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Oxide Synthase (oxide + synthase)
Kinds of Oxide Synthase Terms modified by Oxide Synthase Selected AbstractsRole of Inducible Nitric Oxide Synthase in Skeletal Adaptation to Acute Increases in Mechanical Loading,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2002Makoto Watanuki M.D. Abstract To clarify the role of nitric oxide (NO) in regulation of bone metabolism in response to skeletal loading, we examined inducible NO synthase (iNOS) gene knockout mice in the tail-suspension model. Histomorphometric analyses of proximal tibias revealed that 7 days of tail suspension decreased the bone volume (BV/TV) and bone formation rate (BFR/BS) and increased the osteoclast surface (Oc.S/BS) in mice with all iNOS genotypes. Both iNOS+/+ and iNOS+/, mice responded to subsequent 14-day reloading, with increases in BV/TV and BFR/BS and a decrease in Oc.S/BS, whereas these responses were abolished in iNOS,/, mice. The osteoblasts flattened after tail suspension appeared cuboidal during subsequent reloading. Immunoreactivity for iNOS was detected in these osteoblasts and osteocytes by immunohistochemistry. These defective responses after reloading were rescued in iNOS,/, mice by treatment with an NO donor nitroglycerine (NG). Conversely, the responses in iNOS+/+ mice were inhibited by treatment with an NOS inhibitor aminoguanidine (AG). In bone marrow cell cultures, mineralized nodules derived from iNOS,/, mice after reloading were significantly reduced. Taken together, our results suggest that NO generated by iNOS in osteoblasts plays a critical role in adjusting bone turnover and increasing osteogenic activity in response to the acute increase in mechanical loading after tail suspension. [source] Contribution of Nitric Oxide Synthase to Improved Early Graft Patency in Human Saphenous Vein Graft Harvested by a Novel ,No-Touch' TechniqueJOURNAL OF CARDIAC SURGERY, Issue 6 2002JCS Tsui Aim: Saphenous vein (SV) is the most commonly used conduit in bypass procedures but has a one-year occlusion rate of 15-30%. A new ,no-touch' technique where the SV is harvested with a cushion of surrounding tissue with no distension has led to improved early patency rates of 5% at 18-months. Nitric oxide (NO), synthesised by nitric oxide synthase (NOS) has properties beneficial to graft patency. Our aim was to study the distribution of NOS in SV harvested by this technique and the effect of distension and removal of perivascular tissue on NOS content of SV. Methods: Following ethical committee approval and patients' informed consent, SVs were harvested from ten patients undergoing coronary artery bypass grafting. A segment of vein was harvested by the conventional technique (surrounding tissue stripped and vein distended with saline); another part was stripped but not distended (,control') and the remaining parts harvested by the ,no-touch' technique. Samples of each segment were taken and transverse sections prepared for NOS identification using 3[H]L-NG nitroarginine (NO Arg) autoradiography and NADPH-diaphorase histochemistry. NOS isoforms were studied using standard immunohistochemistry. Endothelial cells and nerves were also identified using immunohistochemistry with CD31 and NF200 respecitvely, to confirm sources of NOS. Morphometric analysis of NADPH-diaphorase staining was carried out to study tissue NOS content. Results: NO Arg binding representing NOS was preserved on the lumen of ,no-touch' vessels whilst that on conventional and control vessels was reduced. NOS was also localised to the medial smooth muscle cells of all vein segments and to the intact adventitia of ,no-touch' segments. This was confirmed by NADPH-diaphorase staining, which revealed a mean reduction of NOS by 19.5% (p < 0.05, ANOVA) in control segments due to stripping of surrounding tissue alone and a reduction of 35.5% (p < 0.01, AVNOVA) in conventional segments due to stripping and distension, compared to ,no-touch' segments. Adventitial NOS sources in ,no-touch' vessels corresponded to vasa vasorum and paravascular nerves. All three NOS isoforms contributed to the preserved NOS in ,no-touch' vessels. Conclusions: Apart from preserved lumenal NOS, NOS sources are also located in the media and adventitia of SV grafts. These are reduced by both adventitial damage and vein distension during conventional vein harvesting. The ,no-touch' technique avoids these procedures, preserving NOS sources. This may result in improved NO availability in SV harvested by this technique, contributing to the improved patency rates reported. [source] Lycopene Inhibits LPS-Induced Proinflammatory Mediator Inducible Nitric Oxide Synthase in Mouse Macrophage CellsJOURNAL OF FOOD SCIENCE, Issue 1 2007Mohamed M. Rafi ABSTRACT:, Lycopene is a fat-soluble red-orange carotenoid found primarily in tomatoes and tomato-derived products, including tomato sauce, tomato paste, and ketchup, and other dietary sources, including dried apricots, guava, watermelon, papaya, and pink grapefruit. In this study, we have demonstrated the molecular mechanism underlying the anti-inflammatory properties of lycopene using a mouse macrophage cell line (RAW 264.7). Treatment with lycopene (10 ,M) inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (40% compared with the control). Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that lycopene treatment decreased LPS-induced inducible nitric oxide synthase (iNOS) protein and mRNA expression in RAW 264.7 cells, respectively. These results suggest that lycopene has anti-inflammatory activity by inhibiting iNOS proteins and mRNA expressions in mouse macrophage cell lines. Furthermore, cyclooxygenase-2 (COX-2) protein and mRNA expression were not affected by treatment with lycopene. [source] Lung eNOS and iNOS are Reoxygenation Time-Dependent Upregulated After Acute HypoxiaTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 6 2010Alma Rus Abstract Nitric oxide plays a critical role in many physiological and physiopathological processes in the lung. Changes in the NO/NOS (Nitric Oxide/Nitric Oxide Synthase) system after hypoxia situations remain controversial in this organ, so that the aim of this work is to perform a complete study of this system in the hypoxic lung after different reoxygenation times ranging from 0 h to 5 days posthypoxia. This is a novel follow-up study carried out in Wistar rats submitted for 30 min to acute hypobaric hypoxia. We measured endothelial and inducible NOS (eNOS, iNOS) mRNA and protein expression, location, and in situ NOS activity as well as nitrated protein expression and location. In addition, NO levels were indirectly quantified (NOx) as well as the apoptosis level. Results showed an increase in eNOS mRNA, protein, activity as well as eNOS positive immunostaining at 0 h posthypoxia, coinciding with raised NOx levels. Contrary, iNOS, nitrated protein expression and apoptosis level augmented during the final reoxygenation times. The lung NO/NOS system provokes two responses to the hypoxia/reoxygenation processes: (i) eNOS is responsible of the immediate response, producing NO, which causes vasodilation and bronchodilation, and (ii) iNOS is related to the second late response, which seems to be involved in some of the deleterious consequences that hypoxia induces in the lung. Anat Rec, 2010. © 2010 Wiley-Liss, Inc. [source] Post-translational Regulation of Endothelial Nitric Oxide Synthase (eNOS) by Estrogens in the Rat VaginaTHE JOURNAL OF SEXUAL MEDICINE, Issue 5 2010Biljana Musicki PhD ABSTRACT Introduction., Estrogens control vaginal blood flow during female sexual arousal mostly through nitric oxide (NO). Although vascular effects of estrogens are attributed to an increase in endothelial NO production, the mechanisms of endothelial NO synthase (eNOS) regulation by estrogens in the vagina are largely unknown. Aims., Our hypothesis was that estrogens regulate eNOS post-translationally in the vagina, providing a mechanism to affect NO bioavailability without changes in eNOS protein expression. Methods., We measured eNOS phosphorylation and eNOS interaction with caveolin-1 and heat shock protein 90 (HSP90) in the distal and proximal vagina of female rats at diestrus, 7 days after ovariectomy and 2 days after replacement of ovariectomized rats with estradiol-17, (15 µg). Main Outcome Measures., Molecular mechanisms of eNOS regulation by estrogen in the rat vagina. Results., We localized phospho-eNOS (Ser-1177) immunohistochemically to the endothelium lining blood vessels and vaginal sinusoids. Estrogen withdrawal decreased phosphorylation of eNOS on its positive regulatory site (Ser-1177) and increased eNOS binding to its negative regulator caveolin-1 (without affecting eNOS/HSP90 interaction), and they were both normalized by estradiol replacement. Protein expressions of phosphorylated Akt (protein kinase B) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) were not affected by estrogen status, suggesting that the effect of estrogens on eNOS (Ser-1177) phosphorylation was not mediated by activated AKT or ERK1/2. eNOS phosphorylation on its negative regulatory site (Ser-114) was increased in the vagina by estrogen withdrawal and normalized by estradiol replacement, implying that the maintenance of low phosphorylation of eNOS on this site by estradiol may limit eNOS interaction with caveolin-1 and preserve the enzyme's activity. Total eNOS, inducible NOS, caveolin-1, and HSP90 protein expressions were not affected by ovariectomy or estradiol replacement in the distal or proximal vagina. Conclusions., These results define novel estrogen signaling mechanisms in the vagina which involve eNOS phosphorylation and eNOS,caveolin-1 interaction. Musicki B, Liu T, Strong TD, Lagoda GA, Bivalacqua TJ, and Burnett AL. Post-translational regulation of endothelial nitric oxide synthase (eNOS) by estrogens in the rat vagina. J Sex Med 2010;7:1768,1777. [source] Age-Related Changes in Phosphorylation of Endothelial Nitric Oxide Synthase in the Rat PenisTHE JOURNAL OF SEXUAL MEDICINE, Issue 3 2005Biljana Musicki PhD ABSTRACT Aim., Aging is associated with erectile dysfunction (ED) attributed to reduced nitric oxide synthase (NOS) activity and nitric oxide bioavailability. However, the mechanism for this effect has not been fully investigated. We evaluated (i) whether age-related ED involves dysregulation of endothelial NOS (eNOS) phosphorylation; and (ii) whether vascular endothelial growth factor (VEGF) exerts erectile effects and operates via eNOS phosphorylation in aged rats. Methods., Male Fischer 344 "young" (4-month-old) and "aged" (19-month-old) rats were used. Electrical stimulation of the cavernous nerve (CNS) was performed to generate penile erection. Erectile response in the presence of rhVEGF165 was evaluated by intracavernosal pressure monitoring 25 minutes after intracavernosal injection of VEGF. Penes were excised at baseline, with or without rhVEGF treatment, and after CNS for Western immunoblot of phospho-eNOS (Ser-1177 and Thr-495), phospho-Akt, and eNOS. Results., Erectile response was significantly reduced in aged rats compared with young rats. Phospho-eNOS (Ser-1177) and phospho-Akt were significantly reduced, while phospho-eNOS (Thr-495) was significantly increased, in the aged penis at baseline and after CNS. rhVEGF significantly improved erection and reversed downregulated Ser-1177, but not upregulated Thr-495 phosphorylation, on eNOS in aged penes. eNOS