Lipid rafts have different sizes depending on membrane composition: a time-resolved fluorescence resonance energy transfer study

The ternary lipid system palmitoylsphingomyelin (PSM)/palmitoyloleoylphosphatidylcholine (POPC)/cholesterol is a model for lipid rafts. Previously the phase diagram for that mixture was obtained, establishing the composition and boundaries for lipid rafts. In the present work, this system is further studied in order to characterize the size of the rafts. For this purpose, a time-resolved fluorescence resonance energy transfer (FRET) methodology, previously applied with success to a well-characterized phosphatidylcholine/cholesterol binary system, is used. It is concluded that: (1) the rafts on the low raft fraction of the raft region are small (below 20 nm), whereas on the other side the domains are larger; (2) on the large domain region, the domains reach larger sizes in the ternary system (> approximately 75-100 nm) than in binary systems phosphatidylcholine/cholesterol (between approximately 20 and approximately 75-100 nm); (3) the raft marker ganglioside G(M1) in small amounts (and excess cholera toxin subunit B) does not affect the general phase behaviour of the lipid system, but can increase the size of the rafts on the small to intermediate domain region. In summary, lipid-lipid interactions alone can originate lipid rafts on very different length scales. The conclusions presented here are consistent with the literature concerning both model systems and cell membrane studies.     

月季切花衰老过程中多胺与膜脂过氧化的关系

以月季切花为材料,研究了月季切花瓶插过程中多胺含量的变化,外源多胺处理对月季药花体内多胺含量的影响以及多胺与膜脂过氧化的关系。结果表明,月季切花瓶插衰老过程中腐胺在前2d略有增加,亚精胺和精胺均呈下降趋势;外源亚精胺和精胺处理均能增加切花体内多胺含量,并能延缓切花衰老和改善切花品质;且亚精胺和精胺处理降低了MDA含量的积累和膜相对透性的上升趋势。     

Generation of oxygen free radicals during the metabolism of cyclosporine A: a cause-effect relationship with metabolism inhibition

A better understanding of the mechanism of lipid peroxidation during the metabolism of cyclosporine A (CsA) might help explain the toxicities of this immunosuppressive drug on various organs. Ourin vitro work used microsomes prepared from livers of phenobarbital-induced male rats. The incubations (total volume 1ml) also contained a NADPH regenerating system and substrate (i.e., CsA, carbon tetrachloride, or aminopyrine) dissolved in ethanol. Lipid peroxidation was inferred from the presence of malondialdehyde (MDA) which was detected by the thiobarbituric acid assay. The formation of CsA hydroxylated metabolites (AM9 and AM1) was monitored by liquid chromatography. The activity of the microsomal incubation was confirmed by measurements of MDA and formaldehyde production caused by increasing concentrations of CsA, carbon tetrachloride, and aminopyrine. The occurrence of hydroxylated metabolites was not coupled to the production of MDA. Aminopyrine could inhibit MDA production by CsA, but CsA could not reduce the formation of formaldehyde by aminopyrine. Erythromycin, a competitor for the binding site of CsA on cytochrome P450, reduced MDA production by CsA, and CsA inhibited formaldehyde production by erythromycin. Interaction studies with SKF 525A, ketoconazole, superoxide dismutase, catalase, -tocopherol, and reduced glutathione confirmed the role of cytochrome P450 and the presence of activated oxygen species as a source of microsomal peroxidation which in return may explain the inhibitory effect of CsA on cytochrome P450 itself.Abbreviations AM9 9hydroxycyclosporine - AM1 1(8)hydroxycyclosporine - AM1c 1hydroxy--cyclo-cyclosporine - AM4N 4N-desmethylcyclosporine     

Effects of phytic acid on the myoglobin-t-butylhydroperoxide-catalysed oxidation of uric acid and peroxidation of erythrocyte membrane lipids

