ET-1 and NOS III gene expression regulation by plaque-free and plaque-prone hemodynamic conditions

Résumé: Les environnements hémodynamiques, favorisant ou protégeant contre la formation de la plaque, induisent tout deux une augmentation de la production d'anion superoxide dans les cellules endothéliales (ECs). Par ailleurs, une régulation différente de l'expression des gènes a été décrite dans les cellules exposées à ces différentes conditions. Dans le but d'investiguer le rôle de l'augmentation du stress oxydatif dans l'expression des gènes régulée par le flux, nous avons d'abord exposé les EC à un flux unidirectionnel, non pulsé. Dans ces conditions, l'état oxydatif des cellules endothéliales est augmenté de façon transitoire. L'expression du gène de l'endothéline 1 (ET-1) est aussi induite de façon transitoire par un tel flux, alors que l'expression du gène de la nitiric oxyde synthase endothéliale (NOS III) est stimulé de façon durable. Au contraire, un flux unidirectionnel pulsé, qui induit une augmentation durable de la production d'anion superoxide, augmente aussi de façon durable l'expression des gènes de ET-1 comme de NOS III. Un flux oscillatoire (favorisant la plaque), qui lui aussi ,a des effets à long terme sur la production d'anion superoxide, a uniquement augmenté l'expression de ET-1. De plus, l'utilisation d'un antioxydant, a seulement partiellement inhibé la stimulation de l'expression du gène NOS III par le flux unidirectionnel pulsé, alors qu'il a complètement abrogé la stimulation de l'expression du gène ET-1 par le flux unidirectionnel pulsé et oscillatoire. Ceci suggère que les forces mécaniques régulent l'expression des gènes dans les EC par un double mécanisme dépendant et indépendant du stress oxidatif des cellules. Par ailleurs, ces résultats supportent ultérieurement l'hypothèse que la balance entre la réponse oxidative et anti-oxidante dans les cellules endothéliales exposées à un environnement hémodynamique est une des clés de la prédisposition à un dysfonctionnement endothélial observé dans des régions exposées à des flux perturbés. Abstract: Both plaque-free and plaque-prone hemodynamic environments induce an increase in the oxidative state of endothelial cells (ECs), whereas differential gene expression regulation was described in cells exposed to these conditions. In order to investigate the role of the increased oxidative state in flow-regulation of gene expression, we first exposed EC to non-pulsed unidirectional shear stress. These conditions only slightly increases ECs oxidative state and endothelin-1 (ET-1) mRNA expression, whereas endothelial nitric oxide synthase (NOS III) mRNA level were significantly up-regulated. On the contrary, both ET-1 and NOS III gene expression were significantly induced in EC exposed to pulsed-unidirectional flow (plaque-free). Only ET-1 gene expression was up-regulated by oscillatory flow (plaque-prone). Moreover, use of an antioxidant only partially inhibited NOS III gene up-regulation by unidirectional flow, whereas it completely abrogated ET-1 gene up-regulation by unidirectional and oscillatory flows. Thus suggesting that mechanical forces regulate gene expression in ECs both via oxidative stress-dependent and -independent mechanisms.

Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells.

To investigate whether caveolin-1 (cav-1) may modulate inducible nitric oxide synthase (iNOS) function in intact cells, the human intestinal carcinoma cell lines HT29 and DLD1 that have low endogenous cav-1 levels were transfected with cav-1 cDNA. In nontransfected cells, iNOS mRNA and protein levels were increased by the addition of a mix of cytokines. Ectopic expression of cav-1 in both cell lines correlated with significantly decreased iNOS activity and protein levels. This effect was linked to a posttranscriptional mechanism involving enhanced iNOS protein degradation by the proteasome pathway, because (i) induction of iNOS mRNA by cytokines was not affected and (ii) iNOS protein levels increased in the presence of the proteasome inhibitors N-acetyl-Leu-Leu-Norleucinal and lactacystin. In addition, a small amount of iNOS was found to cofractionate with cav-1 in Triton X-100-insoluble membrane fractions where also iNOS degradation was apparent. As has been described for endothelial and neuronal NOS isoenzymes, direct binding between cav-1 and human iNOS was detected in vitro. Taken together, these results suggest that cav-1 promotes iNOS presence in detergent-insoluble membrane fractions and degradation there via the proteasome pathway.

