Under flow impedimetric measurements using magnetic particles for label-free detection affinity target

The necessity to adapt sensors based on electrochemical techniques for high throughput analysis control increases the interest to develop new analytical systems able to perform measurements under buffer now. In this report we explored the possibility of employing a new system to make impedimetric measurements to detect the interaction between proteins and small molecules. The well-known biotin-streptavidin interaction was adopted to evaluate the proposed assembly. This system allows us to perform experiments under flow. Magnetic beads functionalized with streptavidin were used and first characterized using AFM and FTIR. Non-faradic impedance spectroscopy allowed the detection of the biotin-streptavidin interaction. Using our new system and under a flow of PBS buffer, 5 10-5 M of biotin was detected with a stable signal. (c) 2007 Elsevier B.V. All rights reserved.

Hydrogen purification for fuel cell using CuO/CeO(2)-Al(2)O(3) catalyst

CuO/CeO(2), CuO/Al(2)O(3) and CuO/CeO(2)-Al(2)O(3) catalysts, with CuO loading varying from 1 to 5 wt.%, were prepared by the citrate method and applied to the preferential oxidation of carbon monoxide in a reaction medium containing large amounts of hydrogen (PROX-CO). The compounds were characterized ex situ by X-ray diffraction, specific surface area measurements, temperature-programmed reduction and temperature-programmed reduction of oxidized surfaces; XANES-PROX in situ experiments were also carried out to study the copper oxidation state under PROX-CO conditions. These analyses showed that in the reaction medium the Cu(0) is present as dispersed particles. On the ceria, these metallic particles are smaller and more finely dispersed, resulting in a stronger metal-support interaction than in CuO/Al(2)O(3) or CuO/CeO(2)-Al(2)O(3) catalysts, providing higher PROX-CO activity and better selectivity in the conversion of CO to CO(2) despite the greater BET area presented by samples supported on alumina. It is also shown that the lower CuO content, the higher metal dispersion and consequently the catalytic activity. The redox properties of the ceria support also contributed to catalytic performance. (C) 2010 Elsevier B.V. All rights reserved.

Valorization of an Industrial Organosolv-Sugarcane Bagasse Lignin: Characterization and Use as a Matrix in Biobased Composites Reinforced With Sisal Fibers

In the present study, the main focus was the characterization and application of the by-product lignin isolated through an industrial organosolv acid hydrolysis process from sugarcane bagasse, aiming at the production of bioethanol. The sugarcane lignin was characterized and used to prepare phenolic-type resins. The analysis confirmed that the industrial sugarcane lignin is of HGS type, with a high proportion of the less substituted aromatic ring p-hydroxyphenyl units, which favors further reaction with formaldehyde. The lignin-formaldehyde resins were used to produce biobased composites reinforced with different proportions of randomly distributed sisal fibers. The presence of lignin moieties in both the fiber and matrix increases their mutual affinity, as confirmed by SEM images, which showed good adhesion at the biocomposite fiber/matrix interface. This in turn allowed good load transference from the matrix to the fiber, leading to biobased composites with good impact strength (near 500 J m(-1) for a 40 wt% sisal fiber-reinforced composite). The study demonstrates that sugarcane bagasse lignin obtained from a bioethanol plant can be used without excessive purification in the preparation of lignocellulosic fiber-reinforced biobased composites displaying high mechanical properties. Biotechnol. Bioeng. 2010;107: 612-621. (C) 2010 Wiley Periodicals, Inc.

Purification and analysis of protein synthetic initiation factors from Volvox Carteri

Volvox carteri, a multi-celled green algae, can grow synchronously given a sixteen hour light period followed by an eight hour dark period, a cycle which is repeated for a 48 hour growth cycle total. Near the end of each light period, reproductive cells divide rapidly resulting in the differentiation of ceIls. When the dark period begins, this differentiation stops and the cells remain dormant with little protein synthesis or differentiation occurring. Immediately after the lights come back on, however, the cells again undergo rapid protein synthesis and complete their differentiation. Previous studies have concluded that Volvox carteri discontinue protein synthesis during the dark phase due to regulation at the translational level and not the transcriptional level. Therefore, the inhibition of protein synthesis does not lie in the transfer of the protein coding sequence from DNA to mRNA, but rather in the transfer of this information from the mRNA to the ribosomes. My research examined this translational regulation to determine the factor(s) causing the discontinuation of protein synthesis during the dark phase. Evidence from other research further suggests that the control of translation lies in the initiation step rather than the elongation step. Eukaryotic initiation factors aid in the binding of the ribosomal subunits to the mRNA to initiate protein synthesis. It is known that initiation factors can be modified by phosphorylation, regulating their activity. Therefore, my study focused upon isolating some of these initiation factors in order to determine whether or not such modifications are responsible for the inhibition of dark phase protein synthesis in Volvox carteri.