Phytic acid stimulated the myoglobin-t-butylhydroperoxide (TBHP)-catalysed oxidation of uric acid, but inhibited the peroxidation of erythrocyte membrane lipids induced by the same system. Butylated hydroxy-toluene, a free radical chain reaction-terminating antioxidant, also suppressed the myoglobin-TBHP-induced lipid peroxidation. Moreover, phytic acid inhibited the hydroxyl radical-induced degradation of deoxyribose, but the extent of inhibition in this system was reduced by increasing the ferric ion concentration, suggesting that these effects of phytic acid on the myoglobin-TBHP-mediated oxidation are more likely attributable to its metal chelating properties rather than to a free radical scavenging action. The effectiveness of phytic acid, a naturally occurring antioxidant, in the inhibition of both iron- (as previously shown) and myoglobin-dependent lipid peroxidation suggests its possible therapeutic application as a non-toxic antioxidant for ameliorating the extent of oxy-radical-mediated myocardial ischemia/reperfusion damage.Abbreviations ASC Ascorbic acid - BHT Butylated Hydroxytoluene - DMSO Dimethyl Sulfoxide - TBHP t-Butylhydroperoxide - TBA Thiobarbituric Acid - TBARS Thiobarbituric Acid-reactive Substances     

Ion selectivity of colicin E1: Modulation by pH and membrane composition

Colicin E1 is a plasmid-encoded bacteriocidal protein which, though water soluble when secreted by its host bacterium, spontaneously interacts with planar lipid bilayers to form voltage-gated ion channels. In asolectin bilayers, the preference for anions over cations exhibited by these channels at low pH can be reversed by raising the pH on either side of the membrane. When incorporated into membranes composed of either of the two zwitterionic lipids, bacterial phosphatidylethanolamine and diphytanoyl phosphatidylcholine, colicin E1 channels were nearly ideally anion selective in the limit of low pH and moderately cation selective at the high pH limit. In phosphatidylcholine membranes, however, the response of these channels to changes in pH exhibited a pattern of behavior peculiar to this lipid. If the side of the membrane on which the protein had been introduced (the cis side) was exposed to pH 4.0, all the channels in the bilayer, whether opened or closed, became refractory to further changes in pH. This irreversibility has been interpreted as evidence that the selectivity of colicin E1 is under the control of a pH-sensitive conformational change. Protonation of groups on the cis side of the membrane appear to be essential to the conversion to the anion-selective state. These groups are rendered kinetically inaccessible to the aqueous phase when the transition takes place in phosphatidylcholine membranes.     

Characteristics of Fe(II)ATP complex-induced damage to the rat liver mitochondrial membrane

It is well established that several iron complexes can induce oxidative damage in hepatic mitochondrial membranes by catalyzing the formation of ·OH radicals and/or by promoting lipid peroxidation. This is a relevant process for the molecular basis of iron overload diseases. The present work demonstrates that Fe(II)ATP complexes (5–50M) promote an oxygen consumption burst in a suspension of isolated rat liver mitochondria (either in the absence or presence of Antimycin A), caused mainly by lipid peroxidation. Fe(II)ATP alone induced small levels of oxygen uptake but no burst. The time course of Fe(II)ATP oxidation to Fe(III)ATP in the extramitochondrial media also reveals a simultaneous burst phase. The iron chelator Desferal (DFO) or the chain-break antioxidant butylated hydroxytoluene (BHT) fully prevented both lipid peroxidation (quantified as oxygen uptake burst) and mitochondrial swelling. DFO and BHT were capable of stopping the ongoing process of peroxidation at any point of their addition to the mitochondrial suspension. Conversely, DFO and BHT only halted the Fe(II)ATP-induced mitochondrial swelling at the onset of the process. Fe(II)ATP could also cause the collapse of mitochondrial potential, which was protected by BHT if added at the onset of the damaging process. These results, as well as correlation studies between peroxidation and mitochondrial swelling, suggest that a two phase process is occurring during Fe(II)ATP-induced mitochondrial damage: one dependent and another independent of lipid peroxidation. The involvement of lipid peroxidation in the overall process of mitochondrial membrane injury is discussed.Abbreviations AA Antimycin A - BHT butylated hydroxytoluene - EGTA ethylene glycol-bis(-aminoethyl ether) - N,N,N,N tetraacetic acid - DFO Desferal - HEPES N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - SOD superoxide dismutase - TPP+ tetraphenylphosphonium bromide - TBARS thiobarbituric acid reactive substances     

