Lipid-phase transitions of the strictly anaerobic bacteria Veillonella parvula and Anaerovibrio lipolytica.

As a basis for physicochemical studies on the membranes of the strictly anaerobic bacteria Veillonella parvula, Anaerovibrio lipolytica, and Megasphaera elsdenii, the fatty acyl and alk-1-enyl moieties on the phosphoglycerides of these organism were characterized. Uncommon is the high proportion of a heptadecenoic acyl and alk-1-enyl moiety in these three lactate-fermenting bacteria. In contrast to V. parvula and A. lipolytica, M. elsdenii contains high amounts of branched-chain acyl and alk-1-enyl moieties. Freeze-etching electron microscopy showed that the lipids of the plasma membranes of V. parvula and A. lipolytica go from the liquid crystalline to the gel state upon lowering of the temperature, indicating that the membrane lipids are predominantly in the fluid state. No lipid-protein segregation could be detected in the plasma membrane of M. elsdenii. This can be explained by the abundance of branched-chain fatty acyl and alk-1-enyl residues in the membranes of this organism which may prevent lipid-protein segregation during the lipid-phase transition.     

Comparative study, using fluorescent methods, of cell membranes and their reconstituted liposomes

Cell plasma membranes and proteoliposomes reconstituted from solubilized plasma membranes of thymocytes and Ehrlich ascites carcinoma cells have been studied by fluorescent methods. It has been shown that proteoliposomes are characterized by greater polarization and rigidity of microsurroundings in membrane proteins and greater microviscosity of membrane lipids. Proteoliposomes from thymocyte membranes contain less membrane proteins and express lower polarity of the lipid bilayer than proteoliposomes from Ehrlich ascites cells.     

Modification of the fatty acid composition of Ehrlich ascites tumor cell plasma membranes

The fatty acyl group composition of Ehrlich ascites tumor cell plasma membranes was modified by feeding the tumor-bearing mice diets rich in either coconut or sunflower oil. When coconut oil was fed, the oleate content of the membrane phospholipids was elevated and the linoleate content reduced. The opposite occurred when sunflower oil was fed. Qualitatively similar changes were observed in the plasma membrane phosphatidylethanolamine, phosphatidylcholine and mixed phosphatidylserine plus phosphatidylinositol fractions. These diets also produced differences in the sphingomyelin fraction, particularly in the palmitic and nervonic acid contents. Unexpectedly, the saturated fatty acid content of the plasma membrane phospholipids was somewhat greater when the highly polyunsaturated sunflower oil was fed. The small quantities of neutral lipids contained in the plasma membrane exhibited changes in acyl group composition similar to those observed in the phospholipids. These fatty acyl group changes were not accompanied by any alteration in the cholesterol or phospholipid contents of the plasma membranes. Therefore, the lipid alterations produced in this experimental model system are confined to the membrane acyl groups.     

Modification of the fatty acid composition of Ehrlich ascites tumor cell plasma membranes.

The fatty acyl group composition of Ehrlich ascites tumor cell plasma membranes was modified by feeding the tumor-bearing mice diets rich in either coconut or sunflower oil. When coconut oil was fed, the oleate content of the membrane phospholipids was elevated and the linoleate content reduced. The opposite occurred when sunflower oil was fed. Qualitatively similar changes were observed in the plasma membrane phosphatidylethanolamine, phosphatidylcholine and mixed phosphatidylserine plus phosphatidylinositol fractions. These diets also produced differences in the sphingomyelin fraction, particularly in the palmitic and nervonic acid contents. Unexpectedly, the saturated fatty acid content of the plasma membrane phospholipids was somewhat greater when the highly polyunsaturated sunflower oil was fed. The small quantities of neutral lipids contained in the plasma membrane exhibited changes in acyl group composition similar to those observed in the phospholipids. These fatty acyl group changes were not accompanied by any alteration in the cholesterol or phospholipid contents of the plasma membranes. Therefore, the lipid alterations produced in this experimental model system are confined to the membrane acyl groups.     

