Microcystin-RR-induced accumulation of reactive oxygen species and alteration of antioxidant systems in tobacco BY-2 cells

Microcystins are cyclic heptapeptide hepatoxins produced by cyanobacteria. It has been shown that microcystins have adverse effects on animals and on plants as well. Previous researches also indicated that microcystins were capable of inducing oxidative damage in animals both in vivo and in vitro. In this study, tobacco BY-2 suspension cell line was applied to examine the effects of microcystin-RR on plant cells. Cell viability and five biochemical parameters including reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxide (GPX) and peroxide dismutase (POD) were investigated when cells were exposed to 50 mg/L microcystin-RR. Results showed that microcystin-RR evoked decline of the cell viability to approximately 80% after treating for 144 h. ROS levels, POD and GPX activities of the treated cells were gradually increased with a time dependent manner. Changes of SOD and CAT activities were also detected in BY-2 cells. After 168 h recovery, ROS contents, POD, GPX and CAT activities returned to normal levels. These results suggest that the microcystin-RR can cause the increase of ROS contents in plant cells and these changes led to oxidant stress, at the same time, the plant cells would improve their antioxidant abilities to combat mirocystin-RR induced oxidative injury. (c) 2005 Elsevier Ltd. All rights reserved.

NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane Subunit gp91(phox) was dose-dependent. Meanwhile, the cytoplasmic subunit p47(phox) was translocated to the cell membrane and localized with p22(phox) and gp91(phox) to form reactive NADPH oxidase. Our data Suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.

Simultaneous and ultrarapid determination of reactive oxygen species and reduced glutathione in apoptotic leukemia cells by microchip electrophoresis

A microchip electrophoresis method coupled with laser-induced fluorescence (LIF) detection was established for simultaneous determination of two kinds of intracellular signaling molecules (reactive oxygen species, ROS, and reduced glutathione, GSH) related to apoptosis and oxidative stress. As the probe dihydrorhodamine-123 (DHR123) can be converted intracellularly by ROS to the fluorescent rhodamine-123 (Rh123), and the probe naphthalene-2,3-dicarboxaldehyde (NDA) can react quickly with GSH to produce a fluorescent adduct, rapid determination of Rh-123 and GSH was achieved on a glass microchip within 27 s using a 20 mm borate buffer (pH 9.2). The established method was tested to measure the intracellular ROS and GSH levels in acute promyelocytic leukemia (APL)-derived NB4 cells. An elevation of intracellular ROS and depletion of GSH were observed in apoptotic N134 cells induced by arsenic trioxide (AS(2)O(3)) at low concentration (1-2 mu m). Buthionine sulfoximine (BSO), in combination with AS(2)O(3) enhanced the decrease of reduced GSH to a great extent. The combined treatment of AS(2)O(3) and hydrogen peroxide (H2O2) led to an inverse relationship between the concentrations of ROS and GSH obtained, showing the proposed method can readily evaluate the generation of ROS, which occurs simultaneously with the consumption of the inherent antioxidant.

Microcystin-RR induced apoptosis in tobacco BY-2 suspension cells is mediated by reactive oxygen species and mitochondrial permeability transition pore status

