Purification and characterization of jararassin-I, a thrombin-like enzyme from Bothrops jararaca snake venom

A thrombin-like serine protease, jararassin-I, was isolated from the venom of Bothrops jararaca. The protein was obtained in high yield and purity by a single chromatographic step using the affinity resin Benzamidine-Sepharose CL-6B. SDS-PAGE and dynamic light scattering analyses indicated that the molecular mass of the enzyme was about 30 kD. The enzyme possessed fibrinogenolytic and coagulant activities. The jararassin-I degraded the Bbeta chain of fibrinogen while the Aalpha chain and gammachain were unchanged. Proteases inhibitors, PMSF and benzamidine inhibited the coagulant activity. These results showed jararassin-I is a serine protease similar to coagulating thrombin-like snake venom proteases, but it specifically cleaves Bbeta chain of bovine fibrinogen. Single crystals of enzyme were obtained (0.2 mmx0.2 mmx0.2 mm) and used for X-ray diffraction experiments.

REEVALUATION OF CHLORIDES REGULATION OF HEMOGLOBIN OXYGEN-UPTAKE - THE NEGLECTED CONTRIBUTION OF PROTEIN HYDRATION IN ALLOSTERISM

We have measured hemoglobin oxygen uptake vs. The partial pressure of oxygen, with independently controlled activities of chloride and water. This control is effected by combining different concentrations of NaCl and sucrose in the bathing solution to achieve: (i) water activities were varied and CI- activity was fixed, (ii) both water and CI- activities were varied with a traditional NaCI titration, or (iii) CI- activities were varied and water activity was fixed by adding compensating sucrose. Within this analysis, the CI--regulated loading of four oxygens can be described by the reaction Hb.CI- + 4 O-2 + 65 H2O reversible arrow Hb.4O(2).65H(2)O + CI-. The dissociation of a neatly integral chloride, rather than the nonintegral 1.6 chlorides inferred earlier from simple salt titration, demonstrates the need to recognize the potentially large contribution from changes in water activity when titrating weakly binding solutes. The single-chloride result might simplify structural considerations of the action of CI- in hemoglobin regulation.

Structural and functional characterization of myotoxin I, a Lys49 phospholipase A(2) homologue from Bothrops moojeni (Caissaca) snake venom

Myotoxin-I (MjTX-I) was purified to homogeneity from the venom of Bothrops moojeni by ion-exchange chromatography on CM-Sepharose. Its molecular weight, estimated by SDS-PAGE, was 13,400 (reduced) or 26,000 (unreduced). The extinction coefficient (E-1.0 cm(1.0 mg/ml)) of MjTX-I was 1.145 at lambda = 278 nm, pH 7.0, and its isoelectric point was 8.2 at ionic strength mu = 0.1. When lyophilized and stored at 4 degrees C, dimeric, trimeric, and pentameric forms of the protein were identified by SDS-PAGE. This heterogeneous sample could be separated into three fractions by gel filtration on Sephadex 6-50. The fractions were analyzed by isoelectric focusing, immunoelectrophoresis, and amino acid composition, which indicated that heterogeneity was the result of different levels of self-association. Protein sequencing indicated that MjTX-I is a Lys49 myotoxin and consists of 121 amino acids (M-r = 13,669), containing a high proportion of basic and hydrophobic residues. It shares a high degree of sequence identity with other Lys49 PLA(2)-like myotoxins, but shows a significantly lower identity with catalytically active Asp49 PLA(2)s. The three-dimensional structure of MjTX-I was modeled based on the crystal structures of three highly homologous Lys49 PLA(2)-like myotoxins. This model showed that the amino acid substitutions are conservative, and mainly the beta-wing region, and the C-terminal extended random coil. MjTX-I displays local myotoxic and edema-inducing activities in mice, and is lethal by intraperitoneal injection, with an LD50 value of 8.5 +/- 0.8 mg/kg, In addition, it is cytotoxic to myoblasts/ myotubes in culture, and disrupts negatively charged liposomes. In comparison with the freshly prepared dimeric sample, the more aggregated forms showed significantly reduced myotoxic activity. However, the edema-inducing activity of MjTX-I was independent of molecular association. Phospholipase A(2) activity on egg yolk, as well as anticoagulant activity, were undetectable both in the native and in the more associated forms. His, Tyr, and Trp residues of the toxin were chemically modified by specific reagents. Although the myotoxic and lethal activities of the modified toxins were reduced by these treatments, neither its edema-inducing or Liposome-disrupting activities were significantly altered. Rabbit antibodies to native MjTX-I cross-reacted with the chemically modified forms, and both the native and modified MjTX-I preparations were recognized by antibodies against the C-terminal region 115-129 of myotoxin II from B. asper, a highly Lys49 PLA(2)-homologue with high sequencial similarity. (C) 2000 Academic Press.

