Oxidase Enzyme System Research Articles

Antioxidant Effects of Neutrophil p29 Peroxiredoxin.

Production of reactive oxygen species through a respiratory burst is critical to the microbicidal activity of the neutrophil. The respiratory burst is initiated by assembly of the components of the NADPH oxidase enzyme system (p47phox, p67phox, p40phox, gp91phox, p22phox and Rac) and expression of the activity of the system to produce superoxide anion (O2 −). We recently identified a neutrophil protein with an approximate MW of 29 kDa which binds to the p67phox, is classified as a peroxiredoxin (Prx) and translocates to the plasma membrane during stimulation of the neutrophil. Additional studies demonstrate that this protein (p29 Prx) increases production of O2 − in a cell-free system of oxidase activity in a specific, stoichiometric manner and that the cysteine residues of p29 Prx, at amino acid positions 47 and 91, are required for this activity. The current studies demonstrate the antioxidant activity of p29 Prx using the technique of small interfering RNA (siRNA) to degrade specific mRNA and decrease the expression of the protein. siRNA probes for p29 Prx were constructed based on standard constraints for unique 19 nucleotide binding sites along with other sequence selection criteria. Nine probes were constructed based on the cDNA sequence of p29 Prx; one resulted in significant knockdown of p29 Prx. A vector for green fluorescent protein was obtained commercially. U293 cell line was cultured under standard conditions and expressed p29 Prx message by RT-PCR and protein by Western blot. U293 cells were transfected with siRNA or GFP with calcium chloride. Cell counts and viability were also determined by standard techniques. For Western blots, proteins from cell lysates were separated on 10% SDS-PAGE and blotted onto nitrocellulose, and specific proteins were detected with polyclonal antibodies to p29 Prx, lamin or actin by chemiluminescent technique. U293 cells were transfected with the siRNA for p29 Prx and the vector for GFPb and the cells were harvested at 24, 48, and 72 hours. A knockdown by one of the 6 siRNAs resulted in decreased levels of p29 Prx by Western blot. Optimum knockdown was achieved by transfection of 1 μg siRNA and the decrease in p29 Prx observed at 24 hours but was maximum at 48 and 72 hours. Greater than 90% transfection was achieved documented by green fluorescence of the cells. Under these conditions, a decrease in p29 Prx >90% detected by Western blot was achieved with no differences in levels of actin or lamin in the cell lysates. In separate experiments, U293 cells were transfected with siRNA for p29 Prx and the vector for GFP and exposed to an oxidant stress (paraquot, 100 μM). The level of p29 Prx, actin, and lamin; cell counts; and viability were determined. Cells transfected with siRNA had lower p29 Prx (<10%) but not actin or lamin. The viability of control cells and siRNA transfected cells after 48 hours was no different (91% and 94%, respectively). Paraquot decreased the viability of nontransfected cells (68%), and knockdown of p29 Prx in the presence of paraquot resulted in poorer viability (49%). The number of cells harvested was slightly decreased in the paraquot treated groups but not in the untreated control or siRNA groups. These results suggest an antioxidant effect of p29 Prx in the cell and support its role in protecting the oxidase enzyme system in the neutrophil.

Resurrection Men Dig through the Drug Graveyard: ARYx Revives Problem Blockbuster Drugs

