Functional characterization by genetic complementation of aroB-Encoded dehydroquinate synthase from Mycobactetium tuberculosis H37Rv and its heterologous expression and purification

The recent recrudescence of Mycobacterium tuberculosis infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in M. tuberculosis, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The aroB-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the aroB gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The Escherichia coli AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant M. tuberculosis dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with M. tuberculosis dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.

Functional XPB/RAD25 redundancy in Arabidopsis genome: characterization of AtXPB2 and expression analysis

The xeroderma pigmentosum complementation group B (XPB) protein is involved in both DNA repair and transcription in human cells. It is a component of the transcription factor IIH (TFIIH) and is responsible for DNA helicase activity during nucleotide (nt) excision repair (NER). Its high evolutionary conservation has allowed identification of homologous proteins in different organisms, including plants. In contrast to other organisms, Arabidopsis thaliana harbors a duplication of the XPB orthologue (AtXPB1 and AtXPB2), and the proteins encoded by the duplicated genes are very similar (95% amino acid identity). Complementation assays in yeast rad25 mutant strains suggest the involvement of AtXPB2 in DNA repair, as already shown for AtXPB1, indicating that these proteins may be functionally redundant in the removal of DNA lesions in A. thaliana. Although both genes are expressed in a constitutive manner during the plant life cycle, Northern blot analyses suggest that light modulates the expression level of both XPB copies, and transcript levels increase during early stages of development. Considering the high similarity between AtXPB1 and AtXPB2 and that both of predicted proteins may act in DNA repair, it is possible that this duplication may confer more flexibility and resistance to DNA damaging agents in thale cress. (C) 2004 Elsevier B.V. All rights reserved.

Eficácia de herbicidas aplicados nas épocas seca e úmida para o controle de Euphorbia heterophylla na cultura da cana-de-açúcar

Objetivou-se estudar o efeito de herbicidas aplicados em pré e pós-emergência, isolados e em combinações nas épocas seca e úmida, para o controle de leiteiro (Euphorbia heterophylla) na cultura de cana-de-açúcar. O experimento foi desenvolvido no período de agosto de 2008 a junho de 2009, em área de produção comercial de cana-de-açúcar localizada no município de Jaboticabal-SP. O delineamento experimental foi o de blocos ao acaso, com quatro repetições, em esquema de parcela subdividida. Na época seca foram avaliados os herbicidas amicarbazone (1.400 g ha-1), imazapic (147 g ha-1) e sulfentrazone (900 g ha-1), aplicados em 1º/8/2008 após a colheita da cana, e testemunha sem manejo prévio das plantas daninhas. Os herbicidas utilizados na época úmida foram: mesotrione isolado (192 g ha-1) e em mistura (120 g ha-1) com ametryn (1.500 gha-1), atrazine (1.500 gha-1) ou diuron + hexazinone (702 + 198 g ha-1), aplicados em 14/11/2008, além de testemunha capinada e outra sem manejo das plantas daninhas. A aplicação de imazapic na época seca foi eficaz no controle de E. heterophylla, dispensando a complementação de manejo na época úmida. No entanto, para os herbicidas amicarbazone e sulfentrazone houve necessidade da aplicação de mesotrione, isolado ou em mistura com ametryn, atrazine ou diuron + hexazinone, para a manutenção do controle de E. heterophylla na época úmida (de 105 a 230 dias após a aplicação na época seca). Quando não foi realizada a aplicação de herbicida na época seca, constatou-se melhor controle de E. heterophylla na época úmida com o herbicida mesotrione associado a ametryn, atrazine ou diuron + hexazinone do que quando aplicado isoladamente. Apesar dos sintomas visuais de fitointoxicação ocasionados pelos tratamentos mesotrione + ametryn e mesotrione + (diuron + hexazinone) pulverizados na época úmida, nenhum dos manejos adotados ou combinações entre eles interferiu no número de colmos viáveis por metro linear, no diâmetro e na altura de colmos de cana-de-açúcar.

