棉花P-type ATPases基因的克隆及表达分析

为了探明棉花P-type ATPases基因在植物不同组织和多种逆境条件下的表达情况,利用棉花黄萎病菌的P-type ATPases蛋白序列,在GenBank中搜索棉花EST序列,得到2个同源的棉花EST片段,结合RACE和Genome walking获得了这个基因的完整读码框序列,其开放阅读框长度为3 570 bp,编码1 190个氨基酸,与毛果杨、蓖麻的同源性达86%左右,将其命名为Gbpatp。洋葱表皮亚细胞定位观察结果显示,Gbpatp编码的蛋白分布在细胞膜上。RT-PCR技术检测组织表达特异性分析表明,P-type ATPases在茎和棉絮中的表达量较高,在种子中低水平表达,在叶片和花蕾中表达量极低,说明Gbpatp可能与棉花较成熟的器官茎、棉絮和种子的生长发育密切相关。在逆境胁迫中发现,干旱处理后Gbpatp的表达强烈,并且随处理时间延长表达量逐渐增加;重金属Cu2+处理24 h后Gbpatp表达量升高,但48 h急剧下降;Gbpatp在低温处理下也有轻微表达。激素诱导后发现,Gbpatp对赤霉素和脱落酸均有响应,随胁迫时间延长其表达量仍能维持在一定水平。

咪唑安定对离体人红细胞膜ATPases及脂质流动性的影响

目的 探讨咪唑安定对离体人红细胞膜ATPases及脂质流动性的影响。方法 将不同浓度咪唑安定 (A组 0浓度 ,B组 4ng/ml,C组 40ng/ml,D组 40 0ng/ml,E组 4μg/ml,F组 40 μg/m)与离体人红细胞孵育后 ,提取红细胞膜 ,测各组红细胞膜ATPases及脂质流动性 ,并进行比较。结果 采用双因素方差分析 ,各组间ATPases活性无显著差异 ,F组膜脂质流动性显著下降 (P <0 .0 1)。结论 咪唑安定在临床使用浓度范围内 ,对离体人红细胞膜ATPases及脂质流动性影响无显著影响 ,从而推断咪唑安定在临床使用中安全范围较广。

稻瘟菌Magnaporthe oryzae P-ATPases基因家族分析

利用TCDB(Transporter Classification Database)网站数据库中的P-ATPases氨基酸序列对稻瘟菌全基因组表达序列(Coding Sequence,CDS)数据库进行搜索和分析,共发现23个P-ATPases基因,进化树分析表明这23个基因分属于4个家族和7个亚家族。构建了本地ESTs数据库,通过P-ATPases基因CDS序列与EST序列比对分析发现,在这些基因中有20个存在EST同源体,另外3个基因没有发现EST同源体,因此这20个基因是真实P-ATPases基因的可能性更高。运用MEME程序分析了这些P-ATPases蛋白结构域的基序,有6种基序在90%以上的基因氨基酸序列中出现,属保守基序。对这23个P-ATPases基因GC含量的分析表明,它们的平均GC含量在0.519-0.628之间,稍高于稻瘟菌整个基因组GC平均含量(0.516),同时这些基因内各区段GC含量变化不大,没有明显的梯度变化。本文结果为下一步深入研究稻瘟菌中P-ATPases基因家族的功能奠定了基础。

非胰岛素依赖型糖尿病微量白蛋白尿患者红细胞膜ATPases活力的变化

测定非胰岛素依赖型糖尿病(NIDDM)微量白蛋白尿患者20例及NIDDM无微量白蛋白尿患者20例和正常人20例红细胞膜ATPases活力。结果NIDDM微量白蛋白尿患者Na^+-K+ATPase,Ca^(2+)+ATPase活力明显低于正常人(P分别<0.05及0.001),Mg^(2+)ATPase活力无明显改变(P>0.05).NIDDM无微量白蛋白尿患者Ca^(2+)ATPase活力也低于正常人(P<0.01),但不及微量白蛋白尿组明显;无白蛋白尿组Na^+-K^+ATPase活力虽有下降,但P>0.05,Mg^(2+)ATPese活力无明显变化(P>0.05).

