SFRR-E Young Investigator Awardee P85 Nitric oxide affects trypomastigote to amastigote differentiation in Trypanosoma cruzi O. Soares Chrislaine a , P. Dias Caroline b , Colli Walter a , Manso Alves M. Julia a a University of Sao Paulo (Institute of Chemistry), Department of Biochemistry, Brazil b Faculdades Oswaldo Cruz (Biological Sciences ), Pharmaceutical Sciences, Brazil Abstract Trypanosoma cruzi is the etiologic agent of Chagas disease. Two main distinct forms are present in the mammalian host: trypomastigote, an infective form, and the amastigote, a typical replicative form. Succeeding the host cell invasion, trypomastigotes differentiate to amastigotes, a process known as amastigogenesis. Amastigogenesis is characterized by parasite body remodeling, with drastic reduction of flagellum, and changes in protein profile and energetic metabolism. Our aim is to explore the role of nitric oxide as a signaling molecule during the amastigogenesis process, which must be strictly regulated. We report herein that acid pH (6.0) is essential for T. cruzi amastigogenesis. Also, during amastigogenesis there is a progressive solubilization of the paraflagellar protein, a flagellum marker. Moreover, the process is dependent on  NO concentra- tion, since it is suppressed by 1 mM SNAP, a  NO donor, and favored by 10 mM L-NAME, a NOS inhibitor. Accordingly, S-nitrosylation of selective proteins occurs in amastigogenesis. Additionally, amastigogenesis is affected by IBMX (PDE inhibitor) treatment, suggesting the importance of cyclic nucleotides signaling. Furthermore, tubulin stability is also affected by the  NO availability. Along amastigogenesis, flagellum disas- sembling is accompanied by changes in α-tubulin tyrosylation and polyglutamylation levels. Taken together, these results suggest  NO participation in trypomastigote differentiation to amastigotes in T. cruzi. http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.817 P86 Cu(II)-disulfide complexes with superoxide dismu- tase- and catalase-like activities protect mitochon- dria and whole cells against oxidative stress Aliaga Margarita E. a , Sandoval-Acuña Cristián b , López- Alarcón Camilo c , Fuentes Jocelyn d , Speisky Hernan e a Pontificia Universidad Católica de Chile (Facultad de Química,), Fisicoquimica, Chile b University of Chile (Nutrition and Food Technology Institute), Antioxidants, Chile c Pontificia Universidad Católica de Chile (Facultad de Química), Farmacia, Chile d University of Chile (Nutrition and Food Technology Institute ), Antioxidants, Chile e University of Chile (Nutrition and Food Technology Institute ), Antioxidants, Chile Abstract Mitochondria are a major subcellular site of superoxide (O 2 - ) formation. Conditions leading to an uncontrolled production, accumulation and/or conversion of O 2 - into hydrogen peroxide result in an increment in the intramitochondrial oxidative tone which, ultimately leads to the loss of cell viability. Recently, we reported on the ability of a series of Cu(II)-disulfide complexes to act simultaneously as SOD- and catalase-like molecules. In the present study, we addressed the potential of such compounds to protect mitochondria and cells against the oxidative stress and the cytolytic damage induced by diclofenac. Exposure of Caco-2 cells to diclofenac (250 mM, 20 min) led to a near 80% inhibition of mitochondrial complex I activity and almost doubled the rate of mitochondrial O 2 - production (assessed by Mitosox). A comparable increment was seen in whole cells when the oxidative tone was assessed through the largely hydrogen peroxide-dependent dichlorofluores- cein (DCFH) oxidation. The increment in mitochondrial O 2 - production was totally and concentration-dependently prevented by the addition of the complexes formed between Cu(II) and the disulfides of glutathione, homo- cysteine, or a-dehydro-lipoic acid (20 mM each); comparatively, the Cu(II)- cystine complex exerted a weaker protection. A comparable protection pattern was seen at the whole cell level, as these complexes were also effective in preventing the increment in DCFH oxidation. The mitochondrial and whole cell antioxidant protection also translated into a full protection against the cytolytic effects of diclofenac (45 min). Results from the present study indicate that the here-tested Cu(II)-disulfides complexes are able to effectively protect cells against the oxidative and the lytic effects of O 2 - -overproducing mitochon- dria, suggesting a potential for these type of compounds to act as SOD- and catalase-like molecules under oxidative-stress conditions. Supported by FONDECYT #1110018. http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.818 P87 Mapping nitro-tyrosine modifications in fibrinogen by mass spectrometry as a biomarker for inflamma- tory disease Meredith Stuart a , Parekh Gita b , Towler Joanna b , Schouten James b , Davis Paul b , Griffiths Helen a , Spickett Corinne a a Aston University (Birmingham), School of Life and Health Sciences, UK b Mologic Ltd (Bedfordshire), Mologic, UK Abstract There is a growing awareness that inflammatory diseases have an oxidative pathology, which can result in speci fic oxidation of amino acids within proteins. It is known that patients with inflammatory disease have higher levels of plasma protein nitro-tyrosine than healthy controls. Fibrinogen is an abundant plasma protein, highly susceptible to such oxidative modifications, and is therefore a potential marker for oxidative protein damage. The aim of this study was to map tyrosine nitration in fibrinogen under oxidative conditions and identify susceptible residues. Fibrinogen was oxidised with 0.25 mM and 1 mM SIN-1, a peroxynitrite generator, and methionine was used to quench excess oxidant in the samples. The carbonyl assay was used to confirm oxidation in the samples. The carbonyl levels were 2.3, 8.72 and 11.5 nmol/ mg protein in 0, 0.25 mM and 1 mM SIN-1 samples respectively. The samples were run on a SDS-PAGE gel and tryptically digested before analysis by HPLC MS-MS. All 3 chains of fibrinogen were observed for all treatment conditions. The overall sequence coverage for fibrinogen determined by Mascot was between 60-75%. The oxidised samples showed increases in oxidative mod- i fications in both alpha and beta chains, commonly methionine sulfoxide and tyrosine nitration, correlating with increasing SIN-1 treatment. Tyrosines that were most susceptible were Tyr135 (tryptic peptide YLQEIYNSNNQK) and Tyr277 (tryptic peptide GGSTSYGTGSETESPR), but several other nitrated tyrosines were also identified with high confidence. Identi fication of these susceptible peptides will allow design of sequences-specific biomarkers of oxidative and nitrative damage to plasma protein in inflammatory conditions. http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.819 P. White et al. / Free Radical Biology and Medicine 75 (2014) S21–S53 S50