Biomedical subjects

M C Dinauer

Publications and source records attributed to M C Dinauer.

At least 37 records · Page 2Linked to original sources

PubMed2000

Impaired superoxide production due to a deficiency in phagocyte NADPH oxidase fails to inhibit atherosclerosis in mice.

Superoxide, the reduced form of molecular oxygen, has been implicated in the genesis of vascular disease. One potential mechanism involves oxidation of low density lipoprotein into an atherogenic particle. A second involves reaction with nitric oxide to generate peroxynitrite, a highly oxidizing intermediate. A third involves regulation of signal transduction in artery wall cells. One well-characterized pathway for superoxide production resides in macrophages, the cellular hallmark of the early atherosclerotic lesion. Macrophages contain a membrane-bound NADPH oxidase that reduces oxygen to superoxide. In the current studies, we used mice that are deficient in the gp91-phox subunit of the NADPH oxidase-a model of chronic granulomatous disease (CGD)-to explore the role of superoxide in atherosclerotic vascular disease. Wild-type and CGD mice on the C57BL/6 background received a high-fat diet for 20 weeks to induce hypercholesterolemia. At the end of this period, the 2 strains of mice had comparable plasma lipid levels, and their atherosclerotic lesions were similar in size. We also crossed CGD mice with apolipoprotein E-deficient (apoE-/-) mice to generate spontaneously hypercholesterolemic animals that lacked functional NADPH oxidase. After 24 weeks, the CGD-apoE-/- animals had lower plasma cholesterol and triglyceride levels than did the apoE-/- animals, but there was no difference in the extent of atherosclerotic plaque. Our findings suggest that superoxide generated by the NADPH oxidase of phagocytes does not promote atherosclerosis in mice with either diet-induced or genetic forms of hypercholesterolemia.

Animals↗

PubMed1999

Functional analysis of NADPH oxidase in granulocytic cells expressing a delta488-497 gp91(phox) deletion mutant.

Chronic granulomatous disease (CGD) is a group of inherited disorders in which phagocytes are unable to generate superoxide (O(2)(-)) due to genetic defects in any 1 of 4 essential NADPH oxidase components. Mutations in the X-linked gene for gp91(phox), the large subunit of the flavocytochrome b(558) heterodimer, account for the majority of CGD. An X-CGD patient in which a splice junction mutation results in an in-frame deletion of 30 nucleotides encoding amino acids 488 to 497 of gp91(phox) (delta488-497 gp91(phox)) has previously been reported. In this study, we generated myeloid PLB-985 cells expressing the mutant triangle delta488-497 gp91(phox) to further characterize its functional properties. These cells mimicked the phenotype of the patient's neutrophils with normal expression of a nonfunctional delta488-497 gp91(phox) flavocytochrome. Translocation of p47(phox) and p67(phox) to delta488-497 gp91(phox) PLB-985 plasma membranes was not affected, as determined both in activated intact cells and in the cell-free system. Furthermore, a synthetic peptide corresponding to residues 488-497 of gp91(phox) was relatively ineffective in inhibiting O(2)(-) production in the cell-free oxidase assay (IC50, approximately 500 micromol/L), suggesting that residues 488-497 of gp91(phox) are not directly involved in oxidase assembly. Mutant delta488-497 gp91(phox) flavocytochrome failed to support iodonitrotetrazolium (INT) reduction, showing a disruption of electron transfer from NADPH to the FAD center of gp91(phox). However, the FAD binding capacity of the mutant flavocytochrome was normal, as measured by equilibrium dialysis. Taken together, these results suggest that the delta488-497 deletion in gp91(phox) disrupts electron transfer to FAD, either due to a defect in NADPH binding or to impaired delivery of electrons from NADPH.

