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Biomedical subjects

D A Webster

Publications and source records attributed to D A Webster.

At least 55 records · Page 3Linked to original sources
PubMed1987

Purification and partial characterization of the membrane-bound cytochrome o(561,564) from Vitreoscilla.

Cytochrome o(561,564) terminal oxidase was solubilized from the membrane fraction of the bacterium Vitreoscilla sp., strain C1, and purified by differential pH dialysis, gel filtration chromatography, and ion-exchange chromatography. Subunit molecular weights, determined on sodium dodecyl sulfate-polyacrylamide gels by the Ferguson plot method, were 49,500 and 23,500. There were two protohemes IX, two coppers, and 45 mol of phosphorus per mole of protomer (73,000). The molecular weight of the cytochrome o complex estimated by chromatography on Sephacryl-400 in deoxycholate was 265,000, which is consistent with the enzyme complex under these conditions being a dimer (146,000) with the remaining molecular weight contribution arising from bound phospholipid, deoxycholate, and possibly other, smaller subunits. Difference spectra of the dithionite-reduced enzyme have split alpha absorption maxima at 561 and 564 nm at room temperature and 558 and 561 nm at 77 K. The CO difference spectrum at room temperature has absorption maxima at 570, 534, and 416 nm. Dissociation constants for CO and cyanide binding to the reduced and oxidized forms of the oxidase are 5.2 microM and 3.5 mM, respectively. The hemes in the cytochrome are one electron accepting centers, both with midpoint potentials around +165 mV at pH 7.0. The enzyme is highly autoxidizable, and its menadiol oxidizing activity is stimulated by phospholipids.

Chromatography, Gel↗
PubMed1987

Identification of b, c, and d cytochromes in the membrane of Vitreoscilla.

Cytochromes b, c, d, and o were identified by spectroscopic analysis of respiratory membrane fragments from Vitreoscilla sp., strain C1. Carbon monoxide difference spectra of the reduced membranes had absorption maxima at 416, 534, and 571 nm (ascribed to cytochrome o) and 632 nm (cytochrome d). Derivative spectra of the pyridine hemochromogen spectra of the membranes identified the presence of b- and c-type cytochromes in Vitreoscilla. The cyanide binding curve of the membranes was biphasic with dissociation constants of 2.14 mM and 10.7 which were assigned to cytochrome o and cytochrome d, respectively. Membranes bound carbon monoxide with dissociation constant 3.9 microM, which was assigned to cytochrome o. Cytochrome c556 and a NADH-p-iodonitrotetrazolium violet reductase component were partially purified from Vitreoscilla membranes.

Carbon Monoxide↗
PubMed1986

Photodissociation of oxygenated cytochrome o(s) (Vitreoscilla) and kinetic studies of reassociation.

Oxygenated cytochrome o(s) from Vitreoscilla was photodissociated by a laser flash but the quantum yield was low. The rebinding of oxygen to the ferrous cytochrome proceeded monophasically, and the second order rate constant was 7.8 X 10(7) M-1 s-1, the off rate constant 5.6 X 10(3) s-1, and the calculated dissociation constant for the oxygenated compound 7.2 X 10(-5) M at pH 7.3 and 25 degrees C. Rapid scanning spectroscopy revealed the formation of chytochrome o-O2 directly from ferrous chytochrome o and oxygen without any evidence for an intermediary formation of Compound D, another type of oxygenated chytochrome o. Photodissociation in solution containing CO/O2 mixtures resulted in rapid binding of oxygen followed by slow replacement by CO. This property as well as the photodissociability of chytochrome o-O2 suggests that the heme iron of the compound is in the ferrous state. In addition, the primary oxygen compound was fairly stable and did not decay further in the absence of CO, in marked contrast with that of mammalian cytochrome oxidase primary oxygen compound which rapidly decayed. This result suggests a possible role of this cytochrome as an oxygen carrier or storage.

Carbon Monoxide↗
PubMed1986

Cyanide- and carbon monoxide-resistant mutants of Vitreoscilla: altered cytochromes and respiratory properties.

Two respiratory mutants of the aerobic bacterium, Vitreoscilla, have been studied: a CO-resistant mutant that can grow in 50% CO-50% oxygen, and a cyanide-resistant mutant that can grow in 1 mM KCN. Wild-type cells are unable to grow under either condition. This report presents evidence that the resistance of the CO mutant is due to an altered membrane-bound cytochrome o [cytochrome o(m)], and that of the cyanide mutant is due to the presence of an increased amount of cytochrome d, which has a lower affinity for cyanide than cytochrome o(m). The evidence was obtained from spectral studies on the three types of intact cells as well as enzymatic and ligand-binding techniques on the cytoplasmic cytochromes o[cytochrome o(s)] and the respiring membrane vesicles isolated from these cells. Carbon monoxide difference spectra of intact cells revealed a 5-nm shift in an absorption maximum of a CO-binding pigment in the CO mutant relative to that of the wild type. The formation of oxygenated cytochrome o(s) and its conversion to the reduced form when the cells became anaerobic due to cellular respiration were inhibited when 1 mM KCN was added to a cell suspension of wild-type cells; the cyanide mutant cells showed resistance to cyanide in this experiment. Cytochrome o(s) purified from all three cell types had identical physical, electron transferring, and ligand binding properties within experimental error. Respiring membrane vesicles isolated from the two mutants showed more resistance to inhibition by cyanide and carbon monoxide than those from the wild type. Carbon monoxide difference spectra of these membrane vesicles revealed that there was a fivefold increase in the amount of cytochrome d in the cyanide mutant relative to the wild type. A CO absorption band of the membrane-bound cytochrome o in the CO mutant membrane vesicles showed a 5-nm shift relative to that of the wild type.

