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Sample records for activate signaling cascades

  1. 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

  2. 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

  3. 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

  4. 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

  5. Signalling pathways: jack of all cascades.

    PubMed

    Cahill, M A; Janknecht, R; Nordheim, A

    1996-01-01

    The transcription factors that bind the c-fos promoter element SRE are targeted by multiple, independent signalling cascades; the identities of these signalling pathways and their modes of activation are being elucidated. PMID:8805215

  6. MAP kinase cascades: scaffolding signal specificity.

    PubMed

    van Drogen, Frank; Peter, Matthias

    2002-01-22

    Scaffold proteins organize many MAP kinase pathways by interacting with several components of these cascades. Recent studies suggest that scaffold proteins provide local activation platforms that contribute to signal specificity by insulating different MAP kinase pathways. PMID:11818078

  7. MAPK Cascades in Guard Cell Signal Transduction

    PubMed Central

    Lee, Yuree; Kim, Yun Ju; Kim, Myung-Hee; Kwak, June M.

    2016-01-01

    Guard cells form stomata on the epidermis and continuously respond to endogenous and environmental stimuli to fine-tune the gas exchange and transpirational water loss, processes which involve mitogen-activated protein kinase (MAPK) cascades. MAPKs form three-tiered kinase cascades with MAPK kinases and MAPK kinase kinases, by which signals are transduced to the target proteins. MAPK cascade genes are highly conserved in all eukaryotes, and they play crucial roles in myriad developmental and physiological processes. MAPK cascades function during biotic and abiotic stress responses by linking extracellular signals received by receptors to cytosolic events and gene expression. In this review, we highlight recent findings and insights into MAPK-mediated guard cell signaling, including the specificity of MAPK cascades and the remaining questions. PMID:26904052

  8. MAPK Cascades in Guard Cell Signal Transduction.

    PubMed

    Lee, Yuree; Kim, Yun Ju; Kim, Myung-Hee; Kwak, June M

    2016-01-01

    Guard cells form stomata on the epidermis and continuously respond to endogenous and environmental stimuli to fine-tune the gas exchange and transpirational water loss, processes which involve mitogen-activated protein kinase (MAPK) cascades. MAPKs form three-tiered kinase cascades with MAPK kinases and MAPK kinase kinases, by which signals are transduced to the target proteins. MAPK cascade genes are highly conserved in all eukaryotes, and they play crucial roles in myriad developmental and physiological processes. MAPK cascades function during biotic and abiotic stress responses by linking extracellular signals received by receptors to cytosolic events and gene expression. In this review, we highlight recent findings and insights into MAPK-mediated guard cell signaling, including the specificity of MAPK cascades and the remaining questions. PMID:26904052

  9. Zinc promotes proliferation and activation of myogenic cells via the PI3K/Akt and ERK signaling cascade

    SciTech Connect

    Ohashi, Kazuya; Nagata, Yosuke; Wada, Eiji; Zammit, Peter S.; Shiozuka, Masataka; Matsuda, Ryoichi

    2015-05-01

    Skeletal muscle stem cells named muscle satellite cells are normally quiescent but are activated in response to various stimuli, such as injury and overload. Activated satellite cells enter the cell cycle and proliferate to produce a large number of myogenic progenitor cells, and these cells then differentiate and fuse to form myofibers. Zinc is one of the essential elements in the human body, and has multiple roles, including cell growth and DNA synthesis. However, the role of zinc in myogenic cells is not well understood, and is the focus of this study. We first examined the effects of zinc on differentiation of murine C2C12 myoblasts and found that zinc promoted proliferation, with an increased number of cells incorporating EdU, but inhibited differentiation with reduced myogenin expression and myotube formation. Furthermore, we used the C2C12 reserve cell model of myogenic quiescence to investigate the role of zinc on activation of myogenic cells. The number of reserve cells incorporating BrdU was increased by zinc in a dose dependent manner, with the number dramatically further increased using a combination of zinc and insulin. Akt and extracellular signal-regulated kinase (ERK) are downstream of insulin signaling, and both were phosphorylated after zinc treatment. The zinc/insulin combination-induced activation involved the phosphoinositide 3-kinase (PI3K)/Akt and ERK cascade. We conclude that zinc promotes activation and proliferation of myogenic cells, and this activation requires phosphorylation of PI3K/Akt and ERK as part of the signaling cascade. - Highlights: • Zinc has roles for promoting proliferation and inhibition differentiation of C2C12. • Zinc promotes activation of reserve cells. • Insulin and zinc synergize activation of reserve cells. • PI3K/Akt and ERK cascade affect zinc/insulin-mediated activation of reserve cells.

  10. Nitric oxide/cGMP/PKG signaling pathway activated by M1-type muscarinic acetylcholine receptor cascade inhibits Na+-activated K+ currents in Kenyon cells.

    PubMed

    Hasebe, Masaharu; Yoshino, Masami

    2016-06-01

    The interneurons of the mushroom body, known as Kenyon cells, are essential for the long-term memory of olfactory associative learning in some insects. Some studies have reported that nitric oxide (NO) is strongly related to this long-term memory in Kenyon cells. However, the target molecules and upstream and downstream NO signaling cascades are not completely understood. Here we analyzed the effect of the NO signaling cascade on Na(+)-activated K(+) (KNa) channel activity in Kenyon cells of crickets (Gryllus bimaculatus). We found that two different NO donors, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-dl-penicillamine (SNAP), strongly suppressed KNa channel currents. Additionally, this inhibitory effect of GSNO on KNa channel activity was diminished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC), and KT5823, an inhibitor of protein kinase G (PKG). Next, we analyzed the role of ACh in the NO signaling cascade. ACh strongly suppressed KNa channel currents, similar to NO donors. Furthermore, this inhibitory effect of ACh was blocked by pirenzepine, an M1 muscarinic ACh receptor antagonist, but not by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) and mecamylamine, an M3 muscarinic ACh receptor antagonist and a nicotinic ACh receptor antagonist, respectively. The ACh-induced inhibition of KNa channel currents was also diminished by the PLC inhibitor U73122 and the calmodulin antagonist W-7. Finally, we found that ACh inhibition was blocked by the nitric oxide synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME). These results suggested that the ACh signaling cascade promotes NO production by activating NOS and NO inhibits KNa channel currents via the sGC/cGMP/PKG signaling cascade in Kenyon cells. PMID:26984419

  11. 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

  12. How sesquiterpenes modulate signaling cascades in cancers.

    PubMed

    Jabeen, S; Qureshi, M Z; Attar, R; Aslam, A; Kanwal, S; Khalid, S; Qureshi, J M; Aras Perk, A; Farooqi, A A; Ismail, M

    2016-01-01

    Data obtained from high-throughput technologies has started to shed light on the interplay between signal transduction cascades and chromatin modifications thus adding another layer of complexity to the already complex regulation of the protein network. Based on the insights gleaned from almost a decade of research, it has now been convincingly revealed that sesquiterpenes effectively modulated different intracellular signaling cascades in different cancers. In this review we summarize how sesquiterpenes mediated Wnt, Shh, Notch and TRAIL induced signaling cascades. PMID:27453282

  13. RNF11 modulates microglia activation through NF-κB signalling cascade

    PubMed Central

    Dalal, Nirjari V.; Pranski, Elaine L.; Tansey, Malu G.; Lah, James J.; Levey, Allan I.; Betarbet, Ranjita S.

    2012-01-01

    Microglia are resident macrophages in the central nervous system (CNS) that play a major role in neuroinflammation and pathogenesis of several neurodegenerative diseases. Upon activation, microglia releases a multitude of pro-inflammatory factors that initiate and sustain an inflammatory response by activating various signalling pathways, including the NF-κB pathway in a feed forward cycle. In microglial cells, activation of NF-κB signalling is normally transient, while sustained NF-κB activation is associated with persistent neuroinflammation. RING finger protein 11 (RNF11), in association with A20 ubiquitin-editing complex, is one of the key negative regulators of NF-κB signalling pathway in neurons. In this study, we have demonstrated and confirmed this role of RNF11 in microglia, the immune cells of the CNS. Coimmunoprecipitation experiments showed that RNF11 and A20 interact in a microglial cell line, suggesting the presence of A20 ubiquitin-editing protein complex in microglial cells. Next, using targeted short hairpin RNA (shRNA) knockdown and over-expression of RNF11, we established that RNF11 expression levels are inversely related to NF-κB activation, as evident from altered expression of NF-κB transcribed genes. Moreover our studies, illustrated that RNF11 confers protection against LPS-induced cell cytotoxicity. Thus our investigations clearly demonstrated that microglial RNF11 is a negative regulator of NF-κB signalling pathway and could be a strong potential target for modulating inflammatory responses in neurodegenerative diseases. PMID:22975135

  14. Plant MAPK cascades: Just rapid signaling modules?

    PubMed Central

    Boudsocq, Marie; Danquah, Agyemang; de Zélicourt, Axel; Hirt, Heribert; Colcombet, Jean

    2015-01-01

    Abscisic acid (ABA) is a major phytohormone mediating important stress-related processes. We recently unveiled an ABA-activated MAPK signaling module constituted of MAP3K17/18-MKK3-MPK1/2/7/14. Unlike classical rapid MAPK activation, we showed that the activation of the new MAPK module is delayed and relies on the MAP3K protein synthesis. In this addendum, we discuss the role of this original and unexpected activation mechanism of MAPK cascades which suggests that MAPKs can regulate both early and long-term plant stress responses. PMID:26313321

  15. Temporal protein expression pattern in intracellular signalling cascade during T-cell activation: a computational study.

