Catalytic mechanism of the glutathione peroxidase-type tryparedoxin peroxidase of Trypanosoma brucei

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three nearly identical genes for cysteine-homologues of the selenocysteine-containing glutathione peroxidases. The enzymes, which are essential for the parasites, lack glutathione peroxidase activity but catalyse the trypanothione/Tpx (tryparedoxin)-dependent reduction of hydroperoxides. Cys47, Gln82 and Trp137 correspond to the selenocysteine, glutamine and tryptophan catalytic triad of the mammalian selenoenzymes. Site-directed mutagenesis revealed that Cys47 and Gln82 are essential. A glycine mutant of Trp137 had 13% of wild-type activity, which suggests that the aromatic residue may play a structural role but is not directly involved in catalysis. Cys95, which is conserved in related yeast and plant proteins but not in the mammalian selenoenzymes, proved to be essential as well. In contrast, replacement of the highly conserved Cys76 by a serine residue resulted in a fully active enzyme species and its role remains unknown. Thr50, proposed to stabilize the thiolate anion at Cys47, is also not essential for catalysis. Treatment of the C76S/C95S but not of the C47S/C76S double mutant with H2O2 induced formation of a sulfinic acid and covalent homodimers in accordance with Cys47 being the peroxidative active site thiol. In the wild-type peroxidase, these oxidations are prevented by formation of an intramolecular disulfide bridge between Cys47 and Cys95. As shown by MS, regeneration of the reduced enzyme by Tpx involves a transient mixed disulfide between Cys95 of the peroxidase and Cys40 of Tpx. The catalytic mechanism of the Tpx peroxidase resembles that of atypical 2-Cys-peroxiredoxins but is distinct from that of the selenoenzymes.     

Combined prenatal and postnatal protein restriction influences adult kidney structure, function, and arterial pressure

The effects of prenatal protein restriction on adult renal and cardiovascular function have been studied in considerable detail. However, little is known about the effects of life-long protein restriction, a common condition in the developing world. Therefore, we determined in rats the effects of combined pre- and postnatal protein restriction on adult arterial pressure and renal function and responses to increased dietary sodium. Nephron number was also determined. Male Sprague-Dawley rats were born to mothers fed a low [8% (wt/wt), LP] or normal [20% (wt/wt), NP] isocaloric protein diet throughout pregnancy and maintained on these diets after birth. At postnatal day 135, nephron number, mean arterial pressure (MAP), and renal function were determined. A high-NaCl [8.0% (wt/wt), high-salt] diet was fed to a subset of rats from weaning. MAP was less in LP than in NP rats (120 +/- 2 vs. 128 +/- 2 mmHg, P < 0.05) and was not significantly altered by increased salt intake. Nephron number was 31% less in LP than in NP rats (P < 0.001). The volume of individual glomeruli was also less in LP than in NP rats, as were calculated effective renal plasma flow and glomerular filtration rate. Glomerular filtration rate, but not effective renal plasma flow, appeared to be increased by high salt intake, particularly in LP rats. In conclusion, protein restriction induced a severe nephron deficit, but MAP was lower, rather than higher, in protein-restricted than in control rats in adulthood. These findings indicate that the postnatal environment plays a key role in determining the outcomes of developmental programming.     

Left ventricular volume measurement in mice by conductance catheter: evaluation and optimization of calibration

The conductance catheter (CC) allows thorough evaluation of cardiac function because it simultaneously provides measurements of pressure and volume. Calibration of the volume signal remains challenging. With different calibration techniques, in vivo left ventricular volumes (V(CC)) were measured in mice (n = 52) with a Millar CC (SPR-839) and compared with MRI-derived volumes (V(MRI)). Significant correlations between V(CC) and V(MRI) [end-diastolic volume (EDV): R(2) = 0.85, P < 0.01; end-systolic volume (ESV): R(2) = 0.88, P < 0.01] were found when injection of hypertonic saline in the pulmonary artery was used to calibrate for parallel conductance and volume conversion was done by individual cylinder calibration. However, a significant underestimation was observed [EDV = -17.3 microl (-22.7 to -11.9 microl); ESV = -8.8 microl (-12.5 to -5.1 microl)]. Intravenous injection of the hypertonic saline bolus was inferior to injection into the pulmonary artery as a calibration method. Calibration with an independent measurement of stroke volume decreased the agreement with V(MRI). Correction for an increase in blood conductivity during the in vivo experiments improved estimation of EDV. The dual-frequency method for estimation of parallel conductance failed to produce V(CC) that correlated with V(MRI). We conclude that selection of the calibration procedure for the CC has significant implications for the accuracy and precision of volume estimation and pressure-volume loop-derived variables like myocardial contractility. Although V(CC) may be underestimated compared with MRI, optimized calibration techniques enable reliable volume estimation with the CC in mice.     

Disease-associated prion protein oligomers inhibit the 26S proteasome

The mechanism of cell death in prion disease is unknown but is associated with the production of a misfolded conformer of the prion protein. We report that disease-associated prion protein specifically inhibits the proteolytic beta subunits of the 26S proteasome. Using reporter substrates, fluorogenic peptides, and an activity probe for the beta subunits, this inhibitory effect was demonstrated in pure 26S proteasome and three different cell lines. By challenge with recombinant prion and other amyloidogenic proteins, we demonstrate that only the prion protein in a nonnative beta sheet conformation inhibits the 26S proteasome at stoichiometric concentrations. Preincubation with an antibody specific for aggregation intermediates abrogates this inhibition, consistent with an oligomeric species mediating this effect. We also present evidence for a direct relationship between prion neuropathology and impairment of the ubiquitin-proteasome system (UPS) in prion-infected UPS-reporter mice. Together, these data suggest a mechanism for intracellular neurotoxicity mediated by oligomers of misfolded prion protein.     

Role of epidermis-type lipoxygenases for skin barrier function and adipocyte differentiation

12R-lipoxygenase (12R-LOX) and epidermis-type LOX-3 (eLOX-3) are novel members of the multigene family of mammalian LOX. A considerable gap exists between the identification of these enzymes and their biologic function. Here, we present evidence that 12R-LOX and eLOX-3, acting in sequence, and eLOX-3 in combination with another, not yet identified LOX are critically involved in terminal differentiation of keratinocytes and adipocytes, respectively. Mutational inactivation of 12R-LOX and/or eLOX-3 has been found to be associated with development of an inherited ichthyosiform skin disorder in humans and genetic ablation of 12R-LOX causes a severe impairment of the epidermal lipid barrier in mice leading to post-natal death of the animals. In preadipocytes, a LOX-dependent PPARgamma activating ligand is released into the cell supernatant early upon induction of differentiation and available evidence indicates that this ligand is an eLOX-3-derived product. In accordance with this data is the observation that forced expression of eLOX-3 enhances adipocyte differentiation.     

Effects of Ethanolic Extract of Cynara cardunculus (Artichoke) Leaves on Neuroinflammatory and Neurochemical Parameters in a Diet-Induced Mice Obesity Model

Neurochemical Research - This study aimed to evaluate the effect of Cynara cardunculus leaf ethanol extract on inflammatory and oxidative stress parameters in the hypothalamus, prefrontal cortex,...