Biomedical subjects

R L Buchanan

Publications and source records attributed to R L Buchanan.

At least 37 records · Page 2Linked to original sources

PubMed1993

Studies on Escherichia coli serotype O157:H7 strains containing a 60-MDa plasmid and on 60-MDa plasmid-cured derivatives.

Seventeen strains of Escherichia coli serotype O157:H7 producing Shiga-like toxin were examined for the presence of plasmids and for the ability to adhere to HEp-2 and Intestine 407 cells. All of the strains possessed a common 60-MDa plasmid. To determine the role of the 60-MDa plasmid, plasmid-cured strains were compared with the parent strains for their ability to produce pili and to adhere to epithelial cells in culture. The total cell lysate protein and outer-membrane protein (OMP) profiles were also compared. Both the parent strains and their plasmid-cured derivatives produced pili. Immunofluorescence assay results indicated that the plasmid-cured and parent strains adhered equally well to HEp-2 and Intestine 407 cells; overall adherence was greater with intestinal cells than HEp-2 cells. SDS-PAGE of polypeptides synthesised in an E. coli system in vitro showed that plasmid DNA encodes c. 35 proteins. SDS-PAGE of OMP preparations demonstrated that the 60-MDa plasmid appears to be involved in the synthesis of a 33-kDa OMP. Two strains cured of the 60-MDa plasmid, one that possessed no plasmids and one that still contained a 2.2-MDa plasmid, produced exopolysaccharide (EPS) when cultured on solid medium at 25 degrees C. Two other strains, which were cured of the 60-MDa plasmid but contained a 4.5-MDa plasmid, did not produce visible amounts of EPS. Gas chromatography analysis showed that the EPS consisted of fucose, glucose and galactose in an approximate molar ratio of 2.0:0.9:1.1 and also had 7% of a uronic acid sugar as part of its structure.

Bacterial Adhesion↗

PubMed1993

Virulence of an Escherichia coli O157:H7 sorbitol-positive mutant.

Virulence and pathogenicity of an Escherichia coli O157:H7 sorbitol-positive mutant were investigated with an infant rabbit animal model as well as a battery of in vitro assays. Total cell lysate protein profiles, outer membrane protein profiles, plasmid profiles, and levels of cytotoxic activity against Vero cells were similar in the wild-type and mutant strains. Both adhered to intestinal epithelial cells in culture and reacted with fluorescein isothiocyanate-labeled antiserum against E. coli O157:H7. The mutant appeared to be similar to the wild type in all respects except in its ability to ferment sorbitol. [14C]sorbitol uptake and sorbitol-6-phosphate dehydrogenase activities were notably increased in the mutant strain. Diarrhea developed in rabbits administered the wild-type strain and in those fed the sorbitol-positive mutant. There was greater bacterial attachment and mucosal damage in the cecum and large intestine than in the small intestine. Scanning electron microscopy revealed bacteria adhering as single cells and as aggregates closely associated with mucus. Mucosal lesions consisted of areas of tissue necrosis with sloughing of epithelial cells. By transmission electron microscopy, electron-dense necrotic epithelial cells were visible in areas where bacteria were present, and epithelial cell debris containing bacteria was observed between the villar luminal surfaces. Light microscopy of epithelial cells of intestinal sections of infected rabbits revealed noticeable vacuolation and spherical, pyknotic nuclei. These data indicate that the sorbitol-negative phenotype is not associated with the pathogenicity of E. coli O157:H7.

Animals↗

PubMed1991

Effect of temperature history on the growth of Listeria monocytogenes Scott A at refrigeration temperatures.

The effect of pre-inoculation temperature on the subsequent growth of Listeria monocytogenes Scott A at 5 degrees C was examined in microbiological medium, UHT milk, canned dog food, and raw ground beef (untreated and irradiation-sterilized). In microbiological medium, the duration of the lag phase was decreased when aerobic and anaerobic cultures were initially grown at less than or equal to 28 and less than or equal to 13 degrees C, respectively. Subsequent exponential growth rates and maximum population densities of the 5 degrees C cultures were not affected by temperature history. Differences in lag phase durations were also observed when L. monocytogenes initially cultured at 19 and 37 degrees C were grown at 5 degrees C in UHT milk and some of the canned dog food varieties. Growth of L. monocytogenes was not observed in either untreated or irradiation-sterilized raw ground beef. While temperature history can affect the growth kinetics of L. monocytogenes at 5 degrees C, it did not account for the lack of growth in raw meat, suggesting that there is an inhibitory condition or component in ground beef that is lost upon cooking.

