Mutomba M C, Wang C C
Department of Pharmaceutical Chemistry, University of California San Francisco 94143-0446, USA.
Mol Biochem Parasitol. 1995 Jun;72(1-2):215-25. doi: 10.1016/0166-6851(95)00081-b.
The bloodstream forms of Trypanosoma brucei monomorphic strain 427 serially passaged in rats can differentiate in vitro equally well in HMI-9, HMI-10, SDM-79 or Cunningham's medium into the insect (procyclic) forms by a simple temperature shift from 37 to 26 degrees C in the presence of citrate and cis-aconitate. The procyclic forms thus generated can also continue to multiply at 26 degrees C without replacing the culture medium. The same strain of T. brucei pre-adapted to grow as bloodstream forms in HMI-10 medium at 37 degrees C is also capable of differentiating showing a similar rate of variant surface glycoprotein (VSG) disappearance and appearance of phosphoenolpyruvate carboxykinase (PEPCK) under the same experimental conditions. However, appearance of both procyclin mRNA and procyclin protein is much delayed and the resulting procyclic forms cannot multiply. The culture-adapted bloodstream forms are capable of infecting rats, and the cells thus harvested from the rats can differentiate but cannot multiply in the same manner as the original culture-adapted bloodstream forms. Apparently, a certain variant has been selected during the adaptation of T. brucei bloodstream forms from rat blood to the culture medium. This variant could be a useful tool for identifying the genes involved in differentiation of T. brucei and multiplication of the procyclic forms. Comparison of the protein profiles between the wild-type and the variant during differentiation showed that a major protein band of about 70 kDa remained in the non-dividing variant procyclic forms but vanished in the rapidly dividing wild type procyclic forms. N-terminal determinations indicated that the 70-kDa protein band consists of bovine serum albumin and fetuin. Presumably these two serum proteins are actively taken up by the bloodstream forms via endocytosis. Since the procyclic forms are incapable of endocytosis, the serum proteins may be rapidly diluted in the growing wild type procyclic cells but remain unchanged in the non-dividing procyclic cells of the variant. Further studies are underway in trying to identify the key distinctions between these two lines of cells at the molecular level.
在大鼠体内连续传代的布氏锥虫单形株427的血流形式，在HMI-9、HMI-10、SDM-79或坎宁安培养基中，通过在柠檬酸盐和顺乌头酸盐存在下从37℃简单地转移至26℃，能够在体外同样良好地分化为昆虫（前循环）形式。由此产生的前循环形式也能够在26℃继续增殖而无需更换培养基。同一株布氏锥虫预先适应于在37℃的HMI-10培养基中作为血流形式生长，在相同实验条件下也能够分化，显示出相似的变异表面糖蛋白（VSG）消失速率和磷酸烯醇式丙酮酸羧激酶（PEPCK）的出现。然而，前环素mRNA和前环素蛋白的出现都大大延迟，并且产生的前循环形式不能增殖。适应培养的血流形式能够感染大鼠，从大鼠收获的细胞能够分化，但不能以前适应培养的血流形式相同的方式增殖。显然，在布氏锥虫血流形式从大鼠血液适应到培养基的过程中选择了某个变体。这个变体可能是鉴定参与布氏锥虫分化和前循环形式增殖的基因的有用工具。分化过程中野生型和变体之间蛋白质谱的比较表明，约70 kDa的主要蛋白条带在不分裂的变体前循环形式中保留，但在快速分裂的野生型前循环形式中消失。N端测定表明，70 kDa蛋白条带由牛血清白蛋白和胎球蛋白组成。推测这两种血清蛋白通过内吞作用被血流形式主动摄取。由于前循环形式不能进行内吞作用，血清蛋白可能在生长的野生型前循环细胞中迅速稀释，但在变体的不分裂前循环细胞中保持不变。正在进行进一步研究以试图在分子水平上确定这两种细胞系之间的关键差异。
