氧化甾醇-磷脂脂质囊泡的制备及特性研究

 氧化甾醇3,19–二羟基-胆甾烷-24-酮（DHCO）和3β,5α,6β-胆甾烷三醇（Triol）替代胆甾醇与大豆磷脂形成脂质囊泡的性质研究。采用乙醇注入法制备脂质囊泡,通过测定脂溶性及水溶性荧光探针在脂质囊泡中的荧光强度变化,考察囊泡膜流动性及通透性;通过测定脂质囊泡中游离DHCO及结合DHCO的浓度考察DHCO与磷脂的结合率;考察DHCO/磷脂比例、超声条件对脂质囊泡粒径大小和分布的影响。结果表明,DHCO、Triol与磷脂形成的脂质囊泡与胆甾烷（CHOL）-磷脂脂质囊泡的膜流动性无明显差异,但膜通透性稍有增大。DHCO与磷脂的结合率为8258%。DHCO、Triol与大豆磷脂经简单工艺即可形成脂质囊泡。可通过调节DHCO/磷脂比例、超声条件获得具理想粒径和外观的脂质囊泡。氧化甾醇数量庞大,其作为脂质囊泡的新型“流动性缓冲剂”有巨大的发展潜力。     

正负离子表面活性剂混合溶液中囊泡的稳定性

水环境中直链烷基羟酸盐与溴代烷基季铵盐１：１混合表面活性剂囊泡可在一定时间和较大温度范围内稳定存在。１－１价型无机盐对混合囊泡稳定性影响较小。但少量高价金属离子与离子、非离子表面活性剂的介入对混合体系形成的囊泡具有较为明显的破坏作用。     

烷基磺酸盐—烷基季铵盐混合溶液中囊泡的形成

采用十六烷基三甲基溴化铵与烷基磺酸盐的混合表面活性剂体系，能在水溶液中通过超声震荡形成具有分子有序组合体形式的单室囊泡。对混合表面活性剂的总浓度、两表面活性剂的混合比以及混合体系中烷基磺酸盐的碳链长度对囊泡形成的影响进行了研究。     

吐温-80与脂质体膜相互作用机理的研究

研究了表面活性剂吐温-80(聚氧乙烯山梨醇脂肪酸酯)与脂质膜间的相互作用机理,运用浊度测定、DSC1、H—NMR等分析手段验证了脂质膜的立体稳定结构.结果表明:吐温-80在水相与脂质相间分配达到饱和时的质量浓度为1.3%,与脂质膜开始增溶成混合胶团时的质量浓度为2.6%.当Re<0.5时,表面活性剂单体在溶液和脂质双层膜中分配,溶液中表面活性剂单体和囊泡并存,脂质双层囊泡膨胀,粒径逐渐增大,形成一个肿胀的脂质囊泡.当0.5相似文献   

表面活性剂缔合结构作为药物载体的研究进展--微乳液、囊泡体系

综合评述了表面活性剂缔合结构,特别是微乳液、囊泡作为药物载体的研究现状.提出了在这一领域进一步开展仿生磷脂囊泡,微乳液凝胶,微乳液/环糊精复合体系研究的设想.     

9,10-二（对烷氧基苯乙烯基）蒽衍生物的合成及其末端基链长对聚集诱导荧光性质的影响

通过4烷氧基苯甲醛和9,10二磷脂基蒽的Witting反应合成了一系列可溶性的9,10-二（对烷氧基苯乙烯基）蒽（DSA-OCn）（n=1～10,12,14,16）衍生物.溶液的紫外吸收和荧光发射光谱随着末端烷基链长的改变没有发生明显的变化,但是其在V（THF）/V（水）=1/9混合溶液中的荧光发射光谱出现了较大的差异.当DSA-OCn系列衍生物的末端烷基链长度较长（n≥7）时它们的荧光光谱峰值在550 nm左右,而末端烷基链较短（n≤6）化合物的荧光光谱峰值出现在500 nm左右.此外,具有较长末端烷基链的化合物其溶液荧光量子效率（鳓较低.结果表明,改变末端烷基链的长度可以有效地调控化合物的聚集行为和光学性质.     

