Melanin synthesis is associated with virulence for the pathogenic fungus Cryptococcus neoformans. Exposure of nonmelanized C. neoformans 24067 cells to 4 M guanidinium isothiocyanate followed by 6 M HCl at 100؇C resulted in complete solubilization of cellular structures. However, exposure of melanized C. neoformans 24067 to the same conditions produced a suspension of black particles. Analysis of black particles with transmission and scanning electron microscopy revealed cell "ghosts" with electron-dense walls. Electron spin resonance (ESR) spectroscopy of suspensions of black particles revealed a signal indicative of a stable free radical population. The magnitude of the ESR signal was increased by illumination, Zn 2؉ ions, and basic pH. Boiling the black particles produced only a small reduction in the magnitude of the ESR signal. These ESR changes are characteristic of melanin. Exposure of melanized black cells to 0.1 or 0.5% NaOCl bleached the cells white and abolished the ESR signal, consistent with melanin disruption. ESR spectroscopy was used to study the rate of stable free radical formation, taken as an index of melanization, of C. neoformans 24067 cells growing in medium with 1.0 mM L-dopa at 30 and 37؇C over the course of 14 days. Melanization was slower at 37؇C, but by day 14, cells grown at 30 and 37؇C had comparable levels of melanin. ESR spectroscopy of seven melanized C. neoformans strains revealed that some strains differed by up to eightfold in melanin content. The ESR spectra of C. neoformans 24067 grown in minimal medium containing either 1.0 mM catechol, dopamine, or L-dopa revealed large differences in signal amplitude. The relative melanin contents were 0.05, 0.45, and 1.0 for C. neoformans grown in media with catechol, dopamine, and L-dopa, respectively. Quantitative analysis revealed that melanin comprised 15.4% of the dry mass of the cell after 10 days of growth in medium containing 1.0 mM L-dopa. The results suggest (i) a useful protocol for isolating C. neoformans melanin, (ii) a role for melanin in maintaining cell wall integrity, (iii) disruption of melanin by NaOCl, (iv) strain differences in melanin content after growth in L-dopa, and (v) quantitative and/or qualitative differences in the melanin produced after growth in various substrates. MATERIALS AND METHODS Chemicals. Glucose, MgSO 4 , KH 2 PO 4 , HCl, guanidinium isothiocyanate, and NaOCl were purchased from Fisher Chemical Inc. (Fair Lawn, N.J.). Vitamin B 1 , L-dopa, catechol (1,2-benzenediol), dopamine (3-hydroxytyramine), sorbitol, and sodium citrate were purchased from Sigma Chemical Co. (Cleveland, Ohio). Novo-zyme 234 was purchased from BioPacific Inc. (Emoryville, Calif.). C. neoformans. Strains were maintained in Sabouraud dextrose agar (Difco Laboratories, Detroit, Mich.). Strain 24067 was obtained from the American Type Culture Collection (Rockville, Md.). Strains SB4A and SB6A were obtained from Eric Spitzer (Stony Brook, N.Y.). Strain NIH371 was obtained from J. E. Bennett (Bethesda, Md.). Strains J9A, J11A, and ...
Ni3N/MCNT (multiwalled carbon nanotube) nanocomposites fabricated by ammonolysis displayed high CO2 electrochemical reduction reactivity with CO Faradaic efficiencies of 89.0% and current density of 6.5 mA cm–2 at −0.73 V vs reversible hydrogen electrode (RHE). The Ni3N/MCNT catalyst could operate under weakly acidic conditions in the pH range of 2.5–7.2. Even at pH 2.5, the CO Faradaic efficiency remained at 50.1%, demonstrating the high selectivity for CO2 reduction. The high catalyst selectivity could be due to increased adsorption of CO2 on the Ni3N surface, which can compete with hydrogen evolution under acidic conditions.
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