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ABSTRACT: 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is a unique derivative of guanosine 3',5'-cyclic monophosphate (cGMP) formed in mammalian and plant cells in response to production of nitric oxide and reactive oxygen species. 8-Nitro-cGMP possesses signaling activity inherited from parental cGMP, including induction of vasorelaxation through activation of cGMP-dependent protein kinase. On the other hand, 8-nitro-cGMP mediates cellular signaling that is not observed for native cGMP, e.g., it behaves as an electrophile and reacts with protein sulfhydryls, which results in cGMP adduction to protein sulfhydryls (protein S-guanylation). Several proteins have been identified as targets for endogenous protein S-guanylation, including Kelch-like ECH-associated protein 1 (Keap1), H-Ras, and mitochondrial heat shock proteins. 8-Nitro-cGMP signaling via protein S-guanylation of those proteins may have evolved to convey adaptive cellular stress responses. 8-Nitro-cGMP may not undergo conventional cGMP metabolism because of its resistance to phosphodiesterases. Hydrogen sulfide has recently been identified as a potent regulator for metabolisms of electrophiles including 8-nitro-cGMP, through sulfhydration of electrophiles, e.g., leading to the formation of 8-SH-cGMP. Better understanding of the molecular basis for the formation, signaling functions, and metabolisms of 8-nitro-cGMP would be useful for the development of new diagnostic approaches and treatment of diseases related to oxidative stress and redox metabolisms.
Nitric Oxide 04/2013; · 3.55 Impact Factor
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ABSTRACT: Abnormal activation of astrocytes (e.g., the overproduction of cytokines and nitric oxide) is relevant to neurodegenerative disease. It is important, therefore, to search for inhibitors of the abnormal activation of astrocytes that can be derived from natural substances. This study focused on the effects of extracts from young fruits of Citrus unshiu on lipopolysaccharide (LPS)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in rat primary astrocytes. The methanol extract of young citrus inhibited NO production in a concentration-dependent manner. After reverse-phase extraction of the extract, we found that polymethoxyflavone, nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeletin inhibited NO production by primary astrocytes. These polymethoxyflavones also inhibited LPS-induced iNOS protein and mRNA expression by suppressing nuclear factor-κB (NF-κB) activation and p38-mitogen-activated protein kinase (MAPK) phosphorylation. To evaluate possible applications of these neuroprotective agents in vivo, we examined the effects of young citrus fruit on delayed neurodegeneration in hippocampal CA1 neurons of the Mongolian gerbil after global ischemia. Oral administration of young citrus fruit significantly suppressed delayed neuronal death in hippocampal CA1 neurons. This suggests a possible application of young citrus fruit as a neuroprotective agent.
Bioscience Biotechnology and Biochemistry 10/2012; 76(10):1843-8. · 1.28 Impact Factor
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Motohiro Nishida,
Tomohiro Sawa,
Naoyuki Kitajima,
Katsuhiko Ono,
Hirofumi Inoue, Hideshi Ihara,
Hozumi Motohashi,
Masayuki Yamamoto,
Makoto Suematsu,
Hitoshi Kurose,
Albert van der Vliet,
Bruce A Freeman,
Takahiro Shibata,
Koji Uchida,
Yoshito Kumagai,
Takaaki Akaike
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ABSTRACT: An emerging aspect of redox signaling is the pathway mediated by electrophilic byproducts, such as nitrated cyclic nucleotide (for example, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP)) and nitro or keto derivatives of unsaturated fatty acids, generated via reactions of inflammation-related enzymes, reactive oxygen species, nitric oxide and secondary products. Here we report that enzymatically generated hydrogen sulfide anion (HS(-)) regulates the metabolism and signaling actions of various electrophiles. HS(-) reacts with electrophiles, best represented by 8-nitro-cGMP, via direct sulfhydration and modulates cellular redox signaling. The relevance of this reaction is reinforced by the significant 8-nitro-cGMP formation in mouse cardiac tissue after myocardial infarction that is modulated by alterations in HS(-) biosynthesis. Cardiac HS(-), in turn, suppresses electrophile-mediated H-Ras activation and cardiac cell senescence, contributing to the beneficial effects of HS(-) on myocardial infarction-associated heart failure. Thus, this study reveals HS(-)-induced electrophile sulfhydration as a unique mechanism for regulating electrophile-mediated redox signaling.
