Anthony M. Roberton

Expression cloning of gastric mucin complementary DNA and localization of mucin gene expression

BACKGROUND & AIMS Secretory mucins play an important role in gastric cytoprotection and are derived from a heterogeneous family of genes. The aim of this study was to determine the specific type and location of mucin gene expression in the human stomach. METHODS Expression cloning was performed by screening a human gastric complementary DNA expression library with antisera against deglycosylated gastric mucin. RNA analysis and immunohistochemistry were used to quantify and localize mucin gene expression. RESULTS Sequencing of positive clones revealed two clones containing tandem repeats. The first contained a 169-amino acid repeat and was named MUC6 (as previously described). The second contained the same 8-amino acid repeat consensus sequence (APTTSTTS) as complementary DNAs previously isolated from a tracheobronchial complementary DNA library and was labeled MUC5 (or MUC5AC). RNA analysis indicated that the gastric epithelium contains high levels of MUC5 and MUC6 messenger RNA with little or no MUC2, MUC3, and MUC4 messenger RNA. Immunohistochemical analysis showed that surface mucous cells of the cardia, fundus, and antrum expressed MUC5 peptide. In contrast, MUC6 peptide expression was limited to mucous neck cells of the fundus, antral-type glands of the antrum and cardia, and Brunners glands of the duodenum. CONCLUSIONS MUC5 and MUC6 represent major secretory mucins in the stomach and are localized to distinct cell types.

Antibacterial actions of fatty acids and monoglycerides against Helicobacter pylori

The bactericidal potencies of saturated and unsaturated fatty acids (FAs) and monoglycerides (MGs) against Helicobacter pylori were determined following short incubations with freshly harvested cells over a range of pHs. FAs and their derivatives with an equivalent-carbon number of 12 were the most potent: lauric acid had a minimum bactericidal concentration (MBC) at pH 7.4 of 1 mM, myristoleic and linolenic acid were the most potent unsaturated FAs (MBCs of 0.5 mM, pH 7.4), and monolaurin was the most potent MG (MBC 0.5 mM). Potencies of saturated FAs were increased sharply by lowering pH, and a decrease of only 0.5 pH units can cause a change from non-lethal to lethal conditions. Conversely, the bactericidal action of monolaurin was not pH-dependent. The bactericidal potencies of unsaturated FAs increased with degree of unsaturation. When more than one FA or FA plus MGs were present, their combined action was additive. Urea and endogenous urease did not protect H. pylori from the bactericidal action of FAs. These results suggest that H. pylori present in the stomach contents (but not necessarily within the mucus barrier) should be rapidly killed by the millimolar concentrations of FAs and MGs that are produced by pre-intestinal lipase(s) acting on suitable triglycerides such as milk fat.

Colorectal mucin histochemistry in health and disease: A critical review

Neoplastic, inflammatory and regenerative processes affecting colorectal mucosa are associated with alterations in structure of epithelial mucin. This review collates mucin‐, lectin‐, and immuno‐histochemical observations on colorectal mucins and introduces recent molecular genetic insights into the structure of the protein backbone of mucins. The numerous structural modifications uncovered by the various technical approaches have been reduced to a few manageable principles that are of relevance to both researcher and diagnostic pathologist. Particular attention is drawn to the need to appreciate the limited specificities of probes, the confounding influences of anatomical site and genetic factors (necessitating the use of appropriate positive and negative control tissues) and the precise location of secretory material. In the past, insufficient attention has been given to the effects of altered differentiation including metaplasia and differing lineage expression in epithelial disorders of growth. It is likely that certain changes loosely ascribed to goblet cell mucin, such as neo‐expression of blood group antigens and anomalous expression of core carbohydrate structures, do not occur at all. Critical examination of available data point to only two consistent and unequivocal changes affecting goblet cell mucin in pathological processes: loss of O‐acetyl substituents at sialic acid C4 and C7,8,9 and increased sialyla‐tion. Furthermore, there are no neoplasia‐specific alterations in mucins documented to date. All neoplasia‐associated changes have been described in non‐neoplastic lesions also.

