Biomedical subjects

J R Reeve

Publications and source records attributed to J R Reeve.

At least 73 records · Page 4Linked to original sources

PubMed1989

Involvement of substance P in noncholinergic excitation of rabbit colonic muscle.

Neurokinins have been implicated as noncholinergic excitatory neurotransmitters in the mammalian gastrointestinal tract. To characterize the myogenic and neurogenic response of colonic muscle to neurokinins we studied the mechanical response of muscle strips from proximal and distal colon and the release of [3H]acetylcholine in response to substance P (SP), neurokinin A (NKA) and neurokinin B (NKB). All neurokinins caused a dose-dependent inotropic response. SP was 80 times more potent in distal compared with proximal longitudinal muscle. The rank order of potencies in proximal longitudinal muscle was NKA greater than SP = NKB and in distal muscle NKA = SP = NKB. Desensitization to SP or pretreatment with a SP antagonist inhibited the mechanical response to SP and the atropine-resistant inotropic off response to electrical stimulation. Only longitudinal muscle from distal colon had an atropine- and hexamethonium-sensitive inotropic component to SP. In contrast, all three peptides were equipotent in releasing [3H]acetylcholine from longitudinal muscle strips preincubated with [3H]choline. These results suggest the following: 1) SP is a potent agonist of rabbit colon with a proximal distal gradient in biological potency; 2) the myogenic response of the distal colon appears to be mediated through a NK-1 receptor; and 3) SP is a major mediator of the noncholinergic component of the off response in distal longitudinal muscle.

Acetylcholine↗

PubMed1988

Identification of serotonin from rabbit upper stomach as a stimulant of in vitro gallbladder contraction.

Using an in vitro rabbit gallbladder bioassay, the distribution and identification of bioactive substances in rabbit gastrointestinal tract were investigated. Comparison of the bioactivities of tissue extracts before and after cholecystokinin was removed by affinity chromatography demonstrated that the distributions of cholecystokinin and non-cholecystokinin substances were different. While cholecystokinin bioactivity per g of tissue was highest in the duodenum, non-cholecystokinin bioactivity was greatest in the upper stomach. The biochemical properties of the non-cholecystokinin substance in the upper stomach could not be distinguished from those of serotonin. These included molecular weights of 176, identical ultraviolet spectra, similar nuclear magnetic resonance spectra, and co-chromatography in HPLC. By weight, serotonin had 1/6th of the bioactivity of cholecystokinin octapeptide. We conclude that the principal gallbladder-contracting substance in rabbit upper stomach is serotonin.

Animals↗

PubMed1988

Autocrine growth of murine lymphoma cells.

By using an assay system in which small numbers of murine T lymphoma cells are stimulated to grow in serum-free medium, we have continued and expanded our previous studies of an autocrine growth factor that we call leukemia-derived growth factor (LDGF). We show that a T lymphoma cell line of immature phenotype, adapted to growth in serum-free medium, produces and responds to LDGF. LDGF activity is distinct from activities of 10 highly purified or recombinant hematopoietic growth factors including IL-1 and IL-2. However, growth-stimulating activity for the murine lymphoma cells is provided by a partially purified human LDGF.

Animals↗

PubMed1988

An amino-terminal fragment of cholecystokinin-58 is present in the gut: evidence for a similar processing site of procholecystokinin in canine gut and brain.

Radioimmunoassays using antibodies specific for the carboxyl terminus of cholecystokinin (CCK) and the midportion of CCK-58 (raised against synthetic canine CCK-33-(1-27] revealed the existence of a CCK fragment in canine gut and brain extracts which lacks the biologically active carboxyl terminal immunoreactivity. This material eluted on Sephadex G-50 gel permeation chromatography in the region of CCK-58, on high-pressure liquid chromatography (HPLC) after CCK-39 and before CCK-58, and on cation-exchange FPLC it eluted after CCK-58. The immunoreactive pattern, the ratio of absorbance at 280-220 nm and the chromatographic elution positions suggest that this large CCK-like molecule represents an amino-terminal fragment of CCK-58. This fragment is present in canine gut and brain. Therefore, a similar processing site of procholecystokinin is suggested in both tissues.

