Biomedical subjects

L Hilsted

Publications and source records attributed to L Hilsted.

46 records · Page 3Linked to original sources

PubMed1988

Neuropeptide Y in guinea pig, rabbit, rat and man. Identical amino acid sequence and oxidation of methionine-17.

Neuropeptide Y (NPY) was isolated and characterised from acid-ethanol extracts of rabbit and guinea pig brain. In both instances the chromatographic purification was a two-step procedure of gel filtration followed by reverse-phase high-performance liquid chromatography. The amino acid sequence of rabbit and guinea pig NPY was found to be identical to human and rat NPY as deduced from the cDNA structures. With the exception of the porcine peptide, all mammalian NPYs characterised to date have a methionine residue in position 17. This methionine residue is readily oxidized as indicated by the high degree of spontaneous oxidation of peptides found in the rabbit and guinea pig brain extracts and in NPY extracted from a rat phaeochromocytoma cell line. It is concluded that NPY is among the most highly conserved peptides and that NPYs containing methionine in position 17 are prone to oxidation.

Amino Acid Sequence↗

PubMed1988

Oxyntomodulin (glicentin-(33-69)): pharmacokinetics, binding to liver cell membranes, effects on isolated perfused pig pancreas, and secretion from isolated perfused lower small intestine of pigs.

The pharmacokinetics of purified synthetic oxyntomodulin were studied after infusing it into euglycaemic pigs at two rates. The elimination of the peptide from plasma was characterized by two components, a fast one (t1/2 7.2 +/- 0.6 min) and a slow one (t1/2 20.4 +/- 3.8 min) (mean +/- S.E.M., n = 7). The metabolic clearance rate was independent of infusion rate (6.96 +/- 0.99 vs 7.44 +/- 0.98 ml/kg . min (mean +/- S.E.M., n = 7). The synthetic peptide bound to pig hepatic glucagon receptors, but with approximately 2% of the affinity of glucagon, and showed insulinotropic and somatostatinotropic effects when infused into isolated perfused pig pancreases at concentrations higher than 10(-10) M. A dose-dependent increase was also shown for pancreatic glucagon output. A naturally occurring peptide, identified as oxyntomodulin by gel filtration and HPLC, was released into the circulation from the pig lower small intestinal mucosa upon intraluminal administration of glucose, and represented 25 +/- 3.8% of the secreted glucagon-like immunoreactivity. 11 +/- 2.3% of the secreted glucagon-like immunoreactivity was indistinguishable from glucagon itself upon gel filtration; thus at least 36% of the glucagon-like immunoreactivity secreted from the intestinal mucosa is already in an active form.

Animals↗

PubMed1988

Cell-specific processing of pro-cholecystokinin and pro-gastrin.

The present review argues that the gastrin-cholecystokinin family is a suitable model for the study of cell-specific processing of pro-hormones. First, the homologous active site of the hormones is a precisely defined tetrapeptide amide, which is well preserved during evolution. Second, the genes of both hormones are translated in a variety of cells (neurons, endocrine cells, paracrine cells, lymphocytes, etc,), but to a varying degree during ontogenesis and pathogenesis of various diseases. Third, each pro-hormone contains multiple processing sites (mono- and dibasic cleavage sites, amidation sites and consensus sequences for seryl phosphorylation and tyrosyl sulfation) leaving ample room for variations in the post-translational processing. The review discusses examples of cell-specific processing that appears to be functionally expedient.

Amino Acid Sequence↗

PubMed1988

Corelease of amidated and glycine-extended antral gastrins after a meal.

Glycine-extended gastrin, the immediate precursor of bioactive (i.e., carboxyamidated) gastrin, was recently identified in antral tissue and in peripheral blood. Studies on pituitary cell lines have shown that although only bioactive peptides are released along the regulated pathway, i.e., stored at high concentrations in secretory granules and released upon appropriate stimulation, the release of precursors is constitutive, i.e., secreted continuously without secretagogue stimulation. To determine whether the secretion of glycine-extended gastrin from the antral G-cell is regulated, meal-induced release was examined. In anesthetized pigs (n = 8) the antral veins were catheterized and blood was collected continuously before and after intragastric instillation of meat extract. Surgical biopsies of antral mucosa were obtained from six animals. Glycine-extended and amidated gastrins were measured and characterized by radioimmunoanalysis and chromatography. The results demonstrate that glycine-extended gastrin is released in a regulated manner from antral G-cells and furthermore the component pattern is identical in mucosa and blood.

Amines↗

PubMed1988

Influence of ascorbic acid on in vivo amidation of alpha-melanocyte stimulating hormone in guinea pig pituitary.

The effect of ascorbic acid depletion on the amidation of alphamelanocyte stimulating hormone (alpha MSH) was studied in vivo in guinea pig pituitary. After four weeks, the concentration of ascorbic acid was 1.20 +/- 0.11 mumol/g tissue (mean +/- SD) in the pituitary and 0.34 +/- 0.07 mumol/g tissue in the cerebral cortex from the depleted animals versus 7.58 +/- 0.08 and 1.51 +/- 0.32 mumol/g tissue, respectively, in the control animals. In the pituitaries from the animals depleted of ascorbate (N = 4), the relative amount of alpha MSH was reduced to approximately half the values obtained in the control group (from 66.5 +/- 4.6% of total ACTH-related peptides to 31.1 +/- 12.2% (P less than 0.0025]. A concomitant increase (from 5.9 +/- 3.1% to 19.4 +/- 4.3% (P less than 0.004] in ACTH (1-14) (the glycine-extended precursor of alpha MSH) immunoreactivity and a smaller increase in ACTH (1-39) immunoreactivity was observed in the depleted guinea pigs. Gel chromatography and reversed-phase high-performance luquid chromatography showed that the alpha MSH and ACTH (1-14) immunoreactivity was of low molecular weight and partly mono- or diacetylated. Depletion of ascorbic acid had no influence on the degree of acetylation of alpha MSH and ACTH (1-14). It is concluded that depletion of ascorbic acid reduces the in vivo amidation of ACTH (1-14) in the guinea pig pituitary.

