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  • Active stabilization of human endothelial nitric oxide synthase mRNA by hnRNP E1 protects against antisense RNA and microRNAs. 23478261

    Human endothelial nitric oxide synthase (eNOS) mRNA is highly stable in endothelial cells (ECs). Posttranscriptional regulation of eNOS mRNA stability is an important component of eNOS regulation, especially under hypoxic conditions. Here, we show that the human eNOS 3' untranslated region (3' UTR) contains multiple, evolutionarily conserved pyrimidine (C and CU)-rich sequence elements that are both necessary and sufficient for mRNA stabilization. Importantly, RNA immunoprecipitations and RNA electrophoretic mobility shift assays (EMSAs) revealed the formation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1)-containing RNP complexes at these 3'-UTR elements. Knockdown of hnRNP E1 decreased eNOS mRNA half-life, mRNA levels, and protein expression. Significantly, these stabilizing RNP complexes protect eNOS mRNA from the inhibitory effects of its antisense transcript sONE and 3'-UTR-targeting small interfering RNAs (siRNAs), as well as microRNAs, specifically, hsa-miR-765, which targets eNOS mRNA stability determinants. Hypoxia disrupts hnRNP E1/eNOS 3'-UTR interactions via increased Akt-mediated serine phosphorylation (including serine 43) and increased nuclear localization of hnRNP E1. These mechanisms account, at least in part, for the decrease in eNOS mRNA stability under hypoxic conditions. Thus, the stabilization of human eNOS mRNA by hnRNP E1-containing RNP complexes serves as a key protective mechanism against the posttranscriptional inhibitory effects of antisense RNA and microRNAs under basal conditions but is disrupted under hypoxic conditions.
    Tipo de documento:
    Referencia
    Referencia del producto:
    17-700
    Nombre del producto:
    Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit

  • Identification of regulatory sites of phosphorylation of the bovine endothelial nitric-oxide synthase at serine 617 and serine 635. 12171920

    Endothelial nitric-oxide synthase (eNOS) is regulated by signaling pathways involving multiple sites of phosphorylation. The coordinated phosphorylation of eNOS at Ser(1179) and dephosphorylation at Thr(497) activates the enzyme, whereas inhibition results when Thr(497) is phosphorylated and Ser(1179) is dephosphorylated. We have identified two further phosphorylation sites, at Ser(617) and Ser(635), by phosphopeptide mapping and matrix-assisted laser desorption ionization time of flight mass spectrometry. Purified protein kinase A (PKA) phosphorylates both sites in purified eNOS, whereas purified Akt phosphorylates only Ser(617). In bovine aortic endothelial cells, bradykinin (BK), ATP, and vascular endothelial growth factor stimulate phosphorylation of both sites. BK-stimulated phosphorylation of Ser(617) is Ca(2+)-dependent and is partially inhibited by LY294002 and wortmannin, phosphatidylinositol 3-kinase inhibitors, suggesting signaling via Akt. BK-stimulated phosphorylation of Ser(635) is Ca(2+)-independent and is completely abolished by the PKA inhibitor, KT5720, suggesting signaling via PKA. Activation of PKA with isobutylmethylxanthine also causes Ser(635), but not Ser(617), phosphorylation. Mimicking phosphorylation at Ser(635) by Ser to Asp mutation results in a greater than 2-fold increase in activity of the purified protein, whereas mimicking phosphorylation at Ser(617) does not alter maximal activity but significantly increases Ca(2+)-calmodulin sensitivity. These data show that phosphorylation of both Ser(617) and Ser(635) regulates eNOS activity and contributes to the agonist-stimulated eNOS activation process.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo

  • The cell-specific expression of endothelial nitric-oxide synthase: a role for DNA methylation. 15180995

