植物体内重要的信号分子--H2O2

越来越多的证据表明,植物体内的H2O2作为信号分子发挥作用.在病原、诱发因子和激素应答中是调节细胞程序性死亡的关键因子.H2O2在环境胁迫防御反应中的信号作用也得到证实.已知H2O2直接调节无数基因的表达,其中有些基因与植物防御和超敏反应有关.H2O2还与其它信号系统特别是激素信号相互作用,是激素介导的信号传导通路上的上游或下游组分;更重要的是H2O2还影响和修饰其它第二信使如钙信号的作用,在H2O2信号和钙信号之间发生众多的交互作用且这两种信号分子都调节植物对多种胁迫的交互耐性.此外,现已广泛地认识到与H2O2相关的氧还状态调节是调整细胞活动的关键因子.本文主要概括和讨论了H2O2在不同生物过程中的信号作用.     

作为叶绿体H2O2分解系统关键酶的抗坏血酸过氧化物酶

介绍叶绿体中H2O2分解系统的共建酶──抗坏血酸过氧化物酶的基本特性、在植物中的分布、分子生物学及其分解H2O2的作用机制。     

植物细胞的氧化猝发和H_2O_2的信号转导

概述了植物细胞氧化猝发的特性、产生机理、生理作用以及H2 O2 信号转导途径及其对基因表达的调控等的研究进展     

拟南芥血红蛋白1(AtGLB1)与过氧化氢的相互作用

拟南芥的血红蛋白1(AtGLB1)属于非共生的血红蛋白。在低氧胁迫中对植物细胞中过氧化氢(H2O2)内稳态的维持起了很重要的作用。为了检测AtGLB1与H2O2能否直接相互作用,我们扩增了拟南芥的AtGLB1基因,并将其克隆到原核表达质粒pET32a中,测序鉴定正确后转化大肠杆菌BL21。IPTG诱导目的蛋白表达后,镍离子亲和层析柱(Ni2+-NTA)纯化了靶蛋白。体外表达的氧合的AtGLB1能与H2O2直接相互作用。因此,与H2O2反应可能是AtGLB1清除低氧胁迫下产生的H2O2的一种方式。     

SB202190 调节蚕豆保卫细胞中SA 诱导H2O2 产生

运用激光共聚焦扫描技术, 在p38 MAP激酶专一抑制剂SB202190处理下, 探索植物促分裂原活化蛋白激酶(mitogenactivated protein kinase, MAP激酶)介导蚕豆(Vicia faba)保卫细胞中H2O2为代表的活性氧(reactive oxygen species, ROS)信号机制, 发现: p38 MAP激酶专一抑制剂SB202190处理没有导致蚕豆保卫细胞中H2O2和Ca2+探针荧光强度增强, 与水杨酸 (salicylic acid, SA) 或脱落酸 (abscisic acid, ABA) 迅速加强2种探针荧光强度形成鲜明对比; 而该抑制剂分别与SA和ABA共同处理, 前者H2O2探针荧光强度没有增加, 而后者荧光强度仍然能够增加; 而进一步使用Ca2+螯合剂BAPTA和SB202190 ＋SA共同处理, H2O2探针荧光强度没有增加。这些结果初步表明: 无论胞质Ca2+浓度高低, SB202190调节蚕豆保卫细胞中SA诱导H2O2产生, 但是不调节植物逆境信使分子ABA 此类的反应。因此推测, 植物细胞中可能有类似动物和酵母细胞中的p38MAP激酶类, 并可能专一调节植物保卫细胞中H2O2信号通路。据我们所知, 这是首次报道SB202190和SA共同调节植物保卫细胞中ROS信号过程。     

