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L Hilsted

Publications and source records attributed to L Hilsted.

At least 19 recordsLinked to original sources
PubMed1992

Expression, but failing maturation of procholecystokinin in cerebellum.

The cerebellum is the only region of the central nervous system which has been found to be devoid of cholecystokinin (CCK). The assays used, however, have been directed against the alpha-amidated C-terminus of fully processed CCK peptides. Using Northern blot analysis and a library of radioimmunoassays specific for different sequences of proCCK in combination with chromatography and enzyme cleavage, we have now examined the expression and processing of proCCK in fetal, neonatal and adult cerebellar tissue from man, pig and rat. In rat cerebellum CCK mRNA was present already in the fetal state. Two weeks after birth the concentrations declined. Also proCCK was found in significant concentrations in the fetal human and rat cerebellum (approximately 20 pmol/g); but already before birth the expression began to decrease towards low concentrations in adults. The adult porcine cerebellum contained 3.2 pmol proCCK and glycine-extended processing intermediates per gram (range less than 0.1-10.4 pmol/g), and 0.8 pmol carboxyamidated CCK per gram (range 0.1-4.1 pmol/g) varying in size from CCK-58 to CCK-5. For comparison, the adult porcine cerebral cortex contained 757 pmol carboxyamidated CCK/g, 20 pmol glycine-extended CCK/g and no proCCK. We conclude that cerebellum expresses proCCK with the highest level of expression in fetal life. In comparison with other regions of the brain, the maturation to transmitter-active, carboxyamidated CCK peptides is, however, attenuated in both fetal and adult cerebellar tissue.

Aging↗
PubMed1992

Ontogeny of procholecystokinin maturation in rat duodenum, jejunum, and ileum.

Expression and processing of procholecystokinin (proCCK) in rat intestine during development were examined using sequence-specific immunoassays, cleavage with processing-like enzymes, and chromatography. Fetal proCCK concentrations were similar in duodenum, jejunum, and ileum, but the maturation to CCK followed different courses: duodenal CCK increased from 14 pmol/g in the fetus to 86 pmol/g 4 days after birth and then declined to 17 pmol/g in the adult. In jejunum, CCK varied from 34 pmol/g in the fetus to 127 pmol/g at day 7, decreased to 54 pmol/g at day 21, and increased again to 93 pmol/g in the adult. Ileal CCK decreased from 20 pmol/g in the fetus to 10 pmol/g postnatally. Whereas duodenal proCCK after birth matured completely to carboxyamidated CCK, jejunoileal proCCK matured only partially. Chromatography showed an increase of tyrosine-sulfation and proteolytic processing of N-terminal sequences. At day 7 jejunal cholecystokinin octapeptide (CCK-8) constituted only a minute fraction of the carboxyamidated CCK, of which less than half was sulfated. However, in the adult jejunum, CCK-8 constituted a significant fraction, which was completely sulfated. It is concluded that the CCK gene is well expressed at propeptide level in the fetal small intestine. Postpartum maturation of proCCK, however, is late and differs in the three parts of the small intestine. The belated maturation supports the hypothesis that factors other than CCK regulate pancreatic growth in fetal and neonatal life.

Amino Acid Sequence↗
PubMed1992

Gastrin and cancer.

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Amino Acid Sequence↗
PubMed1992

Ontogeny of gastric function in the pig: acid secretion and the synthesis and secretion of gastrin.

Gastric acid secretion, gastrin-releasing peptide (GRP)-stimulated gastrin secretion and concentrations of somatostatin in gastric tissues were studied in sucking pigs (n = 48). In addition, gastrin concentrations in plasma and antral tissue were measured in fetal and sucking pigs (n = 66) from 22 days before birth (93 days gestation) to 36 days of age. From 3 days of age littermate pairs were treated twice a day with either saline (n = 20) or adrenocorticotropin [ACTH (1-24); n = 20]. Pentagastrin-stimulated acid secretion per unit stomach weight was 39 +/- 7 mumol H+/g/h at 0-1 day, increased to 194 +/- 15 mumol H+/g/h at 5-7 days and plateaued. Antral gastrin concentration was 0.14 nmol/g 10 days before birth and increased to 2.7 nmol/g at 5 weeks of age. Plasma gastrin was 25 +/- 2 pmol/l at 22 days before birth, increased to 102 +/- 14 pmol/l at birth and decreased during the postnatal period. Somatostatin concentrations were higher in antral than fundic tissues (p < 0.05) and remained constant during the postnatal period. Increased levels of glucocorticoids in plasma following ACTH treatment had no effect on the studied parameters except that it reduced basal (p < 0.07) and GRP-stimulated (p < 0.05) plasma gastrin concentrations at 6-7 days of age. Development of acid secretion and its gastric regulatory peptides in the pig is different from that in the rat in that it occurs at an earlier age and does not appear to be greatly influenced by elevated glucocorticoid levels from 3 days after birth.

