掺入大肠杆菌膜中的磷脂酰胆碱(PC)影响青霉素b-内酰胺酶的分泌

[目的]为了研究磷脂酰胆碱(PC)在原核生物细胞中的生物学作用,探讨PC对细菌膜系统的功能的影响.[方法]使用ptac 85质粒作载体,将螺旋菌pcs基因导入E.coli Top10细胞构建了E.coli Fop10 pcs 菌株,并在特定的条件下培养细菌,使细菌膜磷脂中合成30%左右的磷脂酰胆碱.然后再使用抗生素抗性分析、β-内酰胺酶的酶活测定以及Western blot杂交技术,分析质粒编码的β-内酰胺酶从细胞质到细胞问质的分泌情况.[结果]抗生素抗性分析发现,高浓度的氨苄青霉素抑制E.coliTop10 pcs 细菌的生长的氨苄青霉素剂量低于对照组,其半致死剂量IC50在700～800μg/mL之间.酶活检测显示E.coli Top10 pcs 细菌周质内β-内酰胺酶的酶活性只有对照菌株的1,5,Western blot进一步分析发现周质内β-内酰胺酶的含量也为对照菌株的1/5.由此可见,周质内低含量的β-内酰胺酶是导致E.coli Top10pcs 细菌氨苄青霉素抗性降低的原因.[结论]掺入细菌膜磷脂双分子层的PC影响p.内酰胺酶通过Sec转运途径从细胞质分泌到细菌周质空间内,提示细菌磷脂酰胆碱可能在调节蛋白转运和分泌方面起着重要的作用.     

磷脂酰肌醇转移蛋白

磷脂酰肌醇转移蛋白(phosphatidylinositol/phosphatidylcholine transfer proteins,PITP)普遍存在于真核生物细胞中,PITP能够结合并交换一分子的磷脂酰肌醇(phosphatidylinositol,PI)或磷脂酰胆碱(phosphatidylcholine,PC),并促进这两类脂分子在细胞内膜组分间的转移。PITP对细胞内膜组分间脂类的运输和代谢、分泌囊泡的形成和运输、磷脂酶C(phospholipase,PLC)调节的信号传导以及神经退化等生理生化过程具有重要的影响。综述了近年来PITP的研究进展,并对目前研究中存在的一些问题进行探讨。     

土壤假单胞菌593使用Pcs途径合成磷酯酰胆碱

【目的】原核生物有两条代谢途径N-甲基化途径(Pmt途径)和磷脂酰胆碱合酶途径(Pcs途径)合成磷脂酰胆碱(PC)。本文对土壤细菌Pseudomonas sp.593的磷脂酰胆碱合成进行了研究,试图弄清楚假单胞菌的磷脂酰胆碱的合成途径。【方法】通过氨基酸序列比较,获得已报道的磷脂酰胆碱合酶(Pcs)的氨基酸保守序列,并设计简并引物,从Pseudomonas sp.593总DNA中PCR扩增出磷脂酰胆碱合酶基因(pcs)的片段,然后用扩增的DNA片段作探针,对Pseudomonas sp.593基因组DNA亚克隆文库进行菌落原位杂交,获得pcs全基因序列;利用同源重组原理进行活体突变,获得Pseudomonas sp.593 pcs-突变体;采用薄层层析(TLC)法分析细菌总磷脂,检测PC含量以及pcs基因活性。【结果】TLC分析显示Pseudomonas sp.593细菌仅在添加外源胆碱的M9或LB培养基中生长时才合成PC;从Pseudomonas sp.593细菌中克隆出894 bp的DNA序列,编码的蛋白具有磷脂酰胆碱合酶活性;活体缺失pcs基因后,Pseudomonas sp.593 pcs-突变体在添加或不添加胆碱的条件下都不能合成PC。【结论】Pcs途径是土壤Pseudomonas sp.593乃至其它假单胞菌合成磷脂酰胆碱的唯一途径。     