粉防已碱对大鼠心肌微粒体ATPases的作用

体外在常规反应条件下,粉防已碱(Tet)对大鼠心肌微粒体Na^+,K^+-ATPase活性无明显影响,但浓度依赖性抑制Mg^(2+)-ATPase(IC_(50)=179μmol/L).Tet 10和100μmol/L使哇巴因(Oua)抑制Na^+,K^+-ATPase的量效曲线平行右移.Tet 100μmol/L可显著提高低K^+或高Ca^(2+)浓度时的Na^+,K^+-ATPase活性,但未能明显增加低Na^+浓度时该酶的活性.动力学分析提示Tet 100μmol/L增加Na^+,K^+-ATPase对ATP的亲和力,但不改变其最大反应速度。

吗啡哌替啶芬太尼对离体人红细胞膜ATPases活性的影响

目的：比较研究吗啡、哌替啶、芬太尼对离体人红细胞膜ＡＴＰａｓｅｓ活性的影响。方法：将不同浓度的药物分别与人红细胞孵育，以孔雀绿－磷钼酸复合比色法测定ＡＴＰａｓｅｓ活性。结果：随着药物浓度的增加，吗啡对Ｎａ＋，Ｋ＋－ＡＴＰａｓｅ活性有增强作用（Ｐ＜０．０５），芬太尼则呈明显地抑制作用（Ｐ＜０．０５），哌替啶仅具有轻微抑制作用；吗啡能明显抑制Ｍｇ２＋－ＡＴＰａｓｅ和Ｃａ２＋，Ｍｇ２＋－ＡＴＰａｓｅ活性（Ｐ＜０．０１或Ｐ＜０．０５），但哌替啶和芬太尼对Ｍｇ２＋－ＡＴＰａｓｅ活性均无影响；这两种药物仅轻微地抑制Ｃａ２＋，Ｍｇ２＋－ＡＴＰａｓｅ活性。结论：应用大剂量吗啡或芬太尼时，需注意他们对人红细胞膜ＡＴＰａｓｅｓ活性的影响。

Changes in erythrocyte membrane ATPases and plasma lipid peroxides in upper abdominal surgery under intravenous procaine-balanced anesthesia

AIM To observe the changes in erythrocyte membrane ATPases and plasma lipid peroxides (LPO) patients with in abdominal surgery under intravenous procaine-balanced anesthesia.METHODS By determining the ATPase activities of erythrocyte membrane, effects of upper abdominal surgery under intravenous procaine-balanced anesthesia on the function of erythrocytes were observed in 15 patients undergoing cholecystectomy and gastrectomy (5 males and 10 females, aged 45.9±10.20 years and weighed 60.60kg±11.93kg). All patients were free from severe renal, hepatic, pulmonary, cardiac, metabolic and endocrinological diseases and acute infection for at least 2 weeks before surgery. Patients receiving any drug known to affect carbohydrate metabolism prior to anesthesia were excluded from the study.RESULTS Erythrocyte membrane Na+, K+-ATPase, Mg2+-ATPase, Ca2+, Mg2+-ATPase activities were not significantly changed 60min-90min after incision as compared with 30min before anesthesia, but were decreased markedly 10min and 24 hours after completion of operation (P＜0.01). Plasma lipid peroxides (LPO) were increased significantly 24 hours after surgery (P＜0.01) following an initially marked but transient reduction. Plasma LPO changes were not correlated with erythrocyte membrane ATPase activities, r=-0.0396, -0.0097 and 0.4383, respectively (P＞0.05).CONCLUSION Abdominal surgical trauma under intravenous procaine-balanced anesthesia may be associated with the decreased ATPase activities of erythrocyte membrane and increased LPO in plasma.

