Browsing by Author "Ingleton, P. M."
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- Calcitonin: characterisation and expression in a teleost fish, Fugu rubripesPublication . Clark, M. S.; Bendell, L. A.; Power, Deborah; Warner, S.; Elgar, Greg; Ingleton, P. M.The present report describes the structure and expression of the calcitonin gene in Fugu rubripes. It is composed of 4 exons and 3 introns. Splicing of exons 1, 2 and 3 generates the calcitonin pre-proprotein, while splicing of exons 1, 2 and 4 generates calcitonin gene-related protein (CGRP). Exons 1 and 2 encoding the signal sequence and the N-terminal peptide are common in both the gene products and this gene organisation has been conserved in human, rat, chicken and salmon. The gene environment around calcitonin in Fugu has been poorly conserved when compared with human, apart from a small gene cluster. The calcitonin gene in Fugu has a widespread tissue distribution but it is most highly expressed in the brain. The abundance of gene expression in the ultimobranchial gland and the pituitary indicates that these are important sites of production and that the peptide is probably secreted into the circulation and/or acts as a paracrine or autocrine controlling factor. Whilst the function of calcitonin in fish is still largely unknown, the distribution described here suggests that one of the potential functions may be as a neuropeptide.
- Calcitonin: characterisation and expression in a teleost fish, Fugu rubripesPublication . Clark, Melody; Bendell, L; Power, DM; Warner, S; Elgar, Greg; Ingleton, P. M.The present report describes the structure and expression of the calcitonin gene in Fugu rubripes. It is composed of 4 exons and 3 introns. Splicing of exons 1, 2 and 3 generates the calcitonin pre-proprotein, while splicing of exons 1, 2 and 4 generates calcitonin gene-related protein (CGRP). Exons 1 and 2 encoding the signal sequence and the N-terminal peptide are common in both the gene products and this gene organisation has been conserved in human, rat, chicken and salmon. The gene environment around calcitonin in Fugu has been poorly conserved when compared with human, apart from a small gene cluster. The calcitonin gene in Fugu has a widespread tissue distribution but it is most highly expressed in the brain. The abundance of gene expression in the ultimobranchial gland and the pituitary indicates that these are important sites of production and that the peptide is probably secreted into the circulation and/or acts as a paracrine or autocrine controlling factor. Whilst the function of calcitonin in fish is still largely unknown, the distribution described here suggests that one of the potential functions may be as a neuropeptide.
- Characterization of osteocalcin (BGP) and matrix gla protein (MGP) fish specific antibodies: validation for immunodetection studies in lower vertebratesPublication . Simes, D; Williamson, M. K.; Schaff, Brian J.; Gavaia, Paulo J.; Ingleton, P. M.; Price, P. A.; Cancela, LeonorIn fish species the basic mechanisms of bone development and bone remodeling are not fully understood. The classification of bone tissue in teleosts as cellular or acellular and the presence of transitional states between bone and cartilage and the finding of different types of cartilage in teleosts not previously recognized in higher vertebrates emphasizes the need for a study on the accumulation of the Gla-containing proteins MGP and BGP at the cellular level. In the present study, polyclonal antibodies developed against BGP and MGP from A. regius (a local marine teleost fish) and against MGP from G. galeus (a Pacific Ocean shark), were tested by Western blot for their specificity against BGP and MGP from several other species of teleost fish and shark. For this purpose we extracted and purified both proteins from various marine and freshwater teleosts, identified them by N-terminal amino acid sequence analysis and confirmed the presence of gamma- carboxylation in the proteins with the use of a stain specific for Gla residues. Each antibody recognized either BGP or MGP with no cross-reaction between proteins detected. All purified fish BGPs and MGPs tested were shown to be specifically recognized, thus validating the use of these antibodies for further studies.
- 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.
- Cloning, characterization, and tissue distribution of prolactin receptor in the sea bream (Sparus aurata)Publication . Santos, Cecilia; Ingleton, P. M.; Cavaco, J. E. B.; Kelly, P. A.; Edery, M.; Power, DeborahThe prolactin receptor (PRLR) was cloned and its tissue distribution characterized in adults of the protandrous hermaphrodite marine teleost, the sea bream (Sparus aurata). An homologous cDNA probe for sea bream PRLR (sbPRLR) was obtained by RT-PCR using gill mRNA. This probe was used to screen intestine and kidney cDNA libraries from which two overlapping clones (1100 and 2425 bp, respectively) were obtained.
- Cloning, expression, and tissue localisation of prolactin in adult sea bream (Sparus aurata)Publication . Santos, Cecilia; Brinca, Lilia; Ingleton, P. M.; Power, DeborahA major action of prolactin (PRL) in teleost fish is the maintenance of hydromineral balance in euryhaline species in fresh water. The function of PRL in marine teleosts is less certain and unlike euryhaline teleosts, such as tilapia and salmon, there is relatively little information about protein or gene structure. Associated with studies to determine potential functions of PRL, pituitary prolactin cDNA has been cloned and sequenced from sea bream (Sparus aurata), a marine teleost. The sequence obtained spanned 1349 bp and contained an open reading frame encoding a protein of 212 amino acids composed of a putative signal peptide of 24 residues and a mature protein of 188 amino acids. N-terminal sequencing of the native protein confirmed unambiguously the cleavage site, Ala24, Val25, predicted from alignments of the sea bream PRL cDNA with that of other teleosts. The presence of only one form of PRL in sea bream was supported by identification using Northern blots of only a single transcript of 1.35 kb. Reverse transcription and polymerase chain reaction techniques coupled with Southern blot analysis resulted in the detection of PRL in the pituitary but also in the intestine, liver, ovary, and testes.
- 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.
- 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.
- Distribution of vasoactive intestinal peptide in the brain and hypothalamo-hypophysial system of the sea bream (Sparus aurata)Publication . Power, Deborah; Ingleton, P. M.Vasoactive intestinal polypeptide (VIP) belongs to a family of peptides that show sequence conservation and include glucagon, secretin, glucose-dependent insulin-releasing peptide, growth hormone-releasing factor, peptide having N-terminal histidine and C-terminal isoleucine (PHI) and the reptilian peptides helodermin and the helospectins (TABLE 1). VIP and related peptides have been isolated from several species of teleost and they show a degree of sequence conservation similar to that found within mammalian peptides.