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  • 104 XXIII Meeting of the SBPZ - POSTER

    Bioquimica - Biochemistry

    BQ01 - Acyl-CoA-binding protein expression profile in Rhodnius prolixus

    Alves, M. (Instituto de Bioqúımica Médica, Universidade

    Federal do Rio de Janeiro); Majerowicz, D. (Instituto de

    Bioqúımica Médica, Universidade Federal do Rio de

    Janeiro); Grillo, L. A. M. (Instituto de Bioqúımica

    Médica, Universidade Federal do Rio de Janeiro); Almeida,

    C. B. (Instituto de Bioqúımica Médica, Universidade

    Federal do Rio de Janeiro); Paiva-Silva, G. O. (Instituto

    de Bioqúımica Médica, Universidade Federal do Rio de

    Janeiro); Gondim, K. C. (Instituto de Bioqúımica Médica,

    Universidade Federal do Rio de Janeiro)

    Acyl-CoA esters have many functions in cell metabolism,

    such as energy production and cell signaling. Acyl-CoA-

    binding protein (ACBP), that is a highly conserved 10 kDa

    intracellular protein, binds long straight-chain acyl-CoA es-

    ters with very high affinity and protects acyl-CoA esters

    from hydrolysis. Prediction of three-dimensional structure of

    ACBP from Rhodnius prolixus using CPHmodels 2.0 showed

    that it has four helices in up-down-down-up conformation,

    as it was described for other ACBPs. Using RT-PCR,

    ACBP gene expression was detected in anterior and posterior

    midgut, fat body, ovary, flight muscle and salivary glands,

    and it was highest (5̃ fold) in posterior midgut. Expression

    analysis of ACBP gene in the midgut by Real-Time PCR

    showed a great increase after blood meal, and it was very

    high (7̃-fold increase) on the first day after feeding and de-

    creased after it. In the fat body, expression of ACBP gene

    was highest on the eleventh day after feeding and, in the

    other days, expression was relatively constant. In order to

    investigate the mechanisms involved in the control of ACBP

    gene expression, the possible role of ecdysone was analyzed.

    Injection of 2 ng of 20-hydroxyecdysone into unfed females in-

    hibited the expression of ACBP gene in approximately 30%.

    When females were fed immediately after the injection of 20-

    hydroxyecdysone, the ACBP gene expression was decreased

    by 25%. These results suggest that this hormone may be in-

    volved in the control of ACBP expression, and this is under

    investigation. Supported by CNPq, PIBIC/UFRJ, Faperj

    and CAPES.

    BQ02 - Effect of Ketoconazole on growth, morphology, infectivity and in the pattern

    expression of flagellar glycoproteins in Leishmania (Viannia) braziliensis

    Yoneyama, K. A. G. (Universidade Federal de São

    Paulo); Takahashi, H. K. (Universidade Federal de São

    Paulo); Straus, A. H. (Universidade Federal de São

    Paulo)

    Cholesterol plays an important role in the properties of

    cell membranes in mammalian cells. Leishmania and Try-

    panosoma synthesize ergosterol using a similar biosynthetic

    pathway that pathogenic fungi. Previous work carried out in

    our lab showed that ergosterol from Leishmania (Viannia)

    braziliensis presents an essential role on lipid rafts mainte-

    nance on plasma membrane. In order to better character-

    ize the role of ergosterol on Leishmania, the effect of differ-

    ent concentrations of ketoconazole (an inhibitor of ergosterol

    synthesis) on L. (V.) braziliensis was analyzed. Low keto-

    conazole concentration as 18 nM caused significant decrease

    of parasite growth rate (about 90%) and macrophage infec-

    tivity (84%). Also drastic changes on parasite morphology

    were observed such as rounded body, presence of one or more

    truncated flagella and two or more nucleus, suggesting that

    ketoconazole might inhibit of cytokinesis process. By indirect

    immunofluorescence with monoclonal antibody (mAb) SST-

    3, which recognizes specifically a triplet glycoprotein (200

    to 160 kDa), expressed exclusively in parasite flagellum, it

    was observed that ketoconazole treatment apparently did not

    alter the distribution of this flagellar glycoprotein on trun-

    cated flagellum. On the other hand, by Western blotting it

    was observed a drastic alteration in band profile of flagellar

    glycoproteins recognized by mAb SST-3. Other biochemical

    studies are under investigation in order to better understand

    the role of ergosterol in flagella organization. The present

    data suggest that ketoconazole is a promising drug against

    L. (V.) braziliensis. Supported by FAPESP and CNPq.

    BQ03 - Phosphoproteome during nutritional stress in Trypanosoma cruzi

    Chung, J. (UNIVERSIDADE FEDERAL DE SÃO

    PAULO); Ferreira, L.R.P. (UNIVERSIDADE

    FEDERAL DE SÃO PAULO); Ramos, T.

