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Anjos Guerreiro, Liliana Isabel Tomé

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  • A thyroid hormone regulated asymmetric responsive centre is correlated with eye migration during flatfish metamorphosis
    Publication . Campinho, Marco António; Silva, Nádia; Martins, Gabriel G.; Anjos, Liliana; Florindo, Claudia; Roman-Padilla, Javier; Garcia-Cegarra, Ana; Louro, Bruno; Manchado, Manuel; Power, Deborah
    Flatfish metamorphosis is a unique post-embryonic developmental event in which thyroid hormones (THs) drive the development of symmetric pelagic larva into asymmetric benthic juveniles. One of the eyes migrates to join the other eye on the opposite side of the head. Developmental mechanisms at the basis of the acquisition of flatfish anatomical asymmetry remain an open question. Here we demonstrate that an TH responsive asymmetric centre, determined by deiodinase 2 expression, ventrally juxtaposed to the migrating eye in sole (Solea senegalensis) correlates with asymmetric cranial ossification that in turn drives eye migration. Besides skin pigmentation that is asymmetric between dorsal and ventral sides, only the most anterior head region delimited by the eyes becomes asymmetric whereas the remainder of the head and organs therein stay symmetric. Sub-ocular ossification is common to all flatfish analysed to date, so we propose that this newly discovered mechanism is universal and is associated with eye migration in all flatfish.
  • Vertebrate SLRP family evolution and the subfunctionalization of osteoglycin gene duplicates in teleost fish
    Publication . Costa, Rita; Brazona, Rute Sofia Tavares Martins; Capilla, E.; Anjos, Liliana; Power, Deborah
    Background Osteoglycin (OGN, a.k.a. mimecan) belongs to cluster III of the small leucine-rich proteoglycans (SLRP) of the extracellular matrix (ECM). In vertebrates OGN is a characteristic ECM protein of bone. In the present study we explore the evolution of SLRP III and OGN in teleosts that have a skeleton adapted to an aquatic environment. Results The SLRP gene family has been conserved since the separation of chondrichthyes and osteichthyes. Few gene duplicates of the SLRP III family exist even in the teleosts that experienced a specific whole genome duplication. One exception is ogn for which duplicate copies were identified in fish genomes. The ogn promoter sequence and in vitro mesenchymal stem cell (MSC) cultures suggest the duplicate ogn genes acquired divergent functions. In gilthead sea bream (Sparus aurata) ogn1 was up-regulated during osteoblast and myocyte differentiation in vitro, while ogn2 was severely down-regulated during bone-derived MSCs differentiation into adipocytes in vitro. Conclusions Overall, the phylogenetic analysis indicates that the SLRP III family in vertebrates has been under conservative evolutionary pressure. The retention of the ogn gene duplicates in teleosts was linked with the acquisition of different functions. The acquisition by OGN of functions other than that of a bone ECM protein occurred early in the vertebrate lineage.
  • Evolution and diversity of alpha-carbonic anhydrases in the mantle of the Mediterranean mussel (Mytilus galloprovincialis)
    Publication . Cardoso, João CR; Ferreira, Vinicius; Zhang, Xushuai; Anjos, Liliana; Félix, Rute; Batista, Frederico; Power, Deborah
    The α-carbonic anhydrases (α-CAs) are a large and ancient group of metazoan-specific enzymes. They generate bicarbonate from metabolic carbon dioxide and through calcium carbonate crystal formation play a key role in the regulation of mineralized structures. To better understand how α-CAs contribute to shell mineralization in the marine Mediterranean mussel (Mytilus galloprovincialis) we characterized them in the mantle. Phylogenetic analysis revealed that mollusc α-CA evolution was affected by lineage and species-specific events. Ten α-CAs were found in the Mediterranean mussel mantle and the most abundant form was named, MgNACR, as it grouped with oyster nacreins (NACR). Exposure of the Mediterranean mussel to reduced water salinity (18 vs 37 ppt), caused a significant reduction (p < 0.05) in mantle esterase activity and MgNACR transcript abundance (p < 0.05). Protonograms revealed multiple proteins in the mantle with α-CA hydratase activity and mapped to a protein with a similar size to that deduced for monomeric MgNACR. Our data indicate that MgNACR is a major α-CA enzyme in mantle and that by homology with oyster nacreins likely regulates mussel shell production. We propose that species-dependent α-CA evolution may contribute to explain the diversity of bivalve shell structures and their vulnerability to environmental changes.