Nature Chemical Biology 07/2012; 8(8):714-24. · 14.69 Impact Factor
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ABSTRACT: β-1,3-Xylan was prepared from the green alga, Caulerpa lentillifera, and hydrolyzed to oligosaccharides by a mild acid treatment. The average degree of polymerization was about 5. The oligosaccharides reduced the number of viable human breast cancer MCF-7 cells in a dose-dependent manner, and induced chromatin condensation and degradation of poly ADP-ribose polymerase, indicating that they induced apoptosis in MCF-7 cells.
Bioscience Biotechnology and Biochemistry 05/2012; 76(5):1032-4. · 1.28 Impact Factor
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ABSTRACT: We previously isolated two α-L: -arabinofuranosidases (ABFs), termed AFQ1 and AFS1, from the culture filtrate of Penicillium chrysogenum 31B. afq1 and afs1 complementary DNAs encoding AFQ1 and AFS1 were isolated by in vitro cloning. The deduced amino acid sequences of AFQ1 and AFS1 are highly similar to those of Penicillium purpurogenum ABF 2 and ABF 1, respectively, which belong to glycoside hydrolase (GH) families 51 and 54, respectively. Pfam analysis revealed an "Alpha-L-AF_C" domain in AFQ1 and "ArabFuran-catal" and "AbfB" domains in AFS1. Semi-quantitative RT-PCR analysis indicated that the afq1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was slightly induced with arabinose, arabinitol, arabinan, and arabinoxylan. In contrast, expression of the afs1 gene was strongly expressed by the above four carbohydrates and less strongly induced by galactan. Recombinant enzymes (rAFQ1 and rAFS1) expressed in Escherichia coli were active against both p-nitrophenyl α-L: -arabinofuranoside and polysaccharides with different specificities. (1)H-NMR analysis revealed that rAFS1 degraded arabinofuranosyl side chains that were both singly and doubly linked to the backbones of arabinoxylan and L: -arabinan. On the other hand, rAFQ1 preferentially released arabinose linked to C-3 of single-substituted xylose or arabinose residues in the two polysaccharides.
Applied Microbiology and Biotechnology 03/2012; · 3.42 Impact Factor
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ABSTRACT: The botulinum neurotoxin light chain (BoNT-LC) is a zinc-dependent metalloprotease that cleaves neuronal SNARE proteins such as SNAP-25, VAMP2, and Syntaxin1. This cleavage interferes with the neurotransmitter release of peripheral neurons and results in flaccid paralysis. SNAP, VAMP, and Syntaxin are representative of large families of proteins that mediate most membrane fusion reactions, as well as both neuronal and non-neuronal exocytotic events in eukaryotic cells. Neuron-specific SNARE proteins, which are target substrates of BoNT, have been well studied; however, it is unclear whether other SNARE proteins are also proteolyzed by BoNT. Herein, we define the substrate specificity of BoNT-LC/B, /D, and /F towards recombinant human VAMP family proteins. We demonstrate that LC/B, /D, and /F are able to cleave VAMP1, 2, and 3, but no other VAMP family proteins. Kinetic analysis revealed that all LC have higher affinity and catalytic activity for the non-neuronal SNARE isoform VAMP3 than for the neuronal VAMP1 and 2 isoforms. LC/D in particular exhibited extremely low catalytic activity towards VAMP1 relative to other interactions, which we determined through point mutation analysis to be a result of the Ile present at residue 48 of VAMP1. We also identified the VAMP3 cleavage sites to be at the Gln 59-Phe 60 (LC/B), Lys 42-Leu 43 (LC/D), and Gln 41-Lys 42 (LC/F) peptide bonds, which correspond to those of VAMP1 or 2. Understanding the substrate specificity and kinetic characteristics of BoNT towards human SNARE proteins may aid in the development of novel therapeutic uses for BoNT.
