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Biomedical subjects

J F Rehfeld

Publications and source records attributed to J F Rehfeld.

At least 145 records · Page 8Linked to original sources
PubMed1994

Identification of gastrin component I as gastrin-71. The largest possible bioactive progastrin product.

Gastrin component I is the largest hormonally active form of gastrin. In order to determine its structure, we isolated progastrin-derived peptides from normal human antral tissue. A radioimmunoassay specific for sequence 20-25 of human progastrin was developed to monitor the purifications. After four or five steps of reverse-phase chromatography, the peptides were pure and could be identified by a combination of microsequence, amino acid and mass spectral analysis as well as by a library of sequence-specific immunoassays. In addition to intact progastrin 1-80, fragments 1-71, 1-35, 6-35, 20-35, and 20-36 of progastrin were identified. Only the 71-amino-acid peptide contained at its C-terminus the alpha-amidated bioactive site (Trp-Met-Asp-Phe-NH2). This unoheptacontapeptide amide (gastrin-71) corresponds to component I and is the largest possible bioactive product of progastrin. Its structure shows that progastrin is used in its entirety for biosynthesis of active peptides. The occurrence of fragments 6-35, 20-35, and 20-36 demonstrate that antral progastrin is partially cleaved at two monobasic sites (Arg5 and Arg19) in addition to processing at the three C-terminal dibasic sites. The results show that both the N- and C-terminal parts of antral progastrin undergo extensive processing. The results also suggest that progastrin may follow two different processing pathways of which the less trafficked releases gastrin-71.

Amino Acid Sequence↗
PubMed1994

Ileal expression of gastrin and cholecystokinin. In search of a related hormone.

Antibodies against the common active site of cholecystokinin (CCK) and gastrin stain three endocrine cell-types in the gut: G-cells (that synthesize gastrin) I-cells (that synthesize CCK), and TG-cells (whose product is unknown). In order to examine whether TG-cells either process progastrin or proCCK in a specific manner, or express a novel gastrin-CCK related hormone, we studied the distal porcine ileum, which have far more TG- than G- and I-cells. Ileal CCK and gastrin mRNA corresponded to those of the antroduodenal mucosa. Gel, ion-exchange and reversed-phase chromatography monitored with sequence-specific immunoassays showed that the ileal mucosa on average contain 0.3 and 7.6 pmol/g progastrin and proCCK, respectively; 1.1 and 13.5 pmol/g glycine-extended intermediates; and 1.1 and 24.8 pmol/g of bioactive carboxyamidated gastrin and CCK, respectively. Gastrin was present only as non-sulfated gastrin-34, whereas CCK occurred in forms similar to those of the proximal intestine. Although ileal progastrin processing is tissue specific, the amounts of gastrin and CCK are too small to explain the TG-cells. Moreover, since the ileal extracts were without traces of other peptides having the C-terminus common to gastrin and CCK, the results suggest that TG-cells produce a peptide, which is only weakly related to gastrin and CCK.

Amino Acid Sequence↗
PubMed1994

Insulin-like growth factor II mRNA, peptides, and receptors in a thoracopulmonary malignant small round cell tumor.

Insulin-like growth factor-(IGF) II and IGF-I and IGF-II/mannose 6-phosphate receptors were expressed in a thoracopulmonary malignant small round cell tumor (MSRCT) from a 14-year-old boy. Northern analysis showed that the MSRCT expresses multiple IGF-II mRNA of 6.0, 4.8, 4.2, and 2.2 kilobase from promoters P3 and P4 of the human IGF-II gene. Chromatography and radioimmunoassay revealed two forms of IGF-II with molecular masses of 7.5 kilodalton (kDa) and 10 kDa, corresponding to mature IGF-II and IGF-II with a C-terminal extension, in concentrations of 61 and 41 ng/g/tumor tissue, respectively. By a combined reverse transcription-polymerase chain reaction analysis, the authors also show that the MSRCT expresses IGF-I and IGF-II/mannose 6-phosphate receptor mRNA. The plasma concentration of IGF-II was 600 ng/ml and within the normal range of serum IGF-II. IGF binding proteins (IGFBP) of 41.5, 38.5, 34, 30, and 24 kDa were present in serum. Compared with normal plasma from healthy subjects and an age-matched group of boys, the level of the 41.5, 38.5, and 30 kDa IGFBP were approximately 3-fold elevated. The authors conclude that transcription of the IGF-II gene leads to the production of significant amounts of 10 kDa IGF-II and 7.5 kDa IGF-II. IGF-II may stimulate the proliferation of MSRCT by interaction with IGF-I receptors on the cells.

Adolescent↗
PubMed1994

Plasma gastrin and cholecystokinin responses after pylorus-preserving pancreatoduodenectomy and defunctioned Roux loop pancreaticojejunostomy.

