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Biomedical subjects

R I Scott

Publications and source records attributed to R I Scott.

18 recordsLinked to original sources
PubMed1992

Oxygen utilisation by isopenicillin N synthase from Penicillium chrysogenum.

The enzyme isopenicillin N synthase (IPNS) converts delta-(L-alpha- aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N; an equimolar amount of oxygen is used in this oxidative ring closure reaction. Oxygen uptake rates of the reaction catalysed by partially purified IPNS from Penicillium chrysogenum SC 6140 and P2 were measured using an oxygen electrode. In contrast to published properties of Cephalosporium acremonium IPNS, the enzyme from P. chrysogenum was not stimulated by the addition of glutathione and showed reduced stimulation by Fe2+. The analysis of oxygen uptake rates showed the reaction to be first order with respect to oxygen concentration and the Km for ACV to be 0.4 mmol dm-3. The implications of these results for cell-free reactions using this enzyme and penicillin fermentations are discussed.

Biotechnology↗
PubMed1992

Effects of oxygen on recombinant protein production by suspension cultures of Spodoptera frugiperda (Sf-9) insect cells.

Spodoptera frugiperda (Sf-9) insect cells have been grown in serum-free medium in 250-ml spinner flasks. The maximum cell density obtained in these cultures was dependent on the aeration rate of the culture. Similar yields of uninfected cells were obtained when cultures were stirred in spinner flasks at 80 rev min-1 and in a 4-1 stirred-tank bioreactor and the dissolved oxygen in the bioreactor was controlled at 20% of air saturation. Cells were infected with a recombinant baculovirus at different multiplicities of infection: the timing and maximum level of expression of the recombinant protein were dependent on the multiplicity of infection, the cell density at infection, and on the aeration rate of the culture. Oxygen-limited growth resulted in undetectable levels of recombinant protein (< 6 ng recombinant protein 10(-7) cells). Compared with the maximum yields observed in spinner flask cultures, higher levels of recombinant protein were produced when cells were grown and infected in the bioreactor. The level of dissolved oxygen in the bioreactor was controlled at 50% of air saturation.

Animals↗
PubMed1991

Kinetics of the reactions of trioxodinitrate and nitrite ions with cytochrome d in Escherichia coli.

The rate of reaction of trioxodinitrate with reduced cytochrome oxidase d in membrane particles from Escherichia coli at pH 7 and 25 degrees C depends linearly upon [HN2O3-] over the concentration range studied (up to 0.05 mM) and is also first-order in cytochrome d. The known rate of decomposition of trioxodinitrate to give NO- and NO2- is about 4.5-times faster than the rate of reaction of reduced cytochrome d with trioxodinitrate, implying that cytochrome d reacts directly with NO-, with a trapping ratio of between 0.20 and 0.25, rather than with trioxodinitrate. The implications of the facile formation of the NO(-)-nitrosyl complex of cytochrome d for the mechanism of denitrification are discussed with particular reference to the mechanism of N-N bond formation. The reaction of reduced cytochrome d with nitrite (a decomposition product of trioxodinitrate) under these conditions is much slower than that with trioxodinitrate. The kinetics show a biphasic dependence of initial rate upon nitrite concentration. The rate data at low [NO2-] are consistent with saturation of a high affinity site for nitrite, having Vmax = 4.29.10(-9) M s-1 and Km = 0.034 mM. The existence of two binding sites for nitrite is consistent with the suggestion that the cytochrome bd complex contains two cytochrome d haems.

Cytochrome d Group↗
PubMed1984

The place of odds ratios in the study of place, race and differential occupational opportunities.

During the last few decades the study of racial differences in occupational and economic attainment has progressed rapidly, both in terms of theory and research. The ecological study of the racial and ethnic organization of local labor markets has not. The use of odds ratios and occupation-specific categories may provide a fruitful avenue for future research in the study of inequality across space and time.

Black or African American↗
PubMed1984

Place, race and differential occupational opportunities.

