Browsing by Author "Krieg, Noel R."

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    The 16S rRNA characterization of a novel "microaerophilic" Pseudomonas sp. from the oligotrophic deep subsurface environment
    Lampe, Robert Carl III (Virginia Tech, 1996-11-07)
    A gram negative microaerophilic bacterium, designated Pseudomonas sp. strain MR 100, was isolated from a depth of 463 meters at the Savannah River DOE site and identified using 16S rDNA sequencing and DNA-DNA reassociation. Micro aerophiles from the Middendorf formation were isolated by use of a semi-solid agar assay, and constituted 10% of the plateable microorganisms. Genetic identification involved the isolation of genomic DNA and amplification of the gene encoding 16S rRNA by PCR, using universal primers. The amplified DNA was sequenced and compared to 16S rRNA sequences in Genbank. High sequence similarity (98.5%) was observed with the Pseudomonas mendocina type strain, indicating a similarity to the (Group I) pseudomonads. DNA-DNA reassociation was performed between Pseudomonas sp. strain MR 100 and 11 representative p seudomonads using the S 1 nuclease method. Strain MR 100 was found to be 20% homologous to the Pseudomonas mendocina type strain, 10% homologous to Pseudomonas alcaligenes, and 5% homologous to Pseudomonas aeruginosa. Data from biochemical tests confirm the hypothesis that strain MR 100 is a novel species of Pseudomonas
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    Changes in respiration rates and biomass attributes of epilithon due to extended exposure to zinc
    Colwell, Frederic S. (Virginia Polytechnic Institute and State University, 1986)
    The purpose of this research was to determine the influence of extended dosing of zinc on the carbon cycling and biomass characteristics of freshwater epilithon. Experiments were conducted in artificial streams continuously dosed with 0.00, 0.05, or 1.00 mg Zn liter⁻¹ for 20 to 30 days during summer and fall, 1984 and 1985. Repeated measurement of epilithon structure and function included estimates of ¹⁴C-glucose respiration, ¹⁴C-glutamate respiration, O₂ and CO₂ flux rates, ash-free dry weight (AFDW), protein, carbohydrate, and algal pigment concentrations, and total and zinc-tolerant colony forming units. An increase in epilithic glucose respiration per unit biomass consistently occurred 5 to 10 days after dosing with 1.0 mg Zn liter⁻¹ was started. At the same time significantly lower epilithon biomass occurred in the high dosed streams relative to controls in 3 out of 4 studies. Although algal pigment concentrations were lowest in the high dose streams at the midpoint of the studies, the chlorophyll a-to-pheophytin a ratio remained high, indicating that the minimal algal population was not senescing in situ. After 30 days, the epilithon dosed with 1.0 mg Zn liter⁻¹ had higher AFDW, protein, and carbohydrate concentrations than the other treatments. By 20 days, the high zinc treatment showed evidence of more total and zinc-tolerant colony forming units and lower rates of O₂ and CO₂ flux than epilithon from control streams. The high rates of glucose respiration were characteristic of epilithic communities stressed by 1.0 mg Zn liter⁻¹, and this response was not apparently due to in situ senescence of zinc-sensitive cells; the results suggested that epilithic biomass was washed out of the systems, not being degraded in situ. The development of unique epilithon communities that are acclimated to prolonged zinc exposure is evident in the eventual recolonization of the artificial surfaces, glucose respiration rates that are comparable to controls, and presence of zinc-tolerant heterotrophs.
