RESUMEN
Previous in vivo studies have revealed that resins may generate a persistent inflammation of oral tissues and cell death as well. Apoptosis is an important regulated process that results in rapid cell death. This study tested the hypothesis that the comonomer triethyleneglycol-dimethacrylate (TEGDMA) causes apoptosis. The effects of TEGDMA on proliferation and apoptosis in primary oral fibroblasts were analyzed by light microscopy and flow cytometry (FACS; Annexin V-assay). TEGDMA at 5 and 7.5 mM inhibited proliferation after 24 hrs. No increased frequency of apoptosis or necrosis was observed with 1 mM or 2.5 mM TEGDMA after 24 hrs. Apoptosis and Annexin V-positive cells were observed with 5 mM and 7.5 mM TEGDMA by light microscopy after 24 hrs. A dramatic increase in apoptotic cells was detected by FACS after 24 hrs with 7.5 mM TEGDMA. Thus, TEGDMA was cytotoxic and "apoptotic" in a dose- and time-dependent manner.
Asunto(s)
Apoptosis/efectos de los fármacos , Resinas Compuestas/toxicidad , Materiales Dentales/toxicidad , Fibroblastos/efectos de los fármacos , Encía/efectos de los fármacos , Polietilenglicoles/toxicidad , Ácidos Polimetacrílicos/toxicidad , Análisis de Varianza , Anexina A5 , Técnicas de Cultivo de Célula , División Celular/efectos de los fármacos , Inhibidores Enzimáticos , Citometría de Flujo , Encía/citología , Humanos , Necrosis , Factores de TiempoRESUMEN
Isovaleryl-CoA dehydrogenase (IVD, EC ) catalyzes the third step in the catabolism of leucine in mammals. Deficiency of this enzyme leads to the clinical disorder isovaleric acidemia. IVD has been purified and characterized from human and rat liver, and the x-ray crystallographic structure of purified recombinant human IVD has been reported. Nothing is known about IVD activity in plants, although cDNA clones from Arabidopsis thaliana and partial sequences from Gossypium hirsutum and Oryza sativa have been identified as putative IVDs based on sequence homology and immuno cross-reactivity. In this report we describe the identification and characterization of an IVD from pea, purification of the enzyme using a novel and rapid auxin affinity chromatography matrix, and cloning of the corresponding gene. At the amino acid level, pea IVD is 60% similar to human and rat IVD. The specific activity and abundance of plant IVD was found to be significantly lower than for its human counterpart and exhibits developmental regulation. Substrate specificity of the plant enzyme is similar to the human IVD, and it cross-reacts to anti-human IVD antibodies. Molecular modeling of the pea enzyme based on the structure of human IVD indicates a high degree of structural similarity among these enzymes. Glu-244, shown to function as the catalytic base in human IVD along with most of the amino acids that make up the acyl CoA binding pocket, is conserved in pea IVD. The genomic structure of the plant IVD gene consists of 13 exons and 12 introns, spanning approximately 4 kilobases, and the predicted RNA splicing sites exhibit the extended consensus sequence described for other plant genes.