RESUMEN
A major characteristic of type 2 diabetes mellitus (T2DM) is insulin resistance in skeletal muscle. A growing body of evidence indicates that oxidative stress that results from increased production of reactive oxygen species and/or reactive nitrogen species leads to insulin resistance, tissue damage, and other complications observed in T2DM. It has been suggested that muscular free fatty acid accumulation might be responsible for the mitochondrial dysfunction and insulin resistance seen in T2DM, although the mechanisms by which increased levels of free fatty acid lead to insulin resistance are not well understood. To help resolve this situation, we report that saturated fatty acid palmitate stimulated the expression of inducible nitric oxide (NO) synthase and the production of reactive oxygen species and NO in L6 myotubes. Additionally, palmitate caused a significant dose-dependent increase in mitochondrial DNA (mtDNA) damage and a subsequent decrease in L6 myotube viability and ATP levels at concentrations as low as 0.5 mM. Furthermore, palmitate induced apoptosis, which was detected by DNA fragmentation, caspase-3 cleavage, and cytochrome c release. N-acetyl cysteine, a precursor compound for glutathione formation, aminoguanidine, an inducible NO synthase inhibitor, and 5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrinato iron (III), a peroxynitrite inhibitor, all prevented palmitate-induced mtDNA damage and diminished palmitate-induced cytotoxicity. We conclude that exposure of L6 myotubes to palmitate induced mtDNA damage and triggered mitochondrial dysfunction, which caused apoptosis. Additionally, our findings indicate that palmitate-induced mtDNA damage and cytotoxicity in skeletal muscle cells were caused by overproduction of peroxynitrite.
Asunto(s)
Apoptosis/efectos de los fármacos , ADN Mitocondrial/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Músculo Esquelético/citología , Estrés Oxidativo/fisiología , Palmitatos/farmacología , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/fisiología , Caspasa 3/metabolismo , Núcleo Celular , Células Cultivadas , Citocromos c/metabolismo , Daño del ADN/fisiología , Fragmentación del ADN/efectos de los fármacos , Diabetes Mellitus Tipo 2/patología , Diabetes Mellitus Tipo 2/fisiopatología , Depuradores de Radicales Libres/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Rickettsia prowazekii, the etiologic agent of epidemic typhus, is an obligate, intracytoplasmic, parasitic bacterium. Recently, the transformation of this bacterium via electroporation has been reported. However, in these studies identification of transformants was dependent upon either selection of an R. prowazekii rpoB chromosomal mutation imparting rifampin resistance or expression of the green fluorescent protein and flow cytometric analysis. In this paper we describe the expression in R. prowazekii of the Escherichia coli ereB gene. This gene codes for an erythromycin esterase that cleaves erythromycin. To the best of our knowledge, this is the first report of the expression of a nonrickettsial, antibiotic-selectable gene in R. prowazekii. The availability of a positive selection for rickettsial transformants is an important step in the characterization of genetic analysis systems in the rickettsiae.
Asunto(s)
Antibacterianos/farmacología , Hidrolasas de Éster Carboxílico/genética , Eritromicina/farmacología , Rickettsia prowazekii/genética , Transformación Bacteriana , Animales , Células Cultivadas , Farmacorresistencia Microbiana/genética , Electroporación , Escherichia coli/genética , Fibroblastos/microbiología , Genes Bacterianos , Marcadores Genéticos , Ratones , Selección GenéticaRESUMEN
Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of the eucaryotic host cell. The absence of techniques for genetic manipulation hampers the study of this organism's unique biology and pathogenic mechanisms. To establish the feasibility of genetic manipulation in this organism, we identified a specific mutation in the rickettsial rpoB gene that confers resistance to rifampin and used it to demonstrate allelic exchange in R. prowazekii. Comparison of the rpoB sequences from the rifampin-sensitive (Rifs) Madrid E strain and a rifampin-resistant (Rifr) mutant identified a single point mutation that results in an arginine-to-lysine change at position 546 of the R. prowazekii RNA polymerase beta subunit. A plasmid containing this mutation and two additional silent mutations created in codons flanking the Lys-546 codon was introduced into the Rifs Madrid E strain of R. prowazekii by electroporation, and in the presence of rifampin, resistant rickettsiae were selected. Transformation, via homologous recombination, was demonstrated by DNA sequencing of PCR products containing the three mutations in the Rifr region of rickettsial rpoB. This is the first successful demonstration of genetic transformation of Rickettsia prowazekii and represents the initial step in the establishment of a genetic system in this obligate intracellular pathogen.