protein was significantly increased in aged penes. Conclusions., Age-related ED is associated with eNOS inactivation through a decrease in phosphorylation of its positive regulatory site (Ser-1177) and an increase in phosphorylation of its negative regulatory site (Thr-495) in the penis. Altered phosphorylation/constitutive activation of eNOS by fluid shear stress may be a major determinant of compromised vascular homeostasis of the aged penis. The finding that VEGF rapidly induces erection and partly corrects alterations in eNOS phosphorylation in the aged rat penis suggests impaired eNOS activation by deficient endogenous VEGF and supports the potential for growth factor therapy in the treatment of age-related ED. [source] Detection and Localization of Two Constitutive NOS Isoforms in Bull SpermatozoaANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 6 2003H. Meiser Summary Bull spermatozoa were examined for the presence and localization of constitutive Nitric Oxide Synthase (NOS), as nitric oxide (NO) is involved in calcium-dependent capacitation. In bull spermatozoa, NO generation is enhanced by l -arginine (3 ,m) and abolished by the NOS-inhibitor N -nitro- l -arginine methyl ester (l -NAME). In addition, presence of NOS in bull spermatozoa was verified by immunohistochemistry, revealing the existence of both neuronal NOS (nNOS) and endothelial NOS (eNOS) immunoreaction. These findings were confirmed by Western blot technique, showing immunoreactive bands at 161 kDa (nNOS) and 133 kDa (eNOS). Confocal laser microscopy localized nNOS related immunofluorescence at the acrosome cap of sperms and their flagellum-mainpart. This technique also identified eNOS staining spread over the spermatozoan head. In conclusion, immunohistochemistry, Western blot technique, and NO generation suggest the presence of n- and eNOS in bull spermatozoa. [source] Comparison of Localization of the Neurokinin 1 Receptor and Nitric Oxide Synthase with Calbindin D Labelling in the Rat Spinal CordANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 3 2000M. Nazli Summary A comparison of the localization of the neurokinin 1 (NK1) receptor and nitric oxide synthase with calbindin D labelling in the lumbar spinal cord was carried out in the rat using immunocytochemistry. Considerable regional variations were observed. Application of the antibody to calbindin D resulted in dense staining in laminae I and II and light staining in the other laminae. Occasional scattered cells were seen in the deep laminae and in the lamina X, the ventral horn and the lateral spinal nucleus. The results indicate that neurones expressing calbindin D, NK1 receptor and NOS are three separate populations in the dorsal horn of the lumbar spinal cord. [source] 4-Substituted Indazoles as New Inhibitors of Neuronal Nitric Oxide Synthase.CHEMINFORM, Issue 40 2007Michel Boulouard Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source] Substituted 6-Phenyl-pyridin-2-ylamines: Selective and Potent Inhibitors of Neuronal Nitric Oxide Synthase.CHEMINFORM, Issue 52 2004Deane M. Nason Abstract For Abstract see ChemInform Abstract in Full Text. [source] Novel Lipoic Acid Analogues that Inhibit Nitric Oxide Synthase.CHEMINFORM, Issue 35 2002Jeremiah J. Harnett No abstract is available for this article. [source] ChemInform Abstract: Inhibition of Neuronal Nitric Oxide Synthase by 7-Methoxyindazole and Related Substituted Indazoles.CHEMINFORM, Issue 32 2001Pascale Schumann Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] Combined effect of IL-17 and blockade of nitric oxide biosynthesis on haematopoiesis in miceACTA PHYSIOLOGICA, Issue 1 2010A. Krsti Abstract Aim:, The study was undertaken to extend our investigation concerning both the in vivo activity of interleukin (IL)-17 and the specific role of nitric oxide (NO) in IL-17-induced effects in the process of haematopoiesis. Methods:, CBA mice were simultaneously treated with IL-17 and/or nitric oxide synthase (NOS) inhibitor, l -NAME, for 5 days and changes within various haematopoietic cell lineages in bone marrow, spleen and peripheral blood were analysed. Results:, Findings showed that administration of both IL-17 and l -NAME stimulated increase in net haematopoiesis in normal mice. IL-17-enhanced myelopoiesis was characterized by stimulation of both femoral and splenic haematopoietic progenitor cells and morphologically recognizable granulocytes. Additionally, IL-17 induced alterations in the frequency of erythroid progenitor cells in both bone marrow and spleen, accompanied with their mobilization to the peripheral blood. As a consequence of these changes in the erythroid cell compartments, significant reticulocytosis was observed, which evidenced that in IL-17-treated mice effective erythropoiesis occurred. Exposure of mice to NOS inhibitor also increased the number of both granulocyte-macrophage and erythroid progenitors in bone marrow and spleens, and these alterations were followed by the mobilization of erythroid progenitors and elevated content of reticulocytes in peripheral blood. The specific role of NO in IL-17-induced haematopoiesis was demonstrated only in the IL-17-reducing effect on bone marrow late stage erythroid progenitors, CFU-E. Conclusion:, The results demonstrated the involvement of both IL-17 and NO in the regulation of haematopoietic cell activity in various haematopoietic compartments. They further suggest that IL-17 effects are differentially mediated depending on the haematopoietic microenvironments. [source] Systemic nitric oxide clamping in normal humans guided by total peripheral resistanceACTA PHYSIOLOGICA, Issue 2 2010J. A. Simonsen Abstract Aim:, We wanted to stabilize the availability of nitric oxide (NO) at levels