两相分配法制备甘草根质膜及其纯度鉴定

以甘草（Glycyrrhiza uralensis F．）根为材料,利用差速离心法获取细胞微粒体,通过水溶性聚合物Dextran T-500和PEG4000所构成的两相分配体系制备质膜．标志酶鉴定结果表明,上相中富含质膜,内膜污染较少,质膜纯度较高;下相中富含其它内膜．Western blot分析结果表明,在对应于水通道蛋白分子量大小的位置存在特异条带．实验证明,PEG-Dextran两相分配体系可获得较高纯度的甘草根密实正向型质膜囊泡．     

牛精子体外获能后的超微结构变化

用肝素 咖啡因作获能剂对牛冷冻 解冻精子进行体外获能处理 ,体外培养 1h ,在透射电镜下观察精子获能后的超微结构变化。结果表明 :(1)牛精子顶体内膜和外膜均为双层膜结构 ;(2 )精子获能后顶体囊泡化的形成有多种形式 :质膜与顶体外膜融合 ,双层顶体外膜自身融合 ,顶体外膜内陷或外突打褶 ,双层顶体内膜自身融合 ,顶体内膜外突打褶形成囊泡 ;而且在同一精子的顶体反应中可同时出现多种囊泡化形式 ;(3)精子获能后 ,头部无顶体覆盖的核后区核膜膨胀 ,并与膨胀的质膜融在一起 ;(4 )精子获能后 ,尾部质膜膨胀 ,且主段质膜比中段质膜膨胀明显 ,这可能与超激活运动有关。     

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.     

花生种子活力、ATPase及H~ 分泌初探

线粒体结合ATPase及质膜和液泡膜结合ATPase活性随花生种子吸胀的起始逐渐增加,酶活性与种子活力有正相关性。以萌发一周的花生下胚轴为实验材料,线粒体ATPase,质膜ATPase和液泡膜ATPase的反应最适pH均为9.在pH 6～10范围内随pH的升高H~ 分泌增多。膜ATP酶为二环已烷亚胺抑制,而为KCl激活;NaN_3特异地抑制线粒体ATP酶,而NaVO_3专一地抑制质膜ATP酶,但两者对液泡膜ATP酶均无效。Mg~( )、K~ 激活ATPase,也刺激H~ 分泌。质膜ATPase抑制剂NaVO_3对H~ 9分泌作用不大,而线粒体ATPase抑制剂NaN_3及解偶联剂DNP均显著抑制H~ 分泌。CoⅠ刺激H~ 分泌而C_oⅡ抑制H~ 分泌。似乎线粒体ATP产量对H~ 分泌更重要。     

人老化红细胞囊泡的分离及其特性研究

本文报道了人红细胞在体外老化(37℃。48h)过程中会自然形成并释放的囊泡为不均一的群体,经 Dextran-T70不连续密度梯度离心后可明显分成三种组分。囊泡主要集中在组分Ⅱ和Ⅲ中,而组分Ⅰ除了囊泡外还含有膜和膜碎片。这三种囊泡组分在流动性、封闭度等性质上有明显差异。值得引起注意的是,囊泡的荧光光谱与正常红细胞膜相比变化较大,红移22nm 左右,这提示了囊泡膜脂的结构和组分与红细胞有较大差异。     

人老化红细胞囊泡的分离及其特性研究

本文报道了人红细胞在体外老化(37℃.48h)过程中会自然形成并释放的囊泡为不均一的群体,经Dextran-T70不连续密度梯度离心后可明显分成三种组分.囊泡主要集中在组分Ⅱ和Ⅲ中,而组分Ⅰ除了囊泡外还含有膜和膜碎片.这三种囊泡组分在流动性、封闭度等性质上有明显差异.值得引起注意的是,囊泡的荧光光谱与正常红细胞膜相比变化较大,红移22nm左右,这提示了囊泡膜脂的结构和组分与红细胞有较大差异.     

