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PubMed1996

Evidence for the Saccharomyces cerevisiae ferrireductase system being a multicomponent electron transport chain.

We have studied the relationships between in vivo (whole cells) and in vitro (plasma membranes) ferrireductase activity in Saccharomyces cerevisiae. Isolated plasma membranes were enriched in the product of the FRE1 gene and had NADPH dehydrogenase activity that was increased when the cells were grown in iron/copper-deprived medium. The diaphorase activity was, however, independent of Fre1p, and Fre1p itself had no ferrireductase activity in vitro. There were striking similarities between the yeast ferrireductase system and the neutrophil NADPH oxidase: oxygen could act as an electron acceptor in the ferrireductase system, and Fre1p, like gp91, is a glycosylated hemoprotein with a b-type cytochrome spectrum. The ferrireductase system was sensitive to the NADPH oxidase inhibitor diphenylene iodonium (DPI). DPI inhibition proceeded with two apparent Ki values (high and low affinity binding) in whole wild-type and Deltafre2 cells and with one apparent Ki in Deltafre1 cells (high affinity binding) and in plasma membranes (low affinity binding). These results suggest that the Fre1-dependent ferrireductase system involves at least two components (Fre1p and an NADPH dehydrogenase component) differing in their sensitivities to DPI, as in the neutrophil NADPH oxidase. A third component, the product of the UTR1 gene, was shown to act synergistically with Fre1p to increase the cell ferrireductase activity.

Amino Acid Sequence↗
PubMed2004

A red beet (Beta vulgaris) UDP-glucosyltransferase gene induced by wounding, bacterial infiltration and oxidative stress.

Mechanical wounding, infiltration with P. syringae or A. tumefaciens, and exposure to an H(2)O(2)-generating system (Glc/Glc oxidase) induce betacyanin synthesis in red beet (Beta vulgaris) leaves. These conditions also induced the expression of BvGT, a gene encoding a glucosyltransferase (GT) from Beta vulgaris. BvGT has a high similarity to Dorotheanthus bellidiformis betanidin-5 GT involved in betacyanin synthesis. Furthermore, the transient expression of a BvGT antisense construct resulted in the reduction of BvGT transcript accumulation and betanin synthesis, suggesting a role for this gene product in betacyanin glucosylation. In addition, the NADPH oxidase inhibitor, diphenylene iodonium (DPI), inhibited the accumulation of the BvGT transcript in response to infiltration with Agrobacterium tumefaciens. Hence, this result suggests that ROS produced by a plasma membrane NADPH oxidase may act as a signal to induce BvGT expression, necessary for betanin synthesis after wounding and bacterial infiltration.

Agrobacterium tumefaciens↗
PubMed2005

Oxygen-sensing pathway for SK channels in the ovine adrenal medulla.

1. The intracellular pathways that modulate the opening of oxygen-sensitive ion channels during periods of hypoxia are poorly understood. Different tissues appear to use either NADPH oxidase or a rotenone-sensitive mechanism as an oxygen sensor. The aim of the present study was to identify the oxygen-sensing pathway in the oxygen-sensitive sheep adrenal medullary chromaffin cell (AMCC). 2. The whole-cell patch-clamp technique was used to measure K+ currents in dissociated adult ovine chromaffin cells as well as SK channel currents expressed in the H4IIE cell line. 3. Diphenyliodonium, an inhibitor of NADPH oxidase, had no effect on the hypoxia-evoked closure of K+ channels in primary AMCC, whereas the mitochondrial inhibitor rotenone abolished the hypoxia-evoked response. Both these compounds significantly reduced K+ current amplitude under normoxic conditions. 4. One possible mechanism through which the oxygen sensor may modulate K+ channel activity is by altering the redox state of the cell. In sheep AMCC, altering the redox state by the addition of H2O2 to the extracellular solution increased K+ conductance. 5. The oxygen-sensitive K+ (Ko2) channels in sheep chromaffin cells are from the SK family and the whole-cell conductance of cells expressing mouse SK2 or SK3, but not human SK1, was increased by H2O2 and decreased by the reducing agent dithiothreitol. 6. These studies show that, in sheep AMCC, Ko2 channels are modulated via a rotenone-sensitive mechanism and that alteration of the cellular redox state mimics the change produced by alterations in Po2. In a heterologous expression system, SK2 and SK3 channels, the channels that initiate hypoxia-evoked changes in AMCC function, are modulated appropriately by changes in cellular redox state.

