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Biomedical subjects

William M Nauseef

Publications and source records attributed to William M Nauseef.

At least 19 recordsLinked to original sources
PubMed2006

CFTR Expression in human neutrophils and the phagolysosomal chlorination defect in cystic fibrosis.

Production of hypochlorous acid (HOCl) in neutrophils, a critical oxidant involved in bacterial killing, requires chloride anions. Because the primary defect of cystic fibrosis (CF) is the loss of chloride transport function of the CF transmembrane conductance regulator (CFTR), we hypothesized that CF neutrophils may be deficient in chlorination of bacterial components due to a limited chloride supply to the phagolysosomal compartment. Multiple approaches, including RT-PCR, immunofluorescence staining, and immunoblotting, were used to demonstrate that CFTR is expressed in resting neutrophils at the mRNA and protein levels. Probing fractions of resting neutrophils isolated by Percoll gradient fractionation and free flow electrophoresis for CFTR revealed its presence exclusively in secretory vesicles. The CFTR chloride channel was also detected in phagolysosomes, a special organelle formed after phagocytosis. Interestingly, HL-60 cells, a human promyelocytic leukemia cell line, upregulated CFTR expresssion when induced to differentiate into neutrophils with DMSO, strongly suggesting its potential role in mature neutrophil function. Analyses by gas chromatography and mass spectrometry (GC-MS) revealed that neutrophils from CF patients had a defect in their ability to chlorinate bacterial proteins from Pseudomonas aeruginosa metabolically prelabeled with [(13)C]-l-tyrosine, unveiling defective intraphagolysosomal HOCl production. In contrast, both normal and CF neutrophils exhibited normal extracellular production of HOCl when stimulated with phorbol ester, indicating that CF neutrophils had the normal ability to produce this oxidant in the extracellular medium. This report provides evidence which suggests that CFTR channel expression in neutrophils and its dysfunction affect neutrophil chlorination of phagocytosed bacteria.

Bacterial Proteins↗
PubMed2006

Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity.

The heterodimeric flavocytochrome b558, comprised of the two integral membrane proteins p22phox and gp91phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22phox required for maturation of gp91phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91phox and two other NADPH oxidase subunits, p47phox, and p67phox. The results demonstrate that p22phox-dependent maturation of gp91phox carbohydrate, cell surface expression of gp91phox, and the enzymatic function of flavocytochrome b558 are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22phox are required, as is a hydrophilic region between amino acids 65 and 90. Upon deletion of 54 residues at the C terminus of p22phox (amino acids 142-195), maturation and cell surface expression of gp91phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47phox. Antibody binding studies indicate that the extreme N terminus of p22phox is inaccessible in the absence of cell permeabilization, supporting a model in which both the N- and C-terminal domains of p22phox extend into the cytoplasm, anchored by two membrane-embedded regions.

Amino Acid Sequence↗
PubMed2006

Enzyme-mediated protein haptenation of dapsone and sulfamethoxazole in human keratinocytes: II. Expression and role of flavin-containing monooxygenases and peroxidases.

Arylamine compounds, such as sulfamethoxazole (SMX) and dapsone (DDS), are metabolized in epidermal keratinocytes to arylhydroxylamine metabolites that auto-oxidize to arylnitroso derivatives, which in turn bind to cellular proteins and can act as antigens/immunogens. Previous studies have demonstrated that neither cytochromes P450 nor cyclooxygenases mediate this bioactivation in normal human epidermal keratinocytes (NHEKs). In this investigation, we demonstrated that methimazole (MMZ), a prototypical substrate of the flavin-containing monooxygenases (FMOs), attenuated the protein haptenation observed in NHEKs exposed to SMX or DDS. In addition, recombinant FMO1 and FMO3 were able to bioactivate both SMX and DDS, resulting in covalent adduct formation. Western blot analysis confirmed the presence of FMO3 in NHEKs, whereas FMO1 was not detectable. In addition to MMZ, 4-aminobenzoic acid hydrazide (ABH) also attenuated SMX- and DDS-dependent protein haptenation in NHEKs. ABH did not alter the bioactivation of these drugs by recombinant FMO3, suggesting its inhibitory effect in NHEKs was due to its known ability to inhibit peroxidases. Studies confirmed the presence of peroxidase activity in NHEKs; however, immunoblot analysis and reverse transcription-polymerase chain reaction indicated that myeloperoxidase, lactoperoxidase, and thyroid peroxidase were absent. Thus, our results suggest an important role for FMO3 and yet-to-be identified peroxidases in the bioactivation of sulfonamides in NHEKs.

