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J R Reeve

Publications and source records attributed to J R Reeve.

At least 55 records · Page 3Linked to original sources
PubMed1993

[Proteolytic processing by dipeptidyl aminopeptidase IV generates receptor selectivity for peptide YY (PYY)].

Two receptor subtypes, Y1 and Y2, are known to mediate PYY biological activity. PYY 1-36 binds to Y1 and Y2 receptors with equal affinity, whereas the second endogenous form of PYY, PYY 3-36, selectively binds to Y2 receptors. Dipeptidyl cleavage thus transforms an unselective Y agonist into a highly selective Y2 agonist, PYY 3-36. The enzyme responsible for this processing is unknown. Since PYY has a proline in the penultimate position it is protected from the attack of most unspecific exopeptidases. Only a few exopeptidases are theoretically capable of generating PYY 3-36 from PYY 1-36. Of the enzymes tested only the dipeptidyl aminopeptidase IV (DPP IV, E.C. 3.4.14.5) cleaved Tyr-Pro from PYY 1-36 with high activity. Since DPP IV is found on the endothelial surface and brush border membranes it can be considered a candidate enzyme for generating PYY 3-36 in vivo, thereby regulating the ratio of Y1/Y2 receptor stimulation by PYY.

Dipeptidyl Peptidase 4↗
PubMed1993

[Y1 receptors mediate inhibitory and stimulatory effects of peptide YY in isolated small intestine and large intestine muscles of the rabbit].

Peptide YY has been shown to have stimulatory and inhibitory effects on gastrointestinal motility. However, the receptors mediating these effects are unknown. To determine if specific YY receptor agonists can mediate the effects on gastrointestinal motility we studied the effects of peptide YY (PYY), of Pro34PYY, a selective Y1 agonist, and of PYY 3-36, a selective Y2 agonist, on the motility in isolated smooth muscle strips from rabbit small and large intestine. In strips from distal colon, PYY stimulated spontaneous motility whereas it inhibited spontaneous contractions in circular strips from distal ileum. In distal circular colon maximal inotropic response (10.1 +/- 2.1% of a maximal response to carbachol 10(-5) M) was found at PYY 10(-8) M; (ED50 3.1 +/- 1.2 x 10(-9) M). In distal circular ileum maximal inhibition (by 39 +/- 20% of basal motility index) was found at 10(-7) M; (ID50 6.2 +/- 1.4 x 10(-9) M). PYY caused a dose-dependent inhibition of the on-contraction induced by electrical field stimulation. This inhibition could not be reversed by alpha- or beta-adrenergic blockade. PYY had no influence on the inotropic response evoked by carbachol. Both the stimulatory effect of PYY observed in distal colon and the inhibitory effect in distal ileum could be reproduced by the Y1 agonist Pro34PYY, but not by the Y2 agonist PYY 3-36. In distal circular colon the maximal inotropic response evoked by the Y1 agonist was 10 +/- 1.4%; (ED50 1.2 +/- 0.5 x 10(-8) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Prevention of experimental autoimmune arthritis with a peptide fragment of type II collagen.

Collagen arthritis is induced in inbred rats with the injection of native type II collagen. The pathogenesis of this experimental autoimmune disease is T cell dependent. This study demonstrates that collagen-specific T cells, derived from pathogenic and nonpathogenic rat T cell lines, both recognize the same peptide epitope. The epitope, consisting of amino acids 58-73 of cyanogen bromide fragment 11 of type II collagen, was as effective as whole collagen in stimulating a panel of collagen-specific rat/mouse T cell hybridomas. This peptide may, therefore, constitute a dominant epitope for CD4+ rat T cells in their response to type II collagen. Administration of the peptide to either neonatal or adult rats prevented the subsequent induction of experimental arthritis with whole collagen, demonstrating that the in vivo response to this dominant epitope is, therefore, relevant in the pathogenesis of arthritis. Despite its ability to prevent collagen-induced arthritis, administration of this peptide in incomplete Freund's adjuvant intradermally did not induce disease.

Amino Acid Sequence↗
PubMed1993

Comparison of clearance and metabolism of infused cholecystokinins 8 and 58 in dogs.

