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Biomedical subjects

F Schroeder

Publications and source records attributed to F Schroeder.

195 records · Page 11Linked to original sources
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↗
PubMed1976

Use of beta-parinaric acid, a novel fouorimetric probe, to determine characteristic temperatures of membranes and membrane lipids from cultured animal cells.

A naturally occurring fluorescent compound, beta-parinaric acid, was employed as a probe to measure the effects of temperature changes on plasma membrenes, microsomes, and mitochondria and on their respective lipids after isolation form LM cells grown in suspension culture. A computer-centered spectrofluorimenter simultaneously measured the absorbance, absorbance-corrected fluorescence, and relative fluorescence efficiency of beta-parinaric acid incorporated into the membranes or isolated membrane lipids. These parameters were measured as a function of temperature. The probe revealed five characteristic breaks or changes in slope with both the plasma membranes as well as their extracted lipids. These discontinuities occurred at approximately 18, 23, 31, 38, and 43 degrees. The other isolated subcellular organelles, microsomes, and mitochondria, as well as their respective isolated lipids, exhibited approximately the same characteristic temperatures (+/- 1 degree) as plasma membranes. Thus, these data negate one criterion of the theory that an asymmetric distribution of characteristic temperatures exist across the membranes of LM cells.

Cell Line↗
PubMed1976

Physical properties of membranes isolated from tissue culture cells with altered phospholipid composition.

A choline-requiring strain of mouse fibroblast cells (LM cells) was cultured in suspension with choline, N,N'-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine. These choline analogues were incorporated into membrane phospholipids as phosphatidyl-N,N'-dimethylethanolamine, phosphatidyl-N-monomethylethanolamine, and phosphatidylethanolamine. Plasma membranes, microsomes, mitochondria, and their respective lipids were isolated and the characteristic temperatures were determined by using two types of fluorescent probes: (a) beta-parinaric acid, a naturally occurring molecule, and (b) 8-anilino-1-naphthalene sulfonic acid, a synthetic organic fluorophore. A computer-centered spectrofluorimeter capable of simultaneous measurement of absorbance, absorbance-corrected fluorescence, and relative fluorescence efficiency was utilized for on-line measurement of all fluorescence parameters. Plots of absorbance corrected fluorescence or of relative fluorescence efficiency versus temperature revealed the same five characteristic temperatures with both types of probe. These characteristic temperatures were independent of the phospholipid composition of the LM suspension cell membranes or their extracted lipids. Plasma membranes, microsomes, and mitochondria containing analogue phospholipids had similar (+/- 1 degree) characteristic temperatures. The presence of analogue phopholipids altered the binding characteristics of beta-parinaric acid with plasma membranes and plasma membrane lipids of LM suspension cells. The equilibrium dissociation constant of plasma membranes and plasma membrane lipids was decreased 2- and 5-fold, respectively, when the cells had been supplemented with ethanolamine. The minimum number of phospholipid molecules per probe binding site was approximately constant in the intact plasma membrane but increased (2-fold) in the isolated plasma membrane lipids. The presence of analogue phospholipids also altered the interaction of 8-anilino-1-naphthalene sulfonic acid with LM cell membranes. The equilibrium dissociation constant of this probe interacting with mitochondrial lipid was decreased 40% by ethanolamine supplementation. The fluorescent properties of both probes were sensitive to the degree of methylation of the polar head group. The absolute values of absorbance-corrected fluorescence and relative fluorescence efficiency were different for each type of membrane from LM cells even with the same analogue supplement. Thus, it appears that LM cells maintain the characteristic temperatures which are a measure of the physical properties of their membranes, despite large alterations of the phospholipid polar head group composition.

Anilino Naphthalenesulfonates↗
PubMed1976

Isolation and characterization of subcellular membranes with altered phospholipid composition from cultured fibroblasts.

Plasma membranes, microsomes, and mitochondria were isolated from mouse fibroblast (LM) suspension cells by modification of several established procedures. Choline analogues such as N,N'-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine were incorporated in vivo into phospholipids of all three cell fractions studied, but to varying degrees depending on the type of analogue used. The in vivo incorporation of these bases into membrane phospholipids produced no significant effect on the activities of seven membrane-bound enzymes: (Na+, K+)-ATPase, 5'-nucleotidase (plasma membranes); TPNH-cytochrome c reductase, glucose-6-phosphatase, inosine diphosphatase (microsomes); and succinate cytochrome c reductase (mitochondria). The incorporation of base analogues into phospholipids was accompanied by several compensatory mechanisms. (a) The quantity of both phosphatidylcholine and phosphatidylethanolamine decreased up to 75% and 50% respectively in 3 days. (b) The molar ratio of desmosterol/phospholipid in the plasma membranes of LM cells grown in suspension culture in the presence of choline analogues decreased from 0.65 to 0.45. (c) The percentage of lysophosphatidylcholine increased over 2-fold in the phospholipid of all subcellular fractions studied. The quantity of lysophosphatidylcholine was directly proportional to the number of methyl groups on the nitrogen atom of the base analogue supplemented to the cells. This was a specific effect since the quantity of lysophosphatidylethanolamine, the other major lysophospholipid, remained unchanged. (d) The ratio of zwitterionic phospholipids to acidic phospholipids remained relatively constant in all isolated membrane fractions regardless of analogue supplementation. Neither increase in the degree of unsaturation nor shortening of fatty acid chain length was noted in response to analogue supplementation.

