Effect of methylene blue and illumination on the process of differentiation of the protozoan Herpetomonas samuelpessoai

Herpetomonas samuelpessoai is a non-pathogenic protozoan that shares important antigens with Trypanosoma cruzi (the agent of Chagas' disease) and which shows three developmental stages: promastigote, paramastigote and the highly differentiated form opisthomastigote. Due to the difficulties in obtaining the last form, its physiology and biochemistry are not well understood, and procedures which can induce the process of differentiation promastigote-opisthomastigote are desirable. In this work we show that illumination of H. samuelpessoai with white light in the presence of methylene blue and oxygen (photodynamic effect) triggers the process of differentiation in a very efficient manner (the cultures show up to 70 per cent of the cells in the opisthomastigote form). We also observed that illumination alone and incubation with methylene blue in the dark can trigger the process but in levels markedly lower than illumination in the presence of the dye.     

Characterization of epoxide hydrolase activity in shape Alternaria alternata f. sp. shape lycopersici. Possible involvement in toxin production

Using trans-diphenylpropane oxide (tDPPO) as a substrate, we measured epoxide hydrolase (EH) activity in subcellular fractions of Alternaria alternata f. sp. lycopersici (Aal), a fungus that produces host-specific toxins. The activity was mainly (>99.5%) located in the soluble fraction (100,000 × g supernatant) with the optimum pH at 7.4. An increase of toxin production between days 3 and 9 found in a Aal liquid culture over a 15 days period was concomitant with a period of high EH activity. EH activity remained constant during the same period in an Alternaria alternata culture, a fungus which does not produce toxin. In vivo treatment of Aal culture with the peroxisome proliferator clofibrate stimulated EH activity by 83% and enhanced toxin production 6.3 fold. Both 4-fluorochalcone oxide (4-FCO) and (2S,3S)-(-)-3-(4-nitrophenyl)-glycidol (SS-NPG) inhibited EH activity in vitro with a IM₅₀f 23 ± 1 μM and 72 ± 19 μM, respectively. The possible physiological substrate 9,10-epoxystearic acid was hydrolyzed more efficiently by Aal sEH than the model substrates trans- and cis-stilbene oxide (TSO and CSO) and trans- and cis-diphenylpropane oxide (tDPPO and cDPPO). This revised version was published online in June 2006 with corrections to the Cover Date.