Inducible and endothelial nitric oxide synthase genes polymorphism in inflammatory bowel disease

To assess the influence of inducible and endothelial nitric oxide synthase gene (NOS2A and NOS3) polymorphisms in susceptibility to Crohn's disease (CD) and ulcerative colitis (UC). A total of 505 inflammatory bowel disease (IBD) patients (221 with UC and 284 with CD) and 332 ethnically matched controls were studied. Patients and controls were genotyped by polymerase chain reaction -based techniques for a multiallelic (CCTTT)(n) repeat and biallelic marker (TAAA)(n) in the promoter region of the NOS2A gene and for a T/C polymorphism at position -786 in the promoter region and a polymorphism in exon 7(298Glu/Asp) of the NOS3 gene. There was not association between NOS2A and NOS3 genotypes, alleles or haplotypes frequencies with UC, CD and controls. Our data suggest that NOS2A and NOS3 polymorphisms do not play a major role in IBD predisposition.     

Genetic determinants of rheumatoid arthritis: the inducible nitric oxide synthase (NOS2) gene promoter polymorphism

An association study and a linkage analysis were carried out in parallel in order to investigate the association of the inducible nitric oxide synthase (NOS2) gene promoter polymorphism with rheumatoid arthritis (RA) predisposition and/or outcome including a -954G-C mutation, a di-allelic (TAAA)(n) marker and the highly polymorphic (CCTTT). The -954G-C point mutation occurred non-polymorphic and the di-allelic (TAAA)(n) marker was not associated with RA predisposition. The (CCTTT)(n)locus showed a trend for RA association in the case-control study, however, stratification data by Class II status did not yield significant effect and overall, the family study showed no significant association nor linkage for any of the markers under study using TDT and non-parametric linkage respectively. Finally, no influence was detected regarding any of the clinical parameters tested. After evaluating for the first time the influence of NOS2 promoter polymorphism in RA, it seems to have no major effect on disease susceptibility and/or outcome.     

(CCTTT)n repeat polymorphism in the NOS2 gene promoter is associated with atopy.

BACKGROUND: Several studies have shown that nitric oxide (NO) plays a role in the regulation of the T(H)1/T(H)2 balance, indicating the potential for NO to contribute to the development of atopy and several other allergic diseases, including bronchial asthma. NO synthase 2 (NOS2) is critically involved in the synthesis of NO during several inflammatory states, and the gene encoding NOS2 is located at chromosome 17q11.2-q12, where 2 genome scans have identified a candidate locus for atopy and asthma. OBJECTIVE: The 14-repeat allele of the (CCTTT)(n) repeat polymorphism in the NOS2 promoter region is a powerful enhancer of promoter activity in reporter constructs in vitro. We tested whether this potentially functional allele in the NOS2 gene influences the development of atopy and asthma. METHODS: We studied a total of 497 unrelated Japanese subjects (141 nonatopic healthy controls, 102 atopic healthy controls, 56 nonatopic asthmatic subjects, and 198 atopic asthmatic subjects). The odds ratio (OR) was calculated for atopy and asthma in carriers of the 14-repeat allele through use of logistic regression models. Atopy was defined as a positive specific IgE level to at least 1 of 10 common inhaled allergens. RESULTS: The 14-repeat allele was inversely associated with atopy (OR = 0.42, P < .01). The association remained significant when the model was controlled for asthmatic status (OR = 0.36, P < .01). This allele, however, was associated neither with the development of asthma nor with total serum IgE levels. CONCLUSION: Our findings suggest that the (CCTTT)(n) repeat polymorphism in the promoter of the NOS2 gene that affects promoter activity is a risk factor for the development of atopy, and this genetic effect seems independent of asthma.     