Understanding the mechanisms of antitropical divergence in the seabird White‐faced Storm‐petrel (Procellariiformes: Pelagodroma marina) using a multilocus approach

Analytical methods that apply coalescent theory to multilocus data have improved inferences of demographic parameters that are critical to understanding population divergence and speciation. In particular, at the early stages of speciation, it is important to implement models that accommodate conflicting gene trees, and benefit from the presence of shared polymorphisms. Here, we employ eleven nuclear loci and the mitochondrial control region to investigate the phylogeography and historical demography of the pelagic seabird White‐faced Storm‐petrel (Pelagodroma marina) by sampling subspecies across its antitropical distribution. Groups are all highly differentiated: global mitochondrial Φ_ST = 0.89 (P < 0.01) and global nuclear Φ_ST varies between 0.22 and 0.83 (all P < 0.01). The complete lineage sorting of the mitochondrial locus between hemispheres is corroborated by approximately half of the nuclear genealogies, suggesting a long‐term antitropical divergence in isolation. Coalescent‐based estimates of demographic parameters suggest that hemispheric divergence of P. marina occurred approximately 840 000 ya (95% HPD 582 000–1 170 000), in the absence of gene flow, and divergence within the Southern Hemisphere occurred 190 000 ya (95% HPD 96 000–600 000), both probably associated with the profound palaeo‐oceanographic changes of the Pleistocene. A fledgling sampled in St Helena (tropical South Atlantic) suggests recent colonization from the Northern Hemisphere. Despite the great potential for long‐distance dispersal, P. marina antitropical groups have been evolving as independent, allopatric lineages, and divergence is probably maintained by philopatry coupled with asynchronous reproductive phenology and local adaptation.     

Lipid fluidity and composition of the erythrocyte membrane from healthy dogs and labrador retrievers with hereditary muscular dystrophy

Erythrocyte membranes and their liposomes were prepared from clinically normal dogs and Labrador retrievers with hereditary muscular dystrophy. The static and dynamic components of fluidity of each membrane were then assessed by steady-state fluorescence polarization techniques using limiting hindered fluorescence anisotropy and order parameter values of 1,6-diphenyl-1,3,5-hexatriene (DPH) and fluorescence anisotropy values ofdl-2-(9-anthroyl)-stearic acid anddl-12-(9-anthroyl)-stearic acid, respectively. Membrane lipids were extracted and analyzed by thin-layer chromatography and gas chromatography. The results of these studies demonstrated that the lipid fluidity of erythrocyte membranes, and their liposomes, prepared from dystrophic dogs were found to possess significantly lower static and dynamic components of fluidity than control counterparts. Analysis of the composition of membranes from dystrophic dogs revealed a higher ratio of saturated fatty acyl chain/unsaturated chains (w/w) and lower double-bond index. Alterations in the fatty acid composition such as decrease in levels of linoleic (18:2) and arachidonic (20:4) acids and increase in palmitic (16:0) and stearic (18:0) acids were also observed in the membranes of dystrophic animals. These associated fatty acyl alterations could explain, at least in part, the differences in membrane fluidity between dystrophic and control dogs.     

