伴刀豆球蛋白A与巨噬细胞膜受体结合引起膜蛋白和膜脂分子运动及电荷的变化

本文利用荧光漂白恢复,顺磁共振和细胞电泳等技术研究外源性配体伴刀豆球蛋白A与巨噬细胞膜受体结合后膜蛋白及膜脂分子运动以及细胞表面电荷变化,结果表明,细胞膜表面蛋白分子侧向扩散速度减慢;膜脂分子流动性减慢,烃链有序性增强;细胞电泳速度加快。此等对阐明伴刀豆球蛋白A作为外源信息导致细胞膜分子动力学变化以及电荷改变有重要的生物学意义。     

外源性层粘连蛋白对腹水型肝癌细胞内微丝组装和细胞游动的作用

本文研究外源性层粘连蛋白与癌细胞膜上受体结合调节胞内肌动蛋白微丝组装与改变细胞游动之间的关系,以及影响膜上层粘连蛋白受体的侧向扩散运动及膜脂分子流动性变化之间的关系.用各种荧光技术,如荧光显微米,荧光漂白恢复技术和荧光流式细胞术得到了明显的证据.层粘连蛋白与肝癌细胞膜有结合,使分散的腹水肝癌细胞粘连聚集,并膜下周动蛋白微丝增加,当把癌细胞分开则细胞从原位游动很大距离.如不分离粘连的细胞可减少其脱落和转移.层粘连蛋白与受体结合则受体的侧向扩散系数减小,膜脂流动性降低,使膜上分子运动受影响,对癌的生长不利.     

伴刀豆球蛋白A和层粘连蛋白与膜受体结合下膜分子运动及微丝组装变化的比较

研究伴刀豆球蛋白A和层粘连蛋白分别与小鼠腹腔巨噬细胞膜受体结合下引起细胞膜分子运动的变化和对微丝组装的影响.结果表明,伴刀豆球蛋白A和层粘连蛋白作用下均导致膜表面蛋白分子的侧向扩散速率减慢,膜脂流动性降低,加快膜内微丝组装并使微丝含量增加.两配体作用下引起细胞上述反应有相似性.     

伴刀豆球蛋白A和层粘连蛋白与膜受体结合下膜分子运动及微丝组装变化的比较

研究伴刀豆球蛋白A和层粘连蛋白分别与小鼠腹腔巨噬细胞膜受体结合下引起细胞膜分子运动的变化和对微丝组装的影响.结果表明,伴刀豆球蛋白A和层粘连蛋白作用下均导致膜表面蛋白分子的侧向扩散速率减慢,膜脂流动性降低,加快膜内微丝组装并使微丝含量增加.两配体作用下引起细胞上述反应有相似性.     

层粘连蛋白受体单克隆抗体抗原性质的鉴定

层粘连蛋白受体在癌细胞转移中具有重要作用,ＬＮ－Ｒ的单克隆抗体对于癌转移的基础研究及诊治应用都具有重要意义,本文旨在确定来自人肺巨细胞癌细胞ＬＮ－Ｒ的一种单克隆抗体的抗原性质,经纯化的ＭｃＢ１能与完整细胞表面、细胞质膜提取物及纯化的ＬＮ－Ｒ制品特异性结合,实验证明经亲和层析纯化的ＬＮ－Ｒ制品中含有膜糖脂,用ＳＤＳ－ＰＡＧＥ及转移电泳将其所复合的膜脂去除后,仍具有与ＭｃＢ１结合的活性,表明此ＭｃＢ１     

细胞质膜受体与信息的跨膜传递

(一)膜受体的概念细胞膜受体是位于细胞膜上的糖蛋白、脂蛋白、糖脂蛋白等,它们能有选择地和细胞外环境中的信号物质相结合,并同时产生效应,使细胞的功能和物质代谢朝着一定方向变化,不同的受体接受不同的化学信号,引起不同的生化变化,一般把受体分成3部分:①分辨部(或鉴别器)是受体分子向着细胞外的部分,能识别外界的化学信号,狭义的受体即指分辨部而言。②转换部(或转换器)将分辨部所接受的信号通过蛋白构象的变化传给效应部。③效应部(或效应器)是向着细胞质的部分,它可引起细胞内部产     