    The basis for the endothelial cell-restricted expression of endothelial nitric-oxide synthase (eNOS) is not known. While transgenic promoter/reporter mice demonstrated endothelium cell-specific eNOS expression, we found robust expression of episomal eNOS promoter/reporter constructs in cell types that do not express the native eNOS transcript. To explore the mechanism underlying this differential activity pattern of chromatin-versus episome-based eNOS promoters, we examined the methylation status of 5'-regulatory sequences of the human eNOS gene. DNA methylation differed dramatically between endothelial and nonendothelial cell types, including vascular smooth muscle cells. This same cell type-specific methylation pattern was observed in vivo in endothelial and vascular smooth muscle cells of the mouse aorta at the native murine eNOS promoter. We addressed the functional consequences of methylation on eNOS transcription using transient transfection of in vitro methylated promoter/reporter constructs and found that methylated constructs exhibited a marked decrease in the synergistic action of Sp1, Sp3, and Ets1 on eNOS promoter activity. The addition of methyl-CpG-binding protein 2 further reduced the transcriptional activity of methylated eNOS constructs. Importantly, chromatin immunoprecipitation demonstrated the presence of Sp1, Sp3, and Ets1 at the native eNOS promoter in endothelial cells but not in vascular smooth muscle cells. Finally, robust expression of eNOS mRNA was induced in nonendothelial cell types following inhibition of DNA methyltransferase activity with 5-azacytidine, demonstrating the importance of DNA methylation-mediated repression. This report is the first to show that promoter DNA methylation plays an important role in the cell-specific expression of a constitutively expressed gene in the vascular endothelium.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-107
    Nombre del producto:
    Anti-Sp3 Antibody

  • Simvastatin re-couples dysfunctional endothelial nitric oxide synthase in experimental subarachnoid hemorrhage. 21373645

    Reduced endothelial nitric oxide synthase (eNOS) function has been linked to secondary complications of subarachnoid hemorrhage (SAH). We previously found that there is increased eNOS function after SAH but that it is uncoupled, leading to secondary complications such as vasospasm, microthromboembolism and neuronal apoptosis. Here we test the hypothesis that recoupling eNOS with simvastatin can prevent these complications. SAH was created in mice that were treated with vehicle or simvastatin starting 2 weeks before or 30 minutes after SAH. SAH increased phosphorylated eNOS which was prevented by pre- or post-treatment with simvastatin. Simvastatin pre-treatment also prevented the increase in eNOS monomer formation that was associated with SAH, decreased superoxide anion radical production and increased NO. These changes were associated with decreased vasospasm, microthromboemboli and neuronal injury. The data suggest that simvastatin re-couples eNOS after SAH, leading to decreased secondary complications such as vasospasm, microthromboemboli and neuronal injury.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-039

  • Effect of IBD sera on expression of inducible and endothelial nitric oxide synthase in human umbilical vein endothelial cells. 16586542

    To study the expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) and their role in inflammatory bowel disease (IBD).We examined the effect of sera obtained from patients with active Crohn's disease (CD) and ulcerative colitis (UC) on the function and viability of human umbilical vein endothelial cells (HUVEC). HUVECs were cultured for 0-48 h in the presence of a medium containing pooled serum of healthy controls, or serum from patients with active CD or UC. Expression of eNOS and iNOS was visualized by immunofluorescence, and quantified by the densitometry of Western blots. Proliferation activity was assessed by computerized image analyses of Ki-67 immunoreactive cells, and also tested in the presence of the NOS inhibitor, 10(-4) mol/L L-NAME. Apoptosis and necrosis was examined by the annexin-V-biotin method and by propidium iodide staining, respectively.In HUVEC immediately after exposure to UC, serum eNOS was markedly induced, reaching a peak at 12 h. In contrast, a decrease in eNOS was observed after incubation with CD sera and the eNOS level was minimal at 20 h compared to control (18%+/-16% vs 23%+/-15% Pless than 0.01). UC or CD serum caused a significant increase in iNOS compared to control (UC: 300%+/-21%; CD: 275%+/-27% vs 108%+/-14%, Pless than 0.01). Apoptosis/necrosis characteristics did not differ significantly in either experiment. Increased proliferation activity was detected in the presence of CD serum or after treatment with L-NAME. Cultures showed tube-like formations after 24 h treatment with CD serum.IBD sera evoked changes in the ratio of eNOS/iNOS, whereas did not influence the viability of HUVEC. These involved down-regulation of eNOS and up-regulation of iNOS simultaneously, leading to increased proliferation activity and possibly a reduced anti-inflammatory protection of endothelial cells.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5384
    Nombre del producto:
    Anti-Nitric Oxide Synthase II Antibody

