干旱胁迫对高山离子芥试管苗抗氧化系统及其活性氧代谢的影响

该试验以高山离子芥试管苗(Chorispora bungeana)为试材,采用固液培养法,设置对照(不添加PEG-6000,CK),轻度干旱胁迫(5%PEG-6000)、中度干旱胁迫(20%PEG-6000)、重度干旱胁迫(40%PEG-6000)4个干旱处理水平,分析干旱胁迫对高山离子芥幼苗抗氧化系统、活性氧代谢等部分生理特征的影响,以揭示高山离子芥在干旱胁迫下的生理响应特征,为进一步探讨其对干旱环境的适应机制奠定基础。结果显示:(1)随着干旱胁迫程度的增加以及在各时间胁迫处理下,抗氧化酶SOD活性及可溶性糖含量显著升高,POD活性、丙二醛含量、CAT活性和APX活性均经历了先升后降的过程。(2)超氧阴离子(O-·2)的产生速率和过氧化氢(H2O2)的含量均显著升高;高山离子芥试管苗叶片相对电导率呈现出升-降-升的变化趋势。(3)相关分析结果显示,MDA与相对电导率、可溶性糖、SOD、APX、O-·2及H2O2呈极显著正相关关系,可溶性糖与SOD、POD、O-·2及H2O2呈极显著正相关关系;相对电导率以及保护酶系均与O-·2、H2O2呈极显著正相关关系。研究表明,高山离子芥具有较强的耐旱性,高山离子芥试管苗在响应干旱胁迫过程中,抗氧化酶系、活性氧代谢、脂质过氧化及渗透调节物等共同参与了高山离子芥试管苗对干旱胁迫的综合抗逆性形成,从而积极启动应对外界干旱环境的耐旱响应机制。     

彩叶草对模拟干旱胁迫的生理响应

采用盆栽试验,对彩叶草进行PEG-6000浓度为0(对照)、5%、10%、15%、20%(W/V)模拟干旱胁迫,研究在干旱胁迫下彩叶草的生长、渗透调节能力及抗氧化酶活性的变化。结果表明:与对照相比,随着PEG-6000浓度的增加,鲜质量、干质量、含水量、水势、根系脱氢酶活性、无机离子含量包括K+、Na+、Ca2+、Mg2+等均呈下降趋势;NO-3含量呈先下降后上升趋势;硝酸还原酶活性、可溶性蛋白含量、可溶性糖含量、超氧化物歧化酶活性均呈先上升后下降趋势;脯氨酸含量、游离氨基酸含量、过氧化物酶活性、过氧化氢酶活性、超氧阴离子(O-2·)产生速率、质膜透性则呈上升趋势。因此,模拟干旱胁迫对彩叶草生长有抑制作用,且随着PEG-6000浓度增加,其生长受抑制和水分胁迫程度加重;模拟干旱胁迫下,彩叶草不积累K+、Na+、Ca2+、Mg2+和NO-3等无机离子进行渗透调节,而积累脯氨酸、可溶性蛋白、可溶性糖、游离氨基酸等有机小分子物质进行渗透调节,但这4种小分子物质增加幅度不尽相同;轻度模拟干旱胁迫虽增强彩叶草抗氧化酶活性,但仍表现轻度的氧化伤害;重度模拟干旱胁迫加重彩叶草氧化伤害。研究结果可为彩叶草耐旱生理机制的研究积累资料,也为其节水型栽植和养护提供依据。     