双分子膜中4,4′,4′′,4′′′-四(正丙氧基羰基)酞菁钴(Ⅱ)催化分子氧氧化巯基乙醇的反应

研究了4,4′,4′′,4′′′-四(正丙氧基羰基)酞菁钴(Ⅱ)[CoPc(COOC3H7)4〗分别在由正十六烷基-2-羟基-3-氯丙基磷酸一氢酯(C16-AHCP)所构成的双分子膜泡囊、十二烷基硫酸纳(SDS)胶束、十六烷基三甲基溴化铵(CTAB)胶束、聚乙二醇辛基苯基醚(Triton(x-100))胶束、无水乙醇和无水苯中催化分子氧氧化巯基乙醇的反应.利用微量呼吸检压仪测定体系中的耗氧量来确定催化反应速度,用紫外-可见吸收光谱研究了影响催化反应速度的因素.结果表明,CoPc(COOC3H7)4的催化活性顺序为在双分子膜泡囊无水苯CTAB胶束Triton(x-100)胶束SDS胶束无水乙醇,催化活性与[CoPc(COOC3H7)4在介质中的存在形式有关.     

蛋白质、胆固醇对卵磷脂囊泡稳定性的影响

用卵磷脂、胆固醇和蛋白质所形成的囊泡模拟细胞膜,利用Langmuir膜天平、Zeta电势研究卵磷脂、胆固醇和蛋白质分子之间的相互作用,以及通过停留法和TEM等方法从Gemini(双子)表面活性剂对细胞膜结构破坏方面来探讨不同组分对囊泡的稳定性的影响.实验结果表明,囊泡中的蛋白质、胆固醇和卵磷脂分子之间是相互吸引的.相对于卵磷脂囊泡,混合体系囊泡更加稳定.表面活性剂是通过静电吸引力和疏水效应嵌入囊泡的双分子层中,导致囊泡被破坏.通过动力学实验得到Gemini表面活性剂对囊泡破坏过程的活化能,进一步证明加入蛋白质、胆固醇能够使卵磷脂囊泡更加稳定.     

阴阳离子表面活性剂复配自发形成囊泡的研究

采用阳离子表面活性剂十六烷基三甲基溴化铵分别与阴离子表面活性剂十二烷基苯磺酸钠、 十二烷基磺酸钠及十二烷基硫酸钠复配, 3种体系的水溶液在适当条件下均可自发形成囊泡. 结果表明, 虽然3种阴离子表面活性剂的分子结构相似, 但在与同一种阳离子表面活性剂复配形成囊泡时的复配比例及囊泡的稳定性却有很大区别. 考察了3种体系囊泡自发形成时的复配比例, 并探讨了囊泡的形成机理及其时间稳定性,利用负染TEM技术观察了囊泡的形貌.     

糖脂结构对变色囊泡检测大肠杆菌的影响

研究了3种不同结构的糖分子(双十八烷基甘油基- β-葡萄糖苷(DGG)、1,2-O-双十八烷基-3-O-β-D-麦芽三糖基-甘油醇(DMTG)、3,6,9,12-四氧杂-10-胆甾基-2-乙酰胺-2-脱氧-β-D-吡喃葡萄苷(DL3))对大肠杆菌(E.coli)识别能力的影响.利用比色法测定囊泡对E.coli的识别能力,实验结果表明:检测E.coli 3种体系的灵敏度大小顺序为DL3/TCDA＞DMTG/TCDA＞DGG/TCDA;具有多糖结构和水溶性的-(CH2CH2O)3-结构的糖脂会有利于检测.红外光谱的研究证明了糖脂分子插入了聚联乙炔结构.     

A designed amphiphilic peptide containing the silk fibroin motif as a potential carrier of hydrophobic drugs

The amphiphilic peptide is becoming attractive as a potential drug carrier to improve the dissolvability of hydrophobic drugs in an aqueous system; thus, facilitating drug uptake by target cells. Here, we report a novel designed amphiphilic peptide, Ac-RADAGAGARADAGAGA-NH₂, which was able to stabilize pyrene, a hydrophobic model drug we chose to study in aqueous solution. This designed peptide formed a colloidal suspension by encapsulating pyrene inside the peptide-pyrene complex. Egg phosphatidylcholine (EPC) vesicles were used to mimic cell bilayer membranes. We found that pyrene was released from the peptide coating into the EPC vesicles by mixing the colloidal suspension with EPC vesicles, which was followed by steady fluorescence spectra as a function of time. A calibration curve for the amount of pyrene released into the EPC vesicles at a given time was used to determine the final concentration of pyrene released into the lipid vesicles from the peptide-pyrene complex. The release rate of the peptide-pyrene complex was calculated to quantify the transfer of pyrene into EPC vesicles.     

Reconstitution of a phospholipid flippase from rat liver microsomes

The endoplasmic reticulum is the principal site of synthesis and initial incorporation of membrane lipids in eukaryotic cells; the enzymes of glycerolipid biosynthesis are exclusively located on its cytoplasmic surface. To maintain a phospholipid bilayer in this organelle, newly synthesized phospholipids must be translocated to the lumenal surface. Consistent with this are measurements indicating that movement of phospholipids across microsomal membranes is rapid, with a half-time less than 5 min (refs 3 and 4). Rapid movement of phospholipids has also been detected across the plasma membrane of Bacillus megaterium, another site of de novo lipid biosynthesis. The rapid transmembrane movement of phosphatidylcholine has not been detected, however, in vesicles prepared from microsomal lipids. These latter data suggest involvement in the endoplasmic reticulum of a phospholipid-translocating protein, as was first proposed by Bretscher who called it 'flippase'. Here we report reconstitution of a phospholipid flippase from rat liver microsomes into lipid vesicles.     

氨基酸系列希夫碱的生成与分解研究

合成了甘氨酸(Gly-Schiff base)、DL-丙氨酸(DL-Ala-Schiff base)、L-苯丙氨酸(L-Phe-Schiff base)、L-谷氨酸(L-Glu-Schiff base)、L-天冬氨酸(L-Asp-Schiff base)及L-色氨酸(L-Trp-Schiff base)等6种氨基酸水杨醛希夫碱及其金属铜配合物,研究了其紫外-可见电子吸收光谱.分别在酸碱条件下对氨基酸水杨醛铜配合物进行了分解实验,并选取DL-丙氨酸代替人工信号转导系统中的脂双层膜,与5’-磷酸吡哆醛进行络合形成类似天然磷脂和磷酸吡哆醛的复合体,为下一步构建细胞信号转导过程中的可逆的化学模型作了相关的基础性工作.     

Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension

Lipid bilayer membranes--ubiquitous in biological systems and closely associated with cell function--exhibit rich shape-transition behaviour, including bud formation and vesicle fission. Membranes formed from multiple lipid components can laterally separate into coexisting liquid phases, or domains, with distinct compositions. This process, which may resemble raft formation in cell membranes, has been directly observed in giant unilamellar vesicles. Detailed theoretical frameworks link the elasticity of domains and their boundary properties to the shape adopted by membranes and the formation of particular domain patterns, but it has been difficult to experimentally probe and validate these theories. Here we show that high-resolution fluorescence imaging using two dyes preferentially labelling different fluid phases directly provides a correlation between domain composition and local membrane curvature. Using freely suspended membranes of giant unilamellar vesicles, we are able to optically resolve curvature and line tension interactions of circular, stripe and ring domains. We observe long-range domain ordering in the form of locally parallel stripes and hexagonal arrays of circular domains, curvature-dependent domain sorting, and membrane fission into separate vesicles at domain boundaries. By analysing our observations using available membrane theory, we are able to provide experimental estimates of boundary tension between fluid bilayer domains.     

Curvature of clathrin-coated pits driven by epsin

Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P(2) in conjunction with clathrin polymerization. We have discovered that formation of an amphipathic alpha-helix in epsin is coupled to PtdIns(4,5)P(2) binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is sufficient to facilitate the formation of clathrin-coated invaginations.     

Self-assembling phospholipid filaments

Aqueous dispersions of double-chain phospholipids spontaneously assemble into closed bilayers called vesicles (or liposomes). Although the vesicles are in general topologically spherical, cylindrical and helical liposomes have sometimes been observed. We present here video-enhanced microscopic studies of a diacetylenic phospholipid dispersed in ethanol/water, which reveal the existence of unusual bilayer morphologies. On cooling the dispersion from the isotropic phase, we have observed the formation of long (of the order of hundreds of micrometres), thin (0.2-2 microns) filaments, which fluctuate strongly. When the temperature is decreased further, the filaments rapidly retract into a mass of lipid. At constant temperature, on the other hand, the filaments transform into torus or ring-like vesicles. Such non-spherical structures have been predicted theoretically but not previously observed experimentally.     

The interaction between purple membrane and membrane lipid

Bacteriorhodopsin in purple membrane was reconstituted into different lipid vesicles. The effect of three different lipids on the structure and function of bacteriorhodopsin in lipid vesicles was studied by the observation on freeze-fracture eletron microscopy, the rotational diffusion of bacteriorhodopsin in lipid vesicles, the measurement of absorption spectrum, and the absorbance change with time. For these prepared samples, the results showed that DMPC was the stable lipid environment of bacteriorhodopsin; egg-pc causeed the loss of retinal chromophore of bacteriorhodopsin and it was not reversible change, cholesterol could stabilize the bacteriorhodopsin in lipid environment,but it caused the aggregation of bacteriorhodopsin.     

Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature

Protein coats deform flat lipid membranes into buds and capture membrane proteins to form transport vesicles. The assembly/disassembly cycle of the COPI coat on Golgi membranes is coupled to the GTP/GDP cycle of the small G protein Arf1. At the heart of this coupling is the specific interaction of membrane-bound Arf1-GTP with coatomer, a complex of seven proteins that forms the building unit of the COPI coat. Although COPI coat disassembly requires the catalysis of GTP hydrolysis in Arf1 by a specific GTPase-activating protein (ArfGAP1), the precise timing of this reaction during COPI vesicle formation is not known. Using time-resolved assays for COPI dynamics on liposomes of controlled size, we show that the rate of ArfGAP1-catalysed GTP hydrolysis in Arf1 and the rate of COPI disassembly increase over two orders of magnitude as the curvature of the lipid bilayer increases and approaches that of a typical transport vesicle. This leads to a model for COPI dynamics in which GTP hydrolysis in Arf1 is organized temporally and spatially according to the changes in lipid packing induced by the coat.     

Inositol 1,4,5-trisphosphate activates a channel from smooth muscle sarcoplasmic reticulum

Inositol 1,4,5-trisphosphate (InsP3) can initiate calcium release into the cytoplasm in a variety of cells. From experiments using permeabilized cells, membrane vesicles, and patch-clamp techniques, it has been suggested that InsP3 acts by directly opening calcium channels. Here, we show that InsP3 induced openings of channels in planar lipid bilayers into which vesicles made from aortic muscle sarcoplasmic reticulum (SR) were incorporated. Activation of channels by InsP3 was not observed when vesicles made from SR of cardiac or skeletal muscle were incorporated into planar lipid bilayers. The present study demonstrates for the first time unique properties of an InsP3-gated calcium channel in sarcoplasmic reticulum vesicles from vascular smooth muscle. This InsP3-activated channel from aortic SR differs strikingly from the calcium-gated calcium channel of striated muscle SR in single-channel conductance and pharmacology.     

Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid.

Endophilin I is a presynaptic protein of unknown function that binds to dynamin, a GTPase that is implicated in endocytosis and recycling of synaptic vesicles. Here we show that endophilin I is essential for the formation of synaptic-like microvesicles (SLMVs) from the plasma membrane. Endophilin I exhibits lysophosphatidic acid acyl transferase (LPAAT) activity, and endophilin-I-mediated SLMV formation requires the transfer of the unsaturated fatty acid arachidonate to lysophosphatidic acid, converting it to phosphatidic acid. A deletion mutant lacking the SH3 domain through which endophilin I interacts with dynamin still exhibits LPAAT activity but no longer mediates SLMV formation. These results indicate that endophilin I may induce negative membrane curvature by converting an inverted-cone-shaped lipid to a cone-shaped lipid in the cytoplasmic leaflet of the bilayer. We propose that, through this action, endophilin I works with dynamin to mediate synaptic vesicle invagination from the plasma membrane and fission.