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Spiroplasma membrane lipids.

Membranes of six spiroplasma strains belonging to different Spiroplasma species and subgroups were isolated by a combination of osmotic lysis and sonication in the presence of EDTA to block endogenous phospholipase activity. Analysis of membrane lipids showed that in addition to free and esterified cholesterol the spiroplasmas incorporated exogenous phospholipids from the growth medium. Sphingomyelin was preferentially incorporated from phosphatidylcholine-sphingomyelin vesicles or from the serum used to supplement the growth medium. Palmitate was incorporated better than oleate into membrane lipids synthesized by the organisms during growth. The major phospholipid synthesized by the spiroplasmas was phosphatidylglycerol. The positional distribution of the fatty acids in phosphatidylglycerol of Spiroplasma floricola resembled that found in Mycoplasma species, in which the saturated fatty acids prefer position 2 in the glycerol backbone and not position 1 as found in Acholeplasma species and elsewhere in nature. Electron paramagnetic resonance analysis of spin-labeled fatty acids incorporated into S. floricola membranes exhibited homogeneous single-component spectra without immobilized regions. The S. floricola membranes were more rigid than those of Acholeplasma laidlawii and less rigid than those of Mycoplasma gallisepticum.

Cholesterol↗

Plasma lipids, erythrocyte membrane lipids and blood pressure of hypertensive women after ingestion of dietary oleic acid from two different sources.

OBJECTIVE: To study the effect of a diet rich in mono-unsaturated fatty acids (MUFA), from high-oleic sunflower oil (HOSO) and olive oil, on plasma lipids, erythrocyte membrane lipids (including fatty acid composition) and blood pressure of hypertensive (normocholesterolaemic or hypercholesterolaemic) women. METHODS: There were 16 participants who were hypertensive women aged 56.2 +/- 5.4 years. The participants ate a diet enriched with HOSO or olive oil for two 4-week periods with a 4-week washout period before starting the second type of MUFA diet. At entry and during study of each diet, plasma lipids and apolipoproteins were measured by conventional enzymatic methods. Erythrocyte membrane lipid and fatty acid compositions were analysed by means of the latroscan thin-layer chromatography/flame ionization detection technique and by gas chromatography, respectively. Blood pressure was also measured. The statistical analysis was conducted by using Student's two-tailed paired t-test. RESULTS: In both groups of hypertensive patients, there was a significant increase in plasma high-density lipoprotein (HDL) cholesterol concentration after the HOSO or olive oil diets, with regard to baseline. Additionally, a significant decrease in plasma HDL2 cholesterol concentration and an increase in plasma HDL3 cholesterol concentration were evident. The membrane free-cholesterol concentration increased significantly and the phospholipid concentration decreased significantly in erythrocytes after the olive oil diet, though both MUFA diets produced a significant decrease in the concentration of membrane esterified cholesterol. Therefore, the molar ratio of cholesterol to phospholipids was raised significantly in the erythrocyte membrane of hypertensive women after the dietary olive oil, but not after the HOSO diet. In the hypertensive and normo-cholesterolaemic group the HOSO diet significantly increased the content in the erythrocyte membrane of oleic, eicosenoic, arachidonic and docosapentaenoic acids, whereas the olive oil diet increased the content of palmitoleic acid and long-chain polyunsaturated fatty acids of the n-3 family besides, compared with baseline. A significant decrease in linoleic acid was also evident. In the hypertensive and hypercholesterolaemic group, the HOSO diet resulted in significant increases in palmitoleic, oleic, eicosenoic and behenic acids, whereas the olive oil diet enhanced the content of arachidonic, docosapentaenoic and docosahexaenoic acids besides, with respect to baseline. In addition, there was a significant decrease in stearic acid, but only after dietary olive oil was there a decrease in linoleic acid. The most important differences between the two MUFA diets were the increase in n-3 fatty acids and the decrease in the n-6; n-3 fatty acids ratio after dietary olive oil in the erythrocyte membranes of hypertensive patients. Interestingly, a significant reduction in systolic and diastolic blood pressures was only evident after the ingestion of olive oil. CONCLUSION: These data suggest that the beneficial effects of dietary olive oil on the plasma lipids and lipoprotein profile, lipid and fatty acid composition of erythrocyte membrane, and blood pressure in women with untreated essential hypertension are not found equally for the HOSO-rich diet, despite both vegetable oils providing a similar concentration of MUFA.

