Echinococcus granulosus: Cloning and Functional in Vitro Characterization of an Actin Filament Fragmenting Protein

Cortez-Herrera, Elizabeth; Yamamoto, Ritsu; Rodrigues, Jaqueline J.S.; Farias, Sandra Estrazulas; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

doi:10.1006/expr.2001.4605

Cited by 15 publications

(19 citation statements)

References 40 publications

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“…Based on these results, the mean rPTPgCD specific activity was calculated to be 9 nmol/min/lg suggesting that after purification and thrombin treatment rPTPgCD maintains its native structure and remains intact, with no signs of proteolytic degradation which could occur during the His 6 -tag cleavage procedure. The experimentally determined rPTPgCD specific activity is in the same range as the previously reported activities for PTPl [27], PTPa [28] and for commercial preparations of recombinant PTP-B1 [29] and SHP2 [30]. …”

supporting

confidence: 77%

“…2, shaded). As expected from the building procedure, the model resembles the N-terminal half of the 1D0N structure (domains G1-G3) and suggests that EgAFFP is composed of three independent domains (E1, Phe 49 (30). Each domain adopts a similar geometric fold composed of a core of mixed b-sheet comprising five or six strands sandwiched between a long helix (helix 1) running roughly parallel to the strands in the sheet, and a short helix (helix 2) running approximately perpendicular to the strands.…”

Section: Egaffp Modelingsupporting

confidence: 60%

“…The recombinant EgAFFP protein is recognized by sera of ;69% of human hydatid disease patients (31) and, in vitro, was able to induce actin polymerization and sever actin filaments, confirming that it belongs to the gelsolin superfamily (30). According to sequence analysis, EgAFFP presents three repeated domains and is similar (36% identity) to the gelsolin NH 2 -terminal half (G1-G3).…”

Section: Hre -9-cis-ra + 9-cis-ramentioning

confidence: 92%

“…Our laboratory has previously cloned and functionally characterized a 42 kDa actin filament-fragmenting protein from E. granulosus (EgAFFP) (30). The recombinant EgAFFP protein is recognized by sera of ;69% of human hydatid disease patients (31) and, in vitro, was able to induce actin polymerization and sever actin filaments, confirming that it belongs to the gelsolin superfamily (30).…”

Section: Hre -9-cis-ra + 9-cis-ramentioning

confidence: 98%

“…Sabe-se no entanto que hemocianinas possuem uma variação de estrutura quaternária apreciável, quando comparados seus estados oxi e deoxi (DECKER et al, 1996; ERKER et al, 2005 . (A) SDS-PAGE 15%: coluna PMMarcadores de massa molecular (67,00;45,00;30,00; 22,46; 13,37 kDa). Coluna Af -Após purificação a principal espécie presente é hRXR α∆AB.…”

Section: Reconhecimento De Elementos Responsivosunclassified

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Estudos de complexos macromoleculares por crio-microscopia eletrônica e técnicas biofísicas

Portugal¹

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A Nalva, aos meus pais e irmãos.iii AGRADECIMENTOS Agradeço a todos que estiveram ao meu lado, durante esta etapa da minha vida. Infelizmente, não tenho como citar os nomes de todos que me marcaram ao longo destes anos: amigos, familiares, professores, colegas de grupo e de estudos, funcionários, enfim, todos que de alguma maneira, passaram pela minha vida e deixaram sua marca. Mas posso citar alguns, e através deles agradecer aos outros que não estarei citando aqui.Primeiramente agradeço a Nalva, Companheira, sempre. Aos meus pais, Ceição, João Bosco, Adelina e Roberto, minhas irmãs, Dandan, Marina e Rê, meus irmãos Beto e Caique, e através deles, toda minha família, que mesmo longe, sempre me apoiou e esteve ao meu lado.Ao Igor por ter me orientado neste trabalho e me dado a oportunidade de conhecer diferentes técnicas e laboratórios. Ao Marin e Maurício meus segundos orientadores e amigos. A todos os amigos do grupo, mas em especial à Maria, "mãinha", que além de excelente profissional e professora, sempre foi muito, muito amiga. A todos os amigos aqui do grupo, Niko, Sandra Krau, Nádia, Lucas, João Renato, Fabão, Fred, Alessandro, Wanius, Marcel, Leandro Rizzi, Ribamar, Carol Gatinha, Carol Hoff, Carol Guzzi, Carol Figueira, Ney, Tavin, Suzana, Liliana, Lisandra, Thiago, Dani, Ana Letícia, Lívia, Daniel, Fernando Falvo, Bachega, Augusto, Gê, Mário Minime, Sonoda, Alécio, Caqui, Andressa, Bel, Napoleão, Luciana Ao Dr. Willem Tichelaar para a obtenção de micrografias das amostras de hemocianina e de receptores nucleares, estudados durante o primeiro estágio no CBEM. Ao Gijs, pelo apoio e ensinamentos dados na utilização dos microscópios e ao Tim, Cecília e Aris, pelas discussões e ensinamentos sobre o processamento dos dados. E a todos, todos mesmo, que de alguma maneira contribuíram para que eu esteja aqui hoje, meu muito obrigado! vi RESUMOEste trabalho apresenta o estudo e caracterização de dois complexos moleculares, hRXRα∆AB e hemocianina de Acanthoscurria gomesiana, através de técnicas estruturais e biofísicas. O uso da técnica de crio-microscopia eletrônica para o estudo da hemocianina de Acanthoscurria gomesiana, resultou em um modelo estrutural com resolução de 17 Å -pelo método de Fourier Shell Correlation com o critério de ½ bit. Neste limite de resolução, já é possível observar detalhes estruturais que o mostram como sendo compatível com outros modelos de hemocianinas. Com relação ao estudo de hRXRα∆AB, mostrou-se, através das técnicas de cromatografia analítica de exclusão por tamanho, eletroforese de gel de poliacrilamida e SAXS, que a proteína pode se apresentar no estado dimérico em solução, mesmo na ausência do seu ligante, 9-cis-RA. Também foi estudado a associação de hRXRα∆AB a elementos responsivos: DR1, DR4, F2 e PAL. Suas constantes de dissociação foram calculadas através da técnica de espectroscopia por anisotropia de fluorescência. Os resultados obtidos mostram maior afinidade por DR1 e DR4 e indicam uma origem entrópica para o processo de associação. vii ABSTRACTThis work pres...

