human peroxiredoxins (hPrxs) have been reported: N-acetylation on hPrx I at the C-terminal region, and Nα-termianal acetylation on hPrx II at the N-teminus. Both modifications inhibit overoxidation of each Prx isotypes through different mechanism. Instead of two Nα-acetylated spots of hPrx II on a 2D-PAGE gel, canine Prx II (cPrx II) show multiple (at least four) spot patterns. Asp-N peptide fingerprints of the cPrx II spots reveal native (not acetylated) and Nα-acetylated forms. Metabolic labeling of Madin-Darby Canine Kidney (MDCK) cells with S35, we show shift of the cPrx II spot from native form to the Nα-acetylated form, later than two hours after the S35 pulse, suggesting the acidic migration of the cPrx II spot is due to posttranslational modification rather than cotranslational modification. hPrx II expressed in the MDCK cells show two Nα-acetylated spots, however, cPrx II expressed in the A549 human lung cancer cells show multiple spot patterns, implying posttranslational Nα-acetylation is not a cell type specific but an amino acid sequence specific. Exchanging of penultimate residue of human and canine Prx II, Ala to Thr for hPrx II (A2T-hPrx II) and Thr to Ala for cPrx II (T2A-cPrx II), resulting change of 2D-PAGE spot patterns. A2T-hPrx II show multiple spot pattern like cPrx II and T2A-cPrx II show two spot pattern like hPrx II. Therefore, we suggest posttranslational Nα-acetylation of cPrx II and penultimate amino acid residue is critical for determining cotranslational and posttranslational modifications.

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Antioxidant Therapeutics: From Free­Radical Chemistry To Systems­Biology Mechanisms Vitaly K Koltover1 1Institute of Problems of Chemical Physics, Russian Academy of Sciences Efficiency of natural antioxidants like vitamin E, ascorbic acid or flavonoids and synthetic antioxidants like butylated hydroxytoluene (BHT) to trap active free radicals in cells and tissues in vivo is negligibly low as compared with the natural antioxidant enzymes. For example, the performance of SOD to catch О2

•– exceeds that of ascorbic acid by about 5 order of magnitude. As for HО•, it reacts with any organic molecules so rapidly that there is nothing to intercept this radical in vivo. The so-called “mitochondria-targeted” antioxidants can hardly upgrade the situation without considerable disturbances in mitochondrial nanoreactors. In vivo, antioxidants serve not so much as direct inhibitors of free radical processes but as physiologically active compounds that prevent the production of ROS. For example, flavonoids can exert the preventive antioxidant action by inducing expression of the antioxidant enzymes, SOD and catalase, while BHT, acting upon the endocrine system, can prevent the production of О2

•– in cell mitochondria by optimization of the tissue oxygenation. A similar preventive “antioxidant” effect can be afforded by magnetic isotope of magnesium. Among three stable isotopes, 24Mg, 25Mg and 26Mg with natural abundance 79, 10 and 11 percentages, respectively, only 25Mg has the nuclear spin and, hence, its nucleus has the magnetic field. In mitochondria, 25Mg works as much more effective cofactor of oxidative phosphorylation than the nonmagnetic isotopes 24Mg and 26Mg. It transpires that the reaction of ADP phosphorylation proceeds slower with nonmagnetic nuclei of Mg2+ by comparison with its magnetic nucleus. The retardation of electron transport in the sites of the electron-transport chains which are coupled with phosphorylation of ADP increases probability of the electron leakage onto oxygen with formation of О2

•–. Hence, the yield of О2

•– as the by-product of the mitochondrial respiration is bound to be lower with 25Mg-ADP by comparison with 24Mg-ADP or 26Mg-ADP. Thus, the systems biology approach provides the prospect way toward elucidation of true antioxidant therapeutics mechanisms in vivo. Supported by RFBR, project #10-03-01203.

523 Understanding Protonation and Redox Equilibria of Metal site of Mn Porphyrin­based Cellular Redox Modulators is Essential for Comprehending their ROS­related Chemistry and Biology Tin Weitner1, Ivan Kos1,2, Ines Batinic-Haberle2, and Mladen Biruš1 11University of Zagreb, Zagreb, Croatia, 2Duke University Medical Center, Durham Over last decade, there has been a great interest in cationic Mn(III) N-substituted pyridylporphyrins (MnPs) because of their exceptional ability to dismute O2