BACKGROUND: Cholecystokinin (CCK) 58 is the predominant molecular form of CCK in canine and human intestine and circulating blood. There is no report on the metabolism and clearance rate of CCK-58. The aim of this study was to compare the in vivo half-life and metabolism of CCK-58 with that of synthetic CCK-8. METHODS: CCK-58 was purified from canine intestine by consecutive high-performance liquid chromatographic (HPLC) and fast protein liquid chromatographic steps. The peptides were given to 12 dogs as an intravenous (IV) bolus injection to determine the half-life of circulating CCK. Six dogs were given CCK-58 or CCK-8 as a constant IV infusion to determine plasma clearance rates and stability in circulating blood. Circulating molecular forms of CCK were determined by radioimmunoassay after extraction of CCK from plasma and characterization by HPLC. RESULTS: The half-life of CCK-58 was 4.4 +/- 0.6 minutes compared with 1.3 +/- 0.1 minutes for CCK-8. Less than 5% of CCK-58 could be detected as smaller forms during constant IV infusion. CONCLUSIONS: The longer half-life of CCK-58 compared with CCK-8 and the minimal conversion into smaller forms during constant IV infusion are consistent with the finding that CCK-58 is not only the major stored form but also the circulating form of CCK after endogenous stimulation in dogs.

Animals↗
PubMed1992

Novel generation of hormone receptor specificity by amino terminal processing of peptide YY.

The physiological significance of multiple Y receptors has not been determined since until recently only one form of endogenous agonists was known, namely PYY1-36 and NPY1-36. Recently, a new molecular form of PYY was characterized as des(Tyr-Pro)PYY (PYY3-36 or PYY-II). Its ability to interact at various Y receptors was not characterized. Analytical chromatography of fresh canine colon extracts shows two peaks of immunoreactivity eluting in the positions of PYY-II and PYY1-36 (PYY). PYY-II was about 40% of the total PYY immunoreactivity indicating that it is one of the major forms of PYY expressing its biological activity. It is shown that PYY-II will not displace label from the Y1 receptors found on a human neuroblastoma cell line. It is further shown that PYY-II is as potent as PYY for the inhibition of pancreatic secretion, which must occur through Y2 receptors. The enzymatic removal of Tyr-Pro from PYY to form PYY-II must therefore regulate the relative expression of a non-selective agonist (PYY) to a highly selective Y2 agonist (PYY-II). Amino terminal processing of PYY represents a novel type of regulation of peptide hormone specificity. It has important biological implications for PYY and potential relevance for other peptide hormone receptor systems.

Animals↗
PubMed1992

Gastrin-releasing peptide (GRP) is not mammalian bombesin. Identification and molecular cloning of a true amphibian GRP distinct from amphibian bombesin in Bombina orientalis.

On the basis of structural homology and similar biological activity, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of amphibian bombesin. In this paper we now show this to be incorrect. Chromatography of frog (Bombina orientalis) gut extracts demonstrated two peaks of bombesin-like immunoreactivity (BLI), one similar in size to GRP and one similar in size to amphibian bombesin. These peaks were purified by high pressure liquid chromatography then subjected to mass spectrometric analyses to determine molecular weights and amino acid sequence. Based on the amino acid sequence of the lower molecular weight BLI species, a mixed oligonucleotide probe was prepared and used to screen a B. orientalis stomach cDNA library. Sequence analysis showed that all hybridizing clones encoded a 155-amino acid protein homologous to the mammalian GRP precursor. The mass spectra of the high and low molecular weight peaks of frog gut BLI were consistent with their origin from the processing of the frog GRP (fGRP) precursor into GRP-29 and GRP-10, just like the processing of the rat GRP precursor. Sequence homology showed that the fGRP precursor is more homology showed that the fGRP precursor is more closely related to the mammalian GRP precursors than to either the frog bombesin or frog ranatensin precursors. Northern blot analysis showed that fGRP is encoded by a mRNA of 980 bases, clearly different from the 750-base mRNA which encodes frog bombesin. Northern blot analysis and in situ hybridization showed fGRP mRNA in frog brain and stomach and bombesin mRNA in frog skin, brain, and stomach. That frogs have independent genes for both GRP and bombesin raises the possibility that mammals have an as yet uncharacterized gene encoding a true mammalian bombesin.