    PubMed

    Ganguli, Piyali; Chowdhury, Saikat; Bhowmick, Rupa; Sarkar, Ram Rup

    2015-10-01

    Various T-cell co-receptor molecules and calcium channel CRAC play a pivotal role in the maintenance of cell's functional responses by regulating the production of effector molecules (mostly cytokines) that aids in immune clearance and also maintaining the cell in a functionally active state. Any defect in these co-receptor signalling pathways may lead to an altered expression pattern of the effector molecules. To study the propagation of such defects with time and their effect on the intracellular protein expression patterns, a comprehensive and largest pathway map of T-cell activation network is reconstructed manually. The entire pathway reactions are then translated using logical equations and simulated using the published time series microarray expression data as inputs. After validating the model, the effect of in silico knock down of co-receptor molecules on the expression patterns of their downstream proteins is studied and simultaneously the changes in the phenotypic behaviours of the T-cell population are predicted, which shows significant variations among the proteins expression and the signalling routes through which the response is propagated in the cytoplasm. This integrative computational approach serves as a valuable technique to study the changes in protein expression patterns and helps to predict variations in the cellular behaviour. PMID:26564978

  16. Signal Transduction Cascades Regulating Fungal Development and Virulence

    PubMed Central

    Lengeler, Klaus B.; Davidson, Robert C.; D'souza, Cletus; Harashima, Toshiaki; Shen, Wei-Chiang; Wang, Ping; Pan, Xuewen; Waugh, Michael; Heitman, Joseph

    2000-01-01

    Cellular differentiation, mating, and filamentous growth are regulated in many fungi by environmental and nutritional signals. For example, in response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous growth referred to as pseudohyphal differentiation. Yeast filamentous growth is regulated, in part, by two conserved signal transduction cascades: a mitogen-activated protein kinase cascade and a G-protein regulated cyclic AMP signaling pathway. Related signaling cascades play an analogous role in regulating mating and virulence in the plant fungal pathogen Ustilago maydis and the human fungal pathogens Cryptococcus neoformans and Candida albicans. We review here studies on the signaling cascades that regulate development of these and other fungi. This analysis illustrates both how the model yeast S. cerevisiae can serve as a paradigm for signaling in other organisms and also how studies in other fungi provide insights into conserved signaling pathways that operate in many divergent organisms. PMID:11104818

  17. Fluvoxamine moderates reduced voluntary activity following chronic dexamethasone infusion in mice via recovery of BDNF signal cascades.

    PubMed

    Terada, Kazuki; Izumo, Nobuo; Suzuki, Biora; Karube, Yoshiharu; Morikawa, Tomomi; Ishibashi, Yukiko; Kameyama, Toshiki; Chiba, Koji; Sasaki, Noriko; Iwata, Keiko; Matsuzaki, Hideo; Manabe, Takayuki

    2014-04-01

    Major depression is a complex disorder characterized by genetic and environmental interactions. Selective serotonin reuptake inhibitors (SSRIs) effectively treat depression. Neurogenesis following chronic antidepressant treatment activates brain derived neurotrophic factor (BDNF) signaling. In this study, we analyzed the effects of the SSRI fluvoxamine (Flu) on locomotor activity and forced-swim behavior using chronic dexamethasone (cDEX) infusions in mice, which engenders depression-like behavior. Infusion of cDEX decreased body weight and produced a trend towards lower locomotor activity during darkness. In the forced-swim test, cDEX-mice exhibited increased immobility times compared with mice administered saline. Flu treatment reversed decreased locomotor activity and mitigated forced-swim test immobility. Real-time polymerase chain reactions using brain RNA samples yielded significantly lower BDNF mRNA levels in cDEX-mice compared with the saline group. Endoplasmic reticulum stress-associated X-box binding protein-1 (XBP1) gene expression was lower in cDEX-mice compared with the saline group. However, marked expression of the XBP1 gene was observed in cDEX-mice treated with Flu compared with mice given saline and untreated cDEX-mice. Expression of 5-HT2A and Sigma-1 receptors decreased after cDEX infusion compared with the saline group, and these decreases normalized to control levels upon Flu treatment. Our results indicate that the Flu moderates reductions in voluntary activity following chronic dexamethasone infusions in mice via recovery of BDNF signal cascades. PMID:24582626

  18. Orexin/hypocretin receptor signalling cascades

    PubMed Central

    Kukkonen, JP; Leonard, CS

    2014-01-01

    Orexin (hypocretin) peptides and their two known G-protein-coupled receptors play essential roles in sleep–wake control and powerfully influence other systems regulating appetite/metabolism, stress and reward. Consequently, drugs that influence signalling by these receptors may provide novel therapeutic opportunities for treating sleep disorders, obesity and addiction. It is therefore critical to understand how these receptors operate, the nature of the signalling cascades they engage and their physiological targets. In this review, we evaluate what is currently known about orexin receptor signalling cascades, while a sister review (Leonard & Kukkonen, this issue) focuses on tissue-specific responses. The evidence suggests that orexin receptor signalling is multifaceted and is substantially more diverse than originally thought. Indeed, orexin receptors are able to couple to members of at least three G-protein families and possibly other proteins, through which they regulate non-selective cation channels, phospholipases, adenylyl cyclase, and protein and lipid kinases. In the central nervous system, orexin receptors produce neuroexcitation by postsynaptic depolarization via activation of non-selective cation channels, inhibition of K+ channels and activation of Na+/Ca2+ exchange, but they also can stimulate the release of neurotransmitters by presynaptic actions and modulate synaptic plasticity. Ca2+ signalling is also prominently influenced by these receptors, both via the classical phospholipase C−Ca2+ release pathway and via Ca2+ influx, mediated by several pathways. Upon longer-lasting stimulation, plastic effects are observed in some cell types, while others, especially cancer cells, are stimulated to die. Thus, orexin receptor signals appear highly tunable, depending on the milieu in which they are operating. Linked ArticlesThis article is part of a themed section on Orexin Receptors. To view the other articles in this section visit http://dx.doi.org/10

  19. Domain-Specific Activation of Death-Associated Intracellular Signalling Cascades by the Cellular Prion Protein in Neuroblastoma Cells.

    PubMed

    Vilches, Silvia; Vergara, Cristina; Nicolás, Oriol; Mata, Ágata; Del Río, José A; Gavín, Rosalina

    2016-09-01

    The biological functions of the cellular prion protein remain poorly understood. In fact, numerous studies have aimed to determine specific functions for the different protein domains. Studies of cellular prion protein (PrP(C)) domains through in vivo expression of molecules carrying internal deletions in a mouse Prnp null background have provided helpful data on the implication of the protein in signalling cascades in affected neurons. Nevertheless, understanding of the mechanisms underlying the neurotoxicity induced by these PrP(C) deleted forms is far from complete. To better define the neurotoxic or neuroprotective potential of PrP(C) N-terminal domains, and to overcome the heterogeneity of results due to the lack of a standardized model, we used neuroblastoma cells to analyse the effects of overexpressing PrP(C) deleted forms. Results indicate that PrP(C) N-terminal deleted forms were properly processed through the secretory pathway. However, PrPΔF35 and PrPΔCD mutants led to death by different mechanisms sharing loss of alpha-cleavage and activation of caspase-3. Our data suggest that both gain-of-function and loss-of-function pathogenic mechanisms may be associated with N-terminal domains and may therefore contribute to neurotoxicity in prion disease. Dissecting the molecular response induced by PrPΔF35 may be the key to unravelling the physiological and pathological functions of the prion protein. PMID:26250617

  20. Ouabain triggers preconditioning through activation of the Na+,K+-ATPase signaling cascade in rat hearts

    PubMed Central

    Pierre, Sandrine V.; Yang, Changjun; Yuan, Zhaokan; Seminerio, Jennifer; Mouas, Christian; Garlid, Keith D.; Dos-Santos, Pierre; Xie, Zijian

    2007-01-01

    Objective Because ouabain activates several pathways that are critical to cardioprotective mechanisms such as ischemic preconditioning, we tested if this digitalis compound could protect the heart against ischemia-reperfusion injury through activation of the Na+,K+-ATPase/c-Src receptor complex. Methods and Results In Langendorff-perfused rat hearts, a short (4 min) administration of ouabain 10 μM followed by an 8-minute washout before 30 minutes of global ischemia and reperfusion improved cardiac function, decreased lactate dehydrogenase release and reduced infarct size by 40%. Western blot analysis revealed that ouabain activated the cardioprotective phospholipase Cγ1/protein kinase Cε (PLC-γ1/PKCε) pathway. Pre-treatment of the hearts with the Src kinase family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2) blocked not only ouabain-induced activation of PLC-γ1/PKCε pathway, but also cardiac protection. This protection was also blocked by a PKCε translocation inhibitor peptide (PKCε TIP). Conclusion Short exposure to a low concentration of ouabain protects the heart against ischemia/reperfusion injury. This effect of ouabain on the heart is most likely due to the activation of the Na+,K+-ATPase/c-Src receptor complex and subsequent stimulation of key mediators of preconditioning, namely PLC-γ1 and PKCε. PMID:17157283

  1. Tumor necrosis factor-alpha mediates activation of NF-κB and JNK signaling cascades in retinal ganglion cells and astrocytes in opposite ways

    PubMed Central

    Dvoriantchikova, Galina; Ivanov, Dmitry

    2014-01-01

    Tumor necrosis factor-alpha (TNF) is an important mediator of the innate immune response in the retina. TNF can activate various signaling cascades, including NF-κB, nuclear factor kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways. The harmful role of these pathways, as well as of TNF, has previously been shown in several retinal neurodegenerative conditions including glaucoma and retinal ischemia. However, TNF and TNF-regulated signaling cascades are capable not only of mediating neurotoxicity, but of being protective. We performed this study to delineate the beneficial and detrimental effects of TNF signaling in the retina. To this end, we used TNF-treated primary retinal ganglion cell (RGC) and astrocyte cultures. Levels of expression of NF-κB subunits in RGCs and astrocytes were evaluated by quantitative RT-PCR (qRT-PCR) and Western blot (WB) analysis. NF-κB and JNK activity in TNF-treated cells was determined in a time-dependent manner using ELISA and WB. Gene expression in TNF-treated astrocytes was measured by qRT-PCR. We found that NF-κB family members were present in RGCs and astrocytes at the mRNA and protein levels. RGCs failed to activate NF-κB in the presence of TNF, a phenomenon that was associated with sustained JNK activation and RGC death. However, TNF initiated the activation of NF-κB and mediated transient JNK activation in astrocytes. These events were associated with glial survival and increased expression of neurotoxic pro-inflammatory factors. Our findings suggest that, in the presence of TNF, NF-κB and JNK signaling cascades are activated in opposite ways in RGCs and astrocytes. These events can directly and indirectly facilitate RGC death. PMID:25160799

  2. Laminar shear flow increases hydrogen sulfide and activates a nitric oxide producing signaling cascade in endothelial cells.

