Role of pH on dimeric interactions for DENV envelope protein: an insight from molecular dynamics study

The entry of dengue viruses is mediated by pH triggering in the host cells. Here we have studied the DENV E protein stability and binding of its units at low and normal pH using MD and MM-PB/SA method for the first time. To investigate the role of pH in dissociation of dimeric protein, we have performed a concise study of hydrogen bonding and other interactions between units of dimer at low and normal pH. The Generalized Born calculation connotes that dimeric unit was relatively less stable and less proned for dimerisation at low pH. Our results provide a theoretical verification for previous assumptions of pH triggering mechanism of dengue envelope protein. During the pH alteration, we found a large decrement in salt bridges which were observed at normal pH. We also compared the flexibility of each unit and found that they exhibit different fluctuations during molecular dynamics simulations.     

Mammalian target of rapamycin inhibition in macrophages of asymptomatic HIV+ persons reverses the decrease in TLR-4-mediated TNF-α release through prolongation of MAPK pathway activation

TLR-4-mediated signaling is significantly impaired in macrophages from HIV(+) persons, predominantly owing to altered MyD88-dependent pathway signaling caused in part by constitutive activation of PI3K. In this study we assessed in these macrophages if the blunted increase in TLR-4-mediated TNF-α release induced by lipid A (LA) is associated with PI3K-induced upregulation of mammalian target of rapamycin (mTOR) activity. mTOR inhibition with rapamycin enhanced TLR-4-mediated TNF-α release, but suppressed anti-inflammatory IL-10 release. Targeted gene silencing of mTOR in macrophages resulted in LA-induced TNF-α and IL-10 release patterns similar to those induced by rapamycin. Rapamycin restored MyD88/IL-1R-associated kinase interaction in a dose-dependent manner. Targeted gene silencing of MyD88 (short hairpin RNA) and mTOR (RNA interference) inhibition resulted in TLR-4-mediated 70-kDa ribosomal protein S6 kinase activation and enhanced TNF-α release, whereas IL-10 release was inhibited in both silenced and nonsilenced HIV(+) macrophages. Furthermore, mTOR inhibition augmented LA-induced TNF-α release through enhanced and prolonged phosphorylation of ERK1/2 and JNK1/2 MAPK, which was associated with time-dependent MKP-1 destabilization. Taken together, impaired TLR-4-mediated TNF-α release in HIV(+) macrophages is attributable in part to mTOR activation by constitutive PI3K expression in a MyD88-dependent signaling pathway. These changes result in MAPK phosphatase 1 stabilization, which shortens and blunts MAPK activation. mTOR inhibition may serve as a potential therapeutic target to upregulate macrophage innate immune host defense responsiveness in HIV(+) persons.     

PKCγ is required for ethanol-induced increases in GABA(A) receptor α4 subunit expression in cultured cerebral cortical neurons

Ethanol exposure produces alterations in GABA(A) receptor function and expression associated with CNS hyperexcitability, but the mechanisms of these effects are unknown. Ethanol is known to increase both GABA(A) receptor α4 subunits and protein kinase C (PKC) isozymes in vivo and in vitro. Here, we investigated ethanol regulation of GABA(A) receptor α4 subunit expression in cultured cortical neurons to delineate the role of PKC. Cultured neurons were prepared from rat pups on postnatal day 0-1 and tested after 18?days. GABA(A) receptor α4 subunit surface expression was assessed using P2 fractionation and surface biotinylation following ethanol exposure for 4?h. Miniature inhibitory post-synaptic currents were measured using whole cell patch clamp recordings. Ethanol increased GABA(A) receptor α4 subunit expression in both the P2 and biotinylated fractions, while reducing the decay time constant in miniature inhibitory post-synaptic currents, with no effect on γ2 or δ subunits. PKC activation mimicked ethanol effects, while the PKC inhibitor calphostin C prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression. PKCγ siRNA knockdown prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression, but inhibition of the PKCβ isoform with PKCβ pseudosubstrate had no effect. We conclude that PKCγ regulates ethanol-induced alterations in α4-containing GABA(A) receptors.     

Drug-induced senescence generates chemoresistant stemlike cells with low reactive oxygen species

Tumor recurrence after chemotherapy or radiation remains a major obstacle to successful cancer treatment. A subset of cancer cells, termed cancer stem cells, can elude conventional treatments and eventually regenerate a tumor that is more aggressive. Despite the large number of studies, molecular events that govern the emergence of aggressive therapy-resistant cells with stem cell properties after chemotherapy are poorly defined. The present study provides evidence for the rare escape of tumor cells from drug-induced cell death, after an intermediate stay in a non-cycling senescent stage followed by unstable multiplication characterized by spontaneous cell death. However, some cells appear to escape and generate stable colonies with an aggressive tumor stem cell-like phenotype. These cells displayed higher CD133 and Oct-4 expression. Notably, the drug-selected cells that contained low levels of reactive oxygen species (ROS) also showed an increase in antioxidant enzymes. Consistent with this in vitro experimental data, we observed lower levels of ROS in breast tumors obtained after neoadjuvant chemotherapy compared with samples that did not receive preoperative chemotherapy. These latter tissues also expressed enhanced levels of ROS defenses with enhanced expression of superoxide dismutase. Higher levels of Oct-4 and CD133 were also observed in tumors obtained after neoadjuvant chemotherapy. Further studies provided evidence for the stabilization of Nrf2 due to reduced 26 S proteasome activity and increased p21 association as the driving signaling event that contributes to the transition from a high ROS quiescent state to a low ROS proliferating stage in drug-induced tumor stem cell enrichment.     

