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Developing Countries↗
Biomedical subjects
S Macfarlane
Publications and source records attributed to S Macfarlane.
Starch utilization by Bacteroides ovatus isolated from the human large intestine.
Starch supported growth of continuous cultures of Bacteroides ovatus when this carbohydrate provided the sole source of carbon and energy. Inducible amylase and alpha-glucosidase activities were inversely related to dilution rate in starch-limited and starch-excess chemostats over the dilution rate (D) range D = 0.03/h to D =0.20/h, and were partly repressed during growth under conditions of starch-excess. Preparative isoelectric focusing of B. ovatus cytoplasmic extracts indicated the existence of three distinct starch-hydrolyzing enzymes. Incubation of active fractions from the isoelectric focusing cell with maltose and a variety of low-molecular-weight oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose) identified a single amylase activity, an enzyme with combined beta-amylase and glucoamylase/alpha-glucosidase properties, and also a possible pullulanase. The ability of B. ovatus to synthesize several starch-hydrolyzing enzymes with different specificities and activities may confer a significant competitive advantage to this organism in the colonic ecosystem.
Bacteroides↗
Utilization of starch and synthesis of a combined amylase/alpha-glucosidase by the human colonic anaerobe Bacteroides ovatus.
Bacteroides ovatus preferentially utilized starch and pectin when grown on a mixture of polysaccharides in batch culture, indicating that these carbohydrates are important substrates for the bacterium in the human large intestine. Further studies on starch breakdown showed that continuous cultures grew on the polysaccharide when it provided the sole carbohydrate source, to yield a single hydrolytic product at low dilution rates (D = 0.04 h-1), with an estimated molecular mass of 13 kDa. In contrast, two major types of oligomeric products were formed at higher dilution rates (D = 0.44 h-1), with approximate molecular weights of 11 and 140 kDa. Analysis of cell-associated starch-degrading enzymes produced by Bact. ovatus using ion exchange chromatography and HPLC gel-filtration showed that amylase and alpha-glucosidase activities eluted in the same fractions. The single peak containing amylase and alpha-glucosidase activities obtained by HPLC gel-filtration chromatography corresponded to a molecular mass of approximately 140 kDa, and activity staining of gels for alpha-glucosidase activity after polyacrylamide gel electrophoresis, in the presence of sodium dodecyl sulphate, gave an estimated molecular mass of 70 kDa, indicating this enzyme to be a dimer. After renaturation, the 70 kDa band was cut from the gels and solubilized. The extract hydrolysed gelatinized starch and p-nitrophenyl-alpha-D-glucopyranoside.
Amylases↗
Human colonic microbiota: ecology, physiology and metabolic potential of intestinal bacteria.
In both health and disease, the colonic microbiota plays an important role in several areas of human physiology. This complex assemblage of microorganisms endows great metabolic potential on the large intestine, primarily through its degradative abilities. Many hundreds of different types of bacteria, varying widely in physiology and biochemistry, exist in a multitude of different microhabitats in the lumen of the large gut, the mucin layer and on mucosal surfaces. Both microbiota and host obtain clear benefits from association. For example, growth substrates from diet and body tissues, together with a relatively stable environment for bacteria to proliferate are provided by the host, which in turn has evolved to use butyrate, a bacterial fermentation product, as its principal source of energy for epithelial cells in the distal bowel. The main sources of carbon and energy for intestinal bacteria are complex carbohydrates (starches, non-starch polysaccharides). Carbohydrate metabolism is of great importance in the large intestine, since generically, and in terms of absolute numbers, the vast majority of culturable microorganisms are saccharolytic. The amounts and types of fermentation products formed by colonic bacteria depend on the relative amounts of each substrate available, their chemical structures and compositions, as well as the fermentation strategies (biochemical characteristics and catabolite regulatory mechanisms) of bacteria participating in depolymerization and fermentation of the substrates. Protein breakdown and dissimilatory amino acid metabolism result in the formation of a number of putatively toxic metabolites, including phenols, indoles and amines. Production of these substances is inhibited or repressed in many intestinal microorganisms by a fermentable source of carbohydrate. Owing to the anatomy and physiology of the colon, putrefactive processes become quantitatively more important in the distal bowel, where carbohydrate is more limiting.
