Comparative biochemical studies of myotoxic phospholipase A(2) from Bothrops venom

Venoms from Bothrops jararacussu, Bothrops asper, Bothrops atrox, Bothrops pirajai, Bothrops moojeni, Bothrops alternatus and Bothrops (Bothriopsis) bilineata were fractionated using a simplified procedure based on ion-exchange chromatography on CM-Sepharose at pH 8.0 or reverse phase HPLC. The resulting elution profiles showed important differences in the myotoxin content of these venoms. The venoms from B. alternatus, B. atrox and Bothriopsis bilineata did not contain the major myotoxin found in the other venoms. The amino acid sequence of the first 50 residues of the N-terminal region of the PLA(2)-like myotoxins showed a homology of 90-96% with other bothropic myotoxins. All of the myotoxins isolated induced rat paw edema, increased the level of plasma creatine kinase and produced myonecrosis together with polymorphonuclear cell infiltration.

Purification and some properties of an extracellular acid protease from Aspergillus clavatus

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

Purification and Characterization of Two Extracellular Xylanases from Penicillium sclerotiorum: A Novel Acidophilic Xylanase

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Purification, characterization and crystallization of Jararacussin-I, a fibrinogen-clotting enzyme isolated from the venom of Bothrops jararacussu

A fibrinogen-clotting enzyme, Jararacussin-I, was purified from the venom of Bothrops jararacussu by a combination of ion exchange chromatography using Resource 15S resin and affinity chromatography using Benzamidine Sepharose 6B resin. Jararacussin-I displays a molecular mass of 28 kDa as estimated by sodium dodecyl sulphate-PAGE and possesses an isoetectric point of 5.0. The coagulant specific activity of the enzyme was determined to be 45.8 NIH U/mg using bovine fibrinogen as the substrate and the esterase specific activity was determined to be 258.7 U/mg. The protease inhibitors, benzamidine and DTT inhibited the esterase specific activity by 72.4 and 69.7%, respectively. The optimal temperature and pH for the degradation of both chains of fibrinogen and esterase specific activity were determined to be 37 degreesC and 7.4-8.0, respectively. The enzyme was inactivated at both 4 and 75 T. Single crystals of Jararacussin-I were obtained and complete three-dimensional X-ray diffraction data was collected at the Brazilian National Synchrotron Source (LNLS) to a resolution of 2.4 Angstrom. (C) 2002 Published by Elsevier B.V. Ltd.

Method development for the simultaneous determination of carboxylic acids, phenolic compounds, and sorbic acid in white wines

A reversed phase liquid chromatographic method was developed for the simultaneous determination of carboxylic acids and phenolics in white wines. The samples, diluted, were injected onto a Spherisorb ODS-2 column with a gradient of sulfuric acid (pH 2.5)/methanol as mobile phase. A diode array detector was used which was set at 210nm for carboxylic acids and altered to 278nm, during the run, far phenolics and sorbic acid. The identification of compounds was based on retention time, co-chromatography and UV spectrum. Some clean-up methods (sep-pak C-18 and an ion exchange column) mere tested and did not improve the results.The analysis was simple, with no sample preparation. Application of this method was illustrated by analyses of Brazilian Welchriesling wines.

Ribonuclease production by Aspergillus species

Ribonuclease production by Aspergillus flavipes. A sulphureus and A. fischeri in semisynthetic medium, after 24-144 hours at 30 degrees C under shaking, was studied. After cultivation, the medium was separated from micelia by filtration and the resultant solution was used as enzymatic extract. The highest amount of biomass and RNase was obtained after 96 hours of cultivation. The enzymes produced by three species presented similar characteristics, with optimum temperature at 55 degrees C and two peaks of activity at pH 4.5 and 7.0. A. flavipes RNases were more sensitive to temperature: 50% of the initial activity was lost after 1 hour at 70 degrees C. After this heat treatment, RNase of A. sulphureus lost 30% of this activity and that of A. fischeri only 16%. The nucleotides released by enzimatic hydrolysis of RNA were separated by ion exchange chromatography in a AG-1X8-formiate column and identified by paper chromatography. This procedure indicated that the raw enzymatic extract of Aspergillus flavipes is able to hydrolyze RNA, releasing 3'-nucleotides monophosphate at pH 4.5 and 3' and 5'-nucleotides monophosphate at pH 7.0 and 8.5. This result suggests that this strain produces two different types of RNase, one acidic and other alcaline, with different specificities.