Microbiology and Immunology 01/2012; 56(4):245-53. · 1.30 Impact Factor
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ABSTRACT: Polysaccharides were extracted from Caulerpa lentillifera by treating with water and then purified by size-exclusion chromatography. The purified polysaccharides, termed SP1, were found to be sulfated xylogalactans with a molecular mass of more than 100 kDa. Adding SP1 to murine macrophage RAW 264.7 cells increased the production of nitric oxide (NO) in a dose-dependent manner. NO was found by immunoblotting and RT-PCR analyses to be synthesized by an inducible NO synthase. SP1 caused the degradation of IκB-α and the nuclear translocation of nuclear factor (NF)-κB subunit p65 in macrophage cells. SP1 also increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK). These results demonstrate that SP1 activated macrophage cells via both the NF-κB and p38 MAPK signaling pathways. Moreover, SP1 increased the expression of various genes encoding cytokines, and the phagocytic activity of macrophage cells. These combined results show that SP1 immunostimulated the activity of macrophage cells.
Bioscience Biotechnology and Biochemistry 01/2012; 76(3):501-5. · 1.28 Impact Factor
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ABSTRACT: 8-Nitro-cGMP (8-nitroguanosine 3',5'-cyclic monophosphate) is a nitrated derivative of cGMP, which can function as a unique electrophilic second messenger involved in regulation of an antioxidant adaptive response in cells. In the present study, we investigated chemical and biochemical regulatory mechanisms involved in 8-nitro-cGMP formation, with particular focus on the roles of ROS (reactive oxygen species). Chemical analyses demonstrated that peroxynitrite-dependent oxidation and myeloperoxidase-dependent oxidation of nitrite in the presence of H2O2 were two major pathways for guanine nucleotide nitration. Among the guanine nucleotides examined, GTP was the most sensitive to peroxynitrite-mediated nitration. Immunocytochemical and tandem mass spectrometric analyses revealed that formation of 8-nitro-cGMP in rat C6 glioma cells stimulated with lipopolysaccharide plus pro-inflammatory cytokines depended on production of both superoxide and H2O2. Using the mitochondria-targeted chemical probe MitoSOX Red, we found that mitochondria-derived superoxide can act as a direct determinant of 8-nitro-cGMP formation. Furthermore, we demonstrated that Nox2 (NADPH oxidase 2)-generated H2O2 regulated mitochondria-derived superoxide production, which suggests the importance of cross-talk between Nox2-dependent H2O2 production and mitochondrial superoxide production. The results of the present study suggest that 8-nitro-cGMP can serve as a unique second messenger that may be implicated in regulating ROS signalling in the presence of NO.
Biochemical Journal 10/2011; 441(2):719-30. · 4.90 Impact Factor
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ABSTRACT: The biological significance of nitrated guanine derivatives, especially 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), has become evident. Therefore it is important to determine the presence and relative abundance of 8-nitro-cGMP formed in cells and tissues. In the present study, we performed immunocytochemistry with monoclonal antibodies specific for 8-nitroguanine (clone NO2-52) and 8-nitro-cGMP (clone 1G6) in rat C6 glioma cells and rat primary cultured astrocytes. Immunocytochemistry utilizing the anti-8-nitro-cGMP monoclonal antibody (1G6) indicated that immunostaining increased markedly in C6 cells expressing increased amounts of inducible nitric oxide synthase (iNOS) after treatment with lipopolysaccharide (LPS) plus cytokines. Treatment of C6 cells with inhibitors for NOS and soluble guanylate cyclase (sGC) completely nullified the elevated 1G6 immunoreactivity. These results were consistent with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Immunocytochemistry performed using NO2-52 also showed that treatment of cells with inhibitors for NOS and sGC completely nullified the elevated immunoreactivity; this indicated that 8-nitro-cGMP is a major component of 8-nitroguanine derivatives produced in cells. Similar results were obtained in the primary astrocytes stimulated with LPS plus cytokines. Because immunocytochemistry is a conventional, powerful, and fairly straightforward method for determining the presence, localization, and relative abundance of an antigen of interest in cultured cells, anti-8-nitroguanine (NO2-52) and anti-8-nitro-cGMP (1G6) antibodies could be useful tools for analyzing nitrated guanine nucleotides, especially 8-nitro-cGMP, by means of immunocytochemistry.