Cell Differentiation↗

PubMed1999

Long-term correction of phagocyte NADPH oxidase activity by retroviral-mediated gene transfer in murine X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is an inherited deficiency of the superoxide-generating phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, resulting in recurrent, severe bacterial and fungal infections. The X-linked form of this disorder (X-CGD) results from mutations in the X-linked gene for gp91(phox), the larger subunit of the oxidase flavocytochrome b(558). In this study, we used a murine model of X-CGD to examine the long-term function of retroviral vectors for expression of gp91(phox) based on the murine stem cell virus (MSCV) backbone. NADPH oxidase activity was reconstituted in neutrophils and macrophages for up to 18 to 24 months posttransplantation of transduced X-CGD bone marrow into lethally irradiated syngeneic X-CGD mice. Southern blot analysis and secondary transplant data showed proviral integration in multilineage repopulating cells. Although relatively small amounts of recombinant gp91(phox) (approximately 5% to 10% of wild-type levels) were detected in neutrophils after retroviral-mediated gene transfer, superoxide-generating activity was approximately 20% to 25% of wild-type mouse neutrophils. Expression of gp91(phox) is normally restricted to mature phagocytes. No obvious toxicity was observed in other hematopoietic lineages in transplant recipients, and provirus-marked cells were capable of reconstituting secondary transplant recipients, who also exhibited NADPH oxidase-positive neutrophils. MSCV-based vectors for long-term expression of gp91(phox) may be useful for gene therapy of human CGD targeted at hematopoietic stem cells.

Animals↗

PubMed1999

O2 sensing is preserved in mice lacking the gp91 phox subunit of NADPH oxidase.

The rapid response to hypoxia in the pulmonary artery (PA), carotid body, and ductus arteriosus is partially mediated by O2-responsive K+ channels. K+ channels in PA smooth muscle cells (SMCs) are inhibited by hypoxia, causing membrane depolarization, increased cytosolic calcium, and hypoxic pulmonary vasoconstriction. We hypothesize that the K+ channels are not themselves "O2 sensors" but rather respond to the reduced redox state created by hypoxic inhibition of candidate O2 sensors (NADPH oxidase or the mitochondrial electron transport chain). Both pathways shuttle electrons from donors, down a redox gradient, to O2. Hypoxia inhibits these pathways, decreasing radical production and causing cytosolic accumulation of unused, reduced, freely diffusible electron donors. PASMC K+ channels are redox responsive, opening when oxidized and closing when reduced. Inhibitors of NADPH oxidase (diphenyleneiodonium) and mitochondrial complex 1 (rotenone) both inhibit PASMC whole-cell K+ current but lack the specificity to identify the O2-sensor pathway. We used mice lacking the gp91 subunit of NADPH oxidase [chronic granulomatous disease (CGD) mice] to assess the hypothesis that NADPH oxidase is a PA O2-sensor. In wild-type lungs, gp91 phox and p22 phox subunits are present (relative expression: macrophages > airways and veins > PASMCs). Deletion of gp91 phox did not alter p22 phox expression but severely inhibited activated O2 species production. Nonetheless, hypoxia caused identical inhibition of whole-cell K+ current (in PASMCs) and hypoxic pulmonary vasoconstriction (in isolated lungs) from CGD vs. wild-type mice. Rotenone vasoconstriction was preserved in CGD mice, consistent with a role for the mitochondrial electron transport chain in O2 sensing. NADPH oxidase, though a major source of lung radical production, is not the pulmonary vascular O2 sensor in mice.

Animals↗

PubMed1999

Mutagenesis of an arginine- and lysine-rich domain in the gp91(phox) subunit of the phagocyte NADPH-oxidase flavocytochrome b558.

Site-directed mutagenesis was used to generate a series of mutants harboring point or multiple substitutions within the hydrophilic, polybasic domain of gp91(phox) encompassed by residues 86-102, which was previously identified as a site of interaction with p47(phox) during phagocyte NADPH oxidase assembly. Recombinant wild-type or mutant gp91(phox) was expressed in a human myeloid leukemia cell line in which the endogenous gp91(phox) gene was disrupted by gene targeting. NADPH oxidase activity was measured in a cytochrome c reduction assay following granulocytic differentiation of cells that expressed recombinant gp91(phox). Expression of a gp91(phox) mutant in which amino acids 89-97 were replaced with nine alternate amino acids abolished NADPH oxidase activity. Expression of gp91(phox) mutants R89T, D95A, D95R, R96A, R96E, or K102T did not significantly affect NADPH oxidase activity. However, mutations of individual or paired arginine residues at positions 91 and 92 had substantial effects on superoxide generation. The R91E/R92E mutation completely abolished both NADPH oxidase activity and membrane-translocation of the cytosolic oxidase proteins p47(phox), p67(phox), Rac1, and Rac2. The phorbol 12-myristate 13-acetate-induced rate of superoxide production was reduced by approximately 75% in cells expressing R91T/R92A, R91E, or R92E gp91(phox) along with an increased lag time to the maximal rates of superoxide production relative to cells expressing wild-type gp91(phox). Taken together, these results demonstrate that Arg91 and Arg92 of gp91(phox) are essential for flavocytochrome b558 function in granulocytes and suggest that these residues participate in the interaction of gp91(phox) with the cytosolic oxidase proteins.