Amino Acid Sequence↗
PubMed1991

Characterization of canine intestinal cholecystokinin-58 lacking its carboxyl-terminal nonapeptide. Evidence for similar post-translational processing in brain and gut.

An antibody raised against a synthetic cholecystokinin (CCK) analog, (1-27)-(CCK)-33, corresponding to the midregion of CCK-58, detected immunoreactivity in intestinal extracts which eluted between the positions of CCK-33/39 and CCK-58 on high performance liquid chromatography. This peak, lacking carboxyl-terminal cholecystokinin immunoreactivity, was purified by reverse phase and cation-exchange chromatographies. Amino acid, mass spectral, and microsequence analysis established that it was the amino-terminal desnonapeptide fragment of cholecystokinin-58, (1-49)-CCK-58. It was demonstrated further that CCK-58 has less biological activity than CCK-8, suggesting that the amino terminus either sterically hindered the ability of CCK-58 to exert its biological activity or that its amino terminus acted at another site to inhibit release of amylase from rat pancreatic acini. The desnonapeptide of CCK-58 by itself had no biological activity, nor did it affect CCK-8-stimulated amylase release from isolated rat pancreatic acini, suggesting that the amino terminus shields the carboxyl terminus from expressing its biological activity. Its presence in intestine suggests that it is released into the circulation where it could be detected by midregion antibodies. The presence of high proportions of (1-49)-CCK-58 indicates that most CCK-8 is directly derived from CCK-58. Its occurrence in brain and intestine indicates similar processing for procholecystokinin in both tissues.

Amino Acid Sequence↗
PubMed1991

Purification and structural characterization of progastrin-derived peptides from a human gastrinoma.

Several peptides derived from the gastrin-predicted preprohormone sequence were isolated from a human gastrinoma by gel permeation, anion exchange, and reverse phase chromatography. The peptides were identified and characterized structurally by a combination of radioimmunoassays, mass spectral analysis, and microsequence analysis. The largest peptide, progastrin-(1-35) (cryptagastrin), extends from the putative processing site for the signal peptidase to the double basic residues adjacent to the amino terminus of gastrin 34. A shorter form of this peptide, progastrin-(6-35) (cryptagastrin-(6-35), was also isolated in smaller amounts. In addition, sulfated and nonsulfated gastrin 17 amides (progastrin-(55-71)) and the glycine-extended nonsulfated gastrin 17 (progastrin-(55-72)) were identified by radioimmunoassay, and their structures were confirmed by mass spectral analysis. Isolation of cryptagastrin indicates that the signal peptide of human preprogastrin contains 21 amino acid residues, and progastrin, therefore, contains 80 amino acids. There is minimal processing of the cryptic peptide preceding the sequence of gastrin 34. An amidated gastrin form larger than gastrin 34 could contain 71 amino acids. No evidence was obtained for processing that would produce gastrins containing more than 34 but less than 71 amino acid residues.

Amino Acid Sequence↗
PubMed1991

Development of a biotinylated analog of substance P for use as a receptor probe.

The development of a biotinylated substance P (SP) analog for use as a receptor probe is reported. The lysine in position 3 of SP was substituted by arginine and an amino terminal extension (NTE-SP) was added consisting of Lys-Tyr-Gly-Gly-Gly-Gly-Gly-Gly. Biotinylation of the N-terminal lysine was performed. The biotinylated peptide was purified by high performance liquid chromatography and characterized by mass spectral analysis. Binding studies using human IM-9 lymphoblasts with the biotinylated SP analog (biotin-NTE[Arg3]SP) and native SP yielded dissociation curves which were identical. In addition, the biotinylated SP analog retained functional activity similar to that of native SP in altering intracellular calcium concentration of Fura-2 loaded isolated rabbit colonic myocytes. Applicability of the SP receptor probe was demonstrated by using the streptavidin-peroxidase detection system to identify SP receptors on human IM-9 lymphoblasts. In conclusion, a biotinylated SP analog has been developed which retains the functional characteristics of the native peptide and is a useful and versatile probe for receptor studies.