    PubMed

    Huang, Bin; Chen, Chang-Ting; Chen, Chi-Shia; Wang, Yun-Ming; Hsieh, Hsyue-Jen; Wang, Danny Ling

    2015-09-01

    Laminar shear flow triggers a signaling cascade that maintains the integrity of endothelial cells (ECs). Hydrogen sulfide (H2S), a new gasotransmitter is regarded as an upstream regulator of nitric oxide (NO). Whether the H2S-generating enzymes are correlated to the enzymes involved in NO production under shear flow conditions remains unclear as yet. In the present study, the cultured ECs were subjected to a constant shear flow (12 dyn/cm(2)) in a parallel flow chamber system. We investigated the expression of three key enzymes for H2S biosynthesis, cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), and 3-mercapto-sulfurtransferase (3-MST). Shear flow markedly increased the level of 3-MST. Shear flow enhanced the production of H2S was determined by NBD-SCN reagent that can bind to cysteine/homocystein. Exogenous treatment of NaHS that can release gaseous H2S, ECs showed an increase of phosphorylation in Akt(S473), ERK(T202/Y204) and eNOS(S1177). This indicated that H2S can trigger the NO-production signaling cascade. Silencing of CSE, CBS and 3-MST genes by siRNA separately attenuated the phosphorylation levels of Akt(S473) and eNOS(S1177) under shear flow conditions. The particular mode of shear flow increased H2S production. The interplay between H2S and NO-generating enzymes were discussed in the present study. PMID:26212441

  3. Activation of AMPA receptor promotes TNF-α release via the ROS-cSrc-NFκB signaling cascade in RAW264.7 macrophages

    SciTech Connect

    Cheng, Xiu-Li; Ding, Fan; Li, Hui; Tan, Xiao-Qiu; Liu, Xiao; Cao, Ji-Min; Gao, Xue

    2015-05-29

    The relationship between glutamate signaling and inflammation has not been well defined. This study aimed to investigate the role of AMPA receptor (AMPAR) in the expression and release of tumor necrosis factor-alpha (TNF-α) from macrophages and the underlying mechanisms. A series of approaches, including confocal microscopy, immunofluorescency, flow cytometry, ELISA and Western blotting, were used to estimate the expression of AMPAR and downstream signaling molecules, TNF-α release and reactive oxygen species (ROS) generation in the macrophage-like RAW264.7 cells. The results demonstrated that AMPAR was expressed in RAW264.7 cells. AMPA significantly enhanced TNF-α release from RAW264.7 cells, and this effect was abolished by CNQX (AMPAR antagonist). AMPA also induced elevation of ROS production, phosphorylation of c-Src and activation of nuclear factor (NF)-κB in RAW264.7 cells. Blocking c-Src by PP2, scavenging ROS by glutathione (GSH) or inhibiting NF-κB activation by pyrrolidine dithiocarbamate (PDTC) decreased TNF-α production from RAW264.7 cells. We concluded that AMPA promotes TNF-α release in RAW264.7 macrophages likely through the following signaling cascade: AMPAR activation → ROS generation → c-Src phosphorylation → NF-κB activation → TNF-α elevation. The study suggests that AMPAR may participate in macrophage activation and inflammation. - Highlights: • AMPAR is expressed in RAW264.7 macrophages and is upregulated by AMPA stimulation. • Activation of AMPAR stimulates TNF-α release in macrophages through the ROS-cSrc-NFκB signaling cascade. • Macrophage AMPAR signaling may play an important role in inflammation.

  4. Fangchinoline suppresses the growth and invasion of human glioblastoma cells by inhibiting the kinase activity of Akt and Akt-mediated signaling cascades.

    PubMed

    Guo, Bingyu; Xie, Peng; Su, Jingyuan; Zhang, Tingting; Li, Xiaoming; Liang, Guobiao

    2016-02-01

    Glioblastoma multiforme (GBM) is one of the most palindromic and malignant central nervous system neoplasms, and the current treatment is not effectual for GBM. Research of specific medicine for GBM is significant. Fangchinoline possesses a wide range of pharmacological activities and attracts more attentions due to its anti-tumor effects. In this study, two WHO grade IV human GBM cell lines (U87 MG and U118 MG) were exposed to fangchinoline, and we found that fangchinoline specifically inhibits the kinase activity of Akt and markedly suppresses the phosphorylation of Thr308 and Ser473 of Akt in human GBM cells. We also observed that fangchinoline inhibits tumor cell proliferation and invasiveness and induces apoptosis through suppressing the Akt-mediated signaling cascades, including Akt/p21, Akt/Bad, and Akt/matrix metalloproteinases (MMPs). These data demonstrated that fangchinoline exerts its anti-tumor effects in human glioblastoma cells, at least partly by inhibiting the kinase activity of Akt and suppressing Akt-mediated signaling cascades. PMID:26408176

  5. A2B adenosine receptor contributes to penile erection via PI3K/AKT signaling cascade-mediated eNOS activation

    PubMed Central

    Wen, Jiaming; Grenz, Almut; Zhang, Yujin; Dai, Yingbo; Kellems, Rodney E.; Blackburn, Michael R.; Eltzschig, Holger K.; Xia, Yang

    2011-01-01

    Normal penile erection is under the control of multiple factors and signaling pathways. Although adenosine signaling is implicated in normal and abnormal penile erection, the exact role and the underlying mechanism for adenosine signaling in penile physiology remain elusive. Here we report that shear stress leads to increased adenosine release from endothelial cells. Subsequently, we determined that ecto-5′-nucleotidase (CD73) is a key enzyme required for the production of elevated adenosine from ATP released by shear-stressed endothelial cells. Mechanistically, we demonstrate that shear stress-mediated elevated adenosine functions through the adenosine A2B receptor (A2BR) to activate the PI3K/AKT signaling cascade and subsequent increased endothelial nitric oxide synthase (eNOS) phosphorylation. These in vitro studies led us to discover further that adenosine was induced during sustained penile erection and contributes to PI3K/AKT activation and subsequent eNOS phosphorylation via A2BR signaling in intact animal. Finally, we demonstrate that lowering adenosine in wild-type mice or genetic deletion of A2BR in mutant mice significantly attenuated PI3K/AKT activation, eNOS phosphorylation, and subsequent impaired penile erection featured with the reduction of ratio of maximal intracavernosal pressure to systemic arterial pressure from 0.49 ± 0.03 to 0.41 ± 0.05 and 0.38 ± 0.04, respectively (both P<0.05). Overall, using biochemical, cellular, genetic, and physiological approaches, our findings reveal that adenosine is a novel molecule signaling via A2BR activation, contributing to penile erection via PI3K/AKT-dependent eNOS activation. These studies suggest that this signaling pathway may be a novel therapeutic target for erectile disorders.—Wen, J., Grenz, A., Zhang, Y., Dai, Y., Kellems, R. E., Blackburn, M. R., Eltzschig, H. K., Xia, Y. A2B adenosine receptor contributes to penile erection via PI3K/AKT signaling cascade-mediated eNOS activation. PMID

  6. The p21-activated kinase, PAK2, is important in the activation of numerous pancreatic acinar cell signaling cascades and in the onset of early pancreatitis events.

    PubMed

    Nuche-Berenguer, Bernardo; Ramos-Álvarez, Irene; Jensen, R T

    2016-06-01

    In a recent study we explored Group-1-p21-activated kinases (GP.1-PAKs) in rat pancreatic acini. Only PAK2 was present; it was activated by gastrointestinal-hormones/neurotransmitters and growth factors in a PKC-, Src- and small-GTPase-mediated manner. PAK2 was required for enzyme-secretion and ERK/1-2-activation. In the present study we examined PAK2's role in CCK and TPA-activation of important distal signaling cascades mediating their physiological/pathophysiological effects and analyzed its role in pathophysiological processes important in early pancreatitis. In rat pancreatic acini, PAK2-inhibition by the specific, GP.1.PAK-inhibitor, IPA-3-suppressed cholecystokinin (CCK)/TPA-stimulated activation of focal-adhesion kinases and mitogen-activated protein-kinases. PAK2-inhibition reversed the dual stimulatory/inhibitory effect of CCK/TPA on the PI3K/Akt/GSK-3β pathway. However, its inhibition did not affect PKC activation. PAK2-inhibition protected acini from CCK-induced ROS-generation; caspase/trypsin-activation, important in early pancreatitis; as well as from cell-necrosis. Furthermore, PAK2-inhibition reduced proteolytic-activation of PAK-2p34, which is involved in programmed-cell-death. To ensure that the study did not only rely in the specificity of IPA-3 as a PAK inhibitor, we used two other approaches for PAK inhibition, FRAX597 a ATP-competitive-GP.1-PAKs-inhibitor and infection with a PAK2-dominant negative(DN)-Advirus. Those two approaches confirmed the results obtained with IPA-3. This study demonstrates that PAK2 is important in mediating CCK's effect on the activation of signaling-pathways known to mediate its physiological/pathophysiological responses including several cellular processes linked to the onset of pancreatitis. Our results suggest that PAK2 could be a new, important therapeutic target to consider for the treatment of diseases involving deregulation of pancreatic acinar cells. PMID:26912410