Large pharmaceutical companies have tightened up their screening databases and broadened their patents after incidents of drugs slipping through pharmaceutical patents and being restructured by enterprising startups. Notably in 1996, Sepracor transformed Hoechst Marion Roussel Ltd.'s problematic antihistamine Seldane into a single isomer drug and then licensed it back to HMR as Allegra (fexofenadine/pseudoephedrine). Seldane was subsequently banned by the FDA. “It will be a matter of being very clever at identifying the best candidates, both from the chemical standpoint and from an intellectual property point of view,” John Lazo says. “There are a number of compounds out there that could be reengineered for the benefit of mankind.”ARYx is not the only company in the resurrection business. For example, Jazz Pharmaceuticals in Palo Alto, CA raised $250 million in 2004, the largest second-round financing for a drug company to date. Jazz has no proprietary technology and does not plan to do R&D but rather purports to license, improve, develop, and market known compounds for neurological and psychiatric disorders. ARYx is also competing against pharmaceutical companies themselves, which are developing other drugs to meet the same market needs as the ones they abandoned.“We do the same old lab bench-work medicinal chemistry that everybody else does,” says Druzgala, “and have to establish all the same preclinical data for the FDA. We believe our advantages are that we are not attempting to create new validated targets. The big advantage we have is that we can look at an old drug with many years of research and postmarket surveillance.”ARYx's approach bypasses the long screening stages of drug discovery because it deals with known compounds brought through FDA approval—but the revamped drugs would still have to go through the most expensive parts of phase III human clinical testing and resubmission to the FDA as well as pass post-FDA extended clinical use. Even if a drug has well-characterized structure-activity relationships, redirecting its properties in a complex system, for example, fiddling with fat versus water solubility, can cause other unexpected effects, such as changes in the drug localization within the body, local toxicity of a drug, and its rate of uptake and resulting bioavailability.The company hopes to offload the cost of the clinical phase. “We don't expect to be in partnership with the drug company that made the original drug. Our business is based on entering into a commercial partnership which will work with us under phase III trials and commercialization,” says David Nagler.Resurrection is one thing. Redemption in the market for refurbished drugs known to have a toxic history is another. It remains an open question if doctors will be willing to prescribe drugs that have a bad reputation, even if they have been revived, reworked, and renamed.

Production of pyruvate from lactate using recombinant Pichia pastoris cells as catalyst

Because of the synergistic presence of enzyme systems, whole-cell biocatalysts are an advantageous means of oxidizing α-hydroxy to α-keto carboxylic acids. The effect of different parameters on conversion were determined when whole cells converted l-lactate to pyruvate. The biocatalyst was a double recombinant Pichia pastoris containing a glycolate oxidase and catalase enzyme system. A marked increase in conversion occurred with oxygen compared to air. This indicated that the oxygen concentration was an important factor in the rate-limiting step. Lactate concentrations above 0.5 M showed substrate inhibition. Although temperature played an important role in enzyme stability, whole-cell tranformants were much more stable over time than soluble enzymes.

Oxidase activity in cord blood neutrophils: A balance between increased membrane associated cytochrome b558 and deficient cytosolic components

Newborn infants are prone to develop life-threatening pyogenic infections. Alterations in the function of neonatal phagocytes, including the activity of the neutrophil NADPH oxidase, have been suggested as one cause of increased susceptibility to such infections. In the present study, comprehensive analysis of NADPH oxidase enzyme system was performed in cord blood neutrophils from vaginally and cesarean section (CS) delivered, healthy, full-term infants. Superoxide anion (O(2) (-)) production by intact neutrophils from cord blood in response to soluble stimuli was equal to or increased compared to levels generated by cells from adult controls. In the sodium dodecyl sulfate (SDS) cell-free system, cytosol and plasma membrane from cord blood neutrophils generated O(2) (-) at comparable rates to subcellular fractions from healthy adults. However, mixing experiments demonstrated higher O(2) (-) generation with combination of cytosol from adult controls and membrane from cord blood neutrophils and lower O(2) (-) production with combination of cytosol from cord blood neutrophils and membrane from adult controls. Kinetic parameters for cord blood specimens were no different from those obtained for fractions from adult controls. Quantitative analysis of cytosolic components showed moderately reduced amount of p40-phox, p47-phox, and p67-phox in neutrophils from cord blood. In contrast, cytochrome b(558) content of plasma membrane of cord blood neutrophils was approximately 2-fold higher compared to adult controls. The normal to increased respiratory burst of intact cord blood neutrophils is the result of alterations to oxidase components: increased content of cytochrome b(558) in the plasma membrane and decreased levels of cytosolic components p47-phox, p67-phox, and p40-phox.

Cysteine Residues on Neutrophil p29 Peroxiredoxin Are Required for Enhanced Oxidase Activity.

