Induced rapid phospholipid methylation and arachidonic acid release by dimethyl sulfoxide-treated human promyelocytic leukemic HL-60 cells

The incubation of undifferentiated promyelocytic HL-60 cells with DMSO resulted in the rapid transmethylation of phosphatidyl ethanolamine (PE) into phosphatidylcholine (PC) which was maximal at 60 secs. This rapid generation of PC was followed by a decrease of the methylated phospholipid and the release of arachidonic acid. Thus, the rapid DMSO-induced phospholipid methylation coupled with release of arachidonic acid (precursor for eicosanoids) prior to morphological evidence of cellular differentiation may represent early biochemical events which result in the generation of intracellular chemical signals which may program the promyelocytic cells into a differentiation mode.     

Phospholipid Composition and Metabolism of Micrococcus denitrificans

The phospholipid composition of Micrococcus denitrificans was unusual in that phosphatidyl choline (PC) was a major phospholipid (30.9%). Other phospholipids were phosphatidyl glycerol (PG, 52.4%), phosphatidyl ethanolamine (PE, 5.8%), an unknown phospholipid (5.3%), cardiolipin (CL, 3.2%), phosphatidyl dimethylethanolamine (PDME, 0.9%), phosphatidyl monomethylethanolamine (PMME, 0.6%), phosphatidyl serine (PS, 0.5%), and phosphatidic acid (0.4%). Kinetics of ³²P incorporation suggested that PC was formed by the successive methylations of PE. Pulse-chase experiments with pulses of ³²P or acetate-1-¹⁴C to exponentially growing cells showed loss of isotopes from PMME, PDME, PS, and CL with biphasic kinetics suggesting the same type of multiple pools of these lipids as proposed in other bacteria. The major phospholipids, PC, PG, and PE, were metabolically stable under these conditions. The fatty acids isolated from the complex lipids were also unusual in being a simple mixture of seven fatty acids with oleic acid representing 86% of the total. Few free fatty acids and no non-extractable fatty acids associated with the cell wall or membrane were found.     

The influence of ionic detergents on the phospholipid fatty acid compositions of Porphyridium purpureum

The phospholipid fatty acid composition of Porphyridium purpureum on a solid medium was studied in the presence of sodium dodecyl sulphate (SDS) and cetyl trimethylammonium bromide (CTAB). The most common fatty acids in phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) were palmitic (16:0), stearic (18: 0), linoleic (18:2ω 6), arachidonic (20:4ω 6) and eicosapentaenoic (20:5ω 3) acids, 20:4ω 6 being very abundant. In phosphatidyl glycerol (PG) the most common acids were 16:0, trans-hexadecenoic acid (tr 16:1ω 13), oleic acid (18:1) and 20:4ω 6. Both detergents increased the saturation grade of PC and PE by decreasing the relative amount of the polyunsaturated acids, especially 20:4ω 6. A corresponding increase in the amounts of saturated acids was observed in PC and PE. The changes in PG fatty acid composition were not very significant: a slight increase was observed in the amounts of 16:0 and tr 16:1ω 13 , with a corresponding decrease in the amounts of 20:4ω 6 and 20:5ω 3. Both detergents decreased the PC/PE and the (PC + PE)/PG ratios very markedly, most probably as a result of increases in the amounts of PE and PG. In the presence of CTAB the cells seemed to contain much more phospholipids than in the presence of SDS, perhaps as a result of the mucilage-precipitating effect of CTAB. The significance of the findings is discussed.     

Lipid composition of Friend leukemia cells following induction of erythroid differentiation by dimethyl sulfoxide

