Shigemoto Fujii

Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling

Significance Reactive sulfur-containing compounds, such as l-cysteine hydropersulfide (CysSSH), reportedly form in mammals. However, the biological relevance of these reactive sulfur species remains unclear. We determined that CysSSH was synthesized from cystine by cystathionine β-synthase and cystathionine γ-lyase, which in turn may contribute to high levels of glutathione hydropersulfide (>100 μM) and other CysSSH derivatives of peptides/proteins formed in cells, tissues, and plasma from mice and humans. Compared with glutathione and hydrogen sulfide, CysSSH derivatives were superior nucleophiles and reductants and capable of regulating electrophilic cell signaling mediated by 8-nitroguanosine 3′,5′-cyclic monophosphate. Altogether, it is proposed that reactive Cys persulfides and S-polythiolation have critical regulatory functions in redox cell signaling. Using methodology developed herein, it is found that reactive persulfides and polysulfides are formed endogenously from both small molecule species and proteins in high amounts in mammalian cells and tissues. These reactive sulfur species were biosynthesized by two major sulfurtransferases: cystathionine β-synthase and cystathionine γ-lyase. Quantitation of these species indicates that high concentrations of glutathione persulfide (perhydropersulfide >100 μM) and other cysteine persulfide and polysulfide derivatives in peptides/proteins were endogenously produced and maintained in the plasma, cells, and tissues of mammals (rodent and human). It is expected that persulfides are especially nucleophilic and reducing. This view was found to be the case, because they quickly react with H2O2 and a recently described biologically generated electrophile 8-nitroguanosine 3′,5′-cyclic monophosphate. These results indicate that persulfides are potentially important signaling/effector species, and because H2S can be generated from persulfide degradation, much of the reported biological activity associated with H2S may actually be that of persulfides. That is, H2S may act primarily as a marker for the biologically active of persulfide species.

The critical role of nitric oxide signaling, via protein S-guanylation and nitrated cyclic GMP, in the antioxidant adaptive response

A nitrated guanine nucleotide, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), is formed via nitric oxide (NO) and causes protein S-guanylation. However, intracellular 8-nitro-cGMP levels and mechanisms of formation of 8-nitro-cGMP and S-guanylation are yet to be identified. In this study, we precisely quantified NO-dependent formation of 8-nitro-cGMP in C6 glioma cells via liquid chromatography-tandem mass spectrometry. Treatment of cells with S-nitroso-N-acetylpenicillamine led to a rapid, transient increase in cGMP, after which 8-nitro-cGMP increased linearly up to a peak value comparable with that of cGMP at 24 h and declined thereafter. Markedly high levels (>40 μm) of 8-nitro-cGMP were also evident in C6 cells that had been stimulated to express inducible NO synthase with excessive NO production. The amount of 8-nitro-cGMP generated was comparable with or much higher than that of cGMP, whose production profile slightly preceded 8-nitro-cGMP formation in the activated inducible NO synthase-expressing cells. These unexpectedly large amounts of 8-nitro-cGMP suggest that GTP (a substrate of cGMP biosynthesis), rather than cGMP per se, may undergo guanine nitration. Also, 8-nitro-cGMP caused S-guanylation of KEAP1 in cells, which led to Nrf2 activation and subsequent induction of antioxidant enzymes, including heme oxygenase-1; thus, 8-nitro-cGMP protected cells against cytotoxic effects of hydrogen peroxide. Proteomic analysis for endogenously modified KEAP1 with matrix-assisted laser desorption/ionization time-of-flight-tandem mass spectrometry revealed that 8-nitro-cGMP S-guanylated the Cys434 of KEAP1. The present report is therefore the first substantial corroboration of the biological significance of cellular 8-nitro-cGMP formation and potential roles of 8-nitro-cGMP in the Nrf2-dependent antioxidant response.

