Peroxynitrite: a multifaceted oxidizing and nitrating metabolite.

Peroxynitrite, a short-lived and reactive oxidant, emerges from the diffusion-controlled reaction between the superoxide radical and nitric oxide. Evidence shows that peroxynitrite is a critical mediator in physiological and pathological processes such as the immune response, inflammation, cancer, neurodegeneration, vascular dysfunction, and aging. The biochemistry of peroxynitrite is multifaceted, involving one- or two-electron oxidations and nitration reactions. This minireview highlights recent findings of peroxynitrite acting as a metabolic mediator in processes ranging from oxidative killing to redox signaling. Selected examples of nitrated proteins (i.e., 3-nitrotyrosine) are surveyed to underscore the role of this post-translational modification on cell homeostasis. While accumulated evidence shows that large amounts of peroxynitrite participates of broad oxidation and nitration events in invading pathogens and host tissues, a closer look supports that low to moderate levels selectively trigger signal transduction cascades. Peroxynitrite probes and redox-based pharmacology are instrumental to further understand the biological actions of this reactive metabolite.

Superoxide, nitric oxide and peroxynitrite production by macrophages under different physiological oxygen tensions.

Macrophages count on two O2-consuming enzymes to form reactive radical species: NAPDH oxidase 2 (Nox2) and nitric oxide synthase 2 (inducible isoform, iNOS) that produce superoxide radical (O2•-) and nitric oxide (•NO), respectively. If formed simultaneously, the diffusion-controlled reaction of O2•- and •NO yields peroxynitrite, a potent cytotoxic oxidant. In human tissues and cells, the oxygen partial pressure (pO2) normally ranges within 2-14 %, with a typical average pO2 value for most tissues ca. 5 %. Given that O2 is a substrate for both Nox2 and iNOS, its tissue and cellular concentration can affect O2•- and •NO production. Also, O2 is a modulator of the macrophage adaptative response and may influence iNOS expression in a hypoxia inducible factor 1-α (HIF1α-)-dependent manner. However, most of the reported experiments in cellula, analyzing the formation and effects of O2•- and •NO during macrophage activation and cytotoxicity towards pathogens, have been performed in cells exposed to atmospheric air supplemented with 5 % CO2; under these conditions, most cells are exposed to supraphysiologic oxygen tensions (ca. 20 % O2) which are far from the physiological pO2. Here, the role of O2 as substrate in the oxidative response of J774A.1 macrophages was explored upon exposure to different pO2 and O2•- and •NO formation rates were measured, obtaining a KM of 26 and 42 µM O2 for Nox2 and iNOS, respectively. Consequently, peroxynitrite formation was influenced by pO2, reaching a maximum at ≥ 10 % O2, but even at levels as low as 2 % O2, a substantial formation rate of this oxidant was detected. Indeed, the cytotoxic capacity of immunostimulated macrophages against the intracellular parasite T. cruzi was significant, even at low pO2 values, confirming the role of peroxynitrite as a potent oxidizing cytotoxin within a wide range of physiological oxygen tensions.

De novo sequencing and construction of a unique antibody for the recognition of alternative conformations of cytochrome c in cells.

Cytochrome c (cyt c) can undergo reversible conformational changes under biologically relevant conditions. Revealing these alternative cyt c conformers at the cell and tissue level is challenging. A monoclonal antibody (mAb) identifying a key conformational change in cyt c was previously reported, but the hybridoma was rendered nonviable. To resurrect the mAb in a recombinant form, the amino-acid sequences of the heavy and light chains were determined by peptide mapping-mass spectrometry-bioinformatic analysis and used to construct plasmids encoding the full-length chains. The recombinant mAb (R1D3) was shown to perform similarly to the original mAb in antigen-binding assays. The mAb bound to a variety of oxidatively modified cyt c species (e.g., nitrated at Tyr74 or oxidized at Met80), which lose the sixth heme ligation (Fe-Met80); it did not bind to several cyt c phospho- and acetyl-mimetics. Peptide competition assays together with molecular dynamic studies support that R1D3 binds a neoepitope within the loop 40-57. R1D3 was employed to identify alternative conformations of cyt c in cells under oxidant- or senescence-induced challenge as confirmed by immunocytochemistry and immunoaffinity studies. Alternative conformers translocated to the nuclei without causing apoptosis, an observation that was further confirmed after pinocytic loading of oxidatively modified cyt c to B16-F1 cells. Thus, alternative cyt c conformers, known to gain peroxidatic function, may represent redox messengers at the cell nuclei. The availability and properties of R1D3 open avenues of interrogation regarding the presence and biological functions of alternative conformations of cyt c in mammalian cells and tissues.

