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Biomedical subjects

J R Chipley

Publications and source records attributed to J R Chipley.

At least 19 recordsLinked to original sources
PubMed1990

Purification of Staphylococcus aureus beta-lactamases by using sequential cation-exchange and affinity chromatography.

Boronic acids are active-site inhibitors of serine beta-lactamases, and a phenylboronic acid-agarose affinity column has been used to purify beta-lactamase from crude cell extracts of several bacterial species. We applied phenylboronic acid-agarose chromatography to the purification of Staphylococcus aureus beta-lactamase. Two factors interfered with the success of the previously described single-step chromatographic protocol. First, staphylococcal beta-lactamase exhibited non-active-site-mediated adsorption to the agarose used as a support for the meta-aminophenylborate ligand, preventing the recovery of beta-lactamase from the column. Second, the staphylococcal beta-lactamases exhibited low affinity for meta-aminophenylborate with inhibition constants (Kis) ranging from 8.0 x 10(-3) to 20.0 x 10(-3) M. These problems were resolved by modifying the buffers utilized during chromatography and increasing the dimensions of the affinity column, and a two-stage procedure consisting of cation-exchange chromatography followed by affinity chromatography was used to purify each of the four variants of staphylococcal beta-lactamase. The mean specific activities of the purified type A, B, C, and D beta-lactamases were 44.6, 12.2, 10.6, and 30.8 mumol of nitrocefin hydrolyzed per min/mg of protein, respectively. Dimer formation, presumably from intramolecular cysteine-cysteine cross-linking, was observed with the type D beta-lactamase but not with the type A, B, or C enzyme.

Boronic Acids↗
PubMed1984

Role of biotin in the production of lysine by Brevibacterium lactofermentum.

To investigate the role of biotin in lysine production, Brevibacterium lactofermentum ATCC 21086 was grown in an acid-hydrolysed whey permeate medium with and without added biotin. Added biotin stimulated lysine production and growth of B. lactofermentum. Five micrograms of biotin/100 ml was the optimum level of addition. Biotin increased the uptake of 14C-glucose and affected fatty acid composition of cell wall lipids. Cell walls of test organisms contained less 16:0 and more 18:2 fatty acids than did those from control cells. However, biotin did not substantially affect the phospholipid content of cell walls and whole cells, and the intracellular free lysine level. It was concluded that the promotive effect of biotin in lysine production might be due to the stimulatory effect of biotin on the growth of B. lactofermentum. Alteration of the cell surface caused by biotin did not appear to affect the release or accumulation of lysine.

Biotin↗
PubMed1983

Microbial production of lysine and threonine from whey permeate.

Extracellular accumulation of lysine and threonine was investigated in modified whey permeate by using Brevibacterium lactofermentum ATCC 21086 and Escherichia coli ATCC 21151. Whey permeate was prepared from whey by membrane ultrafiltration, and lactose was hydrolyzed by treating permeate with HCl or beta-galactosidase. The highest amount of lysine (3.3 g/liter) was produced from a mixture of acid-hydrolyzed whey permeate and yeast extract (0.2%). The highest amount of threonine (3.6 g/liter) was produced from a mixture of whey permeate, (NH(4))(2)SO(4) (1.4%), yeast extract (0.1%), and Na(2)CO(3) (0.3%).

Journal Article↗
PubMed1981

Sensory and microbiological qualities of beef loaf in four commissary food service treatments.

Beef loaf prepared according to four treatment options occurring in commissary food service was evaluated for microbiological and sensory quality. General acceptability was scored significantly (p of less than 0.01) higher for beef loaf portions held hot for 1 hour than for those held 2 hours, or those chilled 24 hours and heated in a convection oven. No significant (p of less than 0.01) differences for any quality characteristic scored were found between a loaf held hot for 1 hour and that chilled for 24 hours, heated, and then held hot for 1 hour. Appearance of beef loaves held hot for 1 hour was significantly (p of less than 0.05) better than that of loaves heated in the convection oven but was not significantly better than that of loaves given any of the other treatments. Microbiological analyses revealed the presence of relatively small numbers of microorganisms and no significant differences among treatments. The lowest total plate count was found in loaves baked, chilled for 24 hours, heated in a gas deck oven, and then held in insulated plastic trays for 1 hour. The highest counts were for loaves which were baked, placed immediately in insulated plastic trays, and held for 1 hour. Increases in total microbial counts and clostridial counts were observed as meat was held chilled for 24 hours. But no increases were observed when meat was held for 2 hours in insulated plastic trays. Although these microbiological findings were not significant, data may have important implications for an actual operation, where conditions are not so carefully controlled.

