Estrutura e dinâmica da interação de éteres de celulose com surfactantes

As técnicas de fluorimetria, condutometria, viscosimetria, turbidimetria, espalhamento de luz e espalhamento de raios-X a baixo ângulo (SAXS) foram empregadas no estudo da agregação de diferentes surfactantes aniônicos em presença de soluções aquosas diluídas de (hidroxipropil)celulose (HPC) 0,25% m/m, (hidroxipropilmetil)celulose (HPMC) 0,20% m/m e HPMC 0,10% m/m / NaCl 0,10 mol L-1. Também foram investigadas através de SAXS soluções concentradas de HPC (30, 40 e 50% m/m). Admitindo-se uma faixa geral de concentração, entre 10-4 e 10-1 mol L-1, foram utilizados neste estudo os surfactantes colato de sódio (CS), deoxicolato de sódio (DC), derivados dos sais biliares, e o alquilsintético dodecilsulafato de sódio (SDS). Observou-se que os polímeros contribuem diferentemente no processo de agregação de cada surfactante, evidenciado pela mudança dos valores da concentração de agregação crítica (CAC) em relação à concentração micelar crítica (CMC). Os resultados condutométricos confirmaram a interação éteres de celulose/sais biliares, embora a mesma tenha se mostrado mais fraca em relação a éteres de celulose/SDS. Os dados termodinâmicos demonstraram que a formação de agregados polímero/surfactante apresenta maior estabilidade do que as próprias micelas livres. Os resultados de viscosimetria e turbidimetria evidenciaram as diferenças estruturais entre HPC e HPMC, assim como entre os surfactantes. Através do espalhamento de luz dinâmico, verificou-se a existência de dois modos de correlação, rápido e lento. O primeiro é atribuído à cadeia polimérica isolada, agregados polímero/surfactante intramoleculares ou mesmo a micelas livres. Por sua vez, o modo lento relaciona-se a clusters poliméricos ou agregados polímero/surfactante intermoleculares. Adicionalmente, as curvas de distribuição dos tempos de relaxação demonstraram a influência de cada surfactante sobre a dinâmica dos polímeros. Tal influência é percebida antes mesmo da CAC, contrariando o modelo da interação polímero/surfactante proposto por Cabane. Os resultados de SAXS acusaram a formação de domínios líquido-cristalinos em xx soluções concentradas de HPC, assim como confirmaram a presença de micelas livres a altas concentrações de surfactantes nos sistemas diluídos. Em linhas gerais, os resultados indicaram a interação dos polímeros com SDS mais efetiva do que os mesmos polímeros e os sais biliares. No que tange à natureza do polímero, a HPC mostrou uma maior estabilidade na sua interação com os surfactantes do que a HPMC.

Estudo aerobiológico da cidade do Funchal: detecção bioquímica dos principais aeroalergénios polínicos