Lipid peroxidation and cytotoxicity of Ehrlich ascites tumor cells by ferric nitrilotriacetate

Ferric nitrilotriacetate, which causes in vivo organ injury, induced lipid peroxidation and cell death in Ehrlich ascites tumor cells in vitro. The process was inhibited by butylated hydroxyanisole and enhanced by vitamin C and linolenic acid, indicating a close relationship between cytotoxicity and the lipid peroxidizing ability of Fe3+ NTA. The cytotoxicity was suppressed by glucose and a temperature below 20 degrees C. Lipid peroxidation of Fe3+ NTA-treated cells was greater at 0 degree C than at 37 degrees C, contrary to results with Fe3+ NTA-treated plasma membranes of Ehrlich ascites tumor cell. These results suggested that metabolism and membrane fluidity are important factors in the expression of the Fe3+ NTA-induced cytotoxicity. H2O2 showed a lower cytotoxicity than did Fe3+ NTA but a greater lipid peroxidizing ability. H2O2 appeared to damage the cells less, and was quenched rapidly by cellular metabolism unlike Fe3+ NTA. In transferrin-free medium, Ehrlich ascites tumor cell readily incorporated Fe3+ NTA, and iron uptake was greater than NTA-uptake in Fe3+ NTA-treated cells, suggesting that Ehrlich ascites tumor cell incorporated iron from Fe3+NTA and metabolized it into an inert form such as ferritin.     

Effect of dietary lipids on plasma lipoproteins and fluidity of lymphoid cell membranes in normal and leukemic mice

Mice of the GR/A strain were fed four different isocaloric semipurified diets, enriched in either (1) saturated fatty acids (palm oil), or (2) polyunsaturated fatty acids (corn oil), or (3) palm oil plus cholesterol, or (4) a fat-poor diet containing only a minimal amount of essential fatty acids. We have studied the effects of these dietary lipids on the density profile and composition of the plasma lipoproteins and on the lipid composition and fluidity of (purified) lymphoid cell membranes in healthy mice and in mice bearing a transplanted lymphoid leukemia (GRSL). Tumor development in these mice occurred in the spleen and in ascites. While the fatty acid composition of the VLDL-triacylglycerols still strongly resembled the dietary lipids, the effects of the diets decreased in the order VLDL-triacylglycerols greater than HDL-phospholipids greater than plasma membrane phospholipids. Diet-induced differences in the latter fraction were virtually confined to the content of oleic acid and linoleic acid, and they were too small to affect the membrane fluidity, as measured by fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. Healthy mice were almost irresponsive to dietary cholesterol, but in the tumor bearers, where lipoprotein metabolism has been shown to be disturbed, the cholesterol diet caused a substantial increase in the low- and very-low density regions of both blood and ascites plasma lipoproteins. The cholesterol-rich diet also increased the cholesterol/phospholipid molar ratio and lipid structural order (decreased fluidity) in GRSL ascites cell membranes, but not in the splenic GRSL cell membranes. We conclude that the composition of plasma lipoproteins and cell membrane lipids in GR/A mice is subject to exquisite homeostatic control. However, in these low-responders to dietary lipids the development of an ascites tumor may lead to increased responsiveness to dietary cholesterol. The elevated level of membrane cholesterol thus obtained in GRSL ascites cells did not affect the expression of various cell surface antigens or tumor cell growth.     

Pantothenic acid and its derivatives protect ehrlich ascites tumor cells against lipid peroxidation

Preincubation of Ehrlich ascites tumor cells at 22 or 32°C, but not at 0°C, with pantothenic acid, 4′-phosphopantothenic acid, pantothenol, or pantethine reduced lipid peroxidation (measured by production of thiobarbituric acid-reactive compounds) induced by the Fenton reaction (Fe²⁺ + H₂O₂) and partly protected the plasma membrane against the leakiness to cytoplasmic proteins produced by the same reagent. Pantothenic acid and its derivatives did not inhibit (Fe²⁺ + H₂O₂)-induced peroxidation of phospholipid multilamellar vesicles, thus indicating that their effect on the cells was not due to the scavenging mechanism. Homopantothenic acid and its 4′-phosphate ester (which are not precursors of CoA) neither protected Ehrlich ascites tumor cells against lipid peroxidation nor prevented plasma membrane leakiness under the same conditions. Incubation of the cells with pantothenic acid, 4′-phosphopantothenic acid, pantothenol, or pantethine significantly increased the amount of cellular CoA and potentiated incorporation of added palmitate into phospholipids and cholesterol esters. It is concluded that pantothenic acid and its related compounds protect the plasma membrane of Ehrlich ascites tumor cells against the damage by oxygen free radicals due to increasing cellular level of CoA. The latter compound may act by diminishing propagation of lipid peroxidation and promoting repair mechanisms, mainly the synthesis of phospholipids.     

