外源胆固醇对水稻根端线粒体膜流动性的影响

应用荧光探剂 1,6-二苯基-1,3,5-己三烯(DPH)观察外源胆固醇对水稻极端线粒体膜流动性的影响。结果表明,无论外源胆固醇通过水培根系吸收或是直接添加给予水稻根端线粒体,都能使DPH与线粒体结合后的荧光强度减弱,使荧光偏振度和微粘度降低,增加水稻根端线粒体膜流动性。     

Increasing Age Alters Transbilayer Fluidity and Cholesterol Asymmetry in Synaptic Plasma Membranes of Mice

Abstract: Previous studies examining age differences in membrane fluidity and cholesterol content have reported on the average or total change in membrane structure, respectively. However, a membrane consists of an exofacial leaflet and a cytofacial leaflet that differ in fluidity and cholesterol distribution. The purpose of the present experiments was to determine fluidity and cholesterol distribution of the exofacial and cytofacial leaflets of brain synaptic plasma membranes (SPMs) from 3–4-, 14–15-, and 24–25-month-old C57BL/6NNIA mice by using trinitrobenzenesulfonic acid (TNBS)-quenching techniques and fluorescent probes. The exofacial leaflet of SPMs from young mice was significantly more fluid compared with the cytofacial leaflet. The large difference in fluidity between the two leaflets was abolished in SPMs of the oldest age group. Total SPM cholesterol and the cholesterol-to-phospholipid molar ratio did not differ among the three different age groups of mice. However, considerable differences were observed in the distribution of cholesterol in the two SPM leaflets. The exofacial leaflet contained substantially less cholesterol than did the cytofacial leaflet (13 vs. 87%, respectively) in SPMs of young mice. This asymmetric distribution of cholesterol was significantly modified with increasing age. There was an approximately twofold increase in exofacial leaflet cholesterol in the oldest group compared with the youngest age group. Transbilayer fluidity and cholesterol asymmetry were altered in SPMs of older mice. This approach is a new and different way of viewing how aging modifies membrane structure. Age differences in SPM leaflet structure may be an important factor regulating activity of certain membrane proteins.     

外源胆固醇对水稻幼苗膜脂组成及抗冷力的影响

分析了水稻幼苗低温胁迫前后膜脂和膜脂脂肪酸含量变化。结果表明,经胆固醇处理的幼苗叶和根细胞膜脂中LPC、PS和PG含量比对照下降少,PA含量增加也较少。胆固醇处理的幼苗叶和根棕榈酸(16:0)增加量和亚麻酸(18:3)与IUFA减少量均明显比对照少。试验结果证明,水稻幼苗叶片和根系的抗冷力与PA含量和脂肪酸不饱和程度变化有密切关系。外源胆固醇处理水稻幼苗能阻止低温对膜脂的破坏作用,提高幼苗抗低温胁迫能力。     

巴氏碳球C60光激发对红细胞膜流动性的影响

巴氏碳球Ｃ_（６０）光激发对红细胞膜流动性的影响黄文栋,钱凯先,唐海琼（浙江大学生物科学与技术系,杭州３１００２７）李文铸（渐江大学物理系,杭州３１００２７）关键词Ｃ_（６０）;光激发;红细胞膜;荧光偏振;膜流动性Ｃ６０是Ｋｒｏｔｏ等人［１］于１９８５...     

The composition and fluidity of adipocyte membranes prepared from young and adult rats

Membranes from adipocytes of adult and young rats have been compared. Phospholipid fatty acids from adult rats were more saturated than those from young rats. This difference was associated with a decreased fluidity in the membranes of the adult rats, which was inferred from measurements of fluorescence polarisation of the fluorescent probe, 1,6-diphenylhexa-1,3,5-triene.     

