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- Cartilage acidic protein 1 promotes increased cell viability, cell proliferation and energy metabolism in primary human dermal fibroblastsPublication . Letsiou, Sophia; Félix, Rute; Cardoso, João CR; L, Anjos; Mestre, Ana L G; H, Gomes; Power, DeborahCartilage acidic protein 1 (CRTAC1) is an extracellular matrix protein of human chondrogenic tissue that is also present in other vertebrates, non-vertebrate eukaryotes and in some prokaryotes. The function of CRTAC1 remains unknown but the protein's structure indicates a role in cell-cell or cell-matrix interactions and calcium-binding. The aim of the present study was to evaluate the in vitro effects of hCRTAC1-A on normal human dermal fibroblasts (NHDF). A battery of in vitro assays (biochemical and PCR), immunofluorescence and a biosensor approach were used to characterize the protein's biological activities on NHDF cells in a scratch assay. Gene expression analysis revealed that hCRTAC1-A protein is associated with altered levels of expression for genes involved in the processes of cell proliferation (CXCL12 and NOS2), cell migration (AQP3 and TNC), and extracellular matrix-ECM regeneration and remodeling (FMOD, TIMP1, FN1) indicating a role for hCRTAC1-A in promoting these activities in a scratch assay. In parallel, the candidate processes identified by differential gene transcription were substantiated and extended using Electric cell-substrate impedance sensing (ECIS) technology, immunofluorescence and cell viability assays. Our findings indicate that hCRTAC1-A stimulated cell proliferation, migration and ECM production in primary human fibroblasts in vitro. (C) 2020 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.
- 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, DeborahThe 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.
- Evolution of the glucagon-like system across fishPublication . Cardoso, João CR; Félix, Rute C.; Costa, Carina; PFS, Palma; Canario, Adelino; Power, DeborahIn fishes, including the jawless lampreys, the most ancient lineage of extant vertebrates, plasma glucose levels are highly variable and regulation is more relaxed than in mammals. The regulation of glucose and lipid in fishes in common with mammals involves members of the glucagon (GCG)-like family of gastrointestinal peptides. In mammals, four peptides GCG, glucagon-like peptide 1 and 2 (GLP1 and GLP2) and glucose-dependent insulinotropic peptide (GIP) that activate four specific receptors exist. However, in lamprey and other fishes the glucagon-like family evolved differently and they retained additional gene family members (glucagon-related peptide, gcrp and its receptor, gcrpr) that are absent from mammals. In the present study, we analysed the evolution of the glucagon-like system in fish and characterized gene expression of the family members in the European sea bass (Dicentrarchus labrax) a teleost fish. Phylogenetic analysis revealed that multiple receptors and peptides of the glucagon-like family emerged early during the vertebrate radiation and evolved via lineage specific events. Synteny analysis suggested that family member gene loss is likely to be the result of a single gene deletion event. Lamprey was the only fish where a putative glp1r persisted and the presence of the receptor gene in the genomes of the elephant shark and coelacanth remains unresolved. In the coelacanth and elephant shark, unique proglucagon genes were acquired which in the former only encoded Gcg and Glp2 and in the latter, shared a similar structure to the teleost proglucagon gene but possessed an extra exon coding for Glp-like peptide that was most similar to Glp2. The variable tissue distribution of the gene transcripts encoding the ligands and receptors of the glucagon-like system in an advanced teleost, the European sea bass, suggested that, as occurs in mammals, they have acquired distinct functions. Statistically significant (p < .05) down-regulation of teleost proglucagon a in sea bass with modified plasma glucose levels confirmed the link between these peptides and metabolism. The tissue distribution of members of the glucagon-like system in sea bass and human suggests that evolution of the brain-gut-peptide regulatory loop diverged between teleosts and mammals despite the overall conservation and similarity of glucagon-like family members.