The antimicrobial properties of milkfat after partial hydrolysis by calf pregastric lipase

Studies on the kinetic characteristics of calf pregastric lipase (EC 3.1.1.3) have shown that it preferentially releases short chain fatty acids (SCFAs) from bovine milkfat. The released fatty acids form mixed micelle structures. The aim of this investigation has been to test whether hydrolysed milkfat is antimicrobial, and how the state of the emulsion alters the bactericidal or bacteriostatic effects. Partial hydrolysis of milkfat by pregastric lipase was carried out in two types of emulsion systems, containing either Triton X-100 or casein/lecithin, plus milkfat in citrate/phosphate buffer (pH 5.0-6.0). The concentrations and compositions of fatty acids were determined by gas chromatography. The minimum percentages of hydrolysed milkfat which affected growth and survival of selected Gram-positive and Gram-negative bacteria were measured. The bacterial experiments were repeated using pure fatty acids at similar concentrations. Lauric acid (C12:0) was found to be the most potent bactericidal fatty acid against Enterococcae (Gram-positive), and caprylic acid (C8:0) was the most potent against coliforms (Gram-negative). Use of Triton X-100 for milkfat emulsification provided a more compatible medium for studying bacterial growth in the hydrolysed milkfat than did use of casein/lecithin. The results also show that the antimicrobial effects of individual fatty acids released from hydrolysed milkfat were at least additive and suggest that hydrolysis of milkfat may be a significant factor in controlling growth of organisms imbibed with food in pre-weaned animals. The amount of pregastric catalyzed triglyceride hydrolysis in the digestive tract is sufficient to produce an antibacterial concentration of fatty acids and monoglycerides.

The adsorption of heterocyclic aromatic amines by model dietary fibres with contrasting compositions

It is often recommended that consumption of dietary fibre should in increased to protect against colorectal cancer. However, although more than 95% of dietary fibre is contributed by whole plant cell walls, very little experimental work has been done using whole plant cell walls. These may protect by adsorbing carcinogens, thus lowering their effective concentration in the alimentary tract, and by carrying the carcinogens out of the body in the faeces. However, plant cell walls vary widely in their composition and physical properties, and not all cell walls will necessarily have protective properties. We therefore isolated 4 plant cell-wall preparations with contrasting compositions as models of the types of cell walls that occur in the diet. We investigated the abilities of these preparations to adsorb in vitro 6 heterocyclic aromatic amines (HAAs). HAAs occur in the human diet and several are colon carcinogens, at least in rats. We found that the ability of the HAAs to adsorb to the plant cell walls increased with increasing hydrophobicity of the HAA, measured as the calculated logarithm of the partition coefficient between 1-octanol and water (C logP). A cell-wall preparation containing mainly the walls of parenchyma cells (the most common cell type in food plants) had only poor adsorptive ability. A cell-wall preparation from commercial cork had the best adsorptive ability. This preparation was the most hydrophobic of those examined because the cell walls contained the polymer, suberin, together with associated waxes. The preparation modelled suberized cell walls which occur in the diet, for example in potato skins. The other two cell-wall preparations contained another hydrophobic polymer, lignin, and had intermediate adsorptive abilities which were not significantly different from one another. These preparations modelled lignified cell walls which occur in the diet, for example in wheat bran. Our results indicate that suberized and lignified cell walls may be important in protecting against colorectal cancer.

Roles of endogenous substances and bacteria in colorectal cancer

Because the environment of the human colon is so complex, factors which lead to the development of colorectal cancer are difficult to identify. The effects of 3 endogenous components that affect development of colorectal cancer--colonic bacteria, the mucus layer and bile acids--will be reviewed in this article. The major effects of the bacteria are deconjugation and reduction of bile acids, activation of mutagen precursors, fermentation and production of volatile fatty acids, formation of endogenous mutagens and physical adsorption of hydrophobic chemicals. The mucus layer covering the surface acts as a barrier, and its composition changes in premalignant and malignant colon tissue. Its secretion is elevated by certain plant cell wall components in the diet. Mucus has some hydrophobic properties, and its presence may alter the distribution of hydrophobic molecules. Bile acid concentration in faecal water, rather than the total bile acid concentration, determines the toxicity to epithelial cells and increased concentrations stimulate cell proliferation rates. There is evidence that elevated bile acids in the lumen can activate cellular protein kinase C, which stimulates cell proliferation. These effects are consistent with bile acids acting as tumour promoters.

Cloning of a Mucin-Desulfating Sulfatase Gene from Prevotella Strain RS2 and Its Expression Using a Bacteroides Recombinant System

A gene encoding the mucin-desulfating sulfatase in Prevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into a Bacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.