Clonagem e expressão do gene que codifica o inibidor de quimotripsina de Erythrina velutina WILLD. - caracterização e avaliação de seu potencial farmacológico

Proteinases are enzymes distributed widely founded in several organisms and perform many different functions, from maintaining homeostasis to the worsening of some diseases such as cancer, autoimmune diseases and infections. The proteins responsible of controlling the action of these enzymes are the inhibitors, that are classified based on their target proteases and are founded since simple organisms, such as bacteria, to higher organisms, such as larger plants and mammals. Plant proteinase inhibitors act by reducing or inactivating the activity of target proteases, thus, these proteins have been studied as potential tools in the treatment of diseases related to protease activities. In this context, an inhibitor of chymotrypsin from Erythrina velutina, called EvCI was previously purified and it was observed that this protein plays in vitro anticoagulant activity and anti-inflammatory activity in in vivo model. Aiming to reduce the environmental impact caused by the purification EvCI in high amounts and to facilitate the process of obtaining this protein, the recombinant chymotrypsin inhibitor from Eryhrina velutina was produced after cloning and expression in Escherichia coli. The bacteria were grown in LB medium and after induction of the expression this material was subjected to procedures for cell lysis and the product was applied on Nickel-affinity column. The proteins adsorbed were digested by thrombin and applied on Chymotrypsin-Sepharose affinity column, obtaining the purified inhibitor, named recEvCI. After electrophoresis, the recombinant inhibitor showed an approximately molecular mass of 17 kDa, and reduced the chymotrypsin and elastase activities in vitro. The recombinant inhibitor was sequenced and was found similar amino acids residues when compared to other inhibitors deposited in the database, with some modifications. recEvCI showed high stability under pH variations and reducing conditions, maintaining its activity around 80%. This protein increased the blood coagulation time in vitro by acting on the intrinsic pathway and did not show cytotoxicity against strains of mouse 3T3 fibroblasts and RAW 264.7 macrophages. recEvCI showed microbicide activity related to release of nitric oxide and consequently the activation of macrophages, futhermore having proinflammatory effects assessed by increased release of TNF-α. These results indicate that recEvCI can be biotechnologically used as a new tool in the control of coagulation-related diseases as well as can be an activating agent of the immune system in immunosuppressed individuals

Lectina da esponja marinha Tedania ignis: purificação, caracterização e interação com leishmanias

Lectin obtained from the marine sponge Tedania ignis was purified and characterized by extraction of soluble proteins (crude extract) in 50mM Borax, pH 7.5. The purification procedure was carried out by crude extract precipitation with ammonium sulfate 30% (FI). The precipitated was resuspended in the same buffer and fractionated with acetone 1.0 volume (F1.0). A lectin was purified from this specific fraction by using an affinity chromatography Sepharose 6B. This lectin preferentially agglutinated human erythrocytes from B type previously treated with papain enzyme. The hemagglutinating activity lectin was dependent of divalent Mn2+ cation and was inhibited by the carbohydrates galactose, xylose and fructose. SDS-PAGE analysis indicated a molecular mass of the lectin around 45 kDa. This protein showed stability until 40°C for 1 h. Further, it showed activity between pH 2.5 and 11.5, with an enhanced activity at pH 7.5. Leishmania chagasi promastigotes stained with Coomassie brilliant blue R-250 were agglutinated by F1,0 and in the presence of galactose this interaction was abolished. These results show that this lectin could be implicated in defense procedures and it will can be used as biological tools in studies with this protozoon

Vicilina de sementes da leguminosa selvagem Anadenanthera macrocarpa (AmV): purificação, caracterização, efeito deletério e mecanismo de ação para bruquídeo callosobruchus maculatus

Grains and legume seeds are foods that form the basis of the diets of many cultures around the world, winch contritbute to the daily nutrient requirements of humans. Vicilins (7S globulin) are storage proteins found in legume seeds, and may have an additional function constitutive defense of the embryo against pests and pathogens. In this work the vicilin from Anadenanthera macrocarpa - AmV (red-angico), was purified and partially characterized, its effect on development and larval survival and adult emergence of Callosobruchus maculatus was evaluated by determination of LD50, WD50 and ED50 in system bioassay. Purification of vicilin was initiated by the chitin affinity chromatography and then gel filtration (Superdex 75 Tricorn 10x300 mm) FPLC system followed by reverse phase chromatography (C8 phenomenex) on HPLC system. Bioassays WD50 and LD50 for larvae were 0.32% and 0.33% (w:w) respectively, since the ED50 for adults was 0.096%. The probable mechanism of action was evaluated by testing digestibility of AmV in vitro, and observed for the involvement of two fragments vicilins immunoreactive against polyclonal Anti-vicilin from Erythrina velutina (Anti-EvV) about of 22 and 13 kDa chitin binding. The AmV in its native form has been recognized by the anti-EvV, indicating that there is a conserved region in the vicilin and is probably corresponding to the chitin binding domains. These results point to a new vicilin chitin binding that can subsequently be used as a possible biopesticide protein source, in order to control insect pest C. maculatus and confirm literature findings that demonstrate vicilin in the presence of different kinds of ligands to conserved regions chitin not yet characterized