Genome-Wide Survey and Expression Analysis of P_(1B)-ATPases in Rice, Maize and Sorghum

P1B-type ATPase ion pumps that transport heavy metal ions across cellular membranes are essential for plant growth and development. To date, a genomic comparison overview of the family in rice, maize and sorghum is not yet available. In this study, a total of 31 heavy metal P1B-type ATPase （HMA） genes were identified, including 9 in rice, 11 each from maize and sorghum. They were classified into two distinct subfamilies based on their sequence composition and phylogenetic relationship. Four pairs of HMA genes were expanded via gene duplication with tandemly duplicated. Comprehensive analyses were performed to investigate the expression profiles of HMA genes in various tissues by using quantitative real-time PCR. Some HMA members exhibited abundant and tissue-specific expression patterns. Moreover, most of the genes were found to be differentially expressed under the Cu/Cd treatment. This study will facilitate further studies on P1B-type ATPase family and provide valuable hints for the functional validation in rice, maize and sorghum.

Plasma membrane Ca^(2+)-ATPases in the nervous system during development and ageing

Calcium signaling is used by neurons to control a variety of functions,including cellular differentiation,synaptic maturation,neurotransmitter release,intracellular signaling and cell death.This review focuses on one of the most important Ca2+regulators in the cell,the plasma membrane Ca2+-ATPase(PMCA),which has a high affinity for Ca2+and is widely expressed in brain.The ontogeny of PMCA isoforms,linked to specific requirements of Ca2+ during development of different brain areas,is addressed, as well as their function in the adult tissue.This is based on the high diversity of variants in the PMCA family in brain,which show particular kinetic differences possibly related to specific localizations and functions of the cell. Conversely,alterations in the activity of PMCAs could lead to changes in Ca2+homeostasis and,consequently,to neural dysfunction.The involvement of PMCA isoforms in certain neuropathologies and in brain ageing is also discussed.

Stabilization of membrane bound ATPases and lipid peroxidation by carotenoids from Chlorococcum humicola in Benzo(a)pyrene induced toxicity

Objective:To identify the alleralinn of the membrane polenlial and llie effect of carolenoid extracts from Chlorococcum hnmicola(C.humicola) on membrane hound ATPases and lipid peroxidation.Methods:The lolal carotenoids were extracted from C.humicola.Four groups of Swiss albino mice were treated as control,Benzo(a)pyrene[B(a)P],total carotenoids,B(a)P+ total caralenoids respectively for a period of 60 days.Membrane lipid peroxidation and ATPases(Total ATPases,Ca^(2+)-ATPases.Mg^(2+)-ATPases.Na^+K^+- ATPasei were determined in lung,liver and erythrocyte samples.Results:The activity of lolal ATPase was found to be significantly increased in the B(a)P treated liver and lung tissue.Erythrocyte membrane also showed higher ATPase activity which was significantly reverted on total carolenoid treatment.Conclusions: It can be concluded that the changes in membrane potential favour the functional deterioration of physiological system.The overall findings demonstrates that the animals post treated with carolenoid extract from C.humicola may maintains the alterations in membrane bound ATPase and lipid peroxidation in tissues against the carcinogenic chemical and hence aid in establishing the membrane potential action.Then-fore C.humicola can be further extended to exploits its possible application for various health benefits as neulraceulicals and food additives.

质膜H^(+)-ATPase基因对铝胁迫下水稻硝态氮吸收的 调控作用

为探讨细胞质膜ATP酶(PM H^(+)-ATPase)基因对铝胁迫下水稻(Oryza sativa L.)吸收硝态氮(NO_(3)^(-)-N)的调控,本试验以2个水稻品种峰1A和峰1A优5为研究对象,以无铝处理幼苗为对照,分析铝胁迫下水稻根尖PM H^(+)-ATPase活性、H^(+)-泵活性、H^(+)通量、PM H^(+)-ATPase基因家族(OsA1~OsA10)表达水平、PM H^(+)-ATPase和14-3-3蛋白的互作水平以及NO_(3)^(-)-N吸收量。结果表明,与对照相比,铝胁迫下2个水稻品种根尖PM H^(+)-ATPase活性、H^(+)-泵活性和根尖H^(+)通量下降,PM H^(+)-ATPase和14-3-3蛋白的互作水平下降。铝胁迫抑制了OsA1、OsA5、OsA7和OsA8基因的表达,其中OsA7的表达水平显著下调,铝胁迫下峰1A和峰1A优5的OsA7表达量仅为对照组的21%和39%。NO_(3)^(-)-N吸收量较对照下降,分别为对照的81%和85%。综上,PM H^(+)-ATPase基因的表达水平影响水稻对NO_(3)^(-)-N的吸收能力,OsA1、OsA5、OsA7和OsA8在铝胁迫下水稻吸收NO_(3)^(-)-N过程中起关键的调控作用。本研究结果可为增强酸铝条件下水稻吸收NO_(3)^(-)-N的能力,促进水稻生长及增产提供理论依据。