Animals↗

PubMed1988

Solid phase synthesis and characterization of two canine gut gastrin-releasing peptides.

Two canine gastrin-releasing peptides originally isolated from gut tissue extracts have been synthesized by solid phase methodology and purified by preparative reverse phase high performance liquid chromatography (RP-HPLC). The synthetic gastrin-releasing peptides GRP1-27 and GRP 5-27 were characterized with regard to homogeneity and composition using nine different RP-HPLC systems, mass spectroscopy, amino acid analysis, Edman degradation, methionine oxidation, and peptide mapping with tryptic, Staph. aureus V8 protease and cyanogen bromide cleavage (the latter two systems performed only with GRP 1-27). Although a scarcity of the natural products prevented quantitative biological comparison of the synthetic and natural peptides, they were found to elute identically on RP-HPLC co-chromatography and similar dose dependent biological potencies were observed in canine antral muscle tissue contraction experiments. Indeed, all the peptides containing the bombesin-like carboxyl terminal decapeptide sequence studied to date have similar biological activities.

Amino Acid Sequence↗

PubMed1988

Processing of mammalian preprogastrin-releasing peptide.

The processing of preprogastrin-releasing peptide in mammalian tissues and in cultured cells takes place at discrete sites (Figure 6). Signal peptidase cleaves away the signal peptide from the amino terminus of gastrin-releasing peptide. An exopeptidase activity may remove dipeptides from the amino terminus. The amidation site (not shown in Fig. 6; see Fig. 2) has the same general sequence (Gly-Lys-Lys) seen for other amidated peptides. Cleavage after single basic residues yields gene-related products from Form I or II preproGRP. A unique non-basic cleavage yields a gene-related product from Form III preproGRP. The processing that occurs to form GRP, GRP, and GRP gene-related peptides is shown in Figure 7. ProGRP is cleaved by a series of enzymes to form GRP with an amidated carboxyl-terminal methionine (indicated by an asterisk in Fig. 7). GRP is cleaved to form the decapeptide GRP. The carboxyl-terminal flanking peptides of all three mRNA translation products are cleaved to form several gastrin-releasing peptide gene-related products. Knowledge of the processing of gastrin-releasing peptide and its gene-related products will allow synthesis of duplicates of the stored forms of these peptides, which can then be used for biological testing.

Amino Acid Sequence↗

PubMed1988

Plasma very low density lipoproteins contain a single molecule of apolipoprotein B.

Rat and human very low density lipoproteins (VLDL) were fractionated by zonal ultracentrifugation, yielding sharply defined fractions with narrow sedimentation limits. Sedimentation coefficients for the individual fractions were determined at two densities with the analytical ultracentrifuge, and the results were analyzed to yield buoyant densities and molecular weights for the particles in each fraction. For the rat lipoproteins, the weight concentrations of triglycerides, cholesterol, phospholipid, and protein were determined for each fraction, and their molar concentrations of apolipoprotein B were measured with a radioimmunoassay. For the human lipoproteins the corresponding values were taken from Patsch et al. (Patsch, W., J. R. Patsch, G. M. Kostner, S. Sailer, and H. Braunsteiner. 1978. Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation. J. Biol. Chem. 253:4911-4915). From these data, a ratio of the number of apoB peptides to the number of lipoprotein particles was calculated for each fraction. This ratio was close to 1 for all VLDL fractions, ranging in particle diameter from about 40 to 80 mm and 30 to 50 mm, respectively, for rat and human VLDL. The majority rat VLDL contain B-48 rather than B-100 as their (single) apoB peptide. Based on these data, we proposed that only a single copy of B-48 is required for VLDL assembly in rat liver, unless nascent hepatic VLDL contain additional apoB peptides which are uniformly lost from the plasma VLDL particles when they are analyzed.

Amino Acids↗

PubMed1987

Neurotensin-like immunoreactivity generated by pepsin from human plasma and gastric tissue.

