adjacent to the endothelial cell membrane, forming a pellicle by contacting the first ionic material with a second ionic material, and isolating and purifying the pellicle. The pellicle is then processed to isolate the desired cellular component.

Abstract: The present invention relates to a vehicle for delivery of particles to a sample of cells. The vehicle includes a barrier to retain the particles, which barrier is a dissolvable material. Once released into the sample, the particles are useful in methods to lyse or disrupt cells or in methods to separate cellular components from one another if the cells in the sample are already lysed or disrupted.

Abstract: A method and system for separating a selected molecule from a heterogeneous mixture of molecules in aqueous solution are described. The method comprises (a) providing a separation device comprising a loading reservoir and a receiving reservoir coupled by a channel bearing immobilized microtubules aligned parallel to the longitudinal axis thereof the channel; (b) placing an aqueous solution containing the heterogeneous mixture of molecules in the loading reservoir; (c) adding a motor-ligand composition and ATP to the aqueous solution, wherein the motor-ligand composition comprises a motor protein for attaching to microtubules and moving therealong in the presence of ATP and the ligand is capable of binding the selected molecule, such that the ligand binds the selected molecule to form a complex and the complex moves along the immobilized microtubules to the receiving reservoir; and (d) removing the selected molecule from the receiving chamber.

Abstract: The present invention describes a method for the detection of single base-pair DNA sequence variation in DNA samples isolated from cells with limited ploidy (1.sup..about. 3N). The method can detect variation essentially anywhere in the genome. The method comprises identifying single base-pair polymorphisms or mutations by amplifying a specific region of genomic DNA using a polymerase chain reaction, denaturation of the resultant chains followed by renaturation to form a heteroduplex or hybrid DNA molecule containing one or more single base-pair mismatches. The heteroduplex is then digested with S1 nuclease and the products separated by size with detection by Southern Blot, the use of labeled primers or sensitive gel staining. The method should be generally useful as a simplified approach to identify DNA sequence variants in a variety of samples. It also provides a potentially powerful approach to genetic mapping, DNA fingerprinting, disease detection, and population genetics.

Abstract: Variously pure ribosomal fractions separated from at least one nonphotosynthetic filamentous bacterium are well suited for formulation into a variety of cosmetic/pharmaceutical compositions, for example for the immunostimulation of the immune system of the skin.

Abstract: A process is disclosed for reducing or removing endotoxins from compositions containing therapeutic active substances extracted from natural sources by genetic engineering and/or biotechnology. For that purpose, the compositions are treated with chromatographic materials. The natural sources are disintegrated, the thus obtained fractions are, if required, centrifuged, filtered or treated using affinity chromatography methods, the fractions are pre-incubated in an aqueous salt solution and detergents, are treated with anion exchange materials, then washed with another salt solution. The active substances are eluted from the anion exchanger then further purified in a manner known per se.

Abstract: A plastic is recovered from microorganisms containing it by chemically solubilising non plastic material with an oxidising agent in the presence of a chelating agent.

Abstract: The present invention provides antigenic peptides which bind anti-HCV antibodies for the differential diagnosis of acute and chronic HCV infection.

Abstract: A method for the isolation and sorting of biological materials has been developed. Biological material includes chromosomes, segments of chromosomes, cell organelles, or other minute cellular components. The biological material is separated from the cellular milieu, if necessary, and anchored to a support. Examples of a support are glass coverslips, glass or polymer beads. The anchoring is by means of a reversible polymer and cross-linking system. The supported biological material may then be labelled with compositions capable of binding to said material, and with magnetic particles. Examples of the binding material include nucleic acid probes and antibodies. An example of the antibodies would be those directed to histones. Other labels, for example, fluorescein-biotin-avidin may be used. The material may be released from the support and sorted by a magnetic force.

Abstract: Hybridization method for discriminating between complementarity and partial complementarity of DNA base sequences. Kinetic covalent entrapment of PCR-amplified target DNA is achieved through the use of crosslinkable probes. A crosslinking site is introduced into the target DNA via the PCR amplification process. Problems with renaturation of target DNA in the PCR process and in the hybridization reaction are minimized.

Abstract: A method for observing and determining the size of individual particles and for determining the weight distribution of a sample containing particles of varying size, which involves placing a deformable or nondeformable particle in a medium, subjecting the particle to an external force, thereby causing conformational and/or positional changes, and then measuring these changes. Preferred ways to measure conformational and positional changes include: (1) determining the rate at which a deformable particle returns to a relaxed state after termination of the external force, (2) determining the rate at which a particle becomes oriented in a new direction when the direction of the perturbing force is changed, (3) determining the rate at which a particle rotates, (4) measuring the length of a particle, particularly when it is at least partially stretched, or (5) measuring at least one diameter of a spherical or ellipsoidal particle.

