Sequencing of Spodoptera frugiperda midgut trehalases and demonstration of secretion of soluble trehalase by midgut columnar cells

Both soluble (SfTre1) and membrane-bound (SfTre2) trehalases occur along the midgut of Spodoptera frugiperda larvae. Released SfTre2 was purified as a 67 kDa protein. Its K(m) (1.6 mM) and thermal stability (half life 10 min at 62 degrees C) are different from the previously isolated soluble trehalase (K(m) = 0.47 mM; 100% stable at 62 degrees C). Two cDNAs coding for S. frugiperda trehalases have been cloned using primers based on consensus sequences of trehalases and having as templates a cDNA library prepared from total polyA-containing RNA extracted from midguts. One cDNA codes for a trehalase that has a predicted transmembrane sequence and was defined as SfTre2. The other, after being cloned and expressed, results in a recombinant trehalase with a K(m) value and thermal stability like those of native soluble trehalase. This enzyme was defined as SfTre1 and, after it was used to generate antibodies, it was immunolocalized at the secretory vesicles and at the glycocalyx of columnar cells. Escherichia coli trehalase 3D structure and sequence alignment with SfTre1 support a proposal regarding the residue modulating the pKa value of the proton donor.

Fast batch injection analysis of H(2)O(2) using an array of Pt-modified gold microelectrodes obtained from split electronic chips

A fast and robust analytical method for amperometric determination of hydrogen peroxide (H(2)O(2)) based on batch injection analysis (BIA) on an array of gold microelectrodes modified with platinum is proposed. The gold microelectrode array (n = 14) was obtained from electronic chips developed for surface mounted device technology (SMD), whose size offers advantages to adapt them in batch cells. The effect of the dispensing rate, volume injected, distance between the platinum microelectrodes and the pipette tip, as well as the volume of solution in the cell on the analytical response were evaluated. The method allows the H(2)O(2) amperometric determination in the concentration range from 0.8 mu mol L(-1) to 100 mu mol L(-1). The analytical frequency can attain 300 determinations per hour and the detection limit was estimated in 0.34 mu mol L(-1) (3 sigma). The anodic current peaks obtained after a series of 23 successive injections of 50 mu L of 25 mu mol L(-1) H(2)O(2) showed an RSD < 0.9%. To ensure the good selectivity to detect H(2)O(2), its determination was performed in a differential mode, with selective destruction of the H(2)O(2) with catalase in 10 mmol L(-1) phosphate buffer solution. Practical application of the analytical procedure involved H(2)O(2) determination in rainwater of Sao Paulo City. A comparison of the results obtained by the proposed ampermetric method with another one which combines flow injection analysis (FIA) with spectrophotometric detection showed good agreement. (C) 2011 Elsevier B.V. All rights reserved.

Time Evolution of the Activation Energy in a Batch Chemical Oscillator

The batch-operated bromate/phosphate/acetone/dual catalyst system was studied at four temperatures between 5 and 35 degrees C. The dynamics was simultaneously followed by potential measurements with platinum and bromide selective electrodes, and spectroscopically at two different wavelengths. By simultaneously recording these four time series it was possible to characterize the dynamics of the sequential oscillations that evolve in time. The existence of three sequential oscillatory patterns at each temperature allowed estimating the activation energies in each case. Along with the activation energy of the induction period, it was possible to trace the time evolution of the overall activation energy at four different stages as the reaction proceeds. The study was carried out for two different sets of initial concentrations and it was observed that the overall activation energy increases as reactants turn into products. This finding was propounded as a result of the decrease in the driving force, or the system`s affinity, of the catalytic oxidative bromination of acetone with acidic bromate, as the closed system evolves toward the thermodynamic equilibrium.

Comportamento do reator seqüencial em batelada (RSB) sob estado estacionário dinâmico utilizando idade do lodo como parâmetro de controle operacional

