BackgroundThe 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.ResultsMelanised 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.ConclusionThe NO-trapping ability of F. pedrosoi melanin is an important mechanism to escape the oxidative burst of macrophages.

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 tertBuOOH 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. ß 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

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Paramagnetic 3d9 [Ni(CN)4]3- complexes, with the unpaired electron in a d x 2− y 2 orbital, have been generated from diamagnetic Ni(II) 3d8 cyanide complexes in KCl or NaCl host lattices. The magnetic and quadrupolar hyperfine interactions with the four 14N, hidden in the CW-EPR (continuous wave electron paramagnetic ressonance) line width, are revealed by pulsed EPR and ENDOR (electron nuclear double resonance) angular variation studies. Ab initio embedded UMP2 cluster calculations, which take into account short- and long-range crystal interactions, confirm the unpaired electron orbital assignment and are in agreement with the measured hyperfine values. The trend of 14N A iso values (7.7 MHz for NaCl and 6.8 MHz for KCl) is given by the Ni−CN distance, modified in each host lattice. Small asymmetry factors (about 0.04) for the 14N quadrupolar tensor are obtained both in experiment and in theory. The experimental lines and the calculations indicate spin density at the cations of both lattices. Experimental and theoretical data indicate that lattice chlorine ions near the Ni atom, in axial positions, are not chemically coordinated to Ni. Spin density on these ions arises only from spin polarization of their valence orbitals and of the valence orbitals of the complex.

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