Assignment of the disulfide bridges in bothropstoxin-I, a myonecrotic Lys49 PLA(2) homolog from Bothrops jararacussu snake venom

Bothropstoxin-I (BthTX-1), a Lys49 phospholipase A(2) homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV8) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV8 digests: Cys50-Cys131, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cys105 by determination of the crystal structure, showing that BthTX-1 disulfide bonds follow the normal pattern of group II PLA(2)s.

Changes in plasma protein concentrations in ponies with experimentally induced alimentary laminitis

Objective-To determine whether plasma protein concentrations were altered in ponies with alimentary laminitis.Animals-12 adult ponies.Procedure-Acute laminitis was induced in 6 ponies by oral administration of carbohydrate (85% corn starch, 15% wood flour); the other 6 ponies were used as controls. A physical examination was performed and blood samples were collected immediately before and 4, 8, 12, 24, and 28 hours after administration of carbohydrate. Plasma protein concentrations were determined by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis.Results-19 plasma proteins ranging from a molecular weight of 24,000 to a molecular weight of 350,000 were identified in all 12 ponies. Plasma concentrations of proteins with molecular weights of 350,000 (fibrinogen), 130,000 (ceruloplasmin), 118,000 (c-reactive protein), 67,000 (alpha(1)-antitrypsin I), 65,000 (alpha(1)-antitrypsin II), 50,000 (haptoglobulin), and 45,000 (acid glycoprotein) were significantly increased in ponies with laminitis, compared with concentrations in control ponies.Conclusion-Changes in plasma protein concentrations are detectable within 4 hours after the onset of alimentary laminitis in ponies.Clinical Relevance-Measurement of plasma protein concentrations may be useful in monitoring the progression of laminitis in ponies.

Energy and protein requirements for maintenance and growth of Boer crossbred kids

Meat production by goats has become an important livestock enterprise in several parts of the world. Nonetheless, energy and protein requirements of meat goats have not been defined thoroughly. The objective of this study was to determine the energy and protein requirements for maintenance and growth of 34 3/4 Boer x 1/4 Saanen crossbred, intact male kids (20.5 +/- 0.24 kg of initial BW). The baseline group was 7 randomly selected kids, averaging 21.2 +/- 0.36 kg of BW. An intermediate group consisted of 6 randomly selected kids, fed for ad libitum intake, that were slaughtered when they reached an average BW of 28.2 +/- 0.39 kg. The remaining kids (n = 21) were allocated randomly on d 0 to 3 levels of DMI (treatments were ad libitum or restricted to 70 or 40% of the ad libitum intake) within 7 slaughter groups. A slaughter group contained 1 kid from each treatment, and kids were slaughtered when the ad libitum treatment kid reached 35 kg of BW. Individual body components (head plus feet, hide, internal organs plus blood, and carcass) were weighed, ground, mixed, and subsampled for chemical analyses. Initial body composition was determined using equations developed from the composition of the baseline kids. The calculated daily maintenance requirement for NE was 77.3 +/- 1.05 kcal/kg(0.75) of empty BW (EBW) or 67.4 +/- 1.04 kcal/kg(0.75) of shrunk BW. The daily ME requirement for maintenance (118.1 kcal/g(0.75) of EBW or 103.0 kcal/kg(0.75) of shrunk BW) was calculated by iteration, assuming that the heat produced was equal to the ME intake at maintenance. The partial efficiency of use of ME for NE below maintenance was 0.65. A value of 2.44 +/- 0.4 g of net protein/kg(0.75) of EBW for daily maintenance was determined. Net energy requirements for growth ranged from 2.55 to 3.0 Mcal/kg of EBW gain at 20 and 35 kg of BW, and net protein requirements for growth ranged from 178.8 to 185.2 g/kg of EBW gain. These results suggest that NE and net protein requirements for growing meat goats exceed the requirements previously published for dairy goats. Moreover, results from this study suggest that the N requirement for maintenance for growing goats is greater than the established recommendations.

Magneto Immunoassays for Plasmodium falciparum Histidine-Rich Protein 2 Related to Malaria based on Magnetic Nanoparticles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Polysiloxane-poly(propylene oxide) hybrid discs as solid phase in anti-HCV detection using a recombinant core protein

