Haughan P A, Chance M L, Goad L J
Department of Biochemistry, University of Liverpool, U.K.
Biochem J. 1995 May 15;308 ( Pt 1)(Pt 1):31-8. doi: 10.1042/bj3080031.
Leishmania donovani promastigotes were cultured in the presence of an azasterol (20-piperidin-2-yl-5 alpha-pregnane-3 beta,20-diol) to determine the effects on sterol biosynthesis and cell proliferation. Inhibition of growth increased gradually with azasterol concentrations up to 5 micrograms/ml; concentrations of azasterol exceeding 5 micrograms/ml were lethal. Sterol biosynthesis was affected by the azasterol when administered at concentrations as low as 100 pg/ml. The primary site of action was the alkylation at C-24 of a delta 24-sterol precursor. The 24-alkylated sterols [ergosta-5,7,24(24(1))-trien-3 beta-ol and ergosta-5,7,22-trien-3 beta-ol] of the protozoan were replaced by delta 24-cholesta-type sterols which then accumulated in the cells. Administration of the azasterol together with a bis-triazole inhibitor of the 14 alpha-methylsterol 14-demethylase reaction, which operates in sterol biosynthesis, resulted in depletion of 24-alkylsterols and their replacement with predominantly 14 alpha-methylsterols lacking a 24-alkyl group. Continuous subculture of promastigotes in the presence of the azasterol resulted in gradual depletion of 24-alkylsterols and their complete replacement by delta 24-cholesta-type sterols. Transfer of the azasterol-treated cells to medium lacking azasterol resulted in a gradual restoration, after several subcultures, of the normal 24-alkylsterol pattern. The results indicate that, although 24-alkylsterols are normally produced by the protozoan, it can nevertheless survive with sterols possessing only the cholestane skeleton. Thus there is no absolute requirement for 24-alkylsterols to fulfil some essential 'sparking' role associated with cell growth in promastigotes.
在氮杂甾醇（20-哌啶-2-基-5α-孕甾烷-3β,20-二醇）存在的情况下培养杜氏利什曼原虫前鞭毛体，以确定其对甾醇生物合成和细胞增殖的影响。随着氮杂甾醇浓度高达5微克/毫升，生长抑制逐渐增加；氮杂甾醇浓度超过5微克/毫升则具有致死性。当氮杂甾醇以低至100皮克/毫升的浓度给药时，甾醇生物合成受到影响。主要作用位点是δ24-甾醇前体在C-24位的烷基化。原生动物的24-烷基化甾醇[麦角甾-5,7,24(24(1))-三烯-3β-醇和麦角甾-5,7,22-三烯-3β-醇]被δ24-胆甾烷型甾醇取代，然后在细胞中积累。将氮杂甾醇与在甾醇生物合成中起作用的14α-甲基甾醇14-脱甲基酶反应的双三唑抑制剂一起给药，导致24-烷基甾醇耗竭，并被主要缺乏24-烷基的14α-甲基甾醇取代。在前鞭毛体在氮杂甾醇存在下连续传代培养导致24-烷基甾醇逐渐耗竭，并被δ24-胆甾烷型甾醇完全取代。将经氮杂甾醇处理的细胞转移到不含氮杂甾醇的培养基中，经过几次传代培养后，正常的24-烷基甾醇模式逐渐恢复。结果表明，尽管原生动物通常产生24-烷基甾醇，但它仍然可以在仅具有胆甾烷骨架的甾醇的情况下存活。因此对于前鞭毛体的细胞生长而言，没有绝对需要24-烷基甾醇来发挥某些必需的“启动”作用。
