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.

Biphasic regulation of H2O2 on angiogenesis implicated NADPH oxidase

ROS (reactive oxygen species) take an important signalling role in angiogenesis. Although there are several ways to produce ROS in cells, multicomponent non-phagocytic NADPH oxidase is an important source of ROS that contribute to angiogenesis. In the present work, we examined the effects of H2O2 on angiogenesis including proliferation and migration in HUVECs (human umbilical vein endothelial cells), new vessel formation in chicken embryo CAM (chorioallantoic membrane) and endothelial cell apoptosis, which is closely related to anti-angiogenesis. Our results showed that H2O2 dose-dependently increased the generation of O-2(-) (superoxide anion) in HUVECs, which was suppressed by DPI (diphenylene iodonium) and APO (apocynin), two inhibitors of NADPH oxidase. H2O2 at low concentrations (10 mu M) stimulated cell proliferation and migration, but at higher concentrations, inhibited both. Similarly, H2O2 at 4 nmol/cm(2) strongly induced new vessel formation in CAM, while it suppressed at high concentrations (higher than 4 nmol/cm(2)). Also, H2O2 (200 similar to 500 mu M) could stimulate apoptosis in HUVECs. All the effects of H2O2 on angiogenesis could be suppressed by NADPH oxidase inhibitors, which suggests that NADPH oxidase acts downstream of H2O2 to produce O-2(-) and then to regulate angiogenesis. In summary, our results suggest that H2O2 as well as O-2(-) mediated by NADPH oxidase have biphasic effects on angiogenesis in vitro and in vivo.

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。

Difference in Enzyme Activity and Conformation of Glucose Oxidase Before and After Purification

EEnzyme activity of commercial glucose oxidase was enhanced after purification through a strong anionic exchange resin. In order to get a better insight into this phenomenon, surface pressure–area ( –A) isotherms and surface pressure–time ( –t) isotherms was used to study the interaction and the absorption at different pH values of the subphases between octadecylamine and glucose oxidase purified by a styrene system quaternary ammonium type strongly basic anionic exchange resin. Circular dichroism (CD), electrophoresis and enzyme activity measurements were conducted to study these phenomena. A preliminary hypothesis has been suggested to explain why the enzyme activity of purified glucose oxidase was higher than that of the commercial one. © 2002 Elsevier Science B.V. All rights reserved.