Interaction of Hsp40 with influenza virus M2 protein: implications for PKR signaling pathway

Influenza virus contains three integral membrane proteins: haemagglutinin, neuraminidase, and matrix protein (M1 and M2). Among them, M2 protein functions as an ion channel, important for virus uncoating in endosomes of virus-infected cells and essential for virus replication. In an effort to explore potential new functions of M2 in the virus life cycle, we used yeast two-hybrid system to search for M2-associated cellular proteins. One of the positive clones was identified as human Hsp40/Hdj1, a DnaJ/Hsp40 family protein. Here, we report that both BM2 (M2 of influenza B virus) and A/M2 (M2 of influenza A virus) interacted with Hsp40 in vitro and in vivo. The region of M2-Hsp40 interaction has been mapped to the CTD1 domain of Hsp40. Hsp40 has been reported to be a regulator of PKR signaling pathway by interacting with p58^IPK that is a cellular inhibitor of PKR. PKR is a crucial component of the host defense response against virus infection. We therefore attempted to understand the relationship among M2, Hsp40 and p58^IPK by further experimentation. The results demonstrated that both A/M2 and BM2 are able to bind to p58^IPKin vitro and in vivo and enhance PKR autophosphorylation probably via forming a stable complex with Hsp40 and P58^IPK, and consequently induce cell death. These results suggest that influenza virus M2 protein is involved in p58^IPK-mediated PKR regulation during influenza virus infection, therefore affecting infected-cell life cycle and virus replication.     

Convergence of EU nitrogen surplus,the RDP indicator of water quality

The EU Water Framework Directive (WFD), EU Nitrate Directive and EU Rural Development Policy (RDP) aim to improve water quality. The nutrient content of water can be decreased by reducing nitrogen emission. In this article a novel approach is applied to the evaluation of the impact of Agri Environmental Measures (AEM), which are part of axis 2 of the EU Rural Development Programme. The spending on AEM is linked to the reduction of nitrogen surplus, and hence, to the improvement of water quality. Reduction of nitrogen surplus is considered as a beta convergence process, in which the nitrogen surplus of EU member states converges to a steady state level. The convergence is tested, applying spatial econometrics on a panel data set of EU member states. The development over time of nitrogen surplus is explained applying the conditional beta convergence methodology. To allow for varying steady state nitrogen surpluses, structural variables are added to the analysis. RDP spending on AEM was added as structural variable to evaluate whether they affect the reduction of nitrogen surplus. The fixed effects panel data specification was tested to be the best model and preferred over spatial econometric specifications. A significantly negative effect is found between AEM expenditures and nitrogen surplus. Based on these estimation results it can be concluded that spending on AEM affects the convergence of nitrogen surplus towards a steady state level. A causal relationship cannot be tested with data on EU Member State level and additional analysis at smaller spatial level is warranted.     

4,4,4-三氟乙酰乙酸乙酯羰基还原酶产生菌的筛选及产酶条件研究

对实验室保藏菌种进行筛选,得到一株葡萄汁酵母Saccharomyces uvarum SW-58,对其产酶条件进行优化,其发酵培养基组成:醋酸钠60 g/L,玉米浆30 g/L,KH2PO46 g/L,MgSO4.7H2O 1 g/L;培养条件为:发酵温度30℃,初始pH 6.0,发酵周期36 h。4,4,4-三氟乙酰乙酸乙酯羰基还原酶酶活最高可达388.1 U/L,产物的浓度由优化前的3.5 g/L提高到4.6 g/L,所得产物的光学纯度由优化前的60.8%e.e.提高到85.0%e.e。     

Induction of aromaticl-amino acid decarboxylase mRNA by interleukin-1β and prostaglandin E2 in PC12 cells

Aromatic 1-amino acid decarboxylase (AADC) is involved in the synthesis of the putative neurotransmitters dopamine (DA), norepinephrine (NA) and 5-hydroxytryptamine (5-HT). We report here that the gene expression of AADC can be regulated by interleukin (IL) 1-58j1126/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"> and prostaglandin (PG) E₂ in PC12 cells. The cells were treated with different doses of IL 1-58j1126/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"> and PGE₂ for 3 days. Slot blot hybridization was performed to detect AADC mRNA and Western immunoblot to detect AADC protein. The cDNA probe for rat AADC was generated by the PCR method. IL 1-58j1126/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"> and PGE₂ produced a dose- and time-dependent up-regulation in AADC mRNA levels (up to 200% of the control values) which was followed by a stable increase in AADC protein. The data further support the suggestion that AADC is a regulated enzyme and that the regulation occurs at the level of gene expression. Because IL-1 is synthesized, and acts locally, within the brain to influence neuronal and glial functions, it has been proposed to be a mediator with both beneficial and detrimental responses to inflammation and injury. The regulation of AADC by IL-1 may indicate a possible involvement for AADC in neuronal injury and recovery. Since IL-1 promotes PGE₂ formation, its effects may be occurring by increasing level of PGE₂.Abbreviations AADC aromatic 1-amino acid decarboxylase - IL-1 interleukin 1 - PGE₂ prostaglandin E₂ - GITC guanidinium isothiocyanate - DEPC diethyl pyrocarbonate - MOPS 3-(4-morpholino)propanesulfonic acid - SSPE 0.18M NaCl, 0.001M sodium phosphate, and 0.001M EDTA Special issue dedicated to Dr. Bernard W. Agranoff.     

