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At least 1,711 records · Page 95Linked to original sources
PubMed2002

A 29-kDa protein associated with p67phox expresses both peroxiredoxin and phospholipase A2 activity and enhances superoxide anion production by a cell-free system of NADPH oxidase activity.

Production of toxic oxygen metabolites provides a mechanism for microbicidal activity of the neutrophil. The NADPH oxidase enzyme system initiates the production of oxygen metabolites by reducing oxygen to form superoxide anion (O(2)()). With stimulation of the respiratory burst, cytosolic oxidase components, p47(phox), p67(phox), and Rac, translocate to the phagolysomal and plasma membranes where they form a complex with cytochrome b(558) and express enzyme activity. A 29-kDa neutrophil protein (p29) was identified by co-immunoprecipitation with p67(phox). N-terminal sequence analysis of p29 revealed homology to an open reading frame gene described in a myeloid leukemia cell line. A cDNA for p29 identical to the open reading frame protein was amplified from RNA of neutrophils. Significant interaction between p29 and p67(phox) was demonstrated using a yeast two-hybrid system. A recombinant (rh) p29 was expressed in Sf9 cells resulting in a protein with an apparent molecular weight of 34,000. The rh-p29 showed immunoreactivity with the original rabbit antiserum that detected p47(phox) and p67(phox). In addition, rh-p29 exhibited PLA(2) activity, which was Ca(2+) independent, optimal at low pH, and preferential for phosphatidylcholine substrates. The recombinant protein protected glutathione synthetase and directly inactivated H(2)O(2). By activity and sequence homology, rh-p29 can be classified as a peroxiredoxin. Finally, O(2)() production by plasma membrane and recombinant cytosolic oxidase components in the SDS-activated, cell-free NADPH oxidase system were enhanced by rh-p29. This effect was not inhibited by PLA(2) inhibitors. Thus, p29 is a novel protein that associates with p67 and has peroxiredoxin activity. This protein has a potential role in protecting the NADPH oxidase by inactivating H(2)O(2) or altering signaling pathways affected by H(2)O(2).

Animals↗
PubMed1986

In vitro steady-state levels of hydrogen peroxide after exposure of male F344 rats and female B6C3F1 mice to hepatic peroxisome proliferators.

The hypothesis that hepatocarcinogenesis resulting from treatment of rats and mice with peroxisome proliferators is linked to increased cellular levels of hydrogen peroxide from peroxisomal beta-oxidation was investigated. Male F344 rats and female B6C3F1 mice were treated for 14 days with di(2-ethylhexyl)phthalate (DEHP) or di(2-ethylhexyl)adipate (DEHA), industrial plasticizers, or nafenopin, a hypolipidemic drug. Activities of enzymes responsible for the production [peroxisomal palmitoyl CoA oxidase (PCO)] and degradation [catalase (Cat) and glutathione peroxidase (GSHPx)] of H2O2 were assayed in liver homogenates prepared from treated animals. The activities of the peroxisomal enzymes PCO and Cat were enhanced 5- to 25-fold and 1.5- to 3-fold respectively by treatment with the peroxisome proliferators. The activity of GSHPx, a cytoplasmic enzyme, was decreased 40-60% in liver homogenates prepared from treated animals compared to control animals. A kinetic treatment of the rates of formation of hydrogen peroxide by PCO, and of degradation of hydrogen peroxide by catalase was used to estimate steady-state hydrogen peroxide concentrations ([H2O2]) during peroxisomal oxidation of palmitoyl CoA. Increases in peroxisomal steady-state [H2O2] for the F344 rat liver homogenates correlated well with the carcinogenic potential of these chemicals, determined in previous carcinogenicity studies. Increases in the steady-state [H2O2] were also calculated for liver homogenates prepared from mice treated with these compounds. Decreases in liver lipid peroxidation were observed after treatment with each chemical in both species. The results of these studies are consistent with an involvement of increased peroxisomal hydrogen peroxide in the hepatocarcinogenesis of these compounds.