Cells, Cultured↗
PubMed2006

Neisseria gonorrhoeae delays the onset of apoptosis in polymorphonuclear leukocytes.

Neisseria gonorrhoeae (gonococcus) infection results in recruitment of polymorphonuclear leukocytes (PMNs) to the urethral lumen. Recent work from our laboratory demonstrated that N. gonorrhoeae resists killing and replicates within PMNs. In this study, we examined the effect of gonococci on PMN viability. Using both transmission electron microscopy and light microscopy, we observed nuclear condensation after 6 h in PMNs that were resting or challenged with opsonized zymosan particles (OPZ). In contrast, N. gonorrhoeae delayed nuclear condensation in PMNs for 12 h (13% apoptotic PMNs vs. 90% for resting and 94% for OPZ-stimulated PMNs). Additionally, DNA fragmentation was reduced in PMNs challenged with gonococci for 12 h (28% apoptosis vs. 52% for resting and 98% for OPZ-stimulated PMNs). However, 74% of PMNs challenged with gonococci had condensed nuclei and 67% had fragmented DNA after 24 h. Caspase activity (total caspase, caspase-3/7, caspase-9) was reduced at 4 h and mitochondrial integrity was preserved at 2 h in PMNs challenged with N. gonorrhoeae. Quantitative reverse transcription polymerase chain reaction demonstrated that mRNA levels of X-IAP and cIAP-2 remained high after challenge with gonococci, but were downregulated in OPZ-stimulated PMNs. Collectively, these findings demonstrate that N. gonorrhoeae delayed apoptosis in PMNs, perhaps as a strategy to allow intracellular replication.

Apoptosis↗
PubMed2006

The antibacterial activity of human neutrophils and eosinophils requires proton channels but not BK channels.

Electrophysiological events are of central importance during the phagocyte respiratory burst, because NADPH oxidase is electrogenic and voltage sensitive. We investigated the recent suggestion that large-conductance, calcium-activated K(+) (BK) channels, rather than proton channels, play an essential role in innate immunity (Ahluwalia, J., A. Tinker, L.H. Clapp, M.R. Duchen, A.Y. Abramov, S. Page, M. Nobles, and A.W. Segal. 2004. Nature. 427:853-858). In PMA-stimulated human neutrophils or eosinophils, we did not detect BK currents, and neither of the BK channel inhibitors iberiotoxin or paxilline nor DPI inhibited any component of outward current. BK inhibitors did not inhibit the killing of bacteria, nor did they affect NADPH oxidase-dependent degradation of bacterial phospholipids by extracellular gIIA-PLA(2) or the production of superoxide anion (O(2*)(-)). Moreover, an antibody against the BK channel did not detect immunoreactive protein in human neutrophils. A required role for voltage-gated proton channels is demonstrated by Zn(2+) inhibition of NADPH oxidase activity assessed by H(2)O(2) production, thus validating previous studies showing that Zn(2+) inhibited O(2*)(-) production when assessed by cytochrome c reduction. In conclusion, BK channels were not detected in human neutrophils or eosinophils, and BK inhibitors did not impair antimicrobial activity. In contrast, we present additional evidence that voltage-gated proton channels serve the essential role of charge compensation during the respiratory burst.

Animals↗
PubMed2006

Effect of D-alanylation of (lipo)teichoic acids of Staphylococcus aureus on host secretory phospholipase A2 action before and after phagocytosis by human neutrophils.

