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This study was aimed at exploring the effects of P2X receptor on BV2 microglia cell injury induced by glycoprotein gp120 (gp120) and its underlying mechanisms. We used the MTS method to study the influence of different gp120 concentrations on BV2 microglia cells, and to test the degree of cell injury in each gp120 treatment group; quantitative real-time PCR (qPCR) and Western blot were used to detect the P2X mRNA and receptor protein expressions. Immunocytochemistry and Western blot were used to detect the P2X receptor expression and P65 NF-κB, respectively. We also measured the content of TNFα, IL-1β, nitric oxide (NO) and reactive oxygen species (ROS). We found that the cell survival rate generally decreased as gp120 concentration increased, and the cell survival rate of the gp120 + Brilliant Blue G (BBG) group was higher than that of the gp120 group. Western blot and qPCR results showed that the expressions of P2X receptor protein and mRNA were positively dose-dependent with gp120 concentration; the results of immunocytochemistry and Western blot showed that the expressions of P2X receptor and P65 NF-κB in the gp120 group increased significantly compared to those of the control (Ctrl) group, but those in the gp120+BBG group decreased. Taken together, these results confirmed that the P2X receptor is involved in gp120-induced BV2 microglial cell injury and that the underlying mechanism may be associated with the over-activation of microglia caused by P2X receptor up-regulation, which leads to abundant release of inflammatory factors which exert toxic effects on the cells. [ABSTRACT FROM AUTHOR]

Purpose: To study the molecular mechanisms of colorectal cancer liver metastasis. Methods: Cecal wall implantation was performed in nude mice to subclone a highly liver metastatic human colorectal cancer clone (SW1116-M) from SW1116. In vivo and in vitro assays were adopted to confirm the proliferation and metastasis potential. The human tumor metastasis PCR microarrays were used to analyze the differential gene expressions. The results were confirmed further by real-time quantitative PCR. Results: SW1116-M and SW1116-S5, two human colon cancer cell clones with different metastatic potential, were subcloned from SW1116. In SW1116-M, in vitro invasion, migration and in vivo metastatic potential were higher, and in vitro proliferation rate was lower than SW1116-S5. In tumor metastasis PCR microarray, 24 genes related to cell invading, adhesion, cellular growth and differentiation were found with a twofold difference between SW1116-S5 and SW1116-M. Sixteen of these, including E-cadherins, MTSS1, TRAIL and TRPM1, were up-regulated; eight genes including cathepsin L, EphB2, HGF, MET, MCAM and RORβ were down-regulated. Conclusions: We have established a highly liver metastatic clone. The subsequent metastasis PCR microarray analysis identified a procedure of cellular differentiation and mesenchymal to epithelial transition (MET) in liver metastasis. The colonization to from macrometastasis is not a switch from cell cycle arrest but a result of cell differentiation and MET. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine produced by activated macrophages and lymphocytes and involved in many inflammatory diseases. Preventing the production or action of TNF-α is a potent therapeutic strategy for these inflammatory diseases. Since there is a lack of rapid and effective assay for examining the expression TNF-α in macrophages, we attempt to establish a reporter system to assess TNF-α gene expression through measuring luciferase activity. In this study, mouse macrophage cell line RAW 264.7 was stably transfected with a luciferase reporter pGL3-TNFPro-UTR, which contains TNF-α promoter and 3′-untranslated region (3′-UTR). The TNF-α-luciferase reporter cell line is used for assessing the expression of TNF-α gene induced by LPS in the presence or absence of chemicals that inhibit the biosynthesis of TNF-α such as dexamethasone and emodin, and also for measuring change of expression of TNF-α gene under downregulation of the expression of steroid receptor coactivator-3, a modulator for TNF-α. The luciferase activity correlated well with the ELISA results for TNF-α production, therefore, the TNF-α-luciferase reporter cell line is a sensitive, effective tool for studying the expression of TNF-α gene. [ABSTRACT FROM AUTHOR]