肝源性磷脂酰乙醇胺的分离及其诱导肝癌细胞凋亡的研究

采用氧化铝柱色谱法以95%乙醇-氯仿为洗脱剂分离纯化了肝源性磷脂酰乙醇胺(PE),并采用GF254硅胶板薄层色谱法以氯仿-甲醇-水(65:25:4,体积比)为展开剂检测PE.结果表明,以95%乙醇-氯仿顺序洗脱氧化铝色谱柱中的磷脂时,PE与磷脂酰胆碱(PC)可实现完全分离.采用四甲基偶氮唑蓝比色法(MTT法)测定了在不同时间点25 μmol/L PE对人肝癌SMMC-7721细胞增殖的影响,并与子宫颈癌细胞Hela、正常人肝细胞HL7702做比较,发现肝源性PE对肝癌细胞的生长具有明显的抑制作用,且能诱导其发生凋亡.     

磷脂对琥珀酸-细胞色素c还原酶的作用

琥珀酸细胞色素c还原酶除去90％以上的磷脂后活力丧失约95％。将去脂琥珀酸细胞色素c还原酶与磷脂和辅酶Q_2保温,可恢复其活性。活力恢复程度依赖于磷脂的组成。当磷脂酰胆碱(PC):心磷脂(CL):磷脂酰乙醇胺(PE)=2:2:1时活力恢复最高,比大豆磷脂的效果更为明显,单组分PC,PE或CL恢复活力较差。与酶蛋白紧密结合的CL和PC在活力可逆恢复中有重要作用。     

乳腺癌转移中的磷脂酰胆碱和溶血磷脂酰胆碱分析

我们以前曾报道花生四烯酸（arachidonic acid,AA)代谢产物可以促进乳腺癌细胞增殖和迁移。为了进一步寻找维持高转移乳腺癌细胞中AA高水平代谢的内源机制,深入探求AA代谢促进乳腺癌细胞转移的分子机理,我们应用HPLC/ESI/MSⁿ技术检测和分析了乳腺癌MCF-7和高转移乳腺癌LM-MCF-7细胞中溶血磷脂酰胆碱（lysophosphatidylcholines,LysoPCs）和磷脂酰胆碱（phosphatidylcholines,PCs）的成分和含量。我们发现了10种LysoPC的含量在LM-MCF-7细胞中显著高于MCF-7细胞,有6种PC可水解产生AA,它们在LM-MCF-7细胞中的含量显著低于MCF-7细胞,提示这些溶血磷脂含量的升高和磷脂含量的降低可能与乳腺癌转移相关。在LM-MCF-7细胞中,COX-2抑制剂吲哚美辛（indomethacin,Indo）和LOX抑制剂（nordihydroguaiaretic acid,NDGA）共同作用可明显下调cPLA2的活性,应用HPLC-ESI-MSⁿ技术比较cPLA2活性下调前后LM-MCF-7细胞中LysoPC和PC含量的变化,发现其中4种PC可被cPLA2水解产生AA。我们还发现,细胞内LysoPC与PC的比值可以反映cPLA2的活性。通过以上研究我们进一步证实了由cPLA2活性调节的AA释放及代谢对乳腺癌转移具有重要作用。     

胆碱摄取及磷脂酰胆碱合成的调节研究

木文研究了多种氨基酸、乙醇胺和甲基乙醇胺对细胞摄取胆碱和合成磷脂酰胆碱（ＰＣ）的影响,发现多种氨基酸非竞争性地抑制细胞摄取胆碱。含胆碱代谢物的分析显示胆碱转变成ＣＤＰ－胆碱,随之形成ＰＣ均不受氨基酸影响。乙醇胺竞争性地抑制胆碱摄取,且存在剂量依赖关系。乙醇胺能明显抑制胆碱激酶活性,但细胞内胆碱和磷酸胆碱的代谢池并不改变,提示乙醇胺不影响胆碱转变成磷酸胆碱。根据ＣＤＰ－胆碱和ＰＣ的比放射性分布,乙醇胺也不影响ＰＣ的生物合成。甲基乙醇胺抑制胆碱摄入的程度强于乙醇胺,并抑制胆碱激酶和ＣＴＰ：磷酸胆碱胞苷转移酶活性,含胆碱代谢物以ＣＤＰ－胆碱下降最显著;提示甲基乙醇胺不仅抑制胆碱摄入而且还干扰了ＣＤＰ－胆碱通路。     

