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Biomedical subjects

A M Roberton

Publications and source records attributed to A M Roberton.

At least 37 records · Page 2Linked to original sources

Adsorption of a hydrophobic mutagen to dietary fiber from taro (Colocasia esculenta), an important food plant of the South Pacific.

The incidence of colorectal cancer is lower in Polynesian populations of the South Pacific than in European populations. This difference in incidence of the disease may be, at least partly, related to diet. Dietary fiber is believed to protect against colorectal cancer, and one of the ways it may act is by adsorbing mutagens that are carcinogenic. Very little is known about the chemical composition or the ability to adsorb mutagens of these dietary fibers from South Pacific food plants. In contrast to European food plants, which are mostly dicotyledons, South Pacific food plants are mainly monocotyledons. We isolated cell walls (dietary fiber) from the three edible parts of taro (Colocasia esculenta), which is a monocotyledon and a major South Pacific food plant. The ability of these three unlignified cell-wall preparations to adsorb the hydrophobic environmental mutagen 1,8-dinitropyrene was studied. The greatest adsorption occurred with walls from leaf blade, followed by petiole and corm walls, although the differences were not major. The amount of adsorption was intermediate between the low adsorption previously found with unlignified dicotyledon walls (from the flesh of potato tubers and immature cabbage leaves) and the much higher adsorption found with unlignified walls from monocotyledons of the grass and cereal family (Poaceae) (from leaves of seedling Italian ryegrass). These data are consistent with the monosaccharide compositions of the taro wall preparations, which were more similar to those of unlignified walls of dicotyledons than to unlignified walls of the Poaceae. These findings are consistent with the hypothesis that the composition of the dietary fiber determines its adsorptive properties and that there may be important differences between the major dietary fibers of South Pacific and European food plants.

Adsorption↗

Adsorption of a hydrophobic mutagen to dietary fibre from the skin and flesh of potato tubers.

One of the theories to explain the protective action of some dietary fibres against colon cancer is that certain mutagens and/or cancer promoters are adsorbed to these dietary fibres making the mutagens and/or cancer promoters less available to gut mucosal cells. The abilities of 2 contrasting cell wall preparations (dietary fibre preparations) from potato tubers to adsorb in vitro the hydrophobic mutagen, 1,8-dinitropyrene (DNP), were studied using an incubation mixture containing DNP in phosphate-buffered saline (PBS). Walls from potato skins strongly adsorbed DNP and, at the highest wall concentration tested, only a small porportion of the DNP remained in solution. In marked contrast to the skin walls, potato flesh walls adsorbed only a small proportion of the DNP. Unexpectedly, the flesh walls also caused a large increase in the proportion of DNP found in solution. When flesh walls were pre-extracted with PBS, the ability of the extracted walls to bind DNP increased. The material extracted from the flesh walls was able to maintain DNP in solution, when added to the incubation medium in the absence of cell walls. Pectic polysaccharides appear to be the soluble component responsible for maintaining the DNP in solution. Competition between soluble and insoluble fibre components may have major implications for the availability and distribution of hydrophobic mutagens in the alimentary tract.

Adsorption↗

Adsorption of a hydrophobic mutagen to five contrasting dietary fiber preparations.

The ability of five plant cell wall (dietary fiber) preparations with contrasting compositions to adsorb in vitro the hydrophobic, environmental mutagen, 1,8-dinitropyrene (DNP), was investigated. Many of the fruits and vegetables in Western diets are from dicotyledonous (broad leaved) plants and the dietary fiber from these consists mainly of unlignified cell walls. A representative of this wall type, prepared from immature cabbage leaves, showed little ability to adsorb DNP. Two other cell-wall preparations, representing lignified walls of dicotyledons and unlignified walls of vegetative parts of grasses and cereals (monocotyledons belonging to the family Poaceae), adsorbed DNP much more effectively. However, two further preparations, representing suberized walls of cork cells and lignified walls of vegetative parts of grasses and cereals, were the most effective in adsorbing DNP. Extrapolation of these data to the in vivo situation would indicate that increased consumption of the vegetative parts of grasses or cereals and plant material containing cork cells, for example potato skins, could be effective in removing hydrophobic mutagens from potential contact with colonic mucosal cells.

