Singlet molecular oxygen regulates vascular tone and blood pressure in inflammation.

Singlet molecular oxygen (1O2) has well-established roles in photosynthetic plants, bacteria and fungi1-3, but not in mammals. Chemically generated 1O2 oxidizes the amino acid tryptophan to precursors of a key metabolite called N-formylkynurenine4, whereas enzymatic oxidation of tryptophan to N-formylkynurenine is catalysed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 15. Under inflammatory conditions, this haem-containing enzyme is expressed in arterial endothelial cells, where it contributes to the regulation of blood pressure6. However, whether indoleamine 2,3-dioxygenase 1 forms 1O2 and whether this contributes to blood pressure control have remained unknown. Here we show that arterial indoleamine 2,3-dioxygenase 1 regulates blood pressure via formation of 1O2. We observed that in the presence of hydrogen peroxide, the enzyme generates 1O2 and that this is associated with the stereoselective oxidation of L-tryptophan to a tricyclic hydroperoxide via a previously unrecognized oxidative activation of the dioxygenase activity. The tryptophan-derived hydroperoxide acts in vivo as a signalling molecule, inducing arterial relaxation and decreasing blood pressure; this activity is dependent on Cys42 of protein kinase G1α. Our findings demonstrate a pathophysiological role for 1O2 in mammals through formation of an amino acid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions.

Hmox1 (Heme Oxygenase-1) Protects Against Ischemia-Mediated Injury via Stabilization of HIF-1α (Hypoxia-Inducible Factor-1α).

OBJECTIVE: Hmox1 (heme oxygenase-1) is a stress-induced enzyme that catalyzes the degradation of heme to carbon monoxide, iron, and biliverdin. Induction of Hmox1 and its products protect against cardiovascular disease, including ischemic injury. Hmox1 is also a downstream target of the transcription factor HIF-1α (hypoxia-inducible factor-1α), a key regulator of the body's response to hypoxia. However, the mechanisms by which Hmox1 confers protection against ischemia-mediated injury remain to be fully understood. Approach and Results: Hmox1 deficient (Hmox1-/-) mice had impaired blood flow recovery with severe tissue necrosis and autoamputation following unilateral hindlimb ischemia. Autoamputation preceded the return of blood flow, and bone marrow transfer from littermate wild-type mice failed to prevent tissue injury and autoamputation. In wild-type mice, ischemia-induced expression of Hmox1 in skeletal muscle occurred before stabilization of HIF-1α. Moreover, HIF-1α stabilization and glucose utilization were impaired in Hmox1-/- mice compared with wild-type mice. Experiments exposing dermal fibroblasts to hypoxia (1% O2) recapitulated these key findings. Metabolomics analyses indicated a failure of Hmox1-/- mice to adapt cellular energy reprogramming in response to ischemia. Prolyl-4-hydroxylase inhibition stabilized HIF-1α in Hmox1-/- fibroblasts and ischemic skeletal muscle, decreased tissue necrosis and autoamputation, and restored cellular metabolism to that of wild-type mice. Mechanistic studies showed that carbon monoxide stabilized HIF-1α in Hmox1-/- fibroblasts in response to hypoxia. CONCLUSIONS: Our findings suggest that Hmox1 acts both downstream and upstream of HIF-1α, and that stabilization of HIF-1α contributes to Hmox1's protection against ischemic injury independent of neovascularization.

Effect of chronic mucus hypersecretion on treatment responses to inhaled therapies in patients with chronic obstructive pulmonary disease: Post hoc analysis of the IMPACT trial.

