• International Journal of Oral Biology
• /
• v.41 no.3
• /
• pp.141-147
• /
• 2016

Reactive oxygen species (ROS) and nitrogen species (RNS) are both important signaling molecules involved in pain transmission in the dorsal horn of the spinal cord. Xanthine oxidase (XO) is a well-known enzyme for the generation of superoxide anions ($O_2^{\bullet-}$), while S-nitroso-N-acetyl-DL-penicillamine (SNAP) is a representative nitric oxide (NO) donor. In this study, we used patch clamp recording in spinal slices of rats to investigate the effects of $O_2^{\bullet-}$ and NO on the excitability of substantia gelatinosa (SG) neurons. We also used confocal scanning laser microscopy to measure XO- and SNAP-induced ROS and RNS production in live slices. We observed that the ROS level increased during the perfusion of xanthine and xanthine oxidase (X/XO) compound and SNAP after the loading of 2',7'-dichlorofluorescin diacetate ($H_2DCF-DA$), which is an indicator of intracellular ROS and RNS. Application of ROS donors such as X/XO, ${\beta}-nicotinamide$ adenine dinucleotide phosphate (NADPH), and 3-morpholinosydnomimine (SIN-1) induced a membrane depolarization and inward currents. SNAP, an RNS donor, also induced membrane depolarization and inward currents. X/XO-induced inward currents were significantly decreased by pretreatment with phenyl N-tert-butylnitrone (PBN; nonspecific ROS and RNS scavenger) and manganese(III) tetrakis(4-benzoic acid) porphyrin (MnTBAP; superoxide dismutase mimetics). Nitro-L-arginine methyl ester (NAME; NO scavenger) also slightly decreased X/XO-induced inward currents, suggesting that X/XO-induced responses can be involved in the generation of peroxynitrite ($ONOO^-$). Our data suggest that elevated ROS, especially $O_2^{\bullet-}$, NO and $ONOO^-$, in the spinal cord can increase the excitability of the SG neurons related to pain transmission.

• The Korean Journal of Physiology and Pharmacology
• /
• v.26 no.1
• /
• pp.25-36
• /
• 2022

To identify the effect and mechanism of carbon monoxide (CO) on delayed rectifier K⁺ currents (I_K) of human cardiac fibroblasts (HCFs), we used the wholecell mode patch-clamp technique. Application of CO delivered by carbon monoxidereleasing molecule-3 (CORM3) increased the amplitude of outward K⁺ currents, and diphenyl phosphine oxide-1 (a specific I_K blocker) inhibited the currents. CORM3-induced augmentation was blocked by pretreatment with nitric oxide synthase blockers (L-NG-monomethyl arginine citrate and L-NG-nitro arginine methyl ester). Pretreatment with KT5823 (a protein kinas G blocker), 1H-[1,-2,-4] oxadiazolo-[4,-3-a] quinoxalin-1-on (ODQ, a soluble guanylate cyclase blocker), KT5720 (a protein kinase A blocker), and SQ22536 (an adenylate cyclase blocker) blocked the CORM3 stimulating effect on I_K. In addition, pretreatment with SB239063 (a p38 mitogen-activated protein kinase [MAPK] blocker) and PD98059 (a p44/42 MAPK blocker) also blocked the CORM3's effect on the currents. When testing the involvement of S-nitrosylation, pretreatment of N-ethylmaleimide (a thiol-alkylating reagent) blocked CO-induced I_K activation and DL-dithiothreitol (a reducing agent) reversed this effect. Pretreatment with 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)-21H,23H porphyrin manganese (III) pentachloride and manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (superoxide dismutase mimetics), diphenyleneiodonium chloride (an NADPH oxidase blocker), or allopurinol (a xanthine oxidase blocker) also inhibited CO-induced I_K activation. These results suggest that CO enhances I_K in HCFs through the nitric oxide, phosphorylation by protein kinase G, protein kinase A, and MAPK, S-nitrosylation and reduction/oxidation (redox) signaling pathways.

• Proceedings of the Korean Society of Crop Science Conference
• /
• 2022.10a
• /
• pp.229-229
• /
• 2022