Adult↗
Formation of a dipeptidyl arylamidase by Bacteroides splanchnicus NCTC 10825 with specificities towards glycylprolyl-x and valylalanine-x substrates.
Bacteroides splanchnicus in common with several members of the B. fragilis group constitutively produced a number of protein and peptide hydrolysing enzymes. Amongst the most active was an arylamidase, which specifically hydrolysed the dipeptidyl chromogenic substrates glycylprolyl p-nitroanilide (GPRPNA), glycylprolyl beta-naphthylamide (GP beta NA) and valylalanine p-nitroanilide (VAPNA), and had some proteolytic activity towards azocasein. No activity was detected against proline beta-naphthylamide, glycine, valanine or alanine p-nitroanilides. Physiological studies showed that the enzyme was largely cell-associated during exponential growth in batch culture, but was progressively released by the bacteria before the cells entered stationary phase. Glycylprolyl arylamidase (GPA) was completely cell-bound during growth in continuous culture, where synthesis increased concomitantly with dilution rate (specific growth rate) in both carbon- and nitrogen-limited chemostats. Gel-filtration chromatography of B. splanchnicus cell extracts yielded a single peak of GPA activity, with an apparent molecular mass of c. 160 kDa, while one peak of enzyme activity was eluted by 0.3 M NaCl during cation-exchange chromatography. Activity staining of SDS polyacrylamide gels showed a single GPA band at 80 kDa, suggesting that the enzyme was a dimer. Two fractions of GPA activity were recorded during preparative isoelectric focusing with apparent isoelectric points of pH 3.51 (fraction 3) and 3.95 (fraction 6), indicating the possible existence of GPA isoenzymes. GPRPNA, VAPNA and azocasein were hydrolysed by the major fraction (fraction 3), while only the p-nitroanilide substrates were hydrolysed by fraction 6. Studies with the partially purified enzyme obtained from gel filtration columns showed a relatively broad pH optimum at 7.5-8.2. Inhibition experiments demonstrated that while aspartic (pepstatin A), thiol (iodoacetate) and metalloprotease (EDTA, cysteine) inhibitors had little effect on hydrolysis of glycylproline p-nitroanilide, GPA was strongly inhibited (c. 80%) by 5 mM phenylmethylsulphonyl fluoride (PMSF), indicating it to be a serine enzyme.
Aminopeptidases↗
Consequences of biofilm and sessile growth in the large intestine.
The human colonic ecosystem is an extremely complex environment comprised of several hundred different strains of bacteria. Studies were undertaken to determine whether these organisms formed metabolic or genotypically distinct assemblages in the gut microbiota in relation to polysaccharide fermentation. Measurements of depolymerizing enzymes (4 polysac-charidases, 6 glycosidases) showed that specific amylase and pectinase activities were comparable in bacteria desorbed from the surfaces of food particles and in non-particulate organisms. However, xylanase, beta-xylosidase, arabinogalac-tanase, alpha-arabinofuranosidase, and beta-galacturonidase activities were always significantly greater in particulate bacteria. Short-term in vitro fermentations with both groups of bacteria showed marked differences in relative rates of starch, arabinogalactan, and mucin metabolism, while rates of fermentation product formation with pectin and xylan were broadly comparable. Significant differences were observed with respect to formation of individual fermentation products, especially when mucin or pectin were substrates, where particulate bacteria produced proportionally higher amounts of acetate. Bacteriological studies showed that communities of polymer-degrading bacteria and other groups of intestinal anaerobes growing on particulate matter were essentially similar to those occurring elsewhere in the gut lumen, at genus and species levels. In vitro colonization experiments demonstrated that a variety of polysaccharide-fermenting bifidobacteria and bacteroides--together with other cross-feeding organisms such as peptostreptococci, fusobacteria, and coliforms--rapidly attached to particulate intestinal materials.
Bacteria, Anaerobic↗
Growth, incidence and activities of dissimilatory sulfate-reducing bacteria in the human oral cavity.
