WHITE CELL RESPONSES TO ENVIRONMENTAL TOXINS

该提案的广泛的长期目标是了解 通过聚焦调节中性粒细胞对环境粉尘的反应 人多形核白细胞细胞色素P-450LTB的研究 它调节趋化脂质5S，12R-二羟基的代谢- 6，14-顺-8，10-反式二十碳四烯酸，白三烯(LT)B4。暴露于 有毒的环境粉尘二氧化硅和石棉导致 中性粒细胞募集到肺泡间隙，导致中性粒细胞 肺泡炎继发慢性纤维化。人才招聘的机制 因此，PMN对这些无机粉尘的反应对肺泡是至关重要的 慢性石棉肺和矽肺的发病因素。LTB4具有 建议在这些地区招募PMN方面发挥主要作用 精神错乱。P-450LTB是一种细胞色素P-450，位于猪细小病毒的微粒体中。 PMN通过催化LTB4的活性调节LTB4的生物活性 LTB4的C：20碳的逐步氧化。具体目标是 纯化、克隆P-450LTB基因并研究其在培养细胞中的调控作用 细胞。P-450LTB将用快速蛋白质液相色谱纯化， 或免疫亲和层析。提纯的P-450LTB将被转移到 固定化细胞膜，并测定N-末端氨基酸序列。 还将对可溶性纯化酶或其他酶进行胰酶消化。 转移到硝酸纤维素膜上的酶。胰酶碎片将会是 用高效液相色谱分离，然后进行 氨基酸测序。将构建寡核苷酸探针并 直接利用或构建聚合酶链式反应(PCR)产物 从人外周血多形核细胞中获得的mRNA构建的cDNA文库 慢性粒细胞白血病患者，细胞中有丰富的mRNA。 或者，这个文库将用推测的同源 兔肺前列腺素omega-羟基酶基因(P-450-PGomega)。这个 控制P-450LTB在HL-60细胞中诱导的因素 分化表型成熟的中性粒细胞将被确定。

The broad long-term objectives of the proposal are to understand the regulation of the neutrophil response to environmental dusts, by focusing on the cytochrome P-450LTB of human polymorphonuclear leukocytes (PMN) which regulates the metabolism of the chemotactic lipid 5S,12R-dihydroxy- 6,14-cis-8,10-trans-eicosatetraenoic acid, leukotriene (LT)B4. Exposure to the toxic environmental dusts silica and asbestos results in the recruitment of PMN to the alveolar space, with a resulting neutrophilic alveolitis followed by chronic fibrosis. The mechanism of recruitment of PMN to the alveoli, in response to these inorganic dusts, is thus a crucial factor in the pathogenesis of chronic asbestosis and silicosis. LTB4 has been suggested to play a major role in the recruitment of PMN in these disorders. P-450LTB is the cytochrome P-450 located in the microsomes of PMN which regulates the biological activity of LTB4 by catalyzing the progressive oxidation of the C:20 carbon of LTB4. The specific aims are to purify, clone the cDNA, and study the regulation of P-450LTB in cultured cells. P-450LTB will be purified using Fast Protein Liquid Chromatography, or immunoaffinity chromatography. Purified P-450LTB will be transferred to immobilon membranes, and the N-terminal amino acid sequence determined. Tryptic digestion will also be performed on soluble purified enzyme or on enzyme transferred to nitrocellulose membranes. Tryptic fragments will be isolated by high performance liquid chromatography, and then subjected to amino acid sequencing. Oligonucleotide probes will be constructed and utilized directly or to construct polymerase chain reaction (PCR) products to probe a cDNA library constructed from mRNA obtained from human PMN of patients with chronic myelogenous leukemia, cells abundant in mRNA. Alternatively, this library will be probed with the putatively homologous cDNA of rabbit lung prostaglandin omega-hydroxylase (P-450PGomega). The factors which control the induction of P-450LTB in HL-60 cells induced to differentiate phenotypically mature neutrophils will be determined.