2种玉米幼苗耐旱性生理机制研究

以白种皮(白玉米)和黄种皮(黄玉米)2个玉米栽培品种为材料,在水培条件下进行聚乙二醇(PEG-6000)模拟干旱胁迫处理,分析玉米叶片抗旱性相关生理特性和质膜H+-ATP酶活性的变化,探讨2种玉米幼苗耐旱性生理机制。结果表明:(1)在2%、5%、10%PEG-6000处理条件下,随处理浓度和时间的增加,2种玉米幼苗植株失水率上升,叶片蒸腾速率降低,气孔传导率下降;在所有相同处理条件下,白玉米植株失水率明显小于黄玉米,而叶片蒸腾速率和气孔传导率下降幅度明显大于黄玉米,即白玉米的耐旱性比黄玉米强。(2)在相同浓度PEG-6000处理下,白玉米叶片可溶性蛋白、可溶性糖含量、游离脯氨酸含量均高于黄玉米,它在干旱胁迫下的渗透调节能力强于黄玉米。(3)在抗氧化酶体系中,随着PEG-6000胁迫浓度的升高,2种玉米叶片CAT活性呈下降趋势,但白玉米CAT活性在2%和5%PEG-6000胁迫下均显著高于黄玉米,其叶片中H2O2含量显著低于黄玉米。(4)随着PEG-6000胁迫浓度的升高,白玉米叶片质膜H+-ATPase磷酸化水平及其与14-3-3蛋白的结合受到的抑制作用比黄玉米强,白玉米叶片质膜H+-ATPase活性比黄玉米叶片低,叶片气孔开度小于黄玉米,叶片蒸腾速率和气孔传导率均低于黄玉米,这可能是白玉米耐旱性强于黄玉米的一个重要机制。     

DCPTA对干旱胁迫下玉米幼苗生长和抗氧化酶系统的影响

以玉米自交系‘昌7-2’幼苗为材料,采用水培方式研究了模拟不同干旱胁迫程度(10%、12.5%、15%、17.5%、20%、22.5%、25%PEG-6000)及15%PEG-6000干旱胁迫下不同浓度(5、10、15、20、25、30mg/L)植物生长调节剂2-(3,4-二氯苯氧基)三乙胺(DCPTA)对玉米幼苗生长和抗氧化酶系统的影响,以筛选出玉米苗期抗旱性鉴定的适宜PEG-6000浓度,为玉米自交系苗期的抗旱性鉴定提供依据。结果表明:不同浓度PEG-6000处理后,玉米幼苗地上部和根部的干重、鲜重、叶片相对含水量及叶绿素(SPAD)含量均下降,叶片抗氧化酶SOD、POD、CAT的活性增强,丙二醛(MDA)含量升高,渗透调节物质可溶性蛋白、脯氨酸的积累量增加。且当PEG-6000浓度达15%时,以上各指标变化均与清水对照差异显著;在15%PEG-6000浓度模拟干旱胁迫下,不同浓度DCPTA处理均使玉米幼苗上述抗氧化酶活性增强,渗透调节物质含量增加,叶片相对含水量、叶绿素(SPAD)含量和生物量提高,而MDA含量降低,并以15和20mg/L浓度效果较佳。研究认为,室内水培条件下采用PEG-6000模拟干旱鉴定玉米苗期抗旱性的适宜浓度可初步确定为15%;DCPTA处理可促进干旱胁迫下玉米幼苗的生长,并通过提高抗氧化酶活性和渗透调节物质含量来增强其抗旱性,其适宜浓度为15和20mg/L。     

PEG-6000模拟干旱协迫对大蓟叶片生理特性的影响

以大蓟幼苗为试验材料,采用梯度浓度的聚乙二醇(PEG-6000,浓度为5%、10%、15%、20%、25%、30%)模拟干旱胁迫24h、48h和72h,测定大蓟叶片相对含水量(RWC)、丙二醛(MDA)含量、渗透调节物质含量及保护酶活性随胁迫时间的变化,探讨大蓟的耐旱性和抗旱生理机制。结果表明:(1)随干旱胁迫时间的延长和PEG-6000浓度增加,叶片RWC均呈降低趋势,最大降幅为55.86%,MDA含量均大幅度增加,最大增幅为186.21%。(2)随干旱胁迫时间延长,叶片可溶性糖与游离脯氨酸含量在PEG-6000浓度≤10%时逐渐升高,在大于10%时呈先升高后降低的变化趋势;而随PEG浓度增加,可溶性糖与游离脯氨酸含量在各时间点均呈先上升后下降趋势,可溶性糖峰值在处理24h、48h和72h依次出现在PEG浓度为20%、20%和10%时,游离脯氨酸峰值则依次出现在PEG浓度为20%、15%和15%条件下,两指标的最大增幅均出现在胁迫处理48h时PEG浓度分别为20%和15%,且分别为CK的4.7和10.7倍。(3)随PEG浓度增加,叶片保护性酶(SOD、POD和CAT)活性除SOD在24h时呈逐渐升高趋势外,其余时间点下均呈先升高后下降趋势,3种酶最大增幅依次为370.14%、248.91%和118.78%,前二者均出现在胁迫72h、15%PEG浓度下,后者出现在胁迫48h、10%PEG浓度下。研究认为,长时间(72h)、15%PEG-6000浓度胁迫下,大蓟具有较强的渗透调节能力和较高的酶活性,表现出较强的耐旱能力;若超过此胁迫浓度,大蓟渗透调节能力降低,酶活性减弱,含水量持续降低,MDA持续增加,生理代谢受到明显抑制。     

