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Sample records for cgmp signaling activates

  1. Attenuated vasodilatation in lambs with endogenous and exogenous activation of cGMP signaling: Role of protein kinase G nitration

    PubMed Central

    Aggarwal, Saurabh; Gross, Christine M.; Kumar, Sanjiv; Datar, Sanjeev; Oishi, Peter; Kalka, Gokhan; Schreiber, Christian; Fratz, Sohrab; Fineman, Jeffrey R.; Black, Stephen M.

    2012-01-01

    Pulmonary vasodilation is mediated through the activation of protein kinase G (PKG) via a signaling pathway involving nitric oxide (NO), natriuretic peptides (NP), and cyclic guanosine monophosphate (cGMP). In pulmonary hypertension secondary to congenital heart disease, this pathway is endogenously activated by an early vascular upregulation of NO and increased myocardial B-type NP expression and release. In the treatment of pulmonary hypertension, this pathway is exogenously activated using inhaled NO or other pharmacological agents. Despite this activation of cGMP, vascular dysfunction is present, suggesting that NO-cGMP independent mechanisms are involved and were the focus of this study. Exposure of pulmonary artery endothelial or smooth muscle cells to the NO donor, Spermine NONOate (SpNONOate), increased peroxynitrite (ONOO−) generation and PKG-1α nitration, while PKG-1α activity was decreased. These changes were prevented by superoxide dismutase (SOD) or manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) and mimicked by the ONOO− donor, 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). Peripheral lung extracts from 4-week old lambs with increased pulmonary blood flow and pulmonary hypertension (Shunt lambs with endogenous activation of cGMP) or juvenile lambs treated with inhaled NO for 24h (with exogenous activation of cGMP) revealed increased ONOO− levels, elevated PKG-1α nitration, and decreased kinase activity without changes in PKG-1α protein levels. However, in Shunt lambs treated with L-arginine or lambs administered polyethylene glycol conjugated-SOD (PEG-SOD) during inhaled NO exposure, ONOO− and PKG-1α nitration were diminished and kinase activity was preserved. Together our data reveal that vascular dysfunction can occur, despite elevated levels of cGMP, due to PKG-1α nitration and subsequent attenuation of activity. PMID:21351102

  2. cGMP Signaling, Phosphodiesterases and Major Depressive Disorder

    PubMed Central

    Reierson, Gillian W; Guo, Shuyu; Mastronardi, Claudio; Licinio, Julio; Wong, Ma-Li

    2011-01-01

    Deficits in neuroplasticity are hypothesized to underlie the pathophysiology of major depressive disorder (MDD): the effectiveness of antidepressants is thought to be related to the normalization of disrupted synaptic transmission and neurogenesis. The cyclic adenosine monophosphate (cAMP) signaling cascade has received considerable attention for its role in neuroplasticity and MDD. However components of a closely related pathway, the cyclic guanosine monophosphate (cGMP) have been studied with much lower intensity, even though this signaling transduction cascade is also expressed in the brain and the activity of this pathway has been implicated in learning and memory processes. Cyclic GMP acts as a second messenger; it amplifies signals received at postsynaptic receptors and activates downstream effector molecules resulting in gene expression changes and neuronal responses. Phosphodiesterase (PDE) enzymes degrade cGMP into 5’GMP and therefore they are involved in the regulation of intracellular levels of cGMP. Here we review a growing body of evidence suggesting that the cGMP signaling cascade warrants further investigation for its involvement in MDD and antidepressant action. PMID:22654729

  3. PDE2-mediated cAMP hydrolysis accelerates cardiac fibroblast to myofibroblast conversion and is antagonized by exogenous activation of cGMP signaling pathways.

    PubMed

    Vettel, C; Lämmle, S; Ewens, S; Cervirgen, C; Emons, J; Ongherth, A; Dewenter, M; Lindner, D; Westermann, D; Nikolaev, V O; Lutz, S; Zimmermann, W H; El-Armouche, A

    2014-04-15

    Recent studies suggest that the signal molecules cAMP and cGMP have antifibrotic effects by negatively regulating pathways associated with fibroblast to myofibroblast (MyoCF) conversion. The phosphodiesterase 2 (PDE2) has the unique property to be stimulated by cGMP, which leads to a remarkable increase in cAMP hydrolysis and thus mediates a negative cross-talk between both pathways. PDE2 has been recently investigated in cardiomyocytes; here we specifically addressed its role in fibroblast conversion and cardiac fibrosis. PDE2 is abundantly expressed in both neonatal rat cardiac fibroblasts (CFs) and cardiomyocytes. The overexpression of PDE2 in CFs strongly reduced basal and isoprenaline-induced cAMP synthesis, and this decrease was sufficient to induce MyoCF conversion even in the absence of exogenous profibrotic stimuli. Functional stress-strain experiments with fibroblast-derived engineered connective tissue (ECT) demonstrated higher stiffness in ECTs overexpressing PDE2. In regard to cGMP, neither basal nor atrial natriuretic peptide-induced cGMP levels were affected by PDE2, whereas the response to nitric oxide donor sodium nitroprusside was slightly but significantly reduced. Interestingly, despite persistently depressed cAMP levels, both cGMP-elevating stimuli were able to completely prevent the PDE2-induced MyoCF phenotype, arguing for a double-tracked mechanism. In conclusion, PDE2 accelerates CF to MyoCF conversion, which leads to greater stiffness in ECTs. Atrial natriuretic peptide- and sodium nitroprusside-mediated cGMP synthesis completely reverses PDE2-induced fibroblast conversion. Thus PDE2 may augment cardiac remodeling, but this effect can also be overcome by enhanced cGMP. The redundant role of cAMP and cGMP as antifibrotic meditators may be viewed as a protective mechanism in heart failure. PMID:24531807

  4. ENaC is regulated by natriuretic peptide receptor-dependent cGMP signaling

    PubMed Central

    Guo, Lai-Jing; Alli, Abdel A.; Eaton, Douglas C.

    2013-01-01

    Epithelial sodium channels (ENaCs) located at the apical membrane of polarized epithelial cells are regulated by the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP). The mechanism for this regulation has not been completely characterized. Guanylyl cyclases synthesize cGMP in response to various intracellular and extracellular signals. We investigated the regulation of ENaC activity by natriuretic peptide-dependent activation of guanylyl cyclases in Xenopus 2F3 cells. Confocal microscopy studies show natriuretic peptide receptors (NPRs), including those coupled to guanylyl cyclases, are expressed at the apical membrane of 2F3 cells. Single-channel patch-clamp studies using 2F3 cells revealed that atrial natriuretic peptide (ANP) or 8-(4-chlorophenylthio)-cGMP, but not C-type natriuretic peptide or cANP, decreased the open probability of ENaC. This suggests that NPR-A, but not NPR-B or NPR-C, is involved in the natriuretic peptide-mediated regulation of ENaC activity. Also, it is likely that a signaling pathway involving cGMP and nitric oxide (NO) are involved in this mechanism, since inhibitors of soluble guanylyl cyclase, protein kinase G, inducible NO synthase, or an NO scavenger blocked or reduced the effect of ANP on ENaC activity. PMID:23324181

  5. Nitrite circumvents canonical cGMP signaling to enhance proliferation of myocyte precursor cells.

    PubMed

    Totzeck, Matthias; Schicho, Andreas; Stock, Pia; Kelm, Malte; Rassaf, Tienush; Hendgen-Cotta, Ulrike B

    2015-03-01

    Skeletal muscle tissue has a remarkable high regenerative capacity. The underlying cellular events are governed by complex signaling processes, and the proliferation of skeletal myoblasts is a key initial event. The role of nitric oxide (NO) in cell cycle regulation is well-appreciated. Nitrite, an NO oxidation product, is a stable source for NO-like bioactivity particularly in cases when oxygen shortage compromises NO-synthases activity. Although numerous studies suggest that nitrite effects are largely related to NO-dependent signaling, emerging evidence also implicates that nitrite itself can activate protein pathways albeit under physiological, normoxic conditions. This includes a recently demonstrated cyclic guanosine monophosphate-(cGMP)-independent enhancement of endothelial cell proliferation. Whether nitrite itself has the potential to affect myoblast proliferation and metabolism with or without activation of the canonical NO/cGMP pathway to subsequently support muscle cell regeneration is not known. Here we show that nitrite increases proliferation and metabolic activity of murine cultured myoblasts dose-dependently. This effect is not abolished by the NO scavenger 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimida-zoline-1-oxyl-3 oxide and does not affect intracellular cGMP levels, implicating a cGMP-independent mechanism. Nitrite circumvents the rapamycin induced attenuation of myoblast proliferation and enhances mTOR activity. Our results provide evidence for a novel potential physiological and therapeutic approach of nitrite in skeletal muscle regeneration processes under normoxia independent of NO and cGMP. PMID:25501648

  6. Correlative intravital imaging of cGMP signals and vasodilation in mice

    PubMed Central

    Thunemann, Martin; Schmidt, Kjestine; de Wit, Cor; Han, Xiaoxing; Jain, Rakesh K.; Fukumura, Dai; Feil, Robert

    2014-01-01

    Cyclic guanosine monophosphate (cGMP) is an important signaling molecule and drug target in the cardiovascular system. It is well known that stimulation of the vascular nitric oxide (NO)-cGMP pathway results in vasodilation. However, the spatiotemporal dynamics of cGMP signals themselves and the cGMP concentrations within specific cardiovascular cell types in health, disease, and during pharmacotherapy with cGMP-elevating drugs are largely unknown. To facilitate the analysis of cGMP signaling in vivo, we have generated transgenic mice that express fluorescence resonance energy transfer (FRET)-based cGMP sensor proteins. Here, we describe two models of intravital FRET/cGMP imaging in the vasculature of cGMP sensor mice: (1) epifluorescence-based ratio imaging in resistance-type vessels of the cremaster muscle and (2) ratio imaging by multiphoton microscopy within the walls of subcutaneous blood vessels accessed through a dorsal skinfold chamber. Both methods allow simultaneous monitoring of NO-induced cGMP transients and vasodilation in living mice. Detailed protocols of all steps necessary to perform and evaluate intravital imaging experiments of the vasculature of anesthetized mice including surgery, imaging, and data evaluation are provided. An image segmentation approach is described to estimate FRET/cGMP changes within moving structures such as the vessel wall during vasodilation. The methods presented herein should be useful to visualize cGMP or other biochemical signals that are detectable with FRET-based biosensors, such as cyclic adenosine monophosphate or Ca2+, and to correlate them with respective vascular responses. With further refinement and combination of transgenic mouse models and intravital imaging technologies, we envision an exciting future, in which we are able to “watch” biochemistry, (patho-)physiology, and pharmacotherapy in the context of a living mammalian organism. PMID:25352809

  7. Angiotensin II increases phosphodiesterase 5A expression in vascular smooth muscle cells: A mechanism by which angiotensin II antagonizes cGMP signaling

    PubMed Central

    Kim, Dongsoo; Aizawa, Toru; Wei, Heng; Pi, Xinchun; Rybalkin, Sergei D.; Berk, Bradford C.; Yan, Chen

    2014-01-01

    Angiotensin II (Ang II) and nitric oxide (NO)/natriuretic peptide (NP) signaling pathways mutually regulate each other. Imbalance of Ang II and NO/NP has been implicated in the pathophysiology of many vascular diseases. cGMP functions as a key mediator in the interaction between Ang II and NO/NP. Cyclic nucleotide phosphodiesterase 5A (PDE5A) is important in modulating cGMP signaling by hydrolyzing cGMP in vascular smooth muscle cells (VSMC). Therefore, we examined whether Ang II negatively modulates intracellular cGMP signaling in VSMC by regulating PDE5A. Ang II rapidly and transiently increased PDE5A mRNA levels in rat aortic VSMC. Upregulation of PDE5A mRNA was associated with a time-dependent increase of both PDE5 protein expression and activity. Increased PDE5A mRNA level was transcription-dependent and mediated by the Ang II type 1 receptor. Ang II-mediated activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was essential for Ang II-induced PDE5A upregulation. Pretreatment of VSMC with Ang II inhibited C-type NP (CNP) stimulated cGMP signaling, such as cGMP dependent protein kinase (PKG)-mediated phosphorylation of vasodilator-stimulated-phosphoprotein (VASP). Ang II-mediated inhibition of PKG was blocked when PDE5 activity was decreased by selective PDE5 inhibitors, suggesting that upregulation of PDE5A expression is an important mechanism for Ang II to attenuate cGMP signaling. PDE5A may also play a critical role in the growth promoting effects of Ang II because inhibition of PDE5A activity significantly decreased Ang II-stimulated VSMC growth. These observations establish a new mechanism by which Ang II antagonizes cGMP signaling and stimulates VSMC growth. PMID:15623434

  8. The cyclic nucleotide cGMP is involved in plant hormone signalling and alters phosphorylation of Arabidopsis thaliana root proteins

    PubMed Central

    Isner, Jean Charles; Nühse, Thomas; Maathuis, Frans J. M.

    2012-01-01

    The cyclic nucleotide cGMP has been shown to play important roles in plant development and responses to abiotic and biotic stress. Yet much controversy remains regarding the exact role of this second messenger. Progress in unravelling cGMP function in plants was hampered by laborious and time-consuming methodology to measure changes in cellular [cGMP] but the development of fluorescence-based reporters has removed this disadvantage. This study used the FlincG cGMP reporter to investigate potential interactions between phytohormone and cGMP signalling and found a rapid and significant effect of the hormones abscisic acid (ABA), auxin (IAA), and jasmonic acid (JA) on cytoplasmic cGMP levels. In contrast, brassinosteroids and cytokinin did not evoke a cGMP signal. The effects of ABA, IAA, and JA were apparent at external concentrations in the nanomolar range with EC50 values of around 1000, 300, and 0.03 nmoles for ABA, IAA, and JA respectively. To examine potential mechanisms for how hormone-induced cGMP signals are propagated, the role of protein phosphorylation was tested. A phosphoproteomics analysis on Arabidopsis thaliana root microsomal proteins in the absence and presence of membrane-permeable cGMP showed 15 proteins that rapidly (within minutes) changed in phosphorylation status. Out of these, nine were previously shown to also alter phosphorylation status in response to plant hormones, pointing to protein phosphorylation as a target for hormone-induced cGMP signalling. PMID:22345640

  9. The rhodopsin-guanylyl cyclase of the aquatic fungus Blastocladiella emersonii enables fast optical control of cGMP signaling.

    PubMed

    Scheib, Ulrike; Stehfest, Katja; Gee, Christine E; Körschen, Heinz G; Fudim, Roman; Oertner, Thomas G; Hegemann, Peter

    2015-08-11

    Blastocladiomycota fungi form motile zoospores that are guided by sensory photoreceptors to areas of optimal light conditions. We showed that the microbial rhodopsin of Blastocladiella emersonii is a rhodopsin-guanylyl cyclase (RhGC), a member of a previously uncharacterized rhodopsin class of light-activated enzymes that generate the second messenger cyclic guanosine monophosphate (cGMP). Upon application of a short light flash, recombinant RhGC converted within 8 ms into a signaling state with blue-shifted absorption from which the dark state recovered within 100 ms. When expressed in Xenopus oocytes, Chinese hamster ovary cells, or mammalian neurons, RhGC generated cGMP in response to green light in a light dose-dependent manner on a subsecond time scale. Thus, we propose RhGC as a versatile tool for the optogenetic analysis of cGMP-dependent signaling processes in cell biology and the neurosciences. PMID:26268609

  10. Anxiolytic Effects of Phosphodiesterase-2 Inhibitors Associated with Increased cGMP Signaling

    PubMed Central

    Masood, Anbrin; Huang, Ying; Hajjhussein, Hassan; Xiao, Lan; Li, Hao; Wang, Wei; Hamza, Adel; Zhan, Chang-Guo

    2009-01-01

    Phosphodiesterase (PDE)-2 is a component of the nitric-oxide synthase (NOS)/guanylyl cyclase signaling pathway in the brain. Given recent evidence that pharmacologically induced changes in NO-cGMP signaling can affect anxiety-related behaviors, the effects of the PDE2 inhibitors (2-(3,4-dimethoxybenzyl)-7-det-5-methylimidazo-[5,1-f][1,2,4]triazin-4(3H)-one) (Bay 60-7550) and 3-(8-methoxy-1-methyl-2-oxo-7-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-5-yl)benzamide (ND7001), as well as modulators of NO, were assessed on cGMP signaling in neurons and on the behavior of mice in the elevated plus-maze, hole-board, and open-field tests, well established procedures for the evaluation of anxiolytics. Bay 60-7550 (1 μM) and ND7001 (10 μM) increased basal and N-methyl-d-aspartate- or detanonoate-stimulated cGMP in primary cultures of rat cerebral cortical neurons; Bay 60-7550, but not ND7001, also increased cAMP. Increased cGMP signaling, either by administration of the PDE2 inhibitors Bay 60-7550 (0.5, 1, and 3 mg/kg) or ND7001 (1 mg/kg), or the NO donor detanonoate (0.5 mg/kg), antagonized the anxiogenic effects of restraint stress on behavior in the three tests. These drugs also produced anxiolytic effects on behavior in nonstressed mice in the elevated plus-maze and hole-board tests; these effects were antagonized by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (20 mg/kg). By contrast, the NOS inhibitor Nω-nitro-l-arginine methyl ester (50 mg/kg), which reduces cGMP signaling, produced anxiogenic effects similar to restraint stress. Overall, the present behavioral and neurochemical data suggest that PDE2 may be a novel pharmacological target for the development of drugs for the treatment of anxiety disorders. PMID:19684253

  11. Spatiotemporal cGMP Dynamics in Living Mouse Rods

    PubMed Central

    Gross, Owen P.; Pugh, Edward N.; Burns, Marie E.

    2012-01-01

    Signaling of single photons in rod photoreceptors decreases the concentration of the second messenger, cyclic GMP (cGMP), causing closure of cGMP-sensitive channels located in the plasma membrane. Whether the spatiotemporal profiles of the fall in cGMP are narrow and deep, or broad and shallow, has important consequences for the amplification and the fidelity of signaling. The factors that determine the cGMP profiles include the diffusion coefficient for cGMP, the spontaneous rate of cGMP hydrolysis, and the rate of cGMP synthesis, which is powerfully regulated by calcium feedback mechanisms. Here, using suction electrodes to record light-dependent changes in cGMP-activated current in living mouse rods lacking calcium feedback, we have determined the rate constant of spontaneous cGMP hydrolysis and the longitudinal cGMP diffusion coefficient. These measurements result in a fully constrained spatiotemporal model of phototransduction, which we used to determine the effect of feedback to cGMP synthesis in spatially constricting the fall of cGMP during the single-photon response of normal rods. We find that the spatiotemporal cGMP profiles during the single-photon response are optimized for maximal amplification and preservation of signal linearity, effectively operating within an axial signaling domain of ∼2 μm. PMID:22768933

  12. cGMP Signalling Mediates Water Sensation (Hydrosensation) and Hydrotaxis in Caenorhabditis elegans

    PubMed Central

    Wang, Wei; Qin, Li-Wei; Wu, Tai-Hong; Ge, Chang-Li; Wu, Ya-Qian; Zhang, Qiang; Song, Yan-Xue; Chen, Yuan-Hua; Ge, Ming-Hai; Wu, Jing-Jing; Liu, Hui; Xu, Yao; Su, Chun-Ming; Li, Lan-Lan; Tang, Jing; Li, Zhao-Yu; Wu, Zheng-Xing

    2016-01-01

    Animals have developed the ability to sense the water content in their habitats, including hygrosensation (sensing humidity in the air) and hydrosensation (sensing the water content in other microenvironments), and they display preferences for specific water contents that influence their mating, reproduction and geographic distribution. We developed and employed four quantitative behavioural test paradigms to investigate the molecular and cellular mechanisms underlying sensing the water content in an agar substrate (hydrosensation) and hydrotaxis in Caenorhabditis elegans. By combining a reverse genetic screen with genetic manipulation, optogenetic neuronal manipulation and in vivo Ca2+ imaging, we demonstrate that adult worms avoid the wetter areas of agar plates and hypo-osmotic water droplets. We found that the cGMP signalling pathway in ciliated sensory neurons is involved in hydrosensation and hydrotaxis in Caenorhabditis elegans. PMID:26891989

  13. cGMP inhibition of type 3 phosphodiesterase is the major mechanism by which C-type natriuretic peptide activates CFTR in the shark rectal gland.

    PubMed

    De Jonge, Hugo R; Tilly, Ben C; Hogema, Boris M; Pfau, Daniel J; Kelley, Catherine A; Kelley, Megan H; Melita, August M; Morris, Montana T; Viola, Ryan M; Forrest, John N

    2014-02-15

    The in vitro perfused rectal gland of the dogfish shark (Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride secretion. CNP binds to natriuretic receptors in the basolateral membrane, elevates cellular cGMP, and opens cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in the apical membrane. CNP-provoked chloride secretion was completely inhibitable by the nonspecific protein kinase inhibitor staurosporine and the PKA inhibitor H89 but insensitive to H8, an inhibitor of type I and II isoforms of cGMP-dependent protein kinase (cGKI and cGKII). CNP-induced secretion could not be mimicked by nonhydrolyzable cGMP analogs added alone or in combination with the protein kinase C activator phorbolester, arguing against a role for cGK or for cGMP-induced PKC signaling. We failed to detect a dogfish ortholog of cGKII by molecular cloning and affinity chromatography. However, inhibitors of the cGMP-inhibitable isoform of phosphodiesterase (PDE3) including milrinone, amrinone, and cilostamide but not inhibitors of other PDE isoenzymes mimicked the effect of CNP on chloride secretion in perfused glands and monolayers. CNP raised cGMP and cAMP levels in the SRG epithelial cells. This rise in cAMP as well as the CNP and amrinone-provoked chloride secretion, but not the rise in cGMP, was almost completely blocked by the Gαi-coupled adenylyl cyclase inhibitor somatostatin, arguing against a role for cGMP cross-activation of PKA in CNP action. These data provide molecular, functional, and pharmacological evidence for a CNP/cGMP/PDE3/cAMP/PKA signaling cascade coupled to CFTR in the SRG. PMID:24259420

  14. Receptors and cGMP signalling mechanism for E. coli enterotoxin in opossum kidney

    SciTech Connect

    Forte, L.R.; Krause, W.J.; Freeman, R.H. Harry S. Truman Memorial Veterans Medical Center, Columbia, MO )

    1988-11-01

    Receptors for the heat-stable enterotoxin produced by Escherichia coli were found in the kidney and intestine of the North American opossum and in cultured renal cell lines. The enterotoxin markedly increased guanosine 3{prime},5{prime}-cyclic monophosphate (cGMP) production in slices of kidney cortex and medulla, in suspensions of intestinal mucosa, and in the opossum kidney (OK) and rat kangaroo kidney (PtK-2) cell lines. In contrast, atrial natriuretic factor elicited much smaller increases in cGMP levels of kidney, intestine, or cultured kidney cell lines. The enterotoxin receptors in OK cells had a molecular mass of approximately 120 kDa when measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of receptors crosslinked with {sup 125}I-enterotoxin. The occurrence of receptors for the E. coli peptide in OK implies that these receptors may be involved in the regulation of renal tubular function in the opossum. E. coli enterotoxin caused a much larger increase in urine cGMP excretion than did atrial natriuretic factor when these peptides were injected intravenously into opossums. However, atrial natriuretic factor elicited a marked diuresis, natriuresis, and increased urinary excretion of calcium, phosphate, potassium, and magnesium. In contrast, the enterotoxin did not acutely influence OK fluid and electrolyte excretion. Thus the substantial increase in cGMP synthesis produced by the bacterial peptide in OK cortex and medulla in vitro and the increased renal excretion of cGMP in vivo were not associated with changes in electrolyte or water excretion. Whether cGMP represents a second messenger molecule in the kidney is an interesting question that was raised but not answered in this series of experiments.

  15. Role of local production of endothelium-derived nitric oxide on cGMP signaling and S-nitrosylation

    PubMed Central

    Qian, Jin; Zhang, Qian; Church, Jarrod E.; Stepp, David W.; Rudic, Radu D.; Fulton, David J. R.

    2009-01-01

    Nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), exerts control over vascular function via two distinct mechanisms, the activation of soluble guanylate cyclase (sGC)/cGMP-dependent signaling or through S-nitrosylation of proteins with reactive thiols (S-nitrosylation). Previous studies in cultured endothelial cells revealed that eNOS targeted to the plasma membrane (PM) releases greater amounts of NO compared with Golgi tethered eNOS. However, the significance of eNOS localization to sGC-dependent or -independent signaling is not known. Here we show that PM-targeted eNOS, when expressed in human aortic endothelial cells (HAEC) and isolated blood vessels, increases sGC/cGMP signaling to a greater extent than Golgi-localized eNOS. The ability of local NO production to influence sGC-independent mechanisms was also tested by monitoring the secretion of Von Willebrand factor (vWF), which is tonically inhibited by the S-nitrosylation of N-ethylmaleimide sensitive factor (NSF). In eNOS “knockdown” HAECs, vWF secretion was attenuated to a greater degree by PM eNOS compared with a Golgi-restricted eNOS. Moreover, the PM-targeted eNOS induced greater S-nitrosylation of NSF vs. Golgi eNOS. To distinguish between the amount of NO generated and the intracellular location of synthesis, we expressed Golgi and PM-targeted calcium-insensitive forms of eNOS in HAEC. These constructs, which generate equal amounts of NO regardless of location, produced equivalent increases in cGMP in bioassays and equal inhibition of vWF secretion. We conclude that the greater functional effects of PM eNOS are due to the increased amount of NO produced rather than effects derived from the local synthesis of NO. PMID:19855060

  16. The GTP binding protein-dependent activation and deactivation of cGMP phosphodiesterase in rod photoreceptors

    SciTech Connect

    Yamazaki, Akio.

    1989-01-01

    Cyclic GMP (cGMP) has a crucial role in visual transduction. Recent electrophysiological studies clearly indicate the existence of cGMP-activated conductance in photoreceptor plasma membranes. In darkness, Na{sup +}, Ca{sup ++}, and Mg{sup ++} enter rod outer segments (ROS) through cGMP-activated channels while light closes channels by lowering cGMP concentrations through activation of cGMP phosphodiesterase (PDE). Many excellent reviews reference the mechanism of PDE activation in photoreceptors. However, recent progress in understanding the mechanisms regulating cGMP hydrolysis has raised an important question in the PDE-regulation: how does the three-dimensional movement of a subunit of transducin (retinal G protein) relate to the PDE activation Associated with that question, the mechanism of PDE regulation appears to vary at different stages of evolution, for example, frog and bovine photoreceptors. This review examines recent progress of the cGMP hydrolysis mechanism by focusing on the subunit interactions between transducin and PDE. 36 refs., 2 figs.

  17. Systemic Induction of NO-, Redox-, and cGMP Signaling in the Pumpkin Extrafascicular Phloem upon Local Leaf Wounding.

    PubMed

    Gaupels, Frank; Furch, Alexandra C U; Zimmermann, Matthias R; Chen, Faxing; Kaever, Volkhard; Buhtz, Anja; Kehr, Julia; Sarioglu, Hakan; Kogel, Karl-Heinz; Durner, Jörg

    2016-01-01

    Cucurbits developed the unique extrafascicular phloem (EFP) as a defensive structure against herbivorous animals. Mechanical leaf injury was previously shown to induce a systemic wound response in the EFP of pumpkin (Cucurbita maxima). Here, we demonstrate that the phloem antioxidant system and protein modifications by NO are strongly regulated during this process. Activities of the central antioxidant enzymes dehydroascorbate reductase, glutathione reductase and ascorbate reductase were rapidly down-regulated at 30 min with a second minimum at 24 h after wounding. As a consequence levels of total ascorbate and glutathione also decreased with similar bi-phasic kinetics. These results hint toward a wound-induced shift in the redox status of the EFP. Nitric oxide (NO) is another important player in stress-induced redox signaling in plants. Therefore, we analyzed NO-dependent protein modifications in the EFP. Six to forty eight hours after leaf damage total S-nitrosothiol content and protein S-nitrosylation were clearly reduced, which was contrasted by a pronounced increase in protein tyrosine nitration. Collectively, these findings suggest that NO-dependent S-nitrosylation turned into peroxynitrite-mediated protein nitration upon a stress-induced redox shift probably involving the accumulation of reactive oxygen species within the EFP. Using the biotin switch assay and anti-nitrotyrosine antibodies we identified 9 candidate S-nitrosylated and 6 candidate tyrosine-nitrated phloem proteins. The wound-responsive Phloem Protein 16-1 (PP16-1) and Cyclophilin 18 (CYP18) as well as the 26.5 kD isoform of Phloem Protein 2 (PP2) were amenable to both NO modifications and could represent important redox-sensors within the cucurbit EFP. We also found that leaf injury triggered the systemic accumulation of cyclic guanosine monophosphate (cGMP) in the EFP and discuss the possible function of this second messenger in systemic NO and redox signaling within the EFP. PMID:26904092

  18. Systemic Induction of NO-, Redox-, and cGMP Signaling in the Pumpkin Extrafascicular Phloem upon Local Leaf Wounding

    PubMed Central

    Gaupels, Frank; Furch, Alexandra C. U.; Zimmermann, Matthias R.; Chen, Faxing; Kaever, Volkhard; Buhtz, Anja; Kehr, Julia; Sarioglu, Hakan; Kogel, Karl-Heinz; Durner, Jörg

    2016-01-01

    Cucurbits developed the unique extrafascicular phloem (EFP) as a defensive structure against herbivorous animals. Mechanical leaf injury was previously shown to induce a systemic wound response in the EFP of pumpkin (Cucurbita maxima). Here, we demonstrate that the phloem antioxidant system and protein modifications by NO are strongly regulated during this process. Activities of the central antioxidant enzymes dehydroascorbate reductase, glutathione reductase and ascorbate reductase were rapidly down-regulated at 30 min with a second minimum at 24 h after wounding. As a consequence levels of total ascorbate and glutathione also decreased with similar bi-phasic kinetics. These results hint toward a wound-induced shift in the redox status of the EFP. Nitric oxide (NO) is another important player in stress-induced redox signaling in plants. Therefore, we analyzed NO-dependent protein modifications in the EFP. Six to forty eight hours after leaf damage total S-nitrosothiol content and protein S-nitrosylation were clearly reduced, which was contrasted by a pronounced increase in protein tyrosine nitration. Collectively, these findings suggest that NO-dependent S-nitrosylation turned into peroxynitrite-mediated protein nitration upon a stress-induced redox shift probably involving the accumulation of reactive oxygen species within the EFP. Using the biotin switch assay and anti-nitrotyrosine antibodies we identified 9 candidate S-nitrosylated and 6 candidate tyrosine-nitrated phloem proteins. The wound-responsive Phloem Protein 16-1 (PP16-1) and Cyclophilin 18 (CYP18) as well as the 26.5 kD isoform of Phloem Protein 2 (PP2) were amenable to both NO modifications and could represent important redox-sensors within the cucurbit EFP. We also found that leaf injury triggered the systemic accumulation of cyclic guanosine monophosphate (cGMP) in the EFP and discuss the possible function of this second messenger in systemic NO and redox signaling within the EFP. PMID:26904092

  19. Inhibition of Phosphodiesterase 2 Augments cGMP and cAMP Signaling to Ameliorate Pulmonary Hypertension

    PubMed Central

    Bubb, Kristen J; Trinder, Sarah L; Baliga, Reshma S; Patel, Jigisha; Clapp, Lucie H; MacAllister, Raymond J; Hobbs, Adrian J

    2014-01-01

    Background Pulmonary hypertension (PH) is a life-threatening disorder characterized by increased pulmonary artery pressure, remodeling of the pulmonary vasculature, and right ventricular failure. Loss of endothelium-derived nitric oxide (NO) and prostacyclin (PGI2) contributes to PH pathogenesis and current therapies are targeted to restore these pathways. Phosphodiesterases (PDEs) are a family of enzymes that break down cGMP and cAMP which underpin the bioactivity of NO and PGI2. The PDE5 inhibitor (PDE5i) sildenafil is licensed for PH, but a role for PDE2 in lung physiology and disease has yet to be established. Herein, we investigated whether PDE2 inhibition modulates pulmonary cyclic nucleotide signaling and ameliorates experimental PH. Methods and Results The selective PDE2 inhibitor BAY 60-7550 augmented atrial natriuretic peptide (ANP) and treprostinil -evoked pulmonary vascular relaxation in isolated arteries from chronically hypoxic rats. BAY 60-7550 prevented the onset of both hypoxia- and bleomycin-induced PH, and produced a significantly greater reduction in disease severity when given in combination with a neutral endopeptidase inhibitor (enhances endogenous natriuretic peptides), the PGI2 analogue treprostinil, inorganic nitrate (NO donor), or a PDE5i. Proliferation of pulmonary artery smooth muscle cells from PAH patients was reduced by BAY 60-7550, an effect further enhanced in the presence of ANP, NO and treprostinil. Conclusions PDE2 inhibition elicits pulmonary dilation, prevents pulmonary vascular remodeling, and reduces the RVH characteristic of PH. This favorable pharmacodynamic profile is dependent on natriuretic peptide bioactivity, and is additive with PGI2 analogues, PDE5i, and NO. PDE2 inhibition represents a viable, orally-active therapy for PH. PMID:24899690

  20. Activated G-protein releases cGMP from high affinity binding sites on PDE from toad rod outer segments (ROS)

    SciTech Connect

    Yuen, P.S.T.; Walseth, T.F.; Panter, S.S.; Sundby, S.R.; Graeff, R.M.; Goldberg, N.D.

    1987-05-01

    cGMP binding proteins in toad ROS were identified by direct photoaffinity labeling (PAL) with /sup 32/P-cGMP and quantified by retention of complexes on nitrocellulose filters. By PAL, high affinity sites were present on the ..cap alpha.. and ..beta.. subunits of the cGMP-specific phosphodiesterase (PDE) which have MW/sub app/ of 94 and 90 kDa. A doublet was deduced from its photolabeling properties to represent PDE/sub ..gamma../ photocrosslinked with PDE/sub ..cap alpha../ or PDE/sub ..beta../, respectively. cGMP prebound to these high affinity sites was released by light-activated G-protein or its ..cap alpha.. subunit complexed with GTP..gamma..S; this inhibition of cGMP binding to PDE did not result from decreased cGMP availability due to enhanced hydrolysis. A low affinity cGMP binding component identified by PAL is tightly associated with ROS membranes. Apparent ATP/light-dependent stimulation of cGMP binding was shown to result from light activated cGMP hydrolysis in conjunction with ATP-promoted conversion of GMP to GDP/GTP and increased GDP/GTP binding. These findings coincide with a model for light-related regulation of cGMP binding and metabolism predicted from intact and cellfree kinetic measurements: in the dark state the cGMP hydrolic rate is constrained by the availability of cGMP because of its binding to high affinity sites on PDE. Light activated G-protein releases cGMP from these sites and allows for its redistribution to lower affinity sites represented by PDE catalytic site(s) and possible cGMP-dependent membrane cation channels.

  1. Luteinizing Hormone Causes Phosphorylation and Activation of the cGMP Phosphodiesterase PDE5 in Rat Ovarian Follicles, Contributing, Together with PDE1 Activity, to the Resumption of Meiosis.

    PubMed

    Egbert, Jeremy R; Uliasz, Tracy F; Shuhaibar, Leia C; Geerts, Andreas; Wunder, Frank; Kleiman, Robin J; Humphrey, John M; Lampe, Paul D; Artemyev, Nikolai O; Rybalkin, Sergei D; Beavo, Joseph A; Movsesian, Matthew A; Jaffe, Laurinda A

    2016-05-01

    The meiotic cell cycle of mammalian oocytes in preovulatory follicles is held in prophase arrest by diffusion of cGMP from the surrounding granulosa cells into the oocyte. Luteinizing hormone (LH) then releases meiotic arrest by lowering cGMP in the granulosa cells. The LH-induced reduction of cGMP is caused in part by a decrease in guanylyl cyclase activity, but the observation that the cGMP phosphodiesterase PDE5 is phosphorylated during LH signaling suggests that an increase in PDE5 activity could also contribute. To investigate this idea, we measured cGMP-hydrolytic activity in rat ovarian follicles. Basal activity was due primarily to PDE1A and PDE5, and LH increased PDE5 activity. The increase in PDE5 activity was accompanied by phosphorylation of PDE5 at serine 92, a protein kinase A/G consensus site. Both the phosphorylation and the increase in activity were promoted by elevating cAMP and opposed by inhibiting protein kinase A, supporting the hypothesis that LH activates PDE5 by stimulating its phosphorylation by protein kinase A. Inhibition of PDE5 activity partially suppressed LH-induced meiotic resumption as indicated by nuclear envelope breakdown, but inhibition of both PDE5 and PDE1 activities was needed to completely inhibit this response. These results show that activities of both PDE5 and PDE1 contribute to the LH-induced resumption of meiosis in rat oocytes, and that phosphorylation and activation of PDE5 is a regulatory mechanism. PMID:27009040

  2. Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells.

    PubMed

    Pfannes, Eva K B; Anielski, Alexander; Gerhardt, Matthias; Beta, Carsten

    2013-12-01

    Cyclic GMP (cGMP) is a ubiquitous second messenger in eukaryotic cells. It is assumed to regulate the association of myosin II with the cytoskeleton of motile cells. When cells of the social amoeba Dictyostelium discoideum are exposed to chemoattractants or to increased osmotic stress, intracellular cGMP levels rise, preceding the accumulation of myosin II in the cell cortex. To directly investigate the impact of intracellular cGMP on cytoskeletal dynamics in a living cell, we released cGMP inside the cell by laser-induced photo-cleavage of a caged precursor. With this approach, we could directly show in a live cell experiment that an increase in intracellular cGMP indeed induces myosin II to accumulate in the cortex. Unexpectedly, we observed for the first time that also the amount of filamentous actin in the cell cortex increases upon a rise in the cGMP concentration, independently of cAMP receptor activation and signaling. We discuss our results in the light of recent work on the cGMP signaling pathway and suggest possible links between cGMP signaling and the actin system. PMID:24136144

  3. cAMP and cGMP Play an Essential Role in Galvanotaxis of Cell Fragments.

    PubMed

    Zhu, Kan; Sun, Yaohui; Miu, Anh; Yen, Michael; Liu, Bowei; Zeng, Qunli; Mogilner, Alex; Zhao, Min

    2016-06-01

    Cell fragments devoid of the nucleus and major organelles are found in physiology and pathology, for example platelets derived from megakaryocytes, and cell fragments from white blood cells and glioma cells. Platelets exhibit active chemotaxis. Fragments from white blood cells display chemotaxis, phagocytosis, and bactericidal functions. Signaling mechanisms underlying migration of cell fragments are poorly understood. Here we used fish keratocyte fragments and demonstrated striking differences in signal transduction in migration of cell fragments and parental cells in a weak electric field. cAMP or cGMP agonists completely abolished directional migration of fragments, but had no effect on parental cells. The inhibition effects were prevented by pre-incubating with cAMP and cGMP antagonists. Blocking cAMP and cGMP downstream signaling by inhibition of PKA and PKG also recovered fragment galvanotaxis. Both perturbations confirmed that the inhibitory effect was mediated by cAMP or cGMP signaling. Inhibition of cathode signaling with PI3K inhibitor LY294002 also prevented the effects of cAMP or cGMP agonists. Our results suggest that cAMP and cGMP are essential for galvanotaxis of cell fragments, in contrast to the signaling mechanisms in parental cells. PMID:26517849

  4. Phosphodiesterase 9A Controls Nitric-oxide Independent cGMP and Hypertrophic Heart Disease

    PubMed Central

    Lee, Dong I.; Zhu, Guangshuo; Sasaki, Takashi; Cho, Gun-Sik; Hamdani, Nazha; Holewinski, Ronald; Jo, Su-Hyun; Danner, Thomas; Zhang, Manling; Rainer, Peter P.; Bedja, Djahida; Kirk, Jonathan A.; Ranek, Mark J.; Dostmann, Wolfgang R.; Kwon, Chulan; Margulies, Kenneth B.; Van Eyk, Jennifer E.; Paulus, Walter J.; Takimoto, Eiki; Kass, David A.

    2015-01-01

    Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric oxide (NO) and natriuretic peptide (NP) coupled signaling, stimulating phosphorylation changes by protein kinase G (PKG). Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease1,2. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation3. Furthermore, though PDE5A regulates NO-generated cGMP4,5, NO-signaling is often depressed by heart disease6. PDEs controlling NP-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A7,8 is expressed in mammalian heart including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates NP rather than NO-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neuro-hormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of NO-synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phospho-proteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signaling independent of the NO-pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target. PMID:25799991

  5. cGMP-Dependent Protein Kinases and cGMP Phosphodiesterases in Nitric Oxide and cGMP Action

    PubMed Central

    Busch, Jennifer L.; Corbin, Jackie D.

    2010-01-01

    To date, studies suggest that biological signaling by nitric oxide (NO) is primarily mediated by cGMP, which is synthesized by NO-activated guanylyl cyclases and broken down by cyclic nucleotide phosphodiesterases (PDEs). Effects of cGMP occur through three main groups of cellular targets: cGMP-dependent protein kinases (PKGs), cGMP-gated cation channels, and PDEs. cGMP binding activates PKG, which phosphorylates serines and threonines on many cellular proteins, frequently resulting in changes in activity or function, subcellular localization, or regulatory features. The proteins that are so modified by PKG commonly regulate calcium homeostasis, calcium sensitivity of cellular proteins, platelet activation and adhesion, smooth muscle contraction, cardiac function, gene expression, feedback of the NO-signaling pathway, and other processes. Current therapies that have successfully targeted the NO-signaling pathway include nitrovasodilators (nitroglycerin), PDE5 inhibitors [sildenafil (Viagra and Revatio), vardenafil (Levitra), and tadalafil (Cialis and Adcirca)] for treatment of a number of vascular diseases including angina pectoris, erectile dysfunction, and pulmonary hypertension; the PDE3 inhibitors [cilostazol (Pletal) and milrinone (Primacor)] are used for treatment of intermittent claudication and acute heart failure, respectively. Potential for use of these medications in the treatment of other maladies continues to emerge. PMID:20716671

  6. Sulindac sulfide selectively inhibits growth and induces apoptosis of human breast tumor cells by PDE5 inhibition, elevation of cGMP, and activation of PKG

    PubMed Central

    Tinsley, Heather N.; Gary, Bernard D.; Keeton, Adam B.; Zhang, Wei; Abadi, Ashraf H.; Reynolds, Robert C.; Piazza, Gary A.

    2009-01-01

    Sulindac displays promising antineoplastic activity, but toxicities from cyclooxygenase (COX) inhibition limit its use for chemoprevention. Previous reports suggest that its anticancer properties may be attributed to a COX-independent mechanism, although alternative targets have not been well defined. Here we show that sulindac sulfide (SS) induces apoptosis and inhibits the growth of human breast tumor cells with IC50 values of 60-85 μM. Within the same concentration range, SS inhibited cGMP hydrolysis in tumor cell lysates, but did not affect cAMP hydrolysis. SS did not induce apoptosis of normal human mammary epithelial cells (HMEC), nor did it inhibit PDE activity in HMEC lysates. SS increased intracellular cGMP levels and activated protein kinase G in breast tumor cells, but not HMEC. The guanylyl cyclase (GC) activator, NOR-3, and cGMP PDE inhibitors, trequinsin and MY5445, displayed similar growth inhibitory activity as SS, but the adenylyl cyclase activator, forskolin, and other PDE inhibitors had no effect. Moreover, GC activation increased the sensitivity of tumor cells to SS, while GC inhibition reduced sensitivity. By comparing PDE isozyme profiles in breast tumor cells with HMEC and determining the sensitivity of recombinant PDE isozymes to SS, PDE5 was found to be overexpressed in breast tumor cells and selectively inhibited by SS. The mechanism of SS binding to the catalytic domain of PDE5 was revealed by molecular modeling. These data suggest that PDE5 inhibition is responsible for the breast tumor cell growth inhibitory and apoptosis inducing activity of SS and may contribute to the chemopreventive properties of sulindac. PMID:19996273

  7. From bedside to bench--meeting report of the 7th International Conference on cGMP "cGMP: generators, effectors and therapeutic implications" in Trier, Germany, from June 19th to 21st 2015.

    PubMed

    Friebe, Andreas; Sandner, Peter; Seifert, Roland

    2015-12-01

    During the past decade, our knowledge on the physiology, pathophysiology, basic pharmacology, and clinical pharmacology of the second messenger (cGMP) has increased tremendously. It is now well-established that cGMP, generated by soluble and particulate guanylate cyclases, is highly compartmentalized in cells and regulates numerous body functions. New cGMP-regulated physiological functions include meiosis and temperature perception. cGMP is involved in the genesis of numerous pathologies including cardiovascular, pulmonary, endocrine, metabolic, neuropsychiatric, eye, and tumor diseases. Several new clinical uses of stimulators and activators of soluble guanylate cyclase and of phosphodiesterase inhibitors such as heart failure, kidney failure, cognitive disorders, obesity bronchial asthma, and osteoporosis are emerging. The combination of neprilysin inhibitors-enhancing stimulation of the particulate guanylate cyclase pathway by preventing natriuretic peptide degradation-with angiotensin AT1 receptor antagonists constitutes a novel promising strategy for heart failure treatment. The role of oxidative stress in cGMP signaling, application of cGMP sensors, and gene therapy for degenerative eye diseases are emerging topics. It is anticipated that cGMP research will further prosper over the next years and reach out into more and more basic and clinical disciplines. PMID:26486926

  8. Angiotensin-converting enzyme inhibition prevents myocardial infarction-induced increase in renal cortical cGMP and cAMP phosphodiesterase activities.

    PubMed

    Clauss, François; Charloux, Anne; Piquard, François; Doutreleau, Stéphane; Talha, Samy; Zoll, Joffrey; Lugnier, Claire; Geny, Bernard

    2015-08-01

    We investigated whether myocardial infarction (MI) enhances renal phosphodiesterases (PDE) activities, investigating particularly the relative contribution of PDE1-5 isozymes in total PDE activity involved in both cGMP and cAMP pathways, and whether angiotensin-converting enzyme inhibition (ACEi) decreases such renal PDE hyperactivities. We also investigated whether ACEi might thereby improve atrial natriuretic peptide (ANP) efficiency. We studied renal cortical PDE1-5 isozyme activities in sham (SH)-operated, MI rats and in MI rats treated with perindopril (ACEi) 1 month after coronary artery ligation. Circulating atrial natriuretic peptide (ANP), its second intracellular messenger cyclic guanosine monophosphate (cGMP) and cGMP/ANP ratio were also determined. Cortical cGMP-PDE2 (80.3 vs. 65.1 pmol/min/mg) and cGMP-PDE1 (50.7 vs. 30.1 pmol/min/mg), and cAMP-PDE2 (161 vs. 104.1 pmol/min/mg) and cAMP-PDE4 (307.5 vs. 197.2 pmol/min/mg) activities were higher in MI than in SH rats. Despite increased ANP plasma level, ANP efficiency tended to be decreased in MI compared to SH rats. Perindopril restored PDE activities and tended to improve ANP efficiency in MI rats. One month after coronary ligation, perindopril treatment of MI rats prevents the increase in renal cortical PDE activities. This may contribute to increase renal ANP efficiency in MI rats. PMID:25939307

  9. Cyclic GMP signaling in cardiovascular pathophysiology and therapeutics

    PubMed Central

    Tsai, Emily J.; Kass, David A.

    2009-01-01

    Cyclic guanosine 3′,5′-monophosphate (cGMP) mediates a wide spectrum of physiologic processes in multiple cell types within the cardiovascular system. Dysfunctional signaling at any step of the cascade--- cGMP synthesis, effector activation, or catabolism--- have been implicated in numerous cardiovascular diseases, ranging from hypertension to atherosclerosis to cardiac hypertrophy and heart failure. In this review, we outline each step of the cGMP signaling cascade and discuss its regulation and physiologic effects within the cardiovascular system. In addition, we illustrate how cGMP signaling becomes dysregulated in specific cardiovascular disease states. The ubiquitous role cGMP plays in cardiac physiology and pathophysiology presents great opportunities for pharmacologic modulation of the cGMP signal in the treatment of cardiovascular diseases. We detail the various therapeutic interventional strategies that have been developed or are in development, summarizing relevant preclinical and clinical studies. PMID:19306895

  10. Hydrolysis of N-methyl-D-aspartate receptor-stimulated cAMP and cGMP by PDE4 and PDE2 phosphodiesterases in primary neuronal cultures of rat cerebral cortex and hippocampus.

    PubMed

    Suvarna, Neesha U; O'Donnell, James M

    2002-07-01

    Stimulation of N-methyl-D-aspartate (NMDA) receptors on neurons activates both cAMP and cGMP signaling pathways. Experiments were carried out to determine which phosphodiesterase (PDE) families are involved in the hydrolysis of the cyclic nucleotides formed via this mechanism, using primary neuronal cultures prepared from rat cerebral cortex and hippocampus. The nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) potentiated the ability of NMDA to increase cAMP and cGMP. However, among the family-selective inhibitors, only the PDE4 inhibitor rolipram enhanced the ability of NMDA to increase cAMP in the neurons. In contrast, only the PDE2 inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) enhanced the ability of NMDA to increase cGMP. Neither adenosine nor an adenosine deaminase inhibitor mimicked the effect of EHNA; this suggests that EHNA's inhibition of PDE2, not its effects on adenosine metabolism, mediates its effects on NMDA-stimulated cGMP concentrations. The PDE inhibitor-augmented effects of NMDA on cAMP and cGMP formation were antagonized by 5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate (MK-801), verifying NMDA receptor mediation. In contrast, only NMDA-mediated cGMP formation was affected by altering either nitric oxide signaling or guanylyl cyclase; this suggests that NMDA-induced changes in cAMP are not secondary to altered cGMP concentrations. Overall, the present findings indicate that cAMP and cGMP formed in neurons as a result of NMDA receptor stimulation are hydrolyzed by PDE4 and PDE2, respectively. Selective inhibitors of the two PDE families will differentially affect the functional consequences of activation of these two signaling pathways by NMDA receptor stimulation. PMID:12065724

  11. Improved genetically-encoded, FlincG-type fluorescent biosensors for neural cGMP imaging

    PubMed Central

    Bhargava, Yogesh; Hampden-Smith, Kathryn; Chachlaki, Konstantina; Wood, Katherine C.; Vernon, Jeffrey; Allerston, Charles K.; Batchelor, Andrew M.; Garthwaite, John

    2013-01-01

    Genetically-encoded biosensors are powerful tools for understanding cellular signal transduction mechanisms. In aiming to investigate cGMP signaling in neurones using the EGFP-based fluorescent biosensor, FlincG (fluorescent indicator for cGMP), we encountered weak or non-existent fluorescence after attempted transfection with plasmid DNA, even in HEK293T cells. Adenoviral infection of HEK293T cells with FlincG, however, had previously proved successful. Both constructs were found to harbor a mutation in the EGFP domain and had a tail of 17 amino acids at the C-terminus that differed from the published sequence. These discrepancies were systematically examined, together with mutations found beneficial for the related GCaMP family of Ca2+ biosensors, in a HEK293T cell line stably expressing both nitric oxide (NO)-activated guanylyl cyclase and phosphodiesterase-5. Restoring the mutated amino acid improved basal fluorescence whereas additional restoration of the correct C-terminal tail resulted in poor cGMP sensing as assessed by superfusion of either 8-bromo-cGMP or NO. Ultimately, two improved FlincGs were identified: one (FlincG2) had the divergent tail and gave moderate basal fluorescence and cGMP response amplitude and the other (FlincG3) had the correct tail, a GCaMP-like mutation in the EGFP region and an N-terminal tag, and was superior in both respects. All variants tested were strongly influenced by pH over the physiological range, in common with other EGFP-based biosensors. Purified FlincG3 protein exhibited a lower cGMP affinity (0.89 μM) than reported for the original FlincG (0.17 μM) but retained rapid kinetics and a 230-fold selectivity over cAMP. Successful expression of FlincG2 or FlincG3 in differentiated N1E-115 neuroblastoma cells and in primary cultures of hippocampal and dorsal root ganglion cells commends them for real-time imaging of cGMP dynamics in neural (and other) cells, and in their subcellular specializations. PMID:24068983

  12. Ursolic acid from Agastache mexicana aerial parts produces antinociceptive activity involving TRPV1 receptors, cGMP and a serotonergic synergism.

    PubMed

    Verano, Jazmín; González-Trujano, Ma Eva; Déciga-Campos, Myrna; Ventura-Martínez, Rosa; Pellicer, Francisco

    2013-09-01

    Agastache mexicana is a plant that has long been used in large demands in Mexican folk medicine to treat anxiety, insomnia and pain, among others affections. Chromatographic technique was used to identify ursolic acid (UA), 130.7 mg/g and 20.3 mg/g, as an antinociceptive active compound identified in ethyl acetate and methanol extracts of A. mexicana aerial parts, respectively. Temporal course curves of the antinociceptive response demonstrated a dose-dependent and significant activity of UA (1 to 100 mg/kg, i.p.) with an ED50=2 mg/kg in comparison to the efficacy of diclofenac (1 or 30 to 100 mg/kg, i.p.), a non-steroidal anti-inflammatory drug, with an ED50=11.56 mg/kg. The antinociceptive response consisted in the reduction of abdominal constrictions induced with 1% acetic acid in mice. Similarly, UA at 2 mg/kg produced significant antinociception in the intracolonic administration of 0.3% capsaicin (a TRPV1 agonist) in mice. It has been reported the inhibition produced by UA on the calcium-flux induced by capsaicin on TRPV1 receptor suggesting the antagonistic activity of this receptor. Finally, an ED50=44 mg/kg was calculated in the neurogenic and inflammatory nociception induced in the formalin test in rats. The antinociceptive response of UA in the formalin test was not modified in presence of naloxone, flumazenil or L-arginine. Nevertheless, it was reverted in presence of 1-H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ, an inhibitor of soluble guanylyl cyclase) and increased in presence of N(G)-L-nitro-arginine methyl ester (L-NAME, inhibitor of nitric oxide synthase), theophylline (inhibitor of phosphodiesterase) and WAY100635 (an antagonist of 5-HT1A receptors). Current results provide evidence that the antinociceptive response of A. mexicana depends in part on the presence of UA. Moreover, this triterpene may exerts its antinociceptive effect mediated by the presence of cGMP and an additive synergism with 5HT1A receptors, but also an antagonistic

  13. Antithrombotic activities of ferulic acid via intracellular cyclic nucleotide signaling.

    PubMed

    Hong, Qian; Ma, Zeng-Chun; Huang, Hao; Wang, Yu-Guang; Tan, Hong-Ling; Xiao, Cheng-Rong; Liang, Qian-De; Zhang, Han-Ting; Gao, Yue

    2016-04-15

    Ferulic acid (FA) produces protective effects against cardiovascular dysfunctions. However, the mechanisms of FA is still not known. Here we examined the antithrombotic effects of FA and its potential mechanisms. Anticoagulation assays and platelet aggregation was evaluated in vitro and in vivo. Thromboxane B2 (TXB2), cyclic adenosine monophosphate(cAMP), and cyclic guanosine monophosphate (cGMP) was determined using enzyme immunoassay kits. Nitric oxide (NO) production was measured using the Griess reaction. Protein expression was detected by Western blotting analysis. Oral administration of FA prevented death caused by pulmonary thrombosis and prolonged the tail bleeding and clotting time in mice,while, it did not alter the coagulation parameters, including the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). In addition, FA (50-200µM) dose-dependently inhibited platelet aggregation induced by various platelet agonists, including adenosine diphosphate (ADP), thrombin, collagen, arachidonic acid (AA), and U46619. Further, FA attenuated intracellular Ca(2)(+) mobilization and TXB2 production induced by the platelet agonists. FA increased the levels of cAMP and cGMP and phosphorylated vasodilator-stimulated phosphoprotein (VASP) while decreased phospho-MAPK (mitogen-activated protein kinase) and phosphodiesterase (PDE) in washed rat platelets, VASP is a substrate of cyclic nucleotide and PDE is an enzyme family responsible for hydrolysis of cAMP/cGMP. These results suggest that antithrombotic activities of FA may be regulated by inhibition of platelet aggregation, rather than through inhibiting the release of thromboplastin or formation of thrombin. The mechanism of this action may involve activation of cAMP and cGMP signaling. PMID:26948317

  14. Atrial natriuretic peptide and oxytocin induce natriuresis by release of cGMP

    PubMed Central

    Soares, T. J.; Coimbra, T. M.; Martins, A. R.; Pereira, A. G. F.; Carnio, E. C.; Branco, L. G. S.; Albuquerque-Araujo, W. I. C.; de Nucci, G.; Favaretto, A. L. V.; Gutkowska, J.; McCann, S. M.; Antunes-Rodrigues, J.

    1999-01-01

    Our hypothesis is that oxytocin (OT) causes natriuresis by activation of renal NO synthase that releases NO followed by cGMP that mediates the natriuresis. To test this hypothesis, an inhibitor of NO synthase, l-nitroarginine methyl ester (NAME), was injected into male rats. Blockade of NO release by NAME had no effect on natriuresis induced by atrial natriuretic peptide (ANP). This natriuresis presumably is caused by cGMP because ANP also activates guanylyl cyclase, which synthesizes cGMP from GTP. The 18-fold increase in sodium (Na+) excretion induced by OT (1 μg) was accompanied by an increase in urinary cGMP and preceded by 20 min a 20-fold increase in NO3− excretion. NAME almost completely inhibited OT-induced natriuresis and increased NO3− excretion; however, when the dose of OT was increased 10-fold, a dose that markedly increases plasma ANP concentrations, NAME only partly inhibited the natriuresis. We conclude that the natriuretic action of OT is caused by a dual action: generation of NO leading to increased cGMP and at higher doses release of ANP that also releases cGMP. OT-induced natriuresis is caused mainly by decreased tubular Na+ reabsorption mediated by cGMP. In contrast to ANP that releases cGMP in the renal vessels and the tubules, OT acts on its receptors on NOergic cells demonstrated in the macula densa and proximal tubules to release cGMP that closes Na+ channels. Both ANP- and OT-induced kaliuresis also appear to be mediated by cGMP. We conclude that cGMP mediates natriuresis and kaliuresis induced by both ANP and OT. PMID:9874809

  15. cGMP Is Required for Gibberellic Acid-Induced Gene Expression in Barley Aleurone.

    PubMed Central

    Penson, S. P.; Schuurink, R. C.; Fath, A.; Gubler, F.; Jacobsen, J. V.; Jones, R. L.

    1996-01-01

    The occurrence and roles of cGMP were investigated in aleurone layers and protoplasts isolated from barley (cv Himalaya) grain. Levels of cGMP in freshly isolated barley aleurone layers ranged from 0.065 to 0.08 pmol/g fresh weight of tissue, and cGMP levels increased transiently after incubation in gibberellic acid (GA). Abscisic acid (ABA) did not increase cGMP levels in aleurone layers. LY 83583 (LY), an inhibitor of guanylyl cyclase, prevented the GA-induced increase in cGMP and inhibited GA-induced [alpha]-amylase synthesis and secretion. The inhibitory effects of LY could be overcome by membrane-permeant analogs of cGMP. LY also prevented GA-induced accumulation of [alpha]-amylase and GAMYB mRNAs. cGMP alone was not sufficient to induce the accumulation of [alpha]-amylase or GAMYB mRNA. LY had a less dramatic effect on the accumulation of mRNAs encoding the ABA-responsive gene Rab21. We conclude that cGMP plays an important role in GA, but not ABA, signaling in the barley aleurone cell. PMID:12239379

  16. Insulin, cGMP, and TGF-beta signals regulate food intake and quiescence in C. elegans: a model for satiety.

    PubMed

    You, Young-jai; Kim, Jeongho; Raizen, David M; Avery, Leon

    2008-03-01

    Despite the prevalence of obesity and its related diseases, the signaling pathways for appetite control and satiety are not clearly understood. Here we report C. elegans quiescence behavior, a cessation of food intake and movement that is possibly a result of satiety. C. elegans quiescence shares several characteristics of satiety in mammals. It is induced by high-quality food, it requires nutritional signals from the intestine, and it depends on prior feeding history: fasting enhances quiescence after refeeding. During refeeding after fasting, quiescence is evoked, causing gradual inhibition of food intake and movement, mimicking the behavioral sequence of satiety in mammals. Based on these similarities, we propose that quiescence results from satiety. This hypothesized satiety-induced quiescence is regulated by peptide signals such as insulin and TGF-beta. The EGL-4 cGMP-dependent protein kinase functions downstream of insulin and TGF-beta in sensory neurons including ASI to control quiescence in response to food intake. PMID:18316030

  17. cGMP Binding Sites on Photoreceptor Phosphodiesterase: Role in Feedback Regulation of Visual Transduction

    NASA Astrophysics Data System (ADS)

    Cote, Rick H.; Deric Bownds, M.; Arshavsky, Vadim Y.

    1994-05-01

    A central step in vertebrate visual transduction is the rapid drop in cGMP levels that causes cGMP-gated ion channels in the photoreceptor cell membrane to close. It has long been a puzzle that the cGMP phosphodiesterase (PDE) whose activation causes this decrease contains not only catalytic sites for cGMP hydrolysis but also noncatalytic cGMP binding sites. Recent work has shown that occupancy of these noncatalytic sites slows the rate of PDE inactivation. We report here that PDE activation induced by activated transducin lowers the cGMP binding affinity for noncatalytic sites on PDE and accelerates the dissociation of cGMP from these sites. These sites can exist in three states: high affinity (K_d = 60 nM) for the nonactivated PDE, intermediate affinity (K_d ≈ 180 nM) when the enzyme is activated in a complex with transducin, and low affinity (K_d > 1 μM) when transducin physically removes the inhibitory subunits of PDE from the PDE catalytic subunits. Activation of PDE by transducin causes a 10-fold increase in the rate of cGMP dissociation from one of the two noncatalytic sites; physical removal of the inhibitory subunits from the PDE catalytic subunits further accelerates the cGMP dissociation rate from both sites >50-fold. Because PDE molecules lacking bound cGMP inactivate more rapidly, this suggests that a prolonged cGMP decrease may act as a negative feedback regulator to generate the faster, smaller photoresponses characteristic of light-adapted photoreceptors.

  18. Modulation of cGMP in Heart Failure

    PubMed Central

    Boerrigter, Guido; Lapp, Harald; Burnett, John C.

    2009-01-01

    Heart failure (HF) is a common disease that continues to be associated with high morbidity and mortality warranting novel therapeutic strategies. Cyclic guanosine monophosphate (cGMP) is the second messenger of several important signaling pathways based on distinct guanylate cyclases (GCs) in the cardiovascular system. Both the nitric oxide/soluble GC (NO/sGC) as well as the natriuretic peptide/GC-A (NP/GC-A) systems are disordered in HF, providing a rationale for their therapeutic augmentation. Soluble GC activation with conventional nitrovasodilators has been used for more than a century but is associated with cGMP-independent actions and the development of tolerance, actions which novel NO-independent sGC activators now in clinical development lack. Activation of GC-A by administration of naturally occurring or designer natriuretic peptides is an emerging field, as is the inhibition of enzymes that degrade endogenous NPs. Finally, inhibition of cGMP-degrading phosphodiesterases, particularly phosphodiesterase 5 provides an additional strategy to augment cGMP-signaling. PMID:19089342

  19. Light regulation of cGMP metabolism in toad rod outer segments (ROS) deduced from intact photoreceptor and cellfree kinetics

    SciTech Connect

    Dawis, S.M.; Graeff, R.M.; Heyman, R.A.; Walseth, T.F.; Butz, E.A.

    1987-05-01

    The rate of cGMP hydrolysis by phosphodiesterase (PDE) in intact ROS, monitored in dark-adapted isolated toad retina by the rate of /sup 18/O appearance in guanine nucleotide ..cap alpha..-phosphoryls, is 1/360th of that observed in disrupted ROS at a substrate concentration equivalent to the total (cGMP) in ROS. Low to moderate photic stimuli increase this cGMP hydrolytic rate up to 10-fold in intact ROS with little or no change in total (cGMP). G-protein activation determined in intact ROS by the fraction of GDP labeled with /sup 18/O corresponds with light-related increases in cGMP flux. In contrast, relatively high intensities and extended illumination cause attenuation of maximal cGMP hydrolysis with proportionate reductions in total (cGMP). From these observations combined with the effects of activated G-protein on kinetics and cGMP binding of ROS PDE the following model for light-regulation of cGMP metabolism was deduced: cGMP flux in intact ROS is severely restricted in the dark state because approximately 99% of the cGMP is bound to high affinity sites on the non-stimulated form of PDE. This constraint is relieved when activated G-protein converts the cGMP-binding form of PDE to a high K/sub m/ catalytic form. cGMP is then redistributed to a dynamic pool where it is available to PDE catalytic sites and lower affinity allosteric sites. The (cGMP) in the dynamic pool is maintained or further increased or decreased by modulating the activity of an apparently light-sensitive guanylyl cyclase.

  20. MRP4 Modulation of the Guanylate Cyclase-C/cGMP Pathway: Effects on Linaclotide-Induced Electrolyte Secretion and cGMP Efflux.

    PubMed

    Tchernychev, Boris; Ge, Pei; Kessler, Marco M; Solinga, Robert M; Wachtel, Derek; Tobin, Jenny V; Thomas, Sara R; Lunte, Craig E; Fretzen, Angelika; Hannig, Gerhard; Bryant, Alexander P; Kurtz, Caroline B; Currie, Mark G; Silos-Santiago, Inmaculada

    2015-10-01

    MRP4 mediates the efflux of cGMP and cAMP and acts as an important regulator of these secondary messengers, thereby affecting signaling events mediated by cGMP and cAMP. Immunofluorescence staining showed high MRP4 expression localized predominantly in the apical membrane of rat colonic epithelium. In vitro studies were performed using a rat colonic mucosal layer mounted in an Ussing chamber. Linaclotide activation of the guanylate cyclase-C (GC-C)/cGMP pathway induced a concentration-dependent increase in transepithelial ion current [short-circuit current (Isc)] across rat colonic mucosa (EC50: 9.2 nM). Pretreatment of colonic mucosa with the specific MRP4 inhibitor MK571 potentiated linaclotide-induced electrolyte secretion and augmented linaclotide-stimulated intracellular cGMP accumulation. Notably, pretreatment with the phosphodiesterase 5 inhibitor sildenafil increased basal Isc, but had no amplifying effect on linaclotide-induced Isc. MRP4 inhibition selectively affected the activation phase, but not the deactivation phase, of linaclotide. In contrast, incubation with a GC-C/Fc chimera binding to linaclotide abrogated linaclotide-induced Isc, returning to baseline. Furthermore, linaclotide activation of GC-C induced cGMP secretion from the apical and basolateral membranes of colonic epithelium. MRP4 inhibition blocked cGMP efflux from the apical membrane, but not the basolateral membrane. These data reveal a novel, previously unrecognized mechanism that functionally couples GC-C-induced luminal electrolyte transport and cGMP secretion to spatially restricted, compartmentalized regulation by MRP4 at the apical membrane of intestinal epithelium. These findings have important implications for gastrointestinal disorders with symptoms associated with dysregulated fluid homeostasis, such as irritable bowel syndrome with constipation, chronic idiopathic constipation, and secretory diarrhea. PMID:26216942

  1. Design of CGMP Production of 18F- and 68Ga-Radiopharmaceuticals

    PubMed Central

    Chu, Pei-Chun; Chao, Hao-Yu; Shieh, Wei-Chen; Chen, Chuck C.

    2014-01-01

    Objective. Radiopharmaceutical production process must adhere to current good manufacturing process (CGMP) compliance to ensure the quality of precursor, prodrug (active pharmaceutical ingredient, API), and the final drug product that meet acceptance criteria. We aimed to develop an automated system for production of CGMP grade of PET radiopharmaceuticals. Methods. The hardware and software of the automated synthesizer that fit in the hot cell under cGMP requirement were developed. Examples of production yield and purity for 68Ga-DOTATATE and 18F-FDG at CGMP facility were optimized. Analytical assays and acceptance criteria for cGMP grade of 68Ga-DOTATATE and 18F-FDG were established. Results. CGMP facility for the production of PET radiopharmaceuticals has been established. Radio-TLC and HPLC analyses of 68Ga-DOTATATE and 18F-FDG showed that the radiochemical purity was 92% and 96%, respectively. The products were sterile and pyrogenic-free. Conclusion. CGMP compliance of radiopharmaceuticals has been reviewed. 68Ga-DOTATATE and 18F-FDG were synthesized with high radiochemical yield under CGMP process. PMID:25276810

  2. Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp

    PubMed Central

    Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W.; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

    2015-01-01

    Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s−1). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals. PMID:26345128

  3. Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp.

    PubMed

    Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

    2015-01-01

    Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s(-1)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals. PMID:26345128

  4. Hyperpolarization-activated ion channels as targets for nitric oxide signalling in deep cerebellar nuclei

    PubMed Central

    Wilson, Gary W; Garthwaite, John

    2010-01-01

    Most biological effects of nitric oxide (NO) in the brain are mediated by guanylyl cyclase-coupled NO receptors, whose activation results in increased intracellular cGMP levels. Apart from protein kinase activation little is known about subsequent cGMP signal transduction. In optic nerve axons, hyperpolarization-activated cyclic nucleotide-modulated cation (HCN) channels, which bind cGMP or cAMP directly, were recently suggested to be a target. The aim here was to test this possibility more directly. Neurones of the rat deep cerebellar nuclei were selected for this purpose, their suitability being attested by immunocytochemistry showing that the principal neurones expressed guanylyl cyclase protein and that NO synthase-containing fibres were abundant in the neuropil. Using whole-cell voltage-clamp recording, HCN channels in the neurones were activated in response to isoprenaline and exogenous cAMP but only occasionally did they respond to NO, although exogenous cGMP was routinely effective. With the less invasive sharp microelectrode recording technique, however, exogenous NO modulated the channels reproducibly, as measured by the size of the HCN channel-mediated voltage sag following hyperpolarization. Moreover, NO also blunted the subsequent rebound depolarizing potentials, consistent with it increasing the hyperpolarization-activated current. Optimizing the whole-cell solution to improve the functioning of NO-activated guanylyl cyclase failed to restore NO sensitivity. Minimizing cellular dialysis by using the perforated-patch technique, however, was successful. The results provide evidence that HCN channels are potential downstream mediators of NO signalling in deep cerebellar nuclei neurones and suggest that the more general importance of this transduction pathway may have been overlooked previously because of unsuitable recording methods. PMID:20529121

  5. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum

    SciTech Connect

    Bredt, D.S.; Snyder, S.H. )

    1989-11-01

    Nitric oxide, which mediates influences of numerous neurotransmitters and modulators on vascular smooth muscle and leukocytes, can be formed in the brain from arginine by an enzymatic activity that stoichiometrically generates citrulline. The authors show that glutamate and related amino acids, such as N-methyl-D-aspartate, markedly stimulate arginine-citrulline transformation in cerebellar slices stoichiometrically with enhancement of cGMP levels. N{sup {omega}}-monomethyl-L-arginine blocks the augmentation both of citrulline and cGMP with identical potencies. Arginine competitively reverses both effects of N{sup {omega}}-monomethyl-L-arginine with the same potencies. Hemoglobin, which complexes nitric oxide, prevents the stimulation by N-methyl-D-aspartate of cGMP levels, and superoxide dismutase, which elevates nitric oxide levels, increases cGMP formation. These data establish that nitric oxide mediates the stimulation by glutamate of cGMP formation.

  6. Nitric Oxide Mediates Glutamate-Linked Enhancement of cGMP Levels in the Cerebellum

    NASA Astrophysics Data System (ADS)

    Bredt, David S.; Snyder, Solomon H.

    1989-11-01

    Nitric oxide, which mediates influences of numerous neurotransmitters and modulators on vascular smooth muscle and leukocytes, can be formed in the brain from arginine by an enzymatic activity that stoichiometrically generates citrulline. We show that glutamate and related amino acids, such as N-methyl-D-aspartate, markedly stimulate arginine-citrulline transformation in cerebellar slices stoichiometrically with enhancement of cGMP levels. Nω-monomethyl-L-arginine blocks the augmentation both of citrulline and cGMP with identical potencies. Arginine competitively reverses both effects of Nω-monomethyl-L-arginine with the same potencies. Hemoglobin, which complexes nitric oxide, prevents the stimulation by N-methyl-D-aspartate of cGMP levels, and superoxide dismutase, which elevates nitric oxide levels, increases cGMP formation. These data establish that nitric oxide mediates the stimulation by glutamate of cGMP formation.

  7. Advances and techniques to measure cGMP in intact cardiomyocytes.

    PubMed

    Götz, Konrad R; Nikolaev, Viacheslav O

    2013-01-01

    Förster resonance energy transfer (FRET)-based biosensors are powerful tools for real-time monitoring of signaling events in intact cells using fluorescence microscopy. Here, we describe a highly sensitive method which allows FRET-based measurements of the second messenger cGMP in adult mouse ventricular myocytes. Such measurements have been challenging before, primarily due to relatively low cGMP concentrations in cardiomyocytes and limited sensitivity of the available biosensors. With our new technique, one can reliably measure dynamic changes in cGMP upon stimulation of myocytes with natriuretic peptides and other physiological and pharmacological ligands. PMID:23709029

  8. Modulation of cGMP accumulation by adenosine A1 receptors at the hippocampus: influence of cGMP levels and gender.

    PubMed

    Serpa, André; Sebastião, Ana M; Cascalheira, José F

    2014-12-01

    Adenosine A1 receptor is highly expressed in hippocampus where it inhibits neurotransmitter release and has neuroprotective activity. Similar actions are obtained by increasing cGMP concentration, but a clear link between adenosine A1 receptor and cGMP levels remains to be established. The present work aims to investigate if cGMP formation is modulated by adenosine A1 receptors at the hippocampus and if this effect is gender dependent. cGMP accumulation, induced by phosphodiesterases inhibitors Zaprinast (100 μM) and Bay 60-7550 (10 μM), and cAMP accumulation, induced by Forskolin (20 μM) and Rolipram (50 μM), were quantified in rat hippocampal slices using specific enzymatic immunoassays. N6-cyclopentyladenosine (CPA, 100 nM) alone failed to modify basal cGMP accumulation. However, the presence of adenosine deaminase (ADA, 2 U/ml) unmasked a CPA (0.03-300 nM) stimulatory effect on basal cGMP accumulation (EC50: 4.2±1.4 nM; Emax: 17±0.9%). ADA influence on CPA activity was specific for cGMP, since inhibition of cAMP accumulation by CPA was not affected by the presence of ADA, though ADA inhibited cAMP accumulation in the absence of CPA. Increasing cGMP accumulation, by about four-fold, with sodium nitroprusside (SNP, 100 μM) abolished the CPA (100 nM) effect on cGMP accumulation in males but did not modify the effect of CPA in female rats. This effect was reversed by 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nM), indicating an adenosine A1 receptor mediated effect on cGMP accumulation. In conclusion, adenosine A1 receptors increase intracellular cGMP formation at hippocampus both in males and females under basal conditions, but only in females when cGMP levels are increased by SNP. PMID:25300679

  9. cGMP in mouse rods: the spatiotemporal dynamics underlying single photon responses

    PubMed Central

    Pugh Jr., Edward N.; Burns, Marie E.

    2015-01-01

    Vertebrate vision begins when retinal photoreceptors transduce photons into electrical signals that are then relayed to other neurons in the eye, and ultimately to the brain. In rod photoreceptors, transduction of single photons is achieved by a well-understood G-protein cascade that modulates cGMP levels, and in turn, cGMP-sensitive inward current. The spatial extent and depth of the decline in cGMP during the single photon response (SPR) have been major issues in phototransduction research since the discovery that single photons elicit substantial and reproducible changes in membrane current. The spatial profile of cGMP decline during the SPR affects signal gain, and thus may contribute to reduction of trial-to-trial fluctuations in the SPR. Here we summarize the general principles of rod phototransduction, emphasizing recent advances in resolving the spatiotemporal dynamics of cGMP during the SPR. PMID:25788876

  10. The NO/cGMP pathway inhibits transient cAMP signals through the activation of PDE2 in striatal neurons

    PubMed Central

    Polito, Marina; Klarenbeek, Jeffrey; Jalink, Kees; Paupardin-Tritsch, Danièle; Vincent, Pierre; Castro, Liliana R.V.