O reator seqüencial em batelada (RSB) é uma variante de lodos ativados capaz de promover a remoção da matéria orgânica, a remoção dos nutrientes e a separação da fase sólida da líquida em uma unidade. A valorização das áreas urbanas, a carência de tratamento terciário e a crescente necessidade de redução nas dimensões de estações de tratamento de esgoto devem impulsionar o desenvolvimento de pesquisas sobre RSB em curto espaço de tempo. A partir deste cenário, o presente trabalho teve como objetivo modelar o comportamento do reator seqüencial em batelada a partir da teoria desenvolvida por Marais e colaboradores. Dentro deste contexto, a cinética de oxidação dos compostos orgânicos e do nitrogênio na forma amoniacal foi descrita e modelada. O trabalho experimental foi realizado em duas escalas: bancada e piloto. O experimento em escala de bancada foi dividido em duas fases. Foram utilizados dois RSBs e um sistema de fluxo contínuo. Um reator seqüencial em batelada (RSB1) foi operado com idade de lodo. O outro reator em batelada (RSB2) foi operado em função da relação F/M e o sistema de fluxo contínuo (FC1) por idade de lodo. Estes reatores foram utilizados como controle no monitoramento do RSB1 Na primeira fase, os três sistemas removeram apenas matéria orgânica. Na fase seguinte, removeram matéria orgânica e nitrogênio. A partir dos resultados obtidos em escala de bancada, foi possível concluir que o modelo desenvolvido pode ser aplicado ao reator seqüencial em batelada operando com idade de lodo, permitindo determinar a qualidade do efluente, a produção de lodo e o consumo de oxidante. Além disso, foi possível descrever o comportamento da taxa de consumo de oxigênio em função da oxidação da matéria orgânica biodegradável e da oxidação do nitrogênio na forma amoniacal. O reator seqüencial em batelada operado com idade de lodo (RSB1) alcançou remoção média de matéria orgânica de 90 % nas idades de lodo de 30, 20, 10 e 5 dias. A remoção média de nitrogênio mais elevada foi obtida na idade de lodo de 20 dias e atingiu 87 %. Nas demais idades de lodo a remoção média de nitrogênio variou entre 79 e 42 %. A modelagem do comportamento do reator seqüencial em batelada resultou numa proposta de metodologia para o dimensionamento que tem como finalidade abolir critérios obsoletos e inadequados para o dimensionamento de lodos ativados em batelada No experimento em escala piloto, foram utilizados um reator seqüencial em batelada, denominado RSB, e um sistema de fluxo contínuo com a configuração Bardenpho, denominado FC. Os sistemas de lodos ativados sob investigação foram monitorados em duas idades de lodo: 30 e 10 dias. Os dados do experimento em escala piloto mostraram que os processos físico-químicos e biológicos envolvidos na remoção de matéria orgânica e nitrogênio no RSB foram mais eficientes do que no Bardenpho quando trataram o mesmo esgoto doméstico e foram submetidos às mesmas condições operacionais. No RSB, obteve-se 88 e 89 % de remoção de matéria orgânica nas idades de lodo de 10 e 30 dias, respectivamente. Nesta seqüência das idades de lodo, a eficiência do Bardenpho caiu de 87 para 76 %. O sistema de fluxo contínuo removeu 66 e 52 % do nitrogênio total afluente nas idades de lodo de 10 e 30 dias, respectivamente. A eficiência do RSB na remoção de nitrogênio foi determinada apenas na idade de lodo de 10 dias e alcançou 69 %. A partir dos resultados obtidos em escala de bancada e piloto, constata-se que o reator seqüencial em batelada operando com idade de lodo pode ser utilizado no tratamento de esgoto doméstico e obter eficiência na remoção de matéria orgânica e nitrogênio igual ou superior ao sistema de fluxo contínuo.

Estudo de reatores eletroquímicos para remoção de Cu2+ , Zn2+, Fenol e BTEX em água produzida

The production of water has become one of the most important wastes in the petroleum industry, specifically in the up stream segment. The treatment of this kind of effluents is complex and normally requires high costs. In this context, the electrochemical treatment emerges as an alternative methodology for treating the wastewaters. It employs electrochemical reactions to increase the capability and efficiency of the traditional chemical treatments for associated produced water. The use of electrochemical reactors can be effective with small changes in traditional treatments, generally not representing a significant additional surface area for new equipments (due to the high cost of square meter on offshore platforms) and also it can use almost the same equipments, in continuous or batch flow, without others high costs investments. Electrochemical treatment causes low environmental impact, because the process uses electrons as reagent and generates small amount of wastes. In this work, it was studied two types of electrochemical reactors: eletroflocculation and eletroflotation, with the aim of removing of Cu2+, Zn2+, phenol and BTEX mixture of produced water. In eletroflocculation, an electrical potential was applied to an aqueous solution containing NaCl. For this, it was used iron electrodes, which promote the dissolution of metal ions, generating Fe2+ and gases which, in appropriate pH, promote also clotting-flocculation reactions, removing Cu2+ and Zn2+. In eletroflotation, a carbon steel cathode and a DSA type anode (Ti/TiO2-RuO2-SnO2) were used in a NaCl solution. It was applied an electrical current, producing strong oxidant agents as Cl2 and HOCl, increasing the degradation rate of BTEX and phenol. Under different flow rates, the Zn2+ was removed by electrodeposition or by ZnOH formation, due the increasing of pH during the reaction. To better understand the electrochemical process, a statistical protocol factor (22) with central point was conducted to analyze the sensitivity of operating parameters on removing Zn2+ by eletroflotation, confirming that the current density affected the process negatively and the flow rate positively. For economical viability of these two electrochemical treatments, the energy consumption was calculated, taking in account the kWh given by ANEEL. The treatment cost obtained were quite attractive in comparison with the current treatments used in Rio Grande do Norte state. In addition, it could still be reduced for the case of using other alternative energy source such as solar, wind or gas generated directly from the Petrochemical Plant or offshore platforms