Animals↗
PubMed1980

Comparison of effects of sublethal microwave radiation and conventional heating on the metabolic activity of Staphylococcus aureus.

This study was conducted in an attempt to characterize some of the effects of sublethal microwave radiation on cells of Staphylococcus aureus. Cultures were exposed to microwave radiation for 10, 20, 30, and 40 s. The effects of a conventional heat treatment were also compared by placing flasks containing cultures in a boiling water bath for the amount of time required to reach temperatures equivalent to those found in cultures exposed to microwave radiation. Control, microwave-treated, and conventionally heat-treated cultures were centrifuged, pellets were resuspended in distilled water, and the resulting suspensions were passed through a French pressure cell. Cell lysates and walls were then isolated and assayed for enzymatic activity. Thermonuclease production was also determined at various levels of exposure of cells to microwave radiation. Activities of malate and alpha-ketoglutarate dehydrogenases, cytochrome oxidase, and cytoplasmic adenosine triphosphatase were higher in microwave-treated cells than in control cells. Membrane adenosine triphosphatase, alkaline phosphatase, and lactate dehydrogenase activities were unaffected when cells were exposed to microwave radiation. The activity of glucose-6-phosphate dehydrogenase was decreased by exposure of cells to microwave radiation. In conventionally heated cells, activities of glucose-6-phosphate and malate dehydrogenases and cytoplasmic adenosine triphosphatase increased activities of alpha-ketoglutarate and lactate dehydrogenases decreased, and alkaline phosphatase activity remained unaffected. Increased levels of thermonuclease activity were observed when cells were exposed to microwave radiation for 10 or 20 s. Data indicate that microwave radiation affects S. aureus in a manner which cannot be explained solely by thermal effects.

Adenosine Triphosphatases↗
PubMed1980

Inhibition of Aspergillus growth and aflatoxin release by derivatives of benzoic acid.

A study was conducted to determine the effects of o-nitrobenzoate, p-aminobenzoate, benzocaine (ethyl aminobenzoate), ethyl benzoate, methyl benzoate, salicylic acid (o-hydroxybenzoate), trans-cinnamic acid (beta-phenylacrylic acid), trans-cinnamaldehyde (3-phenylpropenal), ferulic acid (p-hydroxy-3-methoxycinnamic acid), aspirin (o-acetoxy benzoic acid), and anthranilic acid (o-aminobenzoic acid) upon growth and aflatoxin release in Aspergillus flavus NRRL 3145 and A. parasiticus NRRL 3240. A chemically defined medium was supplemented with various concentrations of these compounds and inoculated with spores, and the developing cultures were incubated for 4, 6, and 8 days at 27 degree C in a mechanical shaker. At the beginning of day 8 of incubation, aflatoxins were extracted from cell-free filtrates, separated by thin-layer chromatography, and quantitated by ultraviolet spectrophotometry. The structure of these aromatic compounds appeared to be critically related to their effects on mycelial growth and aflatoxin release. At concentrations of 2.5 and 5.0 mg per 25 ml of medium, methyl benzoate and ethyl benzoate were the most effective in reducing both mycelial growth and aflatoxin release by A. flavus and A. parasiticus. Inhibition of mycelial growth and aflatoxin release by various concentrations of the above-named aromatic compounds may indicate the possibility of their use as fungicides.

Acrolein↗
PubMed1980

Isolation and partial characterization of a glucose-binding protein from Bacillus megaterium.

Attempts were made to use the osmotic shock procedure established for Gram-negative micro-organisms on a Gram-positive micro-organisms, Bacillus megaterium ATCC 19213. The procedure did not appear to shock the cells osmotically. The procedure, in fact, appeared to enhance binding of both glucose and fructose by the organism. The binding of glucose was greater by cells, whether grown on fructose or glucose, than it was for fructose. A procedure was developed for removing and isolating a protein capable of binding glucose. This involved solubilizing the cytoplasmic membrane of the organism with the nonionic detergent Triton X-100, and then isolating it by gel filtration on a Bio-Gel A 1.5 column with 0.05 M pyrophosphate containing 1% (v/v) Triton X-100 as the eluting buffer. This is the first isolation of such a protein in a Gram-positive organism. Isolated protein was demonstrated to have a specificity only for glucose. The protein did not bind to either the monosaccharides fructose and galactose, or the disaccharide sucrose. This was in comparison to whole cells which demonstrated ability to take up fructose. The protein was estimated to have a molecular weight between 24,000 and 30,000 daltons by gel filtration techniques.