Adrenocorticotropic Hormone↗
PubMed1991

Ontogeny of procholecystokinin processing in rat hypothalamus.

The concentration of procholecystokinin (pro-CCK) in the fetal hypothalamus was 126 +/- 41 pmol/g (mean +/- SEM; n = 20), 22 +/- 9 pmol/g at day 7 postpartum and 3 +/- 2 pmol/g in the adult. In contrast, the concentration of bioactive carboxyamidated CCK rose from 6 +/- 2 pmol/g in the fetal hypothalamus to 52 +/- 10 pmol/g in the adult. The concentration of glycine-extended processing intermediates first decreased from 21 +/- 5 pmol/g in the fetus to 5 +/- 1 pmol/g at day 21 postpartum. Subsequently, the concentration rose to 21 +/- 4 pmol/g in the adult. The results show that the CCK gene is well expressed in the fetal hypothalamus. However, only a small fraction of pro-CCK reaches maturation before weaning. We conclude that expression of the CCK gene in the hypothalamus as bioactive peptide to a large degree is regulated at the posttranslational level.

Amino Acid Sequence↗
PubMed1991

The expression of peptide hormones in normal cells and tumour cells.

Insight in the mechanisms of peptide hormone expression has grown explosively by elucidation of gene, mRNA and preprohormone structures for most hormone systems during the 1980s. In addition, information about the structure and substrate specificity of many prohormone processing enzymes is rapidly accumulating in these years. The preprohormones vary considerably in size and organization from poly- to monoprotein structures. According to the structural organization and sequence homology the hormones are grouped in families. The prohormones are processed to bioactive peptides by multiple enzymatic modifications during the intracellular transport from the rough endoplasmatic reticulum to the mature secretory granules. The modifications comprise different proteolytic cleavages and amino acid derivatizations. The same prohormone may be expressed in several different cell types that process the precursor in entirely different ways. Awareness of such cell-specific processing patterns is important for the understanding of ectopic synthesis in neuroendocrine tumours.

Gene Expression Regulation↗
PubMed1990

Alpha-amidation of gastrin is impaired by diethyldithiocarbamate.

The influence of gastrin alpha-amidation of the heavy-metal chelator diethyldithiocarbamate and disulfiram, its disulfide dimer, was studied in rat gastric antrum. Sensitive, sequence-specific immunoassays for glycine-extended and amidated gastrin were used to monitor extractions and chromatography. The results showed that intraperitoneal diethyldithiocarbamate administration (1000 mg/kg body weight) for two days caused a decrease in amidated gastrin from 2.6 +/- 0.4 to 1.4 +/- 0.3 nmol/g tissue (n = 11) with a simultaneous increase in glycine-extended gastrin from 0.84 +/- 0.15 to 2.4 +/- 0.3 nmol/g. Peroral administration of disulfiram (4 mg/kg body weight) for nine days did not change alpha-amidation significantly. The results of the present study demonstrate that the heavy-metal chelating agent diethyldithiocarbamate inhibits alpha-amidation of gastrin in vivo, in agreement with the inhibition of amidating activity observed in vitro. These results are in accordance with the previous observations that the presence of copper ions is necessary for the alpha-amidation to take place.

Amides↗
PubMed1990

Procholecystokinin processing in rat cerebral cortex during development.

Using a library of radioimmunoassays for essential sequences of procholecystokinin (proCCK), we have examined the post-translational processing in the rat cerebral cortex from fetal to adult state. The concentration of proCCK in the fetal cerebral cortex was 43 +/- 7 pmol/g tissue (wet weight; mean +/- S.E.M. (n = 20)). It remained constant until day 21 post partum, after which it decreased to undetectable levels. In contrast, the concentration of fully processed, bioactive CCK peptides (i.e. alpha-carboxyamidated CCK) rose from 2 +/- 1 pmol/g in the fetal cortex to 122 +/- 21 pmol/g in the adult. A particularly steep increase occurred from day 7 post partum (13 +/- 2 pmol/g) to day 21 (108 +/- 11 pmol/g). The concentration of glycine-extended intermediates rose gradually from 8 +/- 1 pmol/g in the fetal brain to 55 +/- 6 pmol/g in the adult. Gel chromatography of cortical extracts from day 7, 21 and 100 confirmed the variable processing at the C-terminal amidation site. The results show that the CCK gene is expressed as proCCK already in the fetal brain. However, the covalent modifications of proCCK follow different time courses so that only a small fraction reaches maturation until the first week post partum. We conclude that expression of transmitter-active CCK peptides in the brain is largely regulated at the post-translational rather than at the transcriptional level.

