RESUMO
During orthodontic tooth movement, bone resorption occurs at the compression site. However, the mechanism underlying resorption remains unclear. Applying compressive force to human osteoblast-like cells grown in a 3D collagen gel, we examined gene induction by using microarray and RT-PCR analysis. Among 43 genes exhibiting significant changes, cyclo-oxygenase-2, ornithine decarboxylase, and matrix metalloproteinase-3 (MMP-3) were up-regulated, whereas membrane-bound interleukin-1 receptor accessory protein was down-regulated. The MMP-3 protein increases were further confirmed by Western blot. To ascertain whether MMP-3 is up-regulated in vivo by orthodontic force, we examined human bone samples at the compressive site by realigning the angulated molars. Immunohistochemical staining revealed MMP-3 distributed along the compressive site of the bony region within 3 days of compression. Since MMP-3 participates in degradation of a wide range of extracellular matrix molecules, we propose that MMP-3 plays an important role in bone resorption during orthodontic tooth movement.
Assuntos
Processo Alveolar/enzimologia , Remodelação Óssea/fisiologia , Metaloproteinase 3 da Matriz/metabolismo , Osteoblastos/enzimologia , Técnicas de Movimentação Dentária , Adaptação Fisiológica , Técnicas de Cultura de Células , Células Cultivadas , Colágeno , Ciclo-Oxigenase 2/metabolismo , Géis , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Ornitina Descarboxilase/metabolismo , Osteoblastos/citologia , Estatísticas não Paramétricas , Ativação TranscricionalRESUMO
The cDNA sequence of beta crystallin B1 was determined from zebrafish (Danio rerio) and compared to the corresponding genes of bovine, rat, chicken, human, and Xenopus. Multispecies comparison of superfamily diversity demonstrated beta crystallin B1 homology between zebrafish, bovine, chicken, and rat, but large distances to beta crystallin B2 and B3. Zebrafish cDNA has a size of 943 nucleotides and encodes a polypeptide of 233 amino acids. Zebrafish beta crystallin B1 shares 71.30, 75.86, and 71.00% similarities with bovine, chicken, and rat beta crystallin B1, respectively. Northern blot analysis revealed a single 0.9-kb beta crystallin B1 transcript which was expressed and progressively increased in the first 20 h of zebrafish embryogenesis. Whole-mount in situ hybridization revealed that the beta crystallin B1 transcript was only specifically expressed in the lens region of the eye. A starvation experiment revealed no variation in mRNA levels after 14 and 21 days. An experiment in which hormone was injected showed that the beta crystallin B1 transcript first increased 24 h after the injection of insulin-like growth factor I, insulin-like growth factor II, or growth hormone, then decreased 48 h after injection. The beta crystallin B1 transcript continuously increased after insulin was injected. Taken together, our results identify the early specific expression of beta crystallin B1 within the lens. Despite small differences, these results indicate that both the structure of the beta crystallin B1 protein and its involvement with regulation by growth factors appear to have been remarkably conserved.
Assuntos
Cristalinas/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Peixe-Zebra , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Cristalinas/química , Cristalinas/metabolismo , Hormônio do Crescimento/farmacologia , Hibridização In Situ , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/farmacologia , Cristalino/efeitos dos fármacos , Cristalino/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Desaturases are enzymes that catalyze double bond formation in fatty acids, which is a critical step in the synthesis of unsaturated fatty acids in organisms. Desaturase cDNA has been cloned from various species. Here we report the cloning of a full-length cDNA of Delta(9)-desaturase from grass carp (Ctenopharyngodon idella), using a combination of PCR techniques: reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The resolved cDNA encompasses 2420 bp, containing an open reading frame corresponding to 324 amino acids. The deduced amino acid sequence shares high homology with those of mammalian desaturases. Northern blot and RT-PCR analyses demonstrated a high abundance of the transcript in liver tissue but low abundance in brain tissue. Furthermore, the structure of the gene has been resolved by screening its cognate genomic DNA library. The analysis shows that this gene is composed of six exons and five introns, encompassing a region of 8.5 kb. In particular, the last exon contains a length of the 3' untranslated region as long as 1382 bp. Although the primary sequence and the genomic organization are phylogenetically conserved between fish and mammals, the regulation of the gene expression appears to be divergent among species.
Assuntos
Carpas/genética , Reação em Cadeia da Polimerase/métodos , Estearoil-CoA Dessaturase/genética , Motivos de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Carpas/metabolismo , Clonagem Molecular , DNA Complementar , Expressão Gênica , Humanos , Dados de Sequência Molecular , RNA/genética , RNA/metabolismo , Alinhamento de Sequência , Estearoil-CoA Dessaturase/química , Estearoil-CoA Dessaturase/metabolismoRESUMO
Cold-shock domain proteins in vertebrates contain a highly conserved domain which is related to the Escherichia coli cold-shock proteins. Here we report the cloning of a cold-shock domain protein from zebrafish embryo. Using the combination of PCR techniques with degenerate primers, 5'RACE and 3'RACE, the full length cDNA of a cold-shock domain protein in the zebrafish embryo was successfully cloned without constructing and screening a library. Determined from the deduced amino acid sequence, this protein is most similar to Xenopus, FRGY1, and this newly cloned zebrafish gene was therefore designated as zfY1.
Assuntos
Proteínas de Ligação a DNA/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , Proteínas de Ligação a DNA/química , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Peixe-Zebra/embriologiaRESUMO
The signalling molecule Sonic hedgehog is involved in a multitude of distinct patterning processes during vertebrate embryogenesis. In the nascent body axis of the zebrafish embryo, sonic hedgehog is co-expressed with axial (HNF3beta in mammals), a transcription regulator of the winged helix family. We show here that misexpression of axial leads to ectopic activation of sonic hedgehog expression in the zebrafish, suggesting that axial is a regulator of sonic hedgehog transcription. The sonic hedgehog gene was cloned from zebrafish and its promoter was characterized with respect to activation by axial. Expression of axial or rat HNF3beta in HeLa cells results in activation of co-transfected sonic hedgehog promoter-CAT fusion genes. This effect is mediated by two Axial (HNF3beta) recognition sequences. We furthermore identified a retinoic acid response element (RARE) in the sonic hedgehog upstream region which can be bound by retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers in vitro and confers retinoic acid inducibility to the sonic hedgehog promoter in the HeLa cell system. Our results suggest that both Axial (HNF3beta) and retinoic acid receptors are direct regulators of the sonic hedgehog gene.