Biomedical subjects

D A Webster

Publications and source records attributed to D A Webster.

At least 19 recordsLinked to original sources

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↗

PubMed1990

Study of Vitreoscilla globin (vgb) gene expression and promoter activity in E. coli through transcriptional fusion.

Bacterial hemoglobin (VtHb) is produced by the gram-negative bacterium, Vitreoscilla, in large quantity in response to hypoxic environmental conditions. The vgb gene coding for VtHb has been cloned in E. coli where it is expressed strongly by its natural promoter. The expression of the vgb gene in Vitreoscilla is transcriptionally regulated by oxygen. When E. coli cells were shifted from 20% to 5% oxygen, vgb specific transcript increased. In E. coli cells with plasmids carrying transcriptional fusions of the vgb gene promoter to either CAT (chloramphenicol acetyl transferase) or xylE (catechol-2,3-dioxygenase) genes, the promoter activity depended on the oxygen level. The concentration of CAT and xylE gene products in cells grown under 5% oxygen was 5-7 times that of aerobically (20% oxygen) grown cells. When the vgb gene promoter was deleted, VtHb was not produced under any conditions. When the promoter was replaced by the E. coli tac promoter, hypoxic oxygen did not affect the level of expression of vgb, but adding IPTG did increase the expression of this gene. These results indicate that the vgb gene promoter is transcriptionally regulated by oxygen even in E. coli, and that microaerobiosis is sufficient to induce vgb expression. The size of S1 nuclease-resistant hybrids, prepared using RNA transcripts protected with restriction enzyme fragments containing the promoter proximal region of vgb, was the same for both Vitreoscilla and E. coli, further evidence that the same promoter is used in both organisms. Transcriptional fusion of the vgb gene promoter to the xylE reporter gene on the broad host range plasmid, pKD-49, was used to demonstrate that the vgb promoter can be expressed in other gram-negative organisms, including Pseudomonas, Azotobacter, and Rhizobium.

Cloning, Molecular↗

PubMed1990

Purification, partial characterization, and possible role of catalase in the bacterium Vitreoscilla.

Vitreoscilla is a gram-negative bacterium that contains a unique bacterial hemoglobin that is relatively autoxidizable. It also contains a catalase whose primary function may be to remove hydrogen peroxide produced by this autoxidation. This enzyme was purified and partially characterized. It is a protein of 272,000 Da with a probable A2B2 subunit structure, in which the estimated molecular size of A is 68,000 Da and that of B, 64,000 Da, and an average of 1.6 molecules of protoheme IX per tetramer. The turnover number for its catalase activity was 27,000 s-1 and the Km for hydrogen peroxide was 16 mM. The peroxidase activity measured using o-dianisidine was 0.6% that of the catalase activity. Cyanide, which inhibited both catalase and peroxidase activities, bound the heme in a noncooperative manner. Azide inhibited the catalase activity but stimulated the peroxidase activity. An apparent compound II was formed by the reaction of the enzyme with ethyl hydrogen peroxide. The enzyme was reducible by dithionite, and the ferrous enzyme reacted with CO. The cellular content of Vitreoscilla hemoglobin varies during the growth cycle and in cells grown under different conditions, but the ratio of hemoglobin to catalase activity remained relatively constant, indicating possible coordinated biosynthesis and supporting the putative role of Vitreoscilla catalase as a scavenger of peroxide generated by Vitreoscilla hemoglobin.

Catalase↗

PubMed1990

Respiratory-driven Na+ electrical potential in the bacterium Vitreoscilla.

Vitreoscilla is a Gram-negative bacterium with unique respiratory physiology in which Na+ was implicated as a coupling cation for the generation of a transmembrane electrical gradient (delta psi). Thus, cells respiring in the presence of 110 mM Na+ generated a delta psi of -142 mV compared to only -42 and -56 mV for Li+ and choline, respectively, and even the -42 and -56 mV were insensitive to the protonophore 3,5-di-tert-butyl-4-hydroxybenzaldehyde (DTHB). The kinetics of delta psi formation and collapse correlated well with the kinetics of Na+ fluxes but not with those of H+ fluxes. Cyanide inhibited respiration, Na+ extrusion, and delta psi formation 81% or more, indicating that delta psi formation and Na+ extrusion were coupled to respiration. Experiments were performed to distinguish among three possible transport systems for this coupling: (1) a Na(+)-transporting ATPase; (2) an electrogenic Na+/H+ antiport system; (3) a primary Na+ pump directly driven by the free energy of electron transport. DCCD and arsenate decreased cellular ATP up to 86% but had no effect on delta psi, evidence against a Na(+)-transporting ATPase. Low concentrations of DTHB had no effect on delta psi; high concentrations transiently collapsed delta psi, but led to a stimulation of Na+ extrusion, the opposite of that expected for a Na+/H+ antiport system. Potassium ion, which collapses delta psi, also stimulated Na+ extrusion. The experimental evidence is against Na+ extrusion by mechanisms 1 and 2 and supports the existence of a respiratory-driven primary Na+ pump for generating delta psi in Vitreoscilla.

