SYNTHESIS AND ROLE OF METALLOTHIONEIN IN THE LUNG

肺中金属硫蛋白的合成和作用

• 批准号: 2153216
• 负责人: BETH A HART
• 金额: $ 17.08万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-07-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2153216
• 关键词: alveolar macrophages; autoradiography; binding proteins; cadmium; cytotoxicity; electron microscopy; environmental adaptation; environmental toxicology; enzyme linked immunosorbent assay; free radical oxygen; gene expression; genetic transcription; genetic translation; hyperoxia; laboratory rat; lung; messenger RNA; metal metabolism; metallothionein; northern blottings; nucleic acid hybridization; protein isoforms

DESCRIPTION (Adapted from the investigator's abstract): Metallothioneins (MTs) are small proteins characterized by low molecular weight, high cysteinyl content; high capacity to bind Cd, Cu or Zn; highly conserved nucleic acid and amino acid sequences; and inducibility by diverse stimuli. Two isoforms exist in rodents. Repeated exposure to Cd aerosols causes a 400-fold augmentation in pulmonary MT. MT also accumulates in lung during exposure to 85 percent oxygen, albeit in smaller amounts. Animals adapt when repeatedly exposed to Cd. Cd-pretreated animals exhibit resistance to acute Cd challenge and are cross-tolerant to hyperoxia. The investigators hypothesize that MTs play a role in metal detoxification and free radical protection in the lung, and will evaluate the amplification of MT-mRNA during repeated Cd exposures by means of Northern analysis and dot blot hybridization techniques. MT isoforms will be separated and quantitated in lung tissue by HPLC in combination with a metal binding assay. The effect of MT transcriptional and translational status on the resistance of animals to acute Cd challenge will be assessed. Cd-induced cross tolerance to hyperoxia will be correlated to MT gene expression and alterations in lung cell populations, as determined by correlative light and electron microscopy and autoradiography. Differential pulmonary MT gene expression in Cd adapted and oxygen adapted rats will be compared. Type II epithelial cells, purified from rat lung by a cell "panning" technique, and a pulmonary vascular endothelial cell line isolated from rat will be used to study mechanisms related to MT expression. MT induction will be assessed using various stimuli. MT gene products will be measured in lung cells using an ELISA procedure and by electrophoresis with Western blotting. MT isoform gene expression in type II cells, isolated from Cd and oxygen exposed animals will be determined. Coordinate induction of Md-SOD in these cells will be tested. The efficacy of MT as a protectant against free radicals will be assessed in vivo. The proposed studies hopefully will help to elucidate the function of MT in health and disease and contribute to our understanding of mechanisms which protect the lung against metal and free radical damage.

描述（改编自调查员的摘要）：金属硫蛋白 （MT）是小蛋白质，其特征是低分子量，高 半胱氨酸含量；高容量结合Cd，Cu或Zn；高度保守 核酸和氨基酸序列；以及各种刺激的诱导性。 啮齿动物中存在两个同工型。 反复接触CD气溶胶会导致A 肺MT中的400倍增强。 MT也在肺中积聚 暴露于85％的氧气，尽管量较小。 动物适应 反复暴露于CD时。 CD预处理的动物表现出对 急性CD挑战，对高氧交叉耐受性。 调查人员 假设MTS在金属排毒和自由基中起作用 在肺部进行保护，并将评估MT-MRNA的扩增 通过北方分析和DOT印迹在重复的CD暴露期间 杂交技术。 MT同工型将分开并定量 HPLC与金属结合测定法结合使用肺组织。 效果 MT的转录和翻译状态在动物的抗性上 将评估急性CD挑战。 CD诱导的交叉耐受性 高氧将与MT基因表达和肺的改变相关 通过相关光和电子确定的细胞种群 显微镜和放射自显影。 鉴定肺MT基因表达 在CD中，将比较适应和氧气的大鼠。 II型上皮 细胞，通过细胞“平移”技术从大鼠肺中纯化，A 从大鼠分离的肺血管内皮细胞系将用于 与MT表达有关的研究机制。 将评估MT归纳 使用各种刺激。 MT基因产物将在肺部细胞中进行测量 使用ELISA程序和通过蛋白质印迹的电泳。 公吨 II型细胞中的同工型基因表达，从CD和氧气分离 将确定暴露的动物。 协调MD-SOD中的诱导 这些细胞将进行测试。 MT作为保护剂的功效 自由基将在体内评估。 拟议的研究希望 将有助于阐明MT在健康和疾病中的功能以及 有助于我们对保护肺的机制的理解 对金属和自由基损害。