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Biomedical subjects

R Ohlsson

Publications and source records attributed to R Ohlsson.

83 records · Page 5Linked to original sources
PubMed1978

Fractionation of microsomal membranes on the basis of their surface properties.

Partition in dextran-poly(ethylene glycol) aqueous-phase systems can be used for both membrane subfractionation and gaining information on membrane surface properties [H. Walter (1977) in Methods of Cell Separation (Catsimpoolas, N., ed.), vol. 1, pp. 307-354, Plenum, New York]. Smooth, light rough and heavy rough rat liver microsome (obtained by sucrose-density-gradient centrifugation) were subjected to countercurrent distribution in such a system. Smooth microsomal membranes had the highest, heavy rough microsomal membranes the lowest and light rough microsomal membranes an intermediate partition coefficient. The separation is based primarily on hydrophobic differences in the membrane surfaces of the three preparations and is thus due to microsomal properties not previously utilized in their fractionation. The method permits additional subfractionations of microsomes.

Animals↗
PubMed1978

A rapid method for the isolation of circular DNA using an aqueous two-phase partition system.

A method of isolating circular plasmid DNA from cleared lysates of E. coli is described. Purification is achieved by virtue of the rapid re-annealing kinetics or supercoiled DNA. After a brief denaturation step, double stranded plasmid DNA is separated from denatured chromosomal DNA and RNA in a two-phase partition system using dextran and polyethylene glycol. The method is much more rapid than the conventional dye-centrifugation technique and plasmid DNA of comparable purity and yield is obtained.

DNA, Bacterial↗
PubMed1977

Preparation of poly (A)-binding proteins from endoplasmic reticulum-containing subfractions of rat liver cells and their use in mRNA purification.

Approximately 2% of the proteins solubilised from rat liver microsomes or rapidly sedimenting endoplasmic reticulum (RS-ER) adsorbed to poly(A)-Sepharose. The adsorption appeared to be selective for a few proteins, and proteins of different apparent molecular weights adsorbed from RS-ER and the microsomes. The proteins from RS-ER with affinity for poly(A) were coupled to Sepharose and used for the purification of mRNA from rabbit mammary glands. A portion of the RNA wihich did not adsorb to poly(U)-Sepharose adsorbed to protein-Sepharose and was active in a cell-free protein synthesis system.

Animals↗
PubMed1977

On the involvement of cytochrome P-450 in the binding of ribosomes to a subfraction of rat-liver rapidly sedimenting endoplasmic reticulum.

Rat liver endoplasmic reticulum has been separated into four ribosome-containing subfractions, two from rapidly sedimentation endoplasmic reticulum and two from the microsomes, by differential centrifugation and sucrose density centrifugation. Ribosomes from one of the rapidly sedimenting subfractions were extracted by Trion X-100 as a complex with cytochrome P-450, optimally at a detergent protein ratio of 2/1 (w/w). Upon extraction approximately 50% of the cytochrome P-450 in the membrane appeared complex-bound to ribosomes, and, maximally, 6-7 subunit molecules of the cytochrome were attached per ribosome. The specific concentration of cytochrome P-450 on these ribosomes was 2.5-times higher than in the parent membrane. Cytochrome b5, glucose-6-phosphatase, NADPH-cytochrome c reductase, NADH-ferricyanide reductase, cytochrome oxidase and phospholipids were present in small or trace amounts on the ribosomes in relation to cytochrome P-450. Ribosomes extracted from other subfractions contained much less bound cytochrome P-450. Phenobarbital treatment induced an increase in the cytochrome P-450 content that was different for the various subfractions. This increase could not be correlated with changes in the amounts of cytochrome-ribosome complexes released by detergent. We propose that cytochrome P-450 is part of a specific binding site in the membrane for a fraction of the ribosomes attached to the endoplasmic reticulum. The ribosomes may be anchored to cytochrome P-450 via nascent chain proteins.

Animals↗
PubMed1972

General ligands in affinity chromatography. Cofactor-substrate elution of enzymes bound to the immobilized nucleotides adenosine 5'-monophosphate and nicotinamide-adenine dinucleotide.

1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD(+)-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD(+)-Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD(+) was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP-Sepharose, N(6)-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD(+) yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP-Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m. A 0.0-0.15m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP-Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5mm-NAD(+)); 80 (lactate+NAD(+)); 95 (lactate+semicarbazide+NAD(+)); 100 (0.5mm-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP-Sepharose.

Adenosine Monophosphate↗
PubMedDate not supplied

Temporal and spatial pattern of cellular myc oncogene expression during human placental development.

The human placental trophoblast component is of embryonic origin and is developmentally regulated; the tissue is highly proliferative and often described as pseudomalignant. Because cellular oncogenes have been implicated in normal cellular proliferation and differentiation processes, we have studied c-myc oncogene expression in relation to the progression of human placental development. The c-myc transcript shows a 20- to 30-fold variation over the course of placental development, with a peak at four to five weeks after conception. A clear decline in placental c-myc transcription is seen before the end of the first trimester of pregnancy. In situ hybridization to [125I]-labelled myc probes demonstrates an unequal distribution of myc transcripts in placenta, with particularly high expression in the cytotrophoblastic shell of the early placenta. The localization of myc transcripts to cytotrophoblast and the temporal pattern of myc expression support a strong correlation between myc transcript abundance and cytotrophoblastic proliferation. These findings are discussed in the light of a possible role for the c-myc gene in proliferation of normal cells.

Female↗