人心肌缺血/再灌注期间红细胞膜分子流动性改变的机理研究

对开心手术病人心肌缺血／再灌注过程中冠状静脉窦血中的红细胞流变性变化和红细胞膜分子结构的改变进行了研究。结果表明,再灌注即刻心脏产生大量自由基,并持续到再灌注第２０分钟时才大幅度回落;于再灌注后的２０分钟的自由基产生时相期间,除红细胞膜蛋白分子和脂质分子的τｐ、τｌ均明显延长外,还伴有血浆ＭＤＡ水平的明显增加和ＳＯＤ活性的显著降低,及红细胞内ＧＳＨ－Ｐｘ活性升高;与此同时其红细胞膜蛋白分子的α螺旋先减少后渐回复和β折叠渐增加并伴有羧基（ＣＯＯＨ）和氨基（ＮＨ３）的减少,而其红细胞膜脂质分子的磷氧双键（Ｐ＝Ｏ）、羰基（Ｃ＝Ｏ）和多不饱和键（Ｃ＝Ｃ）则增加;说明（１）再灌注期间氧自由基分别引起了红细胞膜蛋白分子碳端和氮端的改变及膜磷脂分子亲水区和疏水区化学结构的变化,（２）蛋白分子碳、氮端的变化造成了膜蛋白分子构象的改变,（３）蛋白分子和磷脂分子化学结构及构象的改变是红细胞膜这些分子流动性明显恶化的根本原因。     

人红细胞膜和血浆游离脂肪酸成分分析及其对膜微粘度的影响

采用高效液相色谱（ＨＰＬＣ）和荧光偏振技术测定了４２例正常人红细胞膜和血浆游离脂肪酸（ＦＦＡ）及膜微粘度,并探讨了膜脂肪酸和血浆ＦＦＡ构成与膜微粘度之间的关系。结果表明：正常人红细胞膜主要由廿二碳六烯酸（Ｃ２２∶６）、花生四烯酸（Ｃ２０∶４）、亚油酸（Ｃ１８∶２）、软脂酸（Ｃ１６∶０）、油酸（Ｃ１８∶１）和硬脂酸（Ｃ１８∶０）等六种脂肪酸组成。血浆ＦＦＡ构成与膜脂肪酸相似,但不含Ｃ２２∶６而含十四烷酸（Ｃ１４∶０）。红细胞膜各脂肪酸含量大多与其血浆浓度呈明显正相关。红细胞膜微粘度与膜软脂酸和硬脂酸呈明显正相关,与膜廿二碳六烯酸和花生四烯酸呈明显负相关。提示红细胞膜脂肪酸组成受血浆ＦＦＡ成分影响;而红细胞膜脂肪酸成分对膜微粘度亦有重要影响     

磷脂酶A2对中性粒细胞趋化和粘附的影响

胰源性１４×１０￣３ｕ磷脂酶Ａ＿２（ＰＬＡ＿２）在体外同大鼠中性粒细胞（ＰＭＮ）培养６０ｍｉｎ后,细胞对ＴＮＦ趋化增强,培养１０ｍｉｎ后上清液中血栓素（ＴＸＢ＿２）含量比正常增高（Ｐ＜０．０１）。前列环素（ＰＧＩ＿２）含量不变。ＰＬＡ＿２激动剂Ａ２３１８７也能显著加强中性粒细胞对ＴＮＦ的趋化,并伴有ＴＸＢ＿２释放增多（Ｐ＜０．０１）。此外,ＰＬＡ＿２和Ａ２３１８７还显著增强ＰＭＮ对玻璃珠的粘附活性。使用ＰＬＡ＿２抑制剂二溴苯乙酮（ＰＢＰＢ）和ＰＬＡ＿２多克隆抗体可抑制外源性ＰＬＡ＿２对ＰＭＮ趋化和粘附的增强作用,但对Ａ２３１８７的调节作用无效。以上结果表明ＰＬＡ＿２激活及其代谢产物可能介导ＰＭＮ趋化和粘附作用。     

