Potencial terapêutico de um heparinóide isolado do invertebrado marinho Litopenaeus vannamei

The occurrence of bioactive compounds in marine organisms comes awaking the interest of the pharmaceutical industry. Heparin, a sulfated polysaccharide which presence was already identified in several marine invertebrates, is very attractive due its remarkable functional versatility. Besides to intervene in blood coagulation, this molecule has a great anti-inflammatory potential. However, its strong anticoagulant activity difficult the clinical exploitation of its anti-inflammatory properties. Thus, the aims of this work were to evaluate the effect of a heparin-like compound (heparinoid), isolated from the cephalotorax of the Litopenaeus vannamei shrimp, on the inflammatory response, hemostasia and synthesis of antithrombotic heparan sulfate by endothelial cells, besides studying some aspects concerning its structure. The purified heparinoid was structurally characterized following an analytical boarding, involving electrophoresis and chromatography. The structural analysis have shown that this compound possess a high content of glucuronic acid residues and disulfated disaccharide units. In contrast to mammalian heparin, the heparinoid was incapable to stimulate the synthesis of heparan sulfate by endothelial cells in the tested concentrations, beyond to show reduced anticoagulant activity and hemorrhagic effect. In a model of acute inflammation, the compound isolated from the shrimp reduced more than 50% of the cellular infiltration. Besides reduce the activity of MMP-9 and proMMP-2 of the peritoneal lavage of inflamed animals, the heparinoid also reduced the activity of MMP-9 secreted by activated human leukocytes. These results demonstrate the potential of heparinoid from L. vannamei to intervene in the inflammatory response. For possessing reduced anticoagulant activity and hemorrhagic effect, this compound can serve as a structural model to direct the development of more specific therapeutical agents to the treatment of inflammatory diseases

Avaliação do efeito do composto tipo heparina isolado do caranguejo Chaceon fenneri na hemostasia e na morte celular

Heparin is a pharmaceutical animal widely used in medicine due to its potent anticoagulant effect. Furthermore, it has the ability to inhibit the proliferation, invasion and adhesion of cancer cells to vascular endothelium. However, its clinical applicability can be compromised by side effects such as bleeding. Thus, the search for natural compounds with low bleeding risk and possible therapeutic applicability has been targeted by several research groups. From this perspective, this study aims to evaluate the hemorrhagic and anticoagulant activities and citotoxic effect for different tumor cell lines (HeLa, B16-F10, HepG2, HS-5,) and fibroblast cells (3T3) of the Heparin-like from the crab Chaceon fenneri (HEP-like). The HEP-like was purified after proteolysis, ion-exchange chromatography, fractionation with acetone and characterized by electrophoresis (agarose gel) and enzymatic degradation. Hep-like showed eletroforetic behavior similar to mammalian heparin, and high trisulfated /Nacetylated disaccharides ratio. In addition, HEP-like presented low in vitro anticoagulant activity using aPTT and a minor hemorrhagic effect when compared to mammalian heparin. Furthermore, the HEP-like showed significant cytotoxic effect (p<0.001) on HeLa, HepG2 and B16-F10 tumor cells with IC50 values of 1000 ug/mL, after incubation for 72 hours. To assess the influence of heparin-like on the cell cycle in HeLa cells, analysis was performed by flow cytometry. The results of this analysis showed that HEP-like influence on the cell cycle increasing S phase and decreasing phase G2. Thus, these properties of HEP-like make these compounds potential therapeutic agents

Efeito anti-inflamatório do heparinóide isolado do camarão Litopenaeus vannamei sobre a peritonite aguda

