(Na+/K+)-ATPase研究概况

本文概述(Na⁺/K⁺)-ATPase的一般分子性质。介绍神经元和脂肪细胞中两种不同分子形式(Na⁺/K⁺)-ATPase的分离鉴定和功能性质,以及(Na⁺/K⁺)-ATPase主要功能亚基一级序列和高级结构研究所取得的一些进展。     

Na+/H+逆向转运蛋白和植物耐盐性

Na⁺H⁺逆向转运蛋白对植物耐盐起着重要作用 ,它利用质膜H⁺ATPase或液泡膜H⁺ATPase及Ppiase泵H+产生的驱动力把Na⁺排出细胞或在液泡中区隔化以消除Na⁺的毒害。主要讨论植物中Na⁺H⁺逆向转运蛋白研究在分子水平的最新进展.     

胃(H++K+)-ATPase结构与功能的研究

胃(H⁺+K⁺)-ATPase属于生物膜的第二类质子泵(E₁E₂型),从生理角度它是胃酸分泌的质子泵。本文结合我们初步的研究结果:猪、大白鼠胃粘膜(H⁺+K⁺)-ATPase的纯化以及由消炎痛引起的急性胃粘膜病变与胃粘膜(H⁺+K⁺)-ATPase的关系等,对此酶在近十几年来它的纯化、结构、性质、催化机理,向胃腔分泌盐酸的功能及其调节和胃病变的分子机理等方面进行了简要的综述。     

水稻耐盐相关动态QTL的加性和上位性与环境互作分析

钠离子(Na⁺)、钾离子(K⁺)含量和Na⁺/K⁺值是影响水稻耐盐性的关键指标。水稻的耐盐性由数量性状位点(QTL)控制,目前在水稻苗期已经鉴定了大量Na⁺、K⁺含量和Na⁺/K⁺的QTL,但在田间生长期鉴定的QTL数量较少。该研究以粳稻‘东农425’和‘长白10’杂交衍生的重组自交系(RIL)群体为材料,对田间试验条件下的盐胁迫和对照进行联合分析,在水稻不同发育时期鉴定Na⁺、K⁺含量和Na⁺/K⁺的发育动态QTL,并采用混合线性模型(MCIM)分析各QTL的加性(A)和上位性(AA)及其与环境的互作效应(QE)。结果表明：(1)盐胁迫条件下,亲本和RIL群体的茎Na⁺含量(SNC)、茎Na⁺/K⁺(SN/K)、叶Na⁺含量(LNC)和叶Na⁺/K⁺(LN/K)在各时期均高于对照,茎K⁺含量(SKC)和叶K⁺含量(LKC)均低于对照,对照条件下双亲性状在大多数时期均无显著差异;盐胁迫下两个亲本的多个性状在不同发育时期存在显著差异,其中,SNC和LKC在4个时期差异显著,SN/K和LN/K在3个时期差异显著,SKC和LNC在2个时期差异显著。(2)采用非条件和条件QTL作图方法,共检测到13个加性QTL和11对上位性QTL,其中包括14个非条件QTL和10个条件QTL;而13个加性QTL中有8个,11对上位性QTL中有7个具有环境互作效应。(3)qSKC5 1在水稻4个发育阶段均被检测到,其在调控水稻耐盐性中发挥着重要作用。SNC、SN/K和LN/K的所有QTL均检测到加性×环境互作效应,2对控制SNC的上位性QTL均检测到上位性×环境互作效应,说明这些性状的QTL对盐环境较为敏感。研究发现,田间生长条件下水稻Na⁺、K⁺含量和Na⁺/K⁺的QTL表达与发育时期密切相关;水稻田间生长条件下耐盐性的遗传非常复杂,在利用分子标记辅助选择(MAS)培育耐盐水稻新材料应该考虑上位性和环境互作效应。     

茎瘤固氮根瘤菌ORS571中motA基因的功能分析

[目的] MotA是细菌的鞭毛马达蛋白,是跨膜质子通道的重要组成结构之一,在调控鞭毛运动中具有至关重要的作用。本研究探究了Azorhizobium caulinodans ORS571中鞭毛马达基因motA对菌株表型和植物互作的影响。[方法] 通过同源重组原理和三亲接合转移方法构建突变菌株∆motA,测定野生型与突变体在菌体生长、运动、固氮、胞外多糖合成、生物膜形成及根系定殖能力的差异。[结果] 与野生型相比,突变体菌体生长没有明显差异,但其运动能力完全丧失,固氮、胞外多糖合成、生物膜形成及根系定殖能力减弱。[结论] MotA鞭毛马达蛋白对A.caulinodans ORS571的运动、固氮、胞外多糖合成、生物膜形成及根系定殖能力均有调控作用。     