Ca^(2+)-ATPase参与植物耐盐性调控的研究进展

盐胁迫下细胞质Ca^(2+)浓度升高,细胞会激活Ca^(2+)调节的靶酶或者与Ca^(2+)高度亲和的受体蛋白,其中,与Ca^(2+)高度亲和的受体蛋白中,植物钙泵(Ca^(2+)-ATPase)是P型ATP酶,包含内质网Ca^(2+)-ATPases与质膜Ca^(2+)-ATPas‐es,通过主动运输将Ca^(2+)从细胞质转移到质外体或细胞器。大量研究表明,植物的耐盐性在很大程度上与其维持钙泵即Ca^(2+)-ATPase活性的能力有关。多种植物Ca^(2+)-ATPase对盐胁迫表现出敏感性,并受到外源Ca^(2+)的保护,表明外源钙处理与Ca^(2+)-ATPase活性可能在盐胁迫下的细胞内钙稳态和信号转导中起重要作用。该研究概述了植物Ca^(2+)-ATPase类型、结构与性质,亚细胞定位Ca^(2+)-ATPase及外源钙与亚细胞定位Ca^(2+)-ATPase参与植物耐盐调控研究进展,重点对质膜、液泡膜、核膜、内质网及高尔基体Ca^(2+)-ATPases参与植物耐盐调控的研究进展进行了综述,并提出展望。该研究为了解植物耐盐性生理及分子机制提供帮助,同时为作物耐盐栽培提供新思路。

马氏珠母贝V-ATPase-d基因序列特征及其与耐低温性状的关系

液泡ATP酶(V-ATPase)在生物应对各种环境压力时发挥重要功能,为探究VATPase在马氏珠母贝低温适应过程中的作用,实验克隆了马氏珠母贝的V-ATPase-d基因,采用qRT-PCR技术研究了低温胁迫下Pm-V-ATPase-d表达量的变化,并筛选和比较了该基因在耐低温选育系(low temperature resistant line,R)F_(3)和北部湾野生群体(Beibu Gulf wild population,W)外显子区的SNP位点。结果显示,Pm-V-ATPase-d总长为1473 bp,开放阅读框(ORF)为1140bp,编码379个氨基酸,具有ATP典型的结构域PfamvATPsynt_AC39。Motif分析及三级结构预测结果表明,Pm-V-ATPase-d具有较高的保守性,其在系统进化树中与长牡蛎聚为一支。组织荧光定量结果显示,Pm-V-ATPase-d在所检测的组织中均有表达,在肝胰腺、性腺和鳃组织中表达量较高。在低温胁迫条件下,Pm-VATPase-d基因的表达量随着时间的延长呈现先上升后下降的趋势,且低温组的表达量在5 d内均显著高于对照组,这表明Pm-V-ATPase-d可能参与了马氏珠母贝对温度胁迫的响应;对Pm-V-ATPase-d外显子区的SNP分析共得到35个SNP,其中34个为同义突变,只有一个位点为非同义突变,26个SNP在W和R群体的不同基因型和等位基因之间具有显著差异,单倍型连锁不平衡分析结果显示,Pm-V-ATPase-d基因SNPs可形成6个单倍体块,14种单倍型,其中单倍型GAAT、CGC、TC、TG、AG与马氏珠母贝耐低温性状显著相关。研究表明,Pm-V-ATPase-d可能是参与调节马氏珠母贝低温适应过程中的候选基因,本研究可为马氏珠母贝对低温的适应机制提供研究基础,筛选出的与抗低温性状相关的SNPs及单倍型可应用于分子辅助育种。

Nucleotide Contribution to the Functioning of SERT, Na /K ATPase and GPCR Proteins

