Produção dos fragmentos de anticorpos recombinantes scFv-N e scFv-S1 e suas aplicações na detecção e diferenciação do Vírus da Bronquite Infecciosa

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise estrutural das proteínas da seda da teia da aranha Nephila clavipes por uma abordagem proteômica

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

Influence of parity on concentrations of enzymes, proteins, and minerals in the milk of cows

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

Tepoxalin on renal function and liver enzymes in cats exposed to hypotension with isoflurane

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

Easily handling penicillin G acylase magnetic cross-linked enzymes aggregates: Catalytic and morphological studies

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

The effect of exogenous fibrolytic enzymes and a ferulic acid esterase-producing inoculant on the fibre degradability, chemical composition and conservation characteristics of alfalfa silage

The objective of this study was to determine the effect of applying fibrolytic enzymes at ensiling, either alone or in combination with a ferulic acid esterase-producing bacterial silage inoculant, on the silage conservation characteristics and nutritive value of alfalfa (Medicago sativa L). Second-cut alfalfa (340 g DM/kg fresh crop) was harvested, wilted, chopped and sub-sampled into 24 batches. Samples were randomly allocated in triplicate to one of four enzyme product treatments supplying endoglucanases and xylanases: none (control), EN1, EN2, EN3; applied alone or in combination with a ferulic acid esterase-producing silage inoculant (FAEI). Treatments were arranged in a 4 x 2 factorial design. All enzyme treatments were applied at 2 ml enzyme product/kg herbage DM, and inoculant was applied at 1 x 10(5) cfu/g fresh herbage. Samples were packed into laboratory-scale silos and stored for 7, 27 or 70 days, and analysed for dry matter (DM) losses, aerobic stability, chemical composition and in vitro ruminal degradability. The use of enzymes did not affect (P>0.05) ensilage DM losses or lactic or acetic acid concentrations after 70 days of ensilage, compared to the control silage. Silage produced using EN1 had lesser neutral detergent fibre (aNDF, P=0.046) and acid detergent fibre (ADF; P=0.006) concentrations than control silage. However, no difference (P>0.05) was observed between the control silage and silage produced with EN1 for aNDF or ADF degradability (NDFD, ADFD). Silages produced with FAEI had greater DM losses (P=0.017) and pH (P<0.001) and lesser NDFD (P=0.019), ADFD (P=0.010) and proportion of lactic acid in the total fermentation products (P=0.006) after 70 days of ensilage, compared to uninoculated silages. The use of fibrolytic enzymes did not have a major effect on the ensilage fermentation of alfalfa, either ensiled alone or with an inoculant. No advantage in ruminal DM or fibre degradability was observed for silages produced with fibrolytic enzymes. The use of a ferulic acid esterase-producing inoculant alone did not improve the nutritive value of alfalfa silage, and did not promote any incremental effects when applied in combination with fibrolytic enzyme products. Crown Copyright (C) 2014 Published by Elsevier B.V. All rights reserved.

Vitamin B6-Dependent Enzymes in the Human Malaria Parasite Plasmodium falciparum: A Druggable Target?

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

Taxonomic assessment and enzymes production by yeasts isolated from marine and terrestrial Antarctic samples

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produção de vetores recombinantes para análise das propriedades biológicas e cancerígenas da proteína K1 do herpesvírus associado ao sarcoma de Kaposi (KSHV/HHV-8)

Pós-graduação em Patologia - FMB

Transport of amino acids from milk whey by Caco-2 cell monolayer after hydrolytic action of gastrointestinal enzymes