  • The Akt kinase signals directly to endothelial nitric oxide synthase. 10469573

    Endothelial nitric oxide synthase (eNOS) is an important modulator of angiogenesis and vascular tone [1]. It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion by insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) [2] [3] and is activated by phosphorylation at Ser1177 in the sequence RIRTQS(1177)F (in the single-letter amino acid code) [4]. The protein kinase Akt is an important downstream target of PI 3-kinase [5] [6], regulating VEGF-stimulated endothelial cell survival [7]. Akt phosphorylates substrates within a defined motif [8], which is present in the sequence surrounding Ser1177 in eNOS. Both Akt [5] [6] and eNOS [9] are localized to, and activated at, the plasma membrane. We found that purified Akt phosphorylated cardiac eNOS at Ser1177, resulting in activation of eNOS. Phosphorylation at this site was stimulated by treatment of bovine aortic endothelial cells (BAECs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation with wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induced Akt activity and eNOS phosphorylation. Akt was detected in immunoprecipitates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt directly activates eNOS in endothelial cells. These results strongly suggest that Akt has an important role in the regulation of normal angiogenesis and raise the possibility that the enhanced activity of this kinase that occurs in carcinomas may contribute to tumor vascularization and survival.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-428

  • Src kinase activates endothelial nitric-oxide synthase by phosphorylating Tyr-83. 16123043

    The endothelial nitric-oxide synthase (eNOS) is regulated in part by serine/threonine phosphorylation, but eNOS tyrosine phosphorylation is less well understood. In the present study we have examined the tyrosine phosphorylation of eNOS in bovine aortic endothelial cells (BAECs) exposed to oxidant stress. Hydrogen peroxide and pervanadate (PV) treatment stimulates eNOS tyrosine phosphorylation in BAECs. Phosphorylation is blocked by the Src kinase family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, eNOS and c-Src can be coimmunoprecipitated from BAEC lysates by antibodies directed against either protein. Domain mapping and site-directed mutagenesis studies in COS-7 cells transfected with either eNOS alone and then treated with PV or cotransfected with eNOS and constitutively active v-Src identified Tyr-83 (bovine sequence) as the major eNOS tyrosine phosphorylation site. Tyr-83 phosphorylation is associated with a 3-fold increase in basal NO release from cotransfected cells. Furthermore, the Y83F eNOS mutation attenuated thapsigargin-stimulated NO production. Taken together, these data indicate that Src-mediated tyrosine phosphorylation of eNOS at Tyr-83 modulates eNOS activity in endothelial cells.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Role of immunoexpression of nitric oxide synthases by Hodgkin and Reed-Sternberg cells on apoptosis deregulation and on clinical outcome of classical Hodgkin lymphoma. 18830569

    Hodgkin and Reed-Sternberg (H-RS) cells of classical Hodgkin lymphoma (cHL) present an impaired expression of immunoglobulin genes, but escape apoptotic death. We investigated whether nitric oxide synthases (NOS) are expressed by H-RS cells, studied their association with EBV status and the expression of apoptotic proteins, and investigated their relationship to the clinical outcome of 171 patients. NOS1 and NOS2 were expressed in a large number of cases, whereas NOS3 expression was not detected. Positive associations were found between NOS1 and p53, bax and NOS2, bcl-2 and NOS2, bax and p53, and between bax and fasL. Inverse correlations were established between EBV and NOS2 and between EBV and bcl-2. A shorter overall survival (OS) was associated with strong expression of NOS2. In conclusion, NOS are expressed by H-RS cells of cHL.
    Tipo de documento:
    Referencia
    Referencia del producto:
    04-1128

