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Biomedical subjects

J R Clamp

Publications and source records attributed to J R Clamp.

At least 37 records · Page 2Linked to original sources

The gastric mucosal barrier.

The gastric mucosal barrier is a complex system made up of submucosal, epithelial and mucus elements. The mucus gel layer is a thick tenacious organized layer adherent to the epithelium. Despite these properties it is composed of more than 95% water, the organization being provided by long interacting glycoprotein molecules (mucus glycoprotein or mucin). These molecules are largely made up of carbohydrate which is present in large numbers of relatively small oligosaccharide units packed around the polypeptide core. This structure provides clues to the nature of the protection afforded by the mucus layer. For example, it is relatively resistant to proteolysis in the gastrointestinal tract; it retains water in an unstirred layer; the tangled glycoproteins exclude large molecules and the carbohydrate of the oligosaccharide units mirror that at the surface of the epithelial cell. Few biochemical studies have been carried out on the effect of ulcer-healing drugs on gastric mucus. Normal subjects were, therefore, given two weeks treatment with cimetidine, carbenoxolone or misoprostol and the secretions aspirated from the unstimulated and pentagastrin-stimulated stomach. The volume of secretion and weight and carbohydrate content of non-diffusable glycoconjugates were determined for each specimen, together with the proportion of high molecular mass mucin and qualitative and quantitative analyses of the glycopolypeptide. There were no significant differences between the results for each drug or without drug. This may be because normal subjects were studied who already have an effective mucosal barrier. In addition, it is likely that the process of mucus biosynthesis and secretion in a healthy individual is relatively resistant to the action of ulcer healing drugs.

Gastric Mucosa↗

Degradation by bacterial enzymes of colonic mucus from normal subjects and patients with inflammatory bowel disease: the role of sialic acid metabolism and the detection of a novel O-acetylsialic acid esterase.

1. The activities of enzymes degrading human colonic mucin were examined in faecal specimens from healthy subjects and patients with inflammatory bowel disease. 2. The activity of sialidase was measured using a new physiological substrate related to mucus glycoproteins. In addition, acylneuraminate pyruvate-lyase (N-acetylneuraminate lyase; EC 4.1.3.3.) and a novel O-acetylsialic acid esterase (sialate O-acetylesterase; EC 3.1.1.53) were detected. 3. The O-acetylsialic acid esterase activity was readily detectable in partially purified fractions after Sephadex G-100 chromatography. 4. Patients with inflammatory bowel disease showed significant increases in acylneuraminate pyruvate-lyase and proteinase activity but sialidase activity did not differ from normal. The activity of these enzymes in neutrophils could not account for the differences observed.

Acetylesterase↗

Effects of cimetidine and carbenoxolone on gastric mucus.

The effect of cimetidine on normal human gastric mucus has been compared with that of carbenoxolone, a drug believed to enhance mucus production. Each drug was given for two weeks, the gastric contents aspirated over a timed period and the results assessed in unstimulated and pentagastrin stimulated secretions. The volume, dry weight and the carbohydrate contents of non-diffusable glycoconjugates, high molecular mass glycoproteins and glycopolypeptides were investigated. Both drugs reduced the volumes of stimulated secretions. This was statistically significant after cimetidine. More importantly neither drug affected the amount of non-diffusable glycoconjugates, so that the concentration remained the same or increased. Both drugs reduced the monosaccharide content of the high molecular mass fractions. This reached significance for the stimulated secretion after cimetidine. As the carbohydrate content of the glycopolypeptides was unchanged this indicated the presence of a non-mucin glycoprotein or protein. Overall there was no fundamental difference between the results for cimetidine and carbenoxolone.

Adolescent↗

The gut in the acute phase response: changes in colonic and hepatic enzyme activity in response to dermal inflammation in the rat.

1. Transient mild dermal inflammation was produced in rats by the subcutaneous injection of either carageenan or monosodium urate crystals. The activities of enzymes of the sialic acid metabolic pathway were measured in liver and colon at 8 h, 3 days and 7 days. 2. In both liver and colon the UDP-N-acetyl-D-glucosamine 2-epimerase and asialo-alpha 1-acid glycoprotein sialyltransferase activities increased relative to controls while that of CMP-sialic acid hydrolase decreased. Similar changes occurred for both carageenan and monosodium urate crystals. 3. These results indicate that the acute phase response to a relatively minor inflammatory lesion causes significant changes in a distant and apparently unrelated tissue.

