DNA Barcodes of Rosy Tetras and Allied Species (Characiformes: Characidae: Hyphessobrycon) from the Brazilian Amazon Basin

DNA barcoding can be an effective tool for fast and accurate species-level identification based on sequencing of the mitochondrial cytochrome c oxidase subunit (COI) gene. The diversity of this fragment can be used to estimate the richness of the respective species. In this study, we explored the use of DNA barcoding in a group of ornamental freshwater fish of the genus Hyphessobrycon. We sequenced the COI from 10 species of Hyphessobrycon belonging to the “Rosy Tetra Clade” collected from the Amazon and Negro River basins and combined our results with published data. The average conspecific and congeneric Kimura 2-parameter distances were 2.3% and 19.3%, respectively. Six of the 10 species were easily distinguishable by DNA barcoding (H. bentosi, H. copelandi, H. eques, H. epicharis, H. pulchrippinis, and H. sweglesi), whereas the remaining species (H. erythrostigma, H. pyrrhonotus, H. rosaceus and H. socolofi) lacked reciprocal monophyly. Although the COI gene was not fully diagnostic, the discovery of distinct evolutionary units in certain Hyphessobrycon species under the same specific epithet as well as haplotype sharing between different species suggest that DNA barcoding is useful for species identification in this speciose genus.     

Pure MnTBAP selectively scavenges peroxynitrite over superoxide: Comparison of pure and commercial MnTBAP samples to MnTE-2-PyP in two models of oxidative stress injury,an SOD-specific Escherichia coli model and carrageenan-induced pleurisy

MnTBAP is often referred to as an SOD mimic in numerous models of oxidative stress. We have recently reported that pure MnTBAP does not dismute superoxide, but commercial or poorly purified samples are able to perform O₂^·?dismutation with low-to-moderate efficacy via non-innocent Mn-containing impurities. Herein, we show that neither commercial nor pure MnTBAP could substitute for SOD enzyme in a SOD-deficient Escherichia coli model, whereas MnTE-2-PyP-treated SOD-deficient E. coli grew as well as a wild-type strain. This SOD-specific system indicates that MnTBAP does not act as an SOD mimic in vivo. In another model, carrageenan-induced pleurisy in mice, inflammation was evidenced by increased pleural fluid exudate and neutrophil infiltration and activation: these events were blocked by 0.3 mg/kg MnTE-2-PyP and, to a slightly lesser extent, by 10 mg/kg of either MnTBAP. Also, 3-nitrotyrosine formation, an indication of peroxynitrite existence in vivo, was blocked by both compounds; again MnTE-2-PyP was 33-fold more effective. Pleurisy model data indicate that MnTBAP exerts some protective actions in common with MnTE-2-PyP, which are not O₂^·? related and can be fully rationalized if one considers that the common biological role shared by MnTBAP and MnTE-2-PyP is related to their reduction of peroxynitrite and carbonate radical, the latter arising from ONOOCO₂ adduct. The log k_cat (O₂^·?) value for MnTBAP is estimated to be about 3.16, which is ~ 5 and ~ 6 orders of magnitude smaller than the SOD activities of the potent SOD mimic MnTE-2-PyP and Cu,Zn-SOD, respectively. This very low value indicates that MnTBAP is too inefficient at dismuting superoxide to be of any biological impact, which was confirmed in the SOD-deficient E. coli model. The peroxynitrite scavenging ability of MnTBAP, however, is only ~ 2.5 orders of magnitude smaller than that of MnTE-2-PyP and is not significantly affected by the presence of the SOD-active impurities in the commercial MnTBAP sample (log k_red (ONOO^?) = 5.06 for pure and 4.97 for commercial sample). The reduction of carbonate radical is equally fast with MnTBAP and MnTE-2-PyP. The dose of MnTBAP required to yield oxidative stress protection and block nitrotyrosine formation in the pleurisy model is > 1.5 orders of magnitude higher than that of MnTE-2-PyP, which could be related to the lower ability of MnTBAP to scavenge peroxynitrite. The slightly better protection observed with the commercial MnTBAP sample (relative to the pure MnTBAP) could arise from its impurities, which, by scavenging O₂^·?, reduce consequently the overall peroxynitrite and secondary ROS/RNS levels. These observations have profound biological repercussions as they may suggest that the effect of MnTBAP observed in numerous studies may conceivably relate to peroxynitrite scavenging. Moreover, provided that pure MnTBAP is unable to dismute superoxide at any significant extent, but is able to partially scavenge peroxynitrite and carbonate radical, this compound may prove valuable in distinguishing ONOO^?/CO₃^·? from O₂^·? pathways.     