Amino Acid Sequence↗

PubMed1999

Biosynthesis of flavocytochrome b558 . gp91(phox) is synthesized as a 65-kDa precursor (p65) in the endoplasmic reticulum.

The redox center of the phagocyte NADPH oxidase is flavocytochrome b558, a transmembrane protein with two subunits, gp91(phox) and p22(phox). In this study we investigated the identity, subcellular localization, and maturation of a putative 65-kDa gp91(phox) precursor (p65). Expressing the gp91(phox) cDNA in an in vitro transcription and translation system, we found that synthesis of p65 required endoplasmic reticulum (ER) microsomes. Sucrose density gradient centrifugation of postnuclear supernatants obtained from a PLB-985 derived cell line with a constitutively expressed gp91(phox) transgene demonstrated that p65 co-sedimented with the ER marker protein calreticulin and myeloperoxidase precursors. Unexpectedly, the majority of p22(phox) was found in subcellular compartments containing the mature 91-kDa form of gp91(phox) and not with p65, suggesting that heterodimer formation may occur in a post-ER compartment. The heme synthesis inhibitor, succinyl acetone, reduced the abundance of mature gp91(phox) and p22(phox) but had little or no impact on p65. These studies demonstrate (a) gp91(phox) is synthesized as a glycosylated 65-kDa precursor in the ER, (b) heterodimer formation is not a co-translational process, and (c) heme insertion is a determinant in the formation of a stable heterodimer but does not appear to affect the stability of p65.

Cell Differentiation↗

PubMed1999

Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense.

In mammals, the Rho family GTPase Rac2 is restricted in expression to hematopoietic cells, where it is coexpressed with Rac1. Rac2-deficient mice were created to define the physiological requirement for two near-identical Rac proteins in hematopoietic cells. rac2-/- neutrophils displayed significant defects in chemotaxis, in shear-dependent L-selectin-mediated capture on the endothelial substrate Glycam-1, and in both F-actin generation and p38 and, unexpectedly, p42/p44 MAP kinase activation induced by chemoattractants. Superoxide production by rac2-/- bone marrow neutrophils was significantly reduced compared to wild type, but it was normal in activated peritoneal exudate neutrophils. These defects were reflected in vivo by baseline neutrophilia, reduced inflammatory peritoneal exudate formation, and increased mortality when challenged with Aspergillus fumigatus. Rac2 is an essential regulator of multiple specialized neutrophil functions.

Actins↗

PubMed1999

Are reactive oxygen species involved in the pathogenesis of murine cerebral malaria?

To investigate the involvement of oxidative tissue damage in the pathogenesis of murine cerebral malaria (CM), brain levels of protein carbonyls, 3,4-dihydroxyphenylalanine (DOPA), o-tyrosine, and dityrosine were measured during Plasmodium berghei ANKA (PbA) and P. berghei K173 (PbK) infections. During PbA infection in a CM model, brain levels of the substances were similar to those in uninfected mice. The role of phagocyte-derived reactive oxygen species in the pathogenesis of CM was examined in gp91phox gene knockout mice. The course of CM in these mice was the same as in their wild type counterparts. To examine whether superoxide production in the central nervous system could have occurred via increased xanthine oxidase activity, brain concentrations of urate were measured in CM mice and in mice infected with PbK (which does not cause CM). Brain urate concentration increased significantly in both groups of mice, suggesting that purine breakdown is not specific to CM. These results indicate that reactive oxygen species probably do not contribute to the pathogenesis of murine CM.

Animals↗

PubMed1999

Identification and functional characterization of the murine Rac2 gene promoter.

