Purification and biological effects of a C-type lectin isolated from Bothrops moojeni

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

Renal and vascular effects of the natriuretic peptide isolated from Crotalus durissus cascavella venom

Crotalus durissus cascavella is a snake that is usually found in the scrublands of northeast Brazil. The components of its venom may have effects on the vascular and renal systems. Recently, a new bradykinin inhibitory peptide has been identified in the venom of the Crotalinae family. The aim of the present study was to investigate the renal and vascular effects of the natriuretic peptide isolated from the venom of Crotalus durissus cascavella (NP2_Casca). The chromatographic profile showed the fractionation of substances identified as convulxin, gyroxin, crotoxin and crotamine, as well as fractions V and VI. The electrophoretic profile of fraction V consisted of several bands ranging from approximately 6 kDa to 13 kDa, while fraction VI showed only two main electrophoretic bands with molecular weights of approximately 6 and 14 kDa. Reverse-phase chromatography showed that NP2_Casca corresponds to about 18% of fraction VI and that this fraction is the main natriuretic peptide. NP2_Casca was compared to other natriuretic peptides from other sources of snake venom. All amino acid sequences that were compared showed a consensus region of XGCFGX, XLDRIX and XSGLGCX. The group treated with NP2-Casca showed an increase in perfusion pressure, renal vascular resistance, urinary flow and glomerular filtration rate. The percent of total and proximal tubular transport of sodium was reduced significantly after administration of the peptide. The mean arterial pressure showed a dose-dependent decrease after infusion of NP2_Casca, and an increase in nitrite production. In the aortic ring assay, NP2_Casca caused a relaxant effect in endothelium-intact thoracic aortic rings precontracted with phenylephrine in the presence and absence of isatin. NP2_Casca failed to relax the aortic rings precontracted with an isosmotic potassium Krebs-Henseleit solution. In conclusion, the natriuretic peptide isolated from Crotalus durissus cascavella venom produced renal and vascular effects. NP2_Casca reduced total and proximal sodium tubular transport, leading to an increase in sodium excretion, thereby demonstrating a diuretic action. A hypotensive effect was displayed in an arterial pressure assay, with an increase in nitrite production, suggesting a possible vasoactive action. (C) 2008 Elsevier Ltd. All rights reserved.

Renal- and calcium-dependent vascular effects of Polybia paulista wasp venom

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Renal and cardiovascular effects of Bothrops marajoensis venom and phospholipase A(2)

Bothrops marajoensis is found in the savannah of Marajo Island in the State of Par S and regions of Amapa State, Brazil. The aim of the work was to study the renal and cardiovascular effects of the B. marajoensis venom and phospholipase A(2) (PLA(2)). The venom was fractionated by Protein Pack 5PW. N-terminal amino acid sequencing of sPLA(2) showed amino acid identity with other lysine K49sPLA(2)s of snake venom. B. marajoensis venom (30 mu g/mL) decreased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate and sodium tubular transport. PLA(2) did not change the renal parameters. The perfusion pressure of the mesenteric bed did not change after infusion of venom. In isolated heart, the venom decreased the force of contraction and increased PP but did not change coronary flow. In the arterial pressure, the venom and PLA(2) decreased mean arterial pressure and cardiac frequency. The presence of atrial flutter and late hyperpolarisation reversed, indicating QRS complex arrhythmia and dysfunction in atrial conduction. In conclusion, B. marajoensis venom and PLA(2) induce hypotension and bradycardia while simultaneously blocking electrical conduction in the heart. Moreover, the decrease in glomerular filtration rate, urinary flow and electrolyte transport demonstrates physiological changes to the renal system. (C) 2009 Elsevier Ltd. All rights reserved.

Purification, characterization, crystallization and theoretical molecular modeling of gyroxin fraction from Crotalus durissus terrificus venom (Laurenti, 1768)

