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Dietary Restriction and Rapamycin, Similar or Different Mechanisms on Longevity?
346 Dietary Restriction and Rapamycin, Similar or Different Mechanisms on Longevity? Wilson Chun Yim Fok1, Yiqiang Zhang1, Adam Salmon1, Arunabh Bhattacharya1, Elizabeth Fernandez1, Dean Jones2, Arlan Richardson1, and Viviana Perez1 1 University of Texas Health Science Center at San Antonio, 2 Emory University School of Medicine Rapamycin (rapa) has been found to extend lifespan in yeast, C. elegans, Drosophila, and recently mice. Rapa is a specific inhibitor of the target of rapamycin (TOR), which acts as a key regulatory nexus in the responses of eukaryote cells to nutrients, growth factors, and cellular energy status. Recently, it has been hypothesized that rapa is a dietary restriction (DR) mimetic and that rapa and DR act through a similar mechanism. To test this, we compared the similarities or differences in the mTOR and redox signaling pathways on the intervention of DR, rapa, or combined intervention of DR and rapa in eight month old mice. We saw no differences in body composition, with the exception in fat mass, in which only animals on DR diet had a significant decrease in fat mass. The mTOR signaling pathway was determined by the phosphorylated levels of the downstream targets, 4E-BP1 and S6K1. Our data showed significantly reduced phosphorylation of 4E-BP1 (20%) and S6K1 (60%) by DR and Rapa. Moreover, we observed a more reduced redox state of glutathione (GSH) in DR, but not in any other groups, and striking differences in gene expression of redox signaling pathways such as AP-1, NFκB, and NRF2, between DR and Rapa treated groups. Our preliminary data indicates that DR and Rapa would have similar effects in the mTOR signaling pathway, but have different effects in redox signaling regulation. This work was supported by NIH Grant AG036613. doi: 10.1016/j.freeradbiomed.2010.10.355
347 Role of Chlorinated Nucleosides in the Perturbation of Cellular Function under Inflammatory Conditions Naomi R Stanley1, Kristine CY McGrath1, Alison K Heather2, and Clare L Hawkins1 1 2 Heart Research Institute, Sydney, University of Technology Sydney During inflammation, myeloperoxidase released from activated phagocytes, generates the highly reactive oxidant hypochlorous acid (HOCl). Although HOCl and other chlorinating oxidants such as N-chloramines play an important role in the immune response, they can also induce damage to tissue and contribute to the progression of inflammatory disease. HOCl reacts with cellular DNA inducing strand breaks, mutations, and cross-links. This damage is postulated to involve the formation of chlorinated nucleobases, which have been detected in human inflammatory fluids and tissue. However, whether chlorinated nucleosides are a cause or consequence of inflammation remains uncertain. In this study, we show that exposure of murine macrophage-like cells (J774A.1) to different chlorinated ribose and deoxyribose nucleosides results in a decrease in both cell viability and the rate of cellular proliferation compared to non-treated cells. Evidence was also obtained for the incorporation of 5-chloro-cytidine (5ClC), 8-chloro-adenosine (8ClA) and 8-chloro-guanosine (8ClG) into cellular RNA and 5-chloro-deoxycytidine (5CldC), but not 8chloro-deoxyguanosine (8CldG) or 8-chloro-deoxyadenosine (8CldA), into cellular DNA. The ability of the chlorinated nucleosides to modulate cellular viability and proliferation did not appear to be associated with the extent of incorporation of these species into DNA or RNA, as 8CldG and 8ClG had the most potent cellular effects. PCR array studies showed alterations in the expression of several genes associated with oxidative stress,
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proliferation and growth arrest, inflammation, DNA repair and apoptosis. These results highlight that chlorinated nucleoside formation can be detrimental to cellular function, and as such, these materials may play a novel role in the pathogenesis of inflammatory disease. doi: 10.1016/j.freeradbiomed.2010.10.356