Crystal structure of Trypanosoma cruzi heme peroxidase and characterization of its substrate specificity and compound I intermediate.

The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate-binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.

The superoxide radical switch in the biology of nitric oxide and peroxynitrite.

The free radical nitric oxide (·NO) is a key mediator in different physiological processes such as vasodilation, neurotransmission, inflammation, and cellular immune responses, and thus preserving its bioavailability is essential. In several disease conditions, superoxide radical (O2·-) production increases and leads to the rapid "inactivation" of ·NO by a diffusion-controlled radical termination reaction that yields a potent and short-lived oxidant, peroxynitrite. This reaction not only limits ·NO bioavailability for physiological signal transduction but also can divert and switch the biochemistry of ·NO toward nitrooxidative processes. Indeed, since the early 1990s peroxynitrite (and its secondary derived species) has been linked to the establishment and progression of different acute and chronic human diseases and also to the normal aging process. Here, we revisit an earlier and classical review on the role of peroxynitrite in human physiology and pathology (Pacher P, Beckman J, Liaudet L. Physiol Rev 87: 315-424, 2007) and further integrate, update, and interpret the accumulated evidence over 30 years of research. Innovative tools and approaches for the detection, quantitation, and sub- or extracellular mapping of peroxynitrite and its secondary products (e.g., protein 3-nitrotyrosine) have allowed us to unambiguously connect the complex biochemistry of peroxynitrite with numerous biological outcomes at the physiological and pathological levels. Furthermore, our current knowledge of the ·NO/O2·- and peroxynitrite interplay at the cell, tissue, and organ levels is assisting in the discovery of therapeutic interventions for a variety of human diseases.

Trypanosoma cruzi Mitochondrial Peroxiredoxin Promotes Infectivity in Macrophages and Attenuates Nifurtimox Toxicity.

Trypanosoma cruzi is the causative agent of Chagas disease which is currently treated by nifurtimox (NFX) and benznidazole (BZ). Nevertheless, the mechanism of action of NFX is not completely established. Herein, we show the protective effects of T. cruzi mitochondrial peroxiredoxin (MPX) in macrophage infections and in response to NFX toxicity. After a 3-day treatment of epimastigotes with NFX, MPX content increased (2.5-fold) with respect to control, and interestingly, an MPX-overexpressing strain was more resistant to the drug. The generation of mitochondrial reactive species and the redox status of the low molecular weight thiols of the parasite were not affected by NFX treatment indicating the absence of oxidative stress in this condition. Since MPX was shown to be protective and overexpressed in drug-challenged parasites, non-classical peroxiredoxin activity was studied. We found that recombinant MPX exhibits holdase activity independently of its redox state and that its overexpression was also observed in temperature-challenged parasites. Moreover, increased holdase activity (2-fold) together with an augmented protease activity (proteasome-related) and an enhancement in ubiquitinylated proteins was found in NFX-treated parasites. These results suggest a protective role of MPX holdase activity toward NFX toxicity. Trypanosoma cruzi has a complex life cycle, part of which involves the invasion of mammalian cells, where parasite replication inside the host occurs. In the early stages of the infection, macrophages recognize and engulf T. cruzi with the generation of reactive oxygen and nitrogen species toward the internalized parasite. Parasites overexpressing MPX produced higher macrophage infection yield compared with wild-type parasites. The relevance of peroxidase vs. holdase activity of MPX during macrophage infections was assessed using conoidin A (CA), a covalent, cell-permeable inhibitor of peroxiredoxin peroxidase activity. Covalent adducts of MPX were detected in CA-treated parasites, which proves its action in vivo. The pretreatment of parasites with CA led to a reduced infection index in macrophages revealing that the peroxidase activity of peroxiredoxin is crucial during this infection process. Our results confirm the importance of peroxidase activity during macrophage infection and provide insights for the relevance of MPX holdase activity in NFX resistance.