  • Isolation and characterization of piscine osteonectin and downregulation of Its expression by PTH-related protein
    Publication . Redruello, Begoña; Estêvão, M. Dulce; Rotllant, J.; Guerreiro, P. M.; Anjos, Liliana; Canario, Adelino V. M.; Power, Deborah
    The skeleton is the main source of osteonectin mRNA in adults of the seawater teleost sea bream Sparus auratus. It is expressed by cells forming the basement membrane of calcifying tissue indicating that, as in mammals, it may play a role in osteoblast differentiation. PTHrP induced downregulation of osteonectin mRNA in vitro in scales, a mineralizing tissue with bone-like metabolism. This indicates a means to redirect calcium to activities such as vitellogenesis when this ion is in high demand.
  • Ligand binding and signalling pathways of PTH receptors in sea bream (Sparus auratus) enterocytes
    Publication . Rotllant, J.; Guerreiro, P. M.; Redruello, Begoña; Fernandes, H.; Apolonia, L.; Anjos, Liliana; Canario, Adelino V. M.; Power, Deborah
    Whole animal studies have indicated that Ca2+ uptake by the gastrointestinal tract is regulated by the action of parathyroid hormone-related peptide (PTHrP) in teleost fish. We have characterised PTH receptors (PTHR) in piscine enterocytes and established, by using aminoterminal PTHrP peptides, the amino acid residues important for receptor activation and for stabilising the ligand/receptor complex. Ligand binding of 125I-(1–35tyr) PTHrP to the membrane fraction of isolated sea bream enterocytes revealed the existence of a single saturable high-affinity receptor (KD=2.59 nM; Bmax=71 fmol/mg protein). Reverse transcription/polymerase chain reaction with specific primers for sea bream PTH1R and PTH3R confirmed the mRNA expression of only the later receptor. Fugu (1–34) PTHrP increased cAMP levels in enterocytes but had no effect on total inositol phosphate accumulation. The aminoterminal peptides (2–34)PTHrP, (3–34)PTHrP and (7–34) PTHrP bound efficiently to the receptor but were severely defective in stimulating cAMP in enterocyte cells indicating that the first six residues of piscine (1–34)PTHrP, although not important for receptor binding, are essential for activation of the adenylate cyclase/phosphokinase A (AC-PKA)-receptor-coupled intracellular signalling pathway. Therefore, PTHrP in teleosts acts on the gastrointestinal tract through PTH3R and the AC-PKA intracellular signalling pathway and might regulate Ca2+ uptake at this site. Ligand-receptor binding and activity throughout the vertebrates appears to be allocated to the same amino acid residues of the amino-terminal domain of the PTHrP molecule.
  • Production and characterisation of gilthead sea bream (Sparus auratus) recombinant parathyroid hormone related protein
    Publication . Anjos, Liliana; Rotllant, J.; Guerreiro, P. M.; Hang, X. M.; Canario, Adelino V. M.; Balment, R.; Power, Deborah
    The production and puriWcation of gilthead sea bream recombinant parathyroid hormone related protein [sbPTHrP(1–125)] using an Escherichia coli system and one step puriWcation process with continuous elution gel electrophoresis is reported. The cDNA encoding sbPTHrP(1–125) was cloned into a prokaryotic expression vector pET-11a. The recombinant plasmid was used to transfect E. coli BL21(DE3) pLysS and sbPTHrP(1–125) synthesis was induced by addition of 1mM isopropyl- -D-thiogalactopyranoside. The rapid one step isolation method gave pure sbPTHrP(1–125) as judged by SDS–PAGE and yielded up to 40mg/L of culture medium (3.3mg protein/g of bacteria). The bioactivity of recombinant sbPTHrP(1–125) assessed using an in vitro scale bioassay was found to be equipotent to PTHrP(1–34) in stimulating cAMP accumulation. Assessment of the immunological reactivity of the isolated protein by Western blot revealed it cross-reacts with antisera speciWc for the N-terminal and C-terminal region of PTHrP. In a radioimmunoassay speciWc for piscine N-terminal (1–34 aa) PTHrP, the recombinant sbPTHrP(1–125) was equipotent with PTHrP(1–34) in displacing labelled 125I-PTHrP(1–36) PTHrP from the antisera. The availability of recombinant sbPTHrP will allow the development of region speciWc assays and studies aimed at deWning post-secretory processing of this protein and its biological activity in fish.