    2013-01-01

    The NO-cGMP signaling plays an important role in the regulation of striatal function although the mechanisms of action of cGMP specifically in medium spiny neurons (MSNs) remain unclear. Using genetically encoded fluorescent biosensors, including a novel Epac-based sensor (EPAC-SH150) with increased sensitivity for cAMP, we analyze the cGMP response to NO and whether it affected cAMP/PKA signaling in MSNs. The Cygnet2 sensor for cGMP reported large responses to NO donors in both striatonigral and striatopallidal MSNs, this cGMP signal was controlled partially by PDE2. At the level of cAMP brief forskolin stimulations produced transient cAMP signals which differed between D1 and D2 MSNs. NO inhibited these cAMP transients through cGMP-dependent PDE2 activation, an effect that was translated and magnified downstream of cAMP, at the level of PKA. PDE2 thus appears as a critical effector of NO which modulates the post-synaptic response of MSNs to dopaminergic transmission. PMID:24302895

  11. A sperm-activating peptide controls a cGMP-signaling pathway in starfish sperm.

    PubMed

    Matsumoto, Midori; Solzin, Johannes; Helbig, Annika; Hagen, Volker; Ueno, Sei-ichi; Kawase, Osamu; Maruyama, Yoshinori; Ogiso, Manabu; Godde, Matthias; Minakata, Hiroyuki; Kaupp, U Benjamin; Hoshi, Motonori; Weyand, Ingo

    2003-08-15

    Peptides released from eggs of marine invertebrates play a central role in fertilization. About 80 different peptides from various phyla have been isolated, however, with one exception, their respective receptors on the sperm surface have not been unequivocally identified and the pertinent signaling pathways remain ill defined. Using rapid mixing techniques and novel membrane-permeable caged compounds of cyclic nucleotides, we show that the sperm-activating peptide asterosap evokes a fast and transient increase of the cGMP concentration in sperm of the starfish Asterias amurensis, followed by a transient cGMP-stimulated increase in the Ca(2+) concentration. In contrast, cAMP levels did not change significantly and the Ca(2+) response evoked by photolysis of caged cAMP was significantly smaller than that using caged cGMP. By cloning of cDNA and chemical crosslinking, we identified a receptor-type guanylyl cyclase in the sperm flagellum as the asterosap-binding protein. Sperm respond exquisitely sensitive to picomolar concentrations of asterosap, suggesting that the peptide serves a chemosensory function like resact, a peptide involved in chemotaxis of sperm of the sea urchin Arbacia punctulata. A unifying principle emerges that chemosensory transduction in sperm of marine invertebrates uses cGMP as the primary messenger, although there may be variations in the detail. PMID:12921734

  12. cGMP in ozone and NO dependent responses

    PubMed Central

    Ederli, Luisa; Meier, Stuart; Borgogni, Andrea; Reale, Lara; Ferranti, Francesco; Gehring, Chris

    2008-01-01

    We have recently reported that ozone (O3) can inhibit mitochondrial respiration and induce activation of the alternative oxidase (AOX) pathway and in particular AOX1a in tobacco. While O3 causes mitochondrial H2O2, early leaf nitric oxide (NO) as well as transient ethylene (ET) accumulation, the levels of jasmonic acid and 12-oxo-phytodienoic acid remained unchanged. It was shown that both, NO and ET dependent pathways can induce AOX1a transcription by O3. AOX plays a role in reducing reactive oxygen species (ROS) which in turn are linked to biotic and abiotic plant stresses, much like the second messengers guanosine 3′, 5′-cyclic monophosphate (cGMP). The goal is to unravel specific cGMP signatures and induction pathways downstream from O3 and NO, including transcription of AOX1a. Here we propose that some late (>3 h) responses to NO, e.g., the accumulation of phenylalanine lyase (PAL) transcripts, are critically cGMP dependent, while the early (<2 h) responses, including AOX1a induction are not. PMID:19704720

  13. Biophysical techniques for detection of cAMP and cGMP in living cells.

    PubMed

    Sprenger, Julia U; Nikolaev, Viacheslav O

    2013-01-01

    Cyclic nucleotides cAMP and cGMP are ubiquitous second messengers which regulate myriads of functions in virtually all eukaryotic cells. Their intracellular effects are often mediated via discrete subcellular signaling microdomains. In this review, we will discuss state-of-the-art techniques to measure cAMP and cGMP in biological samples with a particular focus on live cell imaging approaches, which allow their detection with high temporal and spatial resolution in living cells and tissues. Finally, we will describe how these techniques can be applied to the analysis of second messenger dynamics in subcellular signaling microdomains. PMID:23584022

  14. Auxin-induced nitric oxide, cGMP and gibberellins were involved in the gravitropism

    NASA Astrophysics Data System (ADS)

    Cai, Weiming; Hu, Liwei; Hu, Xiangyang; Cui, Dayong; Cai, Weiming

    Gravitropism is the asymmetric growth or curvature of plant organs in response to gravistimulation. There is a complex signal transduction cascade which involved in the differential growth of plants in response to changes in the gravity vector. The role of auxin in gravitropism has been demonstrated by many experiments, but little is known regarding the molecular details of such effects. In our studies before, mediation of the gravitropic bending of soybean roots and rice leaf sheath bases by nitric oxide, cGMP and gibberellins, are induced by auxin. The asymmetrical distribution of nitric oxide, cGMP and gibberellins resulted from the asymmetrical synthesis of them in bending sites. In soybean roots, inhibitions of NO and cGMP synthesis reduced differential NO and cGMP accumulation respectively, which both of these effects can lead to the reduction of gravitropic bending. Gibberellin-induced OsXET, OsEXPA4 and OsRWC3 were also found involved in the gravitropic bending. These data indicated that auxin-induced nitric oxide, cGMP and gibberellins were involved in the gravitropism. More experiments need to prove the more detailed mechanism of them.

  15. Cardioprotective cGMP favors exogenous fatty acid incorporation into tyiglycerides over direct beta-oxidation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While cardiac hypertrophy has been associated with a shift in substrate selection for energy production from fatty acids (FA) to carbohydrates (CHO), it remains controversial whether this shift is adaptive or maladaptive. Since enhanced cGMP signalling can prevent hypertrophy, we hypothesized that t...

  16. Phosphodiesterase 9A regulates central cGMP and modulates responses to cholinergic and monoaminergic perturbation in vivo.

    PubMed

    Kleiman, Robin J; Chapin, Douglas S; Christoffersen, Curt; Freeman, Jody; Fonseca, Kari R; Geoghegan, Kieran F; Grimwood, Sarah; Guanowsky, Victor; Hajós, Mihály; Harms, John F; Helal, Christopher J; Hoffmann, William E; Kocan, Geralyn P; Majchrzak, Mark J; McGinnis, Dina; McLean, Stafford; Menniti, Frank S; Nelson, Fredrick; Roof, Robin; Schmidt, Anne W; Seymour, Patricia A; Stephenson, Diane T; Tingley, Francis David; Vanase-Frawley, Michelle; Verhoest, Patrick R; Schmidt, Christopher J

    2012-05-01

    Cyclic nucleotides are critical regulators of synaptic plasticity and participate in requisite signaling cascades implicated across multiple neurotransmitter systems. Phosphodiesterase 9A (PDE9A) is a high-affinity, cGMP-specific enzyme widely expressed in the rodent central nervous system. In the current study, we observed neuronal staining with antibodies raised against PDE9A protein in human cortex, cerebellum, and subiculum. We have also developed several potent, selective, and brain-penetrant PDE9A inhibitors and used them to probe the function of PDE9A in vivo. Administration of these compounds to animals led to dose-dependent accumulation of cGMP in brain tissue and cerebrospinal fluid, producing a range of biological effects that implied functional significance for PDE9A-regulated cGMP in dopaminergic, cholinergic, and serotonergic neurotransmission and were consistent with the widespread distribution of PDE9A. In vivo effects of PDE9A inhibition included reversal of the respective disruptions of working memory by ketamine, episodic and spatial memory by scopolamine, and auditory gating by amphetamine, as well as potentiation of risperidone-induced improvements in sensorimotor gating and reversal of the stereotypic scratching response to the hallucinogenic 5-hydroxytryptamine 2A agonist mescaline. The results suggested a role for PDE9A in the regulation of monoaminergic circuitry associated with sensory processing and memory. Thus, PDE9A activity regulates neuronal cGMP signaling downstream of multiple neurotransmitter systems, and inhibition of PDE9A may provide therapeutic benefits in psychiatric and neurodegenerative diseases promoted by the dysfunction of these diverse neurotransmitter systems. PMID:22328573

  17. Signal focusing through active transport

    NASA Astrophysics Data System (ADS)

    Godec, Aljaž; Metzler, Ralf

    2015-07-01

    The accuracy of molecular signaling in biological cells and novel diagnostic devices is ultimately limited by the counting noise floor imposed by the thermal diffusion. Motivated by the fact that messenger RNA and vesicle-engulfed signaling molecules transiently bind to molecular motors and are actively transported in biological cells, we show here that the random active delivery of signaling particles to within a typical diffusion distance to the receptor generically reduces the correlation time of the counting noise. Considering a variety of signaling particle sizes from mRNA to vesicles and cell sizes from prokaryotic to eukaryotic cells, we show that the conditions for active focusing—faster and more precise signaling—are indeed compatible with observations in living cells. Our results improve the understanding of molecular cellular signaling and novel diagnostic devices.

  18. Soluble Guanylate Cyclase Stimulators and Activators: Novel Therapies for Pulmonary Vascular Disease or a Different Method of Increasing cGMP?

    PubMed

    Koress, Cody; Swan, Kevin; Kadowitz, Philip

    2016-04-01

    Pulmonary arterial hypertension (PAH) is a progressively worsening disorder characterized by increased pulmonary vascular resistance leading to increased afterload, right ventricular hypertrophy, and ultimately right heart failure and death. Current pharmacologic treatments primarily act to reduce pulmonary vascular resistance (PVR) and provide some benefit but do not cure PAH. Canonical vasodilator therapy involving the nitric oxide (NO)-soluble guanylate cyclase (sGC)-cGMP pathway has demonstrated efficacy, but in pathologic states, endothelial dysfunction within the pulmonary vasculature leads to the reduced synthesis and bioavailability of NO. Acting downstream of NO, sGC stimulators and activators restore the endogenous functions of NO and exploit the positive effects of sGC stimulation on various organ systems, including the heart. Riociguat (BAY 63-2521) is the first agent in a class of sGC stimulators to receive FDA approval for the treatment of PAH and chronic thromboembolic hypertension (CTEPH). Riociguat has demonstrated significant benefit as assessed by 6MWD, PVR, N-terminal pro-brain natriuretic peptide (NT-proBNP) levels, time to clinical worsening, World Health Organization (WHO) functional class, and other quality of life measures in clinical trials as a monotherapy and in combination with endothelin receptor antagonists or non-intravenous prostanoids. Riociguat is the first FDA-approved treatment option for inoperable or persistent CTEPH and adds a new effective drug to available treatment options for pulmonary hypertension (PH). The question of whether riociguat is superior to other available treatment options is unanswered at the present time and requires further study. PMID:27118316

  19. Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.

    PubMed

    Abraham, Nader G; Quan, Shuo; Mieyal, Paul A; Yang, Liming; Burke-Wolin, Theresa; Mingone, Christopher J; Goodman, Alvin I; Nasjletti, Alberto; Wolin, Michael S

    2002-11-01

    Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition. PMID:12376366

  20. Signal transduction in eclosion hormone-induced secretion of ecdysis-triggering hormone.

    PubMed

    Kingan, T G; Cardullo, R A; Adams, M E

    2001-07-01

    Inka cells of insect epitracheal glands (EGs) secrete preecdysis and ecdysis-triggering hormones (PETH and ETH) at the end of each developmental stage. Both peptides act in the central nervous system to evoke the ecdysis behavioral sequence, a stereotype behavior during which old cuticle is shed. Secretion of ETH is stimulated by a brain neuropeptide, eclosion hormone (EH). EH evokes accumulation of cGMP followed by release of ETH from Inka cells, and exogenous cGMP evokes secretion of ETH. The secretory responses to EH and cGMP are inhibited by the broad-spectrum kinase inhibitor staurosporine, and the response to EH is potentiated by the phosphatase inhibitor calyculin A. Staurosporine did not inhibit EH-evoked accumulation of cGMP. Changes in cytoplasmic Ca2+ in Inka cells during EH signaling were monitored via fluorescence ratioing with fura-2-loaded EGs. Cytoplasmic Ca2+ increases within 30-120 s after addition of EH to EGs, and it remains elevated for at least 10 min, corresponding with the time course of secretion. Secretion is increased in dose-dependent manner by the Ca2+-ATPase inhibitor thapsigargin, a treatment that does not elevate glandular cGMP above basal levels. The secretory response to EH is partially inhibited in glands loaded with EGTA, while cGMP levels are unaffected. These findings suggest that EH activates second messenger cascades leading to cGMP accumulation and Ca2+ mobilization and/or influx and that both pathways are required for a full secretory response. cGMP activates a staurosporine-inhibitable protein kinase. We propose that Ca2+ acts via a parallel cascade with a time course that is similar to that for cGMP activation of a cGMP-dependent protein kinase. PMID:11313360

  1. Cyclic GMP is involved in auxin signalling during Arabidopsis root growth and development

    PubMed Central

    Nan, Wenbin; Wang, Xiaomin; Bi, Yurong

    2014-01-01

    The second messenger cyclic guanosine 3′,5′-monophosphate (cGMP) plays an important role in plant development and responses to stress. Recent studies indicated that cGMP is a secondary signal generated in response to auxin stimulation. cGMP also mediates auxin-induced adventitious root formation in mung bean and gravitropic bending in soybean. Nonetheless, the mechanism of the participation of cGMP in auxin signalling to affect these growth and developmental processes is largely unknown. In this report we provide evidence that indole-3-acetic acid (IAA) induces cGMP accumulation in Arabidopsis roots through modulation of the guanylate cyclase activity. Application of 8-bromo-cGMP (a cell-permeable cGMP derivative) increases auxin-dependent lateral root formation, root hair development, primary root growth, and gene expression. In contrast, inhibitors of endogenous cGMP synthesis block these processes induced by auxin. Data also showed that 8-bromo-cGMP enhances auxin-induced degradation of Aux/IAA protein modulated by the SCFTIR1 ubiquitin-proteasome pathway. Furthermore, it was found that 8-bromo-cGMP is unable to directly influence the auxin-dependent TIR1-Aux/IAA interaction as evidenced by pull-down and yeast two-hybrid assays. In addition, we provide evidence for cGMP-mediated modulation of auxin signalling through cGMP-dependent protein kinase (PKG). Our results suggest that cGMP acts as a mediator to participate in auxin signalling and may govern this process by PKG activity via its influence on auxin-regulated gene expression and auxin/IAA degradation. PMID:24591051

  2. Guanylyl cyclase/natriuretic peptide receptor-A signaling antagonizes phosphoinositide hydrolysis, Ca2+ release, and activation of protein kinase C

    PubMed Central

    Pandey, Kailash N.

    2014-01-01

    Thus far, three related natriuretic peptides (NPs) and three distinct sub-types of cognate NP receptors have been identified and characterized based on the specific ligand binding affinities, guanylyl cyclase activity, and generation of intracellular cGMP. Atrial and brain natriuretic peptides (ANP and BNP) specifically bind and activate guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), and C-type natriuretic peptide (CNP) shows specificity to activate guanylyl cyclase/natriuretic peptide receptor-B (GC-B/NPRB). All three NPs bind to natriuretic peptide receptor-C (NPRC), which is also known as clearance or silent receptor. The NPRA is considered the principal biologically active receptor of NP family; however, the molecular signaling mechanisms of NP receptors are not well understood. The activation of NPRA and NPRB produces the intracellular second messenger cGMP, which serves as the major signaling molecule of all three NPs. The activation of NPRB in response to CNP also produces the intracellular cGMP; however, at lower magnitude than that of NPRA, which is activated by ANP and BNP. In addition to enhanced accumulation of intracellular cGMP in response to all three NPs, the levels of cAMP, Ca2+ and inositol triphosphate (IP3) have also been reported to be altered in different cells and tissue types. Interestingly, ANP has been found to lower the concentrations of cAMP, Ca2+, and IP3; however, NPRC has been proposed to increase the levels of these metabolic signaling molecules. The mechanistic studies of decreased and/or increased levels of cAMP, Ca2+, and IP3 in response to NPs and their receptors have not yet been clearly established. This review focuses on the signaling mechanisms of ANP/NPRA and their biological effects involving an increased level of intracellular accumulation of cGMP and a decreased level of cAMP, Ca2+, and IP3 in different cells and tissue systems. PMID:25202235

  3. Ca2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca2+ channels

    PubMed Central

    Qi, Zhi; Verma, Rajeev; Gehring, Chris; Yamaguchi, Yube; Zhao, Yichen; Ryan, Clarence A.; Berkowitz, Gerald A.

    2010-01-01

    A family of peptide signaling molecules (AtPeps) and their plasma membrane receptor AtPepR1 are known to act in pathogen-defense signaling cascades in plants. Little is currently known about the molecular mechanisms that link these signaling peptides and their receptor, a leucine-rich repeat receptor-like kinase, to downstream pathogen-defense responses. We identify some cellular activities of these molecules that provide the context for a model for their action in signaling cascades. AtPeps activate plasma membrane inwardly conducting Ca2+ permeable channels in mesophyll cells, resulting in cytosolic Ca2+ elevation. This activity is dependent on their receptor as well as a cyclic nucleotide-gated channel (CNGC2). We also show that the leucine-rich repeat receptor-like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2-dependent cytosolic Ca2+ elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33) is mediated by the Ca2+ signaling pathway associated with AtPep peptides and their receptor. The work presented here indicates that extracellular AtPeps, which can act as danger-associated molecular patterns, signal by interaction with their receptor, AtPepR1, a plasma membrane protein that can generate cGMP. Downstream from AtPep and AtPepR1 in a signaling cascade, the cGMP-activated channel CNGC2 is involved in AtPep- and AtPepR1-dependent inward Ca2+ conductance and resulting cytosolic Ca2+ elevation. The signaling cascade initiated by AtPeps leads to expression of pathogen-defense genes in a Ca2+-dependent manner. PMID:21088220

  4. Nitric Oxide Participates in Cold-Inhibited Camellia sinensis Pollen Germination and Tube Growth Partly via cGMP In Vitro

    PubMed Central

    Zhu-Ge, Qiang; Jiang, Xin; Wang, Wei-Dong; Fang, Wan-Ping; Chen, Xuan; Li, Xing-Hui

    2012-01-01

    Nitric oxide (NO) plays essential roles in many biotic and abiotic stresses in plant development procedures, including pollen tube growth. Here, effects of NO on cold stress inhibited pollen germination and tube growth in Camellia sinensis were investigated in vitro. The NO production, NO synthase (NOS)-like activity, cGMP content and proline (Pro) accumulation upon treatment with NO scavenger cPTIO, NOS inhibitor L-NNA, NO donor DEA NONOate, guanylate cyclase (GC) inhibitor ODQ or phosphodiesterase (PDE) inhibitor Viagra at 25°C (control) or 4°C were analyzed. Exposure to 4°C for 2 h reduced pollen germination and tube growth along with increase of NOS-like activity, NO production and cGMP content in pollen tubes. DEA NONOate treatment inhibited pollen germination and tube growth in a dose-dependent manner under control and reinforced the inhibition under cold stress, during which NO production and cGMP content promoted in pollen tubes. L-NNA and cPTIO markedly reduced the generation of NO induced by cold or NO donor along with partly reverse of cold- or NO donor-inhibited pollen germination and tube growth. Furthermore, ODQ reduced the cGMP content under cold stress and NO donor treatment in pollen tubes. Meanwhile, ODQ disrupted the reinforcement of NO donor on the inhibition of pollen germination and tube growth under cold condition. Additionally, Pro accumulation of pollen tubes was reduced by ODQ compared with that receiving NO donor under cold or control condition. Effects of cPTIO and L-NNA in improving cold-treated pollen germination and pollen tube growth could be lowered by Viagra. Moreover, the inhibitory effects of cPTIO and L-NNA on Pro accumulation were partly reversed by Viagra. These data suggest that NO production from NOS-like enzyme reaction decreased the cold-responsive pollen germination, inhibited tube growth and reduced Pro accumulation, partly via cGMP signaling pathway in C. sinensis. PMID:23272244

  5. A historical perspective on the lateral diffusion model of GTPase activation and related coupling of membrane signaling proteins

    PubMed Central

    Liebman, Paul A

    2014-01-01

    Aspects of our discovery of lateral diffusion of the G protein coupled receptor (GPCR) rhodopsin and that a single activated rhodopsin can non-covalently catalyze GTP binding to thousands of GTPases per second on rod disk membranes via this diffusion are summarized herein. Rapid GTPase coupling to membrane-bound phosphodiesterase (PDE) further amplifies the signal via cGMP hydrolysis, essential to visual transduction. Important generalizations from this work are that biomembranes can uniquely concentrate, orient for reaction and provide a solvent appropriate to rapid, powerful and appropriately controlled sequential interaction of signaling proteins. Of equal importance to function is timely control and termination of such powerful amplification via receptor phosphorylation (quenching) and arrestin binding. Downstream kinetic modulation by GTPase activating proteins (GAPs) and regulators of G protein signaling (RGS) and related mechanisms as well as limitations set by membrane domain fencing, structural protein binding etc. can be essential in relevant systems. PMID:25279248

  6. Systems Pharmacology and Rational Polypharmacy: Nitric Oxide−Cyclic GMP Signaling Pathway as an Illustrative Example and Derivation of the General Case

    PubMed Central

    Garmaroudi, Farshid S.; Handy, Diane E.; Liu, Yang-Yu; Loscalzo, Joseph

    2016-01-01

    Impaired nitric oxide (NO˙)-cyclic guanosine 3', 5'-monophosphate (cGMP) signaling has been observed in many cardiovascular disorders, including heart failure and pulmonary arterial hypertension. There are several enzymatic determinants of cGMP levels in this pathway, including soluble guanylyl cyclase (sGC) itself, the NO˙-activated form of sGC, and phosphodiesterase(s) (PDE). Therapies for some of these disorders with PDE inhibitors have been successful at increasing cGMP levels in both cardiac and vascular tissues. However, at the systems level, it is not clear whether perturbation of PDE alone, under oxidative stress, is the best approach for increasing cGMP levels as compared with perturbation of other potential pathway targets, either alone or in combination. Here, we develop a model-based approach to perturbing this pathway, focusing on single reactions, pairs of reactions, or trios of reactions as targets, then monitoring the theoretical effects of these interventions on cGMP levels. Single perturbations of all reaction steps within this pathway demonstrated that three reaction steps, including the oxidation of sGC, NO˙ dissociation from sGC, and cGMP degradation by PDE, exerted a dominant influence on cGMP accumulation relative to other reaction steps. Furthermore, among all possible single, paired, and triple perturbations of this pathway, the combined perturbations of these three reaction steps had the greatest impact on cGMP accumulation. These computational findings were confirmed in cell-based experiments. We conclude that a combined perturbation of the oxidatively-impaired NO˙-cGMP signaling pathway is a better approach to the restoration of cGMP levels as compared with corresponding individual perturbations. This approach may also yield improved therapeutic responses in other complex pharmacologically amenable pathways. PMID:26985825

  7. Binding of cGMP to both allosteric sites of cGMP-binding cGMP-specific phosphodiesterase (PDE5) is required for its phosphorylation.

    PubMed Central

    Turko, I V; Francis, S H; Corbin, J D

    1998-01-01

    cGMP-binding phosphodiesterases contain two homologous allosteric cGMP-binding sites (sites a and b) that are arranged in tandem; they constitute a superfamily of mammalian cyclic nucleotide receptors distinct from the cyclic nucleotide-dependent protein kinases/cation channels family. The functional role of each of these two sites in the phosphodiesterases is not known. The cGMP-binding sites of one of these phosphodiesterases, the cGMP-binding cGMP-specific phosphodiesterase (cGB-PDE, PDE5), have been analysed by using site-directed mutagenesis. Mutations that affect cGMP binding to either one or both allosteric sites do not influence cGMP hydrolysis in the catalytic site under the conditions used. However, compared with wild-type enzyme, the D289A, D478A and D289A/D478A mutants, which are defective in cGMP binding to either site a or site b, or both allosteric sites, require much higher cGMP concentrations for the allosteric stimulation of phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. The cGMP effect is on the cGB-PDE rather than on the catalytic subunit of the protein kinase because the latter enzyme does not require cGMP for activity. The D289N mutant, which has higher binding affinity for cGMP than does the wild-type enzyme, is phosphorylated at lower concentrations of cGMP than is the wild-type enzyme. It is concluded that cGMP binding to the allosteric sites of cGB-PDE does not directly affect catalysis, but binding to both of these sites regulates phosphorylation of this enzyme. PMID:9445376

  8. Nitric oxide and cyclic guanosine monophosphate signaling in the eye.

    PubMed

    Murad, Ferid

    2008-06-01

    This brief review describes the components and pathways utilized in nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) signaling. Since the discovery of the effects of NO and cGMP on smooth muscle relaxation about 30 years ago, the field has expanded in many directions such that many, but not all, biochemical and biological effects seem to be regulated by these unique signaling molecules. While many of the effects of NO are due to activation of soluble guanylyl cyclase (sGC) that can be considered the receptor for NO, cGMP, in turn, can activate a cGMP-dependent protein kinase (PKG) to phosphorylate an array of proteins. Some of the effects of cGMP can be independent of PKG and are due to effects on ion channels or cyclic nucleotide phosphodiesterases. Also, some of the effects of NO can be independent of sGC activation. The isoenzymes and macromolecules that participate in these signaling pathways can serve as molecular targets to identify compounds that increase or decrease their activation and thus serve as chemical leads for discovering novel drugs for a variety of diseases. Some examples are given. However, with about 90,000 publications in the field since our first reports in 1977, this brief review can only give the readers a sample of the excitement and opportunities we have found in this cell signaling system. PMID:18443613

  9. Hypergravity differentially modulates cGMP efflux in human melanocytic cells stimulated by nitric oxide and natriuretic peptides

    NASA Astrophysics Data System (ADS)

    Ivanova, K.; Stieber, C.; Lambers, B.; Block, I.; Krieg, R.; Wellmann, A.; Gerzer, R.

    Nitric oxide NO plays a key role in many patho physiologic processes including inflammation and skin cancer The diverse cellular effects of NO are mainly mediated by activation of the soluble guanylyl cyclase sGC isoform that leads to increases in intracellular cGMP levels whereas the membrane-bound isoforms serve as receptors for natriuretic peptides e g ANP In human skin epidermal melanocytes represent the principal cells for skin pigmentation by synthesizing the pigment melanin Melanin acts as a scavenger for free radicals that may arise during metabolic stress as a result of potentially harmful effects of the environment In previous studies we found that long-term exposure to hypergravity stimulated cGMP efflux in normal human melanocytes NHMs and non-metastatic melanoma cells at least partly by an enhanced expression of the multidrug resistance proteins MRP and cGMP transporters MRP4 5 The present study investigated whether hypergravity generated by centrifugal acceleration may modulate the cGMP efflux in NO-stimulated NHMs and melanoma cells MCs with different metastatic potential The NONOates PAPA-NO and DETA-NO were used as direct NO donors for cell stimulation In the presence of 0 1 mM DETA-NO t 1 2 sim 20 h long-term application of hypergravity up to 5 g for 24 h reduced intracellular cGMP levels by stimulating cGMP efflux in NHMs and non-metastatic MCs in comparison to 1 g whereas exposure to 5 g for 6 h in the presence of 0 1 mM PAPA-NO t 1 2 sim 30 min was not effective The hypergravity-stimulated

  10. Changes in cyclic GMP level and phosphodiesterase activity during follicular development in the rat ovary.

    PubMed

    Fu, C Q; Shi, F X; Zhang, Z H; Li, J R; Huang, X H; Wang, Z C

    2014-01-01

    Guanosine 3',5'-cyclic monophosphate (cGMP), as a second messenger, plays potential roles in ovarian functions. To elucidate the role of phosphodiesterase (PDE) in cGMP signaling during ovarian follicular development, the present study was conducted to investigate ovarian cGMP level and cGMP-PDE activity by radioimmunoassay (RIA) in postnatal rats, immature rats during gonadotropin-primed follicular development, ovulation and luteinization, adult rats during normal estrous cycle, and aged rats that spontaneously developed persistent estrus (PE). All four rat models were confirmed by histological examination of one ovary, and the other ovary was used for RIA. In postnatal rats, cGMP level was high at birth and decreased dramatically by Day 5, and then, it increased maximally at Day 10 and declined at Day 21. However, cGMP-PDE activity did not significantly change during Days 1 to 10, but increased significantly on Day 21. In immature female rats, cGMP level markedly decreased upon treatment with equine chorionic gonadotropin (eCG), while cGMP-PDE activity did not show any significant changes; however, ovarian cGMP level and cGMP-PDE activity increased after injection of an ovulatory dose of human chorionic gonadotropin (hCG) for induction of ovulation and luteinization. In adult rats during normal estrous cycle, cGMP level was high on proestrus and metestrus days, while cGMP-PDE activity was high on estrus day. In PE rats, ovarian cGMP level was similar to that in adult rats on estrus and diestrus days but lower than that on proestrus and metestrus days; ovarian cGMP-PDE activity was lower than that on estrus days but similar as the other estrous cycle days. In addition, there was a significant negative correlation between ovarian cGMP level and cGMP-PDE activity during normal estrous cycles in the adult rat (r = -0.7715, N = 16, P < 0.05), but not in the postnatal rat (r = -0.1055, N = 20, P > 0.05). Together, the results of our present study indicated that ovarian

  11. A new nonhydrolyzable reactive cGMP analogue, (Rp)-Guanosine-3′, 5′-cyclic-S-(4-bromo-2, 3-dioxobutyl)monophosphorothioate, which targets the cGMP binding site of human platelet PDE3A

    PubMed Central

    Hung, Su H.; Liu, Andy H.; Pixley, Robin A.; Francis, Penelope; Williams, LaTeeka D.; Matsko, Christopher M.; Barnes, Karine D.; Sivendran, Sharmila; Colman, Roberta F.; Colman, Robert W.

    2008-01-01

    The amino acids involved in substrate (cAMP) binding to human platelet cGMP-inhibited cAMP phosphodiesterase (PDE3A) are identified. Less is known about the inhibitor (cGMP) binding site. We have now synthesized a nonhydrolyzable reactive cGMP analog, Rp-guanosine-3′, 5′-cyclic-S-(4-bromo-2, 3-dioxobutyl)monophosphorothioate (Rp-cGMPS-BDB). Rp-cGMPS-BDB irreversibly inactivates PDE3A (KI = 43.4 ± 7.2 μM and kcart = 0.007 ± 0.0006 min−1). The effectiveness of protectants in decreasing the rate of inactivation by Rp-cGMPS-BDB is: Rp-cGMPS (Kd = 72 μM) > Sp-cGMPS (124), Sp-cAMPS (182) > GMP (1517), Rp-cAMPS (3762), AMP (4370 μM). NAD+, neither a substrate nor an inhibitor of PDE3A, does not protect. Nonhydrolyzable cGMP analogs exhibit greater affinity than the cAMP analogs. These results indicate that Rp-cGMPS-BDB targets favorably the cGMP binding site consistent with a docking model of PDE3A-Rp-cGMPS-BDB active site. We conclude that Rp-cGMPS-BDB is an effective active site-directed affinity label for PDE3A with potential for other cGMP-dependent enzymes. PMID:18394675

  12. An Overgrowth Disorder Associated with Excessive Production of cGMP Due to a Gain-of-Function Mutation of the Natriuretic Peptide Receptor 2 Gene

    PubMed Central

    Miura, Kohji; Namba, Noriyuki; Fujiwara, Makoto; Ohata, Yasuhisa; Ishida, Hidekazu; Kitaoka, Taichi; Kubota, Takuo; Hirai, Haruhiko; Higuchi, Chikahisa; Tsumaki, Noriyuki; Yoshikawa, Hideki; Sakai, Norio; Michigami, Toshimi; Ozono, Keiichi

    2012-01-01

    We describe a three-generation family with tall stature, scoliosis and macrodactyly of the great toes and a heterozygous p.Val883Met mutation in Npr2, the gene that encodes the CNP receptor NPR2 (natriuretic peptide receptor 2). When expressed in HEK293A cells, the mutant Npr2 cDNA generated intracellular cGMP (cyclic guanosine monophosphate) in the absence of CNP ligand. In the presence of CNP, cGMP production was greater in cells that had been transfected with the mutant Npr2 cDNA compared to wild-type cDNA. Transgenic mice in which the mutant Npr2 was expressed in chondrocytes driven by the promoter and intronic enhancer of the Col11a2 gene exhibited an enhanced production of cGMP in cartilage, leading to a similar phenotype to that observed in the patients. In addition, blood cGMP concentrations were elevated in the patients. These results indicate that p.Val883Met is a constitutive active gain-of-function mutation and elevated levels of cGMP in growth plates lead to the elongation of long bones. Our findings reveal a critical role for NPR2 in skeletal growth in both humans and mice, and may provide a potential target for prevention and treatment of diseases caused by impaired production of cGMP. PMID:22870295

  13. Changes in cGMP levels on meiosis reinitiation of starfish oocytes.

    PubMed

    Nemoto, S; Ishida, K

    1983-04-15

    An intracellular level of cGMP (but not cAMP) transiently decreased the reinitiation of oocyte maturation in the starfish, Asterias amurensis. Exogenously applied cGMP inhibited hormone-induced maturation. Methylxanthines inhibited oocyte maturation by suppressing the decrease in cGMP levels. These results suggest that a decrease in cGMP levels is a prerequisite for meiosis reinitiation of starfish oocytes. PMID:6303819

  14. Signaling during platelet adhesion and activation

    PubMed Central

    Li, Zhenyu; Delaney, M. Keegan; O’Brien, Kelly A.; Du, Xiaoping

    2011-01-01

    Upon vascular injury, platelets are activated by adhesion to adhesive proteins like von Willebrand factor and collagen, or by soluble platelet agonists like ADP, thrombin, and thromboxane A2. These adhesive proteins and soluble agonists induce signal transduction via their respective receptors. The various receptor-specific platelet activation signaling pathways converge into common signaling events, which stimulate platelet shape change, granule secretion, and ultimately induce the “inside-out” signaling process leading to activation of the ligand binding function of integrin αIIbβ3. Ligand binding to integrin αIIbβ3 mediates platelet adhesion and aggregation and triggers “outside-in” signaling, resulting in platelet spreading, additional granule secretion, stabilization of platelet adhesion and aggregation, and clot retraction. It has become increasingly evident that agonist-induced platelet activation signals also crosstalk with integrin “outside-in” signals to regulate platelet responses. Platelet activation involves a series of rapid positive feedback loops that greatly amplify initial activation signals, and enable robust platelet recruitment and thrombus stabilization. Recent studies have provided novel insight into the molecular mechanisms of these processes. PMID:21071698

  15. Calcium Feedback to cGMP Synthesis Strongly Attenuates Single-Photon Responses Driven by Long Rhodopsin Lifetimes

    PubMed Central

    Gross, Owen P.; Pugh, Edward N.; Burns, Marie E.

    2013-01-01

    SUMMARY Rod photoreceptors generate amplified, reproducible responses to single photons via a G protein signaling cascade. Surprisingly, genetic perturbations that dramatically alter the deactivation of the principal signal amplifier, the GPCR rhodopsin (R*), do not much alter the amplitude of single-photon responses (SPRs). These same perturbations, when crossed into a line lacking calcium feedback regulation of cGMP synthesis, produced much larger alterations in SPR amplitudes. Analysis of SPRs from rods with and without feedback reveal that the consequences of trial-to-trial fluctuations in R* lifetime in normal rods are also dampened by feedback regulation of cGMP synthesis. Thus, calcium feedback trumps the mechanisms of R* deactivation in determining the SPR amplitude, attenuating responses arising from longer R* lifetimes to a greater extent than those arising from shorter ones. As a result, rod SPRs achieve a more stereotyped amplitude, a characteristic considered important for reliable transmission through the visual system. PMID:23083739

  16. Regulation of the starfish sperm acrosome reaction by cGMP, pH, cAMP and Ca2+.

    PubMed

    Matsumoto, Midori; Kawase, Osamu; Islam, M Sadiqul; Naruse, Masahiro; Watanabe, Shin-Nosuke; Ishikawa, Riho; Hoshi, Motonori

    2008-01-01

    In the starfish, Asterias amurensis, three components in the jelly coat of eggs, namely acrosome reaction-inducing substance (ARIS), Co-ARIS and asterosap, act in concert on homologous spermatozoa to induce the acrosome reaction (AR). Molecular recognition between the sperm surface molecules and the egg jelly molecules must underlie signal transduction events triggering the AR. Asterosap is a sperm-activating molecule, which stimulates rapid synthesis of intracellular cGMP, pH and Ca2+. This transient elevation of Ca2+ level is caused by a K+-dependent Na+/Ca2+ exchanger, and the increase of intracellular pH is sufficient for ARIS to induce the AR. The concerted action of ARIS and asterosap could induce elevate intracellular cAMP levels in starfish sperm and the sustained increase in [Ca2+], which is essential for the AR. The signaling pathway induced by these factors seems to be synergistically regulated to trigger the AR in starfish sperm. PMID:18649265

  17. Effects of tetrandrine on cAMP and cGMP levels in rabbit corpus cavernosum in vitro.

    PubMed

    Chen, Jun; Liu, Jihong; Wang, Tao; Xiao, Hengjun; Yin, Chunping

    2010-07-01

    The aim of this study was to further investigate the relaxation mechanism of tetrandrine (Tet), a bis-benzylisoquinoline alkaloid isolated from the Chinese medicinal herb-root of Stephania tetrandra S Moore, on rabbit corpus cavernosum tissue in vitro. The effects of Tet on the concentrations of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in isolated and incubated rabbit corpus cavernosum tissue were recorded by means of (125)I radioimmunoassay. The basal concentration of cAMP in corpus cavernosum tissue was 5.67 +/- 0.97 pmol mg(-1). Tet increased the cAMP concentration in a dose-dependent manner (p < 0.05), but this effect was not inhibited by an adenylate cyclase inhibitor (cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine, MDL-12, 330A) (p > 0.05). The accumulation of cAMP induced by prostaglandin E(1) (PGE(1), a stimulator of cAMP production) was also augmented by Tet in a dose-dependent manner (p < 0.05). The basal concentration of cGMP in corpus cavernosum tissue is 0.44 +/- 0.09 pmol mg(-1). Tet did not affect this concentration of cGMP, neither in the presence nor the absence of a guanyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, ODQ) (p > 0.05). Further, sodium nitroprusside (SNP, a stimulator of cGMP production)-induced cGMP production was not enhanced by Tet (p > 0.05). Tet, with its relaxation mechanism, can enhance the concentration of cAMP in rabbit corpus cavernosum tissue, probably by inhibiting PDEs activity. PMID:20582806

  18. Defining Specificity Determinants of cGMP Mediated Gustatory Sensory Transduction in Caenorhabditis elegans

    PubMed Central

    Smith, Heidi K.; Luo, Linjiao; O’Halloran, Damien; Guo, Dagang; Huang, Xin-Yun; Samuel, Aravinthan D. T.; Hobert, Oliver

    2013-01-01

    Cyclic guanosine monophosphate (cGMP) is a key secondary messenger used in signal transduction in various types of sensory neurons. The importance of cGMP in the ASE gustatory receptor neurons of the nematode Caenorhabditis elegans was deduced by the observation that multiple receptor-type guanylyl cyclases (rGCs), encoded by the gcy genes, and two presently known cyclic nucleotide-gated ion channel subunits, encoded by the tax-2 and tax-4 genes, are essential for ASE-mediated gustatory behavior. We describe here specific mechanistic features of cGMP-mediated signal transduction in the ASE neurons. First, we assess the specificity of the sensory functions of individual rGC proteins. We have previously shown that multiple rGC proteins are expressed in a left/right asymmetric manner in the functionally lateralized ASE neurons and are required to sense distinct salt cues. Through domain swap experiments among three different rGC proteins, we show here that the specificity of individual rGC proteins lies in their extracellular domains and not in their intracellular, signal-transducing domains. Furthermore, we find that rGC proteins are also sufficient to confer salt sensory responses to other neurons. Both findings support the hypothesis that rGC proteins are salt receptor proteins. Second, we identify a novel, likely downstream effector of the rGC proteins in gustatory signal transduction, a previously uncharacterized cyclic nucleotide-gated (CNG) ion channel, encoded by the che-6 locus. che-6 mutants show defects in gustatory sensory transduction that are similar to defects observed in animals lacking the tax-2 and tax-4 CNG channels. In contrast, thermosensory signal transduction, which also requires tax-2 and tax-4, does not require che-6, but requires another CNG, cng-3. We propose that CHE-6 may form together with two other CNG subunits, TAX-2 and TAX-4, a gustatory neuron-specific heteromeric CNG channel complex. PMID:23695300

  19. Effects of Kaempferia parviflora Wall. Ex. Baker and sildenafil citrate on cGMP level, cardiac function, and intracellular Ca2+ regulation in rat hearts.

    PubMed

    Weerateerangkul, Punate; Palee, Siripong; Chinda, Kroekkiat; Chattipakorn, Siriporn C; Chattipakorn, Nipon

    2012-09-01

    Although Kaempferia parviflora extract (KPE) and its flavonoids have positive effects on the nitric oxide (NO) signaling pathway, its mechanisms on the heart are still unclear. Because our previous studies demonstrated that KPE decreased defibrillation efficacy in swine similar to that of sildenafil citrate, the phosphodiesterase-5 inhibitor, it is possible that KPE may affect the cardiac NO signaling pathway. In the present study, the effects of KPE and sildenafil citrate on cyclic guanosine monophosphate (cGMP) level, modulation of cardiac function, and Ca transients in ventricular myocytes were investigated. In a rat model, cardiac cGMP level, cardiac function, and Ca transients were measured before and after treatment with KPE and sildenafil citrate. KPE significantly increased the cGMP level and decreased cardiac function and Ca transient. These effects were similar to those found in the sildenafil citrate-treated group. Furthermore, the nonspecific NOS inhibitor could abolish the effects of KPE and sildenafil citrate on Ca transient. KPE has positive effect on NO signaling in the heart, resulting in an increased cGMP level, similar to that of sildenafil citrate. This effect was found to influence the physiology of normal heart via the attenuation of cardiac function and the reduction of Ca transient in ventricular myocytes. PMID:22691878

  20. Nitric Oxide-cGMP Signaling Stimulates Erythropoiesis through Multiple Lineage-Specific Transcription Factors: Clinical Implications and a Novel Target for Erythropoiesis

    PubMed Central

    Ikuta, Tohru; Sellak, Hassan; Odo, Nadine; Adekile, Adekunle D.; Gaensler, Karin M. L.

    2016-01-01

    Much attention has been directed to the physiological effects of nitric oxide (NO)-cGMP signaling, but virtually nothing is known about its hematologic effects. We reported for the first time that cGMP signaling induces human γ-globin gene expression. Aiming at developing novel therapeutics for anemia, we examined here the hematologic effects of NO-cGMP signaling in vivo and in vitro. We treated wild-type mice with NO to activate soluble guanylate cyclase (sGC), a key enzyme of cGMP signaling. Compared to untreated mice, NO-treated mice had higher red blood cell counts and total hemoglobin but reduced leukocyte counts, demonstrating that when activated, NO-cGMP signaling exerts hematopoietic effects on multiple types of blood cells in vivo. We next generated mice which overexpressed rat sGC in erythroid and myeloid cells. The forced expression of sGCs activated cGMP signaling in both lineage cells. Compared with non-transgenic littermates, sGC mice exhibited hematologic changes similar to those of NO-treated mice. Consistently, a membrane-permeable cGMP enhanced the differentiation of hematopoietic progenitors toward erythroid-lineage cells but inhibited them toward myeloid-lineage cells by controlling multiple lineage-specific transcription factors. Human γ-globin gene expression was induced at low but appreciable levels in sGC mice carrying the human β-globin locus. Together, these results demonstrate that NO-cGMP signaling is capable of stimulating erythropoiesis in both in vitro and vivo settings by controlling the expression of multiple lineage-specific transcription factors, suggesting that cGMP signaling upregulates erythropoiesis at the level of gene transcription. The NO-cGMP signaling axis may constitute a novel target to stimulate erythropoiesis in vivo. PMID:26727002

  1. Paired inhibitory and activating receptor signals.

    PubMed

    Taylor, L S; Paul, S P; McVicar, D W

    2000-01-01

    The immunological literature has become inundated with reports regarding paired inhibitory receptors. Paired inhibitory receptor systems are highly conserved families that contain receptors involved in either cellular inhibition or activation. In most cases the paired putative biochemical antagonists are co-expressed on a given cell and thought to bind similar, if not identical, ligands making their biological role difficult to understand. Examples of these systems include immunoglobulin (Ig)-like receptors (Killer Ig Receptors, Immunoglobulin-like Transcripts/Leukocyte Ig-like Receptors/Monocyte Macrophage Ig Receptors, and Paired Ig-like Receptors), and type II lectin-like receptor systems (NKG2 and Ly49). General characteristics of these inhibitory receptors include a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). The ITIM is phosphorylated upon engagement and recruits protein tyrosine phosphatases that dephosphorylate cellular substrates that would otherwise mediate activation. In contrast, the activating receptors of these pairs use charged residues within their transmembrane domains to associate with various signal transduction chains including the gamma chain of the receptor for the Fc portion of IgE, DAP12 or DAP10. Once phosphorylated, these chains direct the signal transduction cascade resulting in cellular activation. Here we review the signaling of several paired systems and present the current models for their signal transduction cascades. PMID:11258418

  2. Regulation of cGMP synthesis in cultured podocytes by vasoactive hormones.

    PubMed

    Lewko, B; Gołos, M; Latawiec, E; Angielski, S; Stepinski, J

    2006-12-01

    The podocytes are highly differentiated cells playing a key role in glomerular filtration. Vasoactive factors including angiotensin II (Ang II) and cyclic guanosine 5' monophosphate (cGMP) are synthesized by these cells upon stimulation as well as in the basal state. In this study we have tested whether angiotensin II affects the total synthesis of cGMP in primary culture of rat podocytes. The cells were stimulated with atrial natriuretic peptide (ANP) and/or a nitric oxide (NO) donor, S-nitroso-N-acetyl penicillamine (SNAP), in the absence or presence of Ang II. The cGMP synthesis was determined by radioimmunoassay (RIA). ANP or SNAP alone increased the cGMP synthesis in podocytes although the effects were not additive unless Ang II was present in the medium. Ang II suppressed the ANP-dependent cGMP synthesis whereas SNAP-dependent cGMP production remained unaffected. These effects were prevented by a non-specific antagonist of Ang II receptors (AT), saralasin. Adversely, PD123319, a specific inhibitor of AT2 receptors, augmented inhibition of ANP-dependent and enhanced the NO-dependent cGMP production. Probenecid, an inhibitor of cGMP extrusion from the cells, suppressed the cGMP generation by both ANP and SNAP. We conclude that cGMP synthesis in cultured podocytes is modulated by angiotensin II and that two adversely acting receptors, AT1 and AT2 are involved in this effect. Additionally, production of cGMP might be intrinsically inhibited by cGMP accumulating inside the cells. PMID:17229984

  3. Oxidized LDL activates blood platelets through CD36/NOX2–mediated inhibition of the cGMP/protein kinase G signaling cascade

    PubMed Central

    Magwenzi, Simbarashe; Woodward, Casey; Wraith, Katie S.; Aburima, Ahmed; Raslan, Zaher; Jones, Huw; McNeil, Catriona; Wheatcroft, Stephen; Yuldasheva, Nadira; Febbriao, Maria; Kearney, Mark

    2015-01-01

    Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in dyslipidemic disorders. Although oxLDL stimulates activatory signaling, it is unclear how these events drive accelerated thrombosis. Here, we describe a mechanism for oxLDL-mediated platelet hyperactivity that requires generation of reactive oxygen species (ROS). Under arterial flow, oxLDL triggered sustained generation of platelet intracellular ROS, which was blocked by CD36 inhibitors, mimicked by CD36-specific oxidized phospholipids, and ablated in CD36−/− murine platelets. oxLDL-induced ROS generation was blocked by the reduced NAD phosphate oxidase 2 (NOX2) inhibitor, gp91ds-tat, and absent in NOX2−/− mice. The synthesis of ROS by oxLDL/CD36 required Src-family kinases and protein kinase C (PKC)-dependent phosphorylation and activation of NOX2. In functional assays, oxLDL abolished guanosine 3′,5′-cyclic monophosphate (cGMP)-mediated signaling and inhibited platelet aggregation and arrest under flow. This was prevented by either pharmacologic inhibition of NOX2 in human platelets or genetic ablation of NOX2 in murine platelets. Platelets from hyperlipidemic mice were also found to have a diminished sensitivity to cGMP when tested ex vivo, a phenotype that was corrected by infusion of gp91ds-tat into the mice. This study demonstrates that oxLDL and hyperlipidemia stimulate the generation of NOX2-derived ROS through a CD36-PKC pathway and may promote platelet hyperactivity through modulation of cGMP signaling. PMID:25710879

  4. Cyclic nucleotide signalling in kidney fibrosis

    PubMed Central

    Schinner, Elisabeth; Wetzl, Veronika; Schlossmann, Jens

    2015-01-01

    Kidney fibrosis is an important factor for the progression of kidney diseases, e.g., diabetes mellitus induced kidney failure, glomerulosclerosis and nephritis resulting in chronic kidney disease or end-stage renal disease. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were implicated to suppress several of the above mentioned renal diseases. In this review article, identified effects and mechanisms of cGMP and cAMP regarding renal fibrosis are summarized. These mechanisms include several signalling pathways of nitric oxide/ANP/guanylyl cyclases/cGMP-dependent protein kinase and cAMP/Epac/adenylyl cyclases/cAMP-dependent protein kinase. Furthermore, diverse possible drugs activating these pathways are discussed. From these diverse mechanisms it is expected that new pharmacological treatments will evolve for the therapy or even prevention of kidney failure. PMID:25622251

  5. Notum deacylates Wnts to suppress signalling activity

    PubMed Central

    Howell, Steve; Chang, Tao-Hsin; Liu, Yan; Feizi, Ten; Bineva, Ganka; O’Reilly, Nicola; Snijders, Ambrosius P.; Jones, E. Yvonne; Vincent, Jean-Paul

    2015-01-01

    Signalling by Wnts is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnts from the cell surface. However, this view fails to explain specificity since glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which likely help Notum colocalise with Wnts. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnts and thus constitutes the first known extracellular protein deacylase. PMID:25731175

  6. Constitutive excitation by Gly90Asp rhodopsin rescues rods from degeneration caused by elevated production of cGMP in the dark.

    PubMed

    Woodruff, Michael L; Olshevskaya, Elena V; Savchenko, Andrey B; Peshenko, Igor V; Barrett, Ronald; Bush, Ronald A; Sieving, Paul A; Fain, Gordon L; Dizhoor, Alexander M

    2007-08-15

    Previous experiments indicate that congenital human retinal degeneration caused by genetic mutations that change the Ca(2+) sensitivity of retinal guanylyl cyclase (retGC) can result from an increase in concentration of free intracellular cGMP and Ca(2+) in the photoreceptors. To rescue degeneration in transgenic mouse models having either the Y99C or E155G mutations of the retGC modulator guanylyl cyclase-activating protein 1 (GCAP-1), which produce elevated cGMP synthesis in the dark, we used the G90D rhodopsin mutation, which produces constitutive stimulation of cGMP hydrolysis. The effects of the G90D transgene were evaluated by measuring retGC activity biochemically, by recording single rod and electroretinogram (ERG) responses, by intracellular free Ca(2+) measurement, and by retinal morphological analysis. Although the G90D rhodopsin did not alter the abnormal Ca(2+) sensitivity of retGC in the double-mutant animals, the intracellular free cGMP and Ca(2+) concentrations returned close to normal levels, consistent with constitutive activation of the phosphodiesterase PDE6 cascade in darkness. G90D decreased the light sensitivity of rods but spared them from severe retinal degeneration in Y99C and E155G GCAP-1 mice. More than half of the photoreceptors remained alive, appeared morphologically normal, and produced electrical responses, at the time when their siblings lacking the G90D rhodopsin transgene lost the entire retinal outer nuclear layer and no longer responded to illumination. These experiments indicate that mutations that lead to increases in cGMP and Ca(2+) can trigger photoreceptor degeneration but that constitutive activation of the transduction cascade in these animals can greatly enhance cell survival. PMID:17699662

  7. Amyloid-β impairs, and ibuprofen restores, the cGMP pathway, synaptic expression of AMPA receptors and long-term potentiation in the hippocampus.

    PubMed

    Monfort, Pilar; Felipo, Vicente

    2010-01-01

    Amyloid-β (Aβ) rapidly impairs hippocampal long-term potentiation (LTP) and cognitive function in rats. We hypothesized that: a) Aβ-induced impairment of LTP would be due to impairment of the nitric oxide (NO)-cGMP pathway and AMPA receptor translocation; and b) treatment with the anti-inflammatory drug ibuprofen would restore the NO-cGMP pathway and LTP. The aims of this work were to assess whether ibuprofen prevents and/or rescues Aβ-induced LTP impairments in hippocampal slices and to analyze the role of the altered NO-cGMP-protein kinase G pathway and AMPA receptor phosphorylation and synaptic expression in the mechanisms by which Aβ impairs and ibuprofen restores LTP. Aβ impairs tetanus-induced activation of guanylate cyclase and cGMP increase, preventing protein kinase G activation, phosphorylation of GluR1 in Ser845 and AMPA receptors translocation to synaptic membranes, which is responsible for LTP impairment by Aβ. Ibuprofen prevents LTP impairment by Aβ by restoring guanylate cyclase activation and increase in cGMP and, subsequently, activation of protein kinase G, phosphorylation of GluR1 in Ser845 and synaptic expression of AMPA receptors. Restoration of cGMP levels is enough to restore all this process as indicated by the fact that the cGMP analog 8-Br-cGMP also normalizes the function of this pathway and restores LTP in the presence of Aβ. These results indicate that Aβ impairs LTP by impairing the NO-cGMP pathway and that ibuprofen restores LTP by restoring this pathway. These data suggest that restoring cGMP levels may have therapeutic utility to improve cognitive function impaired by Aβ. PMID:20858955

  8. Investigation of the regulation of transcriptional changes in Ancylostoma caninum larvae following serum activation, with a focus on the insulin-like signalling pathway.

    PubMed

    Datu, Bennett J D; Loukas, Alex; Cantacessi, Cinzia; O'Donoghue, Peter; Gasser, Robin B

    2009-02-01

    The exit from dauer in the free-living nematode Caenorhabditis elegans is under the control of a single amphidial neuron (ASJ) of the insulin-like signalling pathway. Mutations of this pathway have the ability to suppress entry into the dauer stage. It has been postulated that insulin-like signalling plays a significant role in the response to serum stimulation in vitro of the third-stage larvae (L3s) of the canine hookworm Ancylostoma caninum. To test for the possible involvement of the insulin-like signalling cascade in the response to serum stimulation, the effects of two signalling stimulants (8-bromo cGMP and arecoline) and four inhibitors, namely 4,7-phenanthroline, phosphoinositide-3 kinase (PI3K), Akt inhibitor IV and rapamycin on feeding and on levels of selected activation-associated mRNAs in serum-stimulated L3s were explored. L3s of A. caninum were pre-incubated with or without the appropriate inhibitor/agonist. Following serum-stimulation, the feeding activity was assessed. The transcription levels of a number of activation-associated mRNAs linked to particular expressed sequence tags (ESTs) were investigated by reverse transcription, real-time PCR (rtPCR). The treatment of worms with 4,7-phenanthroline completely suppressed feeding and significantly reduced the differential levels of most activation-associated mRNAs, whereas the treatment with cGMP resulted in the resumption of feeding in almost 85% of the L3s and yielded a specific transcriptional profile consistent with that following serum stimulation. The treatment of L3s with arecoline resulted in the resumption of feeding in approximately 85% of L3s, but did not result in a transcriptomic profile consistent with activation. A complete reduction in feeding was recorded in the presence of the PI3K inhibitor LY294002 (1mM) and resulted in a pronounced dampening of differential transcription in response to serum stimulation for the molecules examined. Akt inhibitor IV resulted in a approximately 70

  9. Cellular signaling in eclosion hormone action.

    PubMed

    Morton, David B.; Simpson, P Jeanette

    2002-01-01

    Eclosion hormone (EH) is a 62 amino acid neuropeptide that plays an integral role in triggering ecdysis behavior at the end of each molt. At least three populations of cells are thought to be targets for EH, each of which show an EH-stimulated increase in the intracellular messenger guanosine 3', 5' cyclic monophosphate (cGMP). These EH target cells are believed to include two pairs of neurons in each of the ganglia of the ventral nerve cord (VNC) that contain the neuropeptide crustacean cardioactive peptide (CCAP), the Inka cells of the peripheral epitracheal glands and intrinsic non-neuronal cells in the abdominal transverse nerves. This review describes likely signaling cascades that result in the EH-stimulated cGMP increase. Several lines of evidence suggest the involvement of a novel nitric oxide insensitive soluble guanylyl cyclase (GC). A novel GC with these properties has recently been identified and we also present evidence to suggest that it is activated by EH and describe possible pathways for its activation. In addition, we review our current knowledge on the cellular and molecular events that take place downstream of the increase in cGMP. PMID:12770127

  10. Sleep Loss Activates Cellular Inflammatory Signaling

    PubMed Central

    Irwin, Michael R.; Wang, Minge; Ribeiro, Denise; Cho, Hyong Jin; Olmstead, Richard; Breen, Elizabeth Crabb; Martinez-Maza, Otoniel; Cole, Steve

    2008-01-01

    Background Accumulating evidence suggests that sleep disturbance is associated with inflammation and related disorders including cardiovascular disease, arthritis, and diabetes mellitus. This study was undertaken to test the effects of sleep loss on activation of nuclear factor (NF) -κB, a transcription factor that serves a critical role in the inflammatory signaling cascade. Methods In 14 healthy adults (7 females; 7 males), peripheral blood mononuclear cell NF-κB was repeatedly assessed, along with enumeration of lymphocyte subpopulations, in the morning after baseline sleep, partial sleep deprivation (awake from 23:00 h to 03:00 h), and recovery sleep. Results In the morning after a night of sleep loss, mononuclear cell NF-κB activation was significantly greater compared with morning levels following uninterrupted baseline or recovery sleep, in which the response was found in females but not in males. Conclusions These results identify NF-κB activation as a molecular pathway by which sleep disturbance may influence leukocyte inflammatory gene expression and the risk of inflammation-related disease. PMID:18561896

  11. Heat dissipation guides activation in signaling proteins

    PubMed Central

    Weber, Jeffrey K.; Shukla, Diwakar; Pande, Vijay S.

    2015-01-01

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein–coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  12. Heat dissipation guides activation in signaling proteins.

    PubMed

    Weber, Jeffrey K; Shukla, Diwakar; Pande, Vijay S

    2015-08-18

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein-coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  13. Active voltammetric microsensors with neural signal processing.

    SciTech Connect

    Vogt, M. C.

    1998-12-11

    Many industrial and environmental processes, including bioremediation, would benefit from the feedback and control information provided by a local multi-analyte chemical sensor. For most processes, such a sensor would need to be rugged enough to be placed in situ for long-term remote monitoring, and inexpensive enough to be fielded in useful numbers. The multi-analyte capability is difficult to obtain from common passive sensors, but can be provided by an active device that produces a spectrum-type response. Such new active gas microsensor technology has been developed at Argonne National Laboratory. The technology couples an electrocatalytic ceramic-metallic (cermet) microsensor with a voltammetric measurement technique and advanced neural signal processing. It has been demonstrated to be flexible, rugged, and very economical to produce and deploy. Both narrow interest detectors and wide spectrum instruments have been developed around this technology. Much of this technology's strength lies in the active measurement technique employed. The technique involves applying voltammetry to a miniature electrocatalytic cell to produce unique chemical ''signatures'' from the analytes. These signatures are processed with neural pattern recognition algorithms to identify and quantify the components in the analyte. The neural signal processing allows for innovative sampling and analysis strategies to be employed with the microsensor. In most situations, the whole response signature from the voltammogram can be used to identify, classify, and quantify an analyte, without dissecting it into component parts. This allows an instrument to be calibrated once for a specific gas or mixture of gases by simple exposure to a multi-component standard rather than by a series of individual gases. The sampled unknown analytes can vary in composition or in concentration, the calibration, sensing, and processing methods of these active voltammetric microsensors can detect, recognize, and

  14. Plant Cyclic Nucleotide Signalling

    PubMed Central

    Martinez-Atienza, Juliana; Van Ingelgem, Carl; Roef, Luc

    2007-01-01

    The presence of the cyclic nucleotides 3′,5′-cyclic adenyl monophosphate (cAMP) and 3′,5′-cyclic guanyl monophosphate (cGMP) in plants is now generally accepted. In addition, cAMP and cGMP have been implicated in the regulation of important plant processes such as stomatal functioning, monovalent and divalent cation fluxes, chloroplast development, gibberellic acid signalling, pathogen response and gene transcription. However, very little is known regarding the components of cyclic nucleotide signalling in plants. In this addendum, the evidence for specific mechanisms of plant cyclic nucleotide signalling is evaluated and discussed. PMID:19704553

  15. New Constitutively Active Phytochromes Exhibit Light-Independent Signaling Activity.

    PubMed

    Jeong, A-Reum; Lee, Si-Seok; Han, Yun-Jeong; Shin, Ah-Young; Baek, Ayoung; Ahn, Taeho; Kim, Min-Gon; Kim, Young Soon; Lee, Keun Woo; Nagatani, Akira; Kim, Jeong-Il

    2016-08-01

    Plant phytochromes are photoreceptors that mediate a variety of photomorphogenic responses. There are two spectral photoisomers, the red light-absorbing Pr and far-red light-absorbing Pfr forms, and the photoreversible transformation between the two forms is important for the functioning of phytochromes. In this study, we isolated a Tyr-268-to-Val mutant of Avena sativa phytochrome A (AsYVA) that displayed little photoconversion. Interestingly, transgenic plants of AsYVA showed light-independent phytochrome signaling with a constitutive photomorphogenic (cop) phenotype that is characterized by shortened hypocotyls and open cotyledons in the dark. In addition, the corresponding Tyr-303-to-Val mutant of Arabidopsis (Arabidopsis thaliana) phytochrome B (AtYVB) exhibited nuclear localization and interaction with phytochrome-interacting factor 3 (PIF3) independently of light, conferring a constitutive photomorphogenic development to its transgenic plants, which is comparable to the first constitutively active version of phytochrome B (YHB; Tyr-276-to-His mutant). We also found that chromophore ligation was required for the light-independent interaction of AtYVB with PIF3. Moreover, we demonstrated that AtYVB did not exhibit phytochrome B activity when it was localized in the cytosol by fusion with the nuclear export signal and that AsYVA exhibited the full activity of phytochrome A when localized in the nucleus by fusion with the nuclear localization signal. Furthermore, the corresponding Tyr-269-to-Val mutant of Arabidopsis phytochrome A (AtYVA) exhibited similar cop phenotypes in transgenic plants to AsYVA. Collectively, these results suggest that the conserved Tyr residues in the chromophore-binding pocket play an important role during the Pr-to-Pfr photoconversion of phytochromes, providing new constitutively active alleles of phytochromes by the Tyr-to-Val mutation. PMID:27325667

  16. Role of nitric oxide and cyclic GMP signaling in melanocyte response to hypergravity

    NASA Astrophysics Data System (ADS)

    Ivanova, Krassimira; Lambers, Britta; Tsiockas, Wasiliki; Block, Ingrid; Gerzer, Rupert

    Nitric oxide (NO) has a prominent role in many (patho)physiological processes in the skin including erythema, inflammation, and cancerogenesis. The soluble guanylyl cyclase (sGC), a key transducer in NO signaling, catalyzes the formation of the second messenger guanosine 3´,5´-cyclic monophosphate (cyclic cGMP or cGMP). For human melanocytes, which are responsible for skin pigmentation by synthesizing the pigment melanin, it has been reported that the NO/sGC/cGMP pathway is involved in UVB-induced melanogenesis. Melanin acts as a scavenger for free radicals that may arise during metabolic stress. It may also act as a photosensitizer that generates active oxygen species upon UV irradiation, which may initiate hypopigmentary disorders (e.g., vitiligo) as well as UV-induced oncogene cell transformation. In addition, melanoma, a deadly skin cancer, which arises from transformed melanocytes, is characterized by a resistance to chemotherapy. In our studies we have shown that NO can induce perturbation of melanocyte-extracellular matrix component interactions, which may contribute to loss of melanocytes or melanoma metastasis. Such NO effects appear to be modulated partly via cGMP. Moreover, we found that different guanylyl cyclase isoforms are responsible for cGMP synthesis in melanocytic cells. Normal human melanocytes and nonmetastatic melanoma cells predominantly express sGC, which appears to be associated with melanogenesis, whereas absence of NO-sensitive GC, but up-regulated activities of the natriuretic peptide-sensitive membrane guanylyl cyclase isoforms were found in highly metastatic phenotypes. Due to the growing interest in the regulation of signaling activities in normal and transformed cells under altered gravity conditions, we have further investigated whether the NO/cGMP signaling is involved in melanocyte response to gravitational stress. We found that normal human melanocytes and non-metastatic melanoma cell lines, but not highly metastatic cells

  17. Activation of NO-cGMP Signaling Rescues Age-Related Memory Impairment in Crickets

    PubMed Central

    Matsumoto, Yukihisa; Matsumoto, Chihiro S.; Takahashi, Toshihumi; Mizunami, Makoto

    2016-01-01

    Age-related memory impairment (AMI) is a common feature and a debilitating phenotype of brain aging in many animals. However, the molecular mechanisms underlying AMI are still largely unknown. The cricket Gryllus bimaculatus is a useful experimental animal for studying age-related changes in learning and memory capability; because the cricket has relatively short life-cycle and a high capability of olfactory learning and memory. Moreover, the molecular mechanisms underlying memory formation in crickets have been examined in detail. In the present study, we trained male crickets of different ages by multiple-trial olfactory conditioning to determine whether AMI occurs in crickets. Crickets 3 weeks after the final molt (3-week-old crickets) exhibited levels of retention similar to those of 1-week-old crickets at 30 min or 2 h after training; however they showed significantly decreased levels of 1-day retention, indicating AMI in long-term memory (LTM) but not in anesthesia-resistant memory (ARM) in olfactory learning of crickets. Furthermore, 3-week-old crickets injected with a nitric oxide (NO) donor, a cyclic GMP (cGMP) analog or a cyclic AMP (cAMP) analog into the hemolymph before conditioning exhibited a normal level of LTM, the same level as that in 1-week-old crickets. The rescue effect by NO donor or cGMP analog injection was absent when the crickets were injected after the conditioning. For the first time, an NO donor and a cGMP analog were found to antagonize the age-related impairment of LTM formation, suggesting that deterioration of NO synthase (NOS) or molecules upstream of NOS activation is involved in brain-aging processes. PMID:27616985

  18. Activation of NO-cGMP Signaling Rescues Age-Related Memory Impairment in Crickets.

    PubMed

    Matsumoto, Yukihisa; Matsumoto, Chihiro S; Takahashi, Toshihumi; Mizunami, Makoto

    2016-01-01

    Age-related memory impairment (AMI) is a common feature and a debilitating phenotype of brain aging in many animals. However, the molecular mechanisms underlying AMI are still largely unknown. The cricket Gryllus bimaculatus is a useful experimental animal for studying age-related changes in learning and memory capability; because the cricket has relatively short life-cycle and a high capability of olfactory learning and memory. Moreover, the molecular mechanisms underlying memory formation in crickets have been examined in detail. In the present study, we trained male crickets of different ages by multiple-trial olfactory conditioning to determine whether AMI occurs in crickets. Crickets 3 weeks after the final molt (3-week-old crickets) exhibited levels of retention similar to those of 1-week-old crickets at 30 min or 2 h after training; however they showed significantly decreased levels of 1-day retention, indicating AMI in long-term memory (LTM) but not in anesthesia-resistant memory (ARM) in olfactory learning of crickets. Furthermore, 3-week-old crickets injected with a nitric oxide (NO) donor, a cyclic GMP (cGMP) analog or a cyclic AMP (cAMP) analog into the hemolymph before conditioning exhibited a normal level of LTM, the same level as that in 1-week-old crickets. The rescue effect by NO donor or cGMP analog injection was absent when the crickets were injected after the conditioning. For the first time, an NO donor and a cGMP analog were found to antagonize the age-related impairment of LTM formation, suggesting that deterioration of NO synthase (NOS) or molecules upstream of NOS activation is involved in brain-aging processes. PMID:27616985

  19. Altered cGMP dynamics at the plasma membrane contribute to diarrhea in ulcerative colitis.

    PubMed

    Arora, Kavisha; Sinha, Chandrima; Zhang, Weiqiang; Moon, Chang Suk; Ren, Aixia; Yarlagadda, Sunitha; Dostmann, Wolfgang R; Adebiyi, Adebowale; Haberman, Yael; Denson, Lee A; Wang, Xusheng; Naren, Anjaparavanda P

    2015-10-01

    Ulcerative colitis (UC) belongs to inflammatory bowel disorders, a group of gastrointestinal disorders that can produce serious recurring diarrhea in affected patients. The mechanism for UC- and inflammatory bowel disorder-associated diarrhea is not well understood. The cystic fibrosis transmembrane-conductance regulator (CFTR) chloride channel plays an important role in fluid and water transport across the intestinal mucosa. CFTR channel function is regulated in a compartmentalized manner through the formation of CFTR-containing macromolecular complexes at the plasma membrane. In this study, we demonstrate the involvement of a novel macromolecular signaling pathway that causes diarrhea in UC. We found that a nitric oxide-producing enzyme, inducible nitric oxide synthase (iNOS), is overexpressed under the plasma membrane and generates compartmentalized cGMP in gut epithelia in UC. The scaffolding protein Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) bridges iNOS with CFTR, forming CFTR-NHERF2-iNOS macromolecular complexes that potentiate CFTR channel function via the nitric oxide-cGMP pathway under inflammatory conditions both in vitro and in vivo. Potential disruption of these complexes in Nherf2(-/-) mice may render them more resistant to CFTR-mediated secretory diarrhea than Nherf2(+/+) mice in murine colitis models. Our study provides insight into the mechanism of pathophysiologic occurrence of diarrhea in UC and suggests that targeting CFTR and CFTR-containing macromolecular complexes will ameliorate diarrheal symptoms and improve conditions associated with inflammatory bowel disorders. PMID:26261085

  20. The Gyc76C Receptor Guanylyl Cyclase and the Foraging cGMP-Dependent Kinase Regulate Extracellular Matrix Organization and BMP Signaling in the Developing Wing of Drosophila melanogaster

    PubMed Central

    Schleede, Justin; Blair, Seth S.

    2015-01-01

    The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues. PMID:26440503

  1. Kinase active Misshapen regulates Notch signaling in Drosophila melanogaster.

    PubMed

    Mishra, Abhinava K; Sachan, Nalani; Mutsuddi, Mousumi; Mukherjee, Ashim

    2015-11-15

    Notch signaling pathway represents a principal cellular communication system that plays a pivotal role during development of metazoans. Drosophila misshapen (msn) encodes a protein kinase, which is related to the budding yeast Ste20p (sterile 20 protein) kinase. In a genetic screen, using candidate gene approach to identify novel kinases involved in Notch signaling, we identified msn as a novel regulator of Notch signaling. Data presented here suggest that overexpression of kinase active form of Msn exhibits phenotypes similar to Notch loss-of-function condition and msn genetically interacts with components of Notch signaling pathway. Kinase active form of Msn associates with Notch receptor and regulate its signaling activity. We further show that kinase active Misshapen leads to accumulation of membrane-tethered form of Notch. Moreover, activated Msn also depletes Armadillo and DE-Cadherin from adherens junctions. Thus, this study provides a yet unknown mode of regulation of Notch signaling by Misshapen. PMID:26431585

  2. In vivo cGMP levels in frog photoreceptor cells as a function of light exposure.

    PubMed

    Barbehenn, E K; Klotz, K L; Noelker, D M; Nelson, R; Chader, G J; Passonneau, J V

    1986-11-01

    By employing a combination of highly sensitive radioimmunoassays and histochemical techniques, an in vivo time course of cGMP levels has been determined in the outer segment, photoreceptor cell and outer plexiform layers of frog retina. Frogs (Rana pipiens) were dark-adapted overnight and either frozen rapidly (approximately 3 sec) in liquid nitrogen or exposed to periods of light varying between 0.1 sec and 2 hr before freezing. Frozen retinal sections were cut, freeze-dried, and samples of individual layers dissected out and analysed for cGMP. In the outer plexiform layer, there was a 42% drop in cGMP concentration after 2 sec of light (250 ft candles) followed by a 34% rise after 2 min; a steep concentration gradient formed around the layer after the 2 min exposure. In both the outer-segment layer and photoreceptor-cell layer (which includes outer segments, inner segments and outer nuclear layers), cGMP levels declined from a dark value of 56 mumol kg-1 (dry) to 9 mumol kg-1 (dry) as a result of increasing exposure to several types of light source: levels appear to be primarily a function of total ft candle min. Cyclic GMP concentrations at the longest exposures (2 min with a fiber optic light source or 2 hr with fluorescent room light) reached identical minimum levels. In the outer segments, a 15% decrease in cGMP was observed after 0.1 sec of light exposure. Although the freezing time is too long to be able to say whether the 15% decrease in cGMP at the 0.1 sec exposure is involved in transduction, the low identical levels reached gradually after longer exposures appear to indicate that a light-induced biochemical adjustment in cGMP metabolism occurs over a relatively long time period separate from the msec time course of the transduction process. PMID:3026825

  3. Subcellular trafficking of guanylyl cyclase/natriuretic peptide receptor-A with concurrent generation of intracellular cGMP

    PubMed Central

    Mani, Indra; Garg, Renu; Tripathi, Satyabha; Pandey, Kailash N.

    2015-01-01

    Atrial natriuretic peptide (ANP) activates guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which lowers blood pressure and blood volume. The objective of the present study was to visualize internalization and trafficking of enhanced GFP (eGFP)-tagged NPRA (eGFP–NPRA) in human embryonic kidney-293 (HEK-293) cells, using immunofluorescence (IF) and co-immunoprecipitation (co-IP) of eGFP–NPRA. Treatment of cells with ANP initiated rapid internalization and co-localization of the receptor with early endosome antigen-1 (EEA-1), which was highest at 5 min and gradually decreased within 30 min. Similarly, co-localization of the receptor was observed with lysosome-associated membrane protein-1 (LAMP-1); however, after treatment with lysosomotropic agents, intracellular accumulation of the receptor gradually increased within 30 min. Co-IP assays confirmed that the localization of internalized receptors occurred with subcellular organelles during the endocytosis of NPRA. Rab 11, which was used as a recycling endosome (Re) marker, indicated that ∼20% of receptors recycled back to the plasma membrane. ANP-treated cells showed a marked increase in the IF of cGMP, whereas receptor was still trafficking into the intracellular compartments. Thus, after ligand binding, NPRA is rapidly internalized and trafficked from the cell surface into endosomes, Res and lysosomes, with concurrent generation of intracellular cGMP. PMID:26374856

  4. Subcellular trafficking of guanylyl cyclase/natriuretic peptide receptor-A with concurrent generation of intracellular cGMP.