Selective viral transduction of adult-born olfactory neurons for chronic in vivo optogenetic stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.     

Caprine PrP variants harboring Asp-146, His-154 and Gln-211 alleles display reduced convertibility upon interaction with pathogenic murine prion protein in scrapie infected cells

Scrapie, the prion disease of sheep and goats, is a devastating malady of small ruminants. Due to its infectious nature, epidemic outbreaks may occur in flocks/herds consisting of highly susceptible animals. Field studies identified scrapie-protective caprine PrP variants, harboring specific single amino acid changes (Met-142, Arg-143, Asp-146, Ser-146, His-154, Gln-211 and Lys-222). Their effects are under further evaluation, and aim to determine the most protective allele. We assessed some of these variants (Asp-146, His-154, Gln-211 and Lys-222), after their exogenous expression as murine-caprine chimeras in a scrapie- infected murine cell line. We report that exogenously expressed PrPs undergo conformational conversion upon interaction with the endogenous pathological murine prion protein (PrP^SC), which results in the detection of goat-specific and partially PK-resistant moieties. These moieties display a PK-resistance pattern distinct from the one detected in natural goat scrapie cases. Within this cellular model, distinct conformational conversion potentials were assigned to the tested variants. Molecules carrying the Asp-146, His-154 and Gln-211 alleles showed significantly lower conversion levels compared to wild type, confirming their protective effects against scrapie. Although we utilized a heterologous conversion system, this is to our knowledge, the first study of caprine PrP variants in a cellular context of scrapie, that confirms the protective effects of some of the studied alleles.     

Transglycosylation of a β-galactosyl radical, in the course of enzymic hydrolysis of lactose, in the presence of selected polyhydroxyalcohols

Sorbitol, xylitol, erythritol and lactitol were used as the acceptors of galactosyl radicals, in the process of transgalactosylation accompanying the hydrolysis of lactose, conducted with 58w170036361g03/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-galactosidase (4.0 ml Lactozym 3000 was added to 430 ml 1.45 M lactose with 0.95 M polyhydroxyalcohols). The following concentrations of galactosyl derivatives of polyols were obtained after hydrolysis for 4 h at 4058w170036361g03/xxlarge8201.gif" alt="thinsp" align="MIDDLE" BORDER="0">°C: 0.31 M Gal-erythritol, 0.22 M Gal-xylitol, 0.18 M Gal-sorbitol and 0.14 M Gal-lactitol. A quadruple increase of xylitol content in dry matter of a solution (from 11.5% (w/w) to 44.5% (w/w) brought about a 2.3-fold increase of the product content in the solution (15.2% (w/w) of dry matter).     

Gene expression signature of human HepG2 cell line

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is associated with various clinico-pathological characteristics such as genetic mutations and viral infections. Therefore, numerous laboratories look out for identifying always new putative markers for the improvement of HCC diagnosis/prognosis. Many molecular profiling studies investigated gene expression changes related to HCC. HepG2 represents a pure cell line of human liver carcinoma, often used as HCC model due to the absence of viral infection. In this study we compare gene expression profiles associated with HepG2 (as HCC model) and normal hepatocyte cells by microarray technology. Hierarchical cluster analysis of genes evidenced that 2646 genes significantly down-regulated in HepG2 cells compared to hepatocytes whereas a further 3586 genes significantly up-regulated. By using the Ingenuity Pathway Analysis (IPA) program, we have classified the genes that were differently expressed and studied the functional networks correlating these genes in the complete human interactome. Moreover, to confirm the differentially expressed genes as well as the reliability of our microarray data, we performed a quantitative Real time RT-PCR analysis on 9 up-regulated and 11 down-regulated genes, respectively. In conclusion this work i) provides a gene signature of human hepatoma cells showing genes that change their expression as a consequence of liver cancer in the absence of any genetic mutations or viral infection, ii) evidences new differently expressed genes found in our signature compared to previous published studies and iii) suggests some genes on which to focus future studies to understand if they can be used to improve the HCC prognosis/diagnosis.     

过表达CDK11~(p58)促进人胰腺导管腺癌MIAPaCa-2细胞增殖

CDK11p58属于CDK11/PITSLRE蛋白激酶家族成员,由Cdc2L2编码,是一种重要的细胞周期调控蛋白.为了研究CDK11p58与胰腺癌细胞增殖的关系,我们通过采用脂质体转染真核表达载体及G418筛选的方式,获得了稳定过表达CDK11p58的MIAPaCa-2(人胰腺导管腺癌细胞)单克隆细胞,并通过流式细胞分析、MTT检测及real-time PCR的方法检测了细胞周期、细胞增殖能力及G1/S期相关调控基因的转录水平.结果显示,该单克隆细胞(实验组)与空载体组细胞和空白对照组细胞相比G1期细胞比例明显下降(P0.01),S期细胞比例明显上升(P0.01);细胞增殖能力明显提高(P0.01);cyclin D1、cyclin D3、p21基因mRNA水平较两组对照细胞明显升高(P0.01).提示过表达的CDK11p58通过上调cyclin D1、cyclin D3和p21基因的mRNA水平促进MIAPaCa-2细胞通过细胞增殖的关键限速点G1/S期,加快细胞增殖.