The venom of Crotalus durissus terrificus snakes presents various substances, including a serine protease with thrombin-like activity, called gyroxin, that clots plasmatic fibrinogen and promote the fibrin formation. The aim of this study was to purify and structurally characterize the gyroxin enzyme from Crotalus durissus terrificus venom. For isolation and purification, the following methods were employed: gel filtration on Sephadex G75 column and affinity chromatography on benzamidine Sepharose 6B; 12% SDS-PAGE under reducing conditions; N-terminal sequence analysis; cDNA cloning and expression through RT-PCR and crystallization tests. Theoretical molecular modeling was performed using bioinformatics tools based on comparative analysis of other serine proteases deposited in the NCBI (National Center for Biotechnology Information) database. Protein N-terminal sequencing produced a single chain with a molecular mass of similar to 30 kDa while its full-length cDNA had 714 bp which encoded a mature protein containing 238 amino acids. Crystals were obtained from the solutions 2 and 5 of the Crystal Screen Kit (R), two and one respectively, that reveal the protein constitution of the sample. For multiple sequence alignments of gyroxin-like B2.1 with six other serine proteases obtained from snake venoms (SVSPs), the preservation of cysteine residues and their main structural elements (alpha-helices, beta-barrel and loops) was indicated. The localization of the catalytic triad in His57, Asp102 and Ser198 as well as S1 and S2 specific activity sites in Thr193 and Gli215 amino acids was pointed. The area of recognition and cleavage of fibrinogen in SVSPs for modeling gyroxin B2.1 sequence was located at Arg60, Arg72, Gln75, Arg81, Arg82, Lis85, Glu86 and Lis87 residues. Theoretical modeling of gyroxin fraction generated a classical structure consisting of two alpha-helices, two beta-barrel structures, five disulfide bridges and loops in positions 37, 60, 70, 99, 148, 174 and 218. These results provided information about the functional structure of gyroxin allowing its application in the design of new drugs.

Extração e pré-tratamento químico de frutanos de Yacon, Polymnia sonchifolia

O processo de desintegração de tubérculos de yacon promove a incorporação de oxigênio que favorece a oxidação de compostos fenólicos e decorrente contaminação do extrato aquoso com cores e odores objetáveis que prejudicam a sua utilização. Este estudo buscou verificar uma metodologia simplificada de extração dos carboidratos presentes nos tubérculos de yacon e efetuar uma primeira remoção de resíduos e compostos originários das cores e odores indesejáveis, utilizando processos de coagulação e precipitação, aplicando tecnologias de baixo custo. Após a etapa de trituração e remoção dos restos celulares, o extrato teve seu pH aumentado para 9,5 e a temperatura para 90ºC, seguido de sedimentação. A remoção deste precipitado foi realizada por filtração em papel após a coagulação com diferentes concentrações de sulfato de alumínio comercial. Verificou-se que a melhor concentração de coagulante no extrato foi de 100ppm, removendo 90,6% dos compostos coloridos. O balanço de massa mostrou uma recuperação de 47,5% dos sólidos totais sendo que 35,6% estão na forma de carboidratos, verificado através da concentrção de carbono orgânico. O processo de pré-tratamento químico causou hidrólise nos oligofrutanos, detectado por um aumento na concentração de açúcares redutores totais, diretamente proporcional à concentração de sulfato de alumínio utilizado no tratamento de coagulação. Análises cromatográficas indicaram o perfil dos açúcares no hidrolisado e a extensão desta hidrólise que foi considerada pequena. A metodologia aplicada mostrou-se fácil, de baixo custo, eficiente e aplicável em agroindústrias nas zonas produtoras desta espécie de raiz tropical.

Produção e avaliação de nanoemulsão catiônica como possível vetor não-viral para pIRES2-EGFP

Gene therapy is based on the transfer of exogenous genetic material into cells or tissues in order to correct, supplement or silencing a particular gene. To achieve this goal, efficient vehicles, viral or non-viral, should be developed. The aim of this work was to produce and evaluate a nanoemulsion system as a possible carrier for no-viral gene therapy able to load a plasmid model (pIRES2-EGFP). The nanoemulsion was produced by the sonication method, after been choose in a pseudo-ternary phase diagram build with 5 % of Captex 355®, 1.2 % of Tween 80®, 0.8 % of Span 80®, 0.16% of stearylamine and water (to 100 %). Measurements of droplet size, polydispersity index (PI), zeta potential, pH and conductivity, were performed to characterize the system. Results showed droplets smaller than 200 nm (PI < 0.2) and zeta potential > 30 mV. The formulation pH was near to 7.0 and conductivity was that expected to oil in water systems (70 to 90 μS/s) A scale up study, the stability of the system and the best sterilization method were also evaluated. We found that the system may be scaled up considering the time of sonication according to the volume produced, filtration was the best sterilization process and nanoemulsions were stable by 180 days at 4 ºC. Once developed, the complexation efficiency of the plasmid (pDNA) by the system was tested by agarose gel electrophoresis retardation assay.. The complexation efficiency increases when stearylamine was incorporated into aqueous phase (from 46 to 115 ng/μL); regarding a contact period (nanoemulsion / pDNA) of at least 2 hours in an ice bath, for complete lipoplex formation. The nanoemulsion showed low toxicity in MRC-5 cells at the usual transfection concentration, 81.49 % of survival was found. So, it can be concluded that a nanoemulsion in which a plasmid model was loaded was achieved. However, further studies concerning transfectation efficiency should be performed to confirm the system as non-viral gene carrier