Characterization of changes in the viscosity of lipid membranes with the molecular rotor FCVJ

Membrane viscosity is a key parameter in cell physiology, cell function, and cell signaling. The most common methods to measure changes in membrane viscosity are fluorescence recovery after photobleaching (FRAP) and fluorescence anisotropy. Recent interest in a group of viscosity sensitive fluorophores, termed molecular rotors, led to the development of the highly membrane-compatible (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester (FCVJ). The purpose of this study is to examine the fluorescent behavior of FCVJ in model membranes exposed to various agents of known influence on membrane viscosity, such as alcohols, dimethyl sulfoxide (DMSO), cyclohexane, cholesterol, and nimesulide. The influence of key agents (propanol and cholesterol) was also examined using FRAP, and backcalculated viscosity change from FCVJ and FRAP was correlated. A decrease of FCVJ emission was found with alcohol treatment (with a strong dependency on the chain length and concentration), DMSO, and cyclohexane, whereas cholesterol and nimesulide led to increased FCVJ emission. With the exception of nimesulide, FCVJ intensity changes were consistent with expected changes in membrane viscosity. A comparison of viscosity changes computed from FRAP and FCVJ led to a very good correlation between the two experimental methods. Since molecular rotors, including FCVJ, allow for extremely easy experimental methods, fast response time, and high spatial resolution, this study indicates that FCVJ may be used to quantitatively determine viscosity changes in phospholipid bilayers.     

Transbilayer Distribution of Cholesterol Is Modified in Brain Synaptic Plasma Membranes of Knockout Mice Deficient in the Low-Density Lipoprotein Receptor, Apolipoprotein E, or Both Proteins

Abstract: Both apolipoprotein E (apoE) and the low-density lipoprotein (LDL) receptor are present in brain; however, little is known regarding the function of these proteins in brain, in particular with respect to brain cholesterol. The role of apoE and the LDL receptor in modulating the transbilayer or asymmetric distribution of cholesterol in the exofacial and cytofacial leaflets of synaptic plasma membranes (SPMs) was examined in mutant mice deficient in apoE, the LDL receptor, or both proteins by using the fluorescent sterol dehydroergosterol and fluorescent quenching procedures. Fluidity of the exofacial and cytofacial leaflets was also measured. Cholesterol asymmetry of SPMs was altered in the mutant mice, with the largest effect observed in the LDL receptor-deficient mice. There was an approximately twofold increase in the percent distribution of cholesterol in the exofacial leaflet of the LDL receptor-deficient mice (32%) compared with C57BL/6J mice (15%). Mice deficient in apoE or both proteins also showed a significantly higher percent distribution of cholesterol (23 and 26%, respectively) in the exofacial leaflet compared with the C57BL/6J mice. Although the percent distribution of cholesterol was highest in the exofacial leaflet of the LDL receptor-deficient mice, fluidity of the exofacial leaflet of that group was significantly lower. However, the cholesterol-to-phospholipid ratio of SPMs of the LDL receptor-deficient mice was significantly lower, and this difference was largely the result of a significant increase in the total amount of SPM phospholipid. This study demonstrates for the first time that SPM lipid structure is altered in mice deficient in apoE or the LDL receptor. Although the mechanism that maintains the asymmetric distribution of cholesterol in plasma membranes is not well understood, data of the present experiments indicate that both apoE and the LDL receptor are involved in maintaining the transbilayer distribution of cholesterol.     

Cholesterol is required for the formation of regulated and constitutive secretory vesicles from the trans-Golgi network

We studied the role of cholesterol in regulated protein secretion in neuroendocrine cells by manipulating the cholesterol content of AtT-20 cells. Depletion of cellular cholesterol levels caused a reversible block of immature secretory granule biogenesis at the level of the trans -Golgi-network, whereas increased cholesterol levels promoted immature secretory granule formation. Cholesterol depletion also blocked the formation of constitutive secretory vesicles, but did not inhibit the transport between the endoplasmic reticulum and the Golgi complex. Our results indicate that the assembly of cholesterol-based lipid microdomains is required for the biogenesis of both regulated and constitutive secretory vesicles from the trans -Golgi-network in neuroendocrine cells.     