Adrenal Medulla↗
PubMed1993

Reduction of a tetrazolium salt and superoxide generation in human tumor cells (HeLa).

Experiments have been carried out to explore the use of a tetrazolium salt, MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide in the detection of intracellularly generated superoxide in HeLa cells. From the use of a low molecular weight lipophilic mimic of superoxide dismutase, as well as superoxide dismutase, and inhibitors of superoxide dismutase, it is suggested that at least 20-30% of the intracellular reduction of MTT is due to superoxide. Whilst this may arise from mitochondria another possible intracellular source in HeLa cells may be xanthine oxidase. The overall rate of intracellular MTT reduction in HeLa cells is inversely dependent on levels of serum in the culture medium. Serum components with a modulatory role in this context are those with antioxidant function. Reduced MTT is also detectable extracellularly in cultures of HeLa cells and at least 80% of this is due to superoxide. Use of inhibitors suggest that whilst a small proportion (30%) may arise through an NADPH-oxidase type enzyme, other sources of extracellular superoxide in HeLa cells remain a possibility.

Allopurinol↗
PubMed1981

Aspects of the antibacterial action of diphenyliodonium chloride.

Diphenyliodonium chloride was found to be a broad spectrum antimicrobial agent and its activity was affected by pH and inoculum size. Its uptake by bacterial cells was Langmuirian, and it caused loss of intracellular material. Aerobic glucose metabolism and dehydrogenase activity were inhibited. Low concentrations of the drug affected the membrane-bound ATPase and K transport in Streptococcus faecalis.

Adenosine Triphosphatases↗
PubMed1994

A sensitive detection of neutrophil activation by fluorescence quenching of membrane inserted singlet oxygen probe.

Fluorescence emission intensity of 1,3-diphenylisobenzofuran incorporated in polymorphonuclear granulocytes plasma membranes was investigated in basal conditions and during stimulation with different activators. Phorbol myristate acetate, known as the most effective "oxygen burst" inducer, produced a larger decrease in 1,3-diphenylisobenzofuran fluorescence intensity than 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) and N-formyl-methionyl-leucyl-phenylalanine, known as weak stimulants of oxygen uptake. Diphenyl iodonium an inhibitor of leukocyte NADPH oxidase, and the singlet oxygen selective trap alpha-terpinene inhibited the quenching effect of phorbol myristate acetate on 1,3-diphenylisobenzofuran fluorescence. These data suggest formation of singlet oxygen in activated leukocytes and demonstrate that measurement of 1,3-diphenylisobenzofuran fluorescence intensity provides a new sensitive method of detection of neutrophils activation.

Benzofurans↗
PubMed1992

Volume, conductivity, and scatter changes of activated polymorphonuclear leukocytes: an estimation by Coulter Counter STKS analyzer.

Suspensions of phorbol myristate acetate-activated polymorphonuclear leukocytes were analyzed with a Coulter Counter STKS hematological analyzer. Phorbol myristate acetate activation induced an increase in polymorphonuclear leukocyte volume and conductivity, while scatter was unchanged. Phorbol myristate acetate-activated neutrophils in a suspension containing nitroblue tetrazolium showed increased scatter. The rise in scatter was phorbol myristate acetate dose dependent, completely inhibited by diphenylene iodonium and partially by dimethyl sulfoxide, two inhibitors of NADPH oxidase. Zymosan-activated polymorphonuclear leukocytes were notably larger with a characteristic position on discriminant function 1 display (volume versus scatter) of the analyzer. Volume and conductivity changes were seemingly inexplicable features of phorbol myristate acetate activation. The rise in scatter was produced by cytoplasmic precipitation of reduced nitroblue tetrazolium and thus by O2-generation in phorbol myristate acetate-activated neutrophils. Zymosan phagocytosis was responsible for the notable rise in polymorphonuclear leukocyte volume. The analysis of activated polymorphonuclear leukocytes by Coulter Counter STKS may provide useful information on their activation and a pragmatic approach for studying function.

