Biomedical subjects

J M Conlon

Publications and source records attributed to J M Conlon.

At least 145 records · Page 8Linked to original sources

PubMed1995

Purification and structural characterization of insulin from a caecilian, Typhlonectes natans (Amphibia: Gymnophiona).

Despite the important position of amphibia in phylogeny, efforts at the structural characterization of amphibian neurohormonal peptides have largely been confined to the Anurans (frogs and toads). Insulin was purified from an extract of the pancreas of the caecilian, Typhlonectes natans. The primary structure of the peptide was established as: [formula: see text] This amino acid sequence contains several unusual substitutions (Gln-->Lys at A5, His-->Leu at A8, Gln-->Glu at A15, and Gly -->Ala at B20) that are not present in other amphibian insulins. The structure of insulin appears to be less well conserved among the different orders of amphibia, compared with reptiles and birds.

Amino Acid Sequence↗

PubMed1995

Kallikrein generates angiotensin II but not bradykinin in the plasma of the urodele, Amphiuma tridactylum.

Incubation of heat-denatured plasma from the urodele, Amphiuma tridactylum (three-toed amphiuma) or from the anurans Rana ridibunda (European green frog) and Rana catesbeiana (American bullfrog) with either glass beads, porcine pancreatic kallikrein or trypsin did not generate bradykinin-like immunoreactivity. However, peptides were generated in kallikrein-treated amphiuma plasma that contracted vascular rings from the bullfrog systemic arch and had a spasmogenic action on the bullfrog urinary bladder. These peptides which were not generated in trypsin-treated plasma, were purified to homogeneity by reverse-phase HPLC and their primary structures established as: Asp-Arg-Val-Tyr-Val-His-Pro-Phe ([Asp1,Val5]angiotensin II) and Asn-Arg-Val-Tyr-Val-His-Pro-Phe ([Asn1,Val5]angiotensin II). Incubation of synthetic [Asn1,Val5]angiotensin II with amphiuma plasma resulted in deamidation to [Asp1,Val5]angiotensin II. The data suggest, therefore that amphiuma plasma contains an L-asparagine amidohydrolase (asparaginase), as previously described for the eel. Although bradykinin-related peptides have been isolated from frog skin, this study provides evidence tha the kallikrein-kinin system may be absent from the blood of amphibia.

Amino Acid Sequence↗

PubMed1995

Characterization of the pancreatic hormones from the Brockmann body of the tilapia: implications for islet xenograft studies.

The Brockmann body of the teleost fish, the tilapia (Oreochromis nilotica) has been considered as a potential source of islet xenograft tissue for patients with insulin-dependent diabetes. This study describes the purification from an extract of tilapia Brockmann bodies of insulin and several peptides arising from different pathways of post-translational processing of two proglucagons, two prosomatostatins and proPYY. The primary structure of tilapia insulin is similar to insulins from other teleosts (particularly the anglerfish, Lophius americanus) except that the strongly conserved glutamine residue at position 5 in the A-chain, a residue that is important in the binding of insulin to its receptor, is replaced by glutamic acid. In common with other teleosts, the tilapia Brockmann body expresses two non-allelic glucagon genes. Alternative pathways of post-translational processing lead to glucagons with 29 and 36 amino acid residues derived from proglucagon I and glucagons with 29 and 32 residues derived from proglucagon II. Glucagon-like peptides with 30 and 34 residues derived from proglucagon II were also isolated. In each case, the longer peptide is a C-terminally extended form of the shorter. Tilapia peptide tyrosine-tyrosine (PYY) was isolated in a C-terminally alpha-amidated from with 36 amino acid residues that is structurally similar (89% sequence identity) to anglerfish PYY. A 30-amino acid peptide, representing the C-terminal flanking peptide of PYY, was also isolated that shows only 53% sequence identity with the corresponding anglerfish peptide. Tilapia somatostatin-14 is identical to mammalian somatostatin but the [Tyr7, Gly10] somatostatin-containing peptide derived from prosomatostatin II contains the additional substitution (Phe11-->Leu) compared with the corresponding peptide from other teleosts.

Adipose Tissue↗

PubMed1995

Intracellular degradation of the C-peptide of proinsulin, in a human insulinoma: identification of sites of cleavage and evidence for a role for cathepsin B.

