Percorrer por autor "Guerreiro, P. M."
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- Branchial osmoregulatory response to salinity in the gilthead sea bream,Sparus auratusPublication . Laiz-Carrión, R.; Guerreiro, P. M.; Fuentes, J.; Canario, Adelino V. M.; Martín Del Río, María P.; Mancera, J. M.The branchial osmoregulatory response of gilthead sea bream (Sparus auratus L.) to short-term (2–192 hr) and long-term (2 weeks) exposure to different environmental salinities (5%, 15%, 25%, 38% and 60%) was investigated. A ‘‘U-shaped’’ relationship was observed between environmental salinity and gill Naþ,Kþ-ATPase activity in both long- and short-term exposure to altered salinity, with the increase in activity occurring between 24 and 96 hr after the onset of exposure. Plasma osmolality and plasma ions (sodium, chloride, calcium and potassium) showed a tendency to increase in parallel with salinity. These variables only differed significantly (Po0.05) in fish adapted to 60% salinity with respect to fish adapted to full-strength sea-water (SW). Plasma glucose remained unchanged whereas plasma lactate was elevated at 5% and 60%. Muscle water content (MWC) was significantly lower in fish adapted to 60%. Chloride cells (CC) were only present on the surface of the gill filaments and absent from the secondary lamellae. CC distribution was not altered by external salinity. However, the number and size of CC were significantly increased at salinity extremes (5% and 60%), whereas fish exposed to intermediate salinities (15% and 25%) had fewer and smaller cells. Furthermore, the CC of fish exposed to diluted SW became rounder whereas they were more elongated in fish in full-strength and hypersaline SW. This is consistent with previous reports indicating the existence of two CC types in euryhaline fish. At likely environmental salinities, gilthead sea bream show minor changes in plasma variables and the effective regulation of gill Naþ,Kþ-ATPase. However, at very low salinities both haemodilution and up-regulation of gill Naþ,Kþ-ATPase predict a poor adaptation most likely related to deficiency or absence of specific components of the CC important for ion uptake.
- Calcium balance in sea bream (Sparus aurata): the effect of oestradiol-17 betaPublication . Guerreiro, P. M.; Fuentes, J.; Canario, Adelino V. M.; Power, DeborahIn all teleost fishes vitellogenesis is triggered and maintained by oestradiol-17 (E2) and is accompanied by an increase of blood plasma calcium and phosphate. The action of this hormone on calcium metabolism was investigated by treating fast-growing immature juvenile sea bream (Sparus aurata) with coconut butter implants alone (control) or implants containing 10 μg/g E2. Treatment with E2 induced the production of circulating vitellogenin, a 2·5-fold increase in plasma ionic Ca2+ and a 10-fold increase in plasma total calcium, largely bound to protein. In contrast to freshwater species, which obtain most of their calcium from the environment directly through the gills, the intestinal component of calcium uptake of the salt water-living sea bream represented up to 60–70% of the total uptake. The whole body calcium uptake, expressed as the sum of calcium obtained via intestinal and extra-intestinal (likely branchial) routes increased significantly in response to E2. Combined influx and unchanged efflux rates resulted in a significant 31% increase in net calcium uptake. There was no evidence for an effect of E2 on the calcium and phosphate content of the scales or the tartrate-resistant acid phosphatase activity (an index for bone/scale osteoclast activity). While most freshwater fish appear to rely on internal stores of calcium, i.e. bone and/or scales to increase calcium availability, the marine sea bream accommodates calcium-transporting mechanisms to obtain calcium from the environment and preserve internal stores. These observations suggest that a fundamental difference may exist in the E2-dependent calcium regulation between freshwater and marine teleosts.
- Calcium mobilization from fish scales is mediated by parathyroid hormone related protein via the parathyroid hormone type 1 receptorPublication . Rotllant, J.; Redruello, Begoña; Guerreiro, P. M.; Fernandes, H.; Canario, Adelino V. M.; Power, DeborahThe scales of bony fish represent a significant reservoir of calcium but little is known about their contribution, as well as of bone, to calcium balance and how calcium deposition and mobilization are regulated in calcified tissues. In the present study we report the action of parathyroid hormone-related protein (PTHrP) on calcium mobilization from sea bream (Sparus auratus) scales in an in vitro bioassay. Ligand binding studies of piscine 125I-(1 – 35tyr)PTHrP to the membrane fraction of isolated sea bream scales revealed the existence of a single PTH receptor (PTHR) type. RT-PCR of fish scale cDNA using specific primers for two receptor types found in teleosts, PTH1R, and PTH3R, showed expression only of PTH1R. The signalling mechanisms mediating binding of the N-terminal amino acid region of PTHrP were investigated. A synthetic peptide (10 8 M) based on the N-terminal 1 – 34 amino acid residues of Fugu rubripes PTHrP strongly stimulated cAMP synthesis and [3 H]myo-inositol incorporation in sea bream scales. However, peptides (10 8 M) with N-terminal deletions, such as (2 – 34), (3 – 34) and (7 – 34)PTHrP, were defective in stimulating cAMP production but stimulated [3 H]myo-inositol incorporation. (1 – 34)PTHrP induced significant osteoclastic activity in scale tissue as indicated by its stimulation of tartrate-resistant acid phosphatase. In contrast, (7 – 34)PTHrP failed to stimulate the activity of this enzyme. This activity could also be abolished by the adenylyl cyclase inhibitor SQ-22536, but not by the phospholipase C inhibitor U-73122. The results of the study indicate that one mechanism through which N-terminal (1 – 34)PTHrP stimulates osteoclastic activity of sea bream scales, is through PTH1R and via the cAMP/AC intracellular signalling pathway. It appears, therefore, that fish scales can act as calcium stores and that (1 – 34)PTHrP regulates calcium mobilization from them; it remains to be established if this mechanism contributes to calcium homeostasis in vivo.