自絮凝酵母细胞膜脂肪酸影响乙醇激活 ATP 酶

首次揭示酵母菌细胞膜磷脂脂肪酸组成对影响质膜ATP酶对乙醇刺激的响应.实验结果表明,细胞膜磷脂脂肪酸组成特点,对生长于未添加乙醇条件下的自絮凝酵母的质膜ATP酶活性没有影响,但却明显影响生长于添加乙醇(体积分数为0.01～0.10)的菌体质膜ATP酶对乙醇激活的敏感性.预培养于添加0.6 mmol·L-1棕榈酸、亚油酸或亚麻酸条件下,菌体质膜ATP酶的最大激活水平分别为各自酶的基态水平(未激活)的3.6,1.5和1.2倍,而对照组(预培养于未添加脂肪酸条件下的菌体)的相应值为2.3倍.酶激活后,米氏常数Km、最适pH和对质膜ATP酶特异性抑制剂钒酸钠的敏感性等性质不变,但最大反应速度Vmax明显增加.实验结果还表明,细胞膜磷脂脂肪酸组成特点对提高菌体的耐乙醇能力越有利,其质膜ATP酶被乙醇激活的幅度越大,说明菌体耐乙醇能力的提高,与其质膜ATP酶对乙醇激活的敏感性的增加密切相关.     

植物质膜质子泵H~+——ATPase的活性调节及功能研究进展

植物细胞质膜H~+—ATPase(EC3.6.1.35)是质膜上的插入蛋白,具有利用水解ATP产生的能量,将细胞质膜内侧的H~+泵到质膜外侧的特征,简称为质子泵。它对植物细胞营养物质的吸收、细胞生长、气孔运动和渗透调节等生理过程有重要的调节作用,是植物生命活动的“主宰酶”。自从Hodges等在离体质膜中证实ATPase活性以来,质膜H~+—ATPase的研究受到了广泛的重视。特别是近20年来,随着表面活性剂的应用和分离纯化技术的不断改进,获得了高纯度的酶制剂。本文着重阐述了植物质膜质子泵H~+——ATPase在活性调节及功能方面的研究进展。     

γ-聚谷氨酸合成酶系PgsBCA结构的生物信息学分析

利用ProtParam、TopPred、PredictProtein、PSORT-B Prediction、SWISS-MODEL等软件分别分析蛋白质的理化性质、跨膜区、二级结构、亚细胞定位、三维结构.结果显示:PgsB是亲水不稳定蛋白,通过豆蔻酰锚钩锚定于质膜上,催化作用需与ATP结合提供能量;PgsC是疏水稳定蛋白,通过4个跨膜区和多个豆蔻酰锚钩定位于质膜,具有酰胺化位点;PgsA是亲水稳定蛋白,通过N端一个跨膜区和豆蔻酰锚钩结合于质膜,具有多种磷酸化位点.说明γ-聚谷氨酸(Polyγ-glutamic acid,γ-PGA)合成酶系3个组分蛋白形成复合物定位于质膜上,其中PgsB在胞内催化γ-PGA合成,PgsC固定于质膜,连接PgsB和PgsA组分,PgsA在胞外负责γ-PGA的运输.通过对γ-PGA合成酶系各组分蛋白结构的分析,为日后在谷氨酸高产菌株中的表达奠定了基础.     

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   

CaMBP—10调节生长素应答反应机理初探

前期研究发现ＣａＭＢＰ－１０参与植物细胞对生长素应答反应的调节，但调节机理有待阐明，本文表明ＣａＭＢＰ－１０是由于影响质膜Ｈ－ＡＴＰａｓｅ活性，进而影响了质子外泌和细胞伸长，并进一步证明，它对质膜Ｈ－ＡＴＰａｓｅ活性的调节是通过介导该酶磷酸化而实现的最终调节了细胞对激素的应答。     

大气颗粒物浓度自动监测仪器的研制及性能比对测试

颗粒物污染已成为我国的首要大气污染物,颗粒物污染的来源复杂、危害较大,简要介绍了目前国内外颗粒物监测方法和仪器设备的研究情况,以及所研制的、基于滤膜 称重法的、可同时测量PM₁₀和PM_2.5的DJ3-1型六工位自循环式大气颗粒物浓度自动监测仪。利用具有无限远光学系统的微分干涉相衬显微技术对该系统所采集到的颗粒物进行了显微观察,得到粗颗粒和细颗粒的粒径分布,与Andersen公司240型双通道采样器的切割性能十分 接近。与国内外部分监测仪器进行了同步的颗粒物质量浓度监测的比对测试,结果表明其性能已基本达到国外同类监测仪器的水平。     

