Then, the H2O2 concentration and catalase activity variation in the cells were studied in exponential and stationary phases at various ODs and were compared to those of wild Geneticin type cells and empty vector transformed cells. It was revealed that empty vector introduction and periplasmic recombinant protein expression increased significantly the H2O2 concentration
of the cells. However, the H2O2 concentration in mIL-4 expressing cells was significantly higher than its concentration in empty vector transformed cells, demonstrating more effects of recombinant mIL-4 expression on H2O2 elevation. Likewise, although catalase activity was reduced in foreign DNA introduced cells, it was more lowered following expression of recombinant proteins. Correlation between H2O2 concentration elevation and catalase activity reduction with cell growth depletion is also demonstrated. It was also found that recombinant protein expression results in cell size increase. (C) 2012 Elsevier B.V. All rights reserved.”
“Transforming growth factor (TGF)-beta is a key mediator of proliferative vitreoretinopathy, but the cellular mechanisms
by which TGF-beta induces extracellular matrix protein (ECM) synthesis are not fully understood. This study examined whether the PI3K/Akt pathway is involved in TGF-beta 2-induced collagen expression in human retinal pigment epithelial cells.\n\nHuman Blebbistatin nmr retinal pigment epithelial cells ARPE-19 were cultured and stimulated with TGF-beta 2. The role of the PI3K/Akt pathway was evaluated using the biochemical inhibitor, wortmannin. The effect of wortmannin on the expression of type I collagen mRNA (COL1A1, COL1A2) induced by TGF-beta 2 was evaluated by real-time RT-PCR. The effect of wortmannin on the synthesis of type I collagen induced by TGF-beta 2 was assessed by an immunocytochemical analysis with anti-type I collagen antibody. Luciferase
find more reporter assays were performed to examine the effect of wortmannin on the transcriptional activities of COL1A2. A luciferase assay using a mutation construct of the Smad binding site in COL1A2 promoter (Smad-mut/Luc) was also performed to examine the crosstalk between the Smad pathway and the PI3K/Akt pathway. The effects of wortmannin on the transcriptional activity of Smad3 were also examined using CAGA12-Luc. Moreover, the effect of wortmannin on TGF-beta 2-induced Smad7 mRNA expression was evaluated.\n\nThe biochemical blockade of PI3K/Akt activation inhibited TGF-beta 2-induced type I collagen mRNA expression and type I collagen synthesis. The blockade of PI3K/Akt pathway inhibited the increase in COL1A2 promoter activities when induced by TGF-beta 2 and reduced TGF-beta 2 induction of Smad-mut/Luc promoter activity and CAGA12-Luc activity. Moreover, wortmannin increased the TGF-beta 2-induced Smad7 mRNA expression levels.