Crucial role of cytosolic tryparedoxin peroxidase in Leishmania donovani survival, drug response and virulence

Leishmania donovani, the causative agent of visceral leishmaniasis, uses a cascade of enzymes that include cytosolic tryparedoxin peroxidase (cTXNPx) for detoxification of peroxides, an event pivotal for survival of digenic parasites living in two disparate biological environments. In this study, we observed an increase in promastigote cTXNPx levels after exposure to H2O2 and this group did not show any cell death; however, exposure to a combination of H2O2 and nitric oxide resulted in significant reduction of cTXNPx levels accompanied by high cell death. The protective relationship between higher levels of cTXNPx and survival was further substantiated by the improved ability of L. donovani promastigotes overexpressing cTXNPx to withstand exposure to H2O2 and nitric oxide combination as compared with vector transfectants. In addition, cTXNPx transfectants demonstrated increased virulence, causing higher parasite burden in macrophages as compared with vector transfectants. Interestingly, the cTXNPx transfectants as promastigotes or amastigotes were resistant to clearance by the anti-leishmanial drug antimony, suggesting a cTXNPx link to drug response. Mechanistically, cTXNPx overexpression was protective against changes in Ca2+ homeostasis but not against mitochondrial hyperpolarization brought about by exposure to H2O2 and nitric oxide. Therefore, this study provides a link between cTXNPx expression to survival, virulence and drug response in L. donovani

Determination of gibberellins in fermentation broth produced by Fusarium verticilliodes MTCC 156 by high-performance liquid chromatography tandem mass spectrometry

A method for the detection of gibberellins produced by Fusarium verticilliodes is described using HPLCMS/MS (HPLC tandem MS). A Hypersil (5 μm) octadecylsilane column with methanol/water as eluent in the ratio 3:1 at a flow rate of 0.5 ml/min was used. In the HPLCMS, GA<SUB>3</SUB> (gibberellic acid; m/z 346.3) eluted at retention time t<SUB>r</SUB>=3.08 min, with the corresponding mass chromatogram having peaks at m/z 346.7 and 328.8 corresponding to the M<SUP>+</SUP> and M<SUP>+</SUP>-H<SUB>2</SUB>O ions respectively. The ethyl acetate extract from the broth, subjected to HPLCMS analysis under similar conditions, showed a constituent with t<SUB>r</SUB>=2.13 min, the mass chromatogram of which exhibited peaks at m/z 348.9 and 331.9 corresponding to the MH<SUP>+</SUP> and MH<SUP>+</SUP> -H<SUB>2</SUB>O ions respectively. Comparison of the MS and MS/MS results (direct infusion) of an authentic sample of GA<SUB>3</SUB> and the ethyl acetate extract from the broth revealed the formation of reduced GA<SUB>3</SUB> in the broth. The present study, utilizing HPLCMS/MS, describes an improved methodology for the unambiguous determination and estimation of gibberellins from fermentation broth