Necrosis caused by soybean mosaic virus (SMV) has not been specifically distinguished from susceptible symptoms. The molecular mechanism for the occurrence of necrosis is largely overlooked in soybean genetic research. Field evaluation reveals that SMV disease seriously influences soybean production as indicated by decreasing 22.4% ~ 77.0% and 8.8% ~ 17.0% of yield and quality production, respectively. To expand molecular mechanism behind necrotic reactions, transcriptomic data obtained from the asymptomatic, mosaic, and necrotic pools were assessed. Compared between asymptomatic and mosaic plants, 1689 and 1752 up- and down-regulated differentially expressed genes (DEGs) were specifically found in necrotic plants. Interestingly, the top five enriched pathways with up-regulated DEGs were highly related to the process of the stress response, whereas the top three enriched pathways with down-regulated DEGs were highly related to the process of photosynthesis, demonstrating that defense systems are extensively activated, while the photosynthesis systems were severely destroyed. Further, results of the phylogenetic tree based on gene expression pattern and an amino acid sequence and validation experiments discovered three PR1 genes, Glyma.15G062400, Glyma.15G062500, and Glyma.15G062700, which were especially expressed in necrotic leaves. Meanwhile, exogenous salicylic acid (SA) but not methyl jasmonate (MeJA) could induce the three PR1 gene expressions on healthy leaves. Contrastingly, exogenous SA obviously decreased the expression level of Glyma.15G062400, Glyma.15G062500, and concentration of SMV, but increased Glyma.15G062700 expression in necrotic leaves. These results showed that GmPR1 is associated with the development of SMV-induced necrotic symptoms in soybean. Glyma.15G062400, Glyma.15G062500, and Glyma.15G062700 is up-regulated in necrotic leaves at the transcriptional levels, which will greatly facilitate a better understanding of the mechanism behind necrosis caused by SMV disease. [ABSTRACT FROM AUTHOR]

Lentinan is a Lentinus edodes secondary metabolite that can regulate human immune function, but yields are low. Here, the effects of Ca2+ and Na+ on L. edodes lentinan content were investigated. Metal ion concentrations and induction times were optimized according to mycelial biomass, and intracellular polysaccharide (IPS), extracellular polysaccharide (EPS), and total polysaccharide (TPS) content. The activities and gene expression of phospho-glucose isomerase (PGI), phosphoglucomutase (PGM), and UDP-glcpyrophosphorylase (UGP) were also measured. Ca2+ and Na+ concentration and induction time affected biomass, IPS, and EPS concentrations. Na+ increased EPS, IPS and TPS, while Ca2+ increased biomass, IPS, and TPS. During fermentation, mycelial biomass varied greatly under Ca2+ induction, while IPS, EPS and TPS varied greatly under Na+ induction. PGM and UGP activities increased in the presence of Na+, while PGI increased with Ca2+. Compared to control samples, pgi and pgm expression under Na+ was greater at days 45 and 60, respectively, while under Ca2+, ugp expression was greater at day 45. IPS content correlated significantly with enzyme activity, while EPS correlated with PGM activity. Our data contributes to better understanding how Na+ and Ca2+ affect mycelial growth and secondary metabolite production, and of polysaccharide biosynthesis mechanisms of L. edodes. [ABSTRACT FROM AUTHOR]

This study was designed to detect the expression, detergent resistance, subcellular localization, and channel and hemichannel functions of mutant Cx50 to understand the forming mechanism for inducing congenital cataract by a novel mutation p.S276F in connexin 50 (Cx50) reported previously by us. HeLa and human lens epithelial (HLE) cells were transfected with wild-type Cx50 and mutant Cx50 (S276F). We examined the functional characteristics of mutant Cx50 (S276F) in comparison with those of wild-type Cx50 using immunoblot, confocal fluorescence microscopy, dye transfer analysis and dye uptake assay. The mutant and wild-type Cx50 were expressed in equal levels and could efficiently localize to the plasma membrane without transportation and assembly problems. Scrape loading dye transfer was significantly evident in cells transfected with wild-type Cx50 compared to those in cells transfected with mutant Cx50 and cotransfected with wild-type and mutant Cx50. The dye uptake was found to be significantly lower in cells transfected with mutant Cx50 than in cells transfected with wild-type Cx50 and cells cotransfected with wild-type and mutant Cx50. The transfected HeLa and HLE cell lines showed similar performance in all the experiments. These results indicated that the mutant Cx50 (S276F) might inhibit the function of gap junction channel in a dominant negative manner, but inhibit the hemichannel function in a recessive negative manner. [ABSTRACT FROM AUTHOR]