甲基毒死蜱对红细胞乙酰胆碱酯酶的抑制作用及其与膜脂相互关系

以人红细胞膜为材料,研究了甲基毒死蜱与膜上乙酰胆碱酯酶的相互作用及其与膜脂的关系。结果显示,甲基毒死埤对人红细胞ＡＣｈＥ有明显的抑制作用,与膜温育３０ｍｉｎ,其半数抑制浓度约为０．１０ｍｍｏｌ／Ｌ。动力学分析表明,其抑制作用为非竞争性。０．２％Ｔｒｉｔｏｎ Ｘ－１００并不改变ＡＣｈＥ对甲基毒死蜱的敏感性,亦即ＡＣｈＥ上甲基毒死蜱的作用部位与其所处的脂质微环境无关。     

羟自由基对人红细胞氧化囊泡化的作用

红细胞经氧化处理后,发现红细胞膜区带１、２、２．１及３易形成高聚物,同时Ｈｂ亦有氧化变性。氧化过程中磷脂（ＰＳ、ＰＥ）逐渐减少,根据此结果提出氧化产生囊泡化的机制是：首先Ｈｂ氧化变性,膜骨架蛋白聚集,从而网架松散,膜磷脂脱离骨架的束缚,膜脂质与膜整合蛋白形成小囊泡从红细胞上脱落。     

羟自由基对人红细胞氧化囊泡化的作用

红细胞经氧化处理后,发现红细胞膜区带１、２、２．１及３易形成高聚物,同时Ｈｂ亦有氧化变性。氧化过程中磷脂（ＰＳ、ＰＥ）逐渐减少,根据此结果提出氧化产生囊泡化的机制是：首先Ｈｂ氧化变性,膜骨架蛋白聚集,从而网架松散,膜磷脂脱离骨架的束缚,膜脂质与膜整合蛋白形成小囊泡从红细胞上脱落。     

紫外线B对水稻叶组织中活性氧代谢及膜系统的影响

增强ＵＶ－Ｂ处理下,水稻叶片的Ｏ净产生速率、Ｈ２Ｏ２和ＭＤＡ含量以及膜透性都显著增加,敏感性弱的品种Ｏ２净产生速率和膜伤害程度较低。在ＵＶ－Ｂ处理初期活性氧清除系统的水平增高,但随着处理时间的延长,ＳＯＤ、ＣＡＴ和ＡＰ活性以及ＡＳＡ含量降低,其中ＡＰ和ＳＯＤ活性的下降最为明显,而敏感性弱的品种ＳＯＤ活性始终高于敏感性强的品种。处理１４ｄ后去掉ＵＶ－Ｂ,再经则１４ｄ上述各指标均恢复到与对照相近的水平。根据这些结果推测,水稻的ＵＶ－Ｂ伤害可能主要是由于ＳＯＤ活性的降低而导致Ｏ增生和膜脂过氧化。     

顺铂及其水解产物与红细胞膜相互作用的动力学研究

顺铂（ＤＤＰ）和顺式二水二氨合铂（Ⅱ）（ＡＡＰ）与人红细胞膜的相互作用已经用荧光及ＳＤＳ－ＰＡＧＥ法研究,实验结果表明,ＤＤＰ和ＡＡＰ与人红细胞膜蛋白相互作用属双阶段一段反应动力学,而且ＡＡＰ的反应速率比ＤＤＰ大。ＳＤＳ－ＰＡＧＥ研究表明,ＤＤＰ和ＡＡＰ不仅可引起膜蛋白的交联或聚合,形成新带,也能使某些膜蛋白降解,对于ＤＤＰ,各膜蛋白带结合铂量首先随反应时间增加而下降,而后增加,最后达到饱和结合;     

配体与受体相互作用诱导的膜蛋白构象变化WGA与GPA作用对膜蛋白构象的影响

用ＦＴＩＲ、ＣＤ、微量ＤＳＣ和ＳＴＭ、ＡＦＭ等研究重组脂质体、血影和完整红细胞在配体（ＷＧＡ）与膜受体（ＧＰＡ）相互作用下膜蛋白构象改变以及纳米水平上膜表面蛋白的形貌。结果表明ＷＧＡ可诱导重组脂质体膜、血影膜和完整红细胞膜蛋白发生一定的不同的构象变化、红细胞及其膜的热力学行为变更,以及红细胞膜表面蛋白的可能交联。     

