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Biomedical subjects

S W Edwards

Publications and source records attributed to S W Edwards.

At least 109 records · Page 6Linked to original sources
PubMed1988

Impaired neutrophil killing in a patient with defective degranulation of myeloperoxidase.

A case of recurrent, superficial abscesses in an 18 year old girl, is described. Staphylococcus aureus was the pathogen most often implicated and on several occasions the abscesses required surgical drainage. Defects in humoral immunity, neutrophil chemotaxis or opsonophagocytosis were not observed. However, her neutrophil's ability to kill ingested S. aureus in vitro was impaired. This was associated with impaired luminol-dependent chemiluminescence in response to stimulation by either latex beads, or the chemotactic peptide FMLP plus cytochalasin B. Oxygen uptake and superoxide anion production were normal but release of myeloperoxidase by this patient's neutrophils occurred more slowly and to a lower extent than in control cells. These data suggest that the recurrent infections and diminished in vitro neutrophil bactericidal activity observed in this patient are associated with impaired degranulation of myeloperoxidase.

Abscess↗
PubMed1988

Myeloperoxidase secretion during phagocytosis: a case of a patient with impaired bactericidal activity.

We describe a case of a 5-year-old male patient with prolonged and extensive osteomyelitis of the left femur. Staphylococcus aureus was grown from blood cultures taken upon admission and also from pus drained from an incised hip joint. A defect in immune function was suspected and neutrophil function was assessed. Chemotaxis and phagocytosis were normal, but phagocytosed S. aureus were not killed as efficiently as in control neutrophils. No inherent defect in the ability of these neutrophils to generate reactive oxidants was observed, but an unusual luminol-dependent chemiluminescence response was obtained during phagocytosis of latex beads or opsonized S. aureus: This was characterized by an initial rapid, but transient increase occurring within 1 min of addition of phagocytic stimulus. Whereas during phagocytosis of latex beads by control neutrophils less than 1% of the total myeloperoxidase activity was detected extracellularly, up to 15% was released from the patient's neutrophils. We propose that release of myeloperoxidase from the patient's neutrophils during phagocytosis reduces the intraphagosomal concentration of this enzyme and thus impairs the efficiency of intracellular killing of S. aureus.

Chemotaxis, Leukocyte↗
PubMed1987

Oxidative inactivation of myeloperoxidase released from human neutrophils.

Within 1 min of stimulation of human neutrophils by the chemotactic peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) plus cytochalasin B, myeloperoxidase (together with other granule enzymes) was secreted and detected extracellularly. In contrast with the other granule constituents assayed (vitamin B12-binding protein and beta-glucuronidase), the activity of released myeloperoxidase rapidly decreased, so that, by 10 min after stimulation, only about 5% of the total cellular activity was detected. This inactivation was shown to be dependent on oxidant generation during the respiratory burst, since inactivation was not observed (a) after stimulation of anaerobic suspensions or (b) after release from neutrophils from a patient with chronic granulomatous disease; purified myeloperoxidase was rapidly inactivated after incubation with H2O2, presumably owing to the formation of an inactive enzyme-H2O2 complex. These results show that experiments designed to assess the role of myeloperoxidase in neutrophil functions which utilize assays based on peroxidase activity will grossly underestimate this enzyme if oxidant generation during the respiratory burst has also been activated.

Granulomatous Disease, Chronic↗
PubMed1987

Protein synthesis is activated in primed neutrophils: a possible role in inflammation.

Circulating human neutrophils exhibited low rates of protein biosynthesis, as determined by their ability to incorporate [35S]methionine into TCA-precipitable material. Exposure of cells to the chemotactic peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) increased their rate of protein synthesis, and the maximal stimulation of biosynthesis by this inflammatory agent was observed at 0.1 microM: this concentration of chemotactic peptide "primed" neutrophil activity and only activated the oxidase of these cells by 8% of maximum. The newly-synthesized proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis and compared with those synthesized in control cells. Two classes of proteins were observed in "primed" cells. The first of these comprised proteins whose rate of biosynthesis changed very little upon "priming" whereas the second class comprised proteins whose rate of synthesis increased greatly after exposure to chemotactic peptide. The fMet-Leu-Phe stimulated protein synthesis was inhibited by actinomycin D and cycloheximide showing that this phenomenon required both transcription and translation. We propose that these fMet-Leu-Phe regulated proteins play an important role in the function of neutrophils during an inflammatory response.

