BIOCHEMICAL AND MOLECULAR BIOLOGICAL STUDIES

气管支气管粘液分泌物在肺损伤中起重要的保护作用， 在气道的正常运作中发挥作用。 粘液糖蛋白 在很大程度上， 分泌物的物理特性 在疾病 粘蛋白可能改变了化学和物理性质， 性能，这反过来又可能影响 纤毛上皮的分泌物。 虽然，有一个 大量的信息，寡糖侧链的 气道粘蛋白，结构，包括氨基酸序列， 缺少蛋白质骨架。 本研究的目标 是研究分子大小，氨基酸序列， 气道粘蛋白的蛋白质核心的物理性质。 的 该提案的具体目标是：a）进一步净化和 对天然（非还原）和 来自犬气管粘液分泌物的去糖基化粘蛋白，B） 物理表征天然和去糖基化粘蛋白， 测定分子大小、回转半径、聚集 疏水结构域的行为和存在，c）制备 免疫探针针对天然和去糖基化粘蛋白， 为了检测体外细胞培养过程中产生的特异性脱粘蛋白， 犬气管上皮细胞mRNA的自由翻译 并筛选cDNA中脱粘蛋白的产生 在λ gt11载体中构建的文库，和d）分离和 表征犬气管无粘蛋白的cDNA克隆 上皮细胞，以确定一级氨基酸 序列和氨基酸序列，所述序列和氨基酸序列 处理. 粘蛋白将使用协议进行纯化 建立在这个实验室里。 生化表征将 包括：碳水化合物、硫酸盐、氨基酸 粘蛋白分子的组成、硫醇和二硫化物含量。 分子大小、回转半径与聚集行为 将使用光散射和沉降来确定 平衡方法 多克隆抗体和单克隆抗体 将使用建立的标准制备针对粘蛋白的抗体。 协议. 粘蛋白cDNA的分离、鉴定和检测 克隆和核苷酸测序将使用良好的 分子生物学技术。 知识 从拟议的研究中获得的成果最终将导致更多的 阻塞性肺疾病的有效治疗策略。

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.