大鼠前脂细胞质膜Na+/H+交换同时参与细胞的增殖和分化

以原代培养的大鼠前脂细胞为模型 ,以 2′ ,7′ bis ( 2 carboxyethyl) 5 ( 6 ) carboxyfluorescein (BCECF)作为检测胞内pH(pHi)的荧光探针 ,测定不同生长因子刺激下胞内pH的变化 ,证明大鼠肾周前脂细胞质膜存在Na⁺/H⁺交换活性 ,胎牛血清(FCS)能快速激活Na⁺/H⁺交换 ,导致pHi升高 (约 0 .2pH单位 ) ,并引起DNA合成 .Ethyl isopropyl amiloride (EIPA)抑制Na⁺/H⁺交换与DNA合成 .在无血清条件下 ,胰岛素不刺激DNA合成但引起细胞分化 ,表现为胞内脂滴积累和 3 磷酸 甘油脱氢酶(G₃ PDH酶 )活性增强 ,同时激活Na⁺/H⁺交换活性导致pHi升高 ;EIPA既抑制胰岛素对Na⁺/H⁺交换的激活 ,也抑制G₃ PDH酶活性增强 .结果证明 :Na⁺/H⁺交换的激活不仅与大鼠前脂细胞增殖相关 ,同时也是细胞分化的早期事件 .     

多细胞盐腺离子选择性分泌机理及其成因

为揭示多细胞盐腺对阳离子的选择性分泌机理, 在室内水培条件下, 研究了不同盐分胁迫(NaCl, KCl和NaCl+KCl)对多枝柽柳和短穗柽柳Na⁺, K⁺的分泌和累积, 以及不同盐腺抑制剂(orthovanadate, Ba²⁺, ouabain, tetraethylammonium[TEA]和verapami)对Na⁺和K⁺分泌的抑制及其在体内累积的影响. 结果表明, NaCl处理明显增加了盐腺对Na⁺分泌, 而KCl处理则显著促进K⁺分泌; Na⁺和K⁺的分泌量同累积量的比值表明, Na⁺的比值高于K⁺; 单一盐溶液中添加不同离子成分后, Na⁺和K⁺分泌表现不同: KCl处理中添加NaCl后, K⁺分泌速率显著降低, 但在NaCl处理中添加KCl, Na⁺的分泌则不受影响, 这些结果表明, 柽柳对Na⁺的分泌具有较高的选择性. 此外, 添加盐腺抑制剂后, 柽柳对Na⁺分泌分别受orthovanadate, ouabain, tetraethylammonium[TEA]和verapami的显著抑制, 而K⁺的分泌则分别受ouabain, tetraethylammonium[TEA]和verapami的显著抑制. orthovanadate对Na⁺, K⁺分泌抑制效应的差异, 可能是引起多细胞盐腺分泌Na⁺和K⁺能力不同的主要原因.     

IL-7和胸腺基质细胞系(MTEC5)诱导胸腺CD3- CD4- CD8- 细胞分化

胸腺细胞发育是细胞因子及胸腺细胞与胸腺基质细胞相互作用的结果.观察了IL-7及小鼠胸腺基质上皮细胞系MTEC5对成年小鼠CD3^- CD4^- CD8^- 胸腺细胞发育的影响.IL-7能促进TN细胞增殖.TN细胞经在IL-7条件下培养后表达CD3-TCR分子,其中20％～40％为TCRγδ⁺,60％～80％为TCRαβ⁺,但保持CD4^- CD8^- .该细胞获得对 Con A、抗CD3mAb及抗TCRmAb进行增殖应答的能力,抗CD28mAb能促进这种增殖应答,证明表达的CD3-TCR分子功能成熟.当在该系统中加入MTEC5,部分TN细胞转变为CD3⁺TCRβ⁺CD4⁺CD8^- 和CD3⁺TCRβ⁺CD4^- CD8⁺SP细胞,表明MTEC5可诱导TN细胞分化为表现型成熟的胸腺细胞.     