Abstract: The present invention provides a water insoluble coloring agent containing glucan-containing yeast cell ghosts which contain a proportion of intact yeast cell walls and at least one water soluble color source. The color source is selected to give the desired color in the coloring agent and in any product in which the coloring agent is used and may be any dye or pigment. For uses such as in foodstuffs, pharmaceuticals and cosmetics, the coloring agents must be acceptable for food and pharmaceutical use.

Abstract: A method for the isolation and sorting of biological materials has been developed. Biological material includes chromosomes, segments of chromosomes, cell organelles, or other minute cellular components. The biological material is separated from the cellular milieu, if necessary, and anchored to a support. Example of a support are glass coverslips, glass or polymer beads. The anchoring is by means of a reversible cross-linking system. The supported biological material is then labelled with compositions capable of binding to said material, and with magnetic particles. Examples of the binding material include nucleic acid probes and antibodies. An example of the antibodies would be those directed to histones. Other labels, for example, fluoresceinbiotin-avidin may be used. The material may be released from the support and sorted by a magnetic force.

Abstract: Cellular DNA repair enzyme activity has been found to be an indicator of susceptibility or predisposition of an individual to DNA associated diseases. The activity of the enzyme adenosine diphosphate ribosyl transferase (ADPRT) has been found to be a good indicator as to the susceptibility of an individual to DNA associated diseases, such as cancer.

Abstract: Nucleic acid components in a biological sample are detected and/or quantified utilizing a process wherein the sample is first solubilized with a chaotropic salt solution. In a preferred embodiment, cells and nucleic acid components therein are solubilized in the chaotropic salt solution and the solution is incubated with a labelled nucleic acid probe at 20.degree. to 40.degree. C. in the absence of formamide to cause molecular hybridization between the probe and solubilized nucleic acid components, and the molecular hybridization is detected. The chaotropic salt is selected from quanidine thiocyanate, alkali metal perchlorates, alkali metal iodides, alkali metal trifluoroacetates, alkali metal trichloroacetates and alkali metal thiocyanates. The probe may be in solution or immobilized. RNA detected or quantitated may be ribosomal RNA or genomic RNA, and in one embodiment the RNA is HIV viral RNA. When detecting DNA, the solution containing solubilized cells and DNA is heated to at least 45.degree. C.

Abstract: This invention relates to an improvement in promoting the rate of association for high specificity binding pairs used in a variety of industrial, research and medical applications. These pairs include enzyme/substrate, complementary polynucleotide and antibody/antigen combinations. In one specific embodiment, this invention relates to the acceleration of nucleic acid hybridization by heterogeneous nuclear ribonucleoproteins [hnRNPs]. In another specific embodiment, this invention relates to the acceleration of nucleic acid hybridization by a cationic detergent.

Abstract: Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogenous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include methods for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes.

Abstract: A method of separating cell nuclei from cells comprises: treating a fluid containing whole cells so as to selectively lyse the cytoplasmic membrane together with a small proportion of the nuclear membranes but leaving a large proportion of the cell nuclei intact; applying the treated fluid to a membrane whereby a mesh of DNA from the lysed nuclei is formed on the surface and captures intact cell nuclei. A device for use in the method is described.

Abstract: A crude membrane fraction of leaf sheath tissue of a grass endophytically infected by A. typhinum is produced. The extract exhibits endoproteolytic activity in the presence of detergent or after methanol precipitation. This activity is optimal at 35.degree.-40.degree. C. and pH 10-11 and is exhibited only when a reductant is present. The activity is associated with a first gel electrophoresis band having apparent molecular weight of 205,000 daltons when said extract is electrophoresed without prior boiling; however, when said extract is boiled prior to electrophoresis, there results a band having apparent molecular weight of 34,000 daltons. This band gives rise to polyclonal rabbit antibodies which are not cross react with proteinase K. The preferred source of Acremonium typhinum is ATCC 74228.

Abstract: The cellular binding proteins present in membranes of fleas, especially those which are present in the digestive tract, are useful as screening tools for systemic anti-flea reagents and in the design of vaccine formulations. Particularly useful is the .alpha.-subunit of (Na.sup.+ /K.sup.+)ATPase. The .alpha.-subunit of this protein expressed in recombinant host cells in the presence of the .beta.-subunit is distributed on the membrane and the recombinant cells can thus be used to screen candidates for ability to bind the cells. Secondary screens are used to determine the specificity of the candidate reagent for flea protein as compared to a corresponding protein derived from other sources.