compatible with normal systemic haemodynamics to provide a model for studies of complex regulations in the absence of changes in NO levels. Methods:, Normal volunteers (23,28 years) were infused i.v. with the nitric oxide synthase (NOS) inhibitor NG -nitro- l -arginine methyl ester (l -NAME) at 0.5 mg kg,1 h,1. One hour later, the NO donor sodium nitroprusside (SNP) was co-infused in doses eliminating the haemodynamic effects of l -NAME. Haemodynamic measurements included blood pressure (MABP) and cardiac output (CO) by impedance cardiography. Results:,l -NAME increased MABP and total peripheral resistance (TPR, 1.02 ± 0.05 to 1.36 ± 0.07 mmHg s mL,1, mean ± SEM, P < 0.001). With SNP, TPR fell to a stable value slightly below control (0.92 ± 0.05 mmHg s mL,1, P < 0.05). CO decreased with l -NAME (5.8 ± 0.3 to 4.7 ± 0.3 L min,1, P < 0.01) and returned to control when SNP was added (6.0 ± 0.3 L min,1). A decrease in plasma noradrenaline (42%, P < 0.01) during l -NAME administration was completely reversed by SNP. Plasma renin activity decreased during l -NAME administration and returned towards normal after addition of SNP. In contrast, plasma aldosterone was increased by l -NAME and remained elevated. Conclusions:, Concomitant NOS inhibition and NO donor administration can be adjusted to maintain TPR at control level for hours. This approach may be useful in protocols in which stabilization of the peripheral supply of NO is required. However, the dissociation between renin and aldosterone secretion needs further investigation. [source] The role of caveolin-1 in cardiovascular regulationACTA PHYSIOLOGICA, Issue 2 2009A. Rahman Abstract Caveolae are omega-shaped membrane invaginations present in essentially all cell types in the cardiovascular system, and numerous functions have been ascribed to these structures. Caveolae formation depends on caveolins, cholesterol and polymerase I and transcript release factor-Cavin (PTRF-Cavin). The current review summarizes and critically discusses the cardiovascular phenotypes reported in caveolin-1-deficient mice. Major changes in the structure and function of heart, lung and blood vessels have been documented, suggesting that caveolae play a critical role at the interface between blood and surrounding tissue. According to an emerging paradigm, many of these changes are secondary to uncoupling of endothelial nitric oxide synthase. Thus, nitric oxide synthase not only synthesizes more nitric oxide in the absence of caveolin-1, but also more superoxide with potential pathogenic consequences. It is further argued that the vasodilating drive from increased nitric oxide production in caveolin-1-deficient mice is balanced by changes in the vascular media that favour increased dynamic resistance regulation. Harnessing the therapeutic opportunities buried in caveolae, while challenging, could expand the arsenal of treatment options in cancer, lung disease and atherosclerosis. [source] EndothelinA (ETA) and ETB receptor-mediated regulation of nitric oxide synthase 1 (NOS1) and NOS3 isoforms in the renal inner medullaACTA PHYSIOLOGICA, Issue 4 2007J. C. Sullivan Abstract Aim:, Our laboratory and others have shown that endothelin (ET)-1 directly stimulates nitric oxide (NO) production in inner medullary collecting duct (IMCD) cells. The goal of this study was to determine which NO synthase (NOS) isoforms in IMCD are sensitive to ET-1, and the role of ETA and ETB receptor activation in vivo and in vitro. Methods:, NOS enzymatic activity and NOS isoform protein expression were examined in cultured IMCD-3 cells and isolated renal inner medulla. ETB receptor-deficient homozygous rats (sl/sl) have elevated levels of circulating ET-1 and lack a functional ETB signalling pathway in kidneys, and furthermore provides a unique model to study ETA receptor signalling in the renal inner medulla in vivo. Results:, Incubation of IMCD-3 cells with exogenous ET-1 (50 nm) resulted in ETA -dependent increased NOS1 protein expression in IMCD-3 cells with no effect on NOS2 or NOS3 expression. ETB receptor antagonism has no effect on NOS expression in IMCD-3 cells. Consistent with in vitro results, cytosolic NOS1 protein expression was significantly greater in the renal inner medulla of sl/sl rats compared with heterozygous (sl/+) controls, with no alteration in NOS3 expression. In contrast to protein expression data, NOS1- and NOS3-specific enzymatic activities decreased in the cytosolic fraction from the renal inner medulla of sl/sl compared with sl/+. Conclusion:, These results provide evidence that both ETA and ETB receptors regulate NOS isoform activity in the renal inner medulla and specifically support the hypothesis that ETA receptor activation increases NOS1 expression. [source] Role of neuronal nitric oxide synthase in response to hypertonic saline loading in ratsACTA PHYSIOLOGICA, Issue 4 2004R. Wangensteen Abstract Aims:, This study analyses the influence of neuronal nitric oxide synthase (nNOS) blockade with 7-nitroindazole (7NI) on the haemodynamic and renal response to a hypertonic saline load (HSL). We also evaluated the effects of non-specific NOS inhibitor N, -nitro- l -arginine methyl ester (l -NAME). Methods:, The following groups were used: controls, rats treated with 7NI at 0.5 or 5 mg kg,1, and rats treated with l -NAME at 0.5 or 5 mg kg,1. A further five groups received an isotonic saline load (ISL). Results:, Mean arterial pressure (MAP) was significantly increased in control rats after HSL. MAP was further increased in both 7NI-treated groups, and the l -NAME groups showed marked dose-related pressor responses. During ISL, MAP was only significantly increased in the group treated with 5 mg kg,1 of l -NAME. The pressure,natriuresis