生物膜及其功能的量热学研究

生物膜具多种重要生理功能,因此近十几年,国际上对生物膜的研究已深入到生物学、医学的各个领域,成为当前分子生物学中最活跃的领域之一。七十年代以来,很多物理学和生物学技术都证明膜脂流动性是膜结构的一个基本特征,也是膜行使多种功能的重要体现。膜中各部分的流动性是不均匀的,它与环境及生理状态有关。生物膜的流动性主要体现为膜脂的流动性和膜蛋白的运动性。膜脂的流动性主要是指膜脂中脂肪酸烃链的运动。在正常生理条件下,膜脂大多呈液晶相,当温度降低至某一温度时,     

逆境对真菌膜脂肪酸成分的影响

利用气相色谱对真菌膜脂肪酸在逆境下的变化进行研究,发现低温胁迫时膜上的不饱和脂肪酸较多,低碳源浓度、低氧浓度和高盐浓度胁迫时,膜上不饱和脂肪酸的含量反而出现降低。表明不同逆境胁迫时,真菌中膜的脂肪酸含量不一样。     

南极微生物膜流动性的调节机制

稳定的膜流动性是南极微生物在低温下生存的关键。南极微生物在低温下通过增加不饱和脂肪酸和支链脂肪酸来增加膜的流动性;同时,作为平衡和辅助调节,通过改变细胞质膜中类胡萝卜素的组成来调节膜流动性,从而维持膜的最适流动性。极性类胡萝卜素能降低膜的流动性,而非极性类胡萝卜素增加膜的流动性。因此,通过类胡萝卜素类型(极性／非极性)的转变,细菌可以调节膜的流动性。类胡萝卜素对南极微生物细胞质膜流动性的调节作用及其机制是目前细胞质膜流动性领域新的发现和研究方向。     

外源脂肪酸对莱氏衣原体膜流动性的影响——电子自旋共振与荧光偏振研究

生物膜类脂的物理性质直接影响膜的生理功能,膜的流动动性是反映膜脂物理状态的一个重要特征.本文采用电子自旋共振波谱及荧光偏振技术研究油酸,硬脂酸以及油酸和棕榈酸的混合物渗入莱氏衣原体膜后对膜流动性的影响.结果表明,上述外源脂肪酸均能增加膜的流动性,其中以油酸渗入膜后最为显著.油酸中双键的作用不仅仅局限于双键所在碳原子附近,而且能使整个膜脂双层各个层次上流动性都有增加.对于用荧光偏振和自旋标记顺磁共振二种技术所获得结果的异同也进行了初步讨论.     

NaCl胁迫下大麦根系液泡膜H+-ATPase活性与膜流动性的关系

用50～200 mmol/L NaCl处理2 d后,大麦(Hordeum vulgare L.)品种"滩引2号"(耐盐性强)根的液泡膜H+-ATPase活性增强,600 mmol/L NaCl处理下酶活性下降;"科品7号"(耐盐性弱)在50～100 mmol/L NaCl处理2 d后根的液泡膜H+-ATPase活性增强,200～600 mmol/L NaCl处理下酶活性随盐浓度增加而降低.50～200 mmol/L NaCl处理下"滩引2号"根的液泡膜流动性下降,600 mmol/L NaCl处理下膜流动性明显增大;盐胁迫下液泡膜膜脂脂肪酸不饱和度下降时,膜流动性下降,反之则膜流动性上升.由此推断高盐胁迫下液泡膜膜脂脂肪酸不饱和度上升而引起膜流动性上升可能是引起H+-ATPase活性下降的原因之一.     

外源多胺对铜胁迫下荇菜叶片生物膜的保护作用

研究外源亚精胺和精胺对铜胁迫下荇菜叶片细胞膜透性、膜脂脂肪酸组成、光合、呼吸、铜积累影响的结果表明,50μmol·L^-1CuSO4处理导致膜脂脂肪酸组分中饱和脂肪酸组分增加,不饱和脂肪酸组分及不饱和指数（IUFA）下降,细胞膜透性加大,光合速率下降,呼吸速率迅速上升,铜在叶细胞中大量积累。外施0．1mmol·L^-1亚精胺和精胺可以稳定生物膜的结构和功能,降低铜在细胞中的累积。     