酿酒酵母酚类抑制物耐受性脂质组学研究

通过脂质组学分析方法从细胞膜磷脂分布方面探究适应进化酿酒酵母酚酸耐受性机制。主要利用高效液相色谱-质谱(LC-MS)对酚酸胁迫下适应进化菌株和原始菌株脂质成分检测并进行统计学比较分析。检测出565种脂质代谢物,包含细胞膜磷脂185种。相比初始菌株,适应进化菌株细胞膜中磷脂酰胆碱(PC)、磷脂酰乙醇胺(PE)和磷脂酰肌醇(PI)类磷脂分子相对含量增加,含有长链(C32-C36)和双不饱和脂酰链的磷脂分子含量增加。统计学分析表明显著性差异磷脂分子主要为含有长链不饱和脂酰链的PC和PE类磷脂分子。推测适应进化菌株通过膜磷脂重塑提高细胞膜完整性,对酚类抑制物起到选择性屏障作用,从而保持细胞活性。     

我国健康成人红细胞膜脂类的脂酸组成

 本文采用双向簿层层析分离红细胞膜脂类,继以毛细管气相色谱法分析其脂酸含量,检测了15名我国健康成人红细胞膜脂类的脂酸摩尔百分组成。结果表明:各脂类中,脂酸的类别基本相同,但其含量组成相差甚远。如磷脂酰乙醇胺(PE)富含C_(20:4);磷脂酰胆碱(PC)富含C_(18:2);神经鞘磷脂(SM)主要含C_(16:0);磷脂酰丝氨酸(PS)主要含C_(18:0);而以红细胞糖苷脂(GL)中脂酸含量最少。膜总脂中饱和脂酸与不饱和脂酸的含量大致相等,胆碱磷脂(PC+SM)的脂酸饱和度则明显高于氨基磷脂(PE+PS)。     

磷脂酰丝氨酸外翻和磷脂氧化在凋亡细胞被吞噬细胞清除过程中的协作效应

为探讨磷脂酰丝氨酸(phosphatidylserine,PS)外翻和磷脂氧化在凋亡细胞被吞噬细胞清除中的作用,用脂质体整合的方法将不同的磷脂整合到红细胞上或用N-乙酰马来酰胺(N-ethylmaleimide,NEM)预处理红细胞然后整合磷脂,制备含不同凋亡信号的红细胞模型,测定巨噬细胞对整合不同磷脂信号红细胞的结合率和吞噬率。结果表明,单独整合PS或用NEM处理造成PS外翻,可显著性提高巨噬细胞对红细胞的结合率,但对吞噬率没有影响;同时整合PS和氧化磷脂(氧化PS或氧化磷脂酰胆碱(phosphatidylcholine,PC)),或用NEM处理造成PS外翻后再整合氧化PS或氧化PC,不仅可显著提高巨噬细胞对红细胞的结合率,而且可显著性提高吞噬率。这些结果提示PS外翻可能参与了巨噬细胞对凋亡细胞的结合,而磷脂氧化可能启动了巨噬细胞对凋亡细胞的吞噬,二者协作才可能完成巨噬细胞对凋亡细胞的清除。     

Phorbol esters modulate the turnover of both ether- and ester-linked phospholipids in cultured mammalian cells