A abordagem da doença alérgica, nomeadamente da alergia respiratória, surge reforçada com o contributo da Aerobiologia no conhecimento, evolução e controlo da doença. Por seu turno, a caracterização bioquímica dos aeroalergénios de uma região pemite avaliar a sua composição e potencial alergénico, com particular interesse para espécies vegetais cuja introdução e/ou proximidade a áreas populacionais, potenciam o aparecimento ou o aumento de sensibilização a aeroalergénios. O trabalho apresentado nesta dissertação incidiu em duas vertentes de investigação distintas, porém complementares e interdependentes: numa primeira parte, no estudo da composição aerobiológica da atmosfera da cidade do Funchal no período 2002-2004 (Capítulo I), e numa segunda parte, na caracterização bioquímica dos alergénios polínicos mais frequentes detectados no referido período (Capítulo II). Assim, numa primeira parte, a monitorização aerobiológica (incluindo pólenes, esporos de fungos e demais partículas de origem biológica), foi realizada com um polinómetro do tipo volumétrico, tipo Hirst (Burkard). Os dados obtidos foram correlacionados com os parâmetros meteorológicos e avaliado o seu significado estatístico, permitindo antever a influência das variáveis ambientais em cada tipo particular de pólen e de fungo. Constataram-se algumas diferenças no conteúdo aerobiológico comparativamente ao restante território nacional. No espectro polínico dominaram as Poaceae e Urticaceae, plantas ornamentais (Asteraceae, Boraginaceae, Cupressaceae) e as representativas da faixa norte da cidade (Ericaceae, Myrtaceae e Pinaceae). Comparativamente a outras regiões do País, o espectro polínico foi no cômputo geral semelhante, embora se destaque para esta região, a expressividade do tipo Corylus no total polínico anual. A Primavera e o início de Verão, corresponderam às épocas de maior diversidade e concentração de pólenes. As diferenças encontradas no espectro polínico da cidade do Funchal são explicadas por variáveis intrínsecas desta região, tais como a composição vegetal, a localização da cidade, condições geo-climáticas inerentes, e a influência dos parâmetros meteorológicos, nomeadamente a temperatura e humidade relativa. Verificou-se a ocorrência de um maior número de pólenes quando a humidade se situa entre os 50 e 60 %, com a precipitação e a velocidade do vento a atingir valores mais baixos. A análise da variação intra-diurna observada revelou que há uma maior representação de pólenes entre as 11 e as 16 horas. Este estudo aerobiológico confere dados para o estabelecimento do primeiro calendário polínico da região e a definição de padrões de sazonalidade. Por seu turno, a fenologia dos principais tipos polínicos observados no Funchal permite definir um padrão anual de ocorrências polínicas. Relativamente à aeromicologia, verifica-se que, durante o período de estudo foram observados esporos de fungos sobretudo na Primavera (particularmente em Abril e Maio), início do Verão e no Outono. Os Deuteromicetes representaram a classe predominante, sendo Cladosporium o fungo mais abundante na atmosfera do Funchal, cujas concentrações mais elevadas ocorrem a humidades relativas de 40 a 70%. Tal como os pólenes, os esporos apresentam um dinâmica de variação intra-diurna particular: ocorreram em maior concentração entre as 13 e as 15 horas, surgindo igualmente nas primeiras horas da madrugada e da noite. O coberto vegetal do Funchal poderá afectar a aeromicologia local, na medida em que constitui um substrato importante para o crescimento de fungos, tal como as gramíneas que proliferam em quantidade e variedade ao longo de todo o ano. Constatou-se a existência de correlação entre a ocorrência de alguns esporos de fungos e taxa polínicos mais frequentes na atmosfera do Funchal. A análise de Spearman sugere a existência de correlação entre a ocorrência de Parietaria com a de Alternaria e Drechslera. Os esporos de fungos, incluindo esporos de fetos constituem uma fracção significativa das partículas na atmosfera do Funchal, sendo cerca de 11 vezes superior à dos pólenes. Na segunda parte do trabalho, o estudo bioquímico dos aeroalergénios polínicos implicou a optimização dos procedimentos de extracção, apurando-se três técnicas para a obtenção dos perfis proteicos de extractos de pólen desde a sua fonte natural. Esta análise permitiu detectar e identificar através das técnicas de SDS-PAGE-Imunoblotting, proteínas IgE específicas do pólen de plantas possivelmente relacionadas com a sensibilização alérgica. Em consonância com a monitorização aerobiológica, foram preparados extractos de pólen de 10 espécies de plantas. Os resultados em SDS-PAGE revelaram um elevado polimorfismo proteico em todos os extractos. Obtiveram-se extractos de pólen de 7 plantas acerca das quais não se conheciam estudos desta natureza: Acacia mearnsii, Avena barbata, Carduus squarrosus, Carlina salicifolia, Datura candida, Echium nervosum e Urtica membranosa. Por imunoblotting detectou-se no soro de dois pacientes IgE específica a proteínas de D. candida, com pesos moleculares entre 150,71 ± 0,05 e 58,92 ± 5,67 KDa. O soro de um deles reagiu igualmente com 5 alergénios de P. pinaster com 42,02 ± 0,05; 38,61 ± 0,46 ; 35,70 ± 7,78 ; 31,82 ± 2,11 e 27,45 ± 0,46 KDa. No soro de outro indivíduo foi detectada IgE específica para uma proteína de A. mearnsii com 66,66 ± 0,13 KDa, e outra de C. squarrosus,de 67,53 ± 0,29 KDa. É de destacar a sensibilidade e fiabilidade da técnica de Imunoblotting, e o interesse em incluila na metodologia de diagnóstico complementar da alergia respiratória. A ampla difusão de espécies como Ricinus communis, Urtica spp. ou A. mearnsii e sua proximidade à presença humana, reforçam, por um lado, a importância da vigilância aerobiológica, e por outro lado, requer uma definição do seu carácter alergénico para a população desta região.

