Phillips M A, Wang C C
Mol Biochem Parasitol. 1987 Jan 2;22(1):9-17. doi: 10.1016/0166-6851(87)90064-8.
Procyclic Trypanosoma brucei brucei strain 366D is susceptible to DL-alpha-difluoromethylornithine (DFMO) with an in vitro ED50 value of 225 microM. A mutant of the procyclic strain resistant to 20 mM of DFMO was isolated by serial in vitro passages of the organisms in increasing concentrations of the drug. Drug resistance remains unchanged after at least ten serial passages in the absence of DFMO. The mutant contains the same level of ornithine decarboxylase activity as the wild-type procyclic, and the mutant enzyme exhibits a similar susceptibility toward DFMO as the wild type. Neither the rate of decarboxylation of ornithine, nor the membrane potential in the mutant cell is changed. The only observed change in the mutant is its significantly decreased uptake of DFMO which reaches a saturating level of 18 microM inside the cells; a concentration seven times below the Ki value of DFMO on T. brucei ornithine decarboxylase (130 microM). Apparently, the failure of DFMO uptake in the mutant strain has provided the basis of drug resistance. The results also raise the question on whether the uptake of DFMO by T. brucei is by passive diffusion or by transporter(s) mediation. DFMO does not compete with the uptake of ornithine, arginine or putrescine, and the reverse holds also true. However, the mutant strain cultivated under DFMO for several generations has a greatly enhanced uptake of ornithine and a moderately heightened uptake of putrescine. Both are reduced to the normal level upon further propagations of the mutant strain in the absence of DFMO.
原循环型布氏布氏锥虫366D株对DL-α-二氟甲基鸟氨酸（DFMO）敏感，体外半数有效浓度（ED50）值为225微摩尔。通过在不断增加药物浓度的情况下对该生物体进行连续体外传代，分离出了对20毫摩尔DFMO具有抗性的原循环型菌株突变体。在无DFMO的情况下至少连续传代十次后，耐药性保持不变。该突变体所含鸟氨酸脱羧酶活性水平与野生型原循环型相同，且突变酶对DFMO的敏感性与野生型相似。突变体细胞中鸟氨酸的脱羧速率和膜电位均未改变。在该突变体中唯一观察到的变化是其对DFMO的摄取显著减少，细胞内达到饱和水平18微摩尔；该浓度比DFMO对布氏锥虫鸟氨酸脱羧酶的抑制常数（Ki值，130微摩尔）低七倍。显然，突变菌株中DFMO摄取失败是耐药性产生的基础。这些结果还引发了关于布氏锥虫摄取DFMO是通过被动扩散还是转运蛋白介导的问题。DFMO不与鸟氨酸、精氨酸或腐胺的摄取竞争，反之亦然。然而，在DFMO条件下培养几代的突变菌株对鸟氨酸的摄取有极大增强，对腐胺的摄取有适度提高。在无DFMO的情况下对突变菌株进一步传代时，两者均降至正常水平。
