Browsing by Author "Ortiz-Delgado, J. B."
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- Characterization of specific antibodies for fish osteocalcin and its usefulness to investigate osteocalcin tissue distribution in lower vertebrates.Publication . Simes, D; Gavaia, Paulo J.; Ortiz-Delgado, J. B.; Pinto, Jorge; Cancela, LeonorOsteocalcin (BGP or Bone Gla protein) is a small acidic protein with 46-50 residues (pI»4.0) that belongs to the family of the vitamin K dependent, Gla containing proteins. This protein is the most abundant non-collagenous bone protein in mammals and has been isolated only from bone and dentine suggesting that it may be expressed only in hydroxyapatite-containing bone tissue. Previous studies suggest that in mammals BGP is an ossification regulator, but its mode of action at the molecular level, in particular in non-mammalian organisms, remains unclear
- Gla proteins as markers for studies of skeletal development and malformations in new aquaculture fish species (Pagrus auriga, Diplodus sargo and Scophtalmus maximus)Publication . S B Viegas, Carla; Gavaia, Paulo J.; Ortiz-Delgado, J. B.; Sarasquete, C.; Cancela, LeonorEvaluation of the onset and the development of skeletal structures during larval ontogeny of commercially important species in southern Iberia Peninsula and Mediterranean area (...) is essential for the successful production of high quality fish.
- Matrix gla protein in turbot (Scophthalmus maximus): gene expression analysis and identification of sites of protein accumulationPublication . Roberto, Vania Palma; Cavaco, S.; S B Viegas, Carla; Simes, D; Ortiz-Delgado, J. B.; Sarasquete, C.; Gavaia, Paulo J.; Cancela, LeonorMatrix Gla protein (Mgp) is a secreted vitamin K-dependent extracellular matrix protein and a physiological inhibitor of calcification whose gene structure, amino acid sequence and tissue distribution have been conserved throughout evolution. In the present work, the turbot (Scophthalmus maximus) mgp cDNA was cloned and the sequence of the deduced protein compared to that of other vertebrates. As expected, it was closer to teleosts than to other vertebrate groups but there was a strict conservation of amino-acids thought to be important for protein function. Analysis of mgp gene expression indicated branchial arches as the site with higher levels of expression, followed by heart, vertebra and kidney. These results were confirmed by in situ hybridization with a strong mgp expression in branchial arch chondrocytes. Mgp was found to accumulate in gills where it appeared to be restricted to chondrocytes from branchial filaments, while in vertebrae it was localized in vertebral end plates, in growth zones, in vertebral arches and spines and in notochord cells. In the soft tissues analysed, Mgp was mainly detected in kidney and heart, consistent with previous data and providing further evidence for a role of Mgp as a calcification inhibitor and a modulator of the mineralization process. Our studies provide evidence that turbot, an important new species for aquaculture, is also a useful model to study function and expression of Mgp.
- Osteocalcin and Matrix Gla Protein in developing teleost teeth. Identification of sites of mRNA and protein accumulation at single cell resolutionPublication . Ortiz-Delgado, J. B.; Simes, D; Gavaia, Paulo J.; Sarasquete, C.; Cancela, LeonorIn this study, the tissue distribution and accumulation of osteocalcin or bone Gla protein (BGP) and matrix Gla protein (MGP) were determined during tooth development in a teleost fish, Argyrosomus regius. In this species, the presence ofBGP andMGPmRNAin teethwas revealed by in situ hybridization. mRNA for BGP was detected in the odontoblasts as well as in its cytoplasmic processes emerging through dentinal tubules,whilemRNA for MGP was expressed in the enamel portion within the apical portion of the elongated cell bodies of enameloblasts, adjacent to the root of the teeth as well as in cells within the pulpal space. Immunolocalization of BGP and MGP demonstrated that these proteins accumulate mainly in the mineralized dentin or in enameloblastic processes, confirming in situ hybridization results. In this study, we examined for the first time the localization of both BGP and MGP gene expression and protein accumulation within the different regions of the vertebrate tooth. We clearly demonstrated that although the overall pattern of BGP andMGPgene expression and protein accumulation in A. regius teeth was in general agreement to what is known for other vertebrates such as rats or rodents, our study provided novel information and highlighted some species-differences between fish and higher vertebrates.