Helicobacter cinaedi Cellulitis and Bacteremia in Immunocompetent Hosts after Orthopedic Surgery

ABSTRACT At various times after orthopedic operations (more than a few weeks, with an average of 29.9 days), 11 patients had a sudden onset of high temperature (average 38.9°C) and local cellulitis at different sites on the operated sides. The wounds had completely healed, without complicated infections, when the cellulitis occurred. The clinical picture of cellulitis in all patients was atypical: diffuse salmon-pink skin color, local heat, swelling, spontaneous pain, and tenderness but no eruptions. No patient had any underlying immunocompromising conditions or had been given immunosuppressive agents. Gram-negative spiral bacteria were isolated from blood cultures and were identified as Helicobacter cinaedi on the basis of 16S rRNA gene sequencing and DNA-DNA hybridization using standard strains. By means of phylogenetic analysis, we divided these clinical isolates into two clones. The H. cinaedi strain isolated via fecal cultures from two patients without intestinal symptoms was the same clone as the blood isolate. All isolates were quite susceptible to various antibiotics, and clinical and inflammatory symptoms of bacteremia and cellulitis improved after treatment with penicillins and cephalosporins. A relatively high incidence of recurrence of the same disease was observed, however. Almost all patients responded immunologically to the infection, as evidenced by the production of serum antibody against H. cinaedi. We thus suggest that H. cinaedi should not be regarded as simply an opportunistic pathogen but that it may be a pathogen in immunocompetent hosts and may cause infections together with bacteremia and cellulitis.

l-Arginine Reverses p47phox and gp91phox Expression Induced by High Salt in Dahl Rats

Abstract—Derangements in the production and degradation of reactive oxygen species (ROS) as well as nitric oxide (NO) have been implicated in cardiovascular diseases. We explored how supplementation with l-arginine, an NO synthase substrate, restores such derangements of ROS/NO systems in Dahl salt-sensitive, hypertensive (DS) rats. We detected an increase of NADPH oxidase activity, a key enzyme that produces superoxide, in the membrane fraction of the renal cortex derived from DS rats loaded with high salt for 4 weeks; high salt loading also remarkably increased urinary H2O2, 8-isoprostane, and thromboxane B2 excretion and decreased plasma NO end products. These changes from high salt loading were counteracted by oral l-arginine supplementation. We further examined expression patterns of NADPH oxidase subunits in renal cortex derived from these animals. High salt loading increased gp91phox and p47phox but not p22phox or Rac1 or mRNA abundance, which were counteracted with l-arginine supplementation. Western blot analyses after subcellular fractionation revealed that l-arginine supplementation distinctly decreases membrane localization of p47phox protein, as it decreases total expression of Rac1 protein in DS rats with high salt loading. These results disclose that high salt loading causes a deficiency in available l-arginine amounts for NO synthases and induces NADPH oxidase activation in the renal cortex of DS rats, which l-arginine supplementation markedly restores. Since superoxide rapidly eliminates NO, which inhibits sodium reabsorption in the cortical collecting duct, superoxide production caused by upregulated NADPH oxidase activity in the renal cortex of high salt–loaded DS rats may accelerate sodium reabsorption and hypertension.

Cytoprotective function of heme oxygenase 1 induced by a nitrated cyclic nucleotide formed during murine salmonellosis.

Signaling mechanisms of NO-mediated host defense are yet to be elucidated. In this study, we report a unique signal pathway for cytoprotection during Salmonella infection that involves heme oxygenase 1 (HO-1) induced by a nitrated cyclic nucleotide, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP). Wild-type C57BL/6 mice and C57BL/6 mice lacking inducible NO synthase (iNOS) were infected with Salmonella enterica serovar Typhimurium LT2. HO-1 was markedly up-regulated during the infection, the level being significantly higher in wild-type mice than in iNOS-deficient mice. HO-1 up-regulation was associated with 8-nitro-cGMP formation detected immunohistochemically in Salmonella-infected mouse liver and peritoneal macrophages. 8-Nitro-cGMP either exogenously added or formed endogenously induced HO-1 in cultured macrophages infected with Salmonella. HO-1 inhibition by polyethylene glycol-conjugated zinc-protoporphyrin IX impaired intracellular killing of bacteria in mouse liver and in both RAW 264 cells and peritoneal macrophages. Infection-associated apoptosis was also markedly increased in polyethylene glycol-conjugated zinc-protoporphyrin IX-treated mouse liver cells and cultured macrophages. This effect of HO-1 inhibition was further confirmed by using HO-1 short interfering RNA in peritoneal macrophages. Our results suggest that HO-1 induced by NO-mediated 8-nitro-cGMP formation contributes, via its potent cytoprotective function, to host defense during murine salmonellosis.

