Glutathione reductase fromSaccharomyces cerevisiae undergoes redox interconversionin situ andin vivo

Redox interconversion of glutathione reductase was studiedin situ withS. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 μM NADPH or 100 μM NADH caused 50% inactivation in normal cells in 30 min, 0.75 μM NADPH or 50 μM NADH promoted a similar effect in starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP⁺ concentrations. The inactivation of yeast glutathione reductase has also been studiedin vivo. The activity decreased to 75% after 2 hours of growth with glucono-δ-lactone as carbon source, while NADPH rose to 144% and NADP⁺ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 μM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD⁺ and NADPH/NADP⁺ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-δ-lactone is oxidized promotesin vivo inactivation of glutathione reductase.     

Cardiovascular and ventilatory interactions in the facultative air-breathing teleost Pangasianodon hypophthalmus

Journal of Comparative Physiology B - All vertebrates possess baroreceptors monitoring arterial blood pressure and eliciting reflexive changes in vascular resistance and heart rate in response to...     

Combinatorial peptide library screening for discovery of diverse α-glucosidase inhibitors using molecular dynamics simulations and binary QSAR models

Human α-glucosidase is an enzyme involved in the catalytic cleavage of the glucoside bond and involved in numerous functionalities of the organism, as well as in the insurgence of diabetes mellitus 2 and obesity. Thus, developing chemicals that inhibit this enzyme is a promising approach for the treatment of several pathologies. Small peptides such as di- and tri-peptides may be in natural organism as well as in the GI tract in high concentration, coming from the digestive process of meat, wheat and milk proteins. In this work, we reported the first tentative hierarchical structure-based virtual screening of peptides for human α-glucosidase. The goal of this work is to discover novel and diverse lead compounds that my act as inhibitors of α-glucosidase such as small peptides by performing a computer aided virtual screening and to find novel scaffolds for further development. Thus, in order to select novel candidates with original structure we performed molecular dynamics (MD) simulations among the 12 top-ranked peptides taking as comparison the MD simulations performed on crystallographic inhibitor acarbose. The compounds with the lower RMSD variability during the MD, were reserved for in vitro biological assay. The selected 4 promising structures were prepared on solid phase peptide synthesis and used for the inhibitory assay, among them compound 2 showed good inhibitory activity, which validated our method as an original strategy to discover novel peptide inhibitors. Moreover, pharmacokinetic profile predictions of these 4 peptides were also carried out with binary QSAR models using MetaCore/MetaDrug applications.     

Metabolic efficiency of liver mitochondria in rats with decreased thermogenesis

We have studied changes in hepatic mitochondrial efficiency induced by 24-h fasting or acclimation at 29 degrees C, two conditions of reduced thermogenesis. Basal and palmitate-induced proton leak, which contribute to mitochondrial efficiency, are not affected after 24-h fasting, when serum free triiodothyronine decreases significantly and serum free fatty acids increase significantly. In rats at 29 degrees C, in which serum free triiodothyronine and fatty acids decrease significantly, basal proton leak increases significantly, while no variation is found in palmitate-induced proton leak. The present results indicate that mitochondrial efficiency in the liver is not related to a physiological decrease in whole body thermogenesis.     

Immune Reactions against Gene Gun Vaccines Are Differentially Modulated by Distinct Dendritic Cell Subsets in the Skin

The skin accommodates multiple dendritic cell (DC) subsets with remarkable functional diversity. Immune reactions are initiated and modulated by the triggering of DC by pathogen-associated or endogenous danger signals. In contrast to these processes, the influence of intrinsic features of protein antigens on the strength and type of immune responses is much less understood. Therefore, we investigated the involvement of distinct DC subsets in immune reactions against two structurally different model antigens, E. coli beta-galactosidase (betaGal) and chicken ovalbumin (OVA) under otherwise identical conditions. After epicutaneous administration of the respective DNA vaccines with a gene gun, wild type mice induced robust immune responses against both antigens. However, ablation of langerin⁺ DC almost abolished IgG1 and cytotoxic T lymphocytes against betaGal but enhanced T cell and antibody responses against OVA. We identified epidermal Langerhans cells (LC) as the subset responsible for the suppression of anti-OVA reactions and found regulatory T cells critically involved in this process. In contrast, reactions against betaGal were not affected by the selective elimination of LC, indicating that this antigen required a different langerin⁺ DC subset. The opposing findings obtained with OVA and betaGal vaccines were not due to immune-modulating activities of either the plasmid DNA or the antigen gene products, nor did the differential cellular localization, size or dose of the two proteins account for the opposite effects. Thus, skin-borne protein antigens may be differentially handled by distinct DC subsets, and, in this way, intrinsic features of the antigen can participate in immune modulation.     

Hypoxic cardiorespiratory reflexes in the facultative air-breathing fish jeju (Hoplerythrinus unitaeniatus): role of branchial O2 chemoreceptors

In one series of experiments, heart frequency (f _H), blood pressure (P _a), gill ventilation frequency (f _R ), ventilation amplitude (V _AMP) and total gill ventilation (V _TOT) were measured in intact jeju (Hoplerythrinus unitaeniatus) and jeju with progressive denervation of the branchial branches of cranial nerves IX (glossopharyngeal) and X (vagus) without access to air. When these fish were submitted to graded hypoxia (water PO₂ ~140, normoxia to 17 mmHg, severe hypoxia), they increased f _R , V _AMP, V _TOT and P _a and decreased f _H. In a second series of experiments, air-breathing frequency (f _RA), measured in fish with access to the surface, increased with graded hypoxia. In both series, bilateral denervation of all gill arches eliminated the responses to graded hypoxia. Based on the effects of internal (caudal vein, 150 μg NaCN in 0.2 mL saline) and external (buccal) injections of NaCN (500 μg NaCN in 1.0 mL water) on f _R , V _AMP, V _TOT, P _a and f _H we conclude that the O₂ receptors involved in eliciting changes in gill ventilation and associated cardiovascular responses are present on all gill arches and monitor the O₂ levels of both inspired water and blood perfusing the gills. We also conclude that air breathing arises solely from stimulation of branchial chemoreceptors and support the hypothesis that internal hypoxaemia is the primary drive to air breathing.     

Gill chemoreceptors and cardio-respiratory reflexes in the neotropical teleost pacu, <Emphasis Type="Italic">Piaractus mesopotamicus</Emphasis>

This study examined the location and distribution of O₂ chemoreceptors involved in cardio-respiratory responses to hypoxia in the neotropical teleost, the pacu (Piaractus mesopotamicus). Intact fish and fish experiencing progressive gill denervation by selective transection of cranial nerves IX and X were exposed to gradual hypoxia and submitted to intrabuccal and intravenous injections of NaCN while their heart rate, ventilation rate and ventilation amplitude were measured. The chemoreceptors producing reflex bradycardia were confined to, but distributed along all gill arches, and were sensitive to O₂ levels in the water and the blood. Ventilatory responses to all stimuli, though modified, continued following gill denervation, however, indicating the presence of internally and externally oriented receptors along all gill arches and either in the pseudobranch or at extra-branchial sites. Chemoreceptors located on the first pair of gill arches and innervated by the glossopharyngeal nerve appeared to attenuate the cardiac and respiratory responses to hypoxia. The data indicate that the location and distribution of cardio-respiratory O₂ receptors are not identical to those in tambaqui (Colossoma macropomum) despite their similar habitats and close phylogenetic lineage, although the differences between the two species could reduce to nothing more than the presence or absence of the pseudobranch.