Islam M Khyrul, Miyoshi Takeharu, Yamada Manabu, Alim M Abdul, Huang Xiaohong, Motobu Maki, Tsuji Naotoshi
Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural Research Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.
Acta Trop. 2006 Oct;99(2-3):208-17. doi: 10.1016/j.actatropica.2006.08.007. Epub 2006 Sep 20.
Piperazine (diethylenediamine) is an anthelmintic widely used against animal and bird ascariasis. In this study, we show that treatment with piperazine blocks Ascaris suum larval moulting and development processes and affects larval proteome expression profiles. A. suum lung-stage L3 (LL3) obtained from an infected rabbit's lungs were cultured in RPMI medium in the presence of increasing concentrations of piperazine sulfate (Pzes). Our results showed that Pzes potently inhibited moulting of A. suum LL3 in a dose-dependent manner and that moulting was completely blocked (100%) at 50mM concentrations. We then examined the changes in A. suum LL3 proteome expression patterns following Pzes exposure using two-dimensional (2D) electrophoresis. Pzes exposure inhibited expression of at least 16 major protein spots in unmoulted LL3 out of more than 200 visible protein spots resolved on 2D gels prepared from moulted larvae (i.e., lung-stage L4). Pzes exposure also inhibited expression of 13 immunogenic protein spots in unmoulted LL3. More importantly, Pzes exposure inhibited activity of a moulting-specific enzyme, inorganic pyrophosphatase of A. suum (AsPPase), by 26%. Expression of native AsPPase was also reduced following Pzes exposure as detected by immunoblotting and immunofluorescent staining. Transmission electron microscopy showed that Pzes interfered with growth and ecdysis of the cuticle and caused damage to gut tissues of the larvae. Our results suggest that A. suum LL3 may become a suitable model to screening new-class anthelmintics with antimoulting functions and that A. suum LL3-Pzes may serve as a useful tool for identification of moulting-specific potential proteins in Ascaris roundworms.
哌嗪（二乙二胺）是一种广泛用于治疗动物和鸟类蛔虫病的驱虫药。在本研究中，我们发现哌嗪处理可阻断猪蛔虫幼虫的蜕皮和发育过程，并影响幼虫蛋白质组表达谱。从感染兔子肺部获得的猪蛔虫肺期L3（LL3）在添加不同浓度硫酸哌嗪（Pzes）的RPMI培养基中培养。我们的结果表明，Pzes以剂量依赖的方式有效抑制猪蛔虫LL3的蜕皮，在50mM浓度时蜕皮被完全阻断（100%）。然后，我们使用二维（2D）电泳检测了Pzes处理后猪蛔虫LL3蛋白质组表达模式的变化。Pzes处理抑制了未蜕皮LL3中至少16个主要蛋白点的表达，这些蛋白点在从蜕皮幼虫（即肺期L4）制备的2D凝胶上分辨出的200多个可见蛋白点中。Pzes处理还抑制了未蜕皮LL3中13个免疫原性蛋白点的表达。更重要的是，Pzes处理使猪蛔虫一种蜕皮特异性酶——无机焦磷酸酶（AsPPase）的活性降低了26%。免疫印迹和免疫荧光染色检测显示，Pzes处理后天然AsPPase的表达也降低。透射电子显微镜显示，Pzes干扰了角质层的生长和蜕皮，并对幼虫肠道组织造成损伤。我们的结果表明，猪蛔虫LL3可能成为筛选具有抗蜕皮功能新型驱虫药的合适模型，并且猪蛔虫LL3-Pzes可能作为鉴定蛔虫蜕皮特异性潜在蛋白的有用工具。