旱盐互作对冬小麦幼苗生长及其抗逆生理特性的影响

采用水培方法,以不同浓度的PEG-6000（0、8.3%、12.6%（W/V））和NaCl（0、25、50 mmol·L^-1）溶液模拟不同程度的干旱胁迫及盐胁迫,研究了盐分对干旱胁迫条件下冬小麦沧-6001幼苗生长及其抗逆生理特性的影响.结果表明：在8.3%或12.6% PEG-6000处理条件下,添加25 mmol·L^-1NaCl均使植株干物质积累和植株含水量比单一PEG处理增加,同时叶片可溶性糖和可溶性蛋白质含量增加,丙二醛和脯氨酸含量下降,植株各部位Na⁺含量升高、K⁺含量下降;在12.6% PEG-6000处理条件下,添加50 mmol·L^-1NaCl对植株的胁迫效应高于单一PEG处理.表明在干旱胁迫条件下,加入适量盐分可缓解干旱胁迫对冬小麦幼苗生长的抑制.     

外源亚精胺对Hg2+胁迫下凤眼莲抗氧化系统的影响

通过Hoagland溶液培养实验,研究了外源亚精胺(Spd)(0.1 mmol?L-1)对Hg2+(0、10、20、30和40μmol?L-1)胁迫下凤眼莲叶片细胞内叶绿素、可溶性糖、可溶性蛋白含量及抗氧化系统的调节作用.结果显示,(1)随Hg2+处理浓度的升高,各处理凤眼莲叶片叶绿素a(Chl a)和叶绿素b(Chl b)含量均先升后降,并均在10μmol?L-1时达到最高值,但外源Spd处理组显著高于相应对照.(2)各处理凤眼莲叶片可溶性蛋白含量随Hg2+处理浓度的升高也呈先升后降趋势,而可溶性糖含量则呈持续上升趋势,但外源Spd处理亦明显高于相应对照.(3)随Hg2+处理浓度的升高,抗氧化物质AsA和GSH含量及抗氧化酶SOD、CAT、POD、APX及GR活性也均呈先升后降的变化趋势,而外源Spd处理植株的含量和活性均显著高于相应对照.(4)各处理凤眼莲叶片的H2O2、MDA含量及O2?-产生速率随Hg2+处理浓度的升高均持续上升,但在外源Spd处理后均比对照组下降.研究表明,Hg2+胁迫使凤眼莲生长受到严重伤害,外源Spd可大幅度地提高其抗氧化物质含量和保护酶活性,从而增强凤眼莲抗Hg2+胁迫的能力.     

