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.

Adubação nitrogenada em cobertura no girassol irrigado nas condições de Cassilândia-MS

Nitrogen is the most required nutrient by plants for exerting important functions in the vegetable metabolism. In sunflower crop, its efficiency causes nutritional disorder, being the nutrient that most limits the yield. Besides, high doses may increase the incidence of pests and diseases, affecting the yield. Hereafter, this experiment was carried out with the aim of evaluating the response of the irrigated sunflower crop (Helianthus annuus L.) submitted to different doses of side dressing nitrogen fertilization applied uncovered nitrogen in small parts. A randomized complete block design, with four replications was used. The constituted by four doses of nitrogen (0, 20, 40, and 80 kg ha(-1)), as urea. Side dressing nitrogen fertilization provides increase in all characteristics studied, and the dose of N of maxim suitable technical efficiency to have a good yield is of 55 kg ha(-1) of N for the cultivation of the sunflower irrigated in the region of Cassilandia, Mato Grosso do Sul State.

Phosphorus and potassium balance in soil under crop rotation and fertilization

The use of crop rotation and manure application can provide sustainability for an agricultural production system by improving soil quality and increasing nutrient use efficiency. This study aimed to evaluate the effect of mineral, organic and mineral+organic fertilization on grain yield and on soil phosphorus and potassium balance, in two crop systems under no-till, with and without rotation of cover crops. The experiment was carried out from 2006 to 2008 on a clayey Rhodic Hapludox in Marechal Candido Rondon, Parana State, Brazil. The cropping sequence in the rotation system involving cover crops was black oat + hairy vetch + forage turnip/corn/pigeon pea/wheat/mucuna + brachiaria + sunn hemp, and in the succession system was wheat/corn/wheat/soybean. Organic and mineral+organic fertilizations consisted of the application of solely manure and manure combined with mineral fertilizer, respectively. Soil P and K balances were calculated after the second year of the experiment, up to a depth of 0.40 m. First year corn yields were higher in the crop succession system accompanied by mineral fertilization. In the second year, wheat and soybean yield did not vary between crop systems and nutrient sources, demonstrating the residual effect of crop rotation and manure use. Crop rotation with cover crops resulted in an increase in soil K levels by promoting the recycling of this nutrient in the soil. In both crop systems, the application of mineral and organic fertilizers - either in isolation or in combination - resulted in a negative soil P and K balance in the short term. This represents a threat to the sustainability of the agricultural production system in the long term, due to the depletion of soil nutrient reserves.

Electrochemical study of o-toluidine blue impregnated in mesoporous silica channels

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Banana Peel Applied to the Solid Phase Extraction of Copper and Lead from River Water: Preconcentration of Metal Ions with a Fruit Waste

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

EVALUATION of HEAVY METAL AVAILABILITY IN CONTAMINATED SEDIMENTS FROM THE ILHA SOLTEIRA HYDROELECTRIC DAM on THE PARANA RIVER AT ILHA SOLTEIRA, SP, BRAZIL

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

4-amine-2-mercaptopyrimidine modified silica gel applied in Cd(II) and Pb(II) extraction from an aqueous medium

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

Sediment grain size distribution and heavy metals determination in a dam on the Parana River at Ilha Solteira, Brazil

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

Synthesis, characterization, and application of modified silica in the removal and preconcentration of lead ions from natural river water

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

Satellyptus: analysis and database of microsatellites from ESTs of Eucalyptus

The main goal of our research was to search for SSRs in the Eucalyptus EST FORESTs database (using a software for mining SSR-motifs). With this objective, we created a database for cataloging Eucalyptus EST-derived SSRs, and developed a bioinformatics tool, named Satellyptus, for finding and analyzing microsatellites in the Eucalyptus EST database. The search for microsatellites in the FORESTs database containing 71,115 Eucalyptus EST sequences (52.09 Mb) revealed 20,530 SSRs in 15,621 ESTs. The SSR abundance detected on the Eucalyptus ESTs database (29% or one microsatellite every four sequences) is considered very high for plants. Amongst the categories of SSR motifs, the dimeric (37%) and trimeric ones (33%) predominated. The AG/CT motif was the most frequent (35.15%) followed by the trimeric CCG/CGG (12.81%). From a random sample of 1,217 sequences, 343 microsatellites in 265 SSR-containing sequences were identified. Approximately 48% of these ESTs containing microsatellites were homologous to proteins with known biological function. Most of the microsatellites detected in Eucalyptus ESTs were positioned at either the 5 or 3 end. Our next priority involves the design of flanking primers for codominant SSR loci, which could lead to the development of a set of microsatellite-based markers suitable for marker-assisted Eucalyptus breeding programs.