Several methods can be used to re-establish gastrointestinal continuity after the standard Whipple's Billroth II procedure or Longmire pylorus-preserving pancreatoduodenectomy (PPPD). Standard pancreatoduodenectomy abolishes the postprandial gastrin response whereas the response after Longmire PPPD is similar to that in controls. A novel reconstruction after PPPD has been designed to separate the biliary and pancreatic secretions by restoring continuity in the Billroth I manner, retaining the gastric antrum and with the pancreatic remnant anastomosed to a separate defunctioned Roux loop. Gastrin and cholecystokinin (CCK) responses were measured in the fasting state and after a standard liquid meal of 250 ml containing 15 per cent protein, 30 per cent fat and 55 per cent carbohydrate with an energy value of 525 kJ. Basal gastrin and CCK concentrations were similar in five healthy unoperated controls, and after standard pancreatoduodenectomy (five patients) and isolated Roux loop PPPD (six patients). In controls and after the new PPPD operation, but not after standard pancreatoduodenectomy, mean(s.e.m.) postprandial plasma gastrin values rose from 8.66(0.33) to 13.00(0.33) pmol l-1 at 10 min (controls) and from 7.66(1.56) to 15.00(1.36) pmol l-1 at 10 min (defunctioned Roux loop PPPD); the concentration remained raised for 20 min then fell transiently; a second peak was maintained for 40 min (controls) to 60 min (modified PPPD). In controls, mean(s.e.m.) postprandial plasma CCK concentrations rose from 0.43(0.12) basal to 10.50(1.03) pmol l-1 and returned to basal after 60 min. After standard pancreatoduodenectomy postprandial CCK concentrations rose at a similar rate and remained raised for 80 min. After isolated Roux loop PPPD plasma CCK concentrations remained low (mean(s.e.m.) 0.38(0.29) pmol l-1) after the test meal.

Aged↗
PubMed1994

Localisation of sulfakinin neuronal pathways in the blowfly Calliphora vomitoria.

The distribution of neurones immunoreactive to antisera raised against the undecapeptide C-terminal fragment of drosulfakinin II (DrmSKII), Asp-Gln-Phe-Asp-Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-NH2, has been studied in the blowfly Calliphora vomitoria. Antisera were preabsorbed with combinations of the parent antigen, the tetrapeptide Phe-Met-Arg-Phe-NH2 and cholecystokinin, the vertebrate sulfated octapeptide (CCK-8), Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2, in order to ensure specificity for the sulfakinin peptides of C. vomitoria (the nonapeptide callisulfakinin I is identical to drosulfakinin I and callisulfakinin II differs from DrmSK II only by the presence of -Glu3-Glu4- in place of -Asp3-Asp4-). Only four pairs of sulfakinin-immunoreactive neurons have been visualised in the entire nervous system. These occur in the brain: two pairs of cells situated medially in the caudo-dorsal region close to the roots of the ocellar nerve and two other pairs at the same level but positioned more laterally. Despite the small number of sulfakinin-immunoreactive cells, there are extensive projections to many areas of neuropile in the brain and the thoracic ganglion. The pathway of the medial sulfakinin cells extends into each of the three thoracic ganglia and a metameric arrangement of sulfakinin neuronal projections is also seen in the abdominal ganglia. Neither the dorsal neural sheath of the thoracic ganglion, nor the abdominal nerves contain sulfakinin-immunoreactive material. These observations suggest that the sulfakinins of the blowfly function as neurotransmitters or neuromodulators. They do not appear to have a direct role in gut physiology, as has been shown by in vitro bioassays for the sulfakinins of orthopterans and blattodeans. In addition to the neurones that display specific sulfakinin immunoreactivity, other cells within the brain and thoracic ganglion are immunoreactive to cholecystokinin/gastrin antisera. There are, therefore, at least two types of dipteran neuropeptides with amino acid sequences that are similar to the vertebrate molecules cholecystokinin and gastrin.

Amino Acid Sequence↗
PubMed1994

A processing-independent assay for human procholecystokinin and its products.

In order to develop a processing-independent analysis for procholecystokinin (proCCK) and its products, antibodies were raised against the synthetic fragment 62-71 of human proCCK. All rabbits (n = 8) responded to the immunization. One (No. 89,009) produced antibodies of particularly high titer (1:350,000), homogeneity (Sips' index approximately 1.0) and binding affinity (K0 eff approximately 0.88 x 10(12) l/mol). A radioimmunoassay using this antiserum and [125I]tyrosine-extended fragment 62-71 measured the total CCK mRNA product after tryptic cleavage at Lys61 in normal and neoplastic tissue independent of the degree of precursor processing. In addition to previously known CCK producing tumors, CCK was found also in a thoracic round-cell tumor (Askin tumor) and in brain tumors (gliomas and astrocytomas). These tumors processed proCCK poorly. Thus, they contained 11 and 23 (mean n = 5) pmol/g of proCCK and its products before, versus 71 and 99 (mean) pmol/g after tryptic cleavage, respectively. Accordingly, gel chromatography revealed significant amounts of unprocessed proCCK, large molecular forms of glycine-extended CCKs and the well-known carboxyamidated and tyrosine O-sulfated bioactive CCK-83, -58, -33, -22 and -8. We conclude that monospecific antibodies directed against the N-terminus of sequence 62-71 of human proCCK are suitable for processing-independent analysis (PIA) for proCCK and its products. Moreover, we suggest that such PIA should be used for quantitation of CCK gene expression at peptide level in normal tissue and tumors.