Invading bacteria such as Staphylococcus aureus induce mobilization of professional phagocytes (e.g., neutrophils) and extracellular antibacterial proteins (e.g., group IIA phospholipase A2 (gIIA PLA2)). Accumulation of gIIA PLA2 in inflammatory fluids confers potent extracellular antistaphylococcal activity and at lower concentrations promotes bacterial phospholipid degradation during phagocytosis of S. aureus by human neutrophils. D-alanylation of (lipo) teichoic acids of S. aureus increases bacterial resistance to gIIA PLA2 approximately 100-fold, raising the possibility that the resistance of ingested S. aureus to related gV and gX secretory PLA2 present in human neutrophil granules depends on D-alanylation mediated by the dlt operon. However, we show that isogenic wild-type and dltA S. aureus are equally resistant to gV/X PLA2 during phagocytosis and when exposed to the purified enzymes. The fates of wild-type and dltA S. aureus exposed to serum and human neutrophils differed significantly only when extracellular gIIA PLA2 was also present before phagocytosis. The extreme potency of the gIIA PLA2 toward dltA S. aureus suggests that even small amounts of this extracellular enzyme mobilized early in inflammation could contribute substantially to the overall cytotoxicity of acute inflammatory exudates toward S. aureus when D-alanylation of (lipo)teichoic acids is limiting.

Alanine↗
PubMed2006

Anion channels, including ClC-3, are required for normal neutrophil oxidative function, phagocytosis, and transendothelial migration.

NADPH oxidase activity, phagocytosis, and cell migration are essential functions of polymorphonuclear leukocytes (PMNs) in host defense. The cytoskeletal reorganization necessary to perform these functions has been extensively studied, but the role of cell volume regulation, which is likely dependent upon anion channels, has not been defined. Mice lacking the anion channel ClC-3 (Clcn3(-/-)) died from presumed sepsis following intravascular catheter placement, whereas Clcn3(+/+) littermates survived. We hypothesized that ClC-3 has a critical role in host defense and reasoned that PMN function would be compromised in these mice. Clcn3(-/-) PMNs displayed markedly reduced NADPH oxidase activity in response to opsonized zymosan and modestly reduced activity after phorbol 12-myristate 13-acetate. Human PMNs treated with the anion channel inhibitors niflumic acid or 5-nitro-2-(3-phenylpropylamino)benzoic acid had a very similar defect. ClC-3 protein was detected in the secretory vesicles and secondary granules of resting PMNs and was up-regulated to the phagosomal membrane. Clcn3(-/-) PMNs and human PMNs lacking normal anion channel function both exhibited reduced uptake of opsonized zymosan at 1, 5, and 10 min in a synchronized phagocytosis assay. Niflumic acid-treated PMNs also had impaired transendothelial migration in vitro, whereas migration in vivo was not altered in Clcn3(-/-) PMNs. Selective inhibition of the swelling-activated chloride channel with tamoxifen profoundly reduced PMN migration but had no effect on NADPH oxidase activity. In summary, PMNs lacking normal anion channel function exhibited reduced NADPH oxidase activity, diminished phagocytosis, and impaired migration. ClC-3 was specifically involved in the respiratory burst and phagocytosis.

Animals↗
PubMed2006

Basis for the failure of Francisella tularensis lipopolysaccharide to prime human polymorphonuclear leukocytes.

Francisella tularensis is the intracellular gram-negative coccobacillus that causes tularemia, and its virulence and infectiousness make it a potential agent of bioterrorism. Previous studies using mononuclear leukocytes have shown that the lipopolysaccharide (LPS) of F. tularensis is neither a typical proinflammatory endotoxin nor an endotoxin antagonist. This inertness suggests that F. tularensis LPS does not bind host LPS-sensing molecules such as LPS-binding protein (LBP). Using priming of the polymorphonuclear leukocyte (PMN) oxidase as a measure of endotoxicity, we found that F. tularensis live vaccine strain LPS did not behave like either a classic endotoxin or an endotoxin antagonist in human PMNs, even when the concentration of LBP was limiting. Furthermore, F. tularensis LPS did not compete with a radiolabeled lipooligosaccharide from Neisseria meningitidis for binding to LBP or to the closely related PMN granule protein, bactericidal/permeability-increasing protein. Our results suggest that the inertness of F. tularensis LPS and the resistance of F. tularensis to oxygen-independent PMN killing may result from the inability of F. tularensis LPS to be recognized by these important LPS-sensing molecules of the innate immune system.

Acute-Phase Proteins↗
PubMed2005

Synergy between extracellular group IIA phospholipase A2 and phagocyte NADPH oxidase in digestion of phospholipids of Staphylococcus aureus ingested by human neutrophils.

