RAD51D protects against MLH1-dependent cytotoxic responses to O6-methylguanine

S_N1-type methylating agents generate O⁶-methyl guanine (O⁶-meG), which is a potently mutagenic, toxic, and recombinogenic DNA adduct. Recognition of O⁶-meG:T mismatches by mismatch repair (MMR) causes sister chromatid exchanges, which are representative of homologous recombination (HR) events. Although the MMR-dependent mutagenicity and toxicity caused by O⁶-meG has been studied, the mechanisms of recombination induced by O⁶-meG are poorly understood. To explore the HR and MMR genetic interactions in mammals, we used the Rad51d and Mlh1 mouse models. Ablation of Mlh1 did not appreciably influence the developmental phenotypes conferred by the absence of Rad51d. Mouse embryonic fibroblasts (MEFs) deficient in Rad51d can only proliferate in p53-deficient background. Therefore, Rad51d^?/?Mlh1^?/? Trp53^?/? MEFs with a combined deficiency of HR and MMR were generated and comparisons between MLH1 and RAD51D status were made. To our knowledge, these MEFs are the first mammalian model system for combined HR and MMR defects. Rad51d-deficient MEFs were 5.3-fold sensitive to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) compared to the Rad51d-proficient MEFs. A pronounced G2/M arrest in Rad51d-deficient cells was accompanied by an accumulation of γ-H2AX and apoptosis. Mlh1-deficient MEFs were resistant to MNNG and showed no G2/M arrest or apoptosis at the doses used. Importantly, loss of Mlh1 alleviated sensitivity of Rad51d-deficient cells to MNNG, in addition to reducing γ-H2AX, G2/M arrest and apoptosis. Collectively, the data support the hypothesis that MMR-dependent sensitization of HR-deficient cells is specific for O⁶-meG and suggest that HR resolves DNA intermediates created by MMR recognition of O⁶-meG:T. This study provides insight into recombinogenic mechanisms of carcinogenesis and chemotherapy resulting from O⁶-meG adducts.     

Quantitative determination of formononetin and its metabolite in rat plasma after intravenous bolus administration by HPLC coupled with tandem mass spectrometry

A new simple, rapid, sensitive and accurate quantitative detection method using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for the measurement of formononetin (FMN) and daidzein (DZN) levels in rat plasma is described. Analytes were separated on a Supelco Discovery C18 (4.6 × 50 mm, 5.0 μm) column with acetonitrile: methanol (50:50, v/v) and 0.1% acetic acid in the ratio of 90:10 (v/v) as a mobile phase. The method was proved to be accurate and precise at linearity range of 5–100 ng/mL with a correlation coefficient (r) of ≥0.996. The intra- and inter-day assay precision ranged from 1.66–6.82% and 1.87–6.75%, respectively; and intra- and inter-day assay accuracy was between 89.98–107.56% and 90.54–105.63%, respectively for both the analytes. The lowest quantitation limit for FMN and DZN was 5.0 ng/mL in 0.1 mL of rat plasma. Practical utility of this new LC–MS/MS method was demonstrated in a pharmacokinetic study in rats following intravenous administration of FMN.     

Arsenic affects mineral nutrients in grains of various Indian rice (Oryza sativa L.) genotypes grown on arsenic-contaminated soils of West Bengal

The exposure of paddy fields to arsenic (As) through groundwater irrigation is a serious concern that may not only lead to As accumulation to unacceptable levels but also interfere with mineral nutrients in rice grains. In the present field study, profiling of the mineral nutrients (iron (Fe), phosphorous, zinc, and selenium (Se)) was done in various rice genotypes with respect to As accumulation. A significant genotypic variation was observed in elemental retention on root Fe plaque and their accumulation in various plant parts including grains, specific As uptake (29–167 mg kg^?1 dw), as well as As transfer factor (4–45%). Grains retained the least level of As (0.7–3%) with inorganic As species being the dominant forms, while organic As species, viz., dimethylarsinic acid and monomethylarsonic acid, were non-detectable. In all tested varieties, the level of Se was low (0.05–0.12 mg kg^?1 dw), whereas that of As was high (0.4–1.68 mg kg^?1 dw), considering their safe/recommended daily intake limits, which may not warrant their human consumption. Hence, their utilization may increase the risk of arsenicosis, when grown in As-contaminated areas.     

