Biomedical subjects

D A Webster

Publications and source records attributed to D A Webster.

At least 19 recordsLinked to original sources

PubMed2001

Vitreoscilla hemoglobin. Intracellular localization and binding to membranes.

The obligate aerobic bacterium, Vitreoscilla, synthesizes elevated quantities of a homodimeric hemoglobin (VHb) under hypoxic growth conditions. Expression of VHb in heterologous hosts often enhances growth and product formation. A role in facilitating oxygen transfer to the respiratory membranes is one explanation of its cellular function. Immunogold labeling of VHb in both Vitreoscilla and recombinant Escherichia coli bearing the VHb gene clearly indicated that VHb has a cytoplasmic (not periplasmic) localization and is concentrated near the periphery of the cytosolic face of the cell membrane. OmpA signal-peptide VHb fusions were transported into the periplasm in E. coli, but this did not confer any additional growth advantage. The interaction of VHb with respiratory membranes was also studied. The K(d) values for the binding of VHb to Vitreoscilla and E. coli cell membranes were approximately 5-6 microm, a 4-8-fold higher affinity than those of horse myoglobin and hemoglobin for these same membranes. VHb stimulated the ubiquinol-1 oxidase activity of inverted Vitreoscilla membranes by 68%. The inclusion of Vitreoscilla cytochrome bo in proteoliposomes led to 2.4- and 6-fold increases in VHb binding affinity and binding site number, respectively, relative to control liposomes, suggesting a direct interaction between VHb and cytochrome bo.

Animals↗

PubMed2001

Cell growth and oxygen uptake of Escherichia coli and Pseudomonas aeruginosa are differently effected by the genetically engineered Vitreoscilla hemoglobin gene.

Vitreoscilla hemoglobin is a good oxygen trapping agent and its presence in genetically engineered Escherichia coli helps this bacterium to grow better. Here, the potential use of this hemoglobin, for improving the growth and the oxygen transfer properties of Pseudomonas aeruginosa as well as Escherichia coli, was investigated. To stably maintain it in both bacteria, a broad-host range cosmid vector (pHG1), containing the entire coding sequence for Vitreoscilla hemoglobin gene and its native promoter on a 2.3 kb fragment, was constructed. Though at different levels, both bacteria produced hemoglobin and while the oxygen uptake rates of vgb-bearing strains were 2-3-fold greater than that of non-vgb-bearing strains in both bacteria, the growth advantage afforded by the presence of Vitreoscilla hemoglobin was somewhat varied. As an alternative to the traditional method of the improvement of oxygen transfer properties of the environment in which cells are grown, the genetic manipulation applied here improved the oxygen utilization properties of cells themselves.

Bacterial Proteins↗

PubMed2001

Chromosomal integration of the Vitreoscilla hemoglobin gene in Burkholderia and Pseudomonas for the purpose of producing stable engineered strains with enhanced bioremediating ability.

Using the pUT-miniTn5 vector system developed by the laboratory of K.N. Timmis, the Vitreoscilla hemoglobin gene (vgb) was integrated into the chromosomes of Pseudomonas aeruginosa and Burkholderia cepacia; Vitreoscilla hemoglobin (VHb) was expressed at 8.8 and 0.8 nmol/g wet weight of cells in the respective engineered strains. The vgb-bearing P. aeruginosa outgrew wild-type P. aeruginosa and degraded benzoic acid faster than the latter strain at both normal and low aeration. In contrast, the vgb-bearing B. cepacia strain had a growth advantage over the wild-type strain at ca. 90 ppm, but not at ca. 120 ppm 2,4-dinitrotoluene (DNT); no difference in DNT degradation was seen between the two strains at either normal or low aeration. The results demonstrate the practicality of enhancing bioremediation with vgb stably integrated into the chromosome, but also suggest that a minimal level of VHb expression is required for its beneficial effects to be seen.

Bacterial Proteins↗

PubMed2000

Study of cytochrome bo function in Vitreoscilla using a cyo(-) knockout mutant.

