Browsing by Author "Bendell, L. A."
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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.
- 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.
- Olfactory sensitivity to changes in environmental Ca2 in the freshwater teleost Carassius auratus: an olfactory role for the Ca2+ -sensing receptor?Publication . Hubbard, Peter; Ingleton, P. M.; Bendell, L. A.; Barata, E. N.; Canario, Adelino V. M.Olfactory sensitivity to changes in environmental Ca2+ has been demonstrated in two teleost species; a salmonid (Oncorhynchus nerka) and a marine/estuarine perciform (Sparus aurata). To assess whether this phenomenon is restricted to species that normally experience large fluctuations in external ion concentrations (e.g. moving from sea water to fresh water) or is present in a much wider range of species, we investigated olfactory Ca2+ sensitivity in the goldfish (Carassius auratus), which is a stenohaline, non-migratory freshwater cyprinid. Extracellular recording from the olfactory bulb in vivo by electroencephalogram (EEG) demonstrated that the olfactory system is acutely sensitive to changes in external Ca2+ within the range that this species is likely to encounter in the wild (0.05–3 mmol l–1). The olfactory system responded to increases in external calcium with increasing bulbar activity in a manner that fitted a conventional Hill plot with an apparent EC50 of 0.9±0.3 mmol l–1 (close to both ambient and plasma free [Ca2+]) and an apparent Hill coefficient of 1.1±0.3 (means ± S.E.M., N=6). Thresholds of detection were below 50 mmol l–1. Some olfactory sensitivity to changes in external [Na+] was also recorded, but with a much higher threshold of detection (3.7 mmol l–1). The olfactory system of goldfish was much less sensitive to changes in [Mg2+] and [K+]. Preliminary data suggest that Ca2+ and Mg2+ are detected by the same mechanism, although with a much higher affinity for Ca2+. Olfactory sensitivity to Na+ may warn freshwater fish that they are reaching the limit of their osmotic tolerance when in an estuarine environment. Olfaction of serine, a potent odorant in fish, was not dependent on the presence of external Ca2+ or Na+. Finally, the teleost Ca2+-sensing receptor (Ca-SR) was shown to be highly expressed in a subpopulation of olfactory receptor neurones by both immunocytochemistry and in situ hybridisation. The olfactory sensitivity to Ca2+ (and Mg2+) is therefore likely to be mediated by the Ca-SR. We suggest that olfactory Ca2+ sensitivity is a widespread phenomenon in teleosts and may have an input into the physiological mechanisms regulating internal calcium homeostasis.