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

Phagocytes such as neutrophils play a vital role in host defense against microbial pathogens. The anti-microbial function of neutrophils is based on the production of superoxide anion (O2(.-)), which generates other microbicidal reactive oxygen species (ROS) and release of antimicrobial peptides and proteins. The enzyme responsible for O2(.-) production is called the NADPH oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two transmembrane proteins (p22phox and gp91phox, also called NOX2, which together form the cytochrome b(558)) and four cytosolic proteins (p47phox, p67phox, p40phox and a GTPase Rac1 or Rac2), which assemble at membrane sites upon cell activation. NADPH oxidase activation in phagocytes can be induced by a large number of soluble and particulate agents. This process is dependent on the phosphorylation of the cytosolic protein p47phox. p47phox is a 390 amino acids protein with several functional domains: one phox homology (PX) domain, two src homology 3 (SH3) domains, an auto-inhibitory region (AIR), a proline rich domain (PRR) and has several phosphorylated sites located between Ser303 and Ser379. In this review, we will describe the structure of p47phox, its phosphorylation and discuss how these events regulate NADPH oxidase activation.
*Disease ; Enzyme Activation ; Humans ; Membrane Glycoproteins/chemistry/*metabolism ; NADPH Oxidase/chemistry/genetics/*metabolism ; Phagocytes/cytology/*metabolism ; Phosphorylation ; Protein Conformation

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

BCG Vaccine ; adverse effects ; Child ; Child, Preschool ; Genetic Diseases, X-Linked ; complications ; Granulomatous Disease, Chronic ; complications ; genetics ; Humans ; Infant ; Infant, Newborn ; Lymphadenitis ; etiology ; Male ; Membrane Glycoproteins ; genetics ; NADPH Oxidase 2 ; NADPH Oxidases ; genetics

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

Chronic granulomatous disease (CGD) is a rare hereditary disorder characterized by recurrent life-threatening bacterial and fungal infections. The underlying defect in CGD is an inability of phagocytes to produce reactive oxygen species as a result of defects in NADPH oxidase. Considering that CGD generally affects about 3-4 in 1,000,000 individuals, it is surprising that the prevalence of CGD on Jeju Island is 20.7 in 1,000,000 individuals. We performed genetic analysis on 12 patients from 10 unrelated families and found that all patients had an identical homozygous single-base substitution of C to T in exon 1 (c.7C>T) of the CYBA gene, which was expected to result in a nonsense mutation (p.Q3X). Because Jeju Island has long been a geologically isolated region, the high prevalence of CGD on Jeju Island is presumably associated with an identical mutation inherited from a common ancestor or proband.
Adolescent ; Animals ; Asian Continental Ancestry Group/*genetics ; Base Sequence ; Child ; Child, Preschool ; DNA Mutational Analysis ; Female ; Geography ; Granulomatous Disease, Chronic/*genetics ; Humans ; Infant ; Korea ; Male ; Molecular Sequence Data ; *Mutation ; NADPH Oxidase/*genetics ; Pedigree ; Young Adult

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

Chronic granulomatous disease (CGD) is a rare genetic disease, which is caused by defects in the NADPH oxidase complex (gp91phox, p22phox, p40phox, p47phox, and p67phox) of phagocytes. This defect results in impaired production of superoxide anions and other reactive oxygen species (ROS), which are necessary for killing bacterial and fungal microorganisms and leads to recurrent, life-threatening bacterial and fungal infections and granulomatous inflammation. The dihydrorhodamine (DHR) flow cytometry assay is a useful diagnostic tool for CGD that can detect absent or reduced NADPH oxidase activity in stimulated phagocytes. We report a patient with X-linked CGD carrying a novel mutation of the CYBB gene whose chimerism status following hematopoietic stem cell transplantation (HSCT) has been rapidly determined using the DHR assay. The level of DHR activity correlates well with short tandem repeat PCR analysis. Considering the advantages of this simple, rapid, and cost-effective procedure, serial measurement of DHR assay would facilitate the rapid determination of a patient's engraftment status, as a supplementary monitoring tool of chimerism status following HSCT.
Base Sequence ; *Chimerism ; DNA Mutational Analysis ; Flow Cytometry ; Granulomatous Disease, Chronic/*diagnosis/*enzymology/genetics/surgery ; *Hematopoietic Stem Cell Transplantation ; Homozygote ; Humans ; Infant, Newborn ; Male ; Membrane Glycoproteins/chemistry/*genetics ; Mutation ; NADPH Oxidase/chemistry/*genetics ; Polymerase Chain Reaction ; Rhodamines/chemistry/metabolism

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