RESUMEN
The comet assay (single cell gel electrophoresis) is a cost-effective, sensitive, and simple technique that is traditionally used for analyzing and quantifying DNA damage in individual cells. The aim of this study was to determine whether the comet assay could be modified to detect changes in the levels of DNA methylation in single cells. We used the difference in methylation sensitivity of the isoschizomeric restriction endonucleases HpaII and MspI to demonstrate the feasibility of the comet assay to measure the global DNA methylation level of individual cells. The results were verified with the well-established cytosine extension assay. We were able to show variations in DNA methylation after treatment of cultured cells with 5-azacytidine and succinylacetone, an accumulating metabolite in human tyrosinemia type I.
Asunto(s)
Ensayo Cometa/métodos , Metilación de ADN , Azacitidina/química , Azacitidina/farmacología , Citosina/metabolismo , ADN-Citosina Metilasas/metabolismo , Desoxirribonucleasa HpaII/metabolismo , Células Hep G2 , Heptanoatos/química , Heptanoatos/farmacología , Humanos , Tirosinemias/metabolismoRESUMEN
Mucocutaneous fungal infections are typically found in patients who have no known immune defects. We describe a family in which four women who were affected by either recurrent vulvovaginal candidiasis or onychomycosis had the early-stop-codon mutation Tyr238X in the beta-glucan receptor dectin-1. The mutated form of dectin-1 was poorly expressed, did not mediate beta-glucan binding, and led to defective production of cytokines (interleukin-17, tumor necrosis factor, and interleukin-6) after stimulation with beta-glucan or Candida albicans. In contrast, fungal phagocytosis and fungal killing were normal in the patients, explaining why dectin-1 deficiency was not associated with invasive fungal infections and highlighting the specific role of dectin-1 in human mucosal antifungal defense.
Asunto(s)
Candidiasis/genética , Codón sin Sentido , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Onicomicosis/genética , Animales , Candida albicans/inmunología , Candidiasis/inmunología , Candidiasis Mucocutánea Crónica/genética , Candidiasis Vulvovaginal/genética , Citocinas/biosíntesis , Femenino , Predisposición Genética a la Enfermedad , Humanos , Lectinas Tipo C , Masculino , Mamíferos/genética , Proteínas de la Membrana/inmunología , Proteínas del Tejido Nervioso/inmunología , LinajeRESUMEN
The antioxidant properties of the fruit of the Rosa roxburghii (RR) plant have been associated with several putative health promoting effects. The possible cytotoxic, mutagenic/antimutagenic and genotoxic effects of RR fruit extract were investigated. The effect on antioxidant status and protection against induced oxidative stress were also investigated using primary rat hepatocytes. A RR fruit extract containing 45 g/L total ascorbic acid and 65 g/L total polyphenols was used in this study. Dilutions up to 0.08% (v/v) increased significantly the antioxidant status in primary rat hepatocytes. The glutathione redox state was decreased with RR treatment but was increased in Chang liver cells and MT-2 lymphoblast. No cyto- or genotoxicity were observed at levels of up to 5% (v/v) of the fruit extract. In addition, a significant protection against t-BHP induced oxidative stress was observed in primary rat hepatocytes. The Ames test revealed no mutagenic activity using the Salmonella typhimurium strains TA98, TA100 and TA102. A significant antimutagenic effect of the extract was observed against the metabolic activated mutagens 2-acetylaminofluorene and aflatoxin B1 and to a lesser extent against methyl methanesulfonate. It is concluded that these results support the associated health promoting potential of Rosa Roxburghii fruit and in particular against oxidative stress.