Arsenates↗

PubMed1990

Presence of the bacterial hemoglobin gene improves alpha-amylase production of a recombinant Escherichia coli strain.

A recombinant plasmid (pMK57) was constructed by cloning the Bacillus stearothermophilus alpha-amylase gene into pUC8; plasmid pMK79 was then derived from pMK57 by inserting the bacterial (Vitreoscilla) hemoglobin gene into the latter plasmid. Both pMK57 and pMK79 were transformed into Escherichia coli strain JM 103 to make strains MK57 and MK79, respectively. Both MK57 and MK79 produced alpha-amylase and MK79 produced hemoglobin. MK79 outgrew MK57 in shake flasks in LB medium, the advantage of the former appearing in late log phase. MK79 produced more alpha-amylase than MK57, on both per cell and per volume bases, in both mid and late log phases; the maximum advantage of MK79 (on a per volume basis) occurred in late log phase, at which time it produced 3.3 times as much alpha-amylase as MK57. The numbers of copies per cell of both pMK57 and pMK79 were significantly lower than that of pUC8.

Cloning, Molecular↗

PubMed1990

Variation of oxygen requirement with plasmid size in recombinant Escherichia coli.

We have previously found an inverse relationship between certain cell growth parameters and plasmid size for a series of recombinant Escherichia coli strains containing pUC8 or one of a series of pUC8 recombinant derivatives. To extend these results we investigated whether there was a similar variation among our strains in oxygen requirement, which might be related to the differences in growth. During logarithmic growth in shake flasks, oxygen uptake by E. coli strain JM103 containing an 8.7-kb pUC8 derivative (pBS5) was 2.5 times that of JM103 harboring pUC8 (2.7 kb) and 7.5 times that of plasmid-free JM103. Supplementing the medium with acetate eliminated both the growth disadvantage of and the increased oxygen uptake by the strain harboring pBS5 compared with that containing pUC8. In all cases oxygen consumption decreased drastically as cells began and then continued into stationary phase, and no significant difference was seen among the three strains at these times. When the three strains were grown in a fermentor with continuous monitoring of oxygen levels, plasmid-free JM103 outgrew JM103 containing pUC8 or pBS5 at three levels of aeration. The latter two strains grew identically when aeration was high; their growth curves diverged, however, when aeration was low. In the fermentor experiments the point at which the growth of the three strains diverged was coincident with the point of oxygen depletion in the cultures.

Escherichia coli↗

PubMed1989

Oxygen inhibition of globin gene transcription and bacterial haemoglobin synthesis in Vitreoscilla.

A soluble dimeric haemoprotein, structurally and functionally similar to plant and animal haemoglobins, is found in the Gram-negative aerobic bacterium Vitreoscilla sp., strain C1. Vitreoscilla haemoglobin (VtHb) increases in concentration when the cells are exposed to hypoxic conditions. The globin part of VtHb is encoded by a single gene (vgb). An RNA transcript, approximately 500 bases long, specific for vgb was detected after Northern hybridization. The relative amount of this mRNA increased in cells grown at low levels of oxygen. Two enzymes important for haemoglobin function are delta-aminolaevulinic acid synthase (ALAS), which is necessary for haem biosynthesis, and NADH-methaemoglobin reductase, which is necessary to keep VtHb in the physiologically functional ferrous state. An increase in ALAS specific activity under hypoxic conditions preceded the increased haem production. Cellular reductase content also increased when the VtHb increased in cells grown under hypoxic conditions. The ratio of cellular reductase activity to VtHb content remained relatively constant in cells grown under a variety of conditions. The data suggest that in Vitreoscilla the transcription of the globin gene and the biosynthesis of two enzymes important for VtHb function are regulated by oxygen.

5-Aminolevulinate Synthetase↗

PubMed1989

Proximal humeral replacement for tumor. A preliminary report.

Five patients who underwent prosthetic proximal humeral replacement for tumor were evaluated postoperatively at an average of 52 months. Four patients were reasonably satisfied with their results: they were relieved of pain which existed preoperatively and had hand and elbow function. Shoulder rotation was recovered satisfactorily. However, elevation and abduction were significantly compromised. No radiographic evidence of prosthetic loosening was identified.

Adolescent↗