Purine nucleotides are crucial for the effective operation of cell membrane proteins maintaining the neurotransmitter responses of 5-HT. Major protein targets in the treatment of depression include SERT, N/K ATPase and GPCR. Each protein target is responsive to a specific complement of drugs: antidepressants (SERT), lithium and cardiogenic steroids (N/K ATPase), 5-HT receptor ligands (GPCR). Computational software is useful for comparing molecular similarity within ligand-ligand and ligand-nucleotide structures. Previous studies demonstrate that GPCR ligands of different pharmacologic classes display relative molecular similarity to nucleotide structures. The current study applies this methodology to compound structures modulating SERT and N/K ATPase receptors. Minimum energy conformers of SERT antagonists demonstrate relative molecular similarity to the structural template of GTP nucleotide. GTP template fits of 5-HT and psilocin are similar, whereas a SERT-like fit is one of several for the ketamine structure. Endogenous and pharmaceutical modulators of Na/K ATPase relate to adenine nucleotide. The fits of cardiogenic steroids to a cGMP template demonstrate similarities and differences between compounds. Relative molecular similarity within the structures of hormones, drugs and nucleotides has implications for neurotransmitter transport and cell signal transduction processes.

盐度渐变对日本囊对虾非特异性免疫酶、ATPase酶和抗氧化酶活力的影响

为研究盐度渐变对日本囊对虾的机体免疫的影响,实验设置了4个盐度梯度分别为26、22、18和14,统计了各盐度下日本囊对虾累计死亡率,检测了日本囊对虾体内血清总蛋白(TP)含量、碱性磷酸酶(AKP)和酸性磷酸酶(ACP)、血清ATP酶(Na^(+)/K^(+)-ATPase和T-ATPase)、超氧化物歧化酶(SOD)和多酚氧化酶(PPO)的动态变化。结果显示,盐度渐变胁迫下各盐度组累计死亡率的大小为14>18>22>26,血清蛋白含量随胁迫强度的增加和胁迫时间的延长呈下降趋势,后随着盐度回升而上升。24~72 h ACP和AKP活力表现为盐度组14<18<22<26,且随时间的延长呈下降趋势,Na^(+)/K^(+)-ATPase和T-ATPase活力显示出盐度梯度差异显著,14盐度组显著高于其它盐度组(P<0.05),SOD活力与PPO活力受盐度胁迫强度和胁迫时间的影响显著,盐度胁迫强度越强,SOD活力与对照组的差异越显著(P<0.05),血清PPO活力的变化趋势越明显。日本囊对虾机体免疫受盐度胁迫显著,研究结果为日本囊对虾的健康养殖提供重要的理论指导及借鉴。

Plasma membrane Ca^(2+)-ATPases:Targets of oxidative stress in brain aging and neurodegeneration

The plasma membrane Ca2+-ATPase(PMCA)pumps play an important role in the maintenance of precise levels of intracellular Ca2+[Ca2+]i,essential to the functioning of neurons.In this article,we review evidence showing age-related changes of the PMCAs in synaptic plasma membranes(SPMs).PMCA activity and protein levels in SPMs diminish progressively with increasing age. The PMCAs are very sensitive to oxidative stress and undergo functional and structural changes when exposed to oxidants of physiological relevance.The major signatures of oxidative modification in the PMCAs are rapid inactivation,conformational changes,aggregation, internalization from the plasma membrane and proteolytic degradation.PMCA proteolysis appears to be mediated by both calpains and caspases.The predominance of one proteolytic pathway vs the other,the ensuing pattern of PMCA degradation and its consequence on pump activity depends largely on the type of insult,its intensity and duration.Experimental reduction of PMCA expression not only alters the dynamics of cellular Ca2+ handling but also has a myriad of downstream conse-quences on various aspects of cell function,indicating a broad role of these pumps.Age-and oxidation-related down-regulation of the PMCAs may play an important role in compromised neuronal function in the aging brain and its several-fold increased susceptibility to neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease,and stroke.Therapeutic approaches that protect the PMCAs and stabilize[Ca2+]i homeostasis may be capable of slowing and/or preventing neuronal degeneration.The PMCAs are therefore emerging as a new class of drug targets for therapeutic interventions in various chronic degenerative disorders.