Modelação de diálogos com o Midiki: um gestor de diálogo do tipo Information State Update

Nesta dissertação investigamos a problemática da criação de sistemas e interfaces que permitam a interacção entre pessoas e máquinas através de linguagem natural(LN), recorrendo a Gestores de Diálogo (GD). Esse tipo de interacção concretiza-se através do estabelecimento de diálogos entre uma pessoa (cliente ou utilizador de um serviço) e a máquina, por exemplo, e em particular, através da fala. Quando disponibilizado da forma tradicional, o acesso ao serviço exige um intermediário Humano ou a adaptação da Pessoa a interfaces menos naturais, tais como linhas de comandos num computador, digitadas através de teclado ou o recurso (usual) a janelas, cliques de rato e preenchimento de formulários. Os sistemas que possibilitam a intermediação com esses serviços através de LN chamam-se Sistemas de Diálogo (SD), no núcleo dos quais se encontram os chamados Gestores de Diálogo. A implementação de SDs robustos ainda constitui um desafio, dada a complexidade, problemas e dificuldades que apresenta. Um SD, e em particular um GD, tem de ser configurado para levar a cabo um diálogo em linguagem natural com um Humano, por mais restrito ou mais genérico que seja o domínio (ou tarefa) considerado. Infelizmente, existem poucas metodologias e ferramentas de autoria que possibilitem a modelação fácil e intuitiva de tais diálogos (sobre os GDs). Nesta dissertação apresentamos uma metodologia [Quintal & Sampaio, 2007] e uma ferramenta para a autoria de diálogos com base no Gestor de Diálogo MIDIKI [Burke, 2005b]. A ferramenta de autoria automatiza as partes mais importantes da geração de código com vista à execução de um diálogo nesse GD.

Effect of trypsin inhibitor from Crotalaria pallida seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly)

GOMES, Carlos E. M. et al. Effect of trypsin inhibitor from Crotalaria pallida seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly). Plant Physiology and Biochemistry (Paris), v. 43, n. 12, p. 1095-1102, 2005.ISSN 0981-9428. DOI:10.1016/j.plaphy.2005.11.004.

Purificação e caracterização parcial de uma B-D glicosidade de Artemia franciscana com ação sobre celobiose e lactose

