Structural and Functional Studies of a Bothropic Myotoxin Complexed to Rosmarinic Acid: New Insights into Lys49-PLA2 Inhibition

Snakebite envenoming is an important public health problem in many tropical and subtropical countries, and is considered a neglected tropical disease by the World Health Organization. Most severe cases are inflicted by species of the families Elapidae and Viperidae, and lead to a number of systemic and local effects in the victim. One of the main problems regarding viperidic accidents is prominent local tissue damage whose pathogenesis is complex and involves the combined actions of a variety of venom components. Phospholipases A2 (PLA2s) are the most abundant muscle-damaging components of these venoms. Herein, we report functional and structural studies of PrTX-I, a Lys49-PLA2 from Bothops pirajai snake venom, and the influence of rosmarinic acid (RA) upon this toxin's activities. RA is a known active component of some plant extracts and has been reported as presenting anti-myotoxic properties related to bothopic envenomation. The myotoxic activity of Lys49-PLA2s is well established in the literature and although no in vivo neurotoxicity has been observed among these toxins, in vitro neuromuscular blockade has been reported for some of these proteins. Our in vitro studies show that RA drastically reduces both the muscle damage and the neuromuscular blockade exerted by PrTX-I on mice neuromuscular preparations (by ∼80% and ∼90%, respectively). These results support the hypothesis that the two effects are closely related and lead us to suggest that they are consequences of the muscle membrane-destabilizing activity of the Lys49-PLA2. Although the C-terminal region of these proteins has been reported to comprise the myotoxic site, we demonstrate by X-ray crystallographic studies that RA interacts with PrTX-I in a different region. Consequently, a new mode of Lys49-PLA2 inhibition is proposed. Comparison of our results with others in the literature suggests possible new ways to inhibit bothropic snake venom myotoxins and improve serum therapy

O papel de um instrumento de apoio à comunicação matemática numa turma do 4.º ano

Relatório de Estágio apresentado à Escola Superior de Educação de Lisboa para obtenção de grau de mestre em Ensino do 1.º e 2.º Ciclos do Ensino BásicoA criação de ambientes de sala de aula onde os alunos são agentes efetivos da sua aprendizagem e têm espaço para participar e expor os seus conhecimentos, sabendo que vão ser ouvidos pelo professor e colegas, é, cada vez mais, uma recomendação curricular da atualidade e objeto de estudo. Este relatório dá conta da intervenção realizada numa turma do 4.º ano de escolaridade, com o propósito de promover a aprendizagem, valorizando, sistematicamente, a comunicação. Em simultâneo, explana-se a investigação realizada nesta turma, que envolveu a avaliação do impacto da construção de um instrumento de apoio aos momentos de Comunicação Matemática, no contexto de uma rotina semanal da turma, a Apresentação do Problema da Semana. Com a pretensão de dar resposta às questões (i) quais os contributos de um instrumento de apoio à organização e estruturação dos momentos de comunicação matemática? e (ii) de que forma esse mesmo instrumento contribui para o desenvolvimento de estratégias de resolução de problemas?, investigou-se a evolução da turma quanto à comunicação das resoluções dos problemas e à variedade das estratégias utilizadas, antes e após a construção coletiva do instrumento de apoio. A análise de resultados, feita através da análise das produções dos alunos e das respostas a um questionário que instigou a reflexão sobre o papel do instrumento de apoio ao longo das várias apresentações do problema da semana, permitiu perceber uma crescente variedade de estratégias utilizadas e uma maior preocupação, por parte dos alunos, em organizar e estruturar os momentos de comunicação matemática, bem como em mobilizar um discurso com correção matemática. Este é um estudo que se limita a uma turma, não sendo as suas conclusões generalizáveis, mas que pode contribuir com uma estratégia para melhorar a comunicação matemática dos alunos, de uma forma que os envolve de forma direta e que prevê a sua participação e papel ativo na aprendizagem.ABSTRACT Creating classroom environments where students are effective agents of their learning process and have space to participate and exhibit their skills, knowing that they will be heard by the teacher and classmates, is an increasingly curricular recomendation and object of stdy nowadays. This report gives an account of the intervention carried out on a 4th grade level class, with the purpose of promoting learning, valuing, systematically, the communication. At the same time, explains research performed in this class, which involved the evaluation of the impact of the construction of an instrument in support of Mathematical Communication moments, in the context of a weekly routine of the class, The Presentation of rhe Problem of the Week. With the pretense of responding to questions (i) what are the contributions of an instrument of support to the organization and structuring of moments of mathematical communication? and (ii) how this same instrument contributes to the development of problem solving strategies?, it was investigated the evolution of the class regarding the communication of the resolutions of the problems and the variety of strategies used in problem solving, before and after the collective construction of the instrument. The analysis of results, through the analysis of students’ productions and the replies to a questionnaire that instigated the reflection about the role of the instrument of support along the various presentations of the problem of the week, allowed to realize a growing variety of strategies used and greater concern, on the part of students, to organise and structure the moments of mathematical communication as well as in mobilizing a discourse with mathematical correction. This is a study that is limited to a class, which makes its conclusions not generalizable, but that can contribute with a strategy to improve students’ math communication, in a way that involves them directly and provides their participation and active role in their own learning process.N/

