Resveratrol up-regulates SOD2 and suppresses antimycin A-induced ROS via FOXOs.

<p>(A) C2C12 cells were incubated in the presence or absence of RSV (100 µM) for 6 hours followed by western blotting to analyze the SOD2 protein level. (B) Representative images for SOD2 immunostaining in cultured neonatal rat ventricular myocytes (upper) and quantified data (lower). Cells were incubated in the presence of vehicle (Ctrl), resveratrol (RSV), or nicotinamide (NA) for 48 hours. Scale bar: 10 µm. (C) RT-PCR analyses for FOXO transcription factors in C2C12 cells transfected with negative-control siRNA (Ctrl), or siRNA against FOXO1 (F1), FOXO3a (F3a), or FOXO4 (F4). (D) Quantitative analysis of SOD2 mRNA by real-time RT-PCR in C2C12 cells (N = 6). After being transfected with siRNAs against negative-control (Ctrl), FOXO1 (F1), FOXO3a (F3a), or FOXO4 (F4), the cells were pretreated with resveratrol (RSV, 30 µM) for 3 hours, then incubated with antimycin A (AA, 50 µM) for 24 hours. (E) Immunoblot analysis for SOD2. C2C12 cells transfected with control-siRNA (Ctrl-siRNA) or siRNAs against all three FOXOs (FOXO1, FOXO3a, FOXO4) were preincubated with vehicle or RSV (30 µM) for 3 hours followed by treatment with AA (50 µM) without serum for 24 hours. Total-cell lysates were subjected to immunoblot analysis. (F) Representative images of CM-H₂DCFDA-stained C2C12 cells. Cells transfected with control-siRNA or a mixture of all three FOXO siRNAs (FOXOs-siRNA) were treated with AA (200 µM) for 24 hours after pretreatment with vehicle or RSV (30 µM) for 3 hours. Scale bar: 10 µm. (G) Quantification of CM-H₂DCFDA (DCF) fluorescence. Data are from three independent experiments. (H) Immunoblots for acetyl-FOXO1 and FOXO1 in C2C12 cells. Cells were treated with vehicle, antimycin A (AA, 60 µM), or RSV (30 µM)+AA (left panel), or were incubated with vehicle or nicotinamide (NA, 20 mM) in the presence of trichostatin A (TSA) (right panel). (I) Representative immunoblots for FOXO3a and FOXO4 in C2C12 cells treated with vehicle or AA with or without RSV. ***p<0.001, **p<0.01. n.s. = not significant. a.u. = arbitrary units.</p

FOXOs mediate the anti-apoptotic effect of resveratrol.

<p>(A) (Left) Representative images of nuclear staining with Hoechst33342 in C2C12 cells. Cells transfected with control-siRNA (Ctrl-siRNA) or a mixture of siRNAs against all three FOXOs (FOXO1, FOXO3a, FOXO4) (FOXOs-siRNA) were pretreated with vehicle or resveratrol (RSV, 30 µM) for 3 hours and then incubated with antimycin A (AA, 50 µM) for 24 hours under serum-free conditions. Scale bar: 50 µm. (Right) Quantification of apoptotic cells defined by nuclear condensation. Data are from three independent experiments. (B) (Left) Representative images of immunostaining for BAX (green) in C2C12 cells treated as in A. Scale bar: 10 µm. (Right) Quantification of cells with BAX accumulation. Data are from three independent experiments. (C) Representative immunofluorescence images for acetylated p53 (red) in C2C12 cells treated as in A. The acetylation of p53 was detected by immunostaining using an antibody against acetyl-p53. Data are from three independent experiments. Scale bar: 20 µm. ***p<0.001, **p<0.01, n.s. = not significant.</p

Effects of p53 knockdown on the functions of SIRT1 modulators.