Introduction: Production of reactive oxygen species (ROS) through the respiratory burst is critical to the microbicidal activity of the neutrophil. The respiratory burst is initiated by assembly of an active NADPH oxidase enzyme system. This system has several components including gp91phox and p22phox comprising the membrane associated cytochrome b558 and the cytosolic components p47phox, p67phox, p40phox and rac2. Recently, we identified a novel protein with a molecular weight of 29 kDa which binds p67phox by immunoprecipitation and yeast two-hybrid analysis (Leavey et al J Biol Chem 2002; 277:45181–7). This protein is classified as peroxiredoxin (Prx) by its amino acid sequence and enzyme activity. Prxs oxidize H2O2 through conserved cysteines. The neutrophil p29 Prx has a cysteine at amino acid residue 47 flanked by a consensus sequence shared by other 1-cys Prx proteins and a second cysteine at residue 91. We have also shown that neutrophil p29 Prx translocates to the cell membrane during oxidase activation and enhances oxidase activity in a cell-free assay with recombinant phox cytosolic components and neutrophil plasma membrane. The current studies were undertaken to evaluate requirements of cysteine residue(s) for the effect on the oxidase.Methods: cDNA for p29 Prx was cloned into pBlueBacHis vector and a recombinant protein was expressed in a baculovirus expression system as previously described. Two additional recombinant proteins with ser substituted for cys at residue 47 and 91 were generated using the PCR overlap technique and also expressed in Sf9 cells. The wild type (WT), C47S, and C91S recombinant proteins were purified by affinity chromatography. The effect of these proteins on oxidase activity was determined in a SDS cell-free system of oxidase activity using plasma membrane (1 μg), p47phox (100 nM), p67phox (100 nM), a constitutively active Rac and 1 mM NADPH. O2− production was measured as SOD inhibitable cytochrome c reduction.Results:O2− Production (nmol/min)Concentration (μM)WTC47SC91S07.15 ± 0.46*6.38 ± 0.817.11 ± 0.100.5511.86 ± 1.33**5.42 ± 0.736.57 ± 0.540.8314.58 ± 2.09**7.32 ± 1.027.29 ± 6.701.1017.66 ± 2.50**7.41 ± 1.197.64 ± 0.761.6519.93 ± 2.82**8.37 ± 1.378.14 ± 1.23* Numbers are mean ± SEM, n=3 ** Significantly different from no addition, p<0.05WT p29 Prx increased oxidase activity in a concentration dependent manner. Neither the C47S nor C91S mutants altered oxidase activity to a significant degree. Additional studies varying concentration of NADPH at several concentrations of p29 Prx demonstrated that p29 Prx increased the Vmax without changing the km for NADPH. At a fixed concentration of NADPH, the increase in rate of O2− generation was dependent upon p29 Prx concentration demonstrating saturable kinetics with a km for p29 Prx of 1.72 ± 0.65 μM and a Vmax (increase in velocity) of 15.4 ± 3.0 nmol/min (mean ± SEM for four separate studies). Conclusion: Neutrophil p29 Prx enhances oxidase enzyme activity. The enhancement is specific, exhibiting a saturable effect dependent on the concentration of p29 Prx. However, replacement of either conserved or non-conserved cysteine residues with serines resulted in an inactive protein. Cysteine residues which are required for Prx activity are also critical to the effects of neutrophil p29 Prx on the oxidase.

Serum copper is decreased in premature newborns and increased in newborns with hemolytic jaundice.