The bacterium, Vitreoscilla, produces a delta mu(Na+) across its membrane during respiration. A key enzyme for this function is the cytochrome bo terminal oxidase which, when incorporated into synthetic proteoliposomes, pumps Na(+) across the membrane upon the addition of a substrate. A Vitreoscilla cytochrome bo knock out (cyo(-)) mutant was isolated by transposon mutagenesis using pUT-mini-Tn5Cm. The membranes of this mutant lacked the characteristic 416 nm peak and 432 nm trough in CO difference spectra, which are clearly visible in spectra of the Vitreoscilla wild-type, but peaks at 627, 560, and 530 nm in reduced minus oxidized difference spectra indicate that cytochrome bd is still present. The specific NADH oxidase and ubiquinol-1 oxidase activities of the cyo(-) mutant membranes were less than those of Vitreoscilla wild-type and Escherichia coli membranes, and the stimulation of these activities of the mutant and E. coli membranes by 75 mM NaCl was approximately 50% less than that of Vitreoscilla wild-type membranes. The ubiquinol-1 oxidase activity of the cyo(-) mutant membranes was inhibited by 10 mM KCN to a lesser degree than that of the Vitreoscilla wild-type and E. coli membranes (50, 80, and 85%, respectively). This result is also consistent with the cyo(-) mutant membrane fragments containing only the cytochrome bd terminal oxidase, which is known to be less sensitive to KCN. Although the maximum respiration and growth of the cyo(-) mutant were less than those of the wild-type, this mutant is still capable of growing with cytochrome bd alone.

Cell Division↗

PubMed1999

The effect of hyperbaric oxygen on medial collateral ligament healing in a rat model.

Hyperbaric oxygen has been shown to promote healing in bone and some soft tissues. This study was undertaken to determine its effect on ligamentous healing. Forty-eight Sprague-Dawley rats underwent a standardized surgical laceration of the right (divided) medial collateral ligament, whereas the left (undivided) medial collateral ligament was not surgically lacerated. A control group of 24 rats recovered without intervention. An experimental group consisting of the other 24 rats was exposed to hyperbaric oxygen at 2.8 atmospheres for 1.5 hours a day for 5 days after the surgery. Six rats from each group were euthanized at 2, 4, 6, and 8 weeks. The stiffness and final force to failure were recorded for the divided and undivided medial collateral ligaments. At 4 weeks, a statistically greater force was required to cause failure of the previously divided ligaments that had been exposed to hyperbaric oxygen than those that had not. The stiffness and force to cause failure of previously divided ligaments were statistically greater at 4 weeks than at 2 weeks, whether or not hyperbaric oxygen was used. No additional statistical increases in stiffness or force were observed at 6 weeks.

Animals↗

PubMed1999

Head and face injuries in scholastic women's lacrosse with and without eyewear.

PURPOSE: The use of protective equipment has been absent or inconsistent in scholastic women's lacrosse leading to increasing concern for eye and head injury. There is a paucity of field data, however, on which to base strategic decisions on how best to prevent head injuries in young athletes. METHODS: This study examined the effects of protective eyewear on injury rates in scholastic women's lacrosse in a cohort of approximately 700 varsity and junior varsity players in central New York studied prospectively for 2 yr during a transition from sparse to almost complete eyewear use. RESULTS: The overall head/face injury rate was 0.71 injuries per 1000 exposures (games and practices) and was 16.5% lower in goggle wearers. In games alone, where more aggressive play and stick use prevails, the rate associated with protective eyewear use was markedly lower (51%). Considering specific regions, the rates for peri-orbit and forehead injuries among goggle users were substantially lower than for nonusers (6% and 13%, respectively). Cheek and scalp injury rates tended to be higher among goggle wearers, but not statistically significantly so. Significant compensatory increases with goggle wear at other sites were not observed. Only a few injuries appeared to be mediated by the goggles themselves and potentially could have been more serious if the goggles had not been present. No direct eye (orbit) injuries were reported throughout the study period. CONCLUSION: On balance, then, the use of eyewear in women's lacrosse appears to be beneficial when users are compared with nonusers.

Adolescent↗

PubMed1998

Synthesis and excretion of alpha-amylase in vgb+ and vgb- recombinant Escherichia coli: a comparative study.

