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 H 2 O 2 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 H 2 S can be generated from persulfide degradation, much of the reported biological activity associated with H 2 S may actually be that of persulfides. That is, H 2 S may act primarily as a marker for the biologically active of persulfide species.thiol redox | hydrogen sulfide | electrophilic signaling | polysulfidomics H ydrogen sulfide (H 2 S) has been suggested to be an endogenous small molecule signaling species (1) by unknown mechanisms. Our laboratory recently showed that the presence of hydrogen sulfide anion (HS − ) may be responsible for the regulation and metabolism of various important electrophilic species [e.g., 8-nitroguanosine 3′,5′-cyclic GMP (8-nitro-cGMP)] (2). However, these studies also indicated that reactive intermediates other than HS − likely react with the electrophiles of interest. These previous studies alluded to the generation of a more reactive sulfur species capable of reacting with electrophiles, such as 8-nitro-cGMP. As reported herein, it was determined that reactive sulfur intermediates, such as hydropersulfides (RSSH) and polysulfides [RS(S) n H and RS(S) n SR], are formed in appreciable amounts during sulfur amino acid metabolism and possess important chemical and biological properties. Some of these sulfide species have long been known as sulfane sulfur compounds, which were suggested to exist endogenously in mammalian systems (1,(3)(4)(5). Reports also indicated that a hydropersulfide moiety with the general molecular formula RSSH may be formed on specific protein cysteine (Cys) residues, most typically of sulfur-transferring enzymes (i.e., sulfurtransferases) during enzymatic reactions (1, 5). Although such persulfide chemical reactivity is thought to be involved in the catalytic activity of particular enzymes (e.g., rhodanese, Cys desulfurases, and sulfide:quinone oxidoreductase) (6, 7), the more general physiological function and occurrence of Cys persulfides (CysSSH) and related species in cells and tissues, especially mammals, were unclear. Moreover, the exact chemical nature ...

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.

The signaling pathway of nitric oxide (NO) depends mainly on guanosine 3',5'-cyclic monophosphate (cGMP). Here we report the formation and chemical biology of a nitrated derivative of cGMP, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), in NO-mediated signal transduction. Immunocytochemistry demonstrated marked 8-nitro-cGMP production in various cultured cells in an NO-dependent manner. This finding was confirmed by HPLC plus electrochemical detection and tandem mass spectrometry. 8-Nitro-cGMP activated cGMP-dependent protein kinase and showed unique redox-active properties independent of cGMP activity. Formation of protein Cys-cGMP adducts by 8-nitro-cGMP was identified as a new post-translational modification, which we call protein S-guanylation. 8-Nitro-cGMP seems to regulate the redox-sensor signaling protein Keap1, via S-guanylation of the highly nucleophilic cysteine sulfhydryls of Keap1. This study reveals 8-nitro-cGMP to be a second messenger of NO and sheds light on new areas of the physiology and chemical biology of signal transduction by NO.

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-nitrocGMP and S-guanylation are yet to be identified. In this study, we precisely quantified NO-dependent formation of 8-nitrocGMP in C6 glioma cells via liquid chromatography-tandem mass spectrometry. Treatment of cells with S-nitroso-Nacetylpenicillamine 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 M) 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-flighttandem mass spectrometry revealed that 8-nitro-cGMP S-guanylated the Cys 434 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.Nitric oxide (NO) plays diverse physiological roles in vascular regulation, neuronal transmission, inflammation, and host defense against microbial pathogens. In vascular and neuronal systems, NO performs these functions mainly through a cGMPdependent mechanism (1, 2), but the presence and contribution of other pathways that are not directly linked to cGMP have also been suggested to operate in certain aspects of NO signaling occurring in various cells and tissues in different organisms (3-5). Among these other mechanisms is chemical modification of biomolecules, including nitrosylation and nitration of amino acids, proteins, and lipids, this modification being induced by NO-derived reactive nitrogen oxide species (RNOS), 3 such as peroxynitrite (ONOO Ϫ ) and nitrogen dioxide (NO 2 ) (3-5).RNOS cause nitration of nucleic acids in addition to amino acids, proteins, and lipids. We previously found that nitrated guanine derivatives, including 8-nitroguanine and 8-nitroguanosine, formed in cultured cells and in tissues from murine viral pneumonia and human lung disease (6 -8). An important finding was that 8-nitroguanosine possessed a unique redox activity, which suggested a critical biological rol...