The effects of dimethyl sulfoxide (DMSO)-induced differentiation of Friend leukemia cells in vitro on the lipid composition of these cells have been examined. DMSO had no early effect on the incorporation of either [¹⁴C] glycerol or [³H] methyl choline chloride into the total lipids or individual phospholipids of Friend cells up to 240 min after addition of the inducer. Examination of DMSO-diferentiated Friend cell phospholipids revealed a percentage composition which was similar to control cells, with phosphatidylcholine and phosphatidylethanolamine in both uninduced and differentiated cells accounting for over 75% of the total phospholipid. Sphingomyelin levels were significantly lower in Friend cells than in normal adult mouse erythrocytes, and differentiation of murine erythroleukemia cells resulted in a further lowering of this phospholipid. In contrast, a significant increase in the level of phosphatidylethanolamine occured as a result of maturation. Fatty acid analysis of major lipid classes of differentiated Friend cells showed significant reduction in saturation, but no alteration in chain length in comparison to undifferentiated cells. A pronounced decrease in the cellular content of both free and esterified cholesterol, which resulted in a 45% decrease in the ratio of cholesterol/phospholipids, occurred in cells differentiated by the polar solvent. The findings indicate that erythrodifferentiation induced by DMSO results in a variety of changes in the lipid composition of the membranes of Friend leukemia cells.     

Changes in phospholipids and free fatty acids in the brains of mice preconditioned by hypoxia.

Changes in the composition and contents of phospholipids and free fatty acids were observed and compared in three groups: (A) unpreconditoned normal controls, (B) exposure to 1 run of hypoxia and (C) exposure to 4 runs of hypoxia. In group B, the content of phosphatidyl ethanolamine (PE), phosphatidyl serine (PS) and free fatty acids (FFAs) increased significantly and the content of phosphatidyl choline (PC) and sphingomyelin (SM) decreased significantly. While in group C the content of PE, PS, PC and FFAs changed significantly when compared with that of group B, all phospholipid (except SM) and FFA contents tended to decrease to the level of group A. No new FFA was seen in the brain homogenates in any of the three groups. These results suggest that the changes in the content of mouse brain phospholipids and FFAs may be adaptive and involved in the animals' tolerance to hypoxia.     

Effect of cryopreservation on lipids and some physiological features of spermatozoa from rams pastured in highlands and in valleys

The effect of low temperature preservation on the motility and morphology of acrosomes, acrosomal proteolytic activity, phospholipid and fatty acid composition of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE), and the cholesterol/phospholipid molar ratio in sperm from rams housed in the highlands or in the valleys, were studied. The indices of motility and morphological integrity of sperm from highland rams were much greater compared with those of valley rams. Phosphatidyl choline (PC) of the highland rams was more unsaturated, while PE was more saturated compared with those of valley rams. Cryopreservation of the sperm from highland rams significantly increased the content of choline plasmalogen, accompanied by a slight rise in the levels of lysophosphatidyl choline (LPC) and phosphatidyl inositol (PI) in their sperm. The fatty acid composition altered following cryopreservation. These variations were mainly due to a decrease in the amount of docosahexaenic acid and an increase in the amounts of linoleic and palmitic fatty acids. The results may be indicative of the fact that the alterations in the sperm of the valley rams were more pronounced and they may be attributed to the structural features of the sperm, as well as a reduced concentration of oxygen in the organs and tissues of the highland rams.     

Effects of orally administered cytidine 5'-diphosphate choline on brain phospholipid content

Cytidine, as cytidine 5'-diphosphate choline (CDP-choline), is important for the synthesis of phosphatidylcholine in cell membranes. To investigate whether exogenous CDP-choline could affect brain phospholipid composition, we supplemented the diet of mice with this drug (500 mg/kg/day) for 27 months in 3-month-old mice and for 90, 42, and 3 days in 12-month-old mice, and measured their levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and the content of phosphatidylinositol plus phosphatidic acid in the cerebral cortex. After 27 months of treatment, PC and PE increased significantly by 19% (P < 0.05) and by 20% (P < 0.01), respectively. PS levels increased by 18% (not statistically significant). Similar elevations in PC and PE levels were obtained when older mice were treated for only 3 months (P < 0.05). No changes were observed with shorter treatment periods. These results suggest that chronic administration of CDP-choline can have effects on brain phospholipid composition that may underlie its reported utility in various neurologic disorders.     

