GLYCOSYLATION IN EXPERIMENTAL CARCINOGENESIS

这项研究建议的长期目标是深入了解 N-糖基化在致癌过程中的相关性及其研究进展 较新的抗体和基因探针不仅有助于更好地了解 不仅在癌症的诊断和预后方面，而且在人类癌症的诊断和预后方面也是如此。朝向 为此，我们已经证明了肝脏结节表达了一种独特的 N-乙酰氨基葡萄糖基转移酶III(GnT-III)与 肿瘤组织中N-乙酰氨基葡萄糖(Gn)的生物合成 衍生的糖蛋白，正常肝脏不存在。《公约》的具体目标 目前的建议是：(A)完成GnT-Ill的提纯 在过去的赠款期间启动，(B)准备多克隆和 抗纯化酶的单抗，(C)克隆基因 (D)研究其在实验性肝脏中的表达。 致癌过程。该酶将从肝脏结节中提纯。 以1，2-二甲基肼为模型生成的口香酸 344只雄性Fischer大鼠的引发剂。初步提纯将是 使用我们开发的基于该版本的新程序完成 经磷脂酶C处理后的微粒体膜的活性。 最终的纯化将通过柱层析实现，包括 使用新型亲和层析吸附剂的亲和层析 哪一种UDP-N-乙酰氨基葡萄糖已连接到硫丙基-琼脂糖基 尿嘧啶的5个位置通过5-汞基硫醇键。抗病毒抗体 这种酶将通过标准程序制备。用于克隆 GnT-III基因、肝结节基因表达文库 建造的。将采取两种相辅相成的方法。在第一种方法中 该基因将通过筛选该基因的表达文库进行克隆。 结节和大鼠肾带抗体。在第二种方法中 将在实验确定的基础上合成寡核苷酸 GnT-III的氨基酸序列。寡核苷酸探针将用于 筛选相同的c DNA表达文库。克隆的cdna将被 GnT-III的限制性内切酶图谱和测序以及最终的基因组DNA 将被克隆。

The long term objective of this research proposal is to gain insight on the relevance of N-glycosylation in carcinogenic process with a view to develop newer antibodies and gene probes useful not only for a better understanding of cancer but also in the diagnosis and prognosis of human cancer. Towards this end we have demonstrated that hepatic nodules express an unique N-acetylglucosaminyltransferase III (GnT-III) implicated in the biosynthesis of bisecting N-acetylglucosamine (Gn) detected in cancer derived glycoproteins and not in normal liver. The specific aims of the current proposal will be (a) to complete the purification of the GnT-Ill initiated during the past grant period, (b) to prepare polyclonal and monoclonal antibodies against the purified enzyme, (c) to clone the gene for the enzyme and (d) to study its expression during experimental liver carcinogenic process. The enzyme will be purified from hepatic nodules generated by orotic acid model using 1,2-dimethylhydrazine as the initiating agent in male Fischer 344 rats. Initial purification will be done using a novel procedure developed by us which is based on the release of the activity from the microsomal membrane by phospholipase C treatment. Final purification will be achieved on column chromatography including affinity chromatography using a new affinity chromatography absorbant in which UDP-N-acetylglucosamine has been linked to thiopropyl-sepharose at the 5 position of the uracil via a 5-mercurimercaptide bond. Antibody to the enzyme will be prepared by standard procedures. For the cloning of GnT-III gene, cDNA expression library for hepatic nodule will be constructed. Two complementary methods will be adopted. In the first method the gene will be cloned by screening the cDNA expression libraries of the nodules and rat kidney with the antibody. In the second method oligonucleotide will be synthesized based on the experimentally determined amino acid sequence of GnT-III. The oligonucleotide probe will be used to screen the same cDNA expression libraries. The cloned cDNA will be restriction mapped and sequenced and ultimately genomic DNA for GnT-III will be cloned.