Angiotensin II Regulates Th1 T Cell Differentiation Through Angiotensin II Type 1 Receptor-PKA-Mediated Activation of Proteasome.

BACKGROUND/AIMS: Naive CD4+ T cells differentiate into T helper cells (Th1 and Th2) that play an essential role in the cardiovascular diseases. However, the molecular mechanism by which angiotensin II (Ang II) promotes Th1 differentiation remains unclear. The aim of this study was to determine whether the Ang II-induced Th1 differentiation regulated by ubiquitin-proteasome system (UPS). METHODS: Jurkat cells were treated with Ang II (100 nM) in the presence or absence of different inhibitors. The gene mRNA levels were detected by real-time quantitative PCR analysis. The protein levels were measured by ELISA assay or Western blot analysis, respectively. RESULTS: Ang II treatment significantly induced a shift from Th0 to Th1 cell differentiation, which was markedly blocked by angiotensin II type 1 receptor (AT1R) inhibitor Losartan (LST). Moreover, Ang II significantly increased the activities and the expression of proteasome catalytic subunits (ß1, ß1i, ß2i and ß5i) in a dose- and time-dependent manner. However, Ang II-induced proteasome activities were remarkably abrogated by LST and PKA inhibitor H-89. Mechanistically, Ang II-induced Th1 differentiation was at least in part through proteasome-mediated degradation of IκBα and MKP-1 and activation of STAT1 and NF-κB. CONCLUSIONS: This study for the first time demonstrates that Ang II activates AT1R-PKA-proteasome pathway, which promotes degradation of IκBα and MKP-1 and activation of STAT1 and NF-κB thereby leading to Th1 differentiation. Thus, inhibition of proteasome activation might be a potential therapeutic target for Th1-mediated diseases.

Difference in the Inhibitory Effect of Temozolomide on TJ905 Glioma Cells and Stem Cells.

This study aims to determine the difference in the inhibitory effect of temozolomide (TMZ) on TJ905 glioma cells and stem cells. TJ905 cancer stem cells were isolated. Livin is a member of the inhibitor of apoptosis protein family. The TJ905 cells and cancer stem cells were transfected with a Livin-shRNA and negative-shRNA, respectively, and then treated with TMZ. At 48 h post-transfection, a cell counting kit 8 assay, flow cytometry, and real-time qPCR were performed to detect cell proliferation, the cell cycle, and the expression of the Caspase-3, -7, and -9 mRNAs, respectively. As a result, the suppressive effect of TMZ on TJ905 cells was more significant than its effect on TJ905 cancer stem cells. TMZ exerted an inhibitory effect on the growth of TJ905 glioma cells by arresting them at G0/G1 phase and arresting cancer stem cells at S phase in a dose-dependent manner. TMZ inhibited Livin mRNA expression and increased the expression of the Caspase-3, -7, and -9 mRNAs. Low Livin mRNA expression induced high levels of Caspase-3, -7, and -9 expressions, thus promoting the apoptosis of both TJ905 cells and cancer stem cells in response to TMZ treatment. The TJ905 cells transfected with the Livin-shRNA were more sensitive to TMZ, whereas the TJ905 glioma stem cells transfected with the Livin-shRNA showed no significant changes in their sensitivity to TMZ. In conclusion, the Livin gene may play an important role in the resistance mechanisms of TJ905 glioma cells and cancer stem cells. However, Livin had a more distinct role in TMZ resistance, cell proliferation, and the cell cycle in TJ905 glioma cells than in cancer stem cells.

Effect of Corilagin on the Proliferation and NF-κB in U251 Glioblastoma Cells and U251 Glioblastoma Stem-Like Cells.