In recent years the heparin has been the subject of several studies that aim to expand its use as a therapeutic agent, due to its ability to modulate the activity of various proteins that play important roles in the regulation of pathophysiological processes. In several experiments and preclinical trials, heparin has demonstrated an anti-inflammatory role. However, its clinical use is limited, due to its strong anticoagulant activity and hemorrhagic complications. For this reason, considerable efforts have been employed in discovery of heparin analogous (heparinoid) with reduced side effects, that retain the anti-inflammatory properties of heparin. In this context, a heparinoid obtained from the head of Litopenaeus vannamei shrimp, which presents a structural similarity to heparin, showed, in previous studies, anti-inflammatory activity in a model of acute peritonitis with reduced anticoagulant effect in vitro and low hemorrhagic activity. Thus, the present work had as objective to evaluate the effect the heparinoid of the cephalothorax of gray shrimp on the acute inflammatory response in different times (3 or 6 hours after the induction of inflammatory stimulus), using the model of acute peritonitis induced in mice. It was also analyzed the HL effect over the activity of elastase, an enzyme involved in leukocyte recruitment. Furthermore to check if the different doses of heparin and heparinoid change the hemostatic balance in vivo, was assessed the effect of these compounds on the plasma clotting time in animals submitted to inflammation. The results show that in 3 hours, all doses of heparinoid were able to prevent efficiently in the acute inflammatory process without any anticoagulant effects, unlike the extrapolation dose of heparin, which has induced a large hemorrhage due its high anticoagulant activity. However, 6 hours after induction of inflammation, only the dosages of 0.1 and 1.0 μg/Kg of heparin and 1.0 μg/Kg of heparinoid kept anti-migratory effect, without changing of the hemostatic balance. These results indicate that the anti-migratory effect of theses compounds depends on the dosage and time of inflammatory stimulus. The HL and heparin were also able to inhibit the activity of the enzyme elastase. The discovery of this bioactive compound in the cephalothorax of shrimps can arouse great interest in biotechnology, since this compound could be useful as a structural model interesting for the development of new therapeutic agents for peritonitis

Soroprevalência da infecção pelo Trypanosoma cruzi no município de Caicó, RN, e caracterização genética por RAPD do parasito isolado de humanos e de triatomíneos no semiárido potiguar

Trypanosoma cruzi infection was evaluated in 390 resident individuals in different rural communities of Caicó municipality, State of Rio Grande do Norte (RN). Of 28 investigated communities the soroprevalence of T. cruzi infection was 2.8% in eight rural communities individuals. The epidemiological characteristics of seropositive shown that the age ranged from 22 to 64 years, being significantly raised from 31 years (90.9%). The female gender was predominant and low education degree. Those individuals reported that they never donated blood, but they had direct contact with triatomines bug. The isolation of the parasite was performed by blood culture and xenoculture methods to determine the genetic variability of the samples. Twenty seven T. cruzi isolates were analyzed by RAPD as genetic marker using three random primers (M13-40, gt11-F and L15996). The T. cruzi isolates showed 73.7% of shared bands considering the average obtained with the three primers, and were genetically well correlated. Using this marker it was possible to separate the populations of the parasite in three distinct groups. The first group composed by isolates obtained of triatomines and humans from four different districts (Caicó, Caraúbas, Serra Negra doNorte and Governador Dix-Sept Rosado); the second contained isolates obtained of triatomines of two different species (T. brasiliensis and P. lutzi) captured in Caraúbas and Serra Negra do Norte. The third grouped isolates obtained from humans of Angicos and Caicó municipalities. In different localities of distinct mesoregions, State of RN, a profile genetic well correlated was identified among all isolates and the presence of three distinct groups of the parasite circulating among vertebrate and invertebrate hosts

Aplicação de óleo fúsel isolado e em mistura com glifosato na pós-emergência tardia de plantas daninhas

Este trabalho foi realizado no Centro de Cana IAC, Ribeirão Preto, com o objetivo de avaliar a eficácia do óleo fúsel isolado e em mistura com glifosato, aplicado na pós-emergência tardia de plantas daninhas de uma comunidade natural. O delineamento experimental foi em blocos casualizados (DBC), com 13 tratamentos em quatro repetições, sendo 52 parcelas de 3 x 3 m cada. A aplicação dos tratamentos foi realizada em 22/3/2006, com equipamento costal pressurizado munido de barra com quatro pontas de pulverização Teejet 110.02 TT, regulado para volume de calda de 212 L ha-1. Avaliaram-se a porcentagem de controle aos 14, 21, 28, 35 e 42 dias após a aplicação do tratamento (DAA) e a massa seca, aos 42 DAA. Para a maioria dos tratamentos somente não houve 100% de controle devido à presença de plantas daninhas dos gêneros Commelina e Cyperus spp., que, mesmo com partes amareladas, foram mais tolerantes à aplicação dos produtos. O controle obtido com óleo fúsel aplicado isoladamente não ultrapassou 20%, aos 42 DAA.