  7. Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress

    PubMed Central

    Xing, Yu; Chen, Wei-hua; Jia, Wensuo; Zhang, Jianhua

    2015-01-01

    Superoxide dismutases (SODs) are involved in plant adaptive responses to biotic and abiotic stresses but the upstream signalling process that modulates their expression is not clear. Expression of two iron SODs, FSD2 and FSD3, was significantly increased in Arabidopsis in response to NaCl treatment but blocked in transgenic MKK5-RNAi plant, mkk5. Using an assay system for transient expression in protoplasts, it was found that mitogen-activated protein kinase kinase 5 (MKK5) was also activated in response to salt stress. Overexpression of MKK5 in wild-type plants enhanced their tolerance to salt treatments, while mkk5 mutant exhibited hypersensitivity to salt stress in germination on salt-containing media. Moreover, another kinase, MPK6, was also involved in the MKK5-mediated iron superoxide dismutase (FSD) signalling pathway in salt stress. The kinase activity of MPK6 was totally turned off in mkk5, whereas the activity of MPK3 was only partially blocked. MKK5 interacted with the MEKK1 protein that was also involved in the salt-induced FSD signalling pathway. These data suggest that salt-induced FSD2 and FSD3 expressions are influenced by MEKK1 via MKK5–MPK6-coupled signalling. This MAP kinase cascade (MEKK1, MKK5, and MPK6) mediates the salt-induced expression of iron superoxide dismutases. PMID:26136265

  8. The SH3-SAM adaptor HACS1 is up-regulated in B cell activation signaling cascades.

    PubMed

    Zhu, Yuan Xiao; Benn, Sally; Li, Zhi Hua; Wei, Ellen; Masih-Khan, Esther; Trieu, Young; Bali, Meenakshi; McGlade, C Jane; Claudio, Jaime O; Stewart, A Keith

    2004-09-20

    HACS1 is a Src homology 3 and sterile alpha motif domain-containing adaptor that is preferentially expressed in normal hematopoietic tissues and malignancies including myeloid leukemia, lymphoma, and myeloma. Microarray data showed HACS1 expression is up-regulated in activated human B cells treated with interleukin (IL)-4, CD40L, and anti-immunoglobulin (Ig)M and clustered with genes involved in signaling, including TNF receptor-associated protein 1, signaling lymphocytic activation molecule, IL-6, and DEC205. Immunoblot analysis demonstrated that HACS1 is up-regulated by IL-4, IL-13, anti-IgM, and anti-CD40 in human peripheral blood B cells. In murine spleen B cells, Hacs1 can also be up-regulated by lipopolysaccharide but not IL-13. Induction of Hacs1 by IL-4 is dependent on Stat6 signaling and can also be impaired by inhibitors of phosphatidylinositol 3-kinase, protein kinase C, and nuclear factor kappaB. HACS1 associates with tyrosine-phosphorylated proteins after B cell activation and binds in vitro to the inhibitory molecule paired Ig-like receptor B. Overexpression of HACS1 in murine spleen B cells resulted in a down-regulation of the activation marker CD23 and enhancement of CD138 expression, IgM secretion, and Xbp-1 expression. Knock down of HACS1 in a human B lymphoma cell line by small interfering ribonucleic acid did not significantly change IL-4-stimulated B cell proliferation. Our study demonstrates that HACS1 is up-regulated by B cell activation signals and is a participant in B cell activation and differentiation. PMID:15381729

  9. The monomeric receptor binding domain of tetrameric α2-macroglobulin binds to cell surface GRP78 triggering equivalent activation of signaling cascades.

    PubMed

    Misra, Uma Kant; Payne, Sturgis; Pizzo, Salvatore Vincent

    2013-06-11

    α2-Macroglobulin (α2M) is a broad spectrum proteinase inhibitor that when activated by proteinases (α2M*) undergoes a major conformational change exposing receptor recognition sites in each of its four subunits. These complexes bind to two distinct receptors, namely, the low-density lipoprotein receptor-related protein (LRP) and cell surface glucose-regulated protein [Mr ∼ 78000 (GRP78)]. The latter is a very high affinity receptor (Kd = 50-100 pM) whose ligation triggers pro-proliferative and anti-apoptotic signaling cascades. Despite its four binding sites, Scatchard analysis of binding of α2M* to cells does not yield a cooperative plot. We, therefore, hypothesize that a monomeric cloned and expressed α2M receptor binding domain (RBD) should trigger comparable signaling events. Indeed, RBD or its K1370A mutant that binds to GRP78 but cannot bind to LRP regulates DNA and protein synthesis by human prostate cancer cells in a manner comparable to that of α2M*. Akt and mTORC1 activation and signaling are also comparably upregulated by α2M*, RBD, or mutant K1370A. Antibodies directed against the carboxyl-terminal domain of GRP78 are antagonists that block α2M*-mediated effects on pro-proliferative and anti-apoptotic signaling cascades and protein and DNA synthesis. The effects of RBD and its mutant were similarly blocked by these antibodies. Finally, proteolysis of α2M at pH values from 5.7 to 7.0 causes production of free RBD and RBD-containing fragments. Thus, while α2M* ligates only one GRP78 receptor molecule per α2M*, it may potentially serve as a reservoir for release of up to four binding fragments per molecule. PMID:23721263

  10. Gqalpha-linked phospholipase Cbeta1 and phospholipase Cgamma are essential components of the pheromone biosynthesis activating neuropeptide (PBAN) signal transduction cascade.

    PubMed

    Hull, J J; Lee, J M; Matsumoto, S

    2010-08-01

    Sex pheromone production for most moths is regulated by pheromone biosynthesis activating neuropeptide (PBAN). In Bombyx mori, PBAN binding triggers the opening of store-operated Ca(2+) channels, suggesting the involvement of a receptor-activated phospholipase C (PLC). In this study, we found that PLC inhibitors U73122 and compound 48/80 reduced sex pheromone production and that intracellular levels of (3)H-inositol phosphate species increased following PBAN stimulation. In addition, we amplified cDNAs from pheromone glands corresponding to PLCbeta1, PLCbeta4, PLCgamma and two G protein alpha subunits, Go and Gq. In vivo RNA interference-mediated knockdown analyses revealed that BmPLCbeta1, BmGq1, and unexpectedly, BmPLCgamma, are part of the PBAN signal transduction cascade. PMID:20546038

  11. Genetic Dissection of the Signaling Cascade that Controls Activation of the Shigella Type III Secretion System from the Needle Tip

    PubMed Central

    Murillo, I.; Martinez-Argudo, I.; Blocker, A. J.

    2016-01-01

    Many Gram-negative bacterial pathogens use type III secretion systems (T3SSs) for virulence. The Shigella T3SS consists of a hollow needle, made of MxiH and protruding from the bacterial surface, anchored in both bacterial membranes by multimeric protein rings. Atop the needle lies the tip complex (TC), formed by IpaD and IpaB. Upon physical contact with eukaryotic host cells, T3S is initiated leading to formation of a pore in the eukaryotic cell membrane, which is made of IpaB and IpaC. Through the needle and pore channels, further bacterial proteins are translocated inside the host cell to meditate its invasion. IpaD and the needle are implicated in transduction of the host cell-sensing signal to the T3S apparatus. Furthermore, the sensing-competent TC seems formed of 4 IpaDs topped by 1 IpaB. However, nothing further is known about the activation process. To investigate IpaB’s role during T3SS activation, we isolated secretion-deregulated IpaB mutants using random mutagenesis and a genetic screen. We found ipaB point mutations in leading to defects in secretion activation, which sometimes diminished pore insertion and host cell invasion. We also demonstrated IpaB communicates intramolecularly and intermolecularly with IpaD and MxiH within the TC because mutations affecting these interactions impair signal transduction. PMID:27277624

  12. Genetic Dissection of the Signaling Cascade that Controls Activation of the Shigella Type III Secretion System from the Needle Tip.

    PubMed

    Murillo, I; Martinez-Argudo, I; Blocker, A J

    2016-01-01

    Many Gram-negative bacterial pathogens use type III secretion systems (T3SSs) for virulence. The Shigella T3SS consists of a hollow needle, made of MxiH and protruding from the bacterial surface, anchored in both bacterial membranes by multimeric protein rings. Atop the needle lies the tip complex (TC), formed by IpaD and IpaB. Upon physical contact with eukaryotic host cells, T3S is initiated leading to formation of a pore in the eukaryotic cell membrane, which is made of IpaB and IpaC. Through the needle and pore channels, further bacterial proteins are translocated inside the host cell to meditate its invasion. IpaD and the needle are implicated in transduction of the host cell-sensing signal to the T3S apparatus. Furthermore, the sensing-competent TC seems formed of 4 IpaDs topped by 1 IpaB. However, nothing further is known about the activation process. To investigate IpaB's role during T3SS activation, we isolated secretion-deregulated IpaB mutants using random mutagenesis and a genetic screen. We found ipaB point mutations in leading to defects in secretion activation, which sometimes diminished pore insertion and host cell invasion. We also demonstrated IpaB communicates intramolecularly and intermolecularly with IpaD and MxiH within the TC because mutations affecting these interactions impair signal transduction. PMID:27277624

  13. A p21-activated kinase (PAK1) signaling cascade coordinately regulates F-actin remodeling and insulin granule exocytosis in pancreatic β cells

    PubMed Central

    Kalwat, Michael A.; Yoder, Stephanie M.; Wang, Zhanxiang; Thurmond, Debbie C.

