红细胞膜上结合水的萤光与红外研究

水是生物膜上重要的结构与功能分子。本实验以20℃红细胞膜对水的等温吸附曲线为定量根据,研究了不同水化水平的水化效应。DPH的萤光偏振测量证明:脱水将破坏脂双层的有序结构,大约20%的水化度是维持膜结构所必需的;在膜结构完整的条件下结合水的增加会使膜流动性增大,水分子对双层类脂分子的“松弛效应”和水分子的运动对于膜脂分子的活动性可能有重要贡献。ANS的萤光发射谱说明水化效应增加了膜表面的极性,也提示在高水化水平膜上存在着一部份活动性较大的水分子。红外差示谱表明膜上存在着两类结合水——紧密结合水与松弛结合水,也说明脱水将造成磷脂与胆固醇的相分离现象。     

膜融合诱导因子能使细胞质膜表面水化程度降低

研究了几种膜融合诱导因子,包括介质ｐＨ,温度以及钙离子等对小鼠艾氏腹水癌细胞质膜表面介电常数的影响．结果表明,酸性介质ｐＨ、温度降低或钙离子浓度增加,均可使其膜表面介电常数下降。从而提示,膜表面水化力下降和局部脱水可能是酸诱导膜融合,钙离子诱导膜融合及冰冻融化引起的膜融合等均需经过的关键步骤。     

磷脂酶C诱导膜融合的初步研究

用光散射、电镜和荧光共振能量转移技术研究了ＰＬＣ诱导两种单一膜脂组分的模型膜即二油酸磷脂酰胆碱（ＤＯＰＣ：ｄｉｏｌｅｏｙｌｐｈａｐｈｅｔｉｄｙｌｃｈｏｌｉｎｅ）脂质体和二豆蔻酰磷脂酰胆碱（ＤＭＰＣ：ｄｉｍｙｒｉｓｔｏｙｌｐｈｏｐｈａｔｉｄｙｌｃｈｅｌｏｎｅ）脂质体膜融合的可能性。结果表明：ＰＬＣ可以引起ＤＯＰＣ脂质体的融合。在相同的条件下,未见到ＤＭＰＣ脂质体的融合。这就首次证明了ＰＬＣ诱导单一组分脂质体融合的可能性。结果还表明：ＰＬＣ诱导脂质体膜融合的可能性大小与膜脂结构有关。用大鼠血影膜、人红细胞膜、大鼠巨噬细胞膜和大花萱草花瓣原生质体膜等天然生物膜作为材料,研究了磷脂酶Ｃ（ＰＬＣ：ｐｂｏｓｐｈｏｌｉｐａｓｅＣ）诱导上述各种天然膜融合的可能性,均未观察到膜融合现象。提示ＰＬＣ不易诱导天然细胞膜的融合。     

二氧化硅及柠檬酸铝对红细胞膜结合水影响的研究

本研究应用等温吸附法和付里埃变换红外光谱技术测定了二氧化硅及柠檬酸铝对红细胞膜结合水的不同影响。结果为,二氧化硅明显降低细胞膜水化度,使膜结合水的ν_OH峰位显著红移,表明其可导致细胞膜脱水。而柠檬酸铝对二氧化硅的这一作用有明显的拮抗效应,即通过提高细胞膜的水化度,可维持细胞膜的正常“水结构”。此外,本文还论讨了二氧化硅诱发细胞膜的脱水作用对细胞中毒的意义,以及柠檬酸铝拮抗作用的机理。     

生物膜融合研究进展

生物膜的融合是一个基本的生命过程,在生物的生长发育中有着重要作用.通过融合,两套独立的双层脂分子合二为一,完成一定的生物功能.膜融合分子机制的关键在于其主要成分融合蛋白.I、II类病毒融合蛋白形成"发夹",胞内囊泡与目标膜各提供的融合蛋白形成"类亮氨酸拉链".这些结构将独立的膜拉近,继而促使他们合为一体.细胞与细胞间融合蛋白的作用机制目前还未明确.在各种膜融合中,脂双层的变化可能是类似的,但介导融合的分子机制应该是不同的.目前,对于膜融合很多方面的理解还停留在假说阶段.理解了膜融合的过程和分子机制不仅极大地促进生物学的发展,而且为相关的疾病治疗打下坚实的基础.     

