Production of reactive oxygen species (ROS) is a conserved immune response primarily mediated by NADPH oxidases (NOXs), also known in plants as respiratory burst oxidase homologs (RBOHs). Most microbe-associated molecular patterns (MAMPs) trigger a very fast and transient ROS burst in plants. However, recently, we found that lipopolysaccharides (LPS), a typical bacterial MAMP, triggered a biphasic ROS burst. In this study, we isolated mutants defective in LPS-triggered biphasic ROS burst (delt) in Arabidopsis, and cloned the DELT1 gene that was shown to encode RBOHD. In the delt1-2 allele, the antepenultimate residue, glutamic acid (E919), at the C-terminus of RBOHD was mutated to lysine (K). E919 is a highly conserved residue in NADPH oxidases, and a mutation of the corresponding residue E568 in human NOX2 has been reported to be one of the causes of chronic granulomatous disease. Consistently, we found that residue E919 was indispensable for RBOHD function in the MAMP-induced ROS burst and stomatal closure. It has been suggested that the mutation of this residue in other NADPH oxidases impairs the protein's stability and complex assembly. However, we found that the E919K mutation did not affect RBOHD protein abundance or the ability of protein association, suggesting that the residue E919 in RBOHD might have a regulatory mechanism different from that of other NOXs. Taken together, our results confirm that the antepenultimate residue E is critical for NADPH oxidases and provide a new insight into the regulatory mechanisms of RBOHD.
Agrobacterium tumefaciens/metabolism* ; Alleles ; Arabidopsis/metabolism* ; Arabidopsis Proteins/genetics* ; Gene Expression Regulation, Plant ; Genetic Techniques ; Humans ; Lipopolysaccharides/metabolism* ; Luminescence ; Mutation ; NADPH Oxidase 2/chemistry* ; NADPH Oxidases/genetics* ; Plant Stomata/metabolism* ; Protein Domains ; Reactive Oxygen Species/metabolism* ; Nicotiana/metabolism*

Objective To verify the expressions of genes associated with colorectal cancer with hyperglycemia and evaluate their diagnostic values.Methods Tumor tissues,distal normal intestinal mucosa,and peripheral blood samples were harvested from 109 colorectal cancer patients and peripheral blood samples from 30 diabetes patients and 30 healthy volunteers. The mRNA expressions of glucose regulated protein 78 (GRP78),NADPH oxidase-1 (NOX1),carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5),heat shock protein 60 (HSP60),and histone deacetylase 1(HDAC1) were detected by real-time quantitative polymerase chain reaction. The correlation between the gene expressions and clinicopathological parameters in colorectal cancer patients were analyzed using Pearson's correlation analysis. Diagnostic test accuracy evaluation was used to calculate the sensitivity,specificity,accuracy,predictability,Youden index,and likelihood ratio of serum gene expressions in colorectal cancer patients,and the receiver operating characteristic (ROC) curves were drawn. The area under the ROC curve was calculated to evaluate the diagnostic efficiency of the combined detection of multiple genes.Results The mRNA levels of GRP78 (P=0.001),NOX1 (P=0.022),CEACAM5 (P=0.000),HSP60 (P=0.044),and HDAC1 (P=0.047) were positively correlated with the fasting blood glucose level. The mRNA expressions of NOX1 (P=0.000,P=0.008) and HDAC1 (P=0.000,P=0.037) in tissues and serum were significantly higher in colorectal cancer patients than in patients with normal blood glucose levels. The NOX1 mRNA expression was positively correlated with the diameter of colorectal cancer (P=0.013),and the HDAC1 mRNA expression was significantly correlated with the tumor site (P=0.049),depth of primary tumor invasion (P=0.025),and TNM stage (P=0.042). The areas under the ROC curves of NOX1,CEACAM5,and HDAC1 were 0.931,0.852,and 0.860 respectively (all P=0.000). The specificity,accuracy,and negative predictive value of NOX1,HDAC1 mRNA expression in colorectal cancer patients with hyperglycemia were all above 90%. The diagnostic sensitivity and specificity of the combined detection of NOX1,CEACAM5,and HDAC1 were 98.82% and 99.93%,respectively.Conclusion Combined detection of genes associated with colorectal cancer accompanied by hyperglycemia can improve the diagnostic efficiency of early screening.
Biomarkers, Tumor ; genetics ; Carcinoembryonic Antigen ; genetics ; Case-Control Studies ; Colorectal Neoplasms ; complications ; diagnosis ; genetics ; Diabetes Mellitus ; genetics ; GPI-Linked Proteins ; genetics ; Heat-Shock Proteins ; genetics ; Histone Deacetylase 1 ; genetics ; Humans ; Hyperglycemia ; complications ; diagnosis ; genetics ; NADPH Oxidase 1 ; genetics ; ROC Curve