- Osteocalcin and Matrix Gla Protein in zebrafish (Danio rerio) and Senegal sole (Solea senegalensis): comparative gene and protein expression during larval development through adulthoodPublication . Gavaia, Paulo J.; Simes, D; Ortiz-Delgado, J. B.; S B Viegas, Carla; Pinto, Jorge; Kelsh, R. N.; Sarasquete, C.; Cancela, LeonorBone Gla protein (Bgp or osteocalcin) and matrix Gla protein (Mgp) are important in calcium metabolism and skeletal development, but their precise roles at the molecular level remain poorly understood. Here, we compare the tissue distribution and accumulation of Bgp and Mgp during larval development and in adult tissues of zebrafish (Danio rerio) and throughout metamorphosis in Senegal sole (Solea senegalensis), two fish species with contrasting environmental calcium levels and degrees of skeletal reorganization at metamorphosis. Mineral deposition was investigated in parallel using a modified Alizarin red/Alcian blue protocol allowing sensitive simultaneous detection of bone and cartilage. In zebrafish, bgp and mgp mRNAs were localized in all mineralized tissues during and after calcification including bone and calcified cartilage of branchial arches. Through immunohistochemistry we demonstrated that these proteins accumulate mainly in the matrix of skeletal structures already calcified or under calcification, confirming in situ hybridization results. Interestingly, some accumulation of Bgp was also observed in kidney, possibly due to the presence of a related protein, nephrocalcin. Chromosomal localization of bgp and mgp using a zebrafish radiation hybrid panel indicated that both genes are located on the same chromosome, in contrast to mammals where they map to different chromosomes, albeit in regions showing synteny with the zebrafish location. Results in Senegal sole further indicate that, during metamorphosis, there is an increase in expression of both bgp and mgp, paralleling calcification of axial skeleton structures. In contrast with results obtained for previously studied marine fishes, in zebrafish and Senegal sole Mgp accumulates in both calcified tissues and non-mineralized vessel walls of the vascular system. These results suggest different patterns of Mgp accumulation between fish and mammals.
- Role of the melanocortin system in zebrafish skin physiologyPublication . Leal, E.; Angotzi, A. R.; Gregorio, S. F.; Ortiz-Delgado, J. B.; Rotllant, J.; Fuentes, Juan; Tafalla, C.; Cerdá-Reverter, J. M.The agouti-signaling protein (ASIP) acts as both a competitive antagonist and inverse agonist of melanocortin receptors which regulate dorsal-ventral pigmentation patterns in fish. However, the potential role of ASIP in the regulation of additional physiological pathways in the skin is unknown. The skin plays a crucial role in the immune function, acting as a physical limitation against infestation and also as a chemical barrier due to its ability to synthesize and secrete mucus and many immune effector proteins. In this study, the putative role of ASIP in regulating the immune system of skin has been explored using a transgenic zebrafish model over -expressing the asip1 gene (ASIPzf). Initially, the structural changes in skin induced by asip1 overexpression were studied, revealing that the ventral skin of ASIPzf was thinner than that of wild type (WT) animals. A moderate hypertrophy of mucous cells was also found in ASIPzf. Histochemical studies showed that transgenic animals appear to compensate for the lower number of cell layers by modifying the mucus composition and increasing lectin affinity and mucin content in order to maintain or improve protection against microorganism adhesion. ASIPzf also exhibit higher protein concentration under crowding conditions suggesting an increased mucus production under stressful conditions. Exposure to bacterial lipopolysaccharide (LPS) showed that ASIPzf exhibit a faster pro-inflammatory response and increased mucin expression yet severe skin injures and a slight increase in mortality was observed. Electrophysiological measurements show that the ASIP1 genotype exhibits reduced epithelial resistance, an indicator of reduced tissue integrity and barrier function. Overall, not only are ASIP1 animals more prone to infiltration and subsequent infections due to reduced skin epithelial integrity, but also display an increased inflammatory response that can lead to increased skin sensitivity to external infections.