Aging↗
PubMed1990

Progastrin expression in mammalian pancreas.

Expression and processing of progastrin were examined in fetal, neonatal, and adult pancreatic tissue from five mammalian species (cat, dog, man, pig, and rat). A library of sensitive, sequence-specific immunoassays for progastrin and its products was used to monitor extractions and chromatography before and after cleavage with processing-like enzymes. The results showed that progastrin and its products are expressed in the pancreas of all species in total concentrations varying from 0.3 to 58.9 pmol/g of tissue (medians). The degree of processing was age- and species-dependent. In comparison with adult pancreatic tissue the fetal or neonatal pancreas processed a higher fraction to bioactive, C-terminally amidated gastrin. Nevertheless, the pancreatic processing was always less complete than that of the adult antral mucosa. The moderate level of expression and the attenuated processing in the adult pancreas contribute to explain previous failures to detect gastrin in normal pancreatic tissue. Our results indicate that gastrin-producing tumors in the pancreas are not ectopic, but arise from cells that normally express the gastrin gene.

Aging↗
PubMed1990

Screening for multiple endocrine neoplasia type 1 in patients with recognized pituitary adenoma.

A total of 79 consecutive patients with pituitary tumours were screened for multiple endocrine neoplasia type 1 (MEN-1). The 79 patients included 21 patients with acromegaly, nine with Cushing's disease, 18 with prolactinomas, three with mixed pituitary adenomas (GH and PRL), and 28 patients with no detectable hypersecretion of hormones. The screening consisted of: (1) a family history, (2) a uniform medical history of the patient using a standard questionnaire, and (3) hormonal evaluation including measurements of the serum levels of insulin, gastrin, glucagon, somatostatin, vasoactive intestinal polypeptide and pancreatic polypeptide. Ionized calcium and glucose concentration in serum were also measured. We found no patients with the MEN-1 syndrome. In one patient, we found a transient elevation of serum concentrations of pancreatic polypeptide for which we have no explanation. In another patient, the serum gastrin concentration was elevated secondary to achlorhydria. No other endocrine disorders were found, and no patients had relatives with recognized endocrine pancreatic tumours, primary hyperparathyroidism (HPT), or pituitary adenomas.

Adenoma↗
PubMed1989

Gastrin in human bronchogenic carcinomas: constant expression but variable processing of progastrin.

Using a library of radioimmunoassays against essential sequences of human progastrin and procholecystokinin, we have examined the occurrence of gastrin, cholecystokinin, and their precursors in bronchogenic adenocarcinomas, large-cell, small-cell, and squamous-cell carcinomas (n = 17). Progastrin and some of its bioactive (i.e., alpha-carboxyamidated) products were present in all tumors, irrespective of histological classification. The concentration of progastrin varied from 0.2 to 21.9 pmol/g tissue; glycine-extended intermediates constituted less than 0.1 to 0.5 pmol/g; and bioactive, carboxyamidated gastrin ranged from less than 0.1 to 6.1 pmol/g. Chromatography showed that the bioactive gastrins were exclusively gastrin-17 peptides, half of which were tyrosine O-sulfated. Neither procholecystokinin nor its processing products were found in the tumor extracts. Six samples of nonneoplastic human lung tissue contained traces of progastrin (range, less than 0.1-0.8 pmol/g), but neither bioactive gastrins nor any cholecystokinin. The results show that the gastrin gene is expressed in all classes of bronchogenic carcinomas. Due to incomplete posttranslational processing measurement of progastrin may be necessary to detect such expression.

Base Sequence↗
PubMed1989

Progastrin processing during antral G-cell hypersecretion in humans.