Brassica↗

Use of the ileal conduit as a model for studying human small intestinal mucus glycoprotein secretion.

To establish a human model system for investigating control of mucus secretion by the small intestine, we have studied the output of mucus glycoprotein in the urine of patients with ileal conduits. A highly specific enzyme-linked immunosorbent assay (ELISA) was used to measure mucus glycoprotein with a sensitivity down to 0.3 ng protein of mucus glycoprotein. The assay detects human ileal mucus glycoprotein in the urine of ileal conduit patients; for an individual subject, the amount hardly varied from day to day, although there were substantial differences between individuals in the amounts secreted. Control urine from persons with normal bladders did not contain any mucus glycoprotein that reacted in the ELISA. The model has many advantages over other experimental systems for studying the rate of mucus glycoprotein secretion from the human ileum and can also be used as a reproducible resource of undegraded mucus glycoprotein.

Amino Acids↗

Effects of bile salts on the adsorption of a hydrophobic mutagen to dietary fiber.

The effect of the bile salts, sodium cholate, deoxycholate, glycocholate and taurocholate, on the solubility in aqueous solution of the hydrophobic, environmental mutagen, 1,8-dinitropyrene (DNP), was examined. In the absence of bile salts, the DNP appeared to precipitate out of solution, whereas bile salts at a concentration of greater than or equal to 4 mM maintained the DNP in solution. In the presence of the model dietary fiber, alpha-cellulose, the DNP absorbed to this preferentially. Bile salts reduced this adsorption at low alpha-cellulose levels, but had little effect at high alpha-cellulose levels. The implication of these results is that bile salts have solubilising properties that could affect the distribution of hydrophobic molecules, including mutagens, in the digestive tract.

Adsorption↗

Direct-acting mutagenic properties of some hair dyes used in New Zealand.

Mutagenicity or carcinogenicity data are not publicly available on many hair dyes or dye components commonly used within New Zealand. Representative mid- to dark-warm brown hair dyes of 12 brands supplying more than 1% of the New Zealand market were tested for direct-acting mutagenicity using the bacterial 'Ames' test. Despite recent scientific advances in the development of non-mutagenic dyes, 23 of the 40 products tested gave positive results in one or both of the tester strains used. There appeared to be differences between distributors in the proportion of their hair dyes which were mutagenic. In the case of 6 out of 10 of the above dyes which had tested positive, in vitro mutagenicity or toxicity was enhanced in the presence of verapamil, suggesting that risks from hair-dye exposure may change in the case of persons using this or similar drugs. It is recognised that there are uncertainties regarding human risks from dyes which are shown to be mutagenic in in vitro tests. However, from the above results, it seems possible to produce non-mutagenic hair dyes in this color range; and in the interests of public reassurance, it may be prudent to require that such dyes be used.

Drug Synergism↗

A model system for studying the adsorption of a hydrophobic mutagen to dietary fibre.

The adsorption of 1,8-dinitropyrene (DNP) to alpha-cellulose has been studied as a model system for examining the adsorption of a hydrophobic mutagen to dietary fiber. Most of the DNP rapidly disappeared from an aqueous solution and partitioned between the glass wall of the test tube and the alpha-cellulose. Factors affecting DNP distribution included (i) the time of incubation, (ii) the final concentration of the solvent, dimethyl sulphoxide, in which the DNP has been dissolved, and (iii) the relative concentrations of DNP and alpha-cellulose. We suggest that this model system could be applied to other mutagens, and that alpha-cellulose would provide a useful standard fiber to permit inter-laboratory comparisons.

Adsorption↗

The putative 'link' glycopeptide associated with mucus glycoproteins. Composition and properties of preparations from the gastrointestinal tracts of several mammals.