巴氏醋酸杆菌对发酵中醋酸胁迫的生理应答

【目的】研究Acetobacter pasteurianus CICIM B7003对醋酸发酵形成的酸胁迫环境在细胞形态、生理、代谢方面的响应,初步提出巴氏醋杆菌的动态耐酸机制模型,为高酸度高强度液态深层醋酸发酵提供理论帮助。【方法】在9 L自吸式发酵罐中用A.pasteurianus CICIM B7003发酵醋酸,选取不同生长阶段细胞检测其荚膜多糖含量、膜不饱和脂肪酸含量、耐酸基因转录水平、乙醇呼吸链酶和ATP酶活性,研究醋酸菌形态、生理和代谢随醋酸积累的变化。【结果】醋酸的存在能减少细胞分泌荚膜多糖,发酵中多糖占细胞干重百分比由最初2.5%下降到0.89%;随发酵进行细胞膜不饱和脂肪酸占膜总脂肪酸含量显著提高,致使细胞膜流动性增加;耐酸基因相对转录水平显著提高而提升了细胞对酸性环境的抗性;乙醇呼吸链酶和ATP酶活性随醋酸积累也显著提高,为细胞提供足够的能量以满足耐酸机制对能量的需求。【结论】初步确定A.pasteurianus CICIM B7003主要依靠改变细胞膜脂肪酸组分、激活耐酸基因转录、增强乙醇呼吸链活力及快速产能等机制的协同作用,实现对酸胁迫的制衡。     

外源H_2O_2和·OH对大麦幼苗根系线粒体膜脂和流动性的伤害

以大麦 (H ordeum vulgare L.)为材料 ,研究了外源 H2 O2 和· OH对大麦根系呼吸速率、线粒体膜流动性和膜脂脂肪酸组成的影响。结果表明 ,1 0 mmol/L H2 O2 或· OH处理 4d,大麦幼苗根系呼吸速率和线粒体膜脂不饱和脂肪酸含量及脂肪酸不饱和指数下降 ,线粒体膜脂荧光强度增加 ,膜流动性下降 ,且 H2 O2 或· OH处理浓度 (在 0 .1～ 1 0 mmol/L范围内 )越高 ,膜脂流动性下降越明显。 H2 O2 和· OH处理的同时加入同浓度的抗坏血酸 (As A)和甘露醇 ,膜流动性明显增强或恢复     

巴氏醋酸杆菌对发酵中醋酸胁迫的生理应答

摘要:【目的】研究Acetobacter pasteurianus CICIM B7003 对醋酸发酵形成的酸胁迫环境在细胞形态、生理、代谢方面的响应,初步提出巴氏醋杆菌的动态耐酸机制模型,为高酸度高强度液态深层醋酸发酵提供理论帮助。【方法】在9 L自吸式发酵罐中用A.pasteurianus CICIM B7003发酵醋酸,选取不同生长阶段细胞检测其荚膜多糖含量、膜不饱和脂肪酸含量、耐酸基因转录水平、乙醇呼吸链酶和ATP酶活性,研究醋酸菌形态、生理和代谢随醋酸积累的变化。【结果】醋酸的存在能减少细胞分泌荚膜多糖,发酵中多糖占细胞干重百分比由最初2.5%下降到0.89%;随发酵进行细胞膜不饱和脂肪酸占膜总脂肪酸含量显著提高,致使细胞膜流动性增加;耐酸基因相对转录水平显著提高而提升了细胞对酸性环境的抗性;乙醇呼吸链酶和ATP 酶活性随醋酸积累也显著提高,为细胞提供足够的能量以满足耐酸机制对能量的需求。【结论】初步确定A.pasteurianus CICIM B7003主要依靠改变细胞膜脂肪酸组分、激活耐酸基因转录、增强乙醇呼吸链活力及快速产能等机制的协同作用,实现对酸胁迫的制衡。     

NaCl胁迫下大麦根系液包膜H^＋—ATPase活性与膜流动性的关系

用50－200mmol/L NaCl处理2d后,大麦（Hordeum vulgare L.)品种“滩引2号”（耐盐性强）根的液泡膜H^ -ATPase活性增强,600mmol/L NaCl处理下酶活性下降;“科品7号”（耐盐性弱）在50－100mmol/L NaCl处理2d后根的液泡膜H^ -ATPase活性增强,200－600mmol/L NaCl处理下酶活性随盐浓度增加而降低。50－200mol/L NaCl处理下“滩引2号”根的液泡膜流动性下降,600mmol/L NaCl处理下膜流动性明显增大;盐胁迫下液泡膜膜脂脂肪酸不饱和度下降时,膜流动性下降,反之则膜流动性上升。由此推断高盐胁迫下液泡膜膜脂脂肪酸不饱和度上升而引起膜流动性上升可能是引起H^ -ATPase活性下降的原因之一。     

Fluidization of membrane lipids enhances the tolerance of Saccharomyces cerevisiae to freezing and salt stress

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Delta(9) position. We expressed two sunflower (Helianthus annuus) oleate Delta(12) desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Delta(9,12), the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15 degrees C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp(+) or Trp(-) strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30 degrees C or 15 degrees C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.     

