Caracterização bioquímica e biológica de fosfolipases presentes em veneno de Loxosceles Intermedia e Lonomia Obliqua

Orientador : Prof. Dr. Silvio Sanches VeigaCo-Orientador: Prof. Dr. Andrea Senff Ribeiro, Profa. Dra. Olga Meiri ChaimTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 04/08/2011Bibliografia: fls. 167-198Resumo: No Brasil, existem vários animais de importância médica, as aranhas do gênero Loxosceles e as lagartas do gênero Lonomia em especial apresentam índices alarmantes de acidentes com humanos. Esses animais peçonhentos e traumatizantes podem provocar morbidez importante e algumas vezes mortalidade em humanos. Após a picada da aranha marrom (Loxosceles intermedia), as vítimas apresentam lesões cutâneas necróticas ao redor da picada e, em menor freqüência, sinais sistêmicos tais como insuficiência renal, coagulação intravascular disseminada e hemólise. Enquanto, o contato acidental com as cerdas pontiagudas contendo o veneno da lagarta (Lonomia obliqua) leva a dor em queimação, edema, eritema e em alguns casos hemorragias, hemólise e insuficiência renal. Neste presente trabalho, nós caracterizamos bioquímica e biologicamente seis isoformas recombinantes e uma isoforma mutada de fosfolipase-D presentes no veneno de Loxosceles intermedia. O tratamento de eritrócitos provenientes de sangue humano com as toxinas induziu experimentalmente hemólise direta de modo concentração- e tempo-dependentes. Os eritrócitos expostos à toxina fosfolipase-D recombinante apresentaram alterações morfológicas no tamanho e na forma, agregação dos lipid rafts e externalização de fosfatidilserina. A lise direta dos eritrócitos induzida pela fosfolipase-D recombinante depende da espécie do animal testado, já que eritrócitos obtidos a partir de humanos, de coelhos e de carneiros sofreram hemólise em uma porcentagem muito superior do que a observada com eritrócitos de cavalo. Em ensaios de microscopia confocal e imunofluorescência indireta, bem como em citometria de fluxo, utilizando uma toxina fluorescente recombinante GFPfosfolipase- D mostraram a ligação direta da toxina à membrana dos eritrócitos humanos. Além disso, observou-se que está enzima promove a hidrólise de fosfolipídios como a esfingomielina e lisofosfatidilcolina da membrana dos eritrócitos, formando ácido lisofosfatídico e ceramida-1-fosfato respectivamente, que são capazes de ativar uma série de enzimas intracelulares que culminam na morte e ruptura dos eritrócitos. Além disso, o tratamento com a fosfolipase-D recombinante estimula o influxo de cálcio detectado através de uma sonda fluorescente sensível ao cálcio (Fluo-4). Este influxo de cálcio mostrou ser mediado por canais de cálcio do tipo-L. Fazendo o uso de alguns inibidores sugere-se duas possíveis vias que explicam a hemólise induzida pela fosfolipase-D. A primeira através da formação de ácido lisofosfatídico e ativação dos receptores LPA1/LPA3 acoplados a proteína G e a segunda pela formação de eramida-1-fosfato e da sua interconversão em ceramida, esfingosina e esfingosina-1-fosfato. Desse modo, os resultados aqui descritos fornecem evidências de que as fosfolipases-D do veneno de L. intermedia desencadeiam a hemólise direta sobre de eritrócitos humanos de maneira dependente da atividade catalítica e que a ruptura das células ocorre com a formação de metabolitos bioativos que ativam cascatas de sinalização. Por fim, foi clonada uma nova fosfolipase A2 presente nas cerdas de Lonomia obliqua. Esta nova toxina foi parcialmente caracterizada, porém futuramente será feita uma avaliação mais aprofundada das suas características bioquímicas, estruturais e biológicas. Logo, esse estudo sobre as fosfolipases presente nos venenos de Loxosceles intermedia