The existence of a discrete 'link' peptide in epithelial mucins has been debated for many years. There is evidence that at least some mucins contain a specific 'link' peptide (or glycopeptide) that enhances mucin polymerization by forming disulphide bridges to large mucin glycoprotein subunits. A major difficulty has been to know whether the reported differences in putative 'link' components represent artifacts generated by inter-laboratory differences in technical procedures used in mucin purification. The present paper outlines the results of a collaborative study involving five laboratories and 53 samples of purified gastrointestinal mucins (including salivary, gastric, small-intestinal and colonic mucins) prepared by five techniques from four different animal species. An early step in mucin purification in all cases was the addition of proteinase inhibitors. Representative mucins were analysed for their composition, electrophoretic mobility in SDS/polyacrylamide-gel electrophoresis before and after disulphide-bond reduction, and for their reactivity with monospecific antibodies developed against the 118 kDa putative 'link' glycopeptide isolated from either rat or human small-intestinal mucins. Our results indicate that, despite differences in laboratory techniques, preparative procedures, organs and species, each of the purified mucins contained a 'link' component that was released by disulphide-bond reduction and produced a band on SDS/polyacrylamide-gel electrophoresis at a position of approx. 118 kDa. After electroelution and analyses, the 118 kDa bands from the different mucins were found to have similar amino acid profiles and to contain carbohydrate. It would appear therefore that a 'link' glycopeptide of molecular mass approx. 118 kDa is common to all of the gastrointestinal mucins studied.

Animals↗

Biochemical evidence that high concentrations of the antidepressant amoxapine may cause inhibition of mitochondrial electron transport.

Overdosage with the antidepressant amoxapine causes metabolic acidosis and may lead to brain damage and death. To better understand the metabolic disturbances caused by amoxapine overdose, its effects on three simple systems were studied: growth of Saccharomyces cerevisiae, mitochondrial energy metabolism, and an electron transport system in microsomal membranes. Growth of yeast on all substrates except lactate was inhibited by amoxapine at 50-100 micrograms ml-1. Growth on lactate was observed at 200 micrograms ml-1 of amoxapine. In beef heart mitochondria, amoxapine at 100 micrograms ml-1 inhibited reactions involving large sections of the electron transport chain. Energy-linked reactions in submitochondrial particles were also inhibited. Electron microscopy showed some disruption of the mitochondrial internal structure by amoxapine and a change from orthodox to condensed conformation. Microsomal NADH-cytochrome b5 reductase was inhibited by amoxapine, but at higher amoxapine concentrations than mitochondrial reactions. The results suggest amoxapine disrupts reactions of membrane-associated enzyme complexes, and mitochondrial energy conservation may be one of the first systems affected. We speculate that lactic acid accumulation in patients with amoxapine overdose may be caused by loss of electron acceptor activity in tissues.

Amoxapine↗

Enzyme-linked immunosorbent assay for ileal human goblet cell mucin.

A sandwich ELISA for measuring ileal human goblet cell mucin has been developed with a linear response range 0.2 to 1.5 ng mucin protein. It can be used to quantitate the mucin present in dilute and unpurified samples without interference from other glycoproteins and proteins. Reduction of the mucin decreased the reactivity by only 14% indicating that the assay reacts almost as well with mucin glycopeptides as with native mucin. The assay has the advantages over previously described immunoassays for mucin of giving a result in 6 h, detecting slightly lower concentrations of mucin, and is more sensitive, quantitative and specific than the traditional protein or periodic acid-Schiff assays used for glycoproteins.

Animals↗

Inhibition of attachment of Escherichia coli RDEC-1 to intestinal microvillus membranes by rabbit ileal mucus and mucin in vitro.

Intestinal mucus is postulated to play a role in preventing colonization of the gastrointestinal tract by microbial pathogens. To evaluate the ability of both crude mucus and purified mucin, a glycoprotein of goblet cell origin, to inhibit mucosal adherence of enteric pathogens, we examined whether mucus and mucin derived from rabbit ileum interact with the rabbit enteropathogen Escherichia coli RDEC-1. We examined the manner in which mucus and mucin inhibited adherence of bacteria to rabbit ileal microvillus membranes (MVMs) in vitro. The purity of the mucin preparation was demonstrated by polyacrylamide gel electrophoresis before and after reduction and by showing that an antiserum raised to the mucin localized to goblet cells in rabbit intestine. Using radioactive labeling of bacteria, we quantitated attachment of RDEC-1 to MVMs, mucus, and mucin that had been immobilized on polystyrene microtiter wells. Binding of RDEC-1 to MVMs was also determined after preincubation of organisms with crude ileal mucus and purified mucin. RDEC-1 bound to both crude mucus and purified mucin when they expressed lectinlike adhesions, previously designated attachment factor rabbit 1 pili. Adherence of piliated RDEC-1 to MVMs, mucus, and mucin was significantly greater than when the bacteria were nonpiliated. Binding of piliated RDEC-1 to MVMs was decreased by preincubation of bacteria with both crude mucus (45.6 +/- 4.2% of control) and purified mucin (50.2 +/- 5.8%). These data indicate that the E. coli enteropathogen RDEC-1 can bind to purified glycoproteins of goblet cell origin and that adherence of these bacteria to mucin is mediated by expression of pili. The findings also support a role for intestinal mucus and its principal organic constituent, mucin, in preventing adherence of a known E. coli enteric pathogen to apical MVMs of enterocytes.