Nitric Oxide 05/2011; 25(2):169-75. · 3.55 Impact Factor
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ABSTRACT: We describe here physiological formation of a unique nitrated cyclic nucleotide, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) and its potent antioxidant activity. Our earlier studies revealed the NO-dependent guanine nitration in several types of cells. In fact, we identified physiological formation and functions of 8-nitro-cGMP, which is the first demonstration of a new second messenger derived from cGMP in mammals since the discovery of cGMP more than 40 years ago. Using immunocytochemical methods, we confirmed 8-nitro-cGMP formation in cultured macrophages, hepatocyte-like cells, adipocytes, and endothelial cells, depending on NO production. We further verified 8-nitro-cGMP formation via HPLC plus electrochemical detection and tandem mass spectrometry. 8-Nitro-cGMP as an electrophile reacts efficiently with sulfhydryls of proteins to generate a novel post-translational modification, which we call protein S-guanylation. Particular intracellular proteins can readily undergo S-guanylation by 8-nitro-cGMP. 8-Nitro-cGMP regulates the redox-sensor signaling protein Keap1, via S-guanylation of the highly nucleophilic cysteine sulfhydryls of Keap1. More importantly, we clarified that S-guanylation of Keap1 is involved in potent antioxidant effects mediated by 8-nitro-cGMP, by inducing oxidative stress-response genes such as heme oxygenase-1. Our discovery of 8-nitro-cGMP and its unique antioxidant effects thus shed light on new areas of oxidative stress research. Protein S-guanylation induced by 8-nitro-cGMP thus may have important implications in pharmaceutical chemistry and development of therapeutics for many diseases.
Current topics in medicinal chemistry 04/2011; 11(14):1854-60. · 4.47 Impact Factor
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ABSTRACT: Oxidized and nitrated nucleotides including 8-oxogunanine and 8-nitroguanine derivatives such as 8-nitroguanosine 3',5'-cyclic monophosphate were generated by reactive nitrogen oxides and reactive oxygen species in cultured cells and in tissues. 8-oxoguanine and 8-nitroguanine in DNA and RNA are potentially mutagenic, and the former also induces cell death. Some derivative, 8-nitroguanosine 3',5'-cyclic monophosphate a major nitrated guanine nucleotide, was identified as a novel second messenger. Surprisingly, the amount of 8-nitroguanosine 3',5'-cyclic monophosphate generated was found to be higher than that of guanosine 3',5'-cyclic monophosphate in cells expressing inducible nitric oxide synthase. More important, 8-nitroguanosine 3',5'-cyclic monophosphate is electrophilic and reacted efficiently with sulfhydryls of proteins to produce a novel posttranslational modification (named S-guanylation) via guanosine 3',5'-cyclic monophosphate adduction. For example, 8-nitroguanosine 3',5'-cyclic monophosphate-induced S-guanylation of Kelch-like ECH-associated protein 1 led to NF-E2-related factor activation and induction of antioxidant enzymes. 8-nitroguanosine 3',5'-cyclic monophosphate may thus protect cells against oxidative stress-related cytotoxicity. Therefore, although chemically modified nucleotides produced via oxidative and nitrative stress are regarded simply as endogenous mutagens, the endogenous nucleotides stored in cells per se may serve functionally as a sensing mechanism for reactive nitrogen oxides and oxygen species to induce cellular adaptive responses to oxidative stress.
