Análise do perfil proteico durante o amadurecimento dos frutos da cultivar 'Prata-Anã'.

O amadurecimento da banana é um processo geneticamente coordenado acompanhado pela elevação da produção de etileno e por alterações bioquímicas. O conhecimento deste processo é requisito desejável para o manuseio pós-colheita e melhoramento genético, visando retardar o período de senescência e elevar a qualidade e conservação pós-colheita. O objetivo do presente estudo foi identificar e comparar os perfis proteicos da polpa da variedade Prata-Anã, correspondentes aos estádios de maturação 1, 4 e 6, por gel bidimensional (2DE-PAGE) e cromatografía líquida acoplada à espectrometria de massas (LC/MS/MS)

Purification, characterization and structural determination of UDP-N-acetylglucosamine pyrophosphorylase produced by Moniliophthora perniciosa

The enzyme UDP-N-acetylglucosamine pyrophosphorylase (PyroMp) from Moniliophthora perniciosa (CCMB 0257), a pathogenic fungal strain and the causative agent of the witches' broom disease in Theobroma cacao, was partially purified by precipitation with ammonium sulfate and gel filtration on Sephacryl S-200. The buffer for enzyme extraction was sodium phosphate, 0.050 mol L-1, pH 7.0, containing 1.0 mol L-1 NaCl. Response surface methodology (RSM) was used to determine the optimum pH and temperature conditions. Four different isoenzymes (PyroMp I, PyroMp II, PyroMp III and PyroMp IV) were obtained with optimal pH ranging from 6.9-8.4 and optimum temperature ranging from 28 to 68 °C. The 3D structure of pyrophosphorylase of M. perniciosa was determined by comparative modeling. The model obtained showed a good quality, possessing 78.6% of amino acids in energetically allowed regions. The model was then submitted for DM simulation and showed a good geometric quality (91.1% Ramachandran plot). The active site of the enzyme was found to be extremely well conserved. This model will be useful for developing new inhibitors against witches' broom disease.A enzima UDP-N-acetilglicosamina pirofosforilase de Moniliophthora perniciosa (CCMB 0257), o fungo patogênico causador da doença vassoura-de-bruxa do Theobroma cacao, foi parcialmente purificada por precipitação com sulfato de amônio e cromatografia de gel filtração em Sephacryl S-200. O tampão de extração da enzima foi o fosfato de sódio, 0,050 mol L-1, pH 7,0, contendo 1,0 mol L-1 de NaCl. A metodologia de superfície de resposta (MSR) foi usada para a obtenção do pH e temperatura ótima. Os resultados mostraram quatro diferentes isoenzimas (PyroMp I, PyroMp II, PyroMp III e PyroMp IV) que apresentaram pH ótimo na faixa de 6,9-8,4 e temperatura ótima variando entre 28 a 68 °C. A estrutura 3D de pirofosforilase de M. perniciosa foi obtida por modelagem comparativa. O modelo obtido mostrou uma boa qualidade, possuindo 78,6% de aminoácidos nas regiões energeticamente favoráveis. O modelo foi então submetido a simulações de dinâmica molecular (DM). O modelo apresentou uma boa qualidade geométrica após as simulações de DM (91,1% -gráfico de Ramachandran). A procura pelo sítio ativo da enzima mostrou que este é mantido extremamente conservado. Este modelo pode ser útil para desenvolvimento de inibidores contra a doença vassoura de bruxa.FINEPCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)CNPqFAPESBFIOCRUZ - Programa de Pós-Graduação em Biotecnologia UEF

Purification, characterization and structural determination of UDP-N-acetylglucosamine pyrophosphorylase produced by Moniliophthora perniciosa