In situ differentiation of labile/inert metal species in Brazilian tropical rivers by means of a time-controlled batch-procedure based on TEPHA resin

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

Quali/quantitative characterization of biogas produced in batch digesters supplied with six distinct substrates

Com a finalidade de se analisar quali/quantitativamente o biogás produzido por 6 tipos de substratos, efetuou-se a presente pesquisa no DER/FCAV/UNESP- SP, Brasil. Os substratos utilizados na digestão anaeróbia foram caracterizados como: 1- Esterco de aves de postura (EAP); 2- Esterco de aves de postura triturado (EAPT); 3- Esterco de aves de corte com cama de maravalha (EACM); 4- Esterco de aves de corte com cama de maravalha triturado (EACMT); 5- Esterco de aves de corte com cama de casca de amendoim (EACA), e 6- Esterco de aves de corte com cama de casca de amendoim triturado (EACAT). Dos resultados obtidos, concluiu-se que, em relação à produção acumulada de biogás, o substrato EAPT foi superior aos demais, enquanto EACM apresentou produção acumulada inferior. em geral, os substratos triturados apresentaram maior quantidade de biogás acumulado em relação aos não triturados, exceto o EACAT, com 20,9 m³ inferior ao EACA. O período de produção máxima de biogás teve início entre 45 e 60 dias, começando a decair a partir dos 120 dias. Aos 57 dias após o enchimento dos biodigestores, o gás produzido possuía teores de CH4 superiores a 53%, e a partir dos 99 dias, todos produziram biogás com teores de CH4 superiores a 70%. Os substratos EAP e EAPT apresentaram maiores concentrações de metano no biogás.

Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7

Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO(R) vector which contains thioredoxin and polyhistidine tags at the C-and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 mu g/mL growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.

Sequencing and expression analysis of hepcidin mRNA in donkey (Equus asinus) liver

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

Cloning, sequencing and expression analysis of the equine hepcidin gene by real-time PCR

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

Advantages of using nested collision induced dissociation/post-source decay with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: sequencing of novel peptides from wasp venom

We have examined the applicability of the 'nested' collision induced dissociation/post-source decay (CID/PSD) method to the sequencing of novel peptides from solitary wasps which have neurotoxic venom for paralyzing other insects. The CID/PSD spectrum of a ladder peptide derived from an exopeptidase digest was compared with that of the intact peptide. The mass peaks observed only in the CID/PSD spectrum of a ladder peptide were extracted as C-terminal fragment ions. Assignment of C-terminal fragment ions enabled calculation of N-terminal fragment masses, leading to differentiation between N-terminal fragment ions and internal fragment ions. This methodology allowed rapid and sensitive identification by removing ambiguity in the assignment of the fragment ions, and proved useful for sequencing unknown peptides, in particular those available as natural products with a limited supply. Copyright (C) 2000 John Wiley & Sons, Ltd.

Effect of Conditioning on the Production of Inulinase by Kluyveromyces marxianus var. bulgaricus through Fed-Batch Fermentation

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influence of nitrogen source and sucrose concentration on inulinase production by Kluyveromyces marxianus in fed-batch fermentation

This work studied the influence of nitrogen source and sucrose concentration in the feeding medium for biomass and inulinase production by Kluyveromyces marxianus var. bulgaricus. The results show that the best nitrogen source was a combination of 5 g/L of yeast extract and 10 g/L of peptone. Both cellular growth and enzymatic activity increased with sucrose concentration in the feeding medium (from 200 to 500 g/L). When the sucrose concentration reached 600 g/L, both cellular growth and enzymatic activity decreased.

New primers for the amplification and sequencing of nuclear loci in a taxonomically wide set of reptiles and amphibians

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Suitability of DNA extracted from archival specimens of fruit-eating bats of the genus Artibeus (Chiroptera, Phyllostomidae) for polymerase chain reaction and sequencing analysis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)