Glutathione-S-transferases from chloroquine-resistant and -sensitive strains of Plasmodium falciparum: what are their differences?

Glutathione-S-transferases (GSTs) from chloroquine-resistant (CQR, K1) and -sensitive (CQS, T9/94) strains of Plasmodiumfalciparum were studied. The enzymes from both strains exhibited the optimal pH for enzyme catalysis, at pH 7.5, and were stable at temperatures below 60 degrees C. They also showed the highest specific activities toward CDNB and moderate activities to ethacrynic acid (40% of the activity to CDNB) but little or no activity for other substrates. Km and Vmax values for CDNB and GSH, calculated by Lineweaver-Burk plot from both CQR- and CQS-GSTs, were not statistically different (p<0.05). However, the GSTs activity from CQR appeared to be significantly higher than that from CQS. Therefore, we proposed that GSTs from both malarial strains are identical in their functional domain but different in level of gene expression. Furthermore, protein sequence alignment between P. falciparum GST and GSTs from other organisms suggested that the malarial enzyme is closely similar to other GSTs in Sigma, Alpha, Mu and Pi subclasses, probably most to the Alpha group. Characterization of the purified malarial GST in detail would reveal more precise classification and better understanding of its role in malarial detoxification.

Partial purification of mitochondrial DNA topoisomerase II from Plasmodium falciparum and its sensitivity to inhibitors.

Mitochondria of Plasmodium falciparum (K1 strain) were isolated by differential centrifugation. Mitochondrial DNA topoisomerase II from P. falciparum was partially purified using fast protein liquid chromatography(FPLC). Parasite mitochondria contained approximately 8% of DNA topoisomerase II activity compared with its nuclear fraction. The effects of fluoroquinolones, inhibitors of bacterial DNA topoisomerase II or DNA gyrase, against partially purified P. falciparum mitochondrial DNA topoisomerase II were investigated using a decatenation assay. Minimum inhibitory concentrations (MIC) of ofloxacin, ciprofloxacin and norfloxacin were > 1, 10 and 100 mM, compared with that of >0.5 and 10 mM for eukaryotic DNA topoisomerase II inhibitor etoposide (VP-16) and amsacrine, respectively. The results indicate that partially purified mitochondrial DNA topoisomerase II was insensitive to fluoroquinolones and it is suggested that their inhibitory effects on P. falciparum growth may be directed against plastid DNA topoisomerase II.

In vitro sensitivity of Trichomonas vaginalis to DNA topoisomerase II inhibitors.

Vaginal trichomoniasis is a highly prevalent sexually transmitted disease caused by a microaerophilic protozoan Trichomonas vaginalis. The disease is one of the most common sexually transmitted disease and can augment the predisposition of individuals to human immunodeficiency virus (HIV) infection. Although the disease can be treated with metronidazole and related 5-nitroimidazole, cases of trichomonal vaginitis which are refractory to standard treatment seems to be increasing. Clearly, new antitrichomonad agents are needed and DNA topoisomerase II may acts as a new target for antitrichomonad agents. In this study, in vitro sensitivity of T. vaginalis to DNA topoisomerase II was investigated. Axenic culture of local strain of T. vaginalis was performed. Both eukaryotic and prokaryotic DNA topoisomerase II inhibitors such as ellipticine, amsacrine and fluoroquinolones were tested for effectiveness against T. vaginalis in vitro compared to metronidazole. T. vaginalis was sensitive to metronidazole under aerobic conditions. Minimal inhibitory concentrations (MICs) of eukaryotic DNA topoisomerase II inhibitors, ellipticine and amsacrine, were 6.4 mM and 64 mM, respectively. The MICs of prokaryotic DNA topoisomerase II or DNA gyrase inhibitors; ciprofloxacin, ofloxacin and norfloxacin were 64, 960 and 1,280 mM, respectively. Based on the results, among DNA topoisomerase II inhibitors ellipticine was the most effective drug against T. vaginalis in vitro whereas fluoroquinolones did not show high antitrichomonad activity.

Large scale culture technique for pure Plasmodium falciparum gametocytes.

A large scale technique for pure gametocyte cultures of Plasmodium falciparum was established in culture flasks by using treated erythrocytes and RPMI medium supplemented with 15% human plasma instead of serum. Pure gametocyte cultures were successfully obtained following treatment with 5% sorbitol on day 8 and 9 of cultivation. This method resulted in approximately 97% reduction of asexual parasites and provided pure gametocytes in culture. The highest numbers of gametocytes were obtained from cultures starting with 2% parasitemia and 2% erythrocyte suspension. On day 12 of cultivation, approximately 35 x 10(6) gametocytes per 100 ml of cell suspension could be harvested.

Development of cultivation technique for pure Plasmodium falciparum gametocytes.

Pure gametocyte culture of Plasmodium falciparum, isolate KT3, from Kanchanaburi Province, Thailand, was successfully established by 5% sorbitol treatment on days 9, 10 and 11 following initiation of culture. Using medium supplemented with 15% human plasma and activated erythrocytes, daily medium change was not required during the cultivation. There was 99% reduction in the numbers of asexual parasites in the culture but the numbers of gametocytes were not affected. Furthermore, the gametocytes could undergo their usual morphological development with retention of function as demonstrated by the appearance of exflagellating microgametocytes, macrogametocytes and of oocyst formation in midgut of infected mosquito.