Changes in Glycolipid and Phospholipid Compositions in Cotyledons of Germinating Soybeans

This investigation was conducted to observe changes in the compositions of fatty acids, glycolipids (GL) and phospholipids (PL) in cotyledons of soybean seeds which were germinated either in the dark or the light at 28°C for 8 days. The patterns of changes in lipid composition depended on the germinating conditions tested. In general, non-polar lipids were metabolized at a faster rate than polar lipids. Changes in lipid contents in cotyledons were also observed more clearly with the polar lipids than with the non-polar ones, especially in the light-grown seedlings. The major component of lipid, GL in chloroplasts, appeared rapidly at an earlier stage in the cotyledons of light-grown seedlings. During germination of soybean seeds, acyl sterylglucoside in cotyledons decreased rapidly, but monogalactosyl diglyceride and digalactosyl diglyceride (DGD) increased in the light-grown seedlings, whereas sterylglucoside and DGD increased in the dark-grown seedlings.

The major PL present immediately after immersion were phosphatidyl ethanolamine (PE), phosphatidyl choline (PC) and phosphatidyl inositol (PI). During germination under both conditions, light and dark, PE in cotyledons decreased with PC or PI, while phosphatidic acid increased rapidly, and phosphatidyl glycerol and diphosphatidyl glycerol also increased slightly. These changes in glycolipid and phospholipid compositions during germination seem to occur from the formation of photosynthetic tissues and the metabolic interconversion of phospholipids.     

Specific esterase activity induced in mouse Friend erythroleukemia cells by dimethylsulfoxide.

Three cell lines of mouse erythroleukemia transformed by Friend virus (FLC), namely 745, F4-1, and 3BM-78, were grown for six days in the absence or in the presence of 1.5% (v/v) dimethylsulfoxide (DMSO) and compared cytochemically for naphtol-AS D-chloroacetate esterase (E), alkalinephosphatase (AP), myeloperoxidase (MP) and periodic acid Schiff (PAS) reaction activity. In the absence of inducer only 1–2% of slightly E positive cells could be found. E positivity greatly increased in 3BM-78 and F4-1 but poorly in 745 cells, after treatment with DMSO. Unlike E reaction, AP and MP reactions were positive in about 5% 3BM-78 and F4-1 cells without DMSO, but there were no positive cells after DMSO treatment. All three lines were always PAS negative. Hemoglobin synthesis (benzidine staining) was intensively induced by DMSO in all three lines. Morphologically after DMSO treatment, FLC matured displaying characteristics of basophilic megaloblastoid cells. The emergence of specific esterase activity, a marker of granulocytes, in FLC differentiating along the erythroid pathway, suggests that in these leukemia cells the genetic determinants for leukopoietic differentiation are retained and capable of being expressed phenotypically.     

Phospholipid-transfer proteins in human liver and primary liver carcinoma

Investigations have been carried out on phospholipid-transfer activity of the cytosol and the phospholipid composition of subcellular membranes from human liver and primary liver carcinoma. In both human liver and primary liver carcinoma cytosolic fractions, the transfer activity for phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin has been observed for the first time. The transfer rate of PC and PE in normal human liver was almost equal, whereas sphingomyelin-transfer activity was much slower. In carcinoma cells, the transfer activity for PE and PC was significantly enhanced, while sphingomyelin transfer remained unchanged. Comparative investigations with HepG2 cultured cells have revealed a high PE-transfer activity in this cell line. Parallel with the phospholipid-transfer activity modifications in neoplasic cells, changes in the phospholipid composition of microsomes and mitochondria have been observed. The content of PC and PE in hepatocarcinoma cells was decreased in microsomes, while in the mitochondria it was increased. The possible role of the phospholipid-transfer proteins in the maintenance of membrane composition and structure is discussed.     

Characteristics of ether-linked glycerophospholipids in Friend erythroleukaemia cells differentiated by dimethyl sulphoxide or hexamethylenebisacetamide and in non-inducible clones treated with the inducers.

The present study deals with changes in ether-linked glycerophospholids which accompany differentiation of Friend erythroleukaemia (FEL) cells by dimethyl sulphoxide (DMSO) and hexamethylenebisacetamide (HMBA). We also tested clones of FEL cells non-inducible by DMSO or HMBA for ether-linked lipid changes not related to the differentiation process. FEL cells contained appreciable proportions of alkenylacyl and alkylacyl subfractions in phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Compared with FEL cells, clones non-inducible by DMSO or HMBA had a greater amount of alkenylacyl PE associated with a lack of alkenylacyl PC. The differentiation of FEL cells by DMSO or HMBA was accompanied by a reduction of alkylacyl PE and PC. DMSO- and HMBA-differentiated FEL cells showed changes in alkenyl- and alkyl-chain profiles, some of which were also observed in non-inducible FEL cells treated with DMSO or HMBA.     