Nox2-derived superoxide radical is crucial to control acute Trypanosoma cruzi infection.

Trypanosoma cruzi is a flagellated protozoan that undergoes a complex life cycle between hematophagous insects and mammals. In humans, this parasite causes Chagas disease, which in thirty percent of those infected, would result in serious chronic pathologies and even death. Macrophages participate in the first stages of infection, mounting a cytotoxic response which promotes massive oxidative damage to the parasite. On the other hand, T. cruzi is equipped with a robust antioxidant system to repeal the oxidative attack from macrophages. This work was conceived to explicitly assess the role of mammalian cell-derived superoxide radical in a murine model of acute infection by T. cruzi. Macrophages derived from Nox2-deficient (gp91phox-/-) mice produced marginal amounts of superoxide radical and were more susceptible to parasite infection than those derived from wild type (wt) animals. Also, the lack of superoxide radical led to an impairment of parasite differentiation inside gp91phox-/- macrophages. Biochemical or genetic reconstitution of intraphagosomal superoxide radical formation in gp91phox-/- macrophages reverted the lack of control of infection. Along the same line, gp91phox-/- infected mice died shortly after infection. In spite of the higher lethality, parasitemia did not differ between gp91phox-/- and wt animals, recapitulating an observation that has led to conflicting interpretations about the importance of the mammalian oxidative response against T. cruzi. Importantly, gp91phox-/- mice presented higher and disseminated tissue parasitism, as evaluated by both qPCR- and bioimaging-based methodologies. Thus, this work supports that Nox2-derived superoxide radical plays a crucial role to control T. cruzi infection in the early phase of a murine model of Chagas disease.

Neuronal Parasitism, Early Myenteric Neurons Depopulation and Continuous Axonal Networking Damage as Underlying Mechanisms of the Experimental Intestinal Chagas' Disease.

There is a growing consensus that the balance between the persistence of infection and the host immune response is crucial for chronification of Chagas heart disease. Extrapolation for chagasic megacolon is hampered because research in humans and animal models that reproduce intestinal pathology is lacking. The parasite-host relationship and its consequence to the disease are not well-known. Our model describes the temporal changes in the mice intestine wall throughout the infection, parasitism, and the development of megacolon. It also presents the consequence of the infection of primary myenteric neurons in culture with Trypanosoma cruzi (T. cruzi). Oxidative neuronal damage, involving reactive nitrogen species induced by parasite infection and cytokine production, results in the denervation of the myenteric ganglia in the acute phase. The long-term inflammation induced by the parasite's DNA causes intramuscular axonal damage, smooth muscle hypertrophy, and inconsistent innervation, affecting contractility. Acute phase neuronal loss may be irreversible. However, the dynamics of the damages revealed herein indicate that neuroprotection interventions in acute and chronic phases may help to eradicate the parasite and control the inflammatory-induced increase of the intestinal wall thickness and axonal loss. Our model is a powerful approach to integrate the acute and chronic events triggered by T. cruzi, leading to megacolon.

Cytosolic Fe-superoxide dismutase safeguards Trypanosoma cruzi from macrophage-derived superoxide radical.