Aged↗

Role of membrane lipids and membrane fluidity in thermosensitivity and thermotolerance of mammalian cells.

The role of membrane lipids and membrane fluidity in thermosensitivity of mammalian cells is not well understood. The limited experimental data in the literature have led to conflicting results. A detailed investigation of lipid composition and membrane fluidity of cellular membranes was undertaken to determine their relationship to cell survival after hyperthermia. Ehrlich ascites (EA) cells, mouse fibroblast LM cells, and HeLa S3 cells differed in thermosensitivity as expressed by a D0 of 3.1, 5.2, and 9.7 min, respectively, at 44 degrees C. No correlation with cellular thermosensitivity could be found with respect to the amount of cholesterol and to the cholesterol to phospholipid ratio in the particulate fraction of the cells. By growing the cells for some generations in different media, cholesterol and phospholipid content could be changed in the particulate fraction, but no difference in cell survival was observed. When mouse fibroblasts were grown for 24 hr in a serum-free medium supplemented with arachidonic acid (20:4), all subcellular membranes were about eight times richer in phospholipids containing polyunsaturated acyl (PUFA) chains and membrane fluidity was increased as measured by fluorescence polarization of diphenylhexatriene (DPH). The alterations resulted in a higher thermosensitivity. When mouse fibroblasts were made thermotolerant no change in cholesterol and phospholipid content could be found in the particulate fraction of the cells. The relative weights and the quality of the phospholipids as well as the fatty acid composition of the phospholipids appeared to be the same for normal and thermotolerant cells. Fluidity measurements in whole cells, isolated plasma membranes, and liposomes prepared from phospholipids extracted from the cells revealed no significant differences between normal and thermotolerant fibroblasts when assayed by fluorescence polarization (DPH) and electron spin resonance (5-nitroxystearate). It is concluded that the mechanism of thermal adaptation resulting in differences in lipid composition as reported in the literature differs from the mechanism of the acquisition of thermal tolerance. The lower heat sensitivity of thermotolerant cells, as initiated by a nonlethal triggering heat dose followed by an induction period at 37 degrees C, does not involve changes in lipid composition and membrane fluidity. However, a prompt and clear (also nonlethal) change in membrane fluidity by an increase in PUFA does result in an increased thermosensitivity, probably because of an indirect effect via the lipids in causing disfunctioning of proteins in the membrane and/or the cytoskeleton.

Acclimatization↗

Red blood cell susceptibility to lipid peroxidation, membrane lipid composition, and antioxidant enzymes in continuous ambulatory peritoneal dialysis patients.

OBJECTIVE: To investigate the overall susceptibility of red blood cells (RBC) to lipid peroxidation from patients on continuous ambulatory peritoneal dialysis (CAPD). METHODS: The following parameters were measured: RBC malondialdehyde (MDA) production after oxidative stress with H2O2, RBC antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and RBC membrane lipid composition. The levels of plasma vitamin E and serum selenium were also assayed. PATIENTS: Eleven patients on continuous ambulatory peritoneal dialysis. Twenty-one healthy blood donors of similar age were used as normal controls. RESULTS: The MDA formation after H2O2 stimulation was normal in CAPD patients (0.79 +/- 0.1 mumol/gHb versus 0.78 +/- 0.1 in the control group). RBC from CAPD patients also showed a normal SOD activity, a more than adequate vitamin E status, and a peculiar pattern of membrane lipids, with reduced polyunsaturated fatty acids (p less than 0.001) and increased monounsaturated fatty acids (p less than 0.001). Both RBC GSH-Px activity, a selenium-dependent enzyme, and serum selenium levels were significantly lower in CAPD patients, and a significant positive correlation (r = 0.68; p less than 0.02) between the two parameters was found. CONCLUSIONS: This study found a normal sensitivity to oxidant stress in RBC from a group of CAPD patients, despite an impaired GSH-Px activity. The peculiar lipid pattern of RBC membrane, characterized by reduced PUFA and increased MUFA content, may contribute, in addition to adequate SOD activity and vitamin E status, to normal RBC lipid peroxidation.

Aged↗

Interaction of saponin and digitonin with black lipid membranes and lipid monolayers.