The present study demonstrates precursors of neurotensin-like immunoreactivity (NTLI) endogenous to human gastric tissue and plasma, and the existence of a gastric NTLI-generating enzyme system. The molecular size of the NTLI-precursors in plasma and gastric tissue were estimated by gel permeation chromatography to be ca 50,000-60,000 and 60,000-70,000 Da, respectively. The neurotensin-like peptide generated from the precursor was detected with a carboxyl-terminally directed antiserum but did not cross-react with an amino-terminally directed antiserum. A neurotensin-like peptide isolated from pepsin-treated human plasma was characterized by mass spectrometry and its amino acid sequence determined. This novel nonapeptide, referred to as kinetensin, failed to affect pentagastrin-stimulated acid secretion or blood pressure in the rat. Sequence homologies between neurotensin, kinetensin and proteins of the serum albumin family suggest a common evolutionary origin and raise questions regarding albumin-like proteins as precursors of regulatory peptides.

Amino Acid Sequence↗

PubMed1987

Cholecystokinin-58 is the major circulating form of cholecystokinin in canine blood.

Cholecystokinin-58 (CCK-58) is the largest and most abundant, biologically active form of cholecystokinin in canine intestinal mucosa. Despite the high amounts in mucosa, CCK-58 has not been detected in significant amounts in the circulation. The release of CCK-58 into the peripheral blood in response to an intraduodenal perfusion of sodium oleate (9.0 mmol h-1) was studied in seven conscious dogs. Plasma (50 ml) was obtained before and after endogenous stimulation by a newly developed method that prevents in vitro degradation of large cholecystokinins. The relative abundance of immunoreactive forms of CCK was studied by high pressure liquid chromatography (HPLC) which separated the gastrin and CCK forms. Column eluates were measured with an antibody which recognizes the intact carboxyl terminus of both gastrin and CCK. Cholecystokinin immunoreactivity increased over basal in plasma by 7 fmol/ml after intraduodenal perfusion with sodium oleate. The most abundant form of stimulated cholecystokinin immunoreactivity eluted on HPLC in the position of CCK-58 (63% of total immunoreactivity found). Since CCK-58 is biologically active and is the most abundant circulating form, it should play an important role in the physiology of cholecystokinin.

Animals↗

PubMed1987

Distribution and characterization of calcitonin gene-related peptide immunoreactivity in the digestive system of normal and capsaicin-treated rats.

The distribution and characterization of calcitonin gene-related peptide immunoreactivity in the digestive system of normal, capsaicin-treated, and littermate control rats were studied by radioimmunoassay, chromatography, and immunohistochemistry. The highest concentrations of calcitonin gene-related peptide immunoreactivity were found in the stomach (45 +/- 2.8 pmol/g wet wt, nonsecretory region; 38.7 +/- 4.4 pmol/g wet wt, secretory region) and rectum (30.9 +/- 1.6 pmol/g wet wt). Significant amounts of peptide were also found in the other regions of the gut and in the pancreas. Neonatal treatment with capsaicin, which causes a permanent degeneration of most of the small-diameter sensory neurons, reduced calcitonin gene-related peptide content by greater than 95% in the esophagus and stomach, by 60% in the pancreas, and by less than 50% in the intestine, when compared with littermate controls. Separation of extracts from the gut, pancreas, and brain by chromatography gave major peaks corresponding to the predicted rat calcitonin gene-related peptide and small unidentified peaks, which presumably arise from metabolism of the peptide. Immunohistochemical studies demonstrated that in the esophagus and stomach, calcitonin gene-related peptide immunoreactivity is restricted to nerve fibers, whereas in the intestine it is localized in both nerve fibers and enteric ganglion cells. In capsaicin-treated rats there was a virtually complete elimination of calcitonin gene-related peptide immunoreactive fibers innervating the esophagus and stomach, whereas in the small and large intestine there was a dramatic reduction and often a complete elimination of those associated with blood vessels and a slighter reduction of the nonvascular immunoreactive fibers. The results of this study indicate that calcitonin gene-related peptide immunoreactive nerve fibers innervating the rat digestive system originate from both intrinsic (enteric) and extrinsic (presumably sensory) sources and that both the intrinsic and extrinsic components appear to contain a substance that corresponds to the predicted calcitonin gene-related peptide.