Efeito de concentrações de herbicidas sobre aspectos biológicos de Fusarium sp. (isolado FCAV#940)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Manejo químico de plantas adultas de Digitaria insularis com glyphosate isolado e em mistura com chlorimuronethyl ou quizalofop-p-tefuril em área de plantio direto

O objetivo deste trabalho foi avaliar a eficácia do herbicida glyphosate, isolado e em mistura com chlorimuron-ethyl ou quizalofop-p-tefuril, e o efeito da adição de óleo mineral à calda de glyphosate e chlorimuron-ethy no controle de plantas adultas de Digitaria insularis, em área sob sistema de plantio direto. Foram desenvolvidos dois experimentos, em condições de campo, um de novembro de 2005 a fevereiro de 2006 e outro de novembro de 2006 a janeiro de 2007, em Jaboticabal (SP). O delineamento experimental foi o de blocos ao acaso, em esquema fatorial, com quatro repetições. No primeiro experimento, foram avaliadas oito combinações (Fator A) entre o glyphosate (0,72; 1,44; 2,16 e 2,88 kg ha-1) e o chlorimuron-ethyl (0 e 10 g ha-1), três doses (Fator B) de óleo mineral (0; 0,5 e 1,0 L ha-1) e uma testemunha mantida infestada sem a aplicação de herbicida. No segundo, foram estudadas seis associações (Fator A) entre o glyphosate (1,44; 2,16 e 2,88 kg ha-1) e o quizalofop-p-tefuril (0 e 120 g ha-1) e um tratamento de quizalofop-p-tefuril (120 g ha-1) isolado, combinados com aplicações sequenciais (Fator B) de glyphosate (0; 0,72 e 1,44 kg ha-1), pulverizado 15 dias após a primeira a aplicação, além de uma testemunha infestada sem a aplicação de herbicida. Nas avaliações iniciais, no experimento de 2005/ 2006, em plantas pulverizadas com as maiores doses de glyphosate, isolado ou em mistura ao chlorimuron, havia injúrias visuais severas, com classificação do controle de bom (71% a 80%) a muito bom (81% a 90%). Com o decorrer do tempo, as plantas rebrotaram revelando seu potencial de recuperação e a ineficácia dos herbicidas no controle definitivo desta espécie. A adição de óleo mineral à calda de glyphosate e chlorimuron não contribuiu para o controle de D. insularis. No outro experimento, o quizalofop isolado e a sua associação ao glyphosate, combinados à aplicação sequencial de 1,44 kg ha-1 de glyphosate, resultaram em melhor controle da planta daninha.

Redução do nível protéico da dieta, através da formulação baseada em aminoácidos digestíveis

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

Efeito do hexazinone isolado e em mistura na eficiência fotossintética de Panicum maximum

Esta pesquisa teve como objetivo avaliar a velocidade e intensidade de ação do hexazinone isolado e em mistura com outros inibidores do fotossistema II, através da eficiência fotossintética de Panicum maximum em pós-emergência. O ensaio foi constituído de seis tratamentos: hexazinone (250 g ha-1), tebuthiuron (1,0 kg ha-1), hexazinone + tebuthiuron (125 g ha-1 + 0,5 kg ha-1), diuron (2.400 g ha-1), hexazinone + diuron (125 + 1.200 g ha-1), metribuzin (1.440 g ha-1), hexazinone + metribuzin (125 + 720 g ha-1) e uma testemunha. O experimento foi instalado em delineamento inteiramente casualizado, com quatro repetições. Após a aplicação dos tratamentos, as plantas foram transportadas para casa de vegetação sob condições controladas de temperatura e umidade, onde ficaram durante o período experimental, sendo realizadas as seguintes avaliações: taxa de transporte de elétrons e análise visual de intoxicação. A avaliação com o fluorômetro foi realizada nos intervalos de 1, 2, 6, 24, 48, 72, 120 e 168 horas após a aplicação, e as avaliações visuais, aos três e sete dias após a aplicação. Os resultados demonstraram diferença nos tratamentos, enfatizando a aplicação do diuron, que reduziu lentamente o transporte de elétrons comparado com os outros herbicidas e, em mistura com hexazinone, apresentou efeito sinérgico. Verificou-se com o uso do fluorômetro a intoxicação antecipada em plantas de P. maximum após a aplicação de herbicidas inibidores do fotossistema II de forma isolada e em mistura.

