WHITE CELL RESPONSES TO ENVIRONMENTAL TOXINS

该提案的广泛长期目标是了解 调节中性粒细胞对环境粉尘的反应， 对人多形核白细胞（PMN）细胞色素P-450 LTB的影响 其调节趋化脂质5S，12 R-二羟基- 6，14-顺式-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腔，导致嗜中性粒细胞增多， 肺泡炎，然后是慢性纤维化。 招聘机制 中性粒细胞的肺泡，在响应这些无机粉尘，因此是至关重要的 慢性石棉肺和矽肺发病机制中的一个因素。 LTB 4具有 被建议在这些PMN的招募中发挥重要作用， 紊乱 P-450 LTB是位于小鼠微粒体中的细胞色素P-450， 中性粒细胞通过催化LTB 4的活化来调节LTB 4的生物学活性。 LTB 4的C：20碳的逐渐氧化。 具体目标是 纯化、克隆cDNA并研究P-450 LTB在培养细胞中的调节作用 细胞 P-450 LTB将使用快速蛋白液相色谱法进行纯化， 或免疫亲和层析。 纯化的P-450 LTB将转移至 immobilon膜，并确定N-末端氨基酸序列。 胰蛋白酶消化也将对可溶性纯化酶或对 酶转移到硝酸纤维素膜。 胰蛋白酶片段将 通过高效液相色谱法分离，然后进行 氨基酸测序 将构建寡核苷酸探针， 直接使用或构建聚合酶链式反应（PCR）产物 为了探测从人PMN获得的mRNA构建的cDNA文库， 慢性粒细胞性白血病患者，细胞中mRNA丰富。 或者，该文库将用puronectin同源物探测。 兔肺前列腺素ω-羟化酶（P-450 PG ω）的cDNA。 的 在诱导HL-60细胞中控制P-450 LTB诱导的因素 将确定分化表型成熟的嗜中性粒细胞。

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.