An extract of a neuroendocrine tumor of the human pancreas contained a high concentration of insulin and the C-peptide of proinsulin, as determined by radioimmunoassay, together with somatostatin, calcitonin, and thymosin beta 4. Analysis of the molecular forms of the proinsulin-derived peptides by high-performance liquid chromatography demonstrated that insulin was stored in the tumor as the intact peptide. In contrast, metabolites of C-peptide, representing the (1-21), (1-23), (1-25) and (1-29) N-terminal fragments, were isolated from the extract in addition to intact C-peptide. Generation of these metabolites involves cleavage of Xaa-Leu or Leu-Xaa bonds. Previous immunohistochemical studies have identified cathepsin B in secretory granules and lysosomes of human insulinoma cells. Synthetic human C-peptide was rapidly cleaved by purified human cathepsin B, primarily at the site of leucine residues, to give several metabolites, including the (1-25) and (1-23) fragments. The data indicate that the C-peptide of proinsulin is selectively metabolized in the neoplastic B cell by a mechanism that involves proteolytic cleavages in the C-terminal region of the peptide.

Aged↗

PubMed1995

Characterization of the endogenous intestinal peptide that stimulates the rectal gland of Scyliorhinus canicula.

It has been postulated that gut peptides play a major role in the regulation of rectal gland secretion in elasmobranchs. An isolated perfused rectal gland secretion in elasmobranchs. An isolated perfused rectal gland preparation was developed for Scyliorhinus canicula that responded to dibutyryl 3',5'-cyclic monophosphate plus 3-isobutyl-1-methylxanthine, increasing chloride clearance rates threefold over basal levels. Activity was stimulated by an endogenous peptide, isolated in pur form by reverse-phase high-performance liquid chromatography from the intestine of S. canicula. The primary structure was established as Ser-Pro-Ser-Asn-Ser-Lys-Cys-Pro-Asp-Gly-Pro-Asp-Cys-Phe-Val-Gly-Leu-Met- NH2. This is a sequence identical to that of the tachykinin scyliorhinin II. Perfusion of synthetic scyliorhinin II increased secretion rate in the rectal gland of S. canicula in a dose-dependent manner with a maximal response at 10(-6) M, whereas vasoactive intestinal peptide, a stimulator in the spiny dogfish, Squalus acanthias, had no effect. We propose that scyliorhinin II is the uncharacterized peptide rectin, previously identified from the intestine of S. canicula.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Isolation, localization, and cardiovascular activity of tachykinins from the stomach of the bowfin Amia calva.

The bowfin is an extant representative of an ancient group of ray-finned fish with evolutionary connections to modern teleosts. A peptide with substance P-like immunoreactivity was isolated from an extract of bowfin stomach and its primary structure was established as Ser-Lys-Ser-His-Gln-Phe-Tyr-Gly-Leu-Met-NH2. This amino acid sequence resembles mammalian substance P only in the COOH-terminal region of the peptide. A second tachykinin with neurokinin A-like immunoreactivity isolated from the extract comprises 23 amino acid residues and shows limited structural similarity to mammalian neuropeptide-gamma. A randomly distributed population of cells in the gastric glands of the bowfin were immunostained with an antiserum raised against substance P, but no immunopositive structures were identified in the surface epithelium, lamina propria, or the nerve plexuses of the submucosa. Bolus injections of synthetic bowfin substance P (0.1-10 nmol/kg) into the bulbus arteriosus of unanesthetized bowfin resulted in a significant and dose-dependent rise in vascular resistance and arterial blood pressure (P < 0.01) and a fall in cardiac output (P < 0.05) without change in heart rate. After 5-10 min, arterial pressure and vascular resistance returned to preinjection levels, but cardiac output significantly (P < 0.05) increased over baseline values. The response to the peptide was unaffected by pretreatment of the animals with phentolamine. The study has shown that the stomach of the bowfin synthesizes tachykinins with novel structural features that display cardiovascular activity in this species.

Animals↗

PubMed1995

Effect of ranakinin, a novel tachykinin, on cytosolic free calcium in frog adrenochromaffin cells.