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the metabolism of ester- and ether derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were studied in HeLa and HEL-37 cells. TPA stimulated the incorporation of [3H]choline into diacyl-, alkylacyl- and alkenylacy/PC in HeLa cells, but inhibited the incorporation of [3H]ethanolamine into the corresponding derivatives of PE. TPA also stimulated the incorporation of [3H]ethanolamine into lysoPE and the release of labelled ethanolamine and phosphoethanolamine from HeLa cells prelabelled with [3H]ethanolamine. All responses to TPA were abolished in HeLa cells preincubated with the phorbol ester and which were deficient in protein kinase C. In HEL-37 cells TPA stimulated label incorporation into both ester- and ether-forms of PE. The marked effects of TPA on ether-lipid metabolism raises the possibility that hydrolysis products of this class of lipid are important in transmembrane signalling pathways.     

Phospholipid uptake by Plasmodium knowlesi infected erythrocytes

The uptake of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in Plasmodium knowlesi infected erythrocytes has been studied. Whereas uptake of phospholipids, in the absence of phospholipid transfer proteins, is negligible in control cells, the infected cells can incorporate considerable amounts of added phospholipids. The uptake is enhanced by the presence of lipid transfer proteins. Doubly labeled [3H]oleate, [14C]choline) PC does not undergo any appreciable remodelling following uptake, which strongly suggests that plasma PC is used as such for the biogenesis of the parasite membranes. Transport of extracellularly offered PS and PE towards the intraerythrocytic parasite and utilization of these lipids by the parasite are confirmed by the observation that these lipids are converted into respectively PE and PC. The extent and rate of these conversions depend on the way the phospholipids are introduced into the infected cells.     

Arachidonic acid turnover in peritoneal macrophages is altered in endotoxin-tolerant rats

Peritoneal macrophages from endotoxin-tolerant rats have been found to exhibit depressed metabolism of arachidonic acid (AA) to prostaglandins and thromboxane in response to endotoxin. The effect of endotoxin tolerance on AA turnover in peritoneal macrophages was investigated by measuring [14C]AA incorporation and release from membrane phospholipids. Endotoxin tolerance did not affect the amount of [14C]AA incorporated into macrophages (30 min-24 h). However, the temporal incorporation of [14C]AA into individual phospholipid pools (15 min-24 h) was altered. In endotoxin-tolerant macrophages, [14C]AA incorporation into phosphatidylcholine (PC) (2, 4, 24 h) and phosphatidylethanolamine (PE) (8 h) was increased, while the incorporation into phosphatidylserine (PS) (2-24 h) was reduced (P less than 0.005) compared to control macrophages. There was no change in [14C]AA incorporation into phosphatidylinositol (PI). Following 2 or 24 h of incorporation of [14C]AA, macrophages were incubated (3 h) with endotoxin (50 micrograms/ml) or A23187 (1 microM), and [14C]AA release was measured. Endotoxin-tolerant macrophages released decreased (P less than 0.05) amounts of [14C]AA in response to both endotoxin and the calcium ionophore A23187 compared to controls. Control macrophages in response to endotoxin released [14C]AA from PC, PI and PE. In contrast, tolerant cells released [14C]AA only from PC (P less than 0.05). A23187 released [14C]AA from all four pools in the control cells, but only from PC and PE in the tolerant cells. These data demonstrate that endotoxin tolerance alters the uptake and release of AA from specific macrophage phospholipid pools. These results suggest that changes in AA turnover and/or storage are associated with endotoxin tolerance.     

Regional Membrane Phospholipid Alterations in Alzheimer's Disease

Regional levels of membrane phospholipids [phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylcholine (PC)] were measured in the brain of Alzheimer's disease (AD) and control subjects. The levels of PE-derived and PI-derived total fatty acids were significantly decreased in the hippocampus of AD subjects. Here significant decreases were found in PE-derived stearic, oleic and arachidonic and docosahexaenoic acids, and in PI-derived oleic and arachidonic acids. In the inferior parietal lobule of AD subjects, significant decreases were found only in PE and those decreases were contributed by stearic, oleic and arachidonic acids. In the superior and middle temporal gyri and cerebellum of AD subjects, no significant decreases were found in PC-, PE- and PI-derived fatty acids. The decrease of PE and PI, which are rich in oxidizable arachidonic and docosahexaenoic acids, but not of PC, which contains lesser amounts of these fatty acids, suggests a role for oxidative stress in the increased degradation of brain phospholipids in AD.     