Sistema com aeração, decantação e filtragem para a melhoria da qualidade de água em irrigação localizada

A qualidade da água é muito importante para irrigação por gotejamento, pois ela escoa por pequenos bocais dos emissores, podendo ocorrer obstrução devido à deposição dos sólidos em suspensão. Portanto, antes da instalação do projeto, devem-se avaliar parâmetros de qualidade da água, para adotar medidas preventivas, evitando o risco de entupimento do sistema. Este trabalho teve como objetivo avaliar um sistema composto por aeradores com aspersores, sobre leito de pedra, para a precipitação dos íons Fe+2e Mn+2 em tanque de decantação, e um conjunto de filtragem composto por três filtros de areia e um de disco, em sistema de irrigação localizada. O trabalho foi realizado na Fazenda Alvorada, no município de Nova Granada - SP, no período de março a outubro de 2008. Foram realizadas determinações de variáveis físicas e químicas da água, ao longo do sistema de aeração, decantação e filtragem, o qual foi eficiente para a melhoria da qualidade de água, reduzindo os níveis de risco de entupimento de severo para médio e de médio para baixo. Oxigênio dissolvido, condutividade elétrica, pH, Fe+2e Fe+3 não diferenciaram a qualidade de água entre os pontos do sistema de tratamento, porém a turbidez, sólidos dissolvidos, sólidos em suspensão, ferro total e manganês total reduziram-se significativamente pelo uso do sistema proposto, melhorando a qualidade da água.

Participação da excreção renal de cálcio, fósforo, sódio e potássio na homeostase em cães sadios e cães com doença renal crônica

Na doença renal crônica (DRC) a manutenção da homeostase de água e sódio é o primeiro problema a ser contornado pelo organismo e com o agravamento das lesões renais surgem outros problemas graves relacionados à homeostase de cálcio e fósforo. O presente estudo tem por escopo avaliar a excreção renal de cálcio, fósforo, sódio e potássio, e o perfil sérico destes eletrólitos em cães normais e em cães com DRC naturalmente adquirida. Foram avaliados três grupos de cães adultos, machos ou fêmeas, de raças variadas. Animais normais compuseram o grupo controle (G1) e os cães com DRC foram distribuídos em dois grupos de acordo com os estágios de comprometimento da função renal (G2 e G3, respectivamente, estágios 1-2 e estágios 3-4, descritos pela IRIS 2006 staging CKD). Os cães do G3 apresentaram aumento das concentrações séricas de cálcio ionizado e fósforo, além de diminuição da concentração sérica de sódio. Quanto à excreção renal dos eletrólitos analisados, os animais dos grupos G1 e G2 apresentaram diminuição de carga filtrada e aumento de excreção fracionada, mas as excreções urinárias não variaram significativamente. Os resultados são indicativos de que os rins de cães com DRC podem manter a excreção urinária dos eletrólitos em valores se melhantes aos dos normais. O mecanismo envolve aumento da excreção fracionada na medida em que haja diminuição da filtração glomerular. Esse processo de compensação, entretanto, pode perder a eficiência nos estágios mais avançados da enfermidade no que se refere à manutenção das concentrações séricas de fósforo e sódio.

Inorganic pyrophosphate-phosphohydrolytic activity associated with rat osseous plate alkaline phosphatase

Purified membrane-bound alkaline phosphatase from rat osseous plate hydrolyzed pyrophosphate in the presence of magnesium ions, with a specific activity of 92.7 U/mg. Optimal apparent pH for pyrophosphatase activity was 8.0 and it remained unchanged on increasing the pyrophosphate concentration. In the absence of magnesium ions the enzyme had a K-m = 88 mu M and V = 36.7 U/mg for pyrophosphate and no inhibition by excess substrate was observed. Pyrophosphatase activity was rapidly destroyed at temperatures above 40 degrees C, but magnesium ions apparently protected the enzyme against danaturation. Sodium metavanadate (Ki = 1.0 mM) was a competitive inhibitor of pyrophosphatase activity, while levamisole (Ki = 8.2 mM) and theophylline (Ki = 7.4 mM) were uncompetitive inhibitors. Magnesium ions (K-0.5 = 1.7 mu M) stimulated pyrophosphatase activity, while cobalt (Ki = 48.5 mu M) and zinc (Ki = 22.0 mu M) ions were non-competitive inhibitors. Manganese and calcium ions had no effect on pyrophosphatase activity. The M-w of the pyrophosphatase: protein was 130 kDa by gel filtration, but a value of 65 kDa was obtained by dissociative gel electrophoresis, suggesting that it was a dimer of apparently identical subunits. These results suggested that pyrophosphatase activity stems from the membrane-bound osseous plate alkaline phosphatase and not from a different protein.