Alteration of the fatty acid composition of Ehrlich ascites tumor cell lipids.

The fatty acid composition of Ehrlich ascites tumor lipids was altered markedly $\text{in vivo}$ by changing the type of fat fed to the tumor-bearing mice. As compared with regular chow, large differences were produced in polar and neutral lipid fatty acyl groups when the tumor cells were grown in mice fed coconut oil, sunflower oil or fat deficient diets. Subcellular membrane fractions obtained from these cells exhibited similar variations in fatty acyl composition. This experimental system provides large quantities of malignant cells for study of the relationships between membrane lipid structure and function.     

A method for the quantitative determination of glycerolipids containing O-alkyl and O-alk-1-enyl moieties.

We have developed a spectrophotometric procedure, based on a combination of established methods, for the quantitative determination of aklyl and alk-1-enyl (plasmalogens) ether-linked glycerolipids. It depends upon the release of alkylglycerols and alk-1-enylglycerols from phospholipids by phosphlipase C (Bacillus cereus) followed by saponification or by Vitride reduction the phospholipids; aldehydes are subsequently formed and measured colorimetrically after reacting them with a fuchsin reagent. The total alkyl and alk-1-enyl content of glycerolipids is determined oxidation of the sample withperiodate to form aldehydes and alkylglycolic aldehydes. The O-alk-1-enyl lipid content is determined on a separate sample by measuring the aldehydes produced after acid hydrolysis. The quantity of O-alkyl lipids is calculated from the difference between the values obtained for the total ether-lipid content and that of the O-alk-1enyl lipid content. Alternately, direct determination of alk-1-enylglycerols and alkylglycerols can be made if these hydrolytic products are first separated by thin-layer chromatography.     

Flux of free fatty acids among host tissues, ascites fluid, and Ehrlich ascites carcinoma cells

The role of plasma free fatty acids (FFA) in the transport of fatty acids from host tissues to Ehrlich ascites carcinoma in mice was studied. [9,10-(3)H] Palmitate complexed to mouse serum (albumin) was injected either intraperitoneally or intravenously into unanesthetized tumor-bearing mice. The incorporation of radioactivity into tumor extracellular fluid FFA, tumor cell FFA, neutral lipid, phospholipid, water-soluble material in cells and fluid, plasma FFA, host carcass total lipid fatty acids, and water-soluble (i.e., nonlipid) material was measured. In addition, the quantity of fatty acid in each of the above lipid fractions was determined. The data were analyzed by multicompartmental analysis (SAAM) using a digital computer, and fractional rate constants of FA movement within and out of the host-tumor system were calculated. These rate constants and pool size measurements were used to estimate the corresponding fluxes. Although FFA in the tumor's extracellular fluid were replaced rapidly, almost none of the newly formed fluid FFA was derived from plasma FFA. Moreover, the transfer of FFA from the tumor extracellular fluid FFA to plasma FFA was virtually negligible. We suggest that the net amount of FFA required to replace the fluid FFA utilized for tumor energy and growth may be derived from direct transfer of FFA from host tissues to the ascitic fluid and that plasma FFA is not an intermediate in this transport process. The transport of FFA from the host to tumor cell lipids through the tumor extracellular fluid was about 26-fold greater than that required to account for net lipid accumulation during growth.     