Summary Induction of haem oxygenase-1 (HO-1) as well as nitric oxide (NO) biosynthesis during tumour growth was investigated in an experimental solid tumour model (AH136B hepatoma) in rats. An immunohistochemical study showed that the inducible isoform of NO synthase (iNOS) was localized in monocyte-derived macrophages, which infiltrated interstitial spaces of solid tumour, but not in the tumour cells. Excessive production of NO in the tumour tissue was unequivocally verified by electron spin resonance spectroscopy. Tumour growth was moderately suppressed by treatment with either N ω -nitro-L-arginine methyl ester (L-NAME) or S-methylisothiourea sulphate (SMT). In contrast, HO-1 was found only in tumour cells, not in macrophages, by in situ hybridization for HO-1 mRNA. HO-1 expression in AH136B cells in culture was strongly enhanced by an NO (NO + ) donor S-nitroso-N-acetyl penicillamine. HO-1 mRNA expression in the solid tumour in vivo decreased significantly after treatment with low doses of NOS inhibitors such as L-NAME and SMT (6-20 mg kg -1 ). However, the level of HO-1 mRNA in the solid tumour treated with higher doses of NOS inhibitor was similar to that of the solid tumour without NOS inhibitor treatment. Strong induction of HO-1 was also observed in solid tumours after occlusion or embolization of the tumour-feeding artery, indicating that ischaemic stress which may involve oxidative stress triggers HO-1 induction in the solid tumour. Lastly, it is of great importance that an HO inhibitor, zinc protoporphyrin IX injected intra-arterially to the solid tumour suppressed the tumour growth to a great extent. In conclusion, HO-1 expression in the solid tumour may confer resistance of tumour cells to hypoxic stress as well as to NO-mediated cytotoxicity.

Helicobacter cinaedi was first isolated from rectal cultures from homosexual men in 1984. In the 1980s to mid 1990s, the microorganism was mainly isolated from samples from homosexual men or immunocompromised patients; however, during the last two decades, H. cinaedi has been isolated from immunocompromised and from immunocompetent individuals worldwide. In Japan, the isolation of this microorganism was first reported in 2003. Since then, many cases have been reported in hospitals across the country. Despite many reports, the etiological properties and pathogenicity of H. cinaedi remain elusive; however, we are increasingly able to recognize some of the features and the clinical relevance of infection. In particular, a long incubation period is essential for detection in an automatic blood culture system and many of the recent isolates are resistant to both macrolides and quinolones. Furthermore, there is an association between infection and severe or chronic illnesses, such as meningitis or arteriosclerosis, in addition to mild diseases such as fever, abdominal pain, gastroenteritis, proctitis, diarrhea, erysipelas, cellulitis, arthritis, and bacteremia. In this review, we introduce the current knowledge and our latest findings relating to H. cinaedi.

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.Helicobacter cinaedi, first identified as a Campylobacter species but now known to be an enterohepatic Helicobacter species, is usually found in the intestinal tract and/or liver of humans and other mammals (2,8,28,30,32,33,(35)(36)(37). The last two decades have seen increasing numbers of reports of H. cinaedi infections, mainly in humans and particularly in individuals with underlying immunosuppressive conditions such as AIDS, malignant diseases, and chronic alcoholism (3,11,18,20,23). Vandamme et al. previously documented a few cases of infection with H. cinaedi isolated from feces and blood from an apparently nonimmunocompromised child and adult (36). Other groups have also described invasive infections caused by H. cinaedi and other Campylobacter-like organisms (Helicobacter fennelliae) in patients without underlying diseases (15,26). However, despite these reports, the pathogenicity and etiological properties of this spiral bacterium are only poorly understood. Also, the molecular epidemiology of various strains isolated from humans has not yet been systematically analyzed.We recently observed 11 cases of H. cinaedi bacteremia and cellulitis that occurred consecutively during a particular period in the same hospital. The clinical and epidemiological features of the H. cinaedi infections were investigated in the present study, which may thus provide important insights into the pathogenesis and ep...

The role of superoxide anion (O 2 ؊) and nitric oxide (NO) in the host defense mechanism against Salmonella typhimurium (LT-2) was examined by focusing on xanthine oxidase (XO) as an O 2 ؊-generating system and on inducible NO synthase (iNOS). When ICR mice were infected with a 0.1 50% lethal dose (2 ؋ 10 5 CFU) of S. typhimurium, bacterial growth in the liver reached a peak value 3 days after infection (10 4.32 CFU/g of liver) and decreased thereafter. XO activity in the liver became maximum at 7 days after infection; the value was 34.6 ؎ 1.4 mU/g of liver at 7 days (compared with 11.0 ؎ 1.3 mU/g of liver before infection). The time profile of NO production in the liver as determined by electron spin resonance spectroscopy was consistent with that of XO activity. Histological examination of infected liver showed the formation of multiple microabscesses with granulomatous lesions consisting of polymorphonuclear cells and mononuclear cells, and iNOS-expressing cells were localized in the confined areas of the microabscesses. When XO inhibitors such as allopurinol and 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP) were administered to the infected mice, the mortality of the mice was significantly increased (10 of 21 and 11 of 20 for the allopurinol-and AHPP-treated groups, respectively, versus 2 of 20 for control mice), and bacterial growth was significantly enhanced. A similar exacerbation of the infection was obtained with N-monomethyl-L-arginine (L-NMMA) treatment of the mice. Of considerable importance is that granuloma formation in the liver was poorly developed by treatment with either XO inhibitors or L-NMMA. These results suggest that XO and NO play an important role in the antimicrobial mechanism against S. typhimurium in mice.