Adipates↗
PubMedDate not supplied

Enzyme immunoassays for the detection of infectious antigens in body fluids: current limitations and future prospects.

Enzyme immunoassays are attaining increased usage for the direct detection of microbial antigens in body fluids. Advantages of enzyme immunoassays include a high degree of sensitivity resulting from the inherent magnification of the enzyme-substrate reaction and the use of objective end points without the need for radioactivity. Enzyme immunoassays have been developed for the reliable detection of several important microbial antigens in body fluids, including antigens of rotavirus, hepatitis B virus, and Haemophilus influenzae type b. However, standard enzyme immunoassay techniques are not sufficiently sensitive for the measurement of some antigens from other viruses, bacteria, and parasites in concentrations that commonly occur in body fluids during the course of infectious diseases. This review examines some of the limitations of currently available enzyme immunoassay technology and discusses approaches to increasing the sensitivity and specificity of enzyme immunoassay systems. Methods for improving these assay systems include the use of monoclonal antibodies, improved methods of enzyme-immunoreactant conjugation, more sensitive substrate systems, improved methods of antigen-antibody access, and the direct measurement of microbial enzymes. The use of such techniques should lead to the development of efficient enzyme immunoassay systems for the direct detection of a wide range of bacterial, viral, and parasitic infections.

Absorption↗
PubMed1999

Enhanced expression of neutrophil NADPH oxidase components in intestine of rats after burn injury.

We have evaluated the accumulation of neutrophils in the gut and their infiltration into the intestinal extravascular spaces in rats subjected to a 25% total body surface area scald burn. The accumulation of neutrophils was assessed via measurements of myeloperoxidase (MPO) activity in the intestinal homogenates, and the immunohistochemical localization of neutrophil NADPH oxidase component proteins (p47phox and p67phox) within the intestinal extravascular spaces determined neutrophil tissue infiltration. MPO measurements demonstrated a 12- and 21-fold increase above the control value in the intestinal tissue at day 1 and day 3 post-burn, respectively, suggesting that a substantial total tissue accumulation of neutrophils occurs in the gut after burn injury. The immunohistochemical staining procedures showed both a definitive presence of the neutrophil in the intestinal extravascular spaces and an enhanced immunoreactivity in neutrophils accumulating in intestine after burn injury. There was no evidence of either the presence of neutrophils in the extravascular regions or any significant neutrophil immunoreactivity to NADPH oxidase component proteins in the intestines of sham control rats. These findings indicate that burn injury causes an enhanced migration of circulating neutrophils into the intestinal interstitial spaces and an upregulation of NADPH oxidase activity in the infiltrating neutrophils.

Animals↗
PubMed1977

Subcellular Distributions of Isoenzymes in Fruits of a Normal Cultivar of Tomato and of the rin Mutant at Two Stages of Development.

Fruits of tomato (Lycopersicon esculentum Mill.) cv. Rutgers and of a nearly isogenic stock containing the ripening inhibitor gene rin harvested at green (66% mature) and ripe (107% mature) stages were studied for the subcellular distribution of isoenzymes using isoelectric focusing. The enzymes studied were peroxidases, esterases, phosphatases, phosphorylase, malate dehydrogenases, and IAA oxidases. During ripening of normal fruit the activities in the supernatant fraction of all of these enzymes, except malate dehydrogenase, decreased. In the particulate fractions some enzymes decreased while others increased in activity. The rin gene inhibited only some of the changes which occurred during ripening of normal fruit. It is postulated that changes in the degree to which enzymes are bound to membranes comprise one of the mechanisms by which the activities of enzymes are controlled in tomato pericarp, and that these membranes remain intact during ripening.

Journal Article↗
PubMed1997

The effects of mutation of the anr gene on the aerobic respiratory chain of Pseudomonas aeruginosa.