Acute-Phase Reaction↗

The nature of gastric secretions in a patient with Zollinger-Ellison syndrome without peptic ulceration.

A study has been made of the gastric secretions from a patient with Zollinger-Ellison syndrome without peptic ulceration, before and after treatment with cimetidine. The amount of non-diffusable glycoconjugates was measured as an index of mucin output, and found to be within normal limits. Cimetidine, which greatly reduced the volume of secretions, had no effect on the non-diffusable glycoconjugates. These results may explain the absence of peptic ulceration in certain patients with this syndrome.

Adult↗

Some aspects of the glycoprotein and glycopolypeptide content of human gastric mucus.

The carbohydrate content of non-diffusable, glycoprotein and glycopolypeptide material has been studied in normal human gastric aspirates. Pentagastrin doubles the volume of secretions but has no effect on the amount of non-diffusable material. Only about 40% of the weight of the non-diffusable material is mucin in nature. Gel-permeation chromatography indicated that about half of the mucin survived as high molecular mass glycoprotein. Monosaccharide differences, for example between secretors and non-secretors, only became manifest at the glycopolypeptide stage. These results emphasize the dangers of attempting to assess mucin changes by simple carbohydrate analyses of unfractionated gastric aspirates.

Adolescent↗

Rat colonic mucosal cell sialic acid metabolism in azoxymethane-induced tumours.

Colonic tissue was examined from normal (control) rats and azoxymethane- (carcinogen-) treated animals. Tumour-bearing colons from azoxymethane-treated rats were divided into malignant and non-malignant areas. Mucosal cells were prepared from the three types of colonic tissue and then examined for DNA and protein content and for the activities of ten enzymes involved in sialic acid metabolism. Enzyme activities were related to either the protein or the DNA content of fractions. The DNA content of cell homogenates was significantly different between tumour and non-malignant tissue and between both these tissues and normal mucosa. The protein content of the 100000 X g membrane pellet and supernatant fraction did not vary significantly between normal and non-malignant material but both these tissues differed significantly from tumour tissue. Significant variation between normal control and tumour tissue was detected at all levels of sialic acid metabolism, including N-acetylhexosamine interconversion and phosphorylation, sialic acid formation and activation, CMP-NeuAc breakdown and transfer and sialic acid release from glycoconjugates. The results indicate that major changes at all levels of sialic acid metabolism are associated with malignancy in rat colonic mucosa. Some of these changes are apparent in non-malignant mucosa and may reflect a pre-malignant state.

Animals↗

The metabolism of sialic acids in isolated rat colonic mucosal cells.

The activities of ten enzymes involved in sialic acid metabolism were measured in colonic mucosal cells from rats and compared with those in liver. A methodology was devised that enabled all ten enzyme activities to be evaluated in a single rat colon preparation. Enzyme assays with radioactively labelled substrates were developed for maximum sensitivity, and the identification of substrates and products was carefully checked to assess the contribution of contaminants to enzyme reactions with low activity. The activities of most enzymes involved in the biosynthesis of N-acetyl-D-neuraminic acid (NeuAc) from UDP-N-acetyl-D-glucosamine were found to be more than 20-fold lower than those in liver. The activities of CMP-NeuAc synthase, N-acetyl-D-glucosamine 2-epimerase, N-acetyl-D-glucosamine kinase, sialyltransferase and sialidase were similar to or 2-4-fold lower than in liver. The biosynthesis of NeuAc via its 9-phosphate was demonstrated in the 100 000 g supernatant of colonic-cell homogenates by enzymic assay and precursor experiments with N-acetyl[14C]-mannosamine. No alternative route for NeuAc formation could be detected. The 100 000g supernatant fractions of liver, kidney and colonic mucosal cells utilized N-acetyl[14C]mannosamine with differing efficiencies. Radioactive products identified as sialic acid biosynthetic intermediates amounted to 49%, 0.04% and 5.6% of added precursor in liver, kidney and colon respectively. Catabolism of labelled precursor to non-hexosamine products was high in kidney and colonic mucosal-cell fractions.

