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Biomedical subjects

O Doi

Publications and source records attributed to O Doi.

At least 163 records · Page 9Linked to original sources
PubMed1978

Retention of acyl groups in LM cell fibroblasts with altered phospholipid composition.

Incorporation and loss of membrane lipids were measured in murine fibroblasts grown in suspension culture with [2-14C]acetate in a chemically defined medium devoid of serum or lipid. Greater than 98 and 94% of the 14C present in the phospholipids and neutral glycerides respectively was found in the fatty acyl groups. During a 3-day growth period LM fibroblasts lost 65% of their 14C-labelled acyl groups from the phospholipids and 36% from the neutral lipids. Selective retention of 14C in individual isolated phospholipid and neutral lipid species occurred only in phosphatidylethanolamine. Retention of acyl groups in the phospholipids or neutral glycerides was not a property of LM suspension cultured fibroblasts. Supplementation of the LM cells with choline analogues, such as N,N'-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine instead of choline, decreased the loss of 14C-labelled fatty acids from phospholipid and neutral lipid. Ethanolamine supplementation resulted in increased loss of 14C from desmesterol as well as decreased incorporation of 14C into desmosterol. Thus, polar head group manipulation affected acyl group composition, acyl group retention, and sterol metabolism in suspension cultured LM cells.

Acetates↗
PubMed1978

Manipulation of fatty acid composition of membrane phospholipid and its effects on cell growth in mouse LM cells.

Fatty acid composition of the phospholipids of mouse LM cells grown in suspension culture in serum-free chemically defined medium was modified by supplementing the medium with various fatty acids bound to bovine serum albumin. Following supplementation with saturated fatty acids of longer than 15 carbons (100 micron) profound inhibition of cell growth occurred; this inhibitory effect was completely abolished when unsaturated fatty acids were added at the same concentration. Supplementing with unsaturated fatty acids such as linoleic acid, linolenic acid or arachidonic acid had no effect on the cell growth. Fatty acid composition of membrane phospholipids could be manipulated by addition of different fatty acids. The normal percentage of unsaturated fatty acids in LM cell membrane phospholipids (63%) was reduced to 35--41% following incorporation of saturated fatty acids longer than 15 carbon atoms and increased to 72--82% after addition of unsaturated fatty acids. A good correlation was found between the unsaturated fatty acid content of membrane phospholipids and cell growth. When incorporated saturated fatty acids reduced the percentage of unsaturated fatty acids in membrane phospholipids to less than 50%, severe inhibition of the cell growth was found. Simultaneous addition of an unsaturated fatty acid completely abolished this effect of saturated fatty acids. The results suggest that maintenance of membrane fluidity by unsaturated fatty acids in membrane phospholipids is critical to membrane integrity and cell growth.

Cell Division↗
PubMed1978

Synthesis of acyl phosphatidylglycerol from phosphatidylglycerol in Escherichia coli K-12. Evidence for the participation of detergent-resistant phospholipase A and heat-labile membrane-bound factor(s).

The conversion of phosphatidylglycerol to acyl phosphatidylglycerol by extracts of Escherichia coli K-12 strains was examined under various conditions. The maximum rate of conversion was observed at pH 7.2 in the presence of 50% (v/v) diethyl ether and 10 mM CaCl2. This conversion was found to involve two sequential reactions: (1) The formation of 2-acyl glycerophosphoglycerol and 2-acyl glycerophosphoethanolamine from phosphatidylglycerol and phosphatidylethanolamine, respectively, by detergent-resistant phospholipase A in the presence of Ca2+ and (2) transfer of the acyl group of 2-acyl lysophospholipid to phosphatidylglycerol by a heat-labile factor(s) in the presence of diethyl ether. Neither fatty, acid acyl-CoA nor 1-acyl lysophospholipid could act as an acyl donor for phosphatidylglycerol. The heat-labile factor(s) was found in both the inner membrane and supernatant fractions.

Calcium↗
PubMed1978

Specific inhibition of desmosterol synthesis by ML--236B in mouse LM cells grown in suspension in a lipid-free medium.