Direct measurement of exocytosis and calcium currents in single vertebrate nerve terminals

The release of neurohormone is widely thought to be exocytotic, involving Ca2(+)-dependent fusion of secretory vesicles with the plasma membrane. The inaccessibility of most nerve ending has so far hampered direct time-resolved measurements of neuronal exocytosis in response to brief depolarization. By using 'whole-terminal' patch-clamp and circuit-analysis techniques to measure membrane capacitance, we have now monitored changes in the surface membrane area of individual nerve terminals isolated from the mammalian neurohypophysis. A single depolarizing pulse leading to Ca2+ entry through voltage-gated calcium channels, rapidly and reproducibly increases the membrane area by an amount corresponding to the fusion of 1-100 secretory vesicles. The magnitude of the capacitance increase depends not only on Ca2+ entry and buffering, but also on the pattern of stimulation revealing facilitation, fatigue and recovery of the release process.     

Effect of fatty acids on poly-amine contents and Na+/H+antiport activity in PM vesi-cles isolated from roots ofbarley under salt stress

With 200 mmol/L NaCI treatment on barleycultivar "Jian 4" (Hordeum vulgare L. cv. J4) seedlings for6 d, the contents of covalently and noncovalently conjugatedpolyamines (PAs) and activities of H+-ATPase in plasmamembrane (PM) vesicles isolated from the roots decreasedremarkably. Moreover, the activity of Na+/H+ antiport wasdetected first in PM vesicles. The results showed that thedecrease in the contents of membrane phospholipid, nonco-valently conjugated PAs and activity of H+-ATPase caused byNaCl could be restored partially by application of 1 mmol/Lstearic acid (C16:0) and linoleic acid (C18:2), and C18:2 wasmore effective than C16:0. In addition, a reduction in the con-tents of covalently conjugated PAs was only reversed par-tially in the presence of C18:2. Furthermore, Na+/H+ antiportactivity was strengthened by exogenous C16:0 and C18:2, andC18a was more effective than C16:0. The correlative analysissuggested that, after application of C16:0 and C18:2 under saltstress, there was a significant positive correlation existingamong phospholipid content, noncovalently conjugated PAlevels, H+-ATPase activities and Na+/H+ antiport activities,indicating that one of the mitigative mechanisms of exoge-nous fatty acids on salt injury was to improve membranephospholipid and PA contents, leading to an enhance inmembrane integrity and a change in charge status of PMvesicles, so the activity of membrane-associated enzymeH+-ATPase was increased and synthesis of Na+/H+ antiportprotein was activated.     

Effect of fatty acids on polyamine contents and Na+/H+ antiport activity in PM vesicles isolated from roots of barley under salt stress

With 200 mmol/L NaCl treatment on barley cultivar “Jian 4” (Hordeum vulgare L. cv. J4) seedlings for 6 d, the contents of covalently and noncovalently conjugated polyamines (PAs) and activities of H⁺-ATPase in plasma membrane (PM) vesicles isolated from the roots decreased remarkably. Moreover, the activity of Na⁺/H⁺ antiport was detected first in PM vesicles. The results showed that the decrease in the contents of membrane phospholipid, noncovalently conjugated PAs and activity of H⁺-ATPase caused by NaCl could be restored partially by application of 1 mmol/L stearie acid (C_16:0) and linoleic acid (C_18:2), and C_18:2 was more effective than C_16:0 In addition, a reduction in the contents of covalently conjugated PAs was only reversed partially in the presence of C_18:2 Furthermore, Na⁺/H⁺ antiport activity was strengthened by exogenous C_16:0 and C_18:2 and C_18:2 was more effective than C_16:0. The correlative analysis suggested that, after application of C_16:0 and C_18:2 under salt stress, there was a significant positive correlation existing among phospholipid content, noncovalently conjugated PA levels, H⁺-ATPase activities and Na⁺/H⁺ antiport activities, indicating that one of the mitigative mechanisms of exogenous fatty acids on salt injury was to improve membrane phospholipid and PA contents, leading to an enhance in membrane integrity and a change in charge status of PM vesicles, so the activity of membrane-associated enzyme H⁺-ATPase was increased and synthesis of Na⁺/H⁺ antiport protein was activated.     