ECS1参与调节CO2诱导的拟南芥气孔关闭和H2O2积累

CO2浓度升高可以诱导植物叶片气孔关闭,提高植物对高浓度CO2的适应性.但植物如何感知CO2浓度变化并启动气孔关闭反应的分子机制至今仍不十分清楚.利用高通量、非侵入的远红外成像技术,建立了拟南芥(Arabidopsis thaliana)气孔对CO2浓度变化反应相关的突变体筛选技术,筛选出对环境CO2浓度敏感的拟南芥突变体ecs1.遗传学分析表明,ecs1 为单基因隐性突变体,突变基因ECS1编码一个跨膜钙离子转运蛋白.与野生型拟南芥相比,360 μL·L-1CO2可引起ecs1突变体叶片温度上升和气孔关闭,ecs1突变体对900 μL·L-1CO2长时间处理具有较强的适应性.进一步的实验表明,360 μL·L-1CO2即可诱导ecs1突变体叶片积累较高浓度的H2O2,而900 μL·L-1CO2才能够诱导野生型拟南芥叶片积累H2O2.因此,ECS1可能参与调节高浓度CO2诱导的拟南芥气孔关闭和H2O2产生,H2O2可能作为第二信号分子介导CO2诱导拟南芥气孔关闭的反应.     

植物顺乌头酸酶及其生理功能

介绍了植物顺乌头酸酶酶学特性、分子生物学及其生理功能的研究进展,对其在一氧化氮(NO)介导的植物抗病信号转导和NO、SA以及H2O2对话中的可能作用也作了概述。     

H_2O_2-NOX系统：一种植物体内重要的发育调控与胁迫响应机制

以H2O2为中心的活性氧（reactive oxygen species,ROS）的产生是动植物发育与响应外界生物与非生物胁迫的普遍特征,其在生理和分子2个水平上调控植物的发育和对外界胁迫的响应,并与一系列信号转导过程相关联。作为关键的ROS产生酶,质膜NADPH氧化酶（plasma membrane NADPH oxidase,PM-NOX）在植物应对各种生物和非生物胁迫中具有重要作用,被广泛认为是胁迫条件下植物细胞ROS产生并积累的主要来源。该文简要综述了近年来人们在植物细胞ROS产生、清除、生理功能以及PM-NOX酶的结构特征与功能等方面的研究进展,并认为H2O2-NOX系统是一种植物体内普遍存在的重要发育调控与胁迫响应机制。     

二甲基联苯胺检测植物叶片中H2O2方法的改良

对原二甲基联苯胺(3,3-diaminobenzidine,DAB)为染色剂检测植物叶片H2O2的组织化学方法进行改良研究。结果表明,与原方法相比,改良后的方法主要是在染色后不用体积分数为95%乙醇脱色,而用2%戊二醛 4%多聚甲醛溶液对1cm2左右叶片片段进行固定,固定24h后用冰冻切片机切成12μm薄片,显微镜下观察并拍照。用改良DAB方法对玉米叶片进行观察,结果显示,在水分胁迫4h时,玉米叶片的主脉及叶肉细胞叶绿体上均可观察到H2O2的积累,而原DAB方法观察不到叶肉细胞叶绿体上的H2O2积累。进一步用CeCl3染色的细胞化学方法验证,其结果与改良后的组织化学方法研究结果一致。研究表明,改良后的组织化学方法优于原方法,而且对植物组织中H2O2的化学定位可靠。     

H2O2和水杨酸对陈化马铃薯切片交换呼吸途径影响的比较

外源5.0mmol/L H2O2和0.1mmol/L水杨酸（salicylic acid,SA)处理均可明显提高陈化24h的马铃薯切片的交替呼吸途径容量（Valt)及其与总呼吸的比值（Valt/Vt)。应用交替氧化酶的单克隆抗体进行Western杂交的结果表明,H2O2和SA处理均可明显提高陈化马铃薯切片中交替氧化酶的表达水平。用氧同位素分辨法研究,结果表明：H2O2处理对陈化马铃薯切片中交替呼吸途径的实际运行没有影响,而SA处理对交替呼吸途径的实际运行具有明显的促进作用。上述结果表明,H2O2和SA对植物组织交替呼吸途径的影响存在差异,二均可促进交替氧化酶的表达从而诱导交替呼吸途径容量的发生,但H2O2不影响其实际运行,而SA还可同时诱导其实际运行。     

Hydrogen peroxide as an inductor of uncultivable state of Vibrio cholerae eltor in an experiment

