RESUMO
BACKGROUND: In this study, the mutated phenotypes were produced by treatment of chemical mutagen, N-ethyl-N-nitrosourea (ENU). We analyzed the mutated mice showing the specific phenotype of ectodermal dysplasia (ED) and examined the affected gene. METHODS: Phenotypes, including size, bone formation, and craniofacial morphology of ENU-induced ED mice, were focused. Tooth development and expression of several molecules were analyzed by histologic observations and immunohistochemistry. We carried out genome-wide screening and quantitative real-time PCR to define the affected and related genes. RESULTS: As examined previously in human ectodermal dysplasia, ENU-induced ED mice showed the specific morphologic deformities in tooth, hair, and craniofacial growth. Tooth development in the ENU-induced ED mice ceased at early cap stage. In addition, skeletal staining showed retardation in craniofacial development. Finally, the affected gene, which would be involved in the mechanism of ED, was located between the marker D3Mit14 and D3Mit319 on chromosome 3. CONCLUSIONS: The affected gene in ENU-induced ED mice showed several defects in ectodermal organogenesis and these results indicate that this gene plays an important role in mouse embryogenesis.
Assuntos
Displasia Ectodérmica/induzido quimicamente , Etilnitrosoureia/toxicidade , Anormalidades Dentárias/induzido quimicamente , Animais , Animais Recém-Nascidos , Mapeamento Cromossômico , Cromossomos de Mamíferos , Displasia Ectodérmica/embriologia , Displasia Ectodérmica/genética , Ectodisplasinas/genética , Ectodisplasinas/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Subunidades beta de Inibinas/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Fator 1 de Ligação ao Facilitador Linfoide/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Organogênese/efeitos dos fármacos , Transdução de Sinais/genética , Anormalidades Dentárias/embriologia , Anormalidades Dentárias/genética , Fator de Transcrição RelA/genéticaRESUMO
The coxsackievirus group B (CVB) of the genus Enterovirus and the species human enterovirus B is a nonenveloped virus containing a single-stranded positive-sense RNA genome. Coxsackievirus has icosahedral symmetry and four capsid proteins, VP1, VP2, VP3, and VP4. Specific antibodies against each viral protein are prerequisites for various studies. In this study, we developed seven peptide-derived antibodies directed against coxsackievirus VP1 (NO1-NO5), VP2 (B3), and VP3 (GL3). We developed a type-specific antibody (NO1) and broadly cross-reactive antibodies (NO3 and NO5) to VP1. Anti-VP2 and anti-VP3 antibodies (B3 and GL3, respectively) are also cross-reactive to human enterovirus B such as CVB and echoviruses. Their sensitivities and reactivities are likely to be better than those of the commercial VP1 monoclonal antibody (MAb). The dot-blot analysis also showed that NO5 against VP1 is able to detect less than 1 microg [2x10(6) plaque-forming unit (pfu) of CVB3] of viruses, suggesting that it could be used to develop a diagnostic kit that can directly detect human enterovirus B. The antibodies produced here may allow us to undertake several studies, such as those involving viral trafficking, expression kinetics, and the roles of viral proteins in infection, and the development of diagnostic kits.
Assuntos
Anticorpos Antivirais/biossíntese , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Infecções por Coxsackievirus/diagnóstico , Reações Cruzadas , Fragmentos de Peptídeos/imunologia , Anticorpos Monoclonais , Anticorpos Antivirais/genética , Especificidade de Anticorpos , Antígenos Virais/imunologia , Infecções por Coxsackievirus/genética , Infecções por Coxsackievirus/imunologia , Enterovirus Humano B/imunologia , Enterovirus Humano B/isolamento & purificação , Células HeLa , Humanos , Peptídeos/imunologia , RNA Viral/análiseRESUMO
The 70 kDa heat shock protein (Hsp70) is synthesized in response to a variety of stresses, including ischemia, and is thought to act as a molecular chaperone to prevent protein denaturation and facilitate protein folding. Matrix metalloproteinases (MMPs), a family of serine proteases, are also upregulated by ischemia and are thought to promote cell death and tissue injury. We examined the influence of Hsp70 on expression and activity of MMPs. Astrocyte cultures were prepared from neonatal mice and transfected with retroviral vectors containing hsp70 or lacZ or mock infected, then exposed to oxygen-glucose deprivation followed by reperfusion. Zymograms and Western blots showed that Hsp70 over-expression suppressed MMP-2 and MMP-9. These findings suggest that Hsp70 may protect by regulating MMPs.
Assuntos
Astrócitos/metabolismo , Proteínas de Choque Térmico HSP70/farmacologia , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/biossíntese , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Western Blotting , Células Cultivadas , Proteínas de Choque Térmico HSP70/genética , Óperon Lac/genética , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Endogâmicos ICR , RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
The aberrant protein crosslinks formation during lung injury as results total body irradiation (TBI) and bone marrow transplantation (BMT) therapy has been examined as apossible contributory factor in organ or tissue pathogenesis. Female C3HeB/ FeJ mice were used for an experimental animal. Carbon monoxide uptake (V(CO)) was measured at 1, 2, 3, 4 and 5 months after TBI at respective doses of 12, 14, 16 and 18 Gy 16 h prior to syngeneic BMT. Also as a measure of aberrant protein crosslinking in the inured tissues, transglutaminase (TGase)-activities and crosslinked protein were examined along with thrombin, a protease known to activate TGases. Reductions of VCO were detected following TBI and BMT. Activities of thrombin and TGase 1, and crosslinked protein in bronchoalveolar lavage (BAL) fluid of the mice 1 wk after TBI at 12 Gy and BMT were identified and found to be elevated in the treated animals. These findings suggest that elevated levels of crosslinked proteins and TGase I in the bronchoalveolar larvage during the lung injury could have enhanced the organ pathogenesis following TBI and BMT.