Cycloheximide↗
PubMed1987

CO-reacting haemoproteins of neutrophils: evidence for cytochrome b-245 and myeloperoxidase as potential oxidases during the respiratory burst.

Room temperature, CO-difference spectra of intact rat polymorphonuclear leucocytes (neutrophils) revealed the presence of a number of CO-binding haemoproteins. Absorption maxima at 413, 540 and 570 nm were attributed to the CO-complex of cytochrome b-245 whereas an absorption maximum at 595 nm was assigned to the contribution from a myeloperoxidase complex, since an identical absorption maximum was observed in CO-difference spectra of purified myeloperoxidase in the presence of H2O2. Photochemical action spectra for the relief of CO-inhibited O2 uptake revealed contributions from both cytochrome b-245 and myeloperoxidase. The potential of these two O2- and CO-binding haemoproteins to function as oxidases during the respiratory burst is discussed.

Animals↗
PubMed1987

Oxygen-dependent killing of Staphylococcus aureus by human neutrophils.

Luminol-dependent chemiluminescence was used as a monitor of reactive oxidant generation during phagocytosis of Staphylococcus aureus by human neutrophils. Reactive oxidants play a crucial role in the killing of this organism because: (a) S. aureus was killed most rapidly when the rate of increase of chemiluminescence was greatest; (b) neutrophils which had been activated to generate reactive oxidants by re-aeration of anaerobic suspensions killed this bacterium more efficiently than control suspensions; and (c) neutrophils from a patient with chronic granulomatous disease could neither generate reactive oxidants nor kill S. aureus.

Aerobiosis↗
PubMed1987

Luminol- and lucigenin-dependent chemiluminescence of neutrophils: role of degranulation.

The role of myeloperoxidase in luminol- and lucigenin-dependent chemiluminescence of stimulated human neutrophils has been investigated using purified myeloperoxidase and anti-(human myeloperoxidase) antiserum. This antiserum has been used as a specific enzyme inhibitor to assess myeloperoxidase-dependent neutrophil functions in single preparations of cells, thus overcoming the limitations inherent in other approaches using non-specific haem-inhibitors or myeloperoxidase-deficient neutrophils. The results show that luminol-dependent chemiluminescence is largely dependent on both oxidase activity and degranulation (of myeloperoxidase), while lucigenin monitors oxidase activity independently of the extent of degranulation. Since oxidase activation can occur in the absence of degranulation, assays utilizing luminol-dependent chemiluminescence to measure oxidant generation by stimulated neutrophils should include saturating levels of exogenous myeloperoxidase to overcome this problem.

Acridines↗
PubMed1987

Temperature-compensated ultradian variation in cellular protein content of Acanthamoeba castellanii revisited.

Synchronous cultures of the soil amoeba Acanthamoeba castellanii, established in the laboratory by selection procedures, show oscillations of total cell protein, as well as respiration. Previously, time series analysis by Aspect, a University of Warwick Computer Unit Program, showed an average period of 76 min. A reanalysis by least-squares rhythmometry revealed that, in general, the analyses accompanying the original paper and those in this replication agree very well. The periods extracted by the two approaches differ by only 1 to 5 min. In one case, when the original analysis did not detect a statistically significant period, however, the analysis used here does so. While the incubation temperatures used varied from 20 to 30 degrees C and cell division time increased from 7.8 hr at 30 degrees C to 16 hr at 20 degrees C, the metabolic bioperiodicity appeared to be temperature compensated. Decimation studies indicate that the sampling interval of 5 to 15 min must not be further reduced. It is promising to incorporate the study of ultradian metabolic rhythms into a broad spectral approach, i.e., for a concomitant assessment of ultradian, circadian, and infradian rhythms.

Activity Cycles↗
PubMed1987

Temperature-compensated ultradian rhythms in lower eukaryotes: timers for cell cycles and circadian events?