    PubMed

    Mani, Indra; Garg, Renu; Tripathi, Satyabha; Pandey, Kailash N

    2015-01-01

    Atrial natriuretic peptide (ANP) activates guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which lowers blood pressure and blood volume. The objective of the present study was to visualize internalization and trafficking of enhanced GFP (eGFP)-tagged NPRA (eGFP-NPRA) in human embryonic kidney-293 (HEK-293) cells, using immunofluorescence (IF) and co-immunoprecipitation (co-IP) of eGFP-NPRA. Treatment of cells with ANP initiated rapid internalization and co-localization of the receptor with early endosome antigen-1 (EEA-1), which was highest at 5 min and gradually decreased within 30 min. Similarly, co-localization of the receptor was observed with lysosome-associated membrane protein-1 (LAMP-1); however, after treatment with lysosomotropic agents, intracellular accumulation of the receptor gradually increased within 30 min. Co-IP assays confirmed that the localization of internalized receptors occurred with subcellular organelles during the endocytosis of NPRA. Rab 11, which was used as a recycling endosome (Re) marker, indicated that ∼20% of receptors recycled back to the plasma membrane. ANP-treated cells showed a marked increase in the IF of cGMP, whereas receptor was still trafficking into the intracellular compartments. Thus, after ligand binding, NPRA is rapidly internalized and trafficked from the cell surface into endosomes, Res and lysosomes, with concurrent generation of intracellular cGMP. PMID:26374856

  5. Human ECG signal parameters estimation during controlled physical activity

    NASA Astrophysics Data System (ADS)

    Maciejewski, Marcin; Surtel, Wojciech; Dzida, Grzegorz

    2015-09-01

    ECG signal parameters are commonly used indicators of human health condition. In most cases the patient should remain stationary during the examination to decrease the influence of muscle artifacts. During physical activity, the noise level increases significantly. The ECG signals were acquired during controlled physical activity on a stationary bicycle and during rest. Afterwards, the signals were processed using a method based on Pan-Tompkins algorithms to estimate their parameters and to test the method.

  6. Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

    PubMed Central

    Nemet, Ina; Tian, Guilian; Imanishi, Yoshikazu

    2014-01-01

    A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade. PMID:25616687

  7. Activity Dependent Signal Transduction in Skeletal Muscle

    NASA Technical Reports Server (NTRS)

    Hamilton, Susan L.

    1999-01-01

    The overall goals of this project are: 1) to define the initial signal transduction events whereby the removal of gravitational load from antigravity muscles, such as the soleus, triggers muscle atrophy, and 2) to develop countermeasures to prevent this from happening. Our rationale for this approach is that, if countermeasures can be developed to regulate these early events, we could avoid having to deal with the multiple cascades of events that occur downstream from the initial event. One of our major findings is that hind limb suspension causes an early and sustained increase in intracellular Ca(2+) concentration ([Ca (2+)](sub i)). In most cells the consequences of changes in ([Ca (2+)](sub i))depend on the amplitude, frequency and duration of the Ca(2+) signal and on other factors in the intracellular environment. We propose that muscle remodeling in microgravity represents a change in the balance among several CA(2+) regulated signal transduction pathways, in particular those involving the transcription factors NFAT and NFkB and the pro-apoptotic protein BAD. Other Ca(2+) sensitive pathways involving PKC, ras, rac, and CaM kinase II may also contribute to muscle remodeling.

  8. Platelet hemostasis in patients with metabolic syndrome and type 2 diabetes mellitus: cGMP- and NO-dependent mechanisms in the insulin-mediated platelet aggregation

    PubMed Central

    Suslova, Tatiana E.; Sitozhevskii, Alexei V.; Ogurkova, Oksana N.; Kravchenko, Elena S.; Kologrivova, Irina V.; Anfinogenova, Yana; Karpov, Rostislav S.

    2015-01-01

    Patients with metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM) have high risk of microcirculation complications and microangiopathies. An increase in thrombogenic risk is associated with platelet hyperaggregation, hypercoagulation, and hyperfibrinolysis. Factors leading to platelet activation in MetS and T2DM comprise insulin resistance, hyperglycemia, non-enzymatic glycosylation, oxidative stress, and inflammation. This review discusses the role of nitric oxide (NO) in the regulation of platelet adhesion and aggregation processes. NO is synthesized both in endotheliocytes, smooth muscle cells, macrophages, and platelets. Modification of platelet NO-synthase (NOS) activity in MetS patients can play a central role in the manifestation of platelet hyperactivation. Metabolic changes, accompanying T2DM, can lead to an abnormal NOS expression and activity in platelets. Hyperhomocysteinemia, often accompanying T2DM, is a risk factor for cardiovascular accidents. Homocysteine can reduce NO production by platelets. This review provides data on the insulin effects in platelets. Decrease in a number and sensitivity of the insulin receptors on platelets in T2DM can cause platelet hyperactivation. Various intracellular mechanisms of anti-aggregating insulin effects are discussed. Anti-aggregating effects of insulin are mediated by a NO-induced elevation of cGMP and upregulation of cAMP- and cGMP-dependent pathways. The review presents data suggesting an ability of platelets to synthesize humoral factors stimulating thrombogenesis and inflammation. Proinflammatory cytokines are considered as markers of T2DM and cardiovascular complications and are involved in the development of dyslipidemia and insulin resistance. The article provides an evaluation of NO-mediated signaling pathway in the effects of cytokines on platelet aggregation. The effects of the proinflammatory cytokines on functional activity of platelets are demonstrated. PMID:25601838

  9. Differential regulation of Paramecium ciliary motility by cAMP and cGMP.

    PubMed

    Bonini, N M; Nelson, D L

    1988-05-01

    cAMP and cGMP had distinct effects on the regulation of ciliary motility in Paramecium. Using detergent-permeabilized cells reactivated to swim with MgATP, we observed effects of cyclic nucleotides and interactions with Ca2+ on the swimming speed and direction of reactivated cells. Both cAMP and cGMP increased forward swimming speed two- to threefold with similar half-maximal concentrations near 0.5 microM. The two cyclic nucleotides, however, had different effects in antagonism with the Ca2+ response of backward swimming and on the handedness of the helical swimming paths of reactivated cells. These results suggest that cAMP and cGMP differentially regulate the direction of the ciliary power stroke. PMID:2836435

  10. The Canonical Notch Signaling Pathway: Unfolding the Activation Mechanism

    PubMed Central

    Kopan, Raphael; Ilagan, Ma. Xenia G.

    2009-01-01

    Notch signaling regulates many aspects of metazoan development and tissue renewal. Accordingly, misregulation or loss of Notch signaling underlies multiple human disorders, from developmental syndromes to adult onset diseases and cancer. Notch receptor activation is irreversible as it involves proteolysis-mediated release of the Notch intracellular domain, translocation to the nucleus, and association with a DNA-bound protein. Even though each Notch molecule signals only once without amplification by secondary messenger cascades, Notch signaling is remarkably robust in most tissues. In this review, we highlight the recent studies that reveal new molecular details involved in regulating ligand-mediated activation, receptor proteolysis and target selection. PMID:19379690

  11. Oncocalyxone A inhibits human platelet aggregation by increasing cGMP and by binding to GP Ibα glycoprotein

    PubMed Central

    Ferreira, M A D; do Nascimento, N R F; de Sousa, C M; Pessoa, O D L; de Lemos, T L G; Ventura, J S; Schattner, M; Chudzinski-Tavassi, A M

    2008-01-01

    Background and purpose: Oncocalyxone A (OncoA) has a concentration-dependent anti-platelet activity. The present study aimed to further understand the mechanisms related to this effect. Experimental approach: Human platelet aggregation was measured by means of a turbidimetric method. OncoA (32–256 μM) was tested against several platelet-aggregating agents, such as adenosine diphosphate (ADP), collagen, arachidonic acid (AA), ristocetin and thrombin. Key results: OncoA completely inhibited platelet aggregation with a calculated mean inhibitory concentration (IC50-μM) of 122 for ADP, 161 for collagen, 159 for AA, 169 for ristocetin and 85 for thrombin. The anti-aggregatory activity of OncoA was not inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). OncoA, at a concentration that caused no significant anti-aggregatory activity, potentiated sodium nitroprusside (SNP) anti-aggregatory activity (18.8±2.9%-SNP vs 85.0±8.2%-SNP+OncoA). The levels of nitric oxide (NO) or cAMP were not altered by OncoA while cGMP levels were increased more than 10-fold by OncoA in resting or ADP-activated platelets. Flow cytometry revealed that OncoA does not interact with receptors for fibrinogen, collagen or P-selectin. Nevertheless, OncoA decreased the binding of antibodies to GP Ibα, a glycoprotein that is related both to von Willebrand factor and to thrombin-induced platelet aggregation. Conclusion and implications: OncoA showed anti-aggregatory activity in platelets that was associated with increased cGMP levels, not dependent on NO and with blocking GP Ibα glycoprotein. This new mechanism has the prospect of leading to new anti-thrombotic drugs. PMID:18516074

  12. Wnt Signaling in Neurogenesis during Aging and Physical Activity

    PubMed Central

    Chen, Michael; Do, Huong

    2012-01-01

    Over the past decade, much progress has been made regarding our understanding of neurogenesis in both young and old animals and where it occurs throughout the lifespan, although the growth of new neurons declines with increasing age. In addition, physical activity can reverse this age-dependent decline in neurogenesis. Highly correlated with this decline is the degree of inter and intracellular Wnt signaling, the molecular mechanisms of which have only recently started to be elucidated. So far, most of what we know about intracellular signaling during/following exercise centers around the CREB/CRE initiated transcriptional events. Relatively little is known, however, about how aging and physical activity affect the Wnt signaling pathway. Herein, we briefly review the salient features of neurogenesis in young and then in old adult animals. Then, we discuss Wnt signaling and review the very few in vitro and in vivo studies that have examined the Wnt signaling pathways in aging and physical activity. PMID:24961268

  13. Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling.

    PubMed

    Huang, Pengxiang; Nedelcu, Daniel; Watanabe, Miyako; Jao, Cindy; Kim, Youngchang; Liu, Jing; Salic, Adrian

    2016-08-25

    In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol. PMID:27545348

  14. Activation of endothelial β-catenin signaling induces heart failure

    PubMed Central

    Nakagawa, Akito; Naito, Atsuhiko T.; Sumida, Tomokazu; Nomura, Seitaro; Shibamoto, Masato; Higo, Tomoaki; Okada, Katsuki; Sakai, Taku; Hashimoto, Akihito; Kuramoto, Yuki; Oka, Toru; Lee, Jong-Kook; Harada, Mutsuo; Ueda, Kazutaka; Shiojima, Ichiro; Limbourg, Florian P.; Adams, Ralf H.; Noda, Tetsuo; Sakata, Yasushi; Akazawa, Hiroshi; Komuro, Issei

    2016-01-01

    Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure. PMID:27146149

  15. Activation of endothelial β-catenin signaling induces heart failure.

    PubMed

    Nakagawa, Akito; Naito, Atsuhiko T; Sumida, Tomokazu; Nomura, Seitaro; Shibamoto, Masato; Higo, Tomoaki; Okada, Katsuki; Sakai, Taku; Hashimoto, Akihito; Kuramoto, Yuki; Oka, Toru; Lee, Jong-Kook; Harada, Mutsuo; Ueda, Kazutaka; Shiojima, Ichiro; Limbourg, Florian P; Adams, Ralf H; Noda, Tetsuo; Sakata, Yasushi; Akazawa, Hiroshi; Komuro, Issei

    2016-01-01

    Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure. PMID:27146149

  16. Quantifying bursting neuron activity from calcium signals using blind deconvolution.

    PubMed

    Park, In Jun; Bobkov, Yuriy V; Ache, Barry W; Principe, Jose C

    2013-09-15

    Advances in calcium imaging have enabled studies of the dynamic activity of both individual neurons and neuronal assemblies. However, challenges, such as unknown nonlinearities in the spike-calcium relationship, noise, and the often relatively low temporal resolution of the calcium signal compared to the time-scale of spike generation, restrict the accurate estimation of action potentials from the calcium signal. Complex neuronal discharge, such as the activity demonstrated by bursting and rhythmically active neurons, represents an even greater challenge for reconstructing spike trains based on calcium signals. We propose a method using blind calcium signal deconvolution based on an information-theoretic approach. This model is meant to maximise the output entropy of a nonlinear filter where the nonlinearity is defined by the cumulative distribution function of the spike signal. We tested our maximum entropy (ME) algorithm using bursting olfactory receptor neurons (bORNs) of the lobster olfactory organ. The advantage of the ME algorithm is that the filter can be trained online based only on the statistics of the spike signal, without any assumptions regarding the unknown transfer function characterizing the relation between the spike and calcium signal. We show that the ME method is able to more accurately reconstruct the timing of the first and last spikes of a burst compared to other methods and that it improves the temporal precision fivefold compared to direct timing resolution of calcium signal. PMID:23711821

  17. Activation of Notch-Mediated Protective Signaling in the Myocardium

    PubMed Central

    Gude, Natalie A.; Emmanuel, Gregory; Wu, Weitao; Cottage, Christopher T.; Fischer, Kimberlee; Quijada, Pearl; Muraski, John A.; Alvarez, Roberto; Rubio, Marta; Schaefer, Eric; Sussman, Mark A.

    2013-01-01

    The Notch network regulates multiple cellular processes, including cell fate determination, development, differentiation, proliferation, apoptosis, and regeneration. These processes are regulated via Notch-mediated activity that involves hepatocyte growth factor (HGF)/c-Met receptor and phosphatidylinositol 3-kinase/Akt signaling cascades. The impact of HGF on Notch signaling was assessed following myocardial infarction as well as in cultured cardiomyocytes. Notch1 is activated in border zone cardiomyocytes coincident with nuclear c-Met following infarction. Intramyocardial injection of HGF enhances Notch1 and Akt activation in adult mouse myocardium. Corroborating evidence in cultured cardiomyocytes shows treatment with HGF or insulin increases levels of Notch effector Hes1 in immunoblots, whereas overexpression of activated Notch intracellular domain prompts a 3-fold increase in phosphorylated Akt. Infarcted hearts injected with adenoviral vector expressing Notch intracellular domain treatment exhibit improved hemodynamic function in comparison with control mice after 4 weeks, implicating Notch signaling in a cardioprotective role following cardiac injury. These results indicate Notch activation in cardiomyocytes is mediated through c-Met and Akt survival signaling pathways, and Notch1 signaling in turn enhances Akt activity. This mutually supportive crosstalk suggests a positive survival feedback mechanism between Notch and Akt signaling in adult myocardium following injury. PMID:18369158

  18. A Short History of cGMP, Guanylyl Cyclases, and cGMP-Dependent Protein Kinases

    PubMed Central

    Kots, Alexander Y.; Martin, Emil; Sharina, Iraida G.

    2014-01-01

    Here, we review the early studies on cGMP, guanylyl cyclases, and cGMP-dependent protein kinases to facilitate understanding of development of this exciting but complex field of research encompassing pharmacology, biochemistry, physiology, and molecular biology of these important regulatory molecules. PMID:19089322

  19. Signal peptides are allosteric activators of the protein translocase.

    PubMed

    Gouridis, Giorgos; Karamanou, Spyridoula; Gelis, Ioannis; Kalodimos, Charalampos G; Economou, Anastassios

    2009-11-19

    Extra-cytoplasmic polypeptides are usually synthesized as 'preproteins' carrying amino-terminal, cleavable signal peptides and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA. Most proteins destined for the periplasm and beyond are exported post-translationally by SecA. Preprotein targeting to SecA is thought to involve signal peptides and chaperones like SecB. Here we show that signal peptides have a new role beyond targeting: they are essential allosteric activators of the translocase. On docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, 'triggering' that drives the translocase to a lower activation energy state; second, 'trapping' that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus; and third, 'secretion' during which trapped mature domains undergo several turnovers of translocation in segments. A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases. PMID:19924216

  20. Reduction of endothelial permeability in vitro by cAMP and cGMP

    SciTech Connect

    Kreienberg, P.B.; DeMichele, M.A.; Kowalczyk, P.; Minnear, F.L. )

    1990-02-26

    The cAMP enhancing-vasodilator isoproterenol has been shown previously to decrease endothelial permeability in vitro. This effect may not be unique to cAMP-enhancing agents. The authors have shown that thrombin at a concentration of 2 pM, a level which relaxes aortic vessel strips in association with increased levels of cGMP, reduces endothelial permeability. In this study, the permeability effect of cAMP and cGMP analogues were assessed by measuring the clearance of {sup 125}I-albumin across bovine pulmonary artery endothelial cell monolayers. The experiments were divided into baseline and experimental periods so that each monolayer served as its own control. The cAMP and cGMP analogues, 8-bromo-cAMP (1 mM) and 8-bromo-cGMP (1 mM), decreased clearance form a vehicle control value of 1.3{+-}0.2 (mean {+-}SD of experimental/baseline clearance, n=15 cell monolayers) to 0.7{+-}0.2 and 1.0{+-}0.2, respectively, although cGMP did not decrease clearance from its own baseline value. Coincubation of these analogues with thrombin (0.1 uM) also decreased the thrombin-induced increase in albumin clearance from 2.2{+-}0.5 to 0.8{+-}0.2 (cAMP) and 1.5{+-}0.2 (cGMP). The data indicate that in vitro both cAMP and cGMP-enhancing vasodilators would reduce endothelial permeability and that cAMP-enhancing agents would be more effective.

  1. Optogenetic activation of intracellular adenosine A2A receptor signaling in the hippocampus is sufficient to trigger CREB phosphorylation and impair memory.

    PubMed

    Li, P; Rial, D; Canas, P M; Yoo, J-H; Li, W; Zhou, X; Wang, Y; van Westen, G J P; Payen, M-P; Augusto, E; Gonçalves, N; Tomé, A R; Li, Z; Wu, Z; Hou, X; Zhou, Y; IJzerman, A P; PIJzerman, Ad; Boyden, E S; Cunha, R A; Qu, J; Chen, J-F

    2015-11-01

    Human and animal studies have converged to suggest that caffeine consumption prevents memory deficits in aging and Alzheimer's disease through the antagonism of adenosine A2A receptors (A2ARs). To test if A2AR activation in the hippocampus is actually sufficient to impair memory function and to begin elucidating the intracellular pathways operated by A2AR, we have developed a chimeric rhodopsin-A2AR protein (optoA2AR), which retains the extracellular and transmembrane domains of rhodopsin (conferring light responsiveness and eliminating adenosine-binding pockets) fused to the intracellular loop of A2AR to confer specific A2AR signaling. The specificity of the optoA2AR signaling was confirmed by light-induced selective enhancement of cAMP and phospho-mitogen-activated protein kinase (p-MAPK) (but not cGMP) levels in human embryonic kidney 293 (HEK293) cells, which was abolished by a point mutation at the C terminal of A2AR. Supporting its physiological relevance, optoA2AR activation and the A2AR agonist CGS21680 produced similar activation of cAMP and p-MAPK signaling in HEK293 cells, of p-MAPK in the nucleus accumbens and of c-Fos/phosphorylated-CREB (p-CREB) in the hippocampus, and similarly enhanced long-term potentiation in the hippocampus. Remarkably, optoA2AR activation triggered a preferential p-CREB signaling in the hippocampus and impaired spatial memory performance, while optoA2AR activation in the nucleus accumbens triggered MAPK signaling and modulated locomotor activity. This shows that the recruitment of intracellular A2AR signaling in the hippocampus is sufficient to trigger memory dysfunction. Furthermore, the demonstration that the biased A2AR signaling and functions depend on intracellular A2AR loops prompts the possibility of targeting the intracellular A2AR-interacting partners to selectively control different neuropsychiatric behaviors. PMID:25687775

  2. Activation of the Canonical Wnt Signaling Pathway Induces Cementum Regeneration.

    PubMed

    Han, Pingping; Ivanovski, Saso; Crawford, Ross; Xiao, Yin

    2015-07-01

    Canonical Wnt signaling is important in tooth development but it is unclear whether it can induce cementogenesis and promote the regeneration of periodontal tissues lost because of disease. Therefore, the aim of this study is to investigate the influence of canonical Wnt signaling enhancers on human periodontal ligament cell (hPDLCs) cementogenic differentiation in vitro and cementum repair in a rat periodontal defect model. Canonical Wnt signaling was induced by (1) local injection of lithium chloride; (2) local injection of sclerostin antibody; and (3) local injection of a lentiviral construct overexpressing β-catenin. The results showed that the local activation of canonical Wnt signaling resulted in significant new cellular cementum deposition and the formation of well-organized periodontal ligament fibers, which was absent in the control group. In vitro experiments using hPDLCs showed that the Wnt signaling pathway activators significantly increased mineralization, alkaline phosphatase (ALP) activity, and gene and protein expression of the bone and cementum markers osteocalcin (OCN), osteopontin (OPN), cementum protein 1 (CEMP1), and cementum attachment protein (CAP). Our results show that the activation of the canonical Wnt signaling pathway can induce in vivo cementum regeneration and in vitro cementogenic differentiation of hPDLCs. PMID:25556853

  3. Coco is a dual activity modulator of TGFβ signaling

    PubMed Central

    Deglincerti, Alessia; Haremaki, Tomomi; Warmflash, Aryeh; Sorre, Benoit; Brivanlou, Ali H.

    2015-01-01

    The TGFβ signaling pathway is a crucial regulator of developmental processes and disease. The activity of TGFβ ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGFβ1 signaling. Coco synergizes with TGFβ1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGFβ1 and enhances TGFβ1 binding to its receptor Alk5. Thus, Coco acts as both an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGFβ signaling. PMID:26116664

  4. Cellular phosphatases facilitate combinatorial processing of receptor-activated signals

    PubMed Central

    Kumar, Dhiraj; Dua, Raina; Srikanth, Ravichandran; Jayaswal, Shilpi; Siddiqui, Zaved; Rao, Kanury VS

    2008-01-01

    Background Although reciprocal regulation of protein phosphorylation represents a key aspect of signal transduction, a larger perspective on how these various interactions integrate to contribute towards signal processing is presently unclear. For example, a key unanswered question is that of how phosphatase-mediated regulation of phosphorylation at the individual nodes of the signaling network translates into modulation of the net signal output and, thereby, the cellular phenotypic response. Results To address the above question we, in the present study, examined the dynamics of signaling from the B cell antigen receptor (BCR) under conditions where individual cellular phosphatases were selectively depleted by siRNA. Results from such experiments revealed a highly enmeshed structure for the signaling network where each signaling node was linked to multiple phosphatases on the one hand, and each phosphatase to several nodes on the other. This resulted in a configuration where individual signaling intermediates could be influenced by a spectrum of regulatory phosphatases, but with the composition of the spectrum differing from one intermediate to another. Consequently, each node differentially experienced perturbations in phosphatase activity, yielding a unique fingerprint of nodal signals characteristic to that perturbation. This heterogeneity in nodal experiences, to a given perturbation, led to combinatorial manipulation of the corresponding signaling axes for the downstream transcription factors. Conclusion Our cumulative results reveal that it is the tight integration of phosphatases into the signaling network that provides the plasticity by which perturbation-specific information can be transmitted in the form of a multivariate output to the downstream transcription factor network. This output in turn specifies a context-defined response, when translated into the resulting gene expression profile. PMID:18798986

  5. Nitric oxide synthetic pathway and cGMP levels are altered in red blood cells from end-stage renal disease patients.

    PubMed

    Di Pietro, Natalia; Giardinelli, Annalisa; Sirolli, Vittorio; Riganti, Chiara; Di Tomo, Pamela; Gazzano, Elena; Di Silvestre, Sara; Panknin, Christina; Cortese-Krott, Miriam M; Csonka, Csaba; Kelm, Malte; Ferdinandy, Péter; Bonomini, Mario; Pandolfi, Assunta

    2016-06-01

    Red blood cells (RBCs) enzymatically produce nitric oxide (NO) by a functional RBC-nitric oxide synthase (RBC-NOS). NO is a vascular key regulatory molecule. In RBCs its generation is complex and influenced by several factors, including insulin, acetylcholine, and calcium. NO availability is reduced in end-stage renal disease (ESRD) and associated with endothelial dysfunction. We previously demonstrated that, through increased phosphatidylserine membrane exposure, ESRD-RBCs augmented their adhesion to human cultured endothelium, in which NO bioavailability decreased. Since RBC-NOS-dependent NO production in ESRD is unknown, this study aimed to investigate RBC-NOS levels/activation, NO production/bioavailability in RBCs from healthy control subjects (C, N = 18) and ESRD patients (N = 27). Although RBC-NOS expression was lower in ESRD-RBCs, NO, cyclic guanosine monophosphate (cGMP), RBC-NOS Serine1177 phosphorylation level and eNOS/Calmodulin (CaM)/Heat Shock Protein-90 (HSP90) interaction levels were higher in ESRD-RBCs, indicating increased enzyme activation. Conversely, following RBCs stimulation with insulin or ionomycin, NO and cGMP levels were significantly lower in ESRD- than in C-RBCs, suggesting that uremia might reduce the RBC-NOS response to further stimuli. Additionally, the activity of multidrug-resistance-associated protein-4 (MRP4; cGMP-membrane transporter) was significantly lower in ESRD-RBCs, suggesting a possible compromised efflux of cGMP across the ESRD-RBCs membrane. This study for the first time showed highest basal RBC-NOS activation in ESRD-RBCs, possibly to reduce the negative impact of decreased NOS expression. It is further conceivable that high NO production only partially affects cell function of ESRD-RBCs maybe because in vivo they are unable to respond to physiologic stimuli, such as calcium and/or insulin. PMID:27206740

  6. Activation of cell signaling via optical manipulation of gold-coated liposomes encapsulating signaling molecules

    NASA Astrophysics Data System (ADS)

    Orsinger, Gabriel V.; Leung, Sarah J.; Romanowski, Marek

    2013-02-01

    Many diseases involve changes in cell signaling cascades, as seen commonly in drug resistant cancers. To better understand these intricate signaling events in diseased cells and tissues, experimental methods of probing cellular communication at a single to multi-cell level are required. We recently introduced a general platform for activation of selected signaling pathways by optically controlled delivery and release of water soluble factors using gold-coated liposomes. In the example presented here, we encapsulated inositol trisphosphate (IP3), a ubiquitous intracellular secondary messenger involved in GPCR and Akt signaling cascades, within 100 nm gold-coated liposomes. The high polarizability of the liposome's unique gold pseudo-shell allows stable optical trapping for subcellular manipulation in the presence of cells. We take this optical manipulation further by optically injecting IP3-containing liposomes into the cytosol of a single cell to initiate localized cell signaling. Upon optical injection of liposomal IP3 into a single ovarian carcinoma cell, we observed localized activation as reported by changes in Indo-1 fluorescence intensity. With established gap junctions between the injected cell and neighboring cells, we monitored propagation of this signaling to and through nearby cells.

  7. Correlation of nighttime MF signal strength with solar activity

    NASA Astrophysics Data System (ADS)

    Kohata, Hiroki; Kimura, Iwane; Wakai, Noboru; Ogawa, Tadahiko

    Observations of the signal strength of MF broadcasting signals (774/770 kHz) transmitted from Akita, Japan, on board the Japanese Antarctic ice breaker Fuji, bound from Japan to Syowa station, Antarctica, have revealed an interesting positive correlation between strengths of long distance signals propagating at night and solar activity. It is already known that MF propagation characteristics in North America show a negative correlation with solar activity. The present paper, interprets the results by using the multihop method with full-wave analysis. The difference in correlation with solar activity between the results of Fuji and those in North America can be elucidated if it is assumed that there is a ledge in the electron-density profile around an altitude range of 85 to 90 km and that the density of the ledge is smaller in the North American region than in the equatorial region.

  8. cGMP modulates stem cells differentiation to neurons in brain in vivo.

    PubMed

    Gómez-Pinedo, U; Rodrigo, R; Cauli, O; Herraiz, S; Garcia-Verdugo, J-M; Pellicer, B; Pellicer, A; Felipo, V

    2010-02-17

    During brain development neural stem cells may differentiate to neurons or to other cell types. The aim of this work was to assess the role of cGMP (cyclic GMP) in the modulation of differentiation of neural stem cells to neurons or non-neuronal cells. cGMP in brain of fetuses was reduced to 46% of controls by treating pregnant rats with nitroarginine-methylester (L-NAME) and was restored by co-treatment with sildenafil.Reducing cGMP during brain development leads to reduced differentiation of stem cells to neurons and increased differentiation to non-neuronal cells. The number of neurons in the prefrontal cortex originated from stem cells proliferating on gestational day 14 was 715+/-14/mm(2) in control rats and was reduced to 440+/-29/mm(2) (61% of control) in rats treated with L-NAME. In rats exposed to L-NAME plus sildenafil, differentiation to neurons was completely normalized, reaching 683+/-11 neurons/mm(2). In rats exposed to sildenafil alone the number of cells labelled with bromodeoxyuridine (BrdU) and NeuN was 841+/-16/mm(2). In prefrontal cortex of control rats 48% of the neural stem cells proliferating in gestational day 14 differentiate to neurons, but only 24% in rats exposed to L-NAME. This was corrected by sildenafil, 40% of cells differentiate to neurons. Similar results were obtained for neurons proliferating during all developmental period. Treatment with L-NAME did not reduce the total number of cells labelled with BrdU, further supporting that L-NAME reduces selectively the differentiation of stem cells to neurons. Similar results were obtained in hippocampus. Treatment with L-NAME reduced the differentiation of neural stem cells to neurons, although the effect was milder than in prefrontal cortex. These results support that cGMP modulates the fate of neural stem cells in brain in vivo and suggest that high cGMP levels promote its differentiation to neurons while reduced cGMP levels promote differentiation to non-neuronal cells. PMID:19958812

  9. PDE5A suppression of acute β-adrenergic activation requires modulation of myocyte beta-3 signaling coupled to PKG-mediated troponin I phosphorylation

    PubMed Central

    Lee, Dong I.; Vahebi, Susan; Tocchetti, Carlo Gabriele; Barouch, Lili A.; Solaro, R. John; Takimoto, Eiki

    2010-01-01

    Phosphodiesterase type 5A (PDE5A) inhibitors acutely suppress beta-adrenergic receptor (β-AR) stimulation in left ventricular myocytes and hearts. This modulation requires cyclic GMP synthesis via nitric oxide synthase (NOS)-NO stimulation, but upstream and downstream mechanisms remain un-defined. To determine this, adult cardiac myocytes from genetically engineered mice and controls were studied by video microscopy to assess sarcomere shortening (SS) and fura2-AM fluorescence to measure calcium transients (CaT). Enhanced SS from isoproterenol (ISO, 10 nM) was suppressed ≥50% by the PDE5A inhibitor sildenafil (SIL, 1 µM), without altering CaT. This regulation was unaltered despite co-inhibition of either the cGMP-stimulated cAMP-esterase PDE2 (Bay 60-7550), or cGMP-inhibited cAMP-esterase PDE3 (cilostamide). Thus, the SIL response could not be ascribed to cGMP interaction with alternative PDEs. However, genetic deletion (or pharmacologic blockade) of β3-ARs, which couple to NOS signaling, fully prevented SIL modulation of ISO-stimulated SS. Importantly, both PDE5A protein expression and activity were similar in β3-AR knockout (β3-AR−/−) myocytes as in controls. Downstream, cGMP stimulates protein kinase G (PKG), and we found contractile modulation by SIL required PKG activation and enhanced TnI phosphorylation at S23, S24. Myocytes expressing the slow skeletal TnI isoform which lacks these sites displayed no modulation of ISO responses by SIL. Non-equilibrium isoelectric focusing gel electrophoresis showed SIL increased TnI phosphorylation above that from concomitant ISO in control but not β3-AR−/− myocytes. These data support a cascade involving β3-AR stimulation, and subsequent PKG-dependent TnI S23, S24 phosphorylation as primary factors underlying the capacity of acute PDE5A inhibition to blunt myocardial β-adrenergic stimulation. PMID:20107996

  10. Receptor tyrosine kinases: mechanisms of activation and signaling

    PubMed Central

    Hubbard, Stevan R.; Miller, W. Todd

    2008-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication. These single-pass transmembrane receptors, which bind polypeptide ligands — mainly growth factors — play key roles in processes such as cellular growth, differentiation, metabolism and motility. Recent progress has been achieved towards an understanding of the precise (and varied) mechanisms by which RTKs are activated by ligand binding and by which signals are propagated from the activated receptors to downstream targets in the cell. PMID:17306972

  11. Phosphodiesterase-5 Inhibitors: Action on the Signaling Pathways of Neuroinflammation, Neurodegeneration, and Cognition

    PubMed Central

    Peixoto, Christina Alves; Nunes, Ana Karolina Santana; Garcia-Osta, Ana

    2015-01-01

    Phosphodiesterase type 5 inhibitors (PDE5-Is) have recently emerged as a potential therapeutic strategy for neuroinflammatory, neurodegenerative, and memory loss diseases. Mechanistically, PDE5-Is produce an anti-inflammatory and neuroprotection effect by increasing expression of nitric oxide synthases and accumulation of cGMP and activating protein kinase G (PKG), the signaling pathway of which is thought to play an important role in the development of several neurodiseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). The aim of this paper was to review present knowledge of the signaling pathways that underlie the use of PDE5-Is in neuroinflammation, neurogenesis, learning, and memory. PMID:26770022

  12. Molecular signalling of a novel curcumin derivative versus Tadalafil in erectile dysfunction.

    PubMed

    Abdel Aziz, M T; Rezq, A M; Atta, H M; Fouad, H; Zaahkouk, A M; Ahmed, H H; Sabry, D; Yehia, H M

    2015-08-01

    The efficacy of a novel curcumin derivative (NCD) versus tadalafil in erectile signalling was assessed. Ten control male rats and 50 diabetic male rats were used and divided into the following: diabetic (DM), curcumin (CURC), NCD, tadalafil and NCD combined with tadalafil rat groups. Cavernous tissue gene expression of heme oxygenase-1 (HO-1), Nrf2, NF-B and p38, enzyme activities of heme oxygenase (HO) and nitric oxide synthase (NOS), cGMP and intracavernosal pressure (ICP)/mean arterial pressure (MAP) were assessed. Results showed that 12 weeks after induction of diabetes, erectile dysfunction (ED) was confirmed by the significant decrease in ICP/MAP, a significant decrease in cGMP, NOS, HO enzyme activities, a significant decrease in HO-1 gene and a significant increase in NF-Ҡβ, p38 genes. Administration of all therapeutic interventions led to a significant increase in ICP/MAP, cGMP levels, a significant increase in HO-1 and NOS enzymes, a significant increase in HO-1, and Nrf2 gene expression, and a significant decrease in NF-Ҡβ, p38 gene expression. NCD or its combination with tadalafil showed significant superiority and more prolonged duration of action. In conclusion, a tendency was observed that CURC and NCD have high efficacy and more prolonged duration of action in enhancing erectile function. PMID:25059462

  13. Notch signaling promotes osteoclast maturation and resorptive activity.

    PubMed

    Ashley, Jason W; Ahn, Jaimo; Hankenson, Kurt D

    2015-11-01

    The role of Notch signaling in osteoclast differentiation is controversial with conflicting experimental evidence indicating both stimulatory and inhibitory roles. Differences in experimental protocols and in vivo versus in vitro models may explain the discrepancies between studies. In this study, we investigated cell autonomous roles of Notch signaling in osteoclast differentiation and function by altering Notch signaling during osteoclast differentiation using stimulation with immobilized ligands Jagged1 or Delta-like1 or by suppression with γ-secretase inhibitor DAPT or transcriptional inhibitor SAHM1. Stimulation of Notch signaling in committed osteoclast precursors resulted in larger osteoclasts with a greater number of nuclei and resorptive activity whereas suppression resulted in smaller osteoclasts with fewer nuclei and suppressed resorptive activity. Conversely, stimulation of Notch signaling in osteoclast precursors prior to induction of osteoclastogenesis resulted in fewer osteoclasts. Our data support a mechanism of context-specific Notch signaling effects wherein Notch stimulation inhibits commitment to osteoclast differentiation, but enhances the maturation and function of committed precursors. PMID:25914241

  14. sGC-cGMP signaling: target for anticancer therapy.

    PubMed

    Bian, Ka; Murad, Ferid

    2014-01-01

    The biologic endogenous production of cGMP was reported in the 1960s and followed by the demonstration of guanylyl cyclase activity and the isoforms of soluble and membrane-bound guanylyl cyclases. During the same period, cGMP specific phosphodiesterases also was discovered. Murad's lab established link between the endothelium derived relaxation factor (EDRF) and elevated cGMP concentration in the vascular system. October 12, 1998, the Nobel Assembly awarded the Nobel Prize in Medicine or Physiology to scientists Robert Furchgott, Louis Ignarro, and Ferid Murad for their discoveries concerning nitric oxide (NO) as a signaling molecule in the cardiovascular system. In contrast with the short research history of the enzymatic synthesis of NO, the introduction of nitrate-containing compounds for medicinal purposes marked its 150th anniversary in 1997. Glyceryl trinitrate (nitroglycerin; GTN) is the first compound of this category. Alfred Nobel (the founder of the Nobel Prize) himself had suffered from angina pectoris and was prescribed nitroglycerin for his chest pain while he refused to take due to the induction of headaches. Almost a century after its first chemical use, research in the nitric oxide and 3',5'-cyclic guanosine monophosphate (NO/cGMP) pathway has dramatically expanded and the role of NO/cGMP in physiology and pathology has been extensively studied. Soluble guanylyl cyclase (sGC) is the receptor for NO. The α1β1 heterodimer is the predominant isoform of sGC that is obligatory for catalytic activity. NO binds to the ferrous (Fe(2+)) heme at histidine 105 of the β1 subunit and leads to an increase in sGC activity and cGMP production of at least 200-fold. In this chapter, we reviewed the studies of sGC-cGMP signaling in cell proliferation; introduced our work of targeting sGC-cGMP signaling for cancer therapy; and explored the role of sGC-cGMP signaling in the chromatin-microenvironment. PMID:25015797

  15. Wnt signaling and the activation of myogenesis in mammals.

    PubMed

    Cossu, G; Borello, U

    1999-12-15

    In the amniote embryos, specification of skeletal myoblasts occurs in the paraxial mesoderm in response to a number of signaling molecules produced by neighboring tissues such as neural tube, notochord and dorsal ectoderm. Candidate molecules for this complex signaling activity include Sonic hedgehog, Wnts and Noggin as positive activators and BMP4 as a possible inhibitor. Recently, the receptors and the post-receptor pathways for Sonic hedgehog and Wnts have been characterized, and this has opened up the possibility of linking these signaling events to the activation of myogenic regulatory factor genes such as Myf5 and MyoD and functionally related genes such as Pax3. Here we focus on the role of Wnts, their putative receptors Frizzled and the soluble antagonist Frzb1 in regulating mammalian myogenesis. Although it is becoming evident that the signaling downstream of Frizzled receptors is much more complex than anticipated, it is conceivable that it may lead to transcriptional activation of Myf5 and MyoD and to initiation of myogenesis. However, the fact that both Wnts and Sonic hedgehog have a strong effect on cell proliferation and survival suggests that they may contribute to the overall process of myogenesis by a combination of these different biological activities. PMID:10601008

  16. Wnt signaling and the activation of myogenesis in mammals.

    PubMed Central

    Cossu, G; Borello, U

    1999-01-01

    In the amniote embryos, specification of skeletal myoblasts occurs in the paraxial mesoderm in response to a number of signaling molecules produced by neighboring tissues such as neural tube, notochord and dorsal ectoderm. Candidate molecules for this complex signaling activity include Sonic hedgehog, Wnts and Noggin as positive activators and BMP4 as a possible inhibitor. Recently, the receptors and the post-receptor pathways for Sonic hedgehog and Wnts have been characterized, and this has opened up the possibility of linking these signaling events to the activation of myogenic regulatory factor genes such as Myf5 and MyoD and functionally related genes such as Pax3. Here we focus on the role of Wnts, their putative receptors Frizzled and the soluble antagonist Frzb1 in regulating mammalian myogenesis. Although it is becoming evident that the signaling downstream of Frizzled receptors is much more complex than anticipated, it is conceivable that it may lead to transcriptional activation of Myf5 and MyoD and to initiation of myogenesis. However, the fact that both Wnts and Sonic hedgehog have a strong effect on cell proliferation and survival suggests that they may contribute to the overall process of myogenesis by a combination of these different biological activities. PMID:10601008

  17. Facilitation of corticostriatal transmission following pharmacological inhibition of striatal phosphodiesterase 10A: role of nitric oxide-soluble guanylyl cyclase-cGMP signaling pathways.

    PubMed

    Padovan-Neto, Fernando E; Sammut, Stephen; Chakroborty, Shreaya; Dec, Alexander M; Threlfell, Sarah; Campbell, Peter W; Mudrakola, Vishnu; Harms, John F; Schmidt, Christopher J; West, Anthony R

    2015-04-01

    The striatum contains a rich variety of cyclic nucleotide phosphodiesterases (PDEs), which play a critical role in the regulation of cAMP and cGMP signaling. The dual-substrate enzyme PDE10A is the most highly expressed PDE in striatal medium-sized spiny neurons (MSNs) with low micromolar affinity for both cyclic nucleotides. Previously, we have shown that systemic and local administration of the selective PDE10A inhibitor TP-10 potently increased the responsiveness of MSNs to cortical stimulation. However, the signaling mechanisms underlying PDE10A inhibitor-induced changes in corticostriatal transmission are only partially understood. The current studies assessed the respective roles of cAMP and cGMP in the above effects using soluble guanylyl cyclase (sGC) or adenylate cyclase (AC) specific inhibitors. Cortically evoked spike activity was monitored in urethane-anesthetized rats using in vivo extracellular recordings performed proximal to a microdialysis probe during local infusion of vehicle, the selective sGC inhibitor ODQ, or the selective AC inhibitor SQ 22536. Systemic administration of TP-10 (3.2 mg/kg) robustly increased cortically evoked spike activity in a manner that was blocked following intrastriatal infusion of ODQ (50 μm). The effects of TP-10 on evoked activity were due to accumulation of cGMP, rather than cAMP, as the AC inhibitor SQ was without effect. Consistent with these observations, studies in neuronal NO synthase (nNOS) knock-out (KO) mice confirmed that PDE10A operates downstream of nNOS to limit cGMP production and excitatory corticostriatal transmission. Thus, stimulation of PDE10A acts to attenuate corticostriatal transmission in a manner largely dependent on effects directed at the NO-sGC-cGMP signaling cascade. PMID:25855188

  18. Signaling pathways activated by a protease allergen in basophils

    PubMed Central

    Rosenstein, Rachel K.; Bezbradica, Jelena S.; Yu, Shuang; Medzhitov, Ruslan

    2014-01-01

    Allergic diseases represent a significant burden in industrialized countries, but why and how the immune system responds to allergens remain largely unknown. Because many clinically significant allergens have proteolytic activity, and many helminths express proteases that are necessary for their life cycles, host mechanisms likely have evolved to detect the proteolytic activity of helminth proteases, which may be incidentally activated by protease allergens. A cysteine protease, papain, is a prototypic protease allergen that can directly activate basophils and mast cells, leading to the production of cytokines, including IL-4, characteristic of the type 2 immune response. The mechanism of papain’s immunogenic activity remains unknown. Here we have characterized the cellular response activated by papain in basophils. We find that papain-induced IL-4 production requires calcium flux and activation of PI3K and nuclear factor of activated T cells. Interestingly, papain-induced IL-4 production was dependent on the immunoreceptor tyrosine-based activation motif (ITAM) adaptor protein Fc receptor γ-chain, even though the canonical ITAM signaling was not activated by papain. Collectively, these data characterize the downstream signaling pathway activated by a protease allergen in basophils. PMID:25369937

  19. A Temporal Window for Signal Activation Dictates the Dimensions of a Nodal Signaling Domain

    PubMed Central

    van Boxtel, Antonius L.; Chesebro, John E.; Heliot, Claire; Ramel, Marie-Christine; Stone, Richard K.; Hill, Caroline S.

    2015-01-01

    Summary Morphogen signaling is critical for the growth and patterning of tissues in embryos and adults, but how morphogen signaling gradients are generated in tissues remains controversial. The morphogen Nodal was proposed to form a long-range signaling gradient via a reaction-diffusion system, on the basis of differential diffusion rates of Nodal and its antagonist Lefty. Here we use a specific zebrafish Nodal biosensor combined with immunofluorescence for phosphorylated Smad2 to demonstrate that endogenous Nodal is unlikely to diffuse over a long range. Instead, short-range Nodal signaling activation in a temporal window is sufficient to determine the dimensions of the Nodal signaling domain. The size of this temporal window is set by the differentially timed production of Nodal and Lefty, which arises mainly from repression of Lefty translation by the microRNA miR-430. Thus, temporal information is transformed into spatial information to define the dimensions of the Nodal signaling domain and, consequently, to specify mesendoderm. PMID:26506307

  20. A Temporal Window for Signal Activation Dictates the Dimensions of a Nodal Signaling Domain.

    PubMed

    van Boxtel, Antonius L; Chesebro, John E; Heliot, Claire; Ramel, Marie-Christine; Stone, Richard K; Hill, Caroline S

    2015-10-26

    Morphogen signaling is critical for the growth and patterning of tissues in embryos and adults, but how morphogen signaling gradients are generated in tissues remains controversial. The morphogen Nodal was proposed to form a long-range signaling gradient via a reaction-diffusion system, on the basis of differential diffusion rates of Nodal and its antagonist Lefty. Here we use a specific zebrafish Nodal biosensor combined with immunofluorescence for phosphorylated Smad2 to demonstrate that endogenous Nodal is unlikely to diffuse over a long range. Instead, short-range Nodal signaling activation in a temporal window is sufficient to determine the dimensions of the Nodal signaling domain. The size of this temporal window is set by the differentially timed production of Nodal and Lefty, which arises mainly from repression of Lefty translation by the microRNA miR-430. Thus, temporal information is transformed into spatial information to define the dimensions of the Nodal signaling domain and, consequently, to specify mesendoderm. PMID:26506307

  1. Metabolic signals and innate immune activation in obesity and exercise.

    PubMed

    Ringseis, Robert; Eder, Klaus; Mooren, Frank C; Krüger, Karsten

    2015-01-01

    The combination of a sedentary lifestyle and excess energy intake has led to an increased prevalence of obesity which constitutes a major risk factor for several co-morbidities including type 2 diabetes and cardiovascular diseases. Intensive research during the last two decades has revealed that a characteristic feature of obesity linking it to insulin resistance is the presence of chronic low-grade inflammation being indicative of activation of the innate immune system. Recent evidence suggests that activation of the innate immune system in the course of obesity is mediated by metabolic signals, such as free fatty acids (FFAs), being elevated in many obese subjects, through activation of pattern recognition receptors thereby leading to stimulation of critical inflammatory signaling cascades, like IκBα kinase/nuclear factor-κB (IKK/NF- κB), endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) and NOD-like receptor P3 (NLRP3) inflammasome pathway, that interfere with insulin signaling. Exercise is one of the main prescribed interventions in obesity management improving insulin sensitivity and reducing obesity- induced chronic inflammation. This review summarizes current knowledge of the cellular recognition mechanisms for FFAs, the inflammatory signaling pathways triggered by excess FFAs in obesity and the counteractive effects of both acute and chronic exercise on obesity-induced activation of inflammatory signaling pathways. A deeper understanding of the effects of exercise on inflammatory signaling pathways in obesity is useful to optimize preventive and therapeutic strategies to combat the increasing incidence of obesity and its comorbidities. PMID:25825956

  2. Activation of signalling by the activin receptor complex.

    PubMed Central

    Attisano, L; Wrana, J L; Montalvo, E; Massagué, J

    1996-01-01

    Activin exerts its effects by simultaneously binding to two types of p rotein serine/threonine kinase receptors, each type existing in various isoforms. Using the ActR-IB and ActR-IIB receptor isoforms, we have investigated the mechanism of activin receptor activation. ActR-IIB are phosphoproteins with demonstrable affinity for each other. However, activin addition strongly promotes an interaction between these two proteins. Activin binds directly to ActR-IIB, and this complex associates with ActR-IB, which does not bind ligand on its own. In the resulting complex, ActR-IB becomes hyperphosphorylated, and this requires the kinase activity of ActR-IIB. Mutation of conserved serines and threonines in the GS domain, a region just upstream of the kinase domain in ActR-IB, abrogates both phosphorylation and signal propagation, suggesting that this domain contains phosphorylation sites required for signalling. ActR-IB activation can be mimicked by mutation of Thr-206 to aspartic acid, which yields a construct, ActR-IB(T206D), that signals in the absence of ligand. Furthermore, the signalling activity of this mutant construct is undisturbed by overexpression of a dominant negative kinase-defective ActR-IIB construct, indicating that ActR-IB(T206D) can signal independently of ActR-IIB. The evidence suggests that ActR-IIB acts as a primary activin receptor and ActR-IB acts as a downstream transducer of activin signals. PMID:8622651

  3. TIMP-2 Modulates VEGFR-2 Phosphorylation and Enhances Phosphodiesterase Activity in Endothelial Cells

    PubMed Central

    Lee, Seo-Jin; Tsang, Patricia; Diaz, Tere; Wei, Bei-yang; Stetler-Stevenson, William George

    2010-01-01

    In the present study we examine the effects of tissue inhibitor of metalloproteinases-2 (TIMP-2) on the phosphorylation status of specific phosphotyrosine residues on the vascular endothelial cell growth factor receptor-2 (VEGFR-2) cytoplasmic tail and examine the effects on associated downstream signaling pathways. In order to focus on metalloproteinase-independent mechanisms, we utilized the TIMP-2 analog known as Ala+TIMP-2 that is deficient in matrix metalloproteinase (MMP) inhibitory activity. Our experiments are designed to compare the effects of VEGF-A stimulation with or without Ala+TIMP-2 pretreatment, as well as basal responses in human microvascular endothelial cells. Our results show that Ala+TIMP-2 selectively alters the phosphorylation pattern of VEGFR-2 following VEGF-A stimulation and disrupts the downstream activation of PLC-γ, Ca+2 flux, Akt, and eNOS, as well as decreasing cGMP levels. Moreover, we observed an Ala+TIMP-2-induced reduction in cGMP levels typically elevated by exogenous NO donors implicating Ala+TIMP-2 in the direct activation of an isobutylmethylxanthine (IBMX)-sensitive cGMP phosphodiesterase activity. TIMP-2 suppression of endothelial mitogenesis and angiogenesis involves at least two mechanisms, one mediated by protein tyrosine phosphatase inhibition of VEGFR-2 activation and downstream signaling and a second mechanism involving direct activation of an IBMX-sensitive phosphodiesterase activity. PMID:20084057

  4. Imaging the coordination of multiple signaling activities in living cells

    PubMed Central

    Welch, Christopher M.; Elliott, Hunter; Danuser, Gaudenz; Hahn, Klaus M.

    2013-01-01

    Preface Cellular signal transduction occurs in complex and redundant interaction networks that are best examined at the level of single cells by simultaneously monitoring the activation dynamics of multiple components. Recent advances in biosensor technology have made it possible to visualize and quantify the activation of multiple network nodes in the same living cell. The precision and scope of this approach has been greatly extended by novel computational approaches to determine the relationships between different networks, studied in separate cells. PMID:22016058

  5. The implementation of tissue banking experiences for setting up a cGMP cell manufacturing facility.

    PubMed

    Arjmand, Babak; Emami-Razavi, Seyed Hassan; Larijani, Bagher; Norouzi-Javidan, Abbas; Aghayan, Hamid Reza

    2012-12-01

    Cell manufacturing for clinical applications is a unique form of biologics manufacturing that relies on maintenance of stringent work practices designed to ensure product consistency and prevent contamination by microorganisms or by another patient's cells. More extensive, prolonged laboratory processes involve greater risk of complications and possibly adverse events for the recipient, and so the need for control is correspondingly greater. To minimize the associate risks of cell manufacturing adhering to international quality standards is critical. Current good tissue practice (cGTP) and current good manufacturing practice (cGMP) are examples of general standards that draw a baseline for cell manufacturing facilities. In recent years, stem cell researches have found great public interest in Iran and different cell therapy projects have been started in country. In this review we described the role of our tissue banking experiences in establishing a new cGMP cell manufacturing facility. The authors concluded that, tissue banks and tissue banking experts can broaden their roles from preparing tissue grafts to manufacturing cell and tissue engineered products for translational researches and phase I clinical trials. Also they can collaborate with cell processing laboratories to develop SOPs, implement quality management system, and design cGMP facilities. PMID:21870140

  6. Protein and Signaling Networks in Vertebrate Photoreceptor Cells

    PubMed Central

    Koch, Karl-Wilhelm; Dell’Orco, Daniele

    2015-01-01

    Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination. The photoexcitation and adaptation machinery in photoreceptor cells consists of protein complexes that can form highly ordered supramolecular structures and control the homeostasis and mutual dependence of the secondary messengers cyclic guanosine monophosphate (cGMP) and Ca2+. The visual pigment in rod photoreceptors, the G protein-coupled receptor rhodopsin is organized in tracks of dimers thereby providing a signaling platform for the dynamic scaffolding of the G protein transducin. Illuminated rhodopsin is turned off by phosphorylation catalyzed by rhodopsin kinase (GRK1) under control of Ca2+-recoverin. The GRK1 protein complex partly assembles in lipid raft structures, where shutting off rhodopsin seems to be more effective. Re-synthesis of cGMP is another crucial step in the recovery of the photoresponse after illumination. It is catalyzed by membrane bound sensory guanylate cyclases (GCs) and is regulated by specific neuronal Ca2+-sensor proteins called guanylate cyclase-activating proteins (GCAPs). At least one GC (ROS-GC1) was shown to be part of a multiprotein complex having strong interactions with the cytoskeleton and being controlled in a multimodal Ca2+-dependent fashion. The final target of the cGMP signaling cascade is a cyclic nucleotide-gated (CNG) channel that is a hetero-oligomeric protein located in the plasma membrane and interacting with accessory proteins in highly organized microdomains. We summarize results and interpretations of findings related to the inhomogeneous organization of signaling units in photoreceptor outer segments. PMID:26635520

  7. Protein kinase A activity and Hedgehog signaling pathway.

    PubMed

    Kotani, Tomoya

    2012-01-01

    Protein kinase A (PKA) is a well-known kinase that plays fundamental roles in a variety of biological processes. In Hedgehog-responsive cells, PKA plays key roles in proliferation and fate specification by modulating the transduction of Hedgehog signaling. In the absence of Hedgehog, a basal level of PKA activity represses the transcription of Hedgehog target genes. The main substrates of PKA in this process are the Ci/Gli family of bipotential transcription factors, which activate and repress Hedgehog target gene expression. PKA phosphorylates Ci/Gli, promoting the production of the repressor forms of Ci/Gli and thus repressing Hedgehog target gene expression. In contrast, the activation of Hedgehog signaling in response to Hedgehog increases the active forms of Ci/Gli, resulting in Hedgehog target gene expression. Because both decreased and increased levels of PKA activity cause abnormal cell proliferation and alter cell fate specification, the basal level of PKA activity in Hedgehog-responsive cells should be precisely regulated. However, the mechanism by which PKA activity is regulated remains obscure and appears to vary between cell types, tissues, and organisms. To date, two mechanisms have been proposed. One is a classical mechanism in which PKA activity is regulated by a small second messenger, cAMP; the other is a novel mechanism in which PKA activity is regulated by a protein, Misty somites. PMID:22391308

  8. Activation of B cells by non-canonical helper signals

    PubMed Central

    Cerutti, Andrea; Cols, Montserrat; Puga, Irene

    2012-01-01

    Cognate interaction between T and B lymphocytes of the adaptive immune system is essential for the production of high-affinity antibodies against microbes, and for the establishment of long-term immunological memory. Growing evidence shows that—in addition to presenting antigens to T and B cells—macrophages, dendritic cells and other cells of the innate immune system provide activating signals to B cells, as well as survival signals to antibody-secreting plasma cells. Here, we discuss how these innate immune cells contribute to the induction of highly diversified and temporally sustained antibody responses, both systemically and at mucosal sites of antigen entry. PMID:22868664

  9. Activators of G Protein Signaling in the Kidney

    PubMed Central

    2015-01-01

    Heterotrimeric G proteins play a crucial role in regulating signal processing to maintain normal cellular homeostasis, and subtle perturbations in its activity can potentially lead to the pathogenesis of renal disorders or diseases. Cell-surface receptors and accessory proteins, which normally modify and organize the coupling of individual G protein subunits, contribute to the regulation of heterotrimeric G protein activity and their convergence and/or divergence of downstream signaling initiated by effector systems. Activators of G protein signaling (AGS) are a family of accessory proteins that intervene at multiple distinct points during the activation–inactivation cycle of G proteins, even in the absence of receptor stimulation. Perturbations in the expression of individual AGS proteins have been reported to modulate signal transduction pathways in a wide array of diseases and disorders within the brain, heart, immune system, and more recently, the kidney. This review will provide an overview of the expression profile, localization, and putative biologic role of the AGS family in the context of normal and diseased states of the kidney. PMID:25628392

  10. Molecular hydrogen suppresses activated Wnt/β-catenin signaling.

    PubMed

    Lin, Yingni; Ohkawara, Bisei; Ito, Mikako; Misawa, Nobuaki; Miyamoto, Kentaro; Takegami, Yasuhiko; Masuda, Akio; Toyokuni, Shinya; Ohno, Kinji

    2016-01-01

    Molecular hydrogen (H2) is effective for many diseases. However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/β-catenin signaling by promoting phosphorylation and degradation οf β-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of β-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the β-catenin degradation complex, minimized the suppressive effect of H2 on β-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of β-catenin, as well as the β-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/β-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating β-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/β-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases. PMID:27558955

  11. Spatial Regulation and the Rate of Signal Transduction Activation

    PubMed Central

    Batada, Nizar N; Shepp, Larry A; Siegmund, David O; Levitt, Michael

    2006-01-01

    Of the many important signaling events that take place on the surface of a mammalian cell, activation of signal transduction pathways via interactions of cell surface receptors is one of the most important. Evidence suggests that cell surface proteins are not as freely diffusible as implied by the classic fluid mosaic model and that their confinement to membrane domains is regulated. It is unknown whether these dynamic localization mechanisms function to enhance signal transduction activation rate or to minimize cross talk among pathways that share common intermediates. To determine which of these two possibilities is more likely, we derive an explicit equation for the rate at which cell surface membrane proteins interact based on a Brownian motion model in the presence of endocytosis and exocytosis. We find that in the absence of any diffusion constraints, cell surface protein interaction rate is extremely high relative to cytoplasmic protein interaction rate even in a large mammalian cell with a receptor abundance of a mere two hundred molecules. Since a larger number of downstream signaling events needs to take place, each occurring at a much slower rate than the initial activation via association of cell surface proteins, we conclude that the role of co-localization is most likely that of cross-talk reduction rather than coupling efficiency enhancement. PMID:16699596

  12. Molecular hydrogen suppresses activated Wnt/β-catenin signaling

    PubMed Central

    Lin, Yingni; Ohkawara, Bisei; Ito, Mikako; Misawa, Nobuaki; Miyamoto, Kentaro; Takegami, Yasuhiko; Masuda, Akio; Toyokuni, Shinya; Ohno, Kinji

    2016-01-01

    Molecular hydrogen (H2) is effective for many diseases. However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/β-catenin signaling by promoting phosphorylation and degradation οf β-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of β-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the β-catenin degradation complex, minimized the suppressive effect of H2 on β-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of β-catenin, as well as the β-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/β-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating β-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/β-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases. PMID:27558955

  13. BMP2 Transfer to Neighboring Cells and Activation of Signaling.

    PubMed

    Alborzinia, Hamed; Shaikhkarami, Marjan; Hortschansky, Peter; Wölfl, Stefan

    2016-09-01

    Morphogen gradients and concentration are critical features during early embryonic development and cellular differentiation. Previously we reported the preparation of biologically active, fluorescently labeled BMP2 and quantitatively analyzed their binding to the cell surface and followed BMP2 endocytosis over time on the level of single endosomes. Here we show that this internalized BMP2 can be transferred to neighboring cells and, moreover, also activates downstream BMP signaling in adjacent cells, indicated by Smad1/5/8 phosphorylation and activation of the downstream target gene id1. Using a 3D matrix to modulate cell-cell contacts in culture we could show that direct cell-cell contact significantly increased BMP2 transfer. Using inhibitors of vesicular transport, transfer was strongly inhibited. Interestingly, cotreatment with the physiological BMP inhibitor Noggin increased BMP2 uptake and transfer, albeit activation of Smad signaling in neighboring cells was completely suppressed. Our findings present a novel and interesting mechanism by which morphogens such as BMP2 can be transferred between cells and how this is modulated by BMP antagonists such as Noggin, and how this influences activation of Smad signaling by BMP2 in neighboring cells. PMID:27306974

  14. G Protein Activation Stimulates Phospholipase D Signaling in Plants.

    PubMed Central

    Munnik, T.; Arisz, S. A.; De Vrije, T.; Musgrave, A.

    1995-01-01

    We provide direct evidence for phospholipase D (PLD) signaling in plants by showing that this enzyme is stimulated by the G protein activators mastoparan, ethanol, and cholera toxin. An in vivo assay for PLD activity in plant cells was developed based on the use of a "reporter alcohol" rather than water as a transphosphatidylation substrate. The product was a phosphatidyl alcohol, which, in contrast to the normal product phosphatidic acid, is a specific measure of PLD activity. When 32P-labeled cells were treated with 0.1% n-butanol, 32P-phosphatidyl butanol (32P-PtdBut) was formed in a time-dependent manner. In cells treated with any of the three G protein activators, the production of 32P-PtdBut was increased in a dose-dependent manner. The G protein involved was pertussis toxin insensitive. Ethanol could activate PLD but was itself consumed by PLD as transphosphatidylation substrate. In contrast, secondary alcohols (e.g., sec-butyl alcohol) activated PLD but did not function as substrate, whereas tertiary alcohols did neither. Although most of the experiments were performed with the green alga Chlamydomonas eugametos, the relevance for higher plants was demonstrated by showing that PLD in carnation petals could also be activated by mastoparan. The results indicate that PLD activation must be considered as a potential signal transduction mechanism in plants, just as in animals. PMID:12242371

  15. Luteinizing Hormone Reduces the Activity of the NPR2 Guanylyl Cyclase in Mouse Ovarian Follicles, Contributing to the Cyclic GMP Decrease that Promotes Resumption of Meiosis in Oocytes

    PubMed Central

    Robinson, Jerid W.; Zhang, Meijia; Shuhaibar, Leia C.; Norris, Rachael P.; Geerts, Andreas; Wunder, Frank; Eppig, John J.; Potter, Lincoln R.; Jaffe, Laurinda A.

    2012-01-01

    In preovulatory ovarian follicles of mice, meiotic prophase arrest in the oocyte is maintained by cyclic GMP from the surrounding granulosa cells that diffuses into the oocyte through gap junctions. The cGMP is synthesized in the granulosa cells by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2) in response to the agonist C-type natriuretic peptide (CNP). In response to luteinizing hormone (LH), cGMP in the granulosa cells decreases, and as a consequence, oocyte cGMP decreases and meiosis resumes. Here we report that within 20 minutes, LH treatment results in decreased guanylyl cyclase activity of NPR2, as determined in the presence of a maximally activating concentration of CNP. This occurs by a process that does not reduce the amount of NPR2 protein. We also show that by a slower process, first detected at 2 hours, LH decreases the amount of CNP available to bind to the receptor. Both of these LH actions contribute to decreasing cGMP in the follicle, thus signaling meiotic resumption in the oocyte. PMID:22546688

  16. Purmorphamine induces osteogenesis by activation of the hedgehog signaling pathway.

    PubMed

    Wu, Xu; Walker, John; Zhang, Jie; Ding, Sheng; Schultz, Peter G

    2004-09-01

    Previously, a small molecule, purmorphamine, was identified that selectively induces osteogenesis in multipotent mesenchymal progenitor cells. In order to gain insights into the mechanism of action of purmorphamine, high-density oligonucleotide microarrays were used to profile gene expression in multipotent mesenchymal progenitor cells treated with either purmorphamine or bone morphogenetic protein-4 (BMP-4). In contrast to BMP-4 treatment, purmorphamine activates the Hedgehog (Hh) signaling pathway, resulting in the up- and downregulation of its downstream target genes, including Gli1 and Patched. Moreover, the known Hh signaling antagonists, cyclopamine and forskolin, completely block the osteogenesis and Glimediated transcription induced by purmorphamine. These results demonstrate that purmorphamine is a small molecule agonist of Hedgehog signaling, and it may ultimately be useful in the treatment of bone-related disease and neurodegenerative disease. PMID:15380183

  17. Chemical activation of a food deprivation signal extends lifespan.

    PubMed

    Lucanic, Mark; Garrett, Theo; Yu, Ivan; Calahorro, Fernando; Asadi Shahmirzadi, Azar; Miller, Aaron; Gill, Matthew S; Hughes, Robert E; Holden-Dye, Lindy; Lithgow, Gordon J

    2016-10-01

    Model organisms subject to dietary restriction (DR) generally live longer. Accompanying this lifespan extension are improvements in overall health, based on multiple metrics. This indicates that pharmacological treatments that mimic the effects of DR could improve health in humans. To find new chemical structures that extend lifespan, we screened 30 000 synthetic, diverse drug-like chemicals in Caenorhabditis elegans and identified several structurally related compounds that acted through DR mechanisms. The most potent of these NP1 impinges upon a food perception pathway by promoting glutamate signaling in the pharynx. This results in the overriding of a GPCR pathway involved in the perception of food and which normally acts to decrease glutamate signals. Our results describe the activation of a dietary restriction response through the pharmacological masking of a novel sensory pathway that signals the presence of food. This suggests that primary sensory pathways may represent novel targets for human pharmacology. PMID:27220516

  18. Aversive Behavior in the Nematode C. elegans Is Modulated by cGMP and a Neuronal Gap Junction Network.

    PubMed

    Krzyzanowski, Michelle C; Woldemariam, Sarah; Wood, Jordan F; Chaubey, Aditi H; Brueggemann, Chantal; Bowitch, Alexander; Bethke, Mary; L'Etoile, Noelle D; Ferkey, Denise M

    2016-07-01

    All animals rely on their ability to sense and respond to their environment to survive. However, the suitability of a behavioral response is context-dependent, and must reflect both an animal's life history and its present internal state. Based on the integration of these variables, an animal's needs can be prioritized to optimize survival strategies. Nociceptive sensory systems detect harmful stimuli and allow for the initiation of protective behavioral responses. The polymodal ASH sensory neurons are the primary nociceptors in C. elegans. We show here that the guanylyl cyclase ODR-1 functions non-cell-autonomously to downregulate ASH-mediated aversive behaviors and that ectopic cGMP generation in ASH is sufficient to dampen ASH sensitivity. We define a gap junction neural network that regulates nociception and propose that decentralized regulation of ASH signaling can allow for rapid correlation between an animal's internal state and its behavioral output, lending modulatory flexibility to this hard-wired nociceptive neural circuit. PMID:27459302

  19. Aversive Behavior in the Nematode C. elegans Is Modulated by cGMP and a Neuronal Gap Junction Network

    PubMed Central

    Krzyzanowski, Michelle C.; Wood, Jordan F.; Brueggemann, Chantal; Bowitch, Alexander; Bethke, Mary; L’Etoile, Noelle D.; Ferkey, Denise M.

    2016-01-01

    All animals rely on their ability to sense and respond to their environment to survive. However, the suitability of a behavioral response is context-dependent, and must reflect both an animal’s life history and its present internal state. Based on the integration of these variables, an animal’s needs can be prioritized to optimize survival strategies. Nociceptive sensory systems detect harmful stimuli and allow for the initiation of protective behavioral responses. The polymodal ASH sensory neurons are the primary nociceptors in C. elegans. We show here that the guanylyl cyclase ODR-1 functions non-cell-autonomously to downregulate ASH-mediated aversive behaviors and that ectopic cGMP generation in ASH is sufficient to dampen ASH sensitivity. We define a gap junction neural network that regulates nociception and propose that decentralized regulation of ASH signaling can allow for rapid correlation between an animal’s internal state and its behavioral output, lending modulatory flexibility to this hard-wired nociceptive neural circuit. PMID:27459302

  20. Robust Indoor Human Activity Recognition Using Wireless Signals.

    PubMed

    Wang, Yi; Jiang, Xinli; Cao, Rongyu; Wang, Xiyang

    2015-01-01

    Wireless signals-based activity detection and recognition technology may be complementary to the existing vision-based methods, especially under the circumstance of occlusions, viewpoint change, complex background, lighting condition change, and so on. This paper explores the properties of the channel state information (CSI) of Wi-Fi signals, and presents a robust indoor daily human activity recognition framework with only one pair of transmission points (TP) and access points (AP). First of all, some indoor human actions are selected as primitive actions forming a training set. Then, an online filtering method is designed to make actions' CSI curves smooth and allow them to contain enough pattern information. Each primitive action pattern can be segmented from the outliers of its multi-input multi-output (MIMO) signals by a proposed segmentation method. Lastly, in online activities recognition, by selecting proper features and Support Vector Machine (SVM) based multi-classification, activities constituted by primitive actions can be recognized insensitive to the locations, orientations, and speeds. PMID:26184231

  1. Small molecule activation of NOTCH signaling inhibits acute myeloid leukemia

    PubMed Central

    Ye, Qi; Jiang, Jue; Zhan, Guanqun; Yan, Wanyao; Huang, Liang; Hu, Yufeng; Su, Hexiu; Tong, Qingyi; Yue, Ming; Li, Hua; Yao, Guangmin; Zhang, Yonghui; Liu, Hudan

    2016-01-01

    Aberrant activation of the NOTCH signaling pathway is crucial for the onset and progression of T cell leukemia. Yet recent studies also suggest a tumor suppressive role of NOTCH signaling in acute myeloid leukemia (AML) and reactivation of this pathway offers an attractive opportunity for anti-AML therapies. N-methylhemeanthidine chloride (NMHC) is a novel Amaryllidaceae alkaloid that we previously isolated from Zephyranthes candida, exhibiting inhibitory activities in a variety of cancer cells, particularly those from AML. Here, we report NMHC not only selectively inhibits AML cell proliferation in vitro but also hampers tumor development in a human AML xenograft model. Genome-wide gene expression profiling reveals that NMHC activates the NOTCH signaling. Combination of NMHC and recombinant human NOTCH ligand DLL4 achieves a remarkable synergistic effect on NOTCH activation. Moreover, pre-inhibition of NOTCH by overexpression of dominant negative MAML alleviates NMHC-mediated cytotoxicity in AML. Further mechanistic analysis using structure-based molecular modeling as well as biochemical assays demonstrates that NMHC docks in the hydrophobic cavity within the NOTCH1 negative regulatory region (NRR), thus promoting NOTCH1 proteolytic cleavage. Our findings thus establish NMHC as a potential NOTCH agonist that holds great promises for future development as a novel agent beneficial to patients with AML. PMID:27211848

  2. Activation of a signaling cascade by cytoskeleton stretch.

    PubMed

    Tamada, Masako; Sheetz, Michael P; Sawada, Yasuhiro

    2004-11-01

    Cells sense and respond to mechanical force. However, the mechanisms of transduction of extracellular matrix (ECM) forces to biochemical signals are not known. After removing the cell membrane and soluble proteins by Triton X-100 extraction, we found that the remaining complex (Triton cytoskeletons) activated Rap1 upon stretch. Rap1 guanine nucleotide exchange factor, C3G, was required for this activation; C3G as well as the adaptor protein, CrkII, in cell extract bound to Triton cytoskeletons in a stretch-dependent manner. CrkII binding, which was Cas dependent, correlated with stretch-dependent tyrosine phosphorylation of proteins in Triton cytoskeletons including Cas at the contacts with ECM. These in vitro findings were compatible with in vivo observations of stretch-enhanced phosphotyrosine signals, accumulation of CrkII at cell-ECM contacts, and CrkII-Cas colocalization. We suggest that mechanical force on Triton cytoskeletons activates local tyrosine phosphorylation, which provides docking sites for cytosolic proteins, and initiates signaling to activate Rap1. PMID:15525532

  3. Small molecule activation of NOTCH signaling inhibits acute myeloid leukemia.

    PubMed

    Ye, Qi; Jiang, Jue; Zhan, Guanqun; Yan, Wanyao; Huang, Liang; Hu, Yufeng; Su, Hexiu; Tong, Qingyi; Yue, Ming; Li, Hua; Yao, Guangmin; Zhang, Yonghui; Liu, Hudan

    2016-01-01

    Aberrant activation of the NOTCH signaling pathway is crucial for the onset and progression of T cell leukemia. Yet recent studies also suggest a tumor suppressive role of NOTCH signaling in acute myeloid leukemia (AML) and reactivation of this pathway offers an attractive opportunity for anti-AML therapies. N-methylhemeanthidine chloride (NMHC) is a novel Amaryllidaceae alkaloid that we previously isolated from Zephyranthes candida, exhibiting inhibitory activities in a variety of cancer cells, particularly those from AML. Here, we report NMHC not only selectively inhibits AML cell proliferation in vitro but also hampers tumor development in a human AML xenograft model. Genome-wide gene expression profiling reveals that NMHC activates the NOTCH signaling. Combination of NMHC and recombinant human NOTCH ligand DLL4 achieves a remarkable synergistic effect on NOTCH activation. Moreover, pre-inhibition of NOTCH by overexpression of dominant negative MAML alleviates NMHC-mediated cytotoxicity in AML. Further mechanistic analysis using structure-based molecular modeling as well as biochemical assays demonstrates that NMHC docks in the hydrophobic cavity within the NOTCH1 negative regulatory region (NRR), thus promoting NOTCH1 proteolytic cleavage. Our findings thus establish NMHC as a potential NOTCH agonist that holds great promises for future development as a novel agent beneficial to patients with AML. PMID:27211848

  4. Androgen activates β-catenin signaling in bladder cancer cells.

    PubMed

    Li, Yi; Zheng, Yichun; Izumi, Koji; Ishiguro, Hitoshi; Ye, Bo; Li, Faqian; Miyamoto, Hiroshi

    2013-06-01

    Androgen receptor (AR) signals have been implicated in bladder carcinogenesis and tumor progression. Activation of Wnt/β-catenin signaling has also been reported to correlate with bladder cancer progression and poor patients' outcomes. However, cross talk between AR and β-catenin pathways in bladder cancer remains uncharacterized. In radical cystectomy specimens, we immunohistochemically confirmed aberrant expression of β-catenin especially in aggressive tumors. There was a strong association between nuclear expressions of AR and β-catenin in bladder tumors (P=0.0215). Kaplan-Meier and log-rank tests further revealed that reduced membranous β-catenin expression (P=0.0276), nuclear β-catenin expression (P=0.0802), and co-expression of nuclear AR and β-catenin (P=0.0043) correlated with tumor progression after cystectomy. We then assessed the effects of androgen on β-catenin in AR-positive and AR-negative bladder cancer cell lines. A synthetic androgen R1881 increased the expression of an active form of β-catenin and its downstream target c-myc only in AR-positive lines. R1881 also enhanced the activity of β-catenin-mediated transcription, which was abolished by an AR antagonist hydroxyflutamide. Using western blotting and immunofluorescence, R1881 was found to induce nuclear translocation of β-catenin when co-localized with AR. Finally, co-immunoprecipitation revealed androgen-induced associations of AR with β-catenin or T-cell factor (TCF) in bladder cancer cells. Thus, it was likely that androgen was able to activate β-catenin signaling through the AR pathway in bladder cancer cells. Our results also suggest that activation of β-catenin signaling possibly via formation of AR/β-catenin/TCF complex contributes to the progression of bladder cancer, which may enhance the feasibility of androgen deprivation as a potential therapeutic approach. PMID:23447569

  5. Analysis of signal degradation in an integrated active crossbar switch

    NASA Astrophysics Data System (ADS)

    Probst, David K.; Sodergren, Clifford C.; Krainak, Michael A.

    1996-01-01

    One of the most desirable features for a modern systems architecture is reconfigurability. It facilitates the sharing of various processing and memory resources among many different subsystems, thereby reducing the need for each subsystem to duplicate these resources. Reducing duplication also reduces size, weight, power consumption, and cost; all important considerations, especially in modern military systems. Sensor data requires very-wide- bandwidth, point-to-point connections, which are easily provided by fiber optics with good noise immunity, but the switching of such wide-bandwidth signals is problematic because electrical switches have both limited bandwidth and limited switching times. A wide- bandwidth, reconfigurable optical switch is required that overcomes coupling and splitting losses experienced by the optical signal passing through the switch so that sufficient signal- fidelity is maintained. In this paper, we investigate an integrated, active (i.e. amplifying) photonic crossbar switch to determine the signal degradation incurred for intensity modulation and direct detection using an NRZ data format and an ideal, matched-filter receiver. Various device configurations are analyzed in order to determine which produces the smallest degradation of the signal-to-noise ratio.

  6. Gating Kinetics of the Cyclic-GMP-Activated Channel of Retinal Rods: Flash Photolysis and Voltage-Jump Studies

    NASA Astrophysics Data System (ADS)

    Karpen, Jeffrey W.; Zimmerman, Anita L.; Stryer, Lubert; Baylor, Denis A.