When tobacco BY-2 cells were treated with 60 mu g/mL MC-RR for 5 d, time-dependent effects of MC-RR on the cells were observed. Morphological changes such as abnormal elongation, evident chromatin condensation and margination, fragmentation of nucleus and formation of apoptotic-like bodies suggest that 60 mu g/mL MC-RR induced rapid apoptosis in tobacco BY-2 cells. Moreover, there was a significant and rapid increase of ROS level before the loss of mitochondrial membrane potential (Delta Psi(m)) and the onset of cell apoptosis. Ascorbic acid (AsA), a major primary antioxidant, prevented the increase of ROS generation, blocked the decrease in Delta Psi(m) and subsequent cell apoptosis, indicating a critical role of ROS in serving as an important signaling molecule by causing a reduction of Delta Psi(m) and MC-RR-induced tobacco BY-2 cell apoptosis. In addition, a specific mitochondrial permeability transition pores (PTP) inhibitor, cyclosporin A (CsA), significantly blocked the MC-RR-induced ROS formation, loss of Delta Psi(m), as well as cell apoptosis when the cells were MC-RR stressed for 3 d, suggesting that PTP is involved in 60 mu g/mL MC-RR-induced tobacco cell apoptosis signalling process. Thus, we concluded that the mechanism of MC-RR-induced apoptosis signalling pathways in tobacco BY-2 cells involves not only the excess generation of ROS and oxidative stress, but also the opening of PTP inducing loss of mitochondrial membrane potential. (C) 2007 Published by Elsevier Ltd.

Determination of some reactive oxygen species scavengers based on the peroxidase-oxidase oscillator

This paper reports a new method for detection of ROS scavengers including superoxide dismutase, ascorbic acid and glutathione based on a 'probe' of peroxidase-oxidase biochemical oscillator. The oscillation period and amplitude change with different concentrations of scavengers. The linear ranges of superoxide dismutase, ascorbic acid and glutathione are respectively 1.56 x 10(-4)-1.56 x 10(-3) mg mL(-1), 1.75 x 10(-7) -1.75 x 10(-5) mol L-1 and 9.38 x 10(-7) -7.5 x 10(-5) mol L-1. The selectivity, linearity and precision for superoxide dismutase, ascorbic acid, and glutathione are presented and discussed. The results compared well with other standard methods for determination of superoxide dismutase, ascorbic acid and glutathione. Some possible steps in the overall reaction mechanisms are discussed.

Reactive oxygen species and antioxidant enzymes activity of Anabaena sp PCC 7120 (Cyanobacterium) under simulated microgravity

It was found that reactive oxygen species in Anabaena cells increased under simulated microgravity provided by clinostat. Activities of intracellular antioxidant enzymes, such as superoxide dismutase, catalase were higher than those in the controlled samples during the 7 days' experiment. However, the contents of gluathione, an intracellular antioxidant, decreased in comparison with the controlled samples. The results suggested that microgravity provided by clinostat might break the oxidative/antioxidative balance. It indicated a protective mechanism in algal cells, that the total antioxidant system activity increased, which might play an important role for algal cells to adapt the environmental stress of microgravity. (C) 2004 Elsevier Ltd. All rights reserved.

Stress Resistance and Disease Resistance in Seaweeds:The Role of Reactive Oxygen Metabolism

It has become clear that the last 15-20 years that the immediate effect of a wide range of environmental stresses,and of infection,on vascular plants is to increase the information of reactive oxygen species(ROS) and to impose oxidative stress on the cells.Since 1994,sufficient examples similar responses in a broad range of marine macroalgae have been decribed to show that reactive oxygen metabolism also underlies the mechanisms by which seaweeds respond(and become resistant) to stress and infection.Desiccation,freezing,low temperatures,high light,ultraviolet radiation,and heavy metals all tend to result in a gradual and continued buildup of ROS because photosynthesis is inhibited and excess energy results in the formation of singlet oxygen.The response to other stresses (infection or oligosaccharides which signal that infection is occurring,mechanical stress,hyperosmotic shock) is quite different-a more rapid and intence,but short-lived production of ROS ,discribed as an "oxidative burst"-which is attributed to activation of NADPHoxidases in the plasma membrane.Seaweed species that are able to survive such stresses or resist infection have the capacity to remove the ROS through a high cellular content of antioxidant compounds,or a high activity of antioxidant enzymes.