Carbon Monoxide↗
PubMed1986

Experience with the variable axis knee prosthesis.

In an evaluation of 245 variable axis total knee arthroplasties, no significant deterioration of results were found five to nine years after implantation. The patellar problems originally reported diminished with the use of a patellar button, widening of the flange on the femoral component and the increased use of lateral retinacular release. The local complications were: severe patellar problems, 6.9%; ligamentous laxity, 2.9%; aseptic loosening, 1.2%; deep infections, 2.0%; supracondylar fractures, 1.2%; peroneal nerve palsy, 1.6%.

Aged↗
PubMed1985

Biphasic recombination of photodissociated CO compound of cytochrome o(s) from Vitreoscilla.

The soluble cytochrome o from Vitreoscilla contains two identical subunits and two hemes. The reduced form binds 2 mol of CO in a cooperative manner with a Hill coefficient near 2 (Tyree, B., and Webster, D. A. (1978) J. Biol. Chem. 253, 6988-6991). This carbonyl compound was photolysed with a dye laser and recombination followed at 437 or 420 nm where maximal absorbance changes were registered. Recombination kinetics were biphasic, and the fast phase was approximately 10 times the rate of the slow phase. Apparent rate constants of both phases showed a nonlinear dependence on CO concentration, respectively, in conformity with a reaction scheme which assumes the transient formation of an intermediate species in both slow and fast reactions. A study of temperature dependence of the reactions gave EA = 2.7 kcal/mol for the slow reaction and EA = 3.2 kcal/mol for the fast reaction below 23 degrees C; above this temperature the slope of the Arrhenius plot for the fast reaction became positive. Maximal rates for both phases were around pH 6.5 and fell to approximately 40% of maximal at pH 12. The binding reaction was affected by even a low concentration of sodium dodecyl sulfate (0.0025%), which changed both the kinetic constant of each phase and the relative contribution of each phase to the reaction. A model which assumes the existence of fast and slow reaction conformers in equilibrium is proposed.

Carbon Monoxide↗
PubMed1985

Complications of Variable Axis total knee arthroplasty.

Three hundred seventy-six patients were treated by a Variable Axis total knee arthroplasty between 1974 and 1980 and were reviewed for mechanical and orthopedic complications of their surgery. Sixty-five patients had significant complications. Twenty-eight (7.4%) patients developed significant problems with their patellae, 13 (3.4%) patients required reoperation for patellar realignment, and four (1.0%) patients had a patellar resurfacing for retropatellar pain. Two (0.5%) cases of aseptic loosening of the femoral component required revision surgery. There was no aseptic loosening of the tibial component. Other complications included a ligamentous instability (2.9%), postoperative joint stiffness (1.6%), peroneal nerve palsy (1.3%), supracondylar fractures (1.0%), and postoperative joint infection (1.8%).

Femoral Fractures↗
PubMed1983

Spectral evidence for the existence of a second cytochrome o in whole cells of Vitreoscilla.

Cytochrome o, a protoheme IX pigment, has been proposed as the terminal oxidase of the filamentous bacterium, Vitreoscilla. Aerobic and anaerobic photolysis of CO-liganded whole cells demonstrated the presence of a second CO-reactive pigment, cytochrome o'. At temperatures lower than -100 degrees C, anaerobic photolysis dissociated only about 25% of the total CO-liganded components to reveal the unliganded cytochrome o'. At these temperatures, the photolysis of cytochrome o could not be demonstrated. At warmer temperatures, recombination of CO with the reduced cytochrome o' occurred with an apparent energy of activation of 5.8 kcal/mol. Aerobic photolysis of whole cells demonstrated two oxygen-bound intermediates. At temperatures lower than -95 degrees C, a spectrally distinct compound with absorption maxima at 428, 534, and 564 nm appeared (form I'); the apparent second order rate constant (k+1) for the formation of this intermediate was found to be 9.1 M-1 s-1, the reverse rate (k-1) was 9.9 X 10(-5) s-1, and the equilibrium constant (Kd) was 1.1 X 10(-5) M. This oxygen intermediate of cytochrome o' is spectrally and kinetically similar to the oxygen intermediate of cytochrome o seen in Escherichia coli. At temperatures warmer than -90 degrees C, photolysis of aerobic samples resulted in the immediate formation of a second oxygen-bound intermediate (form I) with absorption maxima at 422, 534, and 564 nm. This second intermediate results from the binding of oxygen to the cytochrome o (oxygenated cytochrome o). These data support the proposal that whole cells of Vitreoscilla contain two alternative pathways of electron transport, one terminating with cytochrome o and the other with cytochrome o'.