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    Characterization and localization of adenylate cyclase during development of Dictyostelium discoideum
    Merkle, Roberta Gayle Kurpit (Virginia Polytechnic Institute and State University, 1982)
    Cyclic AMP functions as the chemotactic signal during aggregation of single-celled amoebae of the cellular slime mold Dictyostelium discoideum. Evidence suggests that cyclic AMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. Biochemical characterization of adenylate cyclase, the cyclic AMP synthetic enzyme, was accomplished using a sensitive radioimmunoassay. The enzyme was found to be pellet-bound. The non-ionic detergents, Triton X-100 and Lubrol PX, were not effective for solubilizing this activity. Magnesium or manganese could serve as the required divalent cation, with the Mn-supported activity over 4-fold greater than the Mg-supported activity. Typical mammalian adenylate cyclase modulators such as guanyl nucleotides, fluoride, and cholera toxin did not activate the Dictyostelium enzyme. Calcium, in conjunction with its protein receptor calmodulin, did not appear to regulate the enzyme. An endogenous extracellular, heat-stable substance was found to inhibit Dictyostelium adenylate cyclase. By use of ultramicrotechniques adenylate cyclase activity was localized in the pre-spore cells of the culminating individual with no activity detected in the pre-stalk region. Lack of detectable activity in the pre-stalk cells may be due to a masking by the endogenous inhibitor. An increasing gradient of activity was found in the pre-spore mass with activity increasing from the uppermost area to the base. No striking localization was seen prior to the pre-culmination stage of development. Two peaks in cyclic AMP levels, as measured in individuals were found during development. One coincided with aggregation, the other occurred at the culmination stage. A gradient of cyclic AMP within the culminating individual paralleled the gradient of adenylate cyclase activity. The tip of the individual had greater levels of cyclic AMP than the middle pre-spore region, and the upper stalks had higher levels than the lower stalks. These data indicate an enzymatic potential for establishing a gradient of cyclic AMP. At the culmination stage of development this molecule could act to direct the chemotactic movements of the pre-stalk cells as well as provide positional information for the terminal differentiation of the pre-spore cells into mature spores.
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    Characterization of DNA-repair potential in deep subsurface bacteria challenged by UV light, hydrogen peroxide, and gamma radiation
    Arrage, Andrew Anthony (Virginia Tech, 1991-08-07)
    Subsurface bacterial isolates obtained through the DOE Subsurface Science Program were tested for resistance to UV light, gamma radiation and H₂0₂. Some deep subsurface bacteria were resistant to UV light, demonstrating ≥1.0% survival at fluences which resulted in a 0.0001% survival level of E. coli B. The percentage of UV resistant aerobic subsurface bacteria and surface soil bacteria were similar; 30.8% and 25.8% respectively. All of the microaerophilic subsurface isolates were UV sensitive as defined in this work; however, subsurface isolates demonstrated UV resistance levels similar to reference bacterial strains of the same Gram reaction. These results were not in agreement with the hypothesis that the resistance of an organism to UV is correlated with the amount of solar radiation in its natural habitat. Evidence for photoreactivation and the presence of an SOS-like mechanism was also detected in subsurface bacteria. The presence of UV resistance and photoreactivation in subsurface bacteria that have been shielded from solar radiation for millions of years may point to a limited rate of evolution in the deep subsurface environment. In subsurface bacteria, there was a relatedness between UV resistance and resistance to gamma radiation and H₂0₂ UV-resistant aerobic subsurface isolates were also gamma and H₂0₂- resistant compared to the microaerophilic isolates tested. Due to the similarities of bacterial responses to UV, H₂0₂ , and gamma radiation, either UV or H₂0₂ may be utilized to model the effects of ionizing radiation on bacterial cultures used for the bioremediation of organic and radioactive waste-containing environments.