Trypanosoma cruzi, the causative agent of Chagas disease (CD), contains exclusively Fe-dependent superoxide dismutases (Fe-SODs). During T. cruzi invasion to macrophages, superoxide radical (O2•-) is produced at the phagosomal compartment toward the internalized parasite via NOX-2 (gp91-phox) activation. In this work, T. cruzi cytosolic Fe-SODB overexpressers (pRIBOTEX-Fe-SODB) exhibited higher resistance to macrophage-dependent killing and enhanced intracellular proliferation compared with wild-type (WT) parasites. The higher infectivity of Fe-SODB overexpressers compared with WT parasites was lost in gp91-phox-/- macrophages, underscoring the role of O2•- in parasite killing. Herein, we studied the entrance of O2•- and its protonated form, perhydroxyl radical [(HO2•); pKa = 4.8], to T. cruzi at the phagosome compartment. At the acidic pH values of the phagosome lumen (pH 5.3 ± 0.1), high steady-state concentrations of O2•- and HO2• were estimated (∼28 and 8 µM, respectively). Phagosomal acidification was crucial for O2•- permeation, because inhibition of the macrophage H+-ATPase proton pump significantly decreased O2•- detection in the internalized parasite. Importantly, O2•- detection, aconitase inactivation, and peroxynitrite generation were lower in Fe-SODB than in WT parasites exposed to external fluxes of O2•- or during macrophage infections. Other mechanisms of O2•- entrance participate at neutral pH values, because the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid decreased O2•- detection. Finally, parasitemia and tissue parasite burden in mice were higher in Fe-SODB-overexpressing parasites, supporting the role of the cytosolic O2•--catabolizing enzyme as a virulence factor for CD.

Reactive species and pathogen antioxidant networks during phagocytosis.

The generation of phagosomal cytotoxic reactive species (i.e., free radicals and oxidants) by activated macrophages and neutrophils is a crucial process for the control of intracellular pathogens. The chemical nature of these species, the reactions they are involved in, and the subsequent effects are multifaceted and depend on several host- and pathogen-derived factors that influence their production rates and catabolism inside the phagosome. Pathogens rely on an intricate and synergistic antioxidant armamentarium that ensures their own survival by detoxifying reactive species. In this review, we discuss the generation, kinetics, and toxicity of reactive species generated in phagocytes, with a focus on the response of macrophages to internalized pathogens and concentrating on Mycobacterium tuberculosis and Trypanosoma cruzi as examples of bacterial and parasitic infection, respectively. The ability of pathogens to deal with host-derived reactive species largely depends on the competence of their antioxidant networks at the onset of invasion, which in turn can tilt the balance toward pathogen survival, proliferation, and virulence over redox-dependent control of infection.

Early Trypanosoma cruzi Infection Triggers mTORC1-Mediated Respiration Increase and Mitochondrial Biogenesis in Human Primary Cardiomyocytes.

Chagasic chronic cardiomyopathy is one of the most frequent and severe manifestations of Chagas disease, caused by the parasite Trypanosoma cruzi. The pathogenic and biochemical mechanisms responsible for cardiac lesions remain not completely understood, although it is clear that hypertrophy and subsequent heart dilatation is in part caused by the direct infection of cardiomyocytes. In this work, we evaluated the initial response of human cardiomyocytes to T. cruzi infection by transcriptomic profiling. Immediately after infection, cardiomyocytes dramatically change their gene expression patterns, up regulating most of the genes encoding for respiratory chain, oxidative phosphorylation and protein synthesis. We found that these changes correlate with an increase in basal and maximal respiration, as well as in spare respiratory capacity, which is accompanied by mitochondrial biogenesis pgc-1α independent. We also demonstrate that these changes are mediated by mTORC1 and reversed by rapamycin, resembling the molecular mechanisms described for the non-chagasic hypertrophic cardiomyopathy. The results of the present work identify that early during infection, the activation of mTORC1, mitochondrial biogenesis and improvement in oxidative phosphorylation are key biochemical changes that provide new insights into the host response to parasite infection and the pathogenesis of chronic chagasic cardiomyopathy. The finding that this phenotype can be reversed opens a new perspective in the treatment of Chagas disease, through the identification of host targets, and the use of combined parasite and host targeted therapies, in order to prevent chagasic cardiomyopathy.