The relationship between cell membrane damage and lipid peroxidation under the condition of hypoxia‐reoxygenation: analysis of the mechanism using antioxidants and electron transport inhibitors

We consecutively observed lipid peroxidation and cell membrane damage under the condition of hypoxia‐reoxygenation (H/R) in cells and analyzed their mechanisms by using electron transport inhibitors and an antioxidant. In H/R experiments, lipid peroxidation and cell membrane damage were observed during the hypoxia phase. In the reoxygenation phase, lipid peroxidation stopped, while cell membrane damage did not. An antioxidant, n‐acetylcystein (NAC), and potassium cyanide (KCN) inhibited lipid peroxidation and cell membrane damage, while rotenone did not inhibit either of them. Although antimycin A did not inhibit lipid peroxidation, it inhibited cell membrane damage during the hypoxia phase but not during the reoxygenation phase. These results suggested that lipid peroxidation can affect cell membrane damage as a trigger during the hypoxia phase and the generation of oxidative stress can vary depending on the inhibition locations in the electron transport system. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of in vitro treatment with ozone on the physical and chemical properties of membranes

Treatment of microsomal membranes from cotyledons of Phaseolus vulgaris with ozone raises the liquid-crystalline to gel lipid phase transition temperature and results in the formation of distinct domains of gel phase lipid in the membranes. Liposomes prepared from the total lipid extracts of ozone-treated membranes undergo phase separations just a few degrees below the transition temperature for intact membranes, indicating that the formation of gel phase lipids is largely attributable to ozone-induced alterations in the membrane lipids. Levels of unsaturated fatty acids as well as the sterol to phospholipid ratio are markedly reduced in the ozone-treated membranes, and the neutral lipid fraction from treated membranes shows, an increased propensity to induce the formation of gel phase phospholipid when incorporated into liposomes of egg phosphatidylcholine. Since gel phase phospholipid also forms in naturally senescing plant membranes and appears to be attributable to changes in the neutral lipid fraction, the effects of natural senescence and ozone on membranes have been compared.     

Superoxide radical formation and associated biochemical alterations in the plasma membrane of brain, heart, and liver during the lifetime of the rat.

Plasma membrane samples from rat brain, heart, and liver were examined for biochemical changes with age. A rise in superoxide radical (SOR) levels was followed by increases in thiobarbituric acid reactive substances and decreases in membrane fluidity with age. The earliest rise in SOR formation appeared in the plasma membrane from the brain. With age, protein synthesis also decreased significantly in tissue homogenates from brain and heart but was unchanged in the liver. Exposure of plasma membrane samples to in vitro-elevated SOR levels stimulated formation of lipid peroxides, as indicated by the thiobarbituric acid test, and resulted in a decrease in membrane fluidity in each tissue and in a decline in protein synthesis in brain and heart. Changes in brain lipid peroxidation and in membrane fluidity in brain and heart as a result of SOR supplementation were further enhanced due to age. In addition, the mechanism of SOR formation was examined in plasma membrane samples from the brain. SOR generation was Ca(2+)-sensitive, blocked by superoxide dismutase or vitamin E and inhibited by both indomethacin, a cyclooxygenase inhibitor, and bromophenacyl bromide, a phospholipase A2 inhibitor. These results show significant increases in SOR formation and biochemical alterations in plasma membranes from brain, heart, and liver in aging rats. SOR formation appears to be enzyme-mediated and elevated levels of this oxygen radical could be involved in membrane breakdown in older rats.     

Effect of laser pretreatment on germination and membrane lipid peroxidation of Chinese pine seeds under drought stress

The germination of laser-irradiated Chinese pine seeds was carried out under drought stress.The activities of superoxide dismutase (SOD) and peroxidase (POD),and the content of malondialdehyde (MDA) were determined.Results showed notably increased germination percentage,root length,vitality index and fresh weight.The SOD and POD protective enzyme system activity of the Chinese pine seedlings obviously rose.It can be concluded that the germination and juvenile resistance of Chinese pine seeds under drought stress are enhanced after laser processing.     