No evidence for association of the inducible nitric oxide synthase promoter polymorphism with Trypanosoma cruzi infection

The purpose of the present study was to address the possible contribution of the (CCTTT)n microsatellite polymorphism in the NOS2 promoter region to the susceptibility to chronic Trypanosoma cruzi infection and to Chagas' disease related cardiomyopathy. We determined the (CCTTT)n genotypes in a sample of 76 serologically positive chagasic individuals and in 78 healthy controls. No statistically significant differences were observed between total chagasic patients and healthy controls with regard to frequency of the (CCTTT)n microsatellite repeat of any given length. Likewise, we found no differences in the distribution of the (CCTTT)n microsatellite repeats between seropositives without manifestations of the disease and those with chagasic cardiomyopathy. Our data suggest that the NOS2 promoter pentanucleotide microsatellite polymorphisms analyzed do not play a major role in the pathogenesis of chronic T. cruzi infection in this Peruvian sample.     

A dinucleotide repeat polymorphism at the poly(ADP-ribose) polymerase gene is not associated with predisposition to type 1 diabetes in French Caucasians

The poly (ADP-ribose) polymerase (PARP) is a nuclear enzyme that detects and binds DNA strand breaks. Excessive PARP activation leads to the death of mice islet beta-cells by depleting cellular energy reserves. On the other hand, PARP-mutant mice are resistant to streptozotocine-induced diabetes, and in the non-obese diabetic (NOD) mouse model, treatment with nicotinamide, a PARP inhibitor, protects islet cells against cytotoxic actions in vitro and results in a decreased incidence of type 1 diabetes. PARP gene in human is located within a recently identified type 1 diabetes-susceptibility region on chromosome 1q41-42, and contains a polymorphic CA dinucleotide repeat in the promoter region. To consider the putative involvement of PARP polymorphism in predisposition to type 1 diabetes, we performed genotyping for the various alleles of the CA dinucleotide repeat in 158 unrelated French Caucasian patients with type 1 diabetes and 193 ethnically-matched healthy controls. We found no significant difference of PARP alleles distribution between patients and controls, even after stratification of the patients according to HLA class II genotype or to age at disease onset. Our results suggest that this PARP polymorphism does not influence susceptibility to type 1 diabetes in French Caucasians.     

Analysis of the inducible nitric oxide synthase gene polymorphisms in Czech patients with atopic diseases

BACKGROUND: Nitric oxide (NO) is an important mediator of physiologic processes in the airways; it plays a significant role in the regulation of the T helper type 1/type 2 balance and contributes to the development of atopic diseases. OBJECTIVE: We analysed several polymorphisms mainly in the promoter region of the inducible NO synthase (NOS2, iNOS) gene and investigated their associations with asthma and/or atopic phenotypes. METHODS: We performed a case-control study in 994 subjects (661 patients with atopic disorders, with subgroups of 304 patients with allergic asthma, and 333 healthy individuals), matched for sex, living in the same geographical area. Screening for polymorphisms was performed by combination of PCR and direct sequencing analysis. RESULTS: We analysed 14 nucleotide sequence variants, seven most common of which were typed in quite large groups of our asthmatic, atopic and control populations. None of these seven frequent polymorphisms was associated with the phenotype bronchial asthma or other atopic diseases. Nevertheless, three from six common promoter polymorphisms showed a significant relation to feather's positivity (P value from 0.01 to 0.03) and the NOS2 608Leu variant was significantly associated with asthma severity [p(corr) = 0.0005; odds ratio (OR) = 5.00, 95% confidence interval (CI): 1.88-13.33]. In haplotype analysis, the most common -2447C/-1659C/-1026G/-0.7del/-277A/Ser608 haplotype was associated with a lower risk of asthma when compared with the common haplotypes with frequency more than 5% (P = 0.01, p(corr) < 0.05; OR = 0.65, 95% CI: 0.56-0.77). CONCLUSION: Our findings suggest that inducible NOS can play a role in atopic disorders, and several polymorphisms in its gene may be important for asthma protection or susceptibility.     

No evidence for involvement of the human inducible nitric oxide synthase (iNOS) gene in susceptibility to typical migraine.