Amino Acid Sequence↗
PubMed1990

Low immunoreactivity of canine cholecystokinin-58.

The immunoreactivity of intact and trypsinized canine cholecystokinin-58 was examined using five C-terminally directed cholecystokinin antisera. Cholecystokinin-58 was nearly as immunoreactive as sulfated, amidated cholecystokinin-8 with one cholecystokinin-specific antiserum and an antiserum with moderate (18%) gastrin cross-reactivity, but two to four times less immunoreactive than the octapeptide with two other cholecystokinin-specific antisera and an antiserum with full cross-reactivity with cholecystokinin and gastrin. The cross-reactivity of cholecystokinin-58 with all antisera was increased by trypsinization, and the magnitude of the increase was related to the degree of trypsinization. These results suggest that it is not possible to measure absolute cholecystokinin levels in tissue and blood with antisera reported to date and that cholecystokinin-58 has a tertiary structure that influences its binding to cholecystokinin antibodies.

Animals↗
PubMed1990

Biological activity of a bombesin-like peptide extracted from the intestine of the ratfish, Hydrolagus colliei.

1. Ratfish (Hydrolagus colliei) intestines were boiled in water to inactivate proteases and then treated with cold 4% trifluoroacetic acid to extract bombesin-like peptides. 2. The extract was fractionated in several steps using reverse-phase and ion exchange HPLC, and bombesin-like immunoreactive peptides were detected by radioimmunoassay using an antiserum specific for the bioactive C-terminal region of bombesin. 3. A highly purified bombesin-like peptide-containing fraction stimulated amylase release in a dose-responsive fashion from rat pancreatic acini; the dose-response curve was parallel to a bombesin standard, and the ratfish peptide stimulated the same maximal rate of amylase secretion as the bombesin standard. 4. A potent, highly selective bombesin receptor antagonist completely abolished the stimulation of amylase release caused by the ratfish peptide, demonstrating the specificity of the response. 5. Estimates of the bombesin-like peptide concentration of this fraction by radioimmunoassay and by bioassay were nearly identical, indicating that ratfish bombesin is very similar biologically and antigenically to frog skin bombesin.

Amylases↗
PubMed1990

Cloning of cDNAs encoding amphibian bombesin: evidence for the relationship between bombesin and gastrin-releasing peptide.

Bombesin is a tetradecapeptide originally isolated from frog skin; its mammalian homologue is the 27-amino acid peptide gastrin-releasing peptide (GRP). cDNAs encoding GRP have been cloned from diverse species, but little is yet known about the amphibian bombesin precursor. Mass spectrometry of HPLC-separated skin exudate from Bombina orientalis was performed to demonstrate the existence of authentic bombesin in the skin of this frog. A cDNA library was prepared from the skin of B. orientalis and mixed oligonucleotide probes were used to isolate cDNAs encoding amphibian bombesin. Sequence analysis revealed that bombesin is encoded in a 119-amino acid prohormone. The carboxyl terminus of bombesin is flanked by two basic amino acids; the amino terminus is not flanked by basic amino acids but is flanked by a chymotryptic-like cleavage site. Northern blot analysis demonstrated similarly sized bombesin mRNAs in frog skin, brain, and stomach. Polymerase chain reaction was used to show that the skin and gut bombesin mRNAs encoded the identical prohormones. Prohormone processing, however, differed between skin and gut. Chromatography showed the presence of only authentic bombesin in skin whereas gut extracts contained two peaks of bombesin immunoreactivity, one consistent in size with bombesin and one closer in size to mammalian GRP. Thus the same bombesin prohormone is processed solely to bombesin in skin but is processed to a peptide similar in size to bombesin and to a peptide similar in size to mammalian GRP in stomach.

Amino Acid Sequence↗
PubMed1990

In-vitro production of precursor peptides for thyrotropin-releasing hormone by human semen.