A Novel Mechanism for Desulfation of Mucin: Identification and Cloning of a Mucin-Desulfating Glycosidase (Sulfoglycosidase) from Prevotella Strain RS2

A novel enzyme which may be important in mucin degradation has been discovered in the mucin-utilizing anaerobe Prevotella strain RS2. This enzyme cleaves terminal 2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sulfate (6-SO3-GlcNAc) residues from sulfomucin and from the model substrate 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sodium sulfate. The existence of this mucin-desulfating glycosidase (sulfoglycosidase) suggests an alternative mechanism by which this bacterium may desulfate sulfomucins, by glycosidic removal of a sulfated sugar from mucin oligosaccharide chains. Previously, mucin desulfation was thought to take place by the action of a specific desulfating enzyme, which then allowed glycosidases to remove desulfated sugar. Sulfate removal from sulfomucins is thought to be a rate-limiting step in mucin degradation by bacteria in the regions of the digestive tract with a significant bacterial flora. The sulfoglycosidase was induced by growth of the Prevotella strain on mucin and was purified 284-fold from periplasmic extracts. Tryptic digestion and sequencing of peptides from the 100-kDa protein enabled the sulfoglycosidase gene to be cloned and sequenced. Active recombinant enzyme was made in an Escherichia coli expression system. The sulfoglycosidase shows sequence similarity to hexosaminidases. The only other enzyme that has been shown to remove 6-SO3-GlcNAc from glycoside substrates is the human lysosomal enzyme beta-N-acetylhexosaminidase A, point mutations in which cause the inheritable, lysosomal storage disorder Tay-Sachs disease. The human enzyme removes GlcNAc from glycoside substrates also, in contrast to the Prevotella enzyme, which acts on a nonsulfated substrate at a rate that is only 1% of the rate observed with a sulfated substrate.

The adsorption of a range of dietary carcinogens by α-cellulose, a model insoluble dietary fiber

Abstract One of the ways dietary fibers may protect against colorectal cancer is by adsorbing carcinogens and carrying them out of the digestive tract, thus lessening interaction of the carcinogens with the colonic tissue. We investigated this mechanism of action by testing in vitro the abilities of a range of carcinogens, including known animal colon carcinogens, to adsorb to α-cellulose, which we have used as a model insoluble dietary fiber. The carcinogens were N-nitroso-N-methylurea (NMU), benzo[a]pyrene (B[a]P) and a number of heterocyclic aromatic amines which have been found in heated foods. It was found that the ability of a carcinogen to adsorb to α-cellulose is strongly related to the hydrophobicity of the carcinogen measured as the calculated logarithm of the partition coefficient between 1-octanol and water (C log P). The hydrophilic carcinogen, NMU, (C log P = −0.204), adsorbed only poorly, whereas the very hydrophobic carcinogen, B[a]P, (C log P = 6.124), adsorbed strongly. Carcinogens with intermediate hydrophobicities showed intermediate abilities to adsorb.

Adsorption of a hydrophobic mutagen to cereal brans and cereal bran dietary fibres

The abilities of brans from the cereals barley, oats, maize, rice, and wheat to adsorb in vitro the hydrophobic, environmental mutagen 1,8-dinitropyrene (DNP) were investigated using a mutagenicity assay. These brans were obtained from known cultivars using defined milling conditions and were chemically characterised. The abilities of total and insoluble dietary fibre preparations obtained from these brans to adsorb DNP were also investigated. The predicted weight of each bran required to adsorb 50% of the added DNP was used to compare the adsorptive abilities of the different brans. The brans were ranked in the order (most effective to least effective): rice, wheat, maize, barley, and oats. The adsorptive abilities of the dietary fibre preparations were not significantly different from the bran from which they were prepared. However, if the dietary fibres (cell walls) were the only components adsorbing the DNP, we would have expected the dietary fibre preparations to have adsorbed more DNP than the equivalent unextracted bran. This suggests that other components, probably starch, also adsorb DNP in the unextracted brans. It is not known why brans from different cereal species differ in adsorptive ability but the lignified cell walls in wheat bran may be important in conferring good adsorptive properties to this bran. The possible relationship between adsorptive ability and ability of the bran from a particular species to protect against colorectal cancer is discussed.