脱水速率对黄皮胚轴脱水敏感性及膜脂过氧化的影响

以黄皮种子离体胚轴为材料,研究了不同干燥速率对胚轴脱水反应和膜脂过氧化的影响.在脱水过程中,胚轴的萌发率、活力指数、电解质渗漏速率,超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性逐渐降低,膜脂过氧化产物MDA的含量不断增加.脱水速率愈快,胚轴的半致死含水量就愈低.快速干燥的胚轴能在较低的含水量下存活是因为缩短了在中间含水量下发生的膜脂过氧化作用的时间,以及保持较高的SOD、POD和CAT活性;缓慢干燥的胚轴当与周围环境达到水分平衡后,生活力的丧失将与保持在水分平衡后的时间有关.因此,脱水速率是一种影响顽拗性种子或者胚轴脱水敏感性的重要因子.     

植物冷驯化的分子机理研究进展

植物冷驯化是一个非常复杂的过程,包括植物将感受到的低温信号转变成生化信号,以激活冷诱导基因的启动子,刺激特定的mRNA的转录,并在特定的组织中合成冷驯化蛋白.冷驯化蛋白通过增强膜脂流动性和阻止胞间冰晶形成等方式,以保护细胞免受低温伤害.冷驯化基因的表达以转录后调控为主,也有转录调控.某些冷诱导基因也可受ABA或其他环境胁迫(如高温、干旱、高盐、脱水等)诱导表达.     

脂滴与其他细胞器互作调控脂稳态的分子机制

随着影像技术的发展,越来越多的研究表明细胞器之间存在广泛的直接相互作用,其主要功能是参与物质运输、细胞器新生与生长、细胞器分裂与融合等.细胞器间的互作主要由定位于这些膜器表面的蛋白质分子相互作用介导,磷脂也在其中发挥作用.脂滴作为储存中性脂的细胞器,是细胞内脂质代谢的中心,同时对机体脂稳态的维持起着至关重要的作用.从脂...     

膜融合机制研究进展

膜的融合是一个基本的生命过程,在生物的生长发育中有着重要作用。通过融合,两套独立的双层脂分子合二为一,完成一定的生物功能。膜融合分子机制的关键在于其主要成分：融合蛋白。Ⅰ、Ⅱ类病毒融合蛋白形成“发夹”,胞内囊泡与目标膜各提供的融合蛋白形成“类亮氨酸拉链”,这些结构将独立的膜拉近,继而促使膜合为一体。细胞与细胞间融合蛋白的作用机制目前还未明确,在各种膜融合中,脂双层的变化可能是类似的,但介导融合的分子机制应该是不同的。目前,对于膜融合很多方面的理解还停留在假说阶段。理解了膜融合的过程和分子机制不仅将极大地促进生物学的发展,更重要是将为相关的疾病治疗打下坚实的基础。     

几种二价阳离子及表面活性剂对元麦叶原生质体电泳率的影响(简报)

原生质体电泳率的研究已应用于了解质膜表面的电荷特性。Ca~( )能影响原生质体质膜表面的电荷状况,在诱导融合时起重要作用。关于二价阳离子对质膜功能的影响,Caldwell报道了Ca~( )、Mg~( )、Mn~( )等多种二价阳离子对质膜ATP酶不同的激活效应。此外,Helenius等指出表面活性剂能改变膜的结构,并与膜脂作用而引起溶胞。为进一步了解不同的二价阳离子对质膜电荷特性的影响,及表面活性剂在改变膜结构这一复杂过程中对质膜电性影响的状况,我们报道Ca~( )、Mg~( )、Mn~( )等二价阳离子及几种表面活性剂对原生质体电泳率影响的结果。     

Low amounts of sucrose are sufficient to depress the phase transition temperature of dry phosphatidylcholine, but not for lyoprotection of liposomes