The enzyme UDP-N-acetylglucosamine pyrophosphorylase (PyroMp) from Moniliophthora perniciosa (CCMB 0257), a pathogenic fungal strain and the causative agent of the witches' broom disease in Theobroma cacao, was partially purified by precipitation with ammonium sulfate and gel filtration on Sephacryl S-200. The buffer for enzyme extraction was sodium phosphate, 0.050 mol L-1, pH 7.0, containing 1.0 mol L-1 NaCl. Response surface methodology (RSM) was used to determine the optimum pH and temperature conditions. Four different isoenzymes (PyroMp I, PyroMp II, PyroMp III and PyroMp IV) were obtained with optimal pH ranging from 6.9-8.4 and optimum temperature ranging from 28 to 68 °C. The 3D structure of pyrophosphorylase of M. perniciosa was determined by comparative modeling. The model obtained showed a good quality, possessing 78.6% of amino acids in energetically allowed regions. The model was then submitted for DM simulation and showed a good geometric quality (91.1% Ramachandran plot). The active site of the enzyme was found to be extremely well conserved. This model will be useful for developing new inhibitors against witches' broom disease22610151023CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFINANCIADORA DE ESTUDOS E PROJETOS - FINEPFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DA BAHIA - FAPESBFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçãoA enzima UDP-N-acetilglicosamina pirofosforilase de Moniliophthora perniciosa (CCMB 0257), o fungo patogênico causador da doença vassoura-de-bruxa do Theobroma cacao, foi parcialmente purificada por precipitação com sulfato de amônio e cromatografia de gel filtração em Sephacryl S-200. O tampão de extração da enzima foi o fosfato de sódio, 0,050 mol L-1, pH 7,0, contendo 1,0 mol L-1 de NaCl. A metodologia de superfície de resposta (MSR) foi usada para a obtenção do pH e temperatura ótima. Os resultados mostraram quatro diferentes isoenzimas (PyroMp I, PyroMp II, PyroMp III e PyroMp IV) que apresentaram pH ótimo na faixa de 6,9-8,4 e temperatura ótima variando entre 28 a 68 °C. A estrutura 3D de pirofosforilase de M. perniciosa foi obtida por modelagem comparativa. O modelo obtido mostrou uma boa qualidade, possuindo 78,6% de aminoácidos nas regiões energeticamente favoráveis. O modelo foi então submetido a simulações de dinâmica molecular (DM). O modelo apresentou uma boa qualidade geométrica após as simulações de DM (91,1% -gráfico de Ramachandran). A procura pelo sítio ativo da enzima mostrou que este é mantido extremamente conservado. Este modelo pode ser útil para desenvolvimento de inibidores contra a doença vassoura de brux

Gene expression, biochemical and physiological activities in evaluating melon seed vigor through ethanol release.

The aim of the present study was to establish parameters to evaluate melon seeds vigor by ethanol release test using a modified ethylometer and correlate with physiological seed quality, gene expression and enzyme activities alterations. To perform this trial, six seeds lots of yellow melon were submitted to determinations such as moisture content, germination assay and vigor tests. 25 and 50 seeds soaked with 0.5 mL of distilled water and tested after 1, 2, 3, 4, 5, 6 and 24 h at 40 °C. After 0, 6 and 24 h of imbibition, the high and low vigor lots were evaluated for the following enzyme activity: superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX) and alcoholic dehydrogenase (ADH). Accumulation of ADH protein was verified using Western Blotting hybridization technique with the primary Anti-ADH antibody. The release of ethanol in 25 seeds, soaked in 0.5 mL of distilled water for 6 h, proved efficient for vigor evaluation and can be related to other traditionally used tests. Enzyme activity varied according to the vigor of the seed lot. For the protein profile, there was variation in the accumulation of proteins of specific molecular weights throughout the soaking periods. There was also variation in expression according to accumulation of ADH protein. The biochemical and molecular tests corroborate with the establishment of the ethanol test as an additional tool to evaluate seed vigor

Evaluation of the Allergenicity Potential of TcPR-10 Protein from Theobroma cacao

