Antitumoural effect of an L-amino acid oxidase isolated from Bothrops jararaca snake venom

An L-amino acid oxidase (BjarLAAO-I) from Bothrops jararaca snake venom was highly purified using a stepwise sequential chromatography on Sephadex G-75, Benzamidine Sepharose and Phenyl Sepharose. Purified BjarLAAO-I showed a molecular weight around 60,000 under reducing conditions and about 125,000 in the native form, when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. BjarLAAO-I is a homodimeric acidic glycoprotein, pI similar to 5.0, and N-terminal sequence showing close structural homology with other snake venom LAAOs. The purified enzyme catalysed the oxidative deamination of L-amino acids, the most specific substrate being L-Phe. Five amino acids, L-Ser, L-Pro, L-Gly, L-Thr and L-Cys were not oxidized, clearly indicating a significant specificity. BjarLAAO-I significantly inhibited Ehrlich ascites tumour growth and induced an influx of polymorphonuclear cells, as well as spontaneous liberation of H(2)O(2) from peritoneal macrophages. Later, BjarLAAO-I induced mononuclear influx and peritoneal macrophage spreading. Animals treated with BjarLAAO-I showed higher survival time.

Avaliação nutricional, em tilápias-do-nilo, de farinhas de sangue bovino obtidas por três métodos de processamento

Avaliaram-se farinhas de sangue obtidas pelos métodos de processamento em tambor, convencional e atomização. As farinhas foram submetidas ao processo de extração e fracionamento da proteína para determinação do perfil do tamanho molecular, que foi comparado ao do sangue bovino in natura. Nas amostras, submetidas ou não ao processo de desengorduramento, foram realizadas análises da digestibilidade in vitro da proteína. Para determinação dos coeficientes de digestibilidade dos nutrientes in vivo, foram confeccionadas quatros rações, sendo uma sem farinha de sangue, denominada ração-referência purificada. Para essa etapa, juvenis de tilápia-do-nilo com peso médio inicial de 100,0±5,0 g foram estocados em aquários de 250 L, em delineamento de blocos casualizados, com quatro repetições e dez peixes/unidade experimental. As rações-teste foram obtidas com a introdução de 30% das farinhas de sangue em estudo. O processamento afetou a estrutura proteica original do sangue in natura em condições de alta temperatura e tempo prolongado, efeito traduzido pela alta proporção de peptídeos de baixo peso molecular e aminoácidos livres, correspondendo a baixos valores de digestibilidade da proteína da farinha de sangue nos testes in vivo e in vitro. A farinha de sangue atomizada e a de tambor são eficientemente utilizadas por tilápias-do-nilo. Na farinha de sangue convencional, a proteína teve valor biológico inferior ao das outras duas farinhas. Na formulação de rações contendo farinha de sangue para tilápias-do-nilo, a isoleucina deve ser considerada o primeiro aminoácido limitante, seguida pela metionina+cistina, arginina e treonina, que foram encontradas em níveis críticos para essa espécie, principalmente na farinha de sangue convencional.

Heparin-binding proteins of canine seminal plasma

Heparin-binding proteins (HBP) from seminal plasma have been expected to participate in modulation of the acrosomal reaction, and have been correlated with fertility in some species. However, they have not been described in the dog. The aim of this study was to document the HBPs of canine seminal plasma. Six pooled samples of seminal plasma from three crossbred dogs were used. The HBPs were isolated by heparin affinity chromatography and the fractions recovered were pooled. One-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on 12 and 18% vertical minigels. The stained gels were scanned and the molecular weight (kDa) values for each band within a lane were calculated by image analysis software. The electrophoresis analysis of the pooled eluded fractions identified 19 bands, with molecular weights varying from 61.5 to 5.2 kDa. Previous studies, using one-dimensional SDS-PAGE, identified two bands (67 and 58.6 kDa), which were positively correlated with some semen parameters (sperm motility, sperm vigor, percentage of morphologically normal sperm and plasma membrane integrity). The 61.5 kDa band detected in the present study apparently corresponded to the 58.6 kDa band identified previously. Canine seminal plasma contained HBP; since HBP modulate the acrosome reaction in other species, they may have the same function in the dog. Further studies are necessary to better characterize this protein and determine if it is associated with fertility in the dog. (c) 2006 Elsevier B.V. All rights reserved.