The influence of hydrogen peroxide on the dynamics of transition into uncultivable state (UCS) and on the reversion of V. cholerae and their subcultures, resistant to hydrogen peroxide, was studied. The transition of the initial cultures in river and distilled water into UCS took place earlier than that in resistant to hydrogen peroxide variants. The capacity for reversion to hydrogen peroxide resistant subcultures preserved, on the average, 2 - 3 times longer. An increase in the level of hydrogen peroxide in uncultivable populations was found to be 2.7 - 4.4 times. Subcultures, resistant to hydrogen peroxide, in the vegetative form had lower characteristics of peroxide concentrations than in uncultivable form (UCF), but somewhat higher than in initial variants. In revertants the concentration of hydrogen peroxide was lower in UCF, but somewhat higher than in vegetative cultures. The dynamics of the formation of UCF by cholera vibrios, with different degree of stability to the action of hydrogen peroxide, the accumulation of hydrogen peroxide in uncultivable populations, the deceleration of transition into uncultivable forms, an accumulation of hydrogen peroxide and an increase in the time of the reversion of clones, resistant to hydrogen peroxide, made it possible to suggest that the accumulation of hydrogen peroxide was possible to make an essential contribution to the formation of UCF of cholera vibrios in an experiment.     

Characterization of an inducible oxidative stress response in Vitreoscilla C1

Vitreoscilla becomes resistant to killing by hydrogen peroxide and heat shock when pretreated with nonlethal levels of hydrogen peroxide. The pretreated Vitreoscilla cells (60 microM hydrogen peroxide for 120 min) significantly increased survival of the lethal dose of 20 mM hydrogen peroxide or heat shock (22 degrees C --> 37 degrees C). This indicates the existence of an adaptive response to oxidative stress. However, cells pretreated with 60 microM hydrogen peroxide became nonresistant to a lethal dose of a menadione. This result shows that hydrogen peroxide does not induce cross-resistance to menadione in Vitreoscilla. Furthermore, Vitreoscilla treated with hydrogen peroxide, heat shock, and menadione showed a change in the protein composition, as monitored by a two-dimensional gel analysis. During adaptation to hydrogen peroxide, 12 proteins were induced. Also, 18 new proteins synthesized in response to heat shock were detected by a 2-D gel analysis. The redox-cycling agents also elicited the synthesis of 6 other proteins that were unseen with hydrogen peroxide.     

The effect of hydrogen peroxide on spores of Clostridium bifermentans.

The effect of hydrogen peroxide on the germination, colony formation and structure of spores of Clostridium bifermentans was examined. Treatment with 0.35 M-hydrogen peroxide increased the germination rate at 25 degrees C but increasing the temperature or concentration of hydrogen peroxide decreased both the germination rate and colony formation. The presence of Cu2+ increased the lethal effect of hydrogen peroxide on colony formation as much as 3000-fold. Pre-incubation of spores with Cu2+ before treatment with hydrogen peroxide produced a similar increase, but this could be eliminated by washing the spores with dilute spores--apparently from the coat--and treatment with dithiothreitol, which also removes spore-coat protein, increased the lethal effect of hydrogen peroxide 500-fold, suggesting that spore-coat protein has a protective effect against hydrogen peroxide.     

A kinetic analysis of the catalase activity of myeloperoxidase

The predominant physiological activity of myeloperoxidase is to convert hydrogen peroxide and chloride to hypochlorous acid. However, this neutrophil enzyme also degrades hydrogen peroxide to oxygen and water. We have undertaken a kinetic analysis of this reaction to clarify its mechanism. When myeloperoxidase was added to hydrogen peroxide in the absence of reducing substrates, there was an initial burst phase of hydrogen peroxide consumption followed by a slow steady state loss. The kinetics of hydrogen peroxide loss were precisely mirrored by the kinetics of oxygen production. Two mols of hydrogen peroxide gave rise to 1 mol of oxygen. With 100 microM hydrogen peroxide and 6 mM chloride, half of the hydrogen peroxide was converted to hypochlorous acid and the remainder to oxygen. Superoxide and tyrosine enhanced the steady-state loss of hydrogen peroxide in the absence of chloride. We propose that hydrogen peroxide reacts with the ferric enzyme to form compound I, which in turn reacts with another molecule of hydrogen peroxide to regenerate the native enzyme and liberate oxygen. The rate constant for the two-electron reduction of compound I by hydrogen peroxide was determined to be 2 x 10(6) M(-1) s(-1). The burst phase occurs because hydrogen peroxide and endogenous donors are able to slowly reduce compound I to compound II, which accumulates and retards the loss of hydrogen peroxide. Superoxide and tyrosine drive the catalase activity because they reduce compound II back to the native enzyme. The two-electron oxidation of hydrogen peroxide by compound I should be considered when interpreting mechanistic studies of myeloperoxidase and may influence the physiological activity of the enzyme.     