- Simultaneous detection of osteocalcin and matrix gla protein in developmental stages of zebra fish (Danio rerio)Publication . Gavaia, Paulo J.; Simes, D; Ortiz-Delgado, J. B.; Sarasquete, C.; Cancela, LeonorOsteocalcin (Bone Gla Protein, BGP) and Matrix Gla Protein (MGP) are gammacarboxylated calcium-binding proteins that have been only recently identified in fish. We have previously purified and characterized the two proteins from zebra fish calcified tissues. Polyclonal antibodies against Argyrosomus regius BGP and MGP were validated by western blot assay. The immunolocalization of BGP and MGP in zebra fish was performed in plastic sections of larvae and juveniles, covering all major developmental stages.
- Solea senegalensis vasa transcripts: molecular characterisation, tissue distribution and developmental expression profilesPublication . Pacchiarini, T.; Cross, I.; Leite, Ricardo; Gavaia, Paulo J.; Ortiz-Delgado, J. B.; Pousão-Ferreira, P.; Rebordinos, L.; Sarasquete, C.; Cabrita, ElsaThe Vasa protein is an RNA helicase belonging the DEAD (Asp-Glu-Ala-Asp)-box family. The crucial role played by the vasa gene in the germ-cell lineage of both vertebrates and invertebrates has made this gene a useful molecular marker for germinal cells and a useful tool in surrogate broodstock production using primordial germ cell transplantation. With the aim of establishing a novel approach to improving Solea senegalensis broodstock management, the vasa gene in this species was characterised. Four S. senegalensis vasa transcripts were isolated: Ssvasa1, Ssvasa2, Ssvasa3 and Ssvasa4. Their phylogenetic relationship with other vasa homologues was determined confirming the high degree of conservation of this helicase throughout evolution. Our qPCR results showed that S. senegalensis vasa transcripts are prevalently expressed in gonads, with ovary-specific expression for Ssvasa3 and Ssvasa4. During embryonic and larval development, a switch between the longest and the shortest transcripts was observed. While Ssvasa1 and Ssvasa2 were maternally supplied, Ssvasa3 and Ssvasa4 depended on the de novo expression program of the growing juveniles, suggesting that vasa mRNA could be involved in Senegalese sole gonad differentiation. In situ hybridisation and immunohistochemical analysis performed in 150-days after hatching (DAH) larvae showed vasa product expression in the germinal region of early gonads. In our work we demonstrated the usefulness of Ssvasa mRNAs as molecular markers for primordial germ cells and germinal cells during embryonic development, larval ontogenesis and gonad differentiation. Furthermore, our results confirmed the potential of vasa to help investigate germinal cell biotechnology for Senegalese sole reproduction.
- Two isoforms of vasa maternal factor in Senegalese sole: Biotechnological applicationsPublication . Pacchiarini, T.; Cabrita, Elsa; Cross, I.; Ortiz-Delgado, J. B.; Leite, Ricardo; Gavaia, Paulo J.; Herráez, M. P.; Rebordinos, L.; Sarasquete, C.Primordial Germ Cells (PGCs) identification and manipulation present considerable potential for hatchery practice and surrogate broodstocks. To carry out the PGCs characterization a specific molecular marker is required. The vasa gene is a good candidate to identify PGCs and others germinal cells (Nagasawa et al., 2009). The aim of this study was the cloning of the Solea senegalensis vasa cDNA and its expression pattern during early development and adulthood.