348 RevERBα Modulates Hyperoxiainduced Trans differentiation of Lipofibroblasts in the Neonatal Mouse Lung Maurice Hinson1, Guang Yang1, Ping La1, and Phyllis Dennery1,2 1 2 Children's Hospital of Philadelphia, University of Pennsylvania Introduction: Lung lipofibroblasts accumulate and mobilize lipids that are then utilized in the production of surfactants by type II cells. Hyperoxia is known to induce the transdifferentiation of lipofibroblasts to myofibroblasts, resulting in a significant change in cytoskeletal structure and loss of ability to accumulate and mobilize lipids. A well-known regulator of the mammalian circadian clock and lipid metabolism is nuclear receptor subfamily 1, group D, member 1 (Rev-ERBα), which also modulates cell proliferation and regulates the differentiation of adipocytes in vitro. Objectives: Given the developmental and characteristic similarities between adipocytes and lipofibroblasts, we hypothesized that Rev-ERBα would also modulate the hyperoxiainduced transdifferentiation of lipofibroblasts. Methods: Neonatal mouse lung fibroblasts were exposed to normoxia (~21% O2, 5% CO2) or hyperoxia (≥ 95% O2, 5% CO2) for 24 hours and lung Rev-ERBα, mRNA steady-state levels were determined by real-time PCR and protein levels by western analysis. Lung steady state mRNA levels of the markers of cellular differentiation CCAAT/enhancer binding protein (C/EBP)α and peroxisome proliferator-activator receptor (PPAR) γ as well as the myofibroblast marker α-smooth muscle actin and the lipofibroblast marker, adipocyte differentiating related protein (ADRP), were assessed and compared between controls and hyperoxia exposed cells. Results: Hyperoxia resulted in a 2-fold increase in Rev-ERBα mRNA but a significant reduction in Rev-ERBα protein, as previously demonstrated in adipocytes. With the hyperoxic modulation of Rev-ERBα, there was a 12-fold and 4-fold increase in C/EBP)α mRNA and PPARγ mRNA, respectively. In addition there was a 5-fold increase in α-smooth muscle actin and a 4-fold decrease in ADRP (p ≤ 0.05, n=3) suggesting a myofibroblast phenotype. Antisense to Rev-ERBα reversed the increase in αsmooth muscle actin. Conclusion: Rev-ERBα is important in the transdifferentiation of lipofibroblasts to myofibroblasts. Speculation: Sustaining Rev-ERBα expression will inhibit hyperoxia-induced transdifferentiation of lipofibroblasts and preserve lipofibroblast function thereby promoting lipid mobilization and enhancing surfactant synthesis in type II cells. doi: 10.1016/j.freeradbiomed.2010.10.357
349 The Association Between Zn Fingers and Forbidden Disulfide Motifs Dhakshinari V Hulugalle1,2, Naomi L Haworth1, Sara Ballouz1,3, Jason Y Liu1, Samuel W Fan1, and Merridee A Wouters1 1 Victor Chang Cardiac Research Institute, Sydney, Australia, 2 School of Medical Sciences, University of New South Wales, 3 Sydney, Australia, School of Computer Science and Engineering, University of New South Wales, Sydney, Australia Here we look at Zinc fingers in protein structures and sequences
SFRBM/SFRRI 2010
347 Role of Chlorinated Nucleosides in the Perturbation of Cellular Function under Inflammatory Conditions Naomi R Stanley1, Kristine CY McGrath1, Alison K Heather2, and Clare L Hawkins1 1 2 Heart Research Institute, Sydney, University of Technology Sydney During inflammation, myeloperoxidase released from activated phagocytes, generates the highly reactive oxidant hypochlorous acid (HOCl). Although HOCl and other chlorinating oxidants such as N-chloramines play an important role in the immune response, they can also induce damage to tissue and contribute to the progression of inflammatory disease. HOCl reacts with cellular DNA inducing strand breaks, mutations, and cross-links. This damage is postulated to involve the formation of chlorinated nucleobases, which have been detected in human inflammatory fluids and tissue. However, whether chlorinated nucleosides are a cause or consequence of inflammation remains uncertain. In this study, we show that exposure of murine macrophage-like cells (J774A.1) to different chlorinated ribose and deoxyribose