CHAPS对M_(412)的动力学和共振拉曼光谱的影响

研究了表面活性剂CHAPS对紫膜中菌紫质在光循环过程中M_(412)的衰减速率及质子泵功能的影响.光循环中间体M_(412)快、慢衰减组分的衰减速率及质子衰减速率均受到CHAPS的影响,综合激光拉曼光谱对M_(412)和其它光循环中间体相对含量的测定,表明CHAPS对紫膜的影响是通过影响其膜脂完整的液晶结构,而使紫膜光循环动力学过程及质子泵功能发生变化的.     

水在菌紫质光循环和质子泵中的作用

用圆二色仪和闪光动力学先谱仪分别测量了空气干燥紫膜薄层的圆二色谱及紫膜LB膜的M_(412)的衰减过程.在于燥紫膜的圆二色谱上出现412nm的正峰,它是光循环中间体M_(412)的特征峰.在无水介质中,紫膜LB膜中的BR仍能进行先化学循环而检测到中间体M_(412),但M_(412)的衰减速度减慢,产生M_(412)的堆积,质子化过程受阻.在有水的介质中,只要有足够的H~+存在,紫膜LB膜中的BR的中间体M_(412)的衰减速度明显加快.说明水介质的H~+是完成正常光化学循和质子化过程必不可少的.     

肼类与蓝膜的相互作用

采用紫外可见光谱、动力学光谱和荧光光谱,研究了肼、甲基肼和偏二甲基肼与蓝膜的相互作用。结果表明这些肼类都可使蓝膜恢复为紫膜,并恢复紫膜原有的光循环方式。但是,中间体(M412)的衰減速率加快,这在金属离子重组紫膜中没有发生。另外,当肼加入蓝膜时,荧光光谱没有明显变化;而去掉金属离子的蓝膜,其荧光強度明显增加。     

紫膜与溶剂的相互作用

本文研究了溶剂正己烷,正十六烷,甲苯和二甲基甲酰胺(DMF dimethyl formamide)与紫膜的相互作用.吸收光谱,园二色谱和紫膜光循环中间产物M412的动力学过程的测量表明,在不同条件下,溶剂与紫膜能相互作用而影响到紫膜的光谱特性和光化学循环动力学过程.结果说明,在制作紫膜LB膜时,正己烷和正十六烷是合适的,使用二甲基甲酰胺时必须防止强光照射,甲苯则不能采用.     

蜂毒对菌紫质(bR)光循环中间体M_(412)和质子泵的影响

蜂毒对菌紫质(bR)光循环中间体M_(412)及质子泵的动力学过程有较大影响.表现在M_(412)慢衰减组分(M_(412~S))和质子的半衰期增大及产出量的减小,说明蜂毒分子的介入抑制了M_(412)的生成和衰减过程,同时也阻碍了紫膜的质子泵功能.但似乎对M_(412)快衰减组分影响不大.以上结果支持了M_(412~S)与质子泵功能有关的说法.实验结果同时反映出蜂毒除与膜脂分子存在强烈作用外还与bR本身直接作用.因此,蜂毒是一种研究膜脂蛋白相互作用及膜蛋白功能的较好材料.     

两种状态细菌视紫红质光循环中间产物与pH的关系

本文主要用微机控制的毫秒级闪光动力学光谱仪研究含三体细菌视紫红质(Bacteriorhodopsin,简称BR)的紫膜碎片和含单体BR的DMPC(dimyristoyl-Phosphatidyl-choline)脂质囊泡在不同pH条件下光循环中间产物M_(412)和O_(640)的变化,研究结果表明:BR单体与其三体状态相比,BR单体的光循环中间产物M_(412)的产量受介质pH变化的影响较大,其慢衰减成份的衰减比三体BR慢3—10倍.说明单体BR的结构状态较易受PH影响,单体BR光循环中间产物O_(640)随pH变化的趋势与三体BR的有很大区别,可能是由于不同状态的BR受pH的影响,但其具有不同的构型,导致光循环途径的变化.     

