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D M Power

Publications and source records attributed to D M Power.

At least 19 recordsLinked to original sources

Identification of estrogen-responsive genes in the testis of sea bream (Sparus auratus) using suppression subtractive hybridization.

There is growing evidence that estrogens play important roles in both normal and xenoestrogen disrupted testis physiology. However, the mechanisms and signaling pathways involved, in particular in fish, are largely unknown. We have used suppression subtractive hybridization to isolate 152 candidate estrogen-responsive genes in the testis of male estradiol (E2)-treated sea bream (Sparus aurata). The E2 up-regulation of some of the genes (e.g., choriogenin L and H, vitellogenin I and II, apolipoprotein A-I, fibrinogen beta and gamma, and thyroid receptor interacting protein 4) was confirmed by reverse transcriptase polymerase chain reaction in fish treated with 0.1-10 mg/kg E2. Many of these genes are typical E2-induced genes in liver, and this is the first report of its up regulation with E2 in testis. Moreover, low levels of expression were also found for nontreated fish. Hepatic differential expression for these genes was also confirmed, although, contrary to testis, fibrinogen beta, and gamma were downregulated. The possible significance of these findings in normal testis physiology and in endocrine disruption is discussed.

Animals↗

Regulation of troponin T expression during muscle development in sea bream Sparus auratus Linnaeus: the potential role of thyroid hormones.

In the sea bream Sparus auratus three stage-specific fast troponin T (fTnT) isoforms have been cloned and correspond to embryonic-, larval- and adult-specific isoforms. Characterisation, using database searches, of the putative genomic organisation of Fugu rubripes and Tetraodon nigroviridis fTnT indicates that alternative exon splicing in the 5 region of the gene generates the different isoforms. Moreover, comparison of teleost fTnTs suggests that alternative splicing of fTnT appears to be common in teleosts. A different temporal expression pattern for each fTnT splice varotnt is found during sea bream development and probably relates to differing functional demands, as a highly acidic embryonic form (pI 5.16) is substituted by a basic larval form (pI 9.57). Thyroid hormones (THs), which play an important regulatory role in muscle development in flatfish and tetrapods, appear also to influence TnT gene expression in the sea bream. However, THs have a divergent action on different sea bream TnT genes and although the slow isoform (sTnT1) is TH-responsive, fTnT, sTnT2 and the itronless isoform (iTnT) are unaffected. The present results taken together with those published for flatfish seem to suggest differences may exist in the regulation of larval muscle development in teleosts.

Aging↗

Ligand binding and signalling pathways of PTH receptors in sea bream (Sparus auratus) enterocytes.

Whole animal studies have indicated that Ca(2+) 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 amino-terminal PTHrP peptides, the amino acid residues important for receptor activation and for stabilising the ligand/receptor complex. Ligand binding of (125)I-(1-35(tyr)) PTHrP to the membrane fraction of isolated sea bream enterocytes revealed the existence of a single saturable high-affinity receptor (K (D)=2.59 nM; B (max)=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 amino-terminal 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 Ca(2+) 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.

Amino Acid Sequence↗

Calcium mobilization from fish scales is mediated by parathyroid hormone related protein via the parathyroid hormone type 1 receptor.

The 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-(1-35(tyr))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 [3H]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 [3H]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.

Animals↗

Identification and analysis of teleost slow muscle troponin T (sTnT) and intronless TnT genes.

In the present study cDNA clones representing two slow skeletal muscle troponin T genes (sTnT1sb and sTnT2sb) in the sea bream (Sparus auratus), an important aquaculture species, were isolated and characterised. A third, intronless, TnT gene (iTnTsb), which is an apparent orthologue of a previously described zebrafish TnT, was also isolated. In adult sea bream sTnT expression was restricted to red muscle and, using northern blotting, a single low abundance transcript was identified for sTnT1sb (1260 nucleotides) and a single high abundance transcript was identified for sTnT2sb (1000 nucleotides). In contrast, iTnTsb is predominantly expressed in adult fast muscle. All three TnT genes are also expressed during larval development. Phylogenetic analysis of sea bream sTnT proteins to identify maximum parsimony showed that iTnTsb, sTnT1sb and sTnT2sb each cluster in independent groups. sTnT1sb clustered with other vertebrate sTnTs, while sTnT2 clustered with a group of fish specific sequences (from Fugu rubripes, Oryzia latipes and Salmo trutta). The teleost sTnT2 and iTnT each constitute new, apparently teleost specific, TnT groups. Analysis of the corresponding Fugu scaffold indicates that sTnT2sb is encoded by a gene with twelve exons. The two sTnT cDNAs isolated in sea bream probably arose by duplication of an ancestral gene, and iTnT by reverse transcription. It remains to be established if the encoded proteins have different structural and mechanistic roles in fish muscle.

