Quantitative assessment of DNA replication to monitor microgametogenesis in Plasmodium berghei
Quantitative assessment of DNA replication to monitor microgametogenesis in Plasmodium berghei
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DOI：
10.1016/j.molbiopara.2009.08.004
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发表时间：
2009-12-01
影响因子：
1.5
通讯作者：
Wengelnik, Kai
中科院分区：
文献类型：
--
作者：
Raabe, Andreas C.;Billker, Oliver;Wengelnik, Kai
针对疟原虫从脊椎动物宿主向蚊子媒介转化的关键步骤，是消除疟疾的一种有希望的途径。蚊子的吸收会在几秒钟内激活配子细胞，在雄性（微型）配子细胞的情况下，会导致DNA的快速复制和8个鞭毛配子的释放。我们开发了一种基于[H-3]次黄嘌呤与DNA结合的灵敏检测方法来监测伯氏小配子细胞的激活。确定了配子体活化的最佳pH范围和黄嘌呤酸浓度，并对DNA复制动力学进行了研究。用柏氏假体突变体证实了该方法的意义，并将该方法应用于蛋白酶抑制剂的效果分析，发现它们的抑制作用存在差异。因此，该方法适用于小配子细胞活化的可重复性测定、中等通量药物筛选和配子发生早期阻滞的深入研究，并将有助于分析化合物的传输阻断活性。(C) 2009 Elsevier B.V.版权所有
Targeting the crucial step of Plasmodium transition from vertebrate host to mosquito vector is a promising approach to eliminate malaria. Uptake by the mosquito activates gametocytes within seconds, and in the case of male (micro) gametocytes leads to rapid DNA replication and the release of eight flagellated gametes. We developed a sensitive assay to monitor P. berghei microgametocyte activation based on [H-3]hypoxanthine incorporation into DNA. Optimal pH range and xanthurenic acid concentrations for gametocyte activation were established and the kinetics of DNA replication investigated. Significance of the method was confirmed using P. berghei mutants and the assay was applied to analyse the effect of protease inhibitors, which revealed differences regarding their inhibitory action. The developed method thus appears suitable for reproducible determination of microgametocyte activation, medium-throughput drug screenings and deeper investigation of early blocks in gametogenesis and will facilitate the analysis of compounds for transmission blocking activities. (C) 2009 Elsevier B.V. All rights reserved.