Synthesis and excretion of alpha-amylase is investigated in batch cultures of Escherichia coli JM103[pMK57] (vgb-) and E. coli JM103[pMK79] (vgb+). While total production and excretion of alpha-amylase were promoted in Luria broth (LB) (excretion being as high as 87%), cell-mass-specific production of the enzyme was promoted in M9 in bioreactor cultures and in LB in shake flask cultures. Low aeration and agitation rates and presence of starch were conducive to alpha-amylase synthesis in E. coli JM103[pMK79]. Two-stage bioreactor operating strategies that will improve alpha-amylase production are proposed. The potential of these strategies is demonstrated via two-stage shake flask cultures.

Bioreactors↗

PubMed1998

Metabolic engineering of Serratia marcescens with the bacterial hemoglobin gene: alterations in fermentation pathways.

Serratia marcescens was transformed with plasmid vector pUC8 or pUC8 containing the bacterial (Vitreoscilla) hemoglobin gene (vgb) on either a 2.3-kb fragment (pUC8:15) or 1.4-kb fragment (pUC8:16) of Vitreoscilla DNA. The vgb-bearing strains were compared with the pUC8 transformant and untransformed S. marcescens with respect to growth in Luria-Bertani (LB) broth supplemented with glucose or casein acid hydrolysate. Growth (on a viable cell basis) was similar to that in unsupplemented LB. Total acid excretion (as estimated by medium pH) was similar for all strains in both LB plus 2% casein acid hydrolysate and LB without additions. Acid excretion in LB plus 2% glucose was somewhat greater at up to 10 h in culture for the two vgb-bearing strains; from 10 to 26 h in culture, the pHs of these cultures continued to decrease (to 4.1-4.2), whereas those of the non-vgb-bearing strains returned to near the starting pH (7.4-7.8). Concomitantly, after 26 h of culture in LB plus 2% glucose, the non-vgb-bearing strains had produced about 15 times as much acetoin and about three to four times as much 2,3-butanediol as the vgb-bearing strains. In general, for all strains, much more acetoin and 2,3-butanediol were produced in LB plus 2% glucose than in unsupplemented LB. The exception was acetoin production by the strain bearing vgb on plasmid pUC8:15; after 26 h of culture in LB without supplementation it was between three and four times that of the other strains, and about 50% higher than its level in LB plus 2% glucose. When grown with the 2% casein acid hydrolysate supplement, the strain bearing vgb on plasmid pUC8:15 produced much more acetoin and 2,3-butanediol than the other strains after 26 hours in culture. The results confirm that vgb can significantly alter carbon metabolism and suggest that the use of vgb technology for directed metabolic engineering may be a complicated process, depending in part on medium composition.

Acetoin↗

PubMed1998

Genetic engineering of Serratia marcescens with bacterial hemoglobin gene: effects on growth, oxygen utilization, and cell size.

The bacterial hemoglobin from Vitreoscilla has been shown to increase growth yield and yield of genetically engineered product in Escherichia coli. To test the generality of this phenomenon, the approximately 560-bp bacterial (Vitreoscilla) hemoglobin gene (vgb) (including the native promoter), cloned into the vector pUC8 in two constructs containing about 1650 and 850 bp, respectively, of Vitreoscilla DNA downstream of vgb, was transformed into Serratia marcescens. After several transfers of the transformants on selective media, both plasmids became stable in this host and the resulting strains produced hemoglobin. Both transformants were compared, regarding growth in liquid Luria-Bertani (LB) medium, with untransformed S. marcescens and S. marcescens transformed with pUC8. The vgb-bearing strains had about 5 times lower maximum viable cell numbers than the strains without hemoglobin, but the former also had late log or early stationary phase cells that were 5-10 times larger than those of the latter. Further, on a dry cell mass basis the presence of vgb inhibited cell growth in liquid media. In contrast, growth of the vgb-bearing strains on LB plates based on cell mass (determined from colony size) was markedly enhanced compared with that of the pUC8 transformant. Respiration of the vgb-bearing strains was lower than that of the strains without vgb on a cell mass basis. These results show that the presence of vgb can have idiosyncratic effects and is not always an aid to cell growth so that its use for genetic engineering must be tested on a case by case basis.