Acute inflammatory responses to invading bacteria such as Staphylococcus aureus include mobilization of polymorphonuclear leukocytes (PMN) and extracellular group IIA phospholipase A2 (gIIA-PLA2). Although accumulating coincidentally, the in vitro anti-staphylococcal activities of PMN and gIIA-PLA2 have thus far been studied separately. We now show that degradation of S. aureus phospholipids during and after phagocytosis by human PMN requires the presence of extracellular gIIA-PLA2. The concentration of extracellular gIIA-PLA2 required to produce bacterial digestion was reduced 10-fold by PMN. The effects of added gIIA-PLA2 were greater when present before phagocytosis but even apparent when added after S. aureus were ingested by PMN. Related group V and X PLA2, which are present within PMN granules, do not contribute to bacterial phospholipid degradation during and after phagocytosis even when added at concentrations 30-fold higher than that needed for action of the gIIA-PLA2. The action of added gIIA-PLA2 required catalytically active gIIA-PLA2 and, in PMN, a functional NADPH oxidase but not myeloperoxidase. These findings reveal a novel collaboration between cellular oxygen-dependent and extracellular oxygen-independent host defense systems that may be important in the ultimate resolution of S. aureus infections.

Cardiolipins↗
PubMed2005

Evaluation of two anti-gp91phox antibodies as immunoprobes for Nox family proteins: mAb 54.1 recognizes recombinant full-length Nox2, Nox3 and the C-terminal domains of Nox1-4 and cross-reacts with GRP 58.

Progress in the study of Nox protein expression has been impeded because of the paucity of immunological probes. The large subunit of human phagocyte flavocytochrome b558 (Cytb), gp91phox, is also the prototype member of the recently discovered family of NADPH oxidase (Nox) proteins. In this study, we have evaluated the use of two anti-gp91phox monoclonal antibodies, 54.1 and CL5, as immunoprobes for Nox family proteins. Sequence alignment of gp91phox with Nox1, Nox3 and Nox4 identified regions of the Nox proteins that correspond to the gp91phox epitopes recognized by mAb 54.1 and CL5. Antibody 54.1 produced positive immunoblots of recombinant C-terminal fragments of these homologous proteins expressed in E. coli. Furthermore, only mAb 54.1 recognized full-length murine and human Nox3 expressed in HEK-293 cells, in immunoblots of alkali-stripped or detergent-solubilized membranes. 54.1 recognized Nox3 expression-specific proteins with Mr 30,000, 50,000, 65,000 and 88,000 for the murine protein and Mr of 38,000-58,000, 90,000, 100,000-130,000 and a broad species of higher than 160,000 for the human protein. We conclude that mAb 54.1 can serve as a probe of Nox3 and possibly other Nox proteins, if precautions are taken to remove GRP 58 and other crossreactive membrane-associated or detergent-insoluble proteins from the sample to be probed.

Amino Acid Sequence↗
PubMed2005

Biosynthesis, processing, and sorting of human myeloperoxidase.

Exclusively synthesized by normal neutrophil and monocyte precursor cells, myeloperoxidase (MPO) functions not only in host defense by mediating efficient microbial killing but also can contribute to progressive tissue damage in chronic inflammatory states such as atherosclerosis. The biosynthetic precursor, apoproMPO, is processed slowly in the ER, undergoing cotranslational N-glycosylation, transient interactions with the molecular chaperones calreticulin and calnexin, and heme incorporation to generate enzymatically active proMPO that is competent for export into the Golgi. After exiting the Golgi the propeptide is removed prior to final proteolytic processing in azurophil granules, resulting in formation of a symmetric MPO homodimer linked by a disulfide bond. Some proMPO escapes granule targeting and becomes constitutively secreted to the extracellular environment. Although the precise mechanism is unknown, the pro-segment is required for normal processing and targeting, as propeptide-deleted MPO precursor is either degraded or constitutively secreted. Characterizing the molecular consequences of naturally occurring mutations that cause inherited MPO deficiency provides unique insight into the structural determinants of MPO involved in biosynthesis, processing and targeting.

