Elucidation Of Cellular Damage During Exposure To Oxidat

在NHLBI生物化学实验室的酶部分的研究，旨在阐明在氧化应激下产生细胞损伤的基本机制，以及这种损伤对衰老和疾病的贡献。为此，我们目前的研究涉及以下几个方面的研究： (A)对细胞凋亡的调控。以往的研究表明，高浓度的锰离子[Mn(II)]通过caspase12介导的细胞色素c非依赖性途径诱导HeLa和NIH3T3细胞凋亡。锰(II)诱导的细胞凋亡与细胞内活性氧(ROS)水平升高、前天冬氨酸氨基转移酶12(Proaspase12)激活和锰超氧化物歧化酶[Mn(II)SOD]上调有关。小鼠caspase 12基因的启动子区域和5‘非翻译区已被分离和测序，caspase基因的功能分析正在进行中。 (B)细胞凋亡在衰老中的作用。以往的研究结果表明，亚砷酸盐处理培养的白血病NB4细胞会导致氧化蛋白水平的增加和细胞凋亡，而抑制凋亡会导致氧化蛋白的积累更多。我们的研究支持了这样一种可能性，即观察到的与年龄相关的氧化蛋白水平的上升部分是由于通过凋亡消除氧化损伤的细胞并用良好的细胞取代它们的能力的丧失，我们的研究表明，用亚砷酸盐处理培养的白血病NB4细胞会导致氧化蛋白水平的增加和凋亡，而抑制凋亡会导致氧化蛋白的积累更多。进一步的研究正在进行中，以确定事实上，衰老是否与诱导细胞凋亡的能力下降有关。 (C)单细胞生物体中的细胞凋亡检查。研究结果表明，酵母含有一个与哺乳动物caspase结构高度同源的基因(YCA1)，该基因能够在酵母中介导细胞程序性死亡，从而支持了单细胞生物发生细胞凋亡的可能性。我们已经开始研究急性氧化应激对含有正常YCA1的野生型酿酒酵母菌株和缺失YCA1的突变纯合子菌株以及杂合子菌株的时间老化的影响，以及这些菌株对氧化应激的敏感性。 (D)核糖核酸(RNA)氧化对翻译效率的影响。RNA的氧化修饰与几种神经疾病有关。为了研究核糖核酸氧化对其翻译效率的影响，将编码荧光素酶基因的RNA用过氧化氢氧化，并检测其在网织红细胞裂解液中产生荧光素酶的能力。从氧化的RNA翻译的荧光素酶蛋白的活性明显低于正常的荧光素酶制剂。进一步的研究表明，在RNA的氧化过程中形成的产物是不同的，这取决于用于产生活性氧物种的体系的组成。除了8-氧鸟嘌呤外，还形成了其他低分子量化合物。这些产品的身份正在调查中。 (E)碳酸氢盐缓冲液对铁催化氧化低密度脂蛋白的影响。众所周知，低密度脂蛋白的氧化与动脉硬化有关。以前的体外尝试是利用非生理性的磷酸盐缓冲系统来阐明各种活性氧产生系统氧化低密度脂蛋白的机制。基于我们早期的研究结果表明，当在由生理浓度的碳酸氢盐和二氧化碳组成的缓冲液中进行反应时，金属催化的蛋白质和氨基酸的氧化大大增强，我们开始研究碳酸氢盐缓冲液对低密度脂蛋白氧化的影响。我们发现，用碳酸氢盐代替磷酸盐缓冲液，可以显著提高低密度脂蛋白的氧化速率，这可以通过丙二醛、蛋白质羰基和蛋氨酸亚砜衍生物的生成来衡量。总而言之，这些研究的结果表明，亚铁、重碳酸盐和各种铁络合剂之间的相互作用刺激了依赖铁+过氧化氢的低密度脂蛋白的氧化，形成了具有有利于Fenton化学的氧化还原电位的络合物。

Research in the Section on Enzymes in the Laboratory of Biochemistry, NHLBI, is directed toward elucidation of basic mechanisms involved in the production of cellular damage during exposure to oxidative stress and the contributions of such damage to aging and disease. To this end, our current research involves studies in the following areas of research: (a) Regulation of apoptosis. Previous studies demonstrated that high concentrations of manganese ions [Mn(II)] induces apoptosis in cultured HeLa and NIH3T3 cells by a caspase 12-mediated cytochochrome c independent pathway. The Mn(II)-induced apoptosis is associated with increases in the cellular levels of reactive oxygen species (ROS), activation of procaspase 12, and up-regulation of manganese superoxide dismutase [Mn(II)SOD]. The promoter region and the 5'-untranslated region of the mouse caspase 12 gene were isolated and sequenced, and the functional analysis of the caspase gene is now under investigation. (b) Role of apoptosis in aging. Results of previous studies demonstrated that treatment of cultured leukemia NB4 cells with arsenite leads to an increase in the levels of oxidized proteins and to apoptosis, and that inhibition of apoptosis leads to an even greater increase in the accumulation of oxidized proteins. The possibility that the observed age-related increase in levels of oxidized proteins is due in part to a loss in ability to eliminate oxidatively damaged cells by apoptosis and replace them with good cells is supported by our studies showing that treatment of cultured leukemia NB4 cells with arsenite leads to an increase in