-D-glucosidase (EC 3.2.1.21) is one of the most interesting glycosidases, especially for hydrolysis cellobiose releasing glucose, is last step degradation of cellulose. This function makes the -D-glucosidase is of great interest as a versatile industrial biocatalyst, being critical to various bio-treatment / biorefinery processes, such as bioethanol production. Hen in the report, a -D-glucosidase was extracts from protein extracted of the invertebrate marine Artemia franciscana was purified and characterized with a combination of precipitation with ammonium sulfate (0 - 30%, 30 to 50%, 50 to 80%), the fraction saturated in the range of 30 to 50% (called F-II) was applied in a molecular exclusion chromatography, in Sephacryl S-200, the fractions corresponding to the first peak of activity of -D-glucosidase were gathered and applied in a chromatography of ion exchange in Mono Q; the third peak this protein obtained chromatography, which coincides with the peak of activity of -D-glucosidase was held and applied in a gel filtration chromatography Superose 12 where the first peak protein, which has activity of -D-glucosidase was rechromatography on Superose 12. This enzyme is probably multimerica, consisting of three subunit molecular mass of 52.7 kDa (determined by SDS-PAGE) with native molecular mass of 157 kDa (determined by gel filtration chromatography on Superose 12 under the system FPLC). The enzyme was purified 44.09 times with a recovery of 1.01%. Using up p-nitrophenyl-β-D-glucopiranoside as substrate obtained a Km apparent of 0.229 mM and a Vmax of 1.109 mM.60min-1.mL-1mM. The optimum pH and optimum temperature of catalysis of the synthetic substrate were 5.0 and 45 °C, respectively. The activity of the -D-glucosidase was strongly, inhibited by silver nitrate and N- etylmaleimide, this inhibition indicates the involvement of radical sulfidrila the hydrolysis of synthetic substrate. The -D-glucosidase of Artemia franciscana presented degradativa action on celobiose, lactose and on the synthetic substrate -nitrophenyl-β-D-glucopiranoside indicating potential use of this enzyme in the industry mainly for the production of bioethanol (production of alcohol from the participating cellulose), and production hydrolysate milk (devoid of milk lactose)

Efeito do consumo de farinha de linhaça (Linum usitatissimum) no crescimento de ratos Wistar e relação com a digestibilidade de globulinas e fatores antinutricionais protéicos

Linseed is an important oilseed consumed raw as nutritional supplement, that although represents a rich source of nutrients, its nutritional value could be impaired due to the presence of antinutritional factors. In this study, protein fractions from raw linseed flour were extracted and isolated being obtained 12% of albumins, 82% of globulins, 5% of glutelins and 1% of prolamins. These proteins were visualized by SDS-PAGE and albumins showed low molecular mass protein bands around 21 kDa and minor bands, similar to that of trypsin inhibitor; Globulins presented protein bands with high molecular masses, which possibly are constituents of multimeric proteins, such as legumins. After determination of the centesimal composition of raw linseed, it was used as exclusive protein source for young rats to evaluate its effect on animal growth. The results showed negative effects on rat growth (weight gain 73% less than the control group) and reduction of intestinal villus (35%), that could be related with in vitro and in vivo globulin digestibility and proteinaceous antinutritional factors (mammalian digestive enzymes inhibitors and lectins) in albumin fraction. Native globulins showed, by SDS-PAGE, low susceptibility in vitro to trypsin and chymotrypsin, however presented high degradation by pancreatin. Thermal treatment of globulins for 5 and 15 minutes at 100ºC improved considerably its digestibility by trypsin and pancreatin. Globulins presented 93.2% in vivo digestibility, similar to the control protein. Albumin fraction had high trypsin inhibition activity (100%) and chymotrypsin inhibition of 28.3%; haemagglutinating activity was not detected. The results of this study indicate the negative action of trypsin inhibitors on animal growth, but can not be discarded its combined action with other antinutritional factors, which could compromise the raw linseed utilization as an alternative food

Uma Lectina D-lactose específica da esponja marinha Cinachyrella apion: purificação, caracterização e interação com Leishmania chagasi

Four different sponge species were screened using Ouchterlony agarose gel and immunodiffusion tests to identify cross-reactivity with the polyclonal antibody IgG anti-deglicosilated CvL, a lectin from Cliona varians. Crude extract from the sponge Cinachyrella apion showed cross-reactivity and also a strong haemmaglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglicosilated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA) gel filtration on a Superose 6 10 300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A and O erythrocytes. The haemagglutinating activity is independent of Ca2+, Mg2+ and Mn2+ ions, and it was strongly inhibited by the disaccharide D-lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass determined by FPLC-AKTA gel filtration on a Superose 12 10 300 column and SDS gel electrophoresis was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was relatively heat- and pH-stable. Leishmania chagasi romastigotes were agglutinated by CaL, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the physiological defense roles of CaL and its possible use in the antibiosis of pathogenic protozoa