一株口腔链球菌新种—寡发酵链球菌产过氧化氢特性的研究

从健康人口腔中分离的寡发酵链球菌(Streptococcus oligofermentans)能够产生大量的过氧化氢,可能具有抑制致病菌的潜力。为了研究该细菌产过氧化氢的特性,检测了其在不同生长时期和从不同底物产过氧化氢的能力。结果表明,寡发酵链球菌从对数生长早期就开始产过氧化氢,在对数生长后期及稳定期过氧化氢产量达到最高,随后下降。在PYG培养基中,寡发酵链球菌所产的过氧化氢主要来源于大豆蛋白胨和酵母提取物;而代谢终产物乳酸也可作为过氧化氢产生的底物。对3种可能与过氧化氢生成有关的氧化酶的酶活测定表明,寡发酵链球菌具有乳酸氧化酶(LOX)及NADH氧化酶(NOX)的活性,说明其过氧化氢的产生主要依赖于这两种酶的活力。     

Intracellular acidification triggered by mitochondrial-derived hydrogen peroxide is an effector mechanism for drug-induced apoptosis in tumor cells

We recently showed that two photoproducts of merocyanine 540, C2 and C5, triggered cytochrome C release; however, C5 was inefficient in inducing caspase activity and apoptosis in leukemia cells, unlike C2. Here we show that HL60 cells acidified upon exposure to C2 but not C5. The intracellular drop in pH and caspase activation were dependent upon hydrogen peroxide production, and were inhibited by scavengers of hydrogen peroxide. On the contrary, caspase inhibitors did not block hydrogen peroxide production. In turn, increased intracellular hydrogen peroxide concentration was downstream of superoxide anion produced within 2 h of exposure to C2. Inhibitor of NADPH oxidase diphenyleneiodonium neither inhibited superoxide production nor caspase activation triggered by C2. However, exposure of purified mitochondria to C2 resulted in significantly increased superoxide production. Furthermore, cytochrome C release from isolated mitochondria induced by C2 was completely inhibited in the presence of scavengers of hydrogen peroxide. Contrarily, scavenging hydrogen peroxide had no effect on the cyclosporin A-sensitive mitochondrial permeability transition induced by C5. Our data suggest a scenario where drug-induced hydrogen peroxide production induces intracellular acidification and release of cytochrome C, independent of the inner membrane pore, thereby creating an intracellular environment permissive for caspase activation.     

黄腐酸引发软骨细胞产生过氧化氢及硒的抑制作用

为探讨黄腐酸（FA）能否直接刺激软骨细胞产生活性氧以及硒能否抑制由此产生的活性氧,采用二氯荧光素双酯（DCFH－DA）作为软骨细胞产生过氧化氢的探针,用流式细胞技术定量地检测了在FA作用下软骨细胞产生的过氧化氢,并同时检测了硒存在时的过氧化氢含量．发现FA不但能够刺激软骨细胞产生过氧化氢（P＜0．05）,并与FA浓度相关,随FA浓度增大,产生过氧化氢增多,当FA浓度为100mg／L时软骨细胞产生过氧化氢达到最大,之后再增大FA浓度,过氧化氢的产生量减少;硒的存在对FA刺激软骨细胞产生过氧化氢有抑制作用．     

Jak2 tyrosine kinase mediates oxidative stress-induced apoptosis in vascular smooth muscle cells

