Avaliação da ação bioinseticida de SBTI e vicilina de Erythrina velutina em enzimas digestivas e membrana peritrófica de larvas de Plodia interpunctella (Lepidoptera: Pyralidae)

Plodia interpunctella (Indian meal moth) is a cosmopolitan pest that attacks not only a wide range of stored grain as well other food products. Due to its economic importance several researches have focused in a method with ability to control this pest with few or no damage to the environment. The study of digestive enzymes inhibitors, lectins and chitin-binding proteins, has often been proposed as an alternative to reduce insect damage. In this study we report the major classes of digestive enzymes during larval growth in P. Interpunctella, being those proteinases actives at pH 9.5 and optimum temperature of 50 oC to both larvae of the 3rd instar and pre-pupal stage of development. In vitro and zymogram assays presented the effects of several inhibitors, such as SBTI, TLCK and PMSF to intestinal homogenate of 3rd instar larvae of 62%, 92% and 87% of inhibition and In pre-pupal stage of 87%, 62 % and 55% of inhibition, respectively. Zymograms showed inhibition of two low molecular masses protein bands by TLCK and that in presence of SBTI were retarded. These results are indicative of predominance of digestive serine proteinases in gut homogenate from Plodia interpunctella larvae. This serine proteinase was then used as a target to evaluate the effect of SBTI on larvae in in vivo assay. Effect of SBTI on mortality and larval mass was not observed at until 4% of concentration (w/w) in diets. Chitin, another target to insecticidal proteins, was observed by chemical method. Moreover, optic microscopy confirmed the presence of a peritrophic membrane. Established this target, in vivo effect of EvV, a chitin binding vicilin, evaluated during the larval development of P. interpunctella and was obtained a LD50 of 0,23% and WD50 of 0,27% to this protein. Mechanism of action was proposed through of the in vivo digestibility of EvV methodology. During the passage through the larval digestive tract was observed that EvV was susceptible to digestive enzymes and a reactive fragment, visualized by Western blotting, produced by digestion was recovered after dissociation of the peritrophic membrane. The bound of EvV to peritrophic membrane was confirmed by immunohystochemical assays that showed strong immunofluorescent signal of EvV-FITC binding and peritrophic membrane. These results are a indicative that vicilins could be utilized as potential insecticide to Plodia interpunctella and a control methods using EvV as bioinsecticide should be studied to reduce lost caused by storage insect pests

Identificação e fatores de virulência de Candida spp isoladas da cavidade bucal de transplantados renais do hospital universitário Onofre Lopes em Natal-RN

Despite Candida species are often human commensals isolated from various oral sites such as: tongue, cheek and palatal mucosa plus subgingival region, there are some properties linked to the organism commonly known as virulence factors which confer them the ability to produce disease. Oral candidiasis is one of the main oral manifestations reported in literature related to kidney transplant patients. The objectives of the present study were to identify and investigate virulence factors of yeasts isolated from the oral cavity of kidney transplant recipients admitted at the Hospital Universitário Onofre Lopes, in Natal RN. Seventy Candida species isolated from 111 kidney transplant recipients were investigated in this study. Identification of the isolates was performed by using the evidence of germ tube formation, hypertonic broth, tolerance to grow at 42°C, micromorphology and biochemical profiles. We observed a high rate of isolation of yeasts from the oral cavity of kidney transplant recipients (63.1%) being C. albicans was the most prevalent species. Oral candidiasis was diagnosed in 14.4% of transplant recipients. We evaluated virulence properties of the isolates regarding to: biofilm formation on polystyrene microplates as well as XTT reduction, adherence to acrylic resin and human buccal epithelial cells and proteinase activity. Most isolates were able to form biofilm by the method of adhesion to polystyrene. All isolates of Candida spp. remained viable during biofilm formation when analyzed by the method of XTT reduction. The number of CFU attached to the acrylic resin suggested high adherence for C. parapsilosis. C. albicans isolates showed higher median adherence to human buccal epithelial cells than non-C. albicans Candida isolates. Nevertheless, this difference was not statistically significant. C. dubliniensis showed low ability to adhere to plastic and epithelial cells and biofilm formation. Proteolytic activity was observed for all the isolates investigated, including the unique isolate of C. dubliniensis. There was a statistically significant association between proteinase production and the presence of oral candidiasis. Studies related to oral candidiasis in renal transplant recipients are limited to clinical and epidemiological data, but investigations concerning Candida spp. virulence factor for this group of individuals are still scarce. We emphasize the importance of studies related to virulence factors of yeasts isolated from this population to contribute to the knowledge of microbiological aspects of oral candidiasis

Extração, caracterização e atividades biológicas de proteínas da espécie cnidoscolus urens (L.) Arthur