Avaliação da determinação da fibra em detergente neutro e da fibra em detergente ácido pelo sistema ANKOM

O objetivo do trabalho foi comparar os valores de fibra em detergente neutro (FDN) e os de fibra em detergente ácido (FDA) obtidos com o equipamento ANKOM e pelo método convencional (Van Soest). No primeiro ensaio foram analisados cinco materiais diferentes (cana-de-açúcar, capim-braquiária, silagem de milho, polpa cítrica e fezes bovina) e testados quatro tipos de saquinhos para filtragem de amostra. O delineamento experimental foi em blocos casualizados, em um fatorial 5x4 (cinco materiais diferentes e quatro tipos de saquinhos de filtragem), com três repetições. As médias obtidas foram comparadas àquelas obtidas com método convencional. No segundo ensaio procurou-se avaliar o efeito da quantidade de amostra por saquinho (0,5; 0,8 e 1,0 g) sobre os teores de FDN e FDA em três tipos de alimentos, utilizando o ANKOM. Neste ensaio, o delineamento experimental foi em blocos casualizados em esquema fatorial 3x3 (três alimentos x três quantidades), com três repetições. Os tipos de saquinhos de filtragem utilizados não influenciaram os teores de FDN nos diferentes alimentos, com exceção das fezes, cujos saquinhos de náilon resultaram em concentrações de FDN inferiores. Não houve diferença entre os valores de FDN e FDA, obtidos pelo equipamento ANKOM ou pelo convencional, para os alimentos estudados, com exceção da polpa cítrica, cujo valor médio de FDA pelo ANKOM foi inferior àquele obtido pelo método convencional. A quantidade não exerceu efeito sobre a concentração de FDN dos alimentos analisados no equipamento ANKOM.

Remoção de óleo da água de produção por flotação em coluna utilizando tensoativos de origem vegetal

In the petroleum industry, water is always present in the reservoir formation together with petroleum and natural gas and this fact provokes the production of water with petroleum, resulting in a great environmental impact. Several methods can be applied for treatment of oily waters, such as: gravitational vases, granulated media filtration systems, flotation process, centrifugation process and the use of hydrocyclones, which can also be used in a combined way. However, the flotation process has showed a great efficiency as compared with other methods, because these methods do not remove great part of the emulsified oil. In this work was investigated the use of surfactants derived from vegetable oils, OSS and OGS, as collectors, using the flotation process in a glass column with a porous plate filter in its base for the input of the gaseous steam. For this purpose, oil/water emulsions were prepared using mechanical stirring, with concentrations around 300 ppm. The air flow rate was set at 700 cm3/min and the porous plate filter used for the generation of the air bubbles has pore size varying from 16 to 40 Pm. The column operated at constant volume (1500mL). A new methodology has been developed to collect the samples, where, instead of collecting the water phase, it was collected the oil phase removed by the process in the top of the flotation column. It has been observed that it is necessary to find an optimum surfactant concentration to achieve enhanced removal efficiency. Being for OSS 1.275 mmol/L and for OGS 0.840 mmol/L, with removal efficiencies of 93% and 99%, respectively, using synthetic solutions. For the produced water, the removal in these concentrations was 75% for OSS and 65% for OGS. It is possible to remove oil from water in a flotation process using surfactants of high HLB, fact that is against the own definition of HLB (Hydrophile-Lipophile Balance). The interfacial tension is an important factor in the oil removal process using a flotation process, because it has direct interference in the coalescence of the oil drops. The spreading of the oil of the air bubble should be considered in the process, and for the optimum surfactant concentrations it reached a maximum value. The removal kinetics for the flotation process using surfactants in the optimum concentration has been adjusted according to a first order model, for synthetic water as for the produced water.