Copper is an active component of several enzyme systems, including cytochrome c oxidase and superoxide dismutase (1), and is essential for the prevention of anemia and leucopenia. It is important for the maturation of collagen and the maintenance of skeletal and vascular integrity because of its involvement in the lysyl oxidase enzyme system (2). Deficiency of copper is associated with an increased incidence of infection, as seen in patients with Menke kinky-hair disease, an inherited disease with low copper concentrations (3). The mechanism by which high copper concentrations interact with genetic control systems, leading to liver damage, is still unknown (4). The normal term infant is born with a generous liver copper store, and copper deficiency in neonates is a rare event (5). Copper deficiency has been associated with anemia, neutropenia, and bone demineralization in both preterm and full-term infants (6)(7)(8). Ceruloplasmin, the major serum copper-transporting protein, is synthesized in the liver, but its precise role in copper metabolism is unclear. Neonatal hepatic ceruloplasmin synthesis occurs after birth and is associated with a gradual increase in plasma concentrations (9). We report the concentrations of copper in healthy and jaundiced full-term and premature infants and their correlations with hemoglobin, bilirubin, and liver enzymes. The study was approved by the Greek Ethical Committee, and parental consent was obtained. The study comprised 303 neonates (full-term and premature). Newborns with obstructive jaundice or with infections or inflammatory process were excluded. All patients underwent routine laboratory tests to determine the etiology of their jaundice (see Table 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue7/) (10)(11). Patients were divided into the following groups (Table 1⇓ ). View this table: Table 1. Copper concentrations and hemolysis related biochemical tests in full-term and premature newborns with …

Hyperresponsiveness of neutrophils from gp 91 phox deficient patients to lipopolysaccharide and serum amyloid A

We demonstrate here that neutrophils from chronic granulomatous disease (CGD) patients release larger amounts of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α) than neutrophils from control subjects. Incremental cytokine production was observed under both basal and stimulated conditions in neutrophils from two CGD (gp 91 phox ) patients. The basal production of IL-8 was over seven-fold greater in CGD patients. The two samples assayed showed 3- and 10-fold increases in TNF-α. Basically, the same magnitude of increment was observed in lypopolysaccharide (LPS) and serum amyloid A protein (SAA)-stimulated cells. We also found that the levels of SAA and IL-8 were higher in the serum of CGD patients than the levels found in the serum of healthy donors. The increased responsiveness of neutrophils from CGD patients may be closely related with a deficiency in the assembly of the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase enzyme system, or it may be due to a frequent inflammatory condition in these patients. In the latter case, the increased serum levels of systemic inflammatory factors, among them SAA, would contribute to the sustained accumulation and activation of phagocytes. Whatever the origin, the excessive production of cytokines may lead to inappropriate activation and tissue injury and even to increased susceptibility to invasive microorganisms, impairing the quality life of CGD patients.

Hyperresponsiveness of neutrophils from gp 91phox deficient patients to lipopolysaccharide and serum amyloid A

We demonstrate here that neutrophils from chronic granulomatous disease (CGD) patients release larger amounts of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α) than neutrophils from control subjects. Incremental cytokine production was observed under both basal and stimulated conditions in neutrophils from two CGD (gp 91 phox ) patients. The basal production of IL-8 was over seven-fold greater in CGD patients. The two samples assayed showed 3- and 10-fold increases in TNF-α. Basically, the same magnitude of increment was observed in lypopolysaccharide (LPS) and serum amyloid A protein (SAA)-stimulated cells. We also found that the levels of SAA and IL-8 were higher in the serum of CGD patients than the levels found in the serum of healthy donors. The increased responsiveness of neutrophils from CGD patients may be closely related with a deficiency in the assembly of the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase enzyme system, or it may be due to a frequent inflammatory condition in these patients. In the latter case, the increased serum levels of systemic inflammatory factors, among them SAA, would contribute to the sustained accumulation and activation of phagocytes. Whatever the origin, the excessive production of cytokines may lead to inappropriate activation and tissue injury and even to increased susceptibility to invasive microorganisms, impairing the quality life of CGD patients.