    1988-02-01

    The gating kinetics of the cGMP-activated cation channel of salamander retinal rods have been studied in excised membrane patches. Relaxations in patch current were observed after two kinds of perturbation: (i) fast jumps of cGMP concentration, generated by laser flash photolysis of a cGMP ester (``caged'' cGMP), and (ii) membrane voltage jumps, which perturb activation of the channel by cGMP. In both methods the speed of activation increased with the final cGMP concentration. The results are explained by a simple kinetic model in which activation involves three sequential cGMP binding steps with bimolecular rate constants close to the diffusion-controlled limit; fully liganded channels undergo rapid open-closed transitions. Voltage perturbs activation by changing the rate constant for channel closing, which increases with hyperpolarization. Intramolecular transitions of the fully liganded channel limit the kinetics of activation at high cGMP concentrations (>50 μ M), whereas at physiological cGMP concentrations (<5 μ M), the kinetics of activation are limited by the third cGMP binding step. The channel appears to be optimized for rapid responses to changes in cytoplasmic cGMP concentration.

  7. Systematic identification of signal-activated stochastic gene regulation.

    PubMed

    Neuert, Gregor; Munsky, Brian; Tan, Rui Zhen; Teytelman, Leonid; Khammash, Mustafa; van Oudenaarden, Alexander

    2013-02-01

    Although much has been done to elucidate the biochemistry of signal transduction and gene regulatory pathways, it remains difficult to understand or predict quantitative responses. We integrate single-cell experiments with stochastic analyses, to identify predictive models of transcriptional dynamics for the osmotic stress response pathway in Saccharomyces cerevisiae. We generate models with varying complexity and use parameter estimation and cross-validation analyses to select the most predictive model. This model yields insight into several dynamical features, including multistep regulation and switchlike activation for several osmosensitive genes. Furthermore, the model correctly predicts the transcriptional dynamics of cells in response to different environmental and genetic perturbations. Because our approach is general, it should facilitate a predictive understanding for signal-activated transcription of other genes in other pathways or organisms. PMID:23372015

  8. Gq signaling causes glomerular injury by activating TRPC6

    PubMed Central

    Wang, Liming; Jirka, Grant; Rosenberg, Paul B.; Buckley, Anne F.; Gomez, Jose A.; Fields, Timothy A.; Winn, Michelle P.; Spurney, Robert F.

    2015-01-01

    Familial forms of focal segmental glomerulosclerosis (FSGS) have been linked to gain-of-function mutations in the gene encoding the transient receptor potential channel C6 (TRPC6). GPCRs coupled to Gq signaling activate TRPC6, suggesting that Gq-dependent TRPC6 activation underlies glomerular diseases. Here, we developed a murine model in which a constitutively active Gq α subunit (GqQ209L, referred to herein as GqQ>L) is specifically expressed in podocytes and examined the effects of this mutation in response to puromycin aminonucleoside (PAN) nephrosis. We found that compared with control animals, animals expressing GqQ>L exhibited robust albuminuria, structural features of FSGS, and reduced numbers of glomerular podocytes. Gq activation stimulated calcineurin (CN) activity, resulting in CN-dependent upregulation of TRPC6 in murine kidneys. Deletion of TRPC6 in GqQ>L-expressing mice prevented FSGS development and inhibited both tubular damage and podocyte loss induced by PAN nephrosis. Similarly, administration of the CN inhibitor FK506 reduced proteinuria and tubular injury but had more modest effects on glomerular pathology and podocyte numbers in animals with constitutive Gq activation. Moreover, these Gq-dependent effects on podocyte injury were generalizable to diabetic kidney disease, as expression of GqQ>L promoted albuminuria, mesangial expansion, and increased glomerular basement membrane width in diabetic mice. Together, these results suggest that targeting Gq/TRPC6 signaling may have therapeutic benefits for the treatment of glomerular diseases. PMID:25844902

  9. Chemical Signaling and Functional Activation in Colloidosome-Based Protocells.

    PubMed

    Sun, Shiyong; Li, Mei; Dong, Faqin; Wang, Shengjie; Tian, Liangfei; Mann, Stephen

    2016-04-13

    An aqueous-based microcompartmentalized model involving the integration of partially hydrophobic Fe(III)-rich montmorillonite (FeM) clay particles as structural and catalytic building blocks for colloidosome membrane assembly, self-directed membrane remodeling, and signal-induced protocell communication is described. The clay colloidosomes exhibit size- and charge-selective permeability, and show dual catalytic functions involving spatially confined enzyme-mediated dephosphorylation and peroxidase-like membrane activity. The latter is used for the colloidosome-mediated synthesis and assembly of a temperature-responsive poly(N-isopropylacrylamide)(PNIPAM)/clay-integrated hybrid membrane. In situ PNIPAM elaboration of the membrane is coupled to a glucose oxidase (GOx)-mediated signaling pathway to establish a primitive model of chemical communication and functional activation within a synthetic "protocell community" comprising a mixed population of GOx-containing silica colloidosomes and alkaline phosphatase (ALP)-containing FeM-clay colloidosomes. Triggering the enzyme reaction in the silica colloidosomes gives a hydrogen peroxide signal that induces polymer wall formation in a coexistent population of the FeM-clay colloidosomes, which in turn generates self-regulated membrane-gated ALP-activity within the clay microcompartments. The emergence of new functionalities in inorganic colloidosomes via chemical communication between different protocell populations provides a first step toward the realization of interacting communities of synthetic functional microcompartments. PMID:26923794

  10. Wnt Signaling Activates Shh Signaling in Early Postnatal Intervertebral Discs, and Re-Activates Shh Signaling in Old Discs in the Mouse

    PubMed Central

    Sinner, Debora; Wylie, Christopher C.; Dahia, Chitra Lekha

    2014-01-01

    Intervertebral discs (IVDs) are strong fibrocartilaginous joints that connect adjacent vertebrae of the spine. As discs age they become prone to failure, with neurological consequences that are often severe. Surgical repair of discs treats the result of the disease, which affects as many as one in seven people, rather than its cause. An ideal solution would be to repair degenerating discs using the mechanisms of their normal differentiation. However, these mechanisms are poorly understood. Using the mouse as a model, we previously showed that Shh signaling produced by nucleus pulposus cells activates the expression of differentiation markers, and cell proliferation, in the postnatal IVD. In the present study, we show that canonical Wnt signaling is required for the expression of Shh signaling targets in the IVD. We also show that Shh and canonical Wnt signaling pathways are down-regulated in adult IVDs. Furthermore, this down-regulation is reversible, since re-activation of the Wnt or Shh pathways in older discs can re-activate molecular markers of the IVD that are lost with age. These data suggest that biological treatments targeting Wnt and Shh signaling pathways may be feasible as a therapeutic for degenerative disc disease. PMID:24892825

  11. Exploiting the yeast stress-activated signaling network to inform on stress biology and disease signaling

    PubMed Central

    Ho, Yi-Hsuan

    2016-01-01

    Healthy cells utilize intricate systems to monitor their environment and mount robust responses in the event of cellular stress. Whether stress arises from external insults or defects due to mutation and disease, cells must be able to respond precisely to mount the appropriate defenses. Multi-faceted stress responses are generally coupled with arrest of growth and cell-cycle progression, which both limits the transmission of damaged materials and serves to reallocate limited cellular resources toward defense. Therefore, stress defense versus rapid growth represent competing interests in the cell. How eukaryotic cells set the balance between defense versus proliferation, and in particular knowledge of the regulatory networks that control this decision, are poorly understood. In this perspective, we expand upon our recent work inferring the stress-activated signaling network in budding yeast, which captures pathways controlling stress defense and regulators of growth and cell-cycle progression. We highlight similarities between the yeast and mammalian stress responses and explore how stress-activated signaling networks in yeast can inform on signaling defects in human cancers. PMID:25957506

  12. Nitric oxide preferentially induces type 1 T cell differentiation by selectively up-regulating IL-12 receptor β2 expression via cGMP

    PubMed Central

    Niedbala, Wanda; Wei, Xiao-qing; Campbell, Carol; Thomson, Duncan; Komai-Koma, Mousa; Liew, Foo Y.

    2002-01-01

    Nitric oxide plays an important role in immune regulation. We have shown that although high concentrations of NO generally were immune-suppressive, low concentrations of NO selectively enhanced the differentiation of T helper (Th)1 cells but not Th2 cells. This finding provided an explanation for the crucial role of NO in defense against intracellular pathogens. However, the mechanism for the selective induction of Th1 cells was unknown. We report here that at low concentrations, NO activates soluble guanylyl cyclase, leading to the up-regulation of cGMP, which selectively induces the expression of IL-12 receptor β2 but has no effect on IL-4 receptor. Because IL-12 and IL-4 are the key cytokines for induction of Th1 and Th2 cells, respectively, these results, therefore, provide the mechanism for the selective action of NO on T cell subset differentiation. Furthermore, this selectivity also applies to CD8+ cytotoxic and human T cells and, thus, demonstrates the general implication of this observation in immune regulation. Our results also provide an example of the regulation of cytokine receptor expression by NO. The selectivity of such action via cGMP suggests that it is amenable to therapeutic intervention. PMID:12451176

  13. Signal integration by Ca2+ regulates intestinal stem cell activity

    PubMed Central

    Deng, Hansong; Gerencser, Akos A.; Jasper, Heinrich

    2015-01-01

    Summary Somatic stem cells (SCs) maintain tissue homeostasis by dynamically adjusting proliferation and differentiation in response to stress and metabolic cues. Here, we identify Ca2+ signaling as a central regulator of intestinal SC (ISC) activity in Drosophila. We find that dietary L-glutamate stimulates ISC division and gut growth. The metabotropic glutamate receptor (mGluR) is required in ISCs for this response and for an associated modulation of cytosolic Ca2+ oscillations that results in sustained high cytosolic Ca2+ concentrations. High cytosolic Ca2+ induces ISC proliferation by regulating Calcineurin and CREB - regulated transcriptional co-activator (CRTC). In response to a wide range of dietary and stress stimuli, ISCs reversibly transition between Ca2+ oscillation states that represent poised or activated modes of proliferation, respectively. We propose that the dynamic regulation of intracellular Ca2+ levels allows effective integration of diverse mitogenic signals in ISCs to tailor their proliferative activity to the needs of the tissue. PMID:26633624

  14. Robust Indoor Human Activity Recognition Using Wireless Signals

    PubMed Central

    Wang, Yi; Jiang, Xinli; Cao, Rongyu; Wang, Xiyang

    2015-01-01

    Wireless signals–based activity detection and recognition technology may be complementary to the existing vision-based methods, especially under the circumstance of occlusions, viewpoint change, complex background, lighting condition change, and so on. This paper explores the properties of the channel state information (CSI) of Wi-Fi signals, and presents a robust indoor daily human activity recognition framework with only one pair of transmission points (TP) and access points (AP). First of all, some indoor human actions are selected as primitive actions forming a training set. Then, an online filtering method is designed to make actions’ CSI curves smooth and allow them to contain enough pattern information. Each primitive action pattern can be segmented from the outliers of its multi-input multi-output (MIMO) signals by a proposed segmentation method. Lastly, in online activities recognition, by selecting proper features and Support Vector Machine (SVM) based multi-classification, activities constituted by primitive actions can be recognized insensitive to the locations, orientations, and speeds. PMID:26184231

  15. Notum deacylates Wnt proteins to suppress signalling activity.

    PubMed

    Kakugawa, Satoshi; Langton, Paul F; Zebisch, Matthias; Howell, Steven A; Chang, Tao-Hsin; Liu, Yan; Feizi, Ten; Bineva, Ganka; O'Reilly, Nicola; Snijders, Ambrosius P; Jones, E Yvonne; Vincent, Jean-Paul

    2015-03-12

    Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase. PMID:25731175

  16. Pathway connectivity and signaling coordination in the yeast stress-activated signaling network

    PubMed Central

    Chasman, Deborah; Ho, Yi-Hsuan; Berry, David B; Nemec, Corey M; MacGilvray, Matthew E; Hose, James; Merrill, Anna E; Lee, M Violet; Will, Jessica L; Coon, Joshua J; Ansari, Aseem Z; Craven, Mark; Gasch, Audrey P

    2014-01-01

    Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowledge of the complete stress-activated regulatory network as well as design principles for signal integration remains incomplete. We developed an experimental and computational approach to integrate available protein interaction data with gene fitness contributions, mutant transcriptome profiles, and phospho-proteome changes in cells responding to salt stress, to infer the salt-responsive signaling network in yeast. The inferred subnetwork presented many novel predictions by implicating new regulators, uncovering unrecognized crosstalk between known pathways, and pointing to previously unknown ‘hubs’ of signal integration. We exploited these predictions to show that Cdc14 phosphatase is a central hub in the network and that modification of RNA polymerase II coordinates induction of stress-defense genes with reduction of growth-related transcripts. We find that the orthologous human network is enriched for cancer-causing genes, underscoring the importance of the subnetwork's predictions in understanding stress biology. PMID:25411400

  17. Angiotensin II activates different calcium signaling pathways in adipocytes.

    PubMed

    Dolgacheva, Lyudmila P; Turovskaya, Maria V; Dynnik, Vladimir V; Zinchenko, Valery P; Goncharov, Nikolay V; Davletov, Bazbek; Turovsky, Egor A

    2016-03-01

    Angiotensin II (Ang II) is an important mammalian neurohormone involved in reninangiotensin system. Ang II is produced both constitutively and locally by RAS systems, including white fat adipocytes. The influence of Ang II on adipocytes is complex, affecting different systems of signal transduction from early Са(2+) responses to cell proliferation and differentiation, triglyceride accumulation, expression of adipokine-encoding genes and adipokine secretion. It is known that white fat adipocytes express all RAS components and Ang II receptors (АТ1 and АТ2). The current work was carried out with the primary white adipocytes culture, and Са(2+) signaling pathways activated by Ang II were investigated using fluorescent microscopy. Са(2+)-oscillations and transient responses of differentiated adipocytes to Ang II were registered in cells with both small and multiple lipid inclusions. Using inhibitory analysis and selective antagonists, we now show that Ang II initiates periodic Са(2+)-oscillations and transient responses by activating АТ1 and АТ2 receptors and involving branched signaling cascades: 1) Ang II → Gq → PLC → IP3 → IP3Rs → Ca(2+) 2) Gβγ → PI3Kγ → PKB 3) PKB → eNOS → NO → PKG 4) CD38 → cADPR → RyRs → Ca(2+) In these cascades, AT1 receptors play the leading role. The results of the present work open a perspective of using Ang II for correction of signal resistance of adipocytes often observed during obesity and type 2 diabetes. PMID:26850364

  18. Purification and physiological evaluation of a guanylate cyclase activating protein from retinal rods.

    PubMed Central

    Gorczyca, W A; Gray-Keller, M P; Detwiler, P B; Palczewski, K

    1994-01-01

    In retinal rods light triggers a cascade of enzymatic reactions that increases cGMP hydrolysis and generates an electrical signal by causing closure of cGMP-gated ion channels in the photoreceptor outer segment. This leads to a decrease in internal Ca, which activates guanylate cyclase and promotes photoresponse recovery by stimulating the resynthesis of cGMP. We report here that the activation of guanylate cyclase by low Ca is mediated by an approximately 20-kDa protein purified from bovine rod outer segments by using DEAE-Sepharose, hydroxylapatite, and reverse-phase chromatographies. In a reconstituted system, this protein restores the Ca-sensitive regulation of guanylate cyclase and when dialyzed into functionally intact lizard rod outer segment decreases the sensitivity, time to peak, and recovery time of the flash response. Images PMID:7909609

  19. The effect of a nitric oxide donor on the synthesis of cGMP in Hymenolepis diminuta: a radiometric study.

    PubMed

    Onufriev, Mikhail V; Gulyaeva, Natalia V; Terenina, Nadezhda B; Tolstenkov, Oleg O; Gustafsson, Margaretha K S

    2005-01-01

    The formation of cGMP in homogenates of the adult rat-tapeworm Hymenolepis diminuta was followed with a radiometric assay during 3 h after stimulation with the nitric oxide donor sodium nitroprusside (SNP) and in the presence of isobutylmethylxanthine (IBMX). The level of cGMP was stable in worms incubated with IBMX during the first hour. After 3 h of incubation, the level of cGMP had declined by 27%. Addition of SNP stimulated the formation of cGMP during the first hour of incubation. After 3 h of incubation, a two-fold decline in cGMP formation was observed. The rate of nitric oxide (NO) release by the worm was determined by a spectrophotometric assay for the accumulation of nitrites and nitrates, the stable degradation products of NO, using the Griess reaction. The results are discussed from the perspective of the current concept on the role of the nitrergic mechanisms in the flatworm nervous system. PMID:15614585

  20. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    PubMed Central

    Maroni, Paul D; Koul, Sweaty; Meacham, Randall B; Koul, Hari K

    2004-01-01

    The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy. PMID:15219238

  1. Digital signaling decouples activation probability and population heterogeneity

    PubMed Central

    Kellogg, Ryan A; Tian, Chengzhe; Lipniacki, Tomasz; Quake, Stephen R; Tay, Savaş

    2015-01-01

    Digital signaling enhances robustness of cellular decisions in noisy environments, but it is unclear how digital systems transmit temporal information about a stimulus. To understand how temporal input information is encoded and decoded by the NF-κB system, we studied transcription factor dynamics and gene regulation under dose- and duration-modulated inflammatory inputs. Mathematical modeling predicted and microfluidic single-cell experiments confirmed that integral of the stimulus (or area, concentration × duration) controls the fraction of cells that activate NF-κB in the population. However, stimulus temporal profile determined NF-κB dynamics, cell-to-cell variability, and gene expression phenotype. A sustained, weak stimulation lead to heterogeneous activation and delayed timing that is transmitted to gene expression. In contrast, a transient, strong stimulus with the same area caused rapid and uniform dynamics. These results show that digital NF-κB signaling enables multidimensional control of cellular phenotype via input profile, allowing parallel and independent control of single-cell activation probability and population heterogeneity. DOI: http://dx.doi.org/10.7554/eLife.08931.001 PMID:26488364

  2. Digital signaling decouples activation probability and population heterogeneity.

    PubMed

    Kellogg, Ryan A; Tian, Chengzhe; Lipniacki, Tomasz; Quake, Stephen R; Tay, Savaş

    2015-01-01

    Digital signaling enhances robustness of cellular decisions in noisy environments, but it is unclear how digital systems transmit temporal information about a stimulus. To understand how temporal input information is encoded and decoded by the NF-κB system, we studied transcription factor dynamics and gene regulation under dose- and duration-modulated inflammatory inputs. Mathematical modeling predicted and microfluidic single-cell experiments confirmed that integral of the stimulus (or area, concentration × duration) controls the fraction of cells that activate NF-κB in the population. However, stimulus temporal profile determined NF-κB dynamics, cell-to-cell variability, and gene expression phenotype. A sustained, weak stimulation lead to heterogeneous activation and delayed timing that is transmitted to gene expression. In contrast, a transient, strong stimulus with the same area caused rapid and uniform dynamics. These results show that digital NF-κB signaling enables multidimensional control of cellular phenotype via input profile, allowing parallel and independent control of single-cell activation probability and population heterogeneity. PMID:26488364

  3. Activity-Based Probe for Histidine Kinase Signaling

    PubMed Central

    Wilke, Kaelyn E.; Francis, Samson; Carlson, Erin E.

    2012-01-01

    Bacterial two-component systems (TCSs) are signaling pathways composed of two proteins, a histidine kinase (HK) and a response regulator (RR). Upon stimulation, the HK autophosphorylates at a conserved histidine. The phosphoryl group is subsequently transferred to an aspartate on a RR, eliciting an adaptive response, often up- or downregulation of gene expression. TCS signaling controls many functions in bacteria including development, virulence and antibiotic resistance, making the proteins involved in these systems potential therapeutic targets. Efficient methods for the profiling of HKs are currently lacking. For direct readout of HK activity, we sought to design a probe that enables detection of the phosphotransfer event; however, analysis of the phosphohistidine species is made difficult by the instability of the P-N bond. We anticipated that use of a γ-thiophosphorylated ATP analog, which would yield a thiophosphorylated histidine intermediate, could overcome this challenge. We determined that the fluorophore-conjugated probe, ATPγS-BODIPY, labels active HK proteins and is competitive for the ATP-binding site. This activity-based probe provides a new strategy for analysis of TCSs and other HK-mediated processes and will facilitate both functional studies and inhibitor identification. PMID:22606938

  4. Cyclic GMP phosphodiesterase activity role in normal and inflamed human dental pulp.

    PubMed

    Spoto, G; Ferrante, M; D'Intino, M; Rega, L; Dolci, M; Trentini, P; Ciavarelli, L

    2004-01-01

    Cyclic GMP phosphodiesterase (cGMP PDE) plays an important role in pulp tissues. High levels of cGMP PDE are found in dental pulp cells. In the present study cGMP PDE activity was analyzed in normal healthy human dental pulps, in reversible pulpitis and in irreversible pulpitis. Enzymatic cGMP PDE control values for normal healthy pulps were 4.74+/-0.32 nmol/mg of proteins. In reversible pulpitis the cGMP PDE activity increased almost 3 times. In irreversible pulpitis specimens the values increased 4.5 times compared with the normal healthy pulps activity. The differences between the groups (control vs. reversible pulpitis and vs. irreversible pulpitis) were statistically significant. These results point to a role of cGMP PDE in the initial pulp response after injury. PMID:16857102

  5. Digital Signal Processing System for Active Noise Reduction

    NASA Astrophysics Data System (ADS)

    Edmonson, William W.; Tucker, Jerry

    2002-12-01

    Over the years there has been a need to improve the comfort of passengers in flight. One avenue for increasing comfort is to reduce cabin noise that is attributed to the engine and the vibration of fuselage panels that radiate sound. High frequency noise can be abated using sound absorbing material. Though, for low frequency noise the sound absorption material would have to very thick, thereby reducing the cabin size. To reduce these low frequency disturbances, active noise control systems (ANC) is being developed that utilizes feedback for cancellation of the disturbance. The active noise control system must be small in size, be a low power device, and operate in real-time. It must also be numerically stable i.e. insensitive to temperature and pressure variations. The ANC system will be a module that consists of digital signal processor (DSP), analog-digital and digital-analog converters, power converters, an actuator and sensors. The DSP will implement the feedback control algorithm that controls the actuators. This module will be attached to panels on the inside of the fuselage for actively eliminating resonant modes of the structure caused by turbulent flow across the fuselage Skin. A hardware prototype of the ANC system must be able to eliminate broadband noise consisting of a bandwidth between 100 Hz and 1500 Hz, which requires a sample rate of 5000 Hz. The analog/digital converters output accuracy is 16 bits with a 2's-compliment format and a very short acquisition time. This will also yield the appropriate dynamic range. Similar specifications are required of the digital/analog converter. The processor section of the system integrates a digital signal processor (TI TMS320C33) with analog/digital (Burr-Brown ADS8320) and digital/analog signal (DAC853 1) converters. The converters with associated power conditioning circuitry and test points reside on a daughter board that sits on top of a Spectrum Digital evaluation module. This will have the ability to test

  6. The role of the Janus kinase family/signal transducer and activator of transcription signaling pathway in fibrotic renal disease

    PubMed Central

    Matsui, Futoshi; Meldrum, Kirstan K.

    2012-01-01

    Over the past several years, a number of cytokines and growth factors including transforming growth factor β1, tumor necrosis factor α, and angiotensin II have been shown to play a crucial role in renal fibrosis. The Janus kinase family (JAK) and signal transducers and activators of transcription (STATs) constitute one of the primary signaling pathways that regulate cytokine expression, and the JAK/STAT signaling pathway has increasingly been implicated in the pathophysiology of renal disease. This review examines the role of the JAK/STAT signaling pathway in fibrotic renal disease. The JAK/STAT signaling pathway is activated in a variety of renal diseases and has been implicated in the pathophysiology of renal fibrosis. Experimental evidence suggests that inhibition of the JAK/STAT signaling pathway, in particular JAK2 and STAT3, may suppress renal fibrosis and protect renal function. However, it is incompletely understood which cells activate the JAK/STAT signaling pathway and which JAK/STAT signaling pathway is activated in each renal disease. Research regarding JAK/STAT signaling and its contribution to renal disease is still ongoing in humans. Future studies are required to elucidate the potential role of JAK/STAT signaling inhibition as a therapeutic strategy in the attenuation of renal fibrosis. PMID:22883438

  7. Role of Calcium Signaling in B Cell Activation and Biology.

    PubMed

    Baba, Yoshihiro; Kurosaki, Tomohiro

    2016-01-01

    Increase in intracellular levels of calcium ions (Ca2+) is one of the key triggering signals for the development of B cell response to the antigen. The diverse Ca2+ signals finely controlled by multiple factors participate in the regulation of gene expression, B cell development, and effector functions. B cell receptor (BCR)-initiated Ca2+ mobilization is sourced from two pathways: one is the release of Ca2+ from the intracellular stores, endoplasmic reticulum (ER), and other is the prolonged influx of extracellular Ca2+ induced by depleting the stores via store-operated calcium entry (SOCE) and calcium release-activated calcium (CRAC) channels. The identification of stromal interaction molecule 1(STIM1), the ER Ca2+ sensor, and Orai1, a key subunit of the CRAC channel pore, has now provided the tools to understand the mode of Ca2+ influx regulation and physiological relevance. Herein, we discuss our current understanding of the molecular mechanisms underlying BCR-triggered Ca2+ signaling as well as its contribution to the B cell biological processes and diseases. PMID:26369772

  8. Serelaxin-mediated signal transduction in human vascular cells: bell-shaped concentration–response curves reflect differential coupling to G proteins

    PubMed Central

    Sarwar, M; Samuel, C S; Bathgate, R A; Stewart, D R; Summers, R J

    2015-01-01

    Background and Purpose In a recently conducted phase III clinical trial, RELAX-AHF, serelaxin infusion over 48 h improved short- and long-term clinical outcomes in patients with acute heart failure. In this study we used human primary cells from the umbilical vasculature to better understand the signalling mechanisms activated by serelaxin. Experimental Approach We examined the acute effects of serelaxin on signal transduction mechanisms in primary human umbilical vascular cells and its chronic actions on markers of cardiovascular function and disease. Key Results The RXFP1 receptor, the cognate serelaxin receptor, was expressed at the cell surface in HUVECs and human umbilical vein smooth muscle cells (HUVSMCs), human umbilical artery smooth muscle cells (HUASMCs) and human cardiac fibroblasts (HCFs), but not human umbilical artery endothelial cells. In HUVECs and HUVSMCs, serelaxin increased cAMP, cGMP accumulation and pERK1/2, and the concentration–response curves (CRCs) were bell-shaped. Similar bell-shaped CRCs for cGMP and pERK1/2 were observed in HCFs, whereas in HUASMCs, serelaxin increased cAMP, cGMP and pERK1/2 with sigmoidal CRCs. Gαi/o and lipid raft disruption, but not Gαs inhibition, altered the serelaxin CRC for cAMP and cGMP accumulation in HUVSMC but not HUASMC. Longer term serelaxin exposure increased the expression of neuronal NOS, VEGF, ETβ receptors and MMPs (gelatinases) in RXFP1 receptor-expressing cells. Conclusions and Implications Serelaxin caused acute and chronic changes in human umbilical vascular cells that were cell background dependent. Bell-shaped CRCs that were observed only in venous cells and fibroblasts involved Gαi/o located within membrane lipid rafts. PMID:25297987

  9. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

    PubMed Central

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

    The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

  10. Biomechanical Signals Suppress TAK1 Activation to Inhibit NF-κB Transcriptional Activation in Fibrochondrocytes

    PubMed Central

    Madhavan, Shashi; Anghelina, Mirela; Sjostrom, Danen; Dossumbekova, Anar; Guttridge, Denis C.; Agarwal, Sudha

    2016-01-01

    Exercise/joint mobilization is therapeutic for inflammatory joint diseases like rheumatoid and osteoarthritis, but the mechanisms underlying its actions remain poorly understood. We report that biomechanical signals at low/physiological magnitudes are potent inhibitors of inflammation induced by diverse proinflammatory activators like IL-1β, TNF-α, and lipopolysaccharides, in fibrochondrocytes. These signals exert their anti-inflammatory effects by inhibiting phosphorylation of TAK1, a critical point where signals generated by IL-1β, TNF-α, and LPS converge to initiate NF-κB signaling cascade and proinflammatory gene induction. Additionally, biomechanical signals inhibit multiple steps in the IL-1β-induced proinflammatory cascade downstream of IκB kinase activation to regulate IκBα and IκBβ degradation and synthesis, and promote IκBα shuttling to export nuclear NF-κB and terminate its transcriptional activity. The findings demonstrate that biomechanical forces are but another important signal that uses NF-κB pathway to regulate inflammation by switching the molecular activation of discrete molecules involved in proinflammatory gene transcription. PMID:17947700

  11. Nitric oxide attenuates matrix metalloproteinase-9 production by endothelial cells independent of cGMP- or NFκB-mediated mechanisms.

    PubMed

    Meschiari, Cesar A; Izidoro-Toledo, Tatiane; Gerlach, Raquel F; Tanus-Santos, Jose E

    2013-06-01

    Cardiovascular diseases involve critical mechanisms including impaired nitric oxide (NO) levels and abnormal matrix metalloproteinase (MMP) activity. While NO downregulates MMP expression in some cell types, no previous study has examined whether NO downregulates MMP levels in endothelial cells. We hypothesized that NO donors could attenuate MMP-9 production by human umbilical vein endothelial cells (HUVECs) as a result of less NFκB activation or cyclic GMP (cGMP)-mediated mechanisms. We studied the effects of DetaNONOate (10-400 μM) or SNAP (50-400 μM) on phorbol 12-myristate 13-acetate (PMA; 10 nM)-induced increases in MMP-9 activity (by gel zymography) or concentrations (by ELISA) as well as on a tissue inhibitor of MMPs' (TIMP)-1 concentrations (by ELISA) in the conditioned medium of HUVECs incubated for 24 h with these drugs. We also examined whether the irreversible inhibitor of soluble guanylyl cyclase ODQ modified the effects of SNAP or whether 8-bromo-cGMP (a cell-permeable analog of cGMP) influenced PMA-induced effects on MMP-9 expression. Total and phospho-NFκB p65 concentrations were measured in HUVEC lysates to assess NFκB activation. Both NO donors attenuated PMA-induced increases in MMP-9 activity and concentrations without significantly affecting TIMP-1 concentrations. This effect was not modified by ODQ, and 8-bromo-cGMP did not affect MMP-9 concentrations. While PMA increased phospho-NFκB p65 concentrations, SNAP had no influence on this effect. In conclusion, this study shows that NO donors may attenuate imbalanced MMP expression and activity in endothelial cells independent of cGMP- or NFκB-mediated mechanisms. Our results may offer an important pharmacological strategy to approach cardiovascular diseases. PMID:23456480

  12. Transsynaptic signaling by activity-dependent cleavage of neuroligin-1.

    PubMed

    Peixoto, Rui T; Kunz, Portia A; Kwon, Hyungbae; Mabb, Angela M; Sabatini, Bernardo L; Philpot, Benjamin D; Ehlers, Michael D

    2012-10-18

    Adhesive contact between pre- and postsynaptic neurons initiates synapse formation during brain development and provides a natural means of transsynaptic signaling. Numerous adhesion molecules and their role during synapse development have been described in detail. However, once established, the mechanisms of adhesive disassembly and its function in regulating synaptic transmission have been unclear. Here, we report that synaptic activity induces acute proteolytic cleavage of neuroligin-1 (NLG1), a postsynaptic adhesion molecule at glutamatergic synapses. NLG1 cleavage is triggered by NMDA receptor activation, requires Ca2+ /calmodulin-dependent protein kinase, and is mediated by proteolytic activity of matrix metalloprotease 9 (MMP9). Cleavage of NLG1 occurs at single activated spines, is regulated by neural activity in vivo, and causes rapid destabilization of its presynaptic partner neurexin-1β (NRX1β). In turn, NLG1 cleavage depresses synaptic transmission by abruptly reducing presynaptic release probability. Thus, local proteolytic control of synaptic adhesion tunes synaptic transmission during brain development and plasticity. PMID:23083741

  13. A novel nuclear export signal in Smad1 is essential for its signaling activity.

    PubMed

    Xiao, Zhan; Brownawell, Amy M; Macara, Ian G; Lodish, Harvey F

    2003-09-01

    To investigate the subcellular distributions of Smad proteins, the intracellular mediators of transforming growth factor-beta family cytokines, we examined their sequences for nuclear export signals (NES). We found a leucine-rich NES-like motif (termed NES2) in the central linker region of the receptor-regulated Smads that is absent from the other two classes of Smads (Co-Smads and I-Smads). In microinjection assays, NES2 peptide caused nuclear export of a fused glutathione S-transferase protein. Mutations in NES2 converted Smad1 from an even distribution throughout the cells into an exclusive nuclear localization in both transiently and stably expressing cell lines, and this nuclear enrichment was more pronounced than that induced by mutations in NES1. Furthermore, overexpression of CRM1, the cellular export receptor, transforms Smad1 into a mostly cytoplasmic profile by enhancing its nuclear export. The Smad1 NES2 mutant but not the Smad1 NES1 mutant is mostly resistant to this cytoplasmic targeting, indicating that NES2, not NES1, is the major target for CRM1 in Smad1. We further confirmed the functionality of NES2 by a heterokaryon assay. The Smad1 NES1 mutant displays good ligand responsiveness and moderately lowered transcriptional activity compared with wild type Smad1. In contrast, the Smad1 NES2 mutant shows a severe disruption in reporter gene activation, minimal response to bone morphogenetic protein stimulation, and significantly lowered bone morphogenetic protein-induced phosphorylation, which may be the reason for its deficient transcription activity. Thus, we have defined a major NES in Smad1 that is essential for its ligand-induced coupling with cell surface receptors and hence, transcriptional activity. Our study, along with recent studies of the nucleocytoplasmic shuttling of Smad2 and Smad3 proteins, demonstrate that continued nucleocytoplasmic shuttling is a common requisite for the active signaling of R-Smads. Although conserved in other R

  14. Signal Transducer and Activator of Transcription-3, Inflammation, and Cancer

    PubMed Central

    Aggarwal, Bharat B.; Kunnumakkara, Ajaikumar B.; Harikumar, Kuzhuvelil B.; Gupta, Shan R.; Tharakan, Sheeja T.; Koca, Cemile; Dey, Sanjit; Sung, Bokyung

    2011-01-01

    Signal transducer and activator of transcription-3 (STAT-3) is one of six members of a family of transcription factors. It was discovered almost 15 years ago as an acute-phase response factor. This factor has now been associated with inflammation, cellular transformation, survival, proliferation, invasion, angiogenesis, and metastasis of cancer. Various types of carcinogens, radiation, viruses, growth factors, oncogenes, and inflammatory cytokines have been found to activate STAT-3. STAT-3 is constitutively active in most tumor cells but not in normal cells. Phosphorylation of STAT-3 at tyrosine 705 leads to its dimerization, nuclear translocation, DNA binding, and gene transcription. The phosphorylation of STAT-3 at serine 727 may regulate its activity negatively or positively. STAT-3 regulates the expression of genes that mediate survival (survivin, bcl-xl, mcl-1, cellular FLICE-like inhibitory protein), proliferation (c-fos, c-myc, cyclin D1), invasion (matrix metalloproteinase-2), and angiogenesis (vascular endothelial growth factor). STAT-3 activation has also been associated with both chemoresistance and radioresistance. STAT-3 mediates these effects through its collaboration with various other transcription factors, including nuclear factor-κB, hypoxia-inducible factor-1, and peroxisome proliferator activated receptor-γ. Because of its critical role in tumorigenesis, inhibitors of this factor’s activation are being sought for both prevention and therapy of cancer. This has led to identification of small peptides, oligonucleotides, and small molecules as potential STAT-3 inhibitors. Several of these small molecules are chemo-preventive agents derived from plants. This review discusses the intimate relationship between STAT-3, inflammation, and cancer in more detail. PMID:19723038

  15. Dynamic neural activity during stress signals resilient coping.

    PubMed

    Sinha, Rajita; Lacadie, Cheryl M; Constable, R Todd; Seo, Dongju

    2016-08-01

    Active coping underlies a healthy stress response, but neural processes supporting such resilient coping are not well-known. Using a brief, sustained exposure paradigm contrasting highly stressful, threatening, and violent stimuli versus nonaversive neutral visual stimuli in a functional magnetic resonance imaging (fMRI) study, we show significant subjective, physiologic, and endocrine increases and temporally related dynamically distinct patterns of neural activation in brain circuits underlying the stress response. First, stress-specific sustained increases in the amygdala, striatum, hypothalamus, midbrain, right insula, and right dorsolateral prefrontal cortex (DLPFC) regions supported the stress processing and reactivity circuit. Second, dynamic neural activation during stress versus neutral runs, showing early increases followed by later reduced activation in the ventrolateral prefrontal cortex (VLPFC), dorsal anterior cingulate cortex (dACC), left DLPFC, hippocampus, and left insula, suggested a stress adaptation response network. Finally, dynamic stress-specific mobilization of the ventromedial prefrontal cortex (VmPFC), marked by initial hypoactivity followed by increased VmPFC activation, pointed to the VmPFC as a key locus of the emotional and behavioral control network. Consistent with this finding, greater neural flexibility signals in the VmPFC during stress correlated with active coping ratings whereas lower dynamic activity in the VmPFC also predicted a higher level of maladaptive coping behaviors in real life, including binge alcohol intake, emotional eating, and frequency of arguments and fights. These findings demonstrate acute functional neuroplasticity during stress, with distinct and separable brain networks that underlie critical components of the stress response, and a specific role for VmPFC neuroflexibility in stress-resilient coping. PMID:27432990

  16. Analysis of nitric oxide-cyclic guanosine monophosphate signaling during metamorphosis of the nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia).

    PubMed

    Bishop, Cory D; Pires, Anthony; Norby, Shong-Wan; Boudko, Dmitri; Moroz, Leonid L; Hadfield, Michael G

    2008-01-01

    The gas nitric oxide (NO), and in some cases its downstream second messenger, cyclic guanosine monophosphate (cGMP) function in different taxa to regulate the timing of life-history transitions. Increased taxonomic sampling is required to foster conclusions about the evolution and function of NO/cGMP signaling during life-history transitions. We report on the function and localization of NO and cGMP signaling during metamorphosis of the nudibranch Phestilla sibogae. Pharmacological manipulation of NO or cGMP production in larvae modulated responses to a natural settlement cue from the coral Porites compressa in a manner that suggest inhibitory function for NO/cGMP signaling. However, these treatments were not sufficient to induce metamorphosis in the absence of cue, a result unique to this animal. We show that induction of metamorphosis in response to the settlement cue is associated with a reduction in NO production. We documented the expression of putative NO synthase (NOS) and the production of cGMP during larval development and observed no larval cells in which NOS and cGMP were both detected. The production of cGMP in a bilaterally symmetrical group of cells fated to occupy the distal tip of rhinophores is correlated with competence to respond to the coral settlement cue. These results suggest that endogenous NO and cGMP are involved in modulating responses of P. sibogae to a natural settlement cue. We discuss these results with respect to habitat selection and larval ecology. PMID:18460091

  17. Accelerated Lactate Dehydrogenase Activity Potentiates Osteoclastogenesis via NFATc1 Signaling

    PubMed Central

    Kim, Jin Man; Kwon, So Hyun; Lee, Seoung Hoon; Lee, Soo Young; Jeong, Daewon

    2016-01-01

    Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role of lactate dehydrogenase (LDH), a tetrameric enzyme consisting of an A and/or B subunit that catalyzes interconversion of pyruvate to lactate, in RANKL-induced osteoclast differentiation is not known. In this study, RANKL treatment induced gradual gene expression and activation of the LDH A2B2 isotype during osteoclast differentiation as well as the LDH A1B3 and B4 isotypes during osteoclast maturation after pre-osteoclast formation. Glucose consumption and lactate production in growth media were accelerated during osteoclast differentiation, together with enhanced expression of H+-lactate co-transporter and increased extracellular acidification, demonstrating that glycolytic metabolism was stimulated during differentiation. Further, oxygen consumption via mitochondria was stimulated during osteoclast differentiation. On the contrary, depletion of LDH-A or LDH-B subunit suppressed both glycolytic and mitochondrial metabolism, resulting in reduced mature osteoclast formation via decreased osteoclast precursor fusion and down-regulation of the osteoclastogenic critical transcription factor NFATc1 and its target genes. Collectively, our findings suggest that RANKL-induced LDH activation stimulates glycolytic and mitochondrial respiratory metabolism, facilitating mature osteoclast formation via osteoclast precursor fusion and NFATc1 signaling. PMID:27077737

  18. Aurora A kinase activity influences calcium signaling in kidney cells.

    PubMed

    Plotnikova, Olga V; Pugacheva, Elena N; Golemis, Erica A

    2011-06-13

    Most studies of Aurora A (AurA) describe it as a mitotic centrosomal kinase. However, we and others have recently identified AurA functions as diverse as control of ciliary resorption, cell differentiation, and cell polarity control in interphase cells. In these activities, AurA is transiently activated by noncanonical signals, including Ca(2+)-dependent calmodulin binding. These and other observations suggested that AurA might be involved in pathological conditions, such as polycystic kidney disease (PKD). In this paper, we show that AurA is abundant in normal kidney tissue but is also abnormally expressed and activated in cells lining PKD-associated renal cysts. PKD arises from mutations in the PKD1 or PKD2 genes, encoding polycystins 1 and 2 (PC1 and PC2). AurA binds, phosphorylates, and reduces the activity of PC2, a Ca(2+)-permeable nonselective cation channel and, thus, limits the amplitude of Ca(2+) release from the endoplasmic reticulum. These and other findings suggest AurA may be a relevant new biomarker or target in the therapy of PKD. PMID:21670214

  19. Signal-to-noise ratio in neuro activation PET studies

    SciTech Connect

    Votaw, J.R.

    1996-04-01

    It has become commonplace to compare scanner sensitivity characteristics by comparing noise equivalent count rate curves. However, because a 20-cm diameter uniform phantom is drastically difference from a human brain, these curves give misleading information when planning a neuro activation PET experiment. Signal-to-noise ratio (SNR) calculations have been performed using measured data (Siemens 921 scanner) from the three-dimensional (3-D) Hoffman brain phantom for the purpose of determining the optimal injection and scanning protocol for [{sup 15}O] labeled activation experiments. Region of interest (ROI) values along with the variance due to prompt (trues plus randoms) and random events were determined for various regions and radioactivity concentrations. Calculated attenuation correction was used throughout. Scatter correction was not used when calculating the SNR in activation studies because the number of scattered events is almost identical in each data acquisition and hence cancels. The results indicate that randoms correction should not be performed and that rather than being limited by the scanner capabilities, neuro activation experiments are limited by the amount of radioactivity that can be injected and the length of time the patient can stay in the scanner.

  20. Role of nitric oxide signaling components in differentiation of embryonic stem cells into myocardial cells

    PubMed Central

    Mujoo, Kalpana; Sharin, Vladislav G.; Bryan, Nathan S.; Krumenacker, Joshua S.; Sloan, Courtney; Parveen, Shanaz; Nikonoff, Lubov E.; Kots, Alexander Y.; Murad, Ferid

    2008-01-01

    Nitric oxide (NO) is involved in number of physiological and pathological events. Our previous studies demonstrated a differential expression of NO signaling components in mouse and human ES cells. Here, we demonstrate the effect of NO donors and soluble guanylyl cyclase (sGC) activators in differentiation of ES cells into myocardial cells. Our results with mouse and human ES cells demonstrate an increase in Nkx2.5 and myosin light chain (MLC2) mRNA expression on exposure of cells to NO donors and a decrease in mRNA expression of both cardiac-specific genes with nonspecific NOS inhibitor and a concomitant increase and decrease in the mRNA levels of sGC α1 subunit. Although sGC activators alone exhibited an increase in mRNA expression of cardiac genes (MLC2 and Nkx2.5), robust inductions of mRNA and protein expression of marker genes were observed when NO donors and sGC activators were combined. Measurement of NO metabolites revealed an increase in the nitrite levels in the conditioned media and cell lysates on exposure of cells to the different concentrations of NO donors. cGMP analysis in undifferentiated stem cells revealed a lack of stimulation with NO donors. Differentiated cells however, acquired the ability to be stimulated by NO donors. Although, 3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo [3,4-b]pyridine (BAY 41-2272) alone was able to stimulate cGMP accumulation, the combination of NO donors and BAY 41-2272 stimulated cGMP levels more than either of the agents separately. These studies demonstrate that cGMP-mediated NO signaling plays an important role in the differentiation of ES cells into myocardial cells. PMID:19020077

  1. PKG-1α mediates GATA4 transcriptional activity.

    PubMed

    Ma, Yanlin; Wang, Jun; Yu, Yanhong; Schwartz, Robert J

    2016-06-01

    GATA4, a zinc-finger transcription factor, is central for cardiac development and diseases. Here we show that GATA4 transcriptional activity is mediated by cell signaling via cGMP dependent PKG-1α activity. Protein kinase G (PKG), a serine/tyrosine specific kinase is the major effector of cGMP signaling. We observed enhanced transcriptional activity elicited by co-expressed GATA4 and PKG-1α. Phosphorylation of GATA4 by PKG-1α was detected on serine 261 (S261), while the C-terminal activation domain of GATA4 associated with PKG-1α. GATA4's DNA binding activity was enhanced by PKG-1α via by both phosphorylation and physical association. More importantly, a number of human disease-linked GATA4 mutants exhibited impaired S261 phosphorylation, pointing to defective S261 phosphorylation in the elaboration of human heart diseases. We showed S261 phosphorylation was favored by PKG-1α but not by PKA, and several other kinase signaling pathways such as MAPK and PKC. Our observations demonstrate that cGMP-PKG signaling mediates transcriptional activity of GATA4 and links defective GATA4 and PKG-1α mutations to the development of human heart disease. PMID:26946174

  2. Bisphenol A (BPA) stimulates the interferon signaling and activates the inflammasome activity in myeloid cells.

    PubMed

    Panchanathan, Ravichandran; Liu, Hongzhu; Leung, Yuet-Kin; Ho, Shuk-mei; Choubey, Divaker

    2015-11-01

    Environmental factors contribute to the development of autoimmune diseases, including systemic lupus erythematosus (SLE), which exhibits a strong female bias (female-to-male ratio 9:1). However, the molecular mechanisms remain largely unknown. Because a feedforward loop between the female sex hormone estrogen (E2) and type I interferon (IFN-α/β)-signaling induces the expression of certain p200-family proteins (such as murine p202 and human IFI16) that regulate innate immune responses and modify lupus susceptibility, we investigated whether treatment of myeloid cells with bisphenol A (BPA), an environmental estrogen, could regulate the p200-family proteins and activate innate immune responses. We found that treatment of murine bone marrow-derived cells (BMCs) and human peripheral blood mononuclear cells with BPA induced the expression of ERα and IFN-β, activated the IFN-signaling, and stimulated the expression of the p202 and IFI16 proteins. Further, the treatment increased levels of the NLRP3 inflammasome and stimulated its activity. Accordingly, BPA-treatment of BMCs from non lupus-prone C57BL/6 and the lupus-prone (NZB×NZW)F1 mice activated the type I IFN-signaling, induced the expression of p202, and activated an inflammasome activity. Our study demonstrates that BPA-induced signaling in the murine and human myeloid cells stimulates the type I IFN-signaling that results in an induction of the p202 and IFI16 innate immune sensors for the cytosolic DNA and activates an inflammasome activity. These observations provide novel molecular insights into the role of environmental BPA exposures in potentiating the development of certain autoimmune diseases such as SLE. PMID:26277401

  3. Lipoicmethylenedioxyphenol Reduces Experimental Atherosclerosis through Activation of Nrf2 Signaling

    PubMed Central

    Ying, Zhekang; Chen, Minjie; Xie, Xiaoyun; Wang, Xiaoke; Kherada, Nisharahmed; Desikan, Rajagopal; Mihai, Georgeta; Burns, Patrick; Sun, Qinghua; Rajagopalan, Sanjay

    2016-01-01

    Objective Oxidative stress is implicated in the pathogenesis of atherosclerosis, and Nrf2 is the transcriptional factor central in cellular antioxidant responses. In the present study, we investigate the effect of a dihydrolipoic acid derivative lipoicmethylenedioxyphenol (LMDP) on the progression of atherosclerosis and test whether its effect on atherosclerosis is mediated by Nrf2. Methods and Results Both magnetic resonance imaging (MRI) scanning and en face analysis reveal that 14 weeks of treatment with LMDP markedly reduced atherosclerotic burden in a rabbit balloon vascular injury model. Myograph analyses show decreased aortic contractile response to phenylephrine and increased aortic response to acetylcholine and insulin in LMDP-treated animals, suggesting that LMDP inhibits atherosclerosis through improving vascular function. A role of Nrf2 signaling in mediating the amelioration of vascular function by LMDP was supported by increased Nrf2 translocation into nuclear and increased expression of Nrf2 target genes. Furthermore, chemotaxis analysis with Boydem chamber shows that leukocytes isolated from LMDP-treated rabbits had reduced chemotaxis, and knock-down of Nrf2 significantly reduced the effect of LMDP on the chemotaxis of mouse macrophages. Conclusion Our results support that LMDP has an anti-atherosclerotic effect likely through activation of Nrf2 signaling and subsequent inhibition of macrophage chemotaxis. PMID:26859892

  4. The plant natriuretic peptide receptor is a guanylyl cyclase and enables cGMP-dependent signaling.

    PubMed

    Turek, Ilona; Gehring, Chris

    2016-06-01

    The functional homologues of vertebrate natriuretic peptides (NPs), the plant natriuretic peptides (PNPs), are a novel class of peptidic hormones that signal via guanosine 3',5'-cyclic monophosphate (cGMP) and systemically affect plant salt and water balance and responses to biotrophic plant pathogens. Although there is increasing understanding of the complex roles of PNPs in plant responses at the systems level, little is known about the underlying signaling mechanisms. Here we report isolation and identification of a novel Leucine-Rich Repeat (LRR) protein that directly interacts with A. thaliana PNP, AtPNP-A. In vitro binding studies revealed that the Arabidopsis AtPNP-A binds specifically to the LRR protein, termed AtPNP-R1, and the active region of AtPNP-A is sufficient for the interaction to occur. Importantly, the cytosolic part of the AtPNP-R1, much like in some vertebrate NP receptors, harbors a catalytic center diagnostic for guanylyl cyclases and the recombinant AtPNP-R1 is capable of catalyzing the conversion of guanosine triphosphate to cGMP. In addition, we show that AtPNP-A causes rapid increases of cGMP levels in wild type (WT) leaf tissue while this response is significantly reduced in the atpnp-r1 mutants. AtPNP-A also causes cGMP-dependent net water uptake into WT protoplasts, and hence volume increases, whereas responses of the protoplasts from the receptor mutant are impaired. Taken together, our results suggest that the identified LRR protein is an AtPNP-A receptor essential for the PNP-dependent regulation of ion and water homeostasis in plants and that PNP- and vertebrate NP-receptors and their signaling mechanisms share surprising similarities. PMID:26945740

  5. Activation of the Extracellular Signal-Regulated Kinase Signaling Is Critical for Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation

    PubMed Central

    Li, Chen-Shuang; Zheng, Zhong; Su, Xiao-Xia; Wang, Fei; Ling, Michelle; Zou, Min; Zhou, Hong

    2016-01-01

    Human umbilical cord mesenchymal stem cells (hUCMSCs) are recognized as candidate progenitor cells for bone regeneration. However, the mechanism of hUCMSC osteogenesis remains unclear. In this study, we revealed that mitogen-activated protein kinases (MAPKs) signaling is involved in hUCMSC osteogenic differentiation in vitro. Particularly, the activation of c-Jun N-terminal kinases (JNK) and p38 signaling pathways maintained a consistent level in hUCMSCs through the entire 21-day osteogenic differentiation period. At the same time, the activation of extracellular signal-regulated kinases (ERK) signaling significantly increased from day 5, peaked at day 9, and declined thereafter. Moreover, gene profiling of osteogenic markers, alkaline phosphatase (ALP) activity measurement, and alizarin red staining demonstrated that the application of U0126, a specific inhibitor for ERK activation, completely prohibited hUCMSC osteogenic differentiation. However, when U0126 was removed from the culture at day 9, ERK activation and osteogenic differentiation of hUCMSCs were partially recovered. Together, these findings demonstrate that the activation of ERK signaling is essential for hUCMSC osteogenic differentiation, which points out the significance of ERK signaling pathway to regulate the osteogenic differentiation of hUCMSCs as an alternative cell source for bone tissue engineering. PMID:26989682

  6. In Vitro Interaction of Tubulin with the Photoreceptor cGMP Phosphodiesterase γ-Subunit

    PubMed Central

    Chu, Uyen B.; Song, Jikui; Mavlyutov, Timur A.; Guo, Lian-Wang

    2010-01-01

    The α and β tubulins compose the microtubule cytoskeleton which is involved in many cellular processes such as vesicular transport. The photoreceptor cells in the retina are neurons specialized for phototransduction. Here we report a novel interaction between tubulin and the photoreceptor cGMP phosphodiesterase (PDE6) gamma subunit (PDEγ). The specificity and molecular details of the PDEγ:tubulin interaction were analyzed through the experiments of pull down, microtubule co-sedimentation, and NMR spectroscopy. The tubulin-interacting site was identified to be in the PDEγ C-terminal I67-G85 region, and the interaction interface appeared to be distinct from those with the other PDEγ targets in phototransduction. We also observed that PDEγ interacted with tubulin in a GTP-dependent manner. Our findings offer implications for non-phototransduction role(s) of PDEγ in the photoreceptor neurons. PMID:20655363

  7. cGMP decreases surface NKCC2 levels in the thick ascending limb: role of phosphodiesterase 2 (PDE2)

    PubMed Central

    Ares, Gustavo R.; Caceres, Paulo; Alvarez-Leefmans, Francisco J.; Ortiz, Pablo A.

    2008-01-01

    NaCl absorption in the medullary thick ascending limb of the loop of Henle (THAL) is mediated by the apical Na/K/2Cl cotransporter (NKCC2). Hormones that increase cGMP, such as nitric oxide (NO) and natriuretic peptides, decrease NaCl absorption by the THAL. However, the mechanism by which cGMP decreases NaCl absorption in THALs is not known. We hypothesized that cGMP decreases surface NKCC2 levels in the THAL. We used surface biotinylation to measure surface NKCC2 levels in rat THAL suspensions. We tested the effect of the membrane-permeant cGMP analog dibutyryl-cGMP (db-cGMP) on surface NKCC2 levels. Incubating THALs with db-cGMP for 20 min decreased surface NKCC2 levels in a concentration-dependent manner (basal = 100%; db-cGMP 100 μM = 77 ± 7%; 500 μM = 54 ± 10% and 1,000 μM = 61 ± 8%). A different cGMP analog 8-bromo-cGMP (8-Br-cGMP) also decreased surface NKCC2 levels by 25%, (basal = 100%; 8-Br-cGMP = 75 ± 5%). Incubation of isolated, perfused THALs with db-cGMP decreased apical surface NKCC2 labeling levels as measured by immunofluorescence and confocal microscopy. cGMP-stimulated phosphodiesterase 2 (PDE2) mediates the inhibitory effect of NO on NaCl absorption by THALs. Thus we examined the role of PDE2 and found that PDE2 inhibitors blocked the effect of db-cGMP on surface NKCC2. Also, a nonstimulatory concentration of db-cAMP blocked the cGMP-induced decrease in surface NKCC2. Finally, db-cGMP inhibited THAL net Cl absorption by 48 ± 4%, and this effect was completely blocked by PDE2 inhibition. We conclude that cGMP decreases NKCC2 levels in the apical membrane of THALs and that this effect is mediated by PDE2. This is an important mechanism by which cGMP inhibits NaCl absorption by the THAL. PMID:18684888

  8. Regulators of G-protein signaling accelerate GPCR signaling kinetics and govern sensitivity solely by accelerating GTPase activity.

    PubMed

    Lambert, Nevin A; Johnston, Christopher A; Cappell, Steven D; Kuravi, Sudhakiranmayi; Kimple, Adam J; Willard, Francis S; Siderovski, David P

    2010-04-13

    G-protein heterotrimers, composed of a guanine nucleotide-binding G alpha subunit and an obligate G betagamma dimer, regulate signal transduction pathways by cycling between GDP- and GTP-bound states. Signal deactivation is achieved by G alpha-mediated GTP hydrolysis (GTPase activity) which is enhanced by the GTPase-accelerating protein (GAP) activity of "regulator of G-protein signaling" (RGS) proteins. In a cellular context, RGS proteins have also been shown to speed up the onset of signaling, and to accelerate deactivation without changing amplitude or sensitivity of the signal. This latter paradoxical activity has been variably attributed to GAP/enzymatic or non-GAP/scaffolding functions of these proteins. Here, we validated and exploited a G alpha switch-region point mutation, known to engender increased GTPase activity, to mimic in cis the GAP function of RGS proteins. While the transition-state, GDP x AlF(4)(-)-bound conformation of the G202A mutant was found to be nearly identical to wild-type, G alpha(i1)(G202A) x GDP assumed a divergent conformation more closely resembling the GDP x AlF(4)(-)-bound state. When placed within Saccharomyces cerevisiae G alpha subunit Gpa1, the fast-hydrolysis mutation restored appropriate dose-response behaviors to pheromone signaling in the absence of RGS-mediated GAP activity. A bioluminescence resonance energy transfer (BRET) readout of heterotrimer activation with high temporal resolution revealed that fast intrinsic GTPase activity could recapitulate in cis the kinetic sharpening (increased onset and deactivation rates) and blunting of sensitivity also engendered by RGS protein action in trans. Thus G alpha-directed GAP activity, the first biochemical function ascribed to RGS proteins, is sufficient to explain the activation kinetics and agonist sensitivity observed from G-protein-coupled receptor (GPCR) signaling in a cellular context. PMID:20351284

  9. Key gravity-sensitive signaling pathways drive T cell activation.

    PubMed

    Boonyaratanakornkit, J B; Cogoli, A; Li, C-F; Schopper, T; Pippia, P; Galleri, G; Meloni, M A; Hughes-Fulford, M

    2005-12-01

    Returning astronauts have experienced altered immune function and increased vulnerability to infection during spaceflights dating back to Apollo and Skylab. Lack of immune response in microgravity occurs at the cellular level. We analyzed differential gene expression to find gravity-dependent genes and pathways. We found inhibited induction of 91 genes in the simulated freefall environment of the random positioning machine. Altered induction of 10 genes regulated by key signaling pathways was verified using real-time RT-PCR. We discovered that impaired induction of early genes regulated primarily by transcription factors NF-kappaB, CREB, ELK, AP-1, and STAT after crosslinking the T-cell receptor contributes to T-cell dysfunction in altered gravity environments. We have previously shown that PKA and PKC are key early regulators in T-cell activation. Since the majority of the genes were regulated by NF-kappaB, CREB, and AP-1, we studied the pathways that regulated these transcription factors. We found that the PKA pathway was down-regulated in vg. In contrast, PI3-K, PKC, and its upstream regulator pLAT were not significantly down-regulated by vectorless gravity. Since NF-kappaB, AP-1, and CREB are all regulated by PKA and are transcription factors predicted by microarray analysis to be involved in the altered gene expression in vectorless gravity, the data suggest that PKA is a key player in the loss of T-cell activation in altered gravity. PMID:16210397

  10. The satiety signaling neuropeptide perisulfakinin inhibits the activity of central neurons promoting general activity.

    PubMed

    Wicher, Dieter; Derst, Christian; Gautier, Hélène; Lapied, Bruno; Heinemann, Stefan H; Agricola, Hans-Jürgen

    2007-01-01

    The metabolic state is one of the determinants of the general activity level. Satiety is related to resting or sleep whereas hunger correlates to wakefulness and activity. The counterpart to the mammalian satiety signal cholecystokinin (CCK) in insects are the sulfakinins. The aim of this study was to resolve the mechanism by which the antifeedant activity of perisulfakinin (PSK) in Periplaneta americana is mediated. We identified the sources of PSK which is used both as hormone and as paracrine messenger. PSK is found in the neurohemal organ of the brain and in nerve endings throughout the central nervous system. To correlate the distributions of PSK and its receptor (PSKR), we cloned the gene coding for PSKR and provide evidence for its expression within the nervous system. It occurs only in a few neurons, among them are the dorsal unpaired median (DUM) neurons which release octopamine thereby regulating the general level of activity. Application of PSK to DUM neurons attenuated the spiking frequency (EC(50)=11pM) due to reduction of a pacemaker Ca(2+) current through cAMP-inhibited pTRPgamma channels. PSK increased the intracellular cAMP level while decreasing the intracellular Ca(2+) concentration in DUM neurons. Thus, the satiety signal conferred by PSK acts antagonistically to the hunger signal, provided by the adipokinetic hormone (AKH): PSK depresses the electrical activity of DUM neurons by inhibiting the pTRPgamma channel that is activated by AKH under conditions of food shortage. PMID:18946521

  11. The Satiety Signaling Neuropeptide Perisulfakinin Inhibits the Activity of Central Neurons Promoting General Activity

    PubMed Central

    Wicher, Dieter; Derst, Christian; Gautier, Hélène; Lapied, Bruno; Heinemann, Stefan H.; Agricola, Hans-Jürgen

    2007-01-01

    The metabolic state is one of the determinants of the general activity level. Satiety is related to resting or sleep whereas hunger correlates to wakefulness and activity. The counterpart to the mammalian satiety signal cholecystokinin (CCK) in insects are the sulfakinins. The aim of this study was to resolve the mechanism by which the antifeedant activity of perisulfakinin (PSK) in Periplaneta americana is mediated. We identified the sources of PSK which is used both as hormone and as paracrine messenger. PSK is found in the neurohemal organ of the brain and in nerve endings throughout the central nervous system. To correlate the distributions of PSK and its receptor (PSKR), we cloned the gene coding for PSKR and provide evidence for its expression within the nervous system. It occurs only in a few neurons, among them are the dorsal unpaired median (DUM) neurons which release octopamine thereby regulating the general level of activity. Application of PSK to DUM neurons attenuated the spiking frequency (EC50=11pM) due to reduction of a pacemaker Ca2+ current through cAMP-inhibited pTRPγ channels. PSK increased the intracellular cAMP level while decreasing the intracellular Ca2+ concentration in DUM neurons. Thus, the satiety signal conferred by PSK acts antagonistically to the hunger signal, provided by the adipokinetic hormone (AKH): PSK depresses the electrical activity of DUM neurons by inhibiting the pTRPγ channel that is activated by AKH under conditions of food shortage. PMID:18946521

  12. Deriving stellar inclination of slow rotators using stellar activity signal

    NASA Astrophysics Data System (ADS)

    Dumusque, Xavier

    2015-01-01

    Stellar inclination is an important parameter for many astrophysical studies. In the context of exoplanets, this allows us to derive the true obliquity of a system if the projected stellar spin-planetary orbit angle can measured via the Rossiter-Mclaughlin effect. Although different techniques allow us to estimate stellar inclination for fast rotators, it becomes much more difficult when stars are rotating slower than 2-2.5 km.s-1. By using the new activity simulation SOAP 2.0 that can reproduce the photometric and spectroscopic variations induced by stellar activity, we are able to fit the activity variation of solar-type stars and derive their inclination. The case of the equator-on star HD189733 will be presented, as well as the case of Alpha Centauri B, which present an inclination of 45+9-19 degrees, implying that the earth-mass orbiting planet is not transiting if aligned with its host star. Other exemples will also demonstrate the power of the technique, that can infer a stellar inclination, even for slow rotators like Alpha Centauri B, that present a projected rotational velocity smaller than 1.15 km.s-1. In addition, the SOAP 2.0 simulation can be used to correct for the effect of activity when one major active region is dominating the RV signal. This could enhance the detection of small mass exoplanets orbiting slightly active stars.This project is funded by ETAEARTH (European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement n. 313014), a transnational collaboration between European countries and the US (the Swiss Space Office, the Harvard Origin of Life Initiative, the Scottish Universities Physics Alliance, the University of Geneva, the Smithsonian Astrophysical Observatory, the Italian National Astrophysical Institute, the University of St. Andrews, Queens University Belfast, and the University of Edinburgh) setup to optimize the synergy between space-and ground-based data whose scientific potential for the characterization of

  13. The effect of resveratrol on beta amyloid-induced memory impairment involves inhibition of phosphodiesterase-4 related signaling.

    PubMed

    Wang, Gang; Chen, Ling; Pan, Xiaoyu; Chen, Jiechun; Wang, Liqun; Wang, Weijie; Cheng, Ruochuan; Wu, Fan; Feng, Xiaoqing; Yu, Yingcong; Zhang, Han-Ting; O'Donnell, James M; Xu, Ying

    2016-04-01

    Resveratrol, a natural polyphenol found in red wine, has wide spectrum of pharmacological properties including antioxidative and antiaging activities. Beta amyloid peptides (Aβ) are known to involve cognitive impairment, neuroinflammatory and apoptotic processes in Alzheimer's disease (AD). Activation of cAMP and/or cGMP activities can improve memory performance and decrease the neuroinflammation and apoptosis. However, it remains unknown whether the memory enhancing effect of resveratrol on AD associated cognitive disorders is related to the inhibition of phosphodiesterase 4 (PDE4) subtypes and subsequent increases in intracellular cAMP and/or cGMP activities. This study investigated the effect of resveratrol on Aβ1-42-induced cognitive impairment and the participation of PDE4 subtypes related cAMP or cGMP signaling. Mice microinfused with Aβ1-42 into bilateral CA1 subregions displayed learning and memory impairment, as evidenced by reduced memory acquisition and retrieval in the water maze and retention in the passive avoidance tasks; it was also significant that neuroinflammatory and pro-apoptotic factors were increased in Aβ1-42-treated mice. Aβ1-42-treated mice also increased in PDE4A, 4B and 4D expression, and decreased in PKA level. However, PKA inhibitor H89, but not PKG inhibitor KT5823, prevented resveratrol's effects on these parameters. Resveratrol also reversed Aβ1-42-induced decreases in phosphorylated cAMP response-element binding protein (pCREB), brain derived neurotrophic factor (BDNF) and anti-apoptotic factor BCl-2 expression, which were reversed by H89. These findings suggest that resveratrol reversing Aβ-induced learning and memory disorder may involve the regulation of neuronal inflammation and apoptosis via PDE4 subtypes related cAMP-CREB-BDNF signaling. PMID:26980711

  14. Evidence of a Ca(2+)-(*)NO-cGMP signaling pathway controlling zoospore biogenesis in the aquatic fungus Blastocladiella emersonii.

    PubMed

    Vieira, André L G; Linares, Edlaine; Augusto, Ohara; Gomes, Suely L

    2009-08-01

    The sporulation stage of the aquatic fungus Blastocladiella emersonii culminates with the formation and release to the medium of a number of zoospores, which are motile cells responsible for the dispersal of the fungus. The presence in the sporulation solution of 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a potent and selective inhibitor of nitric oxide-sensitive guanylyl cyclases, completely prevented biogenesis of the zoospores. In addition, this compound was able to significantly reduce cGMP levels, which increase drastically during late sporulation, suggesting the existence of a nitric oxide-dependent mechanism for cGMP synthesis. Furthermore, increased levels of nitric oxide-derived products were detected during sporulation by fluorescence assays using DAF-2 DA, whose signal was drastically reduced in the presence of the nitric oxide synthase inhibitor Nomega-Nitro-L-arginine methyl ester (L-NAME). These results were confirmed by quantitative chemiluminescent determination of the intracellular levels of nitric oxide-derived products. A putative nitric oxide synthase (NOS) activity was detected throughout sporulation, and this enzyme activity decreased significantly when L-NAME and 1-[2-(Trifluoromethyl)phenyl]imidazole (TRIM) were added to the assays. NOS assays carried out in the presence of EGTA showed decreased enzyme activity, suggesting the involvement of calcium ions in enzyme activation. Additionally, expressed sequence tags (ESTs) encoding putative guanylyl cyclases and a cGMP-phosphodiesterase were found in B. emersonii EST database (http://blasto.iq.usp.br), and the mRNA levels of the corresponding genes were observed to increase during sporulation. Altogether, data presented here revealed the presence and expression of guanylyl cyclase and cGMP phosphodiesterase genes in B. emersonii and provided evidence of a Ca(2+)-(*)NO-cGMP signaling pathway playing a role in zoospore biogenesis. PMID:19393757

  15. The effect of resveratrol on beta amyloid-induced memory impairment involves inhibition of phosphodiesterase-4 related signaling

    PubMed Central

    Wang, Gang; Chen, Ling; Pan, Xiaoyu; Chen, Jiechun; Wang, Liqun; Wang, Weijie; Cheng, Ruochuan; Wu, Fan; Feng, Xiaoqing; Yu, Yingcong; Zhang, Han-Ting; O'Donnell, James M.; Xu, Ying

    2016-01-01

    Resveratrol, a natural polyphenol found in red wine, has wide spectrum of pharmacological properties including antioxidative and antiaging activities. Beta amyloid peptides (Aβ) are known to involve cognitive impairment, neuroinflammatory and apoptotic processes in Alzheimer's disease (AD). Activation of cAMP and/or cGMP activities can improve memory performance and decrease the neuroinflammation and apoptosis. However, it remains unknown whether the memory enhancing effect of resveratrol on AD associated cognitive disorders is related to the inhibition of phosphodiesterase 4 (PDE4) subtypes and subsequent increases in intracellular cAMP and/or cGMP activities. This study investigated the effect of resveratrol on Aβ1-42-induced cognitive impairment and the participation of PDE4 subtypes related cAMP or cGMP signaling. Mice microinfused with Aβ1-42 into bilateral CA1 subregions displayed learning and memory impairment, as evidenced by reduced memory acquisition and retrieval in the water maze and retention in the passive avoidance tasks; it was also significant that neuroinflammatory and pro-apoptotic factors were increased in Aβ1-42-treated mice. Aβ1-42-treated mice also increased in PDE4A, 4B and 4D expression, and decreased in PKA level. However, PKA inhibitor H89, but not PKG inhibitor KT5823, prevented resveratrol's effects on these parameters. Resveratrol also reversed Aβ1-42-induced decreases in phosphorylated cAMP response-element binding protein (pCREB), brain derived neurotrophic factor (BDNF) and anti-apoptotic factor BCl-2 expression, which were reversed by H89. These findings suggest that resveratrol reversing Aβ-induced learning and memory disorder may involve the regulation of neuronal inflammation and apoptosis via PDE4 subtypes related cAMP-CREB-BDNF signaling. PMID:26980711

  16. Hypoxia promotes drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling

    PubMed Central

    Zhao, Changfu; Zhang, Qiao; Yu, Tao; Sun, Shudong; Wang, Wenjun; Liu, Guangyao

    2016-01-01

    Purpose Drug resistance has been recognized to be a major obstacle to the chemotherapy for osteosarcoma. And the potential importance of hypoxia as a target to reverse drug resistance in osteosarcoma has been indicated, though the mechanism underlining such role is not clarified. The present study aims to investigate the role of hypoxia in the drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling. Experimental design We investigated the promotion of the resistance to doxorubicin of osteosarcoma MG-63 and U2-os cells in vitro, and then determined the role of hypoxia-inducible factor-1 (HIF-1)α and HIF-1β, the activation and regulatory role of AMPK in the osteosarcoma U2-os cells which were treated with doxorubicin under hypoxia. Results It was demonstrated that hypoxia significantly reduced the sensitivity of MG-63 and U2-os cells to doxorubicin, indicating an inhibited viability reduction and a reduced apoptosis promotion. And such reduced sensitivity was not associated with HIF-1α, though it was promoted by hypoxia in U2-os cells. Interestingly, the AMPK signaling was significantly promoted by hypoxia in the doxorubicin-treated U2-os cells, with a marked upregulation of phosphorylated AMPK (Thr 172) and phosphorylated acetyl-CoA carboxylase (ACC) (Ser 79), which were sensitive to the AMPK activator, AICAR and the AMPK inhibitor, Compound C. Moreover, the promoted AMPK activity by AICAR or the downregulated AMPK activity by Compound C significantly reduced or promoted the sensitivity of U2-os cells to doxorubicin. Conclusion The present study confirmed the AMPK signaling activation in the doxorubicin-treated osteosarcoma cells, in response to hypoxia, and the chemical upregulation or downregulation of AMPK signaling reduced or increased the chemo-sensitivity of osteosarcoma U2-os cells in vitro. Our study implies that AMPK inhibition might be a effective strategy to sensitize osteocarcoma cells to chemotherapy. PMID

  17. Biomechanical Signals Inhibit IKK Activity to Attenuate NF-κB Transcription Activity in Inflamed Chondrocytes

    PubMed Central

    Dossumbekova, Anar; Anghelina, Mirela; Madhavan, Shashi; He, Lingli; Quan, Ning; Knobloch, Thomas; Agarwal, Sudha

    2016-01-01

    Objective While the effects of biomechanical signals in the form of joint movement and exercise are known to be beneficial to inflamed joints, limited information is available regarding the intracellular mechanisms of their actions. This study was undertaken to examine the intracellular mechanisms by which biomechanical signals suppress proinflammatory gene induction by the interleukin-1-β (IL-1β)–induced NF-κB signaling cascade in articular chondrocytes. Methods Primary rat articular chondrocytes were exposed to biomechanical signals in the form of cyclic tensile strain, and the effects on the NF-κB signaling cascade were examined by Western blot analysis, real-time polymerase chain reaction, and immunofluorescence. Results Cyclic tensile strain rapidly inhibited the IL-1β–induced nuclear translocation of NF-κB, but not its IL-1β–induced phosphorylation at serine 276 and serine 536, which are necessary for its transactivation and transcriptional efficacy, respectively. Examination of upstream events revealed that cyclic tensile strain also inhibited the cytoplasmic protein degradation of IκBβ and IκBα, as well as repressed their gene transcription. Additionally, cyclic tensile strain induced a rapid nuclear translocation of IκBα to potentially prevent NF-κB binding to DNA. Furthermore, the inhibition of IL-1β–induced degradation of IκB by cyclic tensile strain was mediated by down-regulation of IκB kinase activity. Conclusion These results indicate that the signals generated by cyclic tensile strain act at multiple sites within the NF-κB signaling cascade to inhibit IL-1β–induced proinflammatory gene induction. Taken together, these findings provide insight into how biomechanical signals regulate and reduce inflammation, and underscore their potential in enhancing the ability of chondrocytes to curb inflammation in diseased joints. PMID:17907174

  18. Activation of cGMP-Dependent Protein Kinase Stimulates Cardiac ATP-Sensitive Potassium Channels via a ROS/Calmodulin/CaMKII Signaling Cascade

    PubMed Central

    Chai, Yongping; Zhang, Dai-Min; Lin, Yu-Fung

    2011-01-01

    Background Cyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (KATP) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue. Methods and Findings Single-channel recordings of cardiac KATP channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type KATP) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H2O2 scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H2O2 also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of KATP channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions. Conclusion The present study provides novel evidence that PKG exerts dual regulation of cardiac KATP channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H2O2 in

  19. Signal Activation and Inactivation by the Gα Helical Domain: A Long-Neglected Partner in G Protein Signaling

    PubMed Central

    Dohlman, Henrik G.; Jones, Janice C.

    2013-01-01

    Heterotrimeric guanine nucleotide–binding proteins (G proteins) are positioned at the top of many signal transduction pathways. The G protein α subunit is composed of two domains, one that resembles Ras and another that is composed entirely of α helices. Historically, most attention has focused on the Ras-like domain, but emerging evidence reveals that the helical domain is an active participant in G protein signaling. PMID:22649098

  20. CD28 costimulatory signals in T lymphocyte activation: Emerging functions beyond a qualitative and quantitative support to TCR signalling.

    PubMed

    Porciello, Nicla; Tuosto, Loretta

    2016-04-01

    CD28 is one of the most important co-stimulatory receptors necessary for full T lymphocyte activation. By binding its cognate ligands, B7.1/CD80 or B7.2/CD86, expressed on the surface of professional antigen presenting cells (APC), CD28 initiates several signalling cascades, which qualitatively and quantitatively support T cell receptor (TCR) signalling. More recent data evidenced that human CD28 can also act as a TCR-independent signalling unit, by delivering specific signals, which regulate the expression of pro-inflammatory cytokine/chemokines. Despite the enormous progresses made in identifying the mechanisms and molecules involved in CD28 signalling properties, much remains to be elucidated, especially in the light of the functional differences observed between human and mouse CD28. In this review we provide an overview of the current mechanisms and molecules through which CD28 support TCR signalling and highlight recent findings on the specific signalling motifs that regulate the unique pro-inflammatory activity of human CD28. PMID:26970725

  1. Akt1 signaling coordinates BMP signaling and β-catenin activity to regulate second heart field progenitor development.