BACKGROUND AND OBJECTIVE: Chronic mucus hypersecretion (CMH) is a clinical phenotype of COPD. This exploratory post hoc analysis assessed relationship between CMH status and treatment response in IMPACT. METHODS: Patients were randomized to once-daily fluticasone furoate/umeclidinium/vilanterol (FF/UMEC/VI) 100/62.5/25 µg, FF/VI 100/25 µg or UMEC/VI 62.5/25 µg and designated CMH+ if they scored 1/2 in St George's Respiratory Questionnaire (SGRQ) questions 1 and 2. Endpoints assessed by baseline CMH status included on-treatment exacerbation rates, change from baseline in trough forced expiratory volume in 1 second, SGRQ total score, COPD Assessment Test (CAT) score, proportion of SGRQ and CAT responders at Week 52 and safety. RESULTS: Of 10,355 patients in the intent-to-treat population, 10,250 reported baseline SGRQ data (CMH+: 62% [n = 6383]). FF/UMEC/VI significantly (p < 0.001) reduced on-treatment moderate/severe exacerbation rates versus FF/VI and UMEC/VI in CMH+ (rate ratio: 0.87 and 0.72) and CMH- patients (0.82 and 0.80). FF/UMEC/VI significantly (p < 0.05) reduced on-treatment severe exacerbation rates versus UMEC/VI in CMH+ (0.62) and CMH- (0.74) subgroups. Similar improvements in health status and lung function with FF/UMEC/VI were observed, regardless of CMH status. In CMH+ patients, FF/VI significantly (p < 0.001) reduced on-treatment moderate/severe and severe exacerbation rates versus UMEC/VI (0.83 and 0.70). CONCLUSION: FF/UMEC/VI had a favourable benefit: risk profile versus dual therapies irrespective of CMH status. The presence of CMH did not influence treatment response or exacerbations, lung function and/or health status. However, CMH did generate differences when dual therapies were compared and the impact of CMH should be considered in future trial design.

NAD Deficiency, Congenital Malformations, and Niacin Supplementation.

BACKGROUND: Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. METHODS: We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. RESULTS: Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. CONCLUSIONS: Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).

Inhibition of MPO (Myeloperoxidase) Attenuates Endothelial Dysfunction in Mouse Models of Vascular Inflammation and Atherosclerosis.

Objective- Inflammation-driven endothelial dysfunction initiates and contributes to the progression of atherosclerosis, and MPO (myeloperoxidase) has been implicated as a potential culprit. On release by circulating phagocytes, MPO is thought to contribute to endothelial dysfunction by limiting NO bioavailability via formation of reactive oxidants including hypochlorous acid. However, it remains largely untested whether specific pharmacological inhibition of MPO attenuates endothelial dysfunction. We, therefore, tested the ability of a mechanism-based MPO inhibitor, AZM198, to inhibit endothelial dysfunction in models of vascular inflammation. Approach and Results- Three models of inflammation were used: femoral cuff, the tandem stenosis model of plaque rupture in Apoe-/- mice, and C57BL/6J mice fed a high-fat, high-carbohydrate diet as a model of insulin resistance. Endothelial dysfunction was observed in all 3 models, and oral administration of AZM198 significantly improved endothelial function in the femoral cuff and tandem stenosis models only. Improvement in endothelial function was associated with decreased arterial MPO activity, determined by the in vivo conversion of hydroethidine to 2-chloroethidium, without affecting circulating inflammatory cytokines or arterial MPO content. Mechanistic studies in Mpo-/- mice confirmed the contribution of MPO to endothelial dysfunction and revealed oxidation of sGC (soluble guanylyl cyclase) as the underlying cause of the observed limited NO bioavailability. Conclusions- Pharmacological inhibition of MPO is a potential strategy to limit endothelial dysfunction in vascular inflammation. Visual Overview- An online visual overview is available for this article.

Mitochondrial oxidative stress causes insulin resistance without disrupting oxidative phosphorylation.

Mitochondrial oxidative stress, mitochondrial dysfunction, or both have been implicated in insulin resistance. However, disentangling the individual roles of these processes in insulin resistance has been difficult because they often occur in tandem, and tools that selectively increase oxidant production without impairing mitochondrial respiration have been lacking. Using the dimer/monomer status of peroxiredoxin isoforms as an indicator of compartmental hydrogen peroxide burden, we provide evidence that oxidative stress is localized to mitochondria in insulin-resistant 3T3-L1 adipocytes and adipose tissue from mice. To dissociate oxidative stress from impaired oxidative phosphorylation and study whether mitochondrial oxidative stress per se can cause insulin resistance, we used mitochondria-targeted paraquat (MitoPQ) to generate superoxide within mitochondria without directly disrupting the respiratory chain. At ≤10 µm, MitoPQ specifically increased mitochondrial superoxide and hydrogen peroxide without altering mitochondrial respiration in intact cells. Under these conditions, MitoPQ impaired insulin-stimulated glucose uptake and glucose transporter 4 (GLUT4) translocation to the plasma membrane in both adipocytes and myotubes. MitoPQ recapitulated many features of insulin resistance found in other experimental models, including increased oxidants in mitochondria but not cytosol; a more profound effect on glucose transport than on other insulin-regulated processes, such as protein synthesis and lipolysis; an absence of overt defects in insulin signaling; and defective insulin- but not AMP-activated protein kinase (AMPK)-regulated GLUT4 translocation. We conclude that elevated mitochondrial oxidants rapidly impair insulin-regulated GLUT4 translocation and significantly contribute to insulin resistance and that MitoPQ is an ideal tool for studying the link between mitochondrial oxidative stress and regulated GLUT4 trafficking.

