sepharose and metaperiodate

Molecular colonies (also known under names nanocolonies, polonies, RNA or DNA colonies, PCR colonies) form when nucleic acids are amplified in a porous solid or semi-solid medium, such as a gel, which contains a system for the exponential multiplication of RNA or DNA. As an individual colony comprises many copies of a single molecule (a molecular clone), the method can be used for the detection, enumeration, and analysis of individual DNA or RNA molecules, including the products of such rare events as RNA recombinations. Here we describe protocols for the detection of RNA molecules by growing colonies of RNA (in a gel containing Qβ replicase, the RNA-dependent RNA polymerase of phage Qβ) or cDNA (in a gel containing the components of PCR), and visualizing them by hybridization with fluorescent probes directly in the gel, including in real time, or by hybridization with fluorescent or radioactive probes followed by transfer to a nylon membrane.

Topics: Acrylic Resins; DNA; Fluorescent Dyes; Gels; In Situ Hybridization; Molecular Biology; Oligonucleotides; Oxidation-Reduction; Periodic Acid; Radioactivity; Recombination, Genetic; Reverse Transcription; RNA; Sepharose

A water-soluble glucan was obtained from the fruit bodies of an edible mushroom, Pleurotus sajor-caju, by hot water extraction, ethanol precipitation, dialysis, and Sepharose 6B gel filtration. On the basis of total hydrolysis, methylation analysis, periodate oxidation, and NMR studies ((1)H, (13)C, DQF-COSY, TOCSY, NOESY, and HSQC), the structure of the repeating unit of the glucan is determined as [carbohydrate structure: see text].

Topics: Biochemistry; Chromatography; Chromatography, Gas; Chromatography, Gel; Glucans; Hydrolysis; Magnetic Resonance Spectroscopy; Mass Spectrometry; Methylation; Oxygen; Periodic Acid; Pleurotus; Polysaccharides; Sepharose; Water

Two Lactobacillus plantarum strains of human intestinal origin, strains 299 (= DSM 6595) and 299v (= DSM 9843), have proved to be efficient colonizers of the human intestine under experimental conditions. These strains and 17 other L. plantarum strains were tested for the ability to adhere to cells of the human colonic cell line HT-29.L.plantarum 299 and 299v and nine other L. plantarum strains, including all six strains that belong to the same genetic subgroup as L. plantarum 299 and 299v, adhered to HT-29 cells in a manner that could be inhibited by methyl-alpha-D-mannoside. The ability to adhere to HT-29 cells correlated with an ability to agglutinate cells of Saccharomyces cerevisiae and erythrocytes in a mannose-sensitive manner and with adherence to D-mannose-coated agarose beads. L. plantarum 299 and 299v adhered to freshly isolated human colonic and ileal enterocytes, but the binding was not significantly inhibited by methyl-alpha-D-mannoside. Periodate treatment of HT-29 cells abolished mannose-sensitive adherence, confirming that the cell-bound receptor was of carbohydrate nature. Proteinase K treatment of the bacteria also abolished adherence, indicating that the binding involved protein structures on the bacterial cell surface. Thus, a mannose-specific adhesin has been identified in L. plantarum; this adhesin could be involved in the ability to colonize the intestine.

Topics: Adhesins, Bacterial; Animals; Bacterial Adhesion; Cell Line; Colon; Escherichia coli; Hemagglutination; Humans; In Vitro Techniques; Lactobacillus; Mannose; Oxidation-Reduction; Periodic Acid; Saccharomyces cerevisiae; Sepharose

Mild periodate oxidation of certain commercially available crosslinked agarose beads (Sepharose CL-4B and CL-6B) results in the generation of aldehydo groups which were useful for immobilization of amino compounds by reductive amination using pyridine borane. Consumption of periodate ion and production of formaldehyde were only observed with crosslinked Sepharose preparations and were correlated with a binding capacity much greater than that of uncross-linked gels when subjected to the reductive amination reaction. Up to 50 mg (approximately 0.73 mumol) of bovine serum albumin and 30 mumol of glycylglycine were coupled per gram of moist oxidized Sepharose CL-6B. The immobilization reaction was shown to proceed at neutral pH requiring about 12 h for completion and to be relatively insensitive to temperature and pyridine borane concentration. The oxidized gel was shown to be stable for at least 2 months upon storage in 0.1 M acetic acid. This method has proven to be useful for the preparation of a variety of affinity matrices and immobilized enzymes.

Topics: Amination; Amines; Cross-Linking Reagents; Hydrogen-Ion Concentration; Oxidation-Reduction; Periodic Acid; Proteins; Sepharose; Serum Albumin, Bovine; Temperature

beta-Glucuronidase (EC 3.2.1.31) was immobilized on various organic and inorganic carriers by different methods. Optimum coupling conditions have been worked out. The immobilization were characterized and compared to each other. Parameters resulting in most stable preparations with high activities are discussed.

Topics: Cyanogen Bromide; Enzyme Induction; Enzymes, Immobilized; Glass; Glucuronidase; Glutaral; Oxidation-Reduction; Periodic Acid; Sepharose