Superoxide Synthesis Research Articles

Vascular atherosclerotic disease: Evaluation of white blood cell rheology and metabolism after acute intravenous administration of defibrotide

Defibrotide is a single-strand polydeoxyribonucleotide, obtained by controlled depolymerization of DNA extracted from mammalian organs. It stimulates vascular prostacyclin production and secretion (1-3) and enhances fibrinolysis (1, 4-6). Several clinical trials have evidentiated that defibrotide is effective in the prophylaxis of deep vein thrombosis and in the treatment of peripheral obliterative arterial disease (for a review see ref. 7). There are several reports concerning, in vitro and in vivo, the dose-dependent activity of defibrotide on the polymorphonuclear leucocytes (PMN). From these results, the molecule influences the PMN adhesivity (8, 9) and especially PMN activation, with secondary decrease or inhibition of free radical (superoxide anion) synthesis (10, 11). Other authors point out that this molecule interferes with the leucocyte role in the genesis of thrombosis (12). There is, however, no information about the action of defibrotide on white blood cell (WBC) rheology. Considering all these [mdings, we studied the influence of defibrotide on WBC filtration, PMN membrane fluidity and cytosolic Ca2+ content in a group of subjects with vascular atherosclerotic disease (V AD).

Longitudinal analysis of neutrophil superoxide anion generation in patients with septic shock

To examine polymorphonuclear leukocyte respiratory burst function serially in patients with septic shock. Prospective, longitudinal, descriptive study. Adult and pediatric (university hospital) intensive care units. Eight critically ill patients, with septic shock and eight critically ill patients without evidence of infection or sepsis. Severity of patient illness was estimated serially using the Acute Physiology and Chronic Health Evaluation (APACHE II) scoring system. For each patient, neutrophil superoxide anion synthesis was assayed spectrophotometrically in multiple blood samples over a period of 7 to 12 days after clinical identification of septic shock. The initial sample was obtained < 12 hrs after admission. Reaction velocities initially, at 2 to 3 mins, and at 4 to 5 mins (nmol superoxide anion/min/10(6) neutrophils), and extent of reaction at 5 mins (nmol superoxide anion/5 mins/10(6) neutrophils) were determined for each assay. On the day of admission, the mean APACHE II score and initial velocity for the septic shock group were 21.5 +/- 10 and 4.6 +/- 2 nmol superoxide anion/min/10(6) neutrophils, respectively. Over the next 7 to 12 days, as the patients recovered, there was a significant (paired t-test) decrease in APACHE II scores (p < .005) and increase in initial velocity (p < .0005). The increase in initial velocity correlated with the accompanying decrease in APACHE II scores (r2 = .46). Neutrophil superoxide anion generation in the critically ill group was not suppressed compared with the septic shock group and remained normal throughout the evaluation period. In vitro neutrophil respiratory burst function is significantly depressed during early septic shock. As patients improve clinically, as quantitated by decreasing APACHE II scores, neutrophil respiratory burst function recovers, approaching normal values.

Multiple mechanisms for doxorubicin cytotoxicity on glomerular epithelial cells ‘in vitro’

This study was planned to define the metabolic pathways for free radical production by isolated glomeruli and glomerular epithelial cells in vitro after exposure to cytotoxic doses of doxorubicin. A net increment in glomerular superoxide anion (O · 2) synthesis was observed at doxorubicin doses between 10 and 30 μg/ml, a drug level which also induced a parallel increment in uric acid synthesis. Since the synthesis of O · 2 with production of uric acid implies an activity of xanthine oxidase, a few experiments were performed with glomeruli which had been deprived of xanthine oxidase activity. In this case doxorubicin-inducible O · 2 and uric acid synthesis by glomeruli was practically nil. A similar stimulatory effect of O · 2 synthesis was induced by doxorubicin on glomerular epithelial cells and also in this case O · 2 synthesis was suppressed by pre-treating cells with deoxyconformicin, a selective inhibitor of adenosine deaminase. Finally, equimolar amounts of the drug were equally cytotoxic even when kept constantly outside the cell by a stable linkage with an agarose macroporous bed. In summary, these data demonstrate that O · 2 is generated by isolated glomeruli and glomerular epithelial cells ‘in vitro’ when exposed to cytotoxic amounts of doxorubicin and that purine degradation to uric acid furnish the metabolic pathways for glomerular O · 2 generation. However, doxorubicin is comparably cytotoxic on glomerular epithelial cells from outside cells thus suggesting that also a membrane perturbation may activate the series of events leading to cell injury.