In this work, siloxane-poly(propylene oxide) discs (PPO disc) prepared using the sol-gel process were used as solid phase in enzyme-linked immunosorbent assays (ELISA) for the detection of anti-hepatitis C virus (HCV) antibodies. The HCV RNA from serum (genotype 1b) was submitted to the RT-PCR technique and subsequent amplification of the HCV core 408 pb. This fragment was cloned into expression vector pET42a and expressed in Escherichia coli as recombinant protein with glutathione S-transferase (GST). Cell cultures were grown and induced having a final concentration of 0.4 x 10(-3) mol L-1 of IPTG. After induction, the cells were harvested and the soluble fraction was analyzed using polyacrilamide gel 15% showing a band with an approximate molecular weight of 44 kDa, the expected size for this GST-fused recombinant protein. The recombinant protein was purified and continued by immunological detection using HCV-positive serum and showed no cross-reactivity with positive samples for other infectious diseases. An ELISA was established using 1.25 ng of recombinant protein per PPO disc, a dilution of 1: 10,000 and 1:40 for a peroxidase conjugate and serum, respectively, and solutions of hydrogen peroxide and 3,3',5,5'-tetra-methylbenzidine in a ratio of 1: 1. The proposed methodology was compared with the ELISA conventional polystyrene-plate procedure and the performance of the PPO discs as a matrix for immunodetection gave an easy synthesis, good performance and reproducibility for commercial application. (c) 2007 Elsevier B.V. All rights reserved.

A disposable chitosan-modified carbon fiber electrode for dengue virus envelope protein detection

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Protein diets promote the maturation of oocytes and spawning of Piaractus mesopotamicus kept in cages

The objective of this study was to evaluate the effects of balanced diets on the maturation of oocytes and the reproductive performance of P. mesopotamicus in cages. A completely random design with 224 fish in 16 cages measuring 5 m(3) was employed for this purpose. The treatments consisted of diets containing 18, 24, 30, and 36% crude protein (CP) provided ad libitum. The external and internal morphological characteristics of the specimens were examined, as well as: the position of the germinal vesicle, the distribution of oocyte diameters, the fertilization and hatching rates, the number of oocytes released, the total number of oocytes, the remaining weight and total weight of the ovaries, the gonadosomatic index, the condition factor (K), and the histology of the oocytes and ovaries post-spawning and during ovarian regression. The diameters of the oocytes collected before the first hormonal application displayed a unimodal distribution for the lowest protein content and a polymodal distribution for the other treatments. A similar situation was seen during spawning. The lowest fertilization and hatching rates were found as a consequence of the treatment with 30% CP (P < 0.05). The greatest hatching rate occurred in the females fed 18% CP. The greatest total oocyte weight was found in the specimens that received between 30 and 36% CP. The lowest K index was found in the females fed 36% CP. In conclusion, a diet containing 18% CP satisfies the reproductive requirements of females adapted to this system.

NO EVIDENCE of PATHOLOGICAL AUTOIMMUNITY FOLLOWING MYCOBACTERIUM LEPRAE HEAT-SHOCK PROTEIN 65-DNA VACCINATION IN MICE

Heat-shock proteins (HSPs) are currently one of the most promising targets for the development of immunotherapy against tumours and autoimmune disorders. This protein family has the capacity to activate or modulate the function of different immune system cells. They induce the activation of monocytes, macrophages and dendritic cells, and contribute to cross-priming, an important mechanism of presentation of exogenous antigen in the context of MHC class I molecules, These various immunological properties of HSP have encouraged their use in several clinical trials. Nevertheless, an important issue regarding these proteins is whether the high homology among HSPs across different species may trigger the breakdown of immune tolerance and induce autoimmune diseases. We have developed a DNA vaccine codifying the Mycobacterium leprae Hsp65 (DNAhsp65), which showed to be highly immunogenic and protective against experimental tuberculosis. Here, we address the question of whether DNAhsp65 immunization could induce pathological autoimmunity in mice. Our results show that DNAhsp65 vaccination induced antibodies that can recognize the human Hsp60 but did not induce harmful effects in 16 different organs analysed by histopathology up to 210 days after vaccination. We also showed that anti-DNA antibodies were not elicited after DNA vaccination. The results are important for the development of both HSP and DNA-based immunomodulatory agents.

Protein synthesis studies in skeletal muscle of aging rats. II. In vitro studies with 0.5M potassium chloride washed polyribosomes

To investigate further the age-related reduction in muscle protein synthesis activity found previously using a crude polyribosome/pH 5 system (Pluskal et al., 1984), a 0.5M KCl washing procedure was utilized to remove the nonribosomal factors from polyribosomes isolated from male Sprague-Dawley rats in the following age groups: young (1 to 2 months), mature (12 months), and aged (22 to 24 months). Using a common source of enriched elongation factor fraction from young animals, it was not possible to demonstrate any significant difference (p > .05) in protein synthesis between the 0.5M KCl-washed polyribosomes isolated from the various age groups. Using a cell-free system containing young salt washed polyribosomes stimulated by the addition of 0.5M KCl-wash fractions, however, it was shown that the mature and aged salt-wash fractions were less (p < .05) active than material from young animals. Thus, the observed decline in protein synthesis efficiency during aging may be attributed to a reduced capacity to promote initiation/elongation by the nonribosomal salt wash fractions of muscle polyribosomes.