胆固醇对金属离子磷脂酰甘油单分子膜成膜的影响

目的：本文主要研究不同条件胆固醇（Cholesterol,简称Chol）和金属离子（钾、镁离子）对磷脂酰甘油（Phosphatidylglycerols,简称PG）相互作用后形成的单分子膜的影响。方法：首先以金属离子作为亚相,研究胆固醇的量对磷脂酰甘油单分子膜的影响;其次加入同等量的胆固醇量,亚相为不同金属离子时,对磷脂酰甘油单分子膜的影响,最后分析磷脂酰甘油LB膜的π-A曲线,即曲线外扩、相变点、膜压等等变化特征。结果：随着胆固醇的增多,金属离子磷脂酰甘油单分子膜形成π-A曲线变化逐渐明显;当加入同等量的胆固醇时,随着金属离子价态的逐渐增高,磷脂酰甘油单分子膜形成的π-A曲线的变化也逐渐明显。结论：其一：胆固醇对金属离子磷脂酰甘油单分子膜成膜质量是有影响的。其二,金属离子对胆固醇与磷脂酰甘油混合形成的单分子膜同样也是有的影响。     

Cholesterol inhibits the lytic activity of melittin in erythrocytes

Although cell lysis by the hemolytic peptide, melittin, has been extensively studied, the role of specific lipids of the erythrocyte membrane on melittin-induced hemolysis remains unexplored. In this report, we have explored the modulatory role of cholesterol on the hemolytic activity of melittin by specifically depleting cholesterol from rat erythrocytes using methyl-beta-cyclodextrin (MbetaCD). Our results show that the hemolytic activity of melittin is increased by approximately 3-fold upon depletion of erythrocyte membrane cholesterol by approximately 55% without any appreciable loss of phospholipids. This result constitutes the first report demonstrating that the presence of cholesterol inhibits the lytic activity of melittin in its natural target membrane, i.e., the erythrocyte membrane. These results are relevant in understanding the role of cholesterol in the mechanism of action of melittin in the erythrocyte membrane.     

外源NO对铝毒下水稻根系生长和抗氧化系统的影响

以水稻品种‘浙辐802’为材料,采用水培法研究铝毒下外源NO对幼苗根系生长、活性氧产生和抗氧化酶活性的影响,探讨外源NO提高水稻耐铝性的生理生化机制。结果显示:(1)0.05mmol/L Al显著抑制水稻根系生长,促使根尖Al、胼胝质、过氧化氢(H2O2)和超氧阴离子自由基(O-·2)含量显著增加;而外源0.1mmol/L的NO供体亚硝基铁氰化钠(sodium nitroprusside,SNP)预处理能使铝毒下水稻幼苗根相对伸长率及根尖NO含量分别增加34.96%和12.86%,根尖Al和相对胼胝质含量分别下降83.04%和31.93%,表明NO可部分缓解铝毒害,且这种作用与内源NO含量变化有关。(2)外源NO同时使铝毒下水稻幼苗根尖H2O2和O-·2含量分别下降15.43%和12.93%,使超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性显著上升,且外源NO的该种作用可以被0.075mmol/L NO清除剂(carboxy-PTIO,cPTIO)所逆转。研究表明,外源NO在调节活性氧代谢以维持细胞膜结构稳定,进而有效减轻Al对水稻根系的损伤上起着重要作用。     