    PubMed

    Luo, Wen; Zhao, Xia; Jin, Hengwei; Tao, Lichan; Zhu, Jingai; Wang, Huijuan; Hemmings, Brian A; Yang, Zhongzhou

    2015-02-15

    Second heart field (SHF) progenitors exhibit continued proliferation and delayed differentiation, which are modulated by FGF4/8/10, BMP and canonical Wnt/β-catenin signaling. PTEN-Akt signaling regulates the stem cell/progenitor cell homeostasis in several systems, such as hematopoietic stem cells, intestinal stem cells and neural progenitor cells. To address whether PTEN-Akt signaling is involved in regulating cardiac progenitors, we deleted Pten in SHF progenitors. Deletion of Pten caused SHF expansion and increased the size of the SHF derivatives, the right ventricle and the outflow tract. Cell proliferation of cardiac progenitors was enhanced, whereas cardiac differentiation was unaffected by Pten deletion. Removal of Akt1 rescued the phenotype and early lethality of Pten deletion mice, suggesting that Akt1 was the key downstream target that was negatively regulated by PTEN in cardiac progenitors. Furthermore, we found that inhibition of FOXO by Akt1 suppressed the expression of the gene encoding the BMP ligand (BMP7), leading to dampened BMP signaling in the hearts of Pten deletion mice. Cardiac activation of Akt also increased the Ser552 phosphorylation of β-catenin, thus enhancing its activity. Reducing β-catenin levels could partially rescue heart defects of Pten deletion mice. We conclude that Akt signaling regulates the cell proliferation of SHF progenitors through coordination of BMP signaling and β-catenin activity. PMID:25670795

  2. Dermatophytes Activate Skin Keratinocytes via Mitogen-Activated Protein Kinase Signaling and Induce Immune Responses

    PubMed Central

    Achterman, Rebecca R.; Moyes, David L.; Thavaraj, Selvam; Smith, Adam R.; Blair, Kris M.

    2015-01-01

    Dermatophytes cause superficial and cutaneous fungal infections in immunocompetent hosts and invasive disease in immunocompromised hosts. However, the host mechanisms that regulate innate immune responses against these fungi are largely unknown. Here, we utilized commercially available epidermal tissues and primary keratinocytes to assess (i) damage induction by anthropophilic, geophilic, and zoophilic dermatophyte strains and (ii) the keratinocyte signaling pathways, transcription factors, and proinflammatory responses induced by a representative dermatophyte, Trichophyton equinum. Initially, five dermatophyte species were tested for their ability to invade, cause tissue damage, and induce cytokines, with Microsporum gypseum inducing the greatest level of damage and cytokine release. Using T. equinum as a representative dermatophyte, we found that the mitogen-activated protein kinase (MAPK) pathways were predominantly affected, with increased levels of phospho-p38 and phospho-Jun N-terminal protein kinase (JNK) but decreased levels of phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2). Notably, the NF-κB and PI3K pathways were largely unaffected. T. equinum also significantly increased expression of the AP-1-associated transcription factor, c-Fos, and the MAPK regulatory phosphatase, MKP1. Importantly, the ability of T. equinum to invade, cause tissue damage, activate signaling and transcription factors, and induce proinflammatory responses correlated with germination, indicating that germination may be important for dermatophyte virulence and host immune activation. PMID:25667269

  3. Sunitinib activates Axl signaling in renal cell cancer.

    PubMed

    van der Mijn, Johannes C; Broxterman, Henk J; Knol, Jaco C; Piersma, Sander R; De Haas, Richard R; Dekker, Henk; Pham, Thang V; Van Beusechem, Victor W; Halmos, Balazs; Mier, James W; Jiménez, Connie R; Verheul, Henk M W

    2016-06-15

    Mass spectrometry-based phosphoproteomics provides a unique unbiased approach to evaluate signaling network in cancer cells. The tyrosine kinase inhibitor sunitinib is registered as treatment for patients with renal cell cancer (RCC). We investigated the effect of sunitinib on tyrosine phosphorylation in RCC tumor cells to get more insight in its mechanism of action and thereby to find potential leads for combination treatment strategies. Sunitinib inhibitory concentrations of proliferation (IC50) of 786-O, 769-p and A498 RCC cells were determined by MTT-assays. Global tyrosine phosphorylation was measured by LC-MS/MS after immunoprecipitation with the antiphosphotyrosine antibody p-TYR-100. Phosphoproteomic profiling of 786-O cells yielded 1519 phosphopeptides, corresponding to 675 unique proteins including 57 different phosphorylated protein kinases. Compared to control, incubation with sunitinib at its IC50 of 2 µM resulted in downregulation of 86 phosphopeptides including CDK5, DYRK3, DYRK4, G6PD, PKM and LDH-A, while 94 phosphopeptides including Axl, FAK, EPHA2 and p38α were upregulated. Axl- (y702), FAK- (y576) and p38α (y182) upregulation was confirmed by Western Blot in 786-O and A498 cells. Subsequent proliferation assays revealed that inhibition of Axl with a small molecule inhibitor (R428) sensitized 786-O RCC cells and immortalized endothelial cells to sunitinib up to 3 fold. In conclusion, incubation with sunitinib of RCC cells causes significant upregulation of multiple phosphopeptides including Axl. Simultaneous inhibition of Axl improves the antitumor activity of sunitinib. We envision that evaluation of phosphoproteomic changes by TKI treatment enables identification of new targets for combination treatment strategies. PMID:26815723

  4. Conformational transition in signal transduction: metastable states and transition pathways in the activation of a signaling protein.

    PubMed

    Banerjee, Rahul; Yan, Honggao; Cukier, Robert I

    2015-06-01

    Signal transduction is of vital importance to the growth and adaptation of living organisms. The key to understand mechanisms of biological signal transduction is elucidation of the conformational dynamics of its signaling proteins, as the activation of a signaling protein is fundamentally a process of conformational transition from an inactive to an active state. A predominant form of signal transduction for bacterial sensing of environmental changes in the wild or inside their hosts is a variety of two-component systems, in which the conformational transition of a response regulator (RR) from an inactive to an active state initiates responses to the environmental changes. Here, RR activation has been investigated using RR468 as a model system by extensive unbiased all-atom molecular dynamics (MD) simulations in explicit solvent, starting from snapshots along a targeted MD trajectory that covers the conformational transition. Markov state modeling, transition path theory, and geometric analyses of the wealth of the MD data have provided a comprehensive description of the RR activation. It involves a network of metastable states, with one metastable state essentially the same as the inactive state and another very similar to the active state that are connected via a small set of intermediates. Five major pathways account for >75% of the fluxes of the conformational transition from the inactive to the active-like state. The thermodynamic stability of the states and the activation barriers between states are found, to identify rate-limiting steps. The conformal transition is initiated predominantly by movements of the β3α3 loop, followed by movements of the β4α4-loop and neighboring α4 helix region, and capped by additional movements of the β3α3 loop. A number of transient hydrophobic and hydrogen bond interactions are revealed, and they may be important for the conformational transition. PMID:25945797

  5. Linking ligand perception by PEPR pattern recognition receptors to cytosolic Ca2+ elevation and downstream immune signaling in plants

    PubMed Central

    Walker, Robin K.; Zhao, Yichen; Berkowitz, Gerald A.

    2012-01-01

    Little is known about molecular steps linking perception of pathogen invasion by cell surface sentry proteins acting as pattern recognition receptors (PRRs) to downstream cytosolic Ca2+ elevation, a critical step in plant immune signaling cascades. Some PRRs recognize molecules (such as flagellin) associated with microbial pathogens (pathogen-associated molecular patterns, PAMPs), whereas others bind endogenous plant compounds (damage-associated molecular patterns, DAMPs) such as peptides released from cells upon attack. This work focuses on the Arabidopsis DAMPs plant elicitor peptides (Peps) and their receptors, PEPR1 and PEPR2. Pep application causes in vivo cGMP generation and downstream signaling that is lost when the predicted PEPR receptor guanylyl cyclase (GC) active site is mutated. Pep-induced Ca2+ elevation is attributable to cGMP activation of a Ca2+ channel. Some differences were identified between Pep/PEPR signaling and the Ca2+-dependent immune signaling initiated by the flagellin peptide flg22 and its cognate receptor Flagellin-sensing 2 (FLS2). FLS2 signaling may have a greater requirement for intracellular Ca2+ stores and inositol phosphate signaling, whereas Pep/PEPR signaling requires extracellular Ca2+. Maximal FLS2 signaling requires a functional Pep/PEPR system. This dependence was evidenced as a requirement for functional PEPR receptors for maximal flg22-dependent Ca2+ elevation, H2O2 generation, defense gene [WRKY33 and Plant Defensin 1.2 (PDF1.2)] expression, and flg22/FLS2-dependent impairment of pathogen growth. In a corresponding fashion, FLS2 loss of function impaired Pep signaling. In addition, a role for PAMP and DAMP perception in bolstering effector-triggered immunity (ETI) is reported; loss of function of either FLS2 or PEPR receptors impaired the hypersensitive response (HR) to an avirulent pathogen. PMID:23150556

  6. Calcium signaling and the secretory activity of bile duct epithelia.

    PubMed

    Amaya, Maria Jimena; Nathanson, Michael H

    2014-06-01

    Cytosolic calcium (Cai(2+)) is a second messenger that is important for the regulation of secretion in many types of tissues. Bile duct epithelial cells, or cholangiocytes, are polarized epithelia that line the biliary tree in liver and are responsible for secretion of bicarbonate and other solutes into bile. Cai(2+) signaling plays an important role in the regulation of secretion by cholangiocytes, and this review discusses the machinery involved in the formation of Ca(2+) signals in cholangiocytes, along with the evidence that these signals regulate ductular secretion. Finally, this review discusses the evidence that impairments in cholangiocyte Ca(2+) signaling play a primary role in the pathogenesis of cholestatic disorders, in which hepatic bile secretion is impaired. PMID:24612866

  7. Ammonium Activates Ouabain-Activated Signalling Pathway in Astrocytes: Therapeutic Potential of Ouabain Antagonist

    PubMed Central

    Song, Dan; Du, Ting

    2014-01-01

    The causal role of ammonium in hepatic encephalopathy was identified in 1930s. Astroglial cells are primary cellular elements of hepatic encephalopathy which conceptually, can be considered a toxic astrogliopathology. Previously we have reported that acute exposure to ammonium activated ouabain/Na,K-ATPase signalling pathway, which includes Src, EGF receptor, Raf, Ras, MEK and ERK1/2. Chronic incubation of astrocytes with ammonium increased production of endogenous ouabain-like compound. Ouabain antagonist canrenone abolished effects of ammonium on astrocytic swelling, ROS production, and upregulation of gene expression and function of TRPC1 and Cav1.2. However, ammonium induces multiple pathological modifications in astrocytes, and some of them may be not related to this signalling pathway. In this review, we focus on the effect of ammonium on ouabain/Na,K-ATPase signalling pathway and its involvement in ammonium-induced ROS production, cell swelling and aberration of Ca2+ signals in astrocytes. We also briefly discuss Na,K-ATPase, EGF receptor, endogenous ouabain and ouabain antagonist. PMID:25342941

  8. Reduced NOS3 phosphorylation mediates reduced NO/cGMP signaling in mesenteric arteries of deoxycorticosterone acetate-salt hypertensive rats.

    PubMed

    Sasser, Jennifer M; Sullivan, Jennifer C; Elmarakby, Ahmed A; Kemp, Bruce E; Pollock, David M; Pollock, Jennifer S

    2004-05-01

    Salt-sensitive hypertension is associated with impaired NO/cGMP signaling. We hypothesized that increased superoxide production by NADPH oxidase and altered endothelial NO synthase (NOS3) phosphorylation determine endothelial dysfunction in hypertension. Experiments tested if NO/cGMP signaling and NOS3 serine phosphorylation are decreased and NADPH oxidase activity is increased in mesenteric arteries from deoxycorticosterone acetate (DOCA)-salt rats compared with arteries from placebo rats. Concentration response curves to phenylephrine were performed in mesenteric arteries in the presence and absence of Nomega-nitro-L-arginine (LNA) and antioxidants to determine the influence of basal NO and superoxide production on vascular tone. LNA increased phenylephrine sensitivity in arteries from placebo, but not DOCA-salt rats, regardless of antioxidant treatment. To determine basal cGMP production, mesenteric arteries were incubated with 3-isobutyl-1-methylxanthine in the presence or absence of LNA, sodium nitroprusside (SNP), antioxidants, or tetrahydrobiopterin. NOS-dependent cGMP production was reduced in arteries from DOCA-salt rats compared with arteries from placebo rats and was not restored by acute treatment with antioxidants or tetrahydrobiopterin. SNP-induced cGMP production was similar between groups as was NADPH oxidase activity, measured by lucigenin chemiluminescence, in mesenteric arteries. Expression and phosphorylation of NOS3 were examined by Western blotting. Phosphorylation of NOS3 was decreased in arteries from DOCA-salt rats compared with placebo at serine residues 1179 and 635. These findings indicate that diminished NO/cGMP signaling in mesenteric arteries from DOCA-salt rats is caused by reduced phosphorylation of NOS3 at serine 1179 and serine 635, rather than NO scavenging by superoxide. PMID:14993198

  9. New Constitutively Active Phytochromes Exhibit Light-Independent Signaling Activity1[OPEN

    PubMed Central

    Jeong, A-Reum; Lee, Si-Seok; Han, Yun-Jeong; Shin, Ah-Young; Baek, Ayoung; Ahn, Taeho; Kim, Min-Gon; Kim, Young Soon; Lee, Keun Woo; Nagatani, Akira

    2016-01-01

    Plant phytochromes are photoreceptors that mediate a variety of photomorphogenic responses. There are two spectral photoisomers, the red light-absorbing Pr and far-red light-absorbing Pfr forms, and the photoreversible transformation between the two forms is important for the functioning of phytochromes. In this study, we isolated a Tyr-268-to-Val mutant of Avena sativa phytochrome A (AsYVA) that displayed little photoconversion. Interestingly, transgenic plants of AsYVA showed light-independent phytochrome signaling with a constitutive photomorphogenic (cop) phenotype that is characterized by shortened hypocotyls and open cotyledons in the dark. In addition, the corresponding Tyr-303-to-Val mutant of Arabidopsis (Arabidopsis thaliana) phytochrome B (AtYVB) exhibited nuclear localization and interaction with phytochrome-interacting factor 3 (PIF3) independently of light, conferring a constitutive photomorphogenic development to its transgenic plants, which is comparable to the first constitutively active version of phytochrome B (YHB; Tyr-276-to-His mutant). We also found that chromophore ligation was required for the light-independent interaction of AtYVB with PIF3. Moreover, we demonstrated that AtYVB did not exhibit phytochrome B activity when it was localized in the cytosol by fusion with the nuclear export signal and that AsYVA exhibited the full activity of phytochrome A when localized in the nucleus by fusion with the nuclear localization signal. Furthermore, the corresponding Tyr-269-to-Val mutant of Arabidopsis phytochrome A (AtYVA) exhibited similar cop phenotypes in transgenic plants to AsYVA. Collectively, these results suggest that the conserved Tyr residues in the chromophore-binding pocket play an important role during the Pr-to-Pfr photoconversion of phytochromes, providing new constitutively active alleles of phytochromes by the Tyr-to-Val mutation. PMID:27325667

  10. Notch Signaling Activation in Pediatric Low-Grade Astrocytoma

    PubMed Central

    Brandt, William D.; Schreck, Karisa C.; Bar, Eli E.; Taylor, Isabella; Marchionni, Luigi; Raabe, Eric; Eberhart, Charles G.; Rodriguez, Fausto J.

    2014-01-01

    Pilocytic astrocytoma (PA) is the most common primary brain tumor in children; various signaling pathways have been implicated in its biology. The Notch signaling pathway has been found to play a role in development, stem cell biology, and the pathogenesis of several cancers but its role in PA has not been investigated. We studied alterations in Notch signaling components in tumor tissue from 18 patients with PA and 4 with other low-grade astrocytomas to identify much needed therapeutic targets. We found that Notch pathway members were overexpressed at the mRNA (NOTCH1, NOTCH2, HEY1, HEY2) and protein (HES1) levels in PAs at various anatomical sites compared to non-neoplastic brain samples. These changes were not associated with specific BRAF alterations. Inhibiting the Notch pathway in the pediatric low-grade astrocytoma cell lines Res 186 and Res 259 using either RNA interference or a γ-secretase inhibitor resulted in variable but significant reduction in cell growth and migration. This study suggests a potential role for Notch signaling in pediatric low-grade astrocytoma tumorigenesis and that Notch signaling may be a viable pathway therapeutic target. PMID:25575134

  11. Notch signaling activation in pediatric low-grade astrocytoma.

    PubMed

    Brandt, William D; Schreck, Karisa C; Bar, Eli E; Taylor, Isabella; Marchionni, Luigi; Raabe, Eric; Eberhart, Charles G; Rodriguez, Fausto J

    2015-02-01

    Pilocytic astrocytoma (PA) is the most common primary brain tumor in children; various signaling pathways have been implicated in its biology. The Notch signaling pathway has been found to play a role in the development, stem cell biology, and pathogenesis of several cancers, but its role in PA has not been investigated. We studied alterations in Notch signaling components in tumor tissue from 18 patients with PA and 4 with other low-grade astrocytomas to identify much needed therapeutic targets. We found that Notch pathway members were overexpressed at the mRNA (NOTCH1, NOTCH2, HEY1, HEY2) and protein (HES1) levels in PAs at various anatomic sites compared with non-neoplastic brain samples. These changes were not associated with specific BRAF alterations. Inhibiting the Notch pathway in the pediatric low-grade astrocytoma cell lines Res186 and Res259 using either RNA interference or a γ-secretase inhibitor resulted in variable, but significant, reduction in cell growth and migration. This study suggests a potential role for Notch signaling in pediatric low-grade astrocytoma tumorigenesis and that Notch signaling may be a viable pathway therapeutic target. PMID:25575134

  12. Nitrated cyclic GMP modulates guard cell signaling in Arabidopsis.

    PubMed

    Joudoi, Takahiro; Shichiri, Yudai; Kamizono, Nobuto; Akaike, Takaaki; Sawa, Tomohiro; Yoshitake, Jun; Yamada, Naotaka; Iwai, Sumio

    2013-02-01

    Nitric oxide (NO) is a ubiquitous signaling molecule involved in diverse physiological processes, including plant senescence and stomatal closure. The NO and cyclic GMP (cGMP) cascade is the main NO signaling pathway in animals, but whether this pathway operates in plant cells, and the mechanisms of its action, remain unclear. Here, we assessed the possibility that the nitrated cGMP derivative 8-nitro-cGMP functions in guard cell signaling. Mass spectrometry and immunocytochemical analyses showed that abscisic acid and NO induced the synthesis of 8-nitro-cGMP in guard cells in the presence of reactive oxygen species. 8-Nitro-cGMP triggered stomatal closure, but 8-bromoguanosine 3',5'-cyclic monophosphate (8-bromo-cGMP), a membrane-permeating analog of cGMP, did not. However, in the dark, 8-bromo-cGMP induced stomatal opening but 8-nitro-cGMP did not. Thus, cGMP and its nitrated derivative play different roles in the signaling pathways that lead to stomatal opening and closure. Moreover, inhibitor and genetic studies showed that calcium, cyclic adenosine-5'-diphosphate-ribose, and SLOW ANION CHANNEL1 act downstream of 8-nitro-cGMP. This study therefore demonstrates that 8-nitro-cGMP acts as a guard cell signaling molecule and that a NO/8-nitro-cGMP signaling cascade operates in guard cells. PMID:23396828

  13. GSK621 Targets Glioma Cells via Activating AMP-Activated Protein Kinase Signalings

    PubMed Central

    Jiang, Hong; Liu, Wei; Zhan, Shi-Kun; Pan, Yi-Xin; Bian, Liu-Guan; Sun, Bomin; Sun, Qing-Fang; Pan, Si-Jian

    2016-01-01

    Here, we studied the anti-glioma cell activity by a novel AMP-activated protein kinase (AMPK) activator GSK621. We showed that GSK621 was cytotoxic to human glioma cells (U87MG and U251MG lines), possibly via provoking caspase-dependent apoptotic cell death. Its cytotoxicity was alleviated by caspase inhibitors. GSK621 activated AMPK to inhibit mammalian target of rapamycin (mTOR) and downregulate Tetraspanin 8 (Tspan8) in glioma cells. AMPK inhibition, through shRNA knockdown of AMPKα or introduction of a dominant negative (T172A) AMPKα, almost reversed GSK621-induced AMPK activation, mTOR inhibition and Tspan8 degradation. Consequently, GSK621’s cytotoxicity in glioma cells was also significantly attenuated by AMPKα knockdown or mutation. Further studies showed that GSK621, at a relatively low concentration, significantly potentiated temozolomide (TMZ)’s sensitivity and lethality against glioma cells. We summarized that GSK621 inhibits human glioma cells possibly via activating AMPK signaling. This novel AMPK activator could be a novel and promising anti-glioma cell agent. PMID:27532105

  14. PKCδ activated by c-MET enhances infiltration of human glioblastoma cells through NOTCH2 signaling

    PubMed Central

    Kang, Seok-Gu; Kim, Rae-Kwon; Cui, Yan-Hong; Lee, Hae-June; Kim, Min-Jung; Lee, Jae-Seong; Kim, In-Gyu; Suh, Yongjoon; Lee, Su-Jae

    2016-01-01

    Poor prognosis of glioblastoma (GBM) is attributable to the propensity of tumor cells to infiltrate into the brain parenchyma. Protein kinase C (PKC) isozymes are highly expressed or aberrantly activated in GBM. However, how this signaling node translates to GBM cell invasiveness remains unknown. Here, we report that among PKC isoforms, PKCδ is strongly associated with infiltration of GBM cells. Notably, PKCδ enhanced Tyr418 phosphorylation of the non-receptor tyrosine kinase SRC, which in turn activated STAT3 and subsequent NOTCH2 signaling, ultimately leading to GBM cell invasiveness. Furthermore, we showed that PKCδ was aberrantly activated in GBM cells by c-MET, a receptor tyrosine kinase hyperactivated in GBM. In agreement, inhibition either component in the c-MET/PKCδ/SRC/STAT3 signaling axis effectively blocked the NOTCH2 signaling and invasiveness of GBM cells. Taken together, our findings shed a light on the signaling mechanisms behind the constitutive activation of PKCδ signaling in GBM. PMID:26700818

  15. Molecular Mechanisms of Gonadotropin-Releasing Hormone Signaling: Integrating Cyclic Nucleotides into the Network

    PubMed Central

    Perrett, Rebecca M.; McArdle, Craig A.

    2013-01-01

    Gonadotropin-releasing hormone (GnRH) is the primary regulator of mammalian reproductive function in both males and females. It acts via G-protein coupled receptors on gonadotropes to stimulate synthesis and secretion of the gonadotropin hormones luteinizing hormone and follicle-stimulating hormone. These receptors couple primarily via G-proteins of the Gq/ll family, driving activation of phospholipases C and mediating GnRH effects on gonadotropin synthesis and secretion. There is also good evidence that GnRH causes activation of other heterotrimeric G-proteins (Gs and Gi) with consequent effects on cyclic AMP production, as well as for effects on the soluble and particulate guanylyl cyclases that generate cGMP. Here we provide an overview of these pathways. We emphasize mechanisms underpinning pulsatile hormone signaling and the possible interplay of GnRH and autocrine or paracrine regulatory mechanisms in control of cyclic nucleotide signaling. PMID:24312080

  16. Cell signaling through protein kinase C oxidation and activation.

    PubMed

    Cosentino-Gomes, Daniela; Rocco-Machado, Nathália; Meyer-Fernandes, José Roberto

    2012-01-01

    Due to the growing importance of cellular signaling mediated by reactive oxygen species (ROS), proteins that are reversibly modulated by these reactant molecules are of high interest. In this context, protein kinases and phosphatases, which act coordinately in the regulation of signal transduction through the phosphorylation and dephosphorylation of target proteins, have been described to be key elements in ROS-mediated signaling events. The major mechanism by which these proteins may be modified by oxidation involves the presence of key redox-sensitive cysteine residues. Protein kinase C (PKC) is involved in a variety of cellular signaling pathways. These proteins have been shown to contain a unique structural feature that is susceptible to oxidative modification. A large number of scientific studies have highlighted the importance of ROS as a second messenger in numerous cellular processes, including cell proliferation, gene expression, adhesion, differentiation, senescence, and apoptosis. In this context, the goal of this review is to discuss the mechanisms by which PKCs are modulated by ROS and how these processes are involved in the cellular response. PMID:23109817

  17. Now that you want to take your HIV/AIDS vaccine/biological product research concept into the clinic: what are the "cGMP"?

    PubMed

    Sheets, Rebecca L; Rangavajhula, Vijaya; Pullen, Jeffrey K; Butler, Chris; Mehra, Vijay; Shapiro, Stuart; Pensiero, Michael

    2015-04-01

    The Division of AIDS Vaccine Research Program funds the discovery and development of HIV/AIDS vaccine candidates. Basic researchers, having discovered a potential vaccine in the laboratory, next want to take that candidate into the clinic to test the concept in humans, to see if it translates. Many of them have heard of "cGMP" and know that they are supposed to make a "GMP product" to take into the clinic, but often they are not very familiar with what "cGMP" means and why these good practices are so important. As members of the Vaccine Translational Research Branch, we frequently get asked "can't we use the material we made in the lab in the clinic?" or "aren't Phase 1 studies exempt from cGMP?" Over the years, we have had many experiences where researchers or their selected contract manufacturing organizations have not applied an appropriate degree of compliance with cGMP suitable for the clinical phase of development. We share some of these experiences and the lessons learned, along with explaining the importance of cGMP, just what cGMP means, and what they can assure, in an effort to de-mystify this subject and facilitate the rapid and safe translational development of HIV vaccines. PMID:25698494

  18. Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells

    PubMed Central

    Sattu, Kamaraj; Hochgräfe, Falko; Wu, Jianmin; Umapathy, Ganesh; Schönherr, Christina; Ruuth, Kristina; Chand, Damini; Witek, Barbara; Fuchs, James; Li, Pui-Kai; Hugosson, Fredrik; Daly, Roger J; Palmer, Ruth H; Hallberg, Bengt

    2013-01-01

    Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin–ALK and echinoderm microtubule-associated protein-like 4–ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS-based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full-length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription 3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full-length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma. PMID:23889739

  19. AMP-activated Protein Kinase Signaling Activation by Resveratrol Modulates Amyloid-β Peptide Metabolism*

    PubMed Central

    Vingtdeux, Valérie; Giliberto, Luca; Zhao, Haitian; Chandakkar, Pallavi; Wu, Qingli; Simon, James E.; Janle, Elsa M.; Lobo, Jessica; Ferruzzi, Mario G.; Davies, Peter; Marambaud, Philippe

    2010-01-01

    Alzheimer disease is an age-related neurodegenerative disorder characterized by amyloid-β (Aβ) peptide deposition into cerebral amyloid plaques. The natural polyphenol resveratrol promotes anti-aging pathways via the activation of several metabolic sensors, including the AMP-activated protein kinase (AMPK). Resveratrol also lowers Aβ levels in cell lines; however, the underlying mechanism responsible for this effect is largely unknown. Moreover, the bioavailability of resveratrol in the brain remains uncertain. Here we show that AMPK signaling controls Aβ metabolism and mediates the anti-amyloidogenic effect of resveratrol in non-neuronal and neuronal cells, including in mouse primary neurons. Resveratrol increased cytosolic calcium levels and promoted AMPK activation by the calcium/calmodulin-dependent protein kinase kinase-β. Direct pharmacological and genetic activation of AMPK lowered extracellular Aβ accumulation, whereas AMPK inhibition reduced the effect of resveratrol on Aβ levels. Furthermore, resveratrol inhibited the AMPK target mTOR (mammalian target of rapamycin) to trigger autophagy and lysosomal degradation of Aβ. Finally, orally administered resveratrol in mice was detected in the brain where it activated AMPK and reduced cerebral Aβ levels and deposition in the cortex. These data suggest that resveratrol and pharmacological activation of AMPK have therapeutic potential against Alzheimer disease. PMID:20080969

  20. Kurarinol induces hepatocellular carcinoma cell apoptosis through suppressing cellular signal transducer and activator of transcription 3 signaling

    SciTech Connect

    Shu, Guangwen; Yang, Jing; Zhao, Wenhao; Xu, Chan; Hong, Zongguo; Mei, Zhinan; Yang, Xinzhou

    2014-12-01

    Kurarinol is a flavonoid isolated from roots of the medical plant Sophora flavescens. However, its cytotoxic activity against hepatocellular carcinoma (HCC) cells and toxic effects on mammalians remain largely unexplored. Here, the pro-apoptotic activities of kurarinol on HCC cells and its toxic impacts on tumor-bearing mice were evaluated. The molecular mechanisms underlying kurarinol-induced HCC cell apoptosis were also investigated. We found that kurarinol dose-dependently provoked HepG2, Huh-7 and H22 HCC cell apoptosis. In addition, kurarinol gave rise to a considerable decrease in the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) in HCC cells. Suppression of STAT3 signaling is involved in kurarinol-induced HCC cell apoptosis. In vivo studies showed that kurarinol injection substantially induced transplanted H22 cell apoptosis with low toxic impacts on tumor-bearing mice. Similarly, the transcriptional activity of STAT3 in transplanted tumor tissues was significantly suppressed after kurarinol treatment. Collectively, our current research demonstrated that kurarinol has the capacity of inducing HCC cell apoptosis both in vitro and in vivo with undetectable toxic impacts on the host. Suppressing STAT3 signaling is implicated in kurarinol-mediated HCC cell apoptosis. - Highlights: • Kurarinol induces hepatocellular carcinoma (HCC) cell apoptosis. • Kurarinol induces HCC cell apoptosis via inhibiting STAT3. • Kurarinol exhibits low toxic effects on tumor-bearing animals.

  1. Purine 3':5'-cyclic nucleotides with the nucleobase in a syn orientation: cAMP, cGMP and cIMP.

    PubMed

    Řlepokura, Katarzyna Anna

    2016-06-01

    Purine 3':5'-cyclic nucleotides are very well known for their role as the secondary messengers in hormone action and cellular signal transduction. Nonetheless, their solid-state conformational details still require investigation. Five crystals containing purine 3':5'-cyclic nucleotides have been obtained and structurally characterized, namely adenosine 3':5'-cyclic phosphate dihydrate, C10H12N5O6P·2H2O or cAMP·2H2O, (I), adenosine 3':5'-cyclic phosphate 0.3-hydrate, C10H12N5O6P·0.3H2O or cAMP·0.3H2O, (II), guanosine 3':5'-cyclic phosphate pentahydrate, C10H12N5O7P·5H2O or cGMP·5H2O, (III), sodium guanosine 3':5'-cyclic phosphate tetrahydrate, Na(+)·C10H11N5O7P(-)·4H2O or Na(cGMP)·4H2O, (IV), and sodium inosine 3':5'-cyclic phosphate tetrahydrate, Na(+)·C10H10N4O7P(-)·4H2O or Na(cIMP)·4H2O, (V). Most of the cyclic nucleotide zwitterions/anions [two from four cAMP present in total in (I) and (II), cGMP in (III), cGMP(-) in (IV) and cIMP(-) in (V)] are syn conformers about the N-glycosidic bond, and this nucleobase arrangement is accompanied by Crib-H...Npur hydrogen bonds (rib = ribose and pur = purine). The base orientation is tuned by the ribose pucker. An analysis of data obtained from the Cambridge Structural Database made in the context of syn-anti conformational preferences has revealed that among the syn conformers of various purine nucleotides, cyclic nucleotides and dinucleotides predominate significantly. The interactions stabilizing the syn conformation have been indicated. The inter-nucleotide contacts in (I)-(V) have been systematized in terms of the chemical groups involved. All five structures display three-dimensional hydrogen-bonded networks. PMID:27256694

  2. The impact of cGMP compliance on consumer confidence in dietary supplement products.

    PubMed

    Crowley, Richard; FitzGerald, Libby Harvey

    2006-04-01

    The FDA estimates that US citizens spend more than $ 8.5 billion a year on dietary supplements and world wide the market is estimated at more than $ 60 billion. However, although a majority of consumers express confidence in the safety of these products, 74% believe the government should be more involved in ensuring that these products are safe and efficacious. Recent regulatory initiatives such as the imminent adoption of cGMPs for dietary supplements in the US, implementation of cGMPs in Canada and the recent EU dietary supplement initiative represent legislative and industry response to public clamor for more comprehensive oversight of dietary supplements. Regardless of mandated practices, the majority of dietary supplement manufacturers have done an excellent job of protecting the safety and quality of their products. The promulgation of these cGMPs will help ensure consumers that equal standards are followed throughout the industry. For some companies with established processes based on existing food or pharmaceutical cGMP regulations, the transition will be relatively painless while, for many, it will represent a significant increase in the level of documentation and testing. However, consumers deserve and demand that products meet standards for safety and quality and the implementation of cGMPs for these products are an important first step. Although the cGMPs are designed to ensure products are safe from a standpoint of identity, purity, quality, strength and composition, they do not address preclinical or clinical testing of ingredients for safety or efficacy. This would involve ingredients meeting the requirements of Generally Recognized as Safe (GRAS) status or going through the New Dietary Ingredient (NDI) process. PMID:16469425

  3. Msx2 promotes cardiovascular calcification by activating paracrine Wnt signals.

    PubMed

    Shao, Jian-Su; Cheng, Su-Li; Pingsterhaus, Joyce M; Charlton-Kachigian, Nichole; Loewy, Arleen P; Towler, Dwight A

    2005-05-01

    In diabetic LDLR-/- mice, an ectopic BMP2-Msx2 gene regulatory program is upregulated in association with vascular calcification. We verified the procalcific actions of aortic Msx2 expression in vivo. CMV-Msx2 transgenic (CMV-Msx2Tg(+)) mice expressed 3-fold higher levels of aortic Msx2 than nontransgenic littermates. On high-fat diets, CMV-Msx2Tg(+) mice exhibited marked cardiovascular calcification involving aortic and coronary tunica media. This corresponded to regions of Msx2 immunoreactivity in adjacent adventitial myofibroblasts, suggesting a potential paracrine osteogenic signal. To better understand Msx2-regulated calcification, we studied actions in 10T1/2 cells. We found that conditioned media from Msx2-transduced 10T1/2 cells (Msx2-CM) is both pro-osteogenic and adipostatic; these features are characteristic of Wnt signaling. Msx2-CM stimulated Wnt-dependent TCF/LEF transcription, and Msx2-transduced cells exhibited increased nuclear beta-catenin localization with concomitant alkaline phosphatase induction. Msx2 upregulated Wnt3a and Wnt7a but downregulated expression of the canonical inhibitor Dkk1. Dkk1 treatment reversed osteogenic and adipostatic actions of Msx2. Teriparatide, a PTH1R agonist that inhibits murine vascular calcification, suppressed vascular BMP2-Msx2-Wnt signaling. Analyses of CMV-Msx2Tg(+) mice confirmed that Msx2 suppresses aortic Dkk1 and upregulates vascular Wnts; moreover, TOPGAL(+) (Wnt reporter); CMV-Msx2Tg(+) mice exhibited augmented aortic LacZ expression. Thus, Msx2-expressing cells elaborated an osteogenic milieu that promotes vascular calcification in part via paracrine Wnt signals. PMID:15841209

  4. Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling

    PubMed Central

    Alagramam, Kumar N.; Stepanyan, Ruben; Jamesdaniel, Samson; Chen, Daniel H.-C.; Davis, Rickie R.

    2015-01-01

    Noise-induced hearing loss (NIHL) is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL) broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4–8-week-old CBA/CaJ mice were exposed to no noise (control) or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK) signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b, Cacna1g, and Pla2g6, related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway. PMID:25387536

  5. LAF1, a MYB transcription activator for phytochrome A signaling

    PubMed Central

    Ballesteros, María L.; Bolle, Cordelia; Lois, Luisa M.; Moore, James M.; Vielle-Calzada, Jean-Philippe; Grossniklaus, Ueli; Chua, Nam-Hai

    2001-01-01

    The photoreceptor phytochrome (phy) A has a well-defined role in regulating gene expression in response to specific light signals. Here, we describe a new Arabidopsis mutant, laf1 (long after far-red light 1) that has an elongated hypocotyl specifically under far-red light. Gene expression studies showed that laf1 has reduced responsiveness to continuous far-red light but retains wild-type responses to other light wavelengths. As far-red light is only perceived by phyA, our results suggest that LAF1 is specifically involved in phyA signal transduction. Further analyses revealed that laf1 is affected in a subset of phyA-dependent responses and the phenotype is more severe at low far-red fluence rates. LAF1 encodes a nuclear protein with strong homology with the R2R3–MYB family of DNA-binding proteins. Experiments using yeast cells identified a transactivation domain in the C-terminal portion of the protein. LAF1 is constitutively targeted to the nucleus by signals in its N-terminal portion, and the full-length protein accumulates in distinct nuclear speckles. This accumulation in speckles is abolished by a point mutation in a lysine residue (K258R), which might serve as a modification site by a small ubiquitin-like protein (SUMO). PMID:11581165

  6. Signaling-mediated cooperativity between glycoprotein Ib-IX and protease-activated receptors in thrombin-induced platelet activation.

    PubMed

    Estevez, Brian; Kim, Kyungho; Delaney, M Keegan; Stojanovic-Terpo, Aleksandra; Shen, Bo; Ruan, Changgeng; Cho, Jaehyung; Ruggeri, Zaverio M; Du, Xiaoping

    2016-02-01

    Thrombin-induced cellular response in platelets not only requires protease-activated receptors (PARs), but also involves another thrombin receptor, the glycoprotein Ib-IX complex (GPIb-IX). It remains controversial how thrombin binding to GPIb-IX stimulates platelet responses. It was proposed that GPIb-IX serves as a dock that facilitates thrombin cleavage of protease-activated receptors, but there are also reports suggesting that thrombin binding to GPIb-IX induces platelet activation independent of PARs. Here we show that GPIb is neither a passive thrombin dock nor a PAR-independent signaling receptor. We demonstrate a novel signaling-mediated cooperativity between PARs and GPIb-IX. Low-dose thrombin-induced PAR-dependent cell responses require the cooperativity of GPIb-IX signaling, and conversely, thrombin-induced GPIb-IX signaling requires cooperativity of PARs. This mutually dependent cooperativity requires a GPIb-IX-specific 14-3-3-Rac1-LIMK1 signaling pathway, and activation of this pathway also requires PAR signaling. The cooperativity between GPIb-IX signaling and PAR signaling thus drives platelet activation at low concentrations of thrombin, which are important for in vivo thrombosis. PMID:26585954

  7. Mitochondrial Oxidative Stress Corrupts Coronary Collateral Growth by Activating Adenosine Monophosphate Activated Kinase-α Signaling

    PubMed Central

    Pung, Yuh Fen; Sam, Wai Johnn; Stevanov, Kelly; Enrick, Molly; Chen, Chwen-Lih; Kolz, Christopher; Thakker, Prashanth; Hardwick, James P.; Chen, Yeong-Renn; Dyck, Jason R.B.; Yin, Liya; Chilian, William M.

    2015-01-01

    Objective Our goal was to determine the mechanism by which mitochondrial oxidative stress impairs collateral growth in the heart. Approach and Results Rats were treated with rotenone (mitochondrial complex I inhibitor that increases reactive oxygen species production) or sham-treated with vehicle and subjected to repetitive ischemia protocol for 10 days to induce coronary collateral growth. In control rats, repetitive ischemia increased flow to the collateral-dependent zone; however, rotenone treatment prevented this increase suggesting that mitochondrial oxidative stress compromises coronary collateral growth. In addition, rotenone also attenuated mitochondrial complex I activity and led to excessive mitochondrial aggregation. To further understand the mechanistic pathway(s) involved, human coronary artery endothelial cells were treated with 50 ng/ mL vascular endothelial growth factor, 1 µmol/L rotenone, and rotenone/vascular endothelial growth factor for 48 hours. Vascular endothelial growth factor induced robust tube formation; however, rotenone completely inhibited this effect (P<0.05 rotenone versus vascular endothelial growth factor treatment). Inhibition of tube formation by rotenone was also associated with significant increase in mitochondrial superoxide generation. Immunoblot analyses of human coronary artery endothelial cells with rotenone treatment showed significant activation of adenosine monophosphate activated kinase (AMPK)-α and inhibition of mammalian target of rapamycin and p70 ribosomal S6 kinase. Activation of AMPK-α suggested impairments in energy production, which was reflected by decrease in O2 consumption and bioenergetic reserve capacity of cultured cells. Knockdown of AMPK-α (siRNA) also preserved tube formation during rotenone, suggesting the negative effects were mediated by the activation of AMPK-α. Conversely, expression of a constitutively active AMPK-α blocked tube formation. Conclusions We conclude that activation of AMPK

  8. Smoothened regulates activator and repressor functions of Hedgehog signaling via two distinct mechanisms.

    PubMed

    Ogden, Stacey K; Casso, David J; Ascano, Manuel; Yore, Mark M; Kornberg, Thomas B; Robbins, David J

    2006-03-17

    The secreted protein Hedgehog (Hh) plays an important role in metazoan development and as a survival factor for many human tumors. In both cases, Hh signaling proceeds through the activation of the seven-transmembrane protein Smoothened (Smo), which is thought to convert the Gli family of transcription factors from transcriptional repressors to transcriptional activators. Here, we provide evidence that Smo signals to the Hh signaling complex, which consists of the kinesin-related protein Costal2 (Cos2), the protein kinase Fused (Fu), and the Drosophila Gli homolog cubitus interruptus (Ci), in two distinct manners. We show that many of the commonly observed molecular events following Hh signaling are not transmitted in a linear fashion but instead are activated through two signals that bifurcate at Smo to independently affect activator and repressor pools of Ci. PMID:16423832

  9. Prion-like Polymerization Underlies Signal Transduction in Antiviral Immune Defense and Inflammasome Activation

    PubMed Central

    Cai, Xin; Chen, Jueqi; Xu, Hui; Liu, Siqi; Jiang, Qiu-Xing; Halfmann, Randal; Chen, Zhijian J.

    2014-01-01

    SUMMARY Pathogens and cellular danger signals activate sensors such as RIG-I and NLRP3 to produce robust immune and inflammatory responses through respective adaptor proteins MAVS and ASC, which harbor essential N-terminal CARD and PYRIN domains, respectively. Here, we show that CARD and PYRIN function as bona fide prions in yeast and their prion forms are inducible by their respective upstream activators. Likewise, a yeast prion domain can functionally replace CARD and PYRIN in mammalian cell signaling. Mutations in MAVS and ASC that disrupt their prion activities in yeast also abrogate their ability to signal in mammalian cells. Furthermore, fibers of recombinant PYRIN can convert ASC into functional polymers capable of activating caspase-1. Remarkably, a conserved fungal NOD-like receptor and prion pair can functionally reconstitute signaling of NLRP3 and ASC PYRINs in mammalian cells. These results indicate that prion-like polymerization is a conserved signal transduction mechanism in innate immunity and inflammation. PMID:24630723

  10. Integration of light and metabolic signals for stem cell activation at the shoot apical meristem

    PubMed Central

    Pfeiffer, Anne; Janocha, Denis; Dong, Yihan; Medzihradszky, Anna; Schöne, Stefanie; Daum, Gabor; Suzaki, Takuya; Forner, Joachim; Langenecker, Tobias; Rempel, Eugen; Schmid, Markus; Wirtz, Markus; Hell, Rüdiger; Lohmann, Jan U

    2016-01-01

    A major feature of embryogenesis is the specification of stem cell systems, but in contrast to the situation in most animals, plant stem cells remain quiescent until the postembryonic phase of development. Here, we dissect how light and metabolic signals are integrated to overcome stem cell dormancy at the shoot apical meristem. We show on the one hand that light is able to activate expression of the stem cell inducer WUSCHEL independently of photosynthesis and that this likely involves inter-regional cytokinin signaling. Metabolic signals, on the other hand, are transduced to the meristem through activation of the TARGET OF RAPAMYCIN (TOR) kinase. Surprisingly, TOR is also required for light signal dependent stem cell activation. Thus, the TOR kinase acts as a central integrator of light and metabolic signals and a key regulator of stem cell activation at the shoot apex. DOI: http://dx.doi.org/10.7554/eLife.17023.001 PMID:27400267

  11. Integration of light and metabolic signals for stem cell activation at the shoot apical meristem.

    PubMed

    Pfeiffer, Anne; Janocha, Denis; Dong, Yihan; Medzihradszky, Anna; Schöne, Stefanie; Daum, Gabor; Suzaki, Takuya; Forner, Joachim; Langenecker, Tobias; Rempel, Eugen; Schmid, Markus; Wirtz, Markus; Hell, Rüdiger; Lohmann, Jan U

    2016-01-01

    A major feature of embryogenesis is the specification of stem cell systems, but in contrast to the situation in most animals, plant stem cells remain quiescent until the postembryonic phase of development. Here, we dissect how light and metabolic signals are integrated to overcome stem cell dormancy at the shoot apical meristem. We show on the one hand that light is able to activate expression of the stem cell inducer WUSCHEL independently of photosynthesis and that this likely involves inter-regional cytokinin signaling. Metabolic signals, on the other hand, are transduced to the meristem through activation of the TARGET OF RAPAMYCIN (TOR) kinase. Surprisingly, TOR is also required for light signal dependent stem cell activation. Thus, the TOR kinase acts as a central integrator of light and metabolic signals and a key regulator of stem cell activation at the shoot apex. PMID:27400267

  12. High-throughput screening of phosphodiesterase activity in living cells.

    PubMed

    Rich, Thomas C; Karpen, Jeffrey W

    2005-01-01

    Phosphodiesterases (PDEs) hydrolyze the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine 5'-monophosphate (cGMP) and play a crucial role in the termination and spatial segregation of cyclic nucleotide signals. Despite a wealth of molecular information, very little is known about how PDEs regulate cAMP and cGMP signals in living cells because conventional methods lack the necessary spatial and temporal resolution. We present here a sensitive optical method for monitoring cAMP levels and PDE activity near the membrane, using cyclic nucleotide-gated (CNG) ion channels as sensors. These channels are directly opened by the binding of cyclic nucleotides and allow cations to cross the membrane. The olfactory channel A subunit (CNGA2) has been genetically modified to improve its cAMP sensitivity and specificity. Channel activity is assessed by measuring Ca2+ influx using standard fluorometric techniques. In addition to studying PDEs in their native setting, the approach should be particularly useful in high-throughput screening assays to test for compounds that affect PDE activity, as well as the activities of the many G protein-coupled receptors that cause changes in intracellular cAMP. PMID:15988054

  13. Modular activation of Rho1 by GPCR signalling imparts polarized myosin II activation during morphogenesis.

    PubMed

    Kerridge, Stephen; Munjal, Akankshi; Philippe, Jean-Marc; Jha, Ankita; de las Bayonas, Alain Garcia; Saurin, Andrew J; Lecuit, Thomas

    2016-03-01

    Polarized cell shape changes during tissue morphogenesis arise by controlling the subcellular distribution of myosin II. For instance, during Drosophila melanogaster gastrulation, apical constriction and cell intercalation are mediated by medial-apical myosin II pulses that power deformations, and polarized accumulation of myosin II that stabilizes these deformations. It remains unclear how tissue-specific factors control different patterns of myosin II activation and the ratchet-like myosin II dynamics. Here we report the function of a common pathway comprising the heterotrimeric G proteins Gα12/13, Gβ13F and Gγ1 in activating and polarizing myosin II during Drosophila gastrulation. Gα12/13 and the Gβ13F/γ1 complex constitute distinct signalling modules, which regulate myosin II dynamics medial-apically and/or junctionally in a tissue-dependent manner. We identify a ubiquitously expressed GPCR called Smog required for cell intercalation and apical constriction. Smog functions with other GPCRs to quantitatively control G proteins, resulting in stepwise activation of myosin II and irreversible cell shape changes. We propose that GPCR and G proteins constitute a general pathway for controlling actomyosin contractility in epithelia and that the activity of this pathway is polarized by tissue-specific regulators. PMID:26780298

  14. Angiogenic activity of sesamin through the activation of multiple signal pathways

    SciTech Connect

    Chung, Byung-Hee; Lee, Jung Joon; Kim, Jong-Dai; Jeoung, Dooil; Lee, Hansoo; Choe, Jongseon; Ha, Kwon-Soo; Kwon, Young-Geun; Kim, Young-Myeong

    2010-01-01

    The natural product sesamin has been known to act as a potent antioxidant and prevent endothelial dysfunction. We here found that sesamin increased in vitro angiogenic processes, such as endothelial cell proliferation, migration, and tube formation, as well as neovascularization in an animal model. This compound elicited the activation of multiple angiogenic signal modulators, such as ERK, Akt, endothelial nitric oxide synthase (eNOS), NO production, FAK, and p38 MAPK, but not Src. The MEK inhibitor PD98059 and the PI3K inhibitor Wortmannin specifically inhibited sesamin-induced activation of the ERK and Akt/eNOS pathways. These inhibitors reduced angiogenic events, with high specificity for MEK/ERK-dependent cell proliferation and migration and PI3K/Akt-mediated tube formation. Moreover, inhibition of p38 MAPK effectively inhibited sesamin-induced cell migration. The angiogenic activity of sesamin was not associated with VEGF expression. Furthermore, this compound did not induce vascular permeability and upregulated ICAM-1 and VCAM-1 expression, which are hallmarks of vascular inflammation. These results suggest that sesamin stimulates angiogenesis in vitro and in vivo through the activation of MEK/ERK-, PI3K/Akt/eNOS-, p125{sup FAK}-, and p38 MAPK-dependent pathways, without increasing vascular inflammation, and may be used for treating ischemic diseases and tissue regeneration.

  15. Activation of MEK/ERK Signaling by PACAP in Guinea Pig Cardiac Neurons.

    PubMed

    Clason, Todd A; Girard, Beatrice M; May, Victor; Parsons, Rodney L

    2016-06-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling can increase guinea pig cardiac neuron excitability in part through extracellular signal-regulated kinase (ERK) activation. The present study examined the PACAP receptors and signaling cascades that stimulate guinea pig cardiac neuron ERK signaling using confocal microscopy to quantify PACAP-induced neuronal phosphorylated ERK (pERK) immunoreactivity. PACAP and maxadilan, but not vasoactive intestinal polypeptide (VIP), increased cardiac neuron pERK, implicating primary roles for PACAP-selective PAC1 receptor (Adcyap1r1) signaling rather than VPAC receptors (Vipr1 and Vipr2) in the generation of cardiac neuron pERK. The adenylyl cyclase (AC) activator forskolin, but not the protein kinase C (PKC) activator phorbol myristate acetate (PMA), increased pERK. Also, Bim1 did not blunt PACAP activation of pERK. Together, the results suggest PAC1 receptor signal transduction via Gs/adenylyl cyclase (AC)/cAMP rather than Gq/phospholipase C (PLC) generated neuronal pERK. Activator and inhibitor studies suggested that the PACAP-mediated pERK activation was PKA-dependent rather than an exchange protein directly activated by a cAMP (EPAC), PKA-independent mechanism. The PACAP-induced pERK was inhibited by the clathrin inhibitor Pitstop2 to block receptor internalization and endosomal signaling. We propose that the PACAP-mediated MEK/ERK activation in cardiac neurons involves both AC/cAMP/PKA signaling and PAC1 receptor internalization/activation of signaling endosomes. PMID:27194157

  16. Asymmetric activation of Dll4-Notch signaling by Foxn4 and proneural factors activates BMP/TGFβ signaling to specify V2b interneurons in the spinal cord

    PubMed Central

    Misra, Kamana; Luo, Huijun; Li, Shengguo; Matise, Michael; Xiang, Mengqing

    2014-01-01

    During development of the ventral spinal cord, the V2 interneurons emerge from p2 progenitors and diversify into two major subtypes, V2a and V2b, that play key roles in locomotor coordination. Dll4-mediated Notch activation in a subset of p2 precursors constitutes the crucial first step towards generating neuronal diversity in this domain. The mechanism behind the asymmetric Notch activation and downstream signaling events are, however, unknown at present. We show here that the Ascl1 and Neurog basic helix-loop-helix (bHLH) proneural factors are expressed in a mosaic pattern in p2 progenitors and that Foxn4 is required for setting and maintaining this expression mosaic. By binding directly to a conserved Dll4 enhancer, Foxn4 and Ascl1 activate Dll4 expression, whereas Neurog proteins prevent this effect, thereby resulting in asymmetric activation of Dll4 expression in V2 precursors expressing different combinations of proneural and Foxn4 transcription factors. Lineage tracing using the Cre-LoxP system reveals selective expression of Dll4 in V2a precursors, whereas Dll4 expression is initially excluded from V2b precursors. We provide evidence that BMP/TGFβ signaling is activated in V2b precursors and that Dll4-mediated Notch signaling is responsible for this activation. Using a gain-of-function approach and by inhibiting BMP/TGFβ signal transduction with pathway antagonists and RNAi knockdown, we further demonstrate that BMP/TGFβ signaling is both necessary and sufficient for V2b fate specification. Our data together thus suggest that the mosaic expression of Foxn4 and proneural factors may serve as the trigger to initiate asymmetric Dll4-Notch and subsequent BMP/TGFβ signaling events required for neuronal diversity in the V2 domain. PMID:24257627

  17. Choline-releasing glycerophosphodiesterase EDI3 links the tumor metabolome to signaling network activities.

    PubMed

    Marchan, Rosemarie; Lesjak, Michaela S; Stewart, Joanna D; Winter, Roland; Seeliger, Janine; Hengstler, Jan G

    2012-12-15

    Recently, EDI3 was identified as a key factor for choline metabolism that controls tumor cell migration and is associated with metastasis in endometrial carcinomas. EDI3 cleaves glycerophosphocholine (GPC) to form choline and glycerol-3-phosphate (G3P). Choline is then further metabolized to phosphatidylcholine (PtdC), the major lipid in membranes and a key player in membrane-mediated cell signaling. The second product, G3P, is a precursor molecule for several lipids with central roles in signaling, for example lysophosphatidic acid (LPA), phosphatidic acid (PA) and diacylglycerol (DAG). LPA activates intracellular signaling pathways by binding to specific LPA receptors, including membrane-bound G protein-coupled receptors and the intracellular nuclear receptor, PPARγ. Conversely, PA and DAG mediate signaling by acting as lipid anchors that bind and activate several signaling proteins. For example, binding of GTPases and PKC to PA and DAG, respectively, increases the activation of signaling networks, mediating processes such as migration, adhesion, proliferation or anti-apoptosis-all relevant for tumor development. We present a concept by which EDI3 either directly generates signaling molecules or provides "membrane anchors" for downstream signaling factors. As a result, EDI3 links choline metabolism to signaling activities resulting in a more malignant phenotype. PMID:23114620

  18. Suppression of Epithelial Signal Transducer and Activator of Transcription 1 Activation by Extracts of Aspergillus fumigatus

    PubMed Central

    Bhushan, Bharat; Homma, Tetsuya; Norton, James E.; Sha, Quan; Siebert, Jason; Gupta, Dave S.; Schroeder, James W.

    2015-01-01

    Aspergillus fumigatus (AF) is often pathogenic in immune-deficient individuals and can cause life-threatening infections such as invasive aspergillosis. The pulmonary epithelial response to AF infection and the signaling pathways associated with it have not been completely studied. BEAS-2B cells or primary human bronchial epithelial cells were exposed to extracts of AF and challenged with IFN-β or the Toll-like receptor 3 agonist double-stranded RNA (dsRNA). Cytokine release (B-cell activating factor of the TNF family [BAFF], IFN-γ–induced protein-10 [IP-10], etc.) was assessed. AF extract was separated into low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions using ultra 4 centrifugal force filters to characterize the activity. Real-time PCR was performed with a TaqMan method, and protein estimation was performed using ELISA techniques. Western blot was performed to assess phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-β and dsRNA induced messenger RNA (mRNA) expression of BAFF (350- and 452-fold, respectively [n = 3]) and IP-10 (1,081- and 3,044-fold, respectively [n = 3]) in BEAS-2B cells. When cells were pretreated with AF extract for 1 hour and then stimulated with IFN-β or dsRNA for 6 hours, induction of BAFF and IP-10 mRNA was strongly suppressed relative to levels produced by IFN-β and dsRNA alone. When compared with control, soluble BAFF and IP-10 protein levels were maximally suppressed in dsRNA-stimulated wells treated with 1:320 wt/vol AF extract (P < 0.005). Upon molecular size fractionation, a LMW fraction of AF extract had no measurable suppressive effect on IP-10 mRNA expression. However, a HMW fraction of the AF extract significantly suppressed IP-10 expression in BEAS-2B cells that were stimulated with dsRNA or IFN-β. When BEAS-2B cells were pretreated with AF extract and then stimulated with IFN-β, reduced levels of pSTAT1 were observed, with maximum suppression at 4 and 6

  19. Suppression of epithelial signal transducer and activator of transcription 1 activation by extracts of Aspergillus fumigatus.

    PubMed

    Bhushan, Bharat; Homma, Tetsuya; Norton, James E; Sha, Quan; Siebert, Jason; Gupta, Dave S; Schroeder, James W; Schleimer, Robert P

    2015-07-01

    Aspergillus fumigatus (AF) is often pathogenic in immune-deficient individuals and can cause life-threatening infections such as invasive aspergillosis. The pulmonary epithelial response to AF infection and the signaling pathways associated with it have not been completely studied. BEAS-2B cells or primary human bronchial epithelial cells were exposed to extracts of AF and challenged with IFN-β or the Toll-like receptor 3 agonist double-stranded RNA (dsRNA). Cytokine release (B-cell activating factor of the TNF family [BAFF], IFN-γ-induced protein-10 [IP-10], etc.) was assessed. AF extract was separated into low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions using ultra 4 centrifugal force filters to characterize the activity. Real-time PCR was performed with a TaqMan method, and protein estimation was performed using ELISA techniques. Western blot was performed to assess phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-β and dsRNA induced messenger RNA (mRNA) expression of BAFF (350- and 452-fold, respectively [n = 3]) and IP-10 (1,081- and 3,044-fold, respectively [n = 3]) in BEAS-2B cells. When cells were pretreated with AF extract for 1 hour and then stimulated with IFN-β or dsRNA for 6 hours, induction of BAFF and IP-10 mRNA was strongly suppressed relative to levels produced by IFN-β and dsRNA alone. When compared with control, soluble BAFF and IP-10 protein levels were maximally suppressed in dsRNA-stimulated wells treated with 1:320 wt/vol AF extract (P < 0.005). Upon molecular size fractionation, a LMW fraction of AF extract had no measurable suppressive effect on IP-10 mRNA expression. However, a HMW fraction of the AF extract significantly suppressed IP-10 expression in BEAS-2B cells that were stimulated with dsRNA or IFN-β. When BEAS-2B cells were pretreated with AF extract and then stimulated with IFN-β, reduced levels of pSTAT1 were observed, with maximum suppression at 4 and 6

  20. Extracellular signal-regulated kinase 1/2 signalling in SLE T cells is influenced by oestrogen and disease activity.

    PubMed

    Gorjestani, S; Rider, V; Kimler, B F; Greenwell, C; Abdou, N I

    2008-06-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease that occurs primarily in women of reproductive age. The disease is characterized by exaggerated T-cell activity and abnormal T-cell signalling. The mitogen-activated protein kinase (MAPK) pathway is involved in the maintenance of T-cell tolerance that fails in patients with SLE. Oestrogen is a female sex hormone that binds to nuclear receptors and alters the rate of gene transcription. Oestrogen can also act through the plasma membrane and rapidly stimulate second messengers including calcium flux and kinase activation. In this study, we investigated whether oestrogen influences the activation of MAPK signalling through the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in activated SLE T cells. SLE and control T cells were cultured in serum-free medium without and with oestradiol (10(-7) M) for 18 h. The T cells were activated with phorbol 12 myristate 13-acetate and ionomycin for various time points (0-60 min), and the amount of phosphorylated ERK1/2 was measured by immunoblotting. There were no differences in ERK1/2 phosphorylation between SLE and control T cells at 5 and 15 min after the activation stimulus. However, comparison between the amount of phosphorylated ERK1/2 in SLE T cells from the same patients cultured without and with oestradiol showed a significant oestrogen-dependent suppression (P=0.48) of ERK1/2 in patients with inactive/mild systemic lupus erythematosus disease activity index (SLEDAI) (0-2) compared with patients with moderate (4-6) or active (8-12) SLEDAI scores. These results suggest that the suppression of MAPK through ERK1/2 phosphorylation is sensitive to oestradiol in patients with inactive or mild disease, but the sensitivity is not maintained when disease activity increases. Furthermore, studies are now necessary to understand the mechanisms by which oestrogen influences MAPK activation in SLE T cells. PMID:18539708

  1. Asphyxia-activated corticocardiac signaling accelerates onset of cardiac arrest

    PubMed Central

    Li, Duan; Mabrouk, Omar S.; Liu, Tiecheng; Tian, Fangyun; Xu, Gang; Rengifo, Santiago; Choi, Sarah J.; Mathur, Abhay; Crooks, Charles P.; Kennedy, Robert T.; Wang, Michael M.; Ghanbari, Hamid; Borjigin, Jimo

    2015-01-01

    The mechanism by which the healthy heart and brain die rapidly in the absence of oxygen is not well understood. We performed continuous electrocardiography and electroencephalography in rats undergoing experimental asphyxia and analyzed cortical release of core neurotransmitters, changes in brain and heart electrical activity, and brain–heart connectivity. Asphyxia stimulates a robust and sustained increase of functional and effective cortical connectivity, an immediate increase in cortical release of a large set of neurotransmitters, and a delayed activation of corticocardiac functional and effective connectivity that persists until the onset of ventricular fibrillation. Blocking the brain’s autonomic outflow significantly delayed terminal ventricular fibrillation and lengthened the duration of detectable cortical activities despite the continued absence of oxygen. These results demonstrate that asphyxia activates a brainstorm, which accelerates premature death of the heart and the brain. PMID:25848007

  2. Alternative splicing of MALT1 controls signalling and activation of CD4+ T cells

    PubMed Central

    Meininger, Isabel; Griesbach, Richard A.; Hu, Desheng; Gehring, Torben; Seeholzer, Thomas; Bertossi, Arianna; Kranich, Jan; Oeckinghaus, Andrea; Eitelhuber, Andrea C.; Greczmiel, Ute; Gewies, Andreas; Schmidt-Supprian, Marc; Ruland, Jürgen; Brocker, Thomas; Heissmeyer, Vigo; Heyd, Florian; Krappmann, Daniel

    2016-01-01

    MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4+ T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation. PMID:27068814

  3. Shuttle extravehicular activity signal processor pulse amplitude modulation decommutator

    NASA Technical Reports Server (NTRS)

    Noble, D. E.; Conrad, W. M.

    1974-01-01

    To provide data with long-term stability and accuracy, the pulse amplitude modulation (PAM) decommutator was synchronized to the PAM-return to zero wavetrain, and each channel was sampled with a common sample and hold circuit and digitized sequentially. The digital value of each channel was then scaled by the digital value of the calibration channels. The corrected digital value of each channel was stored for one complete frame and then transferred to the multiplexer-demultiplexer at a high rate in one block of serial digital data. A test model was built to demonstrate this design approach taken for the PAM decom and performance data was provided. The accuracies obtained with various signal to noise ratios are shown.

  4. Pleiotropic patterning response to activation of Shh signaling in the limb Apical Ectodermal Ridge

    PubMed Central

    Wang, Chi-Kuang Leo; Tsugane, Mizuyo H.; Scranton, Victoria; Kosher, Robert A.; Pierro, Louis J.; Upholt, William B.; Dealy, Caroline N.

    2012-01-01

    Sonic hedgehog (Shh) signaling in the limb plays a central role in coordination of limb patterning and outgrowth. Shh expression in the limb is limited to the cells of the Zone of Polarizing Activity (ZPA), located in posterior limb bud mesoderm. Shh is not expressed by limb ectoderm or AER, but recent studies suggest a role for AER-Shh signaling in limb patterning. Here, we have examined the effects of activation of Shh signaling in the AER. We find that targeted expression of Shh in the AER activates constitutive Shh signaling throughout the AER and subjacent limb mesoderm, and causes a range of limb patterning defects with progressive severity from mild polydactyly, to polysyndactyly with proximal defects, to severe oligodactyly with phocomelia and partial limb ventralization. Our studies emphasize the importance of control of the timing, level and location of Shh pathway signaling for limb AP, PD and DV patterning. PMID:21465622

  5. GPCR-like signaling mediated by smoothened contributes to acquired chemoresistance through activating Gli

    PubMed Central

    2014-01-01

    Background Smoothened (Smo), which possesses a structural similarity with classic G-protein coupled receptors (GPCR), is the most important molecular target in Hedgehog (Hh) signaling system for developing anticancer drugs; however, whether Smo may transmit GPCR-like signaling to activate the canonical transcriptional factor Gli of Hh signaling system and consequently to be involved in the Gli-dependent biological events remains controversial. Results In this study, using the acquired chemoresistant cancer cell lines and their respective parental cells, we found that Smo may activate Gli through Gαi, Gβγ-JNK signaling axis, thereby promoting the Gli-dependent acquired chemoresistance. These observations were further complementarily strengthened by data obtained from chemosensitive cancer cells with artificially elevated Hh pathway activity. Conclusions Hence, our data demonstrate that GPCR-like signaling mediated by Smo contributes to the acquired chemoresistance through activating the canonical Hh transcriptional factor Gli; therefore improving our knowledge of the nature of the signal transduction of Smo and the molecular mechanisms responsible for the acquired chemoresistance maintained by Hh pathway. Moreover, our data that JNK after activated by Smo-Gβγ signaling axis may stimulate the Gli activity and consequently promotes acquired chemoresistance expose a promising and potential target for developing anti-cancer drugs aimed at Hh pathway and for combating the acquired resistance raised by using of anti-cancer drugs targeting Smo. PMID:24393163

  6. Discovery of Novel Small Molecule Activators of β-Catenin Signaling

    PubMed Central

    Verkaar, Folkert; van der Stelt, Mario; Blankesteijn, W. Matthijs; van der Doelen, Antoon A.; Zaman, Guido J. R.

    2011-01-01

    Wnt/β-catenin signaling plays a major role in embryonic development and adult stem cell maintenance. Reduced activation of the Wnt/β-catenin pathway underlies neurodegenerative disorders and aberrations in bone formation. Screening of a small molecule compound library with a β-galactosidase fragment complementation assay measuring β-catenin nuclear entry revealed bona fide activators of β-catenin signaling. The compounds stabilized cytoplasmic β-catenin and activated β–catenin-dependent reporter gene activity. Although the mechanism through which the compounds activate β-catenin signaling has yet to be determined, several key regulators of Wnt/β-catenin signaling, including glycogen synthase kinase 3 and Frizzled receptors, were excluded as the molecular target. The compounds displayed remarkable selectivity, as they only induced β-catenin signaling in a human osteosarcoma U2OS cell line and not in a variety of other cell lines examined. Our data indicate that differences in cellular Wnt/β-catenin signaling machinery can be exploited to identify cell type-specific activators of Wnt/β-catenin signaling. PMID:21559429

  7. Lipopolysaccharide activated TLR4/NF-κB signaling pathway of fibroblasts from uterine fibroids.

    PubMed

    Guo, Jing; Zheng, Lihua; Chen, Li; Luo, Ning; Yang, Weihong; Qu, Xiaoyan; Liu, Mingmin; Cheng, Zhongping

    2015-01-01

    Uterine fibroids (UF) are the most common benign tumor of the female reproductive tract. The aim of this study was to explore the role of lipopolysaccharide (LPS)-induced activation of TLR4/NF-κB signaling pathway on stromal fibroblasts in the pathogenesis of UF. Here, TLR4/NF-κB signaling pathway was more activated in UF, and UF cells (UFC) and UF derived fibroblasts (TAF) than in smooth muscle tissues, smooth muscle cell (SMC) and myometrial fibroblasts (fib) respectively. After lipopolysaccharide (LPS) stimulation, the activity of fib was enhanced, characterized by the increased expression of fibroblast activation protein (FAP), and increased secretion of collagen I and transforming growth factor-β (TGF-β). Moreover, TLR4 inhibitor (VIPER) and siTLR4 can represses LPS-activated fibroblasts and TLR4/NF-κB signaling transduction pathways in fib and UFC cells. Co-cultured with LPS-activated fibroblast enhanced fibroblast activation and TLR4/NF-κB signaling. In conclusion, LPS treatment activated TLR4/NF-κB signaling pathway on fibroblasts, which may involve in the development of UF. Our study indicated reproductive tract infection may be associated with fibroid pathogenesis through TLR4/NF-κB signaling. Targeting NF-κB with inhibitors may hold promises of treating uterine fibroid. PMID:26617709

  8. Lipopolysaccharide activated TLR4/NF-κB signaling pathway of fibroblasts from uterine fibroids

    PubMed Central

    Guo, Jing; Zheng, Lihua; Chen, Li; Luo, Ning; Yang, Weihong; Qu, Xiaoyan; Liu, Mingmin; Cheng, Zhongping

    2015-01-01

    Uterine fibroids (UF) are the most common benign tumor of the female reproductive tract. The aim of this study was to explore the role of lipopolysaccharide (LPS)-induced activation of TLR4/NF-κB signaling pathway on stromal fibroblasts in the pathogenesis of UF. Here, TLR4/NF-κB signaling pathway was more activated in UF, and UF cells (UFC) and UF derived fibroblasts (TAF) than in smooth muscle tissues, smooth muscle cell (SMC) and myometrial fibroblasts (fib) respectively. After lipopolysaccharide (LPS) stimulation, the activity of fib was enhanced, characterized by the increased expression of fibroblast activation protein (FAP), and increased secretion of collagen I and transforming growth factor-β (TGF-β). Moreover, TLR4 inhibitor (VIPER) and siTLR4 can represses LPS-activated fibroblasts and TLR4/NF-κB signaling transduction pathways in fib and UFC cells. Co-cultured with LPS-activated fibroblast enhanced fibroblast activation and TLR4/NF-κB signaling. In conclusion, LPS treatment activated TLR4/NF-κB signaling pathway on fibroblasts, which may involve in the development of UF. Our study indicated reproductive tract infection may be associated with fibroid pathogenesis through TLR4/NF-κB signaling. Targeting NF-κB with inhibitors may hold promises of treating uterine fibroid. PMID:26617709

  9. Extracellular signal-regulated protein kinases 1 and 2 activation by addictive drugs: a signal toward pathological adaptation.

    PubMed

    Pascoli, Vincent; Cahill, Emma; Bellivier, Frank; Caboche, Jocelyne; Vanhoutte, Peter

    2014-12-15

    Addiction is a chronic and relapsing psychiatric disorder that is thought to occur in vulnerable individuals. Synaptic plasticity evoked by drugs of abuse in the so-called neuronal circuits of reward has been proposed to underlie behavioral adaptations that characterize addiction. By increasing dopamine in the striatum, addictive drugs alter the balance of dopamine and glutamate signals converging onto striatal medium-sized spiny neurons (MSNs) and activate intracellular events involved in long-term behavioral alterations. Our laboratory contributed to the identification of salient molecular changes induced by administration of addictive drugs to rodents. We pioneered the observation that a common feature of addictive drugs is to activate, by a double tyrosine/threonine phosphorylation, the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the striatum, which control a plethora of substrates, some of them being critically involved in cocaine-mediated molecular and behavioral adaptations. Herein, we review how the interplay between dopamine and glutamate signaling controls cocaine-induced ERK1/2 activation in MSNs. We emphasize the key role of N-methyl-D-aspartate receptor potentiation by D1 receptor to trigger ERK1/2 activation and its subsequent nuclear translocation where it modulates both epigenetic and genetic processes engaged by cocaine. We discuss how cocaine-induced long-term synaptic and structural plasticity of MSNs, as well as behavioral adaptations, are influenced by ERK1/2-controlled targets. We conclude that a better knowledge of molecular mechanisms underlying ERK1/2 activation by drugs of abuse and/or its role in long-term neuronal plasticity in the striatum may provide a new route for therapeutic treatment in addiction. PMID:24844603

  10. Neuronal activity regulates remyelination via glutamate signalling to oligodendrocyte progenitors

    PubMed Central

    Gautier, Hélène O. B.; Evans, Kimberley A.; Volbracht, Katrin; James, Rachel; Sitnikov, Sergey; Lundgaard, Iben; James, Fiona; Lao-Peregrin, Cristina; Reynolds, Richard; Franklin, Robin J. M.; Káradóttir, Ragnhildur T

    2015-01-01

    Myelin regeneration can occur spontaneously in demyelinating diseases such as multiple sclerosis (MS). However, the underlying mechanisms and causes of its frequent failure remain incompletely understood. Here we show, using an in-vivo remyelination model, that demyelinated axons are electrically active and generate de novo synapses with recruited oligodendrocyte progenitor cells (OPCs), which, early after lesion induction, sense neuronal activity by expressing AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate receptors. Blocking neuronal activity, axonal vesicular release or AMPA receptors in demyelinated lesions results in reduced remyelination. In the absence of neuronal activity there is a ∼6-fold increase in OPC number within the lesions and a reduced proportion of differentiated oligodendrocytes. These findings reveal that neuronal activity and release of glutamate instruct OPCs to differentiate into new myelinating oligodendrocytes that recover lost function. Co-localization of OPCs with the presynaptic protein VGluT2 in MS lesions implies that this mechanism may provide novel targets to therapeutically enhance remyelination. PMID:26439639

  11. Neuronal activity regulates remyelination via glutamate signalling to oligodendrocyte progenitors.

    PubMed

    Gautier, Hélène O B; Evans, Kimberley A; Volbracht, Katrin; James, Rachel; Sitnikov, Sergey; Lundgaard, Iben; James, Fiona; Lao-Peregrin, Cristina; Reynolds, Richard; Franklin, Robin J M; Káradóttir, Ragnhildur T

    2015-01-01

    Myelin regeneration can occur spontaneously in demyelinating diseases such as multiple sclerosis (MS). However, the underlying mechanisms and causes of its frequent failure remain incompletely understood. Here we show, using an in-vivo remyelination model, that demyelinated axons are electrically active and generate de novo synapses with recruited oligodendrocyte progenitor cells (OPCs), which, early after lesion induction, sense neuronal activity by expressing AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate receptors. Blocking neuronal activity, axonal vesicular release or AMPA receptors in demyelinated lesions results in reduced remyelination. In the absence of neuronal activity there is a ∼6-fold increase in OPC number within the lesions and a reduced proportion of differentiated oligodendrocytes. These findings reveal that neuronal activity and release of glutamate instruct OPCs to differentiate into new myelinating oligodendrocytes that recover lost function. Co-localization of OPCs with the presynaptic protein VGluT2 in MS lesions implies that this mechanism may provide novel targets to therapeutically enhance remyelination. PMID:26439639

  12. The Role of cGMP on Adenosine A1 Receptor-mediated Inhibition of Synaptic Transmission at the Hippocampus

    PubMed Central

    Pinto, Isa; Serpa, André; Sebastião, Ana M.; Cascalheira, José F.

    2016-01-01

    Both adenosine A1 receptor and cGMP inhibit synaptic transmission at the hippocampus and recently it was found that A1 receptor increased cGMP levels in hippocampus, but the role of cGMP on A1 receptor-mediated inhibition of synaptic transmission remains to be established. In the present work we investigated if blocking the NOS/sGC/cGMP/PKG pathway using nitric oxide synthase (NOS), protein kinase G (PKG), and soluble guanylyl cyclase (sGC) inhibitors modify the A1 receptor effect on synaptic transmission. Neurotransmission was evaluated by measuring the slope of field excitatory postsynaptic potentials (fEPSPs) evoked by electrical stimulation at hippocampal slices. N6-cyclopentyladenosine (CPA, 15 nM), a selective A1 receptor agonist, reversibly decreased the fEPSPs by 54 ± 5%. Incubation of the slices with an inhibitor of NOS (L-NAME, 200 μM) decreased the CPA effect on fEPSPs by 57 ± 9% in female rats. In males, ODQ (10 μM), an sGC inhibitor, decreased the CPA inhibitory effect on fEPSPs by 23 ± 6%, but only when adenosine deaminase (ADA,1 U/ml) was present; similar results were found in females, where ODQ decreased CPA-induced inhibition of fEPSP slope by 23 ± 7%. In male rats, the presence of the PKG inhibitor (KT5823, 1 nM) decreased the CPA effect by 45.0 ± 9%; similar results were obtained in females, where KT5823 caused a 32 ± 9% decrease on the CPA effect. In conclusion, the results suggest that the inhibitory action of adenosine A1 receptors on synaptic transmission at hippocampus is, in part, mediated by the NOS/sGC/cGMP/PKG pathway. PMID:27148059

  13. Mapping Neuronal Activation and the Influence of Adrenergic Signaling during Contextual Memory Retrieval

    ERIC Educational Resources Information Center

    Zhang, Wei-Ping; Guzowski, John F.; Thomas, Steven A.