Remoção conjugada de metais e óleo de água produzida

Petroleum can be associated or not with natural gas, but in both cases water is always present in its formation. The presence of water causes several problems, such as the difficulty of removing the petroleum from the reservoir rock and the formation of waterin-oil and oil-in-water emulsions. The produced water causes environmental problems, which should be solved to reduce the effect of petroleum industry in the environment. The main objective of this work is to remove simultaneously from the produced water the dispersed petroleum and dissolved metals. The process is made possible through the use of anionic surfactants that with its hydrophilic heads interacts with ionized metals and with its lipophilic tails interacts with the oil. The studied metals were: calcium, magnesium, barium, and cadmium. The surfactants used in this research were derived from: soy oil, sunflower oil, coconut oil, and a soap obtained from a mixture of 5wt.% coconut oil and 95wt.% animal fat. It was used a sample of produced water from Terminal de São Sebastião, São Paulo. As the concentration of the studied metals in produced water presented values close to 300 mg/L, it was decided to use this concentration as reference for the development of this research. Molecular absorption and atomic absorption spectroscopy were used to determine petroleum and metals concentrations in the water sample, respectively. A constant pressure filtration system was used to promote the separation of solid and liquid phases. To represent the behavior of the studied systems it was developed an equilibrium model and a mathematical one. The obtained results showed that all used surfactants presented similar behavior with relation to metals extraction, being selected the surfactant derived from soy oil for this purpose. The values of the partition coefficients between the solid and liquid phases " D " for the studied metals varied from 0.2 to 1.1, while the coefficients for equilibrium model " K " varied from 0.0002 and 0.0009. The removal percentile for oil with all metals associated was near 100%, showing the efficiency of the process

Utilização De Polímeros à Base De Acrilamida Na Remoção De Cobre De Meio Aquoso

Environmental Laws and Regulations to dump wastewater are increasingly relevant and, together with pressure from environmentalists, provide awareness of academics in search of solutions. In Brazil, federal law, through Resolution No. 357 of 17/03/05 of the National Environmental Council - CONAMA, in Article 24 deals with the disposal of these effluents. Water pollution with heavy metals is concern because of the difficulty of the treatment and removal from the environment. Copper, for example, is a metallic element and in the form of salt is very soluble in water which dificults its removal. In this context, this study aims to evaluate the extraction of copper with acrylamide polymers through the process of assisted flocculation followed by filtration. Therefore, we used acrylamide polymers, produced by SNF Floerger, with varying degrees of ionicity which is the parameter examined on the extraction of copper. We used the FA polymers FA 920 SH, AH 912 SH, AN 905 SH, AN 910 SH, AN 923 SH, AN 945 SH, AN 956 SH and AN 977 SH, which have anionicities different from each other and growing in that order. The parameters temperature, pH, concentration of the copper solution and stirring speed are fixed. The polymer solution was added to a solution of 200 ppm copper, varying the concentration of polymer. After stirring, an assisted flocculation occurred followed by filtration of the effluent. The filtrate was analyzed by atomic absorption spectrophotometer and the percentage removal of copper ranged from 63 % to 97 %, noting that polymers with higher ionic charge were responsible for the highest percentage of copper extraction. The results of this study showed that these polymers can be applied in the treatment of wastewaters containing metals such as copper

Avaliação da eficiência de filtros anaeróbios na remoção de coliformes fecais e ovos de helmintos

The technology of anaerobic reactors for sanitary wastewater treatment has been extensively developed in Brazil, and today it is practically consolidated. They present several advantages, such as low construction and operating costs, and low sludge production, the anaerobic reactors are an attractive alternative to minimize problematic lack of basic sanitation in urban areas, and also of the rural areas. The anaerobic filters have been widely used in Brazil. It produces an effluent with low concentration of organic matter and solids suspended, besides conserving the nutrients, therefore, it is good for use in irrigation, but the practice must be associated with knowledge of the pathogens presence. The main objective of this study was to evaluate the efficiency of anaerobic filters in removal faecal coliforms and helminth eggs, and to verify if the effluent can be used for agricultural purposes, according to the World Organization of Health (WHO, 1989). The protocol used to enumerate helminths eggs was the modified Bailenger method, (Ayres and Mara, 1996) recommended by WHO for evaluation of raw effluent and treated effluent. The membrane filtration method was utilized to determine the concentrations of faecal coliforms. Three different systems of sewer treatment composed by anaerobic filters were analyzed. The results, in a general analysis, showed that all the researched systems reached a larger removal than 93% to helminth eggs, resulting in an effluent with smaller average than 1 egg/L. One of these systems, Sistema RN, reached a larger removal than 99%, confirming the good performance of the anaerobic filters in removal helminths eggs. Even with low concentrations of eggs in the influent, the filters were capable to remove this parameter efficiently. About faecal coliforms, it was observed for all the researched systems an effluent with 106 CFU/100mL. The high concentrations to faecal coliforms in the effluent just allow reuse for restricted irrigation, in agreement with the guidelines of WHO. Although the researched systems have not removed faecal coliforms efficiently, the results indicated a good efficiency of the anaerobic filters in removal helminth eggs