Molecular and cellbiological studies on gametocytogenesis of a malaria parasite, Plasmodium falciparum

利用野生分离株，从细胞生物学和分子生物学两方面研究了恶性疟原虫配子细胞发生的诱导机制。野生分离株配子细胞的诱导：以缅甸和印度尼西亚的野生分离株为研究对象，研究了配子细胞的诱导方法。当寄生虫密度达到5%左右时，可观察到配子细胞发生，然后在比正常培养条件高3-4倍的红细胞浓度下培养。所有适合体外培养的野生菌株均能形成配子体，其转化率可达50 ~ 70%。葡萄糖缺乏诱导配子细胞发生：由于配子细胞发生是在较高红细胞浓度下培养的，因此推测营养缺乏，特别是葡萄糖缺乏可能在触发这一现象中起重要作用。事实上，在培养基中添加葡萄糖抑制了配子细胞发生的诱导。此外，在正常培养条件下，添加2-脱氧葡萄糖可以诱导寄生，但寄生率明显降低。这些结果表明，葡萄糖缺乏可能在诱导配子细胞发生中起重要作用。在已建立的菌株中诱导配子细胞发生的试验：在FCR-3和k - 1菌株中，与野生菌株一样进行了诱导配子细胞发生的试验，但没有形成配子细胞，这表明在这些菌株中可能缺失了诱导配子细胞发生的重要基因。诱导配子细胞发生的主基因的鉴定：利用从诱导和非诱导的寄生虫中分离的mRNA的减法，研究了可能调控配子细胞发生的主基因。然而，减去后得到的所有克隆都是热休克蛋白基因，而预期的基因，如camp依赖性激酶，没有检测到。

Induction mechanism of gametocytogenesis in a malaria parasite, Plasmodium falciparum was investigated cell-biologically and molecularly, using wild isolates.1. Induction of gametocytogenesis in wild isolates : Induction methods of gametocytogenesis was studied, using wild isolates collected from Myanmar and Indonesia.Gametocytogenesis was observed when the parasite density reached to ca. 5% and then they were cultivated at 3-4 times higher concentrations of RBC than normal culture condition. All wild isolates adapted for in vitro cultivation were capable in forming gametocytes, and their transformation rates reached to 50-70%.2. Induction ofgametocytogenesis by glucose deficiency : As gametocytogenesis was induced by cultivation at higher RBC concentration, it was speculated that nutrient deficiency, particularly glucose, may play important role in triggering this phenomenon. In fact, addition of glucose into the medium inhibited the induction of gametocytogenesis. Furthermore, addition of 2-deoxy glucose was capable in inducing it under a normal culture condition, although a great decrease ofparasitaemia was observed. These results indicated that glucose deficiency might play an important role in inducing gametocytogenesis.3. Trials of induction ofgametocytogenesis in established strains : Using FCR-3 and K-l strains, induction of gametocytogenesis was examined as like in wild isolates, but no gametocyte was formed, suggesting that important genes for induction of gametocytogenesis might be deleted in these strains.4. Identification of master gene(s) for induction of gametocytogenesis : Using a subtraction method between mRNA isolated from gametocyogenesis-induced and non-induced parasites, master gene(s) which may regulate gametocytogenesis was investigated. However, all clones obtained after the subtraction were only heat shock protein genes, and expected genes, such as cAMP-dependent kinases, were not detected.