NADPH oxidase activity of neutrophil specific granules: requirements for cytosolic components and evidence of assembly during cell activation

Neutrophils and other phagocytic cells support host defense by ingesting microbes and destroying them with reactive oxygen species or oxygen independent mechanisms. Production of ROS is initiated by the phagocyte NADPH oxidase (phox), an enzyme system composed of several constituents. During activation of the cell cytosolic phox proteins (p47phox, p67phox, p40phox, and Rac2) translocate to the plasma membrane and specific granules fuse with the plasma membrane increasing the amount of flavocytochrome b 558. The resultant assembly of phox components results in formation of a complete complex and expression of activity. In this study, we evaluated the oxidase activity of specific granules. In the SDS cell-free system, specific granules expressed oxidase activity in the presence of cytosol in a manner similar to plasma membrane. In contrast to plasma membrane, activity of specific granules was latent, diminishing rapidly over time. In addition, this subcellular fraction contained an inhibitor, possibly related to contamination with azurophilic granules explaining previously published discrepant results. Experiments with recombinant p47phox, p67phox, and dilute cytosol or fractionated cytosol as a source of Rac demonstrated that specific granules have requirements identical to specific granules for oxidase activity. Finally, analysis of neutrophils stimulated with PMA demonstrated translocation of p47phox and to p67phox to specific granules as well as plasma membrane. Both plasma membrane and specific granules from PMA stimulated cells expressed oxidase activity with addition of NADPH demonstrating an assembled oxidase complex. These studies establish a critical role for specific granules as a site for assembly and activation of the oxidase enzyme system and an important constituent for the microbicidal activity of the neutrophil.

Experimental ecology at deep-sea hydrothermal vents: a perspective

In situ and laboratory experiments conducted over the past quarter of a century have greatly increased our understanding of the ecology of deep-sea hydrothermal systems. Early experiments suggested that chemosynthetic primary production constituted the principal source of organic matter for biological communities associated with vents, although subsequent studies have revealed many complexities associated with interactions between microbes and higher organisms inhabiting these ecosystems. A diversity of host-microbial symbiont relationships has been identified and experimental studies have revealed the exquisite physiological adaptations within the giant tubeworm, Riftia pachyptila, for the uptake, fixation, and assimilation of carbon. In vitro experiments demonstrated the unusual sulfide binding properties of tubeworm hemoglobin that prevent inhibition of the cytochrome- c oxidase enzyme system during transport of sulfide to symbiont-bearing tissues. Studies of respiration and growth of several species of vent organisms conducted over the past two decades transformed earlier views that low metabolism and slow growth are characteristics of all organisms inhabiting all deep-sea environments. Results of recent experiments suggest that metabolic rates correlate with the degree of mobility of the organisms rather than with any specific attribute of the deep-sea environment itself, and growth rates of certain vent organisms (e.g., R. pachyptila) were found to be among the highest in any marine environments. While extreme thermal tolerance has been suggested as characteristic of certain vent fauna (e.g., alvinellid polychaetes and alvinocarid shrimp), the majority of vent metazoans live at temperatures below 20 °C and additional experiments are necessary to reconcile field experiments documenting thermal tolerance in situ, thermal tolerance in vivo, and thermal sensitivity of biochemical constituents of vent organisms. Transplantation and clearance experiments, as well as in situ characterization of vent fluid chemistry, have greatly increased our understanding of organism–environment interactions. Early analyses of metazoan egg size and larval morphology, coupled with in vivo larval culture experiments, available physical oceanographic data, and genetic studies of gene flow, have contributed greatly to our understanding of mechanisms of dispersal between widely separated vent sites. The documentation of invertebrate colonization and succession of new vents following a volcanic eruption, and a series of manipulative field experiments, provide considerable insights into the relative roles of abiotic conditions and biotic interactions in structuring vent communities. Recent and emerging technological developments, such as in situ chemical analyzers, observatory approaches, and laboratory-based pressure culture systems, should provide invaluable new experimental tools for tackling many remaining questions concerning the ecology of deep-sea hydrothermal systems.

Lipid rafts determine efficiency of NADPH oxidase activation in neutrophils

We have investigated the contribution of lipid rafts to activation of the NADPH oxidase enzyme system in neutrophils. Membrane-bound NADPH oxidase subunits are present in the lipid raft compartment of neutrophils. Cytosolic NADPH oxidase components are mainly absent from but are recruited to rafts upon Fcγ receptor activation. In parallel, protein kinase C isotypes are recruited to the rafts. Kinetic analysis of NADPH oxidase activation revealed that rafts determine the onset but not the maximal rate of enzyme activity. Thus lipid rafts serve to physically juxtapose the NADPH oxidase effector, protein kinase C and Fcγ receptor, resulting in efficient coupling.

