RESUMO
OBJECTIVES: To investigate gene expression relevant to osteoclastogenesis in the synovium and bone marrow during the development of collagen-induced arthritis (CIA) in mature rats. METHODS: Total messenger RNAs (mRNAs) were obtained from CIA synovium and bone marrow after immunization. First, reverse transcriptase-polymerase chain reactions (RT-PCR) were carried out to detect the mRNA encoding receptor activator of NF-kappaB (RANK), RANK ligand (RANKL), osteoprotegerin (OPG), tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6 and the osteoclast markers tartrate-resistant acid phosphatase (TRAP) and cathepsin K. Secondly, the genes detected clearly by RT-PCR were quantified using real-time PCR. RESULTS: In the synovium, expression of all genes was confirmed by specific single bands in RT-PCR. In real-time PCR, the expression levels of TNF-alpha, IL-1beta, IL-6, RANKL, TRAP and cathepsin K mRNA increased, whereas the expression levels of RANK and OPG were unchanged and decreased respectively. RANKL expression was highly correlated with the two osteoclast markers. In the bone marrow, RT-PCR did not clearly detect the expression of IL-6, RANKL or OPG mRNA. Quantitative real-time PCR showed that TNF-alpha, RANK and TRAP mRNA expression did not change significantly with time, and that IL-1beta and cathepsin K changed slightly compared with those in the synovium. CONCLUSIONS: In the early stages of arthritis, synovial RANKL is closely involved in osteoclastogenesis, and various changes in synovial cytokines, including down-regulation of OPG, probably accelerate osteoclast formation. In contrast, cytokine mRNA in the bone marrow showed little fluctuation. We suggest that synovial cytokines affect osteoclastogenesis not only in the synovium but in the bone marrow.
Assuntos
Artrite Experimental/patologia , Células da Medula Óssea/patologia , Citocinas/biossíntese , Osteoclastos/patologia , Membrana Sinovial/patologia , Animais , Artrite Experimental/metabolismo , Células da Medula Óssea/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Diferenciação Celular/genética , Citocinas/genética , Citocinas/fisiologia , Feminino , Expressão Gênica , Mediadores da Inflamação/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Reação em Cadeia da Polimerase/métodos , Ligante RANK , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Membrana Sinovial/metabolismoRESUMO
The multisite-specific endonuclease Endo.SceI of yeast mitochondria is unique among endonucleases because its 50-kDa subunit forms a stable dimer with the mitochondrial 70-kDa heat shock protein (mtHSP70), which otherwise fulfills a chaperone function by binding transiently to unfolded proteins. Here we show that the mtHSP70 subunit confers broader sequence specificity, greater stability, and higher activity on the 50-kDa subunit. The 50-kDa subunit alone displayed weaker activity and highly sequence-specific endonuclease activity. The 50-kDa protein exists as a heterodimer with mtHSP70 in vivo, allowing Endo.SceI to cleave specifically at multiple sites on mitochondrial DNA. Endo.SceI may have evolved from a highly specific endonuclease that gained broader sequence specificity after becoming a stable partner of mtHSP70.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Dimerização , Indução Enzimática , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae , Especificidade por SubstratoRESUMO
Three-dimensional (3-D) graphic reconstruction of serial image data of x-ray CT on three dialysis patients was studied to measure peritoneal dialysate (PD) volume. Volume estimation with a surface reconstructed model provided a 16% error; a 35% error was found with a voxel model, although the total time required by the voxel model was 15 times less.