RESUMO
The effects of verapamil modulating collagen biosynthesis have prompted us to study the role of this drug in cultured fibroblasts. In this article, we describe the effects of verapamil on fibroblast behaviour, with special emphasis to phenotypic modifications, reorganisation of actin filaments and secretion of MMP1. Human dermal fibroblasts treated with 50-µM verapamil changed their normal spindle-shaped morphology to stellate. Treated cells showed discrete reorganisation of actin filaments, as revealed by fluorescein isothiocyanate (FITC)-phalloidin staining and confocal microscopy. We hypothesised that these effects would be associated to lower levels of cytosolic Ca(2+). Indeed, short time loading with calcium green confirmed that verapamil-treated fibroblasts exhibited lower intracellular calcium levels compared to controls. We also observed that verapamil increases the secretion of MMP1 in cultured fibroblasts, as demonstrated by zymography, specific substrate assays and immunoblot. The morphological alterations induced by verapamil are neither cytotoxic nor associated with other dramatic cytoskeleton alterations. Thus we may conclude that this drug enhances collagenase secretion and does not disrupt the major tracks necessary to deliver these enzymes in the extracellular space. The present results suggested that verapamil could be used at physiological levels to enhance collagen I breakdown, and may be considered a potential candidate for intralesional therapy of wound healing and fibrocontractive diseases.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Colagenases/metabolismo , Citoesqueleto/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Verapamil/farmacologia , Citoesqueleto de Actina/patologia , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Metaloproteinase 1 da Matriz/biossínteseRESUMO
In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form large and intriguingly organized complexes. A novel nucleolar protein, Nop53p, was isolated by using Nop17p as bait in the yeast two-hybrid system. Nop53p also interacts with a second nucleolar protein, Nip7p. A carbon source-conditional strain with the NOP53 coding sequence under the control of the GAL1 promoter did not grow in glucose-containing medium, showing the phenotype of an essential gene. Under nonpermissive conditions, the conditional mutant strain showed rRNA biosynthesis defects, leading to an accumulation of the 27S and 7S pre-rRNAs and depletion of the mature 25S and 5.8S mature rRNAs. Nop53p did not interact with any of the exosome subunits in the yeast two-hybrid system, but its depletion affects the exosome function. In pull-down assays, protein A-tagged Nop53p coprecipitated the 27S and 7S pre-rRNAs, and His-Nop53p also bound directly 5.8S rRNA in vitro, which is consistent with a role for Nop53p in pre-rRNA processing.
Assuntos
Proteínas Nucleares/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Genes Fúngicos , Humanos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Ribossômico 5,8S/genética , RNA Ribossômico 5,8S/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Técnicas do Sistema de Duplo-HíbridoRESUMO
Saccharomyces cerevisiae was supported on chrysotile, crocidolite and lixiviated chrysotile. Samples of the supported cells and free cells were observed by confocal laser scanning microscopy. After 30 days, the free cells showed no viability when stored at 30 degrees C, and a viability of 40% when stored at 4 degrees C. Supported cells stored at 30 degrees C were more viable than the free cells at early times, but showed no viability after 30 days. Samples stored at 4 degrees C showed that the adhered cells are more viable than the free cells, up to 30 days. Cells supported on chrysotile and lixiviated chrysotile had 80% viability, and on crocidolite 70% viability. Scanning electron microscopy showed that cells supported on lixiviated chrysotile are fully covered by the support, but crocidolite fibers adhere less, since they are stiffer. Fermentation experiments performed after 3 years storage showed that four from the five lixiviated chrysotile samples and one of the three crocidolite samples were active. In all cases, a delay time for the onset of fermentation was observed indicating a state of latency.