<p>(A) RT-PCR analysis for p53 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in C2C12 cells transfected with siRNA against negative control (Ctrl) or p53. (B) C2C12 cells transfected with control- (Ctrl-si) or p53-siRNA (p53-si) were treated with vehicle or AA (50 µM) for 24 hours. Apoptotic cells with condensed nuclei were quantified. Data are from three independent experiments. (C and D) The percentage of nuclear-condensed C2C12 cells was analyzed. C2C12 cells transfected with control- (Ctrl-si) or p53-siRNA (p53-si) were pretreated with vehicle or either 60 µM splitomicin (SP in C) or 10 µM RSV in D for 3 hours followed by incubation with 50 µM antimycin A (AA) for 24 hours. Data are from three independent experiments. (E) RT-PCR analysis for p53 mRNA in wild type (p53+/+) or p53-deficient (p53−/−) HCT116 cells. (F) Quantification of apoptotic cells with nuclear condensation in wild-type (p53+/+) or p53-null (p53−/−) HCT116 cells treated with vehicle or AA for 24 hours. Data are from three independent experiments. (G and H) Analysis of apoptosis in HCT116 cells treated with SIRT1 modulators. Wild-type (p53+/+) or p53-deficient (p53−/−) HCT116 cells were pretreated with 60 µM SP (G) or 10 µM RSV (H) for 3 hours and then incubated with 50 µM AA for 24 hours. Data are from three independent experiments. ***p<0.001, **p<0.01, *p<0.05. (I) Representative immunoblots of acetyl-p53 (K379) and p53 in C2C12 cells treated with vehicle or antimycin A (AA, 50 µM) with or without pretreatment with resveratrol (RSV) or Ex527. All cells were treated in the presence of 50 nM of trichostatin A. (J) Representative images of immunostaining for acetyl-p53 (Lys379) in C2C12 cells treated as in A. Scale bar: 20 µm.</p

The effect of resveratrol on AMPK activity in C2C12 cells.

<p>(A) Representative immunoblots of phospho-AMPK (Thr172), total AMPK, phospho-acetyl-CoA carboxylase (ACC) (Ser79), total ACC, and GAPDH in C2C12 cells treated with vehicle or 60 µM antimycin A (AA) with or without 30 µM resveratrol (RSV). (B) Quantification of phospho-AMPK (Thr172) level normalized to GAPDH (N = 5). (C) Quantification of phospho-ACC (Ser79) level normalized to GAPDH (N = 5). n.s. = not significant.</p

Effects of SIRT1 knockdown on functions of SIRT1 modulators.

<p>(A) Immunoblot analysis for SIRT1 in C2C12 cells transfected with control (Ctrl)- or SIRT1-siRNA. (B) Representative images of CM-H₂DCFDA fluorescence. C2C12 cells transfected with control- (Ctrl-) or SIRT1-siRNA were treated with antimycin A (AA) (100 µM) for 3 hours after pretreatment with vehicle or resveratrol (RSV, 10 µM) for 6 hours. Scale bar: 10 µm. (C) Quantification of CM-H₂DCFDA (DCF) fluorescence. (D and E) Quantification of cells with condensed nuclei. C2C12 cells transfected with control- (Ctrl-) or SIRT1-siRNA were treated with AA (200 µM) for 24 hours after pretreatment with vehicle (Ctrl), 10 µM RSV, or 60 µM splitomicin (SP) for 3 hours. Data are from three independent experiments. **p<0.01, *p<0.05. a.u. = arbitrary unit. n.s. = not significant.</p

Effects of SIRT1 modulators on H₂O₂-induced ROS generation and apoptosis.

<p>C2C12 cells were treated with 50 µM H₂O₂ for 24 hours after their pretreatment with vehicle (Ctrl), 5 mM nicotinamide (NA), 60 µM splitomicin (SP), 10 µM resveratrol (RSV), or 1 mM NAD⁺ for 3 hours. (A) Intracellular levels of reactive oxygen species (ROS) were detected by CM-H₂DCFDA (DCF). Data are from three independent experiments. (B and C) Cell death was analyzed by nuclear condensation (B), or by immunostaining for cleaved (active) caspase-3 (C). Data in each panel are from three independent experiments. **p<0.01, *p<0.05. a.u. = arbitrary unit.</p

RNase T1 mapping of IRES domain III–IV in the presence of aptamer 3-07

<p><b>Copyright information:</b></p><p>Taken from "A hepatitis C virus (HCV) internal ribosome entry site (IRES) domain III–IV-targeted aptamer inhibits translation by binding to an apical loop of domain IIId"</p><p>Nucleic Acids Research 2005;33(2):683-692.</p><p>Published online 28 Jan 2005</p><p>PMCID:PMC548359.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> 5′ end-labeled IRES RNA was digested with RNase U2, RNase T1 and an alkaline condition, respectively, were used as markers (lanes 2, 3 and 4). Cleavage reactions for markers were carried out under optimum buffer conditions for respective RNases. RNase T1 digestion with selection buffer was performed on 5′ end-labeled IRES (lane 5), the aptamer indicated (lanes 6 and 9) or mutants of aptamer 3-07 (lanes 7 and 8). Reactions were stopped and run on an 8% denaturing PAGE. Asterisks represent G triplets of domain IIId. Note that cleavage of G triplets was only protected in the presence of aptamer 3-07