Analysis of Tooth Enamel After Excessive Bleaching: A Study Using Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy

This study assessed alterations on bovine enamel after excessive bleaching. Coronal portions of bovine teeth (n = 30) were sectioned and divided into three groups (n = 10 per group). The coronal parts were further cut incisocervically into two halves. While one half received no bleaching (control), the other half was subjected to either one (group 1), three (group 2), or five bleaching sessions (group 3) with 35% hydrogen peroxide. The enamel surfaces were then analyzed using scanning electron microscopy and energy dispersive x-ray spectroscopy (EDS). Fxcessive bleaching affected the surface morphology and chemistry of the bovine enamel. EDS analysis showed the highest decrease in calcium ion percentages in groups 2 and 3 when compared to their nonbleached halves. Oxygen and phosphorus percentages were comparable on both the control and bleached enamel, regardless of the number of bleaching sessions. Consecutive bleaching sessions with 35% hydrogen peroxide may lead to morphologic and specific elemental changes when performed in a short period of time. Calcium ion percentages may decrease when this bleaching agent is used for more than one session. Int J Prosthodontics 2010;23:29-32.

Caracterização da matéria-prima cerâmica da Mina Tabajara (Limeira, SP)

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

Distribuição de mercúrio em diferentes solos da Bacia do médio Rio Negro-AM: influência da matéria orgânica no ciclo biogeoquímico do mercúrio

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

Label-free DNA detection of hepatitis C virus based on modified conducting polypyrrole films at microelectrodes and atomic force microscopy tip-integrated electrodes

We present a new strategy for the label-free electrochemical detection of DNA hybridization for detecting hepatitis C virus based on electrostatic modulation of the ion-exchange kinetics of a polypyrrole film deposited at microelectrodes. Synthetic single-stranded 18-mer HCV genotype-1-specific probe DNA has been immobilized at a 2,5-bis(2-thienyl)-N-(3-phosphoryl-n-alkyl)pyrrole film established by electropolymerization at the previously formed polypyrrole layer. HCV DNA sequences (244-mer) resulting from the reverse transcriptase-linked polymerase chain reaction amplification of the original viral RNA were monitored by affecting the ion-exchange properties of the polypyrrole film. The performance of this miniaturized DNA sensor system was studied in respect to selectivity, sensitivity, and reproducibility. The limit of detection was determined at 1.82 x 10(-21) mol L-1. Control experiments were performed with cDNA from HCV genotypes 2a/c, 2b, and 3 and did not show any unspecific binding. Additionally, the influence of the spacer length of 2,5-bis(2-thienyl)-N-(3-phosphoryl-n-alkyl)pyrrole on the behavior of the DNA sensor was investigated. This biosensing scheme was finally extended to the electrochemical detection of DNA at submicrometer-sized DNA biosensors integrated into bifunctional atomic force scanning electrochemical microscopy probes. The 18-mer DNA target was again monitored by following the ion-exchange properties of the polypyrrole film. Control experiments were performed with 12-base pair mismatched sequences.

Lability of Cd, Cr, Cu, Mn and Pb complexed by aquatic humic substances

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

Determinação de chumbo em álcool combustível por voltametria de redissolução anódica utilizando um eletrodo de pasta de carbono modificado com resina de troca iônica Amberlite IR 120

Um método envolvendo a pré-concentração e redissolução anódica em condições de voltametria de pulso diferencial empregando um eletrodo de pasta de carbono modificado (EPCM) com uma resina de troca iônica Amberlite IR120 foi proposto para a determinação de íons chumbo em álcool combustível. O procedimento é baseado em um pico de oxidação do analito observado em -0,53 V(vs. Ag/AgCl) em solução de HCl. As melhores condições experimentais encontradas foram: 5% (m/m) da Amberlite IR120 para a construção do eletrodo, solução de HCl 0,1 mol L-1, velocidade de varredura de 10 mVs-1, tempo de pré-concentração de 15 min e amplitude de pulso de 100 mV. Utilizando essas condições, o EPCM apresentou uma resposta linear entre a corrente de pico anódica e a concentração de íons chumbo para o intervalo entre 9,9 x 10-9 e 1,2 x 10-6 mol L-1 e um limite de detecção de 7,2 x 10-9 mol L-1. Valores de recuperação entre 96 % e 102 % foram encontrados para amostras de álcool combustível enriquecidas com Pb2+ em níveis de 10-7 mol L-1. O efeito da presença de outros íons concomitantes sobre a resposta voltamétrica do eletrodo também foi avaliado.

