Establishment and its pharmacological application of novel

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We established an experimental system for measuring the cytosolic free Ca^<2+> concentration ([Ca^<2+>]i) in individual Saccharomyces cerevisiae and Candida albicans cells using fura-2 as a Ca^<2+>-specific fluorescence probe in conjunction with digital image processing and could examine changes in [Ca^<2+>]i in response to nutrient shift/deprivation. Candida albicans is a phathogenic fungus and, when grown in the presence of alcohol, shows dimorphic conversion from vegetative forms to mycelial forms, which are more harmful to host patients. By applying our new method established by present study, the average [Ca^<2+>]i in a single cell of Candida albicans was determined to be at a basal level of 100 nM. We then found that upon addition of alcohol, the [Ca^<2+>]i in induced cells raised 10-fold along with the time course of morphogenic transformation. This specific calcium mobilization is mostly associated with the increase in inositol-1.4.5-3-phosphate. We suggested that this finding is important for future medical and pharmaceutical studies because Ca^<2+>-blockers may be effective inhibitors of dimorphic transformation.The second novel method for measuring the cytosolic free Ca^<2+> and its time-dependent changes in the yeast Saccharomyces cerevisiae was established by using the luminescent protein aequorin as a Ca^<2+>-specific indicator. We constructed a plasmid in which the apoaequorin cDNA was joined downstream from the glyceraldehyde-3-phosphate dehydrogenase gene promoter and introduced into yeast cells. The intracellular concentration of apoaequorin expressed by the cDNA was about 1 mu, which was high enough to detect the cytosolic Ca^<2+>. Aequorin was regenerated effectively by incubating intact cells with coelenterazine at 25ﾟC. We found that glucose added to glucose-starved Go/G1 cells stimulated in increase in extracellular Ca^<2+>-dependent luminescence with maximal intensity occurring 2 min after addition.