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

非低分子蛋白质微生物代谢物有望用于研究细胞生长机制以及降低动物细胞产生的生长因子和激素等蛋白质的生产成本。我们对这类化合物的筛选得到了四种独特的代谢物：从气生链霉菌中分离出了乳糖花青素，从剥离链霉菌中分离出了去叶青酮，从滑行细菌中分离出了间苯二酚，从味萨基链霉菌中分离到了强链霉菌和米沙基霉素。所有这些代谢物都是亲脂性的，并通过溶剂提取和适当的层析进行纯化。经核磁共振波谱分析，确定其结构如下：取代吩嗪类化合物Lavanducyanin，取代吩恶嗪酮Exfoliazone，间苯二酚，2-异十一基-5-异己基-间苯二酚，米沙基霉素，5-hydroxy-2，7-dimethoxy-6-methyl-1，4-naphthoquinone.These衍生物，在较低的血药浓度下，对多种动物细胞有较强的促生长活性。然而，在无血清条件下，没有观察到任何影响。用四甲基偶氮唑盐比色法、细胞数增加法和DNA合成法分析其活性，有效剂量在纳克到微克/毫升范围内。由于它们对RLN-8、NIH3T3和T601等细胞株都有促生长作用，其作用机制似乎是相同的。由于著名的促癌剂TPA在相同的条件下没有活性，因此本工作所分离的化合物的作用机理与TPA不同。

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.