Establishment and its pharmacological application of novel

我们建立了一个实验系统，利用fura-2作为Ca^<2+>特异性荧光探针，结合数字图像处理，测量单个酵母和白色假丝酵母细胞胞浆游离Ca^<2+>浓度（[Ca^<2+>]i），可以检测营养转移/剥夺时[Ca^<2+>]i的变化。白色念珠菌是一种致病性真菌，当在酒精存在下生长时，表现出从营养形态到菌丝形态的二态转化，这对宿主患者更有害。利用本研究建立的新方法，测定了白色念珠菌单细胞的平均[Ca^<2+>]i处于100 nM的基础水平。然后我们发现，在添加酒精后，诱导细胞中的[Ca^<2+>]i随着形态发生转化的时间增加了10倍。这种特定的钙动员主要与肌醇-1.4.5-3-磷酸的增加有关。我们认为这一发现对未来的医学和药物研究很重要，因为Ca^<2+>-阻滞剂可能是二态转化的有效抑制剂。利用发光蛋白aequorin作为Ca^<2+>特异性指示剂，建立了测量酵母胞质游离Ca^<2+>及其随时间变化的第二种新方法。我们构建了一个质粒，将apoaequorin cDNA与甘油醛-3-磷酸脱氢酶基因启动子下游连接并导入酵母细胞。cDNA表达的apoaequorin细胞内浓度约为1 μ，足以检测胞质Ca^<2+>。用coelenterazine在25℃下孵育完整细胞，可有效再生Aequorin。我们发现葡萄糖添加到葡萄糖饥渴的Go/G1细胞中刺激细胞外Ca^<2+>依赖性发光增加，并在添加后2分钟出现最大强度。

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.