SYNERGISTIC STIMULATION AND PRIMING OF NEUTROPHILS

中性粒细胞的协同刺激和启动

• 批准号: 3142793
• 负责人: JOHN A BADWEY
• 金额: $ 16.99万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3142793
• 关键词: antibody formation; binding proteins; blood donor; calcium flux; calcium transporting ATPase; cell membrane; cytoskeleton; eicosanoids; flavins; guinea pigs; human tissue; laboratory rabbit; leukocyte oxidative burst; leukopoietic factor; magnesium; neutrophil; phospholipase A2; phosphoproteins; phosphorylation; protein biosynthesis; protein kinase C; protein sequence; protein signal sequence; superoxides; synthetic peptide; tissue /cell culture

The long term objectives of this proposal are to elucidate the exact sequence of biochemical events that are involved in the priming and stimulation of superoxide (02-) production by neutrophils. Superoxide is a key component of the oxygen-dependent antimicrobial mechanisms of these cells which provide a major defense against infectious organisms. Certain cytokines prime neutrophils to release increased amounts of 02-. Therapeutic value of these factors is now being tested clinically in a variety of cancer and AIDS patients with promising results. Thus, experiments outlined herein are relevant to an understanding of host- defense mechanisms and immune deficiency diseases. Specifically, this project addresses two largely unknown areas. These are: (1) the mechanism(s) of action of the hydroxylated eicosatetraenoic acids (HETE) in modulating 02- production, and (2) the link between protein phosphorylation and 02- generation. Neutrophils produce 5- and 15-HETE under various circumstances. 5-HETE dramatically potentiates 02- release, whereas 15-HETE inhibits it. We propose that 5- HETE primes neutrophils by increasing the flux of Ca2+ across the plasmalemma, perhaps by opening a channel for this cation. This will be measuring the rates of 45Ca2+ uptake by these cells in the presence of different concentrations of 5-HETE. The effects of a variety of calcium channel antagonists on this process will be evaluated. The relationships between the rates of 45Ca2+ uptake and 02- generation will be defined, and possible antagonistic effects of 15-HETE will be sought. With regard to protein phosphorylation, we have observed that stimulation of 02- release by neutrophils is always accompanied by an intense phosphorylation of two proteins with molecular weights of ca. 47 and 49 kDa. Quantitatively, these are the two major proteins which are phosphorylated upon stimulation of these cells. While the 47 kDa protein has been extensively characterized, virtually nothing is known of the 49 kDa species. Experiments are detailed herein to identify, characterize and purify this protein. Techniques of biochemistry and cell biology will be employed. We will determine whether the 49 kDa protein is a component of the 02- generating system, or is involved in some other fashion in the mechanism of stimulation of these cells (e.g., phospholipose D). Changes in the activity of the 49 kDa protein after phosphorylation will be sought to forge a link between this modification reaction and cell stimulation.

该提案的长期目标是阐明确切的 启动涉及的生化事件的顺序 嗜中性粒细胞刺激超氧化物（02-）。 超氧化物是 这些依赖氧的抗菌机制的关键成分 对传染性生物提供了重大防御的细胞。 肯定 细胞因子质量中性粒细胞释放增加的量为02-。 这些因素的治疗价值现在正在临床上在 各种各样的癌症和艾滋病患者具有令人鼓舞的结果。 因此， 本文概述的实验与对宿主的理解有关 防御机制和免疫缺陷疾病。 具体而言，该项目解决了两个很大未知的领域。 这些 为：（1）羟化二十二碳酸酯作用机制 调节02-生产中的酸（Hete），（2） 蛋白质磷酸化和02-生成。 中性粒细胞产生5-和 在各种情况下为15-HETE。 5-Hete急剧 增强02-释放，而15-Hete抑制它。 我们建议5- 通过增加Ca2+的通量，质质质粒细胞遍布整个 血浆膜，也许是通过打开该阳离子的通道。 这将是 在存在的情况下测量这些细胞的45CA2+摄取速率 不同浓度的5-HETE。 各种各样的影响 将评估此过程中的钙通道拮抗剂。 这 45CA2+摄取的速率与02-生成之间的关系 将被定义，15-HETE可能的拮抗作用将是 寻求。 关于蛋白质磷酸化，我们观察到刺激 02-中性粒细胞的释放总是伴随着强烈的 两种蛋白质的磷酸化，分子量为ca。 47和49 KDA。 定量，这是两个主要蛋白质 刺激这些细胞后磷酸化。 而47 kDa蛋白 已经被广泛地描述了，几乎没有什么已知的49 KDA物种。 本文详细介绍了实验，以识别，表征 并纯化该蛋白质。 生物化学和细胞技术 生物学将被使用。 我们将确定49 kDa蛋白是否为 02-生成系统的组成部分，或参与其他一些 这些细胞刺激机制的时尚（例如 磷脂D）。 49 kDa蛋白的活性变化 将寻求磷酸化以在此修饰之间建立联系 反应和细胞刺激。