SMMC-7721肝癌细胞67kD层粘连蛋白受体的分离纯化

分离纯化肝癌细胞的 6 7kD层粘连蛋白受体 (6 7LR) ,以便进一步研究 6 7LR的结构、功能及其在肝癌浸润、转移过程中的作用 .以SMMC 772 1肝癌细胞和L 0 2正常肝细胞为材料 ,采用13 1I标记的层粘连蛋白测定其与细胞的结合能力 ;亲和层析法分离纯化层粘连蛋白受体 ,用SDS PAGE、放射自显影及体外竞争结合实验进行鉴定 .在相同条件下SMMC 772 1肝癌细胞与层粘连蛋白特异结合量为 17 5 4± 0 4 9ng 10 5细胞 ,而L 0 2正常肝细胞与层粘连蛋白的特异结合量为 8 36± 0 4 8ng 10 5细胞 .经过亲和层析 ,从SMMC 772 1肝癌细胞和L 0 2正常肝细胞均可获得纯化受体 ,SDS PAGE显示为单一条带 ,分子量为 6 7kD ,放射自显影及体外竞争结合实验表明其具有较强的与层粘连蛋白结合的活性 .体外竞争结合实验表明 ,SMMC 772 1肝癌细胞层粘连蛋白受体 (772 1LnR)的抑制率可达到 96 2 7± 2 2 9% ,而L 0 2正常肝细胞层粘连蛋白受体 (L 0 2LnR)的抑制率为 4 8 71± 3 79% ,这说明 772 1LnR与层粘连蛋白的亲和力明显高于L 0 2LnR(P <0 0 0 1) .结果表明 ,与L 0 2肝细胞比较 ,SMMC 772 1肝癌细胞具有与层粘连蛋白较强结合能力的特异受体 ,并从肝癌细胞膜上分离纯化到与层粘连蛋白有较强亲和力的 6 7LR     

膜脂—膜蛋白的相互作用(下)

四、膜蛋白对膜脂的影响 1.膜蛋白与膜脂的结合膜蛋白按其不同类型可以多种方式与脂双层结合,现叙述如下: (1)外周蛋白的结合外周蛋白通过静电力(蛋白带电荷)或范德瓦力(蛋白不带电荷)与膜结合。在蛋白与脂的结合面处含有大量的水。 (a)静电结合细胞色素C是外周蛋白通过静电力与细胞膜表面结合的一个典型例子(图3 a)。细胞色素C整个分子具有可观的非     

肝癌细胞67kD层粘连蛋白受体N-糖链结构与功能的变化

探讨肝癌细胞与正常肝细胞 6 7kD层粘连蛋白受体 (6 7LR)N 糖链结构与功能的差异 .采用流式细胞术检测SMMC 772 1肝癌细胞和L 0 2正常肝细胞膜表面 6 7LR的表达 ,并分别从这 2株细胞分离纯化到高亲和力的 6 7LR ,利用凝集素结合分析其糖链结构 ,并用肽 N 糖苷酶水解N 糖链 ,观察糖链在与层粘连蛋白结合过程中的作用 .结果发现 ,L 0 2细胞膜表面 6 7LR表达的阳性率为5 5 3% ,而SMMC 772 1细胞为 34.7% ,这两株细胞 6 7LR与伴刀豆素 (ConA)的结合能力无显著差异 ,但SMMC 772 1细胞的 6 7LR与麦胚凝集素的结合能力明显高于L 0 2细胞的 6 7LR ,说明 2株细胞 6 7LR的糖链结构存在显著差异 .当N 糖链被切除后 ,SMMC 772 1细胞的 6 7LR与层粘连蛋白的结合能力明显下降 ,而L 0 2细胞则没有变化 .这些资料表明 ,SMMC 772 1肝癌细胞和L 0 2正常肝细胞与层粘连蛋白结合能力的差别 ,以及两株细胞的 6 7LR与层粘连蛋白结合能力的不同 ,很可能是由于这两株细胞的层粘连蛋白受体的N 糖链结构不同所引起     

激素作用原理与膜受体

激素作用原理基本上分为两大类:激素通过细胞内受体起作用和激素通过细胞膜受体起作用。本文介绍了近年来有关膜受体及其结合特性、膜受体信息传递的第二信使和G蛋白与信息传递等方面研究的进展。还指出,细胞内的信息传递是多途径的,不同激素与受体结合后所通过的信息传递机制不尽相同,而同一激素与其受体结合后可能通过不只一种细胞内的信息传递机制。这些复杂的关系是激素作用千变万化的基础。     