Myeloperoxidase is a potential molecular imaging and therapeutic target for the identification and stabilization of high-risk atherosclerotic plaque.

Aims: As the inflammatory enzyme myeloperoxidase (MPO) is abundant in ruptured human atherosclerotic plaques, we aimed to investigate the role of MPO as a potential diagnostic and therapeutic target for high-risk plaque. Methods and results: We employed the tandem stenosis model of atherosclerotic plaque instability in apolipoprotein E gene knockout (Apoe-/-) mice. To test the role of MPO, we used Mpo-/-Apoe-/- mice and the 2-thioxanthine MPO inhibitor AZM198. In vivo MPO activity was assessed by liquid chromatography-tandem mass spectrometry detection of 2-chloroethidium generation from hydroethidine and by bis-5HT-DTPA-Gd (MPO-Gd) molecular magnetic resonance imaging (MRI), while plaque phenotype was verified histologically. Myeloperoxidase activity was two-fold greater in plaque with unstable compared with stable phenotype. Genetic deletion of MPO significantly increased fibrous cap thickness, and decreased plaque fibrin and haemosiderin content in plaque with unstable phenotype. AZM198 inhibited MPO activity and it also increased fibrous cap thickness and decreased fibrin and haemosiderin in plaque with unstable phenotype, without affecting lesion monocytes and red blood cell markers or circulating leukocytes and lipids. MPO-Gd MRI demonstrated sustained enhancement of plaque with unstable phenotype on T1-weighted imaging that was two-fold greater than stable plaque and was significantly attenuated by both AZM198 treatment and deletion of the Mpo gene. Conclusion: Our data implicate MPO in atherosclerotic plaque instability and suggest that non-invasive imaging and pharmacological inhibition of plaque MPO activity hold promise for clinical translation in the management of high-risk coronary artery disease.

Assessment of myeloperoxidase activity by the conversion of hydroethidine to 2-chloroethidium.

Oxidants derived from myeloperoxidase (MPO) contribute to inflammatory diseases. In vivo MPO activity is commonly assessed by the accumulation of 3-chlorotyrosine (3-Cl-Tyr), although 3-Cl-Tyr is formed at low yield and is subject to metabolism. Here we show that MPO activity can be assessed using hydroethidine (HE), a probe commonly employed for the detection of superoxide. Using LC/MS/MS, (1)H NMR, and two-dimensional NOESY, we identified 2-chloroethidium (2-Cl-E(+)) as a specific product when HE was exposed to hypochlorous acid (HOCl), chloramines, MPO/H2O2/chloride, and activated human neutrophils. The rate constant for HOCl-mediated conversion of HE to 2-Cl-E(+) was estimated to be 1.5 × 10(5) M(-1)s(-1). To investigate the utility of 2-Cl-E(+) to assess MPO activity in vivo, HE was injected into wild-type and MPO-deficient (Mpo(-/-)) mice with established peritonitis or localized arterial inflammation, and tissue levels of 2-Cl-E(+) and 3-Cl-Tyr were then determined by LC/MS/MS. In wild-type mice, 2-Cl-E(+) and 3-Cl-Tyr were detected readily in the peritonitis model, whereas in the arterial inflammation model 2-Cl-E(+) was present at comparatively lower concentrations (17 versus 0.3 pmol/mg of protein), and 3-Cl-Tyr could not be detected. Similar to the situation with 3-Cl-Tyr, tissue levels of 2-Cl-E(+) were decreased substantially in Mpo(-/-) mice, indicative of the specificity of the assay. In the arterial inflammation model, 2-Cl-E(+) was absent from non-inflamed arteries and blood, suggesting that HE oxidation occurred locally in the inflamed artery. Our data suggest that the conversion of exogenous HE to 2-Cl-E(+) may be a useful selective and sensitive marker for MPO activity in addition to 3-Cl-Tyr.

Heme oxygenase-1 deficiency alters erythroblastic island formation, steady-state erythropoiesis and red blood cell lifespan in mice.