In vascular smooth muscle cells, Jak2 tyrosine kinase becomes activated in response to oxidative stress in the form of hydrogen peroxide. Although it has been postulated that hydrogen peroxide-induced Jak2 activation promotes cell survival, this has never been tested. We therefore examined the role that Jak2 plays in vascular smooth muscle cell apoptosis following hydrogen peroxide treatment. Here, we report that Jak2 tyrosine kinase activation by hydrogen peroxide is required for apoptosis of vascular smooth muscle cells. Upon treatment of primary rat aortic smooth muscle cells with hydrogen peroxide, we observed laddering of genomic DNA and nuclear condensation, both hallmarks of apoptotic cells. However, apoptosis was prevented by either the expression of a dominant negative Jak2 protein or by the Jak2 pharmacological inhibitor AG490. Moreover, expression of the proapoptotic Bax protein was induced following hydrogen peroxide treatment. Again, expression of a dominant negative Jak2 protein or treatment of cells with AG490 prevented this Bax induction. Following Bax induction by hydrogen peroxide, mitochondrial membrane integrity was compromised, and caspase-9 became activated. In contrast, in cells expressing a Jak2 dominant negative we observed that mitochondrial membrane integrity was preserved, and no caspase-9 activation occurred. These data demonstrate that the activation of Jak2 tyrosine kinase by hydrogen peroxide is essential for apoptosis of vascular smooth muscle cells. Furthermore, this report identifies Jak2 as a potential therapeutic target in vascular diseases in which vascular smooth muscle cell apoptosis contributes to pathological progression.     

The effect of hydrogen peroxide on CO2 fixation of isolated intact chloroplasts

Low concentrations of hydrogen peroxide strongly inhibit CO₂ fixation of isolated intact chloroplasts (50% inhibition at 10⁻⁵ M hydrogen peroxide). Addition of catalase to a suspension of intact chloroplasts stimulates CO₂ fixation 2–6 fold, indicating that this process is partially inhibited by endogenous hydrogen peroxide formed in a Mehler reaction.

The rate of CO₂ fixation is strongly increased by addition of Calvin cycle intermediates if the catalase activity of the preparation is low. However, at high catalase activity addition of Calvin cycle intermediates remains without effect. Obviously the hydrogen peroxide formed at low catalase activity leads to a loss of Calvin cycle substrates which reduces the rate of CO₂ fixation.

3-Phosphoglycerate-dependent O₂-evolution is not influenced by hydrogen peroxide at a concentration (5 · 10⁻⁴ M) which inhibits CO₂ fixation almost completely. Therefore the inhibition site of hydrogen peroxide cannot be at the step of 3-phosphoglycerate reduction. Dark CO₂ fixation of lysed chloroplasts in a hypotonic medium is not or only slightly inhibited by hydrogen peroxide (2.5 · 10⁻⁴ M), if ribulose-1,5-diphosphate, ribose 5-phosphate or xylulose 5-phosphate were added as substrates. However, there is a strong inhibition of CO₂ fixation by hydrogen peroxide, if fructose 6-phosphate together with triose phosphate are used as substrates. This indicates that hydrogen peroxide interrupts the Calvin cycle at the transketolase step, leading to a reduced supply of the CO₂-acceptor ribulose 1,5-diphosphate.     

THE DECOMPOSITION OF HYDROGEN PEROXIDE BY LIVER CATALASE

1. The velocity of decomposition of hydrogen peroxide by catalase as a function of (a) concentration of catalase, (b) concentration of hydrogen peroxide, (c) hydrogen ion concentration, (d) temperature has been studied in an attempt to correlate these variables as far as possible. It is concluded that the reaction involves primarily adsorption of hydrogen peroxide at the catalase surface. 2. The decomposition of hydrogen peroxide by catalase is regarded as involving two reactions, namely, the catalytic decomposition of hydrogen peroxide, which is a maximum at the optimum pH 6.8 to 7.0, and the "induced inactivation" of catalase by the "nascent" oxygen produced by the hydrogen peroxide and still adhering to the catalase surface. This differs from the more generally accepted view, namely that the induced inactivation is due to the H₂O₂ itself. On the basis of the above view, a new interpretation is given to the equation of Yamasaki and the connection between the equations of Yamasaki and of Northrop is pointed out. It is shown that the velocity of induced inactivation is a minimum at the pH which is optimal for the decomposition of hydrogen peroxide. 3. The critical increment of the catalytic decomposition of hydrogen peroxide by catalase is of the order 3000 calories. The critical increment of induced inactivation is low in dilute hydrogen peroxide solutions but increases to a value of 30,000 calories in concentrated solutions of peroxide.