During the cell cycles of synchronous cultures of several different yeasts and protozoa, oscillations in the rate of respiration and of total cellular protein content have been demonstrated. Energy supply (from mitochondrial oxidative phosphorylation) and energy demand (biosynthetic reactions, especially protein accumulation) are closely coupled oscillating systems. Phase correspondence between O2 consumption rates, intracellular ADP pool size, and total cellular protein indicates that it is energetic demand that determines mitochondrial activity (respiratory control in vivo). The dynamics of the coupled oscillators indicate that the rate-determining control circuit operates on a time scale expected of epigenetic reactions (transcription and translation). Thus the energy-yielding reactions are enslaved to the slower time constants of biosynthesis. Temperature compensation indicates a timing function, and a common phase reference point makes subcycles commensurate with the cell cycle. It is suggested that cell-cycle timing, by counting subcycles in growing cells, may become dominated by circadian control in slowly growing natural populations and that the same subcycles may be used for circadian timekeeping. The discontinuous nature of growth suggests extensive and rapid turnover of macromolecular cell components organized in alternating temporal compartments of biosynthetic and degradative processes, reminding us that "Structures are slow processes of long duration, functions are quick processes of short duration" (von Bertalanffy).

Activity Cycles↗
PubMed1986

Regulation of superoxide generation by myeloperoxidase during the respiratory burst of human neutrophils.

The role of myeloperoxidase in the regulation of the respiratory burst of human neutrophils activated by the chemotactic peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) plus cytochalasin B was determined by using anti-(human myeloperoxidase) antibody. The respiratory burst activated under these conditions consisted of an initial (1-2 min) phase with high rates of O2 uptake, luminol-dependent chemiluminescence and superoxide radical (O2-.) generation and a second, more sustained, phase of lower magnitude of chemiluminescence and O2 uptake: O2-. generation did not occur during this second phase. In cell suspensions stimulated in the presence of anti-(human myeloperoxidase) antibody, the magnitude of the initial phase of both O2 uptake and O2-. generation was unaffected, but these high rates were maintained over much longer periods than in control suspensions. It is therefore proposed that a product of myeloperoxidase normally regulates the duration of O2-. generation during the respiratory burst, possibly by inhibition of NADPH oxidase.

Antibodies↗
PubMed1986

Formation of myeloperoxidase compound II during aerobic stimulation of rat neutrophils.

We have made simultaneous spectrophotometric and O2 measurements on suspensions of rat neutrophils during activation of the respiratory burst. Under aerobic conditions an absorption increase attributable to myeloperoxidase compound II was observed in parallel with the rapid phase of O2 uptake. Identification of this compound was confirmed by analysis of a spectrum obtained with purified myeloperoxidase and H2O2. Whereas a second addition of stimulus did not increase O2 uptake any further, a second phase of myeloperoxidase release and compound II formation was observed. These results suggest that in vivo myeloperoxidase reacts with H2O2 generated via the respiratory burst to form compound II under conditions in which the chlorination reaction would be the expected major pathway.

Aerobiosis↗
PubMed1985

The Tetrahymena rRNA intron self-splices in E. coli: in vivo evidence for the importance of key base-paired regions of RNA for RNA enzyme function.

We have developed an in vivo RNA splicing assay for the self-splicing rRNA intron of Tetrahymena thermophila using E. coli as the host. A DNA fragment containing the intron sequence has been cloned into M13mp83 so that expression of the beta-galactosidase alpha-fragment is dependent upon intron excision from the mRNA precursor. Plaque phenotypes correlate well with levels of excised intron RNA. Point mutations were made by oligonucleotide-directed mutagenesis in conserved sequences P, Q, and S. All showed reduced splicing, agreeing with mitochondrial genetic data for S and providing the first direct evidence that P and Q are functionally important. The results support the hypothesis that base-pairing of R with S and P with Q is important for intron structure and function.

Base Composition↗
PubMed1984

Oxygen-radical production during inflammation may be limited by oxygen concentration.

The relationship between oxygen-radical production by rat polymorphonuclear leucocytes and O2 concentration was established by the measurement of luminol-dependent chemiluminescence at defined O2 concentrations. The O2 concentration that gave 50% of the maximum stimulated oxygen-radical production was 31 +/- 9 microM for non-opsonized latex beads and 22 +/- 9 microM for chemotactic peptide. The O2 concentration in rheumatoid synovial fluid was approx. 30 microM. It is therefore proposed that radical production at an inflammatory site may be limited by O2 concentration.

Animals↗
PubMed1984

A comparison of the glycosomes (microbodies) isolated from Trypanosoma brucei bloodstream form and cultured procyclic trypomastigotes.