由农杆菌介导将葡萄糖氧化酶基因转入水稻

过氧化氢（Hydrogen peroxide,H2O2）是植物和病原微生物互作中快速合成的一种早期活性氧类（reactive oxygen species, ROS ），它在植物受到病原微生物侵染后引发的一系列防御反应中起着非常重要的作用，因此通过外源基因导入提高植物体内过氧化氢的含量，可以增强植物的广谱抗病性。葡萄糖氧化酶（glucose oxidase, GO）可以催化β-D-葡萄糖氧化生成过氧化氢和葡萄糖酸，此酶已在数种细菌和真菌中检测到，但在植物和动物中仍未发现。为了尝试将此酶应用于水稻广谱抗病基因工程，本研究将葡萄糖氧化酶基因插入具有潮霉素抗性选择标记的双元载体pCAMBIA1301，新构建为水稻高效表达载体pCAG1301。将此质粒导入根癌农杆菌（Agrobacterium tumefaciens ）菌株LBA4404后,转化粳稻(Oryza sativa )品种日本晴(Nipponbare)成熟胚来源的愈伤组织和幼胚，并由筛选出的潮霉素抗性愈伤组织分化再生植株。对所得到的潮霉素抗性植株的Southern杂交分析表明GO基因已整合到受体基因组,为单拷贝或双拷贝插入。利用过氧化氢与淀粉-碘化钾反应显蓝色的特性检测到了转基因植株产生的过氧化氢，证实GO基因表达产生的葡萄糖氧化酶已经在水稻中发挥功能，这是将GO基因转入单子叶植物的首例报道。 基于过氧化氢诱导的植物防御反应没有种属专一性的优点，可以预期所得转基因水稻植株很可能对水稻的多种病原菌具有良好的抗性。已完成的抗病性鉴定表明，所得转基因水稻植株对稻瘟病具有良好的抗性。

外源物质对酵母拮抗菌生活力的影响

　　近年来，酵母拮抗菌在水果采后病害防治中展示了良好的应用前景。然而，在实际应用中,酵母拮抗菌在逆境条件下会因为发生凋亡或细胞损伤而引起生活力的下降，最终导致拮抗菌抑病能力降低。研究酵母拮抗菌生活力下降的规律，提高酵母拮抗菌的生产效率，减少剂型加工过程中的细胞损伤，增强其对逆境条件的耐受力是增加或稳定生防制剂防治效果的有效途径。本文主要研究酵母拮抗菌正常培养过程中生活力下降的规律，筛选剂型加工过程中对酵母拮抗菌具有保护作用的化学物质，并对酵母拮抗菌的培养条件进行了优化。主要研究结果如下： 　　1. 在正常培养过程中，酵母拮抗菌Rhodotorula glutinis和Cryptococcus laurentii中细胞染色质凝集或细胞膜破损的发生一般在6天以后。外源加入的N-乙酰半胱氨酸及硅酸钠等物质在超过一定浓度时会加速酵母菌的死亡。 　　2. 在不同的液体悬浮制剂中，对R. glutinis而言，使用磷酸缓冲液（PBS）悬浮时保护效果最好；而C. laurentii悬浮在NYDB培养基中或海藻糖、乳糖溶液中时的生活力最高。 　　3. 以10 %葡萄糖 + 5 %脱脂牛奶作保护剂，可以有效地保持酵母拮抗菌C. laurentii冻干制剂的生活力，配合使用的保护效果高于它们单独使用时的保护效果。添加1 mM N-乙酰半胱氨酸能更好地保持拮抗菌制剂在常温保存过程中的生活力，这可能与这种还原性物质缓解了细胞内活性氧的积累有关。 　　4. 不同酵母拮抗菌对不同碳、氮源的利用能力有明显差异。在9种不同的碳源和10种不同的氮源中，Pichia membranefaciens能够最有效利用的碳、氮源是葡萄糖、果糖和多价胨，而Candida guilliermondii的最佳碳源和氮源分别是果糖和肉蛋白胨。