Viable counts and activities of sulfate-reducing bacteria were determined in the oral cavities of 12 healthy volunteers. Of these, 10 harboured viable sulfate-reducing bacteria populations. Six separate sites were sampled: the posterior tongue, anterior tongue, mid buccal mucosa, vestibular mucosa, supragingival plaque and subgingival plaque. Sulfate-reducing bacteria occurred in all areas, with the highest incidence in supragingival plaque. Viable counts and sulfate-reducing activities in each of the regions varied from 0 to 10(8) cfu (g wet weight)-1 and from 0 to 50 nmol (g wet weight)-1 h-1, respectively. As sulfate-reducing bacteria can be detected in the oral cavity, they may potentially be involved in terminal oxidative processes carried out by the microflora of the mouth.
Adult↗
Co-culture of Bifidobacterium adolescentis and Bacteroides thetaiotaomicron in arabinogalactan-limited chemostats: effects of dilution rate and pH.
The effects of dilution rate (D = 0.04-0.38/h) and pH (5.0-6.5) on co-cultures of Bifidobacterium adolescentis and Bacteroides thetaiotaomicron were studied in arabinogalactan-limited chemostats. B. thetaiotaomicron outcompeted B. adolescentis at all dilution rates at culture pH values between 5.0 and 6.0, although the bifidobacterium was always detected in the fermenters. At pH 6.5, however, B. adolescentis predominated in co-cultures at dilution rates above 0.24/h. Arabinogalactan degrading enzymes (beta-galactosidase, alpha-arabinofuranosidase) were strongly catabolite repressed in bacteroides at high dilution rates, but were constitutive and growth rate-associated in B. adolescentis. The increased competitiveness of B. adolescentis at high specific growth rates was not related to its ability to synthesise increased levels of depolymerising enzymes. Measurements of residual carbohydrate in pure and mixed culture chemostats showed that the bacteroides extensively digested the galactose backbone of the polymer, and to a lesser degree, the arabinose sidechains. Nevertheless, arabinose monomers and oligosaccharides (d.p. < 10) accumulated in these cultures under all growth conditions. In contrast, the bifidobacterium utilized considerably less arabinogalactan than the bacteroides, and this was reflected in the mixed culture studies. These experiments demonstrate that B. thetaiotaomicron was able to compete most successfully for this plant cell wall polysaccharide under nutritional, physiological and environmental conditions broadly similar to those encountered in the human colon, and indicate the existence of synergistic interactions between the two organisms that were growth rate dependent.
Journal Article↗
Risk-perception: differences between adolescents and adults.
This study investigated age changes in risk perception and unrealistic optimism. Teenagers (n = 376) and parents (n = 160) evaluated the risk of experimental, occasional, and regular involvement in 14 health-related activities (e.g., getting drunk). Respondents also evaluated their comparative changes of encountering the leading causes of morbidity and mortality. Compared with adults, teenagers minimized the perceived risk of experimental and occasional involvement in health-threatening activities. Notably, teenagers were less optimistic about avoiding injury and illness than were their parents, and teenagers at greatest risk for such misfortunes were the least optimistic about avoiding them. These findings do not support traditional explanations of adolescent risk taking. The implications of these findings for understanding and preventing health-damaging behavior among adolescents are discussed.
Adolescent↗
Estimation of the fermentability of dietary fibre in vitro: a European interlaboratory study.
Five European laboratories tested a simple in vitro batch system for dietary fibre fermentation studies. The inoculum was composed of fresh human faeces mixed with a carbonate-phosphate buffer complex supplemented with trace elements and urea. Five dietary fibre sources (cellulose, sugarbeet fibre, soyabean fibre, maize bran and pectin) were used by each laboratory on three occasions to determine pH, residual non-starch polysaccharides (NSP) and short-chain fatty acid production during fermentation. Cellulose and maize bran degradabilities were very low (7.2(SE 10.8) and 6.2 (SE 9.1)% respectively after 24 h), whereas pectin and soyabean fibre were highly degraded (97.4 (SE 4.4) and 91.1 (SE 3.4)% respectively after 24 h). Sugarbeet fibre exhibited an intermediate level of degradability (59.5 (SE 14.9)%). Short-chain fatty acid production was closely related to NSP degradation (r 0.99). Although each variable was ranked similarly by all laboratories, some differences occurred with respect to absolute values. However, the adaptation of donors to the experimental substrates was not an influential factor. Interlaboratory differences could be reduced either by adding less substrate during incubations or using less-diluted inocula. In vitro fermentations with inocula made from human faeces and from rat caecal contents gave similar results. There was a close correspondence between the data obtained in the present experiment and those previously published in in vivo studies in the rat using the same fibres. The in vitro batch system tested during the present study provides a rapid means of obtaining quantitative estimates of the fermentation and the estimation of the energy content of new sources of dietary fibre.
