BIOCHEMICAL AND MOLECULAR BIOLOGICAL STUDIES

生物化学和分子生物学研究

• 批准号: 3358784
• 负责人: GOVERDHAN Pal SACHDEV
• 金额: $ 13.72万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1992-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3358784
• 关键词: bronchial mucus; cell free system; chemical aggregate; complementary DNA; dogs; fluorescent dye /probe; genetic library; glycoproteins; glycosylation; hydropathy; immunologic techniques; light scattering; messenger RNA; molecular cloning; molecular weight; monoclonal antibody; mucins; nucleic acid probes; protein sequence; protein transport; respiratory epithelium

Tracheobronchial mucus secretions play an important protective role in the normal functioning of the airways. Mucus glycoproteins present in the mucus secretions are responsible, to a large extent, for the physical properties of the secretions. In the disease states, the mucins may have altered chemical and physical properties and this in turn may influence the clearance of the secretions by ciliated epithelium. Although, there is a considerable information on the oligosaccharide side chains of airway mucins, the structure, including amino acid sequence of the protein backbone is lacking. The objectives of this research are to investigate the molecular size, amino acid sequence, and physical properties of the protein core of the airway mucins. The specific aims of this proposal are to a) further purify and biochemically characterize the native (non-reduced) and deglycosylated mucins from canine tracheal mucus secretions, b) physically characterize the native and deglycosylated mucins to determine molecular size, radius of gyration, aggregation behavior and presence of hydrophobic domains, c) prepare immunological probes against native and deglycosylated mucins in order to detect specific apomucin(s) generated during in vitro cell free translation of mRNA from canine tracheal epithelial (CTE) cells and to screen the production of apomucin(s) in the cDNA library constructed in the lambda gt11 vector and d) isolate and characterize cDNA clones of apomucin(s) from canine tracheal epithelial cells in order to determine the primary amino acid sequence and amino acid sequences which signal transport and processing. The mucins will be purified using protocols established in this laboratory. Biochemical characterization will include: determination of carbohydrate, sulfate, amino acid composition, thiol and disulfide content of the mucin molecules. The molecular size, radius of gyration and agregation behavior will be determined using light scattering and sedimentation equilibrium methods. Poly- and monoclonal antibodies directed against the mucins will be prepared using standard established protocols. Isolation, characterization, detection of mucin cDNA clones and nucleotide sequencing will be carried out using well established molecular biological techniques. The knowledge gained from the proposed studies should eventually lead to more effective treatment strategies for obstructive lung disease.

气管粘液粘液分泌物发挥重要的保护作用 在气道正常功能中的作用。 粘液糖蛋白 在粘液分泌物中存在，在很大程度上负责 对于分泌物的物理特性。 在疾病中 州，粘蛋白可能改变了化学和物理 属性和这可能会影响清除 纤毛上皮分泌。 虽然，有一个 有关寡糖侧链的大量信息 气道粘蛋白，结构，包括氨基酸序列 缺乏蛋白质主链。 这项研究的目标 正在研究分子大小，氨基酸序列和 气道粘蛋白的蛋白质核心的物理特性。 这 该建议的具体目的是a）进一步净化和 生化表征了本地（未还原）和 犬气管粘液分泌的脱糖性粘蛋白，b） 物理表征天然和脱糖基化的粘蛋白 确定分子大小，回旋半径，聚集 疏水结构域的行为和存在，c）准备 针对天然和脱糖基化粘蛋白的免疫学探针 为了检测体外细胞中产生的特定apomucin 从犬气管上皮（CTE）中免费翻译mRNA 细胞并筛选cDNA中apomucin的产生 在lambda gt11矢量和d）隔离和隔离和 表征来自犬气管的阿哌-蛋白的cDNA克隆 上皮细胞以确定原发性氨基酸 序列和氨基酸序列，这些序列信号转运和 加工。 粘蛋白将使用方案纯化 在这个实验室建立。 生化特征将 包括：确定碳水化合物，硫酸盐，氨基酸 粘蛋白分子的组成，硫醇和二硫键。 分子大小，回旋的半径和一致性行为 将使用光散射和沉降确定 平衡方法。 指导的聚和单克隆抗体 针对粘蛋白将使用已建立的标准准备 协议。 分离，表征，检测粘蛋白cDNA 克隆和核苷酸测序将使用井进行 建立的分子生物学技术。 知识 从拟议的研究中获得的最终应导致更多 阻塞性肺部疾病的有效治疗策略。