It has recently been shown that two novel tachykinins, ranakinin and [Leu3, Ile7]neurokinin A, are present in fibers innervating the frog adrenal gland, and it has been demonstrated that tachykinins stimulate corticosteroid secretion in vitro through activation of chromaffin cells. The purpose of the present study was to investigate the effect of ranakinin on cytosolic free calcium concentrations ([Ca2+]i) and to determine the source of calcium involved. Cultured adrenal cells were loaded with the fluorescent calcium indicator indo-1, and changes in [Ca2+]i were studied using dual emission wavelength microfluorimetry. Administration of a brief pulse of ranakinin (1 microM; 1 sec) in the vicinity of chromaffin cells caused an immediate and transient increase in [Ca2+]i. Repeated pulses of ranakinin resulted in a gradual decline in the [Ca2+]i response, suggesting the occurrence of a desensitization phenomenon. Preincubation of the cells with the calcium channel blockers nifedipine (10 microM) and omega-conotoxin (1 microM) did not alter the response of chromaffin cells to ranakinin. Chelation of extracellular calcium by EGTA (10 mM) caused a marked decrease in the basal [Ca2+]i, but did not suppress the ranakinin-induced [Ca2+]i increase. Conversely, incubation of the cells with thapsigargin (10 microM), an inhibitor of calcium adenosine triphosphatase activity, abolished the stimulatory effect of ranakinin, indicating that the increase in [Ca2+]i can be ascribed to mobilization of calcium from intracellular stores. Preincubation of adrenal cells with the phospholipase C antagonist U-73122 (1 microM; 18 min) or with pertussis toxin (10 microM; 18 h) totally blocked the ranakinin-induced [Ca2+]i rise. Taken together, these data indicate that in frog adrenochromaffin cells, ranakinin causes mobilization of calcium from intracellular stores. The effect of ranakinin is mediated through activation of a phospholipase C via a pertussis toxin-sensitive G protein.

Amino Acid Sequence↗

PubMed1995

Frog vasoactive intestinal polypeptide and galanin: primary structures and effects on pituitary adenylate cyclase.

Vasoactive intestinal polypeptide (VIP) and galanin were isolated in pure form from the stomach of the European green frog, Rana ridibunda. Frog VIP is identical to the previously characterized VIP from chicken and alligator. The primary structure of frog galanin contains only two amino acid substitutions (asparagine for histidine at position 23 and histidine for tyrosine at position 26) compared with porcine galanin. The data indicate that evolutionary pressure to conserve the amino acid sequence of both peptides during the evolution of amphibia to mammals has been strong. Synthetic frog VIP produced a dose-dependent increase in cAMP concentration in frog anterior pituitary fragments. The potency of the peptide (ED50 = 1.2 x 10(-6) M; mean +/- SE; n = 8) was comparable to that of porcine VIP (EC50 = 1.3 x 10(-6) M), but was approximately 10-fold less than that of frog pituitary adenylate cyclase-activating polypeptide [PACAP-(1-38); ED50 = 1.1 x 10(-7) M] in the same system. The increases in cAMP concentrations produced by maximal doses of PACAP (10(-5) M) and VIP (10(-5) M) were not additive. The data suggest that the effects of both peptides are mediated through a common PACAP-preferring receptor that is pharmacologically different from the mammalian PACAP type I receptor. Synthetic frog galanin also produced a dose-dependent increase in the concentration of cAMP in isolated frog anterior pituitary fragments (ED50 = 9.3 x 10(-8) M) consistent with a possible role for the peptide as a hypophysiotropic factor in amphibians.

Adenylyl Cyclases↗

PubMed1995

Evidence for the involvement of chromaffin cells in the stimulatory effect of tachykinins on corticosteroid secretion by the frog adrenal gland.