Effects of Growth Temperature on Fatty Acid and Alk-1-Enyl Group Compositions of Veillonella parvula and Megasphaera elsdenii Phospholipids

Veillonella parvula ATCC 10790, an anaerobic gram-negative coccus, contains diacyl and alk-1-enyl acyl (plasmalogen) forms of phosphatidylethanolamine and phosphatidylserine. We studied the effect of growth temperature on the lipid composition of this strain. There was a small increase in the phosphatidylethanolamine content but no change in the content of plasmalogens at the lower growth temperatures tested. The total acyl chains and the plasmalogen acyl chains contained between 73 and 80% mono-unsaturated fatty acids at all growth temperatures. The plasmalogen alk-1-enyl chains were significantly more unsaturated in cells grown at 30 and 25°C than in cells grown at 37°C. Differential scanning calorimetry of the hydrated phospholipids showed lower phase transition temperatures for the lipids from the cells grown at the lower temperatures. In Megasphaera elsdenii lipids, which are similar in composition to the lipids of V. parvula, the proportion of phosphatidylethanolamine also increased slightly at lower growth temperatures, with no significant change in the content of plasmalogens. M. elsdenii contained cyclopropane fatty acyl and alk-1-enyl chains in addition to the mono-unsaturated and saturated chains previously reported. As cells entered the stationary phase of growth at 30 and 42.5°C, there was a reciprocal increase in the proportion of cyclopropane acyl chains and decrease in the unsaturated moieties. The total proportion of cyclopropane and unsaturated acyl and alk-1-enyl chains was more than 65% at all growth temperatures studied, and there was no discernible increase in the sum of these moieties at the lower growth temperatures.     

Phospholipid aliphatic chain composition modulates lipid class composition, but not lipid asymmetry in Clostridium butyricum

The phospholipid composition of the butyric acid-producing clostridia is responsive to the degree of enrichment of the lipids with cis-unsaturated fatty acids. When Clostridium butyricum and Clostridium beijerinckii are grown on oleic acid in media devoid of biotin, the acyl and alk-1-enyl chains of the phospholipids become highly enriched with 18:1 and C19-cyclopropane. Under these conditions there is a marked increase in the glycerol acetals of the major plasmalogens of these organisms. We have grown both species on mixtures of palmitate and oleate in the absence of biotin. The alk-1-enyl chains were highly enriched with C18-unsaturated and C19-cyclopropane residues at all but the highest ratios of palmitate to oleate (80:20, w/w) added to the medium. At ratios of palmitate to oleate greater than or equal to 40:60, the saturated acid was incorporated predominantly into the phospholipid acyl chains in both organisms. The effects of increasing unsaturation of the acyl chains as the ratio of oleate to palmitate was increased was examined in C. butyricum. In cells grown on mixtures of palmitate and oleate equal to or exceeding 40% palmitate, the ratio of glycerol acetal lipid to total phosphatidylethanolamine (PE) was relatively constant. As the proportion of oleic acid added to the medium was increased, the ratio of glycerol acetal lipid to PE increased from 0.7 to 2.0. Thus the ratio of the polar lipids appears to respond to the content of phospholipids that contain two unsaturated chains. The fraction of PE present as plasmalogen remained relatively stable (0.82 +/- 0.05) at varying ratios of medium oleic and palmitic acids. Both the glycerol acetal of ethanolamine plasmalogen, and ethanolamine plasmalogen, are shown to be 80% or more in the outer monolayer of the cell membrane. These two polar lipids represent approx. 50% of the phospholipids in cells grown on exogenous fatty acid. The bulk of the remainder is polyglycerol phosphatides. We suggest that the ability of both species to grow with highly unsaturated membranes is related to their ability to modulate their polar lipid composition.     

Effects of dietary fat composition on the Ehrlich ascites tumor fluid lipoproteins.