内毒素致大鼠肺损伤时肺组织β受体的变化与膜磷脂代谢的关系

采用放射性配基结合分析法,现察内毒大致大鼠急性肺损伤时肺β－肾上腺素能受体（β－ＡＲ）的变化。分别用荧光偏振法和高效液相色谱测定肺组织细胞膜脂流动性和磷脂含量。结果显示：（１）静脉注射内毒素后４ｈ,大鼠肺β－ＡＲ的最大结合容量明显降低,较对照组减少４７％;（２）内毒素组肺膜脂流动性和磷脂含量均明显降低,同时伴有肺组织磷脂酶Ａ２（ＰＬＡ２）活性升高。提示：（１）β－ＡＲ下调导致其介导的功能减弱,在内毒素诱导的大鼠急性肺损伤发病机理中起一定作用;（２）ＰＬＡ２激活是膜磷脂减少的重要原因,后者可导致膜脂流动性降低,结果引起β－ＡＲ的侧向扩散和旋转运劝减弱,从而减少β－ＡＲ与配基结合的机率,出现β－ＡＫ下调。     

热应激时大鼠肺组织中β—肾上腺素受体的变化与膜磷...

To explore the relationship between the change of beta-adrenoceptor and the metabolism of phospholipids in lung tissue from acute heat stressed rats, the Bmax of beta-adrenoceptors, the activity of phospholipase A2 (PLA2), the content of phosphatidylcholine (PC) and phosphatidylserine (PS), and membrane fluidity in lung tissue of normal and heat stressed rats were investigated. The relevant parameter values mentioned above were 479 +/- 94 fmol/mg protein, 78.5 +/- 8.2 U, 53.5 +/- 1.7 mg/g.wet. w. and 425.1 +/- 68.1 micrograms/g.wet. w. respectively. Whereas in the heat stressed rats with rectal temperature raised to 42 degrees C for 15 min, the Bmax of beta-adrenoceptor was decreased by 43% (P less than 0.01), the activity of PLA2 increased by 83% (P less than 0.01), the contents of PC and PS decreased by 50% and 47% (P less than 0.01) respectively. A lower membrane fluidity in lung tissue for heat stressed rats was also demonstrated. The results suggest that the decreased binding sites of beta-adrenoceptor in lung tissue of rat during hyperthermia may be contributed to the activation of PLA2, which then accelerated the catabolism of phospholipids such as PC and PS in the cell plasma membrane, with a consequent alteration of membrane fluidity.     

Relevance of lipid polar headgroups on boron-mediated changes in membrane physical properties

Using liposomes composed of either brain phosphatidylcholine (PC), or binary mixtures of PC and phosphatidylserine (PS), galactolipids (GL), phosphatidylinositol (PI), cardiolipin (CL), phosphatidic acid (PA), or phosphatidylethanolamine (PE), we investigated the effects of graded amounts of boric acid (B, 0.5-1000 microM) on the following membrane physical properties: (a) surface potential, (b) lipid rearrangement through lateral phase separation, (c) fluidity, and (d) hydration. Incubation of the different populations of vesicles with B was associated with a small, but statistically significant, increase in membrane surface potential in PC, PC:PS, PC:GL, PC:PI, PC:PA, and PC:PE liposomes. B-induced lipid lateral rearrangement through lateral phase separation in PC, PC:PA, and PC:PE liposomes; but had no effects on PC:PS, PC:GL, and PC:PI liposomes. In PC liposomes B affected membrane fluidity at the water-lipid interface without affecting the hydrophobic core of the bilayer. In all the other binary liposomes studied, B increased membrane fluidity in both, the hydrophobic portion of the membrane and in the anionic domains. The above was associated with a decrease in the fluidity of the cationic domains. B (10-1000 microM) decreased membrane hydration regardless the composition of the liposomes. The obtained results demonstrate the ability of B to interact with membranes, and induce changes in membrane physical properties. Importantly, the extent of B-membrane interactions and the consequent effects were dependent on the nature of the lipid molecule; as such, B had greater affinity with lipids containing polyhydroxylated moieties such as GL and PI. These differential interactions may result in different B-induced modulations of membrane-associated processes in cells.     