Animals↗

Effect of pepsin on partially purified pig gastric mucus and purified mucin.

Partially purified native-pig gastric mucus and purified pig gastric mucin, prepared by column chromatography and caesium chloride (CsCl) density-gradient ultracentrifugation, were subjected to pepsin digestion. The products of peptic digestion were chromatographed on Sepharose CL-2B, and fractions were assayed for carbohydrate by the periodic acid-Schiff reaction. The polymeric gastric mucin in the purified mucin samples was readily degraded by pepsin. In sharp contrast, the polymeric mucin in the partially purified mucus was relatively resistant to pepsin digestion. In 45 min, pepsin degraded 40% of the polymeric mucin in the purified samples, whereas it produced no significant degradation (less than 10%) in the partially purified mucus samples. In partially purified gastric mucus, treated with CsCl but not fractionated by ultracentrifugation, digestion with pepsin was also slow and incomplete. This showed that differences in susceptibility between partially purified and purified preparations are not due to the chaotropic effects of CsCl. In addition, the recombination of low-density nonmucin fractions in CsCl ultracentrifugation with the mucin also resisted pepsin digestion. Finally, we have shown that the low-density fractions in mucus exhibited a strong inhibitory effect of peptic activity in vitro. We conclude that under our experimental conditions, pepsin has little effect on partially purified mucus, and our findings indicate an inhibitor of peptic digestion is present in native gastric mucus. It is likely, but unproven, that this inhibitor is a noncovalently bound lipid present in the low-density fraction.

Animals↗

Anaerobic incubation and mutagenicity of nitracrine analogues in Salmonella typhimurium.

Nitracrine [1-nitro-9-(dimethylaminopropylamino)-acridine] is a clinical antitumour agent with hypoxia-selective cytotoxicity and radiosensitizing activity. Developments in tumour therapy using either nitracrine itself or some of its analogues under development are likely to be targeted at anaerobic regions of tumours. We have investigated the effects of anaerobiosis on mutagenic activity of nitracrine and a small series of analogues in three derivatives of Salmonella typhimurium frameshift strain hisC3076. Nitracrine-induced mutagenicity was apparently enhanced by a period of anaerobic incubation followed by an aerobic period, with maximal mutagenic effectiveness (as revertants/nmol) being seen at 24 h anaerobiosis. However, the maximal number of nitracrine-induced revertants was not increased by anaerobic incubation, and the relationship between mutagenicity and toxicity remained similar. Comparisons of the effects of anaerobiosis on mutagenicity of bacterial strains with different DNA repair capacities suggested that anaerobiosis was not simply depressing 'SOS' repair. Comparisons of nitracrine with four of its analogues showed that this effect was not a universal characteristic of either acridine derivatives or of nitracrine analogues. Rather, the dose displacements obtained paralleled hypoxic cell selective cytotoxicity in mammalian cells. If this result extends to other compounds, experiments of this nature using the bacterial strains could provide a novel and useful screening system to identify hypoxia-selective cytotoxic anticancer drugs of potential clinical value.

Aminoacridines↗

The purification and some equilibrium properties of the nitrite reductase of the bacterium Wolinella succinogenes.

The bacterium Wolinella succinogenes produces a nitrite reductase enzyme that can be purified to homogeneity in high yield by a combination of detergent extraction, hydroxyapatite chromatography and Mr fractionation. Nitrite reductase activity is found to be present in both a high- and a low-Mr fraction. The high-Mr fraction has been shown to consist of the low-Mr nitrite reductase enzyme associated with a hydrophobic 'binding protein'. The amino acid composition for both proteins is reported. The nitrite reductase enzyme shows spectral characteristics indicative of the presence of c-type haem groups. Measurements at 610 nm indicate the presence of some high-spin haem groups at neutral pH. This haem subgroup undergoes a pH-linked high-spin - low-spin transition at alkaline pH. Approximately two of the six haem groups present within the enzyme bind CO with low affinity (KD = 0.4 mM). The enzyme also shows a range of redox activities with various inorganic reagents. The enzyme has been shown to exhibit dithionite reductase, oxygen reductase and CO2 reductase activities.