Migraine is a debilitating disorder affecting approximately 12% of Caucasian populations. The disease has a large genetic component, although at present the type and number of genes involved is unclear. Candidate gene studies may be useful strategies for identifying genes involved in complex diseases such as migraine, especially if the gene being examined contributes only a minor effect to the overall phenotype. Nitric oxide (NO) is emerging as a key molecule affecting the pain associated with migraine. Since NO synthase (NOS) enzymes catalyze the synthesis of NO, the genes that code for these enzymes are good candidates for migraine molecular genetic analysis. This study investigated the role of a functionally relevant bi-allelic tetranucleotide polymorphism located in the promoter region of the human inducible nitric oxide synthase (iNOS) gene in migraine etiology. A large group of migraine affected individuals (n = 261) were genotyped and compared with an age- and sex-matched group of unaffected controls (n = 252). Results of a chi-squared analysis indicated that allele distributions for both migraine cases and controls were not significantly different (chi2 = 1.93, P = 0.16). These findings offer no evidence for an allelic association of the tested iNOS polymorphism with the common forms of the disease and therefore do not support a role for this gene in migraine pathogenesis.     

Intron 4 polymorphism of the endothelial nitric oxide synthase eNOS gene and early microangiopathy in type 1 diabetes

Nitric oxide (NO) is an endogenous vasodilator involved in inflammatory and autoimmune response, and in the pathophysiology of diabetic vascular disease. Endothelium‐derived NO is formed from l ‐arginine by endothelial NO synthase (eNOS), and earlier studies have provided evidence for altered NO metabolism and impaired endothelial function in diabetes, probably due to polymorphisms in eNOS gene. In the present study we investigated the association of the eNOS gene intron 4 a/b VNTR polymorphism with diabetic microangiopathy in 61 young individuals with type 1 diabetes (T1D), 35 male and 26 female, aged 5.0–29.1 (mean 15.6) years, and followed up for 3.24–11.4 (mean 7.44) years. Ten patients (16.4%) had developed microalbuminuria, three hypertension and two retinopathy. Wild‐type b/b homozygosity for eNOS gene intron 4 VNTR was found in 37 (60.7%) and a/b polymorphism in 24 (39.3%). No significant relationship was demonstrated between eNOS gene intron 4 polymorphisms and microalbuminuria, hypertension or retinopathy in these young individuals. Our findings suggest that a/b polymorphism of the intron 4 eNOS gene is not associated with early onset diabetic microangiopathy.     

An Inhibitor of Inducible Nitric Oxide Synthase and Scavenger of Peroxynitrite Prevents Diabetes Development in NOD Mice

Peroxynitrite (ONOO(-)) is a highly reactive oxidant produced by the interaction of the free radicals superoxide (O*-2) and nitric oxide (NO(*)). In a previous study, we found that peroxynitrite is formed in islet beta-cells of nonobese diabetic (NOD) mice. Here, we report that guanidinoethyldisulphide (GED), a selective inhibitor of inducible nitric oxide synthase (iNOS) and scavenger of peroxynitrite prevents diabetes in NOD mice. GED treatment of female NOD mice, starting at age 5 weeks, delayed diabetes onset (from age 12 to 22 weeks) and significantly decreased diabetes incidence at 30 weeks (from 80% to 17%). GED did not prevent pancreatic islet infiltration by leukocytes; however, beta-cells that stained positive for nitrotyrosine (a marker of peroxynitrite) were significantly decreased in islets of GED-treated mice (1+/-1%) compared with vehicle-treated mice (30+/-9%). In addition, GED significantly inhibited nitric oxide and nitrotyrosine formation and decreased destruction of beta-cells in NOD mouse islets incubated in vitro with the combination of proinflammatory cytokines interleukin 1-beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). These findings indicate that both superoxide and nitric oxide radicals contribute to islet beta-cell destruction in autoimmune diabetes via peroxynitrite formation in the beta-cells.     