Fluorescence and chemiluminescence approaches for peroxynitrite detection.

In the last two decades, there has been a significant advance in understanding the biochemistry of peroxynitrite, an endogenously-produced oxidant and nucleophile. Its relevance as a mediator in several pathologic states and the aging process together with its transient character and low steady-state concentration, motivated the development of a variety of techniques for its unambiguous detection and estimation. Among these, fluorescence and chemiluminescence approaches have represented important tools with enhanced sensitivity but usual limited specificity. In this review, we analyze selected examples of molecular probes that permit the detection of peroxynitrite by fluorescence and chemiluminescence, disclosing their mechanism of reaction with either peroxynitrite or peroxynitrite-derived radicals. Indeed, probes have been divided into 1) redox probes that yield products by a free radical mechanism, and 2) electrophilic probes that evolve to products secondary to the nucleophilic attack by peroxynitrite. Overall, boronate-based compounds are emerging as preferred probes for the sensitive and specific detection and quantitation. Moreover, novel strategies involving genetically-modified fluorescent proteins with the incorporation of unnatural amino acids have been recently described as peroxynitrite sensors. This review analyzes the most commonly used fluorescence and chemiluminescence approaches for peroxynitrite detection and provides some guidelines for appropriate experimental design and data interpretation, including how to estimate peroxynitrite formation rates in cells.

Cardiomyocyte diffusible redox mediators control Trypanosoma cruzi infection: role of parasite mitochondrial iron superoxide dismutase.

Chagas disease (CD), caused by the protozoa Trypanosoma cruzi, is a chronic illness in which parasites persist in the host-infected tissues for years. T. cruzi invasion in cardiomyocytes elicits the production of pro-inflammatory mediators [TNF-α, IL-1ß, IFN-γ; nitric oxide (·NO)], leading to mitochondrial dysfunction with increased superoxide radical (O2·-), hydrogen peroxide (H2O2) and peroxynitrite generation. We hypothesize that these redox mediators may control parasite proliferation through the induction of intracellular amastigote programmed cell death (PCD). In this work, we show that T. cruzi (CL-Brener strain) infection in primary cardiomyocytes produced an early (24 h post infection) mitochondrial dysfunction with H2O2 generation and the establishment of an oxidative stress evidenced by FoxO3 activation and target host mitochondrial protein expression (MnSOD and peroxiredoxin 3). TNF-α/IL-1ß-stimulated cardiomyocytes were able to control intracellular amastigote proliferation compared with unstimulated cardiomyocytes. In this condition leading to oxidant formation, an enhanced number of intracellular apoptotic amastigotes were detected. The ability of H2O2 to induce T. cruzi PCD was further confirmed in the epimastigote stage of the parasite. H2O2 treatment induced parasite mitochondrial dysfunction together with intra-mitochondrial O2·- generation. Importantly, parasites genetically engineered to overexpress mitochondrial Fe-superoxide dismutase (Fe-SODA) were more infective to TNF-α/IL-1ß-stimulated cardiomyocytes with less apoptotic amastigotes; this result underscores the role of this enzyme in parasite survival. Our results indicate that cardiomyocyte-derived diffusible mediators are able to control intracellular amastigote proliferation by triggering T. cruzi PCD and that parasite Fe-SODA tilts the process toward survival as part of an antioxidant-based immune evasion mechanism.

