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ELUCIDATION OF THE DNA SYNTHETIC CYCLE OF ENTAMOEBA HISTOLYTICA

溶组织内阿米巴 DNA 合成循环的阐明

基本信息

批准号：
06670269
负责人：
KOBAYASHI Seiki
金额：
$ 1.34万
依托单位：
KEIO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06670269/
关键词：
Entamoeba histolytica Entamoeba invadens DNA synthetic cycle flow cytometry mathematical modeling Entamoeba dispar axnic cultivation DNA polymerase Pseudomonas aeruginosa

项目摘要

We developed a method to study the DNA synthetic cycles of Entamoeba histolytica and Entamoeba invadens by flow cytometry (FCM) based on a preparative procedure to reduce both high levels of natural fluorescence and non-specific adsorption of flurochromes. We modeled G_1, S,and G_2 phases as a series of overlapping Gaussian curves. Both E.histolytica and E,invadens displayd G_1, S,and G_2 proportions that are consistent with eukaryotic cell populations in exponential of stationary growth phase. Exponetial phase E.histolytica populations contained a hypodiploid subset with a mass of about 20% less than the diploid value which we estimate by FCM to be 24 x 10^<-14>gDNA/cell.Exponential phase E.invadens populations contained a hypodiploid subset with a mass of about 6% less than the diploid value which estimate by FCM to be 30 x 10^<-14>gDNA/cell.We developed also an axnic cultivation system for Entamoeba dispar which was nonpathogenic amoeba, by using newly developed BCSI-S medium in the presence of Crithidia fasciculata. sterilized by treating with 1% hydrogen peroxide for 24 hours at 4ﾟC.DNA polymerase activity of the ameba which was grown in this culture system, was detected and characterized. DNA polymerase activity was high at pH 2 to pH 6, but at pH 8 and 10 the activity was very low in nuclear extracts from both trophozoites of E.histolytica and E.dispar which were grown axnically.

我们开发了一种通过流式细胞术 (FCM) 研究溶组织内阿米巴和侵入内阿米巴的 DNA 合成循环的方法，该方法基于减少高水平自然荧光和荧光染料非特异性吸附的制备程序。我们将 G_1、S 和 G_2 阶段建模为一系列重叠的高斯曲线。溶组织阿米巴和入侵阿米巴都表现出G_1、S和G_2比例，与稳定生长期指数的真核细胞群一致。指数期 E.histolytica 群体包含的亚二倍体子集，其质量比我们通过 FCM 估计为 24 x 10^<-14>gDNA/细胞的二倍体值小约 20%。指数期 E.invadens 群体包含的亚二倍体子集，其质量比二倍体值小约 6%，通过 FCM 估计为 30 x 10^-14gDNA/细胞。我们还通过在存在Critithia fasciculata的情况下使用新开发的BCSI-S培养基，开发了非致病性阿米巴内阿米巴(Entamoeba dispar)的轴生培养系统。用1%过氧化氢在4℃下处理24小时进行灭菌。检测并表征了在该培养系统中生长的阿米巴的DNA聚合酶活性。在 pH 2 至 pH 6 时，DNA 聚合酶活性较高，但在 pH 8 和 10 时，轴向生长的溶组织内阿米巴和迪斯帕内皮细胞滋养体的核提取物中，DNA 聚合酶活性非常低。