Ethanol-induced changes in the fatty acid composition of Lactobacillus hilgardii, its effects on plasma membrane fluidity and relationship with ethanol tolerance

The effect of environmental ethanol concentration on the fatty acid composition of strains of Lactobacillus hilgardii, differing in their tolerance to ethanol, was determined. A marked increase in the proportion of lactobacillic acid (a cyclopropane fatty acid) and a decrease in oleic and vaccenic acids with increasing ethanol concentration was observed. The amount of lactobacillic acid determined at standard conditions (25°C, 0% ethanol) was found to be proportional to the ethanol tolerance of the strains studied. The effect of this alcohol on plasma membrane fluidity was studied by differential scanning calorimetry. The adaptive response to growth in the presence of high concentrations of ethanol produced membranes which, within the limits of ethanol tolerance, maintained the fluidity and integrity in an environment which tends to increase membrane rigidity. When pre-adapted cells are analysed in the absence of environmental ethanol there is a measurabie increase in fluidity. It is proposed that this phenomenon may be correlated with the increase in the proportion of lactobacillic acid. The existence of a relationship between membrane fluidity and ethanol tolerance is discussed.     

Exogenous 2-aminoethanol can diminish paraquat induced oxidative stress in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Effects of unfavourable environmental conditions (stresses) induce stressor specific and unspecific short- and long-term responses in plants. Long-term responses depend on intensity and duration of the stress. Short-term effects comprise the accumulation of reactive oxygen species (ROS), membrane damages by the oxidation of fatty acids, and the release of amino alcohols. They can incite higher stress tolerance in plants. In the present study, shoots of barley (Hordeum vulgare) were pre-treated with 2-aminoethanol, and, 2 days later, with the oxidative stress inducing herbicide, paraquat. Pre-treatments with 2-aminoethanol increased the stress tolerance in barley by the stabilization of the cell membranes, the enhanced production of superoxide dismutase and catalase, and the stimulation of glutathione metabolism (GSH, GST). These mechanisms of stress tolerance activation by 2-aminoethanol are discussed.     

Lactobacillus casei combats acid stress by maintaining cell membrane functionality

Lactobacillus casei strains have traditionally been recognized as probiotics and frequently used as adjunct culture in fermented dairy products where lactic acid stress is a frequently encountered environmental condition. We have investigated the effect of lactic acid stress on the cell membrane of L. casei Zhang [wild type (WT)] and its acid-resistant mutant Lbz-2. Both strains were grown under glucose-limiting conditions in chemostats; following challenge by low pH, the cell membrane stress responses were investigated. In response to acid stress, cell membrane fluidity decreased and its fatty acid composition changed to reduce the damage caused by lactic acid. Compared with the WT, the acid-resistant mutant exhibited numerous survival advantages, such as higher membrane fluidity, higher proportions of unsaturated fatty acids, and higher mean chain length. In addition, cell integrity analysis showed that the mutant maintained a more intact cellular structure and lower membrane permeability after environmental acidification. These results indicate that alteration in membrane fluidity, fatty acid distribution, and cell integrity are common mechanisms utilized by L. casei to withstand severe acidification and to reduce the deleterious effect of lactic acid on the cell membrane. This detailed comparison of cell membrane responses between the WT and mutant add to our knowledge of the acid stress adaptation and thus enable new strategies to be developed aimed at improving the industrial performance of this species under acid stress.     

Effect of membrane fatty acid substitution and temperature on repair of sublethal damage in mammalian cells

Repair of sublethal radiation damage (SLD) has been investigated as a function of temperature in mouse fibroblast LM cells with different membrane lipid composition. Rigidification or fluidization of the cellular membranes was accomplished by incorporation of myristic acid and arachidonic acid, respectively, in the phospholipids of the membranes. The SLD repair after radiation was essentially the same for the cells with the more rigid (saturated fatty acid) membranes and the cells with the more fluid (polyunsaturated fatty acid) membranes. This observation was made for repair at 37 degrees C as well as for repair at hypothermic temperatures. Incorporation of polyunsaturated fatty acid protected the cells against hypothermic death. These experiments demonstrate that although membranes are likely targets for cell killing by low temperature treatments, membrane lipids are probably not involved in the repair of sublethal radiation damage. It must be concluded that neither the degree of polyunsaturation of the lipids nor the degree of fluidity of the membrane is important for radiation-induced killing of mammalian cells.     

Fluidization of Membrane Lipids Enhances the Tolerance of Saccharomyces cerevisiae to Freezing and Salt Stress

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ⁹ position. We expressed two sunflower (Helianthus annuus) oleate Δ¹² desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ^9,12, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp⁺ or Trp⁻ strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.     

