New Findings r What is the central question of this study?Oxidative stress is known to play an important role in the development and progression of diabetic nephropathy. However, the mechanism of overproduction of reactive oxygen species (ROS) in high-glucose conditions is not completely clear. r What is the main finding and its importance?We demonstrated that high-glucose concentrations induced excessive production of ROS and stimulated the phosphoinositide 3-kinase-Akt-FoxO3a signalling pathway via the upregulation of transforming growth factor-β1, resulting in phosphorylation and inactivation of FoxO3a and a reduction in the expression of its target gene, FoxO3a-dependent manganese superoxide dismutase, and then further excessive production of ROS.Oxidative stress has been shown to play an important role in the development and progression of diabetic nephropathy, and the formation of reactive oxygen species (ROS) is a direct consequence of hyperglycaemia. We hypothesized that hyperglycaemia-induced ROS can activate the transforming growth factor-β1 (TGF-β1)-phosphoinositide 3-kinase (PI3K)-AktFoxO3a signalling pathway, negatively regulating expression of manganese superoxide dismutase (MnSOD), which promotes excessive ROS generation and accelerates the pathological process of diabetic nephropathy. In vitro, in rat mesangial cells, high glucose (30 mmol l −1 ), but not equimolar mannitol, stimulated ROS production, upregulated the levels of TGF-β1, increased the phosphorylated Akt/total Akt and phosphorylated FoxO3a/total FoxO3a protein ratios, altered the subcellular localization of FoxO3a and reduced the levels of MnSOD expression. These highglucose-induced changes further promoted the generation of ROS. In vivo, in db/db mice treated with an inhibitor of TGF-β1 (SB431542) or PI3K (LY294002), the levels of phosphorylated Akt and phosphorylated FoxO3a in the kidney cortices were decreased, the level of MnSOD expression was increased and the level of the lipid peroxidation end-product, malondialdehyde, was reduced. We conclude that overproduction of ROS induced by a high glucose concentration decreases the expression of MnSOD via the PI3K-Akt-FoxO3a pathway and further aggravates oxidative stress in diabetic nephropathy.Q.L. and Y.Z. contributed equally to this work.

No abstract

Aim: To investigate whether NO over-production in rat mesangial cells cultured in high glucose (HG) is related to activation of the TGF-β1/PI3K/Akt pathway. Methods: Rat mesangial cells line (HBZY-1) was exposed to HG (24.44 mmol/L) or H 2 O 2 (10 μmol/L) for 16 h. NO release was quantified using the Griess assay. The TGF-β1 level was measured using ELISA. The protein expression of p-Akt, t-Akt, Bim, and iNOS was examined by Western blotting. The mRNA levels of TGF-β1 and Bim were measured using RT-PCR. The cell proliferation rate was estimated using a BrdU incorporation assay. Results: Treatment of the cells with HG, H 2 O 2 , or TGF-β1 (5 ng/mL) significantly increased the NO level that was substantially inhibited by co-treatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI), TGF-β1 inhibitor SB431542, or PI3K inhibitor LY294002. Both HG and H 2 O 2 significantly increased the protein and mRNA levels of TGF-β1 in the cells, and HG-induced increases of TGF-β1 protein and mRNA were blocked by co-treatment with DPI. Furthermore, the treatment with HG or H 2 O 2 significantly increased the expression of phosphorylated Akt and iNOS and cell proliferation rate, which was blocked by co-treatment with DPI, SB431542, or LY294002. Moreover, the treatment with HG or H 2 O 2 significantly inhibited Bim protein and mRNA expression, which was reversed by co-treatment with DPI, SB431542, or LY294002. Conclusion:The results demonstrate that high glucose causes oxidative stress and NO over-production in rat mesangial cells in vitro via decreasing Bim and increasing iNOS, which are at least partially mediated by the TGF-β1/PI3K/Akt pathway.

Adenylyl Cyclase 3 (AC3) plays an important role in the olfactory sensation-signaling pathway in mice. AC3 deficiency leads to defects in olfaction. However, it is still unknown whether AC3 deficiency affects gene expression or olfactory signal transduction pathways within the main olfactory epithelium (MOE). In this study, gene microarrays were used to screen differentially expressed genes in MOE from AC3 knockout (AC3−/−) and wild-type (AC3+/+) mice. The differentially expressed genes identified were subjected to bioinformatic analysis and verified by qRT-PCR. Gene expression in the MOE from AC3−/− mice was significantly altered, compared to AC3+/+ mice. Of the 41266 gene probes, 3379 had greater than 2-fold fold change in expression levels between AC3−/− and AC3+/+ mice, accounting for 8% of the total gene probes. Of these genes, 1391 were up regulated, and 1988 were down regulated, including 425 olfactory receptor genes, 99 genes that are specifically expressed in the immature olfactory neurons, 305 genes that are specifically expressed in the mature olfactory neurons, and 155 genes that are involved in epigenetic regulation. Quantitative RT-PCR verification of the differentially expressed epigenetic regulation related genes, olfactory receptors, ion transporter related genes, neuron development and differentiation related genes, lipid metabolism and membrane protein transport etc. related genes showed that P75NTR, Hinfp, Gadd45b, and Tet3 were significantly up-regulated, while Olfr370, Olfr1414, Olfr1208, Golf, Faim2, Tsg101, Mapk10, Actl6b, H2BE, ATF5, Kirrrel2, OMP, Drd2 etc. were significantly down-regulated. In summary, AC3 may play a role in proximal olfactory signaling and play a role in the regulation of differentially expressed genes in mouse MOE.

Bacterial glycoproteins have been investigated as vaccine candidates as well as diagnostic biomarkers. However, they are poorly understood in Mycobacterium bovis strain bacille Calmette-Guérin (BCG), a non-pathogenic model of Mycobacterium tuberculosis. To understand the roles of secreted O-mannosylated glycoproteins in BCG, we conducted a ConA lectin-affinity chromatography and mass spectra analysis to identify O-mannosylated proteins in BCG culture filtrate. Subsequent screening of antigens was performed using polyclonal antibodies obtained from a BCG-immunized mouse, with 15 endogenous O-mannosylated proteins eventually identified. Of these, BCG_0470 and BCG_0980 (PstS3) were revealed as the immunodominant antigens. To examine the protective effects of the antigens, recombinant antigens proteins were first expressed in Mycobacterium smegmatis and Escherichia coli, with the purified proteins then used to boost BCG primed-mice. Overall, the treated mice showed a greater delayed-type hypersensitivity response in vivo, as well as stronger Th1 responses, including higher level of IFN-7, TNF-α, and specific-IgG. Therefore, mannosylated proteins BCG_0470 and BCG_0980 effectively amplified the immune responses induced by BCG in mice. Together, our results suggest that the oligosaccharide chains containing mannose are the antigenic determinants of glycoproteins, providing key insight for future vaccine optimization and design.