BACKGROUND: Most cases with congenital hypothyroidism (CH) are usually sporadic, while about 20% of the cases are caused by genetic defects. Little information is available regarding the mutation incidence and genetic heterogeneity of CH in Koreans. We aimed to determine the mutation incidence of CH in newborn screenings (NBS) and to evaluate the frequency and spectrum of mutations underlying CH. METHODS: A total of 112 newborns with thyroid dysfunction were enrolled from 256,624 consecutive NBS. Furthermore, 58 outpatients with primary CH were added from an endocrine clinic. All coding exons of TSHR, PAX8, TPO, DUOX2, DUOXA2, and SCL5A5 were sequenced. RESULTS: The mutation incidence of CH was estimated to be 1 in 6,580 newborns. A total of 36 different mutations were identified in 53 cases. The overall mutation positive rate was 31%. The DUOX2 mutations were the most prevalent in both newborns and outpatients. Seven different recurrent mutations [p.G488R (n=13), p.A649E (n=3), p.R885Q (n=3), p.I1080T (n=2), and p.A1206T (n=2) in DUOX2; p.Y138X (n=9) in DUOXA2; and p.R450H (n=5) in TSHR) were identified as the mutations underlying CH. CONCLUSIONS: The mutation incidence of CH was considerably higher than expected in the Korean newborn population. This study revealed seven different recurrent mutations underlying CH. We conclude that DUOX2 mutations are a frequent cause of CH in the Korean population.
Asian Continental Ancestry Group/*genetics ; Child ; Child, Preschool ; Congenital Hypothyroidism/epidemiology/*genetics/pathology ; Exons ; Female ; Genetic Association Studies ; Genotype ; Humans ; Infant ; Infant, Newborn ; Male ; NADPH Oxidase/*genetics ; Polymorphism, Single Nucleotide ; Republic of Korea/epidemiology ; Sequence Analysis, DNA ; Thyrotropin/blood

OBJECTIVETo explore the relationship between the expression of endothelial nitric oxide synthase (eNOS) and NADPH oxidase (NOX) in the lungs of mice treated by chronic hypoxic exposure.

METHODSThirty male wild-type (WT) C57Bl/6 mice and thirty male eNOS-knockout (KO) C57BL/6 mice were randomly divided into normoxic groups (exposed to normoxia for 7 days or 21 days), hypoxic groups (exposed to 10% oxygen for 7 days or 21 days), and treatment groups (exposed to 10% oxygen and orally administrated 10 mmol/L 4-hydroxy TEMPO in drinking water for 7 days or 21 days) (n=6 in each group). The remodeling of the small pulmonary arteries was evaluated by the percentage of media wall thickness (MT%). The weight ratio of right ventricle to left ventricle plus septum (RV/[LV+S]) was calculated to evaluate the hypertrophy of right ventricle. Real-time PCR was used to measure the mRNA expression of NOX2, NOX4, and eNOS in mouse lungs. ELISA was used to determine the concentration of reactive oxygen species (ROS) in mouse lungs.

RESULTSIn WT mice and KO mice, the hypoxic groups had significantly increased pulmonary vascular remodeling and RV/[LV+S] compared with the normoxic and treatment groups (P<0.05), but there were no significant differences between the normoxic and treatment groups (P>0.05). In WT mice, the hypoxic and treatment groups had significantly lower ROS concentrations than the normoxic group (P<0.05), but there were no significant differences between the hypoxic and treatment groups (P>0.05). In WT mice, the mRNA expression of eNOS, NOX2, and NOX4 was significantly higher in the hypoxic group than in the normoxic group (P<0.05), and 4-hydroxy TEMPO reversed their over-expression. In the normoxic group, the KO mice had significantly higher NOX2 and NOX4 mRNA expression than the WT mice (P<0.05); in KO mice, the hypoxic group showed no significant changes in NOX4 mRNA expression (P>0.05), but had significantly reduced NOX2 mRNA expression (P<0.05), as compared with the normoxic group; the treatment group had reduced expression of NOX2 mRNA expression and increased NOX4 mRNA expression (P<0.05), as compared with the hypoxic group.