Superoxide dismutase depletion and lipid peroxidation in rat liver microsomal membranes: correlation with liver carcinogenesis

The depletion of superoxide dismutase in the liver of rats held on a copper-deficient diet for 8 weeks induces two profound modifications in microsomal membrane characteristics. These membranes show: (1) a low degree of peroxidation induced in vitro by both endogenous (NADPH and tert-butylhydroperoxide) and exogenous sources (xanthine/xanthine oxidase) of oxygen radicals as revealed by malondialdehyde and diene-conjugate production; (2) a strong decrease of polyunsaturated and an increase of monounsaturated fatty acid content. These alterations are similar to those found in microsomal membranes from fast-growing hepatomas which exhibit a pronounced saturation of fatty acid pattern and lack superoxide dismutase. These observations support the hypothesis that during hepatocarcinogenesis the loss of superoxide dismutase causes an oxidative stress that increases cellular membrane lipid peroxidation, as a consequence of which the cell responds by synthesizing more saturated fatty acids that permanently modify cell membrane structure and properties.     

Long-chain fatty acids and ethanol affect the properties of membranes inDrosophila melanogaster larvae

The larval fatty acid composition of neutral lipids and membrane lipids was determined in three ethanol-tolerant strains ofDrosophila melanogaster. Dietary ethanol promoted a decrease in long-chain fatty acids in neutral lipids along with enhanced alcohol dehydrogenase (EC 1.1.1.1) activity in all of the strains. Dietary ethanol also increased the incorporation of¹⁴C-ethanol into fatty acid ethyl esters (FAEE) by two- to threefold and decreased the incorporation of¹⁴C-ethanol into free fatty acids (FFA). When cultured on sterile, defined media with stearic acid at 0 to 5 mM, stearic acid decreased ADH activity up to 33%. In strains not selected for superior tolerance to ethanol, dietary ethanol promoted a loss of long-chain fatty acids in membrane lipids. The loss of long-chain fatty acids in membranes was strongly correlated with increased fluidity in hydrophobic domains of mitochondrial membranes as determined by electron spin resonance and correlated with a loss of ethanol tolerance. In the ethanol-tolerant E2 strain, which had been exposed to ethanol for many generations, dietary ethanol failed to promote a loss of long-chain fatty acids in membrane lipids. We are grateful for the support of National Institutes of Health Grant AA06702 (B.W.G.) and National Science Foundation Grant CHE-891987 (R.G.K.).     

Regulatory role of membrane fluidity in gene expression and physiological functions

Plants, algae, and photosynthetic bacteria experience frequent changes in environment. The ability to survive depends on their capacity to acclimate to such changes. In particular, fluctuations in temperature affect the fluidity of cytoplasmic and thylakoid membranes. The molecular mechanisms responsible for the perception of changes in membrane fluidity have not been fully characterized. However, the understanding of the functions of the individual genes for fatty acid desaturases in cyanobacteria and plants led to the directed mutagenesis of such genes that altered the membrane fluidity of cytoplasmic and thylakoid membranes. Characterization of the photosynthetic properties of the transformed cyanobacteria and higher plants revealed that lipid unsaturation is essential for protection of the photosynthetic machinery against environmental stresses, such as strong light, salt stress, and high and low temperatures. The unsaturation of fatty acids enhances the repair of the damaged photosystem II complex under stress conditions. In this review, we summarize the knowledge on the mechanisms that regulate membrane fluidity, on putative sensors that perceive changes in membrane fluidity, on genes that are involved in acclimation to new sets of environmental conditions, and on the influence of membrane properties on photosynthetic functions.     

Lipids in mammalian hibernation and artificial hypobiosis

Membrane lipids—phospholipids, fatty acids, and cholesterol—participate in thermal adaptation of ectotherms (bacteria, amphibians, reptiles, fishes) mainly via changes in membrane viscosity caused by the degree of fatty acids unsaturation, cholesterol/phospholipids ratio, and phospholipid composition. Studies of thermal adaptation of endotherms (mammals and birds) revealed the regulatory role of lipids in hibernation. Cholesterol and fatty acids participate in regulation of the parameters of torpor, gene expression, and activity of enzymes of lipid metabolism. Some changes in lipid metabolism during artificial and natural hypobiosis, namely, increased concentration of cholesterol and fatty acids in blood and decreased cholesterol concentration in neocortex, are analogous to those observed under stress conditions and coincide with mammalian nonspecific reactions to environmental agents. It is shown that the effects of artificial and natural hypobiosis on lipid composition of mammalian cell membranes are different. Changes in lipid composition cause changes in membrane morphology during mammalian hibernation. The effect of hypobiosis on lipid composition of membranes and cell organelles is specific and seems to be defined by the role of lipids in signaling systems. Comparative study of lipid metabolism in membranes and organelles during natural and artificial hypobiosis is promising for elucidation of adaptation of mammals to low ambient temperatures.