Rapeseed is a crop that is waterlogging sensitive, and it is necessary to breed waterlogging tolerance varieties. Our study presents the comparative transcriptome changes in two rapeseed lines, i.e., waterlogging-tolerant (tJ8634-B-30,) and - sensitive ('EMS26') lines under control and waterlogging stress treatments at the flowering stage. RNA-sequencing analysis revealed 13,279 differentially expressed genes (DEGs) for 'J8634-B-30' and 8,682 DEGs for 'EMS26' under waterlogging stress condition compared to control. Among DEGs of 'J8634-B-30', 6,818 were up-regulated and 6,461 were down-regulated. On the other hand, among the DEGs of 'EMS26', the number of down-regulated genes (5,240) were higher than that of up-regulated genes (3,442). Gene ontology enrichment analysis showed that DEGs related to glucan metabolic, cell wall, and oxidoreductase activity were significantly changed in 'J8634-B-30'. Kyoto Encyclopedia of Genes and Genomes (KEGG)-based analysis in 'J8634-B-30' identified up-regulated DEGs being involved in MAPK signaling pathways. In addition, the DEGs belonging to mechanisms responding to waterlogging stress, i.e., plant hormones, carbon metabolism, Reactive oxygen species (ROS), Nitric oxide (NO) etc. were compared in rapeseed lines. Several DEGs including ethylene-responsive transcription factor (ERF), constitutive triple response (CTR) (in ethylene signaling pathway), monodehydroascorbate Reductase (MDAR), NADPH oxidase (in ROS pathway), cytochrome c oxidase assembly protein (COX) (in NO pathway) up-regulated in 'J8634-B-30'. These outcomes provided the valuable information for further exploring the genetic mechanism of waterlogging tolerance in rapeseed.

• Molecules and Cells
• /
• v.28 no.3
• /
• pp.139-148
• /
• 2009

Cell death has been traditionally classified in apoptosis and necrosis. Apoptosis, known as programmed cell death, is an active form of cell death mechanism that is tightly regulated by multiple cellular signaling pathways and requires ATP for its appropriate process. Apoptotic death plays essential roles for successful development and maintenance of normal cellular homeostasis in mammalian. In contrast to apoptosis, necrosis is classically considered as a passive cell death process that occurs rather by accident in disastrous conditions, is not required for energy and eventually induces inflammation. Regardless of different characteristics between apoptosis and necrosis, it has been well defined that both are responsible for a wide range of human diseases. Glycogen storage disease type I (GSD-I) is a kind of human genetic disorders and is caused by the deficiency of a microsomal protein, glucose-6-phosphatase-${\alpha}$ ($G6Pase-{\alpha}$) or glucose-6-phosphate transporter (G6PT) responsible for glucose homeostasis, leading to GSD-Ia or GSD-Ib, respectively. This review summarizes cell deaths in GSD-I and mostly focuses on current knowledge of the neutrophil apoptosis in GSD-Ib based upon ER stress and redox signaling.

• Journal of Applied Biological Chemistry
• /
• v.59 no.1
• /
• pp.25-29
• /
• 2016

Hepatitis B virus infection can cause hepatic fibrosis leading to cirrhosis and hepatocellular carcinoma. However the mechanism remains poorly understood. In this study, we found that Hepatitis B virus X-protein (HBx) increases vimentin, fibronectin, slug, snail and NOX4 expression. Because NOX4-mediated reactive oxygen species can increase slug and snail, which can induce fibrosis, HBx may be a key regulator of hepatic fibrosis development via NOX4 induction.

• Nutrition Research and Practice
• /
• v.9 no.6
• /
• pp.586-591
• /
• 2015

BACKGROUND/OBJECTIVES: Reactive oxygen species (ROS) formation is closely related to miconazole-induced heart dysfunction. Although rhamnetin has antioxidant effects, it remained unknown whether it can protect against miconazole-induced cardiomyocyte apoptosis. Thus, we investigated the effects of rhamnetin on miconazole-stimulated H9c2 cell apoptosis. MATERIALS/METHODS: Cell morphology was observed by inverted microscope and cell viability was determined using a WelCount$^{TM}$ cell proliferation assay kit. Miconazole-induced ROS production was evaluated by fluorescence-activated cell sorting with 6-carboxy-2',7'-dichlorofluoroscein diacetate ($H_2DCF$-DA) stain. Immunoblot analysis was used to determine apurinic/apyrimidinic endonuclease 1 (APE/Ref-1) and cleaved cysteine-aspartic protease (caspase) 3 expression. NADPH oxidase levels were measured using real-time polymerase chain reaction. RESULTS: Miconazole (3 and $10{\mu}M$) induced abnormal morphological changes and cell death in H9c2 cells. Rhamnetin enhanced the viability of miconazole ($3{\mu}M$)-treated cells in a dose-dependent manner. Rhamnetin (1 and $3{\mu}M$) treatment downregulated cleaved caspase 3 and upregulated APE/Ref-1 expression in miconazole-stimulated cells. Additionally, rhamnetin significantly reduced ROS generation. CONCLUSIONS: Our data suggest that rhamnetin may have cytoprotective effects in miconazole-stimulated H9c2 cardiomyocytes via ROS inhibition. This effect most likely occurs through the upregulation of APE/Ref-1 and attenuation of hydrogen peroxide levels.