Animals↗

Partial characterization of transferrins of catfish (Silurus glanis L.) and pike (Esox lucius L.).

Basic composition and properties of isolated transferrins of Silurus glanis and Esox lucius have been compared. In transferrin of S. glanis carbohydrate is absent, but it is present in transferrin of E. lucius (2.5%). The N-terminal amino acid is alanine in both species. Mol. wts are 68,400 (S. glanis) and 86,800 (E. lucius). Transferrins of the two species are heterogeneous, but genetic polymorphism was not observed.

Amino Acids↗

Glycosylation of hair: possible measure of chronic hyperglycaemia.

To determine whether hair is excessively glycosylated in diabetes mellitus 4 cm hair samples were taken proximally from behind the ear in 50 white non-diabetics and 46 diabetics. Hair glycosylation was assayed by a modification of the thiobarbituric acid reaction. Blood was taken from the diabetics at the same time for measurement of glycosylated haemoglobin concentration. The mean (1 SD) concentration of fructosamine (mumol/100 mg hair) was 0.054 (0.011) for normal hair. Glycosylation was not related to sex, age, or hair colour. The diabetics' hair was more heavily glycosylated (0.097 (0.045] than normal (p less than 0.01) and there was a correlation between hair glycosylation and the concentration of glycosylated haemoglobin in the diabetics (r = 0.71; p less than 0.01). Hair from non-diabetics showed a stable time related increase in glycosylation when incubated with glucose. Glycosylation of hair might provide a stable long term measure of tissue glycosylation, useful in the investigation of microvascular complications of diabetes mellitus.

Adolescent↗

Some non-mucin components of mucus and their possible biological roles.

Non-mucin components have essential roles in the protective functions of mucous secretions. Secretory IgA (SIgA) antibodies probably act by blocking the attachment of pathogenic microorganisms to mucosal cells. In addition SIgA1 may render bacteria more 'mucophilic', possibly by virtue of the 'mucus-like' stretch that the immunoglobulin molecule possesses. Lysozyme will attack cell walls of susceptible bacteria. As the enzyme associates strongly with mucus glycoproteins the mucus layer is provided with powerful bactericidal properties. Lactoferrin, normally unsaturated, sequesters any free iron in secretions, so exerting a bacteriostatic action on iron-requiring microorganisms. In addition it may protect mucus glycoproteins from iron-catalysed active oxygen species. This mucoprotective action would be overcome during infections. Attention is also directed towards a possible copper-mediated limited degradation by hydrogen peroxide. Surfactants and free lipid have long been recognized as components of normal bronchial mucus. For example, some lipid is tightly but non-covalently bound to a hydrophobic region of bronchial mucin. More intriguing is the presence of small amounts of covalently bound lipid in normal human gastric mucin. In addition, normal human gastric mucus contains significant amounts of a galactose-rich polysaccharide. The function of this is not known but it may act as a cross-linking strand in the mucus gel structure or as a renewable cell membrane component, perhaps interacting between glycocalyx and the mucus layer.

Gastric Mucosa↗

Haemopexin in sheep, mouflon and goat: genetic polymorphism, heterogeneity and partial characterization.

Benzidine staining of starch gels after electrophoresis of sera to which haematin was added revealed polymorphism of haemopexin in sheep, mouflon and goat. In sheep three phenotypes were observed, Hpx A, Hpx AB and Hpx B. Pedigree data support the hypothesis of codominant inheritance from a single locus by two alleles, HpxA and HpxB. Neuraminidase treatment of haemopexin preparations showed that Hpx B covered two variants, B1 and B2, thus indicating genetic control by three alleles (HpxA, HpxB1 and HpxB2). In sheep populations the frequency of HpxB is low. In mouflon, in addition to the two variants that are like those of sheep, absence of haemopexin was observed in some animals, by using starch gel electrophoresis as well as immunoelectrophoresis. In goat, three phenotypes were detected, Hpx A, Hpx AB and Hpx B, differing in migration from those of sheep. Haemopexins of the studied species are heterogeneous. Sialic acid is responsible for electrophoretic heterogeneity of sheep haemopexin. Chemical composition (amino acid and carbohydrate), molecular weight (56 060) and N-terminal sequence (Leu-Pro-Pro-) of sheep haemopexin were also determined.