In memoriam: Prof. Dr. José Roberto Giglio and his contributions to toxinology

Submitted by EMERSON LEAL ([email protected]) on 2016-04-04T14:44:51Z No. of bitstreams: 1 In memoriam Prof. Dr. Jose.pdf: 303269 bytes, checksum: 22b9867660d9bf2b01fd2105d6246c9d (MD5)Approved for entry into archive by EMERSON LEAL ([email protected]) on 2016-04-14T14:25:19Z (GMT) No. of bitstreams: 1 In memoriam Prof. Dr. Jose.pdf: 303269 bytes, checksum: 22b9867660d9bf2b01fd2105d6246c9d (MD5)Made available in DSpace on 2016-04-14T14:25:19Z (GMT). No. of bitstreams: 1 In memoriam Prof. Dr. Jose.pdf: 303269 bytes, checksum: 22b9867660d9bf2b01fd2105d6246c9d (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Centro de Estudos de Biomoléculas Aplicadas à Saúde, Porto Velho, RO, Brazil / Universidade Federal de Rondônia. Porto Velho, RO, Brazil.Prof. Dr. José R. Giglio (1934 e 2014) made a highly significant contribution to the field of Toxinology. During 48 years devoted to research and teaching Prof Giglio published more than 160 articles, with more than 4400 citations, in international journals, trained a vast amount of graduate and undergraduate students, and developed an international network of collaborators. Throughout these years, he worked with dedication and deep commitment to science, leaving an immortalized legacy. During his professional career he contributed mostly in the isolation, and biochemical and functional characterization of various protein toxins derived from animal venoms such as snakes, scorpions and spiders, in addition to his studies searching for alternative therapies for poisoning. Even after his departure, his presence and influence remains among his former students and in the outstanding legacy of his scientific contributions

Comparative structural studies on Lys49-phospholipases A(2) from Bothrops genus reveal their myotoxic site

Phospholipases A(2) (PLA(2)s) are membrane-associated enzymes that hydrolyze phospholipids at the sn-2 position, releasing lysophospholipids and free fatty acids. Phospholipase A(2) homologues (Lys49-PLA(2)s) are highly myotoxic and cause extensive tissue damage despite not showing measurable catalytic activity. They are found in different snake venoms and represent one third of bothropic venom composition. The importance of these toxins during envenomation is related to the pronounced local myotoxic effect they induce since this effect is not neutralized by serum therapy. We present herein three structures of Lys49-PLA(2)s from Bothrops genus snake venom crystallized under the same conditions, two of which were grown in the presence of alpha-tocopherol (vitamin E). Comparative structural analysis of these and other Lys49-PLA(2)s showed two different patterns of oligomeric conformation that are related to the presence or absence of ligands in the hydrophobic channel. This work also confirms the biological dimer indicated by recent studies in which both C-termini are in the dimeric interface. In this configuration, we propose that the myotoxic site of these toxins is composed by the Lys 20, Lys115 and Arg118 residues. For the first time, a residue from the short-helix (Lys20) is suggested as a member of this site and the importance of Tyr119 residue to myotoxicity of bothropic Lys49-PLA(2)s is also discussed. These results support a complete hypothesis for these PLA(2)s myotoxic activity consistent with all findings on bothropic Lys49-PLA(2)s studied up to this moment, including crystallographic, bioinformatics, biochemical and biophysical data. (C) 2009 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Crystallization and preliminary X-ray crystallographic studies of a Lys49-phospholipase A(2) homologue from Bothrops pirajai venom complexed with rosmarinic acid