Heme oxygenase-1 is critical for iron recycling during red blood cell turnover, whereas its impact on steady-state erythropoiesis and red blood cell lifespan is not known. We show here that in 8- to 14-week old mice, heme oxygenase-1 deficiency adversely affects steady-state erythropoiesis in the bone marrow. This is manifested by a decrease in Ter-119(+)-erythroid cells, abnormal adhesion molecule expression on macrophages and erythroid cells, and a greatly diminished ability to form erythroblastic islands. Compared with wild-type animals, red blood cell size and hemoglobin content are decreased, while the number of circulating red blood cells is increased in heme oxygenase-1 deficient mice, overall leading to microcytic anemia. Heme oxygenase-1 deficiency increases oxidative stress in circulating red blood cells and greatly decreases the frequency of macrophages expressing the phosphatidylserine receptor Tim4 in bone marrow, spleen and liver. Heme oxygenase-1 deficiency increases spleen weight and Ter119(+)-erythroid cells in the spleen, although α4ß1-integrin expression by these cells and splenic macrophages positive for vascular cell adhesion molecule 1 are both decreased. Red blood cell lifespan is prolonged in heme oxygenase-1 deficient mice compared with wild-type mice. Our findings suggest that while macrophages and relevant receptors required for red blood cell formation and removal are substantially depleted in heme oxygenase-1 deficient mice, the extent of anemia in these mice may be ameliorated by the prolonged lifespan of their oxidatively stressed erythrocytes.

Insulin resistance is a cellular antioxidant defense mechanism.

We know a great deal about the cellular response to starvation via AMPK, but less is known about the reaction to nutrient excess. Insulin resistance may be an appropriate response to nutrient excess, but the cellular sensors that link these parameters remain poorly defined. In the present study we provide evidence that mitochondrial superoxide production is a common feature of many different models of insulin resistance in adipocytes, myotubes, and mice. In particular, insulin resistance was rapidly reversible upon exposure to agents that act as mitochondrial uncouplers, ETC inhibitors, or mitochondrial superoxide dismutase (MnSOD) mimetics. Similar effects were observed with overexpression of mitochondrial MnSOD. Furthermore, acute induction of mitochondrial superoxide production using the complex III antagonist antimycin A caused rapid attenuation of insulin action independently of changes in the canonical PI3K/Akt pathway. These results were validated in vivo in that MnSOD transgenic mice were partially protected against HFD induced insulin resistance and MnSOD+/- mice were glucose intolerant on a standard chow diet. These data place mitochondrial superoxide at the nexus between intracellular metabolism and the control of insulin action potentially defining this as a metabolic sensor of energy excess.

Tryptophan metabolism to kynurenine is a potential novel contributor to hypotension in human sepsis.

OBJECTIVES: To determine whether tryptophan metabolism to kynurenine contributes to the direct regulation of vascular tone in human septic shock. BACKGROUND: Indoleamine 2,3-dioxygenase 1 is an inducible enzyme that converts tryptophan to kynurenine and shares functional similarities with inducible nitric oxide synthase. Recently, kynurenine has been identified as an endothelium-derived relaxing factor produced during inflammation, raising the possibility that this novel pathway may contribute to hypotension in human sepsis. DESIGN: Prospective, matched, single-center, cohort study. SETTINGS: Intensive care unit of a tertiary teaching hospital matched to control subjects from the general medical ward and healthy volunteers. SUBJECTS: Patients (n = 16) with septic shock had indoleamine 2,3-dioxygenase activity assessed as the kynurenine-to-tryptophan ratio, and the severity of hypotension was determined by their inotrope requirements. Healthy and blood pressure-matched nonseptic control subjects were also studied. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Tissues from septic and control patients were stained for the presence of indoleamine 2,3-dioxygenase 1. Indoleamine 2,3-dioxygenase activity increased up to ninefold in patients with septic shock and was significantly higher than in the two control groups (p < .01). Indoleamine 2,3-dioxygenase activity was strongly correlated with inotrope requirements (p < .001). Indoleamine 2,3-dioxygenase protein was expressed in inflamed cardiac tissue as well as in endothelial cells of resistance vessels in hearts and kidneys from subjects who died from sepsis. CONCLUSIONS: : Indoleamine 2,3-dioxygenase 1 is expressed in resistance vessels in human sepsis and Indoleamine 2,3-dioxygenase activity correlates with hypotension in human septic shock. Indoleamine 2,3-dioxygenase 1 is thus a potential novel contributor to hypotension in sepsis.

Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q.

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.

Limited role for the bilirubin-biliverdin redox amplification cycle in the cellular antioxidant protection by biliverdin reductase.