- Cloning and expression of an elongation factor-1α in sea bream ( Sparus aurata ) larvae and adult tissuePublication . Nowell, M. A.; Power, Deborah; Guerreiro, P. M.; Llewellyn, Lynda; Ramsurn, Vimi P.; Wigham, Trevor; Sweeney, Glen E.A clone encoding the polypeptide elongation factor EF-1a was isolated from a complementary DNA library prepared from sea bream (Spartus aurata) larvae 1 to 10 days after hatching. The deduced amino acid sequence is between 82% and 95% similar to EF-1a in other animal species. EF-1a messenger RNA is present at low abundance in sea bream embryos prior to gastrulation, but at around 15 hours postfertilization, there is a 10-fold increase in transcript levels. This increase presumably reflects midblastula transition in this species. In adult sea bream, EF-1a appeared to have a relatively uniform distribution across all the tissues analyzed.
- Cloning of the cDNA for sea bream (Sparus aurata) parathyroid hormone-related proteinPublication . Flanagan, J. A.; Power, Deborah; Bendell, L. A.; Guerreiro, P. M.; Fuentes, J.; Clark, M. S.; Canario, Adelino V. M.; Danks, J. A.; Brown, B. L.; Ingleton, P. M.This paper reports cloning of the cDNA for sea bream (Sparus aurata) parathyroid hormone-related protein (PTHrP). The gene codes for a 125-amino acid mature protein with a 35-residue prepeptide. The total gene sequence is 1.8 kb with approximately 75% noncoding. The N-terminus of the protein resembles mammalian and chicken PTHrP peptides with 12 of the first 21 amino acids identical and for which there is homology with mammalian parathyroid hormone. Toward the C-terminus, the nuclear transporter region between residues 79 and 93 in sea bream is 73% homologous to tetrapod PTHrP, and the RNA binding domain, 96–117, is 50% homologous, moreover starting with the conserved lysine and terminating with the lysine/arginine sequence. Sea bream PTHrP differs significantly from mammalian and chicken PTHrP, having a novel 16-amino acid segment between residues 38 and 54 and completely lacking the terminal domain associated in mammals with inhibition of bone matrix lysis. RT-PCR and in situ hybridization of sea bream tissues show that the gene is expressed widely and the results confirm observations of a PTHrP-like factor in sea bream detected with antisera to human PTHrP.
- Cloning of the cDNA for the putative calcium-sensing receptor and its tissue distribution in sea bream (Sparus aurata)Publication . Flanagan, J. A.; Bendell, L. A.; Guerreiro, P. M.; Clark, M. S.; Power, Deborah; Canario, Adelino V. M.; Brown, B. L.; Ingleton, P. M.The cDNA for the calcium-sensing receptor (CaSR) gene has been cloned from the marine teleost Sparus aurata, the sea bream. The isolated clones were 3.3 kb long with an open reading frame of 2820 bp, a 50 UTR of 240 bp, and 30 UTR of 248 bp. The gene codes for a mature peptide of 940 amino acids which has three principal domains; the extracellular region is more than half the total protein, there is a seven-transmembrane domain, and there is a short intracellular domain. There is considerable sequence identity, 91%, shared between the CaSR of sea bream and puffer fish but overall similarities with mammalian CaSR peptides vary between 44% for rat and mouse and 48% with human CaSR. Nevertheless, the 18 cysteine residues of the extracellular domain are present in all sequences so far analysed of which 9 form a cysteine-rich region in sea bream similar to mammalian CaSR. The distribution of CaSR in sea bream tissues detected by in situ hybridisation showed gene expression in epithelia associated with ion transport or ion regulation including the hind gut, chloride cells of the gills, operculum, gall bladder, pituitary adenohypophysis, and coronet cells of the saccus vasculosus; this distribution was confirmed by RT-PCR. By in situ hybridisation, CaSR gene expression was also present in olfactory nerves and leucocytes.
