Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Endothelial progenitor cells (EPCs) can differentiate from mononuclear cells (MNCs) of adult human peripheral blood, bone marrow, and cord blood during culture. Although MNCs are usually isolated by a Ficoll gradient centrifuge method, this method is time-consuming, and blood is easily contaminated. We developed a novel cell filtration device (StemQuickE, Asahi Kasei Medical, Oita, Tokyo, Japan) to isolate MNCs from human cord blood and examined whether functional EPCs could differentiate from MNCs isolated by this device. Recovery rates of MNCs, CD34(+) and CD133(+) progenitor cells, were significantly greater in the StemQuickE method than in the Ficoll method. During MNC culture, spindle-shaped attaching cells developed, and most of these cells incorporated DiI-acetylated low-density lipoprotein and showed positive binding to fluorescein isothiocyanate-lectin. Reverse transcription-polymerase chain reaction analysis revealed that attaching cells expressed various progenitor and endothelial lineage markers such as KDR, CD31, endothelial cell nitric oxide synthase, CD133, and LOX-1. Culture-expanded EPCs were isolated and labeled with a green fluorescent dye, PKH2-GL, and cocultured with human umbilical vein endothelial cells (HUVECs). EPCs formed angiogenesis-like networks together with HUVECs in 3D matrix gel. Finally, EPCs (3 x 10(5)) were implanted into ischemic hindlimb of nude rats (n = 3), and laser Doppler blood flowmetry (LDBF) revealed that the ratio of ischemic to normal limb LDBF was significantly greater in EPC-transplanted animals compared with controls receiving saline. In conclusion, the novel cell filtration device, StemQuickE, is a useful tool to isolate MNCs from human cord blood. Moreover, MNCs obtained by this filter system can give rise to functional EPCs.
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PMID:Derivation of functional endothelial progenitor cells from human umbilical cord blood mononuclear cells isolated by a novel cell filtration device. 1553 90

Inducible nitric oxide synthase (iNOS) has been implicated in cancer formation because of its vast presence cancer tissues. Studies to support such a role during transformation of human cells are very limited. We have developed a cell culture system, which renders a more transformed epithelial phenotype. The model cells generated from immortalized human gingival mucosal (GM) keratinocytes are consisted of less transformed epithelial-like (EPI) cells and more transformed fibroblast-like (FIB) cells. The latter exhibit anchorage independent growth (AIG). Our data showed that iNOS at mRNA and protein levels was up-regulated in more transformed FIB cells in comparison with less transformed EPI cells. FIB cells at low passages (p<22) were unstable being able to morphologically and functionally revert back to EPI phenotype, while no reversion was observed in FIB cells at high passages (p>43). The morphological reversion of FIB cells was associated with the reversal of vimentin expression as well as AIG. More importantly, these revertants showed reduced levels of iNOS mRNA as well as MAP kinase ERK and phospho-ERK protein expression, while FIB cells without reversion maintained the expression. Furthermore, the MEK1/2 inhibitor U0126 could reduce detectable iNOS mRNA levels suggesting that MAP kinases were upstream regulators of iNOS transcription. U0126 caused both morphological and functional reversion of FIB cells indicating involvement of MAP kinases in these functions. Taken together, we provide evidence for an up-regulation of iNOS in cultured human keratinocytes which exhibit AIG. This up-regulation may reflect progressive transformation which still requires further changes to reach tumorigenic conversion.
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PMID:Human gingival mucosal keratinocytes exhibiting anchorage-independent growth express increased inducible nitric oxide synthase: regulation by MAP kinases. 1556 70

The development of intrinsic ganglia, comprised of neurons and glia cells that innervate airway smooth muscle, is a recognized component of the growing lung. However, the embryological origin of these neurons and glia is unclear. The lung buds develop as an outgrowth of the foregut, which contains migrating neural crest cells (NCC) that ultimately give rise to the enteric nervous system (ENS) along the entire length of the gut. It has therefore been proposed that the intrinsic ganglia of the lung arise from a subset of NCC that leave the gut and migrate into the lung buds during early development. We have tested this hypothesis using quail-chick interspecies grafting to selectively label the hindbrain-derived neural crest cell population that colonizes the gut. In conjunction with antibody labeling and in situ hybridization, we demonstrate that: (i) lung ganglia arise from vagal NCC that migrate from the foregut into the lung buds; (ii) like ENS precursors, these NCC express the transcription factor Sox10, and the receptors EDNRB and RET; (iii) the co-receptor for RET, GFRalpha1, is expressed in the lung mesenchyme and in ganglia; (iv) ganglia persist within the lung throughout development and contain cells immunopositive for the pan-neuronal markers ANNA-1 and PGP9.5, the inhibitory neurotransmitter NO, as shown by NADPH-diaphorase staining, and the glial marker GFAP.
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PMID:Neural crest cell origin for intrinsic ganglia of the developing chicken lung. 1557 40