Effect of the Degree of Hydrogenation of Fish Oil on the Enzymatic Activity and on the Fatty Acid Composition of Hepatic Microsomes from Young and Aged Rats

By modifying the degree of hydrogenation of dietary fat, it is possible to modify the fatty acid composition and the biochemical activity of cellular tissues. The age can be another variable influencing these modifications. The effect of isocaloric diets containing oils with different degrees of hydrogenation: fish oil (FO, 0.3% trans), partially hydrogenated fish oil (PHFO, 29% trans), or highly hydrogenated fish oil (HHFO, 2.3% trans), in the fatty acid composition (cis and trans isomers) of hepatic microsomes from young (70-day-old) and aged (18-month-old) rats, in the microsomal cytochrome P-450 (C-450) content, and in the aminopyrine N-demethylase (AND), aniline hydroxylase (AH), NADPH cytochrome P-450 reductase (NCR), UDP-glucuronyl transferase (UGT), and GSH-S transferase (GST) enzymatic activities were studied. Fatty acid composition and n–6/n–3 ratio of microsomal membranes was modified to a higher extent in young rats. C-450 content and AND activity were reduced when the degree of hydrogenation of dietary fat was increased in the young and the aged rats. AH activity was higher after the PHFO diet in the young rats only. NCR activity was reduced in the young animals when the hydrogenation of the fat was increased. However, in aged rats the enzyme exhibited a higher activity after the PHFO and HHFO diet. UGT and GST activities where not affected by the level of hydrogenation of the dietary fat in both the young and the aged rats. However, UGT activity was higher in the young rats, while GST activity was higher in the aged animals. We conclude that hydrogenation of dietary fat can modify the fatty acid composition of hepatic microsomes, young animals being more sensitive to these changes than aged animals. These effects were also reflected in the amount and/or the activity of some molecular components of the hepatic microsomal mixed-function oxidase enzyme system. Microsomal trans fatty acid composition is not affecting the activity of the enzymes, the age of the animals being the most important factor

Diminished production of anti-inflammatory mediators during neutrophil apoptosis and macrophage phagocytosis in chronic granulomatous disease (CGD).

Genetic defects in the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase enzyme system result in chronic granulomatous disease (CGD). In addition to recurrent, life-threatening infections, patients with CGD frequently present with sterile inflammatory complications, suggesting that NADPH-oxidase deficiency predisposes to these responses in the absence of persistent microbial infection. The mechanisms involved in the aberrant, inflammatory process are unknown. In this study, we have shown that neutrophils isolated from CGD patients, which are more resistant to spontaneous apoptosis in vitro, also produce significantly less of the anti-inflammatory mediator cyclopentenone prostaglandin D(2) (PGD(2)). In addition, during phagocytosis of opsonized and nonopsonized apoptotic targets, CGD macrophages are severely compromised in their ability to produce PGD(2) and transforming growth factor-beta (TGF-beta). We suggest that delayed apoptosis of inflammatory cells, such as neutrophils and deficient production of the anti-inflammatory mediators PGD(2) and TGF-beta during macrophage clearance of apoptotic debris and invading pathogens, contributes to persistence of inflammation in CGD.

A 29-kDa Protein Associated with p67phoxExpresses 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).

Toward the development of a biocatalytic system for oxidation of p-xylene to terephthalic acid: oxidation of 1,4-benzenedimethanol