Triton X-100对细菌视紫红质中间产物M_(412)的效应

本文研究用非离子表面活性剂Triton X-100处理后的细菌视紫红质(BR Bacteriorhodo-psin)光循环中间产物M_(412)动力学过程的变化.实验结果表明,用不同浓度的Triton处理pH=6.5的BR体系时,其中间产物M_(412).快衰减成分的半衰期(τ_(1/2)~f)在Triton浓度为0.05%(w/w)附近突然变慢,随着Triton浓度的加大,τ_(1/2)~f又逐渐加快;慢衰减部分的半衰期(τ_(1/2)~s)则随Tri-ton浓度的增加逐渐变慢.BR的生色团峰发生蓝移.说明不同浓度的Triton在水溶液中聚集状态不同,可不同程度地破坏膜脂的液晶态结构,从而导致镶嵌在其中的BR发生构象的变化,使转运质子的氢键通道受到不同程度的影响,故质子泵转运通道发生改变、致使M_(412)的衰减速率改变.     

细菌视紫红质光循环中间体M412的动力学初探

采用紫外可见吸收光谱技术和闪光光解技术,初步观察了细菌视紫红质(BR)分子在宽pH范围(2.1～12.3)内的特征吸收峰以及M412的相对浓度和M412的慢成分半衰期的变化,并对其结构和光循环功能进行了讨论.紫外可见吸收光谱实验结果显示:pH=5.0～10.0时,BR最大特征吸收峰值约为568 nm;pH＜5.0时,BR最大特征吸收峰发生红移;pH＞10.0时,BR最大特征吸收峰发生蓝移.闪光动力学光谱结果显示:pH为7.3～9.5时,M412的相对浓度(M0)基本稳定在0.038左右;pH＜7.3时,M0逐渐减小;pH＞9.5时,M0明显上升,在pH=11.8时达到最大值0.1355,随后又快速下降.pH为2.1～7.3时,M412的慢成分半衰期(ts1/2)值在(4.1±1.1)ms左右;pH＞7.3时,ts1/2值急剧延长到40 677.4 ms.推测在高pH条件下,BR分子的光循环有新的路径和机理.     

酶切菌紫质(bR)C端对紫膜光循环和质子泵效率的影响

本文研究紫膜悬浮液经低剂量木瓜蛋酶处理去掉菌紫质(Bacteriorhodopsin简写bR)分子C-末端后。其光循环产物和质子泵效率的变化。实验发现经酶切后,M_(412)产物中慢衰减组份M_(412)降低了20％,O_(640)降低了50％,而质子泵效率降低了70％。双光脉冲实验表明酶解作用并不影响光循环周期。这些事实说明了去C-端所引起的质子泵效率降低,不是通过光循环的途径而产生的。介质中离子强度对正常紫膜和酶解紫膜的质子泵效率有明显不同的影响 说明了C端在不同盐浓度中的构象对质子泵行为有很重要的作用。     

离子强度、温度及表面活性剂Triton X-100对紫膜质子泵效率的影响

用毫秒级闪光动力学光谱仪研究了离子强度、温度及表面活性剂Triton X-100对紫膜质子泵效率的影响。结果表明:紫膜溶液中适量离子的加入可使其质子泵效率(H~+/M_(412))显著提高(纯水中,H~+/M_(412)—0.56,而在150mM KCl中可达—1.3),高价离子的影响显然远大于低价离子;在5℃—50℃的较大温度范围内,紫膜的质子泵效率变化不大,但当温度升至60℃以上时,紫膜的质子泵效率迅速下降直至为零;非离子表面活性剂Triton X-100的加入对紫膜的质子泵效率影响不大,仅随Triton量的增加而略微下降。以上实验现象,直接或间接地说明了离子在紫膜质子泵功能中的重要作用。就此对离子及脂在紫膜质子泵中的作用机制进行了进一步的探讨。     

Chloroplast thylakoid membrane fluidity and its sensitivity to temperature

In order to investigate membrane fluidity, the hydrophobic probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), has been incorporated into intact isolated thylakoids and separated granal and stromal lamellae obtained from the chloroplasts of Pisum sativum. The steady-state polarization of DPH fluorescence was measured as a function of temperature and indicated that at physiological values the thylakoid membrane is a relatively fluid system with the stromal lamellae being less viscous than the lamellae of the grana. According to the DPH technique, neither region of the membrane, however, showed a sharp phase transition of its bulk lipids from the liquid-crystalline to the gel state for the temperature range -20° to 50° C. Comparison of intact thylakoids isolated from plants grown at cold (4°/7°C) and warm (14°/17° C) temperatures indicate that there is an adaptation mechanism operating which seems to maintain an optimal membrane viscosity necessary for growth. Using a modified Perrin equation the optimal average viscosity for the thylakoid membrane of the chill-resistant variety used in the study (Feltham First) is estimated to be about 1.8 poise.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - Hepes N-(2-hydroxyethyl)-1-piperazineethanesulphonic acid     

Monoclonal antibodies identify common and differentiation-specific antigens on the plasma membrane of guard cells of Vicia faba L.

Monoclonal antibodies were raised against the plasma membrane of Vicia faba L. guard cells by immunizing either with total membranes from purified guard-cell protoplasts or with sealed, predominantly right-side-out plasma-membrane vesicles prepared from abaxial epidermes of V. faba by aqueous two-phase partitioning. Hybridoma screening was performed by enzyme-linked immunosorbent assay using polystyrene-adsorbed plasma-membrane vesicles as solid phase and by indirect immunofluorescence analysis using unfixed, immobilized protoplasts in a microvolume Terasaki assay. A range of monoclonal antibodies was characterized and is reported here. One monoclonal antibody, G26-6-B2, is guard-cell-specific and does not react with mesophyll-cell protoplasts of the same species. It binds to a periodate-resistant but trypsin-labile epitope, probably a differentiation-specific plasma-membrane protein.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - GCP guard cell protoplast(s) - Ig immunoglobulin - MAB monoclonal antibody - MCP mesophyll-cell protoplast(s) - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Phase transitions and thermal expansion coefficients of plant cuticles

The temperature-induced volume expansion of enzymatically isolated cuticular membranes of twelve plant species was measured. All cuticular membranes exhibited distinct second-order phase transitions in the temperature range of about 40 to 50° C. Increases in the volumes of fruit cuticles (Lycopersicon, Cucumis, Capsicum, Solanum and Malus) were fully reversible, while leaf cuticular membranes (Ficus, Hedera, Nerium, Olea, Pyrus, Picea and Citrus) underwent irreversible structural changes. Below the phase-transition temperature, volumetric expansion coefficients ranged from 0.39·10^–6 m³·kg^–1·K^–1 to 0.62·10^–6 m³·kg^–1·K^–1, and above from 0.60·10⁶ m³·kg^–1·K^\-1 to 1.41· 10^–6 m³·kg^–1·K^–1. Densities of cuticles at 25° C ranged from 1020 kg·m^–3 to 1370 kg·m^–3. Expansion coefficients and phase transitions were characteristic properties of the polymer matrix as a composite material, rather than of cutin alone. It is argued that the sudden increase of water permeability above the transition temperature, is caused by an increase of disorder at the interface between the polymer matrix and the soluble cuticular lipids. Possible ecological and physiological consequences of these results for living plants are discussed.Abbreviations CM Cuticular membrane - CU cutin - MX polymer matrix - SCL soluble cuticular lipids (waxes) The authors greatfully acknowledge stimulating discussions with Drs. H. Gruler (Exp. Physik 3, Universität Ulm, FRG) and M. Riederer (Institut für Botanik und Mikrobiologie, Technische Universität München, München, FRG) and financial support by the Deutsche Forschungsgemeinschaft.     

ECMO在脓毒症中应用的研究进展

体外膜肺氧合(extracorporeal membrane oxygenation,ECMO)是一种心肺支持技术,临床上能够为严重、可逆性的心肺衰竭患者提供循环、呼吸支持.近年来,新生儿、儿童和成人脓毒症患者应用ECMO治疗的成功报道日渐增多,掀起了脓毒症患者应用ECMO治疗的热潮.然而,ECMO在脓毒症患者中的临床应用却一直存在争议.大量回顾性研究表明新生儿及儿童脓毒症患者中应用的成功率相对较高,成人脓毒症患者应用ECMO治疗例数较少,但仍可在其他治疗无效的前提下尽早尝试ECMO治疗.     

Specificity of the accumulation of mRNAs and proteins of the plasma membrane and tonoplast aquaporins in radish organs

Plant aquaporins occur in multiple isoforms and are distributed in both plasma membrane and tonoplast. We cloned cDNAs for plasma-membrane aquaporins (PAQ1, 1b, 1c, 2, 2b, and 2c) of radish (Raphanus sativus L.). The amino acid sequences of the PAQs showed on average 63% sequence identity. Their sequences were 23% identical to those of tonoplast aquaporins (γ- and δ-VM23). A comprehensive investigation of the aquaporin mRNAs, including VM23, in seedlings, plants, flowers and seeds of radish showed a marked accumulation of all the mRNAs in hypocotyls and growing taproots. In other organs, the mRNA level of each isoform varied according to the organ. In petals, stamens, pistils and sepals of flowers, the levels of PAQ1, 1b, 1c and γ-VM23 mRNAs were high, and mRNAs of all aquaporins except for δ-VM23 were detected at high levels in pericarps. The protein levels of aquaporins on the basis of the membrane protein were determined by immunoblotting. Proteins PAQ1 and VM23 were detected in every organ except for the mature petiole. The PAQ2 protein level was especially high in green cotyledons and leaves, but was extremely low in seedling cotyledons and hypocotyls. Proteins PAQ1, PAQ2 and VM23 were highly accumulated in growing pericarps, but not in the immature seeds. These results indicate that the gene expression of the aquaporin isoforms was individually regulated in an organ- and tissue-specific manner, and that the amounts of aquaporin protein, especially PAQ2, are regulated in certain tissues at the translational level and by the rate of protein turnover. Received: 10 February 2000 / Accepted: 30 June 2000     