The bioavailability of amino adds from milk whey protein hydrolysates was evaluated using diffusion of the substances through semi-permeable membranes (dialyzability) and transport by Caco-2 cell cultures. The hydrolysates with low degree of hydrolysis (LDH) and high degree of hydrolysis (HDH) were obtained after 120 min of reaction time at 50 degrees C after the initial addition of pepsin, followed by the addition of trypsin, chymotrypsin and carboxypeptidase-A. The proteins and hydrolysates were further subjected to in vitro digestion with pepsin plus pancreatin. HPLC was used to determine the concentrations of dialyzable amino adds (48.4% of the non-hydrolyzed proteins, 63.2% of the LDH sample and 58.3% of the HDH sample), demonstrating the greater dialyzability of the hydrolysates. The LDH and HDH whey protein hydrolysates prepared with pepsin, trypsin, chymotrypsin and carboxypeptidase-A showed only 14.7% and 20.8% of dialyzable small peptides and amino acids, respectively. The efficiency of absorption was demonstrated by the preferential transport of Ile, Lou and Arg through a layer of cells. In the LDH hydrolysate, Tyr was also transported. Prior high- and low-degree hydrolysis of the whey provided transport by 5.7% and 6.6%, respectively, in comparison with 23% for non-hydrolyzed proteins, considering the total amount of these amino adds that was applied to the cells. (C) 2014 Elsevier Ltd. All rights reserved.

Análise eletroforética de proteínas recombinantes obtidas de Pichia pastoris

A glicerol quinase é uma proteína tetramérica que cataliza a fosforilação do glicerol a glicerol-3-fosfato, composta por quatro subunidades - sua análise eletroforética fornecerá apenas uma banda se for constatada a sua pureza num gel não desnaturante - e sua reação com o glicerol é dependente de magnésio e de ATP. A eletroforese de proteínas utiliza como suporte um gel de poliacrilamida, que é formado pela polimerização de monômeros de acrilamida ao longo da sua cadeia e ligações cruzadas de cadeias pela inclusão de um co-monômero bifuncional apropriado, usualmente a N,N’ – metileno-bis-acrilamida ou apenas Bis . A eletroforese de proteínas mais comum é a que utiliza SDS para um gel de poliacrilamida desnaturante. O SDS é um sal chamado Dodecil Sulfato de Sódio que tem por característica se ligar a cadeia proteína nos resíduos de aminoácidos apolares, deixando com a carga negativa, sendo assim, toda proteína fica com carga total negativa, migrando para o anodo na eletroforese. As técnicas de purificação utilizadas foram ultrafiltração e precipitação com ácido tricloro acético, etanol gelado e sulfato de amônio. A dupla precipitação com etanol resultou na recuperação de maior quantidade de proteínas. A coloração do gel com prata foi mais sensível do que Comassie blue. O gel de eletroforese mostrou quatro bandas, correspondentes às quatro subunidades da glicerol quinase quando revelados com prata em gel de SDS-PAGE.

Produção de vetores recombinantes para expressão de diferentes formas da proteína K1 do herpesvírus associado ao Sarcoma de Kaposi (KSHV)

Kaposi´s sarcoma associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) is a gammaherpesvirus essential for the development of all forms of Kaposi´s sarcoma (KS). The KSHV’s life cycle is basically divided into latent and lytic phases, which have distinct viral gene expression profiles. Some important oncogenic products of KSHV are expressed during the lytic phase, including the viral K1 protein. As an effect of interfer-ence with intracellular signaling, K1 expression increases proliferation and survival of KSHV-infected cells. Due to its high level of genetic variability compared to other re-gions of the viral genome, the K1-encoding ORF (ORF-K1) is commonly evaluated for KSHV genotyping. It remains unclear whether different viral genotypes have particular biological effects that might modify the KSHV oncogenicity. The present study aimed to contribute to the establishment of an experimental in vitro model for evaluation of the K1 protein from common KSHV genotypes. Recombinant expression vectors with the ORF-K1 from KSHV genotypes A, B and C were prepared by genetic cloning. The recombi-nant vectors pKSHVOK1 obtained by cloning were sequenced for structural validation. After that, HEK293 cell line was transfected with the recombinant vectors, and proteins were extracted for expression analysis by Western blot technique, for K1 functional vali-dation. Results showed that ORF-K1 vectors containing KSHV ORF-K1 from the A, B and C genotypes were produced and structurally validated by DNA sequencing. The K1 expression at the protein level was also confirmed by immunoblots using an antibody for FLAG detection, an epitope from the vector that binds to K1. Based on presented re-sults, it´s possible to conclude that the recombinant vectors will be able to be used in future studies of K1 protein biological properties from distinct KSHV genotypes