Rac2, a member of the Rho family of GTPases, is highly expressed in myeloid cells and is a regulator of the NADPH-oxidase complex. A murine genomic clone was isolated that contains the 5' end and putative promoter region of the Rac2 gene. Ribonuclease protection experiments detected 13 transcription initiation sites scattered 50 to 130 bp upstream of the translation initiation site. Transient transfection studies revealed that -7 kb to +31 bp (relative to the strongest transcription initiation site) of the Rac2 gene 5'-flanking region exhibited strong promoter activity in both RAW 264.7 macrophage cells that express the endogenous Rac2 gene and NIH-3T3 fibroblast cells that do not express the endogenous gene. Truncated Rac2 promoter fragments containing as little as the -74 to +31 bp sequence produced full transcriptional activity. However, a -57 to +31 promoter fragment directed significantly less transcription, and a -39 to +31 promoter fragment was transcriptionally inactive. In vitro binding assays revealed sequence-specific and widely expressed DNA-binding activities that interacted within the -74 to -58 Rac2 promoter cis element. Oligonucleotide competition and antibody disruption studies indicated that these complexes contained the transcription factors Spl and Sp3. Specific ablation of the Sp1/Sp3 binding site significantly decreased Rac2 promoter activity in both RAW 264.7 and NIH-3T3 cells. Additional cis elements may be required to restrict Rac2 promoter activity to hematopoietic cells expressing the endogenous gene.

3T3 Cells↗

PubMed1999

Lethality of endotoxin in mice genetically deficient in the respiratory burst oxidase, inducible nitric oxide synthase, or both.

Two classes of oxidants are thought to play a critical role in tissue damage in septic shock: reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Particular importance has been ascribed to peroxynitrite, a product arising from the reaction of nitric oxide with superoxide. A major source of ROI is the respiratory burst oxidase of neutrophils, eosinophils, monocytes, and macrophages. A major source of RNI is inducible nitric oxide synthase (iNOS), an enzyme expressed in leukocytes, hepatocytes, vascular smooth muscle cells, endothelium, and cardiac myocytes during inflammation. In previous studies using various mouse models of endotoxic shock, genetic deficiency of iNOS as a sole intervention did not consistently alter survival. Here, using Salmonella typhimurium endotoxic bacterial lipopolysaccharide (LPS) as a sole challenge, genetic deficiency of iNOS was associated with no protection or a reduction in survival, depending on the dose of LPS. Further, no protection from lethality was observed when LPS was injected into mice genetically deficient in the 91 kDa subunit of the respiratory burst oxidase (gp91phox) nor in mice genetically deficient in both gp91phox and iNOS (gp91phox-/-/NOS2-/- mice). For the latter experiments, mice were challenged either with S. typhimurium LPS alone or with inactivated bacille Calmette-Guerin (BCG) followed by Escherichia coli LPS. Deficiency of gp91phox impaired the inflammatory response to inactivated Propionobacterium acnes, rendering survival studies following priming with P. acnes difficult to interpret. Thus, in two models of endotoxic shock, major reductions in the ability to form nitric oxide or superoxide, alone or in combination, failed to improve survival.

Animals↗

PubMed1999

Glucose 6-phosphate dehydrogenase is required for Salmonella typhimurium virulence and resistance to reactive oxygen and nitrogen intermediates.

Salmonella typhimurium zwf mutants lacking glucose 6-phosphate dehydrogenase (G6PD) activity have increased susceptibility to reactive oxygen and nitrogen intermediates as well as attenuated virulence in mice. Abrogation of the phagocyte respiratory burst oxidase during experimental infection with zwf mutant Salmonella causes a prompt restoration of virulence, while inhibition of inducible nitric oxide synthase results in delayed lethality. These observations suggest that G6PD-dependent bacterial antioxidant defenses play an important pathogenic role during early salmonellosis and additionally may help to antagonize NO-dependent antimicrobial mechanisms later in the course of infection.

Animals↗

PubMed1998

Correction of respiratory burst activity in X-linked chronic granulomatous cells to therapeutically relevant levels after gene transfer into bone marrow CD34+ cells.

Chronic granulomatous disease (CGD) is a disorder of the lymphohematopoietic system, whereby phagocytes of affected patients are unable to kill microorganisms. CGD is caused by a functional defect in the phagocytic nicotinamide adenine dinucleotide phosphatase (NADPH) oxidase (phox) enzyme complex, leading to a lack of microbicidal metabolites. As a therapeutic approach toward the predominant X-linked form of CGD, we have developed a bicistronic retroviral vector containing the coding sequences of gp91-phox and a cytoplasmically truncated version of the human low-affinity receptor for nerve growth factor (deltaLNGFR). Full reconstitution of superoxide-generating activity was achieved with this vector in a gp91-phox-deficient cell line. Using an optimized gene transfer protocol, up to 85% of the CD34+ cells obtained from the bone marrow of X-CGD patients were transduced. CD15+ cells differentiated in vitro from transduced X-CGD CD34+ cells showed correction of NADPH oxidase activity to 45-52% of normal levels whereas deltaLNGFR expression was found in 40-67% of the CD15+ cells. Moreover, immunoblots prepared from extracts of transduced CD15+ cells revealed gp91-phox protein levels similar to those found in neutrophils derived from normal CD34+ cells. Taking into consideration that superoxide production in only 5 to 10% of wild-type neutrophils is sufficient to protect X-CGD heterozygotes from serious infections, the results achieved in this study shows that for X-CGD patients a curative approach based on the genetic modification of hematopoietic stem/progenitor cells is feasible.