- Stanniocalcin-1 protein expression profile and mechanisms in proliferation and cell death pathways in prostate cancerPublication . Costa, Bruna Pasqualotto; Schein, Vanessa; Zhao, R.; Santos, Andressa Schneiders; Kliemann, Lucia Maria; Nunes, Fernanda Bordignon; Cardoso, João CR; C. Félix, Rute; Canario, Adelino; Brum, Ilma Simoni; Branchini, GiseleProstate cancer (PCa) is one of the most prevalent male tumours. Stanniocalcin-1 (STC1) is a glycoprotein and, although the role of STC1 in human cancer is poorly understood, it is suggested to be involved in the development and progression of different neoplasms. This study investigated the protein expression profile of STC1 in PCa and benign prostatic hyperplasia (BPH) samples and STC1 signalling during cell proliferation and cell death in vitro using cell lines. We found higher levels of STC1 in PCa when compared to BPH tissue and that STC1 inhibited forskolin stimulation of cAMP in PC-3 cells. A monoclonal antibody against STC1 was effective in reducing cell proliferation, in promoting cell cycle arrest, and in increasing apoptosis in the same cells. Since STC1 acts as a regulator of prostatic tissue signalling, we suggest that this protein is a novel candidate biomarker for prostate tumour clinical progression and a potential therapeutic target.
- The physiological effect of polystyrene nanoplastic particles on fish and human fibroblastsPublication . Peng, Maoxiao; Félix, Rute C.; Canario, Adelino; Power, DeborahNumerous studies have identified the detrimental effects for the biosphere of large plastic debris, the effect of microplastics (MPs) and nanoplastics (NPs) is less clear. The skin is the first point of contact with NPs, and skin fibroblasts have a vital role in maintaining skin structure and function. Here, a comparative approach is taken using three fibroblast cell lines from the zebrafish (SJD.1), human male newborn (BJ-5ta) and female adult (HDF/TERT164) and their response to polystyrene NP (PS-NPs) exposure is characterized. Cells were exposed to environmentally relevant PS-NP sizes (50, 500 and 1000 nm) and concentrations (0.001 to 10 mu g/ml) and their uptake (1000 nm), and effect on cell viability, proliferation, migration, reactive oxygen species (ROS) production, apoptosis, alkaline phosphatase (ALP) and acid phosphatase (AP) determined. All fibroblasts took up PSNPs, and a relationship between PS-NP particle size and concentration and the inhibition of proliferation and cell migration was identified. The inhibitory effect of PS-NPs on proliferation was more pronounced for human skin fibroblasts. The presence of PS-NPs negatively affected fibroblast migration in a time-, size- and concentration-dependent manner with larger PS-NPs at higher concentrations causing a more significant inhibition of cell migration, with human fibroblasts being the most affected. No major changes were detected in ROS production or apoptosis in NP challenged fibroblasts. While the ALP activity was increased in all fibroblast cell lines, only fish fibroblasts showed a significant increase in AP activity. The heterogeneous response of fibroblasts induced by PS-NPs was clearly revealed by the segregation of HDF, BJ.5ta and SJD.1 fibroblasts in principal component analysis. Our results demonstrate that PS-NP exposure adversely affected cellular processes in a celltype and dose-specific manner in distinct fibroblast cell lines, emphasizing the need for further exploration of NP interactions with different cell types to better understand potential implications for human health.
- Dilution of seawater affects the Ca2 + transport in the outer mantle epithelium of crassostrea gigasPublication . Sillanpää, J. Kirsikka; Cardoso, João CR; Félix, Rute; Anjos, Liliana; Power, Deborah; Sundell, KristinaVarying 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.
- Fish lysozyme gene family evolution and divergent function in early developmentPublication . Li, Lisen; Cardoso, João C. R.; Félix, Rute; Mateus, Ana Patricia; Canario, Adelino; Power, Deborah M.Lysozymes are an ancient group of antimicrobial enzymes of the innate immune system. Here we provide a comparative analysis of the evolution and function of lysozymes during early development in fish, the most speciose vertebrate group. In fishes, lineage and species-specific evolution of both C-type (chicken or conventional) and G-type (goose type) genes occurred. Phylogenetic analysis revealed that the teleost lysozyme G-type members group with the tetrapod homologues but the teleost C-type form three different clusters with the tetrapods. Most of the teleost C-type cluster with tetrapod Lyz but there are some that group with the mammalian Lyzl1/2 and LALBA. This suggests that early in gnathostome evolution these genes already existed and that lyzl1/2 and lalba genes are present in fish and tetrapods. Gene synteny analysis to confirm sequence orthologies failed to identify conserved genome regions between teleosts and other vertebrates lysozyme gene regions suggesting that in the ancestral bony fish genome lyz, lyzl1/2, lalba and lyg precursor genes were transposed to different chromosome regions. The homologue of the mammalian lactalbumin (LALBA) gene was identified for the first time in teleosts and was expressed in skin and during egg and larval development. Lysozyme activity was detected in teleost eggs and varied between species and in the gilthead sea bream lyg and lalba transcript abundance differed in eggs and larvae from different brood stock suggesting differences exist in maternal innate immune protection.