A large body of literature investigating the link between black percentage in the community and occupational differentiation, has found disparities to be greater in places where blacks are in a higher proportion. The present paper discusses first, theoretical perspectives and methodological techniques employed in past research; second, examines the issue in terms of the relative odds of the races for incumbency in a series of occupational groupings; and third, analyzes the effect of percentage black on race-linked occupational differentiation across 124 major American cities. Using log-linear procedures, blacks, relative to whites, are much more likely to be in unskilled and semiskilled manual occupations, while whites, relative to blacks, are far more likely to be in white-collar and upper-status positions. Moreover, blacks' denial of access to higher-status positions, their channeling to lower-status work, and their ability to overflow into specific intermediate-status occupations are found to be greatly influenced both by percentage black as well as other community characteristics. The findings are discussed in light of sociological theories suggesting that the issue cannot be understood solely by the competition hypothesis. One must also consider the overflow and queuing explanations.

Black or African American↗
PubMed1983

Photochemical action spectra indicate that cytochrome a/a3 is the predominant haemoprotein terminal oxidase in Acanthamoeba castellanii.

1. Room-temperature CO-reduced minus reduced difference spectra of intact cells of Acanthamoeba castellanii show the presence of CO-reacting haemoproteins in cells from the early-exponential, late-exponential and stationary phases of growth. 2. The relative rates of reaction with CO of the two haemoproteins differ; that of cytochrome a/a3 with CO is complete within 1 min of bubbling with CO, whereas that of cytochrome b takes longer than 90 min. 3. Photochemical action spectra reveal cytochrome a/a3 as the predominant haemoprotein oxidase at all stages of growth. 4. It is concluded that the alternative oxidases known to be present in these organisms are not cytochromes.

Amoeba↗
PubMed1983

Photochemical action spectra of CO-liganded terminal oxidases using a liquid dye laser.

A method of obtaining photochemical action spectra for the relief of CO inhibition of respiration is described. Continuous readout from a membrane-covered oxygen electrode of dissolved oxygen in a stirred suspension of microorganisms under CO-oxygen gas mixtures in an open reactor enables measurement of increased respiration on illumination. Advantages presented by the use of a liquid dye laser include high intensity of emission and narrow spectral bandwidth; just two dyes (rhodamine 6G and rhodamine 110) are required to match the alpha-absorption maxima of the CO complexes of all known bacterial and mitochondrial terminal oxidases.

Carbon Monoxide↗
PubMed1983

Industrial shifts, female employment, and occupational differentiation: a dynamic model for American cities, 1960-1970.

Sex-linked occupational differentiation has been seen as influenced by both the industrial structure of the economy and the sex composition of the labor force. Here, with a sample of 70 SMSAs, it was found (a) that the odds of men relative to women of joining professional and managerial occupations increased between 1960 and 1970, and (b) that this increase was dependent on the growth of tertiary industries and the greater number of women joining the cash economy. The observed effect of industrial shifts on sex-occupational differentiation, however, is argued to be a spurious consequence of the gender-composition of the work force. Specifically, the development of tertiary industries generates greater demand for female labor. Intensive recruitment of women to the labor force in turn increases occupational differentiation because females, in sex-typed labor markets, are likely to be channeled in disproportionate numbers away from upper-status occupations. The findings demonstrate that traditional modernization theory is unable to account for this. However, the results lend support to expectations derived from a labor market sex-segmentation approach.

Career Choice↗
PubMed1982

Cytochrome a620 in Tetrahymena pyriformis. Reactions with carbon monoxide and oxygen at subzero temperatures and photochemical action spectra.

1. Mitochondria-enriched fractions of the ciliate protozoan Tetrahymena pyriformis ST contained CO-reacting cytochromes b560 and a620. 2. A non-photodissociable oxygen-containing compound of cytochrome a620 was formed in whole cell suspensions at -114 degrees C after photolysis of CO in the presence of 200 microM-O2. 3. Electron transport, indicated by the oxidation of cytochrome a620 and cytochrome c, occurred at temperatures higher than -72 degrees C. 4. Photochemical action spectra for the relief of respiratory inhibition of whole cells by CO obtained by using a liquid dye laser indicate that the only CO-reacting terminal oxidase detectable was cytochrome a620. 5. It is concluded that the alternative electron transport chains in this organism utilize non-cytochrome terminal oxidases.