Mice bearing the Ehrlich ascites tumor were fed diets rich in either coconut oil or sunflower oil. From 20 to 40% less lipid was present in the ascites tumor fluid when the mice were fed the sunflower oil diet. This was associated with a reduction in the amount of very low density lipoproteins (VLDL) and high density lipoproteins (HDL), the main lipoprotein fractions present in the ascites tumor fluid. The VLDL from the mice fed sunflower oil contained more cholesteryl esters and a lower free to esterified cholesterol ratio than those from the mice fed coconut oil. Very little change occurred in the composition of the HDL. All of the lipids contained in both lipoprotein fractions exhibited appreciable differences in fatty acid composition. Much more monoenoic and less polyenoic fatty acid were present in the lipids from the mice fed the coconut oil diet, but no appreciable change in saturated fatty acid content occurred. Similar changes in fatty acid composition were observed in the blood plasma of the tumor-bearing mice. There was no qualitative difference in the apolipoprotein patterns of either the ascites fluid VLDL or HDL. Pyrene fluorescence studies indicated that the fluidity of the VLDL was increased when the mice were fed the sunflower oil diets. No difference in HDL fluidity, however, was observed by this technique. These results indicate that the amount, composition, and physical properties of certain of the lipoproteins contained in the ascites tumor fluid can be modified by changing the composition of the dietary fat fed to mice bearing the Ehrlich ascites tumor.     

Membrane fluidity in Ehrlich ascites tumor cells treated with adriamycin

The incubation of Ehrlich ascites tumor cells with adriamycin resulted in an increase in lipid peroxide content and a decrease in membrane fluidity as measured by electron spin resonance using the paramagnetic probe 5-doxylstearic acid. Coincidently, the incorporation of [3H]thymidine into tumor cells was progressively inhibited as the concentration of adriamycin was increased. The results indicate that adriamycin induces changes in the plasma membrane of Ehrlich ascites tumor cells after exposure to a low, but cytotoxic, level of this agent.     

Characterization and identification of glyceryl ether diesters present in tumor cells

The previously unidentified neutral lipid present in tumor tissues has been isolated from Ehrlich ascites cells and unequivocally identified as a lipid class of glyceryl ether diesters containing various degrees of unsaturation, and ranging in approximate molecular weight from 760 to 990. The glyceryl ether diester fraction was shown to be free from neutral plasmalogens (glyceryl diacyl alk-1'-enyl ethers). The tumor lipid was subjected to saponification, transesterification, and lithium aluminum hydride reduction. The glyceryl monoethers that resulted from deacylation were the 1-isomers ranging in hydrocarbon chain length from C(12) to C(24). The predominant glyceryl ethers were the hexadecyl (49%), octadecyl (21%), and octadecenyl (14%) derivatives. Saturated and mono- and polyunsaturated fatty acids ranging in chain length from C(12) to C(24) carbon atoms were esterified to the glyceryl monoether. Gas-liquid chromatography, thin-layer chromatography, and nuclear magnetic resonance and infrared spectroscopy were used to characterize and identify the intact tumor lipid and its derived products.     

Influenza virus assembly and its defects

The most abundant protein within the influenza virus particles is membrane protein (M protein) which forms an inner virus membrane under a lipid bilayer and plays the role of mediator during the process of assembly of a virus particle on plasma membranes. Ehrlich ascites tumor cells (EAT) when infected with influenza virus, strain WSN, produced virus-like particles containing greatly reduced amounts of M protein. Such particles were extremely fragile and easily lost hemagglutinins. The loss of this glycoprotein was accompanied by a decrease in infectious activity.SDS-PAGE analysis of RNA duplexes formed after hybridization of intracellular labeled mRNAs and unlabeled virion RNA showed that the mRNA for M protein was synthesized in EAT nearly in the same amounts as in productively infected chicken fibroblasts. Accordingly, M protein was readily revealed when the polypeptides of infected EAT were analyzed by SDS-PAGE. Thus, the reduced amount of M protein in virus particles was likely not due to the decrease in its synthesis but rather to its defective structure or to its defective transport and misintegration into plasma membranes of EAT.     

Changes in (Na+ + K+)-ATPase activity of Ehrlich ascites tumor cells produced by alteration of membrane fatty acid composition.