levels of oxidized proteins and to apoptosis, and that inhibition of apoptosis leads to an even greater accumulation of oxidized proteins. Further studies are in progress to determine if, in fact, aging is associated with a decrease in the ability to induce apoptosis. (c) Examination of apoptosis in unicellular organisms. The possibility that apoptosis may occur in unicellular organisms is supported by results of studies showing that yeast contain a gene (YCA1) with high structural homology to the mammalian caspases, and that this gene is able to mediate programmed cell death in yeast. We have initiated studies to determine the effects of acute oxidative stress on the chronological aging of a wild type strain of Saccharomyces cerevisiae containing normal levels of YCA1 and of a mutant homozygous strain that lacks YCA1, as well as a heterozygous strain, and on the susceptibility of these strains to oxidative stress. (d) Effect of ribonucleic acid (RNA) oxidation on translational efficiency. Oxidative modification of RNA is associated with several neurological disorders. To study the effect of ribonucleic acid oxidation on its translational efficiency, RNA encoding the luciferase gene was subjected to oxidation by hydrogen peroxide and its ability to produce luciferase when incubated in reticulocyte lysate was examined. Activity of the luciferase protein translated from oxidized RNA is considerably lower than that of normal luciferase preparations. Further studies have shown that the products formed in the oxidation of RNA vary, depending upon the composition of the system used to generate reactive oxygen species. In addition to 8-oxo-guanine, other low molecular weight compounds are formed. The identity of these products is under investigation. (e) Effect of bicarbonate buffer on the iron-catalyzed oxidation of low density lipoprotein (LDL). It is well established that oxidation of LDL is implicated in arteriosclerosis. Previous in vitro attempts to elucidate mechanisms of LDL oxidation by various reactive oxygen generation systems have been carried out using non-physiological phosphate buffer systems. Based on results of our earlier studies showing that metal-catalyzed oxidation of proteins and amino acids is greatly enhanced when reactions are carried out in buffers composed of physiological concentrations of bicarbonate and carbon dioxide, we initiated studies to examine the effects of bicarbonate buffer on the oxidation of LDL. We found that substitution of bicarbonate for phosphate buffers led to substantial increases in the rates of LDL oxidation as measured by the generation of malondialdehyde, protein carbonyl, and methionine sulfoxide derivatives. Collectively, results of these studies suggest that the iron + peroxide-dependent oxidation of LDL is stimulated by interactions between ferrous iron, bicarbonate, and various iron chelators to form complexes having redox potentials that favor Fenton chemistry.