CONCLUSIONSeNOS plays a key role in the regulation of expression of NOX2 and NOX4 in the lungs exposed to hypoxia. It suggests that NOX and eNOS may physically interact with one another in pulmonary vascular remodeling induced by chronic hypoxia.

Animals ; Chronic Disease ; Hypoxia ; enzymology ; Lung ; enzymology ; Male ; Membrane Glycoproteins ; genetics ; physiology ; Mice ; Mice, Inbred C57BL ; NADPH Oxidase 2 ; NADPH Oxidase 4 ; NADPH Oxidases ; genetics ; physiology ; Nitric Oxide Synthase Type III ; genetics ; physiology ; RNA, Messenger ; analysis

OBJECTIVETo observe the effect of sesamin (Ses) on pulmonary vascular remodeling in rats with monocrotaline ( MCT)-induced pulmonary hypertension (PH).

METHODTotally 48 male Sprague-Dawley (SD) rats were fed adaptively for one week and then divided into the normal control group, the MCT group, the MCT +Ses (50 mg x kg(-1)) group and the MCT + Ses (100 mg x kg(-1)) group, with 12 rats in each group. The PH rat model was induced through the subcutaneous injection with MCT(60 mg x kg(-1)). After the administration for four weeks, efforts were made to measure the right ventricular systolic pressure( RVSP) and mean pulmonary artery pressure (mPAP) through right jugular vein catheterization, and isolate right ventricle( RV) and left ventricle( LV) +septum (S) and measure their length to calculate RV/ ( LV + S) and ratio of RV to tibial length. Pathologic changes in arterioles were observed by HE staining. Masson's trichrome stain was used to demonstrate changes in collagen deposition of arterioles. The alpha-smooth muscle actin (alpha-SMA) expression in pulmonary arteries was measured by immunohistochemisty. The total antioxidative capacity (T-AOC) and malondialdehyde (MDA) content in pulmonary arteries were determined by the colorimetric method. The protein expressions of collagen I, NOX2 and NOX4 were analyzed by Real-time PCR and Western blot.

RESULTAfter the administration for 4 weeks, Ses could attenuate RVSP and mPAP induced by MCT, RV/ (LV + S) and ratio of RV to Tibial length, alpha-SMA and collagen I expressions and remodeling of pulmonary vessels and right ventricle. Meanwhile, Ses could obviously inhibit the expressions of NOX2, NOX4 and MDA content and increase T-AOC.

CONCLUSIONSesamin could ameliorate pulmonary vascular remodeling induced by monocrotaline in PH rats. Its mechanism may be related to expressions of NOX2 and NOX4 expression and reduction in oxidative stress injury.

Animals ; Dioxoles ; administration & dosage ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Hypertension, Pulmonary ; drug therapy ; enzymology ; genetics ; physiopathology ; Lignans ; administration & dosage ; Lung ; blood supply ; enzymology ; metabolism ; Male ; Membrane Glycoproteins ; genetics ; metabolism ; Monocrotaline ; adverse effects ; NADPH Oxidase 2 ; NADPH Oxidase 4 ; NADPH Oxidases ; genetics ; metabolism ; Pulmonary Artery ; drug effects ; metabolism ; physiopathology ; Rats ; Rats, Sprague-Dawley ; Vascular Remodeling ; drug effects

The aim of the present study is to investigate how cytochrome P450 enzymes (CYP) 2C8-derived epoxyeicosatrienoic acids (EETs) regulate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and protect against oxidative stress-induced endothelial injuries in the development and progression of atherosclerosis. In this study, cultured human umbilical vein endothelial cells (HUVECs) were transfected with CYP2C8 or pretreated with exogenous EETs (1 μmol/L) before TNF-α (20 ng/mL) stimulation. Apoptosis and intracellular ROS production were determined by flow cytometry. The expression levels of ROS-associated NAD(P)H subunits gp91 and p47, the anti-oxidative enzyme catalase (CAT), Nrf2, heme oxygenase-1 (HO-1) and endothelial nitric oxide synthase (eNOS) were detected by Western blotting. The results showed that CYP2C8-derived EETs decreased apoptosis of HUVECs treated with TNF-α. Pretreatment with 11, 12-EET also significantly blocked TNF-α-induced ROS production. In addition, 11, 12-EET decreased oxidative stress-induced apoptosis. Furthermore, the ability of 11, 12-EET to protect cells against TNF-α-induced apoptosis via oxidative stress was abrogated by transient transfection with Nrf2-specific small interfering RNA (siRNA). In conclusion, CYP2C8-derived EETs prevented TNF-α-induced HUVECs apoptosis via inhibition of oxidative stress associated with the Nrf2 signaling.
8,11,14-Eicosatrienoic Acid ; analogs & derivatives ; metabolism ; pharmacology ; Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Apoptosis ; drug effects ; Aryl Hydrocarbon Hydroxylases ; genetics ; metabolism ; Atherosclerosis ; genetics ; metabolism ; pathology ; Catalase ; genetics ; metabolism ; Cytochrome P-450 CYP2C8 ; genetics ; metabolism ; Gene Expression Regulation ; Heme Oxygenase-1 ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Membrane Glycoproteins ; genetics ; metabolism ; Models, Biological ; NADPH Oxidase 2 ; NADPH Oxidases ; genetics ; metabolism ; NF-E2-Related Factor 2 ; antagonists & inhibitors ; genetics ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; Reactive Oxygen Species ; antagonists & inhibitors ; metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism ; pharmacology