Topology of polytopic membrane protein subdomains is dictated by membrane phospholipid composition

In Escherichia coli, the major cytoplasmic domain (C6) of the polytopic membrane protein lactose permease (LacY) is exposed to the opposite side of the membrane from a neighboring periplasmic domain (P7). However, these domains are both exposed on the periplasmic side of the membrane in a mutant of E.coli lacking phosphatidylethanolamine (PE) wherein LacY only mediates facilitated transport. When purified LacY was reconstituted into liposomes lacking PE or phosphatidylcholine (PC), C6 and P7 were on the same side of the bilayer. In liposomes containing PE or PC, C6 and P7 were on opposite sides of the bilayer. Only the presence of PE in the liposomes restored active transport function of LacY as opposed to restoration of only facilitated transport function in the absence of PE. These results were the same for LacY purified from PE-containing or PE-lacking cells, and are consistent with the topology and function of LacY assembled in vivo. Therefore, irrespective of the mechanism of membrane insertion, the subdomain topological orientation and function of LacY are determined primarily by membrane phospholipid composition.     

Enhancement by Cytidine of Membrane Phospholipid Synthesis

Cytidine, as cytidine 5'-diphosphate choline, is a major precursor in the synthesis of phosphatidylcholine in cell membranes. In the present study, we examined the relationships between extracellular levels of cytidine, the conversion of [14C]choline to [14C]phosphatidylcholine, and the net syntheses of phosphatidylcholine and phosphatidylethanolamine by PC12 cells. The rate at which cytidine (as [3H]cytidine) was incorporated into the PC12 cells followed normal Michaelis-Menten kinetics (Km = 5 microM; Vmax = 12 x 10(-3) mmol/mg of protein/min) when the cytidine concentrations in the medium were below 50 microM; at higher concentrations, intracellular [3H]cytidine nucleotide levels increased linearly. Once inside the cell, cytidine was converted mainly into cytidine triphosphate. In pulse-chase experiments, addition of cytidine to the medium caused a time- and dose-dependent increase (by up to 30%) in the incorporation of [14C]choline into membrane [14C]-phosphatidylcholine. When the PC12 cells were supplemented with both cytidine and choline for 14 h, small but significant elevations (p less than 0.05) were observed in their absolute contents of membrane phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, all increasing by 10-15% relative to their levels in cells incubated with choline alone. Exogenous cytidine, acting via cytidine triphosphate, can thus affect the synthesis and levels of cell membrane phospholipids.     

The use of newly synthesized phospholipids for assembly into secreted hepatic lipoproteins

We have recently shown (Vance, J.E. (1988) Biochim. Biophys. Acta 963, 70-81) that the percent distribution of molecular species of phosphatidylcholine (PC) derived from [methyl-3H]choline and [3-3H]serine, and phosphatidylethanolamine (PE) derived from [3-3H]serine were different in secreted lipoproteins and in the cultured hepatocytes from which the lipoproteins were produced. The species 1-stearoyl-2-arachidonoyl PC and PE were selectively not secreted. How this selection occurs is not known. One possible explanation is that secreted phospholipids are representative of the newly synthesized pool, whereas the molecular species composition of bulk cellular phospholipids has been altered by selective deacylation or by deacylation-reacylation. This hypothesis has been tested. The percent distribution of radioactivity from [1-3H]ethanolamine, [3-3H]serine and [methyl-3H]choline in nascent cellular and secreted PE and PC molecular species was examined by high-performance liquid chromatography. From [3H]serine labeling, the percent distribution of [3H]PE species in the medium after 4 h resembled closely that in cells 0.5 h, but not 4 h, after labeling. Thus, nascent phosphatidylserine-derived PE was immediately earmarked for secretion before remodeling occurred. Similarly, newly made rather than 'old' PE and PC from alternative biosynthetic sources may be preferred for assembly into lipoproteins. In addition, PE methyltransferase apparently preferred newly made, rather than remodelled, serine-derived PE for methylation to PC. In no instance (i.e., neither for any phospholipid nor any precursor) was there evidence that 'old' rather than 'new' phospholipid was specifically selected for secretion.     