Mechanism of vanadate-induced activation of tyrosine phosphorylation and of the respiratory burst in HL60 cells. Role of reduced oxygen metabolites

Vanadate induces phosphotyrosine accumulation and activates O2 consumption in permeabilized differentiated HL60 cells. NADPH, the substrate of the respiratory burst oxidase, was found to be necessary not only for the increased O2 consumption, but also for tyrosine phosphorylation. The effect of NADPH was not due to reduction of vanadate to vanadyl. Instead, NADPH was required for the synthesis of superoxide, which triggered the formation of peroxovanadyl [V(4+)-OO] and vanadyl hydroperoxide [V(4+)-OOH]. One or both of these species, rather than vanadate itself, appears to be responsible for phosphotyrosine accumulation and activation of the respiratory burst. Accordingly, the stimulatory effects of vanadate and NADPH were abrogated by superoxide dismutase. Moreover, phosphorylation was activated in the absence of NADPH by treatment with V(4+)-OO and/or V(4+)-OOH, generated by treatment of orthovanadate with KO2 or H2O2 respectively. The main source of the superoxide involved in the formation of V(4+)-OO and V(4+)-OOH is the NADPH oxidase. This was shown by the inhibitory effects of diphenylene iodonium and by the failure of undifferentiated cells, which lack oxidase activity, to undergo tyrosine phosphorylation when treated with vanadate and NADPH. By contrast, exogenously generated V(4+)-OO induced marked phosphorylation in the undifferentiated cells, demonstrating the presence of the appropriate tyrosine kinases and phosphatases. A good correlation was found to exist between induction of tyrosine phosphorylation and activation of the respiratory burst, suggesting a causal relationship. Therefore an amplification cycle appears to exist in cells treated with vanadate, whereby trace amounts of superoxide initiate the formation of V(4+)-OO and/or V(4+)-OOH. These peroxides promote phosphotyrosine formation, most likely by inhibition of tyrosine phosphatases. Accumulation of critical tyrosine-phosphorylated proteins then initiates a respiratory burst, with abundant production of superoxide. The newly formed superoxide catalyses the formation of additional V(4+)-OO and/or V(4+)-OOH, thereby magnifying the response. Since vanadium derivatives are ubiquitous in animal tissues, V(4+)-OO and/or V(4+)-OOH could be formed in vivo by reduced O2 metabolites, becoming potential endogenous tyrosine phosphatase inhibitors. Because of their potency, peroxides of vanadate may be useful as probes for the study of protein phosphotyrosine turnover.

Suppression of human neutrophil functions by tetracyclines

Tetracycline inhibition of neutrophil-associated collagenolysis has been the focus of a number of investigations. Evidence has suggested that this inhibition results from the ability of this family of antimicrobial drugs to bind divalent cations such as Ca2+ and Zn2+, two cations that are required for full expression of activity of metalloproteinases such as collagenase and gelatinase. Data presented in this study demonstrate that tetracyclines can also inhibit neutrophil-mediated RBC lysis, superoxide anion synthesis, degranulation and migration. To some extent, tetracycline inhibition of neutrophil functions is mimicked by the Ca2+ binding agents, EDTA and TMB-8. However, Ca2+ enrichment restored full function to EDTA- and TMB-8-treated cells but not to tetracycline-treated neutrophils. This suggests that Ca2+ binding plays a role but is not the critical effect leading to tetracycline suppression of neutrophil functions. It has been suggested that tetracyclines can suppress leukocyte-associated tissue damage. Host tissues are protected from neutrophil-mediated damage by two mechanisms: 1. Neutrophil granule-associated enzymes are secreted in an inactive state; and, 2. tissues are protected from these enzymes by a potent inhibitor shield. Neutrophils can bypass these protective elements by activating enzymes and by destroying the shield through the synthesis of oxygen radicals. Therefore, tetracyclines may suppress neutrophil-mediated tissue damage by inhibiting their migration and degranulation and, potentially more importantly, by suppressing synthesis of oxygen radicals.(ABSTRACT TRUNCATED AT 250 WORDS)

Septic plasma suppresses superoxide anion synthesis by normal homologous polymorphonuclear leukocytes

In vitro exposure of normal O,Rh- PMN to plasma obtained from patients with septic shock results in inhibition of formyl-methionyl-leucyl-phenylalanine stimulated superoxide anion (O2.-) production by 40%. Although all reaction velocities and extent of reaction at 5 min were suppressed, neither lag time preceding O2.- production nor duration of initial velocity linearity was affected. No such inhibition was noted when plasma from healthy controls or nonseptic critically ill patients was utilized in the reaction. These results demonstrate that neutrophils are not only a cause, but also a target of the septic shock host inflammatory response.