Thyrotrophin-releasing hormone (TRH) and related peptides occur in high concentrations in human semen. TRH derives from a 242-amino acid precursor protein, prepro-TRH, with six repetitive sequences of -Lys-Arg-Gln-His-Pro-Gly-Lys/Arg)-Arg- connected by hydrophobic linking sequences. Antibodies to TRH-Gly (pGlu-His-Pro-Gly), a final precursor for TRH formation, were used to detect this tetrapeptide as well as other prepro-TRH fragments which cross-react with these antibodies. The total TRH-Gly immunoreactivity decreased significantly after vasectomy. The TRH-Gly immunoreactivity in semen increased significantly during in-vitro incubation at 0 or 37 degrees C, to a peak value at 5 h, followed by an exponential decline, with t 1/2 equal to 11 h at 37 degrees C. At 60 degrees C, however, the TRH-Gly immunoreactivity rose continuously, attaining, after 20 h, a level 2.2 times that at the start of the incubation (P less than 0.001). Reversed-phase high pressure liquid chromatography (HPLC) revealed both hydrophobic and hydrophilic TRH-Gly immunoreactive peptides in semen with both classes of peptides increasing significantly with heating to 60 degrees C. Cation exchange chromatography of pooled human semen incubated at 60 degrees C revealed a 4.3-fold increase in a TRH-Gly immunoreactive peak which co-eluted with synthetic TRH-Gly, and a 30% increase in another TRH-Gly immunoreactive peak identified as Glu-His-Pro-Gly. A minor, TRH-Gly immunoreactive peak increased 50-fold (P less than 0.001) during 20 h at 60 degrees C. This material co-eluted with Arg-Gln-His-Pro-Gly which is formed by enzymic cleavage of the paired basic residues flanking this sequence in prepro-TRH. When synthetic Arg-Gln-His-Pro-Gly was incubated with fresh semen at 60 degrees C a rapid conversion of most of this peptide to Glu-His-Pro-Gly, Gln-His-Pro-Gly and TRH-Gly occurred within 30 min. These data are consistent with thermal inactivation of the amidation and degrading enzymes at 60 degrees C while the trypsin-like enzymes which cleave the precursor peptide at the paired basic residues remain relatively unaffected. Because other investigators have found the C-terminal amidating enzymes to be associated with secretory vesicles and to be co-secreted with the vesicular contents, we suggest that secretory epithelia of the male reproductive system secrete TRH and TRH-related precursor peptides along with the alpha-amidating enzymes which continue processing of prepro-TRH in the post-ejaculatory seminal fluid.

Amino Acid Sequence↗
PubMed1990

Characterization of the major form of cholecystokinin in human intestine: CCK-58.

Acid extracts of human intestines obtained from surgical samples or from organ donors contain cholecystokinin (CCK) immunoreactivity. From surgical samples, extracted and eluted quickly, greater than 75% of the CCK immunoreactivity eluted in the same region as purified canine CCK-58 during analytical reverse-phase high-pressure liquid chromatography (HPLC). A major portion of the CCK immunoreactivity from donor intestinal extracts also eluted in this region. This immunoreactivity has been purified from human intestinal extracts by a series of several reverse-phase and cation-exchange chromatographies. Amino acid and microsequence analysis showed that this immunoreactivity is human CCK-58. Tryptic digestion of purified human CCK-58 produced another immunoreactive form that eluted in the position of CCK-8 during analytical reverse-phase HPLC. The immunoreactivity of the trypsin-digested material was 2.6-fold higher than that of an identical sample of CCK-58 incubated without trypsin. Thus the carboxyl-terminal antibody used for radioimmunoassay cross-reacts greater than twofold less with human CCK-58. This diminished cross-reactivity would lead to an underestimation of the relative proportions of CCK-58 in tissue and plasma extracts. If CCK-58 is the major circulating form this diminished cross-reactivity would also lead to underestimations of the circulating levels of total CCK. Determination of human CCK-58 structure confirms that one of the major components of human CCK that expresses biological activity is CCK-58.