OBJECTIVETo investigate the mechanism of Panax notoginseng saponins (PNS), an effective component extracted from Panax notoginseng, on atherosclerotic plaque angiogenesis in atherosclerosis-prone apolipoprotein E-knockout (ApoE-KO) mice fed with high-fat, high-cholesterol diet.

METHODSTwenty ApoE-KO mice were divided into two groups, the model group and the PNS group. Ten normal C57BL/6J mice were used as a control group. PNS (60 mg/kg) was orally administered daily for 12 weeks in the PNS group. The ratio of plaque area to vessel area was examined by histological staining. The tissue sample of aortic root was used to detect the CD34 and vascular endothelial growth factor (VEGF) expression areas by immunohistochemistry. The expression of VEGF and nicotinamide adenine dinucleotide phosphate oxidase subunit 4 (NOX4) were measured by reverse transcription polymerase chain reaction and Western blotting respectively.

RESULTSAfter treatment with PNS, the plaque areas were decreased (P<0.05). CD34 expressing areas and VEGF expression areas in plaques were significantly decreased (P<0.05). Meanwhile, VEGF and NOX4 mRNA expression were decreased after treatment with PNS. VEGF and NOX4 protein expression were also decreased by about 72% and 63%, respectively (P<0.01).

CONCLUSIONPNS, which decreases VEGF and NOX4 expression, could alleviate plaque angiogenesis and attenuate atherosclerosis.

Animals ; Down-Regulation ; drug effects ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; NADPH Oxidase 4 ; NADPH Oxidases ; genetics ; metabolism ; Neovascularization, Pathologic ; pathology ; prevention & control ; Panax notoginseng ; chemistry ; Plaque, Atherosclerotic ; pathology ; prevention & control ; Saponins ; pharmacology ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

NADPH oxidase produces a large amount of reactive oxygen species (ROS) in Helicobacter pylori (H. pylori)-induced gastric epithelial cells. Even though ROS mediate apoptotic cell death, direct involvement of NADPH oxidase on H. pylori-induced apoptosis remains unclear. Besides, H. pylori isolates show a high degree of genetic variability. The predominant genotype of H. pylori in Korea has been reported as cagA+, vacA s1b, m2, iceA genotype. Present study aims to investigate whether NADPH oxidase-generated ROS mediate apoptosis in human gastric epithelial AGS cells infected with H. pylori in a Korean isolate. AGS cells were pretreated with or without an NADPH oxidase inhibitor diphenyleneiodonium (DPI) and cultured in the presence of H. pylori at a bacterium/cell ratio of 300:1. Cell viability, hydrogen peroxide level, DNA fragmentation, and protein levels of p53, Bcl-2, and Bax were determined. Results showed that H. pylori inhibited cell viability with the density of H. pylori added to the cells. Inhibition of NADPH oxidase by DPI suppressed H. pylori-induced cell death, increased hydrogen peroxide, DNA fragmentation, and the ratio of Bax/Bcl-2, and p53 induction in AGS cells dose-dependently. The results suggest that targeting NADPH oxidase may prevent the development of gastric inflammation associated with H. pylori infection by suppressing abnormal apoptotic cell death of gastric epithelial cells.
Apoptosis ; Apoptosis Regulatory Proteins/metabolism ; Cell Survival ; Epithelial Cells/metabolism/microbiology ; Gastric Mucosa/metabolism ; Helicobacter Infections/*metabolism/microbiology ; Helicobacter pylori/drug effects/genetics/*isolation & purification ; Humans ; NADPH Oxidase/metabolism ; Onium Compounds/*antagonists & inhibitors/pharmacology ; Oxidative Stress/drug effects ; Reactive Oxygen Species/metabolism ; Republic of Korea ; Stomach/cytology/*metabolism/microbiology