H2-antagonist inhibition of human neutrophil superoxide anion synthesis

In the mmol/L concentration range, cimetidine and ranitidine suppress superoxide anion generation by isolated intact human neutrophils. However, at normal pharmacologic concentrations in the mumol/L range, even prolonged exposure of neutrophils to these agents has no demonstrable effect on toxic oxygen species synthesis. In vitro inhibition does not involve neutrophil activation-densensitization or neutrophil cytotoxicity and is reversible to a great extent by drug washout. In the examination of possible free-radical scavenging action of these drugs, it was demonstrated that both drugs inhibit superoxide anion production by xanthine oxidase but not by chelated iron. This raises the possibility that these agents may bear structural similarities to oxypyrazolopyrimidines such as allopurinol.

FcReceptors Mediate Prostaglandin and Superoxide Synthesis in Cultured Rat Kupffer Cells

Latex beads with covalently bound bovine serum albumin were prepared and coated with anti-BSA immunoglobulin G. These particles were shown to possess on their surfaces a defined quantity of the antibody with the Fc portions exposed to the medium. One homologous and two heterologous antibodies of the G class were used and compared in terms of their binding to the rat Kupffer cells and their ability to elicit the typical phagocytotic responses. These particles were phagocytosed by rat Kupffer cells and elicited synthesis of prostaglandins and superoxide anion radicals. A significant release of superoxide into the medium was observed in the presence of cytochalasin B only. The data presented here suggest that a) Fc-carrying particles can be bound to Kupffer cells and elicit responses via specific receptors; b) coating with the homologous antibody yields the most effective particles; c) superoxide release into the surrounding medium is most abundant when the particle-binding membrane areas are prevented from forming phagocytotic vesicles.

A human embryo fibroblast-derived peptide suppresses the zymosan-induced biochemical activation in human polymorphonuclear leukocytes

Previously, a low-molecular-weight peptide was found in the serum-free culture media conditioned by a human embryo fibroblast cell strain (YH-1), which showed inhibitory effects on the proliferation and functions of B and T lymphocytes (Okai et al. (1987) Zool. Sci. 4, 99-105). In this paper, the inhibitory effect of this peptide on the functional activation in human polymorphonuclear leukocytes (PMNs) is documented. This peptide suppressed the opsonized zymosan-induced generation of chemiluminescence and superoxide anion and concomitant RNA synthesis. However, this peptide did not show significant cytotoxic effects on human PMNs as judged by a cytoplasmic enzyme-releasing assay. These results indicate that a fibroblast-derived peptide reported previously has a suppressive activity on the functional activation of human PMNs. The immunological significance of this finding is discussed.

Evaluation and improvements of a rapid microassay for measuring superoxide anion production by phagocytes: 2. Biochemical aspects

Superoxide anion synthesis by stimulated human polymorphonuclear leukocytes (PMNL) may be measured as superoxide dismutase-inhibitable cytochrome C (Cytc) reduction in a rapid microplate assay. However, when determinations are made over a fixed time interval (15 min) a lack of proportionality is observed between the amount of Cytc reduced and the number of cells stimulated. Examination of the reduction kinetics indicated that depressed superoxide detection at higher cell densities might be a consequence of Cytc re-oxidation by later metabolites in the oxidative burst. This was confirmed using dithionite-reduced Cytc as substrate in the assay. A method of analysis which avoids this source of error in determining superoxide anion synthesis is proposed.

Evaluation and improvement of a rapid microassay for measuring superoxide anion production by phagocytes: 1. Spectrophotometric aspects

Phagocyte synthesis of superoxide anion can be reliably measured as superoxide dismutase-inhibitable reduction of ferricytochrome C (Cytc). the original assay involved separation of the Cytc solution from the cells before estimating the extent of reduction spectrophotometrically. More recently, a rapid microplate assay has been devised, in which the cells are left in situ and reduction is measured on a multi-channel photometer. To date, no direct comparison of the two methods has been reported. The present study, which compares superoxide anion production by human neutrophils in both assays, indicates that different results may be obtained by the two methods and that an important contributory factor to the discrepancy may be the types of photometry employed. The lower values observed in the microplate assay were attributed to the reduced sensitivity of the multi-channel plate reader in detecting the absorbance of reduced Cytc, which in turn could be ascribed to the bandwidth of the interference filter employed in wavelength selection. The association between filter bandwidth and sensitivity is analysed and methods of correction are proposed.

