BIOCHEMISTRY OF CYSTIC FIBROSIS MUCOUS SECRETIONS

这项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控机制 以及这些过程的分泌和与病理的关系 与慢性阻塞性肺疾病相关的高分泌，如 AS，囊性纤维化(CF)。粘液分泌过多，并有改变 粘弹性特性显然是导致临床症状的原因。至 目前，人们对构成粘蛋白的大分子知之甚少。 分泌物；尤其是关于其一级结构的知识 核心蛋白(S)缺乏。这项提议的目的是为了 对人体气管、支气管壁宏观结构组织的洞察 粘蛋白(HTMS)及其基因表达的调控。这个 具体目的是，a)提纯和比较其物理化学性质 从CF和正常肺分泌物中分离的粘蛋白HTM-1和HTM-2，b) 多抗和单抗的制备及鉴定 脱糖粘蛋白，c)制备基于以下物质的合成寡核苷酸探针 筛选粘蛋白的多肽序列，d)构建Lambda的cDNA文库 人气管上皮多聚(A)RNA的GT22A 细胞，e)使用免疫学和人工合成技术筛选文库 寡核苷酸探针，f)分离、鉴定和测序cDNA克隆 编码粘蛋白的蛋白质核心，g)使用这样获得的cDNA， 分离也将被测序的基因组克隆和g)以研究 选择性促分泌剂的作用机制(S)及作用(S) HTM无粘蛋白基因的转录。粘蛋白将被提纯 根据我们实验室制定的协议和他们的 物理化学性质将使用已有的技术来确定。 将从新鲜的hte细胞和培养的分泌hte的粘液中分离出mrna。 细胞。这些准备工作将被用来生成 将使用免疫学和合成寡核苷酸探针进行筛查。 将对插入的cDNA进行测序。克隆人身份将通过以下方式确认 选定的多肽片段的氨基酸序列分析。这些cDNA将 用于分离基因组克隆，这些克隆也将被测序。 促分泌剂(S)对粘蛋白相关基因合成的影响 用粘蛋白基因探针(S)进行Northern印迹分析。这一知识将会 是必要的，以便制定合理的方法来治疗 包括慢性阻塞性肺疾病在内的慢性阻塞性肺疾病最终将 需要有能力控制黏度和分泌物的速度。

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.