Endothelial nitric oxide synthase gene polymorphisms and essential hypertension in Han Chinese

We examined the effect of polymorphisms in the endothelial nitric oxide synthase gene on the risk for essential hypertension in a Han Chinese population through a meta-analysis of data from 15 studies. Associations between increased risk for essential hypertension and 4b/a were obtained in a dominant model and allele contrast (aa + ab vs bb: odds ratio (OR)(FE) = 1.26, 95% confidence interval (CI) = 1.10-1.44; a vs b allele: OR(FE) = 1.23, 95%CI: 1.09-1.40). Four studies with sample sizes over 500 produced similar results. No evidence of publication bias was found. Also, no significant heterogeneity was observed among these studies. When we examined the G894T polymorphism, we found a marginally significant association for allele contrast and the recessive model when all the eligible studies were pooled together. However, there was no evidence for a significant association after the exclusion of two studies deviating from Hardy-Weinberg equilibrium in the control group. Heterogeneity among studies was observed. Results of cumulative and recursive cumulative meta-analysis indicated that more studies are needed to objectively determine the effects of these two polymorphisms.     

Dkk-1在肿瘤中的研究进展

DKK-1是一种分泌型糖蛋白,通过结合细胞表面受体LRP5/6、Kremen1/2在Wnt通路中起负调控作用。Dkk-1的表达受p53、MYCN、β-catenin等基因调控。Dkk-1在细胞内的异位表达能抑制多种肿瘤细胞的增殖,但有时能在促凋亡因子存在时诱发凋亡。Dkk-1在一些肿瘤中低表达,而在另一些肿瘤中高表达。Dkk-1在不同肿瘤的发生、发展以及转移这几个阶段中的表达和功能表现出复杂多重的差异。该文就Dkk-1在肿瘤中表达和功能的研究进展作一综述。     

A Novel Small Molecule Inhibitor of Hepatitis C Virus Entry

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC₅₀ values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.     

Evidence that intracellular stages of Leishmania major utilize amino sugars as a major carbon source

Intracellular parasites, such as Leishmania spp, must acquire suitable carbon sources from the host cell in order to replicate. Here we present evidence that intracellular amastigote stages of Leishmania exploit amino sugars in the phagolysosome of mammalian macrophages as a source of carbon and energy. L. major parasites are capable of using N-acetylglucosamine and glucosamine as primarily carbon sources and contain key enzymes required for conversion of these sugars to fructose-6-phosphate. The last step in this pathway is catalyzed by glucosamine-6-phosphate deaminase (GND), which was targeted to glycosomes via a canonical C-terminal targeting signal when expressed as a GFP fusion protein. Mutant parasites lacking GND were unable to grow in medium containing amino sugars as sole carbohydrate source and rapidly lost viability, concomitant with the hyper-accumulation of hexosamine-phosphates. Expression of native GND, but not a cytosolic form of GND, in Δgnd parasites restored hexosamine-dependent growth, indicating that toxicity is due to depletion of glycosomal pools of ATP. Non-lethal increases in hexosamine phosphate levels in both Δgnd and wild type parasites was associated with a defect in promastigote metacyclogenesis, suggesting that hexosamine phosphate levels may influence parasite differentiation. Promastigote and amastigote stages of the Δgnd mutant were unable to replicate within macrophages and were either completely cleared or exhibited reduced lesion development in highly susceptible Balb/c mice. Our results suggest that hexosamines are a major class of sugars in the macrophage phagolysosome and that catabolism of scavenged amino sugars is required to sustain essential metabolic pathways and prevent hexosamine toxicity.     

Calycosin Promotes Angiogenesis Involving Estrogen Receptor and Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Zebrafish and HUVEC

Background

Angiogenesis plays an important role in a wide range of physiological processes, and many diseases are associated with the dysregulation of angiogenesis. Radix Astragali is a Chinese medicinal herb commonly used for treating cardiovascular disorders and has been shown to possess angiogenic effect in previous studies but its active constituent and underlying mechanism remain unclear. The present study investigates the angiogenic effects of calycosin, a major isoflavonoid isolated from Radix Astragali, in vitro and in vivo.

Methodology

Tg(fli1:EGFP) and Tg(fli1:nEGFP) transgenic zebrafish embryos were treated with different concentrations of calycosin (10, 30, 100 µM) from 72 hpf to 96 hpf prior morphological observation and angiogenesis phenotypes assessment. Zebrafish embryos were exposed to calycosin (10, 100 µM) from 72 hpf to 78 hpf before gene-expression analysis. The effects of VEGFR tyrosine kinase inhibitor on calycosin-induced angiogenesis were studied using 72 hpf Tg(fli1:EGFP) and Tg(fli1:nEGFP) zebrafish embryos. The pro-angiogenic effects of calycosin were compared with raloxifene and tamoxifen in 72 hpf Tg(fli1:EGFP) zebrafish embryos. The binding affinities of calycosin to estrogen receptors (ERs) were evaluated by cell-free and cell-based estrogen receptor binding assays. Human umbilical vein endothelial cell cultures (HUVEC) were pretreated with different concentrations of calycosin (3, 10, 30, 100 µM) for 48 h then tested for cell viability and tube formation. The role of MAPK signaling in calycosin-induced angiogenesis was evaluated using western blotting.

