ERIC KINNOSUKE MARTINS UEDA
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Resumo IPEN-doc 11435 S179D prolactin (PRL) primarity uses the extrinsic pathway and MAPkinase signaling to induced apoptosis in human endothelial cells2006 - UEDA, E.K.; SOARES, C.R.J.; OLIVEIRA, T.L.; HELLER, S.R.; LO, H.L.; WALKER, A.M.; BARTOLINI, P.Resumo IPEN-doc 11434 The use of bacterioophage lambda Psub(L) as a constitutive promoter2006 - HELLER, S.R.; OLIVEIRA, T.L.; UEDA, E.K.; SOUZA, J.M.; OZAKI, N.A.; RIBELA, M.T.C.P.; BARTOLINI, P.; SOARES, C.R.S.Resumo IPEN-doc 11502 Point mutation of serine 179 in the human prolactin (PRL) affects recombinant protein expression, folding and secretion, abolishes PRL nickel (II)-binding and increases heparin binding capacities2006 - UEDA, ERIC; SOARES, CARLOS; WALKER, AMEAE; BARTOLINI, P.Resumo IPEN-doc 11501 Human prolactin (hPRL) and growth hormone (hGH) distinct behavior under bacteriophage lamdda Psub(L) promoter control2006 - SOARES, CARLOS R.J.; UEDA, ERIC K.M.; OLIVEIRA, TAIS L.; HELLER, SUSANA R.; BARTOLINI, PAOLOResumo IPEN-doc 11011 Reversed-phase high performance liquid chromatography (RP-HPLC) analysis and hydrophobicity studies of recombinant human pituitary hormones synthesized in E. coli and CHO cells2005 - RIBELA, M.T.C.P.; CARVALHO, C.M.; HELLER, S.R.; LOUREIRO, R.F.; OLIVEIRA, J.E.; OZAKI, N.A.; PERONI, C.N.; SOARES, C.R.J.; SOUZA, J.M.; UEDA, E.K.; BARTOLINI, P.The synthesis and laboratory production of human growth hormone (hGH) and prolactin (hPRL) have been carried out in genetically modified E. coli, while those of thyroid stimulating hormone (hTSH) and of two analogs antagonists of hPRL (G129RhPRL and S179D-hPRL) in stably transfected CHO cells. Human follicle stimulating hormone (hFSH) and luteinizing hormone (hLH) have not been synthesized yet in our laboratory but their HPLC analytical methodologies are under development. For the purpose of studying and improving synthesis and bioreaction yields and, at the same time, planning and following all subsequent purification steps, novel RP-HPLC methods have been set up for each hormone. For hGH and hPRL, isocratic RP-HPLC methods have been developed that can qualitatively and quantitatively analyze the two hormones directly in osmotic shock fluids, already during fermentation. For hTSH, hFSH and hLH, RP-HPLC gradient elutions have been set for the analysis of these hormones in their purified form and in CHO conditioned medium. For these three glycoproteins hydrophobicities have been compared and the following order established: hLH>hTSH>hFSH. An analogous hydrophobicity index has been also determined for hPRL and its analogs, being G129R-hPRL>hPRL>S179D-hPRL. Still concerning hFSH, for the first time it has been possible to optimize RP-HPLC elution conditions that are able to preserve its undissociated heterodimeric structure. Thanks to this tool it was thus possible to carry out a comparative study on pituitary, urinary and CHO-derived hFSH preparations, revealing differences that are probably due to the carbohydrate moiety, as already observed for hTSH. Classical highperformance size exclusion chromatography (HPSEC) together with MALDI-TOF-MS analysis was also employed along with these studies, to complement physico-chemical characterization of our proteins of interest.Resumo IPEN-doc 06995 A reversed-phase high -performance liquid chromatography (RP-HPLC) method for the determination of human prolactin (hPRL) in osmotic shock fluids and in purified preparations2000 - CAMARGO, I.M.C.; MORGANTI, L.; SOARES, C.R.J.; VIANNA, E.K.G.; UEDA, E.K.M.; OLIVEIRA, J.E.; LEGOUX, R.; FERRARA, P.; BARTOLINI, P.Resumo IPEN-doc 07723 Analysis of recombinant humam prolactin (REC-hPRL) directly in E.coli osmotic shock fluids1999 - CAMARGO, I.M.C.; SOARES, C.R.J.; VIANNA, E.K.G.; UEDA, E.K.M.; OLIVEIRA, J.E.; MORGANTI, L.; BARTOLINI, P.Artigo IPEN-doc 14063 A molecular mimic of phosphorylated prolactin (S179D PRL) secreted by eukaryotic cells has a conformation with an increased positive surface charge compared to that of unmodified prolactin2009 - UEDA, ERIC K.M.; SOARES, CARLOS R.J.; BARTOLINI, PAOLO; GUZMAN, ARIEL de; LORENSON, MARY Y.; WALKER, AMEAE M.Artigo IPEN-doc 11417 Physico-chemical and biological characterizations of two human prolactin analogs exhibiting controversial bioactivity, synthesized in chinese hamster ovary (CHO) cells2006 - SOARES, C.R.J.; GLEZER, A.; OKAZAKI, K.; UEDA, E.K.M.; HELLER, S.R.; WALKER, A.M.; GOFFIN, V.; BARTOLINI, P.The synthesis, puriWcation and characterization of G129R–hPRL and S179D–hPRL, the two better-studied antagonists of human prolactin (hPRL), is described. Both of these have been expressed for the Wrst time, in their authentic form, by a stable CHO cell line, at secretion levels of 7.7 and 4.3 g/106 cells/day, respectively. Previous studies had shown that these hPRL analogs, when produced in bacterial cytoplasm, consistently contained misfolded forms and multimers according to the speciWc denaturation, refolding and puriWcation conditions. These versions also have an N-terminal extra methionine. An extensive physico-chemical characterization was carried out after a practical two-step puriWcation process and included SDS–PAGE and Western blotting analysis, matrix-assisted laser-desorption ionization time-of-Xight mass spectral (MALDI–TOF–MS) analysis, high-performance size-exclusion chromatography (HPSEC) and reversed-phase high-performance liquid chromatography (RP–HPLC). This last technique revealed a considerable diVerence in hydrophobicity due to a single amino acid substitution, with S179D–hPRL less (tRR D 0.85 § 0.010) and G129R–hPRL more (tRR D 1.10§ 0.013) hydrophobic than hPRL, where tRR is the relative retention time. The biological characterization was based on further reWnement of a sensitive proliferation assay using the pro-B murine cell line (Ba/F3) transfected with the long form hPRL receptor cDNA such that the minimal detectable dose was 0.04 ng of hPRL/mL, the Ba/F3-LLP assay. On the basis of this assay, the relative residual agonistic activity of these two products, determined against a hPRL international standard in four independent assays, was 53 £ 10¡3 for S179D–hPRL and 70£ 10¡5 for G129R–hPRL. We believe that the present synthesis and characterization could be extremely helpful for studies of these two proteins, which have been reported to antagonize tumor growth-promoting eVects of hPRL in vivo in animal models of breast and prostate cancer.Artigo IPEN-doc 09785 Periplasmic expression of human growth hormone via plasmid vectors containing the lambdaPsub(L) promoter: use of HPLC for product quantification2003 - SOARES, C.R.J.; GOMIDE, F.I.C.; UEDA, E.K.M.; BARTOLINI, P.