葛根素对糖尿病大鼠白内障过程中晶状体诱导型一氧化氮合酶的影响

目的: 观察大鼠糖尿病性白内障(DC)晶状体诱导型一氧化氮合酶(iNOS)基因表达变化及葛根素对DC的治疗作用。方法: 经腹腔注射链脲佐菌素(STZ)复制大鼠DC模型。实验分为STZ组(STZ,45 mg/kg bw,ip)、葛根素组(STZ+葛根素,140 mg/kg bw ip)和对照组(等量生理盐水,ip),分别于实验第20、40、60 d摘取大鼠晶状体,用逆转录聚合酶链反应(RT-PCR)、蛋白印迹(Western blot)和生化方法检测晶状体iNOS mRNA和蛋白表达及一氧化氮(NO)含量、一氧化氮合酶(NOS)活性变化,观察晶状体损伤的宏观和微观病理变化。 结果:对照组大鼠晶状体透明,iNOS mRNA未见明显表达,蛋白呈微弱表达,NOS活性较低,NO含量较少。在DC形成过程中,晶状体出现混浊、晶状体上皮细胞(LEC)有明显病变,并随病程延长而加重;晶状体iNOS mRNA和蛋白表达明显上调,NOS活性增强、NO生成增加,并呈现时间依赖性。用药40-60 d时葛根素组前述晶状体病理变化轻于STZ组,iNOS mRNA和蛋白表达明显低于STZ组,NOS活性及NO 生成低于STZ组。结论: 在大鼠DC形成过程中晶状体iNOS 基因表达上调、NO含量增加;葛根素可减轻晶状体损伤,机制可能与抑制iNOS 基因表达、减少NO生成有关。     

Association analysis of nitric oxide synthases: NOS1, NOS2A and NOS3 genes,with multiple sclerosis

Abstract

Background: Multiple sclerosis (MS) is a chronic inflammatory autoimmune disorder of the central nervous system.

Aim: To explore the genetic basis of three nitric oxide synthase (NOS) genes: NOS1, NOS2A and NOS3, with susceptibility to MS.

Subjects and methods: A total of 122 MS patients and 118 healthy controls screened for NOS1 (rs2682826, rs41279104), NOS2A (CCTTT)n/(TAAA)n and NOS3 (rs1800783, rs1800779, rs2070744, 27bpVNTR) markers, using TaqMan®SNP Genotyping Assays and fragment analysis were enrolled in this study. QRT-PCR and ELISA were used to analyse the expression of NOS3 mRNA and Nitric Oxide (NO) levels.

Results: Two NOS3 markers were associated with susceptibility to MS and early disease development. The NOS3 rs1800779 G-allele (p?=?0.04) and GG-genotype (p?=?0.02) showed association with susceptibility to MS. Short NOS2 (CCTTT)n (p?=?0.03) and short/long repeat (p?=?0.04) genotypes also showed associations with MS. These associations were intensified by sub-division of patients into Kuwaiti Arabs and Persians (p?<?0.05). The NOS3-27?bp-VNTR a-allele was associated with early MS disease onset ≤26 years (p?=?0.04). The NOS3-27?bp-VNTR a/b-genotype resulted in 23% lower NO production and the NOS3-rs1800779 AA-genotype resulted in lower NOS3 expression. Haplotypes obtained from NOS2A and NOS3 showed increased susceptibility to MS. NOS1 showed no significant association with MS.

Conclusion: This study provides evidence for the association between selected NOS2 and NOS3 markers and MS susceptibility.     

Inducible nitric oxide synthase expression in human urinary bladder cancer

Nitric oxide (NO) is generated by a family of enzymes, nitric oxide synthases (NOS), in a wide range of mammalian cells. NO produced by the inducible NOS isoform (iNOS) has been suggested to play an important role in tumor biology with both tumor promoter and anti-tumor activity. Here, the cellular localization of iNOS in tissue of 100 cases of urinary bladder cancer was assessed immunohistologically using a commercially available antiserum. Positive iNOS immunostaining was detected in all samples of tumor tissue, whereas nonmalignant tissue adjacent to malignant areas did not show any iNOS positivity. The tumor tissue revealed a highly inhomogeneous staining pattern. In addition to uniformly stained tumor specimens, we also found markedly iNOS-positive tumor islets in the midst of unstained tumor tissue and scattered individual tumor cells expressing marked staining. In some cases, the tumor tissue showed no or only weak staining intensity. In some instances, the superficial epithelial layer of papillary carcinomas was extremely immunoreactive, in other cases it was not. Thus we were unable to show a clear correlation to tumor grade or stage. Further studies with a diversity of tumor markers including molecular genetics techniques will be necessary to elucidate how and to what extent NO and bladder cancer of different grades and stages are functionally interrelated.     