Identificação e caracterização de um isolado de Hydrangea ringspot virus em hortênsia no Estado de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Seletividade do halosulfuron isolado ou em mistura com glyphosate para culturas anuais

O objetivo do presente trabalho foi avaliar a seletividade do herbicida halosulfuron isoladamente e na mistura com glyphosate, aplicados em pré e pós-emergência, para culturas de verão (milho, feijão, algodão e soja) e para culturas de inverno (aveia-preta, azevém, centeio, trigo e triticale). Foram instalados dois experimentos em campo, nas Fazendas Experimentais do Lageado e de São Manuel - UNESP - Botucatu-SP - Brasil. Os tratamentos foram constituídos da aplicação isolada do herbicida halosulfuron (100/150 g ha-1), em pré e pós-emergência, e em mistura de tanque halosulfuron + glyphosate NA e WG (100+4.000 g ha-1 e 100+2.000/4.000 g ha¹, respectivamente), em pré-emergência e em diferentes épocas de aplicação (2, 15 e 30 dias antes e 15 e 30 depois da semeadura). A intensidade da fitotoxicidade encontrada nas plantas das culturas de soja, milho, feijão, algodão e azevém foi devido à aplicação do herbicida halosulfuron, que esteve relacionada com dosagens, época e modo de aplicação. Quanto mais próximo da aplicação do halosulfuron em pós-emergência da semeadura das culturas, maiores foram as injúrias encontradas em suas plantas. Todos os tratamentos testados não proporcionaram sintomas de fitotoxicidade nas plantas de aveia-preta, centeio, trigo e triticale.

Avaliação preliminar da viabilidade de produção in vitro de um isolado brasileiro de baculovírus Spodoptera frugiperda MNPV

In vivo production of viral biopesticides is the major source of viral insecticides currently in the marketplace. However, this system presents limitations during production scale-up. For the Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV), the insect used for replication has cannibalistic characteristics, thus production is even more difficult. Insect cells are commonly used for in vitro baculovirus production. Most of these cell lines are derived from Lepidoptera species. The Sf21 cell line is derived from Spodoptera frugiperda caterpillar ovarian tissue, and its clonal isolate Sf9 has been used for biopesticide production due to its ease of growth in suspension cultures. In this work, the in vitro production capabilities of a Brazilian SfMNPV isolate obtained from cornfields was evaluated. Comparison of polyhedra production was carried out using both Sf21 and Sf9 cells, based on volumetric and specific yields. Both cell lines were cultivated in Hyclone medium supplemented with different fetal bovine serum concentrations (2,5 and 5%). The best results were obtained using Sf9 cells supplemented with 5% serum. These results were further confirmed quantitively through kinetic parameter estimation for both cells lines and different serum concentrations. After seven successive passages, this system still presented high specific polyhedra production

Caracterização de um isolado de Bidens mosaic virus proveniente de alface

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

Análise das mudanças no perfil protéico durante o estresse oxidativo in vivo e atuação da MutY-Glicosilase em respostas celulares

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Estratégias de Produção in vitro de Bioinseticida Viral: influências do Isolado, da Cinética e do Modo de operação

Societal concerns about environmental sustainability has lead to the development of ecologically-friendly alternatives to chemical insecticides for crop protection. One such alternative is biological pest control. In particular, baculoviruses are well suited as insect biopesticides due to their narrow host specificity and relative ease of propagation. In Brazil, the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is the main biological control agent employed for the soybean pest, Anticarsia gemmatalis. This baculovirus biopesticide is currently produced using caterpillars, but increasing market demand for the product has encouraged the development of an in vitro manufacturing process, which can be scaled up to much higher virus productivities. In this study, three wild-type AgMNPV isolates (AgMNPV-2D, AgMNPV-MP2 and AgMNPV-MP5) and a recombinant form (vAgEGT-LacZ) were characterised in terms of occlusion body (OB) production and infection kinetics, to enable future optimisation of the in vitro production process. These viruses were propagated using a Spodoptera frugiperda (IPLB-SF21) insect cell line grown in shaker-flask batch cultures. Among the virus isolates tested, AgMNPV-MP5 was found to be the best producer, yielding (5.3±0.85)x108 OB/mL after 8 days post infection. The characterisation of vAgEGT-LacZ propagation in suspension cell cultures has not been previously reported in the literature; hence it became the main focus for this thesis. In particular, it was carried out a study on the effect of the multiplicity of infection (MOI) on OB production. Five successive batches were performed getting a final production (8.9±1.42)x1014 occlusion bodies, considering that production is related for a bioreactor with final volume of 10m3. A low MOI associated with a fed-batch process for vAgEGT-LacZ production was found to support a 3-fold higher OB yield when compared to the default batch process (1.8x107 and 5.3x107 OB/mL, respectively). This yield is competitive with regards to the production process.