STUDIES ON THE ENCYSTATION OF ENTAMOEBA

以入侵内阿米巴（Entamoeba invadens）无菌成环系统为模型，研究了核复制DNA聚合酶特异性抑制剂aphidicolin对溶组织芽胞杆菌成环的影响。阿蚜霉素对胞囊的抑制作用是可逆的。滋养体经药物预处理后，成囊作用几乎完全被抑制。这可以解释为aphidicolin处理后细胞聚集在G1/S边界，这些细胞在胞内培养基中既不能合成DNA也不能分裂，而未经预处理的细胞包括G2和M细胞，它们已经合成了DNA，因此不受aphidicolin的影响。与滋养体相比，囊体中DNA聚合酶活性水平随着囊化的进行而降低。结果表明，环状囊肿的形成伴随着DNA聚合酶活性的降低，这与之前的发现有关，即在没有DNA合成的情况下，环状囊肿的成熟会发生核分裂。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹法检测了侵染大肠杆菌囊特异性蛋白的形态和免疫原性。兔抗囊肿血清与滋养体蛋白和囊肿蛋白反应。最有趣的结果是，兔抗囊肿血清在孵育1天后主要与88-kDa的囊肿蛋白反应。88-kDa蛋白与被滋养体蛋白吸收的抗囊肿血清反应，因此具有囊肿特异性，并且发现该蛋白存在于富含细胞壁碎片的颗粒部分中。结果表明，囊化伴随着囊肿特异性88-kDa糖蛋白的出现，该糖蛋白在孵育1天后在囊肿中具有免疫优势并最丰富地表达，并且似乎与囊肿壁有关。研究了微丝特异性抑制剂细胞松弛素D对侵染大肠杆菌的抑制作用。细胞松弛素D呈剂量依赖性地抑制胞囊化。这是微丝参与胞囊的第一个证据。在细胞松弛素D的作用下，滋养体变成多核，并产生多核包囊。与未施药的滋养体相比，施药后滋养体细胞松弛素D的胞吞受到更大的抑制。因此，细胞松弛素D对滋养体的多核具有抑制作用。少

Using an axenic encystation system of Entamoeba invadens as a model for E. histolytica encystation, effect of aphidicolin, a specific inhibitor of nuclear replicative DNA polymerases, on the encystation was examined. Aphidicolin inhibited the encystation and its effect was reversible. Encystation was almost completely inhibited when trophozoites were pretreated with the drug. This can be explained that treatment with aphidicolin accumulates the cells at G1/S border and those cells can neither synthesize DNA nor divide when placed in encystation medium, whereas the cells without the pretreatment include G2 and M cells which have already synthesized DNA so that they are not affected by aphidicolin. The level of DNA polymerase activity in cysts decreased as encystation proceeded as compared with that of trophozoites. The results indicate that encystation is accompanied by a reduced level of DNA polymerase activity, which correlates with the previous finding that nuclear division occurs du … More ring cyst maturation in the absence of DNA synthesis. The appearance of cyst-specific proteins in encysting E. invadens and their immunogenicity were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. A rabbit antiserum against cysts reacted with the trophozoite proteins as well as cyst proteins. The most interesting result was that the rabbit anticyst serum reacted predominantly with an 88-kDa protein of cysts after 1 day of incubation. The 88-kDa protein reacted with the anticyst serum absorbed with trophozoite proteins and was thus cyst-specific and the protein was found to be present in the particulate fraction, which was rich in cell-wall fragments. The results indicate that encystation is accompanied by appearance of the cyst-specific 88-kDa glycoprotein, which is immunodominant and most abundantly expressed in cysts after 1 day of incubation and appears to be associated with the cyst wall. Effect of cytochalasin D, a specific inhibitor of microfilaments, on the encystation of E. invadens was examined. Encystation was inhibited by cytochalasin D in a dose-dependent manner. This is the first evidence for participation of microfilaments in the encystation. Trophozoites grown with cytochalasin D became multinucleate and produced multinucleate cysts when transferred to encystation medium. Encystation with cytochalasin D of trophozoites grown with the drug was more inhibited than that of trophozoites grown without the drug. Thus multinucleation of trophozoites by cytochalasin D had an inhibitory effect on the encystation. Less