Induction of LOX-1 and iNOS expressions by ischemia-reperfusion of rat kidney and the opposing effect of l-arginine

Lectin‐like oxidized low‐density lipoprotein receptor (LOX‐1) is a newly identified endothelial cell surface major receptor for oxidatively modified low‐density lipoprotein. Progression of arthrosclerosis in the donor organ after organ transplantation is a major problem. We hypothesized that ischemia‐reper‐fusion induces LOX‐1. After 1 h ischemia of bilateral kidneys plus 3, 6, or 12 h reperfusion, we first revealed that LOX‐1 mRNA expression was increased in renal cortex and medulla at 6 h after reperfusion, which was decreased by l‐arginine supplement. Plasma nitric oxide (NO) end‐product nitrite plus nitrate and inducible nitric oxide synthase (NOS) expression were increased after reperfusion of 6 h. However, NOS substrate l‐arginine did not augment but markedly decreased plasma NO end product, because l‐arginine supplement suppressed inducible NOS expression in kidney. We hypothesized that available l‐arginine is depleted by ischemia‐reperfusion, leading to inducible NOS induction. Ischemia decreased l‐arginine levels in kidney and l‐arginine supplement increased NO end products in renal cortex in the earliest phase of reperfusion. These results disclosed for the first time that a deficiency in l‐arginine by ischemia reperfusion causes uncoupling of constitutive NOS, which induces inducible NOS and LOX‐1, implying why l‐arginine is effective for stroke or transplantation in preventing atherosclerotic progress.—Kosaka, H., Yoneyama, H., Zhang, L., Fujii, S., Yamamoto, A., Igarashi, J. Induction of LOX‐1 and iNOS expressions by ischemia‐reperfusion of rat kidney and the opposing effect of L‐arginine. FASEB J. 17, 636–643 (2003)

Identification of and Screening for Human Helicobacter cinaedi Infections and Carriers via Nested PCR

ABSTRACT Helicobacter cinaedi is the most frequently reported enterohepatic Helicobacter species isolated from humans. Earlier research suggested that certain patients with H. cinaedi infection may remain undiagnosed or incorrectly diagnosed because of difficulties in detecting the bacteria by conventional culture methods. Here, we report a nested PCR assay that rapidly detects the cytolethal distending toxin gene (cdt) of H. cinaedi with high specificity and sensitivity. Specificity of the assay was validated by using different species of Helicobacter and Campylobacter, as well as known H. cinaedi-positive and -negative samples. The sensitivity of detection for the cdt gene in the assay was 102 CFU/ml urine or 102 CFU/105 infected RAW 264.7 cells. In an H. cinaedi-infected mouse model, the cdt gene of H. cinaedi was effectively detected via the assay with urine (6/7), stool (2/3), and blood (2/6) samples. Importantly, it detected H. cinaedi in blood, urine, and stool samples from one patient with a suspected H. cinaedi infection and three patients with known infections. The assay was further used clinically to follow up two H. cinaedi-infected patients after antibiotic treatment. Stool samples from these two patients evaluated by nested PCR after antibiotic therapy showed clearance of bacterial DNA. Finally, analysis of stool specimens from healthy volunteers showed occasional positive reactions (4/30) to H. cinaedi DNA, which suggests intestinal colonization by H. cinaedi in healthy subjects. In conclusion, this nested PCR assay may be useful for the rapid diagnosis, antimicrobial treatment evaluation, and epidemiological study of H. cinaedi infection.

Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics

Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate l-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.Cysteine hydropersulfides (CysSSH) are believed to have a cellular redox protective role. Here the authors show that these species can be produced from L-cysteine by cysteinyl-tRNA synthetases and that these enzymes are also involved in mitochondrial biogenesis and bioenergetics regulation.

Effect of oestrogen on reactive oxygen species production in the aortas of ovariectomized Dahl salt-sensitive rats

Objective In the present study, we examined whether ovariectomy increases reactive oxygen species (ROS) and the expression of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase and modulates the scavenger enzymes for ROS in the aortas of Dahl salt-sensitive (DSS) rats fed a high salt diet. Methods DSS female rats were ovariectomized and fed a high salt diet (8% NaCl), or a high salt diet plus oestrogen supplement for 4 weeks. Urinary levels of hydrogen peroxide (H2O2) were measured by using 2′,7′-dichlorofluorescein. The expression of an NADPH oxidase subunit p22phox, extracellular superoxide dismutase (ecSOD), glutathione peroxidase (GPx)1, GPx4 and monocyte chemoattractant protein 1 (MCP-1) messenger RNA was assessed by reverse transcription–polymerase chain reaction. The expression of MCP-1, and macrophage infiltration were examined by immunohistochemical analysis. Results Ovariectomy increased superoxide production and the expression of NADPH oxidase subunit p22phox mRNA and protein in the aortas of DSS rats fed a high salt diet. In contrast, ovariectomy reduced the expression of ecSOD mRNA and protein and the expression of GPx1 and GPx4 mRNA in the aorta. Ovariectomy increased MCP-1 mRNA and protein expression and ED1-positive cells in the aorta. Conclusions Ovariectomy leads to an amplification of oxidative stress in DSS rats fed a high salt diet synergistically by an increase in the ROS-generating system and a decrease in the ROS-eliminating system, as shown in the increase in superoxide production and the urinary excretion of H2O2. Oestrogen supplementation counteracted these alterations, showing how oestrogen is antioxidative.

Nitric oxide produced in Peyer's patches exhibits antiapoptotic activity contributing to an antimicrobial effect in murine salmonellosis

Salmonella species normally infect hosts via the oral‐fecal route. We previously reported that NO had potent host defense functions in murine salmonellosis, not only via a direct antibacterial effect but also because it was cytoprotective for infected host cells. Here, we used an oral route to infect iNOS‐deficient mice infected with S. enterica serovar Typhimurium to further investigate the cytoprotective role of NO in preventing damage caused by Salmonella organisms in PP. Oral bacterial challenge (2 × 105 CFU, or >100 LD50) produced a more severe infection and greater lethality in iNOS‐deficient mice than in iNOS‐competent mice. We used specific antibodies to S. enterica Typhimurium, neutrophils, iNOS, nitrotyrosine, and dendritic cells (CD11c‐positive) in histochemical and immunohistochemical studies to examine infected PP tissues. S. enterica Typhimurium colonization in PP from iNOS‐deficient mice was significantly higher than that in wild‐type mice. Histochemical assays showed extensive cellular damage in PP. We then examined PP tissues for apoptosis by means of in situ TUNEL analysis and by measuring caspase‐3 specific activity in tissue homogenates. Increased numbers of TUNEL‐positive cells and severe granulomatous inflammation with increased infiltration of neutrophils and macrophages were observed during infection in iNOS‐deficient mice compared with wild‐type mice. iNOS‐deficient mice had increased numbers of dendritic cells and significantly higher caspase‐3‐specific activity in PP. These data confirm that NO exerts its protective function not only through direct antibacterial action, but also by preventing apoptosis and thereby contributing to antimicrobial defense during salmonellosis.