Greater diversity of Shiga toxin-encoding bacteriophage insertion sites among Escherichia coli O157:H7 isolates from cattle than in those from humans

Escherichia coli O157:H7, a zoonotic human pathogen for which domestic cattle are a reservoir host, produces a Shiga toxin(s) (Stx) encoded by bacteriophages. Chromosomal insertion sites of these bacteriophages define three principal genotypes (clusters 1 to 3) among clinical isolates of E. coli O157:H7. Stx-encoding bacteriophage insertion site genotypes of 282 clinical and 80 bovine isolates were evaluated. A total of 268 (95.0%) of the clinical isolates, but only 41 (51.3%) of the bovine isolates, belonged to cluster 1, 2, or 3 (P < 0.001). Thirteen additional genotypes were identified in isolates from both cattle and humans (four genotypes), from only cattle (seven genotypes), or from only humans (two genotypes). Two other markers previously associated with isolates from cattle or with clinical isolates showed similar associations with genotype groups within bovine isolates; the tir allele sp-1 and the Q933W allele were under- and overrepresented, respectively, among cluster 1 to 3 genotypes. Stx-encoding bacteriophage insertion site typing demonstrated that there is broad genetic diversity of E. coli O157:H7 in the bovine reservoir and that numerous genotypes are significantly underrepresented among clinical isolates, consistent with the possibility that there is reduced virulence or transmissibility to humans of some bovine E. coli O157:H7 genotypes.     

Increased intrahepatic triglyceride is associated with peripheral insulin resistance: in vivo MR imaging and spectroscopy studies

Recent studies have indicated that the mass/content of intramyocellular lipid (IMCL), intrahepatic triglyceride (IHTG), visceral fat (VF), and even deep abdominal subcutaneous fat (SF) may all be correlated with insulin resistance. Since simultaneous measurements of these parameters have not been reported, the relative strength of their associations with insulin action is not known. Therefore, the goals of this study were 1) to simultaneously measure IMCL, IHTG, VF, and abdominal SF in the same nondiabetic individuals using noninvasive (1)H-magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) and 2) to examine how these fat stores are correlated with systemic insulin sensitivity as measured by whole body glucose disposal (R(d)) during euglycemic-hyperinsulinemic clamp studies. Positive correlations were observed among IMCL, IHTG, and VF. There were significant inverse correlations between whole body R(d) and both IMCL and VF. Notably, there was a particularly tight inverse correlation between IHTG and whole body R(d) (r = -0.86, P < 0.001), consistent with an association between liver fat and peripheral insulin sensitivity. This novel finding suggests that hepatic triglyceride accumulation has important systemic consequences that may adversely affect insulin sensitivity in other tissues.     

Amino acid-based enantiomerically pure 3-substituted 1,4-benzodiazepin-2-ones: a new class of anti-ischemic agents

A series of 3-substituted 1,4-benzodiazepin-2-ones derived from S and R amino acids were evaluated for their anti-ischemic activity in vitro. Treatment with compounds 7h, 16, 9d, and 17 decreased the apoptotic neuronal number, however increased the neuronal viability. The compounds decreasing apoptosis could protect neurons from the ischemic injury. The difference in the activities of 1,4-benzodiazepin-2-ones derived from S- and R-amino acids is discussed and explained on the basis of molecular modeling studies.     

Renal clearance of quantum dots

The field of nanotechnology holds great promise for the diagnosis and treatment of human disease. However, the size and charge of most nanoparticles preclude their efficient clearance from the body as intact nanoparticles. Without such clearance or their biodegradation into biologically benign components, toxicity is potentially amplified and radiological imaging is hindered. Using intravenously administered quantum dots in rodents as a model system, we have precisely defined the requirements for renal filtration and urinary excretion of inorganic, metal-containing nanoparticles. Zwitterionic or neutral organic coatings prevented adsorption of serum proteins, which otherwise increased hydrodynamic diameter by >15 nm and prevented renal excretion. A final hydrodynamic diameter <5.5 nm resulted in rapid and efficient urinary excretion and elimination of quantum dots from the body. This study provides a foundation for the design and development of biologically targeted nanoparticles for biomedical applications.     

Chemical or enzymatic deglycosylation and germination abrogates the inhibitory activity of Cyamopsis tetragonoloba trypsin inhibitor

Journal of Plant Biochemistry and Biotechnology - A glycosylated heat stable trypsin chymotrypsin inhibitor was isolated from Cyamopsis tetragonoloba seeds. It is being reported for the first time...