Biphenyl Compounds↗
PubMed2006

Increased oxidative stress in lambs with increased pulmonary blood flow and pulmonary hypertension: role of NADPH oxidase and endothelial NO synthase.

Although oxidative stress is known to contribute to endothelial dysfunction-associated systemic vascular disorders, its role in pulmonary vascular disorders is less clear. Our previous studies, using isolated pulmonary arteries taken from lambs with surgically created heart defect and increased pulmonary blood flow (Shunt), have suggested a role for reactive oxygen species (ROS) in the endothelial dysfunction of pulmonary hypertension, but in vivo data are lacking. Thus the initial objective of this study was to determine whether Shunt lambs had elevated levels of ROS generation and whether this was associated with alterations in antioxidant capacity. Our results indicate that superoxide, but not hydrogen peroxide, levels were significantly elevated in Shunt lambs. In addition, we found that the increase in superoxide generation was not associated with alterations in antioxidant enzyme expression or activity. These data suggested that there is an increase in superoxide generation rather than a decrease in scavenging capacity in the lung. Thus we next examined the expression of various subunits of the NADPH oxidase complex as a potential source of the superoxide production. Results indicated that the expression of Rac1 and p47(phox) is increased in Shunt lambs. We also found that the NADPH oxidase inhibitor diphenyliodonium (DPI) significantly reduced dihydroethidium (DHE) oxidation in lung sections prepared from Shunt but not Control lambs. As DPI can also inhibit endothelial nitric oxide synthase (eNOS) superoxide generation, we repeated this experiment using a more specific NADPH oxidase inhibitor (apocynin) and an inhibitor of NOS (3-ethylisothiourea). Our results indicated that both inhibitors significantly reduced DHE oxidation in lung sections prepared from Shunt but not Control lambs. To further investigate the mechanism by which eNOS becomes uncoupled in Shunt lambs, we evaluated the levels of dihydrobiopterin (BH(2)) and tetrahydrobiopterin (BH(4)) in lung tissues of Shunt and Control lambs. Our data indicated that although BH(4) levels were unchanged, BH(2) levels were significantly increased. Finally, we demonstrated that the addition of BH(2) produced an increase in superoxide generation from purified, recombinant eNOS. In conclusion our data demonstrate that the development of pulmonary hypertension in Shunt lambs is associated with increases in oxidative stress that are not explained by decreases in antioxidant expression or activity. Rather, the observed increase in oxidative stress is due, at least in part, to increased expression and activity of the NADPH oxidase complex and uncoupled eNOS due to elevated levels of BH(2).

Animals↗
PubMed1979

Preparation and analysis of new sulfonium derivatives from S-adenosyl(5')-3-methylthiopropylamine.

The paper reports the preparation and the chromatographic separation of new deaminated analogs of S-adenosyl(5')-3-methylthiopropylamine, i.e. S-adenosyl(5')-3-methylthiopropanol and S-inosyl(5')(-3-methylthiopropanol. These compounds can be used as specific inhibitors of polyamine biosynthesis. It is also reported the characterization of new sulfonium compounds by U.V. spectrophotometry, thin layer chromatography, high voltage electrophoresis, hydrolysis to known fragments.

Onium Compounds↗
PubMed1995

Micropreparative one- and two-dimensional electrophoresis: improvement with new photopolymerization systems.