Abstract: For the determination of an immunologically detectable substance based on a heterogeneous immunoassay by use of a solid phase on which one of the immunologically active reaction components is bound, a reaction vessel is used as the solid phase on the inner surface of which streptavidin or avidin is bound in such an amount that 0.1 to 2.5 .mu.g are present per ml reaction volume.A suitable reaction vessel for this has optically transparent wall areas which face one another and has avidin or streptavidin coated walls which are at least partially within the inner wall region intended as a receptacle for liquid, wherein the inner space of the container intended as a receptacle for liquid and the respective streptavidin or avidin content of the coating are so matched that 0.1 to 2.5 .mu.g streptavidin or avidin are present per ml reaction volume.

Abstract: Methods for detecting a unique strain of Chlamydia associated with acute respiratory disease are disclosed. These methods utilize monoclonal antibody directed against an antigenic determinant of the TWAR organism, or DNA probes capable of specifically hybridizing to at least a portion of the DNA sequence of the TWAR organism. A method for determining the presence of antibodies to the TWAR organism, utilizing elementary bodies of the TWAR organism as antigen is also disclosed.

Abstract: Methods for detecting a unique strain of Chlamydia associated with acute respiratory disease are disclosed. These methods utilize monoclonal antibody directed against an antigenic determinant of the TWAR organism, or DNA probes capable of specifically hybridizing to at least a portion of the DNA sequence of the TWAR organism. A method for determining the presence of antibodies to the TWAR organism, utilizing elementary bodies of the TWAR organism as antigen is also disclosed.

Abstract: Disclosed are non-histological methods for determining the presence in a sample of a preselected cell type such as a malignant or genetically defective cell, or cell nucleus debris from such cells. The methods involve determination of an intranuclear matrix protein, the mRNA encoding that protein, or matrix protein associated DNA or RNA which acts as a marker for the preselected cell type, the presence of which indicates the presence of the cell type in the test sample. The intranuclear matrix marker protein or nucleotide are detected by hybridization, immunoassay, or other known means.

Abstract: The present invention is directed to a continuous flow method for removing oxygen from a fluid stream. The method comprises the steps of providing a fluid stream containing oxygen, causing the fluid stream to come in contact directly or indirectly, with a sufficient amount of oxygen scavenging cell membrane fragments having an electron transport system which reduces oxygen to water to catalyze the transformation of the oxygen contained in the fluid stream to water. The present invention is also directed to an apparatus for removing oxygen from a fluid stream.

Abstract: The present invention provides a method for rapidly obtaining substantially pure DNA from a biological sample containing cells. The method involves gently lysing the membranes of the cells to yield a lysate containing genomic DNA in a high molecular weight form. The lysate is moved through a porous filter to selectively trap the high molecular weight DNA on the filter. The DNA is released from the filter using an aqueous solution to form a solution containing substantially purified DNA, from which the DNA may analyzed or recovered.

Abstract: The known compound, 13.beta.-13-deoxy-22,23-dihydro-avermectin -B1a/B1b Aglycone is prepared microbiologically from cultures of the novel microorganisms (MA-6762, ATCC 55069, MA-6763 ATCC 55070) or the known microorganism Streptomyces lavendulae MA-6555 (ATCC 14159 or ATCC 55330). The compound is prepared by the biotransformation of 13-deoxy-22,23-dihydro avermectin B1a/B1b aglycone which oxidizes the 13-position and epimerizes the 13-position hydroxy group.

Abstract: A novel method for the isolation of high molecular weight DNA from plants, yeast and bacteria using xanthate-forming compounds such as sodium/potassium ethyl xanthogenate is disclosed. The procedure does not require deproteination and yields clean DNA that is suitable for both PCR and Southern blotting. It can be utilized on a small scale without homogenizing the tissue. These features also facilitate automated screening of plant tissue samples, one of the labor-intensive techniques in molecular biology. This method is also adaptable for use in the field.

Abstract: The present invention provides a method for rapidly obtaining substantially pure DNA from a biological sample containing cells. The method involves gently lysing the membranes of the cells to yield a lysate containing genomic DNA in a high molecular weight form. The lysate is moved through a porous filter to selectively trap the high molecular weight DNA on the filter. The DNA is released from the filter using an aqueous solution to form a solution containing substantially purified DNA, from which the DNA is recovered.