The adrenal gland of the frog is innervated by a network of fibers containing two tachykinins (ranakinin and [Leu3,Ile7]neurokinin A), which both stimulate corticosteroid secretion from frog adrenal tissue. The aim of the present study was to determine the mode of action of tachykinins on the frog adrenal gland. Double immunolabeling of tissue sections with a monoclonal antibody to tyrosine hydroxylase and an antiserum to substance P showed that tachykinin-containing fibers are preferentially apposed onto chromaffin cells. Immunocytochemical labeling at the electron microscope level revealed that tachykinin-immunoreactive fibers establish close contacts only with adrenochromaffin cells. Ranakinin stimulated corticosterone and aldosterone secretion from perifused adrenal slices, but had no stimulative effect on dispersed adrenal cells. Cytoautoradiographic labeling of frog adrenal cells in primary culture with [3H]substance P revealed the existence of specific binding sites located exclusively on chromaffin cells. Microfluorimetric measurement of cytosolic calcium concentrations ([Ca2+]i) in cultured adrenal cells showed that ranakinin induced a dose-dependent increase in [Ca2+]i in chromaffin cells (ED50 = 2 x 10(-7) M). In contrast, ranakinin did not affect [Ca2+]i in adrenocortical cells. The present results indicate that in the frog adrenal gland, tachykinin-containing fibers make preferential contacts with chromaffin cells, and tachykinins directly activate chromaffin cells. These data suggest that the stimulative effect of tachykinins on corticosteroid secretion is mediated via presynaptic activation of adrenochromaffin cells.

Adrenal Cortex Hormones↗

PubMed1994

Differential actions of lamprey peptide methionine-tyrosine at Y1 and Y2 neuropeptide Y receptors.

Peptide methionine-tyrosine (PMY), a peptide of the neuropeptide Y (NPY) superfamily isolated from the brain and intestine of the sea lamprey, had the same maximum effect but was 11-fold less potent than pig NPY in inhibiting field-stimulated contraction of the rat vas deferens, an effect mediated through the Y2 receptor. In contrast, PMY produced a 9-fold greater maximum effect but was 3-fold less potent than pig NPY in contracting the guinea pig mesenteric artery, an effect mediated through the Y1 receptor. Molecular modelling has suggested that the conformation of PMY is appreciably different from NPY only in the beta-turn region of the molecule (residues 9-14). Our data suggest, therefore, that modifications in this region of NPY may useful in the design of receptor selective analogs.

Amino Acid Sequence↗

PubMed1994

Characterization of trout galanin and its distribution in trout brain and pituitary.

Galanin was purified from an extract of the stomach of the rainbow trout, Oncorhynchus mykiss, and its primary structure was established as Gly-Trp-Thr-Leu-Asn-Ser- Ala-Gly-Tyr-Leu10-Leu-Gly-Pro-His-Gly-Ile-Asp-Gly-His-Arg20- Thr-Leu-Ser-Asp- Lys-His-Gly-Leu-Ala. Trout galanin shows six amino acid substitutions compared with pig galanin, but the N-terminal region (residues 1-14) has been fully conserved. The distribution of galanin-immunoreactive (GAL-IR) structures in the trout brain and pituitary was studied via immunohistochemistry. GAL-IR cell bodies were observed only in the caudal telencephalon, the preoptic region, and the mediobasal hypothalamus. GAL-IR fibers, however, are widely distributed throughout the brain, with a much lower density in the midbrain and posterior brain than in the tel- and diencephalon. Particularly dense innervation of the mediobasal hypothalamus, the ventral and supracommissuralis parts of the caudal telencephalon, and the region above and below the anterior commissure was observed. A heavy innervation of the pituitary was consistently detected. GAL-IR fibers were present in neurohypophyseal digitations of both the anterior and intermediate lobes with highest density in the region of the proximal pars distalis, where growth hormone and gonadotropic cells are located. Fibers were also seen in digitations of the rostral pars distalis, in particular between the prolactin follicles. The distribution of GAL-IR neurons in the central nervous system and pituitary of the trout suggests that the peptide may exercise an important role in the regulation of neuroendocrine functions, particularly those related to reproduction.

Amino Acid Sequence↗

PubMed1994

Comparative distribution of neurotensin-like immunoreactivity in the brain of a teleost (Carassius auratus), an amphibian (Hyla meridionalis), and a reptile (Gallotia galloti).

