Biomedical subjects

D A Webster

Publications and source records attributed to D A Webster.

At least 19 recordsLinked to original sources

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↗

PubMed1992

The bacterial hemoglobin from Vitreoscilla can support the aerobic growth of Escherichia coli lacking terminal oxidases.

Two Escherichia coli mutants that lack both cytochrome o and d terminal oxidases are able to grow with glucose as the carbon source but not with the aerobic substrates succinate or lactate. One of these, GV101, is a deletion mutant of cytochrome o and a point mutation of cytochrome d. The other, GK100, is a total deletion mutant of all the genes for both cytochromes. When these mutants were transformed with a plasmid containing the gene for the bacterial hemoglobin from Vitreoscilla, they were capable of growth in the presence of succinate or lactate and showed aerobic respiration in the presence of these substrates, unlike the parent strains. Cells transformed with a plasmid containing the gene for the hemoglobin but lacking the native promoter did not express the hemoglobin and did not respire. Membrane vesicles prepared from the cells consumed oxygen in the presence of succinate. This succinate-supported respiration decreased with successive washings of the vesicles but was restored by adding E. coli cytosol containing the hemoglobin or by adding the hemoglobin purified from Vitreoscilla. This respiration was inhibited by cyanide.

Aerobiosis↗

PubMed1992

Predictions of restoration deterioration.

This study evaluated the application of a mixture model involving a Weibull distribution function to predict the median times for restorations of three dental restorative materials to achieve unsatisfactory rating scores for six clinical factors. The accuracy of the method was assessed graphically against the known actuarial long-term deterioration observations of 1813 amalgam, 1774 anterior resin composite and 474 glass polyalkenoate (ionomer) cement restorations, assessed over periods of up to 20, 18 and 14 years, respectively. Of the six clinical factors investigated (which included marginal fracture), only four had sufficient long-term unsatisfactory rating score data to enable their median times to be predicted. These predicted times were: for amalgam restorations, surface roughness 32.5 years and surface tarnishing 16.0 years; for resin composites, marginal staining 25.4 years and colour mismatch 14.2 years; and for glass polyalkenoate (ionomer) cements, marginal staining 17.6 years and colour mismatch 3.6 years. The known and predictive unsatisfactory rating score results were generally in close agreement. However, it was not possible to predict median times for unsatisfactory rating scores associated with very slowly deteriorating restoration factors. The actual replacement rates of the amalgam restorations were too low to obtain their median survival time. However, for the faster failing resin composites this time was 7.9 +/- 0.5 years, and for the glass polyalkenoate (ionomer) cements 2.2 +/- 0.2 years. The relationship of restoration deterioration to restoration replacement and dental health requires further analysis.

Actuarial Analysis↗

PubMed1992

Family/media approach to HIV prevention: results with a home-based, parent-teen video program.

We describe the first study with a home-based HIV prevention video program for parents and young teenagers. The objectives of the program are to inform parents and teenagers about the causes and prevention of HIV infection and other sexually transmitted diseases, to increase family problem-solving skills, and to increase teen problem-solving and assertiveness skills. The objectives pertain to the goals of increasing skills needed to help teenagers avoid or manage high-risk behaviors and situations. Forty-five families with at least one 12- to 14-year-old were randomly assigned to either experimental (receive video program) or control (no video) conditions in a pretest-posttest design. After 6 months (Follow-Up 1), the experimental and control families were reassessed. The control families next received the video program, and the control families were assessed again (Follow-Up 2). The results indicate increases in parent and teen knowledge and skills only with video viewing. Approaches to improving the video program, particularly with teenagers, are discussed.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Studies on the bacterial hemoglobin from Vitreoscilla. Redox properties and spectroscopic characterization of the different forms of the hemoprotein.

Vitreoscilla contained a homodimeric bacterial hemoglobin (VtHb). The purification of this protein yielded VtmetHb which exhibited electronic and electron paramagnetic resonance (EPR) spectra, showing that it existed predominantly in a high-spin ferric form, both axial and rhombic components being present. The preparations also contained variable amounts of low-spin components. There was no evidence that these high-spin and low-spin forms were in equilibrium. The former were reducible by NADH catalyzed by the NADH-metVtHb reductase, and the latter were not. High ionic strength and high pH led to the formation of low-spin metVtHb; both treatments were reversible. Cyanide and imidazole liganded to VtHb resulted in the conversion of high-spin to low-spin ferric heme centers, each with characteristic electronic and EPR spectra. Some preparations of VtHb exhibited EPR signals consistent with a sulfur ligand bound to the ferric site. When VtHb was treated with NADH plus the reductase in the presence of oxygen, the intensity of the high-spin EPR signals decreased significantly. No reduction occurred in the absence of oxygen, suggesting a possible role for the superoxide anion. Dithionite treatment of VtHb resulted in a slow reduction, but the main product of the reaction of dithionite-reduced VtHb with oxygen was VtmetHb, not VtHbO2. EPR spectra of whole cells of Vitreoscilla exhibited a variety of intense signals at low and high magnetic field, the g-values being consistent with the presence of high-spin ferric heme proteins, in addition to an iron-containing superoxide dismutase (FeSOD) and iron-sulfur proteins. EPR spectra of the cytosol fraction of Vitreoscilla showed the expected resonances for VtmetHb and FeSOD.