- Cortisol and parathyroid hormone-related peptide are reciprocally modulated by negative feedbackPublication . Guerreiro, P. M.; Rotllant, J.; Fuentes, J.; Power, Deborah; Canario, Adelino V. M.In previous in vitro studies, we have shown that the N-terminal region of parathyroid hormone-related protein (PTHrP) can stimulate cortisol production in sea bream, Sparus auratus, interrenal tissue, possibly through a paracrine action. In the current study, the systemic interaction between cortisol and PTHrP was studied in vivo. Sustained elevated blood cortisol levels, induced either by cortisol injection or conWnement stress, suppressed circulating PTHrP 6 and 24-fold,respectively, by comparison to control Wsh.reduced cortisol levels, prevented the decrease of plasma PTHrP observed in conWned Wsh and raised plasma PTHrPrespectively, by comparison to control fish.
- Determination of tissue and plasma concentrations of PTHrP in fish: development and validation of a radioimmunoassay using a teleost 1–34 N-terminal peptidePublication . Rotllant, J.; Worthington, G. P.; Fuentes, J.; Guerreiro, P. M.; Teitsma, C. A.; Ingleton, P. M.; Balment, R.; Canario, Adelino V. M.; Power, DeborahA specific and sensitive radioimmunoassay (RIA) for the N-terminus of sea bream (Sparus auratus) and flounder (Platichthys flesus) parathyroid hormone-related protein (PTHrP) was developed. A (1–34) amino-terminal sequence of flounder PTHrP was synthesized commercially and used as the antigen to generate specific antiserum. The same sequence with an added tyrosine (1– 35Tyr) was used for iodination. Human (1–34) parathyroid hormone (PTH), human (1–34) PTHrP, and rat (1–34) PTHrP did not cross-react with the antiserum or displace the teleost peptide. Measurement of PTHrP in fish plasma was only possible after denaturing by heat treatment due to endogenous plasma binding activity. The minimum detectable concentration of (1–34) PTHrP in the assay was 2.5 pg/tube. The level of immunoreactive (1–34) PTHrP in plasma was 5.2 0.44 ng/ml (mean SEM, n ¼ 20) for flounder and 2.5 0.29 ng/ml (n ¼ 64) for sea bream. Dilution curves of denatured fish plasma were parallel to the assay standard curve, indicating that the activity in the samples was indistinguishable immunologically from (1–34) PTHrP. Immunoreactivity was present, in order of abundance, in extracts of pituitary, oesophagus, kidney, head kidney, gills, intestine, skin, muscle, and liver. The pituitary gland and oesophagus contained the most abundant levels of PTHrP, 37.7 6.1 ng/g wet tissue and 2.3 0.7 ng/g wet tissue, respectively. The results suggest that in fish PTHrP may act in a paracrine and/or autocrine manner but may also be a classical hormone with the pituitary gland as a potential major source of the protein.
- Divergent responsiveness of the dentary and vertebral bone to a selective estrogen-receptor modulator (SERM) in the teleost Sparus auratusPublication . Vieira, Florbela A.; Pinto, Patricia IS; Guerreiro, P. M.; Power, DeborahIn teleosts the regulation of skeletal homeostasis and turnover by estrogen is poorly understood. For this reason raloxifene, a selective estrogen-receptor modulator (SERM), was administered to sea bream (Sparus auratus) and its effect on plasma calcium balance and transcript expression in dentary (dermal bone) and vertebra (perichondral bone) was studied. The concentration of total calcium or phosphorus in plasma was unchanged by raloxifene treatment for 6 days. The activity of alkaline phosphatase (ALP) in dentary bone of raloxifene treated fish was significantly (p < 0.05) higher than control fish but it was not changed in vertebral bone. Transcripts for estrogen receptor (ER) a were in very low abundance in the sea bream dentary and vertebra and were unchanged by raloxifene treatment. In contrast, raloxifene caused significant (p < 0.05) up-regulation of the duplicate ERb transcripts in the dentary but did not affect specific transcripts for osteoclast (TRAP), osteoblast (ALP, Runx2, osteonectin) or cartilage (IGF1, CILP2, FN1a). In the vertebra ERbb was not changed by raloxifene but ERba was significantly (p < 0.05) down-regulated as was the skeletal specific transcripts, TRAP, ALP, CILP2, FN1a. In summary, ERbs regulate estrogen sensitivity of the skeleton in sea bream, which responds in a non uniform manner. In common with mammals raloxifene appears to have an anti-resorptive role (in sea bream vertebra), but also an osteoblast stimulatory role, inducing ALP activity in the dentary of sea bream. Overall, the results indicate bone specific responsiveness to raloxifene in the sea bream. Further work will be required to understand the basis of bone responsiveness and the role of E2 and ERs in teleost bone homeostasis.
- Dynamics of scale regeneration in seawater - and brackish water-acclimated sea bass, Dicentrarchus labraxPublication . Guerreiro, P. M.; Costa, Rita; Power, DeborahScale loss is a common occurrence in both wild fish and those in aquaculture production systems, and regeneration of scales has been described in several freshwater species. Relatively little information exists about this process in marine fish, and in the present study, the chronology of scale regeneration was characterized in juvenile sea bass Dicentrarchus labrax, maintained in full seawater (SW; 36 %, 11.2 mM Ca2?) or brackish water (BW; 3.5 %, 1.1 mM Ca2?). Despite the significant differences in plasma osmolality, plasma calcium (Ca) and phosphorus (P) were similar between SW and BW.
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