The anr gene of Pseudomonas aeruginosa encodes a transcriptional regulator of anaerobic gene expression, homologous to the Fnr protein of Escherichia coli. We report here that Anr has a role in regulating the activity of the aerobic respiratory chain of P. aeruginosa. Strains with internal deletions in their anr gene had lowered levels of membrane bound cytochromes whilst the activity of the cytochrome c oxidase, cytochrome co (likely to be a cytochrome cbb3-type oxidase), and the cyanide-insensitive respiratory pathway was markedly higher than in the wild-type strains. These data, and the finding that provision of multiple copies of the anr gene led to severe repression of these respiratory activities, suggest that Anr is a repressor of aerobic respiratory pathways and possibly the terminal oxidases themselves. In contrast, Anr activated cytochrome c peroxidase, a respiratory chain linked enzyme induced under low oxygen conditions.

Aerobiosis↗
PubMed2001

Antioxidant status and oxidative stress in elite alpine ski racers.

The goal of this study was to access antioxidant status and markers of oxidative damage in elite alpine ski racers during routine training. Subjects included 12 members of the U.S. Men's Alpine Ski Team attending a 10-day summer training camp. Blood draws were collected at rest and after exercise: (a) prior to training, (b) following 2 days of dry land training, and (c) after 4 days of on-snow skiing. Seven measures of antioxidant status were determined using colorimetric and HPLC methods (Trolox equivalent antioxidant capacity, uric oxidase, alpha-tocopherol, total glutathione, cytosolic glutathione peroxidase, and superoxide dismutase). Oxidative stress was assessed using 2 markers of lipid oxidation (malondialdehyde and lipid hydroperoxides) and 2 markers of protein oxidation (carbonylated total proteins and carbonylated hemoglobin). The results of this study suggest that antioxidant status of elite alpine skiers may decline over a period of intense training. However, elevations in markers of oxidative stress were not evident.

Adult↗
PubMed2003

Lysophosphatidylcholines prime the NADPH oxidase and stimulate multiple neutrophil functions through changes in cytosolic calcium.

A mixture of lysophosphatidylcholines (lyso-PCs) are generated during blood storage and are etiologic in models of acute lung injury. We hypothesize that lyso-PCs stimulate polymorphonuclear neutrophils (PMNs) through Ca(2)(+)-dependent signaling. The lyso-PC mix (0.45-14.5 micro M) and the individual lyso-PCs primed formyl-Met-Leu-Phe (fMLP) activation of the oxidase (1.8- to 15.7-fold and 1.7- to 14.8-fold; P<0.05). Labeled lyso-PCs demonstrated a membrane association with PMNs and caused rapid increases in cytosolic Ca(2)(+). Receptor desensitization studies implicated a common receptor or a family of receptors for the observed lyso-PC-mediated changes in PMN priming, and cytosolic Ca(2)(+) functions were pertussis toxin-sensitive. Lyso-PCs caused rapid serine phosphorylation of a 68-kD protein but did not activate mitogen-activated protein kinases or cause changes in tyrosine phosphorylation. With respect to alterations in PMN function, lyso-PCs caused PMN adherence, increased expression of CD11b and the fMLP receptor, reduced chemotaxis, provoked changes in morphology, elicited degranulation, and augmented fMLP-induced azurophilic degranulation (P<0.05). Cytosolic Ca(2)(+) chelation inhibited lyso-PC-mediated priming of the oxidase, CD11b surface expression, changes in PMN morphology, and serine phosphorylation of the 68-kD protein. In conclusion, lyso-PCs affect multiple PMN functions in a Ca(2)(+)-dependent manner that involves the activation of a pertussis toxin-sensitive G-protein.

CD11 Antigens↗
PubMed1987

Increased activity of the respiratory burst in cord blood neutrophils: kinetics of the NADPH oxidase enzyme system in subcellular fractions.