    (UNIVERSIDADE FEDERAL DE SÃO PAULO);

    Schenkman, S. (UNIVERSIDADE FEDERAL DE SÃO

    PAULO)

    Phosphorylation is one of the most common post-

    translational modifications of proteins and is a key event in

    intracellular signaling. Therefore, phosphorylation combined

    with proteomic analysis has been used as important tool to

    elucidate many biological processes. In trypanosomatids a

    large number of protein kinases were predicted by genome

    sequence, suggesting that phosphorylation must be partic-

    ularly important in these organisms. For example, trans-

    formation of Trypanosoma cruzi epimastigotes into meta-

    cyclic trypomastigotes is induced by factors like, nutritional

    stress, differences in temperature and pH variation. Very

    little is known about the signals that induce the process of

    metacyclogenesis. In the present work compared the phos-

    phorylation profiles between epimastigotes that suffered nu-

    tritional stress (incubation at different time points in the

    chemically defined TAU medium) and control parasites (sta-

    tionary phase epimastigotes). 2D gels from were visual-

    ized by specific stains for phosphorylated proteins and to-

    tal proteins (Pro-Q Diamond and Sypro Ruby respectively

    (Molecular Probes). Phosphorylated proteins were also iso-

    lated from T. cruzi total protein extracts after enrichment

  • XXIII Meeting of the SBPZ - POSTER 105

    by affinity chromatography and identified by mass spectrom-

    etry analysis. Among the identified phosphorylated proteins,

    some have their function correlated with cellular nutritional

    stress response, growth and metabolism. Listing two of

    them are Rad23 (UV excision repair RAD23-like protein)

    and cystathionine beta-synthase. Supported by FAPESP

    and FINEP

    BQ04 - PROTEOMIC ANALYSIS OF BENZNIDAZOLE SUSCEPTIBLE AND RESISTENT TRYPANOSOMA CRUZI

    TRYPOMASTIGOTES

    Lopes dos Santos, S. (Instituto René Rachou);

    Silva-Pereira, R.A. (Instituto René Rachou); Romanha,

    A.J. (Instituto René Rachou)

    INTRODUCTION: Chagas‘ disease is a major public health

    problem in Central and South America, where 16-20 mil-

    lion people are infected with Trypanosoma cruzi. Nitrohete-

    rocyclic drugs, benznidazole (Bz) and nifurtimox (Nf), are

    currently used to treat Chagas‘ disease. Both drugs have

    frequent side-effects, very low anti-parasitic activity in long-

    term chronic forms of the disease and variable efficacy ac-

    cording to the geographical area. The existence of strains

    naturally resistant and susceptible to Bz and Nf drugs was

    previously described. Efforts are necessary to provide a bet-

    ter understanding of T. cruzi drug resistance mechanisms,

    which may lead to the identification of new drug targets

    and new chemotherapeutic tools. These efforts must be di-

    rected to T. cruzi human host forms, trypomastigotes and

    amastigotes. Furthermore, proteomic analysis is particularly

    important because the regulation of gene expression in T.

    cruzi occurs by post-transcriptional mechanisms. OBJEC-

    TIVE: The aim of this work is to identify trypomastigote pro-

    teins potentially involved in the Bz resistance mechanisms.

    METODOLOGY: Trypomastigote forms from two popula-

    tions selected in vivo, Bz resistant (BZR) and Bz susceptible

    (BZS) (Murta e Romanha, 1998), were maintained in Vero

    cell monolayers and collected. The resistance/susceptibility

    phenotype was determined by an in vitro biological assay,

    in which trypomastigotes released from cells under the Bz

    activity were counted. RESULTS: Preliminary results have

    shown that BZR population is 1.75-fold more resistant to Bz

    than BZS population. Comparative proteomic analysis be-

    tween BZR and BZS trypomastigote forms is being carried

    out by bi-dimensional electrophoresis and the identification

    of differentially expressed proteins will be performed by mass

    spectrometry and similarity searching against the T. cruzi

    protein databases. Financial support: PDTIS/FIOCRUZ -

    IRR - FAPEMIG

    BQ05 - Proteases activities in recently field-isolated Trypanosoma cruzi strains: identification, expression characterization

    among I and II groups strains.

    Fampa, P. (Fundação Oswaldo Cruz/ RJ); Santos, A.L.S.

    (Universidade Federal do Rio de Janeiro); Ramirez, M.I.

    (Fundação Oswaldo Cruz/ RJ)

    Trypanosoma cruzi presents a heteroxenous life cycle includ-