The distribution of neurotensin (NT) was studied in the brain of three species belonging to the three major classes of cold-blooded vertebrates: teleost fishes (Carassius auratus), anuran amphibians (Hyla meridionalis), and reptiles (Gallotia galloti; Lacertidae). By using antibodies directed against synthetic bovine NT in the three species, immunoreactive cell bodies were discovered mostly in the telencephalon and diencephalon, in particular at the level of the preoptic region the mediobasal hypothalamus, and the thalamus. In the frog and the lizard, additional immunoreactive (ir) structures were observed in the optic tectum and the tegmentum of the mesencephalon. In the goldfish pituitary, an extensive innervation was consistently observed at the level of the rostral pars distalis, whereas in both frog and lizard, positive fibers were only detected in the external layer of the median eminence. In the three species there is a striking overlap between the distribution of the NT-ir cell bodies and that of the target cells for sexual steroids. The results are discussed in relation with those reported in birds and mammals, and with the possible interactions among NT, sexual steroids, and the neuroendocrine control of pituitary hormone release, in particular prolactin and gonadotrophin.

Amino Acid Sequence↗

PubMed1994

Frog diazepam-binding inhibitor: peptide sequence, cDNA cloning, and expression in the brain.

Three peptides derived from diazepam-binding inhibitor (DBI) were isolated in pure form from the brain of the frog Rana ridibunda. The primary structures of these peptides showed that they correspond to mammalian DBI-(1-39), DBI-(58-87), and DBI-(70-87). A set of degenerate primers, whose design was based on the amino acid sequence data, was used to screen a frog brain cDNA library. The cloned cDNA encodes an 87-amino acid polypeptide, which exhibits 68% similarity with porcine and bovine DBI. Frog DBI contains two paired basic amino acids (Lys-Lys) at positions 14-15 and 62-63 and a single cysteine within the biologically active region of the molecule. Northern blot analysis showed that DBI mRNA is expressed at a high level in the brain but is virtually absent in peripheral tissues. The distribution of DBI mRNA and DBI-like immunoreactivity in the frog brain was studied by in situ hybridization and immunocytochemistry. Both approaches revealed that the DBI gene is expressed in ependymal cells and circumventricular organs lining the ventricular cavity. Since amphibia diverged from mammals at least 250 million years ago, the data show that evolutionary pressure has acted to conserve the structure of DBI in the vertebrate phylum. The distribution of both DBI mRNA and DBI-like immunoreactivity indicates that DBI is selectively expressed in glial cells.

Amino Acid Sequence↗

PubMed1994

Characterization of insulins and proglucagon-derived peptides from a phylogenetically ancient fish, the paddlefish (Polyodon spathula).

The North American paddlefish, Polyodon spathula (Order Acipenseriformes) is an extant representative of a group of primitive Actinopterygian (ray-finned) fish that probably shared a common ancestor with present-day teleosts. Two molecular forms of insulin which differ by a single amino acid substitution, His or Asp at position 15 of the A chain, were isolated from the pancreas of the paddlefish. Paddlefish insulins show greatest structural similarity to insulin from the garfish (order Lepisosteiformes) and resemble mammalian insulins more strongly than do insulins from teleost fish. The primary structures of several proglucagon-derived peptides, two molecular forms of glucagon which differ by the single amino acid substitution Arg18-->Ser, and glucagon-like peptide, have been less well conserved during evolution. The paddlefish glucagons contain 31 amino acid residues, rather than the usual 29, and show several structural features, such as Met5, Glu24 and Gly29, not previously observed in glucagons from other species. In spite of considerable differences in structure between paddlefish and mammalian glucagons (10 or 11 amino acid substitutions), both paddlefish glucagons are equally as effective as bovine glucagon in stimulating glycogenolysis in dispersed hepatocytes from the teleost fish Sebastes caurinus (rockfish). However, the substitution Arg18-->Ser in the paddlefish glucagon results in a 6-fold decrease in potency in this system.

Amino Acid Sequence↗

PubMed1994

Purification of a neuropeptide Y-related peptide from the brain of the sea lamprey and its effect on steroidogenesis.