Bacillus megaterium↗
PubMed1980

Heat activation of Bacillus spores by the use of microwave irradiation.

A study was conducted in which microwave irradiation and conventional waterbath treatment were compared as to their efficiency for heat-activating Bacillus spores. Spore suspensions were prepared from B. brevis, B. cereus, B. licheniformis, a lysogenic strain of B. megaterium (NRRL-B-3695), two strains of B. stearothermophilus, and B.Subtilis. Suspensions were either irradiated for 30 sec in a microwave oven, or conventionally heat-treated in the waterbath for 60 min at 60 degrees C, the serially diluted and plated onto nutrient agar. Colonies of each species from each treatment were isolated, and cultures were inoculated into several biochemical media. Spore suspensions heat-activated by microwave irradiation resulted in plate counts that were from 3% to 24% greater than from suspension heat-activated by conventional mean (60 degrees C for 60 min). There were no observed alterations in biochemical activities in any of the representative colonies from either of the two treatments. No induction of bacteriophage from lysogenic B. megaterium NRRL-B-3695 was observed in colonies from either of the two treatments. Microwave irradiation appears to be more efficient, less time-consuming, and at least as effective as heat activation by conventional waterbath treatment for Bacillus spores.

Bacillus↗
PubMed1979

Chemical and electron microscopic studies of factors associated with the release of penicillinase from Staphylococcus aureus.

The effect of various factors such as sodium chloride, sodium citrate, pH, linase by Staphylococcus aureus ATCC 14458 was investigated. Penicillinase was measured at selected time intervals from supernates of cultures grown in Antibiotic Medium 3 broth containing various concentrations of salts or buffers or from supernates of cultures treated with lysostaphin and subsequently disrupted by French press treatment. Incubation of cells with media containing either sodium chloride (5, 10, and 15%), sodium citrate (5 and 10%), or organic buffers (Tris-HCl, 2.5, 5.0, and 7.5%; BES, 10 and 20%) resulted in a significant stimulation of the release of penicillinase when compared to control cells. It was also observed that pH 7.0-7.5 was optimal for penicillinase activity and release. From studies of enzymatic and mechanical disruption of cells, it was observed that an increase in ionic strength of the suspending medium to certain optimal levels appeared to stimulate the conversion of penicillinase to an extracellular form. Electron microscopic studies revealed that a large number of mesosomal vesicles seemed to be present in cells incubated for 4 hours in media containing various concentrations of sodium chloride. It is proposed that either appearance of vesicles or convolution of cell membrane, which may be caused by further synthesis of new membrane, is involved in stimulation of the synthesis and release of membrane-bound penicillinase.

Alkanesulfonates↗
PubMed1979

Conditions for induction of bacteriophage from lysogenic Bacillus megaterium with aflatoxin B1.

The present study was conducted to determine whether or not aflatoxin B1 was an effective inducing agent for lysogenic bacteria and to characterize some of the parameters involved in induction. A lysogenic strain of Bacillus megaterium (NRRL-B-3695) and an indicator strain of this species (NRRL-B-3694) were used. Cultures of the lysogenic strain were incubated for various periods of time in the presence of aflatoxin B1. Plaque-forming units as well as colony-forming units were then determined. Results of the present study indicated that bacteriophage lysogenizing B. megaterium could be induced with aflatoxin B1. The optimum concentration for induction was 25 micrograms of toxin per ml of early-log-phase culture. Evidence suggested that: (i) higher concentrations of aflatoxin B1 formed hydrophobic complexes which would not efficiently induce B. megaterium; (ii) the toxic effect of aflatoxin B1 severely limited the number of cells which could be induced prior to killing action of the toxin; and (iii) concentrations less than 25 micrograms of aflatoxin B1 per ml were not efficient inducers of bacteriophage production nor did they demonstrate the toxic effect observed at higher concentrations.

Aflatoxins↗
PubMed1977

Partial characterization of the mode of action of benzoic acid on aflatoxin biosynthesis.

Aflatoxin production by a toxigenic strain of Aspergillus flavus was greatly reduced by benzoic acid and sodium benzoate in synthetic media. The reduction was accompanied by the appearance of a yellow pigment. Spectral analyses partially characterized this pigment as closely related to an acetyl derivative of a versiconal-type compound. A cell-free extract prepared from A. flavus grown in synthetic media was active in converting this yellow compound into aflatoxin B1 in the presence of reduced nicotinamide adenine dinucleotide phosphate at 25 degrees C (pH 7.4). In the presence of benzoic acid and its salt or autoclaved cell-free extract, conversion of yellow compound to aflatoxin B1 was prevented. These results suggest that the yellow compound is an intermediate in the secondary metabolic cycle involved in aflatoxin B1 production. Benzoic acid, sodium benzoate, or autoclaving the cell-free extract appear to have respectively blocked or denatured an enzymatic step late in the biosynthetic pathway of aflatoxin B1.

Aflatoxins↗
PubMed1976

Production of ochratoxin A by Aspergillus ochraceus NRRL-3174 before and after exposures to 60Co irradiation.

Spores from the toxigenic organism Aspergillus ochraceus NRRL-3174 were exposed to specific levels of gamma irradiation and then allowed to germinate on selected media. Increases in ochratoxin A production by irradiated, compared to non-irradiated, spores were observed after inoculation of spores onto a cracked red wheat or into a synthetic liquid medium. Variations in daily ochratoxin production were also observed for control and irradiated spore-derived cultures developing on both media, with maximum toxin production varying from 7 to 11 days of incubation. The most notable increases in ochratoxin A production occurred from cultures developing from spores having been irradiated with 10, 25, or 50 krad. Exposures to 400 or 600 krad resulted in complete inhibition of spore germination and, consequently, no ochratoxin production. Of the two substrates used, wheat and synthetic, the quantities of ochratoxin A produced were significantly lower in the synthetic media than on the natural substrate. Higher and more rapid toxin production occurred from spores having been irradiated with 10, 25, 50, and 100 krad than occurred from the non-irradiated control spores when grown on synthetic media. Cultures derived from spores having been exposed to 10, 25, 50, and 100 krad produced significantly higher levels of ochratoxin A after 8 days of incubation on natural substrate than did the controls. Analysis of variance revealed that substrate, length of incubation, as well as irradiation levels all affected the time required to produce maximum levels of ochratoxin A.

Aspergillus↗
PubMed1976

Effects of various acids and salts on growth and aflatoxin production by Aspergillus flavus NRRL 3145.

The effects of sodium chloride, sodium acetate, benzoic acid, sodium benzoate, malonic acid, and sodium malonate on growth and aflatoxin production by Aspergillus flavus were investigated in synthetic media. Sodium chloride at concentrations equivalent to or greater than 12 g/100 ml inhibited growth and aflatoxin production, while at 8 g or less/100 ml, growth and aflatoxin production were stimulated. At 2 g or less/100 ml, sodium acetate also stimulated growth and aflatoxin production, but reduction occurred with 4 g or more/100 ml. Malonic acid at 10, 20, 40, and 50 mM reduced growth and aflatoxin production (over 50%) while sodium malonate at similar concentrations but different pH values had the opposite effect. Benzoic acid (pH 3.9) and sodium benzoate (pH 5.0) at 0.4 g/100 ml completely inhibited growth and aflatoxin production. Examination of the effect of initial pH indicated that the extent of inhibitory action of malonic acid and sodium acetate was a function of initial pH. The inhibitory action of benzoic acid and sodium benzoate appeared to be a function of undissociated benzoic acid molecules. Aflatoxin reduction was usually accompanied by an unidentified orange pigment, while aflatoxin stimulation was accompanied by unidentified blue and green fluorescent spots but with lower Rf values that aflatoxins B1, G1, B2, and G2 standards.

Acetates↗
PubMed1975

Cholesterol metabolism by Mycobacterium.

Cholesterol metabolism by Mycobacterium species ATCC Number 19652 was studied in defined media. Whole cells were found to take up 91% of the total cholesterol when incubated five days at 34 degrees C in media of pH 6.8-7.4. Uptake of cholesterol by whole cells could be significantly inhibited by 2,4-dinitrophenol and dicyclohexylcarbodiimide. Growth media supernates as well as isolated microbial cell walls were found to contain cholesterol hydrolysing activity. This activity was extractable by Triton X-100 and appeared to have a molecular weight of approximately 100-200,000.

Cell Wall↗