模拟干旱胁迫下马铃薯StNCED1表达量及与ABA含量的相关性分析

干旱缺水是限制马铃薯产量和品质的关键因素之一。ABA是干旱胁迫应答基因调控网络中的重要组成;9-顺式-环氧类胡萝卜素双加氧酶(NCED)是植物ABA生物合成途径的关键限速酶,该基因的表达模式直接影响ABA代谢。但干旱胁迫下有关马铃薯NCED基因表达与激素、表型间的相关分析的研究尚少。本研究克隆了马铃薯NCED1基因的全长c DNA序列,检测了4个马铃薯种质材料在不同模拟干旱(PEG-6000)胁迫强度处理下NCED1基因表达量、ABA含量及根系长度间的相关性。结果表明,St NCED1全长2181 bp,包含一个1800 bp的完整开放读码框,编码599个氨基酸。5%PEG-6000和15%PEG-6000模拟干旱胁迫4周后,4个品种均表现出随胁迫强度的加重植株生长缓慢、矮小,根系长度显著降低。干旱敏感型材料早大白中根系长度变化最大,ABA含量显著高于其他3个种质材料。CIP478.9、star和米拉3份种质材料中St NCED1表达量与对照植株有显著差异,且随胁迫强度增加而增大;早大白中St NCED1表达量表现出先降低后升高趋势。模拟干旱胁迫下,ABA含量与St NCED1表达量之间呈现出正相关关系(R0.7)。研究结果为解析马铃薯响应干旱胁迫的调节机制及其在抗旱种质资源筛选中的应用提供了基础数据。     

紫穗槐幼苗根系生理特性和解剖结构对PEG-6000模拟干旱的响应

采用PEG-6000模拟干旱胁迫处理,测定了紫穗槐幼苗根系的可溶性糖、可溶性蛋白质、丙二醛、游离脯氨酸含量及SOD、POD酶活性变化以及解剖结构特征,旨在比较不同干旱程度对紫穗槐幼苗根系生理指标、内部解剖结构的影响,探索紫穗槐幼苗对水分胁迫的适应能力,揭示紫穗槐幼苗根系对土壤水分胁迫的响应和调控机制。结果表明:丙二醛含量变化显示当PEG-6000溶液浓度超过50g/L以后,紫穗槐幼苗根的膜系统开始受到损伤,并在PEG-6000溶液浓度达到250g/L受损程度显著增强,达到了对照的1.6倍,同时启动渗透调节作用(游离脯氨酸含量显著增加),达到了对照的3.8倍,在PEG-6000溶液浓度低于200g/L时,紫穗槐幼苗根系中至少没有启动以游离脯氨酸为主的渗透调节过程。可溶性糖和可溶性蛋白质含量及SOD、POD酶活性的变化印证了胞内发生的生理代谢变化,在PEG-6000溶液浓度为200g/L时,可溶性糖含量仅为0.121mg/g,达到最低点,随后上升,当PEG-6000溶液浓度进一步增加到250g/L时,紫穗槐幼苗根系中的可溶性糖含量则迅速回升到0.64mg/g,为对照组的63.37%。可溶性蛋白质含量在低浓度PEG-6000溶液(50g/L)处理下即有明显反应,下降到对照的61.5%,随后呈波动性变化。SOD和POD活性对PEG-6000模拟干旱胁迫的响应规律类似,均对PEG-6000模拟干旱胁迫处理迅速响应且活性增加。当PEG-6000溶液浓度达到50g/L至100g/L时,抗氧化酶的合成量最高,而后活性下降。60d的PEG-6000模拟干旱胁迫处理影响了紫穗槐幼苗根系的生长发育,随着PEG-6000溶液浓度增加,维管柱的直径变大,木质部厚度增大,导管直径变小、但导管密度增加,当PEG-6000溶液浓度达到250g/L时,导管密度比对照组增加了41.3%,木质部厚度比对照组增加了91.5%。以上结果表明,PEG-6000模拟干旱胁迫处理下,不同胁迫程度紫穗槐内部生理和根系解剖结构变化不同,通过改变自身生理代谢和根系内部解剖结构,以适应土壤水分胁迫的逆境条件,来满足自身生长和发育的需求平衡。     