Effect of laser pretreatment on germination and membrane lipid peroxidation of Chinese pine seeds under drought stress

The germination of laser-irradiated Chinese pine seeds was carried out under drought stress. The activities of superoxide dismutase (SOD) and peroxidase (POD), and the content of malondialdehyde (MDA) were determined. Results showed notably increased germination percentage, root length, vitality index and fresh weight. The SOD and POD protective enzyme system activity of the Chinese pine seedlings obviously rose. It can be concluded that the germination and juvenile resistance of Chinese pine seeds under drought stress are enhanced after laser processing. Translated from Journal of Shaanxi Normal University (Natural Science Edition), 2006, 34(2): 40–43 [译自: 陕西师范大学学报 (自然科学版)]     

Differential destabilization of membranes by tryptophan and phenylalanine during freezing: the roles of lipid composition and membrane fusion

The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.     

Lipid Peroxidation and Biogenic Aldehydes: From the Identification of 4-Hydroxynonenal to Further Achievements in Biopathology

The formation, reactivity and toxicity of aldehydes originating from lipid peroxidation of cellular membranes are reviewed. Very reactive aldehydes, namely 4-hydroxyalkenals, were first shown to be formed in autoxidizing chemical systems. It was subsequently shown that 4-hydroxyalkenals are formed in biological conditions, i.e. during lipid peroxidation of liver microsomes incubated in the NADPH-Fe systems. Our studies carried out in collaboration with Hermann Esterbauer which led to the identification of 4-hydroxynonenal (4-HNE) are reported. 4-HNE was the most cytotoxic aldehyde and was then assumed as a model molecule of oxidative stress. Many other aldehydes (alkanals, alk-2-enals and dicarbonyl compounds) were then identified in peroxidizing liver microsomes or hepatocytes. The in vivo formation of aldehydes in liver of animals intoxicated with agents that promote lipid peroxidation was shown in further studies. In a first study, evidence was forwarded for aldehydes (very likely alkenals) bound to liver micro-somal proteins of CCl₄ or BrCCl₃-intoxicated rats. In a second study, 4-HNE and a number of other aldehydes (alkanals and alkenals) were identified in the free (non-protein bound) form in liver extracts from bromoben-zene or ally-1 alcohol-poisoned mice. The detection of free 4-HNE in the liver of CCl₄ or BrCCl₃-poisoned animals was obtained with the use of an electrochemical detector, which greatly increased the sensitivity of the HPLC method. Furthermore, membrane phospho-lipids bearing carbonyl groups were demonstrated in both in vitro (incubation of microsomes with NADPH-Fe) and in vivo (CCl₄ or BrCCl₃ intoxication) conditions. Finally, the results concerned with the histochemical detection of lipid peroxidation are reported. The methods used were based on the detection of lipid peroxidation-derived carbonyls. Very good results were obtained with the use of fluorescent reagents for carbonyls, in particular with 3-hydroxy-2-naphtoic acid hydrazide (NAH) and analysis with confocal scanning fluorescence microscopy with image video analysis. The significance of formation of toxic aldehydes in biological membranes is discussed.     

Free radical-induced endothelial membrane dysfunction at the site of blood-brain barrier: Relationship between lipid peroxidation,Na,K-ATPase activity,and51Cr release

