MOLECULAR BASIS FOR MICROBICIDAL ACTION IN LEUKOCYTES

白细胞杀菌作用的分子基础

• 批准号: 2060299
• 负责人: JAMES K HURST
• 金额: $ 13.76万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-04-01 至 1997-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2060299
• 关键词: MOLECULAR; BASIS; MICROBICIDAL; ACTION; LEUKOCYTES

We have initiated studies to identify the oxidative cytotoxins of stimulated leukocytes and their cytocidal mechanisms. We have shown that the bactericidal reactions of leukocyte peroxidase- generated hypochlorous acid (HOCl) and chemically similar chloramines cause destruction of metabolic energy reserves within the cell and loss of energy-linked functions such as active transport. The locus of oxidative attack is the bacterial plasma membrane; the metabolic dysfunctions leading to massive net ATP hydrolysis can be traced primarily to selective oxidative inactivation of nutrient transport systems and the proton translocating ATP synthase that links respiration to oxidative phosphorylation. We are applying several analytical methods to identify the molecular sites of attack on the ATP synthase F1 complex that give rise to its inactivation. The common response that we observe by several bacteria with disparate metabolic capabilities and the need for all cells to maintain ATP- synthesizing capabilities suggests that these reactions are capable of accounting for the "universal" character of HOCl toxicity towards prokaryotes. In the proposed research, we will investigate inactivation of microbial eukaryotes, whose ATP- synthesizing enzymatic systems are mitochondrial, hence nominally protected from extracellularly-generated HOCl. We will initiate parallel studies to identify the lethal reactions of hydroxyl radical and similar active oxygen species (high-valent metal oxo ions) that might be formed in metal-catalyzed reactions involving H2O2. We will compare loss of viability with specific metabolic functions or individual biochemical components to identify the loci of oxidative attack associated with cellular death. For this purpose, we will use differing methods of forming hydroxyl radical to allow its generation in the extracellular medium, at the surface of the bacterial cell, or intracellulary. Following protocols that we have developed for the studies with HOCl, we will examine the consequences of OH and Fenton-type chemical systems upon bacterial transport and respiratory systems, biosynthetic capabilities and selected cytosolic enzymes, nucleotide phosphorylation, and lesions in nucleic acids. Organisms exhibiting cleanly differing mechanisms of oxidative inactivation by HOCl and OH will then be used to probe for the primacy of one or the other within neutrophil phagosomes. These studies will clarify leukocytic mechanisms comprising host cellular response to infection and should yield insights into the nature of chemotactic and inflammatory responses.

我们已经开始研究以鉴定的氧化细胞毒素 刺激的白细胞及其胞质机制。 我们有 表明白细胞过氧化物酶的杀菌反应 产生的次醛酸（HOCL），化学相似 氯胺会导致代谢能量储备的破坏 电池和能量连锁函数的丧失，例如主动 运输。 氧化攻击的基因座是细菌等离子体 膜;代谢功能障碍导致大量净ATP 水解可以主要追溯到选择性氧化 营养运输系统和质子的灭活 将呼吸与氧化联系起来的易位ATP合酶 磷酸化。 我们正在将几种分析方法应用于 确定对ATP合酶F1攻击的分子位点 复杂引起其失活。 共同的反应 我们通过几种细菌与不同的代谢观察到 能力和所有细胞维持ATP的需求 合成功能表明这些反应是 能够考虑HOCL的“通用”特征 对原核生物的毒性。 在拟议的研究中，我们将 研究微生物真核生物的失活，其ATP- 合成酶系统是线粒体，因此名义上 免受细胞外生成的HOCL的保护。 我们将发起 平行研究以鉴定羟基的致命反应 自由基和类似的活性氧（高价值金属氧 离子）可能在涉及金属催化的反应中形成 H2O2。 我们将比较特定代谢的生存力的丧失 功能或个体生化成分，以识别 与细胞死亡有关的氧化攻击的基因座。 为了 此目的，我们将使用不同的形成羟基的方法 激进以允许其在细胞外介质中产生 细菌细胞的表面或细胞内。 下列的 我们为HOCL研究开发的协议，我们 将检查OH和Fenton型化学的后果 细菌运输和呼吸系统的系统， 生物合成能力和选定的胞质酶， 核苷酸磷酸化和核酸中的病变。 表现出干净不同的氧化机制的生物 然后，HOCL和OH的灭活将用于探测 中性粒细胞中一个或另一种的首要地位。 这些 研究将阐明包含宿主的白细胞机制 细胞对感染的反应，并应对 趋化性和炎症反应的性质。