Amino Acid Sequence↗

Production and characterisation of gilthead sea bream (Sparus auratus) recombinant parathyroid hormone related protein.

The production and purification of gilthead sea bream recombinant parathyroid hormone related protein [sbPTHrP(1-125)] using an Escherichia coli system and one step purification 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-beta-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 specific for the N-terminal and C-terminal region of PTHrP. In a radioimmunoassay specific for piscine N-terminal (1-34aa) PTHrP, the recombinant sbPTHrP(1-125) was equipotent with PTHrP(1-34) in displacing labelled (125)I-PTHrP(1-36) PTHrP from the antisera. The availability of recombinant sbPTHrP will allow the development of region specific assays and studies aimed at defining post-secretory processing of this protein and its biological activity in fish.

Amino Acid Sequence↗

Ontogeny of osteonectin expression in embryos and larvae of sea bream (Sparus auratus).

Osteonectin (OSN) is a glycoprotein which is implicated in development, bone formation and mineralisation, tumorigenesis, angiogenesis, and wound healing. Regulation of its expression by hormones may be one of the mechanisms by which the endocrine system affects bone metabolism. As a first step to understanding OSN function in fish, the gene expression of the recently cloned cDNA for sea bream, Sparus auratus, osteonectin (sbOSN) was characterised during embryonic and larval development. sbOSN mRNA was first detected by semi-quantitative reverse transcription-polymerase chain reaction in embryos at early gastrula and its expression increased continuously until hatch, after which it decreased until 15 days post-hatch (dph), increased transiently until 24 dph and decreased thereafter. In situ hybridisation showed it had a differential tissue distribution which was age dependent. In general, sbOSN mRNA was identified in cartilaginous and calcified structures of both dermal and endochondral origin but its expression was not restricted to the skeleton. sbOSN transcripts were also detected in the skin, perichordal sheath, nerve cord, and kidney tubules.

Aging↗

Sea bream (Sparus auratus) estrogen receptors: phylogeny and tissue distribution.

Most estrogen actions are mediated by two estrogen receptor subtypes (ERalpha and ERbeta). While a single ERbeta appears to be present in higher vertebrates, two forms of ERbeta encoded by different genes have recently been isolated in some teleost fish species. We investigated whether this also applies to the hermaphrodite sparid sea bream (Sparus auratus) and cloned a second ERbeta (sbERbeta2) in this species. We have also compared the tissue distribution of the three receptors as yet identified in sea bream, designated sbERalpha, sbERbeta1, and sbERbeta2.

Animals↗

The secretin G-protein-coupled receptor family: teleost receptors.

Twenty-one members of the secretin family (family 2) of G-protein-coupled receptors (GPCRs) were identified via directed cloning and data-mining of the Fugu Genome Consortium database, representing the most comprehensive description of secretin GPCRs in a teleost fish to date. Duplicated genes were identified for many of the family members, namely the receptors for pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP), calcitonin, calcitonin gene-related peptide (CGRP), growth hormone releasing hormone (GHRH), glucagon receptor/glucagon-like peptide (GLP) and parathyroid hormone-related peptide (PTHrP)/PTH. Mining of other teleost genomes (zebrafish and Tetraodon) revealed that the duplicated genes identified in the Takifugu genome were also present in these fish. Additional database searching of the Escherichia coli, yeast, Drosophila, Caenorhabditis elegans and Ciona genomes revealed that the family 2 of GPCRs were only present in the multicellular organisms. Orthologues of all the human secretin receptors were identified with the exception of secretin itself. Additional database searches in the Fugu Genome Consortium database also failed to reveal a secretin ligand and so it is hypothesised that both the receptor and the ligand evolved after the divergence of teleost/tetrapod lineages. Phylogenetic analysis at both the protein and the DNA level provided strong support for each of the individual receptor family groupings, but weak support between groups, making evolutionary inferences difficult. A more critical analysis of the PACAP/VIP receptor family confirmed previous hypotheses that the vasoactive intestinal peptide receptor (VPAC(1)R) gene is the ancestral form of the receptor.

Animals↗

Developmental ontogeny of prolactin and its receptor in fish.