Cardiomyocyte oxidants production may signal to T. cruzi intracellular development.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, presents a variable clinical course, varying from asymptomatic to serious debilitating pathologies with cardiac, digestive or cardio-digestive impairment. Previous studies using two clonal T. cruzi populations, Col1.7G2 (T. cruzi I) and JG (T. cruzi II) demonstrated that there was a differential tissue distribution of these parasites during infection in BALB/c mice, with predominance of JG in the heart. To date little is known about the mechanisms that determine this tissue selection. Upon infection, host cells respond producing several factors, such as reactive oxygen species (ROS), cytokines, among others. Herein and in agreement with previous data from the literature we show that JG presents a higher intracellular multiplication rate when compared to Col1.7G2. We also showed that upon infection cardiomyocytes in culture may increase the production of oxidative species and its levels are higher in cultures infected with JG, which expresses lower levels of antioxidant enzymes. Interestingly, inhibition of oxidative stress severely interferes with the intracellular multiplication rate of JG. Additionally, upon H2O2-treatment increase in intracellular Ca2+ and oxidants were observed only in JG epimastigotes. Data presented herein suggests that JG and Col1.7G2 may sense extracellular oxidants in a distinct manner, which would then interfere differently with their intracellular development in cardiomyocytes.

Leishmania LABCG1 and LABCG2 transporters are involved in virulence and oxidative stress: functional linkage with autophagy.

BACKGROUND: The G subfamily of ABC (ATP-binding cassette) transporters of Leishmania include 6 genes (ABCG1-G6), some with relevant biological functions associated with drug resistance and phospholipid transport. Several studies have shown that Leishmania LABCG2 transporter plays a role in the exposure of phosphatidylserine (PS), in virulence and in resistance to antimonials. However, the involvement of this transporter in other key biological processes has not been studied. METHODS: To better understand the biological function of LABCG2 and its nearly identical tandem-repeated transporter LABCG1, we have generated Leishmania major null mutant parasites for both genes (ΔLABCG1-2). NBD-PS uptake, infectivity, metacyclogenesis, autophagy and thiols were measured. RESULTS: Leishmania major ΔLABCG1-2 parasites present a reduction in NBD-PS uptake, infectivity and virulence. In addition, we have shown that ΔLABCG1-2 parasites in stationary phase growth underwent less metacyclogenesis and presented differences in the plasma membrane's lipophosphoglycan composition. Considering that autophagy is an important process in terms of parasite virulence and cell differentiation, we have shown an autophagy defect in ΔLABCG1-2 parasites, detected by monitoring expression of the autophagosome marker RFP-ATG8. This defect correlates with increased levels of reactive oxygen species and higher non-protein thiol content in ΔLABCG1-2 parasites. HPLC analysis revealed that trypanothione and glutathione were the main molecules accumulated in these ΔLABCG1-2 parasites. The decrease in non-protein thiol levels due to preincubation with buthionine sulphoximide (a γ-glutamylcysteine synthetase inhibitor) restored the autophagy process in ΔLABCG1-2 parasites, indicating a relationship between autophagy and thiol content. CONCLUSIONS: LABCG1-2 transporters from Leishmania could be considered as phosphatidylserine and non-protein thiol transporters. They probably accomplish transportation in conjunction with other molecules that are involved in oxidative stress, autophagy, metacyclogenesis and infectivity processes. The overall conclusion is that LABCG1-2 transporters could play a key role in Leishmania cell survival and infectivity.

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).

The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H2O2 (k = 2.9 × 107 M-1⋅s-1) and catalytically decomposes H2O2 using Cc as the reducing substrate with higher efficiency than Asc (kcat/Km = 2.1 × 105 versus 3.5 × 104 M-1⋅s-1, respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H2O2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp233•+). Mutation of Trp233 to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp233•+, a Cys222-derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H2O2 addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp233 and Cys222 is possible and likely to participate in the catalytic cycle. Recognizing the ability of TcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). TcAPx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host-parasite interface. TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.

Sensitive detection and estimation of cell-derived peroxynitrite fluxes using fluorescein-boronate.

