Browsing by Author "Santos, C. R. A."
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- Developmental ontogeny of prolactin and prolactin receptor in the sea bream (Sparus aurata)Publication . Santos, C. R. A.; Cavaco, J. E. B.; Ingleton, P. M.; Power, DeborahThe expression of PRL and its receptor (PRLR) were characterised during sea bream embryonic and larval development, by semi-quantitative and quantitative RT-PCR, respectively, until 46 days post-hatch (DPH). Immunocytochemistry with antisera specific for sea bream PRLR was carried out with larval sections from hatching up to 46 DPH. A single transcript of PRL (1.35 Kb) and PRLR (2.8 Kb) identical to the transcripts previously characterised in adult tissue, are present in sea bream embryos and larvae. PRL expression is first detectable at neurula and in all samples collected thereafter. The lowest levels of PRL mRNA are detected in sea bream embryos up until neurula when expression starts to increase. The maximal levels of PRL expression were detected at 24 DPH. PRLR transcripts first appear at 12 h post-fertilisation (0:002qmol=lg total larvae RNA) (blastula) and increase significantly during gastrulation (0:245qmol=lg total larvae RNA) reaching a maximum at 2 DPH (0:281qmol=lg total larvae RNA). After hatching a significant reduction in PRLR expression is observed which reaches a minimum at 4 DPH (0:103qmol=lg total larvae RNA), gradually increasing thereafter. Immunocytochemistry revealed the presence of PRLR in early post-hatching stages of larvae in tissues derived from all three germ layers.
- Evolution of the thyroid hormone-binding protein, transthyretinPublication . Power, Deborah; Elias, N. P.; Richardson, S. J.; Mendes, J.; Soares, M. C.; Santos, C. R. A.Transthyretin (TTR) belongs to a group of proteins, which includes thyroxine-binding globulin and albumin, that bind to and transport thyroid hormones in the blood. TTR is also indirectly implicated in the carriage of vitamin A through the mediation of retinol-binding protein (RBP). It was first identified in 1942 in human serum and cerebrospinal fluid and was formerly called prealbumin for its ability to migrate faster than serum albumin on electrophoresis of whole plasma.
- Isolation of a novel aquaglyceroporin from a marine teleost (Sparus auratus) Function and tissue distributionPublication . Santos, C. R. A.; Estêvão, Dulce; Fuentes, J.; Cardoso, João CR; Fabra, Mercedes; Passos, A. L.; Detmers, F. J.; Deen, P. M. T.; Cerda, J.; Power, DeborahThe aquaporins (formerly called the major intrinsic protein family) are transmembrane channel proteins. The family includes the CHIP group, which are functionally characterised as water channels and the GLP group, which are specialised for glycerol transport. The present study reports the identification and characterisation of a novel GLP family member in a teleost fish, the sea bream Sparus auratus. A sea bream aquaporin (sbAQP) cDNA of 1047·bp and encoding a protein of 298·amino acids was isolated from a kidney cDNA library. Functional characterization of the sbAQP using a Xenopus oocyte assay revealed that the isolated cDNA stimulated osmotic water permeability in a mercury-sensitive manner and also stimulated urea and glycerol uptake. Northern blotting demonstrated that sbAQP was expressed at high levels in the posterior region of the gut, where two transcripts were identified (1.6·kb and 2·kb), and in kidney, where a single transcript was present (2·kb). In situ hybridisation studies with a sbAQP riboprobe revealed its presence in the lamina propria and smooth muscle layer of the posterior region of the gut and in epithelial cells of some kidney tubules. sbAQP was also present in putative chloride cells of the gill. Phylogenetic analysis of sbAQP, including putative GLP genes from Fugu rubripes, revealed that it did not group with any of the previously isolated vertebrate GLPs and instead formed a separate group, suggesting that it may be a novel GLP member.