The effects of the plant glycosides saponin as well as digitonin on the electrical conductance of black lipid membranes and the effect of these agents on the surface pressure of lipid monofilms was investigated. Both saponin and digitonin induced channel-like fluctuations in planar bilayers made either of diphytanoylphosphatidylcholine ( DPhPC ) or of DPhPC and cholesterol 2: 1 (w/w). In cholesterol-free bilayers the amount needed to induce an increase in conductance was 0.3-1 mg/ml for saponin and about 0.2 mg/ml for digitonin. In contrast, in cholesterol-containing bilayers the concentration needed to induce pores was about 10 micrograms/ml for both saponin and digitonin. In cholesterol-containing membranes the fluctuating pores induced by saponin were about 3-times more permeable to K+ than to Cl- and the macroscopic current showed an ohmic behaviour. Surface pressure experiments demonstrate that both glycosides could penetrate into lipid monofilms of pure DPhPC spread at the air/water interface with an initial surface pressure of 30 mN/m. The increase in surface pressure was considerably enhanced in cholesterol-containing films. It is assumed that the channel-like fluctuations induced by saponin as well as digitonin, in both cholesterol-free and cholesterol-rich bilayers are due to the formation of micellar structures within the lipid lattice. Probably the penetration of the glycosides into the lipid bilayer is considerably enhanced by the presence of cholesterol.

Animals↗

Total lipid and membrane lipid analysis of normal animal and human lenses.

Comparisons of lens fiber cell membrane isolation methods were made. Although membrane lipid yield from stirring overnight in hypotonic medium followed by low-speed centrifugation was comparable to that from homogenization and high-speed centrifugation, losses in phosphatidyl ethanolamine were observed with the former method. Lyophilization with vacuum replacement by nitrogen did not exert any deleterious effects. A significant difference in total lens lipid was observed between human and the three species of animals investigated.

Aged↗

Black lipid membranes of tetraether lipids from Thermoplasma acidophilum.

Black lipid membranes were formed of tetraether lipids from Thermoplasma acidophilum and compared to the bilayer forming lipids diphytanoylphosphatidylcholine and diphythanylglucosylglycerol. Bilayer-forming lipids varied in thickness of black lipid membranes due to the organic solvent used. Measurements of the specific membrane capacitance (Cm = 0.744 microF/cm2) showed that the membrane-spanning tetraether lipids from Thermoplasma acidophilum form a monolayer of a constant thickness of 2.5-3.0 nm no matter from which solvent. This finding corresponds to the results of Gliozzi et al. for the lipids of another archaebacterium, Sulfolobus solfataricus. Black lipid membranes were formed at room temperature with a torus from bilayer-forming lipids, however, the torus could also be formed by the tetraether-lipid itself at room temperature and at defined concentration. In these stable black lipid membranes, conductance was measured in the presence of valinomycin, nonactin, and gramicidin. At 10(-7) M concentration, valinomycin mediated higher conductance in membranes from tetraether lipids (200-1200 microS/cm2) than from bilayer-forming lipids (125-480 microS/cm2). Nonactin, at 10(-6) M concentration, mediated a 6-fold higher conductance in a tetraether lipid membrane than in a bilayer, whereas conductance, in the presence of 5 x 10(-11) M gramicidin could reach higher values in bilayers than in tetraether lipid monolayers of comparable thickness. Monensin did not increase the conductance of black lipid membranes from tetraether lipids under all conditions applied in our experiments. Poly(L-lysine) destroyed black lipid membranes. Lipopolysaccharides from Thermoplasma acidophilum were not able to form stable black lipid membranes by themselves. The lipopolysaccharide complexes from Thermoplasma acidophilum and from Escherichia coli decreased the valinomycin-mediated conductance of monolayer and bilayer membranes. This influence was stronger than that of the polysaccharide dextran.

Electric Conductivity↗

Stress tolerance in a yeast lipid mutant: membrane lipids influence tolerance to heat and ethanol independently of heat shock proteins and trehalose.