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    Characterization of plasmids among the three species of Gluconobacter
    Brookman, Lori L. (Virginia Tech, 1995-07-10)
    The genus Gluconobacter consists of acetic acid bacteria which have the ability to generate acidic products from their substrates, particularly acetic acid from ethanol. For this reason, the gluconobacters live in acidic, sugary environments such as flowers, honey bees, fruits, cider, vinegar, wine and beer. The gluconobacters carry out a strictly respiratory type of metabolism using only oxygen as a terminal electron acceptor. They do not completely oxidize a substrate to carbon dioxide. Instead, they partially oxidize the substrate using membrane-bound dehydrogenases and excrete the product into the surrounding growth medium. It is these limited oxidations that make the gtuconobacters industrially useful. Although much is known about the physiology of the limited oxidations in the gluconobacters, little is known of their genetics, particularly, their plasmids. The overall purpose of this dissertation was to determine if Gluconobacter plasmids correlate with oxidative capability and/or antibiotic resistance. To achieve this goal, I first needed a way to screen strains of Gluconobacter for their ability to oxidize many different substrates. 'developed an assay that used an unusual artificial electron acceptor, tetranitroblue tetrazolium (TNBT) and then tested the ability of six strains to oxidize 13 chemical compounds. Although most strains were able to oxidize the 13 compounds tested, they accomplished this with varying extents of oxidation. These differences were noted even with strains representing the same species.
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    Characterization of the carbohydrate receptors of the Clostridium difficile enterotoxin
    Tucker, Kenneth D. (Virginia Tech, 1990)
    Clostridium difficile causes pseudomembranous colitis in humans and a similar ileocecitis in hamsters. This organism can colonize the intestines after antibiotic therapy disrupts the normal intestinal microflora. Once established in the intestines, the organism causes disease by producing two toxins, designated toxin A and toxin B. Only toxin A is active on intestinal epithelium, thus toxin A is the cause of the initial tissue damage in the intestines. In order for a toxin to affect a cell, it must first bind to the cell. Toxin A has been shown to bind to Galα1- 3Galβ 1-4GIcNAc on the intestinal epithelium of hamsters. I provide evidence that toxin A can use this trisaccharide as a functional receptor on cell lines, and that the expression of the carbohydrate receptor increases the sensitivity of the cells to toxin A. Furthermore, the intestinal epithelium of infant hamsters bound less toxin A at 37C than did the adult tissue, and infants are less sensitive to the disease caused by C. difficile than are adults. This provides further evidence that the activity of toxin A is increased by the binding of the toxin to Galα1-3Galβ1- 4GlcNAc. Even though Galα1-3Galβ 1-4GlcNAc was a receptor for toxin A on animal cells, it probably is not a receptor for toxin A in humans, because people do not normally express this carbohydrate. Instead, I found that toxin A bound to the carbohydrate antigens designated I, X, and Y, which are present on the intestinal epithelium of humans. These carbohydrates could be receptors for toxin A. The possible significance of these receptors is discussed.
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    CoA-transferase and 3-hydroxybutyryl-CoA dehydrogenase: acetoacetyl-CoA-reacting enzymes from Clostridium beijerinckii NRRL B593
    Colby, Gary D. (Virginia Tech, 1993-07-05)
    In acetone/butanol-producing clostridia, the metabolic intermediate acetoacetyl-CoA can be directed toward butyrate or butanol formation by the reaction catalyzed by 3-hydroxybutyryl-CoA dehydrogenase, or toward acetone formation by the reaction catalyzed by acetoacetate:acetate/butyrate CoA-transferase. 3-Hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35 or 1.1.1.157) has been purified 45-fold to apparent homogeneity from the solvent-producing anaerobe Clostridium beijerinckii strain NRRL B593. The identities of 34 of the 35 N-terminal amino acid residues have been determined. The enzyme exhibited a native Mr of 213,000 and a subunit Mr of 30,800. It is specific for the (S)-enantiomer of 3-hydroxybutyryl-CoA. Michaelis constants for NADH and acetoacetyl-CoA were 8.6 and 14 µM, respectively. The maximum velocity of the enzyme was 540 µmol/(min mg) for the reduction of acetoacetyl-CoA with NADH. The enzyme could use either NAD(H) or NADP(H) as cosubstrate; however, NAD(H) appeared to be the physiological substrate. In the presence of 9.5 µM NADH, the enzyme was inhibited by acetoacetyl-CoA at concentrations as low as 20 µM, but the inhibition was relieved as the concentration of NADH was increased, suggesting a possible mechanism for modulating the energy efficiency during growth. Acetoacetate:acetate/butyrate CoA-transferase (EC 2.8.3.9) has been purified 308-fold to apparent homogeneity from the same organism. The enzyme exhibited a native Mr of 89,100. The subunits of the enzyme were separated by preparative SDS-PAGE, and exhibited M, values of 28,400 and 25,200. The identities of the 34 N-terminal amino acids of the large subunit and 38 of the 39 N-terminal amino acids of the small subunit were determined. The N-terminal region of the two subunits showed significant similarity with several other CoA transferase enzymes. Michaelis constants for butyrate and acetoacetyl-CoA were 11.7 mM and 107 µM, respectively, while those for acetate and acetoacetyl-CoA were 424 mM and 118 µM, respectively. The value of kcat/Km was approximately 100 times higher with butyrate than with acetate. Implications of the properties of these two enzymes for the acetone-butanol fermentation are discussed, and a model for the induction of the enzymes responsible for solvent production is suggested.