Conclusion

Calycosin was shown to induce angiogenesis in human umbilical vein endothelial cell cultures (HUVEC) in vitro and zebrafish embryos in vivo via the up-regulation of vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 mRNA expression. It was demonstrated that calycosin acted similar to other selective estrogen receptor modulators (SERMs), such as raloxifene and tamoxifen, by displaying selective potency and affinity to estrogen receptors ERα and ERβ. Our results further indicated that calycosin promotes angiogenesis via activation of MAPK with the involvement of ERK1/2 and ER. Together, this study revealed, for the first time, that calycosin acts as a selective estrogen receptor modulator (SERM) to promote angiogenesis, at least in part through VEGF-VEGFR2 and MAPK signaling pathways.     

Prediction of Deleterious Non-Synonymous SNPs Based on Protein Interaction Network and Hybrid Properties

Non-synonymous SNPs (nsSNPs), also known as Single Amino acid Polymorphisms (SAPs) account for the majority of human inherited diseases. It is important to distinguish the deleterious SAPs from neutral ones. Most traditional computational methods to classify SAPs are based on sequential or structural features. However, these features cannot fully explain the association between a SAP and the observed pathophysiological phenotype. We believe the better rationale for deleterious SAP prediction should be: If a SAP lies in the protein with important functions and it can change the protein sequence and structure severely, it is more likely related to disease. So we established a method to predict deleterious SAPs based on both protein interaction network and traditional hybrid properties. Each SAP is represented by 472 features that include sequential features, structural features and network features. Maximum Relevance Minimum Redundancy (mRMR) method and Incremental Feature Selection (IFS) were applied to obtain the optimal feature set and the prediction model was Nearest Neighbor Algorithm (NNA). In jackknife cross-validation, 83.27% of SAPs were correctly predicted when the optimized 263 features were used. The optimized predictor with 263 features was also tested in an independent dataset and the accuracy was still 80.00%. In contrast, SIFT, a widely used predictor of deleterious SAPs based on sequential features, has a prediction accuracy of 71.05% on the same dataset. In our study, network features were found to be most important for accurate prediction and can significantly improve the prediction performance. Our results suggest that the protein interaction context could provide important clues to help better illustrate SAP''s functional association. This research will facilitate the post genome-wide association studies.     

Nitrate respiration protects hypoxic Mycobacterium tuberculosis against acid- and reactive nitrogen species stresses

There are strong evidences that Mycobacterium tuberculosis survives in a non-replicating state in the absence of oxygen in closed lesions and granuloma in vivo. In addition, M. tuberculosis is acid-resistant, allowing mycobacteria to survive in acidic, inflamed lesions. The ability of M. tuberculosis to resist to acid was recently shown to contribute to the bacillus virulence although the mechanisms involved have yet to be deciphered. In this study, we report that M. tuberculosis resistance to acid is oxygen-dependent; whereas aerobic mycobacteria were resistant to a mild acid challenge (pH 5.5) as previously reported, we found microaerophilic and hypoxic mycobacteria to be more sensitive to acid. In hypoxic conditions, mild-acidity promoted the dissipation of the protonmotive force, rapid ATP depletion and cell death. Exogenous nitrate, the most effective alternate terminal electron acceptor after molecular oxygen, protected hypoxic mycobacteria from acid stress. Nitrate-mediated resistance to acidity was not observed for a respiratory nitrate reductase NarGH knock-out mutant strain. Furthermore, we found that nitrate respiration was equally important in protecting hypoxic non-replicating mycobacteria from radical nitrogen species toxicity. Overall, these data shed light on a new role for nitrate respiration in protecting M. tuberculosis from acidity and reactive nitrogen species, two environmental stresses likely encountered by the pathogen during the course of infection.     

Evaluation of D10-Leu metabolic labeling coupled with MALDI-MS analysis in studying the response of the yeast proteome to H2O2 challenge

An efficient D10-Leu metabolic-labeling method combined with isotope-ratio quantitation by MALDI-TOF MS was used to probe the response of the yeast proteome to H2O2. Control cultures correct for effects not associated with H2O2 challenge. A stress-response index to H2O2 (SRIH2O2) is defined, and values are reported for seven proteins at 45-225 min following exposure to 0.4 mM H2O2. The time course of protein accumulation in unstressed cells following the H10- to D10-SCD switch suggests that proteome responses at <45 min could be monitored by addition of excess D10-Leu to H10-cultures.