Using radioimmunoassays specific for essential processing sites of human progastrin in combination with chromatography before and after cleavage with trypsin and carboxypeptidase B, we have examined antral biopsy specimens and serum from 10 hypergastrinemic patients with fundic atrophic gastritis and 7 normal control subjects. Four types of processing were studied: N-terminal proteolysis (at the N-terminus of component I, gastrin 34, and gastrin 17); C-terminal proteolysis (at the C-terminus of the amide donor, glycine93 in preprogastrin); alpha-carboxyamidation (of phenylalanine92); and O-sulfation (of tyrosine87). The results show that progastrin during permanent G-cell hypersecretion is less completely processed with respect to C-terminal proteolysis, alpha-amidation, and tyrosine-sulfation. In contrast, the degree of N-terminal proteolysis is normal. Thus, the processing of progastrin adjacent to the active site of gastrin is more restrictively controlled than N-terminal processing during G-cell hypersecretion associated with pernicious anemia.

Carboxypeptidase B↗
PubMed1989

Cholecystokinin, gastrin and their precursors in pheochromocytomas.

Using sequence-specific radioimmunoassays before and after cleavage with trypsin and carboxypeptidase B, we have examined the occurrence and molecular nature of cholecystokinin (CCK) and gastrin peptides in bioactive (i.e. alpha-carboxyamidated) as well as non-amidated precursor forms in extracts from 13 human pheochromocytomas. All but one tumour contained amidated CCK, but only in moderate amounts (less than or equal to 20 pmol/g tissue). In contrast to the complete sulphation in tissues which normally produce CCK (the brain and small intestine), the amidated adrenal CCK peptides were poorly sulphated (less than or equal to 17%). Four pheochromocytomas, including the one without amidated CCK, contained between 28 and 0.2 pmol amidated gastrin/g, mainly in the form of sulphated gastrin-17. In addition, all tumours contained biosynthetic precursors of both CCK and gastrin. In most extracts there was more precursor than bioactive peptide(s), the progastrin concentration ranging up to 338 pmol/g. The results show that pheochromocytomas synthesize CCK and gastrin. The posttranslational processing differs, however, markedly from that of the principal CCK and gastrin producing tissues, with respect to both proteolytic cleavages and amino acid derivatization. This emphasizes that accurate quantitation in tumours requires assays which measure the translation products irrespective of their degree of processing.

Adrenal Gland Neoplasms↗
PubMed1989

Peptide hormone expression and precursor processing.

Insight in the mechanisms of peptide hormone expression has grown explosively by elucidation of gene, mRNA and preprohormone structures for most hormone systems during the 1980s. The preprohormones vary considerably in size and organization from poly- to mono-protein structures. According to the structural organization and sequence homology the hormones are grouped in families. The prohormones are processed to bioactive peptides by multiple enzymatic modifications during the intracellular transport from the rough endoplasmatic reticulum to the mature secretory granules. The modifications comprise different proteolytic cleavages and amino acid derivatizations. The same prohormone may be expressed in several different cell-types that process the precursor in entirely different ways. Awareness of such cell-specific processing patterns is important for the understanding of ectopic synthesis in neuroendocrine tumours.

Animals↗
PubMed1989

The unique specificity of antibodies in modern radioimmunochemistry--an essay on assays.

The gradual recognition and exploitation of the specificity of antibodies in peptide radioimmunoassays (RIA) during the last three decades are reviewed. From the old fashioned RIA techniques of the sixties through sequence-specific RIA libraries of the seventies to residue-specific immunoassays of the eighties, the RIA technique has to an increasing degree been based on the ability to select strictly monospecific antibodies in high-titered polyclonal antisera. High-avidity monospecific antibodies from polyclonal antisera provide the RIA technology of to-day with unrivalled specificity. This specificity again has increased the utility of RIA as a tool in basic as well as clinical biochemistry.

Amino Acid Sequence↗
PubMed1988

Progastrin maturation during ontogenesis. Accumulation of glycine-extended gastrins in rat antrum at weaning.

The post-translational maturation of antral progastrin was studied in the developing rat. While N-terminal proteolysis remained unchanged and tyrosine O-sulphation varied only slightly during ontogenesis, major changes were observed in the degree of alpha-carboxyamidation. In the third week of life the immediate precursor of amidated gastrin, glycine-extended gastrin, accumulated, and at weaning (day 21) the concentrations exceeded those of amidated gastrin. Our results confirm that weaning is accompanied by an increased synthesis of gastrin and imply that alpha-carboxyamidation is the rate-limiting step during the biosynthetic maturation of gastrin.

Amides↗