H2S对干旱胁迫下高山离子芥的作用研究

以高山冰缘植物高山离子芥（Chorispora bungeana）试管苗为实验材料,研究了0.3 mol·L^-1甘露醇模拟干旱胁迫响应过程中硫化氢（H₂S）调节高山离子芥的膜系统损伤程度、渗透调节物质和抗氧化酶系的作用,以及磷脂酶D（PLD）、活性氧（ROS）与H₂S信号分子在高山离子芥中响应干旱胁迫中的作用和可能存在的信号关系。结果显示：干旱胁迫下,外施H₂S供体NaHS显著降低高山离子芥电解质渗漏率及MDA含量、抑制ROS产生,提高渗透调节物质和抗氧化水平,从而增强高山离子芥的抗旱能力;干旱可诱导PLD活性、H₂S含量、ROS发生显著变化;当分别外施PLD下游产物PA与ROS供体H₂O₂均可促进干旱胁迫下H₂S的释放,当同时外施PA和ROS抑制剂DPI时对干旱胁迫下H₂S含量没有显著影响,当同时外施PLD抑制剂正丁醇与ROS抑制剂DPI则显著抑制干旱胁迫下H₂S含量的产生,表明干旱胁迫下,高山离子芥中ROS位于PLD的下游、H₂S的上游发挥作用。     

ATP/ADP exchange activity of gastric (H+ + K+)-ATPase

The ATP/ADP exchange is shown to be a partial reaction of the $\text{(}\text{H}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to $\text{P}{}^1$, $\text{P}{}^5$ -di(adenosine-5′) pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg²⁺ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration ($\text{K}{}_{0.5}\text{= 116 μ}\text{M}$) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg²⁺ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K⁺ as a function of pH and Mg²⁺.     

AutoFACT: AnAutomaticFunctionalAnnotation andClassificationTool

Background  

Assignment of function to new molecular sequence data is an essential step in genomics projects. The usual process involves similarity searches of a given sequence against one or more databases, an arduous process for large datasets.     

Characterization of cytochrome P-450SCC-containing liposomes

Purified cytochrome $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ from bovine adrenocortical mitochondria was incorporated into liposomes by the cholate-dilution method utilizing either dialysis or Sephadex gel filtration. Among synthetic phospholipids tested, dioleoylglycerophosphocholine showed the best stability during the incorporation of $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}\text{-}\text{containing liposomes}$ showed two major populations on the elution pattern of the Sepharose 4B gel filtration, and were seen at a diameter of 200–600 Å and its aggregated forms. When the cytochrome was incorporated into dioleoylglycerophosphocholine liposomes or cholesterol-free adrenocortical mitochondrial liposomes, $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was less stable than $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$. Liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ required cholesterol for its optimum reduction with adrenodoxin, adrenodoxin reductase, and NADPH in the presence of CO. About 70% of the total heme in the dioleoylglycerophosphocholine liposomes was reduced by the enzymatic reduction in the presence of cholesterol, indicating that 70% of the total molecules are exposed to the surface of the outer monolayer. In order to see the location of the heme in membrane, the dioleoylglycerophosphocholine-liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was subjected to $\text{p-}\text{chloromercuriphenyl sulfonic acid}$ treatment. This reagent destroyed the liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$. These results suggest that the heme is located in the proximity of the $\text{p-}\text{chloromercuriphenyl sulfonic acid}$ reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane.     

Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin

Showdomycin inhibited pig brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na⁺, K⁺, Mg²⁺ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1)$\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$, (2) $\text{Mg}{}^{\mathrm{2+}}$, $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}$ and none, (3) $\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{,}\text{Na}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}$ and $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}$, (4) $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}\text{,}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}$ and $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}\text{, (5)}\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ and ATP. The highest rate was obtained in the presence of Na⁺, Mg²⁺ and ATP. The apparent concentrations of Na⁺, Mg²⁺ and ATP for half-maximum stimulation of inhibition ($\text{K}{}_{0.5}{}^{\mathrm{<mtext>s</mtext>}}$) were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na⁺ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E₁, E₂, ES, E₁-P and E₂-P, i.e., E₂ has higher reactivity with showdomycin than E₁, while E₂-P has almost the same reactivity as E₁-P. We conclude that the reaction of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ proceeds via at least four kinds of enzyme form (E₁, E₂, E₁ · nucleotide and EP), which all have different conformations.     