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    Determination of decomposition rates in selected mid-Atlantic fish species stored under iced and super-chilling temperatures
    Barua, Mala A. (Virginia Tech, 1991-08-18)
    Three different species of fish (sea trout, Spanish mackerel and catfish) were studied. Samples undergoing normal spoilage were compared with samples which had undergone a sanitizing treatment with alcohol. Differential temperature storage studies were conducted at 290 °F (-1.7 °C) and 32 °F (0 °C). Fish quality was assessed by means of microbiological, chemical and sensory analyses. Quality assessment via measurement of proteolytic and lipolytic enzymes was attempted, but these enzyme activities were not detected in any of the samples. It was not possible to differentiate between the contributions of microbial and autolytic spoilage. Alcohol treated samples (reduced numbers of microorganisms) had shelf-lives extended by 6-10 days over untreated samples. The shelf-life of samples stored at 290 °F was extended by 6-10 days over the shelf-life of samples stored at 32 °F. Treated samples stored at 290 °F received highest sensory scores and untreated samples stored at 320 °F received the lowest scores. It was seen that the three fish species studied had different shelf-lives: sea trout-6 days, Spanish mackerel - 10 days and catfish - 16 days. Decomposition rates differed significantly between species and this factor must be taken into account when marketing strategies are developed by firms engaged in fresh fish sales.
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    Development and use of a chemically defined medium for estimating the oxygen tolerance of campylobacter species
    Hodge, Jeffrey Paul (Virginia Tech, 1993-06-05)
    When estimating the degree of oxygen tolerance of Campylobacter spp. on Brucella medium, the brand of tryptone (pancreatic digest of casein) used in the medium greatly influenced the results. In fact, with some brands and batches of tryptone, C. jejuni could grow at 21 % O₂ without any additional scavengers of reactive oxygen intermediates (ROIs), although CO₂ was still required for growth. When the medium was prepared with other brands of tryptone, it did not allow growth of C. jejuni beyond 10% O₂. The dependence of the growth response on different types and brands of tryptone-based media makes it impossible to achieve reproducibility in oxygen tolerance studies. To eliminate such variation in complex media, we developed a chemically defined medium for Campylobacter spp. This medium allows reproducible colony counts to be obtained. The medium was used to assess the effect of ROI scavengers on the oxygen tolerance of various Campylobacter species. Allopurinol, azelaic acid, caffeine, cimetidine, and pyruvate when used singly were the most effective in enhancing oxygen tolerance. When ROI scavengers were combined with dimethyl sulfoxide, the effects of allopurinol, azelaic acid, caffeine, cimetidine, and pyruvate were even more pronounced than when they were used alone. A combination of tetramethylpiperidine-1-oxyl (1EMPOL), a superoxide dis mutase mimic, with pyruvate also enhanced oxygen tolerance effectively. A survey of the literature dealing with the types of ROIs destroyed by scavengers used in our study suggests that hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH.) are the most toxic ROIs for Campylobacter species.