To improve the efficiency of one- and two-dimensional electrophoresis for micropreparative purposes, the use of gels polymerized with other initiators than the standard N,N,N',N'-tetramethylethylenediamine (TEMED)/persulfate systems for sodium dodecyl sulfate electrophoresis has been investigated. We show here that the recently described photoinitiator system, composed of methylene blue, toluene sulfinate and diphenyliodonium chloride, leads to a decreased resolution. Resolution can be restored if methylene blue is replaced by riboflavin. Two-dimensional electrophoresis with mg loadings of proteins has also been evaluated with these systems. Independently of the polymerization system, resolution for the first dimension is low with rod gels, increases with gel strips and is further improved when immobilized pH gradients are used. Here too, only the riboflavin/sulfinate/iodonium system results in a resolution that matches the one obtained with the standard TEMED/persulfate system. Gels polymerized with the riboflavin/sulfinate/iodonium system yield better results upon N-terminal microsequencing after blotting than gels polymerized with the standard TEMED/persulfate system.

Ammonium Sulfate↗
PubMed1994

Endocytosis, partial degradation and release of heparan sulfate by elicited mouse peritoneal macrophages.

Interactions between glycosaminoglycans (GAGs) and low density lipoprotein (LDL) are thought to influence the progression of atherogenesis. In an effort to gauge whether macrophages mediate GAG-LDL interaction by GAG modification, we have investigated the endocytosis, degradation and retro-endocytosis of the GAG heparan sulfate (HS) by mouse peritoneal macrophages. Radiolabelled HS was produced by derivatization with sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate and radio-iodination by the chloramine T method. The amount of 125I-HS internalized by cultures of thioglycollate-elicited macrophages rose over a 24 h time period in proportion to the amount of tracer added to the wells (2-2500 ng ml-1). Analysis of GAG molecular weight was performed using gel filtration chromatography and polyacrylamide gel electrophoresis. After a 24 h pulse period, the 125I-HS in the intracellular fraction of the cultured cells was of smaller molecular weight than for control material. During a 24 h cold chase, fragments of 125I-HS were released into the medium. These fragments had lower affinity for Polybrene-Sepharose but did not appear significantly N-desulfated as determined by low pH nitrous acid treatment. The NADPH oxidase inhibitor diphenylene iodonium, although minimizing basal and phorbol ester-triggered radical output, did not inhibit 125I-HS depolymerization. These data indicate that elicited macrophages can interact with and reduce the polymer length of HS without extensively desulfating the molecule. They are consistent with a mechanism by which the macrophage internalizes and partially degrades HS by endoglucuronidase activity rather than NADPH oxidase-generated free radicals, followed by release of the products into the extracellular milieu.

Animals↗
PubMed1999

Calcium-dependent PAF-stimulated generation of reactive oxygen species in a human keratinocyte cell line.

During inflammation and other pathological states, the lipid mediator platelet-activating factor (PAF) and reactive oxygen species (ROS) are both generated. We have been investigating the effect of exogenous PAF on ROS formation in the human keratinocyte cell line (HaCaT). ROS production, measured using luminol-enhanced chemiluminescence (CL), proved to be rapid, transient, PAF receptor-mediated, and totally dependent on an increase in intracellular Ca2+ ([Ca2+]i) and on the presence of extracellular Ca2+. Repeated administration of PAF resulted in refractoriness to the agonist in terms of both capacities to increase [Ca2+]i and generate ROS. The cells, however, continued to respond fully to other stimulants (bradykinin, epidermal growth factor, thapsigargin). The PAF-induced increases in [Ca2+]i (monitored using the fluorescent probe Fluo-3) were also rapid and transient and paralleled those of ROS generation. Relatively specific inhibitors of potential ROS-producing systems were administered in an attempt to characterize the ROS producing system(s). Inhibitors of xanthine oxidase, phospholipase A2, lipoxygenase, cyclooxygenase and NO synthase did not interfere with PAF evoked ROS. The flavoprotein inhibitor diphenyleneiodonium and the mitochondrial cytochrome oxidase inhibitor KCN, prevented generation of ROS, making NAD(P)H a candidate for the electron source of the ROS and the mitochondria a potential major site of formation.

Aniline Compounds↗
PubMed2000

Reduction of nitrite to nitric oxide catalyzed by xanthine oxidoreductase.