- STC1 interference on calcitonin family of receptors signaling during osteoblastogenesis via adenylate cyclase inhibitionPublication . Terra, Silvia R.; Cardoso, João CR; Félix, Rute; Martins, Leo Anderson M.; Souza, Diogo Onofre G.; Guma, Fatima C. R.; Canario, Adelino; Schein, VanessaStanniocalcin 1 (STC1) and calcitonin gene-related peptide (CGRP) are involved in bone formation/remodeling. Here we investigate the effects of STC1 on functional heterodimer complex CALCRL/RAMP1, expression and activity during osteoblastogenesis. STC1 did not modify CALCRL and ramp 1 gene expression during osteoblastogenesis when compared to controls. However, plasma membrane spatial distribution of CALCRL/RAMP1 was modified in 7-day pre-osteoblasts exposed to either CGRP or STC1, and both peptides induced CALCRL and RAMP1 assembly. CGRP, but not STC1 stimulated cAMP accumulation in 7-day osteoblasts and in CALCRL/RAMP1 transfected HEK293 cells. Furthermore, STC1 inhibited forskolin stimulated cAMP accumulation of HEK293 cells, but not in CALCRL/RAMP1 transfected HEK293 cells. However, STC1 inhibited cAMP accumulation in calcitonin receptor (CTR) HEK293 transfected cells stimulated by calcitonin. In conclusion, STC1 signals through inhibitory G-protein modulates CGRP receptor spatial localization during osteoblastogenesis and may function as a regulatory factor interacting with calcitonin peptide members during bone formation. (C) 2015 Elsevier Ireland Ltd. All rights reserved.
- Extracellular electrophysiological based sensor to monitor cancer cells cooperative migration and cell-cell connectionsPublication . Asgarifar, Sanaz; Mestre, Ana L. G.; Félix, Rute; Inacio, Pedro M. C.; Lurdes S. Cristiano, M.; Medeiros, Maria C. R.; Araújo, Inês; Power, Deborah; Gomes, Henrique L.Herein, we describe an electrophysiological based sensor that reproducibly monitors and quantifies in real-time collective migration and the formation of cell-cell junctions by C6 glioma cells seeded on top of electrodes. The signal amplitude and frequency generated by the migrating cells changed over time and these parameters were used to accurately calculate the migration speed. Electrophysiological measurements could also distinguish individual from collective cell migration. The migration of densely packed cells generated strong signals, while dispersed cells showed weak bioelectrical activity. We propose this electrophysiological technique as a cell-based biosensor to gain insight into the mechanisms of cooperative migration of cancer cells. Possible applications include screening for anti-migratory compounds, which may lead to the development of novel strategies for antineoplastic chemotherapy.
- PACAP system evolution and its role in melanophore function in teleost fish skinPublication . CR Cardoso, Joao; C. Félix, Rute; Martins, Rute; M., Trindade; G Fonseca, Vera; Fuentes, Xoan; Power, DeborahPituitary adenylate cyclase-activating polypeptide (PACAP) administered to tilapia melanophores ex-vivo causes significant pigment aggregation and this is a newly identified function for this peptide in fish. The G-protein coupled receptors (GPCRs), adcyap1r1a (encoding Pac1a) and vipr2a (encoding Vpac2a), are the only receptors in melanophores with appreciable levels of expression and are significantly (p < 0.05) down-regulated in the absence of light. Vpac2a is activated exclusively by peptide histidine isoleucine (PHI), which suggests that Pac1a mediates the melanin aggregating effect of PACAP on melanophores. Paradoxically activation of Pac1a with PACAP caused a rise in cAMP, which in fish melanophores is associated with melanin dispersion. We hypothesise that the duplicate adcyap1ra and vipr2a genes in teleosts have acquired a specific role in skin and that the melanin aggregating effect of PACAP results from the interaction of Pac1a with Ramp that attenuates cAMP-dependent PKA activity and favours the Ca(2+)/Calmodulin dependent pathway.