Animals↗

PubMed1987

CCK-5: sequence analysis of a small cholecystokinin from canine brain and intestine.

The purpose of this study is to purify and to characterize chemically cholecystokinin (CCK)-like peptides present in brain and gut extracts that elute from gel filtration after the octapeptide. Canine small intestinal mucosa and brain were boiled in water and then extracted in cold trifluoroacetic acid, and cholecystokinin-like immunoreactivity was determined by carboxyl-terminal specific radioimmunoassay. Gel permeation chromatography on Sephadex G-50 revealed a form of CCK apparently smaller than CCK-8. This peptide was purified by immunoaffinity chromatography and three successive reverse phase high-performance liquid chromatography steps. Microsequence analysis showed that the amino terminal primary sequence of this small CCK was Gly-Trp-Met-Asp. Immunochemical and chromatographic analysis indicated that the carboxyl-terminal residue was Phe-NH2 and thus the full sequence is Gly-Trp-Met-Asp-Phe-NH2. An antibody that recognizes synthetic CCK-8, CCK-5, and CCK-4 equally did not reveal the presence of significant amounts of CCK-4. These results indicate that CCK-5 is the major CCK form smaller than the octapeptide present in brain (19% of total CCK immunoreactivity) and small intestine (7% of total). This finding, coupled with the demonstration by others that CCK-5 interacts with high-affinity brain CCK receptors, indicates that CCK-5 may play a physiological role in brain function.

Amino Acid Sequence↗

PubMed1987

Detection of cholecystokinin-58 in human blood by inhibition of degradation.

Although cholecystokinin-58 (CCK-58) is a major molecular form stored in the intestine, it has not yet been shown to be released into the circulation. This report describes in vitro degradation of CCK-58 in human blood and plasma and the molecular forms detected when this degradation is inhibited. After incubation of CCK-58 for 150 min between 20 and 24 degrees C, approximately 60% of immunoreactivity recovered was degraded to smaller immunoreactive forms. Storage of the 150-min incubate at -20 degrees C for 3 days greatly increased the observed degradation to 85%. When CCK-58 was added in vitro to blood, similar degradation occurred. Degradation of CCK-58 could be inhibited by addition of acid. Blood was obtained 1 h after a test meal designed to stimulate CCK release. The pH was lowered during collection and processing of blood and plasma to inhibit in vitro degradation of cholecystokinin. This method permitted the detection of significant amounts of CCK-58 in circulation.

Cholecystokinin↗

PubMed1986

Evidence for thyrotropin-releasing hormone (TRH) biosynthesis in rat prostate.

TRH occurs in very high concentration in rat prostate. A species specific protein with repetitive -Gln-His-Pro-Gly- sequences, which are flanked on the N- and C-terminus by paired basic residues, has been shown to be the source of TRH in frog skin and rat hypothalamus. Following cleavage by trypsin-like enzymes, the peptide fragments with N-terminal Gln spontaneously cyclize to pGlu while Gly within the C-terminally extended peptides serves as the -NH2 donor for the alpha-amidation of the proline residue. Because this last step in the biosynthesis of TRH is rate limiting for pGlu-His-Pro-Gly, we have combined several chromatographic and radioimmunoassay techniques to identify this TRH precursor in rat prostate.

Amino Acid Sequence↗

PubMed1986

New molecular forms of cholecystokinin. Microsequence analysis of forms previously characterized by chromatographic methods.

Cholecystokinin previously has been characterized by chromatographic and immunological techniques. Analysis of cDNA has revealed the structure of preprocholecystokinin. The predicted processing sites of preprocholecystokinin cannot account for the multiple forms of cholecystokinin detected. This report details the isolation and characterization of cholecystokinin peptides containing 58, 39, 33, 25, 18, 8, 7, and 5 amino acids. None of the cleavages that occurred to form these peptides was at the carboxyl side of double basic residues. Cholecystokinin 25, 18, and 7 have not previously been isolated and identified by sequence analysis. The processing that forms these peptides includes cleavage after single basic residues for the 58, 39, 33, and 8 amino acid peptides. The 25, 18, 7, and 5 amino acid peptides must be formed by other endopeptidases or combinations of endo- and exopeptidases. The analysis of this series of peptides provides the chemical basis for structural differences in cholecystokinin molecules previously demonstrated by chromatographic and immunological methods. These structures provide insight into tissue-specific processing that occurs for this important regulatory peptide in intestine and brain.

