- Email: [email protected]
Selenomethionine supplementation reduces extracellular trap formation and lesion burden in a mouse model of atherosclerosis
A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
absence of exogenous HOCl, while compounds 2 and 3 do not influence it. Compound 1 in higher concentrations prevents destruction of neutrophils caused by addition of 300 μmol/l HOCl. Thus, compounds 1–3 can be considered as potential cytoprotectors under halogenating stress.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.299
P-153
Nuclear translocation and alternative conformation of cytochrome c after peroxynitrite-mediated challenge to cells 1
1
S91
Nitrated phospholipids (PL) were identified in biological samples using MS based lipidomics. This give new evidence of NO2-PL formation under nitroxidative stress conditions, similar to well-known NO2-FA. Their identification is based on the typical neutral loss of 47 Da (-HNO2), fragmentation of polar head group and fatty acyl chains. However, the reporter ions formed by HNO2 are dependent on the instrumental conditions, which make the identification of nitrated PL a challenge. Determination of the best parameters to obtain the most useful information is a key issue for their identification. We evaluated the influence of normalized collision energy (NCE) in a Q-Orbitrap on the abundance of reporter product ions formed by neutral loss of 47 Da (HNO2), for nitrated phosphatidylcholines synthesized through in vitro mimetic nitration with NO2BF4. Results indicated that lower CID NCE allowed to see typical reporter ions, but they were absent when using higher NCE. Overall, the abundance of reporter ions of NO2-PL is significantly affected by the NCE, which in fact determines the observation of the ions and the successful detection of nitrated PL in biological samples.
E-mail address: [email protected]
2
Florencia Tomasina , Jennyfer Martinez , Luciana Hannibal , Celia Quijano 1, Rafael Radi 1 1
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República and Center for Free Radical Biology and Medicine (CEINBIO), Montevideo, Uruguay 2 Medical Center, University of Freiburg, Center of Pediatrics and Adolescent Medicine, Laboratory of Clinical Biochemistry and Metabolism, Freiburg, Germany
Acknowledgements Funding RNEM (LISBOA-01–0145-FEDER-402–022125), QOPNA (FCT UID/QUI/00062/2013) & BPD/UI51/5388/2017 grant.
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.301
P-155 Cytochrome c (cyt c) is a mitochondrial heme-containing protein that functions as an electron carrier in the mitochondrial respiratory chain and as a pro-apoptotic mediator in the cytosol. A monoclonal antibody (mAb 1D3) was generated that recognizes a region in cyt c surrounding residue Pro44 in an alternative conformation of the protein (such as that present after cyt c nitration Tyr74). Previous studies shown that in a peroxynitrite-treated Hela cell model, cyt c translocate to the cellular nuclei as detected by the 1D3 suggesting its oxidative modification. The molecular interactions of cyt c in this unusual location are not known. The aim of this study was to gain insights into the identification of the possible partners of oxidatively-modified and/or nitrated cyt c with nuclear proteins. Herein, we employed four cell lines (Hela, Vero, BAECs and B16F1), exposed the cultures to peroxynitrite, and evaluated general redox status by flow cytometry, and the presence of nitrated proteins by SILAC technology 24 hours after treatment. In all cases, cyt c was shown in the cellular nuclei. The identification of the cyt c interacting proteins following oxidative modification is under way using cellular fractionation, 2D-gel electrophoresis and mass spectrometry.
E-mail address: fl[email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.300
P-154
Improving the identification of nitrated phospholipids: low vs high energy-dependent fragmentation Bruna Neves 1, Tânia Melo 2, M. Rosário Domingues 2, Pedro Domingues 2 1
Department of Chemistry, University of Trás-os-Montes e Alto Douro, Vila Real, Portugal 2 Mass Spectrometry Centre-QOPNA, Department of Chemistry, University of Aveiro, Aveiro, Portugal
Selenomethionine supplementation reduces extracellular trap formation and lesion burden in a mouse model of atherosclerosis Yunjia Zhang, Siân Cartland, Clare L. Hawkins, Benjamin S. Rayner Heart Research Institute, Newtown, Sydney, NSW, Australia
The release of extracellular traps (ET) by neutrophils in the vessel wall is implicated in atherosclerotic lesion development. In this study the effect of dietary SeMet supplementation was assessed in an apoE-/- high fat diet (HFD) fed mouse model of atherosclerosis. Aortas from mice fed a diet supplemented with 2 mg/kg SeMet (w/w) over 12 weeks exhibited a significant reduction in lesion formation compared to controls in both the normal (0.40% vs 1.26%) and HFD (4.70% vs 6.21%) fed cohorts, as assessed by Oil Red O staining. Furthermore, within lesions, the M1 macrophage marker iNOS was decreased and the M2 macrophage marker CD206 increased, concurrent with increased plasma levels of the anti-inflammatory marker TGF-β in SeMet supplemented mice. In addition, ET formation from bone marrow-derived cells was markedly less pronounced within the SeMet supplemented cohorts with similar results demonstrated in cultured human neutrophils and macrophages exposed to inflammatory stimuli. Together, these data highlight the potential beneficial effect of SeMet supplementation as a therapeutic strategy for the treatment of chronic inflammatory diseases such as atherosclerosis.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.302
absence of exogenous HOCl, while compounds 2 and 3 do not influence it. Compound 1 in higher concentrations prevents destruction of neutrophils caused by addition of 300 μmol/l HOCl. Thus, compounds 1–3 can be considered as potential cytoprotectors under halogenating stress.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.299
P-153
Nuclear translocation and alternative conformation of cytochrome c after peroxynitrite-mediated challenge to cells 1
1
S91
Nitrated phospholipids (PL) were identified in biological samples using MS based lipidomics. This give new evidence of NO2-PL formation under nitroxidative stress conditions, similar to well-known NO2-FA. Their identification is based on the typical neutral loss of 47 Da (-HNO2), fragmentation of polar head group and fatty acyl chains. However, the reporter ions formed by HNO2 are dependent on the instrumental conditions, which make the identification of nitrated PL a challenge. Determination of the best parameters to obtain the most useful information is a key issue for their identification. We evaluated the influence of normalized collision energy (NCE) in a Q-Orbitrap on the abundance of reporter product ions formed by neutral loss of 47 Da (HNO2), for nitrated phosphatidylcholines synthesized through in vitro mimetic nitration with NO2BF4. Results indicated that lower CID NCE allowed to see typical reporter ions, but they were absent when using higher NCE. Overall, the abundance of reporter ions of NO2-PL is significantly affected by the NCE, which in fact determines the observation of the ions and the successful detection of nitrated PL in biological samples.