Effect of environmental temperature on the phospholipid metabolism of gill mitochondria of Carcinus maenas

The different types of phospholipids extracted from gill mitochondria of crab Carcinus maenas have been analysed and it was found that a significant increase of the phosphatidylethanolamine (PE) content and a concomitant decrease of the phosphatidylcholine (PC) amount are present in animals living in low temperatures. The incorporation of [³H]ethanolamine in total phospholipids, PE and PC, was demonstrated in gill mitochondria and a thermal alteration of the in vivo exchange of PE between mitochondria and 10,000 g supernatant is suggested by the kinetics of the incorporation. It is suggested that the conversion of PE to PC by N-methylation is very low in crab gills. There is a marked action of acclimation temperature on the gills-hemolymph exchange of PC and PE. It is postulated that the changes reported at the level of the PE → PC conversion by N-methylation and in phospholipid exchange between hemolymph and gills could be implicated in adapting the organism to seasonal fluctuations of environmental temperatures.     

Differential effects of unsaturated fatty acids on phospholipid synthesis in human leukemia monocytic U937 cells

The biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in monocyte-like leukemia U937 cells was monitored by adding [³H]choline, [¹⁴C]ethanolamine or [¹⁴C]glycerol to the culture media; incorporation into phospholipid (PL) increased with time. The effect of unsaturated fatty acids (UFA) on PC and PE synthesis was investigated by pretreating U937 cells for 72h with 10 μM 18:1 (n –9), 18:2 (n –6), 18:3 (n –3), 20:4 (n –6) and 20:5 (n –3). The UFA caused no alteration in cell growth, as evidenced by light microscopy and the incorporation of [³H]thymidine and [³H]leucine. Total cellular uptake of radioactive precursors remained unaffected by all the treatments. Pretreatment with 20:5 resulted in approximately 25 per cent reduction in the incorporation of [³H]choline into PL, while no significant effect was detected with the other UFAs. 18:3, 20:4 and 20:5 depressed the incorporation of [¹⁴C]ethanolamine into PL by 34 per cent, 28 per cent and 49 per cent respectively. However, there was no redistribution of label with any of the treatments. 18:3, 20:4 and 20:5 also antagonized the stimulatory effect of endotoxin (LPS) on PC and PE synthesis. In addition, the incorporation from [¹⁴C]glycerol into PC and PE was reduced by 18:3, 20:4 and 20:5. Although the PL composition of the cells remained essentially unaffected, our study shows that chronic treatment of U937 cells with n –3 PUFA (20:5) depressed PC and PE synthesis, and 18:3 and 20:4 also caused inhibition of PE synthesis.     

Mobilization of arachidonic acid from phosphatidylethanolamine fraction to phosphatidylcholine fraction in platelets

Rabbit platelets rapidly incorporated methyl groups of [³H] methionine to phosphatidylcholine (PC). Rabbit platelets also incorporated [³H]choline to PC, but the rate of incorporation was far lower than that of [³H]methionine. Further fractionation of labeled PC revealed that a considerable amount of arachidonyl PC was synthesized via the N-methylation pathway. Thrombin stimulation resulted in a release of arachidonic acid from PC, and not from phosphatidylethanolamine (PE). These observations suggest that the N-methylation pathway plays an important role in the intracellular mobilization of arachidonic acid from the PE fraction to the PC fraction, this fraction being more sensitive to the hydrolysis with phospholipase A₂ during platelet activation.