Eficácia de herbicidas aplicados nas épocas seca e úmida para o controle de Merremia aegyptia na cultura da cana-de-açúcar

Objetivou-se estudar o efeito de herbicidas aplicados em pré e pós-emergência, isolados e em combinações nas épocas seca e úmida, para o controle de corda-de-viola (Merremia aegyptia) na cultura de cana-de-açúcar colhida mecanicamente sem queima. O experimento foi desenvolvido no período de julho de 2008 a maio de 2009, em área de produção comercial de cana-de-açúcar localizada no município de Pradópolis, SP. O delineamento experimental foi o de blocos ao acaso, com quatro repetições, em esquema de parcela subdividida. Foram avaliados na época seca os herbicidas amicarbazone (1.400 g ha ¹), clomazone + hexazinone (800 + 200 g ha-1) e imazapic (147 g ha-1), aplicados em 16/7/2008 após a colheita da cana, e o tratamento sem manejo prévio das plantas daninhas nessa época. Os herbicidas estudados na época úmida foram: mesotrione isolado (192 g ha-1) e em mistura (120 g ha-1) com atrazine (1.500 g ha-1), metribuzin (960 g ha-1) ou diuron + hexazinone (702 + 198 g ha-1), aplicados em 6/11/2008, além de testemunha capinada e outra sem manejo das plantas daninhas. Dos herbicidas utilizados na época seca, o amicarbazone promoveu o melhor controle de M. aegyptia. No entanto, para todos eles, a complementação de manejo com a aplicação de herbicidas na época úmida mostrou-se obrigatória. Nesta época, a associação de mesotrione aos herbicidas atrazine, metribuzin e diuron + hexazinone foi mais eficaz no controle de M. aegyptia do que quando aplicado sozinho. Nenhum dos tratamentos com herbicidas interferiu no número de colmos por metro linear, diâmetro e altura de colmos de cana-de-açúcar (variedade SP 81-3250).

Glyphosate e adubação foliar com manganês na cultura da soja transgênica

Com base na hipótese de que a soja transgênica tolerante ao glyphosate necessitaria da adição complementar de manganês devido a alterações na absorção e no metabolismo do elemento pelas plantas, objetivou-se estudar a interação da soja transgênica pulverizada com glyphosate e a adubação foliar com manganês. Foi desenvolvido experimento em campo, no ano agrícola 2007/2008, na Fazenda de Ensino, Pesquisa e Produção da UNESP, campus de Jaboticabal, SP. O delineamento experimental foi o de blocos ao acaso, no esquema fatorial 4 x 4, com quatro repetições. Foram avaliados quatro manejos de plantas daninhas [glyphosate (p.c. Roundup Ready) a 0,72 e 1,20 kg ha-1 de equivalente ácido, fluazifop-p-butyl + fomesafen (p.c. Fusiflex) a 0,25 + 0,25 kg ha-1 e testemunha capinada, sem herbicida] e quatro doses (0, 42, 84 e 126 g ha-1) de manganês em aplicação foliar na soja. Os tratamentos estudados não alteraram significativamente a produtividade de grãos, os teores de manganês no solo, a altura e a matéria seca das plantas de soja. Apenas a mistura fluazifop-p-butyl mais fomesafen ocasionou injúrias visuais nas plantas, porém os sintomas ficaram restritos às folhas que interceptaram o jato de pulverização. Para massa de 100 grãos, os herbicidas estudados não diferiram da testemunha; no entanto, as plantas tratadas com 0,72 kg ha-1 de glyphosate apresentaram menor massa de grãos. A aplicação de manganês não influenciou os teores do elemento nas plantas tratadas com glyphosate e naquelas sem herbicida. Portanto, o glyphosate não prejudicou a absorção ou o metabolismo do manganês pela planta, e o crescimento e desenvolvimento das plantas tratadas foram estatisticamente similares aos das não tratadas com herbicidas.

Identification of Rv3852 as a nucleoid-associated protein in Mycobacterium tuberculosis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The Importance of Bees for Eggplant Cultivations (Hymenoptera: Apidae, Andrenidae, Halictidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biochemical characterization of Neurospora crassa glycogenin (GNN), the self-glucosylating initiator of glycogen synthesis

Glycogenin acts in the initiation step of glycogen biosynthesis by catalyzing a self-glucosylation reaction. In a previous work [de Paula et al., Arch. Biochem. Biophys. 435 (2005) 112-124], we described the isolation of the cDNA gnn, which encodes the protein glycogenin (GNN) in Neurospora crassa. This work presents a set of biochemical and functional studies confirming the GNN role in glycogen biosynthesis. Kinetic experiments showed a very low GNN K-m (4.41 mu M) for the substrate UDP-glucose. Recombinant GNN was produced in Escherichia coli and analysis by mass spectroscopy identified a peptide containing an oligosaccharide chain attached to Tyr196 residue. Site-directed mutagenesis and functional complementation of a Saccharomyces cerevisiae mutant strain confirmed the participation of this residue in the GNN self-glucosylation and indicated the Tyr198 residue as an additional, although less active, glucosylation site. The physical interaction between GNN and glycogen synthase (GSN) was analyzed by the two-hybrid assay. While the entire GSN was required for full interaction, the C-terminus in GNN was more important. Furthermore, mutation in the GNN glucosylation sites did not impair the interaction with GSN. (c) 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Effects of mutations in acetate metabolism on high-cell-density growth of Escherichia coli

