Crystal structure of a truncated urease accessory protein UreF from Helicobacter pylori

Urease plays a central role in the pathogenesis of Helicobacter pylori in humans. Maturation of this nickel metalloenzyme in bacteria requires the participation of the accessory proteins UreD (termed UreH in H. pylori), UreF, and UreG, which form sequential complexes with the urease apoprotein as well as UreE, a metallochaperone. Here, we describe the crystal structure of C‐terminal truncated UreF from H. pylori (residues 1–233), the first UreF structure to be determined, at 1.55 Å resolution using SAD methods. UreF forms a dimer in vitro and adopts an all‐helical fold congruent with secondary structure prediction. On the basis of evolutionary conservation analysis, the structure reveals a probable binding surface for interaction with other urease components as well as key conserved residues of potential functional relevance. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Passiflin,a novel dimeric antifungal protein from seeds of the passion fruit

The intent was to isolate an antifungal protein from seeds of the passion fruit (Passiflora edulis) and to compare its characteristics with other antifungal proteins and bovine β-lactoglobulin in view of its N-terminal amino acid sequence similarity to β-lactoglobulin. The isolation procedure entailed ion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on Phenyl-Sepharose, ion-exchange chromatography on DEAE-cellulose, and FPLC-gel filtration on Superdex 75. The isolated 67-kDa protein, designated as passiflin, exhibited an N-terminal amino acid sequence closely resembling that of bovine β-lactoglobulin. It is the first antifungal protein found to have a β-lactoglobulin-like N-terminal sequence. Its dimeric nature is rarely found in antifungal proteins. It impeded mycelial growth in Rhizotonia solani with an IC₅₀ of 16 μM and potently inhibited proliferation of MCF-7 breast cancer cells with an IC₅₀ of 15 μM. There was no cross-reactivity of passiflin with anti-β-lactoglobulin antiserum. Intact β-lactoglobulin lacks antifungal and antiproliferative activities and is much smaller in molecular size than passiflin. However, it has been reported that hydrolyzed β-lactoglobulin shows antifungal activity. The data suggest that passiflin is distinct from β-lactoglobulin.     

中国环境管理分区:方法与方案

我国生态环境可持续性及其影响因素的区域差异显著,各地区环境管理面临的主要挑战和需要优先解决的生态环境问题不同。进行环境管理分区,根据各地区生态环境特征及其影响因素的差异性,制定有针对性的环境管理政策,将有效促进我国区域生态环境的整体优化。采取定性和定量分析相结合的方法进行我国环境管理分区。首先,在我国3大自然区的基础上,根据我国的自然地理格局和已有的相关区划成果,把我国划分为4个环境管理大区,包括:南部季风区、北部季风区、西北干旱区和青藏高寒区。其次,通过建立的包含13个指标的环境管理分区指标体系,采用一维化欧式距离法分析各环境管理大区下相邻省级行政区环境特征的相似性,把环境特征相似性大的相邻地区划分到同一分区,得到以省级行政区为基本单元的我国环境管理分区方案。然后,结合地区间历史渊源和区域未来发展趋势分析,对基于相似性分析的初步分区方案进行调整,把我国划分为8个以省级行政区为基本单元环境管理区。最后,根据相关调整原则和方法,对以省级行政区为基本单元的分区方案的边界线进行调整,得到以地级行政区为基本单元的分区方案,把我国划分为东北地区、华北平原区、华北山地与高原区、东南沿海地区、长江流域中游地区、西南地区、西北干旱区和青藏高寒区8个环境管理区。     

Activation of human bronchial epithelial cells by inflammatory cytokines IL‐27 and TNF‐α: Implications for immunopathophysiology of airway inflammation

Interleukin (IL)‐27 is a member of IL‐6/IL‐12 family cytokines produced by antigen‐presenting cells in immune responses. IL‐27 can drive the commitment of naive T cells to a T helper type 1 (Th1) phenotype and inhibit inflammation in later phases of infection. Human bronchial epithelial cells have been shown to express IL‐27 receptor complex. In this study, we investigated the in vitro effects of IL‐27, alone or in combination with inflammatory cytokine tumor necrosis factor (TNF)‐α on the pro‐inflammatory activation of human primary bronchial epithelial cells and the underlying intracellular signaling mechanisms. IL‐27 was found to enhance intercellular adhesion molecule 1 (ICAM‐1) expression on the surface of human bronchial epithelial cells, and a synergistic effect was observed in the combined treatment of IL‐27 and TNF‐α on the expression of ICAM‐1. Although IL‐27 did not alter the basal IL‐6 secretion from bronchial epithelial cells, it could significantly augment TNF‐α‐induced IL‐6 release. These synergistic effects on the up‐regulation of ICAM‐1 and IL‐6 were partially due to the elevated expression of TNF‐α receptor (p55TNFR) induced by IL‐27. Further investigations showed that the elevation of ICAM‐1 and IL‐6 in human bronchial epithelial cells stimulated by IL‐27 and TNF‐α was differentially regulated by phosphatidylinositol 3‐OH kinase (PI3K)‐Akt, p38 mitogen‐activated protein kinase, and nuclear factor‐κB pathways. Our results therefore provide a new insight into the molecular mechanisms involved in airway inflammation. J. Cell. Physiol. 223:788–797, 2010. © 2010 Wiley‐Liss, Inc.     