The fatty acid composition of plasma membrane derived from Ehrlich ascites tumor cells was altered in vivo by changing the dietary lipid of the tumor-bearing mice. The activity of (sodium + potassium)-adenosinetriphosphatase ((Na+ + K+ATPase), in partially purified plasma membranes, was measured ass a function of temperature. Arrhenius plots of the data were biphasic. Striking differences, dependent on the membrane fatty acid composition, were observed in the transition temperature and in the energies of activation below the transition temperature. The transition temperatures for the (Na+ + K+)-ATPase of plasma membrane derived from tumor cells grown in mice fed a regular chow diet containing a mixture of fatty acids (PMC), a 16% sunflower oil diet (PMSU), or a 4% tristearin diet (PMTS) were 20, 21, and 13.5 degrees C, respectively...     

Freeze-fracture study of plasma membranes in wild type and daunorubicin-resistant Ehrlich ascites tumor and P388 leukemia cells

The plasma membrane is considered to play a major role in the development and maintenance of the multidrug resistance (MDR) phenotype, a role which may in part be mediated by an inducible 170 kD transmembrane protein (P-170). The present freeze-fracture study of plasma membranes of daunorubicin-resistant Ehrlich ascites and P388 leukemia cells demonstrated a significant increase in the density of intramembrane particles (IMP) in the P-face, but not the E-face, of resistant sublines compared with wild type cells. Furthermore, a three-dimensional histogram plot of the diameters of P-face IMPs in Ehrlich ascites tumor cells showed the emergence of a subpopulation of 9 × 11 nm IMPs not found in wild type cells. The size of these IMPs would be consistent with a MW of approximately 340 kD, thus indicating that P-170, shown to be present in both resistant cell lines by Western blot analysis and immunohistochemical staining, exists as a dimer in the plasma membrane. Incubation with the calcium channel blocker verapamil, in concentrations known to inhibit daunorubicin efflux in resistant cells, showed evidence of membrane disturbance in the form of IMP clustering in both wild type and resistant Ehrlich ascites tumor cells. However, incubation with daunorubicin itself did not alter the freeze-fracture morphology of the plasma membranes.     

Freeze-fracture study of plasma membranes in wild type and daunorubicin-resistant Ehrlich ascites tumor and P388 leukemia cells

The plasma membrane is considered to play a major role in the development and maintenance of the multidrug resistance (MDR) phenotype, a role which may in part be mediated by an inducible 170 kD transmembrane protein (P-170). The present freeze-fracture study of plasma membranes of daunorubicin-resistant Ehrlich ascites and P388 leukemia cells demonstrated a significant increase in the density of intramembrane particles (IMP) in the P-face, but not the E-face, of resistant sublines compared with wild type cells. Furthermore, a three-dimensional histogram plot of the diameters of P-face IMPs in Ehrlich ascites tumor cells showed the emergence of a subpopulation of 9 X 11 nm IMPs not found in wild type cells. The size of these IMPs would be consistent with a MW of approximately 340 kD, thus indicating that P-170, shown to be present in both resistant cell lines by Western blot analysis and immunohistochemical staining, exists as a dimer in the plasma membrane. Incubation with the calcium channel blocker verapamil, in concentrations known to inhibit daunorubicin efflux in resistant cells, showed evidence of membrane disturbance in the form of IMP clustering in both wild type and resistant Ehrlich ascites tumor cells. However, incubation with daunorubicin itself did not alter the freeze-fracture morphology of the plasma membranes.     

Comparison of tyrosine phosphorylation of proteins by membrane fractions from mouse liver, Ehrlich ascites tumor and MH134 hepatoma

When membrane fractions from mouse liver, Ehrlich ascites tumor and MH134 hepatoma were incubated with [gamma-32P]ATP at 0 degree C in the presence of MnCl2, ZnCl2 and NaVO3, proteins were phosphorylated on tyrosines to a larger extent in liver membranes than in tumor membranes. Separation of labelled proteins by SDS-gel electrophoresis showed phosphorylated alkali-resistant bands of 170, 140, 130, 80, 56, 53 and 46 kDa proteins in Ehrlich ascites tumor membranes; liver membranes exhibited more strongly phosphorylated bands of 170, 56, 53 and 46 kDa proteins. Epidermal growth factor stimulated the tyrosine phosphorylation of only a 170 kDa protein, which was more significant in liver membranes. Liver membranes exhibited slightly higher levels of tyrosine protein kinase activity compared to tumor membranes.