Journal of Clinical Biochemistry and Nutrition 01/2011; 48(1):33-9. · 1.98 Impact Factor
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ABSTRACT: An arabinoxylan arabinofuranohydrolase (AXS5) was purified from the culture filtrate of Penicillium chrysogenum 31B. A cDNA encoding AXS5 (axs5) was isolated by in vitro cloning using the N-terminal amino acid sequence of the native enzyme as a starting point. The deduced amino acid sequence of the axs5 gene has high similarities with those of arabinoxylan arabinofuranohydrolases of Aspergillus niger, Aspergillus tubingensis, and Aspergillus sojae. Module sequence analysis revealed that a "Glyco_hydro_62" was present at position 28-299 of AXS5. This is a family of α-L-arabinofuranosidases which are all members of glycoside hydrolase family 62. Recombinant AXS5 (rAXS5) expressed in Escherichia coli was highly active on arabinoxylan but not on branched sugar beet arabinan. (1)H-NMR analysis revealed that the rAXS5 cleaved arabinosyl side-chains linked to C-2 and C-3 of single-substituted xylose residues in arabinoxylan. Semi-quantitative RT-PCR analysis indicated that expression of the axs5 gene in P. chrysogenum 31B was strongly induced by adding D-xylose and arabinoxylan to the culture medium. Moreover, two binding sites of XlnR, a transcriptional activator that regulates the expression of the genes encoding xylanolytic enzymes, are present in the upstream region of the axs5 gene. These results suggest that AXS5 is involved in xylan degradation.
Applied Microbiology and Biotechnology 12/2010; 90(1):137-46. · 3.42 Impact Factor
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ABSTRACT: We recently clarified the physiological formation of 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) and its critical roles in nitric oxide (NO) signal transductions. This discovery of 8-nitro-cGMP is the first demonstration of a nitrated cyclic nucleotide functioning as a new second messenger in mammals since the identification of cGMP more than 40 years ago. By means of chemical analyses, e.g., liquid chromatography-tandem mass spectrometry, we unequivocally identified 8-nitro-cGMP formation, which depended on NO production, in several types of cultured cells, including macrophages and glial cells. Most important, we previously showed that 8-nitro-cGMP as an electrophile reacted with particular sulfhydryls of proteins to generate a unique post-translational modification that we called protein S-guanylation. In fact, certain specific intracellular proteins, such as the redox-sensor protein Keap1, readily underwent S-guanylation induced by 8-nitro-cGMP. 8-Nitro-cGMP activated the Nrf2 signaling pathway by triggering dissociation of Keap1, via S-guanylation of its highly nucleophilic cysteine sulfhydryls. We also determined that S-guanylation of Keap1 was involved in cytoprotective actions of NO and 8-nitro-cGMP by inducing oxidative stress response genes such as heme oxygenase-1. Such unique chemical properties of 8-nitro-cGMP shed light on new areas of NO and cGMP signal transduction. Protein S-guanylation induced by 8-nitro-cGMP may thus have important implications in NO-related physiology and pathology, pharmaceutical chemistry, and development of therapeutics for many diseases.
Nitric Oxide 11/2010; 23(3):166-74. · 3.55 Impact Factor
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Shigemoto Fujii,
Tomohiro Sawa, Hideshi Ihara,
Kit I. Tong,
Tomoaki Ida,
Tatsuya Okamoto,
Ahmed Khandaker Ahtesham,
Yu Ishima,
Hozumi Motohashi,
Masayuki Yamamoto,
Takaaki Akaike
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ABSTRACT: 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.