    2012-01-01

    Human islet studies implicate an important signaling role for the Cdc42 effector protein p21-activated kinase (PAK1) in the sustained/second-phase of insulin secretion. Because human islets from type 2 diabetic donors lack ~80% of normal PAK1 protein levels, the mechanistic requirement for PAK1 signaling in islet function was interrogated. Similar to MIN6 β cells, human islets elicited glucose-stimulated PAK1 activation that was sensitive to the PAK1 inhibitor, IPA3. Given that sustained insulin secretion has been correlated with glucose-induced filamentous actin (F-actin) remodeling, we tested the hypothesis that a Cdc42-activated PAK1 signaling cascade is required to elicit F-actin remodeling to mobilize granules to the cell surface. Live-cell imaging captured the glucose-induced cortical F-actin remodeling in MIN6 β cells; IPA3-mediated inhibition of PAK1 abolished this remodeling. IPA3 also ablated glucose-stimulated insulin granule accumulation at the plasma membrane, consistent with its role in sustained/second-phase insulin release. Both IPA3 and a selective inhibitor of the Cdc42 GTPase, ML-141, blunted the glucose-stimulated activation of Raf-1, suggesting Raf-1 to be downstream of Cdc42→PAK1. IPA3 also inhibited MEK1/2 activation, implicating the MEK1/2→ERK1/2 cascade to occur downstream of PAK1. Importantly, PD0325901, a new selective inhibitor of MEK1/2→ERK1/2 activation, impaired F-actin remodeling and the sustained/amplification pathway of insulin release. Taken together, these data suggest that glucose-mediated activation of Cdc42 leads to activation of PAK1 and prompts activation of its downstream targets Raf-1, MEK1/2 and ERK1/2 to elicit F-actin remodeling and recruitment of insulin granules to the plasma membrane to support the sustained phase of insulin release. PMID:23246867

  14. Regulation of a protein phosphatase cascade allows convergent dopamine and glutamate signals to activate ERK in the striatum.

    PubMed

    Valjent, Emmanuel; Pascoli, Vincent; Svenningsson, Per; Paul, Surojit; Enslen, Hervé; Corvol, Jean-Christophe; Stipanovich, Alexandre; Caboche, Jocelyne; Lombroso, Paul J; Nairn, Angus C; Greengard, Paul; Hervé, Denis; Girault, Jean-Antoine

    2005-01-11

    Many drugs of abuse exert their addictive effects by increasing extracellular dopamine in the nucleus accumbens, where they likely alter the plasticity of corticostriatal glutamatergic transmission. This mechanism implies key molecular alterations in neurons in which both dopamine and glutamate inputs are activated. Extracellular signal-regulated kinase (ERK), an enzyme important for long-term synaptic plasticity, is a good candidate for playing such a role. Here, we show in mouse that d-amphetamine activates ERK in a subset of medium-size spiny neurons of the dorsal striatum and nucleus accumbens, through the combined action of glutamate NMDA and D1-dopamine receptors. Activation of ERK by d-amphetamine or by widely abused drugs, including cocaine, nicotine, morphine, and Delta(9)-tetrahydrocannabinol was absent in mice lacking dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000 (DARPP-32). The effects of d-amphetamine or cocaine on ERK activation in the striatum, but not in the prefrontal cortex, were prevented by point mutation of Thr-34, a DARPP-32 residue specifically involved in protein phosphatase-1 inhibition. Regulation by DARPP-32 occurred both upstream of ERK and at the level of striatal-enriched tyrosine phosphatase (STEP). Blockade of the ERK pathway or mutation of DARPP-32 altered locomotor sensitization induced by a single injection of psychostimulants, demonstrating the functional relevance of this regulation. Thus, activation of ERK, by a multilevel protein phosphatase-controlled mechanism, functions as a detector of coincidence of dopamine and glutamate signals converging on medium-size striatal neurons and is critical for long-lasting effects of drugs of abuse. PMID:15608059

  15. An inside job: hacking into Janus kinase/signal transducer and activator of transcription signaling cascades by the intracellular protozoan Toxoplasma gondii.

    PubMed

    Denkers, Eric Y; Bzik, David J; Fox, Barbara A; Butcher, Barbara A

    2012-02-01

    The intracellular protozoan Toxoplasma gondii is well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype. PMID:22104110

  16. An Inside Job: Hacking into Janus Kinase/Signal Transducer and Activator of Transcription Signaling Cascades by the Intracellular Protozoan Toxoplasma gondii

    PubMed Central

    Bzik, David J.; Fox, Barbara A.; Butcher, Barbara A.

    2012-01-01

    The intracellular protozoan Toxoplasma gondii is well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype. PMID:22104110

  17. Estrogen action and cytoplasmic signaling cascades. Part I: membrane-associated signaling complexes

    PubMed Central

    Segars, James H.; Driggers, Paul H.

    2014-01-01

    Remarkable progress in recent years has suggested that estrogen action in vivo is complex and often involves activation of cytoplasmic signaling cascades in addition to genomic actions mediated directly through estrogen receptors α and β. Rather than a linear response mediated solely through estrogen-responsive DNA elements, in vivo estrogen might simultaneously activate distinct signaling cascades that function as networks to coordinate tissue responses to estrogen. This complex signaling system provides for exquisite control and plasticity of response to estrogen at the tissue level, and undoubtedly contributes to the remarkable tissue-specific responses to estrogens. In part I of this series, we summarize cytoplasmic signaling modules involving estrogen or estrogen receptors, with particular focus on recently described membrane-associated signaling complexes. PMID:12217492

  18. Andrographolide Inhibits Nuclear Factor-κB Activation through JNK-Akt-p65 Signaling Cascade in Tumor Necrosis Factor-α-Stimulated Vascular Smooth Muscle Cells

    PubMed Central

    Chen, Yu-Ying; Hsieh, Cheng-Ying; Lee, Lin-Wen; Sheu, Joen-Rong

    2014-01-01

    Critical vascular inflammation leads to vascular dysfunction and cardiovascular diseases, including abdominal aortic aneurysms, hypertension, and atherosclerosis. Andrographolide is the most active and critical constituent isolated from the leaves of Andrographis paniculata, a herbal medicine widely used for treating anti-inflammation in Asia. In this study, we investigated the mechanisms of the inhibitory effects of andrographolide in vascular smooth muscle cells (VSMCs) exposed to a proinflammatory stimulus, tumor necrosis factor-α (TNF-α). Treating TNF-α-stimulated VSMCs with andrographolide suppressed the expression of inducible nitric oxide synthase in a concentration-dependent manner. A reduction in TNF-α-induced c-Jun N-terminal kinase (JNK), Akt, and p65 phosphorylation was observed in andrographolide-treated VSMCs. However, andrographolide affected neither IκBα degradation nor p38 mitogen-activated protein kinase or extracellular signal-regulated kinase 1/2 phosphorylation under these conditions. Both treatment with LY294002, a phosphatidylinositol 3-kinase/Akt inhibitor, and treatment with SP600125, a JNK inhibitor, markedly reversed the andrographolide-mediated inhibition of p65 phosphorylation. In addition, LY294002 and SP600125 both diminished Akt phosphorylation, whereas LY294002 had no effects on JNK phosphorylation. These results collectively suggest that therapeutic interventions using andrographolide can benefit the treatment of vascular inflammatory diseases, and andrographolide-mediated inhibition of NF-κB activity in TNF-α-stimulated VSMCs occurs through the JNK-Akt-p65 signaling cascade, an IκBα-independent mechanism. PMID:25114952

  19. Mount Rainier active cascade volcano

    NASA Technical Reports Server (NTRS)

    1994-01-01

    Mount Rainier is one of about two dozen active or recently active volcanoes in the Cascade Range, an arc of volcanoes in the northwestern United States and Canada. The volcano is located about 35 kilometers southeast of the Seattle-Tacoma metropolitan area, which has a population of more than 2.5 million. This metropolitan area is the high technology industrial center of the Pacific Northwest and one of the commercial aircraft manufacturing centers of the United States. The rivers draining the volcano empty into Puget Sound, which has two major shipping ports, and into the Columbia River, a major shipping lane and home to approximately a million people in southwestern Washington and northwestern Oregon. Mount Rainier is an active volcano. It last erupted approximately 150 years ago, and numerous large floods and debris flows have been generated on its slopes during this century. More than 100,000 people live on the extensive mudflow deposits that have filled the rivers and valleys draining the volcano during the past 10,000 years. A major volcanic eruption or debris flow could kill thousands of residents and cripple the economy of the Pacific Northwest. Despite the potential for such danger, Mount Rainier has received little study. Most of the geologic work on Mount Rainier was done more than two decades ago. Fundamental topics such as the development, history, and stability of the volcano are poorly understood.

  20. Gqalpha-linked PLCbeta and PLCgamma are essential components of the pheromone biosynthesis activating neuropeptide (PBAN) signal transduction cascade

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sex pheromone production for most moths is regulated by pheromone biosynthesis activating neuropeptide (PBAN). In Bombyx mori, PBAN binding triggers the opening of store-operated Ca2+ channels, suggesting the involvement of a receptor-activated phospholipase C (PLC). In this study, we found that P...

  1. Agonist-mediated activation of Bombyx mori diapause hormone receptor signals to extracellular signal-regulated kinases 1 and 2 through Gq-PLC-PKC-dependent cascade.