Animals↗
Needs assessment in continuing medical education. Its feasibility and value in a seminar about skin cancer for general practitioners.
OBJECTIVE: To investigate the acceptability, feasibility and value of needs assessment in planning and conducting a seminar about skin cancer for general practitioners. METHOD: A self-administered questionnaire was developed to assess general practitioners' educational needs in skin cancer prevention, early detection and management. Registrants for a one-day interactive skin cancer seminar conducted by the NSW Cancer Council were each mailed this questionnaire. Responses received before the seminar were collated and discussed with resource persons involved in the seminar. RESULTS: We achieved an 80% response rate to the pre-seminar needs assessment. Responses indicated gaps in knowledge, widespread differences in views about skin surveillance recommendations and a hierarchy of preferences for practical procedures. The seminar was modified, contributing to high levels of participant satisfaction with its educational content. CONCLUSIONS: It is feasible to conduct a needs assessment in continuing medical education which achieves high response rates and provides vital information to resource persons about the educational needs of participants. Needs assessment holds promise as a strategy to assist general practitioners to identify their own goals for learning. Needs assessment as described here is necessary also to evaluate the outcome of continuing medical education. We recommend needs assessment as an integral part of educational programs for general practitioners although the time, planning and resources required to assess learning needs should not be underestimated.
Adult↗
Factors affecting fermentation reactions in the large bowel.
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Carbohydrate Metabolism↗
Physiology and biochemistry of glycyl-prolyl aminopeptidase formation in Bacteroides splanchnicus.
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Aminopeptidases↗
Degradative activities of gut anaerobes studied in a three-stage continuous culture model of the colon.
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Bacteria, Anaerobic↗
Physiological and nutritional factors affecting synthesis of extracellular metalloproteases by Clostridium bifermentans NCTC 2914.
Extracellular protease production by Clostridium bifermentans NCTC 2914 occurred throughout the growth phase in batch culture. In both glucose-excess and -limited chemostats, protease formation was inversely related to the dilution rate, over the range D = 0.03 to 0.70 h-1. At high dilution rates (D greater than 0.25 h-1), protease activities were greatest under excess glucose conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chemostat culture effluents showed the presence of up to 18 bands of protease activity at low dilution rates, with apparent molecular masses ranging from about 36 to 125 kDa. High-performance liquid chromatography gel filtration of culture supernatants gave four peaks of activity at 34, 42, 60, and 102 kDa. Glucose, peptone, and phosphate stimulated protease formation, but ammonia concentrations up to 10 g liter-1 had little effect on the process. Culture pH in glucose-excess chemostats strongly influenced protease synthesis, which was maximal during growth at pH 6.4. The optimal pH of protease activity was 7.0. Although a wide variety of proteins were hydrolyzed by C. bifermentans proteases, none of the enzymes were collagenolytic. Of 21 different p-nitroanilide, beta-naphthylamide, and N-carbobenzoyl substrates tested, none were hydrolyzed. With the exception of Ca2+, divalent metal ions inhibited proteolysis. Experiments with protease inhibitors demonstrated that 1 mM EDTA inhibited protease activities in culture supernatants by over 90%, indicating that the enzymes were principally of the metalloprotease type.
Chromatography, High Pressure Liquid↗
Characterization of a chondroitin sulfate proteoglycan synthesized by monkey arterial smooth muscle cells in vitro.