The suspended growth of LM cells in a lipid-free chemically defined medium was almost completely inhibited in the presence of 0.1 microgram/ml of ML-236B, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis in mammalian cells. This inhibition was effectively counteracted by adding a small amount of either mevalonate or cholesterol (dispersed in delipidated calf serum) to culture medium. The synthesis of desmosterol, the end product of sterol biosynthesis in LM cells, from [14C]acetate in cultured cells was highly sensitive to ML-236B, being inhibited 35 and 60% at its concentrations of 0.1 and 1 ng/ml, respectively, while the incorporation of [3H]mevalonate into desmosterol was not affected by ML-236B at concentrations up to 0.1 microgram/ml. Synthesis of fatty acids, phospholipids, triglycerides and macromolecules like DNA, RNA and protein were not suppressed by 10 microgram/ml of ML-236B. Desmosterol content of LM cells was reduced by treatment with ML-236B. These results indicate that ML-236B inhibited cell growth via specific interference in the pathway of sterol biosynthesis, presumably on the step catalyzed by HMG-CoA reductase.

Animals↗
PubMed1976

Nature of Escherichia coli mutants deficient in detergent-resistant and/or detergent-sensitive phospholipase A.

1. Escherichia coli K-12 mutants deficient in detergent-resistant (DR) and detergent-sensitive (DS) phospholipases A and deficient in DS phospholipase A were isolated. 2. The growth, compositions of phospholipids and fatty acids and turnover of phospholipids of three mutants (DR-, DS-, DR-DS-) were compared with those of their parent (DR- was isolated in a previous study). 3. Autodegradations of membrane phospholipids of 18,000 X g supernatants and precipitates of the homogenates of these three mutants were also compared with those of the parent, and the effects of various detergents and organic solvents on these activities were examined. 4. We could not identify any significant physiological role for DR or DS phospholipase A.

Detergents↗
PubMed1975

Lysophospholipase of Escherichia coli.

A lysophospholipase from Escherichia coli cells was purified about 1,500-fold to near homogeneity by extraction with Tris-HCl buffer, streptomycin treatment, (NH4)2SO4 fractionation, column chromatographies on Sephadex G-200, DEAE-cellulose and hydroxylapatite-cellulose, and polyacrylamide gel electrophoresis. The final preparation had a molecular weight of 39,500 plus or minus 500. The enzyme hydrolyzes 1-acylglycerylphosphorylethanolamine, 2-acylglycerylphosphorylethanoiamine, and 1-acylglycerylphosphorylglycerol, but does not attack diacylphospholipids with long chain fatty acids, such as phosphatidylethanolamine and phosphatidylglycerol. The enzyme does not show any esterase activity against p-nitrophenyl acetate or palmitate. Although it does not hydrolyze triacylglycerol or diacylglycerol, it hydrolyzes 1-acylglycerol at almost the same rate as 1-acyl-sn-glycerol-3-phosphorylethanolamine. Results indicated that the acyl-hydrolyzing activities toward monoacyl-glycerylphosphorylethanolamine and monoacylglycerol belong to the same enzyme. In general, acidic and nonionic detergents inhibited the reaction. This lysophospholipase preparation hydrolyzes the monomolecular and micellar forms of lysophospholipids as well as of monoacylglycerol. The monomolecular and micellar forms of Triton X-100 both inhibited the hydrolyses of lysophospholipids and monoacylglycerol.

Carbon Radioisotopes↗
PubMed1974

Genetic mapping of the locus for detergent-resistant phospholipase A(pldA) in Escherichia coli K-12.

An Escherichia coli K-12 mutant deficient for detergent-resistant (DR) phospholipase A, a principal enzyme catalyzing the first step in phospholipid degradation, was characterized genetically. The mutation was found to affect the locus pldA (phospholipid degradation), which is cotransducible both with ilv and metE at a frequencies of 13 and 78%, respectively, and shown to lie between the ilv and metE loci on the E. coli chromosome. DR phospholipase A(1) and A(2) activities were simultaneously transduced with pldA(+) by phage P1; therefore it is proposed that DR phospholipase A has both activities.

Carbon Radioisotopes↗