In silico analysis of expression pattern of a Wnt/β-catenin responsive gene ANLN in gastric cancer

Actin-binding protein anillin (ANLN) is primarily involved in the cytokinesis and known to be dysregulated in many cancers including gastric cancer (GC). However, the regulation and clinical significance of ANLN in GC are far less clear. In the present study, we aimed to investigate the clinical significance and possible regulators of ANLN in GC. We have identified the Wnt/β-catenin associated regulation of ANLN by analyzing the in vitro perturbed β-catenin mRNA expression profiles. Investigating the gastric tumors from publicly available genome-wide mRNA expression profiles, we have identified the over expression of ANLN in gastric tumors. Association between ANLN expression and clinical characteristics of GC showed elevated expression in intestinal type GC. Performing a single sample prediction method across GC mRNA expression profiles, we have identified the over expression of ANLN in proliferative type gastric tumors compared to the invasive and metabolic type gastric tumors. In silico pathway prediction analysis revealed the association between Wnt/β-catenin signaling and ANLN expression in gastric tumors. Our results highlight that expression of a Wnt/β-catenin responsive gene ANLN in GC is a molecular predictor of intestinal and proliferative type gastric tumors.     

Age at Menarche and Natural Menopause and Number of Reproductive Years in Association with Mortality: Results from a Median Follow-Up of 11.2 Years among 31,955 Naturally Menopausal Chinese Women

Background

Studies conducted in Western countries suggest that early age at menarche and early age at menopause are both associated with increased total mortality, but limited data are available for Asian populations. We examined associations of age at menarche and natural menopause and duration of the reproductive span with mortality in a population-based cohort study of Chinese women.

Methods

We evaluated the effects of age at menarche, age at natural menopause, and number of reproductive years on total and cause-specific mortality among 31,955 naturally menopausal Chinese women who participated in the Shanghai Women''s Health Study, a population-based, prospective cohort study.

Results

A total of 3,158 deaths occurred during a median follow-up of 11.2 years. Results from Cox proportional hazards models showed that younger age at menopause (<46.64 years) was associated with higher risk of total mortality (P_trend  = 0.02). Younger age at menarche (<14 years) was associated with higher risk of mortality from stroke (P_trend  = 0.03) and diabetes (P_trend = 0.02) but lower risk of mortality from respiratory system cancer (P_trend  = 0.01). Women with a shorter reproductive span had lower risk of mortality from gynecological cancers (P_trend = 0.03).

Conclusions

Our study found that menstrual characteristics are important predictors of mortality, suggesting an important role of sex hormones in biological aging.     

Remediation of Nickel-Contaminated Soil by Brassica juncea L. cv. T-59 and Effect of the Metal on Some Metabolic Aspects of the Plant

Among four cultivars of Brassica juncea L., viz., TM-4, TM-2, RH-30, and T-59, cv. T-59 was relatively more tolerant to nickel (Ni) toxicity based on the growth parameters, seedling vigor index, and metal tolerance index. Nickel application inhibited the activity of the nitrate-assimilating enzyme nitrate reductase in the roots, stem, and leaves, whereas the total organic nitrogen, proline, and activity of a polyamine-metabolizing enzyme, diamine oxidase, increased in this tolerant cultivar (T-59). It accumulated a good amount of Ni from the soil in its root and shoot (i.e., 6.0–6.51 μg Ni g^?1 dry weight) during 2 months of cultivation with an 8.0 mM Ni supply in the soil. The data presented in this paper indicate that Ni tolerance and its removal by Indian mustard from subtropical Indian soil is cultivar dependent, possibly due to different genetic and physiological adaptations of the cultivars.     

Enhanced repair of a cisplatin-damaged reporter chloramphenicol-O-acetyltransferase gene and altered activities of DNA polymerases α and β, and DNA ligase in cells of a human malignant glioma following in vivo cisplatin therapy

Current evidence suggest an important role for increased repair of drug-induced DNA damage as one of the major mechanisms involved in tumor cell resistance to cis-DDP. In this study, we examined the DNA repair capacity and the activities of three DNA repair related proteins, namely, DNA polymerases α and β, and total DNA ligase in cells of a malignant oligodendroglioma obtained from a patient before therapy and compared it with those of a specimen of the tumor acquired after the patient had failed cis-DDP therapy. DNA repair capacity was quantitated as the extent of reactivation of the chloramphenicol-O-acetyltransferase (CAT) gene in a eukaryotic expression vector that has been damaged and inactivated by prior treatment with cis-DDP and then transfected into the tumor cells. The extent of DNA-platinum adduct formation in the expression vector was determined by flameless atomic absorption spectrometry. The level of cis-DDP resistance of cells of the two tumors was determined with the capillary tumor stem cell assay. We observed a 2.8-fold increased capacity to repair Pt-DNA adducts and reactivate the CAT gene in cells of the tumor obtained after cis-DDP therapy, compared to cells of the untreated tumor. This was associated with increases of 9.4-fold and a 2.3-fold, respectively, in DNA polymerase β and total DNA ligase activities in cells of the treated tumor. At 5 μM cis-DDP, there was a 5.9-fold increase in the in vitro cis-DDP resistance of post-therapy tumor cells relative to cells of the untreated tumor. No significant difference in DNA polymerase α activity was observed between the two tumors. These data suggest that the enhanced ability to repair cis-DDP induced DNA damage, mediated, in part, by increased tumor DNA polymerase β and DNA ligase activities, plays an important role in the in vivo acquisition of cis-DDP resistance in human malignant gliomas, and that these proteins and/or their encoding genes may represent critical targets for strategies to overcome such resistance clinically.