Na,K-ATPase activity, membrane lipid peroxidation (TBARM), and membrane leakiness for small molecules were examined in rat cerebromicrovascular endothelial cells (RCEC) following exposure to hydrogen peroxide and xanthine/xanthine oxidase. Whereas short-term (15–30 min) exposure to either oxidant decreased ouabain-sensitive⁸⁶Rb uptake and increased TBARM in a concentration-dependent fashion, significant release of⁵¹Cr (30–40%) from cells was observed only after one hour exposure to the oxidants. By comparison, much longer exposure times (i.e., 4 hours) were needed to induce significant lactate dehydrogenase release from oxidant-treated cells. The oxidant-evoked decrease in Na,K-ATPase activity and increases in TBARM and RCEC permeability were abolished in the presence of the steroid antioxidants U-74500A and U-74389G (5–20 M). Reduced glutathione (4 mM) partially attenuated oxidant-induced changes, whereas ascorbic acid (2 mM) and the disulfide bond-protecting agent, dithiothreitol (1 mM), were ineffective. These results suggest that the oxidant-induced loss of Na,K-ATPase activity in RCEC results primarily from changes in membrane lipids, and implicate both the inhibition of Na,K-ATPase and membrane lipid peroxidation in the mechanism responsible for the delayed free radical-induced increase in RCEC membrane permeability.     

Effects of metabolic rate and sperm competition on the fatty‐acid composition of mammalian sperm

The sperm membrane is a key structure affecting sperm function and thus reproductive success. Spermatozoa are highly specialized and differentiated cells that undergo a long series of processes in the male and female reproductive tracts until they reach the site of fertilization. During this transit, the sperm membrane is prone to damage such as lipid peroxidation. The characteristics and performance of the sperm membrane are strongly determined by the fatty‐acid composition of membrane phospholipids. Polyunsaturated fatty‐acids (PUFAs) are the most prone to lipid peroxidation. Lipid peroxidation and other types of oxidative damage increase with higher metabolism and with higher levels of sperm competition due to the increased ATP production to fuel higher sperm velocities. Consequently, we hypothesized that, in order to avoid oxidative damage, and the ensuing impairment of sperm function, sperm cells exhibit a negative relationship between PUFA content and mass‐specific metabolic rate (MSMR). We also hypothesized that higher sperm competition leads to a reduction in the proportion of sperm PUFAs. We performed a comparative study in mammals and found that high MSMR and high levels of sperm competition both promote a decrease in the proportion of PUFAs that are more prone to lipid peroxidation. The negative relationship between MSMR and these PUFAs in sperm cells is surprising, because a positive relationship is found in all other cell types so far investigated. Our results support the idea that the effects of MSMR and sperm competition on sperm function can operate at very different levels.     

氮肥、钙肥和盐处理在冬小麦融冻胁迫适应中的生理调控作用

采用盆栽法种植冬小麦(烟农19号),在出苗7d后分别用氮肥(60mmol/L NH4CO3)、钙肥(100 mmol/L CaCl2)和NaCl(100 mmol/L)处理,生长30 d后开始冻融处理(15℃,0℃,-15℃,0℃,15℃各处理3h).结果表明,在融冻和冻融胁迫过程中冬小麦叶片细胞膜透性变化和膜脂过氧化程度与温度变化呈显著负相关(R=-0.89**,R=-0.85**).与对照相比,经氮肥、钙肥和盐处理的冬小麦叶片细胞膜透性和丙二醛含量较低,而SOD、CAT和POD活力均较高,这表明施肥能降低细胞膜脂过氧化作用,提高细胞膜抗氧化能力.但不同肥料和盐处理在冬小麦融冻胁迫中对渗透调节物质的调节作用存在差异.施氮肥使冬小麦叶片脯氨酸和可溶性蛋白质含量分别高于对照44.3％和23.6％,可溶性糖含量低于对照(-17.3％);而钙肥和盐处理却使冬小麦叶片可溶性糖含量高于对照(57.5％和37.1％).研究表明,氮肥通过调节氮代谢提高脯氨酸和蛋白质含量,钙肥通过促进糖代谢提高可溶性糖含量.因此,冬季给冬小麦幼苗施一定的混合肥,不仅可促使植物细胞积累较多的渗透调节物质防止细胞结冰,激活体内保护酶系统防止细胞膜脂过氧化,而且施肥还可促使来年春季幼苗的快速返青健壮生长.通过合理施肥改善植物的代谢提高作物的抗融冻能力,这可能是目前投资少,见效快的有效途径.