e de Lonomia obliqua pode auxiliar o melhor entendimento dos efeitos fisiopatológicos desenvolvidos nos acidentes com esses animais, assim como para o esenvolvimento de novas manobras terapêuticas e aplicações biotecnológicas e industriais.Abstract: In Brazil, there are several animals of medical importance, the spider of the genus Loxosceles and caterpillars of the genus Lonomia have particularly alarming rates of accidents with humans. These traumatic and venomous animals may provoke important morbidity and sometimes mortality in humans. After the bite of the brown spider (Loxosceles intermedia), can occur necrotic skin lesions around the bite and,less frequently, systemic signs such as renal failure, disseminated intravascular coag lation and hemolysis. While accidental contact with sharp bristles containing the poison of a caterpillar (Lonomia obliqua) leads to burning pain, swelling, redness and sometimes bleeding, hemolysis and renal failure. In this work, we characterized biochemical and biologically six recombinant isoforms and a mutated isoform of phospholipase-D present in the venom of Loxosceles intermedia. Treatment of ery hrocytes from human blood with toxins experimentally induced hemolysis so concentration-and time-dependent. Erythrocytes exposed to the toxin recombinant phospholipase-D showed morphological changes in size and shape, aggregation of lipid rafts and externalization of phosphatidylserine. The direct lysis of erythrocytes induced by recombinant phospholipase-D depends on the species of animal tested, since red blood cells obtained from human, rabbit and sheep suffered hemolysis in a much higher percentage than that observed with horse erythrocytes. In studies of indirect immunofluorescence and confocal microscopy and flow cytometry, using a recombinant GFP-fluorescent toxin phospholipase-D showed the direct connection of the toxin to the membrane of human erythrocytes. In addition, we observed that this enzyme promotes the hydrolysis of phospholipids such as sphingomyelin and lysophosphatidylcholine erythrocyte membrane, forming ysophosphatidic acid and ceramide-1-phosphate respectively, which are capable of activating a number of intracellular enzymes that culminate in the death and rupture of red blood cells. In addition, treatment with recombinant phospholipase-D stimulates the influx of calcium detected by a calcium-sensitive fluorescent probe (Fluo-4). This influx of calcium was shown to be mediated by calcium channel L-type. Making use of some inhibitors suggests that two possible ways to explain the hemolysis induced by phospholipase- D. The first through the formation of lysophosphatidic acid LPA1/LPA3 and activation of receptors coupled to G protein and second by the formation of ceramide-1- phosphate and its interconversion of ceramide, sphingosine and sphingosine-1- phosphate. Thus, the results described here provide evidence that hospholipase-D from the venom of L. intermedia trigger directly on hemolysis of human erythrocytes in a manner dependent on the catalytic activity and that disruption of the cells occurs with the formation of oactive metabolites that activate signaling cascades. Finally, we cloned a new phospholipase A2 present in Lonomia obliqua bristles. This new toxin has been partially characterized, but the future will be a further evaluation of its biochemical, structural and biological characteristics. Therefore, this study on phospholipases present in the venoms of Loxosceles intermedia and Lonomia obliqua can help better understand the pathophysiological effects developed in accidents with these animals, as well as the development of new therapeutic maneuvers and industrial and biotechnological applications

