Kyoko Miura

Overexpression of L-glutamine:D-fructose-6-phosphate amidotransferase provides resistance to methylmercury in Saccharomyces cerevisiae

To identify novel genes that confer resistance to methylmercury (MeHg), a yeast genomic DNA library was transfected into Saccharomyces cerevisiae. Two functional plasmids were isolated from transfected yeast clones D1 and H5 that exhibited resistance to MeHg. The yeast transfected with plasmid isolated from clone H5 was several‐fold more resistant than yeast transfected with plasmid from clone D1. Functional characterization of the genomic DNA fragment obtained from clone H5 determined that the GFA1 gene conferred resistance to MeHg. GFA1 was reported to encode L‐glutamine:D‐fructose‐6‐phosphate amidotransferase (GFAT) which catalyzes the synthesis of glucosamine‐6‐phosphate from glutamine and fructose‐6‐phosphate. Accumulation of mercury in yeast clone W303B/pGFA1, which contains the transfected GFA1 gene, did not differ from that in control yeast clone W303B/pYES2. The W303B/pGFA1 strain did not show resistance to mercuric chloride, zinc chloride, cadmium chloride or copper chloride, suggesting that the resistance acquired by GFA1 gene transfection might be specific to MeHg. This is the first report of a gene involved in MeHg resistance in eukaryotic cells identified by screening a DNA library.

Glutathione content is correlated with the sensitivity of lines of PC12 cells to cisplatin without a corresponding change in the accumulation of platinum

A study of the involvement of glutathione (GSH) in cellular resistance to cisplatin was performed using methylmercury-resistant sublines (PC12/TM series) of the PC12 line of rat pheochromocytoma cells. The seven clonal sublines of PC12 cells (PC12/TM, PC12/TM2, PC12/TM5, PC12/TM11, PC12/TM15, PC12/TM23, PC12/TM26) used in the study had intracellular levels of GSH that ranged from 8.7–39.9 nmol/mg protein. The intracellular level of GSH was significantly correlated (p < 0.01, r = 0.87) with the sensitivity to cisplatin of PC12 cells and the seven sublines. Among the seven sublines, PC12/TM cells contained the highest concentration of GSH and were the most resistant to cisplatin. Treatment of PC12/TM cells with L-buthionine-SR-sulfoximine, which reduced the level of GSH to that in the parental PC12 cells, significantly reduced the resistance of the cells to cisplatin. The amount of platinum accumulated by resistant PC12/TM cells after treatment with cisplatin was higher than that by sensitive PC12 cells. These results suggest that the intracellular level of GSH might be directly involved in the resistance to cisplatin of these cell lines. However, a high intracellular concentration of GSH does not appear to contribute to a decrease in the accumulation of cisplatin in these cells.

Mechanism of cytotoxicity of methylmercury: With special reference to microtubule disruption

Mechanism of methylmercury cytotoxicity was investigated with special reference to its preferential action on microtubules and protein biosynthesis in cultured cells. The tubulin synthesis analyzed by autoradiography of two-dimensional electropherogram using35S-methionine was inhibited by 50–70% in mouse glioma cells exposed to 5×10−6M methylmercury for 3 h, which almost completely depolymerized microtubules. Total protein synthesis monitored by incorporation of labeled methionine into acid insoluble fraction was decreased slightly but significantly and the protein bands other than tubulin on gradient urea-PAGE gel appeared to remain unchanged under the experimental condition used. These results suggest that the inhibition of protein synthesis observed on exposure to methylmercury can be ascribed, at least partly, to a possible autoregulatory depression in tubulin synthesis owing to the increase in the pool of tubulin subunits resulted from microtubule depolymerization by methylmercury.

Rapid Monitoring of Mercury in Air from an Organic Chemical Factory in China Using a Portable Mercury Analyzer

A chemical factory, using a production technology of acetaldehyde with mercury catalysis, was located southeast of Qingzhen City in Guizhou Province, China. Previous research showed heavy mercury pollution through an extensive downstream area. A current investigation of the mercury distribution in ambient air, soils, and plants suggests that mobile mercury species in soils created elevated mercury concentrations in ambient air and vegetation. Mercury concentrations of up to 600 ng/m3 in air over the contaminated area provided evidence of the mercury transformation to volatile Hg(0). Mercury analysis of soil and plant samples demonstrated that the mercury concentrations in soil with vaporized and plant-absorbable forms were higher in the southern area, which was closer to the factory. Our results suggest that air monitoring using a portable mercury analyzer can be a convenient and useful method for the rapid detection and mapping of mercury pollution in advanced field surveys.