Interaction between the Msh2 and Msh6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-Msh6 Complex

Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes requires ATP binding to only the Msh6 nucleotide-binding site, whereas the formation of Msh2-Msh6 sliding clamps requires ATP binding to both the Msh2 and Msh6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and Msh6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps.     

Acetone-regulated synthesis and degradation of cytochrome P450E1 and cytochrome P4502B1 in rat liver [corrected

The regulation of CYP2E1 and 2B1 was studied by following mRNA levels, catalytic activities and the subcellular distribution of the apoproteins in rat liver 0, 6, 12, 24, 48 and 96 h after a single intragastric dose of acetone. No changes were observed in hepatic CYP2E1 mRNA levels at any time after acetone treatment, whereas rapid rises were observed in the microsomal amount of CYP2E1 protein and CYP2E1-catalyzed 4-nitrophenol hydroxylase and carbon-tetrachloride-initiated lipid-peroxidation activities. However, CYP2E1-dependent catalytic activities declined much faster than the immunodetectable CYP2E1 protein, suggesting that this cytochrome P-450 is inactivated prior to degradation. Similar results were seen in primary hepatocyte cultures. By contrast, concomitant changes in levels of CYP2B1 and CYP2B1-dependent O-depentylation of pentoxyresorufin were observed in the same microsomal preparations. Investigation of the degradative mechanism of both CYP2E1 and CYP2B1 by immunoquantitation of the proteins in lysosomes and by immunohistochemistry indicated their degradation via an autophagic-lysosomal pathway. The data suggest that CYP2E1 is acutely inactivated in the endoplasmic reticulum and that degradation of this isozyme occurs, at least in part, by the lysosomal route. By contrast, CYP2B1 is principally controlled at the level of synthesis.     

Ichthyophthirius multifiliis (Fouquet) in the mirror carp, Cyprinus carpio L.

Mirror carp were infected with Ichthyophthirius multifiliis (Fouquet) under standardized conditions. The size and number of parasites at selected sites on the body were recorded during the course of the infection. Initial exposure to 40 mature parasites resulted in a mild infection with 100% recovery after 18 days. Recovered fish did not appear to be carriers of the parasite. Exposure to 400 parasites resulted in 100% mortality between 22–25 days. The growth rate of the parasite was linear. Parasites were more numerous in the dorsal surface of the fish than in the lateral or ventral surface. The increase in parasite numbers during the disease was greater in the gills than in the skin.     

Age-dependent inhibition of neuronal protein synthesis by hypothyroidism in the developing rat brain cortex

Neuronal perikarya isolated from developing rat brain cortex were employed for studying the effect of hypothyroidism on RNA and protein synthesis in vitro. Neuronal protein synthesis was inhibited by hypothyroidism during the second week of brain development. Thyroxine treatment in vivo stimulated neuronal protein synthesis in hypothyroid rats. The synthesis of neuronal RNA was depressed by hypothyroidism in 7-day old rats. The inhibition of neuronal protein synthesis due to the lack of thyroid hormaones was restricted to membrane-bound ribosomes. The results suggest that the maturation of the neurone is very sensitive to hormonal imbalance during the critical period of brain development.     

Impact of the TCR Signal on Regulatory T Cell Homeostasis,Function, and Trafficking

Signaling through the T cell antigen receptor (TCR) is important for the homeostasis of naïve and memory CD4⁺ T cells. The significance of TCR signaling in regulatory T (Treg) cells has not been systematically addressed. Using an Ox40-cre allele that is prominently expressed in Treg cells, and a conditional null allele of the gene encoding p56^Lck, we have examined the importance of TCR signaling in Treg cells. Inactivation of p56^Lck resulted in abnormal Treg homeostasis characterized by impaired turnover, preferential redistribution to the lymph nodes, loss of suppressive function, and striking changes in gene expression. Abnormal Treg cell homeostasis and function did not reflect the involvement of p56^Lck in CD4 function because these effects were not observed when CD4 expression was inactivated by Ox40-cre.The results make clear multiple aspects of Treg cell homeostasis and phenotype that are dependent on a sustained capacity to signal through the TCR.     

Evidence for Inbreeding and Genetic Differentiation among Geographic Populations of the Saprophytic Mushroom Trogia venenata from Southwestern China

During the past 40 years, more than 400 Sudden Unexplained Deaths (SUDs) have occurred in Yunnan, southwestern China. Epidemiological and toxicological analyses suggested that a newly discovered mushroom called Trogia venenata was the leading culprit for SUDs. At present, relatively little is known about the genetics and natural history of this mushroom. In this study, we analyzed the sequence variation at four DNA fragments among 232 fruiting bodies of T. venenata collected from seven locations. Our ITS sequence analyses confirmed that all the isolates belonged to the same species. The widespread presence of sequence heterozygosity within many strains at each of three protein-coding genes suggested that the fruiting bodies were diploid, dikaryotic or heterokaryotic. Within individual geographic populations, we found significant deviations of genotype frequencies from Hardy-Weinberg expectations, with the overall observed heterozygosity lower than that expected under random mating, consistent with prevalent inbreeding within local populations. The geographic populations were overall genetically differentiated. Interestingly, while a positive correlation was found between population genetic distance and geographic distance, there was little correlation between genetic distance and barium concentration difference for the geographic populations. Our results suggest frequent inbreeding, geographic structuring, and limited gene flow among geographic populations of T. venenata from southwestern China.     

Metabolites in Blood for Prediction of Bacteremic Sepsis in the Emergency Room

A metabolomics approach for prediction of bacteremic sepsis in patients in the emergency room (ER) was investigated. In a prospective study, whole blood samples from 65 patients with bacteremic sepsis and 49 ER controls were compared. The blood samples were analyzed using gas chromatography coupled to time-of-flight mass spectrometry. Multivariate and logistic regression modeling using metabolites identified by chromatography or using conventional laboratory parameters and clinical scores of infection were employed. A predictive model of bacteremic sepsis with 107 metabolites was developed and validated. The number of metabolites was reduced stepwise until identifying a set of 6 predictive metabolites. A 6-metabolite predictive logistic regression model showed a sensitivity of 0.91(95% CI 0.69–0.99) and a specificity 0.84 (95% CI 0.58–0.94) with an AUC of 0.93 (95% CI 0.89–1.01). Myristic acid was the single most predictive metabolite, with a sensitivity of 1.00 (95% CI 0.85–1.00) and specificity of 0.95 (95% CI 0.74–0.99), and performed better than various combinations of conventional laboratory and clinical parameters. We found that a metabolomics approach for analysis of acute blood samples was useful for identification of patients with bacteremic sepsis. Metabolomics should be further evaluated as a new tool for infection diagnostics.