nucleosides results in a decrease in both cell viability and the rate of cellular proliferation compared to non-treated cells. Evidence was also obtained for the incorporation of 5-chloro-cytidine (5ClC), 8-chloro-adenosine (8ClA) and 8-chloro-guanosine (8ClG) into cellular RNA and 5-chloro-deoxycytidine (5CldC), but not 8chloro-deoxyguanosine (8CldG) or 8-chloro-deoxyadenosine (8CldA), into cellular DNA. The ability of the chlorinated nucleosides to modulate cellular viability and proliferation did not appear to be associated with the extent of incorporation of these species into DNA or RNA, as 8CldG and 8ClG had the most potent cellular effects. PCR array studies showed alterations in the expression of several genes associated with oxidative stress,
S130
proliferation and growth arrest, inflammation, DNA repair and apoptosis. These results highlight that chlorinated nucleoside formation can be detrimental to cellular function, and as such, these materials may play a novel role in the pathogenesis of inflammatory disease. doi: 10.1016/j.freeradbiomed.2010.10.356
348 RevERBα Modulates Hyperoxiainduced Trans differentiation of Lipofibroblasts in the Neonatal Mouse Lung Maurice Hinson1, Guang Yang1, Ping La1, and Phyllis Dennery1,2 1 2 Children's Hospital of Philadelphia, University of Pennsylvania Introduction: Lung lipofibroblasts accumulate and mobilize lipids that are then utilized in the production of surfactants by type II cells. Hyperoxia is known to induce the transdifferentiation of lipofibroblasts to myofibroblasts, resulting in a significant change in cytoskeletal structure and loss of ability to accumulate and mobilize lipids. A well-known regulator of the mammalian circadian clock and lipid metabolism is nuclear receptor subfamily 1, group D, member 1 (Rev-ERBα), which also modulates cell proliferation and regulates the differentiation of adipocytes in vitro. Objectives: Given the developmental and characteristic similarities between adipocytes and lipofibroblasts, we hypothesized that Rev-ERBα would also modulate the hyperoxiainduced transdifferentiation of lipofibroblasts. Methods: Neonatal mouse lung fibroblasts were exposed to normoxia (~21% O2, 5% CO2) or hyperoxia (≥ 95% O2, 5% CO2) for 24 hours and lung Rev-ERBα, mRNA steady-state levels were determined by real-time PCR and protein levels by western analysis. Lung steady state mRNA levels of the markers of cellular differentiation CCAAT/enhancer binding protein (C/EBP)α and peroxisome proliferator-activator receptor (PPAR) γ as well as the myofibroblast marker α-smooth muscle actin and the lipofibroblast marker, adipocyte differentiating related protein (ADRP), were assessed and compared between controls and hyperoxia exposed cells. Results: Hyperoxia resulted in a 2-fold increase in Rev-ERBα mRNA but a significant reduction in Rev-ERBα protein, as previously demonstrated in adipocytes. With the hyperoxic modulation of Rev-ERBα, there was a 12-fold and 4-fold increase in C/EBP)α mRNA and PPARγ mRNA, respectively. In addition there was a 5-fold increase in α-smooth muscle actin and a 4-fold decrease in ADRP (p ≤ 0.05, n=3) suggesting a myofibroblast phenotype. Antisense to Rev-ERBα reversed the increase in αsmooth muscle actin. Conclusion: Rev-ERBα is important in the transdifferentiation of lipofibroblasts to myofibroblasts. Speculation: Sustaining Rev-ERBα expression will inhibit hyperoxia-induced transdifferentiation of lipofibroblasts and preserve lipofibroblast function thereby promoting lipid mobilization and enhancing surfactant synthesis in type II cells. doi: 10.1016/j.freeradbiomed.2010.10.357
349 The Association Between Zn Fingers and Forbidden Disulfide Motifs Dhakshinari V Hulugalle1,2, Naomi L Haworth1, Sara Ballouz1,3, Jason Y Liu1, Samuel W Fan1, and Merridee A Wouters1 1 Victor Chang Cardiac Research Institute, Sydney, Australia, 2 School of Medical Sciences, University of New South Wales, 3 Sydney, Australia, School of Computer Science and Engineering, University of New South Wales, Sydney, Australia Here we look at Zinc fingers in protein structures and sequences
SFRBM/SFRRI 2010