西北农林科技大学园艺学院王晓峰教授团队发现BR受体StBRI1通过增强马铃薯PM H+-ATPase活性促进块茎发育

园艺学院王晓峰教授团队近日在《The Plant Cell》期刊发表了题为Brassinosteroid Receptor StBRI1 Promotes Tuber Development by Enhancing Plasma Membrane H+-ATPase Activity in Potato的研究论文,揭示了BR信号通过StBRI1-PM H+-ATPase(PHA2)单元模块调控马铃薯块茎形成的分子机制,这些结果对于马铃薯高产分子育种具有一定的理论价值。

水杨酸对镉胁迫下葡萄根系质膜ATPase和自由基的影响

【目的】研究水杨酸对镉胁迫下葡萄根系特性的影响,探讨缓解葡萄镉伤害的途径。【方法】以‘泽香’葡萄扦插苗为试材,在水培条件下,研究水杨酸预处理对氯化镉胁迫下葡萄根系活力、质膜H+-ATPase和Ca2+-ATPase活性以及活性氧和一氧化氮(NO)生成的影响。【结果】0.10mmol·L-1氯化镉能够提高根系活力和质膜Ca2+-ATPase活性;1.0mmol·L-1氯化镉明显促进根系超氧阴离子()、H2O2和NO的生成,显著抑制根系活力及质膜H+-ATPase和Ca2+-ATPase活性。50μmol·L-1水杨酸显著降低1.0mmol·L-1氯化镉处理下根系、H2O2和NO的生成,阻止根系活力及质膜H+-ATPase和Ca2+-ATPase活性下降,而随着浓度升高至200μmol·L-1,水杨酸的这种作用减弱。【结论】水杨酸缓解葡萄根系镉伤害的适宜浓度为50μmol·L-1;在该浓度下,水杨酸通过降低镉胁迫下自由基的产生而减轻镉对葡萄根系活力和质膜ATPase的损伤。

我国玉米品种更替过程中根系生理特性的演进 Ⅰ.根系活性与ATPase活性的变化

我国 195 0 s～ 1990 s期间主要推广玉米品种 (Zea mays L .)根系活力随播种后天数的变化可用高斯模型作较好地模拟 ,根系活力随生育进程的推进先增后降 ,呈单峰曲线变化 ,在籽粒灌浆期 (播种后 6 7～ 74天 )达到高峰 ;上层 (0～ 2 0 cm )根系活力高于下层。随玉米品种更替根系活力增强 ,在 4 0～ 10 0 cm根层和生育后期更为突出。对根系生长的高斯方程积分 ,求得“根系活力日积”(RVDI) ,当代品种各生育阶段的 RVDI远高于 1970 s和 195 0 s品种 ,且在灌浆期间 (播种后 6 7天至 90天 )这种差别最大。根系中可溶性蛋白含量、 ATPase活性随品种更替呈递增趋势 ,且在距离植株 10～ 15 cm的横向空间内活性提高最大 ,当代品种在 10～ 2 5 cm的横向空间内

高温胁迫对花生幼苗光合速率、叶绿素含量、叶绿体Ca^(2+)-ATPase、Mg^(2+)-ATPase及Ca^(2+)分布的影响

将水培后盆栽的花生幼苗,置于培养箱42℃高温培养,定时测定幼苗叶光合速率、叶绿素含量和叶绿体Ca2+-ATPase、Mg2+-ATPase的相对活性,并观察幼叶细胞内Ca2+分布的变化。试验结果表明:高温胁迫过程中,光合速率及叶绿素含量都随处理时间的延伸而下降,并呈显著正相关;叶绿体Ca2+-ATPase和Mg2+-ATPase高温胁迫过程中相对活性呈先升后降趋势,Ca2+-ATPase热敏性高于Mg2+-ATPase;高温胁迫过程中,Ca2+具有从胞外转运到胞质内和叶绿体中的趋势,Ca2+能够稳定高温胁迫下叶肉细胞膜和叶绿体的超微结构。