Nitric oxide synthase expression in glial cells: suppression by tyrosine kinase inhibitors.

Rat brain glial cells have the capacity to express a calcium-independent form of nitric oxide synthase (iNOS). To test if iNOS induction required tyrosine kinase activity, we made use of genistein, a selective inhibitor of tyrosine kinases. In both primary astrocyte cultures and C6 glioma cells, the presence of genistein prevented both lipopolysaccharide- and cytokine-induced NOS activity in a dose-dependent manner. The presence of tyrphostin-25 (10 microM), which is highly specific for tyrosine kinases, also blocked iNOS induction. Additional characterization showed that genistein blocked iNOS induction in a dose-dependent manner (IC50 of approximately 40 microM), that the continuous presence of genistein was not necessary to observe inhibition, and that preincubation with genistein led to higher levels of inhibition than the simultaneous addition of genistein and inducers. The decrease in iNOS activity due to genistein was accompanied by a decrease in iNOS mRNA level as detected by a specific PCR assay. These results indicate that induction of astroglial iNOS expression requires tyrosine kinase activity.

Clones of the malaria parasite Plasmodium falciparum obtained by microscopic selection: their characterization with regard to knobs, chloroquine sensitivity, and formation of gametocytes.

A culture line of Plasmodium falciparum (FCR-3/Gambia) was used to select and place in culture cells containing a single parasite. The method depends on examination of minute droplets of dilute cell suspensions with oil immersion phase-contrast microscopy. Droplets found to contain a single parasite were maintained under appropriate culture conditions until detectable numbers of parasites were present (generally by day 21). Of nine clones that grew up, seven were knobless and two were knobby. The clones differed somewhat in chloroquine sensitivity. Their 50% inhibition point under one set of conditions in vitro ranged from 0.02 to 0.06 micrograms of base per ml, compared with only 0.003 micrograms for the highly sensitive line FCR-8/West Africa. All three tested clones formed gametocytes under appropriate in vitro conditions.