Gene Expression Regulation, Enzymologic↗
PubMed2005

Neutrophil stimulation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) results in the release of functional soluble TRAIL/Apo-2L.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been used to treat bladder cancer for almost 30 years; however, the effector mechanism of the BCG-induced antitumor response remains enigmatic. Most BCG research has focused on the mononuclear-cell infiltrate, but growing evidence supports a role for neutrophils in the antitumor response. Previously, we demonstrated increased urinary tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo-2L) levels from BCG-responsive patients compared to nonresponders. Interestingly, neutrophils isolated from the urine expressed TRAIL/Apo-2L, leading us to investigate the neutrophil response to BCG. BCG-stimulated neutrophils expressed surface-bound and released functional soluble TRAIL/Apo-2L. Whereas neither interferon alpha (IFN-alpha) nor IFN-gamma directly induced TRAIL/Apo2L expression by neutrophils, IFN-alpha did stimulate TRAIL gene transcription, and IFN-primed neutrophils contained and released more TRAIL/Apo-2L after BCG stimulation than did unprimed neutrophils. In unstimulated neutrophils TRAIL/Apo-2L was present predominantly in the azurophilic granules and plasma-membrane-enriched/secretory-granule fraction. Finally, we observed that killed BCG, Toll-like receptor 2 (TLR2) and TLR4 agonists, and an M tuberculosis cell-wall fraction were each capable of inducing the release of soluble TRAIL/Apo-2L from neutrophils. These results further characterize the potential role neutrophils may play in initiating the antitumor response described with BCG treatment for superficial bladder cancer.

Antineoplastic Agents↗
PubMed2005

Homotypic dimerization of the actin-binding protein p57/coronin-1 mediated by a leucine zipper motif in the C-terminal region.

The actin-binding protein p57/coronin-1, a member of the coronin protein family, is selectively expressed in immune cells, and has been implicated in leucocyte migration and phagocytosis by virtue of its interaction with F-actin (filamentous actin). We previously identified two sites in the N-terminal region of p57/coronin-1 by which it binds actin, and in the present study we examine the role of the leucine zipper motif located in the C-terminal coiled-coil domain in mediating the homotypic association of p57/coronin-1. Recombinant p57/coronin-1 protein in solution formed a homodimer, as analysed by Superose 12 column chromatography and by sucrose density gradient centrifugation. In vivo, a truncated form consisting of the C-terminal coiled-coil domain co-precipitated with full-length p57/coronin-1 when both were co-expressed in COS-1 cells. A chimaeric construct composed of the C-terminal domain of p57/coronin-1 (which lacks the actin-binding sites) fused with green fluorescent protein co-localized with cortical F-actin-rich regions in COS-1 cells only when full-length p57/coronin-1 was expressed simultaneously in the cells, suggesting that the C-terminal region is required for the homotypic association of p57/coronin-1. Furthermore, p57LZ, a polypeptide consisting of the C-terminal 90 amino acid residues of p57/coronin-1, was sufficient for dimerization. When two leucine residues out of the four that constitute the leucine zipper structure in p57LZ or full-length p57 were replaced with alanine residues, the mutants failed to form homodimers. Taken together, these results demonstrate that p57/coronin-1 forms homodimers, that the association is mediated by the leucine zipper structure in the C-terminal region, and that it plays a role in the cross-linking of F-actin in the cell.

Animals↗
PubMed2005

Unusual polyclonal anti-gp91 peptide antibody interactions with X-linked chronic granulomatous disease-derived human neutrophils are not from compensatory expression of Nox proteins 1, 3, or 4.

To obtain topological information about human phagocyte flavocytochrome b558 (Cytb), rabbit anti-peptide antibodies were raised against synthetic peptides mimicking gp91(phox) regions: 1-9 (MGN), 30-44 (YRV), 150-159 (ESY), 156-166 (ARK), 247-257 (KIS-1, KIS-2). Following affinity purification on immobilized peptide matrices, all antibodies but not prebleed controls recognized purified detergent-solubilized Cytb by enzyme-linked immunosorbent assay (ELISA). Affinity-purified antibodies recognizing KIS, ARK and ESY but not YRV, MGN or prebleed IgG specifically detected gp91(phox) in immunoblot analysis. Antibodies recognizing MGN, ESY, ARK and KIS but not YRV or the prebleed IgG fraction labeled intact normal neutrophils. Surprisingly, all antibodies, with the exception of YRV and pre-immune IgG controls, bound both normal and Cytb-negative neutrophils from the obligate heterozygous mother of a patient with X-linked chronic granulomatous disease (X-CGD) and all neutrophils from another patient lacking the gp91(phox) gene. Further immunochemical examination of membrane fractions derived from nine genetically unrelated patients with X-CGD, using an antibody that recognizes other Nox protein family members, suggests that the unusual reactivity observed does not reflect the compensatory expression of gp91(phox) homologs Nox1, 3 or 4. These results suggest that an unusual surface reactivity exists on neutrophils derived from X-linked chronic granulomatous disease patients that most likely extends to normal neutrophils as well. The study highlights the need for caution in interpreting the binding of rabbit polyclonal antipeptide antibodies to human neutrophils in general and, in the specific case of antibodies directed against Cytb, the need for Cytb-negative controls.