Estudo da atividade hemolítica do veneno de Loxosceles intermedia (Aranha Marrom) e seus mecanismos moleculares

Orientador: Silvio Sanches VeigaCo-orientador: Waldemiro GremskiDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 05/11/2008Inclui bibliografia e anexosÁrea de concentração: Biologia Celular e MolecularAs aranhas marrons estão distribuídas mundialmente e os acidentes relacionados são considerados um problema de saúde pública, especialmente na América do Sul, os quais são denominados como Loxoscelismo. Após os acidentes, as vítimas apresentam lesões cutâneas necróticas ao redor da picada e, em menor freqüência, sinais sistêmicos tais como insuficiência renal, coagulação intravascular disseminada e hemólise. Neste presente trabalho, nós estudamos o mecanismo molecular pelo qual uma toxina recombinante purificada e obtida da glândula do veneno, a qual é caracterizada bioquimicamente como uma fosfolipase-D, induz hemólise. O tratamento de eritrócitos provenientes de sangue humano com a toxina induziu experimentalmente hemólise direta de modo concentração- e tempo-dependentes. Os eritrócitos expostos à toxina fosfolipase-D recombinante apresentaram alterações morfológicas no tamanho e na forma das células de modo tempo-dependente. Além disso, a hemólise não foi dependente do grupo ABO e sistema Rh, uma vez que os eritrócitos humanos de diferentes grupos sanguíneos tratados com a toxina recombinante sofreram hemólise de uma maneira semelhante. A lise direta dos eritrócitos induzida pela fosfolipase-D recombinante depende da espécie do animal testado, já que eritrócitos obtidos a partir de humanos, de coelhos e de carneiros sofreram hemólise em uma porcentagem muito superior do que a observada com eritrócitos de cavalo. Em ensaios de microscopia confocal e imunofluorescência indireta, bem como na citometria de fluxo, seja utilizando anticorpos contra a fosfolipase-D, ou uma proteína fusão recombinante GFP-fosfolipase-D mostraram a ligação direta da toxina à membrana dos eritrócitos humanos. Além disso, eritrócitos incubados com a toxina recombinante reagiram com anexina-V e tiveram alteração do seu perfil de distribuição dos microdomínios lipídicos, corroborando com uma mudança no conteúdo de fosfolipídios da membrana tratada, que passou a expor na superfície celular um fosfolipídio carregado negativamente, a fosfatidilserina, mostrando também uma reorganização dos componentes da membrana lipídica. Adicionalmente, três quelantes de íons divalentes (EGTA, EDTA e 1,10-fenantrolina) inibiram significativamente a hemólise induzida pela fosfolipase-D (a qual contém magnésio, no domínio catalítico), em comparação ao PMSF, um inibidor de serino-protease, que não teve efeito sobre hemólise, sugerindo que a catálise está envolvida na hemólise. Finalmente, usando uma fosfolipase-D recombinante com mutação sítio-dirigida no domínio catalítico, na qual foi substituída a histidina da posição 12 por uma alanina, a hemólise e as alterações morfológicas dos eritrócitos foram completamente inibidas. No entanto, a toxina mutada não perdeu a capacidade de ligar-se à superfície dos eritrócitos, o que reforça a idéia do envolvimento da atividade catalítica da enzima no efeito hemolítico e nas alterações celulares, mas não apenas vinculada à ligação da toxina na superfície celular. Desse modo, os resultados aqui descritos fornecem evidências de que a fosfolipase-D do veneno de L. intermedia desencadeia a hemólise direta sobre de eritrócitos humanos de maneira dependente da atividade catalítica.Brown spiders have world-wide distribution and accidents that are considered health problem especially in South America are named loxoscelism. Victims followed accidents present necrotic cutaneous lesions surrounding the bites and in a less intensity systemic signs such as renal failure, disseminated intravascular coagulation, and hemolysis. Here, we studied the molecular way by which a purified recombinant toxin from the venom gland, biochemically characterized as phospholipase-D, causes hemolysis. Toxin treatment of human blood erythrocytes experimentally induced direct hemolysis in a dose-dependent concentration manner and in a time-dependent way. Erythrocytes exposed to the recombinant phospholipase-D toxin showed morphological changes in size and shape of cells in a timedependent manner. Also, hemolysis was not dependent of ABO group and Rhesus systems, since human erythrocytes from all blood group treated by recombinant toxin suffered hemolysis in a similar manner. The direct lysis of erythrocytes evoked by recombinant phospholipase-D depends on animal species tested, since erythrocytes from human, rabbit and sheep suffered hemolysis in higher percentage than horse erythrocytes. Confocal microscopy and immunofluorescence assay using antibodies against phospholipase-D, as well as cell cytometry and confocal microscopy using a recombinant fusion GFP-phospholipase-D protein supported the direct binding of toxin to membrane of human erythrocytes. Moreover, erythrocytes treated by recombinant toxin reacted with annexin-V and had their lipid rafts profile altered, supporting for a changing in the phospholipid membrane contend of treated cells exposing negatively charged phosphatidylserine at the cell surface, and for a reorganization of membrane lipid components. Additionally, three divalent ion chelators (EGTA, EDTA and 1,10-phenanthroline), significantly inhibited hemolysis evoked by phospholipase-D (that has magnesium at the catalytic domain), as compared to PMSF a serine-protease inhibitor that had no effect upon hemolysis, suggesting catalysis as involved upon hemolysis. Finally, by using a site directed mutated recombinant phospholipase-D at the catalytic domain, substituting histidine at position 12 by alanine, the hemolysis and morphologic changes of erythrocytes were completely inhibited, but not the toxin binding on the erythrocytes surface, supporting that the catalytic activity of enzyme is involved on hemolysis and cellular alterations but not on the toxin cell binding. Thus, results described herein provide evidence that phospholipase-D from L. intermedia venom stimulates direct hemolysis upon human blood cells in a catalytic-dependent way

Poder vital e o legado de florence nightingale no processo saúdedoença: revisão integrativa The vital power and the legacy of florence nightingale in the health-disease process: integrative review

Objetivo:.Caracterizar as evidências científicas nacionais dos últimos dez anos sobre Florence Nightingale e o poder vital e sua aplicabilidade na enfermagem, além de identificar os conceitos sobre poder vital. Método:.Revisão integrativa da literatura de artigos nacionais completos, por meio de quatro bases de dados. Foram selecionados sete artigos após a aplicação dos critérios de inclusão e exclusão. Da análise surgiram os temas sobre o legado de Florence e a ação do meio ambiente interno e externo sobre o poder vital. Resultados:.A maioria dos estudos focou o ambiente de inserção do indivíduo como fortalecer do poder vital e esta classificada como força interior. Conclusão:.Os profissionais de enfermagem ocupam um papel imprescindível na observação em manter o ambiente onde o paciente está inserido, favorecendo o fortalecimento do poder vital, destacando a presença do acompanhante como fortalecedor desse poder vital no processo de hospitalização.Descritores: Puerpério, Parto, Enfermagem.