RENEWAL OF FATTY ACIDS IN THE MEMBRANES OF VISUAL CELL OUTER SEGMENTS

The renewal of fatty acids in the visual cells and pigment epithelium of the frog retina was studied by autoradiographic analysis of animals injected with tritiated palmitic, stearic, or arachidonic acids. Most of the radioactive material could be extracted from the retina with chloroform-methanol, indicating that the fatty acids had been esterified in lipids. Analysis of the extracts, after injection of [³H]palmitic acid, revealed that the radioactivity was predominantly in phospholipid. Palmitic acid was initially concentrated in the pigment epithelium, particularly in oil droplets which are storage sites for vitamin A esterified with fatty acid. The cytoplasm, but not the nucleus of these cells, was also heavily labeled. Radioactive fatty acid was bound immediately to the visual cell outer segment membranes, including detached rod membranes which had been phagocytized by the pigment epithelium. This is believed to be due to fatty acid exchange in phospholipid molecules already situated in the membranes. Gradually, the concentration of radioactive material in the visual cell outer segment membranes increased, apparently as a result of the addition of new phospholipid molecules, possibly augmented by the transfer from the pigment epithelium of esterified vitamin A. Injected fatty acid became particularly concentrated in new membranes which are continually assembled at the base of rod outer segments. This localized concentration was short-lived, apparently due to the rapid renewal of fatty acid. The results support the conclusion that rods renew the lipids of their outer segments by membrane replacement, whereas both rods and cones renew the membrane lipids by molecular replacement, including fatty acid exchange and replacement of phospholipid molecules in existing membranes.     

层粘连蛋白促进实体型瘤细胞粘着、铺展及~3H-TdR参人

 恶性肿瘤细胞或粘着于基膜以及在一定的基质上增殖对于肿瘤的浸润转移至关重要。层粘连蛋白(1aminin,LN)是基膜所特有的非胶原糖蛋白。我们研究了LN在肿瘤细胞粘着、铺展及增殖中的作用。通过测定粘着细胞所释放的LDH活性定量分析细胞粘着率。LN可显著增加体外培养的小鼠S180-V肉瘤及B16-MBK黑色素瘤细胞在玻璃及Ⅳ型胶原基质上粘着及铺展。纤维粘连蛋白(fibronectin,FN)亦有类似作用。而非糖蛋白(牛血清清蛋白)或其他糖蛋白(鸡卵清清蛋白或免疫球蛋白)均无类似作用。此外,LN或FN抗体可分别抑制细胞粘着于LN或FN。这些结果表明LN或FN促细胞粘着作用是专一性的。扫描电镜观察表明,粘着在LN敷盖的玻璃表面的瘤细胞呈多突扁平形、膜皱襞及微绒毛十分稀少,而粘着在裸露的玻璃表面者为球形,膜皱襞及微绒毛甚多。再者,~3H-TdR向粘着于LN基质上的瘤细胞群的参入量显著高于粘着在裸露玻璃面者。     

菜豆热激蛋白在生物膜上的定位

选用菜豆 Phaseolus vulgris L. 下胚轴 ,运用35S- Met标记放射自显影和二维电泳技术 ,研究热激蛋白 HSPs 的表达和在生物膜组分中的定位 .实验结果表明 ,盐溶蛋白中主要HSPs为 70 k D HSPs和小分子量 HSPs,而小分子量组 HSPs大量富集在质膜和液泡膜组分中 .     

THE LOCALIZATION OF Mg-Na-K-ACTIVATED ADENOSINE TRIPHOSPHATASE ON RED CELL GHOST MEMBRANES

The lead salt method introduced by Wachstein and Meisel (12) for the cytochemical demonstration of ATPase activity was modified and used to determine sites of activity on red cell ghost membranes. Preliminary studies showed that aldehyde fixation and standard concentrations of the capture reagent Pb(NO₃)₂ resulted in marked inhibition of the ATPase activity of these membranes. By lowering the concentration of Pb²⁺ and incubating unfixed red cell ghosts, over 50% of the total ATPase activity, which included an ouabain-sensitive, Na-K-activated component, could be demonstrated by quantitative biochemical assay. Cytochemical tests, carried out under the same conditions, gave a reaction product localized exclusively along the inner surfaces of the ghost membranes for both Mg-ATPase and Na-K-ATPase. These findings indicate that the ATPase activity of red cell ghosts results in the release of P_i on the inside of the ghost membrane at sites scattered over its inner aspect. There were no deposits of reaction product on the outer surface of the ghost membrane, hence no indication that upon ATP hydrolysis P_i is released outside the ghosts. Nor was there any clear difference in the localization of reaction product of Mg-ATPase as opposed to that of Na-K-ATPase.     

THE INTRACELLULAR MEMBRANES OF BLASTOMYCES DERMATITIDIS

Edwards , George A., and Mercedes R. Edwards . (Div. Labs, and Research, N.Y.S. Dept. of Health, Albany.) The intracellular membranes of Blastomyces dermatitidis. Amer. Jour. Bot. 47 (8): 622–632. Illus. 1960.—The yeast cells of Blastomyces dermatitidis have been studied in thin sections with the electron microscope. The cell is multinucleate, and the nuclei are frequently interconnected by their outer limiting membranes. The cell is bordered by a cell wall and the plasma membrane, which may be seen in direct continuity with the nuclear envelope. The cytoplasm contains numerous mitochondria, many profiles of the endoplasmic reticulum, and few multivesicular bodies. The membranes of all the constant cellular components are interconnected. Mitochondria appear to be formed from any of several membrane systems. The micromorphology of the cell suggests efficiency of communication and cytoplasmic mobility.     