宫颈癌患者阴道微生态变化及其与细胞免疫的相关性探讨

目的探讨宫颈癌患者阴道微生态变化及其与细胞免疫的相关性。方法回顾性分析2018年1月-2020年5月本院收治的且经病理确诊的73例宫颈癌患者(宫颈癌组)和同期65例体检筛查宫颈正常者(正常宫颈组)的临床病例资料。两组均采用直接涂片法、革兰染色法检测阴道分泌物中乳酸杆菌、滴虫、支原体、衣原体、细菌性阴道病、霉菌、淋菌、假丝酵母菌、人乳头瘤病毒(HPV)及阴道唾液酸甘酶活性、白细胞酯酶、过氧化氢等。另采用流式细胞仪检测外周血T淋巴细胞(CD3⁺、CD4⁺、CD8⁺、CD4⁺/CD8⁺)及自然杀伤细胞(CD16⁺CD56⁺)水平。观察各组阴道微生态变化情况及外周血CD3⁺、CD4⁺、CD8⁺、CD4⁺/CD8⁺、CD16⁺CD56⁺水平。另对比宫颈癌组中感染致病微生物者、阴道微生态失调者与未感染致病微生物者、阴道微环境正常者的CD3⁺、CD4⁺、CD8⁺、CD4⁺/CD8⁺、CD16⁺CD56⁺水平,采用Pearson相关性分析法分析宫颈癌组阴道乳酸杆菌定植密度与细胞免疫的相关性。结果宫颈癌组滴虫、支原体、衣原体、霉菌、细菌性阴道病、HPV感染检出率及阴道微生态失调率均高于正常宫颈组,宫颈癌组乳酸杆菌定植密度低于正常宫颈组,差异均有统计学意义(P<0.05),宫颈癌组淋菌、假丝酵母菌感染检出率与正常宫颈组比较差异均无统计学意义(P>0.05)；宫颈癌组CD3⁺、CD4⁺、CD4⁺/CD8⁺、CD16⁺CD56⁺水平均低于正常宫颈组,CD8⁺水平高于正常宫颈组,差异均有统计学意义(P<0.05)；宫颈癌组中滴虫、支原体、衣原体、霉菌、细菌性阴道病、HPV感染者及阴道微生态失调者CD3⁺、CD4⁺、CD4⁺/CD8⁺、CD16⁺CD56⁺均低于未感染者、阴道微环境正常者,CD8⁺高于未感染者、阴道微环境正常者,差异均有统计学意义(P<0.05)；宫颈癌患者乳酸杆菌定植密度与CD3⁺、CD4⁺、CD4⁺/CD8⁺、CD16⁺CD56⁺呈正相关性(P<0.05),与CD8⁺呈负相关性(P<0.05)。结论宫颈癌患者阴道致病微生物感染率较健康人群高,同时细胞免疫功能降低,增加罹患宫颈癌的风险。     

短期NaCl胁迫对不同小麦品种幼苗K+吸收和Na+、K+积累的影响

为研究盐胁迫下小麦幼苗生长及Na⁺、K⁺的吸收和积累规律,以中国春、洲元9369和长武134等3种耐盐性不同小麦品种为材料,采用非损伤微测技术检测盐胁迫2 d后的根系K⁺离子流变化,并对植株体内的Na⁺、K⁺含量进行测定。结果表明:短期(2d)盐胁迫对小麦生长有抑制作用,且对根系的抑制大于地上部,耐盐品种下降幅度小于盐敏感品种。盐胁迫下,小麦根际的 K⁺大量外流,盐敏感品种中国春K⁺流速显著高于耐盐品种长武134,最高可达15倍。小麦幼苗地上部分和根系均表现为Na⁺积累增加,K⁺积累减少,Na⁺/K⁺比随盐浓度增加而上升。中国春限Na⁺能力显著低于长武134,Na⁺/K⁺则显著高于长武134。综上所述,盐胁迫下造成小麦组织器官中Na⁺/K⁺比上升的主要原因是根系K⁺大量外流和Na⁺的过量积累,耐盐性不同的小麦品种间差异显著,并认为根系对K⁺的保有能力可能是作物耐盐性评价的一个重要指标。     

分子发动机研究进展

分子发动机是利用化学能/化学势进行机械作功的生物大分子,包括线性分子发动机与旋转式分子发动机两大类.它们参与了胞质运输、DNA复制、基因转录、ATP合成／水解等一系列重要生命活动过程.目前对于各种分子发动机的结构及作用机制的研究取得了一些重要进展.     