Carbon Monoxide↗
PubMed1982

A re-examination of the cytochromes of Escherichia coli using fourth-order finite difference analysis: their characterization under different growth conditions and accumulation during the cell cycle.

The cytochromes of Escherichia coli K12 have been studied using low-temperature (77 K) difference spectrophotometry. Numerical (i.e. fourth-order finite differences) analysis resolved the alpha band of reduced minus oxidized spectra of whole cells from aerobically growth cultures into five components, with absorption maxima at 548, 551.5, 555.5, 560 and 563 nm. Using the same differencing intervals, numerical analysis of cells grown under oxygen-limited conditions revealed only two components, at 555.5 and 559 nm. Similar analysis of cells grown anaerobically with fumarate as electron acceptor revealed four absorption maxima, at 548, 550.5, 555.5 and 559 nm. Membrane particles from aerobically grown cells showed the same absorption bands as intact cells; preliminary evidence was obtained for a component of the 555.5 nm band that could be relatively easily washed from the membrane. The contribution of cytochrome o or its CO-liganded form to the alpha region could not be determined by numerical analysis. We conclude, in contrast to a previous application of numerical analysis to the cytochromes of E. coli, that growth under anaerobic or oxygen-limited conditions results in the appearance of cytochromes spectrally distinct from those in aerobically grown cells. An attempt has been made to reconcile the presence of multiple components detected in the alpha region by numerical analysis of aerobically grown cells with the diverse components described by others. Quantification of cytochromes revealed by numerical analysis in aerobically grown cells separated into size (and thus age) classes by zonal centrifugation showed that the major components accumulated continuously, probably exponentially, throughout the cell cycle.

Aerobiosis↗
PubMed1981

Respiratory biogenesis during the cell cycle of aerobically grown Escherichia coli K 12. The accumulation and ligand binding of cytochrome o.

A quantitative assay is described for the measurement of cytochrome o in intact cells of E. coli. The procedure involves flash photolysis of the CO-liganded, reduced enzyme in the absence of O2 at temperatures (approx. -100 degrees C) at which the rate of recombination of CO is immeasurably slow. Other CO-binding pigments known to be present, particularly cytochrome d, are excluded from the photodissociation spectrum under these conditions. Measurement of the content of cytochrome o in bacteria separated into size (and thus age) classes by zonal centrifugation shows that the cytochrome accumulates continuously, probably exponentially, throughout the cell cycle and thus constitutes a constant proportion of cell protein during the cycle. The velocity of recombination of CO with cytochrome o at -65 degrees C is invariant over the cell cycle.

Carbon Monoxide↗
PubMed1981

Respiratory biogenesis during the cell cycle of aerobically grown Escherichia coli K12. The accumulation of iron-sulphur clusters and their orientation in the membrane.

The magnitudes of four signals detectable in intact cells by electron paramagnetic resonance spectroscopy, and assigned to membrane-associated protein, increase continuously during the cell cycle of Escherichia coli K12. Studies on membrane multilayers prepared from cells separated according to their age in the cycle suggest that the orientation within the membrane of the ferredoxin-type signal is invariant throughout the cycle.

Cell Cycle↗
PubMed1981

The light-reversible binding of carbon monoxide to cytochrome a1 in Escherichia coli K12.

Difference spectra at 77 K of intact Escherichia coli K 12 grown under oxygen-limited conditions revealed the presence of cytochrome a1. In the presence of CO, the band of the reduced form, observed in both the alpha and gamma regions of the spectrum, was decreased. Dual-wavelength scanning spectrophotometry at sub-zero temperatures revealed a flash-dissociable CO-binding pigment with a broad band around 595 nm, identified as cytochrome alpha 1. Photolysis in the presence of O2 revealed no such band in difference spectra where the reference spectrum was that of the CO-liganded form, a result consistent with the binding of O2 to cytochrome a1. Repeated cycles of photolysis and recombination of the cytochrome with CO were demonstrated at --46 degrees C. The apparent energy of activation for the reaction with CO was 10.9 kcal mol-1 (45.6 kJ mol-1). The results are discussed in relation to previous assumptions and results regarding ligand binding to cytochrome a1 and the function of this cytochrome in bacterial respiration.

Carbon Monoxide↗
PubMed1980

Low-temperature spectral and kinetic properties of cytochromes in Escherichia coli K-12 grown at lowered oxygen tension.