Alleles↗

The presence of polysaccharide in normal human gastric mucus.

Polysaccharide material was found in the proteolysis glycopolypeptide fraction from normal human gastric mucus. The polysaccharide was identified by carbohydrate and amino acid analyses, by elemental analysis and from its behaviour on density-gradient ultracentrifugation. The polysaccharide is polydisperse with a weight-average molecular mass of 300 000 Da. Over 85% of the polysaccharide consists of galactose, and this represents 26% of all the galactose present in the fractions after beta-elimination with reduction of the glycopolypeptide material.

Amino Acids↗

Studies of the limited degradation of mucus glycoproteins. The effect of dilute hydrogen peroxide.

1. The action of dilute H2O2 on a series of ovarian-cyst glycoproteins and glycopolypeptides was investigated. 2. Both native glycoproteins and the glycopolypeptides were carbohydrate-rich, of relatively low molecular weight and of simple structure. 3. At pH 5.6 and 37 degrees C, exposure to H2O2 for a limited time brought about a partial degradation, the molecular weight being decreased by 2-4-fold. 4. Carbohydrate analysis showed very little change in the oligosaccharide moiety, apart from a small decrease in sialic acid in some samples. 5. Amino acid analysis showed minor changes in serine, threonine and proline contents, but almost total loss of histidine. Concomitantly, there was a small gain in aspartic acid. 6. Myosin, examined at both pH 5.7 and 6.7, exhibited generally similar behaviour, there being losses of other amino acid residues as well as histidine: the viscosity was decreased to a low value, and a range of peptides of widely varying size was produced. 7. It is suggested that attack on the histidine residue, with partial conversion into aspartic acid, is accompanied by scission of the histidyl peptide bond.

Amino Acids↗

Incomplete glycosylation of erythrocyte membrane proteins in congenital dyserythropoietic anaemia type II (CDA II).

The alterations in the erythrocyte membrane proteins of individuals with congenital dyserythropoietic anaemia (CDA II) were studied. Alterations were observed in both the erythrocyte sialoglycoproteins and erythrocyte anion transport protein (Band 3). There was a decrease in the apparent molecular weight of the major sialoglycoprotein alpha (glycophorin A) as well as a general reduction in the intensity of staining of all the sialoglycoproteins by the PAS stain. Sialoglycoprotein alpha isolated from CDA II erythrocytes contained 30% less sialic acid than normal alpha. The anion transport protein of CDA II erythrocytes migrated as a band with a lower molecular weight than the normal protein on SDS-gel electrophoresis. The CDA II anion transport protein had a substantially reduced content of N-acetylglucosamine and galactose, which probably reflects a reduction in the number of N-acetyl-lactosamine units carried by the protein. Our results suggest that there is a general defect in glycosylation of the major membrane glycoproteins of CDA II erythrocytes. We suggest that this glycosylation defect is a consequence of bone marrow stress.

Anemia, Dyserythropoietic, Congenital↗

Abnormal small intestinal permeability to sugars in patients with Crohn's disease of the terminal ileum and colon.

The absorption of lactulose and mannitol in 20 patients with Crohn's disease limited to the ileum or colon was studied, and lactulose/mannitol excretion ratios were calculated. The results were compared to those from 16 normal controls and 6 patients with ulcerative colitis. The 13 patients with ileal Crohn's disease had significantly higher lactulose/mannitol excretion ratios than the controls (p less than 0.01) or ulcerative colitics (p less than 0.01). Similarly, the 7 patients with Crohn's disease limited to the colon had significantly higher excretion ratios than the controls (p less than 0.01) or ulcerative colitics (p less than 0.01). The results provide support for the concept that Crohn's disease may be more extensive than is apparent macroscopically.

Adolescent↗