The response of a yeast unsaturated fatty acid auxotroph, defective in delta 9-desaturase activity, to heat and ethanol stresses was examined. The most heat- and ethanol-tolerant cells had membranes enriched with oleic acid (C18:1), followed in order by cells enriched with linoleic (C18:2) and linolenic (C18:3) acids. Cells subjected to a heat shock (25-37 degrees C for 30 min) accumulated trehalose and synthesized typical heat shock proteins. Although there were no obvious differences in protein profiles attributable to lipid supplementation of the mutant, relative protein synthesis as determined by densitometric analysis of autoradiograms suggested that hsp expression was different. However, there was no consistent relationship between the synthesis of heat shock proteins and the acquisition of thermotolerance in the lipid supplemented auxotroph or related wild type. Furthermore, trehalose accumulation was also not closely related to stress tolerance. On the other hand, the data presented indicated a more consistent role for membrane lipid composition in stress tolerance than trehalose, heat shock proteins, or ergosterol. We suggest that the sensitivity of C18:3-enriched cells to heat and ethanol may be attributable to membrane damage associated with increases in membrane fluidity and oxygen-derived free radical attack of membrane lipids.

Electrophoresis, Polyacrylamide Gel↗

Electric field strength of membrane lipids from vertebrate species: membrane lipid composition and Na+-K+-ATPase molecular activity.

Intramembrane electric field strength is a very likely determinant of the activity of ion-transporting membrane proteins in living cells. In the absence of any transmembrane electrical potential or surface potential, its magnitude is determined by the dipole potential of the membrane's lipid components and their associated water of hydration. Here we have used a fluorometric method to quantify the dipole potential of vesicles formed from lipids extracted from kidney and brain of 11 different animal species from four different vertebrate classes. The dipole potential was compared with the fatty acid composition and with the Na(+)-K(+)-ATPase molecular activity of each preparation. The magnitude of the dipole potential was found to be relatively constant across all animal species, i.e., 236-334 mV for vesicles prepared from the total membrane lipids and 223-256 mV for phospholipids alone. The significantly lower value for phospholipids alone is potentially related to the removal of cholesterol and/or other common soluble lipid molecules from the membrane. Surprisingly, no significant dependence of the dipole potential on fatty acid composition was found. This may, however, be due to concomitant compensatory variations in lipid head group composition. The molecular activity of the Na(+)-K(+)-ATPase was found to increase with increasing dipole potential. The fact that the dipole potential is maintained at a relatively constant value over a wide range of animal species suggests that it may play a fundamental role in ensuring correct ion pump conformation and function within the membrane.

Amphibians↗

Does membrane lipid profile explain chilling sensitivity and membrane lipid phase transition of spermatozoa and oocytes?

Ram, fowl and bee spermatozoa, and oocytes of cows and zebrafish were used to study lipid membrane profiles, chilling sensitivity and lipid-phase transitions. The integrity of the membranes was determined by carboxyfluorescein diacetate (CFDA) staining following exposure for 15 minutes to low temperatures. Ram and fowl spermatozoa showed different degrees of loss of membrane integrity. Surprisingly, bee spermatozoa did not show any sensitivity to chilling, and their membranes remained intact down to 0 degree C. In bovine oocytes (at the GV stage) chilling injury was very severe at 16 degree C (membrane integrity decreased by 50%). Lipid phase transition (LPT) and membrane fluidity, which were evaluated by Fourier transform infrared (FTIR) microscopy, and fluorescence polarisation, showed phase transitions at the same temperatures as caused damage (between 30 and 12 degree C). The membrane lipid profiles showed high concentrations of polyunsaturated fatty acids (PUFA) in cold-sensitive ram spermatozoa and zebrafish oocytes, but the ratio between PUFA and saturated fatty acids was highest in cold-resistant bee spermatozoa and lowest in cold-sensitive bovine oocytes. These results suggest a close relationship among cold susceptibility, lipid phase transition and lipids profile in animal gametes.

Journal Article↗

Effects of peroxisomal beta-oxidation antagonist on 2',3'-cyclic-nucleotide 3'-phosphohydrolase, membrane lipid compositions, and membrane fluidity in C-6 glial cells.

In order to understand the relationship between peroxisomal dysfunction and clinical manifestations of peroxisomal disorders, the effect of thioridazine, a peroxisomal beta-oxidation antagonist, on the differentiation, membrane lipid composition and membrane fluidity of C-6 glial cells was examined. In our study, induction of 2',3'-cyclic-nucleotide 3'-phosphohydrolase (CNP), which was considered to be a membrane-associated enzyme closely associated with myelination, was inhibited with supplementation of thioridazine, followed by an increase in the relative concentration of longer chain fatty acids in cell membrane lipids, indicating that thioridazine causes impaired differentiation in the glial stem cell system. Membrane fluidity of C-6 glial cells was examined using a fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The DPH anisotropy value was decreased in the glial cells treated with thioridazine. These results indicate that the alteration of the membrane lipid composition caused by thioridazine affects the differentiation of glial cells via the changes in membrane properties.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗

Effect of haematoporphyrin-induced photosensitization on lipid membranes.

