Melanin in Fonsecaea pedrosoi: a trap for oxidative radicals.

BACKGROUND: The pathogenic fungus Fonsecaea pedrosoi constitutively produces the pigment melanin, an important virulence factor in fungi. Melanin is incorporated in the cell wall structure and provides chemical and physical protection for the fungus.We evaluated the production of nitric oxide (NO) in macrophages, the oxidative burst and the inducible nitric oxide synthase (i-NOS) activity in interactions between activated murine macrophages and F. pedrosoi. Experiments were carried out with or without tricyclazole (TC) treatment, a selective inhibitor of the dihydroxynaphthalene (DHN)-melanin biosynthesis pathway in F. pedrosoi. The paramagnetisms of melanin and the TC-melanin were analysed by electron spin resonance. The fungal growth responses to H2O2 and to S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, were also evaluated. RESULTS: Melanised F. pedrosoi cells were more resistant to both H2O2 and NO. Nitrite was not detected in the supernatant of macrophages incubated with melanised fungal cells. However, i-NOS expression was unaffected by the presence of either untreated control F. pedrosoi or TC-treated F. pedrosoi. In addition, the inhibition of the DHN-melanin pathway by TC improved the oxidative burst capability of the macrophages. CONCLUSION: The NO-trapping ability of F. pedrosoi melanin is an important mechanism to escape the oxidative burst of macrophages.

DFT calculations of EPR parameters in an ionic lattice of [M(CN)4](3-) (M = Ni, Pd, Fe, Ru, Os) complexes.

The electronic g-tensor and hyperfine coupling constants were calculated for cyanide coordination complexes [M(CN)4]3- (M = Ni, Pd, Fe, Ru, Os) in KCl or NaCl host lattices through an embedded calculation approach using the Density Functional Theory and compared with previous experiments. For all tested complexes, the B3LYP functional is in good agreement with the experiments for the hyperfine coupling constants. For the electronic g-tensor calculations, performed using the coupled perturbed SCF theory, some discrepancies were found, and the best agreements with the experimental values were achieved by the B3LYP functional.

Pseudomonas fluorescens dynamics in the soil surface to subsurface transect.

Microbial displacement in the soil is an important process for bioremediation and dispersal of wastewater pathogens. We evaluated cell movement in surface and subsurface red-yellow podzolic soil driven by advection and microbial motility and also survival of a microbial population at high pressure as is prevalent in deep soil layers. Pseudomonas fluorescens Br 12, resistant to rifampycin and kanamycin, was used as a model organism traceable in non-sterile soil. Our results showed that more than 40% of the P. fluorescens population survived under high pressure, and that microbial motility was not a major factor for its displacement in the soil. Cells were adsorbed in similar amounts to surface and subsurface soils, but more viable cells were present in the leachate of surface than in subsurface soils. The nature of this unexpected cell binding to the subsurface soil was studied by EPR, Mossbauer, NMR, and infrared techniques, suggesting iron had a weak interaction with microbes in soil. P. fluorescens movement in soil resulted mainly from convection forces rather than microbial motility. The transport of this bacterium along the transept toward groundwater encountered restricted viability, although it survived under high pressure conditions simulating those in deep soil layers.

On the pro-oxidant effects of haemozoin.

Haemozoin (Hz) is a haem aggregate produced in some blood-feeding organisms. There is a general belief that Hz formation would be a protective mechanism against haem toxicity. Here we show that when aggregated into Hz, haem is less deleterious than its free form. When haem was added to phosphatidylcholine (PC) liposomes, there was an intense stimulation of oxygen consumption, which did not occur when Hz was incubated with the same preparation. Evaluation of oxygen radical attack to lipids, by measurement of thiobarbituric acid reactive substances (TBARS), showed significantly lower levels of lipid peroxidation in samples containing PC liposomes incubated with Hz than with haem. However, TBARS production induced by Hz was much higher when using 2-deoxyribose (2-DR) as substrate, than with PC liposomes. Spin-trapping analysis by electron paramagnetic resonance (EPR) of Hz and tert-butylhydroperoxide (tert-BuOOH) showed that production of methoxyl and tert-butoxyl radicals was only slightly reduced compared to what was observed with haem. Interestingly, when large Hz crystals were used in 2-DR TBARS assays and tert-BuOOH EPR experiments, the pro-oxidant effects of Hz were strongly reduced. Moreover, increasing concentrations of Hz did not induce erythrocyte lysis, as occurred with haem. Thus, the reduced capacity of Hz to impose radical damage seems to result from steric hindrance of substrates to access the aggregated haem, that becomes less available to participate in redox reactions.