荧光碳点的合成及对酿酒酵母的毒性研究

以葡萄糖为碳源采用溶剂热法合成了荧光碳点。紫外吸收光谱、荧光光谱以及透射电镜照片表明,所合成的荧光碳点发光性能优异,分散性好,且无团聚现象。荧光碳点原溶液出现浓度淬灭现象,稀释60倍情况下荧光最强。以酿酒酵母为模型生物,考察了不同生长时期(调整期、对数期早期、对数期中期)的酿酒酵母与不同浓度的荧光碳点共培养后的生长曲线。结果表明,即使荧光碳点浓度在27.75 mmol.L-1条件下也没有影响酵母菌的生长曲线,可认为基本没有细胞毒性。比较了相同荧光强度下的荧光碳点与CdTe量子点对酿酒酵母的细胞毒性,结果表明荧光碳点的毒性显著低于量子点的毒性。     

New concept for a toxicity assay based on multiple indexes from the wave shape of damped metabolic oscillation induced in living yeast cells (part II): application to analytical toxicology

An ideal toxicity assay should utilize multiple indexes obtained from transient changes of metabolic activities. Here, we demonstrate the possibility for a novel toxicity bioassay using the damped glycolytic oscillation phenomenon occurring in starved yeast cells. In a previous study, the phenomenon was characterized in detail. Under optimum conditions to induce the phenomenon, the wave shapes of the damped glycolytic oscillations were changed by the instantaneous addition of both glucose and chemicals and by changing the chemical concentration. We estimated the changes in the oscillation wave shapes as six indexes, i.e., the number of wave cycles, maximum amplitude, oscillation frequency, attenuation coefficient, initial peak height, and non-steady-state time. These index changes were obtained from several kinds of chemicals. The chemicals, especially those for acids (0.01–100 mM HCl and 0.01–50 mM citric acid), bases (0.001–50 mM KOH), heavy metal ions (1–1,000 mg L⁻¹; Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺), respiratory inhibitors (3–500 mg L⁻¹ NaN₃), dissolved oxygen removers (10–300 mg L⁻¹ NaSO₃), surfactants (10–200 mg L⁻¹ benzalkonium chloride), and aldehyde (10–1,000 mg L⁻¹ acetaldehyde), showed characteristic patterns depending on each chemical and its concentration. These significant results demonstrate the possibilities of new methods for both toxicity qualification and quantification. Figure Influences of surfactant on the oscillation wave shape Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

毕赤酵母△~9-脂肪酸脱氢酶基因的克隆和表达

根据真菌Δ9-脂肪酸脱氢酶保守的氨基酸序列设计简并引物进行PCR,获得一个741 bp的毕赤酵母cDNA片段,再根据获得的部分序列设计基因特异性引物,通过cDNA末端扩增技术(RACE)获得该cDNA的3′和5′序列,从而得到全长为1 689 bp的cDNA序列.序列分析结果表明,该序列具有一个长度为1 194 b p、编码397个氨基酸的开放阅读框(PPD9).与报道的Δ9-脂肪酸脱氢酶一样,推测的氨基酸序列具有膜整合脂肪酸脱氢酶特异性的3个组氨酸保守区和疏水结构,在其氨基酸序列的C-末端具有类似于细胞色素b5的血红素结合区.该序列为一个新的编码Δ9-脂肪酸脱氢酶的基因,为了验证其功能,把开放阅读框序列PPD9亚克隆到表达载体pYES 2.0,构建重组表达载体pYPPD9,并转化到酿酒酵母的Δ9-脂肪酸脱氢酶缺陷型菌株DTY-11A中进行表达。通过平板互补实验结果表明,该序列在酿酒酵母中获得表达。所编码的蛋白具有Δ9-脂肪酸脱氢酶活性,能弥补DTY-11A中由于Δ9-脂肪酸脱氢酶的缺失而引起的致死性突变。