In vitro cultured mouse myeloma (Sp-2/0-Ag14) cells and phosphatidylcholine liposomes were used to study the membrane effects of photosensitization with an He-Ne laser activated haematoporphyrin (HP). Lipophilic HP molecules, intercalated between the membrane lipid molecules, caused morphological changes of cell membranes on light activation. Steady state and time-resolved fluorescence spectroscopic studies of membrane-bound HP molecules provide information about the change in membrane lipid dynamics (fluidity). Increased HP fluorescence anisotropy was found after laser irradiation in the case of cell membrane. This finding can be related either to the increased rotation correlation time of the rotating fluorophore (HP) (decreased membrane fluidity) or to the decrease in the angular range of molecular rotation, which corresponds to an increased lipid order after photosensitization. Changes in the ratio of saturated:unsaturated fatty acid content of membrane lipids or other chemical events such as cross-linking of membrane components during the photosensitization process can also account for the observed effects.

Animals↗

Differential responses of three acyl-lipid desaturases to immediate temperature reduction occurring in two lipid membranes of Spirulina platensis strain C1.

In cyanobacteria, the elevation of unsaturated fatty acid levels in membrane lipids has been shown to play a major role in the response to temperature change. The cyanobacterium Spirulina platensis strain C1 contains three desaturases--delta9, delta12 and delta6 desaturases--which are encoded by desC, desA and desD, respectively. In light of the above, a study was conducted of the regulation of desaturase gene expression in response to temperature change in S. platensis strain C1. The two lipid membranes, thylakoid and plasma, were separated, while the expressions of the desaturase genes to the downward shift of growth temperature were studied in the translation level by Western blot analysis. The results revealed that the expression of delta9 desaturase is independent of temperature. In the case of delta12 desaturase, two forms of the enzyme were found, at 45 and 40 kDa. In terms of correlation with the results in the transcription level, it is more likely that the 45 kDa-delta12 desaturase and the 40 kDa-delta12 desaturase are translated from 1.7 kb and 1.5 kb mRNA, respectively. Taken together, the results indicate that the expression of the 40 kDa-delta12 desaturase is temperature independent, whereas, the 45 kDa protein form demonstrates a response to the immediate temperature reduction. Furthermore, the activity of delta6 desaturase in the two lipid membranes is possibly regulated by temperature reduction. However, alteration in the level of gamma-linolenic acid, the product synthesized by delta6 desaturase, was observed in the plasma membrane prior to the thylakoid membrane.

Journal Article↗

Bovine polymorphonuclear cells passively acquire membrane lipids and integral membrane proteins from apoptotic and necrotic cells.

Immune cells can acquire membrane fragments and integral membrane proteins from dead and dying cells or in the case of immature dendritic cells, from live cells. While investigating the possibility that bovine polymorphonuclear cells (PMNs) might present antigen, coculture assays confirmed that integral membrane proteins were transferred rapidly and efficiently to bovine PMNs from a variety of apoptotic and necrotic cells. Specifically, we observed that PMNs rapidly acquired proteins such as major histocompatibility complex (MHC) class II and CD3 from a variety of syngeneic, allogeneic, and xenogeneic cell types. Such acquisition occurred within 40 min of PMN coculture with isolated peripheral blood mononuclear cells, and this acquisition occurred with equal efficiency at 4 degrees C and 37 degrees C. The transfer of murine MHC class II to bovine PMNs precluded the possibility of endogenous protein expression. We also demonstrated the transfer of fluorescently labeled plasma membrane lipids and biotinylated integral membrane proteins. Collectively, these observations support the hypothesis that membrane protein transfer was mediated by the fusion of membrane fragments or microvesicles with the PMN plasma membrane and not by phagocytosis of cell fragments. These observations indicate that phenotypic studies of PMNs must consider circumstances whereby PMNs may passively acquire membrane lipids and a variety of integral membrane proteins from dead or dying cells.

Animals↗