    2005-01-01

    We recently described a critical role for adrenergic signaling in the hippocampus during contextual and spatial memory retrieval. To determine which neurons are activated by contextual memory retrieval and its sequelae in the presence and absence of adrenergic signaling, transcriptional imaging for the immediate-early gene "Arc" was used in…

  14. Cdc42 Regulation of Kinase Activity and Signaling by the Yeast p21-Activated Kinase Ste20

    PubMed Central

    Lamson, Rachel E.; Winters, Matthew J.; Pryciak, Peter M.

    2002-01-01

    The Saccharomyces cerevisiae kinase Ste20 is a member of the p21-activated kinase (PAK) family with several functions, including pheromone-responsive signal transduction. While PAKs are usually activated by small G proteins and Ste20 binds Cdc42, the role of Cdc42-Ste20 binding has been controversial, largely because Ste20 lacking its entire Cdc42-binding (CRIB) domain retains kinase activity and pheromone response. Here we show that, unlike CRIB deletion, point mutations in the Ste20 CRIB domain that disrupt Cdc42 binding also disrupt pheromone signaling. We also found that Ste20 kinase activity is stimulated by GTP-bound Cdc42 in vivo and this effect is blocked by the CRIB point mutations. Moreover, the Ste20 CRIB and kinase domains bind each other, and mutations that disrupt this interaction cause hyperactive kinase activity and bypass the requirement for Cdc42 binding. These observations demonstrate that the Ste20 CRIB domain is autoinhibitory and that this negative effect is antagonized by Cdc42 to promote Ste20 kinase activity and signaling. Parallel results were observed for filamentation pathway signaling, suggesting that the requirement for Cdc42-Ste20 interaction is not qualitatively different between the mating and filamentation pathways. While necessary for pheromone signaling, the role of the Cdc42-Ste20 interaction does not require regulation by pheromone or the pheromone-activated Gβγ complex, because the CRIB point mutations also disrupt signaling by activated forms of the kinase cascade scaffold protein Ste5. In total, our observations indicate that Cdc42 converts Ste20 to an active form, while pathway stimuli regulate the ability of this active Ste20 to trigger signaling through a particular pathway. PMID:11940652

  15. The CLAVATA signaling pathway mediating stem cell fate in shoot meristems requires Ca(2+) as a secondary cytosolic messenger.

    PubMed

    Chou, Hsuan; Zhu, Yingfang; Ma, Yi; Berkowitz, Gerald A

    2016-02-01

    CLAVATA1 (CLV1) is a receptor protein expressed in the shoot apical meristem (SAM) that translates perception of a non-cell-autonomous CLAVATA3 (CLV3) peptide signal into altered stem cell fate. CLV3 reduces expression of WUSCHEL (WUS) and FANTASTIC FOUR 2 (FAF2) in the SAM. Expression of WUS and FAF2 leads to maintenance of undifferentiated stem cells in the SAM. CLV3 binding to CLV1 inhibits expression of these genes and controls stem cell fate in the SAM through an unidentified signaling pathway. Cytosolic Ca(2+) elevations, cyclic nucleotide (cGMP)-activated Ca(2+) channels, and cGMP have been linked to signaling downstream of receptors similar to CLV1. Hence, we hypothesized that cytosolic Ca(2+) elevation mediates the CLV3 ligand/CLV1 receptor signaling that controls meristem stem cell fate. CLV3 application to Arabidopsis seedlings results in elevation of cytosolic Ca(2+) and cGMP. CLV3 control of WUS was prevented in a genotype lacking a functional cGMP-activated Ca(2+) channel. In wild-type plants, CLV3 inhibition of WUS and FAF2 expression was impaired by treatment with either a Ca(2+) channel blocker or a guanylyl cyclase inhibitor. When CLV3-dependent repression of WUS is blocked, altered control of stem cell fate leads to an increase in SAM size; we observed a larger SAM size in seedlings treated with the Ca(2+) channel blocker. These results suggest that the CLV3 ligand/CLV1 receptor system initiates a signaling cascade that elevates cytosolic Ca(2+) , and that this cytosolic secondary messenger is involved in the signal transduction cascade linking CLV3/CLV1 to control of gene expression and stem cell fate in the SAM. PMID:26756833

  16. Fumarate hydratase deficiency and cancer: activation of hypoxia signaling?

    PubMed

    Ratcliffe, Peter J

    2007-04-01

    Molecular genetic analysis of hereditary leiomyomatosis and renal cell cancer (HLRCC) unexpectedly revealed germline defects in the gene encoding the Krebs cycle enzyme fumarate hydratase (FH), stimulating great interest in the underlying mechanism of oncogenesis. It has been proposed that the associated accumulation of fumarate competitively inhibits the 2-oxoglutarate-dependent dioxygenases that regulate hypoxia-inducible factor (HIF), thus activating oncogenic hypoxia pathways. In this issue of Cancer Cell, Pollard and colleagues describe a genetic mouse model of FH deficiency that recapitulates aspects of the human disease, including HIF activation and renal cysts, enabling further insights into this unusual cancer syndrome. PMID:17418405

  17. High Glucose-enhanced Acetylcholine Stimulated CGMP Masks Impaired Vascular Reactivity in Tail Arteries from Short-Term Hyperglycemic Rats

    PubMed Central

    Hamaty, Marwan; Guzmán, Cristina B.; Walsh , Mary F.; Bode, Ann M.; Levy, Joseph

    2000-01-01

    Impaired vascular endothelium-dependent relaxation and augmented contractile responses have been reported in several models of long-term hyperglycemia. However, the effects of short-term ambient hyperglycemia are poorly understood. Since oxidative stress has been implicated as a contributor to impaired vascular function, we investigated the following: Aims: (1) the effects of high glucose exposure in vitro (7 – 10 days) on vascular relaxation to acetylcholine (Ach) and contractility to norepinephrine (NE) and KCl; (2) if NO-dependent cGMP generation is affected under these conditions; and (3) aortic redox status. Methods: Non-diabetic rat tail artery rings were incubated in normal (5mM) (control NG) or high (20mM) glucose buffer (control HG). Vascular responses to Ach, NE and KCl were compared to those of streptozotocin (SZ) diabetic animals in the same buffers (diabetic NG, diabetic HG). Ach stimulated cGMP levels were quantitated as an indirect assessment of endothelial nitric oxide (NO) production and oxidative stress evaluated by measuring vascular glutathione and oxidized glutathione. Results: Rings from diabetic rats in NG showed impaired relaxation to Ach (P = 0.002) but relaxed normally, when maintained in HG. Similarly, contractile responses to NE were attenuated in diabetic rings in NG but similar to controls in HG. HG markedly augmented maximal contraction to KCl compared to control and diabetic vessels in NG (P < 0.0001). Diabetic vessels in a hyperosmolar, but normoglycemic, milieu respond like those in HG. in vitro, HG for 2 hours changed neither relaxation nor contractile responses to NE and KCl in control rings. Basal cGMP levels were lower in aortae from diabetic animals pre-incubated in NG than in HG/LG or in control rings in NG (P < 0.05). cGMP responses to Ach were exaggerated in diabetic vessels in HG (P = 0.035 vs. control NG, P = 0.043 vs. diabetic NG) but not different between control and diabetic rings in NG. Vessels from diabetic animals

  18. Queen signals in a stingless bee: suppression of worker ovary activation and spatial distribution of active compounds

    PubMed Central

    Nunes, Túlio M.; Mateus, Sidnei; Favaris, Arodi P.; Amaral, Mônica F. Z. J.; von Zuben, Lucas G.; Clososki, Giuliano C.; Bento, José M. S.; Oldroyd, Benjamin P.; Silva, Ricardo; Zucchi, Ronaldo; Silva, Denise B.; Lopes, Norberto P.

    2014-01-01

    In most species of social insect the queen signals her presence to her workers via pheromones. Worker responses to queen pheromones include retinue formation around the queen, inhibition of queen cell production and suppression of worker ovary activation. Here we show that the queen signal of the Brazilian stingless bee Friesella schrottkyi is a mixture of cuticular hydrocarbons. Stingless bees are therefore similar to ants, wasps and bumble bees, but differ from honey bees in which the queen's signal mostly comprises volatile compounds originating from the mandibular glands. This shows that cuticular hydrocarbons have independently evolved as the queen's signal across multiple taxa, and that the honey bees are exceptional. We also report the distribution of four active queen-signal compounds by Matrix-assisted laser desorption/ionization (MALDI) imaging. The results indicate a relationship between the behavior of workers towards the queen and the likely site of secretion of the queen's pheromones. PMID:25502598

  19. Ubiquitination in Signaling to and Activation of IKK

    PubMed Central

    Chen, Zhijian J.

    2013-01-01

    A role of polyubiquitination in the activation of IκB kinase (IKK) through a proteasome-independent mechanism was first reported in 1996, but the physiological significance of this finding was not clear until 2000 when TRAF6 was found to be a ubiquitin E3 ligase that catalyzes lysine-63 (K63) polyubiquitination. Since then, several proteins known to regulate IKK have been linked to the ubiquitin pathway. These include the deubiquitination enzymes CYLD and A20 that inhibit IKK, and the ubiquitin binding proteins NEMO and TAB2 which are the regulatory subunits of IKK and TAK1 kinase complexes, respectively. Now accumulating evidence strongly supports a central role of K63 polyubiquitination in IKK activation by multiple immune and inflammatory pathways. Interestingly, recent research suggests that some alternative ubiquitin chains such as linear or K11 ubiquitin chains may also play a role in certain pathways such as the TNF pathway. Here I present a historical narrative of the discovery of the role of ubiquitin in IKK activation, review recent advances in understanding the role and mechanism of ubiquitin-mediated IKK activation, and raise some questions to be resolved in future research. PMID:22435549

  20. Activated AKT/PKB signaling in C. elegans uncouples temporally distinct outputs of DAF-2/insulin-like signaling

    PubMed Central

    Gami, Minaxi S; Iser, Wendy B; Hanselman, Keaton B; Wolkow, Catherine A

    2006-01-01

    Background In the nematode, Caenorhabditis elegans, a conserved insulin-like signaling pathway controls larval development, stress resistance and adult lifespan. AGE-1, a homolog of the p110 catalytic subunit of phosphoinositide 3-kinases (PI3K) comprises the major known effector pathway downstream of the insulin receptor, DAF-2. Phospholipid products of AGE-1/PI3K activate AKT/PKB kinase signaling via PDK-1. AKT/PKB signaling antagonizes nuclear translocation of the DAF-16/FOXO transcription factor. Reduced AGE-1/PI3K signaling permits DAF-16 to direct dauer larval arrest and promote long lifespan in adult animals. In order to study the downstream effectors of AGE-1/PI3K signaling in C. elegans, we conducted a genetic screen for mutations that suppress the constitutive dauer arrest phenotype of age-1(mg109) animals. Results This report describes mutations recovered in a screen for suppressors of the constitutive dauer arrest (daf-C) phenotype of age-1(mg109). Two mutations corresponded to alleles of daf-16. Two mutations were gain-of-function alleles in the genes, akt-1 and pdk-1, encoding phosphoinositide-dependent serine/threonine kinases. A fifth mutation, mg227, located on chromosome X, did not correspond to any known dauer genes, suggesting that mg227 may represent a new component of the insulin pathway. Genetic epistasis analysis by RNAi showed that reproductive development in age-1(mg109);akt-1(mg247) animals was dependent on the presence of pdk-1. Similarly, reproductive development in age-1(mg109);pdk-1(mg261) animals was dependent on akt-1. However, reproductive development in age-1(mg109); mg227 animals required only akt-1, and pdk-1 activity was dispensable in this background. Interestingly, while mg227 suppressed dauer arrest in age-1(mg109) animals, it enhanced the long lifespan phenotype. In contrast, akt-1(mg247) and pdk-1(mg261) did not affect lifespan or stress resistance, while both daf-16 alleles fully suppressed these phenotypes. Conclusion A

  1. Notch2 signaling promotes osteoclast resorption via activation of PYK2.

    PubMed

    Jin, Won Jong; Kim, Bongjun; Kim, Jung-Wook; Kim, Hong-Hee; Ha, Hyunil; Lee, Zang Hee

    2016-05-01

    Notch signaling plays a central role in various cell fate decisions, including skeletal development. Recently, Notch signaling was implicated in osteoclast differentiation and maturation, including the resorption activity of osteoclasts. However, the specific involvement of notch signaling in resorption activity was not fully investigated. Here, we investigated the roles of Notch signaling in the resorption activity of osteoclasts by use of the gamma-secretase inhibitor dibenzazepine (DBZ). Attenuating Notch signaling by DBZ suppressed the expression of NFATc1, a master transcription factor for osteoclast differentiation. However, overexpression of a constitutively active form of NFATc1 did not fully rescue the effects of DBZ. DBZ suppressed the autophosphorylation of PYK2, which is essential for the formation of the podosome belt and sealing zone, with reduced c-Src/PYK2 interaction. We found that RANKL increases PYK2 activation accompanied by increased NICD2 production in osteoclasts. Overexpression of NICD2 in osteoclasts rescued DBZ-mediated suppression of resorption activity with promotion of PYK2 autophosphorylation and microtubule acetylation. Consistent with the in vitro results, DBZ strongly suppressed bone destruction in an interleukin-1-induced bone loss model. Collectively, these results demonstrate that Notch2 in osteoclasts plays a role in the control of resorption activity via the PYK2-c-Src-microtubule signaling pathway. PMID:26829213

  2. Body Morphology, Energy Stores, and Muscle Enzyme Activity Explain Cricket Acoustic Mate Attraction Signaling Variation

    PubMed Central

    Thomson, Ian R.; Darveau, Charles-A.; Bertram, Susan M.

    2014-01-01

    High mating success in animals is often dependent on males signalling attractively with high effort. Since males should be selected to maximize their reproductive success, female preferences for these traits should result in minimal signal variation persisting in the population. However, extensive signal variation persists. The genic capture hypothesis proposes genetic variation persists because fitness-conferring traits depend on an individual's basic processes, including underlying physiological, morphological, and biochemical traits, which are themselves genetically variable. To explore the traits underlying signal variation, we quantified among-male differences in signalling, morphology, energy stores, and the activities of key enzymes associated with signalling muscle metabolism in two species of crickets, Gryllus assimilis (chirper: <20 pulses/chirp) and G. texensis (triller: >20 pulses/chirp). Chirping G. assimilis primarily fuelled signalling with carbohydrate metabolism: smaller individuals and individuals with increased thoracic glycogen stores signalled for mates with greater effort; individuals with greater glycogen phosphorylase activity produced more attractive mating signals. Conversely, the more energetic trilling G. texensis fuelled signalling with both lipid and carbohydrate metabolism: individuals with increased β-hydroxyacyl-CoA dehydrogenase activity and increased thoracic free carbohydrate content signalled for mates with greater effort; individuals with higher thoracic and abdominal carbohydrate content and higher abdominal lipid stores produced more attractive signals. Our findings suggest variation in male reproductive success may be driven by hidden physiological trade-offs that affect the ability to uptake, retain, and use essential nutrients, although the results remain correlational in nature. Our findings indicate that a physiological perspective may help us to understand some of the causes of variation in behaviour. PMID:24608102

  3. Raising the standard: changes to the Australian Code of Good Manufacturing Practice (cGMP) for human blood and blood components, human tissues and human cellular therapy products.

    PubMed

    Wright, Craig; Velickovic, Zlatibor; Brown, Ross; Larsen, Stephen; Macpherson, Janet L; Gibson, John; Rasko, John E J

    2014-04-01

    In Australia, manufacture of blood, tissues and biologicals must comply with the federal laws and meet the requirements of the Therapeutic Goods Administration (TGA) Manufacturing Principles as outlined in the current Code of Good Manufacturing Practice (cGMP). The Therapeutic Goods Order (TGO) No. 88 was announced concurrently with the new cGMP, as a new standard for therapeutic goods. This order constitutes a minimum standard for human blood, tissues and cellular therapeutic goods aimed at minimising the risk of infectious disease transmission. The order sets out specific requirements relating to donor selection, donor testing and minimisation of infectious disease transmission from collection and manufacture of these products. The Therapeutic Goods Manufacturing Principles Determination No. 1 of 2013 references the human blood and blood components, human tissues and human cellular therapy products 2013 (2013 cGMP). The name change for the 2013 cGMP has allowed a broadening of the scope of products to include human cellular therapy products. It is difficult to directly compare versions of the code as deletion of some clauses has not changed the requirements to be met, as they are found elsewhere amongst the various guidelines provided. Many sections that were specific for blood and blood components are now less prescriptive and apply to a wider range of cellular therapies, but the general overall intent remains the same. Use of 'should' throughout the document instead of 'must' allows flexibility for alternative processes, but these systems will still require justification by relevant logical argument and validation data to be acceptable to TGA. The cGMP has seemingly evolved so that specific issues identified at audit over the last decade have now been formalised in the new version. There is a notable risk management approach applied to most areas that refer to process justification and decision making. These requirements commenced on 31 May 2013 and a 12 month

  4. Nandrolone reduces activation of Notch signaling in denervated muscle associated with increased Numb expression

    SciTech Connect

    Liu, Xin-Hua; Yao, Shen; Qiao, Rui-Fang; Levine, Alice C.; Kirschenbaum, Alexander; Pan, Jiangping; Wu, Yong; Qin, Weiping; Bauman, William A.; Cardozo, Christopher P.

    2011-10-14

    Highlights: {yields} Nerve transection increased Notch signaling in paralyzed muscle. {yields} Nandrolone prevented denervation-induced Notch signaling. {yields} Nandrolone induced the expression of an inhibitor of the Notch signaling, Numb. {yields} Reduction of denervation-induced Notch signaling by nandrolone is likely through upregulation of Numb. -- Abstract: Nandrolone, an anabolic steroid, slows denervation-atrophy in rat muscle. The molecular mechanisms responsible for this effect are not well understood. Androgens and anabolic steroids activate Notch signaling in animal models of aging and thereby mitigate sarcopenia. To explore the molecular mechanisms by which nandrolone prevents denervation-atrophy, we investigated the effects of nandrolone on Notch signaling in denervated rat gastrocnemius muscle. Denervation significantly increased Notch activity reflected by elevated levels of nuclear Notch intracellular domain (NICD) and expression of Hey1 (a Notch target gene). Activation was greatest at 7 and 35 days after denervation but remained present at 56 days after denervation. Activation of Notch in denervated muscle was prevented by nandrolone associated with upregulated expression of Numb mRNA and protein. These data demonstrate that denervation activates Notch signaling, and that nandrolone abrogates this response associated with increased expression of Numb, suggesting a potential mechanism by which nandrolone reduces denervation-atrophy.

  5. ASBESTOS-INDUCED ACTIVATION OF CELL SIGNALING PATHWAYS IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Using respiratory epithelial cells transfected with either superoxide dismutase (SOD) or catalase, the authors tested the hypothesis that the activation of the epidermal growth factor (EGF) receptor signal pathway after asbestos exposure involves an oxidative stress. Western blot...

  6. A behavioral switch: cGMP and PKC signaling in olfactory neurons reverses odor preference in C. elegans

    PubMed Central

    Tsunozaki, Makoto; Chalasani, Sreekanth H.; Bargmann, Cornelia I.

    2008-01-01

    Summary Innate chemosensory preferences are often encoded by sensory neurons that are specialized for attractive or avoidance behaviors. Here we show that one olfactory neuron in Caenorhabditis elegans, AWCON, has the potential to direct both attraction and repulsion. Attraction, the typical AWCON behavior, requires a receptor-like guanylate cyclase GCY-28 that acts in adults and localizes to AWCON axons. gcy-28 mutants avoid AWCON–sensed odors; they have normal odor-evoked calcium responses in AWCON, but reversed turning biases in odor gradients. In addition to gcy-28, a diacylglycerol/protein kinase C pathway that regulates neurotransmission switches AWCON odor preferences. A behavioral switch in AWCON may be part of normal olfactory plasticity, as odor conditioning can induce odor avoidance in wild-type animals. Genetic interactions, acute rescue, and calcium imaging suggest that the behavioral reversal results from presynaptic changes in AWCON. These results suggest that alternative modes of neurotransmission can couple one sensory neuron to opposite behavioral outputs. PMID:18817734

  7. Structure–Activity Relationships for Side Chain Oxysterol Agonists of the Hedgehog Signaling Pathway

    PubMed Central

    2012-01-01

    Oxysterols (OHCs) are byproducts of cholesterol oxidation that are known to activate the Hedeghog (Hh) signaling pathway. While OHCs that incorporate hydroxyl groups throughout the scaffold are known, those that act as agonists of Hh signaling primarily contain a single hydroxyl on the alkyl side chain. We sought to further explore how side chain hydroxylation patterns affect oxysterol-mediated Hh activation, by performing a structure–activity relationship study on a series of synthetic OHCs. The most active analogue, 23(R)-OHC (35), demonstrated potent activation of Hh signaling in two Hh-dependent cell lines (EC50 values 0.54–0.65 μM). In addition, OHC 35 was approximately 3-fold selective for the Hh pathway as compared to the liver X receptor, a nuclear receptor that is also activated by endogenous OHCs. Finally, 35 induced osteogenic differentiation and osteoblast formation in cultured cells, indicating functional agonism of the Hh pathway. PMID:24900386

  8. Recognition of forearm muscle activity by continuous classification of multi-site mechanomyogram signals.

    PubMed

    Alves, Natasha; Chau, Tom

    2010-01-01

    Recent studies on identifying multiple activation states from mechanomyogram (MMG) signals for the purpose of controlling switching interfaces have employed pattern recognition methods where MMG signal features from multiple muscle sites are extracted and classified. The purpose of this study is to determine if MMG signal features retain enough discriminatory information to allow reliable continuous classification, and to determine if there is a decline in classification accuracy over short time periods. MMG signals were recorded from two accelerometers attached to the flexor carpi radialis and extensor carpi radialis muscles of 12 able-bodied participants as participants performed three classes of forearm muscle activity. The data were collected over five recording sessions, with a ten-minute interval between each session. The data were spliced into 256 ms epochs, and a comprehensive set of signal features was extracted. A pattern classifier, trained with continuously acquired signal features from the first recording session, was tested with signals recorded from the other sessions. The average classification accuracy over the five sessions was 89 ± 2%. There was no obvious declining trend in classification accuracy with time. These results show that MMG signals recorded at the forearm retain enough discriminatory information to allow continuous recognition of hand motion across multiple (>90) repetitions, and the MMG-classifier does not show short-term degradation. These results indicate the potential of MMG as a multifunction control signal for muscle-machine interfaces. PMID:21097038

  9. Stromal TGF-β signaling induces AR activation in prostate cancer

    PubMed Central

    Yang, Feng; Chen, Yizhen; Shen, Tao; Guo, Dan; Dakhova, Olga; Ittmann, Michael M.; Creighton, Chad J.; Zhang, Yiqun; Dang, Truong D.; Rowley, David R.

    2014-01-01

    AR signaling is essential for the growth and survival of prostate cancer (PCa), including most of the lethal castration-resistant PCa (CRPC). We previously reported that TGF-β signaling in prostate stroma promotes prostate tumor angiogenesis and growth. By using a PCa/stroma co-culture model, here we show that stromal TGF-β signaling induces comprehensive morphology changes of PCa LNCaP cells. Furthermore, it induces AR activation in LNCaP cells in the absence of significant levels of androgen, as evidenced by induction of several AR target genes including PSA, TMPRSS2, and KLK4. SD-208, a TGF-β receptor 1 specific inhibitor, blocks this TGF-β induced biology. Importantly, stromal TGF-β signaling together with DHT induce robust activation of AR. MDV3100 effectively blocks DHT-induced, but not stromal TGF-β signaling induced AR activation in LNCaP cells, indicating that stromal TGF-β signaling induces both ligand-dependent and ligand-independent AR activation in PCa. TGF-β induces the expression of several growth factors and cytokines in prostate stromal cells, including IL-6, and BMP-6. Interestingly, BMP-6 and IL-6 together induces robust AR activation in these co-cultures, and neutralizing antibodies against BMP-6 and IL-6 attenuate this action. Altogether, our study strongly suggests tumor stromal microenvironment induced AR activation as a direct mechanism of CRPC. PMID:25333263

  10. Endocytosis of Seven-Transmembrane RGS Protein Activates G- protein Coupled Signaling in Arabidopsis

    PubMed Central

    Urano, Daisuke; Phan, Nguyen; Jones, Janice C.; Yang, Jing; Huang, Jirong; Grigston, Jeffrey; Taylor, J. Philip; Jones, Alan M.

    2012-01-01

    Signal transduction typically begins by ligand-dependent activation of a concomitant partner which is otherwise in its resting state. However, in cases where signal activation is constitutive by default, the mechanism of regulation is unknown. The Arabidopsis thaliana heterotrimeric Gα protein self-activates without accessory proteins, and is kept in its resting state by the negative regulator, AtRGS1 (Regulator of G protein Signaling 1), which is the prototype of a seven transmembrane receptor fused with an RGS domain. Endocytosis of AtRGS1 by ligand-dependent endocytosis physically uncouples the GTPase accelerating activity of AtRGS1 from the Gα protein, permitting sustained activation. Phosphorylation of AtRGS1 by AtWNK8 kinase causes AtRGS1 endocytosis, required both for G protein-mediated sugar signaling and cell proliferation. In animals, receptor endocytosis results in signal desensitization, whereas in plants, endocytosis results in signal activation. These findings reveal how different organisms rearrange a regulatory system to result in opposite outcomes using similar phosphorylation-dependent endocytosis. PMID:22940907

  11. Active transport improves the precision of linear long distance molecular signalling

    NASA Astrophysics Data System (ADS)

    Godec, Aljaž; Metzler, Ralf

    2016-09-01

    Molecular signalling in living cells occurs at low copy numbers and is thereby inherently limited by the noise imposed by thermal diffusion. The precision at which biochemical receptors can count signalling molecules is intimately related to the noise correlation time. In addition to passive thermal diffusion, messenger RNA and vesicle-engulfed signalling molecules can transiently bind to molecular motors and are actively transported across biological cells. Active transport is most beneficial when trafficking occurs over large distances, for instance up to the order of 1 metre in neurons. Here we explain how intermittent active transport allows for faster equilibration upon a change in concentration triggered by biochemical stimuli. Moreover, we show how intermittent active excursions induce qualitative changes in the noise in effectively one-dimensional systems such as dendrites. Thereby they allow for significantly improved signalling precision in the sense of a smaller relative deviation in the concentration read-out by the receptor. On the basis of linear response theory we derive the exact mean field precision limit for counting actively transported molecules. We explain how intermittent active excursions disrupt the recurrence in the molecular motion, thereby facilitating improved signalling accuracy. Our results provide a deeper understanding of how recurrence affects molecular signalling precision in biological cells and novel medical-diagnostic devices.

  12. Active regulation of receptor ratios controls integration of quorum-sensing signals in Vibrio harveyi

    PubMed Central

    Teng, Shu-Wen; Schaffer, Jessica N; Tu, Kimberly C; Mehta, Pankaj; Lu, Wenyun; Ong, N P; Bassler, Bonnie L; Wingreen, Ned S

    2011-01-01

    Quorum sensing is a chemical signaling mechanism used by bacteria to communicate and orchestrate group behaviors. Multiple feedback loops exist in the quorum-sensing circuit of the model bacterium Vibrio harveyi. Using fluorescence microscopy of individual cells, we assayed the activity of the quorum-sensing circuit, with a focus on defining the functions of the feedback loops. We quantitatively investigated the signaling input–output relation both in cells with all feedback loops present as well as in mutants with specific feedback loops disrupted. We found that one of the feedback loops regulates receptor ratios to control the integration of multiple signals. Together, the feedback loops affect the input–output dynamic range of signal transmission and the noise in the output. We conclude that V. harveyi employs multiple feedback loops to simultaneously control quorum-sensing signal integration and to ensure signal transmission fidelity. PMID:21613980

  13. New Insights into Glomerular Parietal Epithelial Cell Activation and Its Signaling Pathways in Glomerular Diseases

    PubMed Central

    Su, Hua; Chen, Shan; He, Fang-Fang; Wang, Yu-Mei; Bondzie, Philip; Zhang, Chun

    2015-01-01

    The glomerular parietal epithelial cells (PECs) have aroused an increasing attention recently. The proliferation of PECs is the main feature of crescentic glomerulonephritis; besides that, in the past decade, PEC activation has been identified in several types of noninflammatory glomerulonephropathies, such as focal segmental glomerulosclerosis, diabetic glomerulopathy, and membranous nephropathy. The pathogenesis of PEC activation is poorly understood; however, a few studies delicately elucidate the potential mechanisms and signaling pathways implicated in these processes. In this review we will focus on the latest observations and concepts about PEC activation in glomerular diseases and the newest identified signaling pathways in PEC activation. PMID:25866774

  14. Active control of shocks and sonic boom ground signal

    NASA Astrophysics Data System (ADS)

    Yagiz, Bedri

    The manipulation of a flow field to obtain a desired change is a much heightened subject. Active flow control has been the subject of the major research areas in fluid mechanics for the past two decades. It offers new solutions for mitigation of shock strength, sonic boom alleviation, drag minimization, reducing blade-vortex interaction noise in helicopters, stall control and the performance maximization of existing designs to meet the increasing requirements of the aircraft industries. Despite the wide variety of the potential applications of active flow control, the majority of studies have been performed at subsonic speeds. The active flow control cases were investigated in transonic speed in this study. Although the active flow control provides significant improvements, the sensibility of aerodynamic performance to design parameters makes it a nontrivial and expensive problem, so the designer has to optimize a number of different parameters. For the purpose of gaining understanding of the active flow control concepts, an automated optimization cycle process was generated. Also, the optimization cycle reduces cost and turnaround time. The mass flow coefficient, location, width and angle were chosen as design parameters to maximize the aerodynamic performance of an aircraft. As the main contribution of this study, a detailed parametric study and optimization process were presented. The second step is to appraise the practicability of weakening the shock wave and thereby reducing the wave drag in transonic flight regime using flow control devices such as two dimensional contour bump, individual jet actuator, and also the hybrid control which includes both control devices together, thereby gaining the desired improvements in aerodynamic performance of the air-vehicle. After this study, to improve the aerodynamic performance, the flow control and shape parameters are optimized separately, combined, and in a serial combination. The remarkable part of all these

  15. Nitric oxide and salicylic acid signaling in plant defense

    PubMed Central

    Klessig, Daniel F.; Durner, Jörg; Noad, Robert; Navarre, Duroy A.; Wendehenne, David; Kumar, Dhirendra; Zhou, Jun Ma; Shah, Jyoti; Zhang, Shuqun; Kachroo, Pradeep; Trifa, Youssef; Pontier, Dominique; Lam, Eric; Silva, Herman

    2000-01-01

    Salicylic acid (SA) plays a critical signaling role in the activation of plant defense responses after pathogen attack. We have identified several potential components of the SA signaling pathway, including (i) the H2O2-scavenging enzymes catalase and ascorbate peroxidase, (ii) a high affinity SA-binding protein (SABP2), (iii) a SA-inducible protein kinase (SIPK), (iv) NPR1, an ankyrin repeat-containing protein that exhibits limited homology to IκBα and is required for SA signaling, and (v) members of the TGA/OBF family of bZIP transcription factors. These bZIP factors physically interact with NPR1 and bind the SA-responsive element in promoters of several defense genes, such as the pathogenesis-related 1 gene (PR-1). Recent studies have demonstrated that nitric oxide (NO) is another signal that activates defense responses after pathogen attack. NO has been shown to play a critical role in the activation of innate immune and inflammatory responses in animals. Increases in NO synthase (NOS)-like activity occurred in resistant but not susceptible tobacco after infection with tobacco mosaic virus. Here we demonstrate that this increase in activity participates in PR-1 gene induction. Two signaling molecules, cGMP and cyclic ADP ribose (cADPR), which function downstream of NO in animals, also appear to mediate plant defense gene activation (e.g., PR-1). Additionally, NO may activate PR-1 expression via an NO-dependent, cADPR-independent pathway. Several targets of NO in animals, including guanylate cyclase, aconitase, and mitogen-activated protein kinases (e.g., SIPK), are also modulated by NO in plants. Thus, at least portions of NO signaling pathways appear to be shared between plants and animals. PMID:10922045

  16. cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice*

    PubMed Central

    Ma, Hongwei; Butler, Michael R.; Thapa, Arjun; Belcher, Josh; Yang, Fan; Baehr, Wolfgang; Biel, Martin; Michalakis, Stylianos; Ding, Xi-Qin

    2015-01-01

    Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca2+ channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP3R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3−/−/Nrl−/− mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP3R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca2+ channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency. PMID:26124274

  17. cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice.

    PubMed

    Ma, Hongwei; Butler, Michael R; Thapa, Arjun; Belcher, Josh; Yang, Fan; Baehr, Wolfgang; Biel, Martin; Michalakis, Stylianos; Ding, Xi-Qin

    2015-08-21

    Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca(2+) channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP3R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3(-/-)/Nrl(-/-) mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP3R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca(2+) channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency. PMID:26124274

  18. Crosstalk and Signaling Switches in Mitogen-Activated Protein Kinase Cascades

    PubMed Central

    Fey, Dirk; Croucher, David R.; Kolch, Walter; Kholodenko, Boris N.

    2012-01-01

    Mitogen-activated protein kinase (MAPK) cascades control cell fate decisions, such as proliferation, differentiation, and apoptosis by integrating and processing intra- and extracellular cues. However, similar MAPK kinetic profiles can be associated with opposing cellular decisions depending on cell type, signal strength, and dynamics. This implies that signaling by each individual MAPK cascade has to be considered in the context of the entire MAPK network. Here, we develop a dynamic model of feedback and crosstalk for the three major MAPK cascades; extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), c-Jun N-terminal kinase (JNK), and also include input from protein kinase B (AKT) signaling. Focusing on the bistable activation characteristics of the JNK pathway, this model explains how pathway crosstalk harmonizes different MAPK responses resulting in pivotal cell fate decisions. We show that JNK can switch from a transient to sustained activity due to multiple positive feedback loops. Once activated, positive feedback locks JNK in a highly active state and promotes cell death. The switch is modulated by the ERK, p38, and AKT pathways. ERK activation enhances the dual specificity phosphatase (DUSP) mediated dephosphorylation of JNK and shifts the threshold of the apoptotic switch to higher inputs. Activation of p38 restores the threshold by inhibiting ERK activity via the PP1 or PP2A phosphatases. Finally, AKT activation inhibits the JNK positive feedback, thus abrogating the apoptotic switch and allowing only proliferative signaling. Our model facilitates understanding of how cancerous deregulations disturb MAPK signal processing and provides explanations for certain drug resistances. We highlight a critical role of DUSP1 and DUSP2 expression patterns in facilitating the switching of JNK activity and show how oncogene induced ERK hyperactivity prevents the normal apoptotic switch explaining the failure of certain drugs to

  19. K+ efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ signaling1

    PubMed Central

    Katsnelson, Michael A.; Rucker, L. Graham; Russo, Hana M.; Dubyak, George R.

    2015-01-01

    Perturbation of intracellular ion homeostasis is a major cellular stress signal for activation of NLRP3 inflammasome signaling that results in caspase-1 mediated production of IL-1β and pyroptosis. However, the relative contributions of decreased cytosolic [K+] versus increased cytosolic [Ca2+] remain disputed and incompletely defined. We investigated roles for elevated cytosolic [Ca2+] in NLRP3 activation and downstream inflammasome signaling responses in primary murine dendritic cells and macrophages in response to two canonical NLRP3 agonists (ATP and nigericin) that facilitate primary K+ efflux by mechanistically distinct pathways or the lysosome-destabilizing agonist Leu-Leu-O-methyl ester (LLME). The study provides three major findings relevant to this unresolved area of NLRP3 regulation. First, increased cytosolic [Ca2+] was neither a necessary nor sufficient signal for the NLRP3 inflammasome cascade during activation by endogenous ATP-gated P2X7 receptor channels, the exogenous bacterial ionophore nigericin, or the lysosomotropic agent LLME. Second, agonists for three Ca2+-mobilizing G protein-coupled receptors (formyl peptide receptor/FPR; P2Y2 purinergic receptor/P2Y2R; calcium-sensing receptor/CaSR) expressed in murine dendritic cells were ineffective as activators of rapidly induced NLRP3 signaling when directly compared to the K+ efflux agonists. Third, the intracellular Ca2+ buffer, BAPTA, and the channel blocker, 2-aminoethoxydiphenyl borate (2-APB), widely used reagents for disruption of Ca2+-dependent signaling pathways, strongly suppressed nigericin-induced NLRP3 inflammasome signaling via mechanisms dissociated from their canonical or expected effects on Ca2+ homeostasis. The results indicate that the ability of K+ efflux agonists to activate NLRP3 inflammasome signaling can be dissociated from changes in cytosolic [Ca2+] as a necessary or sufficient signal. PMID:25762778

  20. Antibiotic-free production of a herpes simplex virus 2 DNA vaccine in a high yield cGMP process

    PubMed Central

    Nelson, Jared; Rodriguez, Stephen; Finlayson, Neil; Williams, Jim; Carnes, Aaron

    2013-01-01

    Two DNA vaccine plasmids encoding Herpes simplex virus type 2 (HSV-2) glycoprotein D, NTC8485-O2-gD2 and NTC8485-O2-UgD2tr, were produced at large scale under current good manufacturing practice (cGMP) for use in a Phase I human clinical trial. These DNA vaccines incorporate the regulatory agency compliant, minimal, antibiotic-free (AF) NTC8485 mammalian expression vector. Plasmid yields of > 1 g/L were achieved using the HyperGRO™ fed-batch fermentation process, with successful scale up from 10 L process development scale to 320 L culture volume for cGMP production. The DNA vaccines were purified using a low residence time, high shear lysis process and AIRMIXTM technology, followed by chromatographic purification. This combination of optimized plasmid vector, high yield upstream production, and efficient downstream purification resulted in purified HSV-2 DNA vaccines with > 99% total supercoiled plasmid, ≤ 0.2% RNA, ≤ 0.1% host cell genomic DNA, and ≤ 0.1 endotoxin units per mg. PMID:23899469

  1. cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness.

    PubMed

    Wiley, Luke A; Burnight, Erin R; DeLuca, Adam P; Anfinson, Kristin R; Cranston, Cathryn M; Kaalberg, Emily E; Penticoff, Jessica A; Affatigato, Louisa M; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

    2016-01-01

    Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans. PMID:27471043

  2. cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness

    PubMed Central

    Wiley, Luke A.; Burnight, Erin R.; DeLuca, Adam P.; Anfinson, Kristin R.; Cranston, Cathryn M.; Kaalberg, Emily E.; Penticoff, Jessica A.; Affatigato, Louisa M.; Mullins, Robert F.; Stone, Edwin M.; Tucker, Budd A.

    2016-01-01

    Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans. PMID:27471043

  3. Signal processing techniques for damage detection with piezoelectric wafer active sensors and embedded ultrasonic structural radar

    NASA Astrophysics Data System (ADS)

    Yu, Lingyu; Bao, Jingjing; Giurgiutiu, Victor

    2004-07-01

    Embedded ultrasonic structural radar (EUSR) algorithm is developed for using piezoelectric wafer active sensor (PWAS) array to detect defects within a large area of a thin-plate specimen. Signal processing techniques are used to extract the time of flight of the wave packages, and thereby to determine the location of the defects with the EUSR algorithm. In our research, the transient tone-burst wave propagation signals are generated and collected by the embedded PWAS. Then, with signal processing, the frequency contents of the signals and the time of flight of individual frequencies are determined. This paper starts with an introduction of embedded ultrasonic structural radar algorithm. Then we will describe the signal processing methods used to extract the time of flight of the wave packages. The signal processing methods being used include the wavelet denoising, the cross correlation, and Hilbert transform. Though hardware device can provide averaging function to eliminate the noise coming from the signal collection process, wavelet denoising is included to ensure better signal quality for the application in real severe environment. For better recognition of time of flight, cross correlation method is used. Hilbert transform is applied to the signals after cross correlation in order to extract the envelope of the signals. Signal processing and EUSR are both implemented by developing a graphical user-friendly interface program in LabView. We conclude with a description of our vision for applying EUSR signal analysis to structural health monitoring and embedded nondestructive evaluation. To this end, we envisage an automatic damage detection application utilizing embedded PWAS, EUSR, and advanced signal processing.

  4. Nandrolone reduces activation of Notch signaling in denervated muscle associated with increased Numb expression.

    PubMed

    Liu, Xin-Hua; Yao, Shen; Qiao, Rui-Fang; Levine, Alice C; Kirschenbaum, Alexander; Pan, Jiangping; Wu, Yong; Qin, Weiping; Bauman, William A; Cardozo, Christopher P

    2011-10-14

    Nandrolone, an anabolic steroid, slows denervation-atrophy in rat muscle. The molecular mechanisms responsible for this effect are not well understood. Androgens and anabolic steroids activate Notch signaling in animal models of aging and thereby mitigate sarcopenia. To explore the molecular mechanisms by which nandrolone prevents denervation-atrophy, we investigated the effects of nandrolone on Notch signaling in denervated rat gastrocnemius muscle. Denervation significantly increased Notch activity reflected by elevated levels of nuclear Notch intracellular domain (NICD) and expression of Hey1 (a Notch target gene). Activation was greatest at 7 and 35 days after denervation but remained present at 56 days after denervation. Activation of Notch in denervated muscle was prevented by nandrolone associated with upregulated expression of Numb mRNA and protein. These data demonstrate that denervation activates Notch signaling, and that nandrolone abrogates this response associated with increased expression of Numb, suggesting a potential mechanism by which nandrolone reduces denervation-atrophy. PMID:21945932

  5. Activation of Symbiosis Signaling by Arbuscular Mycorrhizal Fungi in Legumes and Rice[OPEN

    PubMed Central

    Sun, Jongho; Miller, J. Benjamin; Granqvist, Emma; Wiley-Kalil, Audrey; Gobbato, Enrico; Maillet, Fabienne; Cottaz, Sylvain; Samain, Eric; Venkateshwaran, Muthusubramanian; Fort, Sébastien; Morris, Richard J.; Ané, Jean-Michel; Dénarié, Jean; Oldroyd, Giles E.D.

    2015-01-01

    Establishment of arbuscular mycorrhizal interactions involves plant recognition of diffusible signals from the fungus, including lipochitooligosaccharides (LCOs) and chitooligosaccharides (COs). Nitrogen-fixing rhizobial bacteria that associate with leguminous plants also signal to their hosts via LCOs, the so-called Nod factors. Here, we have assessed the induction of symbiotic signaling by the arbuscular mycorrhizal (Myc) fungal-produced LCOs and COs in legumes and rice (Oryza sativa). We show that Myc-LCOs and tetra-acetyl chitotetraose (CO4) activate the common symbiosis signaling pathway, with resultant calcium oscillations in root epidermal cells of Medicago truncatula and Lotus japonicus. The nature of the calcium oscillations is similar for LCOs produced by rhizobial bacteria and by mycorrhizal fungi; however, Myc-LCOs activate distinct gene expression. Calcium oscillations were activated in rice atrichoblasts by CO4, but not the Myc-LCOs, whereas a mix of CO4 and Myc-LCOs activated calcium oscillations in rice trichoblasts. In contrast, stimulation of lateral root emergence occurred following treatment with Myc-LCOs, but not CO4, in M. truncatula, whereas both Myc-LCOs and CO4 were active in rice. Our work indicates that legumes and non-legumes differ in their perception of Myc-LCO and CO signals, suggesting that different plant species respond to different components in the mix of signals produced by arbuscular mycorrhizal fungi. PMID:25724637

  6. Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

    PubMed Central

    Olinto, S.C.F.; Adrião, M.G.; Castro-Barbosa, T.; Goulart-Silva, F.; Nunes, M.T.

    2012-01-01

    The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (∼250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (an NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression. PMID:22641416

  7. Hedgehog signal activation coordinates proliferation and differentiation of fetal liver progenitor cells

    SciTech Connect

    Hirose, Yoshikazu; Itoh, Tohru; Miyajima, Atsushi

    2009-09-10

    Hedgehog (Hh) signaling plays crucial roles in development and homeostasis of various organs. In the adult liver, it regulates proliferation and/or viability of several types of cells, particularly under injured conditions, and is also implicated in stem/progenitor cell maintenance. However, the role of this signaling pathway during the normal developmental process of the liver remains elusive. Although Sonic hedgehog (Shh) is expressed in the ventral foregut endoderm from which the liver derives, the expression disappears at the onset of the liver bud formation, and its possible recurrence at the later stages has not been investigated. Here we analyzed the activation and functional relevance of Hh signaling during the mouse fetal liver development. At E11.5, Shh and an activation marker gene for Hh signaling, Gli1, were expressed in Dlk{sup +} hepatoblasts, the fetal liver progenitor cells, and the expression was rapidly decreased thereafter as the development proceeded. In the culture of Dlk{sup +} hepatoblasts isolated from the E11.5 liver, activation of Hh signaling stimulated their proliferation and this effect was cancelled by a chemical Hh signaling inhibitor, cyclopamine. In contrast, hepatocyte differentiation of Dlk{sup +} hepatoblasts in vitro as manifested by the marker gene expression and acquisition of ammonia clearance activity was significantly inhibited by forced activation of Hh signaling. Taken together, these results demonstrate the temporally restricted manner of Hh signal activation and its role in promoting the hepatoblast proliferation, and further suggest that the pathway needs to be shut off for the subsequent hepatic differentiation of hepatoblasts to proceed normally.

  8. Pterygium epithelium abnormal differentiation related to activation of extracellular signal-regulated kinase signaling pathway in vitro

    PubMed Central

    Peng, Juan; Sha, Xiang-Yin; Liu, Yi; Yang, Rui-Ming; Wen, Ye

    2015-01-01

    AIM To investigate whether the abnormal differentiation of the pterygium epithelium is related to the extracellular signal-regulated kinase (ERK) signaling pathway in vitro. METHODS The expression levels of phosphorylated ERK (P-ERK), keratin family members including K19 and K10 and the ocular master control gene Pax-6 were measured in 16 surgically excised pterygium tissues and 12 eye bank conjunctiva. In colony-forming cell assays, the differences in clone morphology and in K10, K19, P-ERK and Pax-6 expression between the head and body were investigated. When cocultured with the ERK signaling pathway inhibitor PD98059, the changes in clone morphology, colony-forming efficiency, differentiated marker K10, K19 and Pax-6 expression and P-ERK protein expression level were examined by immunoreactivity and Western blot analysis. RESULTS The expression of K19 and Pax-6 decreased in the pterygium, especially in the head. No staining of K10 was found in the normal conjunctiva epithelium, but it was found to be expressed in the superficial cells in the head of the pterygium. Characteristic upregulation of P-ERK was observed by immunohistochemistry. The clone from the head with more differentiated cells in the center expressed more K10, and the clone from the body expressed more K19. The P-ERK protein level increased in the pterygium epithelium compared with conjunctiva and decreased when cocultured with PD98059. The same medium with the ERK inhibitor PD98059 was more effective in promoting clonal growth than conventional medium with 3T3 murine feeder layers. It was observed that the epithelium clone co-cultured with the inhibitor had decreased K10 expression and increased K19 and Pax-6 expression. CONCLUSION We suggest ERK signaling pathway activation might play a role in the pterygium epithelium abnormal differentiation. PMID:26682158

  9. MECHANISTIC PATHWAYS AND BIOLOGICAL ROLES FOR RECEPTOR-INDEPENDENT ACTIVATORS OF G-PROTEIN SIGNALING

    PubMed Central

    Blumer, Joe B.; Smrcka, Alan V.; Lanier, S.M.

    2007-01-01

    Signal processing via heterotrimeric G-proteins in response to cell surface receptors is a central and much investigated aspect of how cells integrate cellular stimuli to produce coordinated biological responses. The system is a target of numerous therapeutic agents, plays an important role in adaptive processes of organs, and aberrant processing of signals through these transducing systems is a component of various disease states. In addition to GPCR-mediated activation of G-protein signaling, nature has evolved creative ways to manipulate and utilize the Gαβγ heterotrimer or Gα and Gαβγ subunits independent of the cell surface receptor stimuli. In such situations, the G-protein subunits (Gα and Gαβγ) may actually be complexed with alternative binding partners independent of the typical heterotrimeric Gαβγ. Such regulatory accessory proteins include the family of RGS proteins that accelerate the GTPase activity of Gα and various entities that influence nucleotide binding properties and/or subunit interaction. The latter group of proteins includes receptor independent activators of G-protein signaling or AGS proteins that play surprising roles in signal processing. This review provides an overview of our current knowledge regarding AGS proteins. AGS proteins are indicative of a growing number of accessory proteins that influence signal propagation, facilitate cross talk between various types of signaling pathways and provide a platform for diverse functions of both the heterotrimeric Gαβγ and the individual Gα and Gαβγ subunits. PMID:17240454

  10. Calcium signaling in mammalian egg activation and embryo development: Influence of subcellular localization

    PubMed Central

    Miao, Yi-Liang; Williams, Carmen J.

    2012-01-01

    Calcium (Ca2+) signals drive the fundamental events surrounding fertilization and the activation of development in all species examined to date. Initial studies of Ca2+ signaling at fertilization in marine animals were tightly linked to new discoveries of bioluminescent proteins and their use as fluorescent Ca2+ sensors. Since that time, there has been rapid progress in our understanding of the key functions for Ca2+ in many cell types and the impact of cellular localization on Ca2+ signaling pathways. In this review, which focuses on mammalian egg activation, we consider how Ca2+ is regulated and stored at different stages of oocyte development and examine the functions of molecules that serve as both regulators of Ca2+ release and effectors of Ca2+ signals. We then summarize studies exploring how Ca2+ directs downstream effectors mediating both egg activation and later signaling events required for successful preimplantation embryo development. Throughout this review, we focus attention on how localization of Ca2+ signals influences downstream signaling events, and attempt to highlight gaps in our knowledge that are ripe areas for future research. PMID:22888043

  11. Hyperactivated Wnt signaling induces synthetic lethal interaction with Rb inactivation by elevating TORC1 activities.

    PubMed

    Zhang, Tianyi; Liao, Yang; Hsu, Fu-Ning; Zhang, Robin; Searle, Jennifer S; Pei, Xun; Li, Xuan; Ryoo, Hyung Don; Ji, Jun-Yuan; Du, Wei

    2014-05-01

    Inactivation of the Rb tumor suppressor can lead to increased cell proliferation or cell death depending on specific cellular context. Therefore, identification of the interacting pathways that modulate the effect of Rb loss will provide novel insights into the roles of Rb in cancer development and promote new therapeutic strategies. Here, we identify a novel synthetic lethal interaction between Rb inactivation and deregulated Wg/Wnt signaling through unbiased genetic screens. We show that a weak allele of axin, which deregulates Wg signaling and increases cell proliferation without obvious effects on cell fate specification, significantly alters metabolic gene expression, causes hypersensitivity to metabolic stress induced by fasting, and induces synergistic apoptosis with mutation of fly Rb ortholog, rbf. Furthermore, hyperactivation of Wg signaling by other components of the Wg pathway also induces synergistic apoptosis with rbf. We show that hyperactivated Wg signaling significantly increases TORC1 activity and induces excessive energy stress with rbf mutation. Inhibition of TORC1 activity significantly suppressed synergistic cell death induced by hyperactivated Wg signaling and rbf inactivation, which is correlated with decreased energy stress and decreased induction of apoptotic regulator expression. Finally the synthetic lethality between Rb and deregulated Wnt signaling is conserved in mammalian cells and that inactivation of Rb and APC induces synergistic cell death through a similar mechanism. These results suggest that elevated TORC1 activity and metabolic stress underpin the evolutionarily conserved synthetic lethal interaction between hyperactivated Wnt signaling and inactivated Rb tumor suppressor. PMID:24809668

  12. Sustained nitric oxide (NO)-releasing compound reverses dysregulated NO signal transduction in priapism

    PubMed Central

    Lagoda, Gwen; Sezen, Sena F.; Hurt, K. Joseph; Cabrini, Marcelo R.; Mohanty, Dillip K.; Burnett, Arthur L.

    2014-01-01

    We evaluated the therapeutic potential of a sustained nitric oxide (NO)-releasing compound to correct the molecular hallmarks and pathophysiology of priapism, an important but poorly characterized erectile disorder. 1,5-Bis-(dihexyl-N-nitrosoamino)-2,4-dinitrobenzene (C6′) and an inactive form of the compound [1,5-bis-(dihexylamino)-2,4-dinitrobenzene (C6)] were tested in neuronal cell cultures and penile lysates for NO release (Griess assay) and biological activity (cGMP production). The effect of local depot C6′ or C6 was evaluated in mice with a priapic phenotype due to double neuronal and endothelial NO synthase deletion (dNOS−/−) or human sickle hemoglobin transgenic expression (Sickle). Changes in NO signaling molecules and reactive oxygen species (ROS) surrogates were assessed by Western blot. The physiological response after C6′ treatment was assessed using an established model of electrically stimulated penile erection. C6′ generated NO, increased cGMP, and dose dependently increased NO metabolites. C6′ treatment reversed abnormalities in key penile erection signaling molecules, including phosphodiesterase type 5, phosphorylated endothelial nitric oxide synthase, and phosphorylated vasodilator-stimulated phosphoprotein. In Sickle mice, C6′ also attenuated the increased ROS markers gp91phox, 4-hydroxynonenal, and 3-nitrotyrosine. Finally, C6′ corrected the excessive priapic erection response of dNOS−/− mice. Exogenous sustained NO release from C6′ corrects pathological erectile signaling in mouse models of priapism and suggests novel approaches to human therapy.—Lagoda, G., Sezen, S. F., Hurt, K. J., Cabrini, M. R., Mohanty, D. K., Burnett, A. L. Sustained nitric oxide (NO)-releasing compound reverses dysregulated NO signal transduction in priapism. PMID:24076963

  13. Activation of Akt Signaling in Prostate Induces a TGFβ Mediated Restraint on Cancer Progression and Metastasis

    PubMed Central

    Bjerke, Glen A.; Yang, Chun-Song; Frierson, Henry F.; Paschal, Bryce M.; Wotton, David

    2014-01-01

    Mutations in the PTEN tumor suppressor gene are found in a high proportion of human prostate cancers, and in mice, Pten deletion induces high-grade prostate intra-epithelial neoplasia (HGPIN). However, progression from HGPIN to invasive cancer occurs slowly, suggesting that tumorigenesis is subject to restraint. We show that Pten deletion, or constitutive activation of the downstream kinase AKT, activates the transforming growth factor (TGF) β pathway in prostate epithelial cells. TGFβ signaling is known to play a tumor suppressive role in many cancer types, and reduced expression of TGFβ receptors correlates with advanced human prostate cancer. We demonstrate that in combination either with loss of Pten, or expression of constitutively active AKT1, inactivation of TGFβ signaling by deletion of the TGFβ type II receptor gene relieves a restraint on tumorigenesis. This results in rapid progession to lethal prostate cancer, including metastasis to lymph node and lung. In prostate epithelium, inactivation of TGFβ signaling alone is insufficient to initiate tumorigenesis, but greatly accelerates cancer progression. The activation of TGFβ signaling by Pten loss or AKT activation suggests that the same signaling events that play key roles in tumor initiation also induce the activity of a pathway that restrains disease progression. PMID:23995785

  14. Activation Dynamics and Signaling Properties of Notch3 Receptor in the Developing Pulmonary Artery*

    PubMed Central

    Ghosh, Shamik; Paez-Cortez, Jesus R.; Boppidi, Karthik; Vasconcelos, Michelle; Roy, Monideepa; Cardoso, Wellington; Ai, Xingbin; Fine, Alan

    2011-01-01

    Notch3 signaling is fundamental for arterial specification of systemic vascular smooth muscle cells (VSMCs). However, the developmental role and signaling properties of the Notch3 receptor in the mouse pulmonary artery remain unknown. Here, we demonstrate that Notch3 is expressed selectively in pulmonary artery VSMCs, is activated from late fetal to early postnatal life, and is required to maintain the morphological characteristics and smooth muscle gene expression profile of the pulmonary artery after birth. Using a conditional knock-out mouse model, we show that Notch3 receptor activation in VSMCs is Jagged1-dependent. In vitro VSMC lentivirus-mediated Jagged1 knockdown, confocal localization analysis, and co-culture experiments revealed that Notch3 activation is cell-autonomous and occurs through the physical engagement of Notch3 and VSMC-derived Jagged1 in the interior of the same cell. Although the current models of mammalian Notch signaling involve a two-cell system composed of a signal-receiving cell that expresses a Notch receptor on its surface and a neighboring signal-sending cell that provides membrane-bound activating ligand, our data suggest that pulmonary artery VSMC Notch3 activation is cell-autonomous. This unique mechanism of Notch activation may play an important role in the maturation of the pulmonary artery during the transition to air breathing. PMID:21536678

  15. Analysis of nitric oxide-cyclic guanosine monophosphate signaling during metamorphosis of the nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia)

    PubMed Central

    Bishop, Cory D.; Pires, Anthony; Norby, Shong-Wan; Boudko, Dmitri; Moroz, Leonid L.; Hadfield, Michael G.

    2014-01-01

    SUMMARY The gas nitric oxide (NO), and in some cases its downstream second messenger, cyclic guanosine monophosphate (cGMP) function in different taxa to regulate the timing of life-history transitions. Increased taxonomic sampling is required to foster conclusions about the evolution and function of NO/cGMP signaling during life-history transitions. We report on the function and localization of NO and cGMP signaling during metamorphosis of the nudibranch Phestilla sibogae. Pharmacological manipulation of NO or cGMP production in larvae modulated responses to a natural settlement cue from the coral Porites compressa in a manner that suggest inhibitory function for NO/cGMP signaling. However, these treatments were not sufficient to induce metamorphosis in the absence of cue, a result unique to this animal. We show that induction of metamorphosis in response to the settlement cue is associated with a reduction in NO production. We documented the expression of putative NO synthase (NOS) and the production of cGMP during larval development and observed no larval cells in which NOS and cGMP were both detected. The production of cGMP in a bilaterally symmetrical group of cells fated to occupy the distal tip of rhinophores is correlated with competence to respond to the coral settlement cue. These results suggest that endogenous NO and cGMP are involved in modulating responses of P. sibogae to a natural settlement cue. We discuss these results with respect to habitat selection and larval ecology. PMID:18460091

  16. Carbon nanotube-assisted optical activation of TGF-β signalling by near-infrared light

    NASA Astrophysics Data System (ADS)

    Lin, Liang; Liu, Ling; Zhao, Bing; Xie, Ran; Lin, Wei; Li, He; Li, Yaya; Shi, Minlong; Chen, Ye-Guang; Springer, Timothy A.; Chen, Xing

    2015-05-01

    Receptor-mediated signal transduction modulates complex cellular behaviours such as cell growth, migration and differentiation. Although photoactivatable proteins have emerged as a powerful tool for controlling molecular interactions and signalling cascades at precise times and spaces using light, many of these light-sensitive proteins are activated by ultraviolent or visible light, which has limited tissue penetration. Here, we report a single-walled carbon nanotube (SWCNT)-assisted approach that enables near-infrared light-triggered activation of transforming growth factor β (TGF-β) signal transduction, an important signalling pathway in embryonic development and cancer progression. The protein complex of TGF-β and its latency-associated peptide is conjugated onto SWCNTs, where TGF-β is inactive. Upon near-infrared irradiation, TGF-β is released through the photothermal effect of SWCNTs and becomes active. The released TGF-β activates downstream signal transduction in live cells and modulates cellular behaviours. Furthermore, preliminary studies show that the method can be used to mediate TGF-β signalling in living mice.

  17. Carbon nanotube-assisted optical activation of TGF-β signalling by near-infrared light.

    PubMed

    Lin, Liang; Liu, Ling; Zhao, Bing; Xie, Ran; Lin, Wei; Li, He; Li, Yaya; Shi, Minlong; Chen, Ye-Guang; Springer, Timothy A; Chen, Xing

    2015-05-01

    Receptor-mediated signal transduction modulates complex cellular behaviours such as cell growth, migration and differentiation. Although photoactivatable proteins have emerged as a powerful tool for controlling molecular interactions and signalling cascades at precise times and spaces using light, many of these light-sensitive proteins are activated by ultraviolent or visible light, which has limited tissue penetration. Here, we report a single-walled carbon nanotube (SWCNT)-assisted approach that enables near-infrared light-triggered activation of transforming growth factor β (TGF-β) signal transduction, an important signalling pathway in embryonic development and cancer progression. The protein complex of TGF-β and its latency-associated peptide is conjugated onto SWCNTs, where TGF-β is inactive. Upon near-infrared irradiation, TGF-β is released through the photothermal effect of SWCNTs and becomes active. The released TGF-β activates downstream signal transduction in live cells and modulates cellular behaviours. Furthermore, preliminary studies show that the method can be used to mediate TGF-β signalling in living mice. PMID:25775150

  18. Aldosterone Increases Oxidant Stress to Impair Guanylyl Cyclase Activity by Cysteinyl Thiol Oxidation in Vascular Smooth Muscle Cells*S⃞

    PubMed Central

    Maron, Bradley A.; Zhang, Ying-Yi; Handy, Diane E.; Beuve, Annie; Tang, Shiow-Shih; Loscalzo, Joseph; Leopold, Jane A.

    2009-01-01

    Hyperaldosteronism is associated with impaired endothelium-dependent vascular reactivity owing to increased reactive oxygen species and decreased bioavailable nitric oxide (NO·); however, the effects of aldosterone on vasodilatory signaling pathways in vascular smooth muscle cells (VSMC) remain unknown. Soluble guanylyl cyclase (GC) is a heterodimer that is activated by NO· to convert cytosolic GTP to cGMP, a second messenger required for normal VSMC relaxation. Here, we show that aldosterone (10-9-10-7 mol/liter) diminishes GC activity by activating NADPH oxidase in bovine aortic VSMC to increase reactive oxygen species levels and induce oxidative posttranslational modification(s) of Cys-122, a β1-subunit cysteinyl residue demonstrated previously to modulate NO· sensing by GC. In VSMC treated with aldosterone, Western immunoblotting detected evidence of GC β1-subunit disulfide bonding, whereas mass spectrometry analysis of a homologous peptide containing the Cys-122-bearing sequence exposed to conditions of increased oxidant stress confirmed cysteinyl sulfinic acid (m/z 435), sulfonic acid (m/z 443), and disulfide (m/z 836) bond formation. The functional effect of these modifications was examined by transfecting COS-7 cells with wild-type GC or mutant GC containing an alanine substitution at Cys-122 (C122A). Exposure to aldosterone or hydrogen peroxide (H2O2) significantly decreased cGMP levels in cells expressing wild-type GC. In contrast, aldosterone or H2O2 did not influence cGMP levels in cells expressing the mutant C122A GC, confirming that oxidative modification of Cys-122 specifically impairs GC activity. These findings demonstrate that pathophysiologically relevant concentrations of aldosterone increase oxidant stress to convert GC to an NO·-insensitive state, resulting in disruption of normal vasodilatory signaling pathways in VSMC. PMID:19141618

  19. ACTIVATION OF THE EGF RECEPTOR SIGNALING PATHWAY IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO METALS

    EPA Science Inventory

    We have previously shown that exposure to combustion-derived metals rapidly (within 20 min) activated mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), in the human bronchial epithelial cell line BEAS. To study the mechanisms respons...

  20. Presynaptic spinophilin tunes neurexin signalling to control active zone architecture and function

    PubMed Central

    Muhammad, Karzan; Reddy-Alla, Suneel; Driller, Jan H; Schreiner, Dietmar; Rey, Ulises; Böhme, Mathias A.; Hollmann, Christina; Ramesh, Niraja; Depner, Harald; Lützkendorf, Janine; Matkovic, Tanja; Götz, Torsten; Bergeron, Dominique D.; Schmoranzer, Jan; Goettfert, Fabian; Holt, Mathew; Wahl, Markus C.; Hell, Stefan W.; Scheiffele, Peter; Walter, Alexander M.; Loll, Bernhard; Sigrist, Stephan J.

    2015-01-01

    Assembly and maturation of synapses at the Drosophila neuromuscular junction (NMJ) depend on trans-synaptic neurexin/neuroligin signalling, which is promoted by the scaffolding protein Syd-1 binding to neurexin. Here we report that the scaffold protein spinophilin binds to the C-terminal portion of neurexin and is needed to limit neurexin/neuroligin signalling by acting antagonistic to Syd-1. Loss of presynaptic spinophilin results in the formation of excess, but atypically small active zones. Neuroligin-1/neurexin-1/Syd-1 levels are increased at spinophilin mutant NMJs, and removal of single copies of the neurexin-1, Syd-1 or neuroligin-1 genes suppresses the spinophilin-active zone phenotype. Evoked transmission is strongly reduced at spinophilin terminals, owing to a severely reduced release probability at individual active zones. We conclude that presynaptic spinophilin fine-tunes neurexin/neuroligin signalling to control active zone number and functionality, thereby optimizing them for action potential-induced exocytosis. PMID:26471740

  1. B cell activation involves nanoscale receptor reorganizations and inside-out signaling by Syk

    PubMed Central

    Kläsener, Kathrin; Maity, Palash C; Hobeika, Elias; Yang, Jianying; Reth, Michael

    2014-01-01

    Binding of antigen to the B cell antigen receptor (BCR) initiates a multitude of events resulting in B cell activation. How the BCR becomes signaling-competent upon antigen binding is still a matter of controversy. Using a high-resolution proximity ligation assay (PLA) to monitor the conformation of the BCR and its interactions with co-receptors at a 10–20 nm resolution, we provide direct evidence for the opening of BCR dimers during B cell activation. We also show that upon binding Syk opens the receptor by an inside-out signaling mechanism that amplifies BCR signaling. Furthermore, we found that on resting B cells, the coreceptor CD19 is in close proximity with the IgD-BCR and on activated B cells with the IgM-BCR, indicating nanoscale reorganization of receptor clusters during B cell activation. DOI: http://dx.doi.org/10.7554/eLife.02069.001 PMID:24963139

  2. Presynaptic spinophilin tunes neurexin signalling to control active zone architecture and function.

    PubMed

    Muhammad, Karzan; Reddy-Alla, Suneel; Driller, Jan H; Schreiner, Dietmar; Rey, Ulises; Böhme, Mathias A; Hollmann, Christina; Ramesh, Niraja; Depner, Harald; Lützkendorf, Janine; Matkovic, Tanja; Götz, Torsten; Bergeron, Dominique D; Schmoranzer, Jan; Goettfert, Fabian; Holt, Mathew; Wahl, Markus C; Hell, Stefan W; Scheiffele, Peter; Walter, Alexander M; Loll, Bernhard; Sigrist, Stephan J

    2015-01-01

    Assembly and maturation of synapses at the Drosophila neuromuscular junction (NMJ) depend on trans-synaptic neurexin/neuroligin signalling, which is promoted by the scaffolding protein Syd-1 binding to neurexin. Here we report that the scaffold protein spinophilin binds to the C-terminal portion of neurexin and is needed to limit neurexin/neuroligin signalling by acting antagonistic to Syd-1. Loss of presynaptic spinophilin results in the formation of excess, but atypically small active zones. Neuroligin-1/neurexin-1/Syd-1 levels are increased at spinophilin mutant NMJs, and removal of single copies of the neurexin-1, Syd-1 or neuroligin-1 genes suppresses the spinophilin-active zone phenotype. Evoked transmission is strongly reduced at spinophilin terminals, owing to a severely reduced release probability at individual active zones. We conclude that presynaptic spinophilin fine-tunes neurexin/neuroligin signalling to control active zone number and functionality, thereby optimizing them for action potential-induced exocytosis. PMID:26471740

  3. The regulation and function of the striated muscle activator of rho signaling (STARS) protein

    PubMed Central

    Wallace, Marita A.; Lamon, Séverine; Russell, Aaron P.

    2012-01-01

    Healthy living throughout the lifespan requires continual growth and repair of cardiac, smooth, and skeletal muscle. To effectively maintain these processes muscle cells detect extracellular stress signals and efficiently transmit them to activate appropriate intracellular transcriptional programs. The striated muscle activator of Rho signaling (STARS) protein, also known as Myocyte Stress-1 (MS1) protein and Actin-binding Rho-activating protein (ABRA) is highly enriched in cardiac, skeletal, and smooth muscle. STARS binds actin, co-localizes to the sarcomere and is able to stabilize the actin cytoskeleton. By regulating actin polymerization, STARS also controls an intracellular signaling cascade that stimulates the serum response factor (SRF) transcriptional pathway; a pathway controlling genes involved in muscle cell proliferation, differentiation, and growth. Understanding the activation, transcriptional control and biological roles of STARS in cardiac, smooth, and skeletal muscle, will improve our understanding of physiological and pathophysiological muscle development and function. PMID:23248604

  4. Pituitary Adenylate Cyclase-activating Polypeptide (PACAP)/PAC1HOP1 Receptor Activation Coordinates Multiple Neurotrophic Signaling Pathways

    PubMed Central

    May, Victor; Lutz, Eve; MacKenzie, Christopher; Schutz, Kristin C.; Dozark, Kate; Braas, Karen M.

    2010-01-01

    MAPK and Akt pathways are predominant mediators of trophic signaling for many neuronal systems. Among the vasoactive intestinal peptide/secretin/glucagon family of related peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) binding to specific PAC1 receptor isoforms can engage multiple signaling pathways and promote neuroprotection through mechanisms that are not well understood. Using a primary sympathetic neuronal system, the current studies demonstrate that PACAP activation of PAC1HOP1 receptors engages both MAPK and Akt neurotrophic pathways in an integrated program to facilitate neuronal survival after growth factor withdrawal. PACAP not only stimulated prosurvival ERK1/2 and ERK5 activation but also abrogated SAPK/JNK and p38 MAPK signaling in parallel. In contrast to the potent and rapid effects of PACAP in ERK1/2 phosphorylation, PACAP stimulated Akt phosphorylation in a late phase of PAC1HOP1 receptor signaling. From inhibitor and immunoprecipitation analyses, the PACAP/PAC1HOP1 receptor-mediated Akt responses did not represent transactivation mechanisms but appeared to depend on Gαq/phosphatidylinositol 3-kinase γ activity and vesicular internalization pathways. Phosphatidylinositol 3-kinase γ-selective inhibitors blocked PACAP-stimulated Akt phosphorylation in primary neuronal cultures and in PAC1HOP1-overexpressing cell lines; RNA interference-mediated knockdown of the receptor effectors attenuated PACAP-mediated Akt activation. Similarly, perturbation of endocytic pathways also blocked Akt phosphorylation. Between ERK and Akt pathways, PACAP-stimulated Akt signaling was the primary cascade that attenuated cultured neuron apoptosis after growth factor withdrawal. The partitioning of PACAP-mediated Akt signaling in endosomes may be a key mechanism contributing to the high spatial and temporal specificity in signal transduction necessary for survival pathways. PMID:20093365

  5. Control of macrophage metabolism and activation by mTOR and Akt signaling

    PubMed Central

    Covarrubias, Anthony J.; Aksoylar, H. Ibrahim; Horng, Tiffany

    2015-01-01

    Macrophages are pleiotropic cells that assume a variety of functions depending on their tissue of residence and tissue state. They maintain homeostasis as well as coordinate responses to stresses such as infection and metabolic challenge. The ability of macrophages to acquire diverse, context-dependent activities requires their activation (or polarization) to distinct functional states. While macrophage activation is well understood at the level of signal transduction and transcriptional regulation, the metabolic underpinnings are poorly understood. Importantly, emerging studies indicate that metabolic shifts play a pivotal role in control of macrophage activation and acquisition of context-dependent effector activities. The signals that drive macrophage activation impinge on metabolic pathways, allowing for coordinate control of macrophage activation and metabolism. Here we discuss how mTOR and Akt, major metabolic regulators and targets of such activation signals, control macrophage metabolism and activation. Dysregulated macrophage activities contribute to many diseases, including infectious, inflammatory, and metabolic diseases and cancer, thus a better understanding of metabolic control of macrophage activation could pave the way to the development of new therapeutic strategies. PMID:26360589

  6. Supramolecular organizing centers (SMOCs) as signaling machines in innate immune activation

    PubMed Central

    Qi, QIAO; Hao, WU

    2016-01-01

    Innate immunity offers the first line of defense against infections and other types of danger such as tumorigenesis. Its discovery provides tremendous therapeutic opportunities for numerous human diseases. Delving into the structural basis of signal transduction by innate immune receptors, our lab has recently helped to establish the new paradigm in which innate immune receptors transduce ligand-binding signals through formation of higher-order assemblies containing intracellular adapters, signaling enzymes and their substrates. These large signalosome assemblies may be visible under light microscopy as punctate structures in the μm scale, connecting to the underlying molecular structures in the nm scale. They drive proximity-induced enzyme activation, and provide a mechanism for signaling amplification by nucleated polymerization. These supramolecular signaling complexes also open new questions on their cellular organization and mode of regulation, pose challenges to our methodology, and afford valuable implications in drug discovery against these medically important pathways. PMID:26511517

  7. Supramolecular organizing centers (SMOCs) as signaling machines in innate immune activation.

    PubMed

    Qiao, Qi; Wu, Hao

    2015-11-01

    Innate immunity offers the first line of defense against infections and other types of danger such as tumorigenesis. Its discovery provides tremendous therapeutic opportunities for numerous human diseases. Delving into the structural basis of signal transduction by innate immune receptors, our lab has recently helped to establish the new paradigm in which innate immune receptors transduce ligand-binding signals through formation of higher-order assemblies containing intracellular adapters, signaling enzymes and their substrates. These large signalosome assemblies may be visible under light microscopy as punctate structures in the µm scale, connecting to the underlying molecular structures in the nm scale. They drive proximity-induced enzyme activation, and provide a mechanism for signaling amplification by nucleated polymerization. These supramolecular signaling complexes also open new questions on their cellular organization and mode of regulation, pose challenges to our methodology, and afford valuable implications in drug discovery against these medically important pathways. PMID:26511517

  8. Observed multivariable signals of late 20th and early 21st century volcanic activity

    NASA Astrophysics Data System (ADS)

    Santer, Benjamin D.; Solomon, Susan; Bonfils, Céline; Zelinka, Mark D.; Painter, Jeffrey F.; Beltran, Francisco; Fyfe, John C.; Johannesson, Gardar; Mears, Carl; Ridley, David A.; Vernier, Jean-Paul; Wentz, Frank J.

    2015-01-01

    The relatively muted warming of the surface and lower troposphere since 1998 has attracted considerable attention. One contributory factor to this "warming hiatus" is an increase in volcanically induced cooling over the early 21st century. Here we identify the signals of late 20th and early 21st century volcanic activity in multiple observed climate variables. Volcanic signals are statistically discernible in spatial averages of tropical and near-global SST, tropospheric temperature, net clear-sky short-wave radiation, and atmospheric water vapor. Signals of late 20th and early 21st century volcanic eruptions are also detectable in near-global averages of rainfall. In tropical average rainfall, however, only a Pinatubo-caused drying signal is identifiable. Successful volcanic signal detection is critically dependent on removal of variability induced by the El Niño-Southern Oscillation.