Cytoplasmic pH regulation in phorbol ester-activated human neutrophils

Activation of neutrophils by 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by an initial cytoplasmic acidification, followed by an alkalinizing phase due to Na+-H+ countertransport. The source of the acidification, which is fully expressed by activation with TPA in Na+-free or amiloride-containing media, was investigated. The acidification phase was detected also in degranulated and enucleated cytoplasts, ruling out a major contribution by the nucleus or secretory vesicles. Cytoplasmic acidification was found to be associated with an extracellular acidification, suggesting metabolic generation of H+. Two principal metabolic pathways are stimulated in activated neutrophils: the reduction of O2 by NADPH-oxidase and the hexose monophosphate shunt. A good correlation was found between the activity of these pathways and the changes in cytoplasmic pH. Inhibition of superoxide synthesis prevented the TPA-induced cytoplasmic acidification. Moreover, activation of the hexose monophosphate shunt with permeable NADPH-oxidizing agents (in the absence of TPA) also produced a cytoplasmic acidification. Cytoplasmic acidification was also elicited by exogenous diacylglycerol and by other beta-phorbol diesters, which are activators of the kinase, but not by unesterified phorbol or by alpha-phorbol diesters, which are biologically inactive. The results suggest that the cytoplasmic acidification induced by phorbol esters in neutrophils reflects accumulation of H+ liberated during the metabolic burst that follows activation.

Corrections and additional insights to the synthesis and characterization of tetramethylammonium superoxide [(Me4N)O2

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCorrections and additional insights to the synthesis and characterization of tetramethylammonium superoxide [(Me4N)O2]Kenneth Yamaguchi, Thomas S. Calderwood, and Donald T. SawyerCite this: Inorg. Chem. 1986, 25, 8, 1289–1290Publication Date (Print):April 1, 1986Publication History Published online1 May 2002Published inissue 1 April 1986https://doi.org/10.1021/ic00228a044RIGHTS & PERMISSIONSArticle Views209Altmetric-Citations28LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (252 KB) Get e-Alerts Get e-Alerts

Synthesis and characterization of tetramethylammonium superoxide

Synthese a partir de l'hyperoxyde de potassium et de l'hydroxyde de tetramethylammonium hydrate. Proprietes magnetiques et electrochimiques

Long-term cryopreservation of dog granulocytes

Granulocytes isolated by counterflow centrifugation elutriation (CCE) from leukapheresed dog blood, frozen in liquid nitrogen at −196 °C, were studied. The effects of long-term cryopreservation on cell recovery and in vitro function were detertmined. In seven separate experiments, an average of 1.7 × 10 9 granulocytes were obtained. The white cell differential count was 91% granulocytes and 9% mononuclear cells. There was less than 5% red cells presrent and no platelets. Granulocytes were placed in Hemoflex bags and mixed slowly with equal volumes of sterile ice-cold hyperosmolar cryoprotectant buffer to make a final composition of 5% dimethylsulfoxide (DMS), 6% hydroxyethyl starch (HES), and 4% bovine serum albumin (BSA), pH 7.1. Total volumes of 40 ml were frozen at a cooling rate of 4 °C per minute and stored for periods of 1, 34, 60, 90, and 132 weeks in liquid nitrogen at −196 °C. Thawing was done at a rate of 190 ° per minute to 10 °C. The recovery of cells was 95%, 105%, 100%, 100%, and 88% respectively. Ethidium bromide exclusion, indicative of viable nuclei, was 91%, 81%, 94%, 89%, and 80% respectively. Virtually all thawed cells ingested opsonized Fluolite particles, but the number ingested was approximately one-half that of prefreeze values. Thawed cells also demonstrated superoxide anion synthesis at rates approximating those in unfrozen granulocytes. These results indicate that dog granulocytes obtained by leukapheresis may be preserved in liquid nitrogen at −196 °C with high cellular recovery and at least 50% phagocytic function.

Inhibition of the n-formylmethionyl-leucyl-phenylalanine induced respiratory burst in human neutrophils by adrenergic agonists and prostaglandins of the E series.

Exogenous prostaglandins E1 and E2 and L-isoproterenol potently inhibited the production of superoxide anions by human neutrophils activated in vitro by n-formylmethionyl-leucyl-phenylalanine (FMLP). An estimated ID50 of 50 nM was found for all three agents while L-epinephrine and prostaglandin F2 alpha were 10 and 100 fold, respectively, less active. Inhibition occurred whether these agents were added before, together with, or after the addition of the tripeptide to cell suspensions. Cells treated with dibutyryl adenosine 3',5'-monophosphate also expressed reduced rates of superoxide synthesis thus suggesting that the hormonal inhibitors acted indirectly by stimulating membrane bound adenylate cyclase.

Synthesis of Tetramethylammonium Superoxide

Synthesis of Tetramethylammonium Superoxide