Disaccharides such as sucrose and trehalose play an important role in stabilizing cellular structures during dehydration. In fact, most organisms that are able to survive desiccation accumulate high concentrations of sugars in their cells. The mechanisms involved in the stabilization of cellular membranes in the dry state have been investigated using model membranes, such as phosphatidylcholine liposomes. It has been proposed that the lyoprotection of liposomes depends on the depression of the gel to liquid-crystalline phase transition temperature (T(m)) of the dry membranes below ambient and on the prevention of membrane fusion by sugar glass formation, because both lead to leakage of soluble content from the liposomes. Since fusion is prevented at lower sugar/lipid mass ratios than leakage, it has been assumed that more sugar is needed to depress T(m) than to prevent fusion. Here, we show that this is not the case. In air-dried egg phosphatidylcholine liposomes, T(m) is depressed by >60 degrees C at sucrose/lipid mass ratios 10-fold lower than those needed to depress fusion to below 20%. In fact, T(m) is significantly reduced at mass ratios where no bulk sugar glass phase is detectable by Fourier transform infrared spectroscopy or differential scanning calorimetry. A detailed analysis of the interactions of sucrose with the P=O, C=O, and choline groups of the lipid and a comparison to published data on water binding to phospholipids suggests that T(m) is reduced by sucrose through a "water replacement" mechanism. However, the sucrose/lipid mass ratios necessary to prevent leakage exceed those necessary to prevent both phase transitions and membrane fusion. We hypothesize that kinetic phenomena during dehydration and rehydration may be responsible for this discrepancy.     

Membrane fusion: Lipid vesicles as a model system

In many cellular functions the process of membrane fusion is of vital importance. It occurs in a highly specific and strictly controlled fashion. Proteins are likely to play a key role in the induction and modulation of membrane fusion reactions. Aimed at providing insight into the molecular mechanisms of membrane fusion, numerous studies have been carried out on model membrane systems. For example, the divalent-cation induced aggregation and fusion of vesicles consisting of negatively charged phospholipids, such as phosphatidylserine (PS) or cardiolipin (CL), have been characterized in detail. It is important to note that these systems largely lack specificity and control. Therefore conclusions derived from their investigation can not be extrapolated directly to a seemingly comparable counterpart in biology. Yet, the study of model membrane systems does reveal the general requirements of lipid bilayer fusion. The most prominent barrier to molecular contact between two apposing bilayers appears to be due to the hydration of the polar groups of the lipid molecules. Thus, dehydration of the bilayer surface and fluctuations in lipid packing, allowing direct hydrophobic interactions, are critical to the induction of membrane fusion. These membrane alterations are likely to occur only locally, at the site of intermembrane contact. Current views on the way membrane proteins may induce fusion under physiological conditions also emphasize the notion of local surface dehydration and perturbation of lipid packing, possibly through penetration of apolar amino acid segments into the hydrophobic membrane interior.     

Acyl-chain correlation in membrane fusion intermediates: x-ray diffraction from the rhombohedral lipid phase

We have studied the acyl-chain conformation in stalk phases of model membranes by x-ray diffraction from oriented samples. As an equilibrium lipid phase induced by dehydration, the stalk or rhombohedral phase exhibits lipidic passages (stalks) between adjacent bilayers, representing a presumed intermediate state in membrane fusion. From the detailed analysis of the acyl-chain correlation peak, we deduce the structural parameters of the acyl-chain fluid above, at, and below the transition from the lamellar to rhombohedral state, at the molecular level.     

Effects of phorbol ester and teleocidin on Ca2+-induced fusion of liposomes

The effects of different types of lipid membrane defects on Ca2+-induced fusion of liposomes containing phosphatidylserine (PS) were investigated using fluorescent probes. Teleocidin enhanced the fusion of phospholipid vesicles in an assay system using terbium/dipicolinic acid during mixing of internal aqueous phases of vesicles upon fusion. 12-O-Tetradecanoylphorbol-13-acetate (TPA) suppressed the fusion. This latter phenomenon was also observed by measuring the excitation energy transfer. The promotion of membrane fusion by teleocidin was ascribed to dehydration of the membrane surface, the suppressive effect of TPA to desorption of Ca2+ from the membrane surface. Thus, Ca2+-induced fusion of PS vesicles was shown to be sensitive to defects of the membrane surface, but insensitive to defects of the hydrophobic core of the lipid membrane.     