Phosphatidylethanolamine Is a Key Regulator of Membrane Fluidity in Eukaryotic Cells

Adequate membrane fluidity is required for a variety of key cellular processes and in particular for proper function of membrane proteins. In most eukaryotic cells, membrane fluidity is known to be regulated by fatty acid desaturation and cholesterol, although some cells, such as insect cells, are almost devoid of sterol synthesis. We show here that insect and mammalian cells present similar microviscosity at their respective physiological temperature. To investigate how both sterols and phospholipids control fluidity homeostasis, we quantified the lipidic composition of insect SF9 and mammalian HEK 293T cells under normal or sterol-modified condition. As expected, insect cells show minimal sterols compared with mammalian cells. A major difference is also observed in phospholipid content as the ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) is inverted (4 times higher in SF9 cells). In vitro studies in liposomes confirm that both cholesterol and PE can increase rigidity of the bilayer, suggesting that both can be used by cells to maintain membrane fluidity. We then show that exogenously increasing the cholesterol amount in SF9 membranes leads to a significant decrease in PE:PC ratio whereas decreasing cholesterol in HEK 293T cells using statin treatment leads to an increase in the PE:PC ratio. In all cases, the membrane fluidity is maintained, indicating that both cell types combine regulation by sterols and phospholipids to control proper membrane fluidity.     

Modulation of the susceptibility of human erythrocytes to snake venom myotoxic phospholipases A(2): role of negatively charged phospholipids as potential membrane binding sites.

Cerrophidion (Bothrops) godmani myotoxins I (CGMT-I) and II (CGMT-II), Asp-49 and Lys-49 phospholipases A(2) (PLA2s), which drastically differ in enzymatic activity, were devoid of direct hemolytic effects on erythrocytes (RBC) from different species despite the fact that enzymatically active CGMT-I was able to hydrolyze RBC membrane phospholipids and disrupt liposomes prepared from RBC lipids. Human RBC did not become susceptible to the toxins after treatment with neuraminidase or after altering membrane fluidity with cholesterol or sublytic concentrations of detergent. Unlike normal RBC, significant hemolysis was induced by CGMT-II and another similar Lys-49 isoform, B. asper MT-II (BAMT-II), in RBC enriched with phosphatidylserine (PS). Hemolysis was greater in RBC preincubated with pyridyldithioethylamine (PDA), a potent inhibitor of aminophospholipid transport. RBC enriched with phosphatidic acid (PA) also became susceptible to the myotoxins but was unaffected by PDA. Cells enriched with phosphatidylcholine (PC) remained resistant to the action of the toxins. BAMT-II also induced damage in black lipid membranes prepared with PS but not PC alone. When RBC binding of BAMT-II was measured by enzyme-linked immunosorbent assay, it was observed that PS- and PA-enriched erythrocytes were always able to capture more toxin than normal and PC-enriched RBC. This effect was significantly improved by PDA (in the case of PS) and it was observed either in the presence or in the absence of calcium in the medium. These data suggest that negatively charged lipids in the outer leaflet of cell membranes constitute myotoxic PLA2 binding sites. The scarcity of anionic phospholipids in the outer leaflet of RBC could explain their resistance to the action of these PLA2s.     