Effect of membrane phosphatidylethanolamine-deficiency/phosphatidylcholine-excess on the metabolism of phosphatidylcholine and phosphatidylethanolamine.

Cells of epithelial origin generally require ethanolamine (Etn) to grow in defined culture medium. When such cells are grown without Etn, the membrane phospholipid composition changes drastically, becoming phosphatidylethanolamine (PE)-deficient due to a reduced de novo rate of PE synthesis, and growth stops. We have hypothesized that the cessation of growth occurs because this membrane phospholipid environment is no longer suitable for membrane-associated functions. Phospholipid has long been known to play a role in the transduction of some signals across membranes. In addition to the well-known phosphatidylinositol cycles, hydrolysis of phosphatidylcholine (PC) and PE has recently been shown to play a central role in signal transduction. Using an Etn-requiring rat mammary cell line 64-24, we have studied the metabolism of PC and PE in response to the phorbol ester phorbol 12,13-dibutyrate (PDBu) under conditions where cells have either normal or PE-deficient membrane phospholipid. In cells having normal membrane phospholipid, the synthesis of PC was stimulated by PDBu (approximately fourfold), as was the degradation of PC and PE (by twofold and fourfold, respectively). Product analysis suggested that PDBu stimulated hydrolysis of PC by both phospholipases C and D (PLC and PLD), and of PE by PLD. However, in PE-deficient cells, neither lipid synthesis or degradation were significantly stimulated by PDBu. Analysis of the CDP-choline pathway of PC synthesis indicated that the regulatory enzyme, CTP:phosphorylcholine cytidylyltransferase, was stimulated about twofold by PDBu in cells having normal membrane, but not in PE-deficient cells. These results indicate that the membrane phospholipid environment profoundly affects phospholipid metabolism, which no doubt influences cell growth and regulation.     

Effect of phophatidylcholine and phosphatidylethanolamine enrichment on the structure and function of yeast membrane

The phospholipid composition of yeast plasma membrane was manipulated by two different methods: (i) by using two auxotrophic strains KA101 (cho1) and MC13 (Cho⁺) which required phospholipid bases for growth and (ii) by supplementing Saccharomyces cerevisiae (3059) cells with high concentration of choline or ethanolamine. It was possible to enrich the plasma membrane with phosphatidylcholine (PC) or phosphatidylethanolamine (PE) by both methods. The uptake of amino acids, e.g., glycine, glutamic acid, leucine, lysine methionine, phenylalanine, proline and serine, was significantly reduced in PC- or PE-enriched cells. However, the extent of reduction in transport was variable among different strains. A fluorescent probe, 1-anilino-8-naphthalene sulfonate (ANS), was used to monitor the structural changes induced by altered phospholipid composition. It was observed that the relative fluorescence intensity of bound ANS was decreased as a consequence of PC or PE enrichment. The decrease in fluorescence was probably associated with reduced number of available binding sites (n) and increased apparent dissociation constant (K_d). Furthermore, our results also suggest that a critical level of PE or PC is required for proper functioning of yeast membrane.     

Lipid peroxidation and phospholipase A2 activity in liposomes composed of unsaturated phospholipids: a structural basis for enzyme activation