Escherichia coli K-12 was grown in batch culture in a medium containing succinate as carbon source, supplemented with casein hydrolysate, and with a rate of oxygen supply that resulted in dissolved O2 tension falling to 10% of saturation in the latter stages of growth. Cytochromes in such cells were qualitatively indistinguishable from those present in cells grown under conditions of vigorous aeration where dissolved O2 tensin remained greater than 80% saturation. Spectra recorded at 77 K and their fourth-order finite difference analyses revealed the absence of cytochrome b-558 and only low concentrations of cytochromes a1 and d(a2). At low temperatures, the reaction of cytochrome o with O2 in intact cells, grown under lowered O2 tension, proceeds through the same stage as observed previously in cells grown with vigorous aeration (Poole, R.K., Waring, A.J. and Chance, B. (1979) Biochem. J. 184, 379-389). However, much higher temperatures are required for comparable progress of the reaction in cells grown at lowered O2 tensions. AT 91 degrees C, the reaction with O2 involves ligand binding to give intermediate(s) with spectral characteristics similar to those of the reduced oxidase-CO complex. Temperatures of approx. -79 degrees C are required for the observation of biphasic kinetics and the attainment of an 'end point' in the reaction, features that are seen at temperatures below -98 degrees C in cells from vigorously-aerated cultures. At -32.5 degrees C, oxidation of cytochrome o is observed. The energy of activation for this reaction at low temperatures is 29.9 kJ x mol-1. Binding with CO, in contrast to binding with O2, is characterized by high photolytic reversibility and appears to be less affected by the degree of aeration of cells during growth.

Cold Temperature↗
PubMed1980

The orientation of cytochromes in membrane multilayers prepared from aerobically grown Escherichia coli K12.

Centrifugation of membrane vesicles, prepared from ultrasonically disrupted Escherichia coli K12, on to a planar surface followed by slow, partial dehydration results in a high degree of parallel orientation of the membrane planes with respect to each other and the supporting surface. Rotation of such membrane multilayers about a single axis parallel with the membrane planes within the magnetic field of an electron paramagnetic resonance (e.p.r.) spectrometer allows the orientation of anisotropic paramagnetic centres to be deduced. Computer simulations of the angular dependence of cytochrome e.p.r. spectra show two, or perhaps three, cytochromes, well-oriented with respect to the membrane plane. A low-spin cytochrome is oriented with the normal to its haem plane lying in the membrane plane. One (or perhaps two) high-spin cytochrome(s) lies with its haem plane making an angle of 45 degrees with the membrane plane. The orientation of the low-spin cytochrome haem is thus the same as that of haems in b-type cytochromes and cytochrome oxidases of the a type found in the mitochondria of higher animal and microbial cells and the bacterium Paracoccus denitrificans (Erecińska et al., 1979). The possible identity of this low-spin component as the terminal oxidase, cytochrome o, is discussed.

Computers↗
PubMed1980

Adenosine triphosphatase activity and its sensitivity to ruthenium red oscillate during the cell cycle of Escherichia coli K12.

A procedure has been developed for the large-scale fractionation into size and age classes of bacteria from exponentially growing cultures of Escherichia coli K12 by centrifugation through an equivolumetric gradient of sucrose in a zonal rotor. The resolution attained is superior to that in methods of this type that have been described previously. The activity of adenosine triphosphatase (ATPase) was assayed in extracts from bacteria separated into size classes by this method and from synchronous cultures prepared by size selection. Activity approximately doubled during a cell cycle, but the experimental data did not fit models of either continuously or exponentially increasing activity during the cycle. It is suggested that ATPase activity oscillates during the cell cycle with maxima at about 0.37 and 0.80 of a cycle. The fluctuations in activity greatly exceed the variations due to experimental error and, in the case of synchronous cultures, do not arise from perturbations in growth behaviour following zonal gradient selection. Sensitivity of ATPase activity to 75 micrometer-Ruthenium Red also fluctuates during the cell cycle, with maximum inhibition (60 to 80%) occurring near the middle of the cycle, a time that does not coincide with maximum enzyme activity.

Adenosine Triphosphatases↗