Purificação, caracterização e atividade bioinseticida de um inibidor de tripsina de sementes de Crotalaria pallida

A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulphate fractionation, affinity chromatography on immobilized Trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named ITC, had Mr of 32.5 kDa by SDS-PAGE and was composed by two subunits with 27.7 and 5.6 kDa linked by disulphide bridges, a typical characteristic of Kunitz-Inhibitor family. ITC was stable until 50°C, and at 100°C its residual activity was of about 60%. Also, ITC was stable at pHs 2 to 12. The inhibition of trypsin by ITC was non-competitive, with a Ki of 8,8 x 10-7M. ITC inhibits weakly other serine proteinases such as chymotrypsin and elastase. The inhibition of papain (44% of inhibition), a cysteine proteinase was an indicative of the bi-functionality of ITC. In vitro assays against digestive proteinases from several Lepdoptera, Diptera and Coleoptera pests were made. ITC inhibited in 100% digestive enzymes of Ceratitis capitata (fruit fly), Spodoptera frugiperda and Alabama argillacea, the last one being a cotton pest. It also inhibited in 74.4% Callosobruchus maculatus (bean weevil) digestive enzymes, a Coleoptera pest. ITC, when added in artificial diet models, affected weakly the development of C. capitata larvae and it had a WD50 of 2.65% to C. maculatus larvae

Purificação e caracterização de uma β-glucuronidase extraída do molusco mesogastropoda ampularidade Pomacea sp

This work studies the involved enzymatic way in the metabolism of glycosaminoglycans sulfateds in the mollusc Pomacea sp. Had been identified endoglycosidases and exoglycosidases in the enzymatic extract of the mollusc Pomacea sp by means of hydrolysis activity in condroitim sulphate of whale cartilage and of the p-Nitrofenil-β-glucuronide, respectively. The enzymatic extracts qere obtained of Pomacea sp. being used of 0.1 sodium acetate buffer, pH 5.0 and later centrifugated the 8,000 x g and the presents proteins in the sobrenadante were submitted to the fractionament with two crescents ammonium sulphate concentrations, the visualized activity biggest in the F2 fraction (50-80%). The β-glucuronidase (F3) was isolated in gel chromatography filtration Biogel 1.5m, the purification degree was ratified in Chromatography Liquid of high efficiency (HPLC). The enzyme was purificated 6.362,5 times with 35,6% yield. The β -glucuronidase isolated in this work showed a molecular mass of 100 kDa, determined for eletroforese in poliacrilamida gel . The determination of the ideal kinetic parameters for the catalysis of the p-nitrofenil- β -glucuronide for β-glucuronidase, showed excellent activity in pH 5,0 and temperature 65ºC for 6 hours and apparent Km of 72 x 10-2 mM. It is necessary for the total degradation of 3mM of p-N-β-glucoronide, the amount of 1,2μg of ss-glucuronidase. The BaCl2 increased the activity of ss-glucuronidase, and the activity was inhibited completely by the composites SDS and NaH2PO4

Atividade bioinseticida e mecanismo de ação de vicilinas de sementes Erythrina velutina sobre moscas-das frutas Ceratitis capitata

The fruit fly Ceratitis capitata is considered the most destructive pest of the world fruitculture. Many pest management practices, mainly based on agrochemicals, have been developed to allow the world-wide commerce of fruit. Solutions to decrease the use of synthetic insecticides in agriculture are based on the development of new target-specific compounds which cause less damage to the environment, especially vegetal proteins with insecticidal effects. The aim of this work was to evaluate the deleterious effect of a purified vicilin of E. velutina (EvV) seeds to C. capitata larvae and adult insects and to investigate the mechanisms involved in these effects. EvV was purified, characterized and its deleterious effect was tested in bioassay systems. EvV mechanism of action was determined by immunodetection techniques and fluorescence localization in chitin structures that are present in C. capitata digestory system. EvV is a glycoprotein with affinity to chitin. Its molecular weight, of 216,57 kDa, was determined by gel filtration chromatography in FPLC system. Using SDS-PAGE, it was possible to observe EvV dissociation in two main subunits of 54,8 and 50,8 kDa. When it was submitted to eletrophoresis in native conditions, EvV presented only one band of acid characteristic. The WD50 and LD50 values found in the bioassays were 0,13% and 0,14% (w/w), respectively for the larvae. EvV deleterious effects were related to the binding to chitin structures presented in peritrophic membrane and gut epithelial cells, associated with its low digestibility in C. capitata digestive tract. The results described herein are the first demonstration of the larvicidal effects of plant protein on C. capitata larvae. EvV may be part of the pest management programs, in the toxic bait composition, or an alternative in plant improvement program