Relações solo-paisagem em topossequência de origem basáltica

Variações nos atributos do solo dependem da posição do solo na paisagem e processos de drenagem, erosão e deposição. Este estudo objetivou avaliar os atributos físicos e químicos do solo, em uma topossequência de origem basáltica, na região de Batatais (SP). A área possui relevo aplanado e altitude oscilando entre 740 m e 610 m, em região dominada por basaltos. Foi estabelecido caminhamento de 3.000 m, a partir do espigão da vertente, no seu declive mais suave. As superfícies geomórficas foram identificadas e delimitadas conforme critérios topográficos e estratigráficos, com base em intensas investigações detalhadas de campo. Foram coletadas amostras laterais aos perfis modais representativos das diversas superfícies geomórficas (S.G.) da topossequência (S.G. I = topo; S.G. II = meia encosta e sopé de transporte; S.G. III = ombro e sopé de deposição), totalizando 142 amostras. Além disto, foram abertas trincheiras, nos segmentos de vertente inseridos nas superfícies geomórficas mapeadas. As amostras coletadas foram analisadas quanto à densidade do solo, textura, bases trocáveis (Ca2+, K+ e Mg2+), soma de bases, capacidade de troca catiônica, saturação por bases, pH (água e KCl), SiO2, Al2O3, Fe2O3 (ataque por H2SO4), óxidos de Fe livres extraídos com ditionito-citrato-bicarbonato e Fe mal cristalizado extraído com oxalato de amônio. Os resultados revelaram que os solos oriundos de basalto apresentaram atributos físicos e químicos com comportamento dependente das formas do relevo. Com o uso de técnicas estatísticas multivariadas, foi possível distinguir três diferentes ambientes, que equivalem às três superfícies geomórficas.

Chemical properties of soils treated with biological sludge from gelatin industry

O impacto dos resíduos orgânicos agroindustriais no ambiente pode ser reduzido com o seu uso agrícola. do ponto de vista da fertilidade do solo, o que se deseja com a aplicação dos resíduos é aumentar o teor de matéria orgânica e fornecer nutrientes para as plantas. Neste trabalho, objetivou-se avaliar o efeito do lodo biológico de indústria de gelatina em atributos químicos de dois Argissolos Vermelho-Amarelos (PVA-arenoso e PVA-textura média) e de um Latossolo Vermelho (LV-argiloso). O experimento foi conduzido por 120 dias em laboratório, em delineamento inteiramente casualizado e esquema fatorial combinando os três solos e seis doses de lodo (0, 100, 200, 300, 400 e 500 m³ ha-1), com três repetições. A aplicação de até 500 m³ ha-1 de lodo diminui a acidez do solo e aumenta a CTC efetiva e a disponibilidade de N, Ca, Mg e P, sem ultrapassar o limite de tolerância para Na. O aumento do teor de bases, maior do que o da CTC efetiva, indica que a maior parte dos cátions adicionados pelo lodo permanece em solução e pode ser perdida por lixiviação.

Effects of 60Co gamma radiation on crotamine

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. This has been employed to attenuate animal toxins. Crotamine is a strongly basic polypeptide (pI 10.3) from Crotalus durissus terrificus venom composed of 42 amino acid residues. It induces skeletal muscle spasms leading to a spastic paralysis of hind limbs in mice. The objective of the present study was to carry out a biochemical study and a toxic activity assay on native and irradiated crotamine. Crotamine was purified from C.d. terrificus venom by Sephadex G-100 gel filtration followed by ion-exchange chromatography, and irradiated at 2 mg/ml in 0.15 M NaCl with 2.0 kGy gamma radiation emitted by a 60Co source. The native and irradiated toxins were evaluated in terms of structure and toxic activity (LD50). Irradiation did not change the protein concentration, the electrophoretic profile or the primary structure of the protein although differences were shown by spectroscopic techniques. Gamma radiation reduced crotamine toxicity by 48.3%, but did not eliminate it.