A/California/7/2009与A/California/4/2009病毒感染力比较

目的甲型H1N1流感病毒A/California/7/2009与A/California/4/2009病毒序列比较同源性在99%以上,本实验旨在比较两株病毒感染BALB/c小鼠研究感染力强弱。方法分别将A/California/7/2009（CA7）与A/California/4/2009（CA4）两株病毒分别连续10倍稀释后,对4~6周龄雌性BALB/c小鼠经乙醚麻醉后进行滴鼻攻毒,每个稀释度接种10只实验小鼠,测定CA7 MLD50为101.24/0.05 mL,检测小鼠感染、致病的多项指标,观察期为14 d。结果相同TCID50的CA7和CA4病毒感染小鼠,CA4感染小鼠后14 d内死亡率为20%,而CA7感染小鼠后8 d内死亡率为100%。CA7 106TCID50感染的小鼠病理表现为重度弥漫性间质性肺炎,CA4 106TCID50感染的小鼠病理表现为中度-重度间质性肺炎。结论在相同条件下,CA7感染力明显强于CA4。     

Characterization of partially purified (Na+ + K+)-ATPase from porcine lens

The partial purification of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from pig lens has been achieved by treatment with deoxycholate followed by density gradient centrifugation. The specific activity of the final preparation, ranging from 300 to 500 nmol/h per mg protein, is increased approx. 100-fold compared to the homogenate. A parallel increase in $\text{p-}\text{nitrophenylphosphatase}$ activity is also observed. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals six major protein bands, one of which is the 93 kDa α subunit of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ which can be phosphorylated by reaction with [γ-³²P]ATP. A second band contains a glycoprotein which displays an apparent molecular weight of 51 000 and thus appears to be the β subunit of the enzyme. The enzyme is sensitive to ouabain with the $\text{I}{}_{50}$ for $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and $\text{p-}\text{nitrophenylphosphatase}$ inhibition being 1.2 and 1.3 μM, respectively. Several agents which inhibit $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from other tissues such as oligomycin, Ca²⁺, vanadate, $\text{N-}\text{ethylmaleimide}$, $\text{p-}\text{chloromercuribenzenesulfonic acid}$ (PCMBS) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) also inhibit the lens enzyme. Monovalent cations other than K⁺ are partially effective in activating the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$)-ATPase and $\text{p-}\text{nitrophenylphosphatase}$ activities. The K⁺ congeners were relatively more effective in supporting $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ compared to $\text{p-}\text{nitrophenylphosphatase}$ activity. Other kinetic properties of the lens enzyme are also comparable to those of the enzyme from other tissues. Utilizing the partially purified membrane bound enzyme, discontinuities in Arrhenius plots of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity, $\text{p-}\text{nitrophenylphosphatase}$ activity and fluoresence polarization of the fluidity probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), are observed near the physiological temperature of lens. The possible significance of these observations for the mechanism of cataract formation are discussed.     

An electron spin probe study of (Na+ + K+)-ATPase-Containing membranes

The modulating effect of membrane lipids on enzyme function has been described by several investigators. We have used the spin probe $\text{N-}\text{oxyl}\text{-4′,4′-}\text{dimethyloxazolidine}\text{-12-}\text{keto}$ methyl stearate (M 12-NSE) to study this interaction in ox brain membranes enriched with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. This methyl ester of stearic acid is practically insoluble in aqueous media, and consequently spectra of M 12-NSE-labelled preparations are free of “liquid lines”.At least two types of spectra may be obtained when ox brain microsomes are spin labelled with M 12-NSE, indicating the presence of two distinct binding sites. At one site the spin label is relatively unrestricted and gives rise to an isotropic spectrum. A second spectrum, which is obtained from spin label at another site, is similar to that which is observed after incorporation of M 12-NSE into phospholipid bilayers. This suggests that this latter site is within the core of the microsomal membrane.The two binding sites differ in their affinity for the spin probe. The low affinity site is both more abundant in crude preparations and is more easily removed by detergent treatment; spin labels at this site produce isotropic spectra. The high affinity sites are fewer in number and produce broad spectra. In addition these high affinity sites increase in concentration as the enzyme undergoes purification.The two sites are quite distinct in their sensitivity to ascorbic acid, the low affinity site showing a considerably greater rate of reduction by this agent.This study also demonstrates that the delipidation effects of sodium dodecyl sulfate and sodium deoxycholate on $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase-enriched}$ microsomes from ox brain are not identical.It is suggested that the two spin probe binding sites represent two different lipid domains, one of which is very closely associated with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme and may reflect a protein-directed phospholipid specificity for this enzyme.     