Journal of Biological Chemistry 07/2010; 285(31):23970-23984. · 4.77 Impact Factor
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ABSTRACT: We examined the effects of low protein intake on the development of the remaining kidney in subtotally (5/6) nephrectomized immature rats. Three-week-old rats were kept on a diet containing either 12% protein (Lp rats) or 18% protein (Np rats) for 4 or 8 weeks after subtotal nephrectomy (SUNx). In Western blot analysis, the endothelial NO synthase (eNOS) protein expression of the Lp rats was significantly higher than that of the Np rats at 4 weeks after SUNx. Immunohistochemically, more inducible NO synthase (iNOS)-positive cells were observed in the Np rats than in the Lp rats 4 weeks after SUNx in the distal tubules. In semiquantitative RT-PCR, the expression of renin mRNA was significantly lower in the Lp rats than in the Np rats at 4 and 8 weeks after SUNx. These findings reveal that protein restriction is effective in preventing renal failure of immature rats and that the changes in the expression levels of renin, eNOS, and iNOS is involved in the process of this prevention.
Medical Molecular Morphology 06/2010; 43(2):116-22. · 1.39 Impact Factor
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Shigemoto Fujii,
Tomohiro Sawa, Hideshi Ihara,
Kit I Tong,
Tomoaki Ida,
Tatsuya Okamoto,
Ahmed Khandaker Ahtesham,
Yu Ishima,
Hozumi Motohashi,
Masayuki Yamamoto,
Takaaki Akaike
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ABSTRACT: 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 microm) 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 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.
Journal of Biological Chemistry 05/2010; 285(31):23970-84. · 4.77 Impact Factor
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ABSTRACT: Two distinct extracellular bifunctional proteins with beta-L-arabinopyranosidase/alpha-D-galactopyranosidase activities were purified from the culture filtrate of Fusarium oxysporum 12S. The molecular masses of the enzymes were estimated to be 55 (Fo/AP1) and 73 kDa (Fo/AP2) by SDS-PAGE. They hydrolyzed both p-nitrophenyl beta-L-arabinopyranoside and p-nitrophenyl alpha-D-galactopyranoside with different specificities. Fo/AP1 also showed low activity towards alpha-D-galactopyranosyl oligosaccharides such as raffinose. Interestingly, both enzymes hydrolyzed larch wood arabinogalactan (releasing arabinose) but not carob galactomannan, which has alpha-D-galactopyranosyl side chains. When larch wood arabinogalactan was incubated with excess Fo/AP1 or Fo/AP2, both enzymes released approximately 10% of the total arabinose in the substrate. cDNAs encoding Fo/AP1 and Fo/AP2 (Foap1 and Foap2) were isolated by in vitro cloning. The coding sequences of Foap1 and Foap2 genes were 1,647 and 1,620 bp in length and encode polypeptides of 549 and 540 amino acids, respectively. The N-terminal halves of both proteins had high similarity to putative conserved domains of the melibiase superfamily (Pfam account number 02065). The deduced amino acid sequences of the two enzymes indicate that they belong to glycosyl hydrolase family 27. Moreover, the C-terminal regions of both proteins contain a putative family 35 carbohydrate-binding module.