THE LOCALIZATION OF SPECTRIN ON THE INNER SURFACE OF HUMAN RED BLOOD CELL MEMBRANES BY FERRITIN-CONJUGATED ANTIBODIES

Spectrin, a major protein constituent of mammalian red blood cell membrane preparations, has been localized on the inner surface of human red blood cell membranes by techniques that utilized specific ferritin-conjugated antibodies and fixation of membranes shortly after hemolysis so as to allow penetration of the ferritin-antibody labels. The labeling of spectrin was shown to be specific by the following criteria. (a) Nonhomologous ferritin-conjugated antibodies did not specifically bind to either membrane surface. (b) Blocking the membrane-bound spectrin with excess unconjugated antispectrin antibodies prevented ferritin-antibody labeling. (c) Removal of spectrin by treating the membrane preparation with a low ionic strength buffer containing ethylenediaminetetraacetate and β-mercaptoethanol prevented labeling by specific ferritin-conjugated antibodies.     

ELECTROMAGNETIC PERMEABILIZATION OF YEAST CELL MEMBRANES YIELDING ENHANCED PHOTODYNAMIC ACTION: A SYNERGISM

Electroporative drug delivery (electroloading of cells) takes place effectively by inserting electrodes mediating strong electric pulses. Another possibility is the disturbance of lipid bilayer structures by weak pulsating electromagnetically induced currents (PEMIC) produced by Helmholtz coils. In yeast cells (Saccharomyces cerevisiae) the enhancement of drug (sensitizer) transport through cell membranes by PEMIC results in an increased photodynamic cell killing. Depending on the magnetic flux, a window of 25% killing rate, caused by the photodynamic effect, could be observed at approximately 8 mT with a frequency of 50 Hz.     

THE EFFECTS OF ENUCLEATION ON THE CYTOPLASMIC MEMBRANES OF AMOEBA PROTEUS

The dependence of cytoplasmic membranes upon the nucleus was studied by examining enucleated amebae with the electron microscope at intervals up to 1 wk after enucleation. Amebae were cut into two approximately equal parts, and the fine structure of the enucleated portions was compared with that of the nucleated parts and starved whole cells which had been maintained under the same conditions. Golgi bodies were diminished in size 1 day after enucleation and were not detected in cells enucleated for more than 2 days. The endoplasmic reticulum of enucleated cells appeared to increase in amount and underwent changes in its morphology. The sparsely scattered short tubules of granular endoplasmic reticulum present in unmanipulated amebae from stock cultures were replaced in 1–3-day enucleates by long narrow cisternae. In 3–7-day enucleates, similar cisternae of granular endoplasmic reticulum encircled areas of cytoplasm partially or completely. It was estimated that in most cases hundreds of these areas encircled by two rough membranes were formed per enucleated cell. The number of ribosomes studding the surface of the endoplasmic reticulum decreased progressively with time after enucleation. In contrast, the membranes of nucleated parts and starved whole cells did not undergo these changes. The possible identification of membrane-encircled areas as cytolysomes and their mode of formation are considered. Implications of the observations regarding nuclear regulation of the form of the Golgi apparatus and the endoplasmic reticulum are discussed.     

THE FINE STRUCTURE AND IMMUNOLOGICAL LABELING OF THE ACHROMATIC MITOTIC APPARATUS AFTER DISRUPTION OF CELL MEMBRANES

After treatment of HeLa and L cells with vinblastine sulfate the material of microtubules (tubulin) was reorganized into (a) large paracrystals (PC) of tightly packed tubules; (b) smaller aggregates of tubules with greater diameter whose walls are constituted from well defined, helically arranged morphological subunits; and (c) microtubules associated with helices of polyribosomes of uniform size. All of these structures survived disruption of cellular membranes by means of a nonionic detergent. Following a thorough stripping of membranes there remained a subcellular fraction sedimenting at 1,500 g for 15 min, in which were contained nuclei, centrioles, and the above mentioned microtubular elements, maintained as a complex of organelles by an interconnecting network of 80 Å microfibrils. As a result of membrane disruption it was possible to localize precisely in the electron microscope the binding of ferritin antibody conjugates. Specific labeling at the surface of PC and microtubule aggregates could be demonstrated. This result was substantiated by means of the immunoperoxidase method of labeling the PC. A concentrated deposit of ferritin was also found in the vicinity of centrioles and related structures, the annuli of the nuclear pore complex and the annulate lamellae. However, the specificity of the label on these organelles remains questionable because ferritin, albeit in lower concentration, was also present on them in control preparations reacted with preimmune sera.