PrTX-I, a noncatalytic and myotoxic Lys49-phospholipase A(2) from Bothrops pirajai venom, was crystallized in the presence of the inhibitor rosmarinic acid (RA). This is the active compound in the methanolic extract of Cordia verbenacea, a plant that is largely used in Brazilian folk medicine. The crystals diffracted X-rays to 1.8 angstrom resolution and the structure was solved by molecular-replacement techniques, showing electron density that corresponds to RA molecules at the entrance to the hydrophobic channel. The crystals belong to space group P2(1)2(1)2(1), indicating conformational changes in the structure after ligand binding: the crystals of all apo Lys49-phospholipase A(2) structures belong to space group P3(1)2(1), while the crystals of complexed structures belong to space groups P2(1) or P2(1)2(1)2(1).Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Expression of Human Recombinant Antibody Fragments Capable of Partially Inhibiting the Phospholypase Activity of Crotalus durissus terrificus Venom

Crotoxin is the main toxic component of the South American rattlesnake Crotalus durissus terrificus venom. It is composed of two different subunits: CA, crotapotin, and CB (basic subunit of cortoxin isolated from C. d. terrificus), a weakly toxic phospholipase A(2) with high enzymatic activity. The phospholipases A(2) are abundant in snake venoms and are responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. However, in addition to their normal digestive action, a wide range of pharmacological activities, such as neurotoxic, myotoxic, oedema-inducing, hypotensive, platelet-aggregating, cardiotoxic, and anticoagulant effects have been attributed to venom phospholipases A(2). In this study, we used a non-immune human single-chain fragment variable library, Griffin.1 (Medical Research Council, Cambridge, UK) for selection of recombinant antibodies against antigens present in C. d. terrificus venom and identification of specific antibodies able to inhibit the phospholipase activity. Two clones were identified as capable of inhibiting partially this activity in vitro. These clones were able to reduce in vivo the myotoxic and oedema-inducing activity of CB and the lethality of C. d. terrificus venom and crotoxin, but had no effect on the in vitro anticoagulant activity of CB. These results demonstrate the potential of using recombinant single-chain fragment variable libraries in the production of antivenoms.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Apoio ao Ensino, Pesquisa e Assistencia do Hospital das Clinicas de Ribeirao Preto (FAEPA)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

Effect of treatment with l-amino oxidase fractions, from Bothrops jararaca venom on Ehrlich ascites tumor growth

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Crystallization and preliminary X-ray diffraction studies of BmooPLA(2)-I, a platelet-aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops moojeni venom

Phospholipases A(2) (PLA(2)s) are enzymes that cause the liberation of fatty acids and lysophospholipids by the hydrolysis of membrane phospholipids. In addition to their catalytic action, a wide variety of pharmacological activities have been described for snake-venom PLA(2)s. BmooPLA(2)-I is an acidic, nontoxic and catalytic PLA(2) isolated from Bothrops moojeni snake venom which exhibits an inhibitory effect on platelet aggregation, an immediate decrease in blood pressure, inducing oedema at a low concentration, and an effective bactericidal effect. BmooPLA(2)-I has been crystallized and X-ray diffraction data have been collected to 1.6 angstrom resolution using a synchrotron-radiation source. The crystals belonged to space group C222(1), with unit-cell parameters a = 39.7, b = 53.2, c = 89.2 angstrom. The molecular-replacement solution of BmooPLA(2)-I indicated a monomeric conformation, which is in agreement with nondenaturing electrophoresis and dynamic light-scattering experiments. A comparative study of this enzyme with the acidic PLA(2) from B. jararacussu (BthA-I) and other toxic and nontoxic PLA(2)s may provide important insights into the functional aspects of this class of proteins.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq