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J M Conlon

Publications and source records attributed to J M Conlon.

At least 271 records · Page 15Linked to original sources

Primary structures of three fragments of proglucagon from the pancreatic islets of the daddy Sculpin (Cottus scorpius).

Three peptides isolated from the Brockmann bodies of the daddy sculpin, a teleostean fish, have been identified as fragments of one or more proglucagons. The peptide L Q D A E D S S R F D A D D T L A G E A R E L S T P K represents the NH2 terminus of proglucagon (residues 1-27), H S E G T F S N D Y S K Y L E T R R A Q D F V Q W L K N S represents glucagon and H A D G T F T S D V S S Y L N D Q A I K D F V A K L K S G K V represents the glucagon-like peptide at the COOH terminus of the precursor. The fast-atom bombardment mass spectra of the three peptides were consistent with the proposed structures and demonstrated that further posttranslational modifications of the peptides had not taken place. Sculpin glucagon is identical to anglerfish glucagon II but sculpin proglucagon(1-27) and glucagon-like peptide show stronger homology to the corresponding regions of anglerfish proglucagon I than to proglucagon II. The structures of the peptides are suggestive of the action of trypsin-like and carboxypeptidase-B-like enzymes at the site of pairs of basic amino acid residues in proglucagon. The presence of a COOH-terminal lysyl group in proglucagon(1-27) may indicate, however, that the penultimate prolyl residue partially inhibits the action of the carboxypeptidase-B-like activity.

Amino Acid Sequence↗

Identification of the C-terminally alpha-amidated amino acid in peptides by high-performance liquid chromatography.

A sensitive method for the rapid identification of the C-terminally amidated amino acid in peptides is described. Peptides containing the alpha-amide group at the C-terminus were cleaved with endopeptidases. The fragments released (oligopeptides, amino acids and the C-terminally amidated residue) are coupled to phenylisothiocyanate. The phenylthiocarbamoyl derivative of the amino acid alpha-amide is selectively extracted from the mixture by alkaline butyl acetate and identified by a high-performance liquid chromatography system that enables rapid and complete separation of the derivatives of 17 amino acid amides at a detection limit of 20-50 pmol. The C-terminal alpha-amides of neurokinin-A (Met-NH2), mammalian secretin (Val-NH2), pancreatic polypeptide (Tyr-NH2) and peptide HI (Ile-NH2) are unequivocally determined at a level of 0.5-2 nmol per peptide. This method was used to characterize a crude peptide fraction prepared from porcine brain. Cholecystokinin-58 was identified in this fraction by detection of phenylthiocarbamoyl-phenylalaninamide. The method is suitable for the identification of the C-terminal alpha-amidated residue of purified peptides, but can also be used as a screening strategy to isolate from complex biological extracts novel peptides containing an alpha-amidated amino acid at the C-terminus.

Amides↗

Gastrin-releasing peptide from the intestine of the elasmobranch fish, Scyliorhinus canicula (common dogfish).

The concentration of gastrin-releasing peptide in the intestine of the elasmobranchian fish, Scyliorhinus canicula, measured with an antiserum directed against the COOH-terminal region of porcine gastrin-releasing peptide, was higher than the concentrations measured in mammalian intestines. The immunoreactivity was resolved by gel permeation chromatography into two peaks with the approximate elution volumes of porcine gastrin-releasing peptide and bombesin/neuromedin C. The primary structure of the larger peptide was established as Ala Pro Val Glu Asn Gln Gly Ser Phe Pro Lys Met Phe Pro Arg Ser His (Trp) Ala Val Gly (His Leu Met.NH2). Residues in parentheses are only tentatively assigned. Chromatographic evidence and the presence of the arginyl residue at position 15 in the peptide suggest that the smaller molecular form of gastrin-releasing peptide may be identical to mammalian neuromedin C. Amphibian bombesin was not identified in the dogfish gut.

Animals↗

Tachykinins in the central and peripheral nervous system of the ascidian Ciona intestinalis.

Immunochemical studies were carried out on the ascidian Ciona intestinalis to determine the character and distribution of the tachykinins neurokinin A (NKA) and substance P (SP). Antisera specific for the C-terminus of mammalian SP, and for the N-terminus of mammalian SP and NKA, were used to monitor tissue extracts from Ciona. Parallel immunocytochemical studies assessed the distribution of these tachykinins in the central and peripheral nervous systems as well as their occurrence in endocrine cells. HPLC and radioimmunoassay established the presence of both C-terminal SP and NKA-like material in extracts from neural ganglion and body wall/pharynx. Immunocytochemistry revealed the C-SP material to be present in a population of small neuronal cell bodies and fibers in the ganglion as well as in cell bodies and fibers in the periphery. The NKA-like material was restricted to separate and larger neuronal perikarya in the ganglion while in the periphery its distribution reflected that of the C-SP-like material. Endocrine cells in the pharyngeal epithelium were reactive only with the C-terminal SP antiserum. N-terminal SP antisera were unreactive both in radioimmunoassay and immunocytochemistry. These findings are in accord with the idea that the tachykinin family is represented by at least two of its members at the prevertebrate stage of evolution. Interestingly, the SP-like material shows strong C-terminal homology with the mammalian peptide but little N-terminal similarity. Furthermore, the NKA-like peptide is restricted to the nervous system while SP-like molecules may be found in both central and peripheral neurons as well as endocrine cells in the pharynx.

Chromatography, High Pressure Liquid↗

Primary structure of insulin and glucagon from the flounder (Platichthys flesus).

Insulin and glucagon have been isolated from the Brockmann bodies of the flounder, a teleostean fish, and their primary structures established by automated Edman degradation. The A-chain of flounder insulin shows strong homology to the A-chains from the coho salmon (Oncorhynchus kisutch; 100%) and the anglerfish (Lophius americanus; 95%) but homologies in the B-chain region are weaker (salmon 79%, anglerfish 83%). Flounder insulin B-chain contains the novel sequence Val-Val-Pro-Pro at the NH2 terminus and the highly conserved seryl residue at position 10 (B 9 in mammals) is replaced by an alanyl residue. Flounder glucagon is identical to anglerfish glucagon II but shows four amino acid substitutions compared with salmon glucagon.

Amino Acid Sequence↗

Binding of vasoactive intestinal polypeptide to dispersed enterocytes results in rapid removal of the NH2-terminal histidyl residue.

Specific binding sites for 125I-labelled vasoactive intestinal polypeptide (VIP) (half-maximal inhibition at 1.5 +/- 0.2 nM VIP) were identified on dispersed porcine enterocytes. Radioactivity bound to the cell surface was internalized. At 37 degrees C, a steady state was achieved after 45 min with a ratio of internalized to cell surface-bound radioactivity of approximately 1:2 but at 10 degrees C, no radioactivity appeared intracellularly. Incubation of VIP with cells in the absence of inhibitors of proteolysis for as short a time as 30 s at 37 degrees C led to the formation of [des-His1]VIP by the action of amastatin- and bestatin-sensitive aminopeptidase(s). This metabolite was formed in the presence of sodium azide and when incubations were performed at 10 degrees C suggesting that internalization was not a prerequisite for degradation. As [des-His1]-VIP has only 1% of the bioactivity of VIP, formation of this metabolite will effectivily terminate the action of VIP in the epithelial layer of the intestine.

Amino Acids↗

Xenopsin- and neurotensin-like peptides in gastric juice from patients with duodenal ulcers.

Using an anti-serum directed against the COOH-terminal region of neurotensin and an anti-serum raised xenopsin in radioimmunoassays, the presence of neurotensin- and xenopsin-like immunoreactivity in Sep-pak extracts of human gastric juice was demonstrated. An anti-serum directed against the NH2-terminal to central residues of neurotensin and an anti-serum directed against a conformation, present only in the intact peptide, did not detect immunoreactivity in the juice. Infusions of pentagastrin at doses of 0.75 micrograms kg-1 h-1 and 1.5 micrograms kg-1 h-1 resulted in significant (P less than 0.05) increases in the total amount of xenopsin-like, but not neurotensin-like, immunoreactivity released in the juice compared with basal release. No significant differences in the release of xenopsin- or neurotensin-like immunoreactive were observed between patients with selective proximal vagotomy and patients without operation. Analysis by gel permeation chromatography and high performance liquid chromatography demonstrated that both the neurotensin- and xenopsin-like immunoreactivity was heterogeneous and an increase in molecular complexity on pentagastrin-stimulation was observed. As both neurotensin and xenopsin inhibit gastric exocrine activity, the presence of immunochemically related substances in gastric juice may have physiological relevance.

Chromatography, Gel↗

Primary structure and tissue distribution of guinea pig gastrin-releasing peptide.

The primary structure of gastrin-releasing peptide from the guinea pig stomach has been determined by automated Edman degradation and shown to be identical to porcine gastrin-releasing peptide. Extracts of guinea pig brain and small intestine contained both gastrin-releasing peptide and its COOH-terminal decapeptide (neuromedin C) but the stomach extracts contained only gastrin-releasing peptide. Within the small intestine, highest concentrations of gastrin-releasing peptide-like immunoreactivity were found in extracts of the circular and longitudinal smooth muscle layers.

Amino Acid Sequence↗

Conversion of neuropeptide K to neurokinin A and vesicular colocalization of neurokinin A and substance P in neurons of the guinea pig small intestine.

The highest concentration of neurokinin A-like immunoreactivity and substance P-like immunoreactivity in the guinea pig small intestine was associated with the myenteric plexus-containing longitudinal muscle layer. Chromatographic analysis of extracts of this tissue demonstrated the presence of neurokinin A and neuropeptide K but the probable absence of neurokinin B. A fraction of synaptic vesicles of density 1.133 +/- 0.003 g/ml was prepared from the myenteric plexus-containing tissue by density gradient centrifugation in a zonal rotor and was enriched 29 +/- 12-fold in the concentration of neurokinin A-like immunoreactivity and 43 +/- 13-fold in the concentration of substance P-like immunoreactivity. This fraction was separated from the fraction of vasoactive intestinal peptide-containing vesicles (density, 1.154 +/- 0.009 g/ml). Chromatographic analysis of lysates of the vesicles indicated the presence of neurokinin A but not neuropeptide K. It is postulated that beta-pre-protachykinin is processed to substance P, neurokinin A, and neuropeptide K in the cell bodies of myenteric plexus neurons but that conversion of neuropeptide K to neurokinin A takes place during packaging into storage vesicles for axonal transport. The data are consistent with the proposal that neurokinin A and substance P are stored in the same synaptic vesicle, but the possibility of cosedimentation of different vesicles of very similar density cannot be excluded.

Animals↗

Effect of a transplantable insulinoma upon serotonin concentrations in the intestine of the rat.

Rapid growth of a transplantable insulinoma in rats over a 14 day period results in hyperphagia together with a selective increase in the weight and enteroglucagon (GLI) concentration of the small intestine. Measurement of serotonin concentrations by an HPLC/fluorometric method demonstrated an increase in blood (91%; p less than 0.05) and in extracts of colon (22%; p less than 0.05) but no change in extracts of the small intestine and caecum. The data support the hypothesis that the rise in enteroglucagon is related directly to the growth of the small intestine and is not a non-specific effect of hyperphagia.

Adenoma, Islet Cell↗

Circulating tachykinins (substance P, neurokinin A, neuropeptide K) and the carcinoid flush.

Antisera of defined regional specificity have been used to measure the concentration of substance P-like immunoreactivity (SP-LI) and neurokinin A-like immunoreactivity (NKA-LI) during a meal-induced flush in 10 patients with metastatic carcinoid tumours. Although all patients flushed, NKA-LI levels in five patients and SP-LI in six patients were not elevated relative to healthy subjects (NKA-LI, less than 3 pg/ml; SP-LI, less than 10 pg/ml) both in the fasted state and after food. In the patients with elevated basal plasma tachykinin levels, increases in NKA-LI and SP-LI after food were erratic and did not correspond to a defined digestive phase or the occurrence of the flush. Chromatographic analysis of plasma demonstrated the presence of neuropeptide K and neurokinin A, and the detection of COOH-terminal fragments of substance P is consistent with the higher levels of circulating SP-LI measured with a COOH-terminally directed antiserum compared with an NH2-terminally directed antiserum. Subcutaneous injection of the somatostatin analogue SMS 201-995 (50 micrograms) alleviated symptoms of flush in two of three patients but only partially suppressed NKA-LI and SP-LI concentrations. It is concluded that circulating tachykinins cannot be solely responsible for the meal-induced carcinoid flush.

Adult↗

Characterization of an amidated form of pancreatic polypeptide from the daddy sculpin (Cottus scorpius).

The primary structure of pancreatic polypeptide from the teleostean fish, Cottus scorpius (daddy sculpin) was established as: YPPQPESPGGNASPEDWAKYHAAVRHYVNLITRQRYNH2 The presence of a COOH-terminally alpha-amidated amino acid was established using an HPLC method of general applicability. Although the peptide shows strong homology towards anglerfish pancreatic polypeptide (86%), homology towards porcine peptide YY (PYY) (61%) and porcine neuropeptide Y (NPY) (61%) was greater than towards porcine pancreatic polypeptide (PP) (47%). This result supports suggestions that the gene duplication events which led to PP, NPY and PYY formation took place after the time of divergence of fish and mammals.

Amino Acid Sequence↗

Substance P, neurokinin A, vasoactive intestinal polypeptide and gastrin releasing peptide in the intestine and pancreas of spontaneously obese-diabetic mice.

The concentrations and contents of immunoreactive substance P (SP), neurokinin A (NKA), vasoactive intestinal polypeptide (VIP) and gastrin releasing peptide (GRP) were measured in acid-ethanol extracts of intestine (duodenum-jejunum-ileum) and pancreas of C57BL/KsJ diabetes-obese (db/db) mice, Aston obese-hyperglycaemic (ob/ob) mice, and their respective lean controls. The intestinal concentration of GRP and pancreatic concentrations of VIP and GRP were 36-57% lower in lean Aston mice than lean C57BL/KsJ mice, indicating the influence of genetic background in control mice. Intestinal concentrations of SP and NKA were reduced by 19-33% in the db/db and ob/ob mutants compared with their lean controls, but the intestinal contents of these peptides were normal or greater than normal due to intestinal hypertrophy of the mutant mice. The intestinal VIP concentration was not altered, but the content was increased by 87% and 25% respectively in db/db and ob/ob mice, whereas the intestinal GRP concentration was reduced by 51% in ob/ob mice. Pancreatic concentrations and contents of NKA, VIP and GRP were similar in lean and db/db C57BL/KsJ mice. However, pancreatic concentrations and contents of VIP and GRP were reduced by 51-55% in ob/ob mice compared with their lean controls. The sensitivity of the present assay did not permit accurate determination of the low pancreatic concentrations of SP. The results suggest that the spontaneous ob/ob and db/db syndromes of obesity and diabetes in mice are associated with reduced intestinal concentrations of SP and NKA. The ob/ob mouse also exhibited reductions of intestinal GRP and pancreatic GRP and VIP concentrations. These changes in regulatory peptides may relate to abnormalities of intestinal and possibly pancreatic function in obese and diabetic mutant mice.

Animals↗

The primary structure of ratfish insulin reveals an unusual mode of proinsulin processing.

The primary structure of insulin from the Holocephalan fish, Hydrolagus colliei (the ratfish), has been established by automated Edman degradation as: (Formula: see text). The presence of a COOH-terminal extension to the B-chain is consistent with the occurrence of a single base mutation in the region of the gene encoding one of the dibasic residue processing sites [Arg31(AGA)----Ile* (AUA)] with the result that the ratfish has utilised an alternative cleavage site within the C-peptide region of proinsulin.

Amino Acid Sequence↗

[Ser7]neurotensin: isolation from guinea pig intestine.

Using three antisera to neurotensin of defined regional specificity, a novel neurotensin has been identified in extracts of guinea pig brain and small intestine. The primary structure of the peptide was established as: pGlu Leu Tyr Glu Asn Lys Ser Arg Arg Pro Tyr Ile Leu. Guinea pig neurotensin differs from bovine neurotensin by substitution of a prolyl residue by a seryl residue at position 7. Synthetic [Ser7]neurotensin showed identical chromatographic and immunochemical properties to guinea pig neurotensin. This difference in primary structure may account for some of the anomalous pharmacological effects of bovine neurotensin on guinea pig tissues.

Amino Acid Sequence↗

Scyliorhinin I and II: two novel tachykinins from dogfish gut.

Two peptides with tachykinin-like ability to contract longitudinal muscle from the guinea pig ileum were isolated from the intestine of the common dogfish, Scyliorhinus caniculus. The amino acid sequence of scyliorhinin I was established as Ala-Lys-Phe-Asp-Lys-Phe-Tyr-Gly-Leu-Met-NH2 and this peptide cross-reacted with antisera directed against the C-terminal region fo substance P. The amino acid sequence of scyliorhinin II was established as Ser-Pro-Ser-Asn-Ser-Lys-Cys-Pro-Asp-Gly-Pro-Asp-Cys-Phe-Val-Gly-Leu-Met- NH2 and this peptide cross-reacted with antisera directed against the C-terminal region of neurokinin A. The mammalian peptides substance P and neurokinin A were absent from the dogfish intestinal tissue.

Amino Acid Sequence↗

Conversion of somatostatin-28 to somatostatin-14 during maturation of epithelial cells in the porcine jejunum.

Fractions of isolated epithelial cells were harvested from a segment of porcine jejunum by ten successive incubations with a chelating buffer. The cell fractions showed a progressive decrease in the activity of the brush-border enzymes, alkaline phosphatase and sucrase, with increasing incubation number but a progressive increase in the ability to incorporate labelled thymidine into DNA. Fractions enriched in cells from the crypt region (fractions 9 and 10) contained higher concentrations per mg protein of somatostatin-like immunoreactivity (1.8-fold), glucagon-like immunoreactivity (5.3-fold) and serotonin (3.0-fold) than fractions enriched in cells from the villus tip (fractions 1 and 2). Analysis of extracts of the fractions by gel filtration/radioimmunoassay showed that somatostatin-28 represented the predominant molecular form of somatostatin-like immunoreactivity in all cell fractions but the relative proportion of somatostatin-14 (and related metabolites) to somatostatin-28 was significantly higher (P less than 0.05) in fractions enriched in villus cells (fraction 1 and 2) than in fractions enriched in crypt cells (fractions 5-10). This result suggests that metabolism of somatostatin-28 to somatostatin-14 takes place during migration of the D cell from the crypt base to the villus tip. Heterogeneity in the somatostatin-14 region of the chromatograms indicates that the peptide may be further metabolized by the action of aminopeptidases.

Alkaline Phosphatase↗

Effects of a transplantable insulinoma upon regulatory peptide concentrations in the gastrointestinal tract of the rat.

The rapid growth (0.8 +/- 0.3 g/day) of a transplantable insulinoma, which also contained substance P (2.9 +/- 2.3 pmol/g) and gastrin-releasing peptide (3.2 +/- 2.1 pmol/g), resulted in the development of hyperphagia, hyperinsulinaemia and hypoglycaemia in rats (n = 8). After a 14-day growth period, the insulinoma-bearing rats showed an increase (49%; p less than 0.01) in the weight of the small intestine but no significant change in stomach weight compared with control animals. The content (pmol/organ) of somatostatin, substance P, neurokinin A and vasoactive intestinal peptide in the stomachs of the tumour rats was unchanged. A depletion in the content (53% p less than 0.01) and concentration (57%; p less than 0.01) of gastrin-releasing peptide, however, suggested either hypersecretion, possibly mediated through hypoglycaemia-induced vagal stimulation, or inhibition of synthesis. The concentration and content of glucagon-like immunoreactivity (enteroglucagon) in the small intestine of the insulinoma rats increased markedly (47%; p less than 0.01 and 120%; p less than 0.01). This increase is consistent with a proposed role of this peptide as a factor trophic to the intestinal mucosa. No significant changes in the concentrations of somatostatin, substance P, neurokinin A, vasoactive intestinal peptide and gastrin-releasing peptide in the small intestine were observed. However, the increase in gut weight resulted in a greater content of vasoactive intestinal peptide (40%; p less than 0.01) and substance P (37%; p less than 0.05) in the insulinoma rats.

Adenoma, Islet Cell↗