Amino Acid Sequence↗
PubMed1994

Effect of sulpiride or paroxetine on cerebrospinal fluid neuropeptide concentrations in patients with chronic tension-type headache.

In lumbar cerebrospinal fluid (CSF) obtained from patients with chronic tension-type headache (CTH), the concentrations of beta-endorphin, met-enkephalin, dynorphin, cholecystokinin (CCK), calcitonin gene-related peptide (CGRP), and somatostatin were measured before and after 8 weeks of treatment with sulpiride or paroxetine. We previously reported higher than normal met-enkephalin concentrations in CTH. The present study reveals normal basal concentrations of CCK, CGRP and somatostatin and slightly decreased dynorphin in the same patients. Treatment with sulpiride or paroxetine did not change the concentration of any of the neuropeptides measured. These data suggest central changes in opioid systems but not in other peptide systems (CCK, CGRP, somatostatin) involved in nociceptive processing at the level of the spinal cord dorsal horn/nucleus caudalis of the trigeminal nerve in CTH. Such central changes might be pathophysiologically important or merely secondary to other more important occurrences. The lack of changes in neuropeptide concentrations during drug treatment makes planning of studies involving CSF analysis easier, but also limits the probability of obtaining information on specific neuropeptide systems through CSF analysis.

Adult↗
PubMed1994

Measurement of gut hormone gene expression: mRNA and peptides.

During the past decade numerous methods for measurement of mRNA and peptides have been developed. Since the expression cascade from DNA to protein is regulated at all levels, the methods should be carefully designed to accomplish the purpose of the analysis. Regulation of the nuclear processing, the translational activity and the decay of a particular mRNA changes the proportionality between transcriptional activity and production of prepropeptide. Moreover, the post-translational maturation of the pro-hormones may be attenuated. Detection of mRNA is valuable and feasible because it is easy to generate cDNA probes for most hormones, and because mRNA demonstration unequivocally indicates the cellular site of gene expression. The deduction of preprohormone structures has also made it possible to improve the versatility of radioimmunoassays (RIA). Monospecific antibodies and pure tracers have allowed the development of sequence-specific RIA libraries for bioactive peptides and their precursors. Recently we have introduced a simple processing-independent analysis (PIA) for clinical use, since the post-translational maturation of gut peptides may be changed in gastrointestinal diseases. So far PIA has improved the diagnostic sensitivity for gut hormone tumours.

Animals↗
PubMed1994

Gastrin and somatostatin in Helicobacter pylori infected antral mucosa.

Helicobacter pylori infection is associated with increased meal stimulated gastrin secretion, but the reason for this is unknown. Sequence specific radioimmunoassays were used to measure the concentration of alpha-amidated gastrin, the total progastrin product, and somatostatin in biopsy specimens of human antral mucosa. The antral concentrations of alpha-amidated gastrin and of total progastrin products were significantly higher in H pylori infected patients than in those not infected by this organism. In contrast, the antral somatostatin concentration was significantly decreased in infected patients. Progastrin processing, determined by gel chromatography, seemed unaffected by H pylori infection. The results suggest that the finding of increased gastrin secretion from the antral G cells in H pylori infected patients may be a result of reduced inhibition of G-cell secretion by somatostatin.

Adult↗
PubMed1994

Somatostatin in the boar reproductive system.

Somatostatin (SRIF) is a widely distributed regulatory peptide. Recently, it was shown that human seminal plasma contains high concentrations of SRIF. In order to find the cellular source of seminal SRIF we have examined the presence of SRIF in porcine urogenital tissues. The concentration of SRIF in male accessory reproductive tissues of 3-month-old pigs (N = 4) ranged from 1 to 17 nmol/kg. In the boar, only the prostate gland contained significant amounts of SRIF (median 7 nmol/kg, range 3-18 nmol/kg, N = 4). Testis and semen contained no SRIF. Gel chromatography of extracts of the male accessory sex glands and epididymis showed both SRIF-28 and SRIF-14, whereas urinary bladder contained mainly SRIF-14. In conclusion, the results show a considerable species, tissue and developmental variation in the expression of SRIF in the genitourinary tract.

Amino Acid Sequence↗
PubMed1994

Displaced chromatographic elution of gastrin-17 in normal human serum.

Sulfated gastrin-17 elutes by gel chromatography significantly earlier after incubation in human serum (KD: 0.48) than when added to buffer (KD: 0.62). Also, the elution of non-sulfated gastrin-17 changes after serum incubation although to a lesser extent (KD: 0.68 vs. 0.74). In contrast neither the N-terminal 1-13 nor the sulfated C-terminal hexapeptide fragment of gastrin-17 change elution positions after serum incubation. The earlier elution is concentration dependent. The results suggest that displaced elution of sulfated gastrin-17 may constitute part of the abundance of gastrin-34-like material in normal human serum. Hence, present concepts of gastrin heterogeneity in serum seem to require re-evaluation.

Chromatography, Gel↗