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

Site‐directed spin labeling in combination with paramagnetic relaxation enhancement (PRE) measurements is one of the most promising techniques for studying unfolded proteins. Since the pioneering work of Gillespie and Shortle (J Mol Biol 1997;268:158), PRE data from unfolded proteins have been interpreted using the theory that was originally developed for rotational spin relaxation. At the same time, it can be readily recognized that the relative motion of the paramagnetic tag attached to the peptide chain and the reporter spin such as ¹H^N is best described as a translation. With this notion in mind, we developed a number of models for the PRE effect in unfolded proteins: (i) mutual diffusion of the two tethered spheres, (ii) mutual diffusion of the two tethered spheres subject to a harmonic potential, (iii) mutual diffusion of the two tethered spheres subject to a simulated mean‐force potential (Smoluchowski equation); (iv) explicit‐atom molecular dynamics simulation. The new models were used to predict the dependences of the PRE rates on the ¹H^N residue number and static magnetic field strength; the results are appreciably different from the Gillespie–Shortle model. At the same time, the Gillespie–Shortle approach is expected to be generally adequate if the goal is to reconstruct the distance distributions between ¹H^N spins and the paramagnetic center (provided that the characteristic correlation time is known with a reasonable accuracy). The theory has been tested by measuring the PRE rates in three spin‐labeled mutants of the drkN SH3 domain in 2M guanidinium chloride. Two modifications introduced into the measurement scheme—using a reference compound to calibrate the signals from the two samples (oxidized and reduced) and using peak volumes instead of intensities to determine the PRE rates—lead to a substantial improvement in the quality of data. The PRE data from the denatured drkN SH3 are mostly consistent with the model of moderately expanded random‐coil protein, although part of the data point toward a more compact structure (local hydrophobic cluster). At the same time, the radius of gyration reported by Choy et al. (J Mol Biol 2002;316:101) suggests that the protein is highly expanded. This seemingly contradictory evidence can be reconciled if one assumes that denatured drkN SH3 forms a conformational ensemble that is dominated by extended conformations, yet also contains compact (collapsed) species. Such behavior is apparently more complex than predicted by the model of a random‐coil protein in good solvent/poor solvent.     

β-Endorphin like immunoreactivity of brain,pituitary gland and plasma of rats submitted to postnatal protein malnutrition: Effect of behavioral training

Wistar-derived rats were raised and maintained either on a normal- (25% casein) or on a low-protein (8% casein) diet until the age of 100 to 114 days. Both diets were isocaloric and contained an adequate supply of salts and vitamins. There were gross differences in body, brain and pituitary weight between the two groups. In addition, the brain and pituitary content of β-endorphin like immunoreactivity was lower in the protein malnourished rats, and three different forms of training (50 tone-footshock shuttle avoidance trials; 50 tones alone (habituation); 50 footshocks alone) caused a depletion of brain β-endorphin like immunoreactivity in the normal, but not in the malnourished rats. Footshock stimulation caused, in addition, a pituitary decrease and a plasma increase of β-endorphin like immunoreactivity, also restricted to the normal diet group. Performance in the habituation and in the shuttle avoidance tasks was similar in the two groups, despite the different responsiveness of their brain and pituitary β-endorphin systems to training and/or stimulation. In view of the possible involvement of these systems in learning suggested by these and by previous data, it seems likely that the neurohumoral regulation of habituation and avoidance learning may be different in rats submitted to protein malnutrition when compared to controls.     

Selenium status of patients with Balkan endemic nephropathy

The selenium (Se) contents in common cereals in endemic and nonendemic areas in Serbia are very low. Plasma Se levels of both patients and healthy subjects, were also low, reflecting low Se intakes. Patients with Balkan endemic nephropathy (BEN) had significantly lower (p<0.05) plasma Se levels than healthy individuals, both from regions close to endemic areas, and from Belgrade. Mean plasma Se of BEN patients was slightly but insignificantly higher in samples taken immediately after dialysis than in those taken before, suggesting that very little of the Se present in plasma is dialyzable. Plasma SeGSH-Px activities before and after hemodialysis in both BEN and Nonendemic chronic renal failure (NCRF) patients were not significantly different, but BEN patients had lower enzyme activities than those with NCRF and healthy controls. In BEN patients, a significant correlation between plasma Se and SeGSH-Px activity was found. NCFR patients were with diagnoses: TBC of kidneys, chronic glomerulonephritis, chronic pyelonephritis, and polycystic kidneys.     

Detection of Anaplasma bovis in an undescribed tick species collected from the eastern rock sengi Elephantulus myurus

Ticks are important vectors of numerous pathogens causing illness, fatalities, and economic loss worldwide. Infectious disease episodes are increasing, and novel tick-borne pathogens are described frequently. Identification of novel reservoir hosts and vectors of tick-borne pathogens is essential if control measures are to be successful. In South Africa, the eastern rock sengi, Elephantulus myurus , hosts a number of tick species of veterinary importance. Despite this, there remains a paucity of information regarding the tick fauna of this species, the pathogen associations of ticks that it hosts, and its role as a reservoir host of tick-borne pathogens. The current study documents the tick fauna of E. myurus and sympatric small mammal species in Limpopo Province, South Africa. The pathogen associations of ticks hosted by elephant shrews were also investigated by PCR screening of engorged nymphs for a broad range of bacterial and protozoan tick-borne infections, including Borrelia burgdorferi sensu lato and members of Apicomplexa and the order Rickettsiales. There were marked differences in tick species and abundance among host species. Elephantulus myurus was heavily, and predominantly, parasitized by an as-yet undescribed tick species that we identify as Rhipicephalus sp. near warburtoni. PCR and sequence analysis revealed the presence of Anaplasma bovis in this tick species, which may have consequences for livestock production and conservation efforts in the area where this tick species occurs.