The oxidation of hydrocarbons is an important value-enhancing chemical transformation. Current inorganic catalysts often operate under harsh conditions and produce large amounts of heavy-metal waste. Enzymatic oxidations, on the other hand, operate under relatively mild conditions and produce little if any waste. In the present work, we describe several steps in the oxidative pathway leading from p-xylene to terephthalic acid that are catalyzed by enzymes. Chloroperoxidase (CPO) from Caldariomyces fumago was used to oxidize p-xylene. However, only one of the two aromatic methyl groups was oxidized. To examine the route from 1,4-benzenedimethanol (1,4-BDM) to terephthalic acid, we investigated numerous peroxidase and oxidase enzyme systems. A combination of two enzymes, CPO and xanthine oxidase (XO), was found to produce the highest yield of terephthalic acid from 1,4-BDM. Oxidation of 1,4-BDM to a mixture of predominantly terepthaldicarboxaldehyde, 4-carboxybenzaldehyde, and 4-hydroxymethylbenzaldehyde was carried out by CPO with the continuous addition of hydrogen peroxide as an oxidant. Subsequent addition of XO resulted in a 65% yield of terephthalic acid. A tandem system in which both CPO and XO were present enabled the initial H 2O 2 to be enzymatically regenerated an average of 1.6 times. However, much lower final yields (ca. 2%) of terephthalic acid were obtained.

XANTHINE OXIDASE ACTIVATION IN MILD GESTATIONAL HYPERTENSION

Objective: We hypothesized that activation of the xanthine oxidase (XO) enzyme system is a potential source of free radicals in pregnancy-induced hypertension (PIH). Methods: A prospective observational study was carried out on 16 pregnant women who met the criteria of gestational hypertension [rise in blood pressure (BP) of 30 mm Hg systolic or 15 mm Hg diastolic after 20 weeks gestation or BP>140/90 mm Hg if earlier pressure is unknown] without proteinuria or any signs of renal impairment. Fourteen women with a clinically normal pregnancy matched for maternal age, parity, and gestational age acted as pregnant controls. Nonpregnant control women were members of the laboratory staff (n=15). Main Outcome Measures: Concentrations of free sulfhydryl (SH) groups, purine catabolites, lipid peroxidation products in plasma, and blood carboxyhemoglobin levels were used to follow oxidative stress and potential hemolysis. A noninvasive measurement of functional XO activity was carried out (i.e., the urinary ratio of the two metabolites of caffeine was estimated). Results: A pronounced oxidative stress was demonstrated in plasma samples of patients with hypertension by the elevated concentrations of uric acid and lipid peroxidation products. A reduced level of free sulfhydryl groups and an increased concentration of hypoxanthine (HX) were shown in normotensive pregnant individuals. The XO activity index was substantially higher in overweight pregnant subjects with mild hypertension [0.849±0.096 (p<0.01)] than in normotensive pregnant women or in age-matched nonpregnant subjects [0.596±0.105, 0.542±0.049 (means±SD), respectively]. Conclusions: Our study of mildly hypertensive pregnant subjects provides additional evidence of the putative role of XO activation as a source of free radicals in the early stage of endothelial dysfunction.

Alpha(4)-integrin mediates neutrophil-induced free radical injury to cardiac myocytes.

Previous work has demonstrated that circulating neutrophils (polymorphonuclear leukocytes [PMNs]) adhere to cardiac myocytes via beta(2)-integrins and cause cellular injury via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme system. Since PMNs induced to leave the vasculature (emigrated PMNs) express the alpha(4)-integrin, we asked whether (a) these PMNs also induce myocyte injury via NADPH oxidase; (b) beta(2)-integrins (CD18) still signal oxidant production, or if this process is now coupled to the alpha(4)-integrin; and (c) dysfunction is superoxide dependent within the myocyte or at the myocyte-PMN interface. Emigrated PMNs exposed to cardiac myocytes quickly induced significant changes in myocyte function. Myocyte shortening was decreased by 30-50% and rates of contraction and relaxation were reduced by 30% within the first 10 min. Both alpha(4)-integrin antibody (Ab)-treated PMNs and NADPH oxidase-deficient PMNs were unable to reduce myocyte shortening. An increased level of oxidative stress was detected in myocytes within 5 min of PMN adhesion. Addition of an anti-alpha(4)-integrin Ab, but not an anti-CD18 Ab, prevented oxidant production, suggesting that in emigrated PMNs the NADPH oxidase system is uncoupled from CD18 and can be activated via the alpha(4)-integrin. Addition of exogenous superoxide dismutase (SOD) inhibited all parameters of dysfunction measured, whereas overexpression of intracellular SOD within the myocytes did not inhibit the oxidative stress or the myocyte dysfunction caused by the emigrated PMNs. These findings demonstrate that profound molecular changes occur within PMNs as they emigrate, such that CD18 and associated intracellular signaling pathways leading to oxidant production are uncoupled and newly expressed alpha(4)-integrin functions as the ligand that signals oxidant production. The results also provide pathological relevance as the emigrated PMNs have the capacity to injure cardiac myocytes through the alpha(4)-integrin-coupled NADPH oxidase pathway that can be inhibited by extracellular, but not intracellular SOD.

