BIOCHEMISTRY OF CYSTIC FIBROSIS MUCOUS SECRETIONS

本建议中概述的研究的长期目标是 了解粘蛋白生物合成的调节机制 以及这些过程与 与慢性阻塞性肺疾病相关的高分泌，如 如囊性纤维化（CF）。 粘液分泌过多， 粘弹特性显然有助于临床症状。 到 到目前为止，对构成细胞的粘蛋白大分子知之甚少。 分泌物;尤其是关于其主要结构的知识 核心蛋白缺乏。 本提案的目的是获得 人气管支气管宏观结构的组织学研究 粘蛋白（HTMs），并研究其基因表达的调控。 的 具体目的是，a）纯化和比较 分离自CF和正常肺分泌物的粘蛋白HTM-1和HTM-2，B） 制备和表征抗 去糖基化粘蛋白，c）基于以下制备合成的寡核苷酸探针： 选择粘蛋白的肽序列，d）在λ中构建cDNA文库， gt 22 A来自从人气管上皮（HTE）分离的poly（A）+RNA e）使用免疫学和合成的方法筛选文库， 寡核苷酸探针，f）分离、表征和测序cDNA克隆 编码粘蛋白的蛋白质核心，g）使用由此获得的cDNA， 分离也将测序的基因组克隆，和g）研究 选择性促分泌素调节 HTM脱粘蛋白基因的转录。粘蛋白将被纯化 根据我们实验室建立的协议， 将使用已建立的技术测定物理化学性质。 将从新鲜HTE细胞和分泌HTE的培养粘液中分离mRNA 细胞 这些制备物将用于产生cDNA文库， 将使用免疫学和合成寡核苷酸探针进行筛选。 将对cDNA插入片段进行测序。 克隆身份将由 所选肽片段的氨基酸序列分析。 cDNA将 用于分离也将被测序的基因组克隆。 将检测促分泌素对粘蛋白相关mRNA合成的影响 通过使用粘蛋白cDNA探针的北方印迹分析。 这些知识将 为了制定合理的方法来处理 慢性阻塞性肺疾病，包括CF， 需要控制分泌物的粘度和速率的能力。

The long term goals of the studies outlined in this proposal are to understand the mechanisms involved in the regulation of mucin biosynthesis and secretion and relationship of these processes to the pathology of hypersecretion associated with chronic obstructive pulmonary diseases, such as, cystic fibrosis (CF). Excess mucus secretions with altered viscoelastic properties clearly contributes to the clinical symptoms. To date, little is known about the mucin macromolecules that constitute the secretion; especially knowledge regarding the primary structures of their core protein(s) is lacking. The objectives of this proposal are to gain insights into organization of the macrostructure of human tracheobronchial mucins (HTMs) and to investigate regulation of their gene expression. The specific aims are to, a) purify and compare physicochemical properties of mucins HTM-1 and HTM-2 isolated from CF and normal lung secretions, b) prepare and characterize poly- and monoclonal antibodies against deglycosylated mucins, c) prepare synthetic oligonucleotide probes based on select peptide sequences of the mucins, d) construct cDNA library in lambda gt22A from poly(A) +RNA isolated from human tracheal epithelial (HTE) cells, e) screen the library using immunological and synthetic oligonucleotide probes, f) isolate, characterize and sequence cDNA clones encoding for protein cores of the mucins, g) using cDNAs, thus obtained, isolate genomic clones which will also sequenced and g) to investigate the mechanism(s) and effects of select secretagogue(s) on regulation of transcription of the HTM apomucin gene. The mucins will be purified according to protocols established in our laboratory and their physicochemical properties will be determined using established techniques. mRNA will be isolated from fresh HTE cells and cultured mucus secreting HTE cells. These preparations will be used to generate cDNA libraries which will be screened using immunological and synthetic oligonucleotide probes. The cDNA inserts will be sequenced. Clone identity will be confirmed by amino acid sequence analysis of selected peptide fragments. The cDNAs will be utilized to isolate genomic clones which will also be sequenced. Effects of secretagogue(s) on mucin related mRNA synthesis will be detected by Northern blot analysis using mucin cDNA probe(s). This knowledge will be essential in order to develop a rational approach to the treatment of chronic obstructive lung diseases including CF which will ultimately require an ability to control the viscosity and rate of secretion.