ANG II activation of phospholipase D (PLD) is required for ERK and NAD(P)H oxidase activation, both of which are involved in hypertension. Previous findings demonstrate that ANG II stimulates PLD activity through AT(1) receptors in a RhoA-dependent mechanism. Additionally, endogenous AT(2) receptors in preglomerular smooth muscle cells attenuate ANG II-mediated PLD activity. In the present study, we examined the signal transduction mechanisms used by endogenous AT(2) receptors to modulate ANG II-induced PLD activity through either PLA(2) generation of lysophosphatidylethanolamine or Galpha(i)-mediated generation of nitric oxide (NO) and interaction with RhoA. Blockade of AT(2) receptors, Galpha(i) and NO synthase, but not PLA(2), enhanced ANG II-mediated PLD activity in cells rich in, but not poor in, AT(2) receptors. Moreover, NO donors, a direct activator of guanylyl cyclase and a cGMP analog, but not lysophosphatidylethanolamine, inhibited ANG II-mediated PLD activity, whereas an inhibitor of guanylyl cyclase augmented ANG II-induced PLD activity. AT(2) receptor- and NO-mediated attenuation of ANG II-induced PLD activity was completely lost in cells transfected with S188A RhoA, which cannot be phosphorylated on serine 188. Therefore, our data indicate that AT(2) receptors activate Galpha(i), subsequently stimulating NO synthase and leading to increased soluble guanylyl cyclase activity, generation of cGMP, and activation of a protein kinase, resulting in phosphorylation of RhoA on serine 188. Furthermore, because AT(2) receptors inhibit AT(1) receptor signaling to PLD via modulating RhoA activity, AT(2) receptor signaling can potentially regulate multiple vasoconstrictive signaling systems through inactivating RhoA.
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PMID:AT2 receptors cross talk with AT1 receptors through a nitric oxide- and RhoA-dependent mechanism resulting in decreased phospholipase D activity. 1557 19

Cortical spreading depression (CSD) has been documented to confer ischemic tolerance on brain. Although nitric oxide (NO) is a crucial mediator in preconditioning under certain circumstances, the role of NO in CSD-induced neuroprotection is unclear. We examined the effect of L-NAME, an inhibitor of NO synthase, on CSD-induced tolerance against transient focal cerebral ischemia. A solution of 0.5 M KCl was applied for 2 h on the right hemisphere to induce CSD. Animals received either vehicle or L-NAME (4 mg/kg, iv) 30 min before CSD. Temporary occlusion (120 min) of the right middle cerebral artery was induced 4 days after preconditioning and the infarct volume was measured. Additionally, ERK 1/2 activation and cyclooxygenase-2 (COX-2) expression in the cerebral cortex were examined by Western blotting analysis immediately after cessation of CSD, or at 1, 2, 4, 8, and 24 h after CSD. CSD reduced infarct volume from 275 +/- 15 mm3 (mean +/- SEM) in the non-CSD group to 155 +/- 14 mm3 in the CSD group (P < 0.05). L-NAME abolished this protection (281 +/- 14 mm3; P < 0.05 vs. CSD group). Elevated ERK activation and COX-2 expression were observed immediately after or 8 h after preconditioning, respectively. Those responses are significantly augmented by L-NAME (3-fold for ERK and 4-fold for COX-2). These results suggest a crucial role of NO in the establishment of preconditioning with CSD.
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PMID:The role of nitric oxide in the development of cortical spreading depression-induced tolerance to transient focal cerebral ischemia in rats. 1578 Oct 49

