We searched for F-box proteins that might be related to the mechanism that protects Saccharomyces cerevisiae against the toxic effects of methylmercury. We found that overexpression of Hrt3 and of Ylr224w rendered yeast cells resistant to methylmercury. Yeast cells that overexpressed Hrt3 and Ylr224w were barely resistant to methylmercury in the presence of a proteasome inhibitor. Our results suggest the existence of some protein(s) that enhances the toxicity of methylmercury in yeast cells and, also, that overexpression of Hrt3 or Ylr224w can confer resistance to methylmercury by enhancing the polyubiquitination of this protein(s) and its degradation in proteasomes.
A search was made for genes that confer resistance to methylmercury in yeast using a genomic DNA library derived from Saccharomyces cerevisiae. The genomic library was introduced into yeast and transformants that grew in the presence of a normally toxic concentration of methylmercury were selected. We sequenced the genomic DNA fragment in the plasmid from the clone with the highest resistance to methylmercury and analyzed the sequence for presence of an open reading frame that might confer resistance to methylmercury. We identified a gene, CDC34 (also known as UBC3), that increased resistance to methylmercury when overexpressed in yeast. CDC34 encodes a ubiquitin-conjugating enzyme; such proteins play important roles in the selective targeting of proteins for degradation. Overexpression of UBC4 and of UBC7, two other genes for ubiquitin-conjugating enzymes, also conferred resistance to methylmercury. Yeast strains transformed with the CDC34 gene were resistant not only to methylmercury but also to mercuric chloride and p-chloromercuribenzoate. To our knowledge, this is the first demonstration that overexpression of genes for ubiquitin-conjugating enzymes confers resistance to xenobiotics. Our results suggest that ubiquitination system might be involved in protection against the toxicity of mercury compounds, such as methylmercury, in eukaryotic cells.
-Chronic infection with the hepatitis C virus (HCV) frequently induces steatosis, which is characterized by the accumulation of lipid droplets (LDs) in hepatocytes. Steatosis is a significant risk factor for liver cancer. The HCV structural protein core is distributed on the surface of the endoplasmic reticulum (ER) and in LDs, thereby increasing LD levels. In this work, we attempt to elucidate the effect of the core protein on LD generation using yeast cells. We found that the core localized to the cytosolic surface of the ER in yeast and is able to increase LD levels when overexpressed from an inducible GAL1 promoter for 3 hr. The effect of the core was conserved among three different HCV serotypes: 1b, 2a and 3a. While the ER stress inducer tunicamycin both elicited an unfolded stress response (UPR) and increased LD levels, the core did not induce the UPR. The RNA viral genome changes rapidly due to its high mutation rate in order to replicate under a variety of circumstances. Our observations suggest a functional analogy between core function in hepatocytes and in yeast cells and thus might be applicable to the screening of small molecules that impair the core-ER interaction.
-The expression levels or activities of biological defense factors can fluctuate daily following biological rhythms. We have focused on the relationship between injection timing and the degree of toxicity of cadmium (Cd) to promote the concept of "chronotoxicology," which introduces chronobiology to the field of toxicology. Our previous studies have clearly indicated that Cd may be subject to chronotoxicity. In this report, to confirm the character of the Cd-induced chronotoxicity, we performed multidirectional examinations. Male C57BL/6J mice that received a single intraperitoneal injection of CdCl 2 at ZT6 showed drastic hepatic injury estimated by histopathological analyses, i.e., nuclear condensations, fatty degenerations, and hemorrhages, but showed no injury when injected at ZT18. This difference was supported by several biochemical analyses that were indicators of hepatic injury (levels of alanine aminotransferase, aspartate aminotransferase, and malondialdehyde). The chronotoxicity of Cd was also observed in multiple strains (ICR and Balb/c), in a different injection route (subcutaneous), and in multiple injections (5 injections). Based on these results, we propose that chronotoxicology may provide important information not only for toxicology but also for occupational health, i.e., the importance of injection timing for toxicity evaluation tests, the reproducibility of animal experiments, and the improvement in the quality of risk assessments for night shift workers who may be exposed to toxic substances at various times of the day.
-M1-microglia (neurotoxic microglia) regulate neuronal development and cell death and are involved in many pathologies in the brain. Although organotypic brain slice cultures are widely used to study the crosstalk between neurons and microglia, little is known about the properties of microglia in the mouse cerebral cortex slices. Here, we aimed to optimize the mouse cerebral slice cultures that reflect microglial functions and evaluate the effects of neurotoxic metals on M1-microglial activation. Most microglia in the cerebral slices prepared from postnatal day (P) 7 mice were similar to mature microglia in adult mice brains, but those in the slices prepared from P2 mice were immature, which is a conventional preparation condition. The degree of expression of M1-microglial markers (CD16 and CD32) and inflammatory cytokines (tumor necrosis factor-α and interleukin-1β) by lipopolysaccharide, a representative microglia activator, in the cerebral slices of P7 mice were higher than that in the slices of P2 mice. These results indicate that M1-microglial activation can be evaluated more accurately in the cerebral slices of P7 mice than in those of P2 mice. Therefore, we next examined the effects of various neurotoxic metals on M1-microglial activation using the cerebral slices of P7 mice and found that methylmercury stimulated the activation to M1-microglia, but arsenite, lead, and tributyltin did not induce such activation. Altogether, the optimized mouse cerebral slice cultures used in this study can be a helpful tool to study the influence of various chemicals on the central nervous system in the presence of functionally mature microglia.
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