  9. The Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway as a Discovery Target in Stroke.

    PubMed

    Sun, Jing; Nan, Guangxian

    2016-05-01

    Protein kinases are critical modulators of a variety of intracellular and extracellular signal transduction pathways, and abnormal phosphorylation events can contribute to disease progression in a variety of diseases. As a result, protein kinases have emerged as important new drug targets for small molecule therapeutics. The mitogen-activated protein kinase (MAPK) signaling pathway transmits signals from the cell membrane to the nucleus in response to a variety of different stimuli. Because this pathway controls a broad spectrum of cellular processes, including growth, inflammation, and stress responses, it is accepted as a therapeutic target for cancer and peripheral inflammatory disorders. There is also increasing evidence that MAPK is an important regulator of ischemic and hemorrhagic cerebral vascular disease, raising the possibility that it might be a drug discovery target for stroke. In this review, we discuss the MAPK signaling pathway in association with its activation in stroke-induced brain injury. PMID:26842916

  10. Extraction of motion strength and motor activity signals from video recordings of neonatal seizures.

    PubMed

    Karayiannis, N B; Srinivasan, S; Bhattacharya, R; Wise, M S; Frost, J D; Mizrahi, E M

    2001-09-01

    This paper presents two methods developed to extract quantitative information from video recordings of neonatal seizures in the form of temporal motion strength and motor activity signals. Motion strength signals are extracted by measuring the area of the body parts that move during the seizure and the relative speed of motion using a combination of spatiotemporal subband decomposition of video, nonlinear filtering, and segmentation. Motor activity signals are extracted by tracking selected anatomical sites during the seizure using a modified version of a feature-tracking procedure developed for video, known as the Kanade-Lucas-Tomasi (KLT) algorithm. The experiments indicate that the temporal signals produced by the proposed methods provide the basis for differentiating myoclonic from focal clonic seizures and distinguishing these types of neonatal seizures from normal infant behaviors. PMID:11585212

  11. miR-302 Attenuates Amyloid-β-Induced Neurotoxicity through Activation of Akt Signaling.

    PubMed

    Li, Hsin-Hua; Lin, Shi-Lung; Huang, Chien-Ning; Lu, Fung-Jou; Chiu, Pai-Yi; Huang, Wen-Nung; Lai, Te-Jen; Lin, Chih-Li

    2016-01-01

    Deficiency of insulin signaling has been linked to diabetes and ageing-related neurodegenerative diseases such as Alzheimer's disease (AD). In this regard, brains exhibit defective insulin receptor substrate-1 (IRS-1) and hence result in alteration of insulin signaling in progression of AD, the most common cause of dementia. Consequently, dysregulation of insulin signaling plays an important role in amyloid-β (Aβ)-induced neurotoxicity. As the derivation of induced pluripotent stem cells (iPSC) involves cell reprogramming, it may provide a means for regaining the control of ageing-associated dysfunction and neurodegeneration via affecting insulin-related signaling. To this, we found that an embryonic stem cell (ESC)-specific microRNA, miR-302, silences phosphatase and tensin homolog (PTEN) to activate Akt signaling, which subsequently stimulates nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) elevation and hence inhibits Aβ-induced neurotoxicity. miR-302 is predominantly expressed in iPSCs and is known to regulate several important biological processes of anti-oxidative stress, anti-apoptosis, and anti-aging through activating Akt signaling. In addition, we also found that miR-302-mediated Akt signaling further stimulates Nanog expression to suppress Aβ-induced p-Ser307 IRS-1 expression and thus enhances tyrosine phosphorylation and p-Ser 473-Akt/p-Ser 9-GSK3β formation. Furthermore, our in vivo studies revealed that the mRNA expression levels of both Nanog and miR-302-encoding LARP7 genes were significantly reduced in AD patients' blood cells, providing a novel diagnosis marker for AD. Taken together, our findings demonstrated that miR-302 is able to inhibit Aβ-induced cytotoxicity via activating Akt signaling to upregulate Nrf2 and Nanog expressions, leading to a marked restoration of insulin signaling in AD neurons. PMID:26890744

  12. Cooperativity between MAPK and PI3K signaling activation is required for glioblastoma pathogenesis

    PubMed Central

    Vitucci, Mark; Karpinich, Natalie O.; Bash, Ryan E.; Werneke, Andrea M.; Schmid, Ralf S.; White, Kristen K.; McNeill, Robert S.; Huff, Byron; Wang, Sophie; Van Dyke, Terry; Miller, C. Ryan

    2013-01-01

    Background Glioblastoma (GBM) genomes feature recurrent genetic alterations that dysregulate core intracellular signaling pathways, including the G1/S cell cycle checkpoint and the MAPK and PI3K effector arms of receptor tyrosine kinase (RTK) signaling. Elucidation of the phenotypic consequences of activated RTK effectors is required for the design of effective therapeutic and diagnostic strategies. Methods Genetically defined, G1/S checkpoint-defective cortical murine astrocytes with constitutively active Kras and/or Pten deletion mutations were used to systematically investigate the individual and combined roles of these 2 RTK signaling effectors in phenotypic hallmarks of glioblastoma pathogenesis, including growth, migration, and invasion in vitro. A novel syngeneic orthotopic allograft model system was used to examine in vivo tumorigenesis. Results Constitutively active Kras and/or Pten deletion mutations activated both MAPK and PI3K signaling. Their combination led to maximal growth, migration, and invasion of G1/S-defective astrocytes in vitro and produced progenitor-like transcriptomal profiles that mimic human proneural GBM. Activation of both RTK effector arms was required for in vivo tumorigenesis and produced highly invasive, proneural-like GBM. Conclusions These results suggest that cortical astrocytes can be transformed into GBM and that combined dysregulation of MAPK and PI3K signaling revert G1/S-defective astrocytes to a primitive gene expression state. This genetically-defined, immunocompetent model of proneural GBM will be useful for preclinical development of MAPK/PI3K-targeted, subtype-specific therapies. PMID:23814263

  13. Integrin-Dependent Activation of the JNK Signaling Pathway by Mechanical Stress

    PubMed Central

    Kanger, Johannes S.; Subramaniam, Vinod; Martin-Blanco, Enrique

    2011-01-01

    Mechanical force is known to modulate the activity of the Jun N-terminal kinase (JNK) signaling cascade. However, the effect of mechanical stresses on JNK signaling activation has previously only been analyzed by in vitro detection methods. It still remains unknown how living cells activate the JNK signaling cascade in response to mechanical stress and what its functions are in stretched cells. We assessed in real-time the activity of the JNK pathway in Drosophila cells by Fluorescence Lifetime Imaging Microscopy (FLIM), using an intramolecular phosphorylation-dependent dJun-FRET (Fluorescence Resonance Energy Transfer) biosensor. We found that quantitative FRET-FLIM analysis and confocal microscopy revealed sustained dJun-FRET biosensor activation and stable morphology changes in response to mechanical stretch for Drosophila S2R+ cells. Further, these cells plated on different substrates showed distinct levels of JNK activity that associate with differences in cell morphology, integrin expression and focal adhesion organization. These data imply that alterations in the cytoskeleton and matrix attachments may act as regulators of JNK signaling, and that JNK activity might feed back to modulate the cytoskeleton and cell adhesion. We found that this dynamic system is highly plastic; at rest, integrins at focal adhesions and talin are key factors suppressing JNK activity, while multidirectional static stretch leads to integrin-dependent, and probably talin-independent, Jun sensor activation. Further, our data suggest that JNK activity has to coordinate with other signaling elements for the regulation of the cytoskeleton and cell shape remodeling associated with stretch. PMID:22180774

  14. Evaluation of the effect of signalment and body conformation on activity monitoring in companion dogs

    PubMed Central

    Brown, Dorothy Cimino; Michel, Kathryn E.; Love, Molly; Dow, Caitlin

    2015-01-01

    Objective To evaluate the effect of signalment and body conformation on activity monitoring in companion dogs. Animals 104 companion dogs. Procedures While wearing an activity monitor, each dog was led through a series of standard activities: lying down, walking laps, trotting laps, and trotting up and down stairs. Linear regression analysis was used to determine which signalment and body conformation factors were associated with activity counts. Results There was no significant effect of signalment or body conformation on activity counts when dogs were lying down, walking laps, and trotting laps. However, when dogs were trotting up and down stairs, there was a significant effect of age and body weight such that, for every 1-kg increase in body weight, there was a 1.7% (95% confidence interval, 1.1% to 2.4%) decrease in activity counts and for every 1-year increase in age, there was a 4.2% (95% confidence interval, 1.4% to 6.9%) decrease in activity counts. Conclusions and Clinical Relevance When activity was well controlled, there was no significant effect of signalment or body conformation on activity counts recorded by the activity monitor. However, when activity was less controlled, older dogs and larger dogs had lower activity counts than younger and smaller dogs. The wide range in body conformation (eg, limb or body length) among dogs did not appear to significantly impact the activity counts recorded by the monitor, but age and body weight did and must be considered in analysis of data collected from the monitors. PMID:20187834

  15. Signaling Interactions in the Adrenal Cortex

    PubMed Central

    Spät, András; Hunyady, László; Szanda, Gergő

    2016-01-01

    The major physiological stimuli of aldosterone secretion are angiotensin II (AII) and extracellular K+, whereas cortisol production is primarily regulated by corticotropin (ACTH) in fasciculata cells. AII triggers Ca2+ release from internal stores that is followed by store-operated and voltage-dependent Ca2+ entry, whereas K+-evoked depolarization activates voltage-dependent Ca2+ channels. ACTH acts primarily through the formation of cAMP and subsequent protein phosphorylation by protein kinase A. Both Ca2+ and cAMP facilitate the transfer of cholesterol to mitochondrial inner membrane. The cytosolic Ca2+ signal is transferred into the mitochondrial matrix and enhances pyridine nucleotide reduction. Increased formation of NADH results in increased ATP production, whereas that of NADPH supports steroid production. In reality, the control of adrenocortical function is a lot more sophisticated with second messengers crosstalking and mutually modifying each other’s pathways. Cytosolic Ca2+ and cGMP are both capable of modifying cAMP metabolism, while cAMP may enhance Ca2+ release and voltage-activated Ca2+ channel activity. Besides, mitochondrial Ca2+ signal brings about cAMP formation within the organelle and this further enhances aldosterone production. Maintained aldosterone and cortisol secretion are optimized by the concurrent actions of Ca2+ and cAMP, as exemplified by the apparent synergism of Ca2+ influx (inducing cAMP formation) and Ca2+ release during response to AII. Thus, cross-actions of parallel signal transducing pathways are not mere intracellular curiosities but rather substantial phenomena, which fine-tune the biological response. Our review focuses on these functionally relevant interactions between the Ca2+ and the cyclic nucleotide signal transducing pathways hitherto described in the adrenal cortex. PMID:26973596

  16. Biased signalling from the glucocorticoid receptor: Renewed opportunity for tailoring glucocorticoid activity.

    PubMed

    Keenan, Christine R; Lew, Michael J; Stewart, Alastair G

    2016-07-15

    Recent landmark studies applying analytical pharmacology approaches to the glucocorticoid receptor (GR) have demonstrated that different ligands can cause differential activation of distinct GR-regulated genes. Drawing on concepts of signalling bias from the field of G protein-coupled receptor (GPCR) biology, we speculate that ligand-dependent differences in GR signalling can be considered analogous to GPCR biased signalling, and thus can be quantitatively analysed in a similar way. This type of approach opens up the possibility of using rational structure-based drug optimisation strategies to improve the therapeutic selectivity of glucocorticoid drugs to maximise their efficacy and minimise adverse effects. PMID:26898958

  17. The functional activity of hypothalamic signaling systems in rats with neonatal diabetes mellitus treated with metformin.

    PubMed

    Derkach, K V; Sukhov, I B; Kuznetsova, L A; Buzanakov, D M; Shpakov, A O

    2016-03-01

    The effect of the two-month metformin treatment (200 mg/kg/day) of rats with the neonatal model of type 2 diabetes mellitus on the functional activity of hypothalamic signaling systems was studied. It was shown that metformin treatment restored the sensitivity of hypothalamic adenylyl cyclase signaling system to agonists of the type 4 melanocortin receptor and the type 2 dopamine receptor but did not influence significantly the functions of the insulin signaling system. These data suggest new targets and mechanisms of metformin action in the CNS, which may mediate its restoring effect on energy homeostasis impaired in diabetic pathology. PMID:27193707

  18. Nitrated Cyclic GMP Modulates Guard Cell Signaling in Arabidopsis[W

    PubMed Central

    Joudoi, Takahiro; Shichiri, Yudai; Kamizono, Nobuto; Akaike, Takaaki; Sawa, Tomohiro; Yoshitake, Jun; Yamada, Naotaka; Iwai, Sumio

    2013-01-01

    Nitric oxide (NO) is a ubiquitous signaling molecule involved in diverse physiological processes, including plant senescence and stomatal closure. The NO and cyclic GMP (cGMP) cascade is the main NO signaling pathway in animals, but whether this pathway operates in plant cells, and the mechanisms of its action, remain unclear. Here, we assessed the possibility that the nitrated cGMP derivative 8-nitro-cGMP functions in guard cell signaling. Mass spectrometry and immunocytochemical analyses showed that abscisic acid and NO induced the synthesis of 8-nitro-cGMP in guard cells in the presence of reactive oxygen species. 8-Nitro-cGMP triggered stomatal closure, but 8-bromoguanosine 3′,5′-cyclic monophosphate (8-bromo-cGMP), a membrane-permeating analog of cGMP, did not. However, in the dark, 8-bromo-cGMP induced stomatal opening but 8-nitro-cGMP did not. Thus, cGMP and its nitrated derivative play different roles in the signaling pathways that lead to stomatal opening and closure. Moreover, inhibitor and genetic studies showed that calcium, cyclic adenosine-5′-diphosphate-ribose, and SLOW ANION CHANNEL1 act downstream of 8-nitro-cGMP. This study therefore demonstrates that 8-nitro-cGMP acts as a guard cell signaling molecule and that a NO/8-nitro-cGMP signaling cascade operates in guard cells. PMID:23396828

  19. A Gaussian process framework for modelling stellar activity signals in radial velocity data

    NASA Astrophysics Data System (ADS)

    Rajpaul, V.; Aigrain, S.; Osborne, M. A.; Reece, S.; Roberts, S.

    2015-09-01

    To date, the radial velocity (RV) method has been one of the most productive techniques for detecting and confirming extrasolar planetary candidates. Unfortunately, stellar activity can induce RV variations which can drown out or even mimic planetary signals - and it is notoriously difficult to model and thus mitigate the effects of these activity-induced nuisance signals. This is expected to be a major obstacle to using next-generation spectrographs to detect lower mass planets, planets with longer periods, and planets around more active stars. Enter Gaussian processes (GPs) which, we note, have a number of attractive features that make them very well suited to disentangling stellar activity signals from planetary signals. We present here a GP framework we developed to model RV time series jointly with ancillary activity indicators (e.g. bisector velocity spans, line widths, chromospheric activity indices), allowing the activity component of RV time series to be constrained and disentangled from e.g. planetary components. We discuss the mathematical details of our GP framework, and present results illustrating its encouraging performance on both synthetic and real RV data sets, including the publicly available Alpha Centauri B data set.

  20. Fn14, a Downstream Target of the TGF-β Signaling Pathway, Regulates Fibroblast Activation

    PubMed Central

    Yang, Min; Lai, Wen; Ye, Litong; Chen, Jing; Hou, Xinghua; Ding, Hong; Zhang, Wenwei; Wu, Yueheng; Liu, Xiaoying; Huang, Shufang; Yu, Xiyong; Xiao, Dingzhang

    2015-01-01

    Fibrosis, the hallmark of human injuries and diseases such as serious burns, is characterized by excessive collagen synthesis and myofibroblast accumulation. Transforming growth factor-β (TGF-β), a potent inducer of collagen synthesis, has been implicated in fibrosis in animals. In addition to TGF-β, fibroblast growth factor-inducible molecule 14 (Fn14) has been reported to play an important role in fibrotic diseases, such as cardiac fibrosis. However, the function and detailed regulatory mechanism of Fn14 in fibrosis are unclear. Here, we investigated the effect of Fn14 on the activation of human dermal fibroblasts. In normal dermal fibroblasts, TGF-β signaling increased collagen production and Fn14 expression. Furthermore, Fn14 siRNA blocked extracellular matrix gene expression; even when TGF-β signaling was activated by TGF-β1, fibroblast activation remained blocked in the presence of Fn14 siRNA. Overexpressing Fn14 increased extracellular matrix gene expression. In determining the molecular regulatory mechanism, we discovered that SMAD4, an important TGF-β signaling co-mediator, bound to the Fn14 promoter and activated Fn14 transcription. Taken together, these results indicate that the TGF-β signaling pathway activates Fn14 expression through the transcription factor SMAD4 and that activated Fn14 expression increases extracellular matrix synthesis and fibroblast activation. Therefore, Fn14 may represent a promising approach to preventing the excessive accumulation of collagen or ECM in skin fibrosis. PMID:26625141

  1. Subcellular optogenetic activation of Cdc42 controls local and distal signaling to drive immune cell migration

    PubMed Central

    O’Neill, Patrick R.; Kalyanaraman, Vani; Gautam, N.

    2016-01-01

    Migratory immune cells use intracellular signaling networks to generate and orient spatially polarized responses to extracellular cues. The monomeric G protein Cdc42 is believed to play an important role in controlling the polarized responses, but it has been difficult to determine directly the consequences of localized Cdc42 activation within an immune cell. Here we used subcellular optogenetics to determine how Cdc42 activation at one side of a cell affects both cell behavior and dynamic molecular responses throughout the cell. We found that localized Cdc42 activation is sufficient to generate polarized signaling and directional cell migration. The optically activated region becomes the leading edge of the cell, with Cdc42 activating Rac and generating membrane protrusions driven by the actin cytoskeleton. Cdc42 also exerts long-range effects that cause myosin accumulation at the opposite side of the cell and actomyosin-mediated retraction of the cell rear. This process requires the RhoA-activated kinase ROCK, suggesting that Cdc42 activation at one side of a cell triggers increased RhoA signaling at the opposite side. Our results demonstrate how dynamic, subcellular perturbation of an individual signaling protein can help to determine its role in controlling polarized cellular responses. PMID:26941336

  2. Subcellular optogenetic activation of Cdc42 controls local and distal signaling to drive immune cell migration.

    PubMed

    O'Neill, Patrick R; Kalyanaraman, Vani; Gautam, N

    2016-05-01

    Migratory immune cells use intracellular signaling networks to generate and orient spatially polarized responses to extracellular cues. The monomeric G protein Cdc42 is believed to play an important role in controlling the polarized responses, but it has been difficult to determine directly the consequences of localized Cdc42 activation within an immune cell. Here we used subcellular optogenetics to determine how Cdc42 activation at one side of a cell affects both cell behavior and dynamic molecular responses throughout the cell. We found that localized Cdc42 activation is sufficient to generate polarized signaling and directional cell migration. The optically activated region becomes the leading edge of the cell, with Cdc42 activating Rac and generating membrane protrusions driven by the actin cytoskeleton. Cdc42 also exerts long-range effects that cause myosin accumulation at the opposite side of the cell and actomyosin-mediated retraction of the cell rear. This process requires the RhoA-activated kinase ROCK, suggesting that Cdc42 activation at one side of a cell triggers increased RhoA signaling at the opposite side. Our results demonstrate how dynamic, subcellular perturbation of an individual signaling protein can help to determine its role in controlling polarized cellular responses. PMID:26941336

  3. Recombinant TCR ligand induces early TCR signaling and a unique pattern of downstream activation.

    PubMed

    Wang, Chunhe; Mooney, Jeffery L; Meza-Romero, Roberto; Chou, Yuan K; Huan, Jianya; Vandenbark, Arthur A; Offner, Halina; Burrows, Gregory G

    2003-08-15

    Recombinant TCR ligands (RTLs) consisting of covalently linked alpha(1) and beta(1) domains of MHC class II molecules tethered to specific antigenic peptides represent minimal TCR ligands. In a previous study we reported that the rat RTL201 construct, containing RT1.B MHC class II domains covalently coupled to the encephalitogenic guinea pig myelin basic protein (Gp-MBP(72-89)) peptide, could prevent and treat actively and passively induced experimental autoimmune encephalomyelitis in vivo by selectively inhibiting Gp-MBP(72-89) peptide-specific CD4(+) T cells. To evaluate the inhibitory signaling pathway, we tested the effects of immobilized RTL201 on T cell activation of the Gp-MBP(72-89)-specific A1 T cell hybridoma. Activation was exquisitely Ag-specific and could not be induced by RTL200 containing the rat MBP(72-89) peptide that differed by a threonine for serine substitution at position 80. Partial activation by RTL201 included a CD3zeta p23/p21 ratio shift, ZAP-70 phosphorylation, calcium mobilization, NFAT activation, and transient IL-2 production. In comparison, anti-CD3epsilon treatment produced stronger activation of these cellular events with additional activation of NF-kappaB and extracellular signal-regulated kinases as well as long term increased IL-2 production. These results demonstrate that RTLs can bind directly to the TCR and modify T cell behavior through a partial activation mechanism, triggering specific downstream signaling events that deplete intracellular calcium stores without fully activating T cells. The resulting Ag-specific activation of the transcription factor NFAT uncoupled from the activation of NF-kappaB or extracellular signal-regulated kinases constitutes a unique downstream activation pattern that accounts for the inhibitory effects of RTL on encephalitogenic CD4(+) T cells. PMID:12902496

  4. Fatty acid-induced NLRP3-PYCARD inflammasome activation interferes with insulin signaling

    PubMed Central

    Wen, Haitao; Gris, Denis; Lei, Yu; Jha, Sushmita; Zhang, Lu; Huang, Max Tze-Han; Brickey, Willie June; Ting, Jenny P.-Y.

    2014-01-01

    High-fat diet (HFD) and inflammation are key contributors to insulin resistance and type 2 diabetes (T2D). Interleukin (IL)-1β plays a role in insulin resistance; yet, how IL-1β is induced by fatty acid with HFD, and how this alters insulin signaling is unclear. We show that the saturated fatty acid, palmitate, but not unsaturated oleate, induces the activation of NLRP3-PYCARD inflammasome, causing caspase-1, IL-1β, and IL-18 production. This involves mitochondrial reactive oxygen species and the AMP-activated protein kinase and ULK1 autophagy signaling cascade. Inflammasome activation in hematopoietic cells impairs insulin signaling in several target tissues to reduce glucose tolerance and insulin sensitivity. Furthermore, IL-1β affects insulin sensitivity via TNF-independent and dependent pathways. These findings provide insights into the association of inflammation, diet and T2D. PMID:21478880

  5. Phagocyte respiratory burst activates macrophage erythropoietin signalling to promote acute inflammation resolution.

    PubMed

    Luo, Bangwei; Wang, Jinsong; Liu, Zongwei; Shen, Zigang; Shi, Rongchen; Liu, Yu-Qi; Liu, Yu; Jiang, Man; Wu, Yuzhang; Zhang, Zhiren

    2016-01-01

    Inflammation resolution is an active process, the failure of which causes uncontrolled inflammation which underlies many chronic diseases. Therefore, endogenous pathways that regulate inflammation resolution are fundamental and of wide interest. Here, we demonstrate that phagocyte respiratory burst-induced hypoxia activates macrophage erythropoietin signalling to promote acute inflammation resolution. This signalling is activated following acute but not chronic inflammation. Pharmacological or genetical inhibition of the respiratory burst suppresses hypoxia and macrophage erythropoietin signalling. Macrophage-specific erythropoietin receptor-deficient mice and chronic granulomatous disease (CGD) mice, which lack the capacity for respiratory burst, display impaired inflammation resolution, and exogenous erythropoietin enhances this resolution in WT and CGD mice. Mechanistically, erythropoietin increases macrophage engulfment of apoptotic neutrophils via PPARγ, promotes macrophage removal of debris and enhances macrophage migration to draining lymph nodes. Together, our results provide evidences of an endogenous pathway that regulates inflammation resolution, with important implications for treating inflammatory conditions. PMID:27397585

  6. PAK1 is a breast cancer oncogene that coordinately activates MAPK and MET signaling

    PubMed Central

    Shrestha, Yashaswi; Schafer, Eric J.; Boehm, Jesse S.; Thomas, Sapana R.; He, Frank; Du, Jinyan; Wang, Shumei; Barretina, Jordi; Weir, Barbara A.; Zhao, Jean J.; Polyak, Kornelia; Golub, Todd R.; Beroukhim, Rameen; Hahn, William C.

    2011-01-01

    Activating mutations in the RAS family or BRAF frequently occur in many types of human cancers but are rarely detected in breast tumors. However, activation of the RAS-RAF-MEK-ERK Mitogen-Activated Protein Kinase (MAPK) pathway is commonly observed in human breast cancers, suggesting that other genetic alterations lead to activation of this signaling pathway. To identify breast cancer oncogenes that activate the MAPK pathway, we screened a library of human kinases for their ability to induce anchorage-independent growth in a derivative of immortalized human mammary epithelial cells (HMLE). We identified PAK1 as a kinase that permitted HMLE cells to form anchorage-independent colonies. PAK1 is amplified in several human cancer types, including 33% of breast tumor samples and cancer cell lines. The kinase activity of PAK1 is necessary for PAK1-induced transformation. Moreover, we show that PAK1 simultaneously activates MAPK and MET signaling; the latter via inhibition of Merlin. Disruption of these activities inhibits PAK1-driven anchorage-independent growth. These observations establish PAK1 amplification as an alternative mechanism for MAPK activation in human breast cancer and credential PAK1 as a breast cancer oncogene that coordinately regulates multiple signaling pathways, the cooperation of which leads to malignant transformation. PMID:22105362

  7. Ras-activated Dsor1 promotes Wnt signaling in Drosophila development.

    PubMed

    Hall, Eric T; Verheyen, Esther M

    2015-12-15

    Wnt/Wingless (Wg) and Ras-MAPK signaling both play fundamental roles in growth and cell fate determination, and when dysregulated, can lead to tumorigenesis. Several conflicting modes of interaction between Ras-MAPK and Wnt signaling have been identified in specific cellular contexts, causing synergistic or antagonistic effects on target genes. We find novel evidence that the Drosophila homolog of the dual specificity kinases MEK1/2 (also known as MAP2K1/2), Downstream of Raf1 (Dsor1), is required for Wnt signaling. Knockdown of Dsor1 results in loss of Wg target gene expression, as well as reductions in stabilized Armadillo (Arm; Drosophila β-catenin). We identify a close physical interaction between Dsor1 and Arm, and find that catalytically inactive Dsor1 causes a reduction in active Arm. These results suggest that Dsor1 normally counteracts the Axin-mediated destruction of Arm. We find that Ras-Dsor1 activity is independent of upstream activation by EGFR, and instead it appears to be activated by the insulin-like growth factor receptor to promote Wg signaling. Taken together, our results suggest that there is a new crosstalk pathway between insulin and Wg signaling that is mediated by Dsor1. PMID:26542023

  8. A high-content screening assay in transgenic zebrafish identifies two novel activators of fgf signaling.

    PubMed

    Saydmohammed, Manush; Vollmer, Laura L; Onuoha, Ezenwa Obi; Vogt, Andreas; Tsang, Michael

    2011-09-01

    Zebrafish have become an invaluable vertebrate animal model to interrogate small molecule libraries for modulators of complex biological pathways and phenotypes. We have recently described the implementation of a quantitative, high-content imaging assay in multi-well plates to analyze the effects of small molecules on Fibroblast Growth Factor (FGF) signaling in vivo. Here we have evaluated the capability of the assay to identify compounds that hyperactivate FGF signaling from a test cassette of agents with known biological activities. Using a transgenic zebrafish reporter line for FGF activity, we screened 1040 compounds from an annotated library of known bioactive agents, including FDA-approved drugs. The assay identified two molecules, 8-hydroxyquinoline sulfate and pyrithione zinc, that enhanced FGF signaling in specific areas of the brain. Subsequent studies revealed that both compounds specifically expanded FGF target gene expression. Furthermore, treatment of early stage embryos with either compound resulted in dorsalized phenotypes characteristic of hyperactivation of FGF signaling in early development. Documented activities for both agents included activation of extracellular signal-related kinase (ERK), consistent with FGF hyperactivation. To conclude, we demonstrate the power of automated quantitative high-content imaging to identify small molecule modulators of FGF. PMID:21932436

  9. Differential Signaling by Protease-Activated Receptors: Implications for Therapeutic Targeting

    PubMed Central

    Sidhu, Tejminder S.; French, Shauna L.; Hamilton, Justin R.

    2014-01-01

    Protease-activated receptors (PARs) are a family of four G protein-coupled receptors that exhibit increasingly appreciated differences in signaling and regulation both within and between the receptor class. By nature of their proteolytic self-activation mechanism, PARs have unique processes of receptor activation, “ligand” binding, and desensitization/resensitization. These distinctive aspects have presented both challenges and opportunities in the targeting of PARs for therapeutic benefit—the most notable example of which is inhibition of PAR1 on platelets for the prevention of arterial thrombosis. However, more recent studies have uncovered further distinguishing features of PAR-mediated signaling, revealing mechanisms by which identical proteases elicit distinct effects in the same cell, as well as how distinct proteases produce different cellular consequences via the same receptor. Here we review this differential signaling by PARs, highlight how important distinctions between PAR1 and PAR4 are impacting on the progress of a new class of anti-thrombotic drugs, and discuss how these more recent insights into PAR signaling may present further opportunities for manipulating PAR activation and signaling in the development of novel therapies. PMID:24733067

  10. Ionizing Radiation Shifts the PAI-1/ID-1 Balance and Activates Notch Signaling in Endothelial Cells

    SciTech Connect

    Scharpfenecker, Marion; Kruse, Jacqueline; Sprong, Debbie; Russell, Nicola S.; Dijke, Peter ten; Stewart, Fiona A.

    2009-02-01

    Purpose: Transforming growth factor-{beta} (TGF-{beta}) and Notch signaling pathways are important regulators of vascular homeostasis and vessel remodeling; mutations in these pathways can lead to vascular disorders. Similar vascular phenotypes develop in the normal tissues of cancer patients as a long-term effect of radiotherapy. Irradiation most severely affects the capillaries, which become leaky and dilated and might eventually rupture. To investigate the mechanism of such capillary damage, we studied the effect of TGF-{beta} and Notch signaling in microvascular endothelial cells. Methods and Materials: Human microvascular endothelial cells were irradiated with 5 or 10 Gy and activation of TGF-{beta} and Notch signaling pathways was assessed by biochemical methods and a cell migration assay. Results: Ionizing radiation induced Smad2 phosphorylation and nuclear translocation and increased mRNA and protein expression of the activin-like kinase 5 (ALK5) target gene plasminogen activator inhibitor-1 (PAI-1). At the same time, we observed diminished Smad1/5/8 activation and downregulation of the ALK1 downstream target, inhibitor of DNA binding-1 (ID-1). We also measured an upregulation of the Notch ligand Jagged-1 and the target gene Hey1. Decreased inhibitor of DNA binding-1 levels coincided with a reduced ability of the cells to migrate. Conclusion: Ionizing radiation shifts the balance from ALK1 to ALK5 signaling and activates the Notch pathway in endothelial cells. This combination of anti-angiogenic signals contributes to reduced cell migration after irradiation.

  11. Head direction cell activity in mice: robust directional signal depends on intact otolith organs.

    PubMed

    Yoder, Ryan M; Taube, Jeffrey S

    2009-01-28

    The head direction (HD) cell signal is a representation of an animal's perceived directional heading with respect to its environment. This signal appears to originate in the vestibular system, which includes the semicircular canals and otolith organs. Preliminary studies indicate the semicircular canals provide a necessary component of the HD signal, but involvement of otolithic information in the HD signal has not been tested. The present study was designed to determine the otolithic contribution to the HD signal, as well as to compare HD cell activity of mice with that of rats. HD cell activity in the anterodorsal thalamus was assessed in wild-type C57BL/6J and otoconia-deficient tilted mice during locomotion within a cylinder containing a prominent visual landmark. HD cell firing properties in C57BL/6J mice were generally similar to those in rats. However, in C57BL/6J mice, landmark rotation failed to demonstrate dominant control of the HD signal in 36% of the sessions. In darkness, directional firing became unstable during 42% of the sessions, but landmark control was not associated with HD signal stability in darkness. HD cells were identified in tilted mice, but directional firing properties were not as robust as those of C57BL/6J mice. Most HD cells in tilted mice were controlled by landmark rotation but showed substantial signal degradation across trials. These results support current models that suggest otolithic information is involved in the perception of directional heading. Furthermore, compared with rats, the HD signal in mice appears to be less reliably anchored to prominent environmental cues. PMID:19176815

  12. IL-1 signaling modulates STAT activation to antagonize retinoic acid signaling and control Th17–iTreg balance

    PubMed Central

    Basu, Rajatava; Whitley, Sarah K.; Bhaumik, Suniti; Zindl, Carlene L.; Schoeb, Trenton R.; Benveniste, Etty N.; Pear, Warren S.; Hatton, Robin D.; Weaver, Casey T.

    2016-01-01

    Interleukin 17 (IL-17)-producing helper (TH17) and inducible regulatory CD4+ T (iTreg) cells emerge from an overlapping developmental program. In the intestines, the vitamin A metabolite retinoic acid (RA) is produced at steady state and acts as an important cofactor to induce iTreg cell development while potently inhibiting TH17 development. Here, we found that IL-1 was required to fully override RA-mediated Foxp3 expression and induce protective TH17 responses. Through induction of an NF-κB-dependent repression of SOCS3 expression, IL-1 increased the amplitude and duration of STAT3 phosphorylation induced by TH17-polarizing cytokines, leading to an altered balance of STAT3–STAT5 binding to shared consensus sequences in developing T cells. Thus, IL-1 signaling differentially modulated STAT activation downstream of cytokine receptors to control TH17–iTreg developmental fate. PMID:25642823

  13. Canonical Wnt Signaling Activity in Early Stages of Chick Lung Development

    PubMed Central

    daMota, Paulo; Correia-Pinto, Jorge

    2014-01-01

    Wnt signaling pathway is an essential player during vertebrate embryonic development which has been associated with several developmental processes such as gastrulation, body axis formation and morphogenesis of numerous organs, namely the lung. Wnt proteins act through specific transmembrane receptors, which activate intracellular pathways that regulate cellular processes such as cell proliferation, differentiation and death. Morphogenesis of the fetal lung depends on epithelial-mesenchymal interactions that are governed by several growth and transcription factors that regulate cell proliferation, fate, migration and differentiation. This process is controlled by different signaling pathways such as FGF, Shh and Wnt among others. Wnt signaling is recognized as a key molecular player in mammalian pulmonary development but little is known about its function in avian lung development. The present work characterizes, for the first time, the expression pattern of several Wnt signaling members, such as wnt-1, wnt-2b, wnt-3a, wnt-5a, wnt-7b, wnt-8b, wnt-9a, lrp5, lrp6, sfrp1, dkk1, β-catenin and axin2 at early stages of chick lung development. In general, their expression is similar to their mammalian counterparts. By assessing protein expression levels of active/total β-catenin and phospho-LRP6/LRP6 it is revealed that canonical Wnt signaling is active in this embryonic tissue. In vitro inhibition studies were performed in order to evaluate the function of Wnt signaling pathway in lung branching. Lung explants treated with canonical Wnt signaling inhibitors (FH535 and PK115-584) presented an impairment of secondary branch formation after 48 h of culture along with a decrease in axin2 expression levels. Branching analysis confirmed this inhibition. Wnt-FGF crosstalk assessment revealed that this interaction is preserved in the chick lung. This study demonstrates that Wnt signaling is crucial for precise chick lung branching and further supports the avian lung as a good

  14. 17β-Oestradiol enhances the expansion and activation of myeloid-derived suppressor cells via signal transducer and activator of transcription (STAT)-3 signalling in human pregnancy.

    PubMed

    Pan, T; Zhong, L; Wu, S; Cao, Y; Yang, Q; Cai, Z; Cai, X; Zhao, W; Ma, N; Zhang, W; Zhang, H; Zhou, J

    2016-07-01

    During a successful pregnancy, the maternal immune system plays a critical role in maintaining immunotolerance towards semi-allogeneic fetal antigens. Recent studies have indicated that myeloid-derived suppressor cells (MDSCs) are active players in establishing fetal-maternal tolerance; however, the underlying mechanism remains poorly understood. In this study, we observed a significant expansion of monocytic MDSCs (M-MDSCs) in the peripheral blood of pregnant women, which suppressed T cell responses in a reactive oxygen species-dependent manner and required cell-cell contact. The number of M-MDSCs correlated positively with serum oestrogen and progesterone levels. Administration of 17β-oestradiol, but not progesterone, enhanced both the expansion and suppressive activity of M-MDSCs through signal transducer and activator of transcription (STAT)-3. Pretreatment with STAT-3 inhibitor JSI-124 almost completely abrogated the effects of 17β-oestradiol on MDSCs. Collectively, these results demonstrate that 17β-oestradiol-induced STAT-3 signalling plays an important role in both the expansion and activation of MDSCs during human pregnancy, which may benefit the development of novel therapeutic strategies for prevention of immune-related miscarriage. PMID:26969967

  15. Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

    NASA Astrophysics Data System (ADS)

    House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

    2015-06-01

    Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

  16. A novel reporter of notch signalling indicates regulated and random notch activation during vertebrate neurogenesis

    PubMed Central

    2011-01-01

    Background Building the complex vertebrate nervous system involves the regulated production of neurons and glia while maintaining a progenitor cell population. Neurogenesis starts asynchronously in different regions of the embryo and occurs over a long period of time, allowing progenitor cells to be exposed to multiple extrinsic signals that regulate the production of different cell types. Notch-mediated cell-cell signalling is one of the mechanisms that maintain the progenitor pool, however, little is known about how the timing of Notch activation is related to the cell cycle and the distinct modes of cell division that generate neurons. An essential tool with which to investigate the role of Notch signalling on cell by cell basis is the development a faithful reporter of Notch activity. Results Here we present a novel reporter for Notch activity based on the promoter of the well characterised Notch target chick Hes5-1, coupled with multiple elements that confer instability, including a destabilized nuclear Venus fluorescent protein and the 3' untranslated region (UTR) of Hes5-1. We demonstrate that this reporter faithfully recapitulates the endogenous expression of Hes5-1 and that it robustly responds to Notch activation in the chick neural tube. Analysis of the patterns of Notch activity revealed by this reporter indicates that although Notch is most frequently activated prior to mitosis it can be activated at any time within the cell cycle. Notch active progenitors undergoing mitosis generate two daughters that both continue to experience Notch signalling. However, cells lacking Notch activity before and during mitosis generate daughters with dissimilar Notch activity profiles. Conclusions A novel Notch reporter with multiple destabilisation elements provides a faithful read-out of endogenous Notch activity on a cell-by-cell basis, as neural progenitors progress through the cell cycle in the chick neural tube. Notch activity patterns in this cell population

  17. Process development and production of cGMP grade Melan-A for cancer vaccine clinical trials.

    PubMed

    Bardliving, Cameron L; Lowe, Adam J; Huang, Chung-Jr; Manley, Laura; Ritter, Gerd; Old, Lloyd; Batt, Carl A

    2013-12-01

    Melan-A is a cancer testis antigen commonly found in melanoma, and has been shown to stimulate the body's immune response against cancerous cells. We have developed and executed a process utilizing current good manufacturing practices (cGMP) to produce the 6 times-His tagged protein in C41DE3 Escherichia coli for use in Phase I clinical trials. Approximately 11 g of purified Melan-A were produced from a 20 L fed-batch fermentation. Purification was achieved through a three column process utilizing immobilized metal affinity, anion exchange, and cation exchange chromatography with a buffer system optimized for low-solubility, high LPS binding capacity proteins. The host cell proteins, residual DNA, and endotoxin concentration were well below limits for a prescribed dose with a final purity level of 91%. PMID:24045055

  18. Aurora A drives early signalling and vesicle dynamics during T-cell activation

    PubMed Central

    Blas-Rus, Noelia; Bustos-Morán, Eugenio; Pérez de Castro, Ignacio; de Cárcer, Guillermo; Borroto, Aldo; Camafeita, Emilio; Jorge, Inmaculada; Vázquez, Jesús; Alarcón, Balbino; Malumbres, Marcos; Martín-Cófreces, Noa B.; Sánchez-Madrid, Francisco

    2016-01-01

    Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3ζ-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal. PMID:27091106

  19. Aurora A drives early signalling and vesicle dynamics during T-cell activation.

    PubMed

    Blas-Rus, Noelia; Bustos-Morán, Eugenio; Pérez de Castro, Ignacio; de Cárcer, Guillermo; Borroto, Aldo; Camafeita, Emilio; Jorge, Inmaculada; Vázquez, Jesús; Alarcón, Balbino; Malumbres, Marcos; Martín-Cófreces, Noa B; Sánchez-Madrid, Francisco

    2016-01-01

    Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3ζ-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal. PMID:27091106

  20. Gastrointestinal pain: unraveling a novel endogenous pathway through uroguanylin/guanylate cyclase-C/cGMP activation.

    PubMed

    Silos-Santiago, Inmaculada; Hannig, Gerhard; Eutamene, Helene; Ustinova, Elena E; Bernier, Sylvie G; Ge, Pei; Graul, Christopher; Jacobson, Sarah; Jin, Hong; Liong, Elaine; Kessler, Marco M; Reza, Tammi; Rivers, Samuel; Shea, Courtney; Tchernychev, Boris; Bryant, Alexander P; Kurtz, Caroline B; Bueno, Lionel; Pezzone, Michael A; Currie, Mark G

    2013-09-01

    The natural hormone uroguanylin regulates intestinal fluid homeostasis and bowel function through activation of guanylate cyclase-C (GC-C), resulting in increased intracellular cyclic guanosine-3',5'-monophosphate (cGMP). We report the effects of uroguanylin-mediated activation of the GC-C/cGMP pathway in vitro on extracellular cGMP transport and in vivo in rat models of inflammation- and stress-induced visceral hypersensitivity. In vitro exposure of intestinal Caco-2 cells to uroguanylin stimulated bidirectional, active extracellular transport of cGMP into luminal and basolateral spaces. cGMP transport was significantly and concentration dependently decreased by probenecid, an inhibitor of cGMP efflux pumps. In ex vivo Ussing chamber assays, uroguanylin stimulated cGMP secretion from the basolateral side of rat colonic epithelium into the submucosal space. In a rat model of trinitrobenzene sulfonic acid (TNBS)-induced visceral hypersensitivity, orally administered uroguanylin increased colonic thresholds required to elicit abdominal contractions in response to colorectal distension (CRD). Oral administration of cGMP mimicked the antihyperalgesic effects of uroguanylin, significantly decreasing TNBS- and restraint stress-induced visceromotor response to graded CRD in rats. The antihyperalgesic effects of cGMP were not associated with increased colonic spasmolytic activity, but were linked to significantly decreased firing rates of TNBS-sensitized colonic afferents in rats in response to mechanical stimuli. In conclusion, these data suggest that the continuous activation of the GC-C/cGMP pathway along the intestinal tract by the endogenous hormones guanylin and uroguanylin results in significant reduction of gastrointestinal pain. Extracellular cGMP produced on activation of GC-C is the primary mediator in this process via modulation of sensory afferent activity. PMID:23748116

  1. Activation of aryl hydrocarbon receptor signaling by extracts of teak and other wood dusts.

    PubMed

    Wilson, Mark J; Sabbioni, Gabriele; Rando, Roy; Miller, Charles A

    2015-12-01

    Wood dusts, as a group, are categorized as known human carcinogens, but the risks of exposure to specific types of wood dusts and the carcinogenic chemicals they contain are not well studied. Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is linked to the carcinogenic action of specific classes of chemicals. Here we examined whether chemicals in various wood dusts had the potential to activate AhR signaling as a potential toxic mechanism of action. We found that methanol extracts of teak, walnut, mahogany, and poplar dusts contained a wide range of AhR ligand activity, whereas extracts of oak, pine, and other softwoods did not contain appreciable activity. Teak dust extract, being particularly potent, was subjected to chemical analysis. The 2-methylanthraquinone (2-MAQ) accounted for the AhR ligand activity and was present at an average concentration of 0.27 parts per hundred in teak dust. Pure 2-MAQ potently induced AhR signaling (EC50 115 nM), confirming that this was the active ligand. Aqueous extracts of teak dust made using yeast or mammalian cell culture medium also contained robust AhR activity, suggesting the 2-MAQ ligand is soluble at bioactive concentrations in physiologically relevant fluids. The high concentration and potency of 2-MAQ in teak wood suggest it may mediate toxic effects through activation of AhR signaling in exposed wood workers. PMID:24898320

  2. Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: improving the efficiency of cell line generation.

    PubMed

    Porter, Alison J; Racher, Andrew J; Preziosi, Richard; Dickson, Alan J

    2010-01-01

    Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide-ranging transfectant population. This identification process is on the critical path for first-in-human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One-hundred and seventy-five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines. PMID:20623584

  3. Clicks, whistles and pulses: Passive and active signal use in dolphin communication

    NASA Astrophysics Data System (ADS)

    Herzing, Denise L.

    2014-12-01

    The search for signals out of noise is a problem not only with radio signals from the sky but in the study of animal communication. Dolphins use multiple modalities to communicate including body postures, touch, vision, and most elaborately sound. Like SETI radio signal searches, dolphin sound analysis includes the detection, recognition, analysis, and interpretation of signals. Dolphins use both passive listening and active production to communicate. Dolphins use three main types of acoustic signals: frequency modulated whistles (narrowband with harmonics), echolocation (broadband clicks) and burst pulsed sounds (packets of closely spaced broadband clicks). Dolphin sound analysis has focused on frequency-modulated whistles, yet the most commonly used signals are burst-pulsed sounds which, due to their graded and overlapping nature and bimodal inter-click interval (ICI) rates are hard to categorize. We will look at: 1) the mechanism of sound production and categories of sound types, 2) sound analysis techniques and information content, and 3) examples of lessons learned in the study of dolphin acoustics. The goal of this paper is to provide perspective on how animal communication studies might provide insight to both passive and active SETI in the larger context of searching for life signatures.

  4. Boehmenan, a lignan from Hibiscus ficulneus, showed Wnt signal inhibitory activity.

    PubMed

    Shono, Takumi; Ishikawa, Naoki; Toume, Kazufumi; Arai, Midori A; Ahmed, Firoj; Sadhu, Samir K; Ishibashi, Masami

    2015-07-15

    The Wnt signal pathway modulates numerous biological processes, and its aberrant activation is related to various diseases. Therefore, inhibition of the Wnt signal may provide an effective (or efficient) strategy for these diseases. Cell-based luciferase assay targeting the Wnt signal (TOP assay) revealed that Hibiscus ficulneus extract inhibited the Wnt signal. The activity-guided isolation of the MeOH extract of H. ficulneus stems yielded four known (1-4) lignans along with myriceric acid (5). Compounds 1-4 potently inhibited the Wnt signal with TOPflash IC50 values of 1.0, 4.5, 6.3, and 1.9 μM, respectively. Compound 1 exhibited cytotoxicity against both Wnt-dependent (HCT116) and Wnt-independent (RKO) cells. Western blot analysis showed that 1 decreased the expression of full, cytosolic and nuclear β-catenin along with c-myc in STF/293 cells. Our results suggested that 1 may have inhibited the Wnt signal by decreasing β-catenin levels. PMID:26026364

  5. Hedgehog signaling activation induces stem cell proliferation and hormone release in the adult pituitary gland

    PubMed Central

    Pyczek, Joanna; Buslei, Rolf; Schult, David; Hölsken, Annett; Buchfelder, Michael; Heß, Ina; Hahn, Heidi; Uhmann, Anja

    2016-01-01

    Hedgehog (HH) signaling is known to be essential during the embryonal development of the pituitary gland but the knowledge about its role in the adult pituitary and in associated tumors is sparse. In this report we investigated the effect of excess Hh signaling activation in murine pituitary explants and analyzed the HH signaling status of human adenopituitary lobes and a large cohort of pituitary adenomas. Our data show that excess Hh signaling led to increased proliferation of Sox2+ and Sox9+ adult pituitary stem cells and to elevated expression levels of adrenocorticotropic hormone (Acth), growth hormone (Gh) and prolactin (Prl) in the adult gland. Inhibition of the pathway by cyclopamine reversed these effects indicating that active Hh signaling positively regulates proliferative processes of adult pituitary stem cells and hormone production in the anterior pituitary. Since hormone producing cells of the adenohypophysis as well as ACTH-, GH- and PRL-immunopositive adenomas express SHH and its target GLI1, we furthermore propose that excess HH signaling is involved in the development/maintenance of hormone-producing pituitary adenomas. These findings advance the understanding of physiological hormone regulation and may open new treatment options for pituitary tumors. PMID:27109116

  6. Hedgehog signaling activation induces stem cell proliferation and hormone release in the adult pituitary gland.

    PubMed

    Pyczek, Joanna; Buslei, Rolf; Schult, David; Hölsken, Annett; Buchfelder, Michael; Heß, Ina; Hahn, Heidi; Uhmann, Anja

    2016-01-01

    Hedgehog (HH) signaling is known to be essential during the embryonal development of the pituitary gland but the knowledge about its role in the adult pituitary and in associated tumors is sparse. In this report we investigated the effect of excess Hh signaling activation in murine pituitary explants and analyzed the HH signaling status of human adenopituitary lobes and a large cohort of pituitary adenomas. Our data show that excess Hh signaling led to increased proliferation of Sox2(+) and Sox9(+) adult pituitary stem cells and to elevated expression levels of adrenocorticotropic hormone (Acth), growth hormone (Gh) and prolactin (Prl) in the adult gland. Inhibition of the pathway by cyclopamine reversed these effects indicating that active Hh signaling positively regulates proliferative processes of adult pituitary stem cells and hormone production in the anterior pituitary. Since hormone producing cells of the adenohypophysis as well as ACTH-, GH- and PRL-immunopositive adenomas express SHH and its target GLI1, we furthermore propose that excess HH signaling is involved in the development/maintenance of hormone-producing pituitary adenomas. These findings advance the understanding of physiological hormone regulation and may open new treatment options for pituitary tumors. PMID:27109116

  7. TRIM5 Retroviral Restriction Activity Correlates with the Ability To Induce Innate Immune Signaling

    PubMed Central

    Lascano, Josefina; Uchil, Pradeep D.; Mothes, Walther

    2015-01-01

    ABSTRACT Host restriction factor TRIM5 inhibits retroviral transduction in a species-specific manner by binding to and destabilizing the retroviral capsid lattice before reverse transcription is completed. However, the restriction mechanism may not be that simple since TRIM5 E3 ubiquitin ligase activity, the proteasome, autophagy, and TAK1-dependent AP-1 signaling have been suggested to contribute to restriction. Here, we show that, among a panel of seven primate and Carnivora TRIM5 orthologues, each of which has potential for potent retroviral restriction activity, all activated AP-1 signaling. In contrast, TRIM family paralogues most closely related to TRIM5 did not. While each primate species has a single TRIM5 gene, mice have at least seven TRIM5 homologues that cluster into two groups, Trim12a, -b, and -c and Trim30a, -b, -c, and -d. The three Trim12 proteins activated innate immune signaling, while the Trim30 proteins did not, though none of the murine Trim5 homologues restricted any of a panel of cloned retroviruses. To determine if any mouse TRIM5 homologues had potential for restriction activity, each was fused to the human immunodeficiency virus type 1 (HIV-1) CA binding protein cyclophilin A (CypA). The three Trim12-CypA fusions all activated AP-1 and restricted HIV-1 transduction, whereas the Trim30-CypA fusions did neither. AP-1 activation and HIV-1 restriction by the Trim12-CypA fusions were inhibited by disruption of TAK1. Overall then, these experiments demonstrate that there is a strong correlation between TRIM5 retroviral restriction activity and the ability to activate TAK1-dependent innate immune signaling. IMPORTANCE The importance of retroviruses for the evolution of susceptible host organisms cannot be overestimated. Eight percent of the human genome is retrovirus sequence, fixed in the germ line during past infection. Understanding how metazoa protect their genomes from mutagenic retrovirus infection is therefore of fundamental importance to

  8. Signal transduction pathways in mast cell granule-mediated endothelial cell activation.

    PubMed Central

    Chi, Luqi; Stehno-Bittel, Lisa; Smirnova, Irina; Stechschulte, Daniel J; Dileepan, Kottarappat N

    2003-01-01

    BACKGROUND: We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8. AIMS: The objective of the present study was to identify candidate molecules and signal transduction pathways involved in the synergy between mast cell granules and lipopolysaccharide on endothelial cell activation. METHODS: Human umbilical vein endothelial cells were incubated with rat mast cell granules in the presence and absence of lipopolysaccharide, and IL-6 production was quantified. The status of c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2 activation, nuclear factor-kappaB translocation and intracellular calcium levels were determined to identify the mechanism of synergy between mast cell granules and lipopolysaccaride. RESULTS: Mast cell granules induced low levels of interleukin-6 production by endothelial cells, and this effect was markedly enhanced by lipopolysaccharide. The results revealed that both serine proteases and histamine present in mast cell granules were involved in this activation process. Mast cell granules increased intracellular calcium, and activated c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2. The combination of lipopolysaccharide and mast cell granules prolonged c-Jun amino-terminal kinase activity beyond the duration of induction by either stimulant alone and was entirely due to active proteases. However, both proteases and histamine contributed to calcium mobilization and extracellular signal-regulated kinase 1/2 activation. The nuclear translocation of nuclear factor-kappaB proteins was of greater magnitude in endothelial cells treated with the combination of mast cell granules and lipopolysaccharide. CONCLUSIONS:Mast cell granule serine proteases and histamine can amplify lipopolysaccharide-induced endothelial cell activation, which involves calcium mobilization, mitogen-activated

  9. ASPP1 and ASPP2 bind active RAS, potentiate RAS signalling and enhance p53 activity in cancer cells

    PubMed Central

    Wang, Y; Godin-Heymann, N; Dan Wang, X; Bergamaschi, D; Llanos, S; Lu, X

    2013-01-01

    RAS mutations occur frequently in human cancer and activated RAS signalling contributes to tumour development and progression. Apart from its oncogenic effects on cell growth, active RAS has tumour-suppressive functions via its ability to induce cellular senescence and apoptosis. RAS is known to induce p53-dependent cell cycle arrest, yet its effect on p53-dependent apoptosis remains unclear. We report here that apoptosis-stimulating protein of p53 (ASPP) 1 and 2, two activators of p53, preferentially bind active RAS via their N-terminal RAS-association domains (RAD). Additionally, ASPP2 colocalises with and contributes to RAS cellular membrane localisation and potentiates RAS signalling. In cancer cells, ASPP1 and ASPP2 cooperate with oncogenic RAS to enhance the transcription and apoptotic function of p53. Thus, loss of ASPP1 and ASPP2 in human cancer cells may contribute to the full transforming property of RAS oncogene. PMID:23392125

  10. False alarm reduction in BSN-based cardiac monitoring using signal quality and activity type information.

    PubMed

    Tanantong, Tanatorn; Nantajeewarawat, Ekawit; Thiemjarus, Surapa

    2015-01-01

    False alarms in cardiac monitoring affect the quality of medical care, impacting on both patients and healthcare providers. In continuous cardiac monitoring using wireless Body Sensor Networks (BSNs), the quality of ECG signals can be deteriorated owing to several factors, e.g., noises, low battery power, and network transmission problems, often resulting in high false alarm rates. In addition, body movements occurring from activities of daily living (ADLs) can also create false alarms. This paper presents a two-phase framework for false arrhythmia alarm reduction in continuous cardiac monitoring, using signals from an ECG sensor and a 3D accelerometer. In the first phase, classification models constructed using machine learning algorithms are used for labeling input signals. ECG signals are labeled with heartbeat types and signal quality levels, while 3D acceleration signals are labeled with ADL types. In the second phase, a rule-based expert system is used for combining classification results in order to determine whether arrhythmia alarms should be accepted or suppressed. The proposed framework was validated on datasets acquired using BSNs and the MIT-BIH arrhythmia database. For the BSN dataset, acceleration and ECG signals were collected from 10 young and 10 elderly subjects while they were performing ADLs. The framework reduced the false alarm rate from 9.58% to 1.43% in our experimental study, showing that it can potentially assist physicians in diagnosing a vast amount of data acquired from wireless sensors and enhance the performance of continuous cardiac monitoring. PMID:25671512

  11. False Alarm Reduction in BSN-Based Cardiac Monitoring Using Signal Quality and Activity Type Information

    PubMed Central

    Tanantong, Tanatorn; Nantajeewarawat, Ekawit; Thiemjarus, Surapa

    2015-01-01

    False alarms in cardiac monitoring affect the quality of medical care, impacting on both patients and healthcare providers. In continuous cardiac monitoring using wireless Body Sensor Networks (BSNs), the quality of ECG signals can be deteriorated owing to several factors, e.g., noises, low battery power, and network transmission problems, often resulting in high false alarm rates. In addition, body movements occurring from activities of daily living (ADLs) can also create false alarms. This paper presents a two-phase framework for false arrhythmia alarm reduction in continuous cardiac monitoring, using signals from an ECG sensor and a 3D accelerometer. In the first phase, classification models constructed using machine learning algorithms are used for labeling input signals. ECG signals are labeled with heartbeat types and signal quality levels, while 3D acceleration signals are labeled with ADL types. In the second phase, a rule-based expert system is used for combining classification results in order to determine whether arrhythmia alarms should be accepted or suppressed. The proposed framework was validated on datasets acquired using BSNs and the MIT-BIH arrhythmia database. For the BSN dataset, acceleration and ECG signals were collected from 10 young and 10 elderly subjects while they were performing ADLs. The framework reduced the false alarm rate from 9.58% to 1.43% in our experimental study, showing that it can potentially assist physicians in diagnosing a vast amount of data acquired from wireless sensors and enhance the performance of continuous cardiac monitoring. PMID:25671512

  12. Spinal Neuronal NOS Signaling Contributes to Morphine Cardioprotection in Ischemia Reperfusion Injury in Rats.

    PubMed

    Jiang, Lingling; Hu, Jun; He, Shufang; Zhang, Li; Zhang, Ye

    2016-09-01

    Morphine has been widely used as rescue treatment for heart attack and failure in humans for many decades. Relatively little has been known about the role of spinal opioid receptors in morphine cardioprotection. Recent studies have shown that intrathecal injection of morphine can reduce the heart injury caused by ischemia (I)/reperfusion (R) in rats. However, the molecular and cellular mechanisms underlying intrathecal morphine cardioprotection has not been determined. Here, we report that intrathecal morphine postconditioning (IMPOC) rescued mean artery pressure (MAP) and reduced myocardial injury in I/R. Pretreatment with either naloxone (NAL), a selective mu-opioid receptor antagonist, or nitric oxide synthase (NOS) inhibitors via intrathecal delivery completely abolished IMPOC cardioprotection, suggesting that the spinal mu-opioid receptor and its downstream NOS signaling pathway are involved in the mechanism of the morphine-induced effect. Consistent with this, IMPOC significantly enhanced spinal neural NOS phosphorylation, nitric oxide, and cGMP content in a similar time course. Intrathecal application of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a specific inhibitor of guanylate cyclase, completely ablated IMPOC-induced enhancement of cardioprotection and spinal cGMP content. IMPOC rescue of MAP and ischemic injury is correlated with IMPOC enhancement of NOS signaling. Collectively, these findings strengthen the concept of spinal mu-opioid receptors as a therapeutic target that mediates morphine-induced cardioprotection. We also provide evidence suggesting that the activation of spinal NOS signaling is essential for morphine cardioprotection. PMID:27358482

  13. Effects of anesthesia on BOLD signal and neuronal activity in the somatosensory cortex.

    PubMed

    Aksenov, Daniil P; Li, Limin; Miller, Michael J; Iordanescu, Gheorghe; Wyrwicz, Alice M

    2015-11-01

    Most functional magnetic resonance imaging (fMRI) animal studies rely on anesthesia, which can induce a variety of drug-dependent physiological changes, including depression of neuronal activity and cerebral metabolism as well as direct effects on the vasculature. The goal of this study was to characterize the effects of anesthesia on the BOLD signal and neuronal activity. Simultaneous fMRI and electrophysiology were used to measure changes in single units (SU), multi-unit activity (MUA), local field potentials (LFP), and the blood oxygenation level-dependent (BOLD) response in the somatosensory cortex during whisker stimulation of rabbits before, during and after anesthesia with fentanyl or isoflurane. Our results indicate that anesthesia modulates the BOLD signal as well as both baseline and stimulus-evoked neuronal activity, and, most significantly, that the relationship between the BOLD and electrophysiological signals depends on the type of anesthetic. Specifically, the behavior of LFP observed under isoflurane did not parallel the behavior of BOLD, SU, or MUA. These findings suggest that the relationship between these signals may not be straightforward. BOLD may scale more closely with the best measure of the excitatory subcomponents of the underlying neuronal activity, which may vary according to experimental conditions that alter the excitatory/inhibitory balance in the cortex. PMID:26104288

  14. Cadmium Induces Apoptosis in Freshwater Crab Sinopotamon henanense through Activating Calcium Signal Transduction Pathway

    PubMed Central

    Wang, Jinxiang; Zhang, Pingping; Liu, Na; Wang, Qian; Luo, Jixian; Wang, Lan

    2015-01-01

    Calcium ion (Ca2+) is one of the key intracellular signals, which is implicated in the regulation of cell functions such as impregnation, cell proliferation, differentiation and death. Cadmium (Cd) is a toxic environmental pollutant that can disturb cell functions and even lead to cell death. Recently, we have found that Cd induced apoptosis in gill cells of the freshwater crab Sinopotamon henanense via caspase activation. In the present study, we further investigated the role of calcium signaling in the Cd-induced apoptosis in the animals. Our data showed that Cd triggered gill cell apoptosis which is evidenced by apoptotic DNA fragmentation, activations of caspases-3, -8 and -9 and the presence of apoptotic morphological features. Moreover, Cd elevated the intracellular concentration of Ca2+, the protein concentration of calmodulin (CaM) and the activity of Ca2+-ATPase in the gill cells of the crabs. Pretreatment of the animals with ethylene glycol-bis-(b-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), Ca2+ chelator, inhibited Cd-induced activation of caspases-3, -8 and -9 as well as blocked the Cd-triggered apoptotic DNA fragmentation. The apoptotic morphological features were no longer observed in gill cells pretreated with the Ca2+ signaling inhibitors before Cd treatment. Our results indicate that Cd evokes gill cell apoptosis through activating Ca2+-CaM signaling transduction pathway. PMID:26714174

  15. Calmodulin activity regulates group I metabotropic glutamate receptor-mediated signal transduction and synaptic depression.

    PubMed

    Sethna, Ferzin; Zhang, Ming; Kaphzan, Hanoch; Klann, Eric; Autio, Dawn; Cox, Charles L; Wang, Hongbing

    2016-05-01

    Group I metabotropic glutamate receptors (mGluR), including mGluR1 and mGluR 5 (mGluR1/5), are coupled to Gq and modulate activity-dependent synaptic plasticity. Direct activation of mGluR1/5 causes protein translation-dependent long-term depression (LTD). Although it has been established that intracellular Ca(2+) and the Gq-regulated signaling molecules are required for mGluR1/5 LTD, whether and how Ca(2+) regulates Gq signaling and upregulation of protein expression remain unknown. Through pharmacological inhibition, we tested the function of the Ca(2+) sensor calmodulin (CaM) in intracellular signaling triggered by the activation of mGluR1/5. CaM inhibitor N-[4-aminobutyl]-5-chloro-2-naphthalenesulfonamide hydrochloride (W13) suppressed the mGluR1/5-stimulated activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p70-S6 kinase 1 (S6K1) in hippocampal neurons. W13 also blocked the mGluR1/5 agonist-induced synaptic depression in hippocampal slices and in anesthetized mice. Consistent with the function of CaM, inhibiting the downstream targets Ca(2+) /CaM-dependent protein kinases (CaMK) blocked ERK1/2 and S6K1 activation. Furthermore, disruption of the CaM-CaMK-ERK1/2 signaling cascade suppressed the mGluR1/5-stimulated upregulation of Arc expression. Altogether, our data suggest CaM as a new Gq signaling component for coupling Ca(2+) and protein upregulation and regulating mGluR1/5-mediated synaptic modification. PMID:26864654

  16. Surfactant protein A integrates activation signal strength to differentially modulate T cell proliferation.

    PubMed

    Mukherjee, Sambuddho; Giamberardino, Charles; Thomas, Joseph; Evans, Kathy; Goto, Hisatsugu; Ledford, Julie G; Hsia, Bethany; Pastva, Amy M; Wright, Jo Rae

    2012-02-01

    Pulmonary surfactant lipoproteins lower the surface tension at the alveolar-airway interface of the lung and participate in host defense. Previous studies reported that surfactant protein A (SP-A) inhibits lymphocyte proliferation. We hypothesized that SP-A-mediated modulation of T cell activation depends upon the strength, duration, and type of lymphocyte activating signals. Modulation of T cell signal strength imparted by different activating agents ex vivo and in vivo in different mouse models and in vitro with human T cells shows a strong correlation between strength of signal (SoS) and functional effects of SP-A interactions. T cell proliferation is enhanced in the presence of SP-A at low SoS imparted by exogenous mitogens, specific Abs, APCs, or in homeostatic proliferation. Proliferation is inhibited at higher SoS imparted by different doses of the same T cell mitogens or indirect stimuli such as LPS. Importantly, reconstitution with exogenous SP-A into the lungs of SP-A(-/-) mice stimulated with a strong signal also resulted in suppression of T cell proliferation while elevating baseline proliferation in unstimulated T cells. These signal strength and SP-A-dependent effects are mediated by changes in intracellular Ca(2+) levels over time, involving extrinsic Ca(2+)-activated channels late during activation. These effects are intrinsic to the global T cell population and are manifested in vivo in naive as well as memory phenotype T cells. Thus, SP-A appears to integrate signal thresholds to control T cell proliferation. PMID:22219327

  17. Two Drosophila suppressors of cytokine signaling (SOCS) differentially regulate JAK and EGFR pathway activities

    PubMed Central

    Rawlings, Jason S; Rennebeck, Gabriela; Harrison, Susan MW; Xi, Rongwen; Harrison, Douglas A

    2004-01-01

    Background The Janus kinase (JAK) cascade is an essential and well-conserved pathway required to transduce signals for a variety of ligands in both vertebrates and invertebrates. While activation of the pathway is essential to many processes, mutations from mammals and Drosophila demonstrate that regulation is also critical. The SOCS (Suppressor Of Cytokine Signaling) proteins in mammals are regulators of the JAK pathway that participate in a negative feedback loop, as they are transcriptionally activated by JAK signaling. Examination of one Drosophila SOCS homologue, Socs36E, demonstrated that its expression is responsive to JAK pathway activity and it is capable of downregulating JAK signaling, similar to the well characterized mammalian SOCS. Results Based on sequence analysis of the Drosophila genome, there are three identifiable SOCS homologues in flies. All three are most similar to mammalian SOCS that have not been extensively characterized: Socs36E is most similar to mammalian SOCS5, while Socs44A and Socs16D are most similar to mammalian SOCS6 and 7. Although Socs44A is capable of repressing JAK activity in some tissues, its expression is not regulated by the pathway. Furthermore, Socs44A can enhance the activity of the EGFR/MAPK signaling cascade, in contrast to Socs36E. Conclusions Two Drosophila SOCS proteins have some overlapping and some distinct capabilities. While Socs36E behaves similarly to the canonical vertebrate SOCS, Socs44A is not part of a JAK pathway negative feedback loop. Nonetheless, both SOCS regulate JAK and EGFR signaling pathways, albeit differently. The non-canonical properties of Socs44A may be representative of the class of less characterized vertebrate SOCS with which it shares greatest similarity. PMID:15488148

  18. Role of Nongenomic Signaling Pathways Activated by Aldosterone During Cardiac Reperfusion Injury.

    PubMed

    Ashton, Anthony W; Le, Thi Y L; Gomez-Sanchez, Celso E; Morel-Kopp, Marie-Christine; McWhinney, Brett; Hudson, Amanda; Mihailidou, Anastasia S

    2015-08-01

    Aldosterone (Aldo) activates both genomic and nongenomic signaling pathways in the cardiovascular system. Activation of genomic signaling pathways contributes to the adverse cardiac actions of Aldo during reperfusion injury; however, the extent nongenomic signaling pathways contribute has been difficult to identify due to lack of a specific ligand that activates only nongenomic signaling pathways. Using a pegylated aldosterone analog, aldosterone-3-carboxymethoxylamine-TFP ester conjugated to methoxypegylated amine (Aldo-PEG), we are able for the first time to distinguish between nongenomic and genomic cardiac actions of Aldo. We confirm Aldo-PEG activates phosphorylation of ERK1/2 in rat cardiomyocyte H9c2 cells similar to Aldo and G protein-coupled receptor 30 (GPR30 or GPER) agonist G1. GPER antagonist, G36, but not mineralocorticoid receptor (MR) antagonist spironolactone, prevented ERK1/2 phosphorylation by Aldo, Aldo-PEG, and G1. The selective nongenomic actions of Aldo-PEG are confirmed, with Aldo-PEG increasing superoxide production in H9c2 cells to similar levels as Aldo but having no effect on subcellular localization of MR. Striatin serves as a scaffold for GPER and MR, with GPER antagonist G36, but not spironolactone, restoring MR-striatin complexes. Aldo-PEG had no effect on MR-dependent transcriptional activation, whereas Aldo increased transcript levels of serum-regulated kinase 1 and plasminogen activator inhibitor-1. Using our ex vivo experimental rat model of myocardial infarction, we found aggravated infarct size and apoptosis by Aldo but not Aldo-PEG. Our studies confirm that in the heart, activation of nongenomic signaling pathways alone are not sufficient to trigger the deleterious effects of aldosterone during myocardial reperfusion injury. PMID:26121234

  19. Swelling-Activated Ca2+ Channels Trigger Ca2+ Signals in Merkel Cells

    PubMed Central

    Haeberle, Henry; Bryan, Leigh A.; Vadakkan, Tegy J.; Dickinson, Mary E.; Lumpkin, Ellen A.

    2008-01-01

    Merkel cell-neurite complexes are highly sensitive touch receptors comprising epidermal Merkel cells and sensory afferents. Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results. To test this model in a simplified system, we asked whether purified Merkel cells are directly activated by mechanical stimulation. Cell shape was manipulated with anisotonic solution changes and responses were monitored by Ca2+ imaging with fura-2. We found that hypotonic-induced cell swelling, but not hypertonic solutions, triggered cytoplasmic Ca2+ transients. Several lines of evidence indicate that these signals arise from swelling-activated Ca2+-permeable ion channels. First, transients were reversibly abolished by chelating extracellular Ca2+, demonstrating a requirement for Ca2+ influx across the plasma membrane. Second, Ca2+ transients were initially observed near the plasma membrane in cytoplasmic processes. Third, voltage-activated Ca2+ channel (VACC) antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs. Finally, emptying internal Ca2+ stores attenuated transients by 80%, suggesting Ca2+ release from stores augments swelling-activated Ca2+ signals. To identify candidate mechanotransduction channels, we used RT-PCR to amplify ion-channel transcripts whose pharmacological profiles matched those of hypotonic-evoked Ca2+ signals in Merkel cells. We found 11 amplicons, including PKD1, PKD2, and TRPC1, channels previously implicated in mechanotransduction in other cells. Collectively, these results directly demonstrate that Merkel cells are activated by hypotonic-evoked swelling, identify cellular signaling mechanisms that mediate these responses, and support the hypothesis that Merkel cells contribute to touch reception

  20. Low-cost detection of RC-IED activation signals in VHF band

    NASA Astrophysics Data System (ADS)

    Camargo Suarez, Victor Hugo; Marulanda B., Jose Ignacio

    2014-05-01

    The proliferation of Radio Controlled Improvised Explosive Devices (RC-IED) is a growing threat around the world. The ease of construction and low cost of these devices are transforming common things in lethal tramps. The fight against this threats normally involves the use of sophisticated and expensive equipment of Electronic Warfare based on high speed DSP systems, just to detect the presence of detonation signals. In this work is showed how to find activation signals based on the characteristic of the power in a specific band and the previous knowledge about the detonation signals. As proof of concept we have taken the information about the RC-IEDs used in the Colombian conflict and develop an algorithm to find detonation signals based on the measured power in frequencies between 136 MHz and 174 MHz (2 meter civil band)

  1. Opposing activities of Notch and Wnt signaling regulate intestinal stem cells and gut homeostasis.

    PubMed

    Tian, Hua; Biehs, Brian; Chiu, Cecilia; Siebel, Christian W; Wu, Yan; Costa, Mike; de Sauvage, Frederic J; Klein, Ophir D

    2015-04-01

    Proper organ homeostasis requires tight control of adult stem cells and differentiation through the integration of multiple inputs. In the mouse small intestine, Notch and Wnt signaling are required both for stem cell maintenance and for a proper balance of differentiation between secretory and absorptive cell lineages. In the absence of Notch signaling, stem cells preferentially generate secretory cells at the expense of absorptive cells. Here, we use function-blocking antibodies against Notch receptors to demonstrate that Notch blockade perturbs intestinal stem cell function by causing a derepression of the Wnt signaling pathway, leading to misexpression of prosecretory genes. Importantly, attenuation of the Wnt pathway rescued the phenotype associated with Notch blockade. These studies bring to light a negative regulatory mechanism that maintains stem cell activity and balanced differentiation, and we propose that the interaction between Wnt and Notch signaling described here represents a common theme in adult stem cell biology. PMID:25818302

  2. Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling

    PubMed Central

    Holland, Sabrina M.; Collura, Kaitlin M.; Ketschek, Andrea; Noma, Kentaro; Ferguson, Toby A.; Jin, Yishi; Gallo, Gianluca; Thomas, Gareth M.

    2016-01-01

    Dual leucine-zipper kinase (DLK) is critical for axon-to-soma retrograde signaling following nerve injury. However, it is unknown how DLK, a predicted soluble kinase, conveys long-distance signals and why homologous kinases cannot compensate for loss of DLK. Here, we report that DLK, but not homologous kinases, is palmitoylated at a conserved site adjacent to its kinase domain. Using short-hairpin RNA knockdown/rescue, we find that palmitoylation is critical for DLK-dependent retrograde signaling in sensory axons. This functional importance is because of three novel cellular and molecular roles of palmitoylation, which targets DLK to trafficking vesicles, is required to assemble DLK signaling complexes and, unexpectedly, is essential for DLK’s kinase activity. By simultaneously controlling DLK localization, interactions, and activity, palmitoylation ensures that only vesicle-bound DLK is active in neurons. These findings explain how DLK specifically mediates nerve injury responses and reveal a novel cellular mechanism that ensures the specificity of neuronal kinase signaling. PMID:26719418

  3. The Viral Oncoprotein LMP1 Exploits TRADD for Signaling by Masking Its Apoptotic Activity

    PubMed Central

    Schneider, Frank; Neugebauer, Julia; Griese, Janine; Liefold, Nicola; Kutz, Helmut; Briseño, Cinthia; Kieser, Arnd

    2008-01-01

    The tumor necrosis factor (TNF)-receptor 1–associated death domain protein (TRADD) mediates induction of apoptosis as well as activation of NF-κB by cellular TNF-receptor 1 (TNFR1). TRADD is also recruited by the latent membrane protein 1 (LMP1) oncoprotein of Epstein-Barr virus, but its role in LMP1 signaling has remained enigmatic. In human B lymphocytes, we have generated, to our knowledge, the first genetic knockout of TRADD to investigate TRADD's role in LMP1 signal transduction. Our data from TRADD-deficient cells demonstrate that TRADD is a critical signaling mediator of LMP1 that is required for LMP1 to recruit and activate I-κB kinase β (IKKβ). However, in contrast to TNFR1, LMP1-induced TRADD signaling does not induce apoptosis. Searching for the molecular basis for this observation, we characterized the 16 C-terminal amino acids of LMP1 as an autonomous and unique virus-derived TRADD-binding domain. Replacing the death domain of TNFR1 by LMP1′s TRADD-binding domain converts TNFR1 into a nonapoptotic receptor that activates NF-κB through a TRAF6-dependent pathway, like LMP1 but unlike wild-type TNFR1. Thus, the unique interaction of LMP1 with TRADD encodes the transforming phenotype of viral TRADD signaling and masks TRADD's pro-apoptotic function. PMID:18198944

  4. A Phosphatase Activity of Sts-1 Contributes to the Suppression of TCR Signaling

    SciTech Connect

    Mikhailik,A.; Ford, B.; Keller, J.; Chen, Y.; Nassar, N.; Carpino, N.

    2007-01-01

    Precise signaling by the T cell receptor (TCR) is crucial for a proper immune response. To ensure that T cells respond appropriately to antigenic stimuli, TCR signaling pathways are subject to multiple levels of regulation. Sts-1 negatively regulates signaling pathways downstream of the TCR by an unknown mechanism(s). Here, we demonstrate that Sts-1 is a phosphatase that can target the tyrosine kinase Zap-70 among other proteins. The X-ray structure of the Sts-1 C terminus reveals that it has homology to members of the phosphoglycerate mutase/acid phosphatase (PGM/AcP) family of enzymes, with residues known to be important for PGM/AcP catalytic activity conserved in nature and position in Sts-1. Point mutations that impair Sts-1 phosphatase activity in vitro also impair the ability of Sts-1 to regulate TCR signaling in T cells. These observations reveal a PGM/AcP-like enzyme activity involved in the control of antigen receptor signaling.