Inhibitory effect of Artemisia capillaris extract on cytokine-induced nitric oxide formation and cytotoxicity of RINm5F cells

Cytokines produced by immune cells infiltrating pancreatic islets are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. Cytokines stimulate an inducible form of nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, leading to insulin insufficiency. In the present study, the effects of Artemisia capillaris extract (ACE) on cytokine-induced beta-cell damage were examined. Treatment of RINm5F (RIN) rat insulinoma cells with interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) induced cell damage. ACE completely protected IL-1beta and IFN-gamma-mediated cytotoxicity in a concentration-dependent manner. Incubation with ACE resulted in a significant reduction in IL-1beta and IFN-gamma-induced NO production, a finding that correlated well with reduced levels of the iNOS mRNA and protein. The molecular mechanism by which ACE inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. The IL-1beta and IFN-gamma-stimulated RIN cells showed increases in NF-kappaB binding activity and p65 subunit levels in the nucleus, and IkappaBalpha degradation in cytosol compared to unstimulated cells. Furthermore, ACE restored the cytokine-induced inhibition of insulin release from isolated islets. These results suggest that ACE protects beta-cells by suppressing NF-kappaB activation.     

Association of a microsatellite in FASL to type II diabetes and of the FAS-670G>A genotype to insulin resistance

Type II diabetes is caused by a failure of the pancreatic beta-cells to compensate for insulin resistance leading to hyperglycaemia. There is evidence for an essential role of an increased beta-cell apoptosis in type II diabetes. High glucose concentrations induce IL-1beta production in human beta-cells, Fas expression and concomitant apoptosis owing to a constitutive expression of FasL. FASL and FAS map to loci linked to type II diabetes and estimates of insulin resistance, respectively. We have tested two functional promoter polymorphisms, FAS-670 G>A and FASL-844C>T as well as a microsatellite in the 3' UTR of FASL for association to type II diabetes in 549 type II diabetic patients and 525 normal-glucose-tolerant (NGT) control subjects. Furthermore, we have tested these polymorphisms for association to estimates of beta-cell function and insulin resistance in NGT subjects. We found significant association to type II diabetes for the allele distribution of the FASL microsatellite (P-value 0.02, Bonferroni corrected). The FAS-670G>A was associated with homeostasis model assessment insulin resistance index and body mass index (P-values 0.02 and 0.02). We conclude that polymorphisms of FASL and FAS associate with type II diabetes and estimates of insulin resistance in Danish white subjects.     

Imidazoline-induced amplification of glucose- and carbachol-stimulated insulin release includes a marked suppression of islet nitric oxide generation in the mouse

Aim: The role of islet nitric oxide (NO) production in insulin‐releasing mechanisms is unclear. We examined whether the beneficial effects of the imidazoline derivative RX 871024 (RX) on β‐cell function might be related to perturbations of islet NO production. Methods: Experiments were performed with isolated islets or intact mice challenged with glucose or carbachol with or without RX treatment. Insulin was determined with radioimmunoassay, NO generation with high‐performance liquid chromatography and expression of inducible NO synthase (iNOS) with confocal microscopy. Results: RX treatment, in doses lacking effects on basal insulin, greatly amplified insulin release stimulated by the NO‐generating secretagogues glucose and carbachol both in vitro and in vivo. RX also improved the glucose tolerance curve. Islets incubated at high glucose levels (20 mmol L^?1) displayed increased NO production derived from both neuronal constitutive NO synthase (ncNOS) and iNOS. RX abrogated this glucose‐induced NO production concomitant with amplification of insulin release. Confocal microscopy revealed abundant iNOS expression in β cells after incubation of islets at high but not low glucose levels. This was abolished after RX treatment. Similarly, islets cultured for 24 h at high glucose levels showed intense iNOS expression in β cells. This was abrogated with RX and followed by an amplified glucose‐induced insulin release. Conclusion: RX effectively counteracts the negative impact of β‐cell NO generation on insulin release stimulated by glucose and carbachol suggesting imidazoline compounds by virtue of NOS inhibitory properties being of potential therapeutic value for treatment of β‐cell dysfunction in hyperglycaemia and type 2 diabetes.