The effect of lipid peroxidation on membrane structure and phospholipase A2 activity was studied using liposomes composed of bovine liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The phospholipids were mixed at set ratios and sonicated to yield small unilamellar vesicles. The liposome preparations were subjected to lipid peroxidation as induced by cumene hydroperoxide and hematin. Under these conditions, a sharp increase in lipid peroxidation was noted over a 30 min incubation period and was accompanied by loss of polyunsaturated fatty acids (PUFA). Liposomes enriched in PE were most extensively peroxidized with a preferred oxidation of this phospholipid. The extent of PC oxidation was also greater in liposomes containing the largest proportions of PE. Analysis of liposome anisotropy, via steady-state fluorescence polarization of diphenylhexatriene indicated that progressive increases in either PE content or the level of lipid peroxidation increased the apparent microviscosity of the vesicles. Moreover, lipid peroxidation increased anisotropy more effectively than variations in the ratios of PE vs. PC. Thus, peroxidation of 5-10% of the phospholipids produced the same anisotropy increase as a 20% increase in the ratio of PE vs. PC. Analysis of vesicle turbidity suggested that fusion was also more readily achieved through lipid peroxidation. When liposomes were incubated with 0.4 U/ml of snake venom phospholipase A2, a direct correlation was found between the degree of lipid peroxidation and the extent of phospholipid hydrolysis. The more unsaturated phospholipid, PE, was most extensively hydrolyzed following peroxidation. Increasing the proportion of PE also resulted in more extensive phospholipid hydrolysis. These findings indicate that lipid peroxidation produces a general increase in membrane viscosity which is associated with vesicle instability and enhanced phospholipase A2 attack. A structural basis for membrane phospholipase A2 activation as a consequence of lipid peroxidation is discussed in light of these findings.     

Temperature Dependence of Membrane Lipid Composition in Early Blastula Embryos of Lytechinus pictus: Selective Sorting of Phospholipids into Nascent Plasma Membranes

Lytechinus pictus eggs were fertilized and incubated at 10, 16, and 23°C until the early blastula stage of embryonic development. The phospholipid composition of the embryos and control unfertilized eggs remain identical and unchanged as incubating temperatures are varied; thus, neither incubating temperature, fertilization nor membrane assembly affect their total phospholipid composition. This result agrees with metabolic studies by others, using only a single incubation temperature, and indicates that embryonic development to the early blastula stage occurs with little, if any, de novo phospholipid biosynthesis. However, as in all poikilotherms, the phospholipid composition of the nascent plasma membranes varies with the incubation temperature. Thus, until the blastula stage of embryonic development, the lipids of these newly formed plasma membranes are derived from lipid pools within the embryo whose phospholipid composition is static. The variation of plasma membrane composition is primarily reflected in an increase in the phosphatidylethanolamine (PE): phosphatidylcholine (PC) ratio as incubating temperatures decrease; this is achieved by an exchange of PE for PC. Several mechanisms are considered for the specificity of the selective sorting and assembly of these phospholipids into the nascent plasma membranes. Received: 16 March 1999/Revised: 15 May 1999     

Alterations in the phospholipid composition of Escherichia coli B during growth at different temperatures

The major phospholipid classes of Escherichia coli B, phosphatidyl ethanolamine, cardiolipin, and phosphatidyl glycerol, were quantitated at different stages of the growth cycle. The organisms were incubated at both 27 and 37 C. Significant differences were observed both in the amounts of total lipid phosphorus per gram (dry weight) of cells and in the relative percentages of the individual phospholipids. At 37 C the total amount of lipid phosphorus decreased significantly throughout the growth cycle. However, at 27 C total lipid phosphorus accumulated. The patterns of the three major phospholipid classes of Escherichia coli exhibited complex quantitative changes. In addition, some evidence based on glycerol to phosphate molar ratios indicated that phosphatidyl glycerolphosphate replaced phosphatidyl glycerol during the late growth stages of E. coli B when grown at 27 C. A comparative analysis of phospholipid and fatty acid patterns led to a hypothesis attempting to explain some reported variations in the lipid composition of E. coli under different conditions of growth.     

Comparative metabolism of phospholipids of osmoregulation effector organs in European eel (Anguilla anguilla)]

The phospholipid composition from various organs of the fresh water eel, such as gill, kidney, gut, liver and muscle, were determined by thin-layer chromatography. The major phosphatides found in these tissues were PC, PE and SPH and minor constituents PS, PI, DPG, AP and also LPC in the gut. A greater percentage of PS and SPH occurs in the osmoregulatory effector organs such as gill, kidney, and gut. From in vivo comparative kinetic studies of the 32P incorporation into the phospholipids, between 6 and 48 hours, certain remarkable features of phospholipid metabolism have been found in these tissues. A low uptake of inorganic 32P into the tissue lipid phosphorus was observed in the eel at 15 degrees C. The specific activity of the lipid phosphorus increased continuously in all tissues during 48 hours after 32P injection. During this experimental period, phosphatidic acid and phosphatidyl inositol fractions were labelled most rapidly in gill, kidney and gut, while the specific activity of the phosphatidyl choline fraction remained low in these organs. In liver, the rate of PC formation appears to be faster than the PI and PE biosynthesis. In gill and gut, the PE showed greater turnover than the PC as measured by 32P incorporation. In the eel, an euryhalin fish, the DPG of osmoregulatory effector organs has a high specific activity at all times. PS showed only a high specific activity in the gill. Labelling of SPH occured slowly in the various tissues only becoming evident after 24 hours. The results are compared with those published for other poikilotherm and homeotherm vertebrates. Relative differences between the turnover of various tissue phosphatides are discussed with of reference to the general scheme on phospholipid biosynthesis and to the physiological functions of the various organs.     