Aerobiosis↗

PubMed1990

Cisplatin resistance and mechanism in a viral test system: SV40 isolates that resist inhibition by the antitumor drug have lost regulatory DNA.

Isolates of SV40 that have enhanced ability to survive inhibition by the antitumor drug cisplatin were selected by serial drug challenge in vivo. These mutant viruses have acquired specific deletions within the repeated regulatory motif (GGGCGG)6 or GC box. This DNA element was shown previously to be a strong target of drug attack by cisplatin and other anticancer drugs in vitro and is an important viral and cellular DNA control sequence. Thus, drug resistance in this viral test system is dependent on the loss of important target DNA sequences. The results also indicate that drug efficacy may be related to the ability of certain anticancer drugs to attack regulatory DNA sequences containing strings of guanosines.

Animals↗

PubMed1990

Programmed factor binding to simian virus 40 GC-box replication and transcription control sequences.

Nuclear footprinting revealed a temporal program involving factor binding to the repetitive GC-box DNA elements present in the simian virus 40 regulatory region. This program specified ordered and directional binding to these tandem regulatory sequences in vivo during the late phase of infection. The program was interrupted by the DNA replication inhibitor aphidicolin or by inactivation of the viral replication factor simian virus 40 T antigen, suggesting a link between viral DNA replication and new factor binding. Measurements of DNA accumulation in viruses lacking either the distal or proximal halves of the GC-box region suggested that the region has a dual role in replication control. Overall, the data point to important relationships between DNA replication and factor binding to the GC-box DNA, a multifunctional regulatory region.

Animals↗

PubMed1989

Comparison of lithium chloride-phenylethanol-moxalactam and modified Vogel Johnson agars for detection of Listeria spp. in retail-level meats, poultry, and seafood.

The effectiveness of Modified Vogel Johnson agar and lithium chloride-phenylethanol-moxalactam agar for detection of Listeria spp. in foods was compared by using the media to analyze retail-level meat, poultry, and seafood both by direct plating and in conjunction with a three-tube most-probable-number enrichment. The most-probable-number protocol detected Listeria species, including Listeria monocytogenes, in a substantial portion of the fresh meat and seafood samples. In most instances the Listeria levels were less than 2 CFU/g, which precluded detection by direct plating. Modified Vogel Johnson agar performed as well as did lithium chloride-phenylethanol-moxalactam agar and was considerably easier to use because of its ability to differentiate Listeria spp. from other microorganisms.

Animals↗

PubMed1989

Characterization of the Aeromonas hydrophila group isolated from retail foods of animal origin.

During a recent survey of retail fresh foods of animal origin (fish and seafood, raw milk, poultry, and red meats) for organisms of the Aeromonas hydrophila group, we isolated representative strains from the various foods. In this study, we sought to characterize these isolates for biochemical properties and virulence-associated factors and to compare the food isolates with clinical isolates. We identified all food and clinical isolates as A. hydrophila and found that all isolates were typical in their biochemical reactions. Examination of the isolates for various virulence-associated factors indicated that most food and clinical isolates were serum resistant, beta-hemolytic, cytotoxin positive (against Y1 adrenal cells), hemagglutinin positive, Congo red positive, elastase positive, and staphylolysin positive. Mouse 50% lethal doses were log10 8 to 9 CFU for most isolates. All isolates had biotypes identical to those of enterotoxin-positive strains. The public health significance of these organisms in foods is not known at present, although their widespread occurrence and ability to grow competitively in foods kept at 5 degrees C represents a potential hazard.

Aeromonas↗

PubMed1987

Effect of miconazole on growth and aflatoxin production by Aspergillus parasiticus.

At 5 microM, miconazole prevented the growth of Aspergillus parasiticus Speare in a number of media. Sensitivity to miconazole was increased approximately 10-fold in a medium containing glycerol. At sub-inhibitory concentrations, miconazole stimulated aflatoxin synthesis on media which normally support toxin formation. Miconazole inhibited respiration and altered mitochondrial ultrastructure, suggesting that miconazole inhibits growth and stimulates aflatoxin production by depressing mitochondrial activity.

Aflatoxins↗

PubMed1987

Inhibition of Clostridium botulinum 62A by fumarates and maleates and relationship of activity to some physicochemical constants.

A series of n-monoalkyl maleates and n-mono-, di-, and methyl n-alkyl fumarates were prepared, 18 esters of each, with R = CH3 to C18H37. Their activity against Clostridium botulinum was determined in culture medium. The n-monoalkyl maleates and fumarates possessed significant activity, particularly those esterified with higher C13 to C18 alcohols. Somewhat lower activity was exhibited by methyl n-alkyl fumarates, while symmetrical esters, di-n-alkyl fumarates, were almost inactive. An attempt was made to correlate the activity of n-monoalkyl maleates and fumarates with chain length, solubility in water, apparent dissociation constant (pKa'), and infrared and UV absorption frequencies. The active esters may have potential as preservatives in foods.