•– and reduce ONOO–, affect redox-based cellular signaling events, and in turn suppress excessive inflammatory and immune responses. The most studied MnTE-2-PyP5+, MnTnHex-2-PyP5+, and MnTDE-2-ImP5+ were efficacious in animal models of cancer, central nervous system disorders, radiation injury, diabetes, morphine tolerance, ischemia/reperfusion injuries etc. Recently, we showed that 10-fold enhanced lipophilicity of meta isomers relative to ortho species outbalances their somewhat inferior SOD-like activity; therefore, in an E. coli SOD-deficient model of oxidative stress, both ortho and meta MnPs, MnTE-2-PyP5+ and MnTE-3-PyP5+ were equally efficacious; therefore, they both bear therapeutic potential. Biologically relevant chemistry that happens at Mn porphyrin center, depends upon its electron density; i.e. its ability to give and accept electrons. Understanding protolytic and redox equilibria of Mn site is essential for understanding MnPs redox-based interactions with reactive species and redox-sensitive signaling proteins. In vivo, Mn can adopt four oxidation states, +2 +3, +4 and +5. At least 3 of them (+2, +3 and +4) are heavily involved in MnPs reactivity in vivo. Upon protonation/ deprotonation and reduction/oxidation, porphyrin charge is being changed, which affects its solvation, thus lipophilicity, shape and bulkiness, and therefore its cellular and subcellular accumulation. Hence, MnP in vivo efficacy is greatly affected also. Therefore, a comprehensive study was conducted to explore the protolytic and electrochemical behavior of MnTE-2-PyP5+ and MnTE-3-PyP5+ in aqueous solutions in a broad pH range. Protonation constants and formal reduction potentials were determined and relevant thermodynamic parameters were calculated. At physiological pH (7.4) MnTE-2-PyP5+ and MnTE-3-PyP5+ have two axially coordinated waters on Mn site. In vivo, they get easily reduced with cellular reductants from +3 to +2 Mn oxidation state, whereby they lose single charge from Mn site. Indeed the study found that reduced compounds bear only one axially coordinated molecule of water; consequently they are up to 2 orders of magnitude more lipophilic than the oxidized analogues. Gain in lipophilicity enhances their cellular accumulation and thus efficacy U19AI0677989, MZOŠ grant : 006-0061247-0009

524 Molecular Modeling of Dual Function Anticancer/Antioxidant Compounds Brian Rivera1, Selvendiran Karuppaiyah1, Kalman Hideg2, and Periannan Kuppusamy1 1The Ohio State University, 2The University of Pecs Introduction and Background: Our group has been investigating the anticancer efficacy of a class of curcumin analogs called diarylidenylpiperidones (DAPs). We have reported that DAPs are effective in promoting apoptosis in several types of cancer cells, and promote an increase in free-radical formation in exposed cells. Antioxidant-promoting DAPs containing an N-hydroxypyrroline moiety appear to selectively protect normal cells from damage while eliminating cancer cells. We have also reported that DAPs appear to inhibit the activation of signal transducer and activator of transcription 3 (STAT3). The objective

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of this study was to use in silico molecular modeling to investigate the binding affinity of DAP compounds with the STAT3 molecule. Materials and Methods: The free software AutoDock 4;2 (The Scripps Institute) was used for the in silico docking simulations. Minimized 3-dimensional topographies of the DAP compounds were obtained using the Dundee PRODRG2 server. Curcumin, and the DAPs H-4073, HO-3867, HO-4138, and HO-4200 were used. The murine STAT3 molecule (PDB ID: 3CWG) was downloaded from the RCSB Protein Data Bank. Blind docking was performed with 0.625 Å grid spacing with 126 points in the X, Y, and Z directions covering the majority of the SH2, linker, and DNA-binding domains of the STAT3 dimer. The analysis was refined by focusing upon the preferred binding location using 0.375 Å grid spacing with 100 points in the X, Y, and Z directions. Results: The DAP compounds exhibited preferential binding to a previously unreported site located in the DNA-binding domain of the STAT3 dimer, not the SH2 domain that is the most common target for STAT3 inhibition. In order of lowest to highest binding energy (kcal/mol), the compounds ranked as follows: HO-3867 < HO-4200 << H-4073 < Curcumin < HO-4138, indicating the antioxidant-promoting DAPs HO-3867 and HO-4200 have greater STAT3 inhibitory potential than the compounds lacking the N-hydroxypyrroline moiety. Conclusions: We have identified a potential new target site for STAT3 inhibition using in silico molecular modeling, and have shown that antioxidant-promoting DAP compounds have high binding affinity for this site.