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supporting

confidence: 77%

Section: Egaffp Modelingsupporting

confidence: 60%

Section: Hre -9-cis-ra + 9-cis-ramentioning

confidence: 92%

Section: Hre -9-cis-ra + 9-cis-ramentioning

confidence: 98%

Section: Reconhecimento De Elementos Responsivosunclassified

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Estudos de complexos macromoleculares por crio-microscopia eletrônica e técnicas biofísicas

Portugal¹

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Response ofAlvinella pompejanato variable oxygen stress: A proteomic approach

et al. 2010

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Alvinella pompejana is one of the most emblematic species of the animal communities colonizing the deep-sea hydrothermal vents of the East Pacific Rise. This extreme environment is characterized by high temporal variability of its physical-chemical parameters. Among these, the variation in concentration of available oxygen should lead to a specific physiological adaptive response of the animal. To evaluate the mechanisms of this response at a molecular level, a classical 2-DE-based proteomic approach has been implemented. After collection (Garrett-18S and -17S vent sites) animals were reconditioned in a high-pressure chamber pressurized at 260 bar and then the oxygen concentration was regulated to a constant value corresponding to hypoxia, normoxia or hyperoxia for 7 h. The soluble proteins from gills were then analyzed by 2-DE. The protein content of spots showing specific changes following oxygen concentration variation was determined based on comparison of MS/MS sequence data with a recently established A. pompejana ESTs database. Fifteen proteins, belonging mainly to three families, cytoskeleton protein, enzymes of energetic metabolism and heat shock proteins, have been identified as potentially involved in the response to the change in oxygen concentration. The significance of the relatively small set of proteins modulated by oxygen variations is discussed in the context of a potential universal cellular response to stress.

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Characterization of Various Recombinant Antigens from Echinococcus multilocularis for Use in the Immunodiagnosis

et al. 2005

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Using four clones isolated from Echinococcus multilocularis cDNA library with alveolar echinococcosis (AE) patient sera, various antigens were expressed as ThioHis tag-fused protein. Recombinant EmII/3 antigen was produced as the five fragments divided into the N-terminal (#5 and #5s), the central (#6 and #6s) and the C-terminal domain (#7). Immunoblot analysis revealed that the #7 showed significant reactivity whereas those of #5 and #5s were relatively low. The #6 and #6s also showed lower reactivity than that of #7, although the two minor bands of #6 reacted with every serum. These results suggested that an immunodominant region of EmII/3 locate within the C-terminal one third. The #8s recombinant antigen, Ser23-Glu176 of actin filament fragmenting protein (AFFP), apparently reacted with the AE patient sera, while the #1 antigen synthesized as a full-length antigen BI did not show such high reactivity. Thus, #7 and #8s antigens showed significant potential for use in immunodetection of AE. In addition, the specific antibodies against #7 and #8s reacted with specific antigens in crude extract of E. multilocularis cyst, indicating that these antigens retained antigenicity common to native EmII/3 and AFFP, respectively.

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Echinococcus granulosus: Cloning and Functional in Vitro Characterization of an Actin Filament Fragmenting Protein

Cited by 15 publications

References 40 publications

Estudos de complexos macromoleculares por crio-microscopia eletrônica e técnicas biofísicas

Estudos de complexos macromoleculares por crio-microscopia eletrônica e técnicas biofísicas

Response ofAlvinella pompejanato variable oxygen stress: A proteomic approach

Characterization of Various Recombinant Antigens from Echinococcus multilocularis for Use in the Immunodiagnosis

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