Na+-driven bacterial flagellar motors

Bacterial flagellar motors are the reversible rotary engine which propels the cell by rotating a helical flagellar filament as a screw propeller. The motors are embedded in the cytoplasmic membrane, and the energy for rotation is supplied by the electrochemical potential of specific ions across the membrane. Thus, the analysis of motor rotation at the molecular level is linked to an understanding of how the living system converts chemical energy into mechanical work. Based on the coupling ions, the motors are divided into two types; one is the H⁺-driven type found in neutrophiles such asBacillus subtilis andEscherichia coli and the other is the Na⁺-driven type found in alkalophilicBacillus and marineVibrio. In this review, we summarize the current status of research on the rotation mechanism of the Na⁺-driven flagellar motors, which introduces several new aspects in the analysis.     

Functional role of a conserved aspartic acid residue in the motor of the Na-driven flagellum from Vibrio cholerae

The flagellar motor consists of a rotor and a stator and couples the flux of cations (H⁺ or Na⁺) to the generation of the torque necessary to drive flagellum rotation. The inner membrane proteins PomA and PomB are stator components of the Na⁺-driven flagellar motor from Vibrio cholerae. Affinity-tagged variants of PomA and PomB were co-expressed in trans in the non-motile V. cholerae pomAB deletion strain to study the role of the conserved D23 in the transmembrane helix of PomB. At pH 9, the D23E variant restored motility to 100% of that observed with wild type PomB, whereas the D23N variant resulted in a non-motile phenotype, indicating that a carboxylic group at position 23 in PomB is important for flagellum rotation. Motility tests at decreasing pH revealed a pronounced decline of flagellar function with a motor complex containing the PomB-D23E variant. It is suggested that the protonation state of the glutamate residue at position 23 determines the performance of the flagellar motor by altering the affinity of Na⁺ to PomB. The conserved aspartate residue in the transmembrane helix of PomB and its H⁺-dependent homologs might act as a ligand for the coupling cation in the flagellar motor.     

Forced rotation of Na-driven flagellar motor in a coupling ion-free environment

Rotational characteristics of Na⁺-driven flagellar motor in the presence and absence of coupling ion were analyzed by electrorotation method. The motor rotated spontaneously in the presence of Na⁺, and the rotation accelerated or decelerated following the direction of the applied external torque. The spontaneous motor rotation was inhibited by removal of external Na⁺, however, the motor could be forcibly rotated by relatively small external torque applied by the electrorotation apparatus. The observed characteristic of the motor was completely different from that of ATP-driven motor systems, which form rigor bond when their energy source, ATP, is absent. The internal resistance of the flagellar motor increased significantly when the coupling ion could not access the inside of the motor, suggesting that the interaction between the rotor and the stator is changed by the binding of the coupling ion to the internal sites of the motor.     

Contribution of Many Charged Residues at the Stator-Rotor Interface of the Na+-Driven Flagellar Motor to Torque Generation in Vibrio alginolyticus

In torque generation by the bacterial flagellar motor, it has been suggested that electrostatic interactions between charged residues of MotA and FliG at the rotor-stator interface are important. However, the actual role(s) of those charged residues has not yet been clarified. In this study, we systematically made mutants of Vibrio alginolyticus whose charged residues of PomA (MotA homologue) and FliG were replaced by uncharged or charge-reversed residues and characterized the motilities of those mutants. We found that the members of a group of charged residues, 7 in PomA and 6 in FliG, collectively participate in torque generation of the Na⁺-driven flagellar motor in Vibrio. An additional specific interaction between PomA-E97 and FliG-K284 is critical for proper performance of the Vibrio motor. Our results also reveal that more charged residues are involved in the PomA-FliG interactions in the Vibrio Na⁺-driven motor than in the MotA-FliG interactions in the H⁺-driven one. This suggests that a larger number of conserved charged residues at the PomA-FliG interface contributes to the robustness of the Vibrio motor against mutations. The interaction surfaces of the stator and rotor of the Na⁺-driven motor seem to be more complex than those previously proposed in the H⁺-driven motor.     

Exchange of rotor components in functioning bacterial flagellar motor

The bacterial flagellar motor is a rotary motor driven by the electrochemical potential of a coupling ion. The interaction between a rotor and stator units is thought to generate torque. The overall structure of flagellar motor has been thought to be static, however, it was recently proved that stators are exchanged in a rotating motor. Understanding the dynamics of rotor components in functioning motor is important for the clarifying of working mechanism of bacterial flagellar motor. In this study, we focused on the dynamics and the turnover of rotor components in a functioning flagellar motor. Expression systems for GFP-FliN, FliM-GFP, and GFP-FliG were constructed, and each GFP-fusion was functionally incorporated into the flagellar motor. To investigate whether the rotor components are exchanged in a rotating motor, we performed fluorescence recovery after photobleaching experiments using total internal reflection fluorescence microscopy. After photobleaching, in a tethered cell producing GFP-FliN or FliM-GFP, the recovery of fluorescence at the rotational center was observed. However, in a cell producing GFP-FliG, no recovery of fluorescence was observed. The transition phase of fluorescence intensity after full or partially photobleaching allowed the turnover of FliN subunits to be calculated as 0.0007 s⁻¹, meaning that FliN would be exchanged in tens of minutes. These novel findings indicate that a bacterial flagellar motor is not a static structure even in functioning state. This is the first report for the exchange of rotor components in a functioning bacterial flagellar motor.     

Mutations inmotBSuppressible by Changes in Stator or Rotor Components of the Bacterial Flagellar Motor

Five proteins (MotA, MotB, FliG, FliM and FliN) may be involved in energizing flagellar rotation inEscherichia coli. To study interactions between the Mot proteins, and between them and the three Fli proteins of the switch-motor complex, we have isolated extragenic suppressors of dominant and partially dominantmotBmissense mutations. Four of the 13motBmutations yielded partially allele-specific suppressors. Of the suppressing mutations, 57 are in themotAgene, eight are infliG, and one is infliM; no suppressor was identified infliN. The prevalence of suppressors infliGsuggests that FliG interacts rather directly with the Mot proteins. The behaviour of cells in tethering and swarm assays indicates that themotAsuppressors are more efficient than thefliGorfliMsuppressors. Some of the suppressing mutations themselves confer distinctive phenotypes inmotB⁺cells. We propose a model in which mutations affecting residues in or near the putative peptidoglucan-binding region of MotB misalign the stator relative to the rotor. We suggest that most of the suppressors restore motility by introducing compensatory realignments in MotA or FliG.     

Steps in the Bacterial Flagellar Motor

The bacterial flagellar motor is a highly efficient rotary machine used by many bacteria to propel themselves. It has recently been shown that at low speeds its rotation proceeds in steps. Here we propose a simple physical model, based on the storage of energy in protein springs, that accounts for this stepping behavior as a random walk in a tilted corrugated potential that combines torque and contact forces. We argue that the absolute angular position of the rotor is crucial for understanding step properties and show this hypothesis to be consistent with the available data, in particular the observation that backward steps are smaller on average than forward steps. We also predict a sublinear speed versus torque relationship for fixed load at low torque, and a peak in rotor diffusion as a function of torque. Our model provides a comprehensive framework for understanding and analyzing stepping behavior in the bacterial flagellar motor and proposes novel, testable predictions. More broadly, the storage of energy in protein springs by the flagellar motor may provide useful general insights into the design of highly efficient molecular machines.     

Functional Transfer of an Essential Aspartate for the Ion-binding Site in the Stator Proteins of the Bacterial Flagellar Motor

Rotation of the bacterial flagellar motor exploits the electrochemical potential of the coupling ion (H⁺ or Na⁺) as its energy source. In the marine bacterium Vibrio alginolyticus, the stator complex is composed of PomA and PomB, and conducts Na⁺ across the cytoplasmic membrane to generate rotation. The transmembrane (TM) region of PomB, which forms the Na⁺-conduction pathway together with TM3 and TM4 of PomA, has a highly conserved aspartate residue (Asp24) that is essential for flagellar rotation. This residue contributes to the Na⁺-binding site. However, it is not clear whether residues other than Asp24 are involved in binding the coupling ion. We examined the possibility that loss of the negative charge of Asp24 can be suppressed by introduction of negatively charged residues in TM3 or TM4 of PomA. The motility defect associated with the D24N substitution in PomB could be rescued only by a N194D substitution in PomA. This result suggests that there must be a negatively charged ion-binding pocket in the stator complex but that the presence of a negatively charged residue at position 24 of PomB is not essential. A tandemly fused PomA dimer containing the N194D mutation either in its N-terminal or C-terminal half with PomB-D24N was functional, suggesting that PomB-D24N can form an ion-binding pocket with either subunit of PomA dimer. The findings obtained in this study provide important clues to the mechanism of ion binding in the stator complex.