and the Ellison Medical Foundation.

doi:10.1016/j.freeradbiomed.2011.10.424 173 Cytochrome C Peroxidase Deletion Delays Yeast Aging by Repressing Catalase Activity and Inducing Superoxide Dismutase Activity Dorival Martins1, Meena Kathiresan1, and Ann M English1 1Concordia University, Department of Chemistry and Biochemistry, Montreal, QC, Canada Control of ROS depends on the concerted activity of several antioxidant enzymes, including superoxide dismutases, catalases and peroxidases. Synchronization of these antioxidant enzymes is critical in antioxidant defence and the heme enzyme, cytochrome c peroxidase (CcP), is reported to be a mitochondrial redox sensor that coordinates signaling in the yeast Saccharomyces cerevisiae. We are investigating CcP’s role in modulating intracellular concentrations of O2

•– and H2O2, the key ROS implicated in aging of eukaryotic cells. Compared to isogenic wild-type strain, CcP-null yeast cells (∆ccp1) exhibit depressed catalase activity and accumulate up to 10-fold more H2O2 over 10 days, which surprisingly leads to chronological lifespan extension. However, these ∆ccp1 cells exhibit increased MnSOD activity and diminished levels of O2

•–, which are characteristics of mitohormesis, an adaptive response to H2O2. in contrast to ∆ccp1, a strain expressing the catalytically inactive CcPW191F variant accumulates less H2O2 but more O2

•– relative to wild-type cells and has a shorter chronological lifespan. Thus, lifespan correlates directly with H2O2 levels but inversely with O2

•– levels. Elevated O2

•– accelerates loss of aconitase activity (a critical TCA cycle enzyme) and increases carbonylation of mitochondrial proteins, which likely accelerate aging. Clearly, our yeast strains that separate CcP’s redox signaling and peroxidase activities define a fruitful system for detailed biochemical analysis of oxidative damage as a determinant of the aging process.

doi:10.1016/j.freeradbiomed.2011.10.425

174 Redox-control of Senescence Associated IL-1alpha Donald McCarthy1,2, Nadine Hempel2, Sita Subbaram1, and J. Andres Melendez2 1Albany Medical College, 2SUNY Albany College of Nanoscale Science and Engineering Interleukin-1 alfa (IL-1α) is a key senescence associated (SA) pro-inflammatory cytokine synthesized as a precursor protein. Proteolytic processing produces a C-terminal mature fragment (aa114-271) that interacts with its cognate cell surface receptor. a unique feature of the remaining N-terminal propiece (aa1-113) is its nuclear localization and ability to augment pro-inflammatory cytokine gene transcription. We establish that shifts in steady state H2O2 production resulting from enforced antioxidant enzyme expression or replicative senescence control IL-1α expression and nuclear localization. Inhibition of the Ca2+-dependent protease, calpain, results in cytoplasmic retention of IL-1α and ablation of its redox-dependent nuclear distribution. H2O2 treatment initiates extracellular Ca2+ influx that precedes IL-1α nuclear localization. IL-1α’s interaction with the histone acetyl transferase (HAT) p300 and its ability to activate NF-κB-driven gene expression is also H2O2-dependent. Collectively, these findings link the cellular redox-state to IL-1α expression, nuclear localization, and regulation of the senescence-associated pro-

inflammatory phenotype.

doi:10.1016/j.freeradbiomed.2011.10.426 175 Mechanisms for the Differential Expression of Endothelial Argininosuccinate Synthetase 1 Due To Endothelial Cell Senescence and Laminar Shear Stress Gyeong in Mun1, and Yong Chool Boo1 1Department of Molecular Medicine, Kyungpook National University School of Medicine,S. Korea Endothelial argininosuccinate synthetase 1 (ASS1) regulates the provision of L-arginine to nitric oxide synthase 3. We previously demonstrated that endothelial ASS1 expression levels were increased by laminar shear stress (LSS) while decreased by cellular senescence [1] and that this enzyme plays a role in maintaining anti-inflammatory microenvironments [2]. the mechanisms for differential expression of ASS1 due to LSS and cellular senescence were investigated in the present study. First, the regulatory mechanism for ASS1 expression by LSS was examined focusing on Kruppel-like factors (KLFs) that are known to coordinate endothelial gene expressions in response to LSS. the basal and stimulated expression levels of KLF2 and KLF4 were similar in young versus senescent endothelial cells. Regulatory roles of KLF2 and KLF4 in LSS-induced expression of ASS1 were confirmed by pre-treating young cells with small interfering RNAs. Then, it was examined whether epigenetic mechanisms are involved in the suppression of ASS1 expression in senescent endothelial cells. the in silico analysis of ASS1 promoter region using a CpG plot software identified two putative CpG islands comprising the regions between -217/+56 and +278/+764. Treatment of senescent endothelial cells with 5’-aza-2’-deoxycytidine to inhibit cytidine methylation led to significant increases of ASS1 expression levels under both static and LSS conditions. This study indicated that endothelial ASS1 expression levels may be regulated by the epigenetic change of cis-acting control elements as well as the activity of trans-acting factors, KLFs, in response to endothelial senescence and LSS. This work was supported by the Brain Korea 21 Project in 2011 and grants from the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology, Republic of Korea (Grant 2009-0071171). [1] Free Radic. Biol. Med. 47: 291–299 [2] J. Biol. Chem. 286: 2536-2542 Key words: Argininosuccinate synthetase 1, Endothelial cell senescence, Laminar shear stress, Gene expression. doi:10.1016/j.freeradbiomed.2011.10.427

176 Contribution of ROS-Metabolism in Tissue Homogenates and Dermal Fibroblasts of Long-Lived Insulin Receptor Substrate 1 (Irs1) Knockout Mice Melissa M Page1, Dominic J Withers2, and Colin Selman1 1University of Aberdeen, 2Imperial College London Disruption of insulin/insulin-like growth factor signalling (IIS) has been shown to extend lifespan in a range of model organisms. for example, we have previously shown that both male and female mice globally null for insulin receptor substrate 1 (Irs1-/-) are long-lived relative to wild type controls (Irs1+/+). However, the precise mechanism underlying this extended lifespan is unknown. in this study we examine whether Irs1-/- mice had enhanced resistance to oxidative damage by reactive oxygen species (ROS). We firstly determined the activity levels of a range of antioxidant enzymes

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