膜脂的物理状态对G_s激活AC的影响

用生物膜的拆离与重建方法将从牛脑皮层膜中纯化的激活型ＧＴＰ结合蛋白（Ｇｓ）和腺苷酸环化酶（ＡＣ）在含有不同极性头部或不同脂肪酸侧链的磷脂组成的脂质体上重建形成脂酶体,测定脂酶体中ＡＣ的基础活力及Ｇｓ激活ＡＣ的活力。实验结果表明,磷脂影响ＡＣ的基础活力和Ｇｓ激活ＡＣ活力的顺序依次为：ＰＥ＞ＰＳ＞ＰＣ;含不同脂肪酸侧链的混合磷脂对Ｇｓ的激活活力的影响大于含单一脂肪酸侧链的纯磷脂,如ＰＥＤＰＰＥ,ＰＳＤＰＰＳ,ＰＣＤＰＰＣ。含不同脂肪酸侧链的磷脂影响Ｇｓ的活力的顺序为ＤＬＰＣ＞ＤＭＰＣ＞ＤＰＰＣ。用反映磷脂分子的堆积程度的荧光探剂ＭＣ５４０和脂双层的流动性变化的ＤＰＨ以及专一性标记蛋白质巯基（－ＳＨ）基团的荧光探剂ａｃｒｙｌｏｄａｎ的测定结果表明,不同磷脂影响Ｇｓ的活力的差异主要是由于脂质物理状态的不同所致。     

NaCl-induced changes in plasma membrane lipids and proteins of <Emphasis Type="Italic">Zea mays</Emphasis> L. cultivars differing in their response to salinity

Two contrasting maize (Zea mays L.) cultivars, i.e., Giza 2 (salt tolerant) and Trihybrid 321 (salt sensitive), were grown hydroponically to study NaCl effect (100 mM) on root plasma membrane (PM) lipid and protein alterations. The PM total sterols of Trihybrid 321 were decreased while that of Giza 2 was increased in response to salt. Salt imposition had no significant effect on PM total glycolipids and proteins of both cultivars. The PM total phospholipids were increased in Trihybrid 321 but it did not change significantly in Giza 2 after salinity stress. Molecular percentage of PM phospholipids and fatty acids of both cultivars was different in absence (0 mM) and presence (100 mM) of salt. The most abundant phospholipids in untreated Trihybrid 321 PM were phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS), which changed into PG, PS, phosphatidylinositol (PI) and PC after salt treatment. However, the dominant phospholipids of the control PM of Giza 2 were PC, PE, PS and PG, which changed into PG, PE, PS and diphosphatidylglycerol (DPG) after salt imposition. Over 60% of the total fatty acids were saturated in control and salinized PM of both cultivars, which was increased after salt stress. The predominant fatty acid in the control and salinized PM of Trihybrid 321 was C18:1 and C17:0, respectively. However, in control and treated PM of Giza 2, the predominant fatty acid was C17:0 and C20:0, respectively. Qualitative and quantitative differences in PM protein patterns were found in both cultivars with and without salt. PM lipid changes enhanced membrane integrity, reflected in different ion accumulation (Mansour et al. 2005), and hence salt tolerance of Giza 2.     

Cerebral Phospholipid Content and Na+, K+-ATPase Activity During Ischemia and Postischemic Reperfusion in the Mongolian Gerbil

Using bilateral carotid artery occlusion in adult gerbils we examined the effects of ischemia and ischemia/reperfusion on cerebral phospholipid content and Na+,K+-ATPase (EC 3.6.1.3) activity. In contrast to the large changes in phospholipid content and membrane-bound enzyme activity that have been observed in liver and heart tissues, we observed relatively small changes in the cerebral content of total phospholipid, phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE) following ischemic intervals of up to 240 min. Following 15 min of ischemia the cerebral content of sphingomyelin (SM) was decreased to less than 50% of control values but returned to near-normal levels with longer ischemic periods. Significant decreases in the cerebral content of phosphatidylinositol (PI) and phosphatidic acid (PA) were observed following shorter intervals of ischemia (15-45 min). Na+,K+-ATPase activity of cerebral homogenates prepared from the brains of gerbils subjected to 30-240 min of ischemia was decreased but significantly different from control activity only after 30 min of ischemia (-29%, p less than or equal to 0.05). With the exception of PS, reperfusion for 60 min following 60 min of ischemia resulted in marked increases in cerebral phospholipid content with PC, SM, PI, and PA levels exceeding and PE levels equal to preischemic values. Longer periods of reperfusion (180 min) resulted in decreases in cerebral phospholipid content toward (PC, SM, PI, and PA) or below (PE) preischemic levels. In contrast, the cerebral content of PS significantly decreased during reperfusion (-51% at 60 min, p less than or equal to 0.05) and remained below preischemic values even after 180 min of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ethanol on the incorporation of free fatty acids into cerebral membrane phospholipids