Anaerobiosis↗
PubMed1983

Mechanical and functional properties of implanted freeze-dried flexor tendons.

Tendons harvested from donor dogs were freeze-dried and implanted as free allografts in the paws of recipient experimental animals; the tendons were subsequently tested for mechanical properties, clinical function, and histologic appearance. Free tendon autografts, normal tendons in the operated experimental paw, and normal tendons in the unoperated paw were used as controls and compared with the allografts. Mechanically, the free tendon allografts and autografts were similar. The ultimate strength of the two grafts was statistically the same but significantly less, about one-third, than that of normal tendons. The implanted allografts appeared to be tolerated well by the host and to allow flexor tendon function similar to that allowed by autografts. The histologic appearance of the allograft and autograft tendons was similar at three and six months. Freeze-dried flexor tendon allografting is a satisfactory alternative to free tendon autografting.

Animals↗
PubMed1983

Freeze-dried flexor tendons in anterior cruciate ligament reconstruction.

Flexor tendons were harvested from donor dogs, freeze-dried, and reimplanted into six mongrel dogs that were made surgically anterior cruciate ligament (ACL) deficient. After one month of immobilization the dogs were allowed to run free for eight months. At this time all ACL substitutes were found to be intact and repopulated by host cells. Mechanical testing showed the freeze-dried allograft ACL to be significantly weaker than the normal control ligament but similar in strength to implanted tendon autograft ligament substitutes.

Animals↗
PubMed1981

The variable-axis knee prosthesis. Two-year follow-up study.

This study reports the clinical results following knee replacement with the variable-axis knee prosthesis. All patients have been followed for a minimum of two years. The first consecutive fifty-two patients, comprising a total of fifty-five knee replacements, were personally reviewed by an independent evaluator. The results on a subjective basis consisted of 79 per cent good to excellent results, 15 per cent fair results, and 6 per cent failures. Failures were related to infection or continued pain. Fair results reflected some patellar discomfort or instability. There was no detectable clinical loosening of the components in any patient. To date, the design of the variable-axis prosthesis appears to offer promise in the control of the complication of prosthetic loosening.

Adult↗
PubMed1980

Effect of growth conditions on yield and heme content of Vitreoscilla.

Vitreoscilla, a gliding bacterium in the Beggiatoaceae, is an obligate aerobe in which cytochrome o functions as the terminal oxidase. Protoheme IX is the only heme type present in this organism. The yield and heme content of Vitreoscilla cells grown in yeast extract, peptone, and acetate were dependent on growth conditions. Cells harvested in early stationary phase contained roughly three times as much heme as cells in early log phase. There was an optimal shaking rate for maximum heme content of cells harvested in stationary phase at fixed initial nutrient concentration. The heme content of cells grown at a fixed shaking rate increased from 5 nmol/g (wet weight) in media which had low nutrient concentration to a maximum of 45 nmol/g (wet weight) in media which had high nutrient concentration, and there was a corresponding sixfold increase in cytochrome o content and an eightfold increase in respiratory rate, evidence that some of the additional heme was incorporated into respiratory pigments. Heme content may be controlled jointly by competition for oxygen and availability of nutrients. Temperature and initial pH affected the growth rate but not the final yield or heme content. Growth rate was optimal at pH 8.0 to 8.5. A defined medium for Vitreoscilla, which is based on glutamate as the carbon source, is described; the other organic components of this medium are acetate, tryptophan, thiamine, biotin, and riboflavin.

Aerobiosis↗
PubMed1979

Silver polymethyl methacrylate antibacterial bone cement.

An improved antibacterial bone cement was sought based on the addition of low concentrations of inorganic silver compounds to polymethyl methacrylate. Composites with AgCl, Ag-AgCl, Ag2O, Ag2SO4 and Ag3PO4 in concentrations of 0.05% to 1% by weight, were tested in vitro against bacterial cultures. All were effective, but Ag2SO4 was especially so, even after 7 weeks of incubation in normal saline. Compressive strength of the cement was not affected by these additions, except in the case of Ag2O. Biocompabibility tests in rabbit muscle for up to 12 weeks showed no significant difference between the Ag -PMM and plain PMM in tissue reactivity, both being minimal. These features, coupled with the broad spectrum of antibacterial activity and low allergic potential of silver, make Ag-PMM an attractive alternative to conventional organic antibiotic/bone cement composites.

Acrylic Resins↗