Regulation of lipocalin prostaglandin-D synthase expression by interleukin-1β in human chondrocytes

L'arthrose est une maladie multifactorielle complexe. Parmi les facteurs impliqués dans sa pathogénie, les certains prostaglandines exercent un rôle inflammatoire et d’autres un rôle protecteur. La prostaglandine D2 (PGD2) est bien connue comme une PG anti-inflammatoire, qui est régulée par l’enzyme «Lipocalin prostaglandine D-synthase». Avec l’inflammation de l'arthrose, les chondrocytes essaient de protéger le cartilage en activant certaines voies de récupération dont l'induction du gène L-PGDS. Dans cette étude, nous étudions la voie de signalisation impliquée dans la régulation de l'expression du (L-PGDS) sur les chondrocytes traités avec différents médiateurs inflammatoires. Le but de projet: Nous souhaitons étudier la régulation de la L-PGDS dans le but de concevoir des approches thérapeutiques qui peuvent activer la voie intrinsèque anti-inflammatoire. Méthode et conclusions: In vivo, l'arthrose a été suivie en fonction de l’âge chez la souris ou chirurgicalement suivant une intervention au niveau des genoux de souris. Nous avons confirmé les niveaux d’expression de L-PGDS histologiquement et par immunohistochimie. In vitro, dans les chondrocytes humains qui ont été traités avec différents médiateurs de l'inflammation, nous avons observé une augmentation de l’expression de la L-PGDS dose et temps dépendante. Nous avons montré, in vivo et in vitro que l’inflammation induit une sécrétion chondrocytaire de la L-PGDS dans le milieu extracellulaire. Enfin, nous avons observé la production de différentes isoformes de la L-PGDS en réponse à l'inflammation.

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

Altered reactivity of gastric fundus smooth muscle in the mouse with targeted disruption of the kinin B(1) receptor gene

Relaxing action of sodium nitroprusside (SNP) was significantly reduced in the stomach fundus of mice lacking the kinin B(1) receptor (B(1)(-/-)). Increased basal cGMP accumulation was correlated with attenuated SNP induced dose-dependent relaxation in B(1)(-/-) when compared with wild type (WT) control mice. These responses to SNP were completely blocked by the guanylate cyclase inhibitor ODQ(10 mu M). It was also found that Ca(2+)-dependent, constitutive nitric oxide synthase (cNOS) activity was unchanged but the Ca(2+)-independent inducible NOS (iNOS) activity was greater in B(1)(-/-) mice than in WT animals. Zaprinast (100 mu M), a specific phosphodiesterase inhibitor, increased the nitrergic relaxations and the accumulation of the basal as well as the SNP-stimulated cGMP in WT but not in B(1)(-/-) stomach fundus. From these findings it is concluded that the inhibited phosphodiesterase activity and high level of cGMP reduced the resting muscle tone, impairing the relaxant responses of the stomach in B(1)(-/-) mice. In addition, it can be suggested that functional B(2) receptor might be involved in the NO compensatory mechanism associated with the deficiency of kinin B(1) receptor in the gastric tissue of the transgenic mice. (C) 2009 Elsevier Inc. All rights reserved.

Role of the different isoforms of cyclooxygenase and nitric oxide synthase during gastric ulcer healing in cyclooxygenase-1 and -2 knockout mice

Traditional NSAIDs, selective cyclooxygenase (COX)-2 inhibitors, and inhibitors of nitric oxide synthase (NOS) impair the healing of preexisting gastric ulcers. However, the role of COX-1 (with or without impairment of COX-2) and the interaction between COX and NOS isoforms during healing are less clear. Thus we investigated healing and regulation of COX and NOS isoforms during ulcer healing in COX-1 and COX-2 deficiency and inhibition mouse models. In this study, female wild-type COX-1(-/-) and COX-2(-/-) mice with gastric ulcers induced by cryoprobe were treated intragastrically with vehicle, selective COX-1 (SC-560), COX-2 (celecoxib, rofecoxib, and valdedoxib), and unselective COX (piroxicam) inhibitors. Ulcer healing parameters, mRNA expression, and activity of COX and NOS were quantified. Gene disruption or inhibition of COX-1 did not impair ulcer healing. In contrast, COX-2 gene disruption and COX-2 inhibitors moderately impaired wound healing. More severe healing impairment was found in dual (SC-560 + rofecoxib) and unselective (piroxicam) COX inhibition and combined COX impairment (in COX-1(-/-) mice with COX-2 inhibition and COX-2(-/-) mice with COX-1 inhibition). In the ulcerated repair tissue, COX-2 mRNA in COX-1(-/-) mice, COX-1 mRNA in COX-2(-/-) mice, and, remarkably, NOS-2 and NOS-3 mRNA in COX-impaired mice were more upregulated than in wild-type mice. This study demonstrates that COX-2 is a key mediator in gastric wound healing. In contrast, COX-1 has no significant role in healing when COX-2 is unimpaired but becomes important when COX-2 is impaired. As counterregulatory mechanisms, mRNA of COX and NOS isoforms were increased during healing in COX-impaired mice.

Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-d-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol

In Escherichia coli, 1-deoxy-d-xylulose (or its 5-phosphate, DXP) is the biosynthetic precursor to isopentenyl diphosphate [Broers, S. T. J. (1994) Dissertation (Eidgenössische Technische Hochschule, Zürich)], thiamin, and pyridoxol [Himmeldirk, K., Kennedy, I. A., Hill, R. E., Sayer, B. G. & Spenser, I. D. (1996) Chem. Commun. 1187–1188]. Here we show that an open reading frame at 9 min on the chromosomal map of E. coli encodes an enzyme (deoxyxylulose-5-phosphate synthase, DXP synthase) that catalyzes a thiamin diphosphate-dependent acyloin condensation reaction between C atoms 2 and 3 of pyruvate and glyceraldehyde 3-phosphate to yield DXP. We have cloned and overexpressed the gene (dxs), and the enzyme was purified 17-fold to a specific activity of 0.85 unit/mg of protein. The reaction catalyzed by DXP synthase yielded exclusively DXP, which was characterized by 1H and 31P NMR spectroscopy. Although DXP synthase of E. coli shows sequence similarity to both transketolases and the E1 subunit of pyruvate dehydrogenase, it is a member of a distinct protein family, and putative DXP synthase sequences appear to be widespread in bacteria and plant chloroplasts.

Regulation of Oleoresinosis in Grand Fir (Abies grandis)1 : Differential Transcriptional Control of Monoterpene, Sesquiterpene, and Diterpene Synthase Genes in Response to Wounding

Grand fir (Abies grandis Lindl.) has been developed as a model system for the study of wound-induced oleoresinosis in conifers as a response to insect attack. Oleoresin is a roughly equal mixture of turpentine (85% monoterpenes [C10] and 15% sesquiterpenes [C15]) and rosin (diterpene [C20] resin acids) that acts to seal wounds and is toxic to both invading insects and their pathogenic fungal symbionts. The dynamic regulation of wound-induced oleoresin formation was studied over 29 d at the enzyme level by in vitro assay of the three classes of synthases directly responsible for the formation of monoterpenes, sesquiterpenes, and diterpenes from the corresponding C10, C15, and C20 prenyl diphosphate precursors, and at the gene level by RNA-blot hybridization using terpene synthase class-directed DNA probes. In overall appearance, the shapes of the time-course curves for all classes of synthase activities are similar, suggesting coordinate formation of all of the terpenoid types. However, closer inspection indicates that the monoterpene synthases arise earlier, as shown by an abbreviated time course over 6 to 48 h. RNA-blot analyses indicated that the genes for all three classes of enzymes are transcriptionally activated in response to wounding, with the monoterpene synthases up-regulated first (transcripts detectable 2 h after wounding), in agreement with the results of cell-free assays of monoterpene synthase activity, followed by the coordinately regulated sesquiterpene synthases and diterpene synthases (transcription beginning on d 3–4). The differential timing in the production of oleoresin components of this defense response is consistent with the immediate formation of monoterpenes to act as insect toxins and their later generation at solvent levels for the mobilization of resin acids responsible for wound sealing.

Molecular mechanisms of dexamethasone inhibition of nitric oxide synthase expression in interleukin 1 beta-stimulated mesangial cells: evidence for the involvement of transcriptional and posttranscriptional regulation.

Inducible nitric oxide synthase (iNOS; EC 1.14.13.39) is expressed in rat glomerular mesangial cells upon exposure to the inflammatory cytokine interleukin 1 beta (IL-1 beta). We have reported that nanomolar concentrations of dexamethasone suppress IL-1 beta-induced iNOS protein expression and production of nitrite, the stable end product of NO formation, without affecting IL-1 beta-triggered increase in iNOS mRNA levels. We now have studied the mechanisms by which dexamethasone suppresses IL-1 beta-stimulated iNOS expression in mesangial cells. Surprisingly, nuclear run-on transcription experiments demonstrate that dexamethasone markedly attenuates IL-1 beta-induced iNOS gene transcription. However, this is counteracted by a prolongation of the half-life of iNOS mRNA from 1 h to 2.5 h by dexamethasone. Moreover, dexamethasone drastically reduces the amount of iNOS protein by reduction of iNOS mRNA translation and increased degradation of iNOS protein. These results indicate that glucocorticoids act at multiple levels to regulate iNOS expression, thus providing important insights into the treatment of inflammatory diseases.