    PubMed

    Jiang, Xue; Yang, Jingwen; Shen, Zhangfei; Chen, Yajie; Shi, Liangen; Zhou, Naiming

    2016-08-01

    Diapause is a developmental strategy adopted by insects to survive in challenging environments such as the low temperatures of a winter. This unique process is regulated by diapause hormone (DH), which is a neuropeptide hormone that induces egg diapause in Bombyx mori and is involved in terminating pupal diapause in heliothis moths. An G protein-coupled receptor from the silkworm, B. mori, has been identified as a specific cell surface receptor for DH. However, the detailed information on the DH-DHR system and its mechanism(s) involved in the induction of embryonic diapause remains unknown. Here, we combined functional assays with various specific inhibitors to elucidate the DHR-mediated signaling pathways. Upon activation by DH, B. mori DHR is coupled to the Gq protein, leading to a significant increase of intracellular Ca(2+) and cAMP response element-driven luciferase activity in an UBO-QIC, a specific Gq inhibitor, sensitive manner. B. mori DHR elicited ERK1/2 phosphorylation in a dose- and time-dependent manner in response to DH. This effect was almost completely inhibited by co-incubation with UBO-QIC and was also significantly suppressed by PLC inhibitor U73122, PKC inhibitors Gö6983 and the Ca(2+) chelator EGTA. Moreover, DHR-induced activation of ERK1/2 was significantly attenuated by treatment with the Gβγ specific inhibitors gallein and M119K and the PI3K specific inhibitor Wortmannin, but not by the Src specific inhibitor PP2. Our data also demonstrates that the EGFR-transactivation pathway is not involved in the DHR-mediated ERK1/2 phosphorylation. Future efforts are needed to clarify the role of the ERK1/2 signaling pathway in the DH-mediated induction of B. mori embryonic diapause. PMID:27318251

  2. Targeting Mitogen-Activated Protein Kinase/Extracellular Signal–Regulated Kinase Kinase in the Mutant (V600E) B-Raf Signaling Cascade Effectively Inhibits Melanoma Lung Metastases

    PubMed Central

    Sharma, Arati; Tran, Melissa A.; Liang, Shile; Sharma, Arun K.; Amin, Shantu; Smith, Charles D.; Dong, Cheng; Robertson, Gavin P.

    2009-01-01

    Malignant melanoma has a high propensity for metastatic spread, making it the most deadly form of skin cancer. B-RAF has been identified as the most mutated gene in these invasive cells and therefore an attractive therapeutic target. However, for uncertain reasons, chemotherapy inhibiting B-Raf has not been clinically effective. This has raised questions whether this pathway is important in melanoma metastasis or whether targeting a protein other than B-Raf in the signaling cascade could more effectively inhibit this pathway to reduce lung metastases. Here, we investigated the role played by V600EB-Raf in melanoma metastasis and showed that targeting this signaling cascade significantly reduces lung metastases. Small interfering RNA (siRNA)–mediated inhibition was used in mice to reduce expression (activity) of each member of the signaling cascade and effects on metastasis development were measured. Targeting any member of the signaling cascade reduced metastasis but inhibition of mitogen-activated protein kinase/extracellular signal–regulated kinase kinase (Mek) 1 and Mek 2 almost completely prevented lung tumor development. Mechanistically, metastatic inhibition was mediated through reduction of melanoma cell extravasation through the endothelium and decreased proliferative capacity. Targeting B-Raf with the pharmacologic inhibitor BAY 43-9006, which was found ineffective in clinical trials and seems to act primarily as an angiogenesis inhibitor, did not decrease metastasis, whereas inhibition of Mek using U0126 decreased cellular proliferative capacity, thereby effectively reducing number and size of lung metastases. In summary, this study provides a mechanistic basis for targeting Mek and not B-Raf in the mutant V600EB-Raf signaling cascade to inhibit melanoma metastases. PMID:16912199

  3. Centrally administered CDP-choline induced cardiovascular responses are mediated by activation of the central phospholipase-prostaglandin signaling cascade.

    PubMed

    Topuz, Bora B; Altinbas, Burcin; Ilhan, Tuncay; Yilmaz, Mustafa S; Erdost, Hatice; Saha, Sikha; Savci, Vahide; Yalcin, Murat

    2014-05-14

    The present study was designed to determine the involvement of central prostaglandin synthesis on the pressor and bradycardic effect of cytidine 5'-diphosphocholine (CDP-choline). Intracerebroventricular (i.c.v.) administration of CDP-choline was made and blood pressure and heart rate were recorded in male Sprague Dawley rats throughout this study. Microdialysis and immunohistochemical studies were performed to measure extracellular total prostaglandin concentration and to show cyclooxygenase-1 and -2 (COX-1 and -2) immunoreactivities, respectively, in the posterior hypothalamic area. Moreover, rats were pretreated (i.c.v) with mepacrine [a phospholipase A2 (PLA2) inhibitor], ibuprofen [a nonselective COX inhibitor], neomycine [a phospholipase C (PLC) inhibitor] or furegrelate [a thromboxane A2 (TXA2) synthesis inhibitor] 5 min prior to the injection of CDP-choline to determine the effects of these inhibitors on cardiovascular responses to CDP-choline. Control rats were pretreated (i.c.v) with saline. CDP-choline caused a dose- and time-dependent increase in blood pressure and decrease in heart rate. Immunohistochemical studies showed that CDP-choline increased COX-1 and -2 immunoreactivities in the posterior hypothalamic area. CDP-choline also elevated hypothalamic extracellular total prostaglandin concentration by 62%, as shown in microdialysis studies. Mepacrine or ibuprofen pretreatments almost completely blocked the pressor and bradycardic responses to CDP-choline while neomycine or furegrelate partially attenuated the drug-induced cardiovascular effects. The results suggest that CDP-choline may stimulate prostaglandin synthesis through the activation of PLA2, cyclooxygenases (COX-1 and -2) and prostaglandins and at least TXA2, may mediate the drug׳s cardiovascular effects. PMID:24704528

  4. Prion protein induced signaling cascades in monocytes

    SciTech Connect

    Krebs, Bjarne; Dorner-Ciossek, Cornelia; Vassallo, Neville; Herms, Jochen; Kretzschmar, Hans A. . E-mail: Hans.Kretzschmar@med.uni-muenchen.de

    2006-02-03

    Prion proteins play a central role in transmission and pathogenesis of transmissible spongiform encephalopathies. The cellular prion protein (PrP{sup C}), whose physiological function remains elusive, is anchored to the surface of a variety of cell types including neurons and cells of the lymphoreticular system. In this study, we investigated the response of a mouse monocyte/macrophage cell line to exposure with PrP{sup C} fusion proteins synthesized with a human Fc-tag. PrP{sup C} fusion proteins showed an attachment to the surface of monocyte/macrophages in nanomolar concentrations. This was accompanied by an increase of cellular tyrosine phosphorylation as a result of activated signaling pathways. Detailed investigations exhibited activation of downstream pathways through a stimulation with PrP fusion proteins, which include phosphorylation of ERK{sub 1,2} and Akt kinase. Macrophages opsonize and present antigenic structures, contact lymphocytes, and deliver cytokines. The findings reported here may become the basis of understanding the molecular function of PrP{sup C} in monocytes and macrophages.

  5. Signalling in the Yeasts: An Informational Cascade with Links to the Filamentous Fungi

    PubMed Central

    Banuett, Flora

    1998-01-01

    All cells, from bacteria and yeasts to mammalian cells, respond to cues from their environment. A variety of mechanisms exist for the transduction of these external signals to the interior of the cell, resulting in altered patterns of protein activity. Eukaryotic cells commonly transduce external cues via a conserved module composed of three protein kinases, the mitogen-activated protein kinase (MAPK) cascade. This module can then activate substrates, some of which include transcriptional activators. Multiple MAPK signalling pathways coexist in a cell. This review considers different MAPK cascade signalling pathways that govern several aspects of the life cycle of budding and fission yeasts: conjugation and meiosis by the pheromone response pathway, stress response by the high-osmolarity sensing pathway, cell wall biosynthesis in response to activation of the low-osmolarity and heat-sensing pathway, and pseudohyphal growth in response to activation of a subset of the components of the pheromone response pathway. Because the MAPK cascade components are highly conserved, a key question in studies of these pathways is the mechanism by which specificity of response is achieved. Several other issues to be addressed in this review concern the nature of the receptors used to sense the external signals and the mechanism by which the receptors communicate with other components leading to activation of the MAPK cascade. Recently, it has become apparent that MAPK cascades are important in governing the pathogenicity of filamentous fungi. PMID:9618441

  6. A Complex Signaling Cascade Governs Pristinamycin Biosynthesis in Streptomyces pristinaespiralis

    PubMed Central

    Guezguez, Jamil; Handel, Franziska; Schinko, Eva

    2015-01-01

    Pristinamycin production in Streptomyces pristinaespiralis Pr11 is tightly regulated by an interplay between different repressors and activators. A γ-butyrolactone receptor gene (spbR), two TetR repressor genes (papR3 and papR5), three SARP (Streptomyces antibiotic regulatory protein) genes (papR1, papR2, and papR4), and a response regulator gene (papR6) are carried on the large 210-kb pristinamycin biosynthetic gene region of Streptomyces pristinaespiralis Pr11. A detailed investigation of all pristinamycin regulators revealed insight into a complex signaling cascade, which is responsible for the fine-tuned regulation of pristinamycin production in S. pristinaespiralis. PMID:26187956

  7. Interaction of LRRK2 with kinase and GTPase signaling cascades

    PubMed Central

    Boon, Joon Y.; Dusonchet, Julien; Trengrove, Chelsea; Wolozin, Benjamin

    2014-01-01

    LRRK2 is a protein that interacts with a plethora of signaling molecules, but the complexity of LRRK2 function presents a challenge for understanding the role of LRRK2 in the pathophysiology of Parkinson’s disease (PD). Studies of LRRK2 using over-expression in transgenic mice have been disappointing, however, studies using invertebrate systems have yielded a much clearer picture, with clear effects of LRRK2 expression, knockdown or deletion in Caenorhabditis elegans and Drosophila on modulation of survival of dopaminergic neurons. Recent studies have begun to focus attention on particular signaling cascades that are a target of LRRK2 function. LRRK2 interacts with members of the mitogen activated protein kinase (MAPK) pathway and might regulate the pathway action by acting as a scaffold that directs the location of MAPK pathway activity, without strongly affecting the amount of MAPK pathway activity. Binding to GTPases, GTPase-activating proteins and GTPase exchange factors are another strong theme in LRRK2 biology, with LRRK2 binding to rac1, cdc42, rab5, rab7L1, endoA, RGS2, ArfGAP1, and ArhGEF7. All of these molecules appear to feed into a function output for LRRK2 that modulates cytoskeletal outgrowth and vesicular dynamics, including autophagy. These functions likely impact modulation of α-synuclein aggregation and associated toxicity eliciting the disease processes that we term PD. PMID:25071441