Histological evaluation of chitosan-based biomaterials used for the correction of critical size defects in rat's calvaria

Chitosan, a biopolymer obtained from chitin, and its derivates, such as chitosan hydrochloride, has been reported as wound healing accelerators and as possible bone substitutes for tissue engineering, and therefore these Substances could be relevant in dentistry and periodontology. The purpose of this investigation was to make a histological evaluation of chitosan and chitosan hydrochloride biomaterials (gels) used in the correction of critical size bone defects made in rat's calvaria. Bone defects of 8 mm in diameter were surgically created in the calviria of 50 Holtzman (Rattus norvegicus) rats and filled with blood clot (control), low molecular weight chitosan, high molecular weight chitosan, low molecular weight chitosan hydrochloride, and high molecular weight chitosan hydrochloride, numbering 10 animals, divided into two experimental periods (15 and 60 days), for each biomaterial. The histological evaluation was made based on the morphology of the new-formed tissues in defect's region, and the results indicated that there was no statistical difference between the groups when the new bone formation in the entire defect's area were compared (p > 0.05) and, except in the control groups, assorted degrees of inflammation Could be Seen. In Conclusion, chitosan and chitosan hydrochloride biomaterials used in this study were not able to promote new bone formation in critical size defects made in rat's calvaria. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res 93A: 107-114, 2016

Bothrops moojeni myotoxin-II, a Lys49-phospholipase A(2) homologue: An example of function versatility of snake venom proteins

MjTX-II, a myotoxic phospholipase A(2) (PLA(2)) homologue from Bothrops moojeni venom, was functionally and structurally characterized. The MjTX-II characterization included: (i) functional characterization (antitumoral, antimicrobial and antiparasitic effects); (ii) effects of structural modifications by 4-bromophenacyl bromide (BPB), cyanogen bromide (CNBr), acetic anhydride and 2-nitrobenzenesulphonyl fluoride (NBSF); (iii) enzymatic characterization: inhibition by low molecular weight heparin and EDTA; and (iv) molecular characterization: cDNA sequence and molecular structure prediction. The results demonstrated that MjTX-II displayed antimicrobial activity by growth inhibition against Escherichia coli and Candida albicans, antitumoral activity against Erlich ascitic tumor (EAT), human breast adenocarcinoma (SK-BR-3) and human T leukemia cells (JURKAT) and antiparasitic effects against Schistosoma mansoni and Leishmania spp., which makes MjTX-II a promising molecular model for future therapeutic applications, as well as other multifunctional homologous Lys49-PLA(2)S or even derived peptides. This work provides useful insights into the structural determinants of the action of Lys49-PLA2 homologues and, together with additional strategies, supports the concept of the presence of others bioactive sites distinct from the catalytic site in snake venom myotoxic PLA(2)s. (c) 2005 Elsevier B.V. All rights reserved.

Structural and functional characterization of BnSP-7, a Lys49 myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis Venom

BnSP-7, a Lys49 myotoxic phospholipase A, homologue from Bothrops neuwiedi pauloensis venom, was structurally and functionally characterized. Several biological activities were assayed and compared with those of the chemically modified toxin involving specific amino acid residues, the cDNA produced from the total RNA by RT-PCR contained approximately 400 bp which codified its 121 amino acid residues with a calculated pi and molecular weight of 8.9 and 13,727, respectively. Its amino acid sequence showed strong similarities with several Lys49 phospholipase A, homologues from other Bothrops sp, venoms. By affinity chromatography and gel diffusion, it was demonstrated that heparin formed a complex with BnSP-7, held at least in part by electrostatic interactions. BnSP-7 displayed bactericidal activity and promoted the blockage of the neuromuscular contraction of the chick, biventer cervicis muscle. In addition to its in vivo myotoxic and edema-inducing activity, it disrupted artificial membranes, Both BnSP-7 and the crude venom released creatine kinase from the mouse gastrocnemius muscle and induced the development of a dose-dependent edema. His, Tyr, and Lys residues of the toxin were chemically modified by 4-bromophhenacyl bromide (BPB), 2-nitrobenzenesulfonyl fluoride (NBSF), and acetic anhydride (AA), respectively. Cleavage of its N-terminal octapeptide was achieved with cyanogen bromide (CNBr), the bactericidal action of BnSP-7 on Escherichia coli was almost completely abolished by acetylation or cleavage of the N-terminal octapeptide, the neuromuscular effect induced by BnSP-7 was completely inhibited by heparin, BPB, acetylation, and CNBr treatment. The creatine kinase releasing and edema-inducing effects were partially inhibited by heparin or modification by BPB and almost completely abolished by acetylation or cleavage of the N-terminal octapeptide, the rupture of liposomes by BnSP-7 and crude venom was dose and temperature dependent. Incubation of BnSP-7 with EDTA did not change this effect, suggesting a Ca2+-independent membrane lytic activity. BnSP-7 cross-reacted with antibodies raised against B. moojeni (MjTX-II), B. jararacussu (BthTX-I), and B. asper (Basp-II) myotoxins as well as against the C-terminal peptide (residues 115-129) from Basp-II. (C) 2000 Academic Press.

Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The promoter of a gene encoding an isoflavone reductase-like protein in coffee (Coffea arabica) drives a stress-responsive expression in leaves

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Comparative cytogenetic studies of Bufo ictericus, B. paracnemis (Amphibia, Anura) and an intermediate form in sympatry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Proteomic analysis reveals suppression of bark chitinases and proteinase inhibitors in citrus plants affected by the citrus sudden death disease

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Purification and physicochemical properties of a ribonuclease produced by Aspergillus flavipes (IZ : 1501)

A RNase of Aspergillus flavipes (IZ:1501) was purified from culture medium by chromatography on DEAE-cellulose and Sephadex G50 columns, after 96 h of cultivation. The molecular weight of the RNase was estimated to be 15 kD by gel filtration using Sephadex G100, and the optimum pH and temperature were 4.0 and 55 degrees C, respectively. Catalytic activity was inhibited by Hg2+, Ag+, Fe3+, Co2+ and Zn2+. The enzyme showed guanosine specificity producing only 3'-GMP from yeast RNA.

Purification and characterization of the exopolygalacturonase produced by Aspergillus giganteus in submerged cultures

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Purification and characterization of an exo-polygalacturonase produced by Penicillium viridicatum RFC3 in solid-state fermentation

The pectinolytic enzyme obtained from Penicillium viridicatum RFC by solid-state fermentation was purified to homogeneity by pretreatment with kaolin (40 mg mL(-1) ) and ultrafiltration. followed by chromatography on a Sephadex G50 column. The apparent molecular weight of the enzyme was 24 kDa. Maximal activity occurred at pH 6.0 and at 60 degrees C. The enzyme proved to be an exo-polygalacturonase, releasing galacturonic acid by hydrolysis of highly esterified pectin. The presence of 10 mM Ba2+ increased the enzyme activity by 96% and its thermal stability by 30%. besides increasing its stability at acid pH. The apparent K-m with apple pectin as substrate was 1.82 mg mL(-1) and the V-max was 81 mu mol min(-1). (c) 2007 Elsevier Ltd. All rights reserved.

DNA Psi-condensation and reentrant decondensation: Effect of the PEG degree of polymerization

psi-Condensation of DNA fragments of about 4 kbp was induced by poly(ethylene glycol) (PEG), with degrees of polymerization ranging from 45 to 182, and univalent salt (NaCl). Using circular dichroism spectroscopy, we were able to accurately determine the critical amount of PEG needed to induce condensation, as a function of the NaCl concentration. A significant dependence on the PEG degree of polymerization was found. Phase boundaries determined for the multimolecular condensation were very similar to those observed previously for the monomolecular collapse, with two asymptotic regimes at low and high salt concentrations. We analyze our data using a theoretical model that properly takes into account both the polyelectrolyte nature of the DNA and the liquid crystallinity of the condensed phase. The model assumes that all PEG is excluded from the condensates and shows reentrant decondensation only at low salt. We also systematically study reentrant decondensation and find a very strong dependence on PEG molecular weight. At low PEG molecular weight, decondensation occurs at relatively low concentrations of PEG, and over a wide range of salt concentrations. This suggests that in the reentrant decondensation the flexible polymers used are not completely excluded from the condensed phase.

Purification, biochemical and functional characterization of miliin, a new thiol-dependent serine protease isolated from the latex of Euphorbia milii

Miliin, a new thiol-dependent serine protease purified from the latex of Euphorbia milii possesses a molecular weight of 79 kDa, an isoelectric point of 4.3 and is optimally active at 60 degrees C in the pH range of and 7.5-11.0. Activity tests indicate that milliin is a thiol-dependent serine protease.