Roles of membrane structure and phase transition on the hyperosmotic stress survival of Geobacter sulfurreducens

Geobacter sulfurreducens is a delta-proteobacterium bacteria that has biotechnological applications in bioremediation and as biofuel cells. Development of these applications requires stabilization and preservation of the bacteria in thin porous coatings on electrode surfaces and in flow-through bioreactors. During the manufacturing of these coatings the bacteria are exposed to hyperosmotic stresses due to dehydration and the presence of carbohydrates in the medium. In this study we focused on quantifying the response of G. sulfurreducens to hyperosmotic shock and slow dehydration to understand the hyperosmotic damage mechanisms and to develop the methodology to maximize the survival of the bacteria. We employed FTIR spectroscopy to determine the changes in the structure and the phase transition behavior of the cell membrane. Hyperosmotic shock resulted in greatly decreased membrane lipid order in the gel phase and a less cooperative membrane phase transition. On the other hand, slow dehydration resulted in increased membrane phase transition temperature, less cooperative membrane phase transition and a small decrease in the gel phase lipid order. Both hyperosmotic shock and slow dehydration were accompanied by a decrease in viability. However, we identified that in each case the membrane damage mechanism was different. We have also shown that the post-rehydration viability could be maximized if the lyotropic phase change of the cell membrane was eliminated during dehydration. On the other hand, lyotropic phase change during re-hydration did not affect the viability of G. sulfurreducens. This study conclusively shows that the cell membrane is the primary site of injury during hyperosmotic stress, and by detailed analysis of the membrane structure as well as its thermodynamic transitions it is indeed possible to develop methods in a rational fashion to maximize the survival of the bacteria during hyperosmotic stress.     

The effect of fructan on the phospholipid organization in the dry state

Fructans, a family of oligo- and polyfructoses, are implicated to play a drought-protecting role in plants. Inulin-type fructan is able to preserve the membrane barrier during dehydration. However, whether other fructans would be able to perform this function is unknown. In addition, almost nothing is known about the organization of these systems, which could give insight into the protective mechanism. To get insight into these questions the effect of different fructans on phosphatidylcholine-based model systems under conditions of dehydration was analyzed. Using a vesicle leakage assay, it was found that both levan- and inulin-type fructans protected the membrane barrier. This suggests that fructans in general would be able to protect the membrane barrier function. Furthermore, both fructan-types inhibited vesicle fusion to a large extent as measured using a lipid-mixing assay. Using x-ray diffraction, it was found that in the presence of both inulin- and levan-type fructans the lamellar repeat distance increased considerably. From this it was concluded that fructans are present between the lipid bilayers during drying. Furthermore, they stabilize the L(alpha) phase. In contrast to fructans, dextran did not increase the lamellar repeat distance and it even promoted L(beta) phase formation. These data support the hypothesis that fructans can have a membrane-protecting role during dehydration, and give insight into the mechanism of protection.     

Effects of Al(3+) and related metals on membrane phase state and hydration: correlation with lipid oxidation

The aim of the present study was to further understand how changes in membrane organization can lead to higher rates of lipid oxidation. We previously demonstrated that Al(3+), Sc(3+), Ga(3+), Be(2+), Y(3+), and La(3+) promote lipid packing and lateral phase separation. Using the probe Laurdan, we evaluated in liposomes if the higher rigidity of the membrane caused by Al(3+) can alter membrane phase state and/or hydration, and the relation of this effect to Al(3+)-stimulated lipid oxidation. In liposomes of dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylserine, Al(3+) (10-100 microM) induced phase coexistence and displacement of T(m). In contrast, in liposomes of brain phosphatidylcholine and brain phosphatidylserine, Al(3+) (10-200 microM) did not affect membrane phase state but increased Laurdan generalized polarization (GP = -0. 04 and 0.09 in the absence and presence of 200 microM Al(3+), respectively). Sc(3+), Ga(3+), Be(2+), Y(3+), and La(3+) also increased GP values, with an effect equivalent to a decrease in membrane temperature between 10 and 20 degrees C. GP values in the presence of the cations were significantly correlated (r(2) = 0.98, P < 0.001) with their capacity to stimulate Fe(2+)-initiated lipid oxidation. Metal-promoted membrane dehydration did not correlate with ability to enhance lipid oxidation, indicating that dehydration of the phospholipid polar headgroup is not a mechanism involved in cation-mediated enhancement of Fe(2+)-initiated lipid oxidation. Results indicate that changes in membrane phospholipid phase state favoring the displacement to gel state can facilitate the propagation of lipid oxidation.     