relationship during the experimental period after the HSL was reduced in the 7NI group treated with 5 mg kg,1 and severely attenuated in both l -NAME groups. The increase in plasma sodium was significantly greater after the HSL in both 7NI groups and both l -NAME groups compared with controls. Conclusions:, The present results suggest that nNOS and other NOS isozymes play a counter-regulatory role in the pressor response to HSL. Moreover, the blockade of nNOS with the higher dose of 7NI produces a blunted pressure,natriuresis relationship in response to the HSL. Finally, it is concluded that nNOS participates in the homeostatic cardiovascular and renal response to hypertonic saline loading by attenuating the blood pressure increase and hypernatremia, and facilitating natriuresis. [source] Expression of zebrafish nos2b surrounds oral cavityDEVELOPMENTAL DYNAMICS, Issue 6 2008Kar-Lai Poon Abstract Inducible nitric oxide synthase (NOS2) catalyzes the production of nitric oxide (NO), and is one of the factors establishing innate immunity. In zebrafish, Nos2 is represented by nos2a and nos2b. Here, we report the cloning and expression pattern of the zebrafish nos2b gene, which does not seem to participate in induced immune response. nos2b was mapped to zebrafish linkage group 15. The spatial and temporal expression pattern of nos2b in embryonic zebrafish was analyzed by whole-mount in situ hybridization. nos2b is expressed constitutively in two primordia located along the ventral midline. The first group of cells contributes to the neurohypophysis. Initially at the level of the ventral hindbrain, the second group of cells migrates closely with the thyroid primordium to its final position at the basihyal by 3 dpf. Thus, the analysis of expression pattern of nos2b reveals complex morphogenetic movements resulting in its expression surrounding the oral cavity. Developmental Dynamics 237:1662,1667, 2008. © 2008 Wiley-Liss, Inc. [source] Oxidative stress, nitric oxide, and the mechanisms of cell death in Lurcher Purkinje cellsDEVELOPMENTAL NEUROBIOLOGY, Issue 8 2007Rebecca McFarland Abstract Oxidative stress is postulated to play a role in cell death in many neurodegenerative diseases. As a model of neonatal neuronal cell death, we have examined the role of oxidative stress in Purkinje cell death in the heterozygous Lurcher mutant (+/Lc). Lurcher is a gain of function mutation in the ,2 glutamate receptor (GluR,2) that turns the receptor into a leaky membrane channel, resulting in chronic depolarization of +/Lc Purkinje cells starting around the first week of postnatal development. Virtually, all +/Lc Purkinje cells die by the end of the first postnatal month. To investigate the role of oxidative stress in +/Lc Purkinje cell death, we have examined nitric oxide synthase (NOS) activity and the expression of two markers for oxidative stress, nitrotyrosine and manganese super oxide dismutase (MnSOD), in wild type and +/Lc Purkinje cells at P10, P15, and P25. The results show that NOS activity and immunolabeling for nitrotyrosine and MnSOD are increased in +/Lc Purkinje cells. To determine whether peroxynitrite formation is a prerequisite for +/Lc Purkinje cell death, +/Lc mutants were crossed with an ,-nNOS knockout mutant (nNOS,,/,) to reduce the production of NO. Analysis of the double mutants showed that blocking ,-nNOS expression does not rescue +/Lc Purkinje cells. However, we present evidence for sustained NOS activity and nitrotyrosine formation in the GluR,2+/Lc:nNOS,/, double mutant Purkinje cells, which suggests that the failure to rescue GluR,2+/Lc:nNOS,/, Purkinje cells may be explained by the induction of alternative nNOS isoforms. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007. [source] Splice-isoform specific immunolocalization of neuronal nitric oxide synthase in mouse and rat brain reveals that the PDZ-complex-building nNOS, ,-finger is largely exposed to antibodiesDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2007Kristina Langnaese Abstract Knock out mice deficient for the splice-isoform ,, of neuronal nitric oxide synthase (nNOS,,) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, ,, and ,,, we generated isoform-specific anti-peptide antibodies against the nNOS,, specific ,,-finger motif involved in PDZ domain scaffolding and the nNOS,, specific N-terminus. The nNOS,, ,,-finger-specific antibody clearly recognized the 160-kDa band of recombinant nNOS,, on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild-type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan-nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ-domain proteins via the ,,-finger motif. This conclusion was confirmed by the inhibition of PSD-95/nNOS interaction by the nNOS,, ,,-finger antibody in pull-down assays. By contrast, nNOS,, ,,-finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOS,, knock out mice, nNOS,, was not detectable, whereas the pan-nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting ,,/,,-isoforms in these cells. The nNOS,, antibody clearly detected bacterial expressed nNOS,, fusion protein and nNOS,, in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild-type mice, indicating an upregulation of nNOS,, in nNOS,, deficient animals. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007 [source] Expression of a dominant negative form of Daxx in vivo rescues motoneurons from Fas (CD95)-induced cell deathDEVELOPMENTAL NEUROBIOLOGY, Issue 2 2005Cedric Raoul Abstract Fas-induced death of motoneurons in vitro has been shown to involve two signaling cascades that act together to execute the death program: a Fas-Daxx-ASK-1-p38 kinase-nNOS branch, which controls transcriptional and post-translational events, and the second classical Fas-FADD-caspase-8 branch. To analyze the role of Daxx in the developmental motoneuron cell death, we studied Fas-dependent cell death in motoneurons from transgenic mice that overexpress a dominant-negative form of Daxx. Motoneurons purified from these transgenic mice are resistant to Fas-induced death. This protective effect is specific to Fas because ultraviolet irradiation-triggered death is not affected by the transgene. The Daxx and the FADD pathways work in parallel because only Daxx, but not FADD, is involved in the transcriptional control of neuronal nitric oxide synthase and nitric oxide production. Nevertheless, we do not observe involvement of Daxx in developmental motoneuronal cell death, as the pattern of naturally occurring programmed cell death in vivo is normal in transgenic mice overexpressing the dominant negative form of Daxx, suggesting that Daxx-independent pathways are used during development. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005 [source] Endothelially Derived Nitric Oxide Affects the Severity of Early Acetaminophen-induced Hepatic Injury in MiceACADEMIC EMERGENCY MEDICINE, Issue 5 2006Steven D. Salhanick MD Abstract Objectives: The precise mechanism of hepatocellular toxicity following acetaminophen (APAP) poisoning remains unclear. Nitric oxide is implicated in APAP toxicity as an inflammatory signaling molecule and as a precursor to the free radical peroxynitrate. The effects of inducible nitric oxide synthase (iNOS)-derived NO in APAP toxicity are known; however, the role of endothelial nitric oxide synthase (eNOS)-derived NO is unknown. The authors sought to evaluate the effect of eNOS-derived NO during APAP toxicity. Methods: C57BL6/J mice deficient in eNOS (eNOS KO) or iNOS (iNOS KO) and wild-type mice (WT) were treated with 300 mg/kg APAP. Alanine aminotransferase levels and plasma nitrate and nitrite levels were measured. Hypoxia inducible factor (HIF)-1, and Glucose Transporter 1 (Glut-1) levels were determined by Western blot. Results: Alanine aminotransferase levels were significantly elevated in all treated animals. Alanine aminotransferase levels were significantly lower in eNOS KO and iNOS KO than in treated WT animals. Plasma nitrate/nitrite levels were significantly higher in WT animals than in iNOS KO and eNOS KO animals. HIF-1, expression was increased in WT mice and decreased in iNOS KO mice. Glut-1 is a downstream, indirect marker of HIF function. Glut-1 expression was increased in WT and eNOS KO mice. Conclusions: Deficiency of either iNOS or eNOS results in decreased NO production and is associated with reduced hepatocellular injury following APAP poisoning. HIF-1, and Glut-1 levels are increased following APAP poisoning, implying that HIF-1, is functional during the pathogenic response to APAP poisoning. [source] C-peptide makes a comebackDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2003John Wahren Proinsulin C-peptide was for long considered to be without biological activity of its own. New findings demonstrate, however, that it is capable of eliciting both molecular and physiological effects, suggesting that C-peptide is in fact a bioactive peptide. When administered in replacement doses to animal models or to patients with type 1 diabetes, C-peptide ameliorates diabetes-induced functional and structural changes in both the kidneys and the peripheral nerves. It augments blood flow in a number of tissues, notably skeletal muscle, myocardium, skin and nerve. These effects are thought to be mediated via a stimulatory influence on Na+,K+ -ATPase and on endothelial nitric oxide synthase. Specific binding of C-peptide to cell membranes of intact cells and to detergent-solubilized cellular components has been demonstrated, indicating the existence of cell-surface binding sites for C-peptide. A number of intracellular responses are elicited by C-peptide, including a rise in Ca2+ concentration and activation of MAP-kinase signaling pathways. Many but not all of C-peptide's intracellular effects can be inhibited by pertussis toxin, supporting the notion that C-peptide may interact via a G-protein-coupled receptor. Additional data suggest that C-peptide may interact synergistically also in the insulin signaling pathway. Combined, the available observations show conclusively that C-peptide is biologically active, even though its molecular mechanism of action is not as yet fully understood. The possibility that replacement of C-peptide in patients with type 1 diabetes may serve to retard or prevent the development of long-term complications should be evaluated. Copyright © 2003 John Wiley & Sons, Ltd. [source] Association analysis of genes in the renin-angiotensin system with subclinical cardiovascular disease in families with Type 2 diabetes mellitus: The Diabetes Heart StudyDIABETIC MEDICINE, Issue 3 2006K. P. Burdon Abstract Aims Cardiovascular disease (CVD) is a major complication of Type 2 diabetes mellitus. The renin-angiotensin system (RAS) and nitric oxide production are both important regulators of vascular function and blood pressure. Genes encoding proteins involved in these pathways are candidates for a contribution to CVD in diabetic patients. We have investigated variants of the angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin type 1 receptor (AT1R) and endothelial nitric oxide synthase (NOS3) genes for association with subclinical measures of CVD in families with Type 2 diabetes mellitus (T2DM). Methods Atherosclerosis was measured by carotid intima-media thickness and calcification of the carotid and coronary arteries in 620 European Americans and 117 African Americans in the Diabetes Heart Study. Because of the role of these systems in blood pressure