Circulating leukocyte heat shock protein 70 (HSP70) and oxidative stress markers in rats after a bout of exhaustive exercise

A novel method to measure oxidative stress resulting from exhaustive exercise in rats is presented. In this new procedure we evaluated the erythrocyte antioxidant enzymes, catalase ( CAT) and glutathione reductase (GR), the plasma oxidative attack markers, reactive carbonyl derivatives (RCD) and thiobarbituric reactive substances (TBARS). Muscular tissue damage was evaluated by monitoring plasma creatine kinase (CK) and plasma taurine ( Tau) concentrations. Also, we monitored total sulphydryl groups (TSG) and uric acid (UA), and the level of the 70 kDa heat shock protein (HSP70) in leukocytes as a marker of oxidative stress. In the study we found a correspondence between erythrocyte CAT and GR activities and leukocyte HSP70 levels, principally 3 h after the acute exercise, and this suggested an integrated mechanism of antioxidant defense. The increase in levels of plasma Tau was coincident with the increasing plasma levels of CK and TBARS, principally after two hours of exercise. Thus tissue damage occurred before the expression of any anti-oxidant system markers and the monitoring of Tau, CK or TBARS may be important for the estimation of oxidative stress during exhaustive exercise. Furthermore, the integrated analyses could be of value in a clinical setting to quantify the extent of oxidative stress risk and reduce the need to perform muscle biopsies as a tool of clinical evaluation.

Characterization of a new platelet aggregating factor from crotoxin Crotalus durissus cascavella venom

In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA(2), crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 mu M/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA(2), thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.

Free- and peptide-based dietary arginine supplementation for the South American fish pacu (Piaractus mesopotamicus)

Arginine was hypothesized to be a model compound in the present study on molecular forms of indispensable amino acid (IAA) dietary supplementation. Juvenile South American pacu (Piaractus mesopotamicus) were fed diets containing arginine in a protein base (casein-wheat gluten or casein-gelatin), or the casein-wheat gluten base supplemented with dipeptide or free arginine at two levels (5 and 10 g kg(-1)). Growth and protein efficiency ratios were significantly affected by diets, but not by arginine molecular form. Three free dispensable amino acids (DAA) and four IAA in plasma were affected by diet, but plasma arginine concentrations did not differ. Plasma urea concentrations, being very low in the pacu, and hepatic arginase activities, were not affected by diet (P = 0.10-0.11), but together with plasma ornithine, mirrored the growth data. Molecular form of arginine supplementation, free or dipeptide, significantly changed several free IAA (Phe, Leu, Ile, His) and urea, with a higher mean plasma concentration in dipeptide fed fish. The dietary treatments, or molecular form of the arginine supplementation, did not change proximate composition, except that calcium levels decreased with higher dietary arginine supplementation level. The present study indicates that dipeptides can provide IAA to pacu, and that arginine supplemented in this form is utilized as efficiently as in free form.

Amino acid composition of processed fish silage using different raw materials

The objective was to evaluate amino acid composition of silages produced from three raw materials. Commercial marine fish waste, commercial freshwater fish waste, and tilapia filleting residue were used to produce fish silage by acid digestion (20 ml/kg formic acid and 20 ml/kg sulfuric acid) and anaerobic fermentation (50 g/kg Lactobacillus plantarum, 150 g/kg sugar cane molasses). Protein content and amino acid composition were determined for raw materials and silage. Marine fish waste had higher crude protein content (776.7 g/kg) compared to freshwater fish waste (496.2 g/kg) and tilapia filleting residue (429.9 g/kg). All silages lacked up to three amino acids for each product according to FAO standards for essential amino acids. However, considering as the limiting factor only the amino acids below the 30% minimum requirement for fish in general, all products were satisfactory with respect to essential amino acids. Therefore, the results suggest that all products investigated are appropriate for use in balanced fish diets. (C) 2003 Elsevier B.V. B.V. All rights reserved.