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    Development of a reporter system for the study of gene expression for solvent production in Clostridium beijerinckii NRRL B592 and Clostridium acetobutylicum ATCC 824
    Li, Guang-Shan (Virginia Tech, 1998-09-23)
    To study the regulation of gene expression, a good reporter system is very useful. The lack of a good reporter system for the solvent-producing clostridia hindered the progress of research in this area. The objective of this study was to develop a reporter system to facilitate the elucidation of the control mechanism for the expression of solvent-producing genes. A potential reporter gene was found in Clostridium beijerinckii NRRL B593, which contains an adh gene encoding a primary-secondary alcohol dehydrogenase and this adh gene is not present in Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NRRL B592. The adh gene was cloned into the E. coli -Clostridium shuttle vectors to generate plasmids. An electro-transformation procedure was developed for C. beijerinckii NRRL B592. Shuttle plasmids were transformed into C. beijerinckii NRRL B592 or C. acetobutylicum ATCC 824. The copy number of the plasmids in C. beijerinckii was 4. Isopropanol production suggested that the adh gene was expressed in transformants of C. acetobutylicum ATCC 824 and C. beijerinckii NRRL B592. Northern analysis indicated that the expression of the adh gene was regulated at the transcriptional level in the transformants of C. beijerinckii. The transcriptional start site for the adh gene was identified by the primer extension method. A promoter-probing vector was constructed and tested with the promoter from the ferredoxin(fer) gene. The expression of the adh gene under the control of the fer promoter was at a low and similar level during acidogenesis and solventogenesis. The expression pattern of the adh gene under the control of the promoter of the adh gene differed from that under the control of the promoter of the fer gene.
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    Development of defined media for motility and for growth of Spirillum volutans, with special reference to biological monitoring of pollutants and to obligate microaerophilism of bacteria
    Bowdre, Jean Handy (Virginia Tech, 1975-05-05)
    Two investigations were pursued in this study. One was a possible application of the motility of Spirillum volutans to detection of pollutants in industrial effluent for the purpose of in-plant biological monitoring. The other was a nutritional study of the organism with emphasis on its obligate microaerophilism. In the pollution monitoring project, flagellar uncoordination in S. volutans was studied as a bioindicator of toxicity. Uncoordination is produced by a variety of agents. Cells displaying normal motility show frequent reversal of direction, accompanied by reversal of orientation of their bipolar fascicles of flagella. In uncoordinated cells, the flagella at opposite poles oppose each other and the cell cannot swim, although the flagella remain active. A defined motility test medium was devised in an attempt to maximize the sensitivity of the uncoordination response to potential pollutants. Suspensions of S. volutans in this medium responded to a variety of agents, including metal ions (1-3 ppm), cetyl pyridinium chloride (1 ppm), hydrazine (10 ppm), i-naphthol (3 ppm), and others. Effective concentrations ranged up to several per cent for the alcohols tested. The response was immediate.
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    Diversity of limited oxidations accomplished by gluconobacter oxydans
    Edwards, Deborah Elizabeth (Virginia Tech, 1990-11-16)
    Gluconobacter oxydans is characterized by the ability to carry out rapid, single-step oxidations of many different hydroxyl-containing compounds. These oxidations are believed to be catalyzed by the membrane-bound NAD(P)-independent dehydrogenases. Experiments were designed to use G. oxydans ATCC strain 621 to determine the contribution of these dehydrogenases in whole-cell oxidations and to determine the range of substrates that can be oxidized by the membrane fraction of these cells when grown on a single substrate. My first hypothesis was that the membranes would accomplish these oxidations at the same rate as an equivalent number of whole cells. Oxidative activity data obtained from using both oxygen uptake and tetranitroblue tetrazolium assays, however, did not support this hypothesis. I attribute this to the probability that the membranes were damaged during isolation of the membrane fraction and, therefore, were unable to exhibit full oxidative potential. My second hypothesis was that the membranes from cells grown on one substrate would oxidize many other substrates. Potassium fenicyanide was used to assay the oxidative activity of the membrane fraction of cells grown on glycerol. Of 41 substrates tested all were significantly oxidized. I concluded from these data, therefore, that the enzyme(s) responsible for the oxidation of these substrates are synthesized constitutively. Unfortunately, one cannot draw any conclusions as to whether or not these enzymes are highly substrate-specific. I speculate that one or a few enzymes show a broad range of substrate specificity, as it would be inefficient for the cell to consecutively synthesize more than forty different substrate-specific enzymes for substrates it may never encounter.
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    The effect of bioxidation on the coagulation of dispersed color
    Laing, Gary Thomas (Virginia Tech, 1967-08-15)
    This thesis shows how the first plan of the Air War Plans Division (AWPD-l) became the most important document in the development of American strategic bombing doctrine in World War II. This plan was not only the first in the Air War Plans Division. but it was the first of its kind in the world. Beyond the history and importance of the plan itself, this study testifies to the success of a handful of dedicated airmen who believed in the power of a strong air force. General Henry H. Arnold deserves tribute for having had the wisdom and foresight to pick a former Air Corps Tactical School (ACTS) instructor to head the AWPD. Finally, the unofficial acceptance of AWPD-l was a triumph for the ACTS itself. Significantly, all four officers ultimately responsible for completing A WPD-l had been instructors at the ACTS.
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    Effect of temperature-induced membrane lipid phase transition in recipient cells on conjugation in Escherichia coli K-12
    Smith, Richard Thomas (Virginia Polytechnic Institute and State University, 1983)
    For the unsaturated fatty acid auxotroph (fabB⁻) strain K1059, conditions have been found which allow for maintenance of viability following inhibition of cell growth by.a temperature-induced membrane lipid phase transition. In matings with a F-like, R-factor donor, strain K1059 demonstrated a measurably reduced recipient ability at temperatures below 35.5 C when growth medium was supplemented with elaidate versus oleate. K1059 parent strain, Ymel, did not show variability. in recipient ability when the growth medium was supplemented with either of these two unsaturated fatty acids. Membrane phase transition induced inhibition of recipient cell function is reversible; elaidate-grown cells of strain K1059 held at a temperature of 33.5 C for 30 minutes demonstrating the same mating proficiency as cells remaining at the growth temperature of 39.5 C. Neither pair formation nor maintenance of plasmid once stably inherited appear to be affected by the onset of a membrane lipid phase transition. Furthermore, transfer of ³H-thymidine labeled DNA from donor to recipient cell is only minimally affected by the change in recipient cell membrane lipid phase. Analysis of the results suggest that DNA transferred to recipient cells in possession of ordered phase membrane lipids is either in a fonn which will not permit formation of a stable plasmid or is trapped in a region outside of cytoplasmic membrane.
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    Effects of subtherapeutic doses of antibiotics on poultry intestinal bacteria
    Kelley, Roger W. (Virginia Polytechnic Institute and State University, 1982)
    Supplementation of the diet with low concentrations of antibiotics stimulates the growth of poultry by affecting the intestinal flora. The bacterial flora of the small intestine of turkey poults was extensively analyzed in an attempt to correlate changes in populations with growth response. Lactobacillus species comprised almost 100% of the duodenal flora of two-week-old poults but there was no difference in species associated with antibiotic (zinc bacitracin, 55 ppm) treatment. The ileal flora also was predominantly lactobacilli (average 75% of the flora). The most common lactobacilli from the turkey intestinal tract were several previously undescribed Lactobacillus species followed by L. acidophilus, L. salivarius subsp. salivarius, L. fermentum, and L. plantarum. Antibiotic treatment resulted in a shift in the proportions of several of the unnamed Lactobacillus sp. Preliminary feed trials using two strains of lactobacilli that belonged to species that increased in numbers with antibiotic treatment did not stimulate growth when one-day-old birds were colonized with the strains. A probable explanation for the increase in growth is the effect of antibiotic treatment on the multiplication of bacteria in the small intestine. As the digesta move from the gizzard to lower ileum an average 16-fold increase in bacteria occurs in untreated birds. In antibiotic-treated birds the increase was only 2-fold. This inhibition of growth is not due strictly to cell lysis because there are no significant differences in microscopic counts, but the viable counts do decrease. As a corollary there is significantly less lactic acid in the lower ileum of antibiotic-fed birds. Antibiotics did not affect total microscopic or viable counts in the crop or ceca. The above experiments were all done with zinc bacitracin; however, the inhibition of bacterial multiplication was also observed with procaine penicillin. The conclusion from my data is that zinc bacitracin, and probably procaine penicillin, stimulate the growth of turkey poults by a general suppression of the small intestinal flora rather than by an effect on any individual bacterial species.
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    Enzymology of butanol formation in Clostridium Beijerinckii
    Yan, Run-Tao (Virginia Tech, 1991)
    The present study encompasses an investigation of the expression of solvent forming enzymes and purification and characterization of butanol-forming enzymes. More sensitive and accurate procedures for the determination of acids and solvents in cultures have been developed, which led to the recognition of the onset of solvent production at the mid-exponential phase, about two h earlier than previously reported. Activities of solvent-forming enzymes started to increase about one h before the onset of measurable solvent production and the activities of solvent-forming enzymes did not increase simultaneously. CoA-acylating aldehyde dehydrogenase (ALDH) was purified to near homogeneity. The ALDH showed a native M.. of 100,000, and a subunit Mr of 55,000. ALDH could use either NAD(H) or NADP(H) as the coenzyme. ALDH was oxygenlabile. The O₂-inactivated enzyme could be reactivated by incubating the enzyme with CoA. Both NADH- and NADPH-dependent alcohol dehydrogenase activities were present in crude extracts. The ratio of NADPH-dependent activity to NADH-dependent activity (the PID ratio) varied in crude extracts. The PID ratio was affected by O~ ionic strength, pH, growth stage of cell, Fe in culture medium and temperature. Two ADHs have been identified in crude extracts. The NADPH-dependent ADH (P-ADH) could be separated from the NADH/NADPH-dependent ADH (D/P-ADH). The D/P-ADH has been extensively purified. The D/P-ADH showed a native Mr of 70,000 and subunits with Mr of 45,300 and 40,000. The D/P-ADH activity could be inactivated by a,a' -dipyridyl and restored by Fe2+.
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    Epidemiology of Mycobacterium avium complex infecting AIDS patients
    Eaton, Twilla (Virginia Tech, 1993-12-06)
    Organisms of the Mycobacterium avium complex cause disseminated infections in 25 to 50 % of patients with AIDS. To assess the likelihood of exposure to M avium, we attempted to recover M. avium complex from environmental samples in geographical areas (Boston, Massachusetts; Hanover, New Hampshire; Helsinki, Finland; Nairobi, Kenya; and Kinsasha, Zaire) located near M. avium infected AIDS patients. Although M. avium was recovered from environmental samples at all sites, it was found more frequently in water supply systems in the United States and Finland (8/25, 32 %) compared to water supply samples from Africa (0/14, 0%). To determine if M. avium isolates recovered from the same geographical area as AIDS patients shared phenotypic and genetic characteristics with clinical AIDS M. avium isolates (recovered by collaborating laboratories), the ability to grow at 43°C, cadmium-and streptomycin-resistance, and the presence of plasmids were used as epidemiological markers. We found that environmental isolates in this study shared similar characteristics with the clinical AIDS M. avium isolates. Compared to developed countries, the prevalence of M. avium infections among AIDS patients in developing countries (i.e., Africa) is very low. To determine if M. avium was absent in the African environment, we attempted to recover the organisms from water and soil in Kampala, Uganda. M. avium was recovered from 43 % of environmental samples, and these isolates shared similar phenotypic and genetic characteristics with M. avium isolates from the United States. Cigararette smoking was identified as a possible risk factor for HIV infected individuals. M avium isolates were recovered from several brands of cigarettes, suggesting that cigarettes are a possible source of infection.
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    Evaluation of a Monoclonal-based EIA for the Detection of Giardia lamblia and the Identification of the Antigen
    Boone, James Hunter M.S. (Virginia Tech, 1998-05-05)
    I. A number of commercial enzyme immunoassay (EIA) tests are available for the diagnosis of giardiasis. In a time of rising health-care costs, there is a need for diagnostic tests that are rapid, specific, sensitive and inexpensive. In the first phase of this study, I developed a monoclonal-based EIA, the GIARDIA TEST, with these qualities in mind. This assay's performance characteristics were determined by a comparison study using conventional ova and parasite examination, immunofluorescence antibody test (IFA) and other commercial EIA tests. Studies were done in-house at TechLab, Inc. and at various U.S. medical facilities. Results were statistically analyzed to determine sensitivity (ability of the assay to detect a positive result), specificity (amount of cross-reactivity), predictive positive value (the confidence in a positive result), predictive negative value (the confidence in a negative result) and overall correlation with the reference assay. II. There remain many questions to be answered about the various antigens produced by Giardia lamblia and how they can be utilized as diagnostic markers. In the second phase of this study, I identified and partially characterized the antigen (Ct7 Ag) that reacts with the Ct7 monoclonal antibody (MAb). This MAb is an IgM class mouse immunoglobin that is utilized by the GIARDIA TEST and by an immunofluorence antibody test (IFA) which detects Giardia cysts in water and feces. The results of this study will provide physicians and researchers with detailed information about the Ct7 Ag and why it is a useful marker for giardiasis.
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    Experimental anaerobic bacterial infections in mice
    Walker, Clay B. (Virginia Tech, 1977-06-15)
    The development of a technique for producing a pure Bacteroides fragilis infection in mice is described. The infection produces large subcutaneous abscesses at the site of bacterial injection in approximately 90% of the mice. The abscess can be seen as an obvious swelling within 5-7 days post-injection. The infection was initiated by injection of pure cultures grown in a semisolid medium. Similar infections were also produced with pure cultures of B. distasonis, B. ovatus, B. thetaiotaomicron, and B. vulgatus.
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    Formulation of improved media for isolation and cultivation of Campylobacter fetus
    George, Hugh A. (Virginia Tech, 1977-12-15)
    Campylobacter fetus, a microaerophilic, Gram-negative rod, is a well-known cause of contagious abortion and infertility in cattle and sheep and is gaining increasing recognition as an opportunistic human pathogen. In the past, the unusual oxygen requirements of the organism have complicated its recovery from clinical sources; optimum recovery necessitates the use of special gas mixtures, vacuum pumps, etc., not routinely used in most laboratories. In this study, the stimulatory effects of compounds found to enhance aerotolerance and growth of C. fetus were tested for 62 strains of C. fetus, representing each subspecies, to test the desirability of supplementing conventional media with these additives. Brucella agar supplemented with 0.025% (each) FeS04·7H20, sodium bisulfite, and pyruvic acid (FBPA agar) supported growth of 82% of the strains tested under simulated candle jar condtions. Brucella broth supplemented with 0.2% FeS04 ·7H20, 0.025% sodium bisulfite, and 0.050% pyruvic acid (FBPB broth) supported growth of 61 of 62 strains at 21% 02, 2.5% CO2 with static incubation. Therefore, FBPA agar and FBPB broth are recommended for the isolation and cultivation of C. fetus. Although isolation from clinical sources is still dictated to some extent by the oxygen tension used for cultivation, improved recovery may be expected regardless of equipment or facilities available. Another compound found to enhance aerotolerance of C. fetus was SOD. This finding supports the hypothesis that the stimulatory effect of the media additives results from a direct action on the culture medium by degrading toxic derivatives of oxygen, such as the superoxide radical and hydrogen peroxide.