Cell Division↗
PubMedDate not supplied

Expression of rat L-FABP in mouse fibroblasts: role in fat absorption.

Fatty acid-binding proteins (FABP) are abundant cytosolic proteins whose levels is responsive to nutritional, endocrine, and a variety of pathological states. Although FABPs have been investigated in vitro for several decades, little is known of their physiological function. Liver L-FABP binds both fatty acids and cholesterol. Competitive binding analysis and molecular modeling studies of L-FABP indicate the presence of two ligand binding pockets that accommodate one fatty acid each. One fatty acid binding site is identical to the cholesterol binding site. To test whether these observations obtained in vitro were physiologically relevant, the cDNA encoding L-FABP was transfected into L-cells, a cell line with very low endogenous FABP and sterol carrier proteins. Uptake of both ligands did not differ between control cells and low expression clones. In contrast, both fatty acid uptake and cholesterol uptake were stimulated in the high expression cells. In high expression cells, uptake of fluorescent cis-parinaric acid was enhanced more than that of trans-parinaric acid. This is consistent with the preferential binding of cis-fatty acids to L-FABP but in contrast to the preferential binding of trans-parinaric acid to the L-cell plasma membrane fatty acid transporter (PMFABP). These data show that the level of cytosolic fatty acids in intact cells can regulate both the extent and specificity of fatty acid uptake. Last, sphingomyelinase treatment of L-cells released cholesterol from the plasma membrane to the cytoplasm and stimulated microsomal acyl-CoA: cholesteryl acyl transferase (ACAT). This process was accelerated in high expression cells. These observations show for the first time in intact cells that L-FABP, a protein most prevalent in liver and intestine where much fat absorption takes place, may have a role in fatty acid and cholesterol absorption.

Animals↗
PubMedDate not supplied

Chronic ethanol consumption alters transbilayer distribution of phosphatidylcholine in erythrocytes of Sinclair (S-1) miniature swine.

Effects of chronic ethanol consumption on transbilayer distribution of phospholipids in the exofacial and cytofacial leaflets of erythrocytes from chronic ethanol-consuming Sinclair (S-1) miniature swine were examined. Phosphatidylcholine (PC) was predominantly located in the exofacial leaflet and phosphatidylethanolamine (PE) and phosphatidylserine (PS) located primarily in the cytofacial leaflet. Chronic ethanol consumption significantly increased PC content in the exofacial leaflet without changing bulk membrane PC composition. Ethanol-induced changes in PC distribution were specific for PC and not detected in PE or PS. There was also a significant decrease in sphingomyelin in the ethanol group. Sphingomyelin is primarily an exofacial phospholipid. The specific ethanol-induced changes in the exofacial leaflet are consistent with recent studies showing that the exofacial membrane leaflet is more susceptible to effects of ethanol as compared to the cytofacial leaflet. Such specificity of action provides a new way of viewing how ethanol alters membrane structure and function.

Animals↗
PubMedDate not supplied

Cholesterol domains in biological membranes.

Membrane cholesterol is distributed asymmetrically both within the cell or within cellular membranes. Elaboration of intracellular cholesterol trafficking, targeting and intramembrane distribution has been spurred by both molecular and structural approaches. The expression of recombinant sterol carrier proteins in L-cell fibroblasts has been especially useful in demonstrating for the first time that such proteins actually elicit intracellular and intraplasma membrane redistribution of sterol. Additional advances in the use of native fluorescent sterols allowed resolution of transbilayer and lateral cholesterol domains in plasma membranes from cultured fibroblasts, brain synaptosomes and erythrocytes. In all three cell surface membranes, cholesterol is enriched in the inner, cytofacial leaflet. Up to three different cholesterol domains have been identified in the lateral plane of the plasma membrane: a fast exchanging domain comprising less than 10% of cholesterol, a slowly exchanging domain comprising about 30% of cholesterol, and a very slowly or non-exchangeable sterol domain comprising 50-60% of plasma membrane cholesterol. Factors modulating plasma membrane cholesterol domains include polyunsaturated fatty acids, expression of intracellular sterol carrier proteins, drugs such as ethanol, and several membrane pathologies (systemic lupus erythematosus, sickle cell anaemia and aging). Disturbances in plasma membrane cholesterol domains alter transbilayer fluidity gradients in plasma membranes. Such changes are associated with decreased Ca(2+)-ATPase and Na+, K(+)-ATPase activity. Thus, the size, dynamics and distribution of cholesterol domains within membranes not only regulate cholesterol efflux/influx but also modulate plasma membrane protein functions and receptor-effector coupled systems.

Adult↗