RESUMO
This study sought to characterize the reduced glutathione (GSH)/oxidized GSSG ratio during osteoclast differentiation and determine whether changes in the intracellular redox status regulate its differentiation through a RANKL-dependent signaling pathway. A progressive decrease of the GSH/GSSG ratio was observed during osteoclast differentiation, and the phenomenon was dependent on a decrease in total glutathione via downregulation of expression of the gamma-glutamylcysteinyl synthetase modifier gene. Glutathione depletion by L-buthionine-(S,R)-sulfoximine (BSO) was found to inhibit osteoclastogenesis by blocking nuclear import of NF-kappaB and AP-1 in RANKL-propagated signaling and bone pit formation by increasing BSO concentrations in mature osteoclasts. Furthermore, intraperitoneal injection of BSO in mice resulted in an increase in bone density and a decrease of the number of osteoclasts in bone. Conversely, glutathione repletion with either N-acetylcysteine or GSH enhanced osteoclastogenesis. These findings indicate that redox status decreases during osteoclast differentiation and that this modification directly regulates RANKL-induced osteoclastogenesis.
Assuntos
Diferenciação Celular/fisiologia , Núcleo Celular/metabolismo , Osteoclastos/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Butionina Sulfoximina/farmacologia , Proteínas de Transporte/farmacologia , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Immunoblotting , Glicoproteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , NF-kappa B/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Oxirredução/efeitos dos fármacos , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição AP-1/metabolismoAssuntos
Bacteriólise , Biotecnologia/métodos , DNA Bacteriano/isolamento & purificação , Mycobacterium/genética , Acetilglucosaminidase , Desoxirribonucleases de Sítio Específico do Tipo II , Eletroforese em Gel de Campo Pulsado , Temperatura Alta , Muramidase , Mycobacterium avium/genética , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Sefarose , Fatores de TempoRESUMO
OBJECTIVE: To establish the usefulness of DNA fingerprinting for the epidemiology of Mycobacterium tuberculosis isolated from Korean tuberculosis patients. METHODS: Comparison of restriction fragment length polymorphism (RFLP) patterns produced by southern hybridization of PvuII-digested chromosomal DNA. RESULTS: IS6110-associated banding patterns of 41 isolates varied considerably, containing 1-13 copies. The RFLP pattern of the epidemiologically related M. tuberculosis isolates was identical in 8 of 10 groups of close contact patients. No noticeable differences in RFLP were observed between drug-sensitive and drug-resistant isolates. IS1081-containing restriction fragment analysis of 52 isolates showed 6 different banding patterns, and the C type was found dominant in Korea. Identification of G type M. tuberculosis, which has a 8.0 kb IS1081-containing PvuII fragment, is unusual because it has been observed only in M. bovis BCG so far. CONCLUSIONS: IS6110 was a very useful tool for tracing the transmission route of tuberculosis; IS1081 was also useful for subdividing M. tuberculosis into several groups.
Assuntos
Técnicas de Tipagem Bacteriana , Impressões Digitais de DNA , Mycobacterium tuberculosis/classificação , Tuberculose Pulmonar/microbiologia , Adolescente , Adulto , Sequência de Bases , Southern Blotting , DNA Bacteriano/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Polimorfismo de Fragmento de Restrição , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/transmissãoRESUMO
Bordetella pertussis coordinately regulates expression of its virulence factors in response to changing environmental conditions. These factors include pertussis toxin, adenylate cyclase toxin, and the filamentous hemagglutinin (FHA). The vir (or bvg) locus has been shown genetically to be required for this coordinate regulation. We have attempted to study the biochemical basis for coordinate regulation. DNA promoter deletion studies from other laboratories have shown that two tandem 20-bp repeats -157 to -117 bp upstream from the pertussis toxin promoter are essential for transcription. A similar 20-bp tandem repeat was found at the same site in the upstream region of the adenylate cyclase toxin promoter but is not present in the FHA or vir promoter region. Gel retardation revealed protein from virulent strains (able to express the virulence genes) but not from avirulent strains (unable to express the virulence genes) bound to the promoter region of the pertussis toxin gene, and this binding could be abolished by competition with an excess of oligonucleotides corresponding to either tandem repeat. The protein was determined to be 23 kDa by Southwestern (DNA-protein) analysis and could bind to either 20-bp oligonucleotide from the pertussis toxin promoter and either 20-bp oligonucleotide from the adenylate cyclase toxin promoter. BvgA, a 23-kDa protein encoded in the vir locus, has been reported to bind to a 14-bp inverted repeat in the FHA promoter which is not present in the pertussis toxin or adenylate cyclase promoter. We could not demonstrate binding of BvgA to the pertussis toxin promoter region. These data suggest that we have identified a second 23-kDa protein, distinct from BvgA but regulated by the vir operon, that binds to DNA sequences required for transcription of some, but not all, vir-regulated genes.