Antigens, CD34↗

PubMed1998

Gp91(phox) is the heme binding subunit of the superoxide-generating NADPH oxidase.

The phagocyte NADPH oxidase flavocytochrome b558 is a membrane-bound heterodimer comprised of a glycosylated subunit, gp91(phox), and a nonglycosylated subunit, p22(phox). It contains two nonidentical heme groups that mediate the final steps of electron transfer to molecular oxygen (O2), resulting in the generation of superoxide ion (O2-). However, the location of the hemes within the flavocytochrome heterodimer remains controversial. In this study, we have used transgenic COS7 cell lines expressing gp91(phox), p22(phox), or both polypeptides to examine the relative role of each flavocytochrome b558 subunit in heme binding and O2- formation. A similar membrane localization was observed when gp91(phox) and p22(phox) were either expressed individually or coexpressed, as analyzed by confocal microscopy and immunoblotting of subcellular fractions. Spectral analysis of membranes prepared from COS7 cell lines expressing either gp91(phox) or both gp91(phox) and p22(phox) showed a b-type cytochrome with spectral characteristics identical to those of human neutrophil flavocytochrome b558. In contrast, no heme spectrum was detected in wild-type COS7 membranes or those containing only p22(phox). Furthermore, redox titration studies suggested that two heme groups were contained in gp91(phox) expressed in COS7 membranes, with midpoint potentials of -264 and -233 mV that were very similar to those obtained for neutrophil flavocytochrome b558. These results provide strong support for the hypothesis that gp91(phox) is the sole heme binding subunit of flavocytochrome b558. However, coexpression of gp91(phox) and p22(phox) in COS7 membranes was required to support O2- production in combination with neutrophil cytosol, indicating that the functional assembly of the active NADPH oxidase complex requires both subunits of flavocytochrome b558.

Animals↗

PubMed1998

Probing the role of the carboxyl terminus of the gp91phox subunit of neutrophil flavocytochrome b558 using site-directed mutagenesis.

Site-directed mutagenesis was used to generate a series of substitutions and deletions in the carboxyl-terminal 11 residues of gp91phox, the 91-kDa subunit of the phagocyte NADPH oxidase flavocytochrome b558. This region encompasses 559RGVHFIF565, implicated as a contact point for the cytosolic oxidase subunit p47phox during oxidase activation, and a carboxyl-terminal phenylalanine (Phe570), which corresponds in position to a highly conserved aromatic residue that interacts with the flavin group in the ferredoxin-NADP+ reductase flavoenzyme family, of which gp91phox is a member. Mutant proteins were expressed in human myeloid leukemia cells which lack expression of endogenous gp91phox due to targeted disruption of the X-linked gp91phox gene. Although specific residues within 559RGVHFIF565 had previously been identified by alanine scanning as essential for peptide inhibition of oxidase activity in a cell-free assay, comparable substitutions in the gp91phox polypeptide had either no or only a modest effect on oxidase activity in whole cells. Replacement of nonpolar with polar or charged residues had greater effects on oxidase activity, but were also associated with decreased gp91phox expression, suggesting that overall protein structure was perturbed. No stable gp91phox protein was detected upon deletion of the terminal 11 amino acids. Alanine substitution or deletion of the carboxyl-terminal Phe570 in gp91phox resulted in a 2-fold reduction in superoxide production. This contrasts with a approximately 300-800-fold reduction reported for comparable mutations in pea ferredoxin-NADP+ reductase, which suggests that structural or functional differences exist between the carboxyl terminus of gp91phox and other ferredoxin-NADP+ reductases.

Amino Acid Sequence↗

PubMed1998

X-Linked chronic granulomatous disease: mutations in the CYBB gene encoding the gp91-phox component of respiratory-burst oxidase.