Background: The pathogenesis related protein PR10 (TcPR-10), obtained from the Theobroma cacao-Moniliophthora perniciosa interaction library, presents antifungal activity against M. perniciosa and acts in vitro as a ribonuclease. However, despite its biotechnological potential, the TcPR-10 has the P-loop motif similar to those of some allergenic proteins such as Bet v 1 (Betula verrucosa) and Pru av 1 (Prunus avium). The insertion of mutations in this motif can produce proteins with reduced allergenic power. The objective of the present work was to evaluate the allergenic potential of the wild type and mutant recombinant TcPR-10 using bioinformatics tools and immunological assays. Methodology/Principal Findings: Mutant substitutions (T10P, I30V, H45S) were inserted in the TcPR-10 gene by sitedirected mutagenesis, cloned into pET28a and expressed in Escherichia coli BL21(DE3) cells. Changes in molecular surface caused by the mutant substitutions was evaluated by comparative protein modeling using the three-dimensional structure of the major cherry allergen, Pru av 1 as a template. The immunological assays were carried out in 8-12 week old female BALB/c mice. The mice were sensitized with the proteins (wild type and mutants) via subcutaneous and challenged intranasal for induction of allergic airway inflammation. Conclusions/Significance: We showed that the wild TcPR-10 protein has allergenic potential, whereas the insertion of mutations produced proteins with reduced capacity of IgE production and cellular infiltration in the lungs. On the other hand, in vitro assays show that the TcPR-10 mutants still present antifungal and ribonuclease activity against M. perniciosa RNA. In conclusion, the mutant proteins present less allergenic potential than the wild TcPR-10, without the loss of interesting biotechnological properties. (Résumé d'auteur

The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco

The ER-resident molecular chaperone BiP (binding protein) was overexpressed in soybean. When plants growing in soil were exposed to drought (by reducing or completely withholding watering) the wild-type lines showed a large decrease in leaf water potential and leaf wilting, but the leaves in the transgenic lines did not wilt and exhibited only a small decrease in water potential. During exposure to drought the stomata of the transgenic lines did not close as much as in the wild type, and the rates of photosynthesis and transpiration became less inhibited than in the wild type. These parameters of drought resistance in the BiP overexpressing lines were not associated with a higher level of the osmolytes proline, sucrose, and glucose. It was also not associated with the typical drought-induced increase in root dry weight. Rather, at the end of the drought period, the BiP overexpressing lines had a lower level of the osmolytes and root weight than the wild type. The mRNA abundance of several typical drought-induced genes [NAC2, a seed maturation protein (SMP), a glutathione-S-transferase (GST), antiquitin, and protein disulphide isomerase 3 (PDI-3)] increased in the drought-stressed wild-type plants. Compared with the wild type, the increase in mRNA abundance of these genes was less (in some genes much less) in the BiP overexpressing lines that were exposed to drought. The effect of drought on leaf senescence was investigated in soybean and tobacco. It had previously been reported that tobacco BiP overexpression or repression reduced or accentuated the effects of drought. BiP overexpressing tobacco and soybean showed delayed leaf senescence during drought. BiP antisense tobacco plants, conversely, showed advanced leaf senescence. It is concluded that BiP overexpression confers resistance to drought, through an as yet unknown mechanism that is related to ER functioning. The delay in leaf senescence by BiP overexpression might relate to the absence of the response to drought

Detecting Network Communities: An Application to Phylogenetic Analysis

This paper proposes a new method to identify communities in generally weighted complex networks and apply it to phylogenetic analysis. In this case, weights correspond to the similarity indexes among protein sequences, which can be used for network construction so that the network structure can be analyzed to recover phylogenetically useful information from its properties. The analyses discussed here are mainly based on the modular character of protein similarity networks, explored through the Newman-Girvan algorithm, with the help of the neighborhood matrix . The most relevant networks are found when the network topology changes abruptly revealing distinct modules related to the sets of organisms to which the proteins belong. Sound biological information can be retrieved by the computational routines used in the network approach, without using biological assumptions other than those incorporated by BLAST. Usually, all the main bacterial phyla and, in some cases, also some bacterial classes corresponded totally (100%) or to a great extent (>70%) to the modules. We checked for internal consistency in the obtained results, and we scored close to 84% of matches for community pertinence when comparisons between the results were performed. To illustrate how to use the network-based method, we employed data for enzymes involved in the chitin metabolic pathway that are present in more than 100 organisms from an original data set containing 1,695 organisms, downloaded from GenBank on May 19, 2007. A preliminary comparison between the outcomes of the network-based method and the results of methods based on Bayesian, distance, likelihood, and parsimony criteria suggests that the former is as reliable as these commonly used methods. We conclude that the network-based method can be used as a powerful tool for retrieving modularity information from weighted networks, which is useful for phylogenetic analysis