Previous studies with neutrophils from newborn infants compared to neutrophils from healthy adults have documented increased respiratory burst activity including enhanced superoxide anion (O2-) production, nitroblue tetrazolium dye reduction, and hexose monophosphate shunt activity. To investigate the biochemical basis for these observations, we examined oxidative metabolism in membrane-rich fractions of neutrophils. Neutrophils from cord blood of vaginally delivered term infants or healthy adults were disrupted by nitrogen cavitation and subcellular fractions collected on discontinuous sucrose density gradients. Subcellular fractions of newborn neutrophils separated in a fashion identical with samples from healthy adults. Activity of alkaline phosphatase, a plasma membrane marker, was increased 4- to 5-fold in disrupted cells free from nuclei (postnuclear supernatant) as well as plasma membrane fractions from newborn samples compared to those from healthy adults. Content of lactoferrin, a specific granule marker, was decreased in postnuclear supernatants but equivalent in specific granule fractions of newborn cells compared to those from adults. No differences were noted in myeloperoxidase content of postnuclear supernatants or any other subcellular fraction. Plasma membrane fractions from phorbol myristate acetate-stimulated cord blood neutrophils made significantly more O2- than samples from adults (newborn 32.9 +/- 8.1 nmol O2-/min/mg protein mean +/- SEM, n = 3 versus adult 10.8 +/- 4.2, n = 3; p less than 0.05). Plasma membrane-rich fractions were also collected by the technique of differential centrifugation and kinetic parameters of the NADPH-dependent oxidase enzyme(s) were measured for vaginally delivered newborn and adult samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1996

Oxygen toxicity to the developing lung of the mouse: role of reactive oxygen species.

Since the description of bronchopulmonary dysplasia (BPD) in premature infants, the supplemental oxygen administered has been suspect in the etiology of BPD. This has prompted studies on the effect of hyperoxia on lung growth in neonatal animals. So far, these have not led to a treatment which either prevents or mitigates BPD. Another approach to investigate the effect of hyperoxia on the immature lung is to use lung explants from 12-d gestation mouse fetuses. Exposing explants to different concentrations of oxygen for 48 h, we found that exposures to oxygen both below (10%) and above (35% or greater) normoxia adversely affected branching morphogenesis and growth. The effect was irreversible at exposures of 50% oxygen and greater. To determine the role of reactive oxygen species (ROS) in the effect of hyperoxia, antioxidants and inhibitors of ROS formation were added to the incubating explants, and their influence on reducing the adverse effect of 50% oxygen was assessed. The combination of CuZn superoxide dismutase (SOD) and catalase, manganese SOD, manganese-3-tetrakis(1-methyl-4-pyridyl)porphorin, a low molecular weight SOD mimetic, and to a lesser extent, deferoximine, an antioxidant and inhibitor of hydroxyl radical formation, were successful in reducing the effect of 50% oxygen on morphogenesis. Not successful were N-nitro-L-arginine methyl ester (an inhibitor of nitric oxide synthase); allopurinol (an inhibitor of xanthine oxidase); N-acetylcysteine and ebselen (a glutathione peroxidase mimetic); Trolox (a synthetic tocopherol); catalase, and CuZnSOD used alone. These results provide evidence that superoxide anion and possibly hydroxyl radical are the ROS most likely responsible for the growth effects of hyperoxia on mouse fetal lung morphogenesis.

Analysis of Variance↗
PubMed1995

A tobacco soluble protoporphyrinogen-oxidizing enzyme similar to plant peroxidases in their amino acid sequences and immunochemical reactivity.

Partial amino acid sequences of a soluble protoporphyrinogen-oxidizing enzyme (PPO) purified from tobacco cultured cells were identified. The sequences of two regions of the soluble PPO corresponded to the acid/base catalysis and heme binding regions of plant peroxidases. Anti-soluble PPO IgG cross-reacted with these plant peroxidases. Thus, the soluble PPO seems to be a kind of peroxidase.

Amino Acid Sequence↗