Navegando por Palavras-chave "CHO cells"
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- ItemSomente MetadadadosAliphatic amino acids in helix VI of the AT(1) receptor play a relevant role in agonist binding and activity(Elsevier B.V., 2002-06-15) Corrêa, Silvana Aparecida Alves [UNIFESP]; Zalcberg, H.; Han, S. W.; Oliveira, L.; Costa-Neto, C. M.; Paiva, ACM; Shimuta, S. I.; Universidade Federal de São Paulo (UNIFESP)Angiotensin II (AII) AT(1) receptor mutants with replacements of aliphatic amino acids in the distal region of helix VI and the adjoining region of the third extracellular loop (EC-3) were expressed in Chinese hamster ovary (CHO) cells to determine their role in ligand binding and activation. the triple mutant [L262D, L265D, L268D]AT(1) (L3D) showed a marked reduction in affinity for All and for non-peptide (losartan) and peptide ([Sar(1)Leu(8)]All) antagonists; in functional assays using inositol phosphate (IP) accumulation, the relative potency and the maximum effect of All were reduced in L3D. Replacement of Leu(268) (in EC-3) and Leu(262) (in the transmembrane domain) by aspartyl residues did not cause significant changes in the receptor's affinity for the ligands and in IP production. in contrast, the point mutation L265D, at helix VI, markedly decreased affinity and ability to stimulate phosphatidylinositol turnover. Molecular modeling of the AT(1) receptor based on a recent crystal structure of rhodopsin, suggests that the side chain of Leu(265) but not that of Leu(262) is facing a cleft between helices V and VI and interacts with the lipid bilayer, thus helping to stabilize the receptor structure near the Lys(199) residue of helix V in the agonist binding site which is necessary for full activity. (C) 2002 Elsevier Science B.V. All rights reserved.
- ItemSomente MetadadadosDetermination of angiotensin I-converting enzyme activity in cell culture using fluorescence resonance energy transfer peptides(Elsevier B.V., 2007-04-15) Sabatini, R. A.; Bersanetti, P. A.; Farias, S. L.; Juliano, L.; Juliano, M. A.; Casarini, D. E.; Carmona, A. K.; Paiva, A. C. M.; Pesquero, J. B.; Universidade Federal de São Paulo (UNIFESP)An assay using fluorescence resonance energy transfer peptides was developed to assess angiotensin I-converting enzyme (ACE) activity directly on the membrane of transfected Chinese hamster ovary cells (CHO) stably expressing the full-length somatic form of the enzyme. the advantage of the new method is the possibility of using selective substrates for the two active sites of the enzyme, namely Abz-FRK(Dnp)P-OH for somatic ACE, Abz-SDK(Dnp)P-OH for the N domain, and Abz-LFK(Dnp)-OH for the C domain. Hydrolysis of a peptide bond between the donor/acceptor pair (Abz/Dnp) generates detectable fluorescence, allowing quantitative measurement of the enzymatic activity. the kinetic parameter K-m for the hydrolysis of the three substrates by ACE in this system was also determined and the values are comparable to those obtained using the purified enzyme in solution. the specificity of the activity was demonstrated by the complete inhibition of the hydrolysis by the ACE inhibitor lisinopril. Therefore, the results presented in this work show for the first time that determination of ACE activity directly on the surface of intact CHO cells is feasible and that the method is reliable and sensitive. in conclusion. we describe a methodology that may represent a new tool for the assessment of ACE activity which will open the possibility to study protein interactions in cells in culture. (c) 2007 Elsevier Inc. All rights reserved.
- ItemAcesso aberto (Open Access)A fucan from the brown seaweed Spatoglossum schröederi inhibits Chinese hamster ovary cell adhesion to several extracellular matrix proteins(Associação Brasileira de Divulgação Científica, 2001-05-01) Rocha, Hugo Alexandre Oliveira [UNIFESP]; Franco, Celia Regina Cavichlolo [UNIFESP]; Trindade, Edvaldo da Silva [UNIFESP]; Carvalho, L.c.m.; Veiga, Silvio Sanches [UNIFESP]; Leite, Edda Lisboa [UNIFESP]; Dietrich, Carl Peter [UNIFESP]; Nader, Helena Bonciani [UNIFESP]; Universidade Federal do Rio Grande do Norte Departamento de Bioquímica; Universidade Federal do Paraná Departamento de Biologia Celular; Universidade Federal de São Paulo (UNIFESP)Fucans, a family of sulfated polysaccharides present in brown seaweed, have several biological activities. Their use as drugs would offer the advantage of no potential risk of contamination with viruses or particles such as prions. A fucan prepared from Spatoglossum schröederi was tested as a possible inhibitor of cell-matrix interactions using wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). The effect of this polymer on adhesion properties with specific extracellular matrix components was studied using several matrix proteins as substrates for cell attachment. Treatment with the polymer inhibited the adhesion of fibronectin to both CHO-K1 (2 x 10(5))()and CHO-745 (2 x 10(5) and 5 x 10(5)) cells. No effect was detected with laminin, using the two cell types. On the other hand, adhesion to vitronectin was inhibited in CHO-K1 cells and adhesion to type I collagen was inhibited in CHO-745 cells. In spite of this inhibition, the fucan did not affect either cell proliferation or cell cycle. These results demonstrate that this polymer is a new anti-adhesive compound with potential pharmacological applications.