Adrenocorticotropic Hormone↗

PubMed1987

Alpha-carboxyamidation of antral progastrin. Relation to other post-translational modifications.

Using radioimmunoassays for amidated and glycine-extended gastrin before and after trypsin-carboxypeptidase B cleavage and chromatography, alpha-carboxyamidation of porcine antral progastrin has been related to tyrosine-O-sulfation and proteolytic cleavages. Corresponding to the sequence at the proteolysis and amidation site, -Gly-Arg-Arg-, antrum contained three COOH-terminally extended precursor types. The glycine-extended gastrins were present in the highest concentrations (241 +/- 58 pmol/g). The degree of tyrosine-O-sulfation was identical for amidated and precursor gastrins irrespective of component size, whereas the component size differed for glycine-extended and amidated forms. For instance, gastrin-34-Gly constituted 54% of the glycine-extended gastrins, while gastrin-34 comprised 8% of the amidated gastrins. The results indicate that tyrosine-O-sulfation occurs prior to NH2-terminal cleavages, which again precede carboxyamidation; but a significant correlation between tyrosine-O-sulfation and proteolytic cleavages or alpha-carboxy-amidation of antral gastrin could not be demonstrated. Furthermore, our results suggest that the immediate precursor of the principal hormonal form, gastrin-17, is gastrin-17-Gly rather than gastrin-34 as previously believed.

Amides↗

PubMed1987

Immunochemical analysis of neuropeptides.

Measurement of neuropeptides requires assays that take into account the basic characteristics of bioactive peptides, i.e. the structural homology ("families"); the molecular heterogeneity of a given neuropeptide system; the widespread synthesis in different neurons and cells; and cellspecific processing of the primary translationproduct. Development of libraries of sensitive radioimmunoassays (RIAs), each of which is monospecific for essential sequences of propeptides, comply with some of the needs. Processing-site specific RIAs have proven particularly useful in combination with chromatography and enzymography.

Antibodies↗

PubMed1986

Impaired alpha-carboxyamidation of gastrin in vitamin C-deficient guinea pigs.

The biosynthetic activation of many hormonal peptides requires enzymatic alpha-carboxyamidation via glycine-extended intermediate forms. By specific radioimmunochemical measurement on extracts of guinea pig antra, we found that vitamin C deficiency induced a 30-fold increase in glycine-extended (i.e. inactive) gastrins. The results indicate that ascorbic acid is necessary for alpha-carboxyamidation in vivo, and thus, that the biosynthetic activation of hormonal peptides can be modified by simple dietary means.

Animals↗

PubMed1986

Measurement of precursors for alpha-amidated hormones by radioimmunoassay of glycine-extended peptides after trypsin-carboxypeptidase B cleavage.

Using fragment 5-17 of human gastrin-17 extended with glycine at the C-terminus as hapten, three of six rabbits produced high-titer, high-avidity antisera specific for glycine-extended gastrins. In combination with trypsin and carboxypeptidase B cleavage, radioimmunoassays based on these antisera measured progastrins in some extra-antral tissues and certain malignant tumors. The results show that sequential cleavage with trypsin and carboxypeptidase B followed by radioimmunoassay of glycine-extended peptides is a rapid and accurate procedure for measurement of biosynthetic precursors of alpha-amidated peptide hormones. Moreover, the procedure seems promising in the search for tumor markers.

Animals↗

PubMed1982

On the accuracy of radioimmunological determination of somatostatin in plasma.

We performed the following experiments to evaluate the accuracy of our newly developed radioimmunoassay for somatostatin: (1) Recovery of synthetic somatostatin added to human, porcine, and canine plasma with or without extraction with 67% acetone or 76% ethanol, using 3 different region-specific antibodies and, where applicable, 125I-labelled Tyr-1- or Tyr-11-substituted somatostatin or 125I-N-Tyr-somatostatin as tracers. The recovery of somatostatin corrected for losses inherent in the extraction procedure was close to 100%, and independent of species, antibody and tracer. Somatostatin 1-28 was extracted slightly less efficiently. Unextracted plasma interfered massively in the assay. (2) Pharmacokinetic experiments with infusion of somatostatin into 14 pigs and determination of metabolic clearance rate (MCR) and T-1/2. MCR was 27-38 ml/kg per min, independent of infusion rate (6.1 or 13 pmol/kg per min), extraction procedure or tracer. T-1/2 was 1.9 min. The infused somatostatin was not measurable in unextracted plasma. (3) Characterization of endogenous and exogenous, labelled and unlabelled somatostatin 1-14 in human plasma, using Sephadex G-50 columns at pH 7.5 and 9.0. Human plasma showed excess immunoreactivity eluting at the void volume whereas synthetic somatostatin was recovered quantitatively at the position of marker somatostatin when added to the plasma. The immunoreactivity of the tracers was decreased (125I-Tyr-11-somatostatin) or abolished (125I-N-Tyr- or 125I-Tyr-1-somatostatin) after incubation with plasma or void volume fractions of plasma subjected to gel filtration. Extracted plasma did not contain void volume immunoreactivity, but like whole plasma, small amounts of components which coeluted with intact somatostatin.

Animals↗