LptB-LptF coupling mediates the closure of the substrate-binding cavity in the LptB2FGC transporter through a rigid-body mechanism to extract LPS

Lipopolysaccharides (LPS) are essential envelope components in many Gram-negative bacteria and provide intrinsic resistance to antibiotics. LPS molecules are synthesized in the inner membrane and then transported to the cell surface by the LPS transport (Lpt) machinery. In this system, the ATP-binding cassette (ABC) transporter LptB₂FGC extracts LPS from the inner membrane and places it onto a periplasmic protein bridge through a poorly understood mechanism. Here, we show that residue E86 of LptB is essential for coupling the function of this ATPase to that of its partners LptFG, specifically at the step where ATP binding drives the closure of the LptB dimer and the collapse of the LPS-binding cavity in LptFG that moves LPS to the Lpt periplasmic bridge. We also show that defects caused by changing residue E86 are suppressed by mutations altering either LPS structure or transmembrane helices in LptG. Furthermore, these suppressors also fix defects in the coupling helix of LptF, but not of LptG. Together, these results support a transport mechanism in which the ATP-driven movements of LptB and those of the substrate-binding cavity in LptFG are bi-directionally coordinated through the rigid-body coupling, with LptF’s coupling helix being important in coordinating cavity collapse with LptB dimerization.     

Membrane fouling in a membrane bioreactor (MBR): sludge cake formation and fouling characteristics

A submerged membrane bioreactor (MBR) with a working volume of 1.4 L and a hollow fiber microfiltration membrane was used to treat a contaminated raw water supply at a short hydraulic retention time (HRT) of approximately 1 h. Filtration flux tests were conducted regularly on the membrane to determine various fouling resistances, and confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were employed to characterize the biofouling development and sludge cake formation on the membrane. The experimental results demonstrate that the MBR is highly effective in drinking water treatment for the removal of organic pollutants, ammonia, and UV absorbance. During the MBR operation, the fouling materials were not uniformly distributed on the entire surface of all of the membrane fibers. The membrane was covered partially by a static sludge cake that could not be removed by the shear force of aeration, and partially by a thin sludge film that was frequently washed away by aeration turbulence. The filtration resistance coefficients were 308.4 x 10(11) m(-1) on average for the sludge cake, 32.5 x 10(11) m(-1) on average for the dynamic sludge film, and increased from 10.5 x 10(11) to 59.7 x 10(11) m(-1) for the membrane pore fouling after 10 weeks of MBR operation at a filtration flux of 0.5 m3/m2 x d. Polysaccharides and other biopolymers were found to accumulate on the membrane, and hence decreased membrane permeability. More important, the adsorption of biopolymers on the membrane modified its surface property and led to easier biomass attachment and tighter sludge cake deposition, which resulted in a progressive sludge cake growth and serious membrane fouling. The sludge cake coverage on the membrane can be minimized by the separation, with adequate space, of the membrane filters, to which sufficient aeration turbulence can then be applied.     

茉莉酸甲酯和脱落酸对绿豆下胚轴质膜H+-ATPase水解活性的影响

茉莉酸甲酯(MeJA)促进绿豆下胚轴质膜H+-ATPase水解活性.活体条件下,50μmol·L-1 MeJA处理7 h的酶活性提高30%;离体条件下,10 μmol·L-1 MeJA处理2 h的酶活性最大,即提高30%.壳梭孢素(FC)和MeJA在离体条件下对H+-ATPase活性的促进效应相同,均提高30%左右,无协同效应;活体条件下,FC促进质膜H+-ATPase水解活性可达70%,而MeJA仅为30%.离体条件下,脱落酸(ABA)对H+-ATPase水解活性无明显促进;而活体条件下则有一定的抑制.