Further investigation is necessary to define the structure of fungal melanins

and describe putative chemical reactions that could occur in the infection environment, the products of such reactions and possible target sites for the development of new drugs. Methods Microorganism and reagents A human isolate of F. pedrosoi (5VLP) [37] PLX3397 was inoculated in modified Czapek Dox (CD) liquid media (Sucrose 30 g/L, NaNO3 2 g/L, KH2PO4 1 g/L, MgSO4.7H2O 0.5 g/L, KCl 0.5 g/L, ammoniacal iron citrate 0.01 g/L), pH 5.5, with shaking at 28°C for five days. TC (kindly provided by Dow AgroSciences, Indianapolis, USA) was dissolved in dimethylsulphoxide (DMSO) and added to cultures at a final concentration of 16 μg/ml to block the DHN-melanin biosynthesis pathway. All other reagents were acquired from Sigma-Aldrich (Brazil), unless otherwise specified. Saccharomyces cerevisiae (INCQS 40001, ATCC 2601) was donated by Coleção de Culturas de Fungos of Instituto Oswaldo

Cruz, Rio de Janeiro, Brazil. Melanin isolation F. pedrosoi melanins were isolated from fungal cultures following incubation with 16 μg/ml of TC (TC-melanin) or without the drug (control-melanin) by an alkali-acid extraction method described elsewhere [6]. Electron Spin Resonance After isolation, melanins (10 mg) from F. pedrosoi cultures were thoroughly triturated manually in a solid marble buy CFTRinh-172 mortar with a pestle. The trituration was Isotretinoin a necessary step CYT387 price in order to diminish the grain size, which otherwise could lead to preferential orientations and to the observation of artifacts in the ESR spectra. The pigments were analysed by ESR spectroscopy coupled to a spin-trapping analysis. The spectra were acquired at room temperature in quartz tubes on a Bruker ESP 380-E CW/FT spectrometer (Bruker, Germany) operating at X-Band (9.5 GHz). The amplitude modulation was kept constant at 3.0 gauss and low power

microwaves were used to avoid saturation. The microwave power saturation experiments were measured between 0.02-200 mW, while all others parameters remained the same. The g factors (the ESR quantity analogous to the chemical shift in nuclear magnetic ressonance spectroscopy), which are related to the magnetic field, were measured upon a diphenylpicrylhydrazyl radical (DPPH) standard, g = 2.0023 [38]. Conidia Isolation F. pedrosoi cells with or without a treatment of 16 μg/ml of TC were filtered in a 40-60G porous plate filter, followed by conidia recovery by centrifugation (13,600 g, 30 min, 4°C). Peritoneal Macrophages Peritoneal washes with Hanks’ Balanced Salt Solution were performed in 2-3-week-old Swiss male mice. Resident macrophages were seeded on glass coverslips in 24-well plates or in Petri dishes for 1 h at 37°C in a 5% CO2 atmosphere. Cells were then washed and cultured for 24 h in DMEM containing 10% foetal bovine serum.