GLYCOLIPID METABOLISM IN TUMOR AND TRANSFORMED CELLS

拟议研究的近期目标有六个方面： 1.乳清蛋白的分离、鉴定和纯化 GalNAcT-2(UDP-GalNAc:GbOse3Cer(Betal-3)N-acetylgalactosaminyltransferase) 和 GalNAcT-3(UDP-GalNAc:GbOse4Cer(Alphal-3)N-acetyl-galactosaminyltransferase) 豚鼠肿瘤洗涤剂增溶上清液中的存在 细胞系104CL(致癌)和106B(非致瘤)。 2.《福斯曼》中确切化学键的测定 104Cl和106B GalNAcT-3催化反应的生物合成产物。 3.GalNAcT-1(UDP-GalNAc：GM3或 豚鼠骨中的Lac-Cer(Betal-4)N-acetyl-galactosaminyltransferase) 骨髓。 4.衣霉素和胰岛素对GalNAcT-2和 GalNAcT-3在104CL和106B豚鼠肿瘤细胞中的活性 5.肿瘤特异性抗原的体外生物合成 神经母细胞瘤IMR-32细胞中唾液酸化Alpha2-3Le-x、唾液酸化Alpha2-3Le-a GD3和Fuc-GM1在大鼠前列腺PA-I/II细胞中的表达。 6.对 GalT-5(UDP-Gal:nLcOse4Cer(Alphal-3)galactosyltransferase)发件人 神经母细胞瘤IMR-32细胞及其在不同生长状态下的活性研究 细胞周期中的条件和分化的存在 试剂((但)2cAMP和HMBA)。 我们的总体目标是加深对 豚鼠三种肿瘤细胞系糖脂糖基转移酶的研究 人类起源。衣霉素抑制糖共轭生物合成。然而， 抑制糖基转移酶的糖基化是一个新的观察结果。 在该项目结束时，将尝试建立一个 银杏叶提取物对糖基转移酶(GalNAcT-2)的抑制作用 衣霉素与肿瘤特异性抗原Forssman GSL的变化 ~(125)I-Helix pomatia和~(125)I-Helix pomatia在104CL和106B细胞表面的研究 扁豆凝集素和SIGA毒素。小鼠基因组的筛选 AJ小鼠基因组DNA的EcoRI部分酶切文库 (Charon 4a中的猎枪)与GalNAcT-2的32P标记的cDNA插入将被 已执行。用125I-单抗和多克隆抗体翻译 还将对特定的104ClGalNAcT-2 DNA序列的产物进行筛选。

The immediate objectives of the proposed research are sixfold: 1. Separation, characterization, and purification of GalNAcT-2(UDP-GalNAc:GbOse3Cer(Betal-3)N-acetylgalactosaminyltransferase) and GalNAcT-3(UDP-GalNAc:GbOse4Cer(Alphal-3)N-acetyl-galactosaminyltransferase) present in the detergent solubilized supernatants from guinea pig tumor cell lines 104Cl (tumorigenic) and 106B(nontumorigenic), respectively. 2. Determination of the exact chemical linkages in the Forssman biosynthetic products of the 104Cl and 106B GalNAcT-3-catalyzed reactions. 3. Purification and kinetic studies of GalNAcT-1(UDP-GalNAc:GM3 or Lac-Cer(Betal-4)N-acetyl-galactosaminyltransferase) from guinea pig bone marrow. 4. Determination of the effects of tunicamycin and insulin on GalNAcT-2 and GalNAcT-3 activities in 104Cl and 106B guinea pig tumor cells. 5. Biosynthesis in vitro of the tumor-specific antigens sialosylAlpha2-3Le-x, sialosylAlpha2-3Le-a in neuroblastoma IMR-32 cells and GD3 and Fuc-GM1 in rat prostate PA-I/II cells. 6. Characterization of GalT-5(UDP-Gal:nLcOse4Cer(Alphal-3)galactosyltransferase) from neuroblastoma IMR-32 cells and study of its activity under various growth conditions during the cell cycle and in the presence of differentiating agent ((But)2cAMP and HMBA). Our overall goal is to develop an understanding of the specificities of glycolipid:glycosyltransferases in three tumor cell lines of guinea pig and human origin. Tunicamycin inhibits glycoconjugate biosynthesis. However, inhibition of glycosylation of glycosyltransferase is a novel observation. At the end of this project an attempt will be made to establish a correlation between inhibition of a glycosyltransferase (GalNAcT-2) by tunicamycin and the change of a tumor-specific antigen (Forssman GSL) on the surface of 104Cl and 106B cells using 125I-labeled Helix pomatia and Dolichos biflorus lectins and Siga toxin. Screening of the mouse genomic library, prepared from a partial EcoRI digestion of AJ mouse genomic DNA (Shotgun in Charon 4a) with a 32P-labeled cDNA insert of GalNAcT-2 will be performed. Using 125I-mono and polyclonal antibodies the translated products for particular 104Cl GalNAcT-2 DNA sequences will also be screened.