The aim of the present study was to evaluate the protective effect of catalpol on vascular endothelial function in STZ-induced type 2 diabetes mellitus (T2DM) rats. 40 high-fat diet with STZ-induced diabetes rats were randomly divided into model group, catalpol low-dose, middle-dose and high-dose group (10, 50, 100 mg x kg(-1) x d(-1)), 10 normal Wistar rats were used as the normal group. The normal and model groups were given an equivalent amount of saline. All reagents were administered by oral gavage for 6 weeks. After 6 weeks, blood glucose and lipids were detected by an automatic biochemical analyzer. The endothelium-dependent vasodilation response of thoracic aortar was detected. The pathological changes of the thoracic aorta were observed by HE staining. Ser- um nitric oxide (NO), 8-iso prostaglandin F2α (8-iso-PGF2α) and superoxide dismutase (SOD) were detected by ELISA. Reactive oxygen species (ROS) level of thoracic aorta was detected by fluorescence method. The expression of Nox4 and p22phox mRNA and protein in aortic tissue were detected by RT-PCR and Western-blot respectively. After catalpol treatment, endothelial damage of thoracic aorta was attenuated significantly; ROS level of thoracic aorta and serum level of 8-iso-PGF2α were decreased significantly; serum NO and SOD levels were remarkably elevated; expression of Nox4, p22phox mRNA and protein in thoracic aorta were significantly reduced (P < 0.05). Therefore, catalpol has protective effect on endothelial of T2DM, its mechanism may be associated with the down-regulation of Nox4 and p22phox expression, inhibiting oxidative stress reaction response.
Animals ; Blood Glucose ; metabolism ; Diabetes Mellitus, Experimental ; pathology ; Diabetes Mellitus, Type 2 ; pathology ; Dinoprost ; analogs & derivatives ; metabolism ; Endothelium, Vascular ; drug effects ; metabolism ; pathology ; Enzyme Inhibitors ; pharmacology ; Gene Expression Regulation ; drug effects ; Iridoid Glucosides ; pharmacology ; Male ; NADPH Oxidase 4 ; NADPH Oxidases ; antagonists & inhibitors ; genetics ; metabolism ; Nitric Oxide ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism

We aimed to elucidate the effect of bilirubin on dyslipidemia and nephropathy in a diabetes mellitus (DM) type I animal model. Sprague-Dawley rats were separated into control, DM, and bilirubin-treated DM (Bil) groups. The Bil group was injected intraperitoneally with 60 mg/kg bilirubin 3 times per week and hepatoma cells were cultured with bilirubin at a concentration of 0.3 mg/dL. The Bil group showed lower serum creatinine levels 5 weeks after diabetes onset. Bilirubin treatment also decreased the amount of mesangial matrix, lowered the expression of renal collagen IV and transforming growth factor (TGF)-beta1, and reduced the level of apoptosis in the kidney, compared to the DM group. These changes were accompanied by decreased tissue levels of hydrogen superoxide and NADPH oxidase subunit proteins. Bilirubin decreased serum total cholesterol, high-density lipoprotein cholesterol (HDL-C), free fatty acids, and triglycerides (TGs), as well as the TG content in the liver tissues. Bilirubin suppressed protein expression of LXRalpha, SREBP-1, SCD-1, and FAS, factors involved in TG synthesis that were elevated in the livers of DM rats and hepatoma cells under high-glucose conditions. In conclusion, bilirubin attenuates renal dysfunction and dyslipidemia in diabetes by suppressing LXRalpha and SREBP-1 expression and oxidative stress.
Animals ; Bilirubin/pharmacology/*therapeutic use ; Cell Line, Tumor ; Creatine/blood ; Diabetes Mellitus, Experimental/chemically induced/complications/*pathology ; Diabetic Nephropathies/*drug therapy/etiology ; Disease Models, Animal ; Kidney/pathology ; Lipoproteins, HDL/blood ; Liver/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; NADPH Oxidase/metabolism ; Orphan Nuclear Receptors/antagonists & inhibitors/genetics/metabolism ; Oxidative Stress/drug effects ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism ; Streptozocin/toxicity ; Triglycerides/analysis/*biosynthesis/blood