Bacterial Proteins↗

PubMed1991

Prediction of amalgam restoration longevity.

The purpose of the present study was to assess predictions of longevity for amalgam restorations using a fitted Weibull distribution against base estimates using actuarial methods. The 1345 restorations involved were from 100 members of the Royal Australian Air Force whose dental conditions had been monitored regularly over a minimum period of 10 years. Based on maximum likelihood estimates of the parameters of a Weibull distribution, Weibull curves could be determined and were found to be very close to actuarial survival curves established from the same data set. However, Weibull estimates based on survival experience of less than 5-6 years failed to predict long-term survival. Using the 6 year actuarial survival data, the predicted and observed survival curves disagreed by less than 10 per cent for every time period. Twenty-five per cent of restorations had failed for the 6 year data, compared to 22 per cent in these first 6 years for the full 17 year data set.

Dental Amalgam↗

PubMed1991

Survival predictions of amalgam restorations.

The purpose of this study was to evaluate survival predictions made for four different amalgam alloy restorations, using a mixture model involving the standard Weibull function. The amalgam alloys were placed by students and staff in patients attending a dental hospital, and 1680 restorations were examined over periods of up to 18 years. Based on maximum likelihood estimations of the parameters of the mixture model distribution, predictive survival distributions were generated and found to match closely the actuarial survival estimates established from the same data. The 13-year restoration survivals of one low-copper alloy could be predicted accurately from the 6-year survival results. However, another low-copper alloy and two high-copper alloys with much lower restoration failure rates required 18 years of data for accurate long-term survival predictions.

Actuarial Analysis↗

PubMed1991

Survival predictions of four types of dental restorative materials.

The present study assessed the survival predictions made for four different types of dental restorative materials, using a mixture model involving the standard Weibull distribution function. A large number of amalgam, anterior resin, glass polyalkenoate (ionomer) cement, and pit and fissure sealant restorations were examined over varying periods of up to 18 years. The materials had been placed by numerous staff and students at a teaching hospital. Based on maximum likelihood estimations of the parameters of the mixture model distribution, survival curves were generated and found to agree closely with the actuarial survival curves estimated from the same data. As the years of data used to fit the mixture model curves decreased, then the fitted curves started to exhibit obvious divergences from the actuarial curves at 12-13 years for amalgams, 3-4 years for anterior resins and sealants, and 1-2 years for glass polyalkenoate (ionomer) cements. At least 5 per cent of restorations needed to have failed over any period to allow close agreement of the two curves, with the slower failing materials requiring longer observation periods.

Composite Resins↗

PubMed1991

Large database management in clinical dental research.

Previously the management and analysis of large databases for longitudinal clinical dental research has been severely restricted by the costs of custom-made software and access to suitable computing equipment. However, the recent availability of powerful personal computers and the use of the Scientific Information Retrieval Database Management System (SIR/DBMS) in association with BMDP Statistical Software has now created an enormously powerful tool for extensive and fast data manipulation requiring relatively few commands and the capability to easily perform detailed statistical analyses.

Computer Graphics↗

PubMed1990

A cytochrome that can pump sodium ion.

Previous studies have shown that the bacterium, Vitreoscilla, generates a respiratory-driven delta psi Na+. Two major respiratory electron transport proteins, NADH dehydrogenase (NADH:Quinone oxidoreductase), and cytochrome o terminal oxidase are candidates for the electrogenic Na+ pumping that mediates the delta psi Na+ formation. The NADH oxidase activity of the membranes was enhanced more by Na+ than by Li+. The NADH:Quinone oxidoreductase activity in the respiratory chain was enhanced by Na+ and Li+, whereas the quinol oxidase activity of cytochrome o was enhanced specifically by Na+, and not by Li+, K+, or choline. Purified cytochrome o, reconstituted into Na(+)-loaded liposomes in the right-side-out orientation, catalyzed a net Na+ extrusion when energized with Q1H2(1). In nonloaded inside-out proteoliposomes, this cytochrome catalyzed a net uptake of 22Na+ when energized with ascorbate/TMPD. Both Na(+)-pumping activities were inhibited by CN-. These results are consistent with the Vitreoscilla cytochrome o being a redox-driven Na+ pump.

Bacteria, Aerobic↗