A monoclonal antibody against arterial smooth muscle cell chondroitin sulfate proteoglycan has been developed. Incubation of [35S]-methionine labeled proteoglycans with MAb 941 quantitatively immunoprecipitated all the chondroitin sulfate proteoglycan (CSPG) synthesized by these cells. Digestion of the immunoprecipitate with chondroitin AC lyase revealed one major protein band (Mr 420,000) and two minor bands (Mr 509,000 and 390,000) on SDS-PAGE that are composed of very similar peptides when analyzed by limited peptide digestion by S. aureus V8 protease. Additional studies demonstrated that this monoclonal antibody recognized an epitope on the chondroitin sulfate chains. However, only a minor subpopulation (5-12%) of the alkaline-borohydride released glycosaminoglycan chains was immunoprecipitated and this subset of chains was slightly larger than the non-immunoprecipitated chains. High pressure liquid chromatography analysis of the disaccharides generated from the immunoprecipitated glycosaminoglycan chains demonstrated that these chains were enriched in chondroitin-6-sulfate relative to chondroitin-4-sulfate (2:1) while that of the non-immunoprecipitated chains had a ratio of 1:1. These studies indicate that at least two distinct pools of chondroitin sulfate chains are present on all the chondroitin sulfate proteoglycan synthesized by arterial smooth muscle cells: a major population (89-95%) containing 6-sulfate and 4-sulfate in relatively equal proportion and a minor population (5-12%) which is hydrodynamically larger with a 6-sulfate to 4-sulfate ratio of 2:1.
Animals↗
Effect of different carbohydrates on growth, polysaccharidase and glycosidase production by Bacteroides ovatus, in batch and continuous culture.
Bacteroides ovatus was grown in batch culture on 12 different carbon sources (five polysaccharides, seven monosaccharides and disaccharides). Specific growth rates were determined for each substrate together with polysaccharidase and glycosidase activities. Growth rates on polymerized carbohydrates were as fast or faster than on corresponding simple sugars, demonstrating that the rate of polysaccharide depolymerization was not a factor limiting growth. Bacteroides ovatus synthesized a large range of polymer-degrading enzymes. These polysaccharidases and glycosidases were generally repressed during growth on simple sugars, but arabinose was required for optimal production of alpha-arabinofuranosidase. Polysaccharidase and glycosidase activities were measured in continuous cultures grown with either xylan or guar gum under putative carbon limitation. With the exception of beta-xylosidase, activities of the polymer-degrading enzymes were inversely related to growth rate. This correlated with polysaccharide utilization which was greatest at low dilution rates. These results show that Bact. ovatus is highly adapted for growth on polymerized carbohydrate in the human colon and confirm that the utilization of polysaccharides is partly regulated at the level of enzyme synthesis.
Bacteroides↗
Influence of retention time on degradation of pancreatic enzymes by human colonic bacteria grown in a 3-stage continuous culture system.
Hydrolytic enzymes were measured in gut contents from four sudden death victims. Pancreatic amylase and total protease activities decreased distally from the small bowel to the sigmoid/rectum region of the large intestine, showing that considerable breakdown or inactivation of the enzymes occurred during gut transit. To determine whether pancreatic enzymes were substrates for the gut microflora, mixed populations of bacteria were grown in a 3-stage continuous culture system on a medium that contained pancreatic extract as the sole nitrogen source. The multichamber system (MCS) was designed to reproduce in vitro, the low pH, high nutrient, fast growth conditions of the caecum and right colon and the neutral pH, low nutrient, slow growth conditions of the left colon. Results showed that pancreatic amylase was resistant to breakdown by intestinal bacteria compared with the peptide hydrolases in pancreatic secretions. Leucine aminopeptidase, trypsin and to a lesser degree, chymotrypsin, were easily degraded by gut bacteria, but pancreatic elastase was comparatively resistant to breakdown. Protein degradation in the MCS, as determined by enzyme activities, protein concentration and ammonia and phenol production, increased concomitantly with system retention time over the range 24-69 h. These results suggest that intestinal bacteria play an important role in the breakdown of hydrolytic enzymes secreted by the pancreas and that this process and protein fermentation in general, is likely to occur maximally in individuals with extended colonic retention times.
Amylases↗