Bacterial Proteins↗

PubMed1998

Site-directed mutagenesis of bacterial hemoglobin: the role of glutamine (E7) in oxygen-binding in the distal heme pocket.

The bacterial and yeast hemoglobins have a glutamine instead of histidine in the E7 position of the distal heme pocket. The recently determined crystal structure of Vitreoscilla hemoglobin (VHb) indicates that this residue is oriented out of the heme pocket and may not ligand the bound oxygen. This is in contrast to elephant myoglobin which also has a Gln(E7) but which does ligand the bound oxygen. This residue was changed in VHb using site-directed mutagenesis to leucine (VHbL) or to histidine (VHbH). Spectral and kinetic studies of the binding of oxygen and CO to VHbL showed that this substitution had little effect on the ligand-binding properties of this protein, evidence that Gln(E7) does not H-bond the bound ligand, in agreement with the findings of the crystallographic study of VHb. In contrast, the functional properties of VHbH were drastically altered in a way suggesting that the E7His may itself be liganded to the heme iron. These studies are further evidence that the distal heme pocket in VHb and related microbial hemoglobins differs from that in mammalian hemoglobins and may resemble in some ways the heme pocket in cytochrome b5.

Bacteria↗

PubMed1996

Na(+)-translocating cytochrome bo terminal oxidase from Vitreoscilla: some parameters of its Na+ pumping and orientation in synthetic vesicles.

Vitreoscilla cytochrome bo ubiquinol oxidase is similar in some properties to the Escherichia coli enzyme, but unlike the latter, the Vitreoscilla oxidase functions as a primary Na+ pump. When purified Vitreoscilla cytochrome bo is incorporated into liposomes made from Vitreoscilla phospholipids and energized with a quinol substrate, it translocates Na+, not H+, across the vesicle membrane. Since protonophores CCCP (carbonyl cyanide m-chlorophenylhydrazone) and DTHB (3,5-di-tert-butyl-4-hydroxybenzaldehyde) stimulated the Na+ pumping, it is unlikely that it is a secondary effect due to the presence of Na+/H+ antiporter activity in the preparations. The efficiency of the Na+ pumping was 3.93 Na+ pumped per O2 consumed when ascorbate/TMPD was used as the substrate. The cytochrome has a K(m) and Kcat for Na+ of 2.9 mM and 277 s-1, respectively. When ferricytochrome c was entrapped within liposomes prepared from Vitreoscilla phospholipids, it was reduced by Q1H2 (ubiquinol-1) but not by ascorbate/TMPD (N,N,N',N'-tetramethyl-1,4-phenylenediamine). Although Q1H2 was oxidized by cytochrome bo in solution at a rate approximately 14 times that of the latter substrate, the rate of accumulation of Na+ within cytochrome bo vesicles driven by the membrane impermeable ascorbate/TMPD was 1.23 times that of the membrane permeable ubiquinol. These data allowed a calculation that in these synthetic proteoliposomes the cytochrome bo molecules are only 51% directed inward; a value of 61% inward-directed was estimated by measuring the ascorbate/TMPD oxidase activity of the proteoliposomes before and after disrupting them with Triton X-100. A random orientation of the E. coli cytochrome bo oxidase in proteoliposomes has also been reported.

Ascorbic Acid↗

PubMed1996

Effects of cognitive coping skills training on coping strategies and experimental pain sensitivity in African American adults with sickle cell disease.

The present study examined whether training in cognitive coping skills would enhance pain coping strategies and alter pain perception in adults with sickle cell disease (SCD). Sixty-four African Americans with SCD were randomly assigned to either a cognitive coping skills condition (three 45-min sessions in which patients were trained to use 6 cognitive coping strategies) or a disease-education control condition (three 45-min didactic-discussion sessions about SCD). Pain sensitivity to calibrated noxious stimulation was measured at pre- and posttesting, as were cognitive coping strategies, clinical pain, and health behaviors. Results indicated that, compared with the randomly assigned control condition, brief training in cognitive coping skills resulted in increased coping attempts, decreased negative thinking, and lower tendency to report pain during laboratory-induced noxious stimulation.

Adaptation, Psychological↗

PubMed1994

Sequence of the region downstream of the Vitreoscilla hemoglobin gene: vgb is not part of a multigene operon.

The 1668 base pairs (bp) downstream of the Vitreoscilla hemoglobin gene were sequenced in the hope of finding related genes that might be part of an operon. Instead, a sequence was found that constituted an open reading frame (ORF) of 569 amino acids (apparently the carboxy-terminal part of a larger ORF), in the direction opposite to the hemoglobin gene. This sequence was found to have 64% similarity with the 1685 bp at the 3' end of the Escherichia coli uvrA gene. The inferred amino acid sequence of the Vitreoscilla DNA has 69% similarity with the corresponding sequence of the E. coli uvrA protein, with similarities of 90, 100, and 85% in the helix-turn-helix, C-terminal ATP binding, and C-terminal zinc finger domains, respectively. The distance between the 3' ends of the Vitreoscilla hemoglobin and uvrA genes is 63 bp.

Adenosine Triphosphatases↗

PubMed1993

Chromosomal radiosensitivity in common variable immune deficiency.

From more than 500 tumours reported in human primary immune deficiencies a majority has been observed in two disorders: ataxia telangiectasia (A-T) and common variable immune deficiency (CVID). Since both diseases have an increased risk of lymphomas/leukaemias and gastrointestinal tumours, suggesting a common risk factor, and the cells derived from A-T patients exhibit an increased chromosomal radiosensitivity we analysed chromosome damage in the G2 lymphocytes of 24 CVID patients and 21 controls after X-irradiation in vitro. There was a significant difference in mean aberration yields between patients and controls. Three CVID patients had yields higher than the mean + 3SD of the controls. Six patients but only one control had yields higher than the mean + 2SD of controls. The patient with the highest chromosomal radiosensitivity subsequently developed a lymphoma. Repeat assays on the same blood sample, with a 24-h delay in setting up the second culture, showed as much variability for control donors as the variation between control donors although for CVID patients inter-individual variation was greater than the difference between results of repeat samples. There was a weak positive correlation between radiosensitivity and age of donor. Chromosomal radiosensitivity of five patients with X-linked hypogammaglobulinaemia was not different from healthy donors. The mean mitotic index (MI) for unirradiated samples from CVID patients was significantly lower than for controls and there was an inverse relationship between MI and aberration yields in the patients, but not in controls. We suggest that the defect in CVID patients that reduces response to mitogenic stimuli may have mechanism(s) in common with those involved in cellular repair processes.

Adult↗

PubMed1993

Predictions of resin-bonded bridge survivals, comparing two statistical models.

The purpose of this study was to compare the predictive accuracy of the standard Weibull distribution model with that of a modification, or mixture model, for resin-bonded etched-metal bridge survivals. A total of 189 bridges placed in adult patients treated by students and staff at a teaching hospital was evaluated over periods of up to 8 years. Survival predictions based on the actual survival data accumulated in 1989 were compared with the later survival data updated in 1992. Comparison of the two fitted Weibull predictive curves with the life table survival estimates from the 1992 updated data demonstrated clearly the improved accuracy of the mixture model for predicting survivals of the resin-bonded bridges in this study.

Denture, Partial, Fixed, Resin-Bonded↗

PubMed1993

Efficacy of a home-based human immunodeficiency virus prevention video program for teens and parents.

Family-based communication about sexual behavior and sexuality is a predominate theme in the key risk reduction and service objectives for human immunodeficiency virus (HIV) prevention and related issues in Healthy People 2000. These recommendations are particularly poignant for younger adolescents who are not yet sexually active and whose social orientation is primarily to family and parents. A family-centered, home-based, video program to foster parent and teen knowledge and communication regarding issues related to HIV prevention and to increase family and teen communication skills was used in an intensive field test of program efficacy. Sixty-nine families with at least one adolescent age 12 to 14 years were recruited through their primary-care physicians and were randomly assigned to either experimental (video with information and skills training) or control (video with information only) conditions in a pre-posttest with a 4-month follow-up design. Both versions of the video program resulted in substantial increases in HIV-related knowledge for parents and teens, but only the skill-training (experimental) version produced increases in knowledge of communication skills and behavioral demonstrations of family problem solving. Approaches for a broader and more effective parent-training video program and for dissemination through primary-care physicians are discussed.

Adolescent↗

PubMed1992

NADH-dependent methemoglobin reductase from the obligate aerobe Vitreoscilla: improved method of purification and reexamination of prosthetic groups.

The NADH-dependent methemoglobin reductase from the bacterium Vitreoscilla was purified using hydrophobic chromatography on a phenyl-Sepharose column. The new procedure resulted in a purer protein and increased the overall yield of the enzyme by a factor of approximately three. The active site of the enzyme was investigated by ultraviolet/visible, fluorescence, Mössbauer, and electron paramagnetic resonance spectroscopy (EPR) at 9.4 GHz. Prosthetic group analysis revealed the presence of one FAD per active enzyme molecule but no iron in contrast to earlier reports. The NADH-methemoglobin reductase activity of the pure enzyme was in the range of 1.1-1.25 units; its electronic and fluorescence spectra were typical of metal-free flavoproteins. No EPR signals were detected between 5 and 150 K over a field range 0.05-0.5 T, and there was no Mössbauer signal, consistent with the absence of iron. Methemoglobin reductase from Vitreoscilla was reduced by dithionite, NADH, and deazaflavin/EDTA upon illumination. The main species observed during these anaerobic oxidation-reduction experiments was the blue semiquinone radical with an EPR signal at g = 2.005, linewidth 1.5 mT. The fully reduced state of the enzyme, FlredH3, was also observed in the reaction with NADH. The reduction was fully reversible with ferricyanide. The observations reported here are consistent with a redox enzyme interacting both with a two-electron donating agent such as NADH and a one-electron accepting center such as the Fe(III)/Fe(II) couple of Vitreoscilla hemoglobin.

Aerobiosis↗

PubMed1992

Sodium-coupled ATP synthesis in the bacterium Vitreoscilla.

The bacterium Vitreoscilla generates an electrical potential gradient due to sodium ion (delta psi Na+) across its membrane via respiratory-driven primary Na+ pump(s). The role of the delta psi Na+ as a driving force for ATP synthesis was, therefore, investigated. In respiring starved cells pulsed with 100 mM external Na+ [( Na+]o) there was a 167% net increase in cellular ATP concentration over basal levels compared with 0, 56, 78, and 78% for no addition, choline, Li+, and K+ controls, respectively. Doubling the [Na+]o to 200 mM boosted the net increase to 244% but a similar doubling of the choline caused only an increase to 78%. When the initial condition was intracellular Na+ ([Na+]i) = [Na+]o = 100 mM, there was a 94% net increase in cellular ATP compared with only 18 and 11% for Li+ and K+ controls, respectively, indicating that Nai+ may be the only cation tested that the cells extruded to generate the electrochemical gradient required to drive ATP synthesis. The Na(+)-dependent ATP synthesis was inhibited completely by monensin (12 microM), but only transiently by the protonophore 3,5-di-tert-butyl-4-hydroxybenzaldehyde (100 microM), further evidence that the Na+ gradient and not a H+ gradient was driving the ATP synthesis. ATP synthesis in response to an artificially imposed H+ gradient (delta pH approximately 3) in the absence of an added cation, or in the presence of Li+, K+, or choline, yielded similar delta ATP/delta pH ratios of 0.98-1.22. In the presence of Na+, however, this ratio dropped to 0.23, indicating that Na+ inhibited H(+)-coupling to ATP synthesis and possibly that H+ and Na+ coupling to ATP synthesis share a common catalyst. The above evidence adds to previous findings that under normal growth conditions Na+ is probably the main coupling cation for ATP synthesis in Vitreoscilla.

Adenosine Triphosphatases↗