Poder vital e o legado de florence nightingale no processo saúdedoença: revisão integrativa The vital power and the legacy of florence nightingale in the health-disease process: integrative review

Objetivo:.Caracterizar as evidências científicas nacionais dos últimos dez anos sobre Florence Nightingale e o poder vital e sua aplicabilidade na enfermagem, além de identificar os conceitos sobre poder vital. Método:.Revisão integrativa da literatura de artigos nacionais completos, por meio de quatro bases de dados. Foram selecionados sete artigos após a aplicação dos critérios de inclusão e exclusão. Da análise surgiram os temas sobre o legado de Florence e a ação do meio ambiente interno e externo sobre o poder vital. Resultados:.A maioria dos estudos focou o ambiente de inserção do indivíduo como fortalecer do poder vital e esta classificada como força interior. Conclusão:.Os profissionais de enfermagem ocupam um papel imprescindível na observação em manter o ambiente onde o paciente está inserido, favorecendo o fortalecimento do poder vital, destacando a presença do acompanhante como fortalecedor desse poder vital no processo de hospitalização.Descritores: Puerpério, Parto, Enfermagem.

Dysbiotic oral microbiota contributes to alveolar bone loss associated with obesity in mice

Periodontal diseases (PD) are inflammatory conditions that affect the teeth supporting tissues. Increased body fat tissues may contribute to activation of the systemic inflammatory response, leading to comorbidities. Some studies have shown that individuals with obesity present higher incidence of PD than eutrophics. Objective: To investigate the impact of obesity on periodontal tissues and oral microbiota in mice. Methodology: Two obesity mice models were performed, one using 12 weeks of the dietary protocol with a high-fat (HF) diet in C57BL/6 mice and the other using leptin receptor-deficient mice (db/db-/-), which became spontaneously obese. After euthanasia, a DNA-DNA hybridization technique was employed to evaluate the microbiota composition and topical application of chlorhexidine (CHX), an antiseptic, was used to investigate the impact of the oral microbiota on the alveolar bone regarding obesity. Results: Increased adipose tissue may induce alveolar bone loss, neutrophil recruitment, and changes in the oral biofilm, similar to that observed in an experimental model of PD. Topical application of CHX impaired bone changes. Conclusion: Obesity may induce changes in the oral microbiota and neutrophil recruitment, which are associated with alveolar bone loss

Structural basis of nSH2 regulation and lipid binding in PI3Kα

We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP2, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP2 molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant

The transcription factor PAX8 promotes angiogenesis in ovarian cancer through interaction with SOX17.

PAX8 is a master transcription factor that is essential during embryogenesis and promotes neoplastic growth. It is expressed by the secretory cells lining the female reproductive tract, and its deletion during development results in atresia of reproductive tract organs. Nearly all ovarian carcinomas express PAX8, and its knockdown results in apoptosis of ovarian cancer cells. To explore the role of PAX8 in these tissues, we purified the PAX8 protein complex from nonmalignant fallopian tube cells and high-grade serous ovarian carcinoma cell lines. We found that PAX8 was a member of a large chromatin remodeling complex and preferentially interacted with SOX17, another developmental transcription factor. Depleting either PAX8 or SOX17 from cancer cells altered the expression of factors involved in angiogenesis and functionally disrupted tubule and capillary formation in cell culture and mouse models. PAX8 and SOX17 in ovarian cancer cells promoted the secretion of angiogenic factors by suppressing the expression of SERPINE1, which encodes a proteinase inhibitor with antiangiogenic effects. The findings reveal a non-cell-autonomous function of these transcription factors in regulating angiogenesis in ovarian cancer

Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purposes

Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5–40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the discovery and identification of a great number of novel toxins. The brown spider phospholipase-D family is undoubtedly the most investigated and characterized, although other important toxins, such as low molecular mass insecticidal peptides, metalloproteases and hyaluronidases have also been identified and featured in literature. The molecular pathways of the action of these toxins have been reported and brought new insights in the field of biotechnology. Herein, we shall see how recent reports describing discoveries in the area of brown spider venom have expanded biotechnological uses of molecules identified in these venoms, with special emphasis on the construction of a cDNA library for venom glands, transcriptome analysis, proteomic projects, recombinant expression of different proteic toxins, and finally structural descriptions based on crystallography of toxins

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt