BRONCHIAL SECRETIONS--PHYSICAL AND CHEMICAL STUDIES

支气管分泌物——物理和化学研究

• 批准号: 3344756
• 负责人: Mary C Rose
• 金额: $ 13.62万
• 依托单位: CHILDREN'S NATIONAL MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-03-01 至 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3344756
• 关键词: acylation; autoradiography; bacteriophage lambda; bronchial mucus; bronchus disorder; carbohydrate sequence; chemical structure function; cystic fibrosis; electron microscopy; emphysema; fatty acids; gel electrophoresis; genetic library; genetic manipulation; genetic recombination; glycoproteins; histochemistry /cytochemistry; human tissue; immunochemistry; molecular pathology; mucins; nucleic acid sequence; oligosaccharides; protein sequence; protein structure; radiotracer; respiratory epithelium; secretion; tissue /cell culture

The long term objective is to elucidate the role of mucous glycoproteins (mucins) in health and in disease. Mucins are a major component of the mucosal layer lining the respiratory tract and are over-produced in chronic obstructive pulmonary diseases including cystic fibrosis (CF). Since mucins are the macromolecules responsible for the viscoelastic properties of mucus, alterations in mucin structure may effect the physiological behavior of mucus. It has become increasingly evident that the complex structure and physical properties of human mucins almost preclude obtaining definitive information on the polypeptide backbone of normal and pathological mucins by biochemical studies alone. Thus, in order to compare mucin polypeptides and determine the role of glycosylated and non-glycosylated domains in regulating the physical properties of mucins, this laboratory has begun studies designed to isolate cDNA that codes for tracheobronchial mucin (TBM). The specific aims are (1) to prepare, purify and sequence TBM peptide fragments. The resultant primary sequences will be useful for generating polynucleotide probes to identify and confirm clones containing TBM cDNA and to confirm or correct the primary structure of human TBM deduced from the nucleotide sequence of; (2) to isolate cDNA that encodes TBM. mRNA isolated from respiratory tract tissue will be used to synthesize cDNA for the construction of recombinant libraries in the bacteriophage lambda gt11. The cDNA libraries will be screened with antibodies specific to the mucin polypeptide backbone and with polynucleotide probes generated from amino acid sequence data; (3) to sequence sufficient number of cDNA clones to deduce the primary sequence of TBM including the glycosylated and non-glycosylated segments. (4) In addition, the effect of fatty-acid acylation on respiratory tract mucin and the question of whether CF respiratory tract mucin, like gastric mucin is over-acylated will be examined. If increased covalently bound-lipid has relevance to the pathogenesis or molecular defect of CF, increased acylation should also be manifested by non-gastric mucins. Recent studies indicate that respiratory tract mucins are fatty acid-acylated. We will further examine this finding and determine whether CF mucins are over-acylated, whether lipid is attached to glycosylated or non-glycosylated mucin domains, and whether covalently found fatty acid protects respiratroy tract mucin against proteolysis.

长期目标是阐明粘液糖蛋白的作用 （粘蛋白）在健康和疾病中。 粘蛋白是免疫球蛋白的主要成分。 粘膜层内衬呼吸道，并在慢性 阻塞性肺病，包括囊性纤维化（CF）。 以来 粘蛋白是负责粘弹性的大分子 粘液，粘蛋白结构的改变可能会影响生理 粘液的行为。 越来越明显的是， 人类粘蛋白的结构和物理性质几乎排除了获得 关于正常的和 病理性粘蛋白单独生化研究。 因而为了 比较粘蛋白多肽，并确定糖基化和 非糖基化结构域在调节粘蛋白的物理性质中的作用， 这个实验室已经开始研究分离编码 气管支气管粘蛋白（TBM）。 具体目的是（1）制备、纯化和测序TBM肽 片段 所得到的一级序列将用于生成 鉴定和确认含有TBM cDNA的克隆的多核苷酸探针 并确认或校正由以下推导出的人TBM的一级结构： 的核苷酸序列;（2）分离编码TBM的cDNA。 mRNA 从呼吸道组织中分离的cDNA将用于合成 在λ噬菌体中构建重组文库 gt11. cDNA文库将用特异性针对该蛋白的抗体进行筛选。 粘蛋白多肽骨架和由其产生的多核苷酸探针 氨基酸序列数据;（3）对足够数量的cDNA克隆进行测序 推导出包括糖基化和 非糖基化片段。 (4)此外，脂肪酸的作用 呼吸道粘蛋白的酰化以及CF是否 呼吸道粘蛋白，像胃粘蛋白是过度酰化， 考察 如果增加的共价结合的脂质具有相关性， CF的发病机制或分子缺陷，增加酰化也应该是 表现为非胃粘蛋白。 最近的研究表明， 道粘蛋白是脂肪酸酰化的。 我们将进一步研究这一发现 并确定CF粘蛋白是否过度酰化，脂质是否过度酰化 连接到糖基化或非糖基化粘蛋白结构域， 共价发现的脂肪酸保护尿道粘蛋白免受 蛋白水解