In mammalian cells, heme is degraded by heme oxygenase to biliverdin, which is then reduced to bilirubin by biliverdin reductase (BVR). Both bile pigments have reducing properties, and bilirubin is now generally considered to be a potent antioxidant, yet it remains unclear how it protects cells against oxidative damage. A presently popular explanation for the antioxidant function of bilirubin is a redox cycle in which bilirubin is oxidized to biliverdin and then recycled by BVR. Here, we reexamined this putative BVR-mediated redox cycle. We observed that lipid peroxidation-mediated oxidation of bilirubin in chloroform, a model of cell membrane-bound bilirubin, did not yield biliverdin, a prerequisite for the putative redox cycle. Similarly, H(2)O(2) did not oxidize albumin-bound bilirubin to biliverdin, and in vitro oxidation of albumin or ligandin-bound bilirubin by peroxyl radicals gave modest yields of biliverdin. In addition, decreasing cellular BVR protein and activity in HeLa cells using RNA interference did not alter H(2)O(2)-mediated cell death, just as BVR overexpression failed to enhance protection of these cells against H(2)O(2)-mediated damage, irrespective of whether bilirubin or biliverdin were added to the cells as substrate for the putative redox cycle. Similarly, transformation of human BVR into hmx1 (heme oxygenase) mutant yeast did not provide protection against H(2)O(2) toxicity above that seen in hmx1 mutant yeast expressing human heme oxygenase-1. Together, these results argue against the BVR-mediated redox cycle playing a general or important role as cellular antioxidant defense mechanism.

Serotonin as a physiological substrate for myeloperoxidase and its superoxide-dependent oxidation to cytotoxic tryptamine-4,5-dione.

During inflammatory events, neutrophils and platelets interact to release a variety of mediators. Neutrophils generate superoxide and hydrogen peroxide, and also discharge the haem enzyme myeloperoxidase. Among numerous other mediators, platelets liberate serotonin (5-hydroxytryptamine), which is a classical neurotransmitter and vasoactive amine that has significant effects on inflammation and immunity. In the present study, we show that serotonin is a favoured substrate for myeloperoxidase because other physiological substrates for this enzyme, including chloride, did not affect its rate of oxidation. At low micromolar concentrations, serotonin enhanced hypochlorous acid production by both purified myeloperoxidase and neutrophils. At higher concentrations, it almost completely blocked the formation of hypochlorous acid. Serotonin was oxidized to a dimer by myeloperoxidase and hydrogen peroxide. It was also converted into tryptamine-4,5-dione, especially in the presence of superoxide. This toxic quinone was produced by stimulated neutrophils in a reaction that required myeloperoxidase. In plasma, stimulated human neutrophils oxidized serotonin to its dimer using the NADPH oxidase and myeloperoxidase. We propose that myeloperoxidase will oxidize serotonin at sites of inflammation. In doing so, it will impair its physiological functions and generate a toxic metabolite that will exacerbate inflammatory tissue damage. Consequently, oxidation of serotonin by myeloperoxidase may profoundly influence inflammatory processes.

Oxidation of methionine to dehydromethionine by reactive halogen species generated by neutrophils.

During infection and inflammation, neutrophils and eosinophils produce hypochlorous acid, hypobromous acid, chloramines, and bromamines. These reactive halogen species preferentially oxidize methionine and thiols. It is commonly assumed that they convert methionine to methionine sulfoxide. However, iodine and organic chloramines are known to convert methionine to dehydromethionine, which is a cyclic azasulfonium salt. The potential for this reaction to occur in biologically relevant situations has so far been neglected. Therefore, we investigated the oxidation of methionine and N-terminal methionine residues by biologically relevant reactive halogen species and neutrophils. When hypochlorous acid reacted with methionine, two major products in addition to methionine sulfoxide were formed. They both had molecular masses two mass units lower than that of methionine and were identified as the diastereomers of dehydromethionine. Hypochlorous acid and chloramines converted methionine to a mixture of approximately 25% dehydromethionine and 75% methionine sulfoxide. Hypobromous acid and bromamines produced upward of 50% dehydromethionine. When methionine was present on the N-termini of peptides, reactive halogen species oxidized them to dehydromethionine with yields as high as 80%. Formylated N-terminal methionines and non-N-terminal methionine residues gave stoichiometric production of the corresponding sulfoxides only. Purified myeloperoxidase used hydrogen peroxide and chloride to catalyze the oxidation of N-terminal methionines to dehydromethionine. Neutrophils oxidized extracellular methionine to 30% dehydromethionine and 70% methionine sulfoxide. They also oxidized their intracellular methionine to dehydromethionine as well as methionine sulfoxide. We propose that reactive halogen species will produce dehydromethionine and form azasulfonium cations on the N-termini of peptides and proteins during inflammatory events.

