Browsing by Author "Pombinho, A. R."
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- Characterization of Sparus aurata osteonectin cDNA and in silico analysis of protein conserved features: Evidence for more than one osteonectin in SalmonidaePublication . Laizé, Vincent; Pombinho, A. R.; Cancela, LeonorOsteonectin is a matricellular protein involved in various cellular mechanisms but its exact function remains unclear despite numerous studies. We present here the cloning of Sparus aurata partial osteonectin cDNA and the reconstruction of 15 other sequences from both vertebrates and invertebrates, almost doubling the set of available sequences (a total of 35 sequences is now available). Taking advantage of the resulting large amount of data, we have created multiple sequence alignments and identified osteonectin putative conserved features (intra- and inter-disulfide bonds, collagen- and calcium-binding domains and phosphorylation sites) likely to be important for protein structure and function. This work also provides the first evidence for the presence of more than one osteonectin in some species. Finally, S. aurata osteonectin gene expression has been shown to initiate during larval development shortly after gastrulation, and to be high in bone-derived cell lines while down-regulated during extracellular matrix mineralization, further emphasizing the important role of osteonectin in skeletal development and bone formation.
- Development of two bone-derived cell lines from the marine teleost Sparus aurata; evidence for extracellular matrix mineralization and cell-type-specific expression of matrix Gla protein and osteocalcinPublication . Pombinho, A. R.; Laizé, Vincent; Molha, D. M.; Marques, S.; Cancela, LeonorA growing interest in the understanding of the ontogeny and mineralization of fish skeleton has emerged from the recent implementation of fish as a vertebrate model, particularly for skeletal development. Whereas several in vivo studies dealing with the regulation of bone formation in fish have been published, in vitro studies have been hampered because of a complete lack of fish-bone-derived cell systems. We describe here the development and the characterization of two new cell lines, designated VSa13 and VSa16, derived from the vertebra of the gilthead sea bream. Both cell types exhibit a spindle-like phenotype and slow growth when cultured in Leibovitz’s L-15 medium and a polygonal phenotype and rapid growth in Dulbecco’s modified Eagle medium (D-MEM). Scanning electron microscopy and von Kossa staining have revealed that the VSa13 and VSa16 cells can only mineralize their extracellular matrix when cultured in D-MEM under mineralizing conditions, forming calcium-phosphate crystals similar to hydroxyapatite. We have also demonstrated the involvement of alkaline phosphatase, a marker of bone formation in vivo, and Gla proteins (osteocalcin and matrix Gla protein, MGP) in the process of mineralization. Finally, we have shown that VSa13 and VSa16 cell lines express osteocalcin and MGP in a mutually exclusive manner. Thus, both cell lines are capable of mineralizing in vitro and of expressing genes found in chondrocyte and osteoblast cell lineages, emphasizing the suitability of these new cell lines as valuable tools for analyzing the expression and regulation of cartilage- and bone-specific genes.
- Proteínas gla do osso e cartilagem: importância dos anfíbios e peixes como modelos biológicos para elucidação da sua função e evolução - gla proteins in bone and cartilage: the importance of fish and amphibian models to understand their function and evolutionPublication . Cancela, Leonor; Laizé, Vincent; Simes, D; Gavaia, Paulo J.; Conceição, N.; S B Viegas, Carla; Pombinho, A. R.; Marques, S.; Fidalgo, J.; Braga, D.Gla proteins, as the name indicates, undergo a post-translation modification where specific glutamic acid residues are γ-carboxylated through the action of the ubiquitous enzyme γ-carboxylase and using vitamin K as cofactor. Therefore, these proteins are also called vitamin K dependent proteins or VKD [1, 2]. We can assign VKDs to essentially four different groups: 1) those involved in blood coagulation (such as prothrombin and various coagulation factors; the first group to be discovered), 2) those involved in tissue mineralization (bone and matrix Gla proteins), 3) a nerve growth factor (gas6), and 4) those of unknown function (the latest group to be discovered). The carboxylase enzyme is present in fly and worm but not in yeast, indicating that γ-carboxylation is likely a feature appearing in multicellular eukaryotes.