Purification and renal effects of phospholipase A(2) isolated from Bothrops insularis venom

Bothrops insularis venom contains a variety of substances presumably responsible for several pharmacological effects. We investigated the biochemical and biological effects of phospholipase A(2) protein isolated from B. insularis venom and the chromatographic profile showed 7 main fractions and the main phospholipase A(2) (PLA(2)) enzymatic activity was detected in fractions IV and V. Fraction IV was submitted to a new chromatographic procedure on ion exchange chromatography, which allowed the elution of 5 main fractions designated as lV-1 to IV-5, from which lV-4 constituted the main fraction. The molecular homogeneity of this fraction was characterized by high-performance liquid chromatography (HPLC) and demonstrated by mass spectrometry (MS), which showed a molecular mass of 13984.20 Da; its N-terminal sequence presented a high amino acid identity (up to 95%) with the PLA(2) of Bothrops jararaca and Bothrops asper. Phospholipase A(2) isolated from B. insularis (Bi PLA(2)) venom (10 mu g/mL) was also studied as to its effect on the renal function of isolated perfused kidneys of Wistar rats (n = 6). Bi PLA(2) increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa+) and chloride tubular reabsorption (%TCl-) decreased at 120 min, without alteration in potassium transport. In conclusion, PLA(2) isolated from B. insularis venom promoted renal alterations in the isolated perfused rat kidney. (c) 2007 Elsevier Ltd. All rights reserved.

Purification and characterization of the biological effects of phospholipase A(2) from sea anemone Bunodosoma caissarum

Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA(2) isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the Sao Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA(2) proteins (named BcPLA(2)1, BCPLA(2)2 and BcPLA(2)3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA(2)1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA(2)1 showed high amino acid sequence identity with PLA(2) group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA(2) and 1POC_A). In addition, BcPLA(2)1 also showed significant overall homology to bee PLA(2). The enzymatic activity induced by native BCPLA(2)1 (20 mu g/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA(2)1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA(2) from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA(2)1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA(2), a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration. (C) 2009 Elsevier Ltd. All rights reserved.

Purification and preliminary crystallographic analysis of a new Lys49-PLA2 from B-jararacussu

BjVIII is a new myotoxic Lys49-PLA2 isolated from Bothrops jararacussu venom that exhibits atypical effects on human platelet aggregation. To better understand the mode of action of BjVIII, crystallographic studies were initiated. Two crystal forms were obtained, both containing two molecules in the asymmetric unit (ASU). Synchrotron radiation diffraction data were collected to 2.0 angstrom resolution and 1.9 angstrom resolution for crystals belonging to the space group P2(1)2(1)2(1) (a = 48.4 angstrom, b = 65.3 angstrom, c = 84.3 angstrom) and space group P3(1)21 (a = b = 55.7 angstrom, c = 127.9 angstrom), respectively. Refinement is currently in progress and the refined structures are expected to shed light on the unusual platelet aggregation activity observed for BjVIII.

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.

Purification and characterization of β-Fructosidase with inulinase activity from Aspergillus niger - 245

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

Purification and structural characterisation of (1 -> 3 ; 1 -> 6)-beta-D-glucans (botryosphaerans) from Botryosphaeria rhodina grown on sucrose and fructose as carbon sources: a comparative study

Two botryosphaerans, exopolysaccharides (EPS) secreted by the ascomyceteous fungus Botryosphaeria rhodina, when grown on sucrose and fructose as sole carbon sources, were structurally compared after their isolation from the culture medium. Both EPS were submitted to trypsin digestion, and eluted as a single peak on gel filtration. Total acid hydrolysis yielded only glucose, and data from methylation analysis and Smith degradation indicated that both EPS constituted a main chain of glucopyranosyl beta(1 -> 3) linkages substituted at O-6. The products obtained after partial acid hydrolysis demonstrated side chains consisting of glucosyl- and gentiobiosyl- linked beta(1 -> 6) residues. C-13-NMR spectroscopy studies showed that all glucosidic linkages were of the beta-configuration. The carbon source affected the side chain structures of botryosphaeran but not the main chain makeup. Sucrose produced less branching (21%) than fructose (31%). (c) 2005 Published by Elsevier Ltd.

New insights into trypanosomatid U5 small nuclear ribonucleoproteins

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

Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon source, This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6 HR 10/30 columns. The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30, Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity, the optimal hydrolysis temperature for sucrose, raffinose and inulin was 55 degrees C and the optimal pH for sucrose was 4.75, the apparent K-m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively, Thin layer chromatography showed that inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides, the results obtained suggest the hypothesis that enzyme production was constitutive.