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An Insertion/Deletion Polymorphism in the Glutathione Biosynthesis Pathway Is Associated with Earlier Onset of Alzheimer’s Disease and Increased Susceptibility to Oxidant Injury In Vitro Truc M. Le1, Alecia S. Willis2, Jonathan L. Haines3, Marshall L. Summar4, and David N. Cornfield1 1Stanford University Medical Center, 2Baylor College Of Medicine, 3Vanderbilt University Medical Center, 4Children's National Medical Center Background: Glutamate cysteine ligase (GCL) is the rate-limiting enzyme for de novo glutathione synthesis. Polymorphisms within the genes encoding GCL can lead to decreased glutathione synthesis and increased host susceptibility to oxidant injury. We hypothesize that an insertion/deletion polymorphism, located within the 3’-UTR of the gene that encodes the catalytic subunit of the GCL enzyme, predisposes to oxidant injury. Methods: Patients with Alzheimer’s disease were genotyped using the Taqman Allelic Discrimination Assay and age of disease onset was correlated with each genotype group. Second, normal human fibroblasts were exposed to H2O2, and cellular viability (determined using a MTT-based assay) was correlated with genotype. Results: Alzheimer’s disease patients homozygous for the deletion allele (Del/Del genotype) had an earlier median age of onset (69 vs 75 years) than patients homozygous or heterozygous for the insertion allele (p < 0.05). In vitro, the Del/Del genotype was associated with significantly decreased fibroblast viability following exposure to H2O2 as compared tofibroblasts homozygous or heterozygous for the insertion allele. Conclusion: Alzheimer’s patients with the Del/Del genotype have an earlier onset of disease, suggesting an increased susceptibility to oxidant injury. Further evidence in support of this paradigm includes in vitro data demonstrating that oxidant stress causes greater cellular injury in human fibroblasts with the Del/Del genotype. Patients with this genotype may be uniquely vulnerable to oxidant injury and may benefit most from strategies designed to mitigate oxidant injury, thereby providing rationale for a genomic-based approach to the prevention of oxidant injury.

526 Protecting Effects of the Self­assembling Trolox­chitosan Nanoparticles Against t­BHP­induced Cell Apoptosis Yang Liu1, Lu Han1, Libo Du1, Hongying Jia1, and Qiu Tian1 1Institute of Chemistry, Chinese Academy of Sciences Chitosan nanoparticles, because of their excellent loading capacity, have been extensively studied for delivery of genes, proteins, and drugs with low biocompatibility. In this work, to futher improve the biocompatibility even for the water-soluble antioxidants, such as Trolox C, a new self-assembling trolox-chitosan nanoparticle with very small size (~30nm) has been prepared. Protection effects of the trolox and the nano-antioxidant on the t-BHP-induced apoptisis in RAW 264.7 cell have been comparatively examined by annexin V-FITC/PI stain and DNA ladder, as well as MTT assay. The results indicate that the nano-antioxidant can more efficiently suppress the apoptosis than trolox dose. Additionally, we have found that the trolox-chitosan nanoparticles can be efficiently taken up into cells, probably by endocytosis. To verify the inhibition mechanism, some apoptosis-related proteins, such as bax, bcl-2, caspase-3, PARP and bid, have been determined by Western blot. Meanwhile, the mitochondrial membrane potential (ΔΨm), which is considered as an initial and irreversible step towards apoptosis, has been monitored by JC-1 dye. It was found that the nanoparticles more evidently attenuated disruption of ΔΨm, increased bcl-2 and pro-caspase-3 expressions and decreased bax, bid and PARP expressions than trolox dose. Taken together, our results proposed that the nano-antioxidants inhibited the t-BHP-induced apoptosis of RAW264.7 cells via the mitochondria-mediated apoptosis pathway.

527 Phenolic Antioxidant tert­Butylhydroquinone and Alzheimer’s Disease Treatment Rui-Ming Liu1, Hasina Akhter1, and Ashwini Katre1 1University of Alabama at Birmingham Alzheimer’s disease (AD) is a major cause of dementia in the elderly with no effective treatment. Tert butyl hydroquinone (TBHQ) is a phenolic antioxidant and highly effective preservative used in a wide range of food and cosmetic products. In this study, we show for the first time that feeding transgenic mice that overexpress mutated human amyloid peptide precursor protein (APP) and presenilin1 genes (APP/PS1 mice), a well established mouse model of AD, with a diet containing 1% TBHQ for 6 weeks dramatically reduces amyloid beta peptide (Aβ) burden in the brain, associated with an increase in the concentration of glutathione (GSH), the most abundant intracellular free thiol and an important antioxidant. Western analyses show that TBHQ feeding decreases the amount of beta-C-terminal fragment but has no significant effect on the level of alpha-C-terminal fragment or full-length APP, suggesting that TBHQ feeding inhibits the expression/activity of β-secretase, a redox sensitive enzyme. Furthermore, we show that TBHQ feeding inhibits the expression and activity of plasminogen activator inhibitor-1 (PAI-1), a physiological inhibitor of tissue type and urokinase type plasminogen activators (tPA and uPA), which convert plasminogen into plasmin, a serine protease playing a critical role in Aβ degradation. Together with our previous findings which show that knockout of the PAI-1 gene reduces brain Aβ burden in APP/PS1 mice and that PAI-1 expression is redox regulated, it is suggested that TBHQ feeding reduces brain Aβ burden by increasing brain antioxidant capacity and thereby downregulating the expression and activity of PAI-1 and β-secretase, which leads to increased degradation and decreased synthesis of Aβ. As

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