E-mail address: [email protected]
2
Florencia Tomasina , Jennyfer Martinez , Luciana Hannibal , Celia Quijano 1, Rafael Radi 1 1
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República and Center for Free Radical Biology and Medicine (CEINBIO), Montevideo, Uruguay 2 Medical Center, University of Freiburg, Center of Pediatrics and Adolescent Medicine, Laboratory of Clinical Biochemistry and Metabolism, Freiburg, Germany
Acknowledgements Funding RNEM (LISBOA-01–0145-FEDER-402–022125), QOPNA (FCT UID/QUI/00062/2013) & BPD/UI51/5388/2017 grant.
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.301
P-155 Cytochrome c (cyt c) is a mitochondrial heme-containing protein that functions as an electron carrier in the mitochondrial respiratory chain and as a pro-apoptotic mediator in the cytosol. A monoclonal antibody (mAb 1D3) was generated that recognizes a region in cyt c surrounding residue Pro44 in an alternative conformation of the protein (such as that present after cyt c nitration Tyr74). Previous studies shown that in a peroxynitrite-treated Hela cell model, cyt c translocate to the cellular nuclei as detected by the 1D3 suggesting its oxidative modification. The molecular interactions of cyt c in this unusual location are not known. The aim of this study was to gain insights into the identification of the possible partners of oxidatively-modified and/or nitrated cyt c with nuclear proteins. Herein, we employed four cell lines (Hela, Vero, BAECs and B16F1), exposed the cultures to peroxynitrite, and evaluated general redox status by flow cytometry, and the presence of nitrated proteins by SILAC technology 24 hours after treatment. In all cases, cyt c was shown in the cellular nuclei. The identification of the cyt c interacting proteins following oxidative modification is under way using cellular fractionation, 2D-gel electrophoresis and mass spectrometry.
E-mail address: fl[email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.300
P-154
Improving the identification of nitrated phospholipids: low vs high energy-dependent fragmentation Bruna Neves 1, Tânia Melo 2, M. Rosário Domingues 2, Pedro Domingues 2 1
Department of Chemistry, University of Trás-os-Montes e Alto Douro, Vila Real, Portugal 2 Mass Spectrometry Centre-QOPNA, Department of Chemistry, University of Aveiro, Aveiro, Portugal
Selenomethionine supplementation reduces extracellular trap formation and lesion burden in a mouse model of atherosclerosis Yunjia Zhang, Siân Cartland, Clare L. Hawkins, Benjamin S. Rayner Heart Research Institute, Newtown, Sydney, NSW, Australia
The release of extracellular traps (ET) by neutrophils in the vessel wall is implicated in atherosclerotic lesion development. In this study the effect of dietary SeMet supplementation was assessed in an apoE-/- high fat diet (HFD) fed mouse model of atherosclerosis. Aortas from mice fed a diet supplemented with 2 mg/kg SeMet (w/w) over 12 weeks exhibited a significant reduction in lesion formation compared to controls in both the normal (0.40% vs 1.26%) and HFD (4.70% vs 6.21%) fed cohorts, as assessed by Oil Red O staining. Furthermore, within lesions, the M1 macrophage marker iNOS was decreased and the M2 macrophage marker CD206 increased, concurrent with increased plasma levels of the anti-inflammatory marker TGF-β in SeMet supplemented mice. In addition, ET formation from bone marrow-derived cells was markedly less pronounced within the SeMet supplemented cohorts with similar results demonstrated in cultured human neutrophils and macrophages exposed to inflammatory stimuli. Together, these data highlight the potential beneficial effect of SeMet supplementation as a therapeutic strategy for the treatment of chronic inflammatory diseases such as atherosclerosis.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.302