Moderate Alcohol Use and Cardiovascular Disease from Mendelian Randomization

Background

Observational studies show moderate alcohol use negatively associated with ischemic heart disease (IHD) and cardiovascular disease (CVD). However, healthier attributes among moderate users compared to never users may confound the apparent association. A potentially less biased way to examine the association is Mendelian randomization, using alcohol metabolizing genes which influence alcohol use.

Methods

We used instrumental variable analysis with aldehyde dehydrogenase 2 (ALDH2) genotypes (AA/GA/GG) as instrumental variables for alcohol use to examine the association of alcohol use (10 g ethanol/day) with CVD risk factors (blood pressure, lipids and glucose) and morbidity (self-reported IHD and CVD) among men in the Guangzhou Biobank Cohort Study.

Results

ALDH2 genotypes were a credible instrument for alcohol use (F-statistic 74.6). Alcohol was positively associated with HDL-cholesterol (0.05 mmol/L per alcohol unit, 95% confidence interval (CI) 0.02 to 0.08) and diastolic blood pressure (1.15 mmHg, 95% CI 0.23 to 2.07) but not with systolic blood pressure (1.00 mmHg, 95% CI -0.74 to 2.74), LDL-cholesterol (0.03 mmol/L, 95% CI -0.03 to 0.08), log transformed triglycerides (0.03 mmol/L, 95% CI -0.01 to 0.08) or log transformed fasting glucose (0.01 mmol/L, 95% CI -0.006 to 0.03), self-reported CVD (odds ratio (OR) 0.98, 95% CI 0.76 to 1.27) or self-reported IHD (OR 1.10, 95% CI 0.83 to 1.45).

Conclusion

Low to moderate alcohol use among men had the expected effects on most CVD risk factors but not fasting glucose. Larger studies are needed to confirm the null associations with IHD, CVD and fasting glucose.     

Regulating the reapers: activating metacaspases for programmed cell death

Research during the past two decades has revealed that specialized cysteine proteases act as conserved initiators or executioners for programmed cell death (PCD) in eukaryotes. Caspases were first identified as common regulators of PCD in metazoans, whereas the role of metacaspases (MCs) as regulators of cellular suicide in plants has only been shown genetically in the past several years. Together with recent biochemical and molecular characterizations of some of the representative MCs from different model systems, multiple mechanisms that can mediate the post-translational regulation of these proteases are beginning to emerge. Further elucidation of these regulatory pathways and definition of the downstream degradomes targeted by MCs should lead to a better understanding of cell death control in plants, protozoans, and fungi.     

Development of a selective pseudosubstrate-based peptide inhibitor of pp60c-src protein tyrosine kinase

Summary Using a combinatorial peptide library method, we identified YIYGSFK as an efficient and specific peptide substrate for pp60^c-src protein tyrosine kinase (PTK) [Lam et al., Int. J. Pept. Protein Res., 45 (1995) 587]. Employing YIYGSFK as a template, we synthesized and evaluated a series of pseudosubstrate-based inhibitors for pp60^c-src. We found that the efficiency of a given inhibitor was highly dependent on the specific tyrosine analog used at the phosphorylation site of the substrate. One of these pseudosubstrate inhibitors, YI(2-Nal)GSFK, selectively inhibited the kinase activity of pp60^c-src, with a K_i of 24 M. This peptide inhibitor exhibited selectivity for pp60^c-src as compared to other PTKs tested, such as c-Abl and Bcr-Abl. Our results suggest that selective inhibitors for a specific PTK can be developed when the structure of a specific and efficient small peptide substrate for this PTK can be used as a template for structure modification.Abbreviations 1-Nal l-1-naphthylalanine - 2-Nal l-2-naphthylalanine - BOP benzotriazolyl-N-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate - BSA bovine serum albumin - cAPK cyclic AMP-dependent protein kinase - DIEA diisopropylethylamine - EGFR epidermal growth factor receptor - Fmoc fluorenylmethoxycarbonyl - HOBt 1-hydroxybenzotriazole - MES 2-[N-morpholino]ethanesulfonic acid - PBS phosphate-buffered salts - pCl l-p-chlorophenylalanine - pF l-p-fluorophenylalanine - PTK protein tyrosine kinase - TLC thin-layer chromatography     

The extracellular domain of p185/neu is released from the surface of human breast carcinoma cells, SK-BR-3.

The human breast carcinoma cell line SK-BR-3, expresses the neu oncogene product, p185, which is a receptor tyrosine kinase. Using a double monoclonal antibody capture enzyme-linked immunosorbent assay for p185, activity was detected in conditioned media from cultures of SK-BR-3 cells. Two monoclonal antibodies specific for the extracellular domain of p185/neu immunoprecipitated a protein with a molecular mass of approximately 105 kDa. p105 was further shown to compete with p185 for binding to monoclonal antibodies and pulse-chase experiments indicate that it was generated by post-translational processing. Peptide maps showed that p105 and p185 are related polypeptides. Since p105 is close to the predicted size for the extracellular domain of p185/neu, we propose that SK-BR-3 cells specifically process and release this portion of the receptor into the medium. The release of the extracellular domain may have implications in oncogenesis and its detection could prove useful as a cancer diagnostic.