Xanthine oxidase (XO) was shown to catalyze the reduction of nitrite to nitric oxide (NO), under anaerobic conditions, in the presence of either NADH or xanthine as reducing substrate. NO production was directly demonstrated by ozone chemiluminescence and showed stoichiometry of approximately 2:1 versus NADH depletion. With xanthine as reducing substrate, the kinetics of NO production were complicated by enzyme inactivation, resulting from NO-induced conversion of XO to its relatively inactive desulfo-form. Steady-state kinetic parameters were determined spectrophotometrically for urate production and NADH oxidation catalyzed by XO and xanthine dehydrogenase in the presence of nitrite under anaerobic conditions. pH optima for anaerobic NO production catalyzed by XO in the presence of nitrite were 7.0 for NADH and </=6.0 for xanthine. Involvement of the molybdenum site of XO in nitrite reduction was shown by the fact that alloxanthine inhibits xanthine oxidation competitively with nitrite. Strong preference for Mo=S over Mo=O was shown by the relatively very low NADH-nitrite reductase activity shown by desulfo-enzyme. The FAD site of XO was shown not to influence nitrite reduction in the presence of xanthine, although it was clearly involved when NADH was the reducing substrate. Apparent production of NO decreased with increasing oxygen tensions, consistent with reaction of NO with XO-generated superoxide. It is proposed that XO-derived NO fulfills a bactericidal role in the digestive tract.

Anaerobiosis↗
PubMed2001

Oxidative protein cross-linking reactions involving L-tyrosine in transforming growth factor-beta1-stimulated fibroblasts.

The mechanisms by which ligand-stimulated generation of reactive oxygen species in nonphagocytic cells mediate biologic effects are largely unknown. The profibrotic cytokine, transforming growth factor-beta1 (TGF-beta1), generates extracellular hydrogen peroxide (H2O2) in contrast to intracellular reactive oxygen species production by certain mitogenic growth factors in human lung fibroblasts. To determine whether tyrosine residues in fibroblast-derived extracellular matrix (ECM) proteins may be targets of H2O2-mediated dityrosine-dependent cross-linking reactions in response to TGF-beta1, we utilized fluorophore-labeled tyramide, a structurally related phenolic compound that forms dimers with tyrosine, as a probe to detect such reactions under dynamic cell culture conditions. With this approach, a distinct pattern of fluorescent labeling that seems to target ECM proteins preferentially was observed in TGF-beta1-treated cells but not in control cells. This reaction required the presence of a heme peroxidase and was inhibited by catalase or diphenyliodonium (a flavoenzyme inhibitor), similar to the effect on TGF-beta1-induced dityrosine formation. Exogenous addition of H2O2 to control cells that do not release extracellular H2O2 produced a similar fluorescent labeling reaction. These results support the concept that, in the presence of heme peroxidases in vivo, TGF-beta1-induced H2O2 production by fibroblasts may mediate oxidative dityrosine-dependent cross-linking of ECM protein(s). This effect may be important in the pathogenesis of human fibrotic diseases characterized by overexpression/activation of TGF-beta1.

Biphenyl Compounds↗
PubMed1993

Polymorphonuclear leukocyte-derived O2-reactive species activate primed platelets in human whole blood.

The activation of human platelets by polymorphonuclear leukocytes (PMN) was investigated in human whole blood challenged with "priming" concentrations of arachidonic acid or collagen in the presence or absence of N-formyl-Met-Leu-Phe (FMLP), a selective activator of PMN. With the use of arachidonic acid or collagen alone at priming concentrations or FMLP alone, no platelet response was observed. In contrast, FMLP in combination with arachidonic acid or collagen caused irreversible platelet aggregation with thromboxane A2 production. Platelet response to FMLP-activated PMN was enhanced by superoxide dismutase and blocked by catalase or the NADPH oxidase inhibitor diphenyliodonium, suggesting a role for the O2-.-H2O2 system in this cellular interaction. This was corroborated by experiments with exogenously added H2O2, which mimicked FMLP effects in the activation of primed platelets in whole blood. The present investigation indicates that platelets primed with minute amounts of arachidonic acid or collagen can be activated, in human whole blood, by oxygen-reactive species released by PMN.

Arachidonic Acid↗