regulation, blood pressure was also investigated. Results Compelling evidence of association was not detected with any of the SNPs with any outcome measures after adjustments for covariates despite sufficient power to detect relatively small differences in traits for specific genotype combinations. Conclusions Genetic variation of the RAS and NOS3 genes do not appear to strongly influence subclinical cardiovascular disease or blood pressure in this diabetic population. [source] Gestational diabetes affects platelet behaviour through modified oxidative radical metabolismDIABETIC MEDICINE, Issue 1 2004L. Mazzanti Abstract Aims Patients with Type 1 and Type 2 diabetes mellitus show altered platelet function including decreased nitric oxide synthase (NOS) activity and increased peroxynitrite production. Gestational diabetes mellitus (GDM) is a clinical condition which is ideal for evaluating short-term effects of impaired glucose metabolism, ruling out the possibility that the platelet abnormalities are a consequence of diabetic complications. The aim of the present work was to study NO metabolism in platelets from pregnant women with GDM. The production of peroxides was also studied as it is strongly involved in peroxynitrite formation. Methods Platelet NOS activity and peroxynitrite production, levels of hydroperoxides and thiobarbituric acid reactive substances (TBARS) in platelet membranes in the basal state and after in vitro peroxidative stress with phenylhydrazine were determined in 40 pregnant women with GDM, 40 healthy pregnant women (pregnant controls) of comparable age and gestational age, and 15 healthy non-pregnant women (controls). Results NOS activity was significantly increased in both groups of pregnant women compared with non-pregnant ones, and in GDM women compared with pregnant controls. Production of peroxynitrite was higher in GDM women than in pregnant controls, who also had significantly reduced production compared with non-pregnant women. Basal levels of peroxidation of the platelet membranes evaluated either by hydroperoxide content and TBARS levels or the susceptibility to peroxidation were increased in GDM patients in comparison with both control groups. Conclusions We have shown a modification in platelet NO and peroxynitrite production and an increase in platelet indicators of oxidative stress in GDM women compared with healthy pregnant women which might be at the basis of a cellular dysfunction. [source] Evidence for a widespread involvement of NO in control of photogenesis in bioluminescent fishACTA ZOOLOGICA, Issue 4 2010Jenny Krönström Abstract Krönström, J. and Mallefet, J. 2009. Evidence for a widespread involvement of NO in control of photogenesis in bioluminescent fish. ,Acta Zoologica (Stockholm) 91: 474,483. The presence of nitric oxide synthase (NOS) and nerve fibres in the photophores of seven bioluminescent fish species (Hygophum benoiti, Myctophum punctatum, Electrona risso, Cyclothone braueri, Vinciguerria attenuata, Maurolicus muelleri and Porichthys notatus) with endogenous photocytes, were investigated. Antibodies directed against neuronal and inducible NOS (n and iNOS respectively) and NADPH-diaphorase activity were used to reveal the locations of NOS, while antibodies directed against acetylated tubulin were used to visualize nerve fibres. The nNOS antibody labelled structures in all investigated photophores except in the organs from P. notatus. The photocytes of P. notatus showed NADPH-diaphorase activity. In the myctophid species, NOS-like immunoreactivity was found in small intracellular structures of the photocytes and in nerve fibres reaching the photocytes. nNOS-positive fibres were also found among lens/filter cells in V. attenuata, and in M. muelleri the cytoplasm of lens/filter cells contained NOS-like material. In C. braueri, a cell type located at a collecting chamber for luminous products in the photophore contained NOS-like material. All photophores received an innervation reaching the photocytes, as well as other components including lens/filter areas. The results of this study comply with an involvement of nitric oxide in the control of bioluminescence in several fish species. [source] Assessment of protein-incorporated arginine methylation in biological specimens by CZE UV-detectionELECTROPHORESIS, Issue 23 2007Angelo Zinellu Dr. Abstract Protein arginine methyltransferases methylate post-translationally arginine residues in proteins to synthesize monomethylarginine (MMA), asymmetric dimethylarginine (ADMA), or symmetric dimethylarginine. Protein arginine methylation is involved in the regulation of signal transduction, RNA export, and cell proliferation. Moreover, upon proteolysis, arginines are released into the cytosol in which they exert important biological effects. Both MMA and ADMA are inhibitors of nitric oxide synthase and especially elevated levels of ADMA are associated with endothelial dysfunction and cardiovascular disease. Quantification of these analytes is commonly performed by HPLC after sample cleanup and derivatization. We propose a CE method in which these steps have been avoided and the procedure for sample preparation has been simplified. After acidic hydrolysis of proteins, samples were dried, resuspended in water, and directly injected in CE. A baseline separation of analytes was reached in a 60 cm×75,,m id uncoated silica capillary, by using a Tris-phosphate run buffer at pH,2.15. This method allows an accurate assessment of protein arginine methylation degree in different biological samples such as whole blood, plasma, red blood cells, cultured cells, and tissue. Moreover, its good sensitivity permits to evaluate the methylation of a single protein type after the opportune