The specific and sensitive detection of peroxynitrite (ONOO-/ONOOH) in biological systems is a great challenge due to its high reactivity towards several biomolecules. Herein, we validated the advantages of using fluorescein-boronate (Fl-B) as a highly sensitive fluorescent probe for the direct detection of peroxynitrite under biologically-relevant conditions in two different cell models. The synthesis of Fl-B was achieved by a very simply two-step conversion synthetic route with high purity (>99%) and overall yield (∼42%). Reactivity analysis of Fl-B with relevant biological oxidants including hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and peroxynitrite were performed. The rate constant for the reaction of peroxynitrite with Fl-B was 1.7×106M-1s-1, a million times faster than the rate constant measured for H2O2 (k=1.7M-1s-1) and 2,700 faster than HOCl (6.2×102M-1s-1) at 37°C and pH 7.4. The reaction of Fl-B with peroxynitrite was significant even in the presence of physiological concentrations of CO2, a well-known peroxynitrite reactant. Experimental and simulated kinetic analyses confirm that the main oxidation process of Fl-B takes place with peroxynitrite itself via a direct bimolecular reaction and not with peroxynitrite-derived radicals. Fl-B was successfully applied for the detection of endogenously-generated peroxynitrite by endothelial cells and in macrophage-phagocyted parasites. Moreover, the generated data allowed estimating the actual intracellular flux of peroxynitrite. For instance, ionomycin-stimulated endothelial cells generated peroxynitrite at a rate of ∼ 0.1µMs-1, while immunostimulated macrophages do so in the order of ∼1µMs-1 inside T. cruzi-infected phagosomes. Fl-B revealed not to be toxic in concentrations up to 1mM for 24h. Cellular peroxynitrite detection was achieved by conventional laboratory fluorescence-based methods including flow cytometry and epi-fluorescence microscopy. Fl-B was shown to be more sensitive than the coumarin boronate due to a higher molar absorption coefficient and quantum yield. Overall, our results show that Fl-B is a kinetically selective and highly sensitive probe for the direct detection of cell-derived peroxynitrite.

Structural and molecular basis of the peroxynitrite-mediated nitration and inactivation of Trypanosoma cruzi iron-superoxide dismutases (Fe-SODs) A and B: disparate susceptibilities due to the repair of Tyr35 radical by Cys83 in Fe-SODB through intramolecular electron transfer.

Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 10(4) M(-1) s(-1) and 4.3 ± 0.4 × 10(4) M(-1) s(-1) at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr(35). Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys(83) mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys(83) present in Fe-SODB acts as an electron donor that repairs Tyr(35) radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.

A preliminary longitudinal volumetric MRI study of amygdala and hippocampal volumes in autism.

BACKGROUND: Previous studies suggest that amygdala volume, when compared with healthy controls, is increased in young children with autism, is unchanged in cohorts of older youth, and is smaller in adults. Hippocampal volume, however, does not appear to have age-related changes, and it is unclear whether individuals with autism have volumetric differences in this structure. The goal of this pilot investigation is to characterize the developmental trajectories of the amygdala and hippocampus in children with autism between the ages of 8 and 14years and to examine clinical correlates of volume change. METHODS: Twenty-three children with autism and 23 controls between the ages of 8 and 12 underwent a magnetic resonance imaging procedure of the brain (T1-weighted) at two time points. Nine children with autism and 14 controls had good quality scans from both time points; however, all usable scans from all subjects (15 children with autism and 22 controls) were included in a mixed effect analysis. Regression models were used to estimate group differences in amygdala and hippocampal volumes. Changes in amygdala and hippocampal volumes (Time 2-Time 1) were correlated with clinical severity measures. RESULTS: Amygdala volume changes with time were similar between the two groups. Within the autism group, right amygdala volume change was correlated with the ability to establish appropriate eye contact. Right hippocampal volume was significantly increased in the autism group when compared with controls. Differences in right hippocampal volume change with time between the two groups approached significance. CONCLUSION: This study provides preliminary evidence of normalization of amygdala volumes in late childhood and adolescence. It also suggests that hippocampal volumetric differences may exist in autism in late childhood and adolescence.