Applied Microbiology and Biotechnology 11/2009; 86(4):1115-24. · 3.42 Impact Factor
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ABSTRACT: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(2) is a classic glycolytic enzyme that also mediates cell death by its nuclear translocation under oxidative stress. Meanwhile, we previously presented that oxidative stress induced disulfide-bonded GAPDH aggregation in vitro. Here, we propose that GAPDH aggregate formation might participate in oxidative stress-induced cell death both in vitro and in vivo. We show that human GAPDH amyloid-like aggregate formation depends on the active site cysteine-152 (Cys-152) in vitro. In SH-SY5Y neuroblastoma, treatment with dopamine decreases the cell viability concentration-dependently (IC(50) = 202 microM). Low concentrations of dopamine (50-100 microM) mainly cause nuclear translocation of GAPDH, whereas the levels of GAPDH aggregates correlate with high concentrations of dopamine (200-300 microM)-induced cell death. Doxycycline-inducible overexpression of wild-type GAPDH in SH-SY5Y, but not the Cys-152-substituted mutant (C152A-GAPDH), accelerates cell death accompanying both endogenous and exogenous GAPDH aggregate formation in response to high concentrations of dopamine. Deprenyl, a blocker of GAPDH nuclear translocation, fails to inhibit the aggregation both in vitro and in cells but reduced cell death in SH-SY5Y treated with only a low concentration of dopamine (100 microM). These results suggest that GAPDH participates in oxidative stress-induced cell death via an alternative mechanism in which aggregation but not nuclear translocation of GAPDH plays a role. Moreover, we observe endogenous GAPDH aggregate formation in nigra-striatum dopaminergic neurons after methamphetamine treatment in mice. In transgenic mice overexpressing wild-type GAPDH, increased dopaminergic neuron loss and GAPDH aggregate formation are observed. These data suggest a critical role of GAPDH aggregates in oxidative stress-induced brain damage.
Journal of Biological Chemistry 10/2009; 284(49):34331-41. · 4.77 Impact Factor
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ABSTRACT: Staphylococcal enterotoxin H (SEH) is predicted to be involved in staphylococcal food poisoning. To characterize SEH-producing Staphylococcus aureus isolates from staphylococcal food poisoning cases in Japan, we investigated the relationship between SEH production and coagulase serotype, which is an epidemiological marker, and compared the properties of SEH production with those of staphylococcal enterotoxins A (SEA) and B (SEB). SEH production was determined by a newly developed sandwich enzyme-linked immunosorbent assay. Eighty-six (59.7%) of 144 isolates from staphylococcal food poisoning cases produced SEH. Seventy-one of the SEH-producing isolates simultaneously produced SEA, SEB, or both. All SEH-producing isolates belonged to coagulase type VII, which was the predominant type, representing 99 (68.8%) of 144 isolates. The amount of SEH produced in brain heart infusion was almost the same as the amount of SEA and approximately 10-fold lower than that of SEB. SEH and SEA were produced mainly during the late exponential phase of growth, whereas SEB was produced mostly during the stationary phase. The production levels of SEH and SEA were gradually affected by decreases in water activity, but the production of SEB was greatly reduced under conditions of low water activity. These findings indicate that SEH-producing S. aureus isolates are of high prevalence in staphylococcal food poisoning cases. Given the unique epidemiological characteristic of these isolates, SEH and SEA probably are responsible for food poisoning.
Journal of food protection 10/2008; 71(9):1855-60. · 1.94 Impact Factor
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ABSTRACT: The binding of Bacillus thuringiensis delta-endotoxin to brush border membrane vesicles (BBMVs) from the target insect larval midgut comprises with not only a reversible but also an irreversible component. The irreversible binding of delta-endotoxin is thought to be a pathologically important factor. Here, we studied the irreversible binding of Cry1Aa to the BBMVs of Bombyx mori. The (125)I-labeled Cry1Aa bound to the solubilized brush border membrane (BBM) through rapid dissociation only, unlike the binding to BBMVs, indicating that the toxin bound to the solubilized BBM through only a reversible process. Low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the toxin bound irreversibly to BBMVs formed an oligomer of 220kDa, whereas that bound reversibly to the solubilized BBM did not oligomeraize. When the (125)I-labeled Cry1Aa bound irreversibly to the BBMVs was digested by proteinase K, approximately 40% of the toxin observed to be resistant to proteinase K. The molecular mass of the toxin resistant to proteinase K was 60kDa, suggesting that the irreversible binding comprise two forms. These results support the notion that the irreversible binding of the toxin to BBMVs is due to the insertion of the toxin into the lipid bilayers and oligomerization to form channels.
Journal of Invertebrate Pathology 07/2008; 98(2):177-83. · 2.06 Impact Factor