脂筏极化参与活性氧介导的白杄花粉管顶端生长

质膜上存在一种富含甾醇物质的液相有序膜脂微区，被称作脂筏 (lipid rafts或lipid microdomains)。这种小的膜微区可以通过在质膜上的侧向移动，聚集形成较大的片状结构，而与微区相关联的蛋白可以通过脂筏的这种聚合作用而凝聚分布于特定的亚细胞结构上。脂筏区域在真菌和动物质膜上具极性分布，并参与细胞的极性形态建成和运动。最近，通过生物化学研究证实，脂筏也存在于植物细胞，然而迄今为止，脂筏与植物细胞极性生长相关联的直接功能证据尚未见报道。 NADPH氧化酶 (NOX，在植物中又称为 Rboh) 产生的活性氧 (Reactive oxygen species, ROS) 可能是调控植物细胞（包括花粉管、根毛和墨角藻合子等）极性生长的通用信号机制。花粉管作为研究细胞极性控制的一种理想模式系统，已被许多信号转导调控研究所采用。在本研究中，我们使用一种能螯合甾醇类物质的多烯类抗生素filipin破坏脂筏结构，以探讨脂筏极化对ROS介导的白杄花粉管极性生长的作用。 我们首次在白杄 (Picea meyeri) 花粉管上应用一种全新的苯乙烯基染料di-4-ANEPPDHQ，成功地在活体细胞上观察到脂筏在花粉管生长顶端的极性分布模式。通过脂筏和甾醇在质膜上的相似定位清楚表明：在花粉管极性生长过程中，存在富含甾醇类物质的质膜微区在花粉管生长顶端的极化现象。 氮蓝四唑（NBT）的还原和二氯二氢荧光素（H2DCF）的氧化均显示，在活跃生长的花粉管顶端区域存在一个以顶端为基底的陡峭ROS梯度，从而进一步验证了ROS在细胞极性生长过程中的信号作用。此外，我们还发现在生长花粉管的亚顶端位置有另一类性质的活性氧组分存在，该ROS组分与线粒体的能量代谢相关。研究结果首次揭示，在快速生长的花粉管中同时存在两类性质不同的ROS组分。 ROS是一种寿命很短而且容易扩散的分子，NADPH氧化酶产生的ROS信号在细胞伸长位点的准确定位是调控极性生长的必要条件。免疫共定位实验显示，NOX成簇极化分布于花粉管的生长顶端。使用filipin进行甾醇的螯合会破坏膜的异质性，干扰NOX簇在生长顶端的定位，减少了顶端的ROS形成，消弱了胞质Ca2+ 浓度梯度，进而抑制了花粉管的顶端生长。 在纯化质膜的基础上，我们使用Triton去垢剂处理结合Optiprep密度梯度离心，分离纯化了抗去垢剂抽提的质膜微区 (Detergent-resistant microdomains, DRMs)。通过免疫印迹分析证实，NADPH氧化酶部分地存在于DRMs中。非变性胶活性实验证明，该酶需要脂筏定位来保持酶活性。因此我们认为，在正常的细胞极性生长中，脂筏招募并运载NADPH氧化酶到花粉管的生长顶端，并为NOX及其活性亚基的有效互作提供了适宜的微环境，由此保证了NOX蛋白产生ROS的较高酶活性，进而维持花粉管的极性顶端生长。 总之，甾醇螯合对白杄花粉管生长影响的研究，为脂筏极化在花粉管极性生长中的作用提供了证据。基于以上生物化学和细胞生物学的结果，我们针对花粉管中富含甾醇的脂筏微区和NOX功能之间的联系，提出了一种假说模式：(1) 植物细胞质膜上的脂筏为信号分子ROS在特定位点的聚集提供了物理载体；(2) 脂筏的完整性和甾醇依赖性对NOX的定位和活性是必要的，并为花粉管细胞极性产生和维持所必需。上述研究结果表明，脂筏在花粉管顶端的极化，以及作为关键生长因子的NOX在质膜脂筏中的定位，对花粉管的高度极性生长具有重要作用。