Chromatography, High Pressure Liquid↗

PubMed1987

Regulation of aflatoxin biosynthesis: assessment of the role of cellular energy status as a regulator of the induction of aflatoxin production.

Possible relationships among cellular energy status and the induction and initiation of aflatoxin synthesis were studied by using replacement culture techniques in conjunction with aflatoxin-supporting and-nonsupporting media. Transcription and translation processes associated with the induction of aflatoxin synthesis occurred 3 to 6 and 6 to 10 h, respectively, after mycelia were transferred to glucose-containing media. From adenylate energy charge determinations and in situ 31P nuclear magnetic resonance analyses, a relationship between overall energy status and the induction or initiation of aflatoxin synthesis could not be identified; however, electron microscopic evaluations indicated that aflatoxin synthesis occurred in association with a glucose-mediated inactivation of mitochondria. The results suggest that aflatoxin synthesis is not regulated by the overall energy status of the fungal cell but may be controlled by the energy status of specific subcellular compartments.

Aflatoxins↗

PubMed1987

Effect of NaCl, pH, temperature, and atmosphere on growth of Salmonella typhimurium in glucose-mineral salts medium.

The interactions of pH (5.0, 6.0, and 7.0), temperature (19, 28, and 37 degrees C), and atmosphere (aerobic versus anaerobic) with NaCl (0, 1, 2, 3, 4, and 5%) on the growth of Salmonella typhimurium ATCC 14028 in defined glucose-mineral salts culture medium were evaluated. Response surface methodology was used to develop equations describing the response of S. typhimurium to environmental changes. The response to an increasing concentration of NaCl at any temperature tested was nonlinear. The maximum growth was predicted to occur at an NaCl concentration of 0.5%, a temperature of 19 degrees C, and an initial pH of 7.0 under aerobic growth conditions. The relative amounts of aerobic growth at 19 degrees C, pH 7.0, and NaCl concentrations of 0, 0.5, 1, 2, 3, 4, and 5% were predicted to be 99.2, 100.0, 98.8, 90.2, 73.5, 48.6, and 15.6%, respectively. Anaerobic growth conditions repressed the amount of growth relative to that under aerobic conditions, and the effects of NaCl and pH were additive at low salt concentrations; however, at higher salt levels anaerobiosis provided protection against the effects of NaCl.

Aerobiosis↗

PubMed1987

Factor interactions at simian virus 40 GC-box promoter elements in intact nuclei.

Primer extension footprinting was used to probe late simian virus 40 regulatory elements in intact infected cell nuclei. Specific protection was observed over the viral "GC-box" transcription elements. The participation of the bound templates in gene activation is addressed by quantitation that shows that their abundance greatly exceeds that of transcription complexes but is comparable to that of open chromatin.

Animals↗

PubMed1987

Determination of glucose level needed to induce aflatoxin production in Aspergillus parasiticus.

Previous studies have established that aflatoxin production is induced by high levels of glucose (or other compatible carbohydrates). However, the minimal amount of glucose needed to achieve this effect had not been determined. Aspergillus parasiticus was induced to produce aflatoxin when incubated for 18 h in the presence of greater than or equal to 0.1 M glucose before addition of cycloheximide. Toxin formation was not induced at any glucose concentration in the mycelia incubated for 8 h. These results suggest that aflatoxin synthesis would not be expected in substrate containing less than 0.1 M of a carbohydrate source.

Aflatoxins↗

PubMed1986

Enterotoxin A production in Staphylococcus aureus: inhibition by glucose.

In this study, we investigated the relationship between carbohydrate metabolism and repression of staphylococcus enterotoxin A (SEA) in Staphylococcus aureus 196E and a pleiotrophic mutant derived from strain 196E. The mutant, designated at strain 196E-MA, lacked a functional phosphoenolpyruvate phosphotransferase system (PTS). The mutant produced acid, under aerobic conditions, from only glucose and glycerol. The parent strain contained an active PTS, and aerobically produced acid from a large number of carbohydrates. Prior growth in glucose led to repression of SEA synthesis in the parent strain; addition to the casamino acids enterotoxin production medium (CAS) led to more severe repression of toxin synthesis. The repression was not related to pH decreases produced by glucose metabolism. When S. aureus 196E was grown in the absence of glucose, there was inhibition of toxin production as glucose level was increased in CAS. The inhibition was related to pH decrease and was unlike the repression observed with glucose-grown strain 196E. The inhibition of SEA synthesis in mutant strain 196E-MA was approximately the same in cells grown with or without glucose and was pH related. Repression of SEA synthesis similar to that seen with glucose-grown S. aureus 196E could not be demonstrated in the mutant. In addition, glucose-grown S. aureus 196E neither synthesized beta-galactosidase nor showed respiratory activity with certain tricarboxylic acid (TCA) cycle compounds. Glucose-grown strain 196E-MA, however, did not show suppressed respiration of TCA cycle compounds; beta-galactosidase was not synthesized because the mutant lacked a functional PTS.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate Metabolism↗