Background. This study is to explore the effect of corilagin on the proliferation and NF-κB signaling pathway in U251 glioblastoma cells and U251 glioblastoma stem-like cells. Methods. CD133 positive U251 glioblastoma cells were separated by immunomagnetic beads to isolate glioblastoma stem-like cells. U251 cells and stem-like cells were intervened by different corilagin concentrations (0, 25, 50, and 100 µg/mL) for 48 h, respectively. Cell morphology, cell counting kit-8 assay, flow cytometry, dual luciferase reporter assay, and a western blot were used to detect and analyze the cell proliferation and cell cycle and investigate the expression of IKBα protein in cytoplasm and NF-κB/p65 in nucleus. Results. Corilagin inhibited the cell proliferation of U251 cells and their stem-like cells and the inhibition role was stronger in U251 stem-like cells (P < 0.05). The cell cycle was arrested at G2/M phase in the U251 cells following corilagin intervention; the proportion of cells in G2/M phase increased as the concentration of corilagin increased (P < 0.05). The U251 stem-like cells were arrested at the S phase following treatment with corilagin; the proportion of cells in the S phase increased as the concentration of corilagin increased (P < 0.05). The ratio of dual luciferase activities of U251 stem-like cells was lower than that of U251 cells in the same corilagin concentration. With increasing concentrations of corilagin, the IKBα expression in cytoplasm of U251 cells and U251 stem-like cells was increased, but the p65 expression in nucleus of U251 cells and U251 stem-like cells was decreased (P < 0.05). Conclusion. Corilagin can inhibit the proliferation of glioblastoma cells and glioblastoma stem-like cells; the inhibition on glioblastoma stem-like cell proliferation is stronger than glioblastoma cells. This different result indicates that the effect of corilagin on U251 cells and U251 stem-like cells may have close relationships with mechanism of cell cycle and NF-κB signaling pathway; however, the real antitumor mechanism of corilagin is not yet clear and requires further study.

[Advances in functional studies of nonstructural proteins and development of antiviral agents for enterovirus 71].

Human enterovirus 71 (EV71) is one of the major etiological agents for the hand, foot, and month disease (HFMD) and is causing frequent, widespread occurrence in the mainland of China. The single positive-stranded RNA genome of EV71 is translated into a single polyprotein which is autocleavaged into structural and nonstructural proteins. The functions of many nonstructural proteins characterized in the life cycle of virus are potential targets for blocking viral replication. This article reviews the studies of the structures and functions of nonstructural proteins of EV71 and the anti-enterovirus 71 drugs targeting on these nonstructural proteins.

[Elucidating the structure of two cyclotides of Viola tianshanica maxim by MALDI TOF/TOF MS analysis].

The cyclotides are a family of cyclic "mini" proteins that occur in Violaceae, Rubiaceae and Cucurbitaceae plant families and contain a head-to-tail cyclic backbone and a cystine knot arranged by three disulfide bonds. To study the natural cyclotides of V tianshanica, dried herb was extracted with 50% ethanol, and the concentrated aqueous extract was subjected to a solvent-solvent partitioning between water and hexane, ethyl acetate and n-butanol, separately. The n-butanol extract containing cyclotides was subjected to column chromatography over Sephadex LH-20, eluted with 30% methanol. The subfractions were directly reduced by DTT and analyzed by reverse-phase HPLC. The peaks with different retention times were shown on the profile of RP-HPLC and collected. The cyclotides were speculated based on masses range from 3 000 to 3 500 Da. The purified cyclotides were reduced with DTT, alkylated with iodoacetamide, and then were cleaved with endoproteinase Glu-C, endoproteinase Lys-C and Trypsin, separately. The digested peptides were purified on RP-HPLC and analyzed on MALDI TOF/TOF analyzer. A new cyclotide, cycloviolacin T1 and a reported cyclotide varv E were systemically determined using MALDI TOF/TOF system. So the method for the isolation and characterization of cyclotides was quickly built up in succession.

[cDNA cloning, heterologous overexpression and activity analysis of cytochrome P450 reductase of Taxus chinensis].

NADPH-cytochrome P450 reductase (CPR), a partner for P450 monooxygenases, serves as the electron donor to almost all eukaryotic cytochrome P450s. One cDNA (TchCPR) encoding cytochrome P450 reductase of T. chinensis was isolated from callus cells. The cDNA contains an open reading frame of 2154 nucleotides which encodes a protein of 717 amino acid residues. The TchCPR has higher similarity to other CPRs of gumnosperms (>82%) than that of angiosperms (<74%). The recombinant full-length TchCPR and a series of N-terminal truncated constructs with N-terminal fusion of His Tag were obtained and induced to express in E. coli B121(DE3), and then purified using affinity chromatography. The truncated forms of N-terminal more than 61 amino acid residues could be efficiently expressed while the truncated mutant of N-terminal 48 amino acid residues and the wild-type TchCPR were not successfully expressed in E. coli cells. The activity of the truncated TchCPR was assayed by measuring the reduction of cytochrome C. The electron transfer activity of the recombinantly purified CPRT61 was 1.6057 nmol of cytochrome C reduced per min per microg TchCPR reductase, and it is higher than that of the other four truncated forms.