Inhibitors of Xanthine Oxidase and Scavengers of Superoxide Anions from &lt;I&gt;Cryptolepis sanguinolenta&lt;/I&gt; (Lindl.) Schlechter (Periplocaceae)

Cryptolepis sanguinolenta (Lindl.) Schlechter (Periplocaceae) is a medicinal plant used in some African countries against infectious and parasitic diseases. Six of its constituent alkaloids have been isolated from the root bark, identified spectroscopically as cryptoquindolinine (1), quindoline (2), neocryptolepine (3), cryptolepine (4), 11-hydroxycryptolepine (5) and biscryptolepine (6), and their interaction with the xanthine-xanthine oxidase enzyme system has been tested. Compound 5 has been shown to inhibit xanthine oxidase and act as a scavenger of superoxide anions. Alkaloids 1–4 and 6 were devoid of effect in both assays at the highest test concentration of 100 mM. These findings suggest the importance, in the manifestation of both activities, of the hydroxyl group present in 5 but not in the other alkaloids.

Superoxide Dismutase Biosensors for Superoxide Radical Analysis

ABSTRACT Some basic work has been performed on the development and optimisation of superoxide dismutase (SOD) biosensors for superoxide radical analysis. Initially we studied the possibility of obtaining a SOD biosensor using the Clark electrode as indicating sensor. However, the best results were obtained using as indicator a classical amperometric electrode for H2O2. In both cases the superoxide radical was generated in situ using the xanthine/xanthine oxidase (XOD) enzyme system, while the SOD was immobilised in kappa-carrageenan gel. The first application was realised by studying the effects in vitro on the superoxide radical of some molecules commonly accepted as radical scavengers.

Isolation and some properties of cytochrome c oxidase purified from a bisulfite ion resistant Thiobacillus ferrooxidans strain, OK1-50.

Sulfite ion (HSO3-) is one of the products when elemental sulfur is oxidized by the hydrogen sulfide:ferric ion oxidoreductase of Thiobacillus ferrooxidans AP19-3. Under the conditions in which HSO3- is accumulated in the cells, the iron oxidase of this bacterium was strongly inhibited by HSO3-. Since cytochrome c oxidase is one of the most important components of the iron oxidase enzyme system in T. ferrooxidans, effects of HSO3- on cytochrome c oxidase activity were studied with the plasma membranes of HSO3(-)-resistant and -sensitive strains of T. ferrooxidans, OK1-50 and AP19-3. The enzyme activity of AP19-3 compared with OK1-50 was strongly inhibited by HSO3-. To investigate the inhibition mechanism of HSO3- in T. ferrooxidans, cytochrome c oxidases were purified from both strains to an electrophoretically homogeneous state. Cytochrome c oxidase activity of a purified OK1-50 enzyme was not inhibited by 5 mM HSO3-. In contrast, the same concentration of HSO3- inhibited the enzyme activity of AP19-3 50%, indicating that the cytochrome c oxidase of OK1-50 was more resistant to HSO3- than that of AP19-3. Cytochrome c oxidases purified from both strains were composed of three subunits. However, the molecular weight of the largest subunit differed between OK1-50 and AP19-3. Apparent molecular weights of the three subunits of cytochrome c oxidases were 53,000, 24,000, and 19,000 for strain AP19-3 and 55,000, 24,000, and 19,000 for strain OK1-50, respectively.