Regulation of lipocalin prostaglandin-D synthase expression by interleukin-1β in human chondrocytes

L'arthrose est une maladie multifactorielle complexe. Parmi les facteurs impliqués dans sa pathogénie, les certains prostaglandines exercent un rôle inflammatoire et d’autres un rôle protecteur. La prostaglandine D2 (PGD2) est bien connue comme une PG anti-inflammatoire, qui est régulée par l’enzyme «Lipocalin prostaglandine D-synthase». Avec l’inflammation de l'arthrose, les chondrocytes essaient de protéger le cartilage en activant certaines voies de récupération dont l'induction du gène L-PGDS. Dans cette étude, nous étudions la voie de signalisation impliquée dans la régulation de l'expression du (L-PGDS) sur les chondrocytes traités avec différents médiateurs inflammatoires. Le but de projet: Nous souhaitons étudier la régulation de la L-PGDS dans le but de concevoir des approches thérapeutiques qui peuvent activer la voie intrinsèque anti-inflammatoire. Méthode et conclusions: In vivo, l'arthrose a été suivie en fonction de l’âge chez la souris ou chirurgicalement suivant une intervention au niveau des genoux de souris. Nous avons confirmé les niveaux d’expression de L-PGDS histologiquement et par immunohistochimie. In vitro, dans les chondrocytes humains qui ont été traités avec différents médiateurs de l'inflammation, nous avons observé une augmentation de l’expression de la L-PGDS dose et temps dépendante. Nous avons montré, in vivo et in vitro que l’inflammation induit une sécrétion chondrocytaire de la L-PGDS dans le milieu extracellulaire. Enfin, nous avons observé la production de différentes isoformes de la L-PGDS en réponse à l'inflammation.

Association Of Single Nucleotide Polymorphisms In The Gene Encoding Glut1 And Diabetic Nephropathy In Brazilian Patients With Type 1 Diabetes Mellitus.

Mesangial cells subject to high extracellular glucose concentrations, as occur in hyperglycaemic states, are unable to down regulate glucose influx, resulting in intracellular activation of deleterious biochemical pathways. A high expression of GLUT1 participates in the development of diabetic glomerulopathy. Variants in the gene encoding GLUT1 (SLC2A1) have been associated to this diabetic complication. The aim of this study was to test whether polymorphisms in SLC2A1 confer susceptibility to diabetic nephropathy (DN) in Brazilian type 1 diabetes patients. Four polymorphisms (rs3820589, rs1385129, rs841847 and rs841848) were genotyped in a Brazilian cohort comprised of 452 patients. A prospective analysis was performed in 155 patients. Mean duration of follow-up was 5.6±2.4years and the incidence of renal events was 18.0%. The rs3820589 presented an inverse association with the prevalence of incipient DN (OR: 0.36, 95% CI: 0.16 - 0.80, p=0.01) and with progression to renal events (HR: 0.20; 95% CI: 0.03 - 0.70; p=0.009). AGGT and AGAC haplotypes were associated with the prevalence of incipient DN and the AGAC haplotype was also associated with the prevalence of established/advanced DN. In conclusion, rs3820589 in the SLC2A1 gene modulates the risk to DN in Brazilian patients with inadequate type 1 diabetes control.

Genetic Diversity on the Integrase Region of the pol Gene among HIV Type 1-Infected Patients Naive for Integrase Inhibitors in Sao Paulo City, Brazil

The presence of mutations associated with integrase inhibitor (INI) resistance among INI-naive patients may play an important clinical role in the use of those drugs Samples from 76 HIV-1-infected subjects naive to INIs were submitted to direct sequencing. No differences were found between naive (25%) subjects and subjects on HAART (75%). No primary mutation associated with raltegravir or elvitegravir resistance was found. However, 78% of sequences showed at least one accessory mutation associated with resistance. The analysis of the 76 IN sequences showed a high polymorphic level on this region among Brazilian HIV-1-infected subjects, including a high prevalence of aa substitutions related to INI resistance. The impact of these findings remains unclear and further studies are necessary to address these questions.