  • Partial eNOS deficiency causes spontaneous thrombotic cerebral infarction, amyloid angiopathy and cognitive impairment. 26104027

    Cerebral infarction due to thrombosis leads to the most common type of stroke and a likely cause of age-related cognitive decline and dementia. Endothelial nitric oxide synthase (eNOS) generates NO, which plays a crucial role in maintaining vascular function and exerting an antithrombotic action. Reduced eNOS expression and eNOS polymorphisms have been associated with stroke and Alzheimer's disease (AD), the most common type of dementia associated with neurovascular dysfunction. However, direct proof of such association is lacking. Since there are no reports of complete eNOS deficiency in humans, we used heterozygous eNOS(+/-) mice to mimic partial deficiency of eNOS, and determine its impact on cerebrovascular pathology and perfusion of cerebral vessels.Combining cerebral angiography with immunohistochemistry, we found thrombotic cerebral infarctions in eNOS(+/-) mice as early as 3-6 months of age but not in eNOS(+/+) mice at any age. Remarkably, vascular occlusions in eNOS(+/-) mice were found almost exclusively in three areas: temporoparietal and retrosplenial granular cortexes, and hippocampus this distribution precisely matching the hypoperfused areas identified in preclinical AD patients. Moreover, progressive cerebral amyloid angiopaphy (CAA), blood brain barrier (BBB) breakdown, and cognitive impairment were also detected in aged eNOS(+/-) mice.These data provide for the first time the evidence that partial eNOS deficiency results in spontaneous thrombotic cerebral infarctions that increase with age, leading to progressive CAA and cognitive impairments. We thus conclude that eNOS(+/-) mouse may represent an ideal model of ischemic stroke to address early and progressive damage in spontaneously-evolving chronic cerebral ischemia and thus, study vascular mechanisms contributing to vascular dementia and AD.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-1401
    Nombre del producto:
    Anti-GLUT-1 Antibody, CT

  • Endothelial nitric oxide synthase is expressed in amacrine cells of developing human retinas. 16639026

    To examine the expression and cellular distribution pattern of endothelial nitric oxide synthase (eNOS) in the developing human retina and to compare its expression with that in rats.Expression of eNOS was examined by immunohistochemistry in retinas of humans ranging from 8.5 to 28 weeks of gestation (WG) and of rats.In the developing human retina, eNOS expression was first detected in the proximal margin of the neuroblastic layer in the incipient fovea-surrounding area at 12 WG. At 17 to 28 WG, eNOS-immunoreactive cells were located in the innermost part of the inner nuclear layer and in the ganglion cell layer, expanding to both temporal and nasal retinas and the processes projecting into the inner plexiform layer. These eNOS-positive cells coexpressed syntaxin and glutamate decarboxylase, and are probably GABAergic amacrine cells. The onset of eNOS expression in developing amacrine cells, however, preceded the invasion of retinal vasculature, long before vascular function involving these cells can be expected, suggesting that eNOS has a role not only in vasoregulation but also in retinal development. From 20 WG on, eNOS was also detected in the photoreceptors adjacent to the fovea. eNOS expression in amacrine cells and photoreceptors was observed in the central-to-peripheral and temporal-to-nasal gradients. However, in the developing rat retina, eNOS was expressed exclusively in the vascular endothelial cells.The results support that eNOS plays a role, not only in the regulation of vascular function but also in the process of retinal development in humans.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB377
    Nombre del producto:
    Anti-NeuN Antibody, clone A60