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Biochemical studies on cell growth stimulating substances of low molecular weight produced by microorganisms

微生物产生的低分子量细胞生长刺激物质的生化研究

基本信息

批准号：
03453141
负责人：
SETO Haruo
金额：
$ 0.96万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

None proteinous microbial metabolites with low molecular weight are expected to be useful for studying cell growth mechanisms as well as for reducing production cost of proteins such as growth factors and hormones produced by animal cells. Our screening for such compounds resulted in isolation of four unique metabolites; lavanducyanin from Streptomyces aeriouvifer, exfoliazone from Streptomyces exfoliatus, resorcinin from a gliding bacterium, Cytophaga johnsonae and misakimycin from Streptomyces misakiensis. All of these metabolites were lipophilic and purified by solvent extraction followed by appropriate chromatographies. Their structures were determined mainly based on NMR spectral analysis to be as follows; lavanducyanin, a phenazine derivative with an alkyl substituent; exfoliazone, a substituted phenoxazinone; resorcinin, 2-isoundecy1-5-isohexyl-resorcinol; misakimycin, 5-hydroxy-2,7-dimethoxy-6-methyl-1,4-naphthoquinone.These derivatives showed strong growth promoting activities to some kind of animal cell lines at lower serum concentrations. However, no effect was observed under serum-free conditions. Their activities were analyzed by MTT assay and by the increases of cell number and DNA synthesis, and their effective doses were determined at the range of nanogram to microgram/ml.Since the growth stimulating effect were observed with same kinds of cell lines including RLN-8, NIH 3T3 and T601, their action mechanisms seem to be identical. Since the famous tumor promoter TPA was inactive under the same condition, the action mechanisms of the compounds isolated by this work are concluded to be different from that of TPA.

具有低分子量的非蛋白质微生物代谢物预期可用于研究细胞生长机制以及用于降低由动物细胞产生的蛋白质如生长因子和激素的生产成本。我们对这些化合物的筛选导致了四种独特的代谢产物的分离;从Streptomyces aeriouvifer中分离出lavanducyanin，从Streptomyces exfoliatus中分离出exfoliazone，从滑行细菌中分离出间苯二酚，从Streptomyces misakiensis中分离出Cytophaga johnsonae和misakimycin。所有这些代谢产物均为亲脂性，并通过溶剂提取和适当的色谱法进行纯化。通过核磁共振谱分析，确定了它们的结构：lavanducyanin，一种含烷基取代的吩嗪衍生物; exfoliazone，一种取代的吩恶嗪酮; resorcinin，2-isundecyl-5-isohexyl-resorcinol; misakimycin，5-hydroxy-2，7-dimethoxy-6-methyl-1，4-naphthoquinone。然而，在无血清条件下没有观察到任何效果。用MTT法、细胞计数法和DNA合成法测定了它们的活性，并确定了它们的有效剂量范围为ng ~ μ g/ml。由于它们对同种细胞系RLN-8、NIH 3 T3和T601均有促生长作用，因此它们的作用机制似乎是一致的。由于著名的肿瘤促进剂TPA在相同条件下无活性，因此本工作所分离的化合物的作用机制与TPA不同。