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The chemical composition of the mycocidal complex (formerly known as microcin) secreted by Cryptococcus humicola was investigated by chemical, mass spectrometric and nuclear magnetic resonance methods. The results indicate that the mycocidal complex is composed of glycolipids with a highly acetylated (up to five acetyl groups) cellobiose backbone [beta-D-Glcp-(1'-->4)-beta-D-Glcp] linked to the omega-hydroxyl group of alpha,omega-dihydroxy palmitate [16:0-alpha,omega-di-OH] with an unsubstituted carboxyl group. The acyl chain forming aglycon can be replaced by [18:0-(alpha,omega-di-OH)], [18:0-(alpha,omega-1,omega-tri-OH)], and [18:0-(alpha,omega-2,omega-tri-OH)]. The complex has a comparatively high surface activity; 0.5 mg/ml of it reduced the surface tension of 0.1 M NaHCO(3) from 71 mN/m to 37 mN/m and interfacial tension against n-hexadecane from 39 mN/m to 10 mN/m. The critical micelle concentration of the complex at pH 4.0, determined by the fluorometric method with N-phenyl-1-naphthylamine as fluorescent probe and by the De Nouy ring method, was 2 x 10(-5) M (taking the average molecular mass of the complex to be 750); it did not depend on the presence of 100 mM KCl and was an order of magnitude higher at pH 7.0. By fluorescence resonance energy transfer spectroscopy with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine as energy donor and N-(rhodamine B sulfonyl)-phosphatidylethanolamine as energy acceptor the complex was shown to intercalate into the liposomal lipid matrix. Primary lesions caused by the complex in planar lipid bilayers were revealed as short-living current fluctuations of a broad spectrum of amplitudes. The mycocidal effect of the complex is suggested to be associated with its detergent-like properties.

In the vacuoles of Saccharomyces cerevisiae yeast cells, vividly moving insoluble polyphosphate complexes (IPCs) <1 µm size, stainable by a fluorescent dye, 4 ,6-diamidino-2-phenylindole (DAPI), may appear under some growth conditions. The aim of this study was to quantitatively characterize the movement of the IPCs and to evaluate the viscosity in the vacuoles using the obtained data. Studies were conducted on S. cerevisiae cells stained by DAPI and fluorescein isothyocyanate-labelled latex microspheres, using fluorescence microscopy combined with computer image analysis (ImageJ software, NIH, USA). IPC movement was photorecorded and shown to be Brownian motion. On latex microspheres, a methodology was developed for measuring a fluorescing particle's two-dimensional (2D) displacements and its size. In four yeast cells, the 2D displacements and sizes of the IPCs were evaluated. Apparent viscosity values in the vacuoles of the cells, computed by the Einstein-Smoluchowski equation using the obtained data, were found to be 2.16 ± 0.60, 2.52 ± 0.63, 3.32 ± 0.9 and 11.3 ± 1.7 cP. The first three viscosity values correspond to 30-40% glycerol solutions. The viscosity value of 11.3 ± 1.7 cP was supposed to be an overestimation, caused by the peculiarities of the vacuole structure and/or volume in this particular cell. This conclusion was supported by the particular quality of the Brownian motion trajectories set in this cell as compared to the other three cells.

This study was undertaken to evaluate the apparent viscosity within the vacuoles of single Saccharomyces cerevisiae cells by steady‐state fluorescence anisotropy measurements of quinacrine, using wide‐field fluorescence polarization microscopy combined with computer image analysis. Quinacrine was shown to be rather specifically accumulated within the vacuoles of the cells. This accumulation was effectively reversed by ATP depletion of the cells, with no detectable binding of the dye within the vacuoles. Quinacrine fluorescence anisotropy in the sucrose solutions of various viscosities obeyed the Perrin equation. The fluorescence anisotropy of quinacrine was measured in the vacuoles of 39 cells. From cell to cell, this parameter changed in the range 0.032–0.086. Using the Perrin plot as a calibration curve, apparent viscosity values of the vacuolar milieu were calculated for each cell. The population of the cells studied was heterogeneous with regard to vacuolar viscosity, which was in the range 3.5 ± 0.4–14.06 ± 0.64 cP. There was a characteristic distribution of the frequencies of cells with apparent viscosities within certain limits, and cells with viscosity values in the range 5–6 cP were the most frequent. No relationship was found between the sizes of the vacuoles and their apparent viscosities. Copyright © 2012 John Wiley & Sons, Ltd.

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