Highly purified glycosomes were isolated from Trypanosoma brucei bloodstream forms and cultured procyclic trypomastigotes. A comparison of the specific activities of glycosomal enzymes revealed that glycosomes from insect stages had decreased levels of hexokinase, phosphoglucose isomerase, phospho-fructokinase, fructose-bisphosphate aldolase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase, but contained increased levels of adenylate kinase, malate dehydrogenase and phosphoenolpyruvate carboxykinase. Glycosomes from bloodstream forms were almost totally devoid of the latter two activities. Comparison of the two types of glycosomes by sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed that bloodstream form glycosomes contained 3 prominent polypeptides (64, 46 and 40 kDa) which were hardly detectable in insect stage glycosomes, whereas the latter contained 3 insect stage specific bands with molecular weight of 34 000, 61 000 and 77 000 and 4 additional bands with molecular weights between 94 000 and 110 000. Both types of glycosome contained the phospholipids phosphatidylcholine and phosphatidylethanolamine. Insect stage glycosomes contained in addition also phosphatidylinositol and some phosphatidylserine.

Animals↗
PubMed1984

Purification, morphometric analysis, and characterization of the glycosomes (microbodies) of the protozoan hemoflagellate Trypanosoma brucei.

Trypanosoma brucei glycosomes (microbodies containing nine enzymes involved in glycolysis) have been purified to near homogeneity from bloodstream-form trypomastigotes for the purpose of morphologic and biochemical analysis. Differential centrifugation followed by two isopycnic centrifugations in an isotonic Percoll and in a sucrose gradient, respectively, resulted in 12- to 13-fold purified glycosomes with an overall yield of 31%. These glycosomes appeared to be highly pure and contained less than 1% mitochondrial contamination as judged by morphometric and biochemical analyses. In intact cells, glycosomes displayed a remarkably homogeneous size distribution centered on an average diameter of 0.27 micron with a standard deviation of 0.03 micron. The size distribution of isolated glycosomes differed only slightly from that measured in intact cells. One T. brucei cell contained on average 230 glycosomes, representing 4.3% of the total cell volume. The glycosomes were surrounded by a single membrane and contained as phospholipids only phosphatidyl choline and phosphatidyl ethanolamine in a ratio of 2:1. The purified glycosomal fraction had a very low DNA content of 0.18 microgram/mg protein. No DNA molecules were observed that could not have been derived from contaminating mitochondrial or nuclear debris.

Animals↗
PubMed1983

Complement-mediated lysis of pigeon erythrocyte ghosts analysed by flow cytometry. Evidence for the involvement of a 'threshold' phenomenon.

Flow-cytometric analysis of complement-mediated lysis of antibody-coated pigeon erythrocyte ghosts containing fluorescein was carried out to determine whether lysis involved a gradual release of fluorescein or a 'threshold' release from individual cells. Antibody-coated ghosts were comprised of three subpopulations identified by fluorescence and scatter (size). These were: (a) highly fluorescent, medium scatter, (b) medium fluorescence, high scatter, and (c) low (or zero) fluorescence, low scatter. Lysed ghosts and isolated nuclei were identified by fluorescence microscopy and scanning electron microscopy. Fluorescence distributions analysed by flow cytometry indicated that, after complement attack, those ghosts remaining intact retained all their fluorescent label. A time course of changes in ratios of the three subpopulations indicated that once lysis of an individual ghost was initiated, release of label was complete within 1 min; no stages of intermediary fluorescence appeared, and those ghosts remaining at the end of the experiment retained the same fluorescence intensity as control ghosts. The results supported the hypothesis that complement-mediated cell lysis is a 'threshold' phenomenon; a submaximal response by a cell population representing a complete response by only some of the cells rather than a partial response by all of the cells.

Animals↗
PubMed1983

Decrease in apparent Km for oxygen after stimulation of respiration of rat polymorphonuclear leukocytes.

The respiratory burst of polymorphonuclear leukocytes, induced by the addition of chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine) and cytochalasin B was found to consist of two phases. The first phase of very rapid oxygen uptake lasted 1-3 min. and was followed by a second more prolonged phase of lower magnitude. The apparent Km for oxygen of unstimulated cells was 9.6 +/- 0.67 microM, while that of the second phase of stimulation was 3.7 +/- 1.6 microM oxygen. The possibility that lowered oxygen concentrations may regulate polymorphonuclear leukocyte activity in some pathological conditions is discussed.

Animals↗