- Quantification of prolactin (PRL) and PRL receptor messenger RNA in gilthead Seabream (Spares aurata) after treatment with Estradiol-17 betaPublication . Cavaco, J. E. B.; Santos, C. R. A.; Ingleton, P. M.; Canario, Adelino V. M.; Power, DeborahProlactin (PRL) in fish is considered to be an osmoregulatory hormone, although some studies suggest that it may influence the production of steroid hormones in the gonads. The objective of the present study was to establish if PRL is involved in reproduction of the gilthead seabream—a protandrous hermaphrodite. Adult and juvenile gilthead seabream received implants of estradiol-17b (E2) for 1 wk during the breeding season, and the mRNA expressions of PRL and PRL receptor (sbPRLR) were determined. Northern blot analysis revealed a single pituitary PRL transcript, the expression of which was significantly reduced by E2 treatment in adults but significantly increased in juvenile fish. In adult gonads, four sbPRLR transcripts of 1.1, 1.3, 1.9, and 2.8 kilobases were observed. A competitive reverse transcription-polymerase chain reaction was developed and used to determine how E2 treatment alters expression of the gonadal sbPRLR gene. Seabream PRLR was detectable in all samples analyzed by this assay. Levels of sbPRLR mRNA increased significantly (50-fold) after E2 treatment in adults, but a 24-fold decrease was measured in juveniles. Immunohistochemistry using specific polyclonal antibodies raised against an oligopeptide from the extracellular domain of sbPRLR detected the receptor in spermatogonia and oocytes. Taken together, the preceding results suggest that in the seabream, PRL may act on both testis and ovary via its receptor and that the stage of maturity influences this process. The full characterization and relative importance of the different transcripts of sbPRLR in eliciting the action of PRL in the gonads remain to be elucidated.
- Regulation of transthyretin by thyroid hormones in WshPublication . Morgado, Isabel; Santos, C. R. A.; Jacinto, R.; Power, DeborahTransthyretin (TTR) is a thyroid hormone-binding protein (THBP) which in its tetrameric form transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. The principal site of production of TTR is the liver but in the sea bream TTR mRNA is also present in the heart, intestine and brain. The regulation of TTR is unstudied in Wsh and the normal circulating level of this THBP is unknown. The aim of the present study was to establish factors which regulate TTR production in Wsh. As a Wrst step a number of tools were generated; sea bream recombinant TTR (sbrTTR) and speciWc sbrTTR antisera which were used to establish an ELISA (enzyme-linked immunosorbent assay) for measuring TTR plasma levels. Subsequently, an experiment was conducted to determine the inXuence of THs on TTR production. Circulating physiological levels of TTR in sea bream determined by ELISA are approximately 3.8 gml¡1. Administration of T3 and T4 to sea bream signiWcantly increased (p< 0.001 and p<0.005, respectively) the concentration of circulating TTR (V11.5 gml¡1) in relation to control Wsh, but did not change gene transcription in the liver. Methimazol (MMI) an antithyroid agent, failed to signiWcantly reduce circulating THs below control levels but signiWcantly increased (p < 0.005) plasma TTR levels (approximately 10.8 gml¡1) and decreased (p< 0.05) transcription in the liver. Future studies will aim to elucidate in more detail these regulatory pathways.
- The effect of food deprivation and refeeding on the liver, thyroid hormones and transthyretin in sea breamPublication . Power, Deborah; Melo, J.; Santos, C. R. A.There was a significant reduction in body weight of sea bream Sparus aurata and the hepatosomatic index after 3 weeks food deprivation. Liver biochemical indices and morphology were altered by food deprivation, there was a loss of eosin staining in hepatocyte cytoplasm and the appearance of large depleted vacuoles. Cell and nuclear area were significantly reduced (P<0·001) and did not return to control values after 1 week of refeeding. The water, lipid, glycogen and protein content of the liver was significantly reduced by 3 weeks fasting but recovered rapidly after 1 week refeeding. Food restriction also had a marked effect on circulating thyroid hormones and the concentration of plasma T3 (33·98 12·47 ng ml 1) and T4 (16·54 9·5 ng ml 1) was significantly (P<0·001) lower than the control (66·52 13·4 and 56·83 7·3, respectively). Refeeding for 1 week restored circulating T3 (P<0·05) close to control levels but did not significantly affect the concentration of T4. Northern blotting with an homologous probe for transthyretin (TTR) demonstrated clearly the expression of a single mRNA transcript of 0·7 kb for this protein in the liver. The level of TTR message was substantially reduced below control levels in the liver of fasted fish and 1 week refeeding failed to restore expression.