Inhibition analysis of IRES-mediated translation by aptamers

<p><b>Copyright information:</b></p><p>Taken from "A hepatitis C virus (HCV) internal ribosome entry site (IRES) domain III–IV-targeted aptamer inhibits translation by binding to an apical loop of domain IIId"</p><p>Nucleic Acids Research 2005;33(2):683-692.</p><p>Published online 28 Jan 2005</p><p>PMCID:PMC548359.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Luciferase activity in the absence of an aptamer was used as a control (100%). Values represent results from at least three independent experiments. , and are equivalent to the groups in

Accelerated Recovery of Mitochondrial Membrane Potential by GSK-3β Inactivation Affords Cardiomyocytes Protection from Oxidant-Induced Necrosis

<div><p>Loss of mitochondrial membrane potential (ΔΨ_m) is known to be closely linked to cell death by various insults. However, whether acceleration of the ΔΨ_m recovery process prevents cell necrosis remains unclear. Here we examined the hypothesis that facilitated recovery of ΔΨ_m contributes to cytoprotection afforded by activation of the mitochondrial ATP-sensitive K⁺ (mK_ATP) channel or inactivation of glycogen synthase kinase-3β (GSK-3β). ΔΨ_m of H9c2 cells was determined by tetramethylrhodamine ethyl ester (TMRE) before or after 1-h exposure to antimycin A (AA), an inducer of reactive oxygen species (ROS) production at complex III. Opening of the mitochondrial permeability transition pore (mPTP) was determined by mitochondrial loading of calcein. AA reduced ΔΨ_m to 15±1% of the baseline and induced calcein leak from mitochondria. ΔΨ_m was recovered to 51±3% of the baseline and calcein-loadable mitochondria was 6±1% of the control at 1 h after washout of AA. mK_ATP channel openers improved the ΔΨ_m recovery and mitochondrial calcein to 73±2% and 30±7%, respectively, without change in ΔΨ_m during AA treatment. Activation of the mK_ATP channel induced inhibitory phosphorylation of GSK-3β and suppressed ROS production, LDH release and apoptosis after AA washout. Knockdown of GSK-3β and pharmacological inhibition of GSK-3β mimicked the effects of mK_ATP channel activation. ROS scavengers administered at the time of AA removal also improved recovery of ΔΨ_m. These results indicate that inactivation of GSK-3β directly or indirectly by mK_ATP channel activation facilitates recovery of ΔΨ_m by suppressing ROS production and mPTP opening, leading to cytoprotection from oxidant stress-induced cell death.</p></div

IgA Nephropathy Caused by Unusual Polymerization of IgA1 with Aberrant N-Glycosylation in a Patient with Monoclonal Immunoglobulin Deposition Disease

<div><p>Immunoglobulin A nephropathy (IgAN) is a form of chronic glomerulonephritis characterized by the deposition of IgA immune complexes in the glomerular region. The cause of IgAN is unknown, but multiple mechanisms have been suggested. We previously reported a rare case of mesangioproliferative glomerulonephritis in a patient with monoclonal immunoglobulin deposition disease associated with monoclonal IgA1. In this study, we performed the detailed analyses of serum IgA1 from this patient in comparison with those from patients with mIgA plasma cell disorder without renal involvement and healthy volunteers. We found unusual polymerization of IgA1 with additional <i>N</i>-glycosylation distinctive in this patient, which was different from known etiologies. Glycan profiling of IgA1 by the lectin microarray revealed an intense signal for <i>Wisteria floribunda</i> agglutinin (WFA). This signal was reduced by disrupting the native conformation of IgA1, suggesting that the distinct glycan profile was reflecting the conformational alteration of IgA1, including the glycan conformation detected as additional <i>N</i>-glycans on both the heavy and light chains. This unusually polymerized state of IgA1 would cause an increase of the binding avidity for lectins. WFA specifically recognized highly polymerized and glycosylated IgA1. Our results of analysis in the rare case of a patient with monoclonal immunoglobulin deposition disease suggest that the formation of unusually polymerized IgA1 is caused by divergent mechanisms including multiple structural alterations of glycans, which contributes to IgA1 deposition and mesangium proliferation.</p></div