- ItemSomente MetadadadosFucan inhibits Chinese hamster ovary cell (CHO) adhesion to fibronectin by binding to the extracellular matrix(Georg Thieme Verlag Kg, 2005-07-01) Rocha, Hugo Alexandre de Oliveira [UNIFESP]; Franco, Celia Regina Cavichiolo; Trindade, Edvaldo da Silva; Veiga, Silvio Sanches; Leite, Edda Lisboa; Nader, Helena Bonciani [UNIFESP]; Dietrich, Carl Peter [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Fed Rio Grande Norte; Univ Fed ParanaIn recent years, sulfated fucans have emerged as an important class of natural biopolymers. in this study, the anti-adhesive activity of a fucan from the brown seaweed Spatoglossum schroederi was analyzed using tumorigenic cells: wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). Fibronectin (FN) was used as substrate for cell attachment. for both cell types, this fucan has shown a dose-dependent anti-adhesive effect, reaching saturation at around 400 mu g/mL. This effect was abolished by desulfation of the fucan. in addition, this polymer exhibited the highest inhibitory effect in comparison to other sulfated polysaccharides. the fucan was biotinylated and used as a probe to identify its action sites. Biotinylated fucan was detected in the extracellular matrix environment by confocal microscopy and flow cytometric analysis, but not at the cell surface. the results suggest that the fucan shows anti-adhesive activity by binding directly to FN, and blocking FN sites that are recognized by cell surface ligands, possibly the integrin family.
- ItemAcesso aberto (Open Access)Heparan sulfate and control of cell division: adhesion and proliferation of mutant CHO-745 cells lacking xylosyl transferase(Associação Brasileira de Divulgação Científica, 2001-08-01) Franco, Celia Regina Cavichlolo [UNIFESP]; Rocha, Hugo Alexandre Oliveira [UNIFESP]; Trindade, Edvaldo da Silva [UNIFESP]; Santos, Isabel Anunciacao Neves dos [UNIFESP]; Leite, Edda Lisboa [UNIFESP]; Veiga, Silvio Sanches [UNIFESP]; Nader, Helena Bonciani [UNIFESP]; Dietrich, Carl Peter [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade Federal do Rio Grande do Norte Departamento de Bioquímica; Universidade Federal do Paraná Departamento de Biologia CelularWe have examined the role of cell surface glycosaminoglycans in cell division: adhesion and proliferation of Chinese hamster ovary (CHO) cells. We used both wild-type (CHO-K1) cells and a mutant (CHO-745) which is deficient in the synthesis of proteoglycans due to lack of activity of xylosyl transferase. Using different amounts of wild-type and mutant cells, little adhesion was observed in the presence of laminin and type I collagen. However, when fibronectin or vitronectin was used as substrate, there was an enhancement in the adhesion of wild-type and mutant cells. Only CHO-K1 cells showed a time-dependent adhesion on type IV collagen. These results suggest that the two cell lines present different adhesive profiles. Several lines of experimental evidence suggest that heparan sulfate proteoglycans play a role in cell adhesion as positive modulators of cell proliferation and as key participants in the process of cell division. Proliferation and cell cycle assays clearly demonstrate that a decrease in the amount of glycosaminoglycans does not inhibit the proliferation of mutant CHO-745 cells when compared to the wild type CHO-K1, in agreement with the findings that both CHO-K1 and CHO-745 cells take 8 h to enter the S phase.
- ItemSomente MetadadadosLycopene activity against chemically induced DNA damage in Chinese hamster ovary cells(Elsevier B.V., 2007-08-01) Scolastici, Clarissa; Lima, Rodrigo Otavio Alves de; Barbisan, Luis Fernando; Ferreira, Ana Lucia dos Anjos; Ribeiro, Daniel Araki [UNIFESP]; Salvadori, Daisy Maria Favero; São Paulo State Univ; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)Lycopene is a natural pigment synthesized by plants and microorganisms, and it is mainly found in tomatoes. It is an acyclic isomer of P-carotene and one of the most potent antioxidants. Several studies have demonstrated the ability of lycopene to prevent chemically induced DNA damage; however, the mechanisms involved are still not clear. in the present study, we investigated the antigenotoxic/antimutagenic effects of lycopene in Chinese Hamster Ovary Cells (CHO) treated with hydrogen peroxide, methylmethanesulphonate (MMS), or 4-nitroquinoline-1-oxide (4-NQO). Lycopene (97%), at final concentrations of 10, 25, and 50 M, was tested under three different protocols: before, simultaneously, and after the treatment with the mutagens. Comet and cytokinesis-block micronucleus assays were used to evaluate the level of DNA damage. Data showed that lycopene reduced the frequency of micronucleated cells induced by the three mutagens. However, this chemopreventive activity was dependent on the concentrations and treatment schedules used. Similar results were observed in the comet assay, although some enhancements of primary DNA damage were detected when the carotenoid was administered after the mutagens. in conclusion, our findings confirmed the chemopreventive activity of lycopene, and showed that this effect occurs under different mechanisms. (c) 2007 Elsevier B.V. All rights reserved.