Amino Acids↗

Degradation of pig gastric and colonic mucins by bacteria isolated from the pig colon.

Mucin degradation was studied with one Clostridium (RS42) and two Bacteroides (RS2 and RS13) strains isolated from the pig colon mucosa. Mucins from pig colon and stomach were prepared in their subunit forms for use as growth substrates, and the loss of the individual sugars from the mucins was measured after bacterial growth. Colonic mucin was more resistant to degradation than gastric mucin. The strains differed in their competence in degrading the mucins. Carbohydrate plus sulfate removal from gastric mucin varied from 63 to 76% for RS2, 37 to 46% for RS13, and 37 to 53% for RS42. All three strains removed more fucose (67 to 87%) and less sulfate (22 to 63%) than the average carbohydrate plus sulfate loss. Under the same conditions of growth, a mixed pig fecal culture removed 78% of sulfate and 96% of each sugar. Of the two major glycoprotein types present in the subunit pig gastric mucin preparation (R. A. Stanley, S. P. Lee, and A. M. Roberton, Biochim. Biophys. Acta 760:262-269, 1983), the less highly sulfated mucin was more susceptible to RS42 degradation. The degradation of gastric mucin by RS2 was not affected by glucose or high sulfate concentrations in the growth medium. The results show that the three strains of colon bacteria are capable of significant hydrolysis of mucin carbohydrate and that the extent of degradation seen with pure cultures is determined in part by the subunit glycoprotein type(s) present in the mucin.

Animals↗

Intra-caecal short chain fatty acids are altered by dietary pectin in the rat.

When Dark Agouti rats were changed from a conventional pellet diet containing 3.3% crude fibre and 4% fat to the experimental diet containing 5% pectin and 17% fat, the levels of n-butyrate in the caecum rapidly decreased more than fivefold. Such a change could be important as n-butyrate is known to be a major energy source for colonocytes, and to affect colon tumour cell development in vitro. Striking decreases in the caecal concentrations of the short chain fatty acids isobutyrate, n-valerate and isovalerate occurred when Wistar rats were fed an experimental diet containing 5% pectin as the only dietary fibre, compared to rats fed the experimental diet containing no dietary fibre. These large changes in the short chain fatty acid profile, caused by pectin supplementation, have important implications for the metabolism and health of the colon.

Animals↗

Heterogeneity in gastrointestinal mucins.

Pig digestive tract mucins have often been used as model mucins for studying mucin structure, function and metabolism. In the present study pig gastric mucin and pig colonic mucin in the subunit form have been characterised and compared. Following Sepharose 4B or 2B-CL gel chromatography, the mucin eluant fractions were assayed colorimetrically by both the periodic acid-Schiff and the Alcian blue binding assays. Subunit colonic mucin eluted as a single unimodel peak that was easily detected by both assays. In contrast, subunit gastric mucin gave a peak primarily detected by periodic acid-Schiff that was overlapped by, but partially separated from, another peak primarily detected by Alcian blue. Subunit gastric mucin was separated into two periodic acid-Schiff staining spots when electrophoresed on cellulose acetate. Cetylpyridinium chloride (CPC) was able to precipitate only about half the subunit gastric mucin. The CPC-precipitable subunit gastric mucin corresponded to the faster running spot on electrophoresis, and the subunit gastric mucin in the CPC supernatant (which may have more than one subunit mucin type) to the slower spot(s). The former had a higher sulphate content and stained with Alcian blue. The latter had a lower sulphate content and showed very little Alcian blue reactivity. These results indicate that subunit pig gastric mucin is heterogeneous with respect to both size and charge. The differences between the types may be important in biological and physiochemical behaviour of gastric mucin. It seems likely that different laboratories may have worked on one or other of the pig gastric mucin types or a mixture, depending on the preparation method.

Animals↗