The influence of fatty acids on model cholesterol/phospholipid membranes

The aim of this work was to verify the influence of the saturated (SFA) (stearic acid) and the unsaturated (UFA) (oleic and alpha-linolenic) fatty acids on model cholesterol/phospholipid membranes. The experiments were based on the Langmuir monolayer technique. Cholesterol and phospholipid were mixed in the molar ratio that corresponds to the proportion of these lipids in the majority of natural human membranes. Into the binary cholesterol/phospholipid monolayers, various amounts of fatty acids were incorporated. Our investigations were based on the analysis of the interactions between molecules in ternary (cholesterol/phospholipids/fatty acid) mixtures, however, also binary (cholesterol/fatty acid and phospholipids/fatty acid) mixed system were examined. It was concluded that the influence of the fatty acids on model cholesterol/phospholipid membrane is closely connected with the shape of the fatty acid molecule, resulting from the saturation degree of the hydrocarbon chain. It was found that the saturated fatty acid makes the model membrane more rigid, while the presence of unsaturated fatty acid increases its fluidity. The increasing amount of stearic acid gradually destabilizes model membrane, however, this effect is the weakest at low content of SFA in the mixed monolayer. Unsaturated fatty acids in a small proportion make the membrane thermodynamically more stable, while higher content of UFA decreases membrane stability. This explains low proportion of the free fatty acids to other lipids in natural membrane.     

Cholesterol sulphate sulphohydrolase from human placenta microsomes--purification and properties of the dephosphorylated form of enzyme

The procedure for purification of cholesterol sulphate sulphohydrolase (ChS-ase) from human placenta microsomes was elaborated. The highy purified enzyme preparation (specific activity 2000 nmol×min⁻¹×mg protein⁻¹) exhibited optimal activity at pH 9.0. The K_m value was established to be 1.5±0.85×10⁻⁵ M. The high molecular weight form (200 kDa) and the low molecular weight form (20 kDa) of the enzyme were separated. The interconversion of the high molecular weight variant into the low one occurs under the influence of dephosphorylation. Both forms exhibited typical Michaelis–Menten saturation kinetics. The effect of different compounds on the enzyme activity was tested.     

水稻根质膜蛋白质组双向电泳水化液的优化

以水稻(Oryza sativa L.)苗期幼嫩根尖作为材料,利用葡聚糖-聚乙二醇两相分配法纯化得到纯度达90%的质膜组分,使用4种不同的水化液溶解质膜蛋白,进行IEF/SDS-PAGE双向电泳和MALDI-TOF/TOF质谱分析.结果显示,4种水化液中,以7 mol/L Urea2、mol/L Thiourea、4%CHAPS、20 mmol/L DTE、1%ASB14的条件对膜蛋白的溶解效果和双向电泳分离效果最好;16个被鉴定蛋白中有9个为质膜相关蛋白,5个为未知蛋白,来自其它细胞器的蛋白仅有2个.研究表明,在常用水化液中添加磺基甘氨酸三甲内盐ASB14有利于植物细胞质膜蛋白质组的分析,并且该优化条件下的双向电泳适合分离水稻质膜中亲水性相对较高的膜附着蛋白.     

qPCR array检测高糖对胆固醇合成基因表达的影响

目的:高血糖易引起胆固醇在体内积聚,增加糖尿病合并动脉粥样硬化性心血管疾病的患病风险。本文通过建立稳定的实时定量PCR芯片(Real-time quantitative polymerasechain reaction array,qPCR array)检测方案,研究高糖对小鼠肝癌细胞Hepa1-6胆固醇合成基因表达的影响,探讨胆固醇合成基因在糖尿病大血管并发症发展中的作用机制。方法:以不同浓度葡萄糖(5、15、30mmo/L)和不同时间(0、6、12、18、24 h),刺激肝癌细胞Hepa1-6,利用qPCR array检测其胆固醇合成基因的表达差异。结果:与5mmol/L相比,高糖组(15、30 mmo/L)处理细胞18 h后,胆固醇合成基因CYP51、EBP、NSDHL、SQLE、FDFT1和PMVK的表达上调(P0.05),呈现剂量依赖性。与0 h相比,15 mmol/L高糖处理细胞12 h,CYP51、EBP和SQLE mRNA表达量上调(P0.01)。至24 h,CYP51、EBP降至0 h水平,而SQLE的表达量继续增加;NSDHL在12 h表达无差异,至18 h表达量发生上调(P0.05)。结论:该qPCR array检测方案能特异性检测胆固醇合成基因的表达量。高糖能够促进胆固醇合成基因的表达,使细胞内胆固醇积聚,这可能是糖尿病患者容易发生动脉粥样硬化的原因。这提示我们将胆固醇合成基因作为药物靶点可能延缓糖尿病动脉粥样硬化进展。     