Blastocyst implantation is a critical process in the establishment of pregnancy in eutherian mammals and requires a harmonious symbiosis between the developing conceptus and the differentiating maternal uterus. A better understanding of this symbiotic relationship will provide novel approaches and interventions for realizing anti-implantation strategies for effective fertility regulation and reproductive health care management. We have been using the rhesus monkey (Macaca mulatta) as a nonhuman primate model to this end. In the present study, the process of progesterone-mediated regulation of endometrial receptivity for blastocyst implantation has been targeted by the use of mifepristone as an emergency contraceptive agent. Furthermore, based on cell-specific, temporal and spatial distribution of vasotropic cytokines and mediators in the "receptive" and periimplantation periods, the pregnancy interceptive potentials of (a) monoclonal antibody (MAb) to leukemia inhibitory factor (LIF); (b) inhibitors of nitric oxide synthase [e.g., N6-nitro-l-arginine (l-NAME) and aminoguanidine]; and (c) MAb to vascular endothelial growth factor (VEGF) were examined. LIF is a progesterone-responsive pleiotropic cytokine that functions as a proinflammatory cytokine, together with interleukins 1 and 6, during the process of implantation-placentation in primates, and its immunoneutralization with MAb resulted in inhibition (p<.04) of pregnancy establishment in the rhesus monkey. However, timed administration of l-NAME or aminoguanidine failed to inhibit blastocyst implantation in a significant manner. Also, no synergistic antinidatory action of antiprogestin combined with l-NAME was detected in the rhesus monkey. The application of MAb to VEGF during the periimplantation period, on the other hand, led to significant (p<.04) prevention of pregnancy without influencing steroid hormone levels in the circulation. Our data lend support to the hypothesis that VEGF is essential for pregnancy establishment and that trophoblast-derived VEGF, acting via its specific receptors Flt-1 and KDR, is necessary for blastocyst implantation. The use of cDNA-based expression arrays followed by differential display analysis has provided preliminary understanding of the nature of gene cluster networks operative in the receptive endometrium of potential conception cycles in the rhesus monkey. This knowledge may, in the future, lead to further innovative anti-implantation strategies for targeted pregnancy interception.
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PMID:Target-oriented anti-implantation approaches for pregnancy interception: experiences in the rhesus monkey model. 1579 48

Oxygen enhancement of tumor radiosensitivity is attributed to DNA damage by reactive oxygen species. The mechanism remains unclear but may involve mitochondria as major sources of oxygen and nitrogen radicals as well as central effectors of energy homeostasis and apoptosis. Here we used dihydrorhodamine and 2',7'-dichlorodihydrofluorescein to compare mitochondrial and total cell generation, respectively, of reactive oxygen or nitrogen species in cells irradiated at 5 Gy. Irradiation in the presence of oxygen selectively stimulated mitochondrial radical production in HeLa and MeWo cells, but in MCF7 cells radical production was more generalized. In all three cell lines oxygen impaired cell proliferation as measured by resazurin reduction 7 days after irradiation. Antioxidants N-acetylcysteine, ascorbic acid, and melatonin largely prevented dye oxidation during normoxic irradiation yet had no effect on oxygen-dependent irradiation injury. However, NO synthase inhibitor N(G)-monomethyl-L-arginine protected HeLa and MCF7 though not MeWo cells, consistent with their different levels of constitutive NO generation. SB203580 inhibition of p38 MAPK appreciably protected HeLa and marginally protected MCF7 cells against oxygen-dependent irradiation injury, while the less specific JNK/SAPK inhibitor SP600125 and ERK inhibitor U0126 had no effect. None of the inhibitors affected MeWo radiosensitivity. Therefore oxygen-enhanced radiosensitivity in these tumor cell lines does not depend on extensive production of oxygen radicals and is cell-type dependent. NO mediates oxygen-dependent injury in HeLa and MCF7 cells, by p38-dependent and MAPK-independent mechanisms, respectively. In MeWo cells this oxygen-enhanced radiosensitivity is independent of both NO and MAPK signaling.
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PMID:Radical mediators and mitogen-activated protein kinase signaling in oxygen-dependent radiosensitivity of human tumor cell lines. 1596 10

L-arginine is metabolized to nitric oxide (NO) by NO synthase (NOS), or to urea and L-ornithine by arginase. L-ornithine contributes to vascular remodeling in pulmonary hypertension via metabolism to polyamines and proline. Previously we found that cytokines upregulate both NOS and arginase in pulmonary arterial endothelial cells. We hypothesized that cytokine-induced arginase I and II expression depend on epidermal growth factor (EGF) receptor (EGFR) activity. Bovine pulmonary arterial endothelial cells were treated with lipopolysaccharide and tumor necrosis factor-alpha (L/T). L/T treatment resulted in a substantial increase in urea production, and this increase in urea production was potently inhibited by both genistein and AG1478, inhibitors of EGFR. Levels of arginase I protein and arginase II mRNA were increased in response to L/T treatment, and genistein prevented the L/T-induced elevations in both arginase I protein and arginase II mRNA levels. L/T treatment increased production of nitrites and inducible NOS mRNA accumulation, and genistein and AG1478 had little effect on these changes. EGF (50 ng/ml) treatment resulted in enhanced urea production. Finally, a 170-kD protein was phosphorylated upon treatment with either EGF or L/T. Our results indicate that arginase induction by L/T depends in part on EGFR activity. We speculate that EGFR inhibitors may attenuate vascular remodeling without affecting NO release, and thus may represent novel therapeutic modalities for pulmonary hypertensive disorders.
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PMID:Cytokine-induced endothelial arginase expression is dependent on epidermal growth factor receptor. 1599 32

