Millard P J, Roth B L, Thi H P, Yue S T, Haugland R P
Molecular Probes, Inc., Eugene, Oregon 97402, USA.
Appl Environ Microbiol. 1997 Jul;63(7):2897-905. doi: 10.1128/aem.63.7.2897-2905.1997.
A new family of fluorescent probes has been developed for assessing the viability and metabolic activity of yeasts. This class of halogenated unsymmetric cyanine dyes is exemplified by the FUN-1 [2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)- methylidene)-1-phenylquinolinium iodide] stain, a membrane-permeant nucleic acid-binding dye that has been found to give rise to cylindrical intravacuolar structures (CIVS) in Saccharomyces cerevisiae. Biochemical processing of the dye by active yeasts yielded CIVS that were markedly red shifted in fluorescence emission and therefore spectrally distinct from the nucleic acid-bound form of the dye. The formation of CIVS occurred under both aerobic and anaerobic conditions and was highly temperature dependent. Treatment of yeasts with the nonmetabolizable glucose analog 2-deoxy-D-glucose reduced cellular ATP levels approximately 6-fold and completely inhibited CIVS formation. Under aerobic conditions, the formation of CIVS was abrogated by the cytochrome oxidase inhibitors azide and cyanide; however, the H+ transport uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited CIVS formation under both aerobic and anaerobic conditions. Depletion of cellular thiols, including glutathione, with millimolar concentrations of N-ethylmaleimide, iodoacetamide, or allyl alcohol completely inhibited CIVS production. Marked reduction in the formation of CIVS by ethacrynic acid and sulfobromophthalein, inhibitors of glutathione S-transferase, suggested that dye processing can involve enzyme-mediated formation of glutathione conjugates. The conversion of FUN-1 by S. cerevisiae was studied quantitatively by using several techniques, including fluorometry, flow cytometry, and wide-field and confocal laser scanning fluorescence microscopy.
已开发出一类新的荧光探针，用于评估酵母的活力和代谢活性。这类卤化不对称花青染料以FUN-1[2-氯-4-(2,3-二氢-3-甲基-(苯并-1,3-噻唑-2-基)-亚甲基)-1-苯基喹啉碘化物]染色剂为例，它是一种可透过细胞膜的核酸结合染料，已发现其在酿酒酵母中会产生圆柱形液泡内结构（CIVS）。活性酵母对该染料的生化处理产生了荧光发射明显红移的CIVS，因此在光谱上与染料的核酸结合形式不同。CIVS的形成在有氧和无氧条件下均会发生，且高度依赖温度。用不可代谢的葡萄糖类似物2-脱氧-D-葡萄糖处理酵母可使细胞ATP水平降低约6倍，并完全抑制CIVS的形成。在有氧条件下，细胞色素氧化酶抑制剂叠氮化物和氰化物可消除CIVS的形成；然而，H⁺转运解偶联剂羰基氰化物间氯苯腙在有氧和无氧条件下均抑制CIVS的形成。用毫摩尔浓度的N-乙基马来酰亚胺、碘乙酰胺或烯丙醇耗尽包括谷胱甘肽在内的细胞硫醇会完全抑制CIVS的产生。谷胱甘肽S-转移酶抑制剂依他尼酸和磺溴酞对CIVS形成的显著减少表明，染料处理可能涉及酶介导的谷胱甘肽共轭物的形成。通过使用多种技术，包括荧光测定法、流式细胞术以及宽场和共聚焦激光扫描荧光显微镜，对酿酒酵母对FUN-1的转化进行了定量研究。