Annular and non-annular binding sites on the (Ca2+ + Mg2+)-ATPase

Quenching of the fluorescence of the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.     

Comparison of parameters estimated from A/Ci and A/Cc curve analysis

The parameters estimated from traditional A/C _i curve analysis are dependent upon some underlying assumptions that substomatal CO₂ concentration (C _i) equals the chloroplast CO₂ concentration (C _c) and the C _i value at which the A/C _i curve switches between Rubisco- and electron transport-limited portions of the curve (C _i-t) is set to a constant. However, the assumptions reduced the accuracy of parameter estimation significantly without taking the influence of C _i-t value and mesophyll conductance (g _m) on parameters into account. Based on the analysis of Larix gmelinii’s A/C _i curves, it showed the C _i-t value varied significantly, ranging from 24 Pa to 72 Pa and averaging 38 Pa. t-test demonstrated there were significant differences in parameters respectively estimated from A/C _i and A/C _c curve analysis (p<0.01). Compared with the maximum ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation rate (V_cmax), the maximum electron transport rate (J_max) and J_max/V_cmax estimated from A/C _c curve analysis which considers the effects of g _m limit and simultaneously fits parameters with the whole A/C _c curve, mean V_cmax estimated from A/C _i curve analysis (V_cmax-C _i) was underestimated by 37.49%; mean Jmax estimated from A/C _i curve analysis (J_max-C _i) was overestimated by 17.8% and (J_max-C _i)/(V_cmax-C _i) was overestimated by 24.2%. However, there was a significant linear relationship between V_cmax estimated from A/C _i curve analysis and V_cmax estimated from A/C _c curve analysis, so was it J_max (p<0.05).     

Regulation of rat brain (Na+ + K+)-ATPase activity by cyclic AMP

The interaction between the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and the adenylate cyclase enzyme systems was examined. Cyclic AMP, but not 5′-AMP, cyclic GMP or 5′-GMP, could inhibit the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme present in crude rat brain plasma membranes. On the other hand, the cyclic AMP inhibition could not be observed with purified preparations of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme. Rat brain synaptosomal membranes were prepared and treated with either NaCl or cyclic AMP plus NaCl as described by Corbin, J., Sugden, P., Lincoln, T. and Keely, S. ((1977) J. Biol. Chem. 252, 3854–3861). This resulted in the dissociation and removal of the catalytic subunit of a membrane-bound cyclic AMP-dependent protein kinase. The decrease in cyclic AMP-dependent protein kinase activity was accompanied by an increase in $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity. Exposure of synaptosomal membranes containing the cyclic AMP-dependent protein kinase holoenzyme to a specific cyclic AMP-dependent protein kinase inhibitor resulted in an increase in $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme activity. Synaptosomal membranes lacking the catalytic subunit of the cyclic-AMP-dependent protein kinase did not show this effect. Reconstitution of the solubilized membrane-bound cyclic AMP-dependent protein kinase, in the presence of a neuronal membrane substrate protein for the activated protein kinase, with a purified preparation of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$, resulted in a decrease in overall $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity in the presence of cyclic AMP. Reconstitution of the protein kinase alone or the substrate protein alone, with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has no effect on $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity in the absence or presence of cyclic AMP. Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme, there appeared to be a decrease in the Na⁺-dependent phosphorylation of the Na⁺-ATPase enzyme, while the K⁺-dependent dephosphorylation of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ was unaffected.