脱落酸对绿豆下胚轴不定根发生的影响

以10^-4 mol/L脱落酸(ABA)处理绿豆种子24 h,在幼苗下胚轴长6 cm时,切除根部作为插条,研究ABA对插条不定根发生及插条基部细胞周期时相的影响。结果表明,ABA可促进下胚轴插条不定根发生,增加生根数和生根范围;ABA提高插条基部细胞色氨酸转氨酶、吲哚丙酮酸脱羧酶和吲哚乙醛脱氢酶的比活性,增加吲哚乙酸含量,同时进入细胞周期S期的基部细胞数目增加,促进DNA合成,有利于不定根的发生。     

Photochemical changes of fluorescent probes in membranes and their effect on the observed fluorescence anisotropy values

The fluorescence intensity of diphenylhexatriene (DPH) and of trimethylammonium-diphenylhexatriene (TMA-DPH) is measured when these probes are embedded in vesicles of dipalmitoyl- and dioleoylphosphatidylcholine (DPPC and DOPC), in mixtures of these vesicles as well as in vesicles of the mixed phospholipids, in trout intestinal brush border membranes and in mitoplasts of rat liver cells. The intensity in DOPC vesicles is found to be significantly higher than in DPPC vesicles. When these systems are irradiated with strong ultraviolet light radiation, a decrease in the fluorescence intensity is observed; this effect is much stronger in DOPC than in DPPC vesicles. The fluorescence anisotropy values in the mixture of vesicles as well as in the membranes show an initial increase with irradiation which is followed by a significant decrease. A transfer of DPH molecules between DPPC and DOPC vesicles is observed. For TMA-DPH this transfer takes place only from DPPC to DOPC vesicles, but not vice-versa. These results are related to intensity and anisotropy measurements of these probes in cell cultures.     

氧化修饰HDL对培养人主动脉平滑肌细胞胆固醇流出的影响

大量研究显示,高密度脂蛋白(highdensitylipoprotein,HDL)具有抗动脉粥样硬化(atherosclerosis,AS)作用.这是由于HDL能够促进外周组织如血管壁内皮细胞、平滑肌细胞(smoothmusclecell,SMC)及巨噬细胞储集的胆固醇流出,并将其转移到肝脏通过胆汁分泌而排出体外[1],这一过程...     

Effect of Ca2+ on Structure and Fluidity of Microvillus Membranes of Human Placenta

This investigation shows the effect of a Ca²⁺ addition on the structural and physicochemical properties of microvillus plasma membranes obtained from human placenta. Ca²⁺ addition induces an increase in microviscosity, as shown by the increase of order parameter and rotational correlation time of 5-and 16-doxylsterate derivatives and by the increase of fluorescence polarization of diphenylhexatriene. All the effects were obtained in a wide temperature range. The morphometric analysis of the ultrastructural images shows that the vesicle profiles of syncytiotrophoblast membranes decrease both area and form factor (FF) in the presence of Ca²⁺ with respect to the controls. The freeze-fracture results also show that Ca²⁺ induces an enhanced tendency to IMP clusterization. The Ca²⁺-induced changes were observed in both E and P faces. Our results underline the important role of Ca²⁺ in the cell membrane structure per se and in modulating interactions between cytoplasmic and extracellular microenvironments. The results of morphometric analysis of the ultrastructural images agree with biochemical data showing an increased stability induced by calcium on plasma membranes.