Chronic granulomatous disease (CGD) is a hereditary disorder of host defense due to absent or decreased activity of phagocyte NADPH oxidase. The X-linked form of the disease derives from defects in the CYBB gene, which encodes the 91-kD glycoprotein component (termed "gp91-phox") of the oxidase. We have identified the mutations in the CYBB gene responsible for X-linked CGD in 131 consecutive independent kindreds. Screening by SSCP analysis identified mutations in 124 of the kindreds, and sequencing of all exons and intron boundary regions revealed the other seven mutations. We detected 103 different specific mutations; no single mutation appeared in more than seven independent kindreds. The types of mutations included large and small deletions (11%), frameshifts (24%), nonsense mutations (23%), missense mutations (23%), splice-region mutations (17%), and regulatory-region mutations (2%). The distribution of mutations within the CYBB gene exhibited great heterogeneity, with no apparent mutational hot spots. Evaluation of 87 available mothers revealed X-linked carrier status in all but 10. The heterogeneity of mutations and the lack of any predominant genotype indicate that the disease represents many different mutational events, without a founder effect, as is expected for a disorder with a previously lethal phenotype.

DNA Mutational Analysis↗

PubMed1997

Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase.

Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide. Periplasmic localization of bacterial Cu,Zn-SOD has suggested a role of this enzyme in defense against extracellular phagocyte-derived reactive oxygen species. Sequence analysis of regions flanking the Salmonella typhimurium sodC gene encoding Cu,Zn-SOD demonstrates significant homology to lambda phage proteins, reflecting possible bacteriophage-mediated horizontal gene transfer of this determinant among pathogenic bacteria. Salmonella deficient in Cu,Zn-SOD has reduced survival in macrophages and attenuated virulence in mice, which can be restored by abrogation of either the phagocyte respiratory burst or inducible nitric oxide synthase. Moreover, a sodC mutant is extremely susceptible to the combination of superoxide and nitric oxide. These observations suggest that SOD protects periplasmic or inner membrane targets by diverting superoxide and limiting peroxynitrite formation, and they demonstrate the ability of the respiratory burst and nitric oxide synthase to synergistically kill microbial pathogens in vivo.

Animals↗

PubMed1997

Biosynthesis of the phagocyte NADPH oxidase cytochrome b558. Role of heme incorporation and heterodimer formation in maturation and stability of gp91phox and p22phox subunits.

The NADPH oxidase cytochrome b558 is a membrane heterodimer comprised of a glycosylated 91-kDa subunit, gp91(phox), and a nonglycosylated 22-kDa subunit, p22(phox). The role of heme in cytochrome b558 biosynthesis was studied using succinyl acetone, an inhibitor of heme synthesis, in PLB-985 myeloid cells undergoing granulocytic differentiation. Succinyl acetone markedly reduced expression of p22(phox) and the mature 91-kDa form of gp91(phox) but not its 65-kDa high mannose precursor, in association with a profound reduction in NADPH oxidase activity. Expression of non-heme-containing cytosolic oxidase components was unaffected. The reduction in cytochrome b558 expression and NADPH oxidase activity was prevented by adding exogenous heme and was reversible upon removal of succinyl acetone. Transgenic expression of gp91(phox) in monkey COS-7 and murine 3T3 cells, both of which lacked endogenous p22(phox) mRNA, demonstrated that p22(phox) was not required for maturation of gp91(phox) carbohydrate to complex oligosaccharides. However, coexpression of transgenic p22(phox) increased the abundance of the mature gp91(phox) glycoprotein. These results suggest that heme incorporation plays an important role in cytochrome b558 assembly and provide further support for the concept that stability of p22(phox) and the mature gp91(phox) subunit is increased by heterodimer formation.

3T3 Cells↗

PubMed1997

Mice lacking reduced nicotinamide adenine dinucleotide phosphate oxidase activity show increased susceptibility to early infection with Listeria monocytogenes.

Mice lacking the gp91 protein of reduced nicotinamide adenine dinucleotide phosphate oxidase showed heightened susceptibility to Listeria infection. The enhanced susceptibility was noted 2 days after infection, the usual peak time of the neutrophil-dependent lesion in the liver. Infected gp91phox -/- mice had an increase of approximately 30-fold in Listeria in the liver and an increase in the number of microabscesses. The study of the gp91phox mice underscores the early role of the neutrophil and of an oxidative burst in this infection with an intracellular pathogen. These results contrast with others showing that the late macrophage-dependent stage of the infection is dependent on nitric oxide.

Animals↗