Cationic triple-chain amphiphiles facilitate vesicle fusion compared to double-chain or single-chain analogues

Cationic, triple-chain amphiphiles promote vesicle fusion more than structurally related double-chain or single-chain analogues. Two types of vesicle fusion experiments were conducted, mixing of oppositely charged vesicles and acid-triggered self-fusion of vesicles composed of cationic amphiphile and anionic cholesteryl hemisuccinate (CHEMS). Vesicle fusion was monitored by standard fluorescence assays for intermembrane lipid mixing, aqueous contents mixing and leakage. Differential scanning calorimetry was used to show that triple-chain amphiphiles lower the lamellar-inverse hexagonal (L(alpha)-H(II)) phase transition temperature for dipalmitoleoylphosphatidylethanolamine. The triple-chain amphiphiles may enhance vesicle fusion because they can stabilize the inversely curved membrane surfaces of the fusion intermediates, however, other factors such as extended conformation, packing defects, chain motion, or surface dehydration may also contribute. From the perspective of drug delivery, the results suggest that vesicles containing cationic, triple-chain amphiphiles (and cationic, cone-shaped amphiphiles in general) may be effective as fusogenic delivery capsules.     

Association between Membrane Phase Properties and Dehydration Injury in Soybean Axes

Axes of soybean seeds are tolerant to dehydration at 6 hours of imbibition, but susceptible to dehydration injury if dried at 36 hours of imbibition. Smooth microsomal membranes were isolated from axes imbibed for 6 hours (dehydration tolerant state) and 36 hours (dehydration susceptible state) before and after dehydration treatment. The phase properties and the lipid composition of the membrane fraction were investigated. Wide angle x-ray diffraction patterns of microsomal membranes from axes imbibed for 6 or 36 hours indicated a liquid-crystalline to gel phase transition at approximately 7°C. Membranes from axes dehydrated at 6 or 36 hours of imbibition and rehydrated for 2 hours exhibited a phase transition at 7°C and 47°C, respectively. Changes in fatty acid saturation did not account for the changes in phase properties. However, the increased phase transition temperature of the membranes from dehydration injured axes was associated with an increase in free fatty acid:phospholipid molar ratio and a decrease in phospholipid:sterol ratio. These results suggests that dehydration prompted a deesterification of the linkage between glycerol and fatty acid side chains of the phospholipid molecules in the membrane. The resultant increase in free fatty acid content in the membrane is thought to alter the fluidity and phase properties of the membrane and contribute to dehydration injury.     

Cationic triple-chain amphiphiles facilitate vesicle fusion compared to double-chain or single-chain analogues

Cationic, triple-chain amphiphiles promote vesicle fusion more than structurally related double-chain or single-chain analogues. Two types of vesicle fusion experiments were conducted, mixing of oppositely charged vesicles and acid-triggered self-fusion of vesicles composed of cationic amphiphile and anionic cholesteryl hemisuccinate (CHEMS). Vesicle fusion was monitored by standard fluorescence assays for intermembrane lipid mixing, aqueous contents mixing and leakage. Differential scanning calorimetry was used to show that triple-chain amphiphiles lower the lamellar-inverse hexagonal (L_α-H_II) phase transition temperature for dipalmitoleoylphosphatidylethanolamine. The triple-chain amphiphiles may enhance vesicle fusion because they can stabilize the inversely curved membrane surfaces of the fusion intermediates, however, other factors such as extended conformation, packing defects, chain motion, or surface dehydration may also contribute. From the perspective of drug delivery, the results suggest that vesicles containing cationic, triple-chain amphiphiles (and cationic, cone-shaped amphiphiles in general) may be effective as fusogenic delivery capsules.