Myocardial dysfunction with increased ventricular compliance in volume overload hypertrophy

The aim this study was to evaluate systolic and diastolic function in volume overload induced myocardial hypertrophy in rats.Volume overload myocardial hypertrophy was induced in thirteen male Wistar rats by creating infrarenal arteriovenous fistula (AVF). The results were compared with a SHAM operated group (n = 11). Eight weeks after surgery, tail-cuff blood pressure was recorded, then rats were sacrificed for isolated heart studies using Langendorffs preparation.AVF rats presented increased left and right ventricular weights, compared to controls. The increased normalized ventricular volume (V0/LVW, 0.141 +/- 0.035 mL/g vs. 0.267 +/- 0.071 mL/g, P < 0.001) in the AVF group indicated chamber dilation. Myocardial hydroxyproline concentration remained unchanged. There was a significant decrease in +dP/dt (3318 +/- 352 mm Hg s(-1) vs. 2769 +/- 399 mm Hg s(-1); P=0,002), end-systolic pressure-volume relation (246 +/- 56 mm Hg mL(-1) vs. 114 +/- 63 mm Hg mL(-1);, P < 0,001), and -dP/dt (1746 +/- 240 min Hg s(-1) vs. 1361 +/- 217 mm Hg s(-1), P < 0.001) in the AVF group, which presented increased ventricular compliance (Delta V-25: SHAM=0.172 +/- 0.05 mL vs. AVF=0.321 +/- 0.072 mL, P < 0.001) with preserved myocardial passive stiffness (Strain(25): SHAM=13.5 +/- 3.0% vs. AVF=12.3 +/- 1.9%, P > 0.05).We conclude that volume-overload induced hypertrophy causes myocardial systolic and diastolic dysfunction with increased ventricular compliance. These haemodynamic features help to explain the long-term compensatory phase of chronic volume overload before transition to overt congestive heart failure. (c) 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity.

Pump excited state absorption in holmium-doped fluoride glass

The primary excited state absorption processes relating to the (5)I(6) -> (5)I(7) 3 mu m laser transition in singly Ho(3+)-doped fluoride glass have been investigated in detail using time-resolved fluorescence spectroscopy. Selective laser excitation of the (5)I(6) and (5)I(7) energy levels established the occurrence of two excited state absorption transitions from these energy levels that compete with previously described energy transfer upconversion processes. The (5)I(7) -> (5)I(4) excited state absorption transition has peak cross sections at 1216 nm (sigma(esa)=2.8x10(-21) cm(2)), 1174 nm (sigma(esa)=1x10(-21) cm(2)), and 1134 nm (sigma(esa)=7.4x10(-22) cm(2)) which have a strong overlap with the (5)I(8) -> (5)I(6) ground state absorption. on the other hand, it was established that the excited state absorption transition (5)I(6) -> (5)S(2) had a weak overlap with ground state absorption. Using numerical solution of the rate equations, we show that Ho(3+)-doped fluoride fiber lasers employing pumping at 1100 nm rely on excited state absorption from the lowest excited state of Ho(3+) to maintain a population inversion and that energy transfer upconversion processes compete detrimentally with the excited state absorption processes in concentrated Ho(3+)-doped fluoride glass. (c) 2008 American Institute of Physics.

Efeito de diferentes substratos na produção de mudas de mangabeira (Hancornia speciosa)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Sazonalidade na produção de raízes e teor de β-ecdisona em acessos de fáfia

As raízes de espécies de Pfaffia são utilizadas há muito na medicina popular no Brasil, especialmente como tônicas, afrodisíacas e antidiabéticas. Nas margens e ilhas do Rio Paraná, vegeta naturalmente a espécie Pfaffia glomerata (Spreng.) Pedersen, que corre risco de extinção pela coleta intensiva de suas raízes. Para preservá-la, é preciso desenvolver sistemas que permitam seu uso sustentável. Nesse contexto, foi avaliada a sazonalidade da produção de biomassa e o teor de β-ecdisona em raízes de diferentes acessos de P. glomerata. A coleta dos acessos foi realizada ao longo dos rios Paraná (A1 e A3), Ivaí (A2) e Paranapanema (A4). Mudas produzidas a partir desses acessos foram plantadas experimentalmente em área de ocorrência natural da espécie. O delineamento utilizado foi blocos casualizados, com parcelas subdivididas (acessos nas parcelas; épocas de colheita nas subparcelas), quatro repetições e subparcelas com seis plantas na área útil. Foram realizadas quatro colheitas: oito, dez, doze e quatorze meses após o plantio. As maiores produtividades em massa seca (MS) de raízes foram alcançadas na terceira colheita, com 38,41 g planta-1. Já o acesso A4 foi significativamente mais produtivo que os demais, tanto para massa fresca (94,67 g planta-1), quanto para MS de raízes (26,39 g planta-1). A relação MF/MS das raízes, com média de 26,9% (3,7:1), foi pouco influenciada pela origem dos acessos, embora tenha se alterado significativamente ao longo das colheitas (de 4,1:1, na primeira colheita, a 3,3:1, na terceira colheita, que correspondeu ao valor máximo). O teor de β-ecdisona foi estatisticamente similar nas três primeiras colheitas, variando de 0,26 a 0,38%, com redução drástica na última colheita (0,16%). Os acessos A2 e A3 foram aqueles com teor de β-ecdisona mais altos, respectivamente 0,36 e 0,30%. Considerando os resultados obtidos, sugere-se que a terceira colheita, que corresponde a doze meses após o transplante, como a mais adequada para colheita das raízes.