As reported in [32]

As reported in [32] blog of sinaling pathways and shown in Table 1, the hGrx1-roGFP2 biosensor is not sensitive to direct interaction with micromolar H2O2 concentrations. However, millimolar H2O2 might lead to a direct oxidation of the probe. Since, however, H2O2 is likely to be at least partially detoxified by the antioxidative defense systems of the parasite-host cell unit, an oxidizing effect on the glutathione system also needs to be considered. The long and steady increase in the ratio after treatment with THBP might be associated to the fact that TBHP is, in contrast to H2O2, not detoxified by red blood cell catalase and is therefore likely to have more pronounced and longer-lasting effects. Induction of nitrosative stress using the peroxynitrite generator SIN-1 also led to a rapid ratio change.

Therefore, we assume that ROS and RNS do affect EGSH in P. falciparum and that hGrx1-roGFP2 seems to be a suitable tool to monitor these changes in living cells. However, when using the redox probe, one has to take into account that some compounds, independent from glutathione, might also directly interact with the probe. Thus, before testing the effects of antimalarial compounds on the ratio of the probe in living parasites, we characterized their direct in vitro interaction with recombinant hGrx1-roGFP2. Table 1 and Figs. 3 and S5 summarize the data for different concentrations and incubation times. Whereas high concentrations of oxidizing agents and redox cyclers such as GSSG, diamide, H2O2, MB, and PYO led to direct interactions with the probe, antimalarial drugs including the quinolines and artemisinin derivatives did not cause a major increase in the fluorescence ratio even at concentrations up to 100 ��M and after 24 h incubation.

These data can of course only be indirectly compared to the situation in vivo, where a direct interaction is hindered by multiple cell membranes, degradation of the stressors, and binding to other proteins. However, the data are important when interpreting the results described in the next paragraph. Whereas compounds such as MB might induce changes in the probe via direct interaction and by acting on the cellular redox metabolism, the direct interactions of the other drugs with the probe seem to be negligible. Furthermore, effects induced by diamide or H2O2 might recover more rapidly than those induced by potent redox cyclers such as MB and pyocyanin.

Effects of antimalarial drugs on the cytosolic glutathione redox potential In order to investigate whether hGrx1-roGFP2 can be used to monitor changes in EGSH after the treatment of cell cultures with antimalarial drugs, we incubated the Plasmodium falciparum strains 3D7 and Dd2 with different concentrations of MB, quinoline, and artemisinin derivatives Brefeldin_A in short-term (5 min), medium-term (4 h), and long-term (24 h) experiments (Table 2, Figs. 4�C7).

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>