  8. Network Features and Pathway Analyses of a Signal Transduction Cascade

    PubMed Central

    Yanashima, Ryoji; Kitagawa, Noriyuki; Matsubara, Yoshiya; Weatheritt, Robert; Oka, Kotaro; Kikuchi, Shinichi; Tomita, Masaru; Ishizaki, Shun

    2008-01-01

    The scale-free and small-world network models reflect the functional units of networks. However, when we investigated the network properties of a signaling pathway using these models, no significant differences were found between the original undirected graphs and the graphs in which inactive proteins were eliminated from the gene expression data. We analyzed signaling networks by focusing on those pathways that best reflected cellular function. Therefore, our analysis of pathways started from the ligands and progressed to transcription factors and cytoskeletal proteins. We employed the Python module to assess the target network. This involved comparing the original and restricted signaling cascades as a directed graph using microarray gene expression profiles of late onset Alzheimer's disease. The most commonly used method of shortest-path analysis neglects to consider the influences of alternative pathways that can affect the activation of transcription factors or cytoskeletal proteins. We therefore introduced included k-shortest paths and k-cycles in our network analysis using the Python modules, which allowed us to attain a reasonable computational time and identify k-shortest paths. This technique reflected results found in vivo and identified pathways not found when shortest path or degree analysis was applied. Our module enabled us to comprehensively analyse the characteristics of biomolecular networks and also enabled analysis of the effects of diseases considering the feedback loop and feedforward loop control structures as an alternative path. PMID:19543432

  9. Signal Propagation in Multi-Layer DNAzyme Cascades using Structured Chimeric Substrates**

    PubMed Central

    Brown, Carl W.; Lakin, Matthew R.; Horwitz, Eli K.; Fanning, M. Leigh; West, Hannah E.

    2014-01-01

    Signal propagation through enzyme cascades is a critical component of information processing in cellular systems. Although such systems have potential as biomolecular computing tools, rational design of synthetic protein networks remains infeasible. DNA strands with catalytic activity (DNAzymes) are an attractive alternative, enabling rational cascade design through predictable base-pair hybridization principles. Here we report multi-layered DNAzyme signaling and logic cascades. We achieve signaling between DNAzymes using a structured chimeric substrate (SCS) that releases a downstream activator after cleavage by an upstream DNAzyme. The SCS can be activated by various upstream DNAzymes, can be coupled to DNA strand displacement devices, and is highly resistant to interference from background DNA. This work enables rational design of synthetic DNAzyme regulatory networks, with potential applications in biomolecular computing, biodetection, and autonomous theranostics. PMID:24890874

  10. A respiratory chain controlled signal transduction cascade in the mitochondrial intermembrane space mediates hydrogen peroxide signaling

    PubMed Central

    Patterson, Heide Christine; Gerbeth, Carolin; Thiru, Prathapan; Vögtle, Nora F.; Knoll, Marko; Shahsafaei, Aliakbar; Samocha, Kaitlin E.; Huang, Cher X.; Harden, Mark Michael; Song, Rui; Chen, Cynthia; Kao, Jennifer; Shi, Jiahai; Salmon, Wendy; Shaul, Yoav D.; Stokes, Matthew P.; Silva, Jeffrey C.; Bell, George W.; MacArthur, Daniel G.; Ruland, Jürgen; Meisinger, Chris; Lodish, Harvey F.

    2015-01-01

    Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) govern cellular homeostasis by inducing signaling. H2O2 modulates the activity of phosphatases and many other signaling molecules through oxidation of critical cysteine residues, which led to the notion that initiation of ROS signaling is broad and nonspecific, and thus fundamentally distinct from other signaling pathways. Here, we report that H2O2 signaling bears hallmarks of a regular signal transduction cascade. It is controlled by hierarchical signaling events resulting in a focused response as the results place the mitochondrial respiratory chain upstream of tyrosine-protein kinase Lyn, Lyn upstream of tyrosine-protein kinase SYK (Syk), and Syk upstream of numerous targets involved in signaling, transcription, translation, metabolism, and cell cycle regulation. The active mediators of H2O2 signaling colocalize as H2O2 induces mitochondria-associated Lyn and Syk phosphorylation, and a pool of Lyn and Syk reside in the mitochondrial intermembrane space. Finally, the same intermediaries control the signaling response in tissues and species responsive to H2O2 as the respiratory chain, Lyn, and Syk were similarly required for H2O2 signaling in mouse B cells, fibroblasts, and chicken DT40 B cells. Consistent with a broad role, the Syk pathway is coexpressed across tissues, is of early metazoan origin, and displays evidence of evolutionary constraint in the human. These results suggest that H2O2 signaling is under control of a signal transduction pathway that links the respiratory chain to the mitochondrial intermembrane space-localized, ubiquitous, and ancient Syk pathway in hematopoietic and nonhematopoietic cells. PMID:26438848

  11. A respiratory chain controlled signal transduction cascade in the mitochondrial intermembrane space mediates hydrogen peroxide signaling.

    PubMed

    Patterson, Heide Christine; Gerbeth, Carolin; Thiru, Prathapan; Vögtle, Nora F; Knoll, Marko; Shahsafaei, Aliakbar; Samocha, Kaitlin E; Huang, Cher X; Harden, Mark Michael; Song, Rui; Chen, Cynthia; Kao, Jennifer; Shi, Jiahai; Salmon, Wendy; Shaul, Yoav D; Stokes, Matthew P; Silva, Jeffrey C; Bell, George W; MacArthur, Daniel G; Ruland, Jürgen; Meisinger, Chris; Lodish, Harvey F

    2015-10-20

    Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) govern cellular homeostasis by inducing signaling. H2O2 modulates the activity of phosphatases and many other signaling molecules through oxidation of critical cysteine residues, which led to the notion that initiation of ROS signaling is broad and nonspecific, and thus fundamentally distinct from other signaling pathways. Here, we report that H2O2 signaling bears hallmarks of a regular signal transduction cascade. It is controlled by hierarchical signaling events resulting in a focused response as the results place the mitochondrial respiratory chain upstream of tyrosine-protein kinase Lyn, Lyn upstream of tyrosine-protein kinase SYK (Syk), and Syk upstream of numerous targets involved in signaling, transcription, translation, metabolism, and cell cycle regulation. The active mediators of H2O2 signaling colocalize as H2O2 induces mitochondria-associated Lyn and Syk phosphorylation, and a pool of Lyn and Syk reside in the mitochondrial intermembrane space. Finally, the same intermediaries control the signaling response in tissues and species responsive to H2O2 as the respiratory chain, Lyn, and Syk were similarly required for H2O2 signaling in mouse B cells, fibroblasts, and chicken DT40 B cells. Consistent with a broad role, the Syk pathway is coexpressed across tissues, is of early metazoan origin, and displays evidence of evolutionary constraint in the human. These results suggest that H2O2 signaling is under control of a signal transduction pathway that links the respiratory chain to the mitochondrial intermembrane space-localized, ubiquitous, and ancient Syk pathway in hematopoietic and nonhematopoietic cells. PMID:26438848

  12. Mitogen-activated protein kinase cascades in Vitis vinifera

    PubMed Central

    Çakır, Birsen; Kılıçkaya, Ozan

    2015-01-01

    Protein phosphorylation is one of the most important mechanisms to control cellular functions in response to external and endogenous signals. Mitogen-activated protein kinases (MAPK) are universal signaling molecules in eukaryotes that mediate the intracellular transmission of extracellular signals resulting in the induction of appropriate cellular responses. MAPK cascades are composed of four protein kinase modules: MAPKKK kinases (MAPKKKKs), MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In plants, MAPKs are activated in response to abiotic stresses, wounding, and hormones, and during plant pathogen interactions and cell division. In this report, we performed a complete inventory of MAPK cascades genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with MAPK, MAPK kinases, MAPK kinase kinases and MAPK kinase kinase kinase kinase members of Arabidopsis thaliana, we revealed the existence of 14 MAPKs, 5 MAPKKs, 62 MAPKKKs, and 7 MAPKKKKs in Vitis vinifera. We identified orthologs of V. vinifera putative MAPKs in different species, and ESTs corresponding to members of MAPK cascades in various tissues. This work represents the first complete inventory of MAPK cascades in V. vinifera and could help elucidate the biological and physiological functions of these proteins in V. vinifera. PMID:26257761

  13. The "ups and downs" of signaling cascades in addiction.

    PubMed

    Ron, Dorit; Jurd, Rachel

    2005-11-01

    Drug addiction is a chronic disease characterized by compulsive drug use despite the severe negative consequences associated with it. Repeated exposure to drugs of abuse results in molecular adaptations in neuronal signaling pathways, which eventually manifest in the complex behavioral alterations that characterize addiction. These include tolerance, sensitization, dependence, drug craving, and relapse. In this Review, we focus on recent studies highlighting signaling cascades initiated by cocaine, as a representative of a drug of abuse with a defined site of action, and alcohol, as a drug with an undefined primary site of action. Specifically, we describe recent studies that emphasize the role of protein-protein interactions, phosphorylation, and compartmentalization in the molecular mechanisms that result in the cellular and behavioral adaptations that underlie addiction. Signaling cascades that contribute to addiction, as well as those that protect or delay the development of addiction, are presented. PMID:16278489

  14. Hard-metal (WC-Co) particles trigger a signaling cascade involving p38 MAPK, HIF-1α, HMOX1, and p53 activation in human PBMC.