Chronic ethanol exposure is known to affect deacylation-reacylation of membrane phospholipids (PL). In our earlier studies we have demonstrated that chronic exposure to ethanol (EtOH) leads to a progressive increase in membrane phospholipase A2 (PLA2) activity. In the current study, we investigated the effects of chronic EtOH exposure on the incorporation of different free fatty acids (FFAs) into membrane PL. The results suggest that the incorporation of fatty acids into four major PL varied from 9.6 fmol/min/mg protein for docosahexaenoic acid (DHA) into phosphatidylinositol (PI) to 795.8 fmol/min/mg protein for linoleic acid (LA) into phosphatidylcholine (PC). These results also suggest a preferential incorporation of DHA into PC; arachidonic acid (AA) into PI; oleic acid into phosphatidylethanolamine (PE) and PC; LA into PC and stearic acid into PE. Chronic EtOH exposure affected the incorporation of unsaturated fatty acid into PI, phosphatidylserine (PS) and PC. However, EtOH did not affect significantly the incorporation of any of the fatty acids (FA) studied into PE. No significant differences were observed with the stearic acid. It is suggested that acyltransferases may play an important role in the membrane adaptation to the injurious effects of EtOH.     

The response of plasma membrane lipid composition in callus of the halophyte Spartina patens (Poaceae) to salinity stress

Callus cultures of the salt marsh grass Spartina patens were examined to determine changes and consistencies in membrane lipid composition in response to salt. Major membrane lipid classes remained stable at all salinity levels (0, 170, 340 mmol/L). However, the membrane protein to lipid ratio decreased significantly in response to elevated NaCl. Callus plasma membrane (PM) consisted predominantly of sterols, about 60% (mol%) of the total lipids. Glycolipid was the second largest lipid class, making up about 20% (mol%) of the total. With increasing salinity, the relative percentage of sitosterol decreased, while that of campesterol increased. The phospholipid species detected were phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI). When callus was grown at 340 mmol/L NaCl, PC increased significantly. PI and PS were also significantly elevated in salinity treatments. Only 24-32% of the PM fatty acids were common plant membrane fatty acids, C16, C18, C20, and C22, while over 60% were the less common fatty acids, C11 and C14. Membrane fluidity remained stable in response to growth medium salinity. The findings on membrane responses to salinity will facilitate a better understanding of this halophyte's tactics for salt tolerance.     

Mathematical modelling of lipid transbilayer movement in the human erythrocyte plasma membrane

A model is presented to simulate transverse lipid movement in the human erythrocyte membrane. The model is based on a system of differential equations describing the time-dependence of phospholipid redistribution and the steady state distribution between the inner and outer membrane monolayer. It takes into account several mechanisms of translocation: (i) ATP-dependent transport via the aminophospholipid translocase; (ii) protein-mediated facilitated and (iii) carrier independent transbilayer diffusion. A reasonable modelling of the known lipid asymmetry could only be achieved by introducing mechanism (iii). We have called this pathway the compensatory flux, which is proportional to the gradient of phospholipids between both membrane leaflets. Using realistic model parameters, the model allows the calculation of the transbilayer motion and distribution of endogenous phospholipids of the human erythrocyte membrane for several biologically relevant conditions. Moreover, the model can also be applied to experiments usually performed to assess phospholipid redistribution in biological membranes. Thus, it is possible to simulate transbilayer motion of exogenously added phospholipid analogues in erythrocyte membranes. Those experiments have been carried out here in parallel using spin labeled lipid analogues. The general application of this model to other membrane systems is outlined.Abbreviations PBS phosphate buffered saline - DFP diisopropyl fluorophosphate - ESR electron spin resonance - RBC red blood cells - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - SM sphingomyelin - (0,2)PC 1-palmitoyl-2(4doxylpentanoyl)-PC - (0,2)PE 1-palmitoyl-2(4-doxylpentanoyl)-PE - (0,2) PS 1-palmitoyl-2(4-doxylpentanoyl)-PS