Effects of linoleic acid and mitogenic stimulation on the fatty acid composition of human lymphocytes

Perturbation of the fatty acid composition of human lymphocytes in vitro was investigated by addition of linoleic acid complexed to bovine serum albumin (BSA-LA) and by mitogenic stimulation with phytohaemagglutinin (PHA). BSA-LA resulted in a 45% increase in linoleic acid in phosphatidylethanolamine (PE) and over 100% in phosphatidylcholine (PC) in peripheral blood cells. Supplementation with BSA-LA in PHA-stimulated lymphocytes produced even greater changes: 100% increase in linoleic acid content for PE and over 300% for PC. There was a large decrease in oleic acid: 40% for PE and almost 100% in PC. Significant decreases in arachidonic acid occurred in both phospholipid fractions. PHA alone also altered membrane phospholipid fatty acid composition, with reductions in palmitic, stearic and linoleic acid for PE and increases in oleic acid and arachidonic acid (almost 100%). For PC, there were large decreases in stearic (40%), linoleic (30%) and arachidonic (40%) acids, together with an increase in oleic acid (65%). Cells supplemented with linoleic acid grown in the presence of PHA, compared with those grown in linoleic acid-supplemented medium alone, showed a 40% decrease in palmitic acid and a 55% increase in arachidonic acid in PE. For PC, there were large decreases in stearic acid (40%) and arachidonic acid (57%). Antibody-induced redistribution of surface molecules ('capping') was inhibited by some 14% after incubation with BSA-LA. However, no consistent alterations in PHA-induced cell proliferation were observed. These data suggest that profound alterations of membrane fatty acid composition occur spontaneously during the mitotic cycle, and may be further induced by experimental manipulation, without gross perturbation of cell function.     

The relationship between plasma membrane lipid composition and physical-chemical properties. III. Detailed physical and biochemical analysis of fatty acid-substituted EL4 plasma membranes

Murine leukemia EL4 cells were modified by supplementation of culture media with fatty acids for 24 h. A plasma membrane-enriched fraction was prepared from substituted and normal cells. Analyses were performed to determine fatty acyl composition, phospholipid headgroup composition and cholesterol content. The two major membrane phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were isolated by thin-layer chromatography and ESR measurements were done on liposomes prepared from these lipids as well as on the intact plasma membrane preparations. Slight perturbations in overall plasma membrane lipid composition were observed when EL4 cells were supplemented with a single exogenous fatty acid. This may be consistent with the idea that the incorporation of exogenous fatty acid induces compensatory changes in membrane lipid composition. On the other hand, we observed no significant difference in two ESR motional parameters between the unsubstituted control and various fatty acid-substituted plasma membranes. ESR measurements carried out on PE and PC liposomes derived from 17:0- and 18:2c-substituted membranes also failed to detect major differences between these liposomes and those made from normal EL4 phospholipids. In the case of liposomes prepared from 18:2t,-substituted membranes, the order parameter was significantly changed from the normal. However, the change was in opposite directions in PE and PC, perhaps accounting for the fact that no change parameter is seen in intact 18:2t-substituted plasma membrane. Measurements of order parameter (S) in mixed lipid vesicles showed that at up to 50 mol% mixture of a synthetic PC with plasma membrane PC, the value of S was only marginally different from that of the plasma membrane PC vesicles. We interpret these data as an indication that the two ESR parameters used are not sufficiently sensitive to detect changes due to modifications of the acyl chain composition of a complex biological membrane.