Vascular endothelial growth factor (VEGF) is a potent mitogen with angiogenic and vasoactive properties. VEGF can bind to two types of receptors. VEGF receptor 2 (VEGFR2) is mainly responsible for the dilator response to VEGF through nitric oxide (NO) release, whereas VEGFR1 may sequestrate the ligand. We hypothesized that in neonatal hypoxia-induced pulmonary hypertension, VEGF vasodilation is reduced. The dilator response to VEGF was assessed in isolated perfused lung of 1-d-old piglets that were exposed to either normoxia or hypoxia (fraction of inspired oxygen 0.10) for 14 d. The plasma and pulmonary artery concentration of VEGF was measured by quantitative sandwich enzyme immunoassay in piglets that were exposed to either normoxia or hypoxia for 1, 3, 7, or 14 d. The expression of VEGFR1, VEGFR2, and endothelial NO synthase in pulmonary artery was measured in the same study groups using Western blot analysis. VEGF (10(-12)-10(-9) M) induces a dose-dependent relaxation in 14-d normoxic piglets, whereas vasodilation is abolished after 14 d of hypoxia. VEGF tissue concentration is increased by hypoxia. VEGFR1 expression is dramatically increased after 1, 3, and 7 d of hypoxia compared with normoxia and returns to normal afterward. VEGFR2 expression is reduced by hypoxia at 14 d. However, endothelial NO synthase expression is not affected by hypoxia compared with normoxia. In neonatal hypoxia-induced pulmonary hypertension, VEGF is increased, whereas vasodilation to VEGF is abolished. This reduced vasodilation may be due to decreased VEGFR2 expression. We speculate that sequestration by VEGFR1 may also limit, to some extent, the vascular protecting effect of VEGF, thus contributing to the pathophysiologic changes seen in neonatal hypoxia-induced pulmonary hypertension.
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PMID:Changes in expression of vascular endothelial growth factor and its receptors in neonatal hypoxia-induced pulmonary hypertension. 1600 32

In muscle, physiologic hyperinsulinemia, presumably acting on endothelial cells (ECs), dilates arterioles and regulates both total blood flow and capillary recruitment, which in turn influences glucose disposal. In cultured ECs, however, supraphysiological (e.g. >or=10 nM) insulin concentrations are typically used to study insulin receptor (IR) signaling pathways and nitric oxide generation. IGF-I receptors (IGF-IRs) are more abundant than IR in ECs, and they also respond to high concentrations of insulin. To address whether IR mediates responses to physiologic insulin stimuli, we examined the insulin concentration dependence of IR and IGF-IR-mediated insulin signaling in bovine aortic ECs (bAECs). We also assessed whether insulin/IGF-I hybrid receptors were present in bAECs. Insulin, at 100-500 pM, significantly stimulated the phosphorylation of IRbeta, Akt1, endothelial isoform of nitric oxide synthase, and ERK 1/2 but not the IGF-IRbeta subunit. At concentrations 1-5 nm or greater, insulin dose-dependently enhanced the tyrosine phosphorylation of IGF-IRbeta, and this was inhibited by IGF-IR neutralizing antibody. In addition, immunoprecipitation of IRbeta pulled down the IGF-IRbeta, and the IRbeta immunocytochemically colocalized with IGF-IRbeta, suggesting that ECs have insulin/IGF-I hybrid receptors. We conclude that: 1) insulin at physiological concentrations selectively activates IR signaling in bAECs; 2) bAECs express IGF-IR and insulin/IGF-I hybrid receptors in addition to IR; 3) high concentrations of insulin (>or=1-5 nM) activate IGF-IR and hybrid receptors as well as IR; and 4) this crossover activation can confound interpretation of studies of insulin action in ECs when high insulin concentrations are used.
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PMID:Insulin at physiological concentrations selectively activates insulin but not insulin-like growth factor I (IGF-I) or insulin/IGF-I hybrid receptors in endothelial cells. 1609 60


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