Prolactin (PRL) is a member of a family of structurally similar proteins which includes growth hormone (GH) and somatolactin (SL) in teleost fish. The genes encoding these proteins are expressed principally in the pituitary gland and sequence analysis reveals they share considerable similarity. GH, PRL, and SL bring about their physiological action by binding to specific receptors localised in the membrane of cells in target tissue. The PRL receptor (PRLR) and GH receptor (GHR) have been identified in a number of teleosts but the SL receptor remains to be characterised. On hormone binding, receptors dimerise, and signal transduction occurs via the JAK/STAT signalling pathway. The principal action of PRL in fish is freshwater osmoregulation, although it has also been implicated in reproduction, behaviour, growth, and immunoregulation. The role of PRL in early development and metamorphosis is well established, respectively, in mammals and amphibians, although its role in fish is not so well known. Studies have shown that PRL mRNA and protein are restricted to the developing pituitary gland in fish embryos and larvae. PRLR mRNA and protein is also present in fish embryos and has a widespread tissue distribution in larvae. The levels of PRLR and PRL mRNA vary throughout embryonic and early larval development. The potential role of PRL in fish embryos and larvae is considered in relation to their physiological status.

Aging↗

Stimulation of cortisol release by the N terminus of teleost parathyroid hormone-related protein in interrenal cells in vitro.

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.

Adenylyl Cyclases↗

Isolation of a novel aquaglyceroporin from a marine teleost (Sparus auratus): function and tissue distribution.

The 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.

Amino Acid Sequence↗

Duplicated receptors for VIP and PACAP (VPAC1R and PAC1R) in a teleost fish, Fugu rubripes.

Two principal groups of receptors orthologous with human PAC1R and VPAC1R and were identified and characterised at the genomic level in the teleost fish Fugu rubripes. An additional group orthologous with VPAC2R was also identified and partially characterised. In Fugu, gene duplication of each of the PAC1Rs, VPAC1Rs and VPAC2Rs appears to have occurred. The topology of the tree surrounding the Fugu duplications and other isolated piscine sequences indicates that the duplication events for these six genes clearly preceded the speciation event leading to the Cypriniformes and Tetraodontiformes and is probably teleost-specific. Overall, the combined pattern of gene expression for each pair of duplicated genes mirrored the expression in other vertebrates. However, within each pair of duplicates further specialisation had occurred, with each demonstrating differential tissue distribution profiles suggesting they that may be responsible for the divergent action of the ligands, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP). The Fugu VPAC1R gene regions showed conserved synteny with human chromosome 3p21.3 and also C. elegans chromosome X, indicating that the putative ancestral human chromosome 3 region may be equivalent to chromosome X in Caenorhabditis elegans.

Amino Acid Sequence↗

Developmental ontogeny of prolactin and prolactin receptor in the sea bream (Sparus aurata).

The 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 12h post-fertilisation (0.002 rho mol/microg total larvae RNA) (blastula) and increase significantly during gastrulation (0.245 rho mol/microg total larvae RNA) reaching a maximum at 2 DPH (0.281 rho mol/microg total larvae RNA). After hatching a significant reduction in PRLR expression is observed which reaches a minimum at 4 DPH (0.103 rho mol/microg 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.

Aging↗

The regulatory action of estrogen and vasoactive intestinal peptide on prolactin secretion in sea bream (Sparus aurata, L.).

The effect of estradiol-17beta (E(2)) implants on the in vitro secretion of prolactin (PRL) and its modulation by vasoactive intestinal peptide (VIP) in a marine teleost, sea bream (Sparus aurata L.), was determined. Experiments were conducted during winter and spring. During winter, fish (n=130, body weight 50-70 g) were randomly divided into 2 groups; control and E(2) treated (10 mg/kg, wet weight). Fish were sacrificed after 7 days treatment and in vitro pituitary cultures in Ringer bicarbonate supplemented with increasing doses (0-200 nM) of VIP were carried out for 18 h. Culture medium was analysed by PAGE and secreted PRL quantified by densitometry. Fish treated with E(2) secreted significantly more PRL (P<0.05) in vitro than control fish. In E(2) primed fish VIP caused a dose-dependent inhibition of PRL secretion in vitro. VIP had no detectable effect on the secretion of PRL from control pituitaries. Treatment with E(2) had a different effect during spring; PRL secretion was significantly decreased (P<0.01) compared with the control fish. Anatomical evidence of abundant VIP immunoreactive nerve fibres in neurohypophysial (NH) tissue penetrating the rostral pars distalis provide further evidence supporting an action for VIP in the regulation of PRL cells. In conclusion, the responsiveness of PRL in the pituitary gland varied with season. Moreover, in the sea bream VIP appears to modulate PRL secretion from E(2) primed pituitary glands.

Animals↗

Determination of tissue and plasma concentrations of PTHrP in fish: development and validation of a radioimmunoassay using a teleost 1-34 N-terminal peptide.

A 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 (1-35(Tyr)) 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=20) for flounder and 2.5+/-0.29 ng/ml (n=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.

Animals↗

Cloning of the cDNA for the putative calcium-sensing receptor and its tissue distribution in sea bream (Sparus aurata).

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 5' UTR of 240 bp, and 3' 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.

Amino Acid Sequence↗