The extraction, chemical and structural characterization of a wide variety of compounds derived from plants has been a major source of bioactive molecules. Several proteases have been isolated in the plant kingdom, with numerous pharmacological and biotechnological applications. Among the proteases isolated from plants, are the fibrinogenolytic, with relevant application in the treatment of disorders in the coagulation cascade, in addition to potential use as a tool in clinical laboratories. In this study, in addition to evaluating the effects of the protein extract of Cnidoscolus urens (L.) Arthur (Euphorbiaceae) in the coagulation cascade also investigates the presence of antimicrobial activity and characterizes the proteolytic activity detected in this extract, aiming to determine their potential pharmacological and biotechnological application. In this way, crude protein extracts obtained from the leaves of C. urens in Tris-HCl 0.05M, NaCl 0.15M, pH 7.5, were precipitated in different concentrations of acetone, and assessed for the presence of proteolytic activity in azocaseína and fibrinogen. The most active fraction (F1.0) in these tests was chosen for assessment of biological activity and biochemical characterization. The Aα chain and Bβ of fibrinogen were completely cleaved at a concentration of 0.18 μg/μL of protein fraction in 4 minutes. Fibrinogenolytic activity presented total inhibition in the presence of E-64 and partial in the presence of EDTA. The fraction demonstrated coagulant activity in plasm and reduced the APTT, demonstrating acting on the factors coagulation of the intrinsic pathway and common, not exerting effects on the PT. Fibrinolytic activity on plasma clot was detected only in SDS-PAGE in high concentrations of fraction, and there were no defibrinating. Although several proteases isolated from plants and venomous animals are classically toxic, the fraction F1.0 of C. urens not expressed hemorrhagic nor hemolytic activities. Fraction F1.0 also showed no antimicrobial activity. In proteolytic activity on the azocasein, the optimal pH was 5.0 and optimum temperature of 60ºC. The enzyme activity has been shown to be sensitive to the presence of salts tested, with inhibition for all compounds. The surfactant triton did not influence the enzyme activity, but the tween-20 and SDS inhibited the activity. In the presence of reducing agents increase in enzyme activity occurred, a typical feature of enzymes belonging to the class of cysteine proteases. Several bands with proteolytic activity were detected in zymogram, in the region of high-molecular-weight, which were inhibited by E-64. In this study, we found that C. urens presents in its constitution cysteine proteases with fibrinogenolytic and procoagulant activity, which may be isolated, with potential application in treatment of bleeding disorders, thrombolytic and clinical laboratory

Effect of chickpea (Cicer arietinum L.) germination on the major globulin content and in vitro digestibility

A germinação das sementes de grão-de-bico foi acompanhada por um período de 6 dias, no qual pequenas variações nos teores de nitrogênio e globulina total foram registradas. A globulina majoritária (tipo 11 S) apresentou maiores variações após o quarto dia de germinação. A natureza e distribuição da fração globulina majoritária isolada na cromatografia em Sepharose CL-6B mostrou pequenas modificações ao final do período de germinação. A eletroforese em gel de poliacrilamida com dodecilssulfato de sódio do pico eluído na cromatografia em Sepharose CL-6B demonstra modificações nas bandas de proteínas entre os pesos moleculares de 20 e 30 kDa e acima de 60 kDa, indicando degradação protéica durante o período. Atividade proteolítica foi detectada na fração albumina da semente que aumentou até o quarto dia, seguido de queda até o sexto dia de germinação, quando da utilização de globulina total isolada da semente e caseína como substratos. Farinha de grão-de-bico, frações albumina e globulina total isoladas não apresentaram aumento na digestibilidade in vitro; entretanto, a fração globulina majoritária isolada foi mais suscetível à hidrólise após germinação.

Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

O presente estudo consiste em uma caracterização preliminar da atividade proteolítica de frações de proteínas purificadas a partir de lisados de trofozoítos de cepa isolada e axenizada no Brasil. Frações obtidas por cromatografia líquida (FPLC) foram analisadas quanto ao perfil eletroforético em géis de poliacrilamida (SDS-PAGE) e a atividade proteolítica foi avaliada em géis contendo gelatina como substrato. A caracterização das enzimas foi realizada a partir da análise do efeito de inibidores sintéticos de cisteína-proteases (E-64, IAA), serina-proteases (PMSF), serina e cisteína-proteases (TPCK, TLCK, elastatinal), metalo-proteases (EDTA) e aspartil proteases (pepstatina) sobre a degradação do substrato. Entre 30 frações eluídas, bandas de proteínas foram observadas em oito delas, entretanto, atividade proteolítica foi detectada apenas nas frações 23, 24, 25 e 26. O perfil eletroforético das proteínas revelou poucas bandas distribuídas na faixa de 45 a 18 kDa. Os zimogramas revelaram zonas de proteólise na faixa de aproximadamente 62 a 35 kDa, entretanto destacaram-se as bandas de hidrólise de 62, 55, 53, 50, 46 e 40 kDa. Nos ensaios de inibição, a proteólise foi marcantemente inibida por E-64, TPCK, TLCK e elastatinal. Redução discreta da proteólise foi observada com IAA e PMSF, enquanto que EDTA e pepstatina não promoveram alteração dos perfis de hidrólise. Estas observações são relevantes, especialmente se considerarmos que para elucidar o envolvimento das proteases na relação parasita-hospedeiro, a purificação dessas moléculas é um requisito importante.

Caracterização bioquímica de uma serino-protease produzida pelo fungo termofílico Myceliophthora sp

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Análise bioquímica dos produtos de excreção/secreção de larvas de Dermatobia hominis

Pós-graduação em Medicina Veterinária - FMVZ

Variação sexual, ontogenética e ambiental do veneno de Crotalus durissus terrificus da região de Botucatu - São Paulo: caracterização enzimática, bioquímica e farmacológica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Identificação e caracterização dos produtos de excreção/secreção de trofozóitos de Giardia duodenalis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Detecção das metaloproteinases-2 e -9 no plexo coróide e no liquor de cães naturalmente infectados por Leishmania chagasi

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

Fermentação, purificação e caracterização da protease produzida pelo fungo Aspergillus fumigatus Fresenius

Pós-graduação em Microbiologia - IBILCE

Identificação e caracterização da microbiota lática isolada de queijo mussarela de búfala

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

Abordagem bioanalítica e físico-química da qualidade de carne em bovinos Nelore (Bos indicus) selecionados para produção

Pós-graduação em Zootecnia - FMVZ

Produção, purificação e caracterização da protease de Thermomucor indicae seudaticae N31 e avaliação de sua aplicação na fabricação do queijo maturado

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Papel do receptor tipo-2 ativado por protease (PAR-2) na doença periodontal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)