Actin ring formation around the cell nucleus of long-neck yeast.

The unique long-neck yeast Fellomyces fuzhouensis has F-actin cables and cortical patches. Here, we describe a new F-actin structure present in fungi, a perinuclear F-actin collar ring around the cell nucleus. This F-actin structure can be visualized by fluorescent microscopic imaging of rhodamine-phalloidin-stained F-actin in cells treated with the mitotic drug isopropyl N-(3-chlorophenyl) carbamate or the microtubule inhibitor thiabendazol or when cells were grown in cut dried radish medium or yeast extract pepton dextrose (YEPD) medium. In contrast, these structures were absent in cells treated with Latrunculin A. The hypothetical functions of the F-actin ring are discussed.

Secondary cell wall formation in Cryptococcus neoformans as a rescue mechanism against acid-induced autolysis.

Growth of the opportunistic yeast pathogen Cryptococcus neoformans in a synthetic medium containing yeast nitrogen base and 1.0-3.0% glucose is accompanied by spontaneous acidification of the medium, with its pH decreasing from the initial 5.5 to around 2.5 in the stationary phase. During the transition from the late exponential to the stationary phase of growth, many cells died as a consequence of autolytic erosion of their cell walls. Simultaneously, there was an increase in an ecto-glucanase active towards beta-1,3-glucan and having a pH optimum between pH 3.0 and 3.5. As a response to cell wall degradation, some cells developed an unusual survival strategy by forming 'secondary' cell walls underneath the original ones. Electron microscopy revealed that the secondary cell walls were thicker than the primary ones, exposing bundles of polysaccharide microfibrils only partially masked by an amorphous cell wall matrix on their surfaces. The cells bearing secondary cell walls had a three to five times higher content of the alkali-insoluble cell wall polysaccharides glucan and chitin, and their chitin/glucan ratio was about twofold higher than in cells from the logarithmic phase of growth. The cell lysis and the formation of the secondary cell walls could be suppressed by buffering the growth medium between pH 4.5 and 6.5.

Analyses to clarify rich fractions in hepatic progenitor cells from human umbilical cord blood and cell fusion.

Umbilical cord blood (UCB) is a source of hematopoietic stem cells and other stem cells, and human UCB cells have been reported to contain transplantable hepatic progenitor cells. However, the fractions of UCB cells in which hepatic progenitor cells are rich remain to be clarified. In the present study, first, the fractionated cells by CD34, CD38, and c-kit were transplanted via portal vein of NOD/SCID mice, and albumin mRNA expression was examined in livers at 1 and 3 months posttransplantation. At 1 and 3 months, albumin mRNA expression in CD34+UCB cells-transplanted livers was higher than that in CD34- cells-transplanted livers. Albumin mRNA expression in CD34+CD38+ cells-transplanted livers was higher than that in CD34+CD38- cells-transplanted [corrected] liver at 1 month. However, it was much higher [corrected] in CD34+CD38- cell-transplanted livers at 3 months. Similar expression of albumin mRNA was obtained between CD34+CD38+c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, and between CD34+CD38-c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, respectively. Second, fluorescence in situ hybridization and immunohistochemistry were performed to examine whether UCB cells really transdifferentiated into hepatocytes or they only fused with mouse hepatocytes. In mouse liver sections, of 1.2% cells which had human chromosomes, 0.9% cells were due to cell fusion, whereas 0.3% cells were transdifferentiated into human hepatocytes. These results suggest that CD34+UCB cells are rich fractions in hepatic progenitor cells, and that transdifferentiation from UCB cells into hepatocytes as well as cell fusion simultaneously occur in this situation.

Intra-strain variability of Cryptococcus neoformans can be detected on phloxin B medium.

A method was devised for easy detection of intra-strain variability of the human pathogenic yeast Cryptococcus neoformans. Cultivation of strains on a medium containing Phloxin B resulted in different coloured colonies. Generally, colonies were either pink or red; however there were also several colony-colour segregant in which both colours could be observed. A number of these segregants were isolated and analysed. Virulence factors such as the cell and capsule sizes were measured; further temperature sensitivity, growth rates, mating-types and melanin production were also studied. Segregants were examined by random amplified polymorphic DNA (RAPD) fingerprinting and electrophoretic karyotyping by pulsed-field gel electrophoresis (CHEF). They showed both phenotypic and genotypic differences. The main differences appeared in phenotypic characters and RAPD patterns; while the chromosomal patterns remained unchanged. Reversion frequency analysis revealed that the reason for this segregation could be due to phenotypic switching. The physiological reason for the colour changes was also investigated and was attributed to the differential ability of the cells to accumulate Phloxin B either into their capsules or into their cells. The method described here is potentially applicable for the detection of strain heterogeneity in both basic and clinical microbiology laboratories.