purification steps. A method applicability concerns both clinical laboratories, where the evaluation of blood protein from numerous samples could be rapidly performed, and research laboratories where the factors affecting the arginine protein methylation degree could be easily studied. [source] Nitric oxide and p53 in cancer-prone chronic inflammation and oxyradical overload disease,ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2004Julie E. Goodman Abstract Nitric oxide (NO·), which is generated under chronic inflammatory conditions that predispose individuals to cancer, has paradoxical effects. NO· can activate p53, which can result in anti-carcinogenic effects, or it can be mutagenic and increase cancer risk. We explored the mechanisms by which NO· induced p53 activation in vitro and found that NO· induced p53 accumulation and phosphorylation, particularly at ser-15, via ATM and ATR kinases, which then led to cell cycle arrest at G2/M. We next examined proteins in these pathways in both inflamed and normal human colon tissue. Inducible nitric oxide synthase (iNOS) levels and p53-P-ser15 levels were positively correlated with the degree of inflammation and with each other. Additionally, the p53 targets, HDM-2 and p21 (WAF1), were present in ulcerative colitis (UC) colon, but undetectable in normal colon, consistent with activated p53. We also found higher p53 mutant frequencies of both G:C , A:T transitions at the CpG site of codon 248 and C:G , T:A transitions at codon 247 in lesional colon tissue from UC cases versus nonlesional tissue from these cases or colon tissue from normal adult controls. Consistent with nitrosative stress and the deamination of 5-methylcytosine, p53 mutations were also detected in sporadic colon cancer tissue and were associated with iNOS activity in these tissues. These studies identified a potential mechanistic link between NO· and p53 in UC and sporadic colon cancer. Environ. Mol. Mutagen. 44:3,9, 2004. Published 2004 Wiley-Liss, Inc. [source] Suppression of inflammatory responses by celastrol, a quinone methide triterpenoid isolated from Celastrus regeliiEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 9 2009D. H. Kim Abstract Background, Celastrol, a quinone methide triterpenoid isolated from the Celastraceae family, exhibits various biological properties, including chemopreventive, antioxidant and neuroprotective effects. In this study, we showed that celastrol inhibits inflammatory reactions in macrophages and protects mice from skin inflammation. Materials and methods, Anti-inflammatory effects of celastrol (0,1 ,M) were examined in lipopolysaccharide (LPS)-stimulated RAW 264·7 macrophages. To investigate the effects of celastrol (0,50 ,g per mice) in vivo, activation of myeloperoxidase (MPO) and histological assessment were examined in the 12- O -tetradecanoyl-phorbol-13-acetate (TPA)-induced mouse ear oedema model. Results, Our in vitro experiments showed that celastrol suppressed not only LPS-stimulated generation of nitric oxide and prostaglandin E2, but also expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW264·7 cells. Similarly, celastrol inhibited LPS-induced production of inflammatory cytokines, including tumour necrosis factor-, and interleukin-6. In an animal model, celastrol protected mice from TPA-induced ear oedema, possibly by inhibiting MPO activity and production of inflammatory cytokines. Conclusions, Our data suggest that celastrol inhibits the production of inflammatory mediators and is a potential target for the treatment of various inflammatory diseases. [source] Genetic influence in antithrombotic actions of atorvastatin in hypercholesterolaemiaEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 1 2008L. Puccetti ABSTRACT Background, Recent data indicate that statins could offer coronary artery disease (CAD) benefit even by mechanisms beyond lipid lowering. Genetic influence has been shown for some antithrombotic actions of statins via oxidized-low-density lipoprotein cholesterol (ox-LDL) receptors and nitric oxide synthase (NOS) activity modulation. The present study was designed to evaluate the influence of ox-LDL lectin-like receptor-1 (LOX-1) and NOS polymorphisms in the incidence of cardiovascular events in pure hypercholesterolaemic subjects during statin treatment. Materials and methods, A prospective 4-year study involving 1039 event-free subjects (643 males, 396 females) treated with atorvastatin (10,40 mg day,1) to reach the appropriate Adult Treatment Panel-III LDL target of 3·36 mmol L,1. Enrolled subjects were evaluated every 6 months or at a clinical event. LOX-1 3,UTR/T-C and NOS G894T polymorphisms were detected by allelic discrimination assays (polymerase chain reaction), lipid profile by enzymatic-colorimetric method, ox-LDL by enzyme linked immunosorbent assay, platelet activation by P-selectin (P-sel) expression (FACScan), NOS activity (by intracellular citrullin recovery) and homocysteine (high performance liquid chromatography), C-reactive protein (CRP) by sensitive nephelometric technique. Results, LOX-1 3,UTR/T showed the strongest association with events in the whole cohort with respect to each other variable including LDL reduction and NOS G894T (OR 4·90, 95% CI 3·19,6·98, P < 0·00001). Smoking influenced events in LDL-targeted subjects (P < 0·0001). Ox-LDL and P-sel were better indicators than LDL or other variables according to 3,UTR/C genotype regardless of the magnitude of LDL reduction (OR 4·21, 95% CI 2·29,6·70 P < 0·0001). Conclusions, LOX-1 polymorphisms could influence statin effectiveness in CAD prevention by induction of sensitivity to antithrombotic mechanisms such as antiplatelet activity. [source] | |||||||||||||||||||||||||||||||||||||