  • Dilution of seawater affects the Ca2 + transport in the outer mantle epithelium of crassostrea gigas
    Publication . Sillanpää, J. Kirsikka; Cardoso, João CR; Félix, Rute; Anjos, Liliana; Power, Deborah; Sundell, Kristina
    Varying salinities of coastal waters are likely to affect the physiology and ion transport capabilities of calcifying marine organisms such as bivalves. To investigate the physiological effect of decreased environmental salinity in bivalves, adult oysters (Crassostrea gigas) were exposed for 14 days to 50% seawater (14) and the effects on mantle ion transport, electrophysiology and the expression of Ca2+ transporters and channels relative to animals maintained in full strength sea water (28) was evaluated. Exposure of oysters to a salinity of 14 decreased the active mantle transepithelial ion transport and specifically affected Ca2+ transfer. Gene expression of the Na+/K+-ATPase and the sarco(endo)plasmic reticulum Ca2+-ATPase was decreased whereas the expression of the T-type voltage-gated Ca channel and the Na+/Ca2+-exchanger increased compared to animals maintained in full SW. The results indicate that decreased environmental salinities will most likely affect not only osmoregulation but also bivalve biomineralization and shell formation.
  • Proteomics of fish white muscle and Western blotting to detect putative allergens
    Publication . Anjos, Liliana; Loukissas, A. Ζ.; Power, Deborah
    A detailed workflow is provided for preparation from teleost fish white muscle of extracts for proteomics analysis. The protocol generates samples that can be analyzed by SWATH (Sequential Window data independent Acquisition of the Total High-resolution-Mass Spectra), a modern MS-based quantitative label free technology. The main steps for the extraction of three independent protein fractions, (1) soluble sarcoplasmic, (2) soluble myofibrillar, and (3) insoluble material, from fish white muscle are detailed. Coupled to the protein extraction protocol a Western blotting approach is outlined for detection of common fish allergens, in this case β-parvalbumin, in the white muscle sarcoplasmic protein fraction.
  • Stimulation of cortisol release by the N terminus of teleost parathyroid hormone-related protein in interrenal cells in vitro
    Publication . Rotllant, J.; Guerreiro, P. M.; Anjos, Liliana; Redruello, Begoña; Canario, Adelino V. M.; Power, Deborah
    The mode of action of PTHrP in the regulation of sea bream (Sparus auratus) interrenal cortisol production was studied in vitro using a dynamic superfusion system. Piscine (1–34)PTHrP (10−6–10−11 m) stimulated cortisol production in a dose-dependent manner. The ED50 of (1–34)PTHrP was 2.8 times higher than that of (1–39)ACTH, and maximum increase in cortisol production in response to 10−8 m of (1–34)PTHrP was approximately 7-fold lower than for 10−8 m of (1–39)ACTH. In contrast to (1–34)PTHrP, piscine (10–20)PTHrP, (79–93)PTHrP, and (100–125)PTHrP (10−9–10−7 m) did not stimulate cortisol production. The effect of piscine (1–34)PTHrP on cortisol production was abolished by N-terminal peptides in which the first amino acid (Ser) was absent and by simultaneous addition of inhibitors of the adenylyl cyclase-protein kinase A and phospholipase C-protein kinase C intracellular pathways but not by each separately. The PTHrP-induced signal transduction was further investigated by measurements of cAMP production and [H3]myo-inositol incorporation in an interrenal cell suspension. Piscine (1–34)PTHrP increased cAMP and total inositol phosphate accumulation, which is indicative that the mechanism of action of PTHrP in interrenal tissue involves the activation of both the adenylyl cyclase-cAMP and phospholipase C-inositol phosphate signaling pathways. These results, together with the expression of mRNA for PTHrP and for PTH receptor (PTHR) type 1 and PTHR type 3 receptors in sea bream interrenal tissue, suggest a specific paracrine or autocrine steroidogenic action of PTHrP mediated by the PTHRs.
  • Cartilage acidic protein a novel therapeutic factor to improve skin damage repair?
    Publication . Félix, Rute; Anjos, Liliana; Costa, Rita; Letsiou, Sophia; Power, Deborah Mary
    Fish skin has been gaining attention due to its efficacy as a human-wound-treatment product and to identify factors promoting its enhanced action. Skin fibroblasts have a central role in maintaining skin integrity and secrete extra cellular matrix (ECM) proteins, growth factors and cytokines to rapidly repair lesions and prevent further damage or infection. The effects on scratch repair of the ubiquitous but poorly characterized ECM protein, cartilage acidic protein 1 (CRTAC1), from piscine and human sources were compared using a zebrafish SJD.1 primary fibroblast cell line. A classic in vitro cell scratch assay, immunofluorescence, biosensor and gene expression analysis were used. Our results demonstrated that the duplicate sea bass Crtac1a and Crtac1b proteins and human CRTAC-1A all promoted SJD.1 primary fibroblast migration in a classic scratch assay and in an electric cell impedance sensing assay. The immunofluorescence analysis revealed that CRTAC1 enhanced cell migration was most likely caused by actin-driven cytoskeletal changes and the cellular transcriptional response was most affected in the early stage (6 h) of scratch repair. In summary, our results suggest that CRTAC1 may be an important factor in fish skin promoting damage repair.