• BMB Reports
• /
• v.49 no.4
• /
• pp.232-237
• /
• 2016

Mulberry tree twigs (Ramulus mori) contain large amounts of oxyresveratrols and have traditionally been used as herbal medicines because of their anti-inflammatory properties. However, the signaling mechanism by which R. mori exerts its anti-inflammatory action remains to be elucidated. In this study, we observed that R. mori ethanol extracts (RME) exerted an inhibitory effect on the lipopolysaccharide (LPS)-induced production of the pro-inflammatory cytokine interleukin-6 (IL-6) in Raw264.7 macrophage cells. Additionally, RME inhibited IL-6 production by blocking the leukotriene B₄ receptor-2 (BLT2)-dependent-NADPH oxidase 1 (NOX1)-reactive oxygen species (ROS) cascade, leading to anti-inflammatory activity. Finally, RME suppressed the production of the BLT2 ligands LTB4 and 12(S)-HETE by inhibiting the p38 kinase-cytosolic phospholipase A₂-5-/12-lipoxygenase cascade in LPS-stimulated Raw264.7 cells. Overall, our results suggest that RME inhibits the 'BLT2 ligand-BLT2'-linked autocrine inflammatory axis, and that this BLT2-linked cascade is one of the targets of the anti-inflammatory action of R. mori.

• The Plant Pathology Journal
• /
• v.29 no.4
• /
• pp.427-434
• /
• 2013

2R,3R-Butanediol, a volatile compound produced by certain rhizobacteria, is involved in induced drought tolerance in Arabidopsis thaliana through mechanisms involving stomatal closure. In this study, we examined the involvement of nitric oxide and hydrogen peroxide in induced drought tolerance, because these are signaling agents in drought stress responses mediated by abscisic acid (ABA). Fluorescence-based assays showed that systemic nitric oxide and hydrogen peroxide production was induced by 2R,3R-butanediol and correlated with expression of genes encoding nitrate reductase and nitric oxide synthase. Co-treatment of 2R,3R-butanediol with an inhibitor of nitrate reductase or an inhibitor of nitric oxide synthase lowered nitric oxide production and lessened induced drought tolerance. Increases in hydrogen peroxide were negated by co-treatment of 2R,3R-butanediol with inhibitors of NADPH oxidase, or peroxidase. These findings support the volatile 2R,3R-butanediol synthesized by certain rhizobacteria is an active player in induction of drought tolerance through mechanisms involving nitric oxide and hydrogen peroxide production.

• Biomolecules & Therapeutics
• /
• v.7 no.2
• /
• pp.112-120
• /
• 1999

The protective actions of ambroxol, rutin, glutathione and harmaline on oxidative damages of various tissue components were compared. The mechanisms by which they prevent oxidative tissue damages were explored. Lipid peroxidation of liver microsomes induced by combinations of $Fe^{2+}$ and ascorbate or $Fe^{+3}$, ADP and NADPH was inhibited by $50\; \muM$ of rutin, ambroxol, harmaline and glutathione. Ambroxol ($100\; \muM$) inhibited the degradation of hyaluronic acid by $Fe^{2+}$, $H_2O$_2$ and ascorbate, and it was greater than that of harmaline, whereas hyaluronic acid degradation was not prevented by rutin and glutathione. The compounds used ($100\; \muM$) did not protect the degradation of cartilage collagen by xanthine and xanthine oxidase. Rutin, glutathione and harmaline decreased the degradation of IgG by xanthine and xanthine oxidate, while ambroxol did not attenuate degradation of IgG. Glutathione showed a scavenging action on $H_2O_2$. The compounds all showed scavenging actions on hydroxyl radical. Ambroxol and harmaline exhibited quenching effects en singlet oxygen. In conclusion, ambroxol, rutin, glutathione and harmaline may exert protective effects differently on tissue components against oxidative attack depend on kind of tissue component and free radical.

• Mycobiology
• /
• v.44 no.1
• /
• pp.38-47
• /
• 2016

The ascomycete fungus Mycosphaerella graminicola (synonym Zymoseptoria tritici) is an important pathogen of wheat causing economically significant losses. The primary nutritional mode of this fungus is thought to be hemibiotrophic. This pathogenic lifestyle is associated with an early biotrophic stage of nutrient uptake followed by a necrotrophic stage aided possibly by production of a toxin or reactive oxygen species (ROS). In many other fungi, the genes CREA and AREA are important during the biotrophic stage of infection, while the NOXa gene product is important during necrotrophic growth. To test the hypothesis that these genes are important for pathogenicity of M. graminicola, we employed an over-expression strategy for the selected target genes CREA, AREA, and NOXa, which might function as regulators of nutrient acquisition or ROS generation. Increased expressions of CREA, AREA, and NOXa in M. graminicola were confirmed via quantitative real-time PCR and strains were subsequently assayed for pathogenicity. Among them, the NOXa over-expression strain, NO2, resulted in significantly increased virulence. Moreover, instead of the usual filamentous growth, we observed a predominance of yeast-like growth of NO2 which was correlated with ROS production. Our data indicate that ROS generation via NOXa is important to pathogenicity as well as development in M. graminicola.