To study the role played by acetate metabolism during high-cell-density growth of Escherichia coli cells, we constructed isogenic null mutants of strain W3100 deficient for several genes involved either in acetate metabolism or the transition to stationary phase. We grew these strains under identical fed-batch conditions to the highest cell densities achievable in 8 h using a predictive-plus-feedback-controlled computer algorithm that maintained glucose at a set-point of 0.5 g/l, as previously described. Wild-type strains, as well as mutants lacking the sigma(s) subunit of RNA polymerase (rpoS), grew reproducibly to high cell densities (44-50 g/l dry cell weights, DCWs). In contrast, a strain lacking acetate kinase (ackA) failed to reach densities greater than 8 g/l. Strains lacking other acetate metabolism genes (pta, acs, poxB, iciR, and fadR) achieved only medium cell densities (15-21 g/l DCWs). Complementation of either the acs or the ackA mutant restored wild-type high-cell-density growth, on a dry weight basis, poxB and fadR strains produced approximately threefold more acetate than did the wild-type strain. In contrast, the pta, acs, or rpoS strains produced significantly less acetate per cell dry weight than did the wild-type strain. Our results show that acetate metabolism plays a critical role during growth of E. coli cultures to high cell densities. They also demonstrate that cells do not require the sigma(s) regulon to grow to high cell densities, at least not under the conditions tested.

GNN is a self-glucosylating protein involved in the initiation step of glycogen biosynthesis in Neurospora crassa

The initiation of glycogen synthesis requires the protein glycogenin, which incorporates glucose residues through a self-glucosylation reaction, and then acts as substrate for chain elongation by glycogen synthase and branching enzyme. Numerous sequences of glycogenin-like proteins are available in the databases but the enzymes from mammalian skeletal muscle and from Saccharomyces cerevisiae are the best characterized. We report the isolation of a cDNA from the fungus Neurospora crassa, which encodes a protein, GNN, which has properties characteristic of glycogenin. The protein is one of the largest glycogenins but shares several conserved domains common to other family members. Recombinant GNN produced in Escherichia coli was able to incorporate glucose in a self-glucosylation reaction, to trans-glucosylate exogenous substrates, and to act as substrate for chain elongation by glycogen synthase. Recombinant protein was sensitive to C-terminal proteolysis, leading to stable species of around 31 kDa, which maintained all functional properties. The role of GNN as an initiator of glycogen metabolism was confirmed by its ability to complement the glycogen deficiency of a S. cerevisiae strain (glg1 glg2) lacking glycogenin and unable to accumulate glycogen. Disruption of the gnn gene of N. crassa by repeat induced point mutation (RIP) resulted in a strain that was unable to synthesize glycogen, even though the glycogen synthase activity was unchanged. Northern blot analysis showed that the gnn gene was induced during vegetative growth and was repressed upon carbon starvation. (C) 2004 Elsevier B.V. All rights reserved.

A putative twin-arginine translocation system in the phytopathogenic bacterium Xylella fastidiosa

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Clinicopathological significance of ERCC1 expression in breast cancer

The excision repair cross-complementation 1 (ERCC1) enzyme plays an essential role in the nucleotide excision repair pathway and is associated with resistance to platinum-based chemotherapy in different types of cancer. The aim of the present study was to evaluate the clinicopathological significance of ERCC1 expression in breast cancer patients. We analyzed the immunohistochemical expression of ERCC1 in a tissue microarray from 135 primary breast carcinomas and correlated the immunohistochemical findings with clinicopathological factors and outcome data. ERCC1 expression analysis was available for 109 cases. In this group, 58 (53.2%) were positive for ERCC1. ERCC1-positive expression was correlated with smaller tumor size (P=0.007) and with positivity for estrogen receptor (P=0.040), but no correlation was found with other clinicopathological features. Although not statistically significant, triple negative breast cancers were more frequently negative for ERCC1 (61.5% of the cases) compared to the non-triple negative breast cancer cases (41.5%). In conclusion, ERCC1 expression correlated significantly with favorable prognostic factors, such as smaller tumor size and ER-positivity, suggesting a possible role for ERCC1 as a predictive and/or prognostic marker in breast cancer. © 2013 Elsevier GmbH.

Extração semi-automática de rodovias no espaço-objeto: uso integrado de um estéreo par de imagens aéreas e um MDT

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Tricomas secretores de Lippia stachyoides Cham. (Verbenaceae): estrutura, ontogênese e secreção

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