PubMed1985

Effect of 2-deoxyglucose, alpha-methylglucoside, and glucosamine on aflatoxin production by Aspergillus parasiticus.

The effects of 2-deoxyglucose (2-DOG), alpha-methylglucoside (alpha-MG), and glucosamine (GA) on aflatoxin production by Aspergillus parasiticus were studied using conidia-initiated and replacement cultures. In conidia-initiated, 2-DOG, alpha-MG, and GA supported varying amounts of growth when employed as sole carbon sources. In both conidia-initiated and replacement cultures, 2-DOG, but not alpha-MG nor GA, as sole carbon sources support toxin formation. None of the compounds inhibited aflatoxin production when used in combination with glucose. It appears that neither 2-DOG, alpha-MG, nor GA can be considered nonmetabolizable analogs of glucose in A. parasiticus.

Aflatoxins↗

PubMed1985

Starch-Ampicillin Agar for the Quantitative Detection of Aeromonas hydrophila.

Interest in Aeromonas hydrophila as a food-borne and human pathogen is increasing. Isolation media from the clinical laboratory were evaluated for food use and either did not give quantitative recovery of A. hydrophila or did not permit ready differentiation of A. hydrophila from the background microflora. A new medium was developed which permitted quantitative recovery of A. hydrophila from foods. The medium consisted of phenol red agar base (Difco Laboratories), soluble starch (10 g/liter), and ampicillin (10 mg/liter). All foods surveyed contained A. hydrophila. Foods sampled included red meats, chicken, raw milk, and seafood (fish, shrimp, scallops, crab, and oysters). The count of A. hydrophila at the time of purchase ranged from 1 x 10/g (lower limit of detection) to 5 x 10/g. In most instances, the count of A. hydrophila increased during 1 week of storage at 5 degrees C. The starch-ampicillin agar developed permitted rapid quantitative recovery of A. hydrophila from foods in the presence of very large numbers of competing microflora.

Journal Article↗

PubMed1984

Caffeine inhibition of aflatoxin synthesis: probable site of action.

Aflatoxin production by pregrown cultures of Aspergillus parasiticus was completely inhibited by incorporation of 2 mg of caffeine per ml into the medium. This was accompanied by a decrease in glucose utilization and an inhibition of oxygen uptake and carbon dioxide evolution. Enzyme analyses indicated no significant differences in specific activities on glucose-6-phosphate dehydrogenase, mannitol dehydrogenase, phosphofructokinase, fructose 1,6-diphosphatase, pyruvate kinase, or malate dehydrogenase. Glucose uptake kinetics indicated a linear dose-related inhibition of glucose uptake. It appears likely that caffeine inhibits aflatoxin synthesis by restricting the uptake of carbohydrates which are ultimately used by the mold to synthesize this family of mycotoxins.

Aflatoxins↗

PubMed1984

Regulation of aflatoxin biosynthesis: effect of glucose on activities of various glycolytic enzymes.

Catabolism of carbohydrates has been implicated in the regulation of aflatoxin synthesis. To characterize this effect further, the activities of various enzymes associated with glucose catabolism were determined in Aspergillus parasiticus organisms that were initially cultured in peptone-mineral salts medium and then transferred to glucose-mineral salts and peptone-mineral salts media. After an initial increase in activity, the levels of glucose 6-phosphate dehydrogenase, mannitol dehydrogenase, and malate dehydrogenase were lowered in the presence of glucose. Phosphofructokinase activity was greater in the peptone-grown mycelium, but fructose diphosphatase was largely unaffected by carbon source. Likewise, carbon source had relatively little effect on the activities of pyruvate kinase, malic enzyme, isocitrate-NADP dehydrogenase, and isocitrate-NAD dehydrogenase. The results suggest that glucose may, in part, regulate aflatoxin synthesis via a carbon catabolite repression of NADPH-generating and tricarboxylic acid cycle enzymes.

Aflatoxins↗