Purificação, caracterização e análise da atividade bioinseticida, de um inibidor de tripsina em sementes de catanduva (Piptadenia moniliformis)

One Kunitz-type trypsin inhibitors (PmTI) was purified from Piptadenia moniliformis seeds, a tree of the sub-family Mimosoideae, by TCA precipitation, affinity chromatography on immobilized trypsin-Sepharose, DEAE cellulose (ion exchange) and Superose 12 (molecular exclusion) column FPLC/AKTA. The inhibitor has Mr of 25 kDa by SDS-PAGE and chromatography molecular exclusion. The N-terminal sequence of this inhibitor showed high homology with other family Kunitz inhibitors. This also stable variations in temperature and pH and showed a small decrease in its activity when incubated with DDT in the concentration of 100mM for 120 minutes. The inhibition of trypsin by PmTI was competitive, with Ki of 1.57 x10-11 M. The activity of trypsin was effectively inhibited by percentage of inhibition of 100%, among enzymes tested, was not detected inhibition for the bromelain, was weak inhibitor of pancreatic elastase (3.17% of inhibition) and inhibited by 76.42% elastase of neutrophils, and inhibited in a moderate, chymotrypsin and papain with percentage of inhibition of 42.96% and 23.10% respectively. In vitro assays against digestive proteinases from Lepidoptera, Diptera and Coleoptera pests were carried out. Several degrees of inhibition were found. For Anthonomus grandis and Ceratitis capitata the inhibition was 89.93% and 70.52%, respectively, and the enzymes of Zabrotes subfasciatus and Callosobruchus maculatus were inhibited by 5.96% and 9.41%, respectively, and the enzymes of Plodia. interpunctella and Castnia licus were inhibited by 59.94% and 23.67, respectively. In vivo assays, was observed reduction in the development of larvae in 4rd instar of C. capitata, when PmTI was added to the artificial diet, getting WD50 and LD50 of 0.30% and 0.33%, respectively. These results suggest that this inhibitor could be a strong candidate to plant management programs cross transgenic

Purificação, caracterização e avaliação da atividade antiproliferativa de um inibidor de quimotripsina tipo kunitz de semente de Eryhrina velutina

A chymotrypsin inhibitor was purified from Erythrina velutina seeds by ammonium sulphate fractionation, affinities chromatographies on Trypsin-Sepharose, Quimotrypsin-Sepharose and reversed phase C-18 FPLC/AKTA system. The inhibitor, named EvCI, shown molecular mass of 17 kDa, as determined by SDSPAGE. 2D-PAGE showed four isoinhibitors with pI values of 4,42, 4,63, 4,83 and 5,06, with molecular mass of 17 kDa each. The aminoacid sequence of EvCI was determined by MALDI-TOF-MS and showed a high similarity with other Kunitz-type inhibitor of Erythrina variegata. EvCI competitively inhibited chymotrypsin, with Ki of 4 x10-8 M, but did not inhibited trypsin, pancreatic elastase, bromelain and papain. The inhibitory activity of EvCI was stable over wide pH and temperature ranges. In the presence of DTT 100 mM for 120 min, EvCI lost 50 % of activity. Cytotoxicity was studied in HeLa, MDA, HepG2, K562 and PC3 cells after 72-h incubation period. EvCl inhibited HeLa cells growth with an IC50 value of 50 μg/ml. Subsequent studies in HeLa cells analysis of cell death by annexin V/PI double-staining and cell cycle, using flow cytometry. The results provide evidence for a cytostatic activity of EvCl and support further studies on potential application of this inhibitors as an antiproliferative agent in combined therapy against cervical cancer