  5. Focal Activation of Cells by Plasmon Resonance Assisted Optical Injection of Signaling Molecules

    PubMed Central

    2015-01-01

    Experimental methods for single cell intracellular delivery are essential for probing cell signaling dynamics within complex cellular networks, such as those making up the tumor microenvironment. Here, we show a quantitative and general method of interrogation of signaling pathways. We applied highly focused near-infrared laser light to optically inject gold-coated liposomes encapsulating bioactive molecules into single cells for focal activation of cell signaling. For this demonstration, we encapsulated either inositol trisphosphate (IP3), an endogenous cell signaling second messenger, or adenophostin A (AdA), a potent analogue of IP, within 100 nm gold-coated liposomes, and injected these gold-coated liposomes and their contents into the cytosol of single ovarian carcinoma cells to initiate calcium (Ca2+) release from intracellular stores. Upon optical injection of IP3 or AdA at doses above the activation threshold, we observed increases in cytosolic Ca2+ concentration within the injected cell initiating the propagation of a Ca2+ wave throughout nearby cells. As confirmed by octanol-induced inhibition, the intercellular Ca2+ wave traveled via gap junctions. Optical injection of gold-coated liposomes represents a quantitative method of focal activation of signaling cascades of broad interest in biomedical research. PMID:24877558

  6. Graded boosting of synaptic signals by low-threshold voltage-activated calcium conductance.

    PubMed

    Carbó Tano, Martín; Vilarchao, María Eugenia; Szczupak, Lidia

    2015-07-01

    Low-threshold voltage-activated calcium conductances (LT-VACCs) play a substantial role in shaping the electrophysiological attributes of neurites. We have investigated how these conductances affect synaptic integration in a premotor nonspiking (NS) neuron of the leech nervous system. These cells exhibit an extensive neuritic tree, do not fire Na(+)-dependent spikes, but express an LT-VACC that was sensitive to 250 μM Ni(2+) and 100 μM NNC 55-0396 (NNC). NS neurons responded to excitation of mechanosensory pressure neurons with depolarizing responses for which amplitude was a linear function of the presynaptic firing frequency. NNC decreased these synaptic responses and abolished the concomitant widespread Ca(2+) signals. Coherent with the interpretation that the LT-VACC amplified signals at the postsynaptic level, this conductance also amplified the responses of NS neurons to direct injection of sinusoidal current. Synaptic amplification thus is achieved via a positive feedback in which depolarizing signals activate an LT-VACC that, in turn, boosts these signals. The wide distribution of LT-VACC could support the active propagation of depolarizing signals, turning the complex NS neuritic tree into a relatively compact electrical compartment. PMID:25972583

  7. Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death

    PubMed Central

    Graham, Nicholas A; Tahmasian, Martik; Kohli, Bitika; Komisopoulou, Evangelia; Zhu, Maggie; Vivanco, Igor; Teitell, Michael A; Wu, Hong; Ribas, Antoni; Lo, Roger S; Mellinghoff, Ingo K; Mischel, Paul S; Graeber, Thomas G

    2012-01-01

    The altered metabolism of cancer can render cells dependent on the availability of metabolic substrates for viability. Investigating the signaling mechanisms underlying cell death in cells dependent upon glucose for survival, we demonstrate that glucose withdrawal rapidly induces supra-physiological levels of phospho-tyrosine signaling, even in cells expressing constitutively active tyrosine kinases. Using unbiased mass spectrometry-based phospho-proteomics, we show that glucose withdrawal initiates a unique signature of phospho-tyrosine activation that is associated with focal adhesions. Building upon this observation, we demonstrate that glucose withdrawal activates a positive feedback loop involving generation of reactive oxygen species (ROS) by NADPH oxidase and mitochondria, inhibition of protein tyrosine phosphatases by oxidation, and increased tyrosine kinase signaling. In cells dependent on glucose for survival, glucose withdrawal-induced ROS generation and tyrosine kinase signaling synergize to amplify ROS levels, ultimately resulting in ROS-mediated cell death. Taken together, these findings illustrate the systems-level cross-talk between metabolism and signaling in the maintenance of cancer cell homeostasis. PMID:22735335

  8. Mechanical Stretch-Induced Activation of ROS/RNS Signaling in Striated Muscle

    PubMed Central

    Ward, Christopher W.; Prosser, Benjamin L.

    2014-01-01

    Significance: Mechanical activation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) occurs in striated muscle and affects Ca2+ signaling and contractile function. ROS/RNS signaling is tightly controlled, spatially compartmentalized, and source specific. Recent Advances: Here, we review the evidence that within the contracting myocyte, the trans-membrane protein NADPH oxidase 2 (Nox2) is the primary source of ROS generated during contraction. We also review a newly characterized signaling cascade in cardiac and skeletal muscle in which the microtubule network acts as a mechanotransduction element that activates Nox2-dependent ROS generation during mechanical stretch, a pathway termed X-ROS signaling. Critical Issues: In the heart, X-ROS acts locally and affects the sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptors) and tunes Ca2+ signaling during physiological behavior, but excessive X-ROS can promote Ca2+-dependent arrhythmias in pathology. In skeletal muscle, X-ROS sensitizes Ca2+-permeable sarcolemmal “transient receptor potential” channels, a pathway that is critical for sustaining SR load during repetitive contractions, but when in excess, it is maladaptive in diseases such as Duchenne Musclar dystrophy. Future Directions: New advances in ROS/RNS detection as well as molecular manipulation of signaling pathways will provide critical new mechanistic insights into the details of X-ROS signaling. These efforts will undoubtedly reveal new avenues for therapeutic intervention in the numerous diseases of striated muscle in which altered mechanoactivation of ROS/RNS production has been identified. Antioxid. Redox Signal. 20, 929–936. PMID:23971496

  9. Activation of the wnt/β-Catenin Signaling Pathway in Polymyositis, Dermatomyositis and Duchenne Muscular Dystrophy

    PubMed Central

    Liu, Fuchen; Liang, Zonglai; Xu, Jingwen; Li, Wei; Zhao, Dandan; Zhao, Yuying

    2016-01-01

    Background and Purpose The wnt/β-catenin signaling pathway plays a critical role in embryonic development and adult-tissue homeostasis. Recent investigations implicate the importance of wnt/β-catenin signaling in normal wound healing and its sustained activation being associated with fibrogenesis. We investigated the immunolocalization and activation of wnt/β-catenin in polymyositis (PM), dermatomyositis (DM), and Duchenne muscular dystrophy (DMD). Methods Immunofluorescence staining and Western blot analysis of β-catenin were performed in muscle specimens from 6 PM, 8 DM, and 6 DMD subjects. The β-catenin/Tcf4 DNA-binding activity in muscle was studied using an electrophoretic mobility shift assay (EMSA), and serum wnt/β-catenin/Tcf transcriptional activity was measured using a luciferase reporter gene assay. Results Immunoreactivity for β-catenin was found in the cytoplasm and nuclei of muscle fibers in PM, DM, and DMD. The protein level of β-catenin was elevated, and EMSA analysis confirmed the activation of wnt/β-catenin signaling. The transcriptional activities of β-catenin/Tcf in the circulation were increased in patients with PM, DM, and DMD, especially in those with interstitial lung disease, and these transcriptional activities decreased when PM or DM patients exhibited obvious clinical improvements. Conclusions Our findings indicate that wnt/β-catenin signaling is activated in PM, DM, and DMD. Its activation in muscle tissue and the circulation may play a role in modulating muscle regeneration and be at least partly involved in the process of muscle and pulmonary fibrosis. PMID:27165423

  10. Rapid estrogen signaling negatively regulates PTEN activity through phosphorylation in endometrial cancer cells

    PubMed Central

    Scully, Melanie M.; Palacios-Helgeson, Leslie K.; Wah, Lah S.; Jackson, Twila A.

    2014-01-01

    Hyperestrogenicity is a risk factor for endometrial cancer. 17β-estradiol (E2) is known to stimulate both genomic and nongenomic estrogen receptor-α (ERα) actions in a number of reproductive tissues. However, the contributions of transcription-independent ERα signaling on normal and malignant endometrium are not fully understood. Phosphatase and tensin homolog (PTEN) is a tumor suppressor that decreases cellular mitosis primarily through negative regulation of the phosphoinositide 3-kinase/AKT signaling axis. PTEN levels are elevated during the E2 dominated, mitotically active, proliferative phase of the menstrual cycle, indicating possible hormonal regulation of PTEN in the uterus. In order to determine if rapid E2 signaling regulates PTEN, we used ERα positive, PTEN positive, endometrial cells. We show that cytosolic E2/ERα signaling leads to increased phosphorylation of PTEN at key regulatory residues. Importantly, E2 stimulation decreased PTEN lipid phosphatase activity and caused consequent increases in phospho-AKT. We further demonstrate that cytosolic ERα forms a complex with PTEN in an E2-dependent manner, and that ERα constitutively complexes with protein kinase2-α (CK2α), a kinase previously shown to phosphorylate the C-terminal tail of PTEN. These results provide mechanistic support for an E2-dependent, ERα cytosolic signaling complex that negatively regulates PTEN activity through carboxy terminus phosphorylation. Using an animal model, we show that sustained E2 signaling results in increased phospho-PTEN (S380, T382, T383), total PTEN and phospho-AKT (S473). Taken together, we provide a novel mechanism in which transcription-independent E2/ERα signaling may promote a pro-tumorigenic environment in the endometrium. PMID:24844349

  11. Alternative Activation Mechanisms of Protein Kinase B Trigger Distinct Downstream Signaling Responses.

    PubMed

    Balzano, Deborah; Fawal, Mohamad-Ali; Velázquez, Jose V; Santiveri, Clara M; Yang, Joshua; Pastor, Joaquín; Campos-Olivas, Ramón; Djouder, Nabil; Lietha, Daniel

    2015-10-01

    Protein kinase B (PKB/Akt) is an important mediator of signals that control various cellular processes including cell survival, growth, proliferation, and metabolism. PKB promotes these processes by phosphorylating many cellular targets, which trigger distinct downstream signaling events. However, how PKB is able to selectively target its substrates to induce specific cellular functions remains elusive. Here we perform a systematic study to dissect mechanisms that regulate intrinsic kinase activity versus mechanisms that specifically regulate activity toward specific substrates. We demonstrate that activation loop phosphorylation and the C-terminal hydrophobic motif are essential for high PKB activity in general. On the other hand, we identify membrane targeting, which for decades has been regarded as an essential step in PKB activation, as a mechanism mainly affecting substrate selectivity. Further, we show that PKB activity in cells can be triggered independently of PI3K by initial hydrophobic motif phosphorylation, presumably through a mechanism analogous to other AGC kinases. Importantly, different modes of PKB activation result in phosphorylation of distinct downstream targets. Our data indicate that specific mechanisms have evolved for signaling nodes, like PKB, to select between various downstream events. Targeting such mechanisms selectively could facilitate the development of therapeutics that might limit toxic side effects. PMID:26286748

  12. Phosphatidylserine enhances IKBKAP transcription by activating the MAPK/ERK signaling pathway.

    PubMed

    Donyo, Maya; Hollander, Dror; Abramovitch, Ziv; Naftelberg, Shiran; Ast, Gil

    2016-04-01

    Familial dysautonomia (FD) is a genetic disorder manifested due to abnormal development and progressive degeneration of the sensory and autonomic nervous system. FD is caused by a point mutation in the IKBKAP gene encoding the IKAP protein, resulting in decreased protein levels. A promising potential treatment for FD is phosphatidylserine (PS); however, the manner by which PS elevates IKAP levels has yet to be identified. Analysis of ChIP-seq results of the IKBKAP promoter region revealed binding of the transcription factors CREB and ELK1, which are regulated by the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) signaling pathway. We show that PS treatment enhanced ERK phosphorylation in cells derived from FD patients. ERK activation resulted in elevated IKBKAP transcription and IKAP protein levels, whereas pretreatment with the MAPK inhibitor U0126 blocked elevation of the IKAP protein level. Overexpression of either ELK1 or CREB activated the IKBKAP promoter, whereas downregulation of these transcription factors resulted in a decrease of the IKAP protein. Additionally, we show that PS improves cell migration, known to be enhanced by MAPK/ERK activation and abrogated in FD cells. In conclusion, our results demonstrate that PS activates the MAPK/ERK signaling pathway, resulting in activation of transcription factors that bind the promoter region of IKBKAP and thus enhancing its transcription. Therefore, compounds that activate the MAPK/ERK signaling pathway could constitute potential treatments for FD. PMID:26769675

  13. Signal transducer and activator of transcription 5 is implicated in disease activity in adult and juvenile onset systemic lupus erythematosus.

    PubMed

    Meshaal, Safa; El Refai, Rasha; El Saie, Ahmed; El Hawary, Rabab

    2016-06-01

    The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is one of a handful of pleiotropic cascades used to transduce a multitude of signals for development and homeostasis in humans. It is the principal signaling mechanism for a wide array of cytokines and growth factors. Dysregulated cytokine action on immune cells plays an important role in the initiation and progress of systemic lupus erythematosus (SLE). In this study, we tried to assess the role of STAT5 in systemic lupus erythematosus and correlate its phosphorylation level with the disease activity. The activation of the STAT5 was assessed by measuring the level of expression of phosphorylated STAT5 (pSTAT5) using flow cytometry on the peripheral blood T and B cells in 58 SLE patients (40 adult and 18 juvenile onset) and on 23 healthy age- and sex-matched controls for both groups. Serum prolactin level was also assessed in the patients and control by ELISA. The study revealed that the level of pSTAT5 was higher in adult SLE patients than in healthy control (p = 0.001) and in juvenile-onset SLE patients versus age-matched control (p = 0.031). A positive correlation existed between the pSTAT5 levels and Systemic Lupus Activity Measure (SLAM) score and also with multiple clinical manifestations indicating a potential role of STAT5 signaling in pathogenesis SLE. The pSTAT5 signaling is implicated in the disease activity of SLE and may be a useful target of therapy by correcting the dysregulation of cytokines involved in the disease pathogenesis. PMID:27041383

  14. Signaling Pathways That Control Rho Kinase Activity Maintain the Embryonic Epicardial Progenitor State

    PubMed Central

    Artamonov, Mykhaylo V.; Jin, Li; Franke, Aaron S.; Momotani, Ko; Ho, Ruoya; Dong, Xiu Rong; Majesky, Mark W.; Somlyo, Avril V.

    2015-01-01

    This study identifies signaling pathways that play key roles in the formation and maintenance of epicardial cells, a source of progenitors for coronary smooth muscle cells (SMCs). After epithelial to mesenchymal transition (EMT), mesenchymal cells invade the myocardium to form coronary SMCs. RhoA/Rho kinase activity is required for EMT and for differentiation into coronary SMCs, whereas cAMP activity is known to inhibit EMT in epithelial cells by an unknown mechanism. We use outgrowth of epicardial cells from E9.5 isolated mouse proepicardium (PE) explants, wild type and Epac1 null E12.5 mouse heart explants, adult rat epicardial cells, and immortalized mouse embryonic epicardial cells as model systems to identify signaling pathways that regulate RhoA activity to maintain the epicardial progenitor state. We demonstrate that RhoA activity is suppressed in the epicardial progenitor state, that the cAMP-dependent Rap1 GTP exchange factor (GEF), Epac, known to down-regulate RhoA activity through activation of Rap1 GTPase activity increased, that Rap1 activity increased, and that expression of the RhoA antagonistic Rnd proteins known to activate p190RhoGAP increased and associated with p190RhoGAP. Finally, EMT is associated with increased p63RhoGEF and RhoGEF-H1 protein expression, increased GEF-H1 activity, with a trend in increased p63RhoGEF activity. EMT is suppressed by partial silencing of p63RhoGEF and GEF-H1. In conclusion, we have identified new signaling molecules that act together to control RhoA activity and play critical roles in the maintenance of coronary smooth muscle progenitor cells in the embryonic epicardium. We suggest that their eventual manipulation could promote revascularization after myocardial injury. PMID:25733666

  15. Continuous tooth generation in mouse is induced by activated epithelial Wnt/β-catenin signaling

    PubMed Central

    Järvinen, Elina; Salazar-Ciudad, Isaac; Birchmeier, Walter; Taketo, Makoto M.; Jernvall, Jukka; Thesleff, Irma

    2006-01-01

    The single replacement from milk teeth to permanent teeth makes mammalian teeth different from teeth of most nonmammalian vertebrates and other epithelial organs such as hair and feathers, whose continuous replacement has been linked to Wnt signaling. Here we show that mouse tooth buds expressing stabilized β-catenin in epithelium give rise to dozens of teeth. The molar crowns, however, are typically simplified unicusped cones. We demonstrate that the supernumerary teeth develop by a renewal process where new signaling centers, the enamel knots, bud off from the existing dental epithelium. The basic aspects of the unlocked tooth renewal can be reproduced with a computer model on tooth development by increasing the intrinsic level of activator production, supporting the role of β-catenin pathway as an upstream activator of enamel knot formation. These results may implicate Wnt signaling in tooth renewal, a capacity that was all but lost when mammals evolved progressively more complicated tooth shapes. PMID:17121988

  16. Using activation status of signaling pathways as mechanism-based biomarkers to predict drug sensitivity

    PubMed Central

    Amadoz, Alicia; Sebastian-Leon, Patricia; Vidal, Enrique; Salavert, Francisco; Dopazo, Joaquin

    2015-01-01

    Many complex traits, as drug response, are associated with changes in biological pathways rather than being caused by single gene alterations. Here, a predictive framework is presented in which gene expression data are recoded into activity statuses of signal transduction circuits (sub-pathways within signaling pathways that connect receptor proteins to final effector proteins that trigger cell actions). Such activity values are used as features by a prediction algorithm which can efficiently predict a continuous variable such as the IC50 value. The main advantage of this prediction method is that the features selected by the predictor, the signaling circuits, are themselves rich-informative, mechanism-based biomarkers which provide insight into or drug molecular mechanisms of action (MoA). PMID:26678097

  17. Continuous tooth generation in mouse is induced by activated epithelial Wnt/beta-catenin signaling.

    PubMed

    Järvinen, Elina; Salazar-Ciudad, Isaac; Birchmeier, Walter; Taketo, Makoto M; Jernvall, Jukka; Thesleff, Irma

    2006-12-01

    The single replacement from milk teeth to permanent teeth makes mammalian teeth different from teeth of most nonmammalian vertebrates and other epithelial organs such as hair and feathers, whose continuous replacement has been linked to Wnt signaling. Here we show that mouse tooth buds expressing stabilized beta-catenin in epithelium give rise to dozens of teeth. The molar crowns, however, are typically simplified unicusped cones. We demonstrate that the supernumerary teeth develop by a renewal process where new signaling centers, the enamel knots, bud off from the existing dental epithelium. The basic aspects of the unlocked tooth renewal can be reproduced with a computer model on tooth development by increasing the intrinsic level of activator production, supporting the role of beta-catenin pathway as an upstream activator of enamel knot formation. These results may implicate Wnt signaling in tooth renewal, a capacity that was all but lost when mammals evolved progressively more complicated tooth shapes. PMID:17121988

  18. Ultraviolet radiation-induced inflammation activates β-catenin signaling in mouse skin and skin tumors.

    PubMed

    Prasad, Ram; Katiyar, Santosh K

    2014-04-01

    UVB-induced inflammation, in particular the overexpression of cyclooxygenase-2 (COX-2) and prostaglandin (PG) E2, has been implicated in photocarcinogenesis. UVB-induced COX-2 has been associated with β-catenin signaling in keratinocytes. However, a definitive role for COX-2 in the activation of β-catenin signaling as well as its role in UVB-induced skin tumors has not been established. We report that exposure of the skin to UVB resulted in a time- and dose-dependent activation of β-catenin in C3H/HeN mice. This response was COX-2-dependent as UVB-exposed COX-2-deficient mice exhibited significantly lower levels of UVB-induced activation of β-catenin. Moreover, treatment of mice with indomethacin, a COX-2 inhibitor, and an EP2 antagonist inhibited UVB-induced β-catenin signaling. Exposure of SKH-1 hairless mice to UVB radiation (180 mJ/cm2) 3 times a week for 24 weeks resulted in activation of β-catenin signaling in UVB-irradiated skin as well as UVB-induced skin tumors. Concomitantly, the levels of CK1α and GSK-3β, which are responsible for β-catenin signaling, were reduced while the levels of c-Myc and cyclin D1, which are downstream targets of β-catenin, were increased. To further verify the role of UVB-induced inflammation in activation of β-catenin signaling, a high-fat-diet model was used. Administration of high-fat diet exacerbated UVB-induced inflammation. Administration of the high-fat diet enhanced β-catenin signaling and the levels of its downstream targets (c-Myc, cyclin D1, cyclin D2, MMP-2 and MMP-9) in UVB-exposed skin and skin tumors in SKH-1 mice. These data suggest that UV-induced COX-2/PGE2 stimulates β-catenin signaling, and that β-catenin activation may contribute to skin carcinogenesis. PMID:24481495

  19. Wnt signaling activation in adipose progenitors promotes insulin-independent muscle glucose uptake

    PubMed Central

    Zeve, Daniel; Seo, Jin; Suh, Jae Myoung; Stenesen, Drew; Tang, Wei; Berglund, Eric D.; Wan, Yihong; Williams, Linda J.; Lim, Ajin; Martinez, Myrna J.; McKay, Renée M.; Millay, Douglas P.; Olson, Eric N.; Graff, Jonathan M.

    2012-01-01

    SUMMARY Adipose tissues provide circulating nutrients and hormones. We present in vivo mouse studies highlighting roles for Wnt signals in both aspects of metabolism. β-catenin activation in PPARγ–expressing fat progenitors (PBCA) decreased fat mass and induced fibrotic replacement of subcutaneous fat specifically. In spite of lipodystrophy, PBCA mice did not develop the expected diabetes and hepatosteatosis, but rather exhibited improved glucose metabolism and normal insulin sensitivity. Glucose uptake was increased in muscle independently of insulin, associated with cell surface translocation of glucose transporters and AMPK activation. Ex vivo assays showed these effects were likely secondary to blood-borne signals since PBCA sera or conditioned media from PBCA fat progenitors enhanced glucose uptake and activated AMPK in muscle cultures. Thus, adipose progenitor Wnt activation dissociates lipodystrophy from dysfunctional metabolism and highlights a fat-muscle endocrine axis, which may represent a potential therapy to lower blood glucose and improve metabolism. PMID:22482731

  20. Chemical Signals of Synthetic Disaccharide Derivatives Dominate Rhamnolipids at Controlling Multiple Bacterial Activities.

    PubMed

    Singh, Nischal; Shetye, Gauri S; Zheng, Hewen; Sun, Jiayue; Luk, Yan-Yeung

    2016-01-01

    Microbes secrete molecules that modify their environment. Here, we demonstrate a class of synthetic disaccharide derivatives (DSDs) that mimics and dominates the activity of naturally secreted rhamnolipids by Pseudomonas aeruginosa. The DSDs exhibit the dual function of activating and inhibiting the swarming motility through a concentration-dependent activity reversal that is characteristic of signaling molecules. Whereas DSDs tethered with a saturated farnesyl group exhibit inhibition of both biofilm formation and swarming motility, with higher activities than rhamnolipids, a saturated farnesyl tethered with a sulfonate group only inhibits swarming motility but promote biofilm formation. These results identified important structural elements for controlling swarming motility, biofilm formation, and bacterial adhesion and suggest an effective chemical approach to control intertwined signaling processes that are important for biofilm formation and motilities. PMID:26511780

  1. Identification of key residues involved in the activation and signaling properties of dopamine D3 receptor.

    PubMed

    Kota, Kokila; Kuzhikandathil, Eldo V; Afrasiabi, Milad; Lacy, Brett; Kontoyianni, Maria; Crider, A Michael; Song, Daniel

    2015-09-01

    The dopamine D3 receptor exhibits agonist-dependent tolerance and slow response termination (SRT) signaling properties that distinguish it from the closely-related D2 receptors. While amino acid residues important for D3 receptor ligand binding have been identified, the residues involved in activation of D3 receptor signaling and induction of signaling properties have not been determined. In this paper, we used cis and trans isomers of a novel D3 receptor agonist, 8-OH-PBZI, and site-directed mutagenesis to identify key residues involved in D3 receptor signaling function. Our results show that trans-8-OH-PBZI, but not cis-8-OH-PBZI, elicit the D3 receptor tolerance and SRT properties. We show that while both agonists require a subset of residues in the orthosteric binding site of D3 receptors for activation of the receptor, the ability of the two isomers to differentially induce tolerance and SRT is mediated by interactions with specific residues in the sixth transmembrane helix and third extracellular loop of the D3 receptor. We also show that unlike cis-8-OH-PBZI, which is a partial agonist at the dopamine D2S receptor and full agonist at dopamine D2L receptor, trans-8-OH-PBZI is a full agonist at both D2S and D2L receptors. The different effect of the two isomers on D3 receptor signaling properties and D2S receptor activation correlated with differential effects of the isomers on agonist-induced mouse locomotor activity. The two isomers of 8-OH-PBZI represent novel pharmacological tools for in silico D3 and D2 receptor homology modeling and for determining the role of D3 receptor tolerance and SRT properties in signaling and behavior. PMID:26116441

  2. Reduced endogenous Ca2+ buffering speeds active zone Ca2+ signaling.

    PubMed

    Delvendahl, Igor; Jablonski, Lukasz; Baade, Carolin; Matveev, Victor; Neher, Erwin; Hallermann, Stefan

    2015-06-01

    Fast synchronous neurotransmitter release at the presynaptic active zone is triggered by local Ca(2+) signals, which are confined in their spatiotemporal extent by endogenous Ca(2+) buffers. However, it remains elusive how rapid and reliable Ca(2+) signaling can be sustained during repetitive release. Here, we established quantitative two-photon Ca(2+) imaging in cerebellar mossy fiber boutons, which fire at exceptionally high rates. We show that endogenous fixed buffers have a surprisingly low Ca(2+)-binding ratio (∼ 15) and low affinity, whereas mobile buffers have high affinity. Experimentally constrained modeling revealed that the low endogenous buffering promotes fast clearance of Ca(2+) from the active zone during repetitive firing. Measuring Ca(2+) signals at different distances from active zones with ultra-high-resolution confirmed our model predictions. Our results lead to the concept that reduced Ca(2+) buffering enables fast active zone Ca(2+) signaling, suggesting that the strength of endogenous Ca(2+) buffering limits the rate of synchronous synaptic transmission. PMID:26015575

  3. Single Particle Tracking Reveals that EGFR Signaling Activity Is Amplified in Clathrin-Coated Pits

    PubMed Central

    Ibach, Jenny; Radon, Yvonne; Gelléri, Márton; Sonntag, Michael H.; Brunsveld, Luc; Bastiaens, Philippe I. H.; Verveer, Peter J.

    2015-01-01

    Signaling from the epidermal growth factor receptor (EGFR) via phosphorylation on its C-terminal tyrosine residues requires self-association, which depends on the diffusional properties of the receptor and its density in the plasma membrane. Dimerization is a key event for EGFR activation, but the role of higher order clustering is unknown. We employed single particle tracking to relate the mobility and aggregation of EGFR to its signaling activity. EGFR mobility alternates between short-lived free, confined and immobile states. In the immobile state, EGFR tends to aggregate in clathrin-coated pits, which is further enhanced in a phosphorylation-dependent manner and does not require ligand binding. EGFR phosphorylation is further amplified by cross-phosphorylation in clathrin-coated pits. Because phosphorylated receptors can escape from the pits, local gradients of signaling active EGFR are formed. These results show that amplification of EGFR phosphorylation by receptor clustering in clathrin-coated pits supports signal activation at the plasma membrane. PMID:26575183

  4. Jasmonate signaling in plant stress responses and development - active and inactive compounds.

    PubMed

    Wasternack, Claus; Strnad, Miroslav

    2016-09-25

    Jasmonates (JAs) are lipid-derived signals mediating plant responses to biotic and abiotic stresses and in plant development. Following the elucidation of each step in their biosynthesis and the important components of perception and signaling, several activators, repressors and co-repressors have been identified which contribute to fine-tuning the regulation of JA-induced gene expression. Many of the metabolic reactions in which JA participates, such as conjugation with amino acids, glucosylation, hydroxylation, carboxylation, sulfation and methylation, lead to numerous compounds with different biological activities. These metabolites may be highly active, partially active in specific processes or inactive. Hydroxylation, carboxylation and sulfation inactivate JA signaling. The precursor of JA biosynthesis, 12-oxo-phytodienoic acid (OPDA), has been identified as a JA-independent signaling compound. An increasing number of OPDA-specific processes is being identified. To conclude, the numerous JA compounds and their different modes of action allow plants to respond specifically and flexibly to alterations in the environment. PMID:26581489

  5. ASBESTOS-INDUCED ACTIVATION OF SIGNALING PATHWAYS IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Title: Asbestos-Induced Activation of Signaling Pathways in Human
    Bronchial Epithelial Cells

    X. Wang, MD 1, J. M. Samet, PhD 2 and A. J. Ghio, MD 2. 1 Center for
    Environmental Medicine, Asthma and Lung Biology, University of North
    Carolina, Chapel Hill, NC, Uni...

  6. 78 FR 28801 - Foreign-Trade Zone 117-Orange, TX, Authorization of Production Activity, Signal International...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-16

    ... notice in the Federal Register inviting public comment (78 FR 4383, 1-22-2013). The FTZ Board has... Foreign-Trade Zones Board Foreign-Trade Zone 117--Orange, TX, Authorization of Production Activity, Signal International Texas GP, LLC (Shipbuilding), Orange, TX On January 10, 2013, the Foreign Trade Zone of...

  7. Epigenetic regulator Lid maintains germline stem cells through regulating JAK-STAT signaling pathway activity

    PubMed Central

    Tarayrah, Lama; Li, Yuping; Gan, Qiang; Chen, Xin

    2015-01-01

    ABSTRACT Signaling pathways and epigenetic mechanisms have both been shown to play essential roles in regulating stem cell activity. While the role of either mechanism in this regulation is well established in multiple stem cell lineages, how the two mechanisms interact to regulate stem cell activity is not as well understood. Here we report that in the Drosophila testis, an H3K4me3-specific histone demethylase encoded by little imaginal discs (lid) maintains germline stem cell (GSC) mitotic index and prevents GSC premature differentiation. Lid is required in germ cells for proper expression of the Stat92E transcription factor, the downstream effector of the Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling pathway. Our findings support a germ cell autonomous role for the JAK-STAT pathway in maintaining GSCs and place Lid as an upstream regulator of this pathway. Our study provides new insights into the biological functions of a histone demethylase in vivo and sheds light on the interaction between epigenetic mechanisms and signaling pathways in regulating stem cell activities. PMID:26490676

  8. 78 FR 4383 - Foreign-Trade Zone 117-Orange, Texas; Notification of Proposed Production Activity; Signal...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-22

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF COMMERCE Foreign-Trade Zones Board Foreign-Trade Zone 117--Orange, Texas; Notification of Proposed Production Activity; Signal International Texas GP, LLC (Shipbuilding), Orange, TX The Foreign Trade Zone of Southeast Texas, Inc., grantee of FTZ 117, submitted...

  9. Reduced endogenous Ca2+ buffering speeds active zone Ca2+ signaling

    PubMed Central

    Delvendahl, Igor; Jablonski, Lukasz; Baade, Carolin; Matveev, Victor; Neher, Erwin; Hallermann, Stefan

    2015-01-01

    Fast synchronous neurotransmitter release at the presynaptic active zone is triggered by local Ca2+ signals, which are confined in their spatiotemporal extent by endogenous Ca2+ buffers. However, it remains elusive how rapid and reliable Ca2+ signaling can be sustained during repetitive release. Here, we established quantitative two-photon Ca2+ imaging in cerebellar mossy fiber boutons, which fire at exceptionally high rates. We show that endogenous fixed buffers have a surprisingly low Ca2+-binding ratio (∼15) and low affinity, whereas mobile buffers have high affinity. Experimentally constrained modeling revealed that the low endogenous buffering promotes fast clearance of Ca2+ from the active zone during repetitive firing. Measuring Ca2+ signals at different distances from active zones with ultra-high-resolution confirmed our model predictions. Our results lead to the concept that reduced Ca2+ buffering enables fast active zone Ca2+ signaling, suggesting that the strength of endogenous Ca2+ buffering limits the rate of synchronous synaptic transmission. PMID:26015575

  10. Endogenous heparan sulfate and heparin modulate bone morphogenetic protein-4 signaling and activity.

    PubMed

    Khan, Shaukat A; Nelson, Matthew S; Pan, Chendong; Gaffney, Patrick M; Gupta, Pankaj

    2008-06-01

    Bone morphogenetic proteins (BMPs) and their endogenous antagonists are important for brain and bone development and tumor initiation and progression. Heparan sulfate (HS) proteoglycans (HSPG) modulate the activities of BMPs and their antagonists. How glycosaminoglycans (GAGs) influence BMP activity in various malignancies and in inherited abnormalities of GAG metabolism, and the structural features of GAGs essential for modulation of BMP signaling, remain incompletely defined. We examined whether chemically modified soluble heparins, the endogenous HS in malignant cells and the HS accumulated in Hurler syndrome cells influence BMP-4 signaling and activity. We show that both exogenous (soluble) and endogenous GAGs modulate BMP-4 signaling and activity, and that this effect is dependent on specific sulfate residues of GAGs. Our studies suggest that endogenous sulfated GAGs promote the proliferation and impair differentiation of malignant human cells, providing the rationale for investigating whether pharmacological agents that inhibit GAG synthesis or function might reverse this effect. Our demonstration of impairment of BMP-4 signaling by GAGs in multipotent stem cells in human Hurler syndrome identifies a mechanism that might contribute to the progressive neurological and skeletal abnormalities in Hurler syndrome and related mucopolysaccharidoses. PMID:18385288

  11. Electrochemical detection of protein kinase activity based on carboxypeptidase Y digestion triggered signal amplification.

    PubMed

    Yin, Huanshun; Wang, Xinxu; Guo, Yunlong; Zhou, Yunlei; Ai, Shiyun

    2015-04-15

    An effective assay method for monitoring protein kinase activity and screening inhibitors is greatly beneficial to kinase-related drug discovery, early diagnosis of diseases, and therapeutic effect evaluation. Herein, we develop a simple electrochemical method for detecting the activity of casein kinase II (CK2) based on phosphorylation against carboxypeptidase Y (CPY) digestion triggered signal amplification, where CK2 catalyzed phosphorylation event protects the substrate peptide from the digestion of CPY, maintains the repulsive force of the substrate peptide towards the redox probe, and results in a weak electrochemical signal. Whereas, without phosphorylation, the substrate peptide is digested by CPY and a strong electrochemical signal is obtained. The detection feasibility is demonstrated for the assay of CK2 activity with low detection limit of 0.047unit/mL. Moreover, the biosensor was used for the analysis of kinase inhibition. Based on the electrochemical signal dependent inhibitor concentration, the IC50 value of ellagic acid was estimated to be 39.77nM. The proposed method is also successfully applied to analyze CK2 activity in cell lysates, proving the applicability in complex biological samples. PMID:25460885

  12. Phyllostachys edulis extract induces apoptosis signaling in osteosarcoma cells, associated with AMPK activation

    PubMed Central

    Chou, Chi-Wen; Cheng, Ya-Wen; Tsai, Chung-Hung

    2014-01-01

    Objective Bamboo is distributed worldwide, and its different parts are used as foods or as a traditional herb. Recently, antitumoral effects of bamboo extracts on several tumors have been increasingly reported; however, antitumoral activity of bamboo extracts on osteosarcoma remains unclear. In the present study, we investigated effects of an aqueous Phyllostachys edulis leaf extract (PEE) on osteosarcoma cells and the underlying mechanism of inhibition. Methods The growth of human osteosarcoma cell lines 143B and MG-63 and lung fibroblast MRC-5 cells was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Apoptosis was demonstrated using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay and flow cytometric analysis. Phosphorylation and protein levels were determined by immunoblotting. Results After treatment with PEE, viability of 143B and MG-63 cells was dose-dependently reduced to 36.3%±1.6% of control values, which were similar to AICAR (5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside) treatments. In parallel, ratios of apoptotic cells and cells in the sub-G1 phase were significantly increased. Further investigation showed that PEE treatments led to activation of caspase cascades and changes of apoptotic mediators Bcl2, Bax, and p53. Consistently, our results revealed that PEE activated adenosine monophosphate-activated protein kinase (AMPK) signaling, and the AMPK activation was associated with the induction of apoptotic signaling. Conclusion Our results indicated that PEE suppressed the growth of 143B and MG-63 cells but moderately affected MRC-5 cells. PEE-induced apoptosis may attribute to AMPK activation and the following activation of apoptotic signaling cascades. These findings revealed that PEE possesses antitumoral activity on human osteosarcoma cells by manipulating AMPK signaling, suggesting that PEE alone or combined with regular antitumor drugs may be beneficial as osteosarcoma

  13. S-nitrosothiols dilate the mesenteric artery more potently than the femoral artery by a cGMP and L-type calcium channel-dependent mechanism.

    PubMed

    Liu, Taiming; Schroeder, Hobe J; Zhang, Meijuan; Wilson, Sean M; Terry, Michael H; Longo, Lawrence D; Power, Gordon G; Blood, Arlin B

    2016-08-31

    S-nitrosothiols (SNOs) are metabolites of NO with potent vasodilatory activity. Our previous studies in sheep indicated that intra-arterially infused SNOs dilate the mesenteric vasculature more than the femoral vasculature. We hypothesized that the mesenteric artery is more responsive to SNO-mediated vasodilation, and investigated various steps along the NO/cGMP pathway to determine the mechanism for this difference. In anesthetized adult sheep, we monitored the conductance of mesenteric and femoral arteries during infusion of S-nitroso-l-cysteine (L-cysNO), and found mesenteric vascular conductance increased (137 ± 3%) significantly more than femoral conductance (26 ± 25%). Similar results were found in wire myography studies of isolated sheep mesenteric and femoral arteries. Vasodilation by SNOs was attenuated in both vessel types by the presence of ODQ (sGC inhibitor), and both YC-1 (sGC agonist) and 8-Br-cGMP (cGMP analog) mediated more potent relaxation in mesenteric arteries than femoral arteries. The vasodilatory difference between mesenteric and femoral arteries was eliminated by antagonists of either protein kinase G or L-type Ca(2+) channels. Western immunoblots showed a larger L-type Ca(2+)/sGC abundance ratio in mesenteric arteries than in femoral arteries. Fetal sheep mesenteric arteries were more responsive to SNOs than adult mesenteric arteries, and had a greater L-Ca(2+)/sGC ratio (p = 0.047 and r = -0.906 for correlation between Emax and L-Ca(2+)/sGC). These results suggest that mesenteric arteries, especially those in fetus, are more responsive to SNO-mediated vasodilation than femoral arteries due to a greater role of the L-type calcium channel in the NO/cGMP pathway. PMID:27235767

  14. Activation of MAP kinase signaling pathway in the mussel Mytilus galloprovincialis as biomarker of environmental pollution.

    PubMed

    Châtel, A; Hamer, B; Talarmin, H; Dorange, G; Schröder, H C; Müller, W E G

    2010-03-01

    Stimulation of MAP kinase signal transduction pathway by various stressful stimuli was investigated in the marine bivalve Mytilus galloprovincialis. Analyses were performed in animals exposed in laboratory to selected pollutants and in mussels collected in winter and summer along the eastern Adriatic coast (Croatia). Effects of oxidative stress, induced by tributyltin, hydrogen peroxide and water soluble fraction of diesel fuel on the activation/phosphorylation of the three Mitogen-Activated Protein Kinases (MAPKs) p38, JNK and ERK using a newly developed ELISA procedure were evaluated. MAP kinase activation was analyzed 1h after exposure of mussels to chemical agents, and after recovery periods of 6 and 24h. Our results clearly indicated that pollutants generated different patterns of induction of the MAPK phosphorylation. Indeed, only pp38 and pJNK were activated with 11, 33 and 100 microg/L TBT, reaching a maximum activation after 6h in seawater following treatment of mussels with 11 microg/L TBT. Treatment with 0.074 and 0.222 mM H2O2 enhanced activation of both p38 and ERK. These two kinases were activated after 1h exposure, followed by a diminution after 6h of recovery in seawater and a reactivation after 24h. The levels of phosphorylated P38 and JNK were increased after mussel exposure with 7.5, 15 and 30% of water soluble fraction of diesel oil. P38 was activated concentration dependently at 1h exposure. Additionally, field study pointed out seasonal differences in MAP kinases activation as mussels collected during summer had a higher enzyme activation state than in winter, as well as sampling site differences which could be correlated to the industrial/tourism activity and environmental stresses (salinity). All the results converge towards MAP kinase signaling pathway being induced by various pollutants in M. galloprovincialis. This signaling cascade should be considered as a possible biomarker of environmental stress and pollution. PMID:19948362

  15. Active site coupling in PDE:PKA complexes promotes resetting of mammalian cAMP signaling.

    PubMed

    Krishnamurthy, Srinath; Moorthy, Balakrishnan Shenbaga; Xin Xiang, Lim; Xin Shan, Lim; Bharatham, Kavitha; Tulsian, Nikhil Kumar; Mihalek, Ivana; Anand, Ganesh S

    2014-09-16

    Cyclic 3'5' adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (KD ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and

  16. Cadmium Activates Multiple Signaling Pathways That Coordinately Stimulate Akt Activity to Enhance c-Myc mRNA Stability

    PubMed Central

    Tsai, Jia-Shiuan; Chao, Cheng-Han; Lin, Lih-Yuan

    2016-01-01

    Cadmium is a known environmental carcinogen. Exposure of Cd leads to the activation of several proto-oncogenes in cells. We investigated here the mechanism of c-Myc expression in hepatic cells under Cd treatment. The c-Myc protein and mRNA levels increased in dose- and time-dependent manners in HepG2 cells with Cd treatment. This increase was due to an increase in c-Myc mRNA stability. To explore the mechanism involved in enhancing the mRNA stability, several cellular signaling factors that evoked by Cd treatment were analyzed. PI3K, p38, ERK and JNK were activated by Cd. However, ERK did not participate in the Cd-induced c-Myc expression. Further analysis revealed that mTORC2 was a downstream factor of p38. PI3K, JNK and mTORC2 coordinately activated Akt. Akt was phosphorylated at Thr450 in the untreated cells. Cd treatment led to additional phosphorylation at Thr308 and Ser473. Blocking any of the three signaling factors resulted in the reduction of phosphorylation level at all three Akt sites. The activated Akt phosphorylated Foxo1 and allowed the modified protein to translocate into the cytoplasm. We conclude that Cd-induced accumulation of c-Myc requires the activation of several signaling pathways. The signals act coordinately for Akt activation and drive the Foxo1 from the nucleus to the cytoplasm. Reduction of Foxo1 in the nucleus reduces the transcription of its target genes that may affect c-Myc mRNA stability, resulting in a higher accumulation of the c-Myc proteins. PMID:26751215

  17. mTOR signaling promotes stem cell activation via counterbalancing BMP-mediated suppression during hair regeneration.

    PubMed

    Deng, Zhili; Lei, Xiaohua; Zhang, Xudong; Zhang, Huishan; Liu, Shuang; Chen, Qi; Hu, Huimin; Wang, Xinyue; Ning, Lina; Cao, Yujing; Zhao, Tongbiao; Zhou, Jiaxi; Chen, Ting; Duan, Enkui

    2015-02-01

    Hair follicles (HFs) undergo cycles of degeneration (catagen), rest (telogen), and regeneration (anagen) phases. Anagen begins when the hair follicle stem cells (HFSCs) obtain sufficient activation cues to overcome suppressive signals, mainly the BMP pathway, from their niche cells. Here, we unveil that mTOR complex 1 (mTORC1) signaling is activated in HFSCs, which coincides with the HFSC activation at the telogen-to-anagen transition. By using both an inducible conditional gene targeting strategy and a pharmacological inhibition method to ablate or inhibit mTOR signaling in adult skin epithelium before anagen initiation, we demonstrate that HFs that cannot respond to mTOR signaling display significantly delayed HFSC activation and extended telogen. Unexpectedly, BMP signaling activity is dramatically prolonged in mTOR signaling-deficient HFs. Through both gain- and loss-of-function studies in vitro, we show that mTORC1 signaling negatively affects BMP signaling, which serves as a main mechanism whereby mTORC1 signaling facilitates HFSC activation. Indeed, in vivo suppression of BMP by its antagonist Noggin rescues the HFSC activation defect in mTORC1-null skin. Our findings reveal a critical role for mTOR signaling in regulating stem cell activation through counterbalancing BMP-mediated repression during hair regeneration. PMID:25609845

  18. Toll-like receptor 9 trafficking and signaling for type I interferons requires PIKfyve activity.

    PubMed

    Hayashi, Kachiko; Sasai, Miwa; Iwasaki, Akiko

    2015-09-01

    Toll-like receptors (TLRs) traffic to distinct membranes for signaling. TLR7 and TLR9 recognize viral nucleic acids in the endosomes and induce robust anti-viral program. Signaling from these TLRs bifurcate at the level of distinct endosomal compartments, namely VAMP3(+) and LAMP(+) endosomes, to mediate the induction of cytokine and type I interferon (IFN) genes, respectively. The formation of the TLR9 endosome competent for IFNs induction requires AP-3. Phosphoinositides (PIs) mark distinct subcellular membranes and control membrane trafficking. However, their role in TLR trafficking and signaling in different dendritic cell (DC) subsets remains unclear. Here, we examined the role of phosphatidylinositol 3P 5-kinase, PIKfyve, in TLR9 trafficking and signaling. We demonstrate that inhibition of PIKfyve activity preferentially blocks TLR9 signaling for type I IFN induction in FLT3L-bone marrow-derived DCs. By confocal microscopy using RAW264.7 cells, we show that trafficking of both TLR9 and CpG to the LAMP1(+) compartment was blocked by PIKfyve inhibitor treatment, whereas their trafficking to the VAMP3(+) endosome remained intact. Further, AP-3 recruitment to TLR9 endosomes was impaired by PIKfyve inhibition. These data indicate that PIKfyve provides critical PIs necessary for the formation of endosome from which TLR9 signals to induce type I IFNs. PMID:25925170

  19. An apodized Kepler periodogram for separating planetary and stellar activity signals

    NASA Astrophysics Data System (ADS)

    Gregory, Philip C.

    2016-05-01

    A new apodized Keplerian (AK) model is proposed for the analysis of precision radial velocity (RV) data to model both planetary and stellar activity (SA) induced RV signals. A symmetrical Gaussian apodization function with unknown width and centre can distinguish planetary signals from SA signals on the basis of the span of the apodization window. The general model for m AK signals includes a linear regression term between RV and the SA diagnostic log (R'hk), as well as an extra Gaussian noise term with unknown standard deviation. The model parameters are explored using a Bayesian fusion Markov chain Monte Carlo code. A differential version of the generalized Lomb-Scargle periodogram that employs a control diagnostic provides an additional way of distinguishing SA signals and helps guide the choice of new periods. Results are reported for a recent international RV blind challenge which included multiple state-of-the-art simulated data sets supported by a variety of SA diagnostics. In the current implementation, the AK method achieved a reduction in SA noise by a factor of approximately 6. Final parameter estimates for the planetary candidates are derived from fits that include AK signals to model the SA components and simple Keplerians to model the planetary candidates. Preliminary results are also reported for AK models augmented by a moving average component that allows for correlations in the residuals.

  20. An apodized Kepler periodogram for separating planetary and stellar activity signals

    PubMed Central

    Gregory, Philip C.

    2016-01-01

    A new apodized Keplerian (AK) model is proposed for the analysis of precision radial velocity (RV) data to model both planetary and stellar activity (SA) induced RV signals. A symmetrical Gaussian apodization function with unknown width and centre can distinguish planetary signals from SA signals on the basis of the span of the apodization window. The general model for m AK signals includes a linear regression term between RV and the SA diagnostic log (R′hk), as well as an extra Gaussian noise term with unknown standard deviation. The model parameters are explored using a Bayesian fusion Markov chain Monte Carlo code. A differential version of the generalized Lomb–Scargle periodogram that employs a control diagnostic provides an additional way of distinguishing SA signals and helps guide the choice of new periods. Results are reported for a recent international RV blind challenge which included multiple state-of-the-art simulated data sets supported by a variety of SA diagnostics. In the current implementation, the AK method achieved a reduction in SA noise by a factor of approximately 6. Final parameter estimates for the planetary candidates are derived from fits that include AK signals to model the SA components and simple Keplerians to model the planetary candidates. Preliminary results are also reported for AK models augmented by a moving average component that allows for correlations in the residuals. PMID:27346979

  1. Zinc Chloride Transiently Maintains Mouse Embryonic Stem Cell Pluripotency by Activating Stat3 Signaling

    PubMed Central

    Hu, Jing; Yang, Zhiyong; Wang, Jinbo; Yu, Jia; Guo, Jing; Liu, Shiying; Qian, Chunmei; Song, Liwen; Wu, Yi; Cheng, Jiajing

    2016-01-01

    An improved understanding of the pluripotency maintenance of embryonic stem (ES) cells is important for investigations of early embryo development and for cell replacement therapy, but the mechanism behind pluripotency is still incompletely understood. Recent findings show that zinc, an essential trace element in humans, is critically involved in regulating various signaling pathways and genes expression. However, its role in ES cell fate determination remains to be further explored. Here we showed that 2μM zinc chloride (ZnCl2) transiently maintained mouse ES cell pluripotency in vitro. The cultured mouse ES cells remained undifferentiated under 2μM ZnCl2 treatment in leukemia inhibitory factor (LIF) withdrawal, retinoic acid (RA) or embryoid bodies (EBs) differentiation assays. In addition, ZnCl2 increased pluripotency genes expression and inhibited differentiation genes expression. Further mechanistic studies revealed that ZnCl2 transiently activated signal transducers and activators of transcription 3 (Stat3) signaling through promoting Stat3 phosphorylation. Inhibition of Stat3 signaling abrogated the effects of ZnCl2 on mouse ES cell pluripotency. Taken together, this study demonstrated a critical role of zinc in the pluripotency maintenance of mouse ES cells, as well as an important regulator of Stat3 signaling. PMID:26910359

  2. Dishevelled attenuates the repelling activity of Wnt signaling during neurite outgrowth in Caenorhabditis elegans.

    PubMed

    Zheng, Chaogu; Diaz-Cuadros, Margarete; Chalfie, Martin

    2015-10-27

    Wnt proteins regulate axonal outgrowth along the anterior-posterior axis, but the intracellular mechanisms that modulate the strength of Wnt signaling in axon guidance are largely unknown. Using the Caenorhabditis elegans mechanosensory PLM neurons, we found that posteriorly enriched LIN-44/Wnt acts as a repellent to promote anteriorly directed neurite outgrowth through the LIN-17/Frizzled receptor, instead of controlling neuronal polarity as previously thought. Dishevelled (Dsh) proteins DSH-1 and MIG-5 redundantly mediate the repulsive activity of the Wnt signals to induce anterior outgrowth, whereas DSH-1 also provides feedback inhibition to attenuate the signaling to allow posterior outgrowth against the Wnt gradient. This inhibitory function of DSH-1, which requires its dishevelled, Egl-10, and pleckstrin (DEP) domain, acts by promoting LIN-17 phosphorylation and is antagonized by planar cell polarity signaling components Van Gogh (VANG-1) and Prickle (PRKL-1). Our results suggest that Dsh proteins both respond to Wnt signals to shape neuronal projections and moderate its activity to fine-tune neuronal morphology. PMID:26460008

  3. Quorum activation at a distance: spatiotemporal patterns of gene regulation from diffusion of an autoinducer signal

    NASA Astrophysics Data System (ADS)

    Dilanji, Gabriel; Langebrake, Jessica; Deleenheer, Patrick; Hagen, Stephen J.

    2012-02-01

    Bacteria in colonies coordinate gene regulation through the exchange of diffusible signal molecules known as autoinducers (AI). This ``quorum signaling'' often occurs in physically heterogeneous and spatially extended environments such as biofilms. Under these conditions the space and time scales for diffusion of the signal limit the range and timing of effective gene regulation. We expect that spatial and temporal patterns of gene expression will reflect physical environmental constraints as well as nonlinear transcriptional activation and feedback within the gene regulatory system. We have combined experiments and modeling to investigate how these spatiotemporal patterns develop. We embed engineered plasmid/GFP quorum sensor strains or wild type strains in a long narrow agar lane, and then introduce AI signal at one terminus of the lane. Diffusion of the AI initiates reporter expression along the length of the lane, extending to macroscopic distances of mm-cm. Resulting patterns are captured quantitatively by a mathematical model that incorporates logistic growth of the population, diffusion of AI, and nonlinear transcriptional activation. Our results show that a diffusing quorum signal can coordinate gene expression over distances of order 1cm on time scales of order 10 hrs.

  4. Dishevelled attenuates the repelling activity of Wnt signaling during neurite outgrowth in Caenorhabditis elegans

    PubMed Central

    Zheng, Chaogu; Diaz-Cuadros, Margarete; Chalfie, Martin

    2015-01-01

    Wnt proteins regulate axonal outgrowth along the anterior–posterior axis, but the intracellular mechanisms that modulate the strength of Wnt signaling in axon guidance are largely unknown. Using the Caenorhabditis elegans mechanosensory PLM neurons, we found that posteriorly enriched LIN-44/Wnt acts as a repellent to promote anteriorly directed neurite outgrowth through the LIN-17/Frizzled receptor, instead of controlling neuronal polarity as previously thought. Dishevelled (Dsh) proteins DSH-1 and MIG-5 redundantly mediate the repulsive activity of the Wnt signals to induce anterior outgrowth, whereas DSH-1 also provides feedback inhibition to attenuate the signaling to allow posterior outgrowth against the Wnt gradient. This inhibitory function of DSH-1, which requires its dishevelled, Egl-10, and pleckstrin (DEP) domain, acts by promoting LIN-17 phosphorylation and is antagonized by planar cell polarity signaling components Van Gogh (VANG-1) and Prickle (PRKL-1). Our results suggest that Dsh proteins both respond to Wnt signals to shape neuronal projections and moderate its activity to fine-tune neuronal morphology. PMID:26460008

  5. Organ-specific adaptive signaling pathway activation in metastatic breast cancer cells

    PubMed Central

    Burnett, Riesa M.; Craven, Kelly E.; Krishnamurthy, Purna; Goswami, Chirayu P.; Badve, Sunil; Crooks, Peter; Mathews, William P.; Bhat-Nakshatri, Poornima; Nakshatri, Harikrishna

    2015-01-01

    Breast cancer metastasizes to bone, visceral organs, and/or brain depending on the subtype, which may involve activation of a host organ-specific signaling network in metastatic cells. To test this possibility, we determined gene expression patterns in MDA-MB-231 cells and its mammary fat pad tumor (TMD-231), lung-metastasis (LMD-231), bone-metastasis (BMD-231), adrenal-metastasis (ADMD-231) and brain-metastasis (231-BR) variants. When gene expression between metastases was compared, 231-BR cells showed the highest gene expression difference followed by ADMD-231, LMD-231, and BMD-231 cells. Neuronal transmembrane proteins SLITRK2, TMEM47, and LYPD1 were specifically overexpressed in 231-BR cells. Pathway-analyses revealed activation of signaling networks that would enable cancer cells to adapt to organs of metastasis such as drug detoxification/oxidative stress response/semaphorin neuronal pathway in 231-BR, Notch/orphan nuclear receptor signals involved in steroidogenesis in ADMD-231, acute phase response in LMD-231, and cytokine/hematopoietic stem cell signaling in BMD-231 cells. Only NF-κB signaling pathway activation was common to all except BMD-231 cells. We confirmed NF-κB activation in 231-BR and in a brain metastatic variant of 4T1 cells (4T1-BR). Dimethylaminoparthenolide inhibited NF-κB activity, LYPD1 expression, and proliferation of 231-BR and 4T1-BR cells. Thus, transcriptome change enabling adaptation to host organs is likely one of the mechanisms associated with organ-specific metastasis and could potentially be targeted therapeutically. PMID:25926557

  6. Organ-specific adaptive signaling pathway activation in metastatic breast cancer cells.

    PubMed

    Burnett, Riesa M; Craven, Kelly E; Krishnamurthy, Purna; Goswami, Chirayu P; Badve, Sunil; Crooks, Peter; Mathews, William P; Bhat-Nakshatri, Poornima; Nakshatri, Harikrishna

    2015-05-20

    Breast cancer metastasizes to bone, visceral organs, and/or brain depending on the subtype, which may involve activation of a host organ-specific signaling network in metastatic cells. To test this possibility, we determined gene expression patterns in MDA-MB-231 cells and its mammary fat pad tumor (TMD-231), lung-metastasis (LMD-231), bone-metastasis (BMD-231), adrenal-metastasis (ADMD-231) and brain-metastasis (231-BR) variants. When gene expression between metastases was compared, 231-BR cells showed the highest gene expression difference followed by ADMD-231, LMD-231, and BMD-231 cells. Neuronal transmembrane proteins SLITRK2, TMEM47, and LYPD1 were specifically overexpressed in 231-BR cells. Pathway-analyses revealed activation of signaling networks that would enable cancer cells to adapt to organs of metastasis such as drug detoxification/oxidative stress response/semaphorin neuronal pathway in 231-BR, Notch/orphan nuclear receptor signals involved in steroidogenesis in ADMD-231, acute phase response in LMD-231, and cytokine/hematopoietic stem cell signaling in BMD-231 cells. Only NF-κB signaling pathway activation was common to all except BMD-231 cells. We confirmed NF-κB activation in 231-BR and in a brain metastatic variant of 4T1 cells (4T1-BR). Dimethylaminoparthenolide inhibited NF-κB activity, LYPD1 expression, and proliferation of 231-BR and 4T1-BR cells. Thus, transcriptome change enabling adaptation to host organs is likely one of the mechanisms associated with organ-specific metastasis and could potentially be targeted therapeutically. PMID:25926557

  7. Brazilian Red Propolis Attenuates Inflammatory Signaling Cascade in LPS-Activated Macrophages

    PubMed Central

    Bueno-Silva, Bruno; Kawamoto, Dione; Ando-Suguimoto, Ellen S.; Alencar, Severino M.; Rosalen, Pedro L.; Mayer, Marcia P. A.

    2015-01-01

    Although previous studies suggested an anti-inflammatory property of Brazilian red propolis (BRP), the mechanisms involved in the anti-inflammatory effects of BRP and its activity on macrophages were still not elucidated. This study aimed to evaluate whether BRP attenuates the inflammatory effect of LPS on macrophages and to investigate its underlying mechanisms. BRP was added to RAW 264.7 murine macrophages after activation with LPS. NO production, cell viability, cytokines profile were evaluated. Activation of inflammatory signaling pathways and macrophage polarization were determined by RT-qPCR and Western blot. BRP at 50 μg/ml inhibited NO production by 78% without affecting cell viability. Cd80 and Cd86 were upregulated whereas mrc1 was down regulated by BRP indicating macrophage polarization at M1. BRP attenuated the production of pro-inflammatory mediators IL-12, GM-CSF, IFN-Ɣ, IL-1β in cell supernatants although levels of TNF- α and IL-6 were slightly increased after BRP treatment. Levels of IL-4, IL-10 and TGF-β were also reduced by BRP. BRP significantly reduced the up-regulation promoted by LPS of transcription of genes in inflammatory signaling (Pdk1, Pak1, Nfkb1, Mtcp1, Gsk3b, Fos and Elk1) and of Il1β and Il1f9 (fold-change rate > 5), which were further confirmed by the inhibition of NF-κB and MAPK signaling pathways. Furthermore, the upstream adaptor MyD88 adaptor-like (Mal), also known as TIRAP, involved in TLR2 and TLR4 signaling, was down- regulated in BRP treated LPS-activated macrophages. Given that BRP inhibited multiple signaling pathways in macrophages involved in the inflammatory process activated by LPS, our data indicated that BRP is a noteworthy food-source for the discovery of new bioactive compounds and a potential candidate to attenuate exhacerbated inflammatory diseases. PMID:26660901

  8. Accumulated source imaging of brain activity with both low and high-frequency neuromagnetic signals

    PubMed Central

    Xiang, Jing; Luo, Qian; Kotecha, Rupesh; Korman, Abraham; Zhang, Fawen; Luo, Huan; Fujiwara, Hisako; Hemasilpin, Nat; Rose, Douglas F.

    2014-01-01

    Recent studies have revealed the importance of high-frequency brain signals (>70 Hz). One challenge of high-frequency signal analysis is that the size of time-frequency representation of high-frequency brain signals could be larger than 1 terabytes (TB), which is beyond the upper limits of a typical computer workstation's memory (<196 GB). The aim of the present study is to develop a new method to provide greater sensitivity in detecting high-frequency magnetoencephalography (MEG) signals in a single automated and versatile interface, rather than the more traditional, time-intensive visual inspection methods, which may take up to several days. To address the aim, we developed a new method, accumulated source imaging, defined as the volumetric summation of source activity over a period of time. This method analyzes signals in both low- (1~70 Hz) and high-frequency (70~200 Hz) ranges at source levels. To extract meaningful information from MEG signals at sensor space, the signals were decomposed to channel-cross-channel matrix (CxC) representing the spatiotemporal patterns of every possible sensor-pair. A new algorithm was developed and tested by calculating the optimal CxC and source location-orientation weights for volumetric source imaging, thereby minimizing multi-source interference and reducing computational cost. The new method was implemented in C/C++ and tested with MEG data recorded from clinical epilepsy patients. The results of experimental data demonstrated that accumulated source imaging could effectively summarize and visualize MEG recordings within 12.7 h by using approximately 10 GB of computer memory. In contrast to the conventional method of visually identifying multi-frequency epileptic activities that traditionally took 2–3 days and used 1–2 TB storage, the new approach can quantify epileptic abnormalities in both low- and high-frequency ranges at source levels, using much less time and computer memory. PMID:24904402

  9. Selenium Deficiency Attenuates Chicken Duodenal Mucosal Immunity via Activation of the NF-κb Signaling Pathway.

    PubMed

    Liu, Zhe; Qu, Yanpeng; Wang, Jianfa; Wu, Rui

    2016-08-01

    Selenium (Se) deficiency can cause intestinal mucosal inflammation, which is related to activation of nuclear transcription factor kappa-B (NF-κB) signaling pathway. However, the mechanism of inflammatory response in chicken duodenal mucosa caused by Se deficiency and its relationship with the NF-κB signaling pathway remain elusive. In this study, we firstly obtained Se-deficient chickens bred with 0.01 mg/kg Se and the normal chickens bred with 0.4 mg/kg Se for 35 days. Then, NF-κB signaling pathway, secretory immunoglobulin A (SIgA), inflammatory cytokines, oxidized glutathione, glutathione peroxidase, and glutathione activities were determined. The results showed that Se deficiency obviously enhanced p50, p65, and p65 DNA-binding activities. The phosphorylation of IκB-α and phosphorylation of kappa-B kinase subunit alpha (IKKα) and IKKα were elevated, but IκB-α was decreased (P < 0.05). Moreover, Se deficiency reduced SIgA amount in the duodenal mucosa but increased the level of interleukin-1β (IL-1β), IL-17A, tumor necrosis factor-α (TNF-α), and interferon gamma (IFN-γ). In contrast, anti-inflammatory cytokines, such as TGF-β1 and IL-10, were significantly suppressed. Additionally, Se deficiency increased oxidized glutathione activity, whereas decreased glutathione peroxidase and glutathione activities (P < 0.05), suggesting that Se deficiency affected the regulation function of redox. Taken together, our results demonstrated that Se deficiency attenuated chicken duodenal mucosal immunity via activation of NF-κB signaling pathway regulated by redox activity, which suggested that Se is a crucial host factor involved in regulating inflammation. PMID:26728795

  10. Convergence of dopamine and glutamate signaling onto striatal ERK activation in response to drugs of abuse

    PubMed Central

    Cahill, Emma; Salery, Marine; Vanhoutte, Peter; Caboche, Jocelyne

    2014-01-01

    Despite their distinct targets, all addictive drugs commonly abused by humans evoke increases in dopamine (DA) concentration within the striatum. The main DA Guanine nucleotide binding protein couple receptors (GPCRs) expressed by medium-sized spiny neurons of the striatum are the D1R and D2R, which are positively and negatively coupled to cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling, respectively. These two DA GPCRs are largely segregated into distinct neuronal populations, where they are co-expressed with glutamate receptors in dendritic spines. Direct and indirect interactions between DA GPCRs and glutamate receptors are the molecular basis by which DA modulates glutamate transmission and controls striatal plasticity and behavior induced by drugs of abuse. A major downstream target of striatal D1R is the extracellular signal-regulated kinase (ERK) kinase pathway. ERK activation by drugs of abuse behaves as a key integrator of D1R and glutamate NMDAR signaling. Once activated, ERK can trigger chromatin remodeling and induce gene expression that permits long-term cellular alterations and drug-induced morphological and behavioral changes. Besides the classical cAMP/PKA pathway, downstream of D1R, recent evidence implicates a cAMP-independent crosstalk mechanism by which the D1R potentiates NMDAR-mediated calcium influx and ERK activation. The mounting evidence of reciprocal modulation of DA and glutamate receptors adds further intricacy to striatal synaptic signaling and is liable to prove relevant for addictive drug-induced signaling, plasticity, and behavior. Herein, we review the evidence that built our understanding of the consequences of this synergistic signaling for the actions of drugs of abuse. PMID:24409148

  11. Cyclic GMP signaling in cardiomyocytes modulates fatty acid trafficking and prevents triglyceride accumulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While the balance between carbohydrates and fatty acids for energy production appears to be crucial for cardiac homeostasis, much remains to be learned about the molecular mechanisms underlying this relationship. Given the reported benefits of cGMP signaling on the myocardium, we investigated the im...

  12. Resistance of Cancer Cells to Targeted Therapies Through the Activation of Compensating Signaling Loops.

    PubMed

    von Manstein, Viktoria; Yang, Chul Min; Richter, Diane; Delis, Natalia; Vafaizadeh, Vida; Groner, Bernd

    2013-12-01

    The emergence of low molecular weight kinase inhibitors as "targeted" drugs has led to remarkable advances in the treatment of cancer patients. The clinical benefits of these tumor therapies, however, vary widely in patient populations and with duration of treatment. Intrinsic and acquired resistance against such drugs limits their efficacy. In addition to the well studied mechanisms of resistance based upon drug transport and metabolism, genetic alterations in drug target structures and the activation of compensatory cell signaling have received recent attention. Adaptive responses can be triggered which counteract the initial dependence of tumor cells upon a particular signaling molecule and allow only a transient inhibition of tumor cell growth. These compensating signaling mechanisms are often based upon the relief of repression of regulatory feedback loops. They might involve cell autonomous, intracellular events or they can be mediated via the secretion of growth factor receptor ligands into the tumor microenvironment and signal induction in an auto- or paracrine fashion. The transcription factors Stat3 and Stat5 mediate the biological functions of cytokines, interleukins and growth factors and can be considered as endpoints of multiple signaling pathways. In normal cells this activation is transient and the Stat molecules return to their non-phosphorylated state within a short time period. In tumor cells the balance between activating and de-activating signals is disturbed resulting in the persistent activation of Stat3 or Stat5. The constant activation of Stat3 induces the expression of target genes, which cause the proliferation and survival of cancer cells, as well as their migration and invasive behavior. Activating components of the Jak-Stat pathway have been recognized as potentially valuable drug targets and important principles of compensatory signaling circuit induction during targeted drug treatment have been discovered in the context of kinase

  13. Epigenetic Activation of Wnt/β-Catenin Signaling in NAFLD-Associated Hepatocarcinogenesis.

    PubMed

    Tian, Yuan; Mok, Myth T S; Yang, Pengyuan; Cheng, Alfred S L

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD), characterized by fat accumulation in liver, is closely associated with central obesity, over-nutrition and other features of metabolic syndrome, which elevate the risk of developing hepatocellular carcinoma (HCC). The Wnt/β-catenin signaling pathway plays a significant role in the physiology and pathology of liver. Up to half of HCC patients have activation of Wnt/β-catenin signaling. However, the mutation frequencies of CTNNB1 (encoding β-catenin protein) or other antagonists targeting Wnt/β-catenin signaling are low in HCC patients, suggesting that genetic mutations are not the major factor driving abnormal β-catenin activities in HCC. Emerging evidence has demonstrated that obesity-induced metabolic pathways can deregulate chromatin modifiers such as histone deacetylase 8 to trigger undesired global epigenetic changes, thereby modifying gene expression program which contributes to oncogenic signaling. This review focuses on the aberrant epigenetic activation of Wnt/β-catenin in the development of NAFLD-associated HCC. A deeper understanding of the molecular mechanisms underlying such deregulation may shed light on the identification of novel druggable epigenetic targets for the prevention and/or treatment of HCC in obese and diabetic patients. PMID:27556491

  14. Linear models of activation cascades: analytical solutions and coarse-graining of delayed signal transduction.

    PubMed

    Beguerisse-Díaz, Mariano; Desikan, Radhika; Barahona, Mauricio

    2016-08-01

    Cellular signal transduction usually involves activation cascades, the sequential activation of a series of proteins following the reception of an input signal. Here, we study the classic model of weakly activated cascades and obtain analytical solutions for a variety of inputs. We show that in the special but important case of optimal gain cascades (i.e. when the deactivation rates are identical) the downstream output of the cascade can be represented exactly as a lumped nonlinear module containing an incomplete gamma function with real parameters that depend on the rates and length of the cascade, as well as parameters of the input signal. The expressions obtained can be applied to the non-identical case when the deactivation rates are random to capture the variability in the cascade outputs. We also show that cascades can be rearranged so that blocks with similar rates can be lumped and represented through our nonlinear modules. Our results can be used both to represent cascades in computational models of differential equations and to fit data efficiently, by reducing the number of equations and parameters involved. In particular, the length of the cascade appears as a real-valued parameter and can thus be fitted in the same manner as Hill coefficients. Finally, we show how the obtained nonlinear modules can be used instead of delay differential equations to model delays in signal transduction. PMID:27581482

  15. Redundant canonical and noncanonical Caenorhabditis elegans p21-activated kinase signaling governs distal tip cell migrations.

    PubMed

    Peters, Eldon C; Gossett, Andrea J; Goldstein, Bob; Der, Channing J; Reiner, David J

    2013-02-01

    p21-activated kinases (Paks) are prominent mediators of Rac/Cdc42-dependent and -independent signaling and regulate signal transduction and cytoskeletal-based cell movements. We used the reproducible migrations of the Caenorhabditis elegans gonadal distal tip cells to show that two of the three nematode Pak proteins, MAX-2 and PAK-1, function redundantly in regulation of cell migration but are regulated by very different mechanisms. First, we suggest that MAX-2 requires CED-10/Rac function and thus functions canonically. Second, PIX-1 and GIT-1 function in the same role as PAK-1, and PAK-1 interaction with PIX-1 is required for PAK-1 activity; thus, PAK-1 functions noncanonically. The human Pak-Pix-Git complex is central to noncanonical Pak signaling and requires only modest Rac/CDC-42 input. Unlike the human complex, our results suggest that the C. elegans Pak-Pix-Git complex requires PAK-1 kinase domain activity. This study delineates signaling network relationships in this cell migration model, thus providing potential further mechanistic insights and an assessment of total Pak contribution to cell migration events. PMID:23390595

  16. F-actin polymerization and retrograde flow drive sustained PLCγ1 signaling during T cell activation

    PubMed Central

    Babich, Alexander; Li, Shuixing; O'Connor, Roddy S.; Milone, Michael C.; Freedman, Bruce D.

    2012-01-01

    Activation of T cells by antigen-presenting cells involves assembly of signaling molecules into dynamic microclusters (MCs) within a specialized membrane domain termed the immunological synapse (IS). Actin and myosin IIA localize to the IS, and depletion of F-actin abrogates MC movement and T cell activation. However, the mechanisms that coordinate actomyosin dynamics and T cell receptor signaling are poorly understood. Using pharmacological inhibitors that perturb individual aspects of actomyosin dynamics without disassembling the network, we demonstrate that F-actin polymerization is the primary driver of actin retrograde flow, whereas myosin IIA promotes long-term integrity of the IS. Disruption of F-actin retrograde flow, but not myosin IIA contraction, arrested MC centralization and inhibited sustained Ca2+ signaling at the level of endoplasmic reticulum store release. Furthermore, perturbation of retrograde flow inhibited PLCγ1 phosphorylation within MCs but left Zap70 activity intact. These studies highlight the importance of ongoing actin polymerization as a central driver of actomyosin retrograde flow, MC centralization, and sustained Ca2+ signaling. PMID:22665519

  17. Linear models of activation cascades: analytical solutions and coarse-graining of delayed signal transduction

    PubMed Central

    Desikan, Radhika

    2016-01-01

    Cellular signal transduction usually involves activation cascades, the sequential activation of a series of proteins following the reception of an input signal. Here, we study the classic model of weakly activated cascades and obtain analytical solutions for a variety of inputs. We show that in the special but important case of optimal gain cascades (i.e. when the deactivation rates are identical) the downstream output of the cascade can be represented exactly as a lumped nonlinear module containing an incomplete gamma function with real parameters that depend on the rates and length of the cascade, as well as parameters of the input signal. The expressions obtained can be applied to the non-identical case when the deactivation rates are random to capture the variability in the cascade outputs. We also show that cascades can be rearranged so that blocks with similar rates can be lumped and represented through our nonlinear modules. Our results can be used both to represent cascades in computational models of differential equations and to fit data efficiently, by reducing the number of equations and parameters involved. In particular, the length of the cascade appears as a real-valued parameter and can thus be fitted in the same manner as Hill coefficients. Finally, we show how the obtained nonlinear modules can be used instead of delay differential equations to model delays in signal transduction. PMID:27581482

  18. Periostin Responds to Mechanical Stress and Tension by Activating the MTOR Signaling Pathway

    PubMed Central

    Rosselli-Murai, Luciana K.; Galindo-Moreno, Pablo; Padial-Molina, Miguel; Volk, Sarah L.; Murai, Marcelo J.; Rios, Hector F.; Squarize, Cristiane H.; Castilho, Rogerio M.

    2013-01-01

    Current knowledge about Periostin biology has expanded from its recognized functions in embryogenesis and bone metabolism to its roles in tissue repair and remodeling and its clinical implications in cancer. Emerging evidence suggests that Periostin plays a critical role in the mechanism of wound healing; however, the paracrine effect of Periostin in epithelial cell biology is still poorly understood. We found that epithelial cells are capable of producing endogenous Periostin that, unlike mesenchymal cell, cannot be secreted. Epithelial cells responded to Periostin paracrine stimuli by enhancing cellular migration and proliferation and by activating the mTOR signaling pathway. Interestingly, biomechanical stimulation of epithelial cells, which simulates tension forces that occur during initial steps of tissue healing, induced Periostin production and mTOR activation. The molecular association of Periostin and mTOR signaling was further dissected by administering rapamycin, a selective pharmacological inhibitor of mTOR, and by disruption of Raptor and Rictor scaffold proteins implicated in the regulation of mTORC1 and mTORC2 complex assembly. Both strategies resulted in ablation of Periostin-induced mitogenic and migratory activity. These results indicate that Periostin-induced epithelial migration and proliferation requires mTOR signaling. Collectively, our findings identify Periostin as a mechanical stress responsive molecule that is primarily secreted by fibroblasts during wound healing and expressed endogenously in epithelial cells resulting in the control of cellular physiology through a mechanism mediated by the mTOR signaling cascade. PMID:24349533

  19. Analysis of G Protein and β-Arrestin Activation in Chemokine Receptors Signaling.

    PubMed

    Vacchini, Alessandro; Busnelli, Marta; Chini, Bice; Locati, Massimo; Borroni, Elena Monica

    2016-01-01

    Chemokines are key regulators of leukocyte migration and play fundamental roles in immune responses. The chemokine system includes a set of over 40 ligands which engage in a promiscuous fashion a panel of over 25 receptors belonging to a distinct family of 7 transmembrane-domain receptors (7TM) widely expressed on a variety of cells. Although responses evoked by chemoki