Amino Acid Sequence↗

PubMed1986

The amino acid sequence of kinetensin, a novel peptide isolated from pepsin-treated human plasma: homology with human serum albumin, neurotensin and angiotensin.

A novel nonapeptide with neurotensin-like immunoreactivity was isolated from pepsin-treated human plasma by dialysis, ion-exchange chromatography and high performance reversed-phase liquid chromatography. The amino acid sequence was determined by automated gas-phase sequence analysis as Ile-Ala-Arg-Arg-His-Pro-Tyr-Phe-Leu. Sequence homology with human serum albumin and with the biologically active peptides neurotensin and angiotensin is demonstrated. The name proposed for this peptide is kinetensin.

Amino Acid Sequence↗

PubMed1986

Partial purification of a novel stimulant of gastric acid secretion from canine non-antral mucosa.

A novel stimulant of gastric acid secretion was extracted and purified from the non-antral gastric mucosa of the canine stomach and some of its biological properties were examined. Tissue was boiled in water and extracted in 2% trifluoroacetic acid. The stimulatory activity was purified by a combination of reverse-phase high pressure liquid chromatography (HPLC) and gel filtration. Fractions were assayed for a stimulation of basal, pentagastrin- and histamine-stimulated gastric acid secretion in the anaesthetized rat. Stimulatory activity was eluted from reverse-phase HPLC columns with acetonitrile and its elution from Sephadex G-10 and G-50 columns suggested a molecular weight of 1,000 to 3,000. The highly purified extracts enhanced basal, pentagastrin- and histamine-stimulated acid secretion in the rat. A stimulatory fraction was purified which was devoid of immunoreactive gastrin and gastrin-releasing peptide and contained only small amounts of histamine. Its chromatographic properties differed from those of histamine and acetylcholine. On two occasions the stimulant was purified to homogeneity and found to contain amino acids. Insufficient pure material was obtained for full characterization. The stimulant has been tentatively called oxyntin.

Amino Acids↗

PubMed1986

Isolation and characterization of a neurotensin-like decapeptide from a canine upper small intestinal extract.

Neurotensin-like immunoreactivity can be detected in extracts of canine upper gastrointestinal mucosa when measured by carboxyl terminal but not by amino terminal antibodies to neurotensin. The nature of this immunoreactive material was characterized by complete purification on gel filtration and HPLC followed by peptide microsequence analysis. The structure obtained was Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-(Leu), identical in structure to the carboxyl terminal decapeptide of neurotensin. It cannot, however, be excluded that this neurotensin decapeptide was generated from a larger neurotensin-like peptide during the extraction procedure by a physiological or artificial enzymatic process. Since carboxyl terminal neurotensin fragments containing eight or more residues have full biological activity, this peptide may be responsible for neurotensin-like biological activities within the mucosa of, or after release from, the upper gut.

Animals↗

PubMed1986

Homology of lipoprotein lipase to pancreatic lipase.

Bovine milk lipoprotein lipase was subjected to amino acid sequence analysis. The first 19 amino-terminal residues were Asp-Arg-Ile-Thr-Gly-Gly-Lys-Asp-Phe-Arg-Asp-Ile-Glu-Ser-Lys-Phe-Ala-Leu- Arg. In addition, reversed-phase high-performance liquid chromatography of a tryptic digest of reduced and alkylated lipase resolved a number of peptides, five of which contained cysteine. Sequence analysis of the tryptic peptides revealed in most instances a close homology to porcine pancreatic lipase. Based on this homology, the relative alignment of the sequenced lipoprotein lipase peptides can be made. In addition, a potential binding site for the triacylglycerol substrate and a carbohydrate-binding domain for lipoprotein lipase are postulated.

Amino Acid Sequence↗