Purificação, caracterização e efeitos imunomodulatório e antiproliferativo de uma lectina do fungo Clavaria cristata (Holmsk.) Pers

A 140,0 kDa lectin was purified and characterized from the mushroom Clavaria cristata. The purification procedures from the crude extract of the mushroom comprised gel filtration chromatography on Sephacryl s200 and ion exchange on Resource Q column. The purified lectin agglutinated all types of human erythrocytes with preference for trypsinized type O erythrocytes. The haemagglutinating activity is dependent of Ca 2+ ions and was strongly inhibited by the glycoprotein bovine submaxillary mucin (BSM) up to the concentration of 0, 125 mg/mL. The C. cristata lectin (CcL) was stable in the pH range of 2,5-11,5 and termostable up to 80 °C. CcL molecular mass determined by gel filtration on a Superose 6 10 300 column was approximately 140,3 kDa. SDS polyacrilamide gel electrophoresis revealed a single band with a molecular mass of approximately 14,5 kDa, when the lectin was heated at 100 ⁰C in the presence or absence of β-mercaptoethanol. CcL induced activation of murine peritoneal macrophages in vitro resulting in the release of nitric oxide (NO), reaching the maximum production at 24 h. In experimental paw oedema model in mice, CcL showed proinflammatory activity being able to induce oedema formation. Cell viability of HepG2, MDA 435 e 3T3 cell lines was examined after 72 h of incubation with CcL in different concentrations (0,5-50 μg/mL). CcL inhibited HepG2 cells growth with an IC50 value of 50 μg/mL. In the present work, the observed immunomodulatory and antiproliferative effects indicate CcL as a possible immunomodulator compound, interfering in the macrophages immune response, taking possible anti-parasitic, anti-tumoral effects or diagnostic and/or therapeutic

Avaliação das atividades anti-inflamatória, anticoagulante e antiproliferativa do inibidor de quimotripsina das sementes de erythrina velutina (EvCI)

Studies indicate that several components were isolated from medicinal plants, which have antibacterial, antifungal, antitumor and anti-inflammatory properties. Sepsis is characterized by a systemic inflammation which leads to the production of inflammatory mediators exacerbated by excessive activation of inflammatory cells and disseminated intravascular coagulation (DIC), in which the human neutrophil elastase plays an important role in its pathogenesis. Several epidemiological studies suggest that components of plants, especially legumes, can play a beneficial role in reducing the incidence of different cancers. A chymotrypsin inhibitor of Kunitz (Varela, 2010) was purified from seeds of Erythrina velutina (Mulungu) by fractionation with ammonium sulfate, affinity chromatography on Trypsin-Sepharose, Chymotrypsin-Sepharose and ion exchange chromatography on Resource Q 1 ml (GE Healthcare) in system FPLC / AKTA. The inhibitor, called EvCI, had a molecular mass of 17 kDa determined by SDS-PAGE. The purified protein was able to inhibit human neutrophil elastase (HNE), with an IC50 of 3.12 nM. The EvCI was able to inhibit both pathways of HNE release stimulated by PAF and fMLP (75.6% and 65% respectively). The inhibitor also inhibited leukocyte migration in septic mice about 87% and prolonged the time of coagulation and inhibition factor Xa. EvCI showed neither hemolytic activity nor cytotoxicity. EvCI showed a selective antiproliferative effect to HepG2 cell lines with IC50 of 0.5 micrograms per milliliter. These results suggest EvCI as a molecule antagonist of PAF / fMLP and a potential use in fighting inflammation related disorders, disseminated intravascular coagulation (DIC) and cancer

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