A peptide with neuropeptide Y-like immunoreactivity was identified by radioimmunoassay in an extract of the brain of the sea lamprey, Petromyzon marinus using an antiserum raised against the conserved COOH-terminal region of mammalian neuropeptide Y. Purification of the peptide and determination of its primary structure showed that it was identical to peptide methionine-tyrosine (PMY), previously isolated from the intestine of the same species. Intraperitoneal injection of synthetic PMY (0.15 micrograms/g) into female lampreys undergoing final maturation before spawning produced a significant (P < 0.05) decrease in plasma concentrations of estradiol compared with control lampreys injected with vehicle only. These data suggest the hypothesis that the observed decrease in the concentration of PMY-containing cells in the intestines of lampreys during upstream migration may correlate with the increase in circulating estradiol concentrations and final maturational processes.

Amino Acid Sequence↗

PubMed1994

Effects of dogfish urotensin II on lipid mobilization in the fasted dogfish, Scyliorhinus canicula.

Previous studies, both in vitro and in vivo, have shown that urotensin II stimulates hepatic release of nonesterified fatty acids in teleost fish, but the effects of the peptide on lipid mobilization in elasmobranchs are unknown. Infusions of synthetic dogfish urotensin II (40 pmol/min/kg for 30 min followed by 400 pmol/min/kg for 30 min) into the first afferent gill artery of the fasted, unrestrained dogfish, Scyliorhinus canicula (n = 6), produced no significant change in the circulating concentrations of acetoacetate, 3-hydroxybutyrate, and triacylglycerols. Circulating nonesterified fatty acids were undetectable (< 0.1 mmol/liter) both before and after the urotensin II infusions. There was no significant change in the circulating concentrations of glucose and lactate during either the low- or the high-dose infusions. The data support the hypothesis that lipid is converted primarily to ketone bodies in the livers of elasmobranch fish and do not indicate a role for urotensin II in regulating either lipid or glycogen mobilization.

3-Hydroxybutyric Acid↗

PubMed1994

Generation of [Ala1,Thr6]bradykinin in the plasma of a snake, the reticulated python.

Incubation of heat-denatured plasma from the reticulated python, Python reticulatus with trypsin generated bradykinin-like immunoreactivity which was detected by radioimmunoassay using an antiserum raised against mammalian bradykinin. The primary structure of python bradykinin was established as Ala-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg. This amino acid sequence contains two substitutions (Arg1-->Ala and Ser6-->Thr) compared with mammalian bradykinin. Treatment of python plasma with glass beads under conditions previously shown to generate [Thr6]bradykinin in the plasma of a turtle and an alligator did not generate bradykinin-like immunoreactivity. The data indicate that the plasma of a lepidosaur contains a kininogen but, unlike the plasma of chelonians and crocodilians, does not contain a prekallikrein activator related to Factor XII.

Amino Acid Sequence↗

PubMed1994

Spasmogenic actions of frog urotensin II on the bladder and ileum of the frog, Rana catesbeiana.

The effects of synthetic frog urotensin II on the motility of isolated smooth muscle strips of urinary bladder and ileum from the bullfrog Rana catesbeiana were investigated. The strips of bladder exhibited strong spontaneous contractions. Frog urotensin II produced a concentration-dependent increase in the frequency of contraction (EC50 = 8.1 x 10(-9) M) but had no significant effect on tension. Maximum stimulation (4 times the rate of spontaneous contractions) was produced by 10(-7) M peptide. The ileal smooth muscle strips exhibited only weak spontaneous contractions but frog urotensin II produced concentration-dependent increases in both the frequency (EC50 = 1.3 x 10(-9) M) and the strength (EC50 = 3.6 x 10(-9) M) of contraction. The peptide had no effect on the motility of strips of rat urinary bladder and ileum. In both the frog bladder and ileum preparations, the effect of urotensin II was not affected by preincubation of the strips with atropine, tetrodotoxin, and somatostatin-14 but indomethacin produced a significant (P < 0.05) decrease in the strength of the contractions of the bladder (to 21 +/- 6% of control values) and ileum (to 32 +/- 13%). Arachidonic acid produced an increase in the frequency of contraction of the bladder and an increase in both the frequency and the strength of contraction of the ileum. The data demonstrate that urotensin II exercises a spasmogenic effect on amphibian smooth muscle that is similar to that of the peptide on teleost tissues. An involvement of prostaglandin synthesis in the spasmogenic action of urotensin II on frog bladder and ileum is indicated.

Animals↗