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.

Hyperbaric oxygenation applied immediately after coronary occlusion reduces myocardial necrosis and acute mortality in rats.

1. Because in ischaemia there is a critical lack of O2, it has been reasoned that increasing O2 delivery to the ischaemic myocardium could serve as adjunctive therapy for acute myocardial infarction (MI). Accordingly, in the present study, the effect of early hyperbaric oxygenation (HBO) on mortality and MI size after coronary occlusion was examined in rats. 2. After coronary occlusion, male Wistar rats were randomly assigned to receive either HBO for 1 h in a hyperbaric chamber (100% O(2) at 253 kPa; n = 106) or ambient O2 as the control (n = 111). The extent of myocardial necrosis was assessed (triphenyltetrazolium) immediately after treatment in the HBO (n = 50) and control (n = 47) groups. The remaining rats were evaluated 24 h after occlusion to enable calculation of MI size and mortality. 3. Immediately after therapy, the size of the MI was significantly greater in the control group compared with that in the HBO group (40 +/- 3 vs 27 +/- 2% of the left ventricle (LV), respectively; P < 0.001). The 24 h mortality of control rats was higher than that of HBO rats (34 vs 16%, respectively; P = 0.02). Control rats that survived 24 h had a larger MI than did HBO rats that survived 24 h (40 +/- 4 vs 29 +/- 3% of the LV, respectively; P = 0.005). Furthermore, large necrotic areas (> 40% of the LV) were more frequent in control than HBO rats (55 vs 27% of infarcted hearts, respectively; P = 0.01). There was less pulmonary congestion observed in HBO rats compared with control rats. 4. In conclusion, early therapy with HBO during the onset of an acute ischaemic event decreases the necrotic area and reduces acute mortality. These data support further investigation of HBO as an adjuvant therapy for acute MI.

En búsqueda de la esencia de la etonografía / In search for the essence of ethnography

In june 16, 2003 Harry Wolcott gave The Bornemouth talk revisited for Medellín lecture. The talk deals with the search for the essence of ethnography and answers the question what makes a study ethnographic? The lecturer in his carreer has identified twelve attributes that can reasonably be expected in a ethnographic report. Therefore ethnography is holistic, comparative, of first-hand experience, conducted in natural setting, requires intimate and long-term acquaintance, is non evaluative, basically descriptive, specific, adaptive, corroborative and finally idiosyncratic and individualistic.All those attributes are discussed in the lecture with the warning that there is no absolute set of attributes. Then he could conclude that anyone can borrow the research techniques, and we all draw our data from everyday experience, both our own and of others. The result is far more likely to produce ethnography when a researcher sets out to create ethnography and has a clear idea of what is involved.