Amino Acid Sequence↗
PubMed1990

Molecular variants of cholecystokinin after endogenous stimulation in humans: a time study.

The time-dependent release of molecular variants of cholecystokinin (CCK) into the circulation was studied before and 1, 2, and 4 h after a test meal in six healthy volunteers. At each time period, 100 ml of blood were drawn in a manner to inhibit CCK degradation. Plasma was formed and CCK concentrated by Sep-Pak C18 cartridge chromatography. Molecular variants of CCK and gastrin were well separated from each other by high-performance liquid chromatography (HPLC). Molecular forms of CCK and gastrin were measured by radioimmunoassay using an antibody that requires the presence of the carboxyl-terminal phenylalanine amide for full recognition, implying that biologically active forms were detected. HPLC elution positions of gastrin forms were determined using a gastrin-specific antibody. Chromatographic separation of CCK from gastrin forms was complete, allowing separate integration of gastrin and CCK forms. Therefore no subtraction of gastrin-like immunoreactivity from CCK-like immunoreactivity (CCK-LI) was necessary and CCK-LI could be directly determined. Peaks of CCK-LI were integrated in the column eluates and the plasma concentrations were calculated. Total plasma CCK-LI rose from a value of 2.4 +/- 0.6 pM before the test meal to 6.4 +/- 0.8, 6.6 +/- 0.9, and 5.8 +/- 1.2 pM 1, 2, and 4 h postprandially. The major molecular forms released into the circulation eluted on HPLC in the position of CCK-58 and CCK-39 (which coelutes with CCK-33). Minor amounts were detected in the position of CCK-8. There was no significant difference in the relative proportions of the molecular forms released at the different time periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Characterization of the carboxyl terminal flanking peptide of rat progastrin.

A peptide identical in structure to the carboxyl-terminal flanking nonapeptide of rat progastrin, predicted by cDNA sequence, was synthesized. The synthetic peptide was used for production of a rabbit antiserum. This antiserum was used to develop a radioimmunoassay specific for rat carboxyl terminal flanking peptide. This assay was used to monitor the purification of immunoreactivity from rat antral extracts. Gel permeation, anion exchange and reverse phase chromatography steps resulted in a single absorbance peak associated with the carboxyl terminal flanking peptide immunoreactivity. The purified peptide eluted in the same position as the synthetic peptide during all three types of chromatography. This material was shown to be identical in mass to Ser-Ala-Glu-Glu-Glu-Asp-Gln-Tyr-Asn, the predicted sequence of the carboxyl terminal nonapeptide of rat progastrin.

Amino Acid Sequence↗
PubMed1989

Multiple gastrin-releasing peptide gene-associated peptides are produced by a human small cell lung cancer line.

Products of the gastrin-releasing peptide gene were isolated from culture medium supernatant of a small cell lung cancer line, NCI-H345, by several (high performance liquid chromatography) HPLC steps. The column eluates were monitored by immunoassay and absorbance profiles. Gastrin-releasing peptide was identified in HPLC eluates by a specific radioimmunoassay. Two carboxyl-terminal gastrin-releasing peptide gene-associated peptides were identified by a radioimmunoassay specific for their predicted carboxyl terminus. The amino termini of these two peptides were determined by microsequence analysis. The shorter peptide was revealed to be a fragment of the larger peptide. Expression of an alternate mRNA was shown by isolation and characterization of a novel tetradecapeptide. Amino acid analysis, microsequence analysis, and mass spectral analysis confirmed that the structure was Ser-Leu-Leu-Gln-Val-Leu-Asn-Val-Lys-Glu-Gly-Thr-Pro-Ser. This peptide represents the carboxyl terminus of a peptide resulting from alternate processing of gastrin releasing peptide mRNA. This mRNA contains a 19-base deletion, creating a frame shift. A radioiodinated synthetic analog of this peptide (Tyr-Leu-Val-Asp-Ser-Leu-Leu-Gln-Val-Leu-Asn-Val-Lys-Glu-Gly-Thr-Pro-Ser ) bound specifically to a small cell cancer line with high affinity, suggesting possible biological activity of the isolated peptide.

Amino Acid Sequence↗