[Mathematical modeling of kinetics of the tegumental-socket gastric epithelium].

With the aim of formalization of gastric epithelium kinetics estimation the morphometric analysis of gastric mucosa bioptates was performed in 26 patients without endoscopic signs of stomach pathology. During step estimation of gastric pits at different depth authors analyzed changes of cells size, nucleocytoplasmic ratio, coefficient of nucleus elongation in immature cells of pit bottom (11 zone), differentiating cells in pit intermediate zone (L2), and on mucosa surface (L3 zone). Authors used claster and neuronet analysis (by Statistica and MedStat) to make the mathematical model which permit to determine the proportion of functional zones in normal pit (L--33 +/- 3%; L2--27 +/- 3%; L1--40 +/- 3% of gastric pit depth) and to develop the system of cell standards for each zone. These results can be used to diagnostic of gastric epithelium differentiation changes under pathology and to interpret the barrier properties of gastric epithelium.

Bothrops insularis venomics: a proteomic analysis supported by transcriptomic-generated sequence data

A joint transcriptomic and proteomic approach employing two-dimensional electrophoresis, liquid chromatography and mass spectrometry was carried out to identify peptides and proteins expressed by the venom gland of the snake Bothrops insularis, an endemic species of Queimada Grande Island, Brazil. Four protein families were mainly represented in processed spots, namely metalloproteinase, serine proteinase, phospholipase A2 and lectin. Other represented families were growth factors, the developmental protein G10, a disintegrin and putative novel bradykinin-potentiating peptides. The enzymes were present in several isoforms. Most of the experimental data agreed with predicted values for isoelectric point and Mr of proteins found in the transcriptome of the venom gland. The results also support the existence of posttranslational modifications and of proteolytic processing of precursor molecules which could lead to diverse multifunctional proteins. This study provides a preliminary reference map for proteins and peptides present in Bothrops insularis whole venom establishing the basis for comparative studies of other venom proteomes which could help the search for new drugs and the improvement of venom therapeutics. Altogether, our data point to the influence of transcriptional and post-translational events on the final venom composition and stress the need for a multivariate approach to snake venomics studies.

BnP1, a novel P-I metalloproteinase from Bothrops neuwiedi venom: biological effects benchmarking relatively to jararhagin, a P-III SVMP.

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding.

Chronic dynamic exercise increases apolipoprotein A-I expression in rabbit renal cortex as determined by proteomic technology.

OBJECTIVE: We have shown previously that exercise training enhances endothelium-dependent and endothelium-independent vascular relaxation in rabbit kidney. This study aimed to investigate protein expression changes in the rabbit renal cortex induced by chronic dynamic exercise. DESIGN: Kidneys were obtained from New Zealand rabbits either confined to pens (n = 8) or trained on a treadmill (0% grade) for 5 days/week at a speed of 18 m/min for 60-min periods over 12 weeks (n = 8). Expression of proteins in the renal cortex was determined by colloidal Coomassie blue staining after two-dimensional polyacrylamide gel electrophoresis. Differential protein spots were excised and digested with trypsin, and peptides were sequenced by electrospray ionization-ion trap mass spectrometry. RESULTS: Two pairs of matching differentially stained spots displayed an approximate threefold increase in trained compared with sedentary animals. These four spots presented a molecular mass of 23 kDa but different pI values. Mass spectrometric analyses revealed the pairs of matching spots as being rabbit apolipoprotein A-I. CONCLUSION: Chronic dynamic exercise increases apolipoprotein A-I expression in the rabbit renal cortex. This fact could be involved in the alterations observed in the renal circulation after exercise training.

Cytosolic glutathione peroxidase from liver of pacu (Piaractus mesopotamicus), a hypoxia-tolerant fish of the Pantanal.

Pacu (Piaractus mesopotamicus Holmberg, 1887, Characiformes) dwells in waters of Pantanal, in which it has adapted for alternate concentrations of dissolved oxygen. Intracellular antioxidant protection should be vital for such an adaptation. Accordingly, we found that cytosol from liver of pacu has the highest antioxidant glutathione peroxidase activity so far reported for fish and murine species. To clarify whether this activity was due to a selenium independent glutathione S-transferase or to a glutathione peroxidase, we purified it and studied its kinetics. The substrates cumene hydroperoxide and hydrogen peroxide were promptly reduced by the enzyme, but peroxidized phosphatidylcholine had to undergo previous fatty acid removal with phospholipase A(2). Augmenting concentrations (from 2 to 6 mM) of reduced glutathione activated the pure enzyme. Curves of velocity versus different micromolar concentrations of hydrogen peroxide in the presence of 2, 4 or 8 mM reduced glutathione indicated that at least 2.5 mM reduced glutathione should be available in vivo for an efficient continuous destruction of micromolar concentrations of hydrogen peroxide by this peroxidase. Molecular exclusion HPLC and SDS-polyacrylamide gel electrophoresis indicated that the purified peroxidase is a homotetramer. Data from internal sequences showed selenocysteine in its primary structure and that the enzyme was a homologue of the type-1 glutathione peroxidase found in rat, bull, trout, flounder and zebra fish. Altogether, our data establish that in liver cells of pacu, a hypoxia-tolerant fish from South America, there are high levels of a cytosolic GPX-1 capable of quenching hydrogen peroxide and fatty acid peroxides, providing an effective antioxidant action.