A phosphatidylcholine-BODIPY 581/591 conjugate allows mapping of oxidative stress in P. falciparum-infected erythrocytes.

The chromophore, BODIPY 581/591, has an extended conjugated system that reacts with oxygen centered-radicals leading to changes in its spectral characteristics. Fatty acid-conjugated BODIPY 581/591 transfers readily between membrane bilayers and can be used as a sensor of oxidative stress in cell populations. We report here the use of a phosphatidylcholine (PC) derivative of BODIPY 581/591, which transfers much less rapidly between membranes. This allows the analysis of oxidative stress in individual cells and in different compartments within cells. Quantitative imaging and flow cytometry were used to measure the ratio of fully conjugated to oxidized probe in model systems and in Plasmodium falciparum-infected erythrocytes. We observed an increase in the oxidation of the parasite-associated BODIPY 581/591-PC as the intraerythrocytic parasite matures. By contrast, BODIPY 581/591-PC associated with the erythrocyte membrane experiences a low level of oxidation even in the later stages of parasite development. Treatment with a pro-oxidant compound caused increased oxidation of the probe in the parasite compartment, but less so in the host cell membrane. Conversely, treatment with ferricyanide increases oxidation of the probe in the erythrocyte cell membrane but does not inhibit parasite growth. Chromatographic analysis of the lipids in infected erythrocytes shows no evidence for loss of alpha-tocopherol or the accumulation of lipid hydroperoxides indicating that, despite the increased oxidative stress, the parasite membranes remain protected from substantial lipid oxidation. We have established BODIPY 581/591-PC as a useful probe of the spatial distribution of oxidative stress in P. falciparum-infected erythrocytes; however, the probe appears to be more sensitive to oxidative damage than endogenous lipids.

Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells.

Cancer cells without mitochondrial DNA (mtDNA) do not form tumors unless they reconstitute oxidative phosphorylation (OXPHOS) by mitochondria acquired from host stroma. To understand why functional respiration is crucial for tumorigenesis, we used time-resolved analysis of tumor formation by mtDNA-depleted cells and genetic manipulations of OXPHOS. We show that pyrimidine biosynthesis dependent on respiration-linked dihydroorotate dehydrogenase (DHODH) is required to overcome cell-cycle arrest, while mitochondrial ATP generation is dispensable for tumorigenesis. Latent DHODH in mtDNA-deficient cells is fully activated with restoration of complex III/IV activity and coenzyme Q redox-cycling after mitochondrial transfer, or by introduction of an alternative oxidase. Further, deletion of DHODH interferes with tumor formation in cells with fully functional OXPHOS, while disruption of mitochondrial ATP synthase has little effect. Our results show that DHODH-driven pyrimidine biosynthesis is an essential pathway linking respiration to tumorigenesis, pointing to inhibitors of DHODH as potential anti-cancer agents.

Absence of the biliverdin reductase-a gene is associated with increased endogenous oxidative stress.

Bilirubin, a byproduct of heme catabolism, has been shown to be an effective lipid-soluble antioxidant in vitro. Bilirubin is able to inhibit free radical chain reactions and protects against oxidant-induced damage in vitro and ex vivo. However, direct evidence for bilirubin's antioxidant effects in vivo remains limited. As bilirubin is formed from biliverdin by biliverdin reductase, we generated global biliverdin reductase-a gene knockout (Bvra-/-) mice to assess the contribution of bilirubin as an endogenous antioxidant. Bvra-/- mice appear normal and are born at the expected Mendelian ratio from Bvra+/- x Bvra+/- matings. Compared with corresponding littermate Bvra+/+ and Bvra+/- animals, Bvra-/- mice have green gall bladders and their plasma concentrations of biliverdin and bilirubin are approximately 25-fold higher and 100-fold lower, respectively. Naïve Bvra-/- and Bvra+/+ mice have comparable plasma lipid profiles and low-molecular weight antioxidants, i.e., ascorbic acid, α-tocopherol and ubiquinol-9. Compared with wild-type littermates, however, plasma from Bvra-/- mice contains higher concentrations of cholesteryl ester hydroperoxides (CE-OOH), and their peroxiredoxin 2 (Prx2) in erythrocytes is more oxidized as assessed by the extent of Prx2 dimerization. These data show that Bvra-/- mice experience higher oxidative stress in blood, implying that plasma bilirubin attenuates endogenous oxidative stress.