Dissemination of bla(IMP-1)-carrying integron In86 among Klebsiella pneumoniae isolates harboring a new trimethoprim resistance gene dfr23

The genetic context of the bla(IMP-1) gene was evaluated in 9 Klebsiella pneumoniae isolates recovered from 2 hospitals in Sao Paulo, Brazil. All isolates harbored a copy of In86 carrying bla(Imp-1), aac(6`)-31, and aadAl. Eight strains from the same hospital also carried another class I integron harboring a new trimethoprim resistance gene (dfr23) that was chromosomally embedded. In86 was likely to be in a 30-kb nontransferable plasmid and was flanked upstream by a sequence identical to one identified in an IMP-1-producing Pseudomonas putida isolate. The bla(IMP-1)-carrying integron In86 was recently reported from nonfermentative bacilli isolated in Sao Paulo. These isolates appear to be the Source of this integron now acquired by K. pneumoniae strains from different hospitals in the same city. Metallo-beta-lactamase production is still rare among Enterobacteriaceae isolates in Brazil, but the acquisition of genetic structures carrying these mobile resistance determinants is worrisome and could lead to an increase in the prevalence of these phenotypes of resistance. (C) 2008 Elsevier Inc. All rights reserved.

Functional polymorphism of the human arylamine N-acetyltransferase type 1 gene caused by (CT)-T-190 and G(560)A mutations

Human N-acetyltransferase type 1 (NAT1) catalyses the N- or O-acetylation of various arylamine and heterocyclic amine substrates and is able to bioactivate several known carcinogens. Despite wide inter-individual variability in activity, historically, NAT1 was considered to be monomorphic in nature. However, recent reports of allelic variation at the NAT1 locus suggest that it may be a polymorphically expressed enzyme. In the present study, peripheral blood mononuclear cell NAT1 activity in 85 individuals was found to be bimodally distributed with approximately 8% of the population being slow acetylators. Subsequent sequencing of the individuals having slow acetylator status showed all to have either a (CT)-T-190 or G(560)A base substitution located in the protein encoding region of the NAT1 gene. The (CT)-T-190 base substitution changed a highly conserved Arg(64), which others have shown to be essential for fully functional NAT1 protein. The (CT)-T-190 mutation has not been reported previously and we have named it NAT1*17. The G(560)A mutation is associated with the base substitutions previously observed in the NAT1*10 allele and this variant (NAT1*14) encodes for a protein with reduced acetylation capacity. A novel method using linear PCR and dideoxy terminators was developed for the detection of NAT1*14 and NAT1*17. Neither of these variants was found in the rapid acetylator population. We conclude that both the (CT)-T-190 (NAT1*17) and G(560)A (NAT1*14) NAT1 structural variants are involved in a distinct NAT1 polymorphism. Because NAT1 can bioactivate several carcinogens, this polymorphism may have implications for cancer risk in individual subjects. (C) 1998 Chapman & Hall Ltd.

Febrile seizures and generalized epilepsy associated with a mutation in the Na+-channel beta 1 subunit gene SCN1B

Febrile seizures affect approximately 3% of all children under six years of age and are by far the most common seizure disorder(1). A small proportion of children with febrile seizures later develop ongoing epilepsy with afebrile seizures(2). Segregation analysis suggests the majority of cases have complex inheritance(3) but rare families show apparent autosomal dominant: inheritance. Two putative loci have been mapped (FEB1 and FEB2), but specific genes have not yet been identified(4,5). We recently described a clinical subset, termed generalized epilepsy with febrile seizures plus (GEFS(+)), in which many family members have seizures with fever that may persist beyond six years of age or be associated with afebrile generalized seizures(6). We now report linkage, in another large GEFS(+) family, to chromosome region 19q13.1 and identification of a mutation in the voltage-gated sodium (Na+)-channel beta 1 subunit gene (SCN1B). The mutation changes a conserved cysteine residue disrupting a putative disulfide bridge which normally maintains an extracellular immunoglobulin-like fold. Go-expression of the mutant pr subunit with a brain Na+-channel alpha subunit in Xenopus laevis oocytes demonstrates that the mutation interferes with the ability of the subunit to modulate channel-gating kinetics consistent with a loss-of-function allele. This observation develops the theme that idiopathic epilepsies are a family of channelopathies and raises the possibility of involvement of other Na+-channel subunit genes in febrile seizures and generalized epilepsies with complex inheritance patterns.