Amino Acid Sequence↗
PubMed2005

Interactions of Neisseria gonorrhoeae with adherent polymorphonuclear leukocytes.

Neisseria gonorrhoeae causes severe exudative urethritis. The exudates from infected individuals contain large numbers of polymorphonuclear leukocytes (PMN) with ingested gonococci. The fate of N. gonorrhoeae within PMN has been a topic of debate for years. In this study, we examined the interactions of N. gonorrhoeae with PMN adherent to surfaces as a system that better models events during clinical disease. Using chemiluminescence to measure reactive oxygen species (ROS), we found that N. gonorrhoeae stimulated PMN to produce a respiratory burst. Different kinetics were seen when PMN were stimulated with opsonized zymosan particles. In addition, ROS were produced predominantly inside the PMN in response to gonococci. Laser scanning confocal microscopy and transmission electron microscopy showed that N. gonorrhoeae rapidly associated with PMN under these experimental conditions and was internalized. Some gonococci were cleared in the first 30 to 60 min after phagocytosis, but a majority of the population persisted for 6 h after phagocytosis. Quantification of viable organisms showed that a significant portion of the population resisted killing. The viability of this subpopulation remained unchanged for 2 h after phagocytosis. A significant increase of viable gonococci from 1 to 6 h was also observed, suggesting intracellular replication. Four different N. gonorrhoeae strains demonstrated the same capacity to resist PMN-mediated killing, whereas Escherichia coli was rapidly killed by PMN under the same conditions. Taken together, these findings suggest that a subpopulation of N. gonorrhoeae resists killing and replicates within PMN phagosomes in spite of NADPH oxidase activation.

Cell Adhesion↗
PubMed2004

Myeloperoxidase potentiates nitric oxide-mediated nitrosation.

Nitrosation is an important reaction elicited by nitric oxide (NO). To better understand how nitrosation occurs in biological systems, we assessed the effect of myeloperoxidase (MPO), a mediator of inflammation, on nitrosation observed during NO autoxidation. Nitrosation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ; 10 mum) to 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) was monitored by HPLC. Using the NO donor spermine NONOate at pH 7.4, MPO potentiated N-NO-IQ formation. The minimum effective quantity of necessary components was 8.5 nm MPO, 0.25 mum H(2)O(2)/min, and 0.024 mum NO/min. Autoxidation was only detected at >/=1.2 mum NO/min. MPO potentiation was not affected by a 40-fold excess flux of H(2)O(2) over NO or less than a 2.4-fold excess flux of NO over H(2)O(2). Potentiation was due to an 8.8-fold increased affinity of MPO-derived nitrosating species for IQ. Autoxidation was inhibited by azide, suggesting involvement of the nitrosonium ion, NO(+). MPO potentiation was inhibited by NADH, but not azide, suggesting oxidative nitrosylation with NO(2)(.) or an NO(2)(.)-like species. MPO nonnitrosative oxidation of IQ with 0.3 mm NO(2)(-) at pH 5.5 was inhibited by azide, but not NADH, demonstrating differences between MPO oxidation of IQ with NO compared with NO(2)(-). Using phorbol ester-stimulated human neutrophils, N-NO-IQ formation was increased with superoxide dismutase and inhibited by catalase and NADH, but not NaN(3). This is consistent with nitrosation potentiation by MPO, not peroxynitrite. Increased N-NO-IQ formation was not detected with polymorphonuclear neutrophils from two unrelated MPO-deficient patients. Results suggest that the highly diffusible stable gas NO could initiate nitrosation at sites of neutrophil infiltration.

Chromatography, High Pressure Liquid↗