Red cell sodium fluxes catalysed by the sodium pump in the absence of K+ and ADP

In the absence of extracellular Na+ or K+, the sodium pump catalyses an ouabain-sensitive "uncoupled" Na+ efflux1-4. With red cell ghosts Glynn and Karlish5 showed that this Na+ efflux is accompanied by ATP hydrolysis and that extracellular sodium ions, at low concentrations, inhibit this efflux as well as the associated ATP hydrolysis. At higher concentrations, extracellular sodium ions restore the hydrolysis of ATP3,6 but it is not known whether there is an associated increase in Na+ efflux and, perhaps, an influx. To answer this question we have used inside-out red cell membrane vesicles which are specially suitable for controlling the composition of the medium at the two membrane surfaces while measuring 22Na+ fluxes in both directions. We report here that the sodium pump can operate in a mode in which influx and efflux of sodium are associated with ATP hydrolysis. This mode is different from the Na-Na exchange described by Garrahan and Glynn7, and Glynn and Hoffman8, which requires ADP as well as ATP9 and is probably associated with ADP-ATP exchage rather than ATP hydrolysis10,11.     

STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis

Synaptic transmission is mediated by neurotransmitters that are stored in synaptic vesicles and released by exocytosis upon activation. The vesicle membrane is then retrieved by endocytosis, and synaptic vesicles are regenerated and re-filled with neurotransmitter. Although many aspects of vesicle recycling are understood, the fate of the vesicles after fusion is still unclear. Do their components diffuse on the plasma membrane, or do they remain together? This question has been difficult to answer because synaptic vesicles are too small (approximately 40 nm in diameter) and too densely packed to be resolved by available fluorescence microscopes. Here we use stimulated emission depletion (STED) to reduce the focal spot area by about an order of magnitude below the diffraction limit, thereby resolving individual vesicles in the synapse. We show that synaptotagmin I, a protein resident in the vesicle membrane, remains clustered in isolated patches on the presynaptic membrane regardless of whether the nerve terminals are mildly active or intensely stimulated. This suggests that at least some vesicle constituents remain together during recycling. Our study also demonstrates that questions involving cellular structures with dimensions of a few tens of nanometres can be resolved with conventional far-field optics and visible light.     

PM10浓度的时间序列模型及预测.doc

利用统计软件Eviews3.1对四川省宜宾市2004年7月1日到8月31日的PM10浓度时间序列数据进行了分析,建立时间序列模型,并对PM10浓度进行预测。结果表明,预测模型精度较高,残差最大值小于10%,预测结果与实际状况基本相符。     

Exposure of an antigen of chromaffin granules on cell surface during exocytosis

The synthesis rate of the membrane proteins of the catecholamine-storing vesicles (chromaffin granules) of the adrenal medulla is lower than that of the secretory proteins of the contents. Based on these results we proposed that after exocytosis the membranes of chromaffin granules are retrieved and are re-used for several secretion cycles (see also ref. 4). This concept of re-use of granule membranes has been further strengthened by the finding that exogenous markers which are taken up by secretory cells during stimulation can be traced to the Golgi region and to immature secretory organelles. However, one basic question remains: are the membranes of secretory organelles specifically and completely removed from the plasma membrane and if so, how fast is this process? By using an antiserum against a membrane glycoprotein of chromaffin granules we have now obtained quantitative data which demonstrate that during exocytosis this antigen becomes exposed on the cell surface and disappears again to a large degree within 30 min.     

上海市2002年以来空气中可吸入颗粒物浓度变化特征

用时间序列的时域分析和频域分析法，探讨2002～2004年空气中可吸入颗粒物（PMIO）浓度的频谱和趋势。结果表明PMIO的浓度呈周期性变化，而且是由三个周期成分构成；即每年有一次周期性升高的慢周期变化，每周有变化一次的周期和每周变化二次的周期。其浓度呈波动下降的趋势；在2002年较高，2003有一定下降，而在2004年又有呈上升波动的趋势，但其值仍远低于2002年的水平，由此可以说明PM10浓度已得到了较好的控制。