    PubMed

    Lombaert, Noömi; Castrucci, Eleonora; Decordier, Ilse; Van Hummelen, Paul; Kirsch-Volders, Micheline; Cundari, Enrico; Lison, Dominique

    2013-02-01

    Hard-metals are made of tungsten carbide (WC) and metallic cobalt (Co) particles and are important industrial materials produced for their extreme hardness and high wear resistance properties. While occupational exposure to metallic Co alone is apparently not associated with an increased risk of cancer, the WC-Co particle mixture was shown to increase the risk of lung cancer in exposed workers. We have previously shown that WC-Co specifically induces a burst of reactive oxygen species (ROS) and in vitro mutagenic/apoptogenic effects in human peripheral blood mononucleated cells (PBMC) used as a validated experimental model. In the present study, PBMCs were treated during a short period (15 min) to focus on the very rapid ROS burst induced by WC-Co. We investigated by microarray the response to WC-Co versus Co(2+) ions (CoCl(2)) after 15 min exposure and found that the oxidative stress response HMOX1 gene was highly expressed in WC-Co-treated samples. This result was confirmed by qRT-PCR, and western blotting was carried out to analyze translational and post-translational regulation of genes belonging to the HMOX1 pathway. We show here that WC-Co, and metallic Co particles although with slower kinetics, but not CoCl(2) or WC alone, induced a temporally ordered cascade of events. This cascade implies p38/MAP kinase activation, HIF-1α stabilization, HMOX1 transcriptional activation, and ATM-independent p53 stabilization. These events, and in particular HIF-1α stabilization, could contribute to the carcinogenic activity of WC-Co dusts. PMID:23052192

  15. Thermally Induced Osteocyte Damage Initiates a Remodelling Signaling Cascade

    PubMed Central

    Dolan, Eimear B.; McNamara, Laoise M.

    2015-01-01

    Thermal elevations experienced by bone during orthopaedic procedures, such as cutting and drilling, exothermal reactions from bone cement, and thermal therapies such as tumor ablation, can result in thermal damage leading to death of native bone cells (osteocytes, osteoblasts, osteoclasts and mesenchymal stem cells). Osteocytes are believed to be the orchestrators of bone remodeling, which recruit nearby osteoclast and osteoblasts to control resorption and bone growth in response to mechanical stimuli and physical damage. However, whether heat-induced osteocyte damage can directly elicit bone remodelling has yet to be determined. This study establishes the link between osteocyte thermal damage and the remodeling cascade. We show that osteocytes directly exposed to thermal elevations (47°C for 1 minute) become significantly apoptotic and alter the expression of osteogenic genes (Opg and Cox2). The Rankl/Opg ratio is consistently down-regulated, at days 1, 3 and 7 in MLO-Y4s heat-treated to 47°C for 1 minute. Additionally, the pro-osteoblastogenic signaling marker Cox2 is significantly up-regulated in heat-treated MLO-Y4s by day 7. Furthermore, secreted factors from heat-treated MLO-Y4s administered to MSCs using a novel co-culture system are shown to activate pre-osteoblastic MSCs to increase production of the pro-osteoblastic differentiation marker, alkaline phosphatase (day 7, 14), and calcium deposition (day 21). Most interestingly, an initial pro-osteoclastogenic signaling response (increase Rankl and Rankl/Opg ratio at day 1) followed by later stage pro-osteoblastogenic signaling (down-regulation in Rankl and the Rankl/Opg ratio and an up-regulation in Opg and Cox2 by day 7) was observed in non-heat-treated MLO-Y4s in co-culture when these were exposed to the biochemicals produced by heat-treated MLO-Y4s. Taken together, these results elucidate the vital role of osteocytes in detecting and responding to thermal damage by means of thermally induced apoptosis

  16. Thermally induced osteocyte damage initiates a remodelling signaling cascade.

    PubMed

    Dolan, Eimear B; Haugh, Matthew G; Voisin, Muriel C; Tallon, David; McNamara, Laoise M

    2015-01-01

    Thermal elevations experienced by bone during orthopaedic procedures, such as cutting and drilling, exothermal reactions from bone cement, and thermal therapies such as tumor ablation, can result in thermal damage leading to death of native bone cells (osteocytes, osteoblasts, osteoclasts and mesenchymal stem cells). Osteocytes are believed to be the orchestrators of bone remodeling, which recruit nearby osteoclast and osteoblasts to control resorption and bone growth in response to mechanical stimuli and physical damage. However, whether heat-induced osteocyte damage can directly elicit bone remodelling has yet to be determined. This study establishes the link between osteocyte thermal damage and the remodeling cascade. We show that osteocytes directly exposed to thermal elevations (47°C for 1 minute) become significantly apoptotic and alter the expression of osteogenic genes (Opg and Cox2). The Rankl/Opg ratio is consistently down-regulated, at days 1, 3 and 7 in MLO-Y4s heat-treated to 47°C for 1 minute. Additionally, the pro-osteoblastogenic signaling marker Cox2 is significantly up-regulated in heat-treated MLO-Y4s by day 7. Furthermore, secreted factors from heat-treated MLO-Y4s administered to MSCs using a novel co-culture system are shown to activate pre-osteoblastic MSCs to increase production of the pro-osteoblastic differentiation marker, alkaline phosphatase (day 7, 14), and calcium deposition (day 21). Most interestingly, an initial pro-osteoclastogenic signaling response (increase Rankl and Rankl/Opg ratio at day 1) followed by later stage pro-osteoblastogenic signaling (down-regulation in Rankl and the Rankl/Opg ratio and an up-regulation in Opg and Cox2 by day 7) was observed in non-heat-treated MLO-Y4s in co-culture when these were exposed to the biochemicals produced by heat-treated MLO-Y4s. Taken together, these results elucidate the vital role of osteocytes in detecting and responding to thermal damage by means of thermally induced apoptosis

  17. Forecasting Social Unrest Using Activity Cascades.

    PubMed

    Cadena, Jose; Korkmaz, Gizem; Kuhlman, Chris J; Marathe, Achla; Ramakrishnan, Naren; Vullikanti, Anil

    2015-01-01

    Social unrest is endemic in many societies, and recent news has drawn attention to happenings in Latin America, the Middle East, and Eastern Europe. Civilian populations mobilize, sometimes spontaneously and sometimes in an organized manner, to raise awareness of key issues or to demand changes in governing or other organizational structures. It is of key interest to social scientists and policy makers to forecast civil unrest using indicators observed on media such as Twitter, news, and blogs. We present an event forecasting model using a notion of activity cascades in Twitter (proposed by Gonzalez-Bailon et al., 2011) to predict the occurrence of protests in three countries of Latin America: Brazil, Mexico, and Venezuela. The basic assumption is that the emergence of a suitably detected activity cascade is a precursor or a surrogate to a real protest event that will happen "on the ground." Our model supports the theoretical characterization of large cascades using spectral properties and uses properties of detected cascades to forecast events. Experimental results on many datasets, including the recent June 2013 protests in Brazil, demonstrate the effectiveness of our approach. PMID:26091012

  18. Forecasting Social Unrest Using Activity Cascades

    PubMed Central

    Cadena, Jose; Korkmaz, Gizem; Kuhlman, Chris J.; Marathe, Achla; Ramakrishnan, Naren; Vullikanti, Anil

    2015-01-01

    Social unrest is endemic in many societies, and recent news has drawn attention to happenings in Latin America, the Middle East, and Eastern Europe. Civilian populations mobilize, sometimes spontaneously and sometimes in an organized manner, to raise awareness of key issues or to demand changes in governing or other organizational structures. It is of key interest to social scientists and policy makers to forecast civil unrest using indicators observed on media such as Twitter, news, and blogs. We present an event forecasting model using a notion of activity cascades in Twitter (proposed by Gonzalez-Bailon et al., 2011) to predict the occurrence of protests in three countries of Latin America: Brazil, Mexico, and Venezuela. The basic assumption is that the emergence of a suitably detected activity cascade is a precursor or a surrogate to a real protest event that will happen “on the ground.” Our model supports the theoretical characterization of large cascades using spectral properties and uses properties of detected cascades to forecast events. Experimental results on many datasets, including the recent June 2013 protests in Brazil, demonstrate the effectiveness of our approach. PMID:26091012

  19. Future Antidepressant Targets: Neurotrophic Factors and Related Signaling Cascades

    PubMed Central

    Schmidt, Heath D.; Banasr, Mounira; Duman, Ronald S.

    2009-01-01

    Preclinical and clinical studies demonstrate that neurotrophic factors play critical roles in the etiology and treatment of depression. While the mechanisms underlying the therapeutic efficacy of antidepressants remain unknown, increasing evidence supports a role for increased trophic support in the treatment of depression. Furthermore, antidepressants block or reverse stress-induced down regulation of neurotrophic factor expression in limbic and cortical nuclei involved in the underlying pathophysiology of depression. Thus, components of neurotrophic factor-mediated signaling cascades or the signal transduction pathways that regulate neurotrophic factor expression may provide additional targets for the development of novel, more efficacious antidepressant drugs. PMID:19802372

  20. Galanin stimulates cortisol secretion from human adrenocortical cells through the activation of galanin receptor subtype 1 coupled to the adenylate cyclase-dependent signaling cascade.

    PubMed

    Belloni, Anna S; Malendowicz, Ludwik K; Rucinski, Marcin; Guidolin, Diego; Nussdorfer, Gastone G

    2007-12-01

    Previous studies showed that galanin receptors are expressed in the rat adrenal, and galanin modulates glucocorticoid secretion in this species. Hence, we investigated the expression of the various galanin receptor subtypes (GAL-R1, GAL-R2 and GAL-R3) in the human adrenocortical cells, and the possible involvement of galanin in the control of cortisol secretion. Reverse transcription-polymerase chain reaction detected the expression of GAL-R1 (but not GAL-R2 and GAL-R3) in the inner zones of the human adrenal cortex. The galanin concentration dependently enhanced basal, but not ACTH-stimulated secretion of cortisol from dispersed inner adrenocortical cells (maximal effective concentration, 10(-8) M). The cortisol response to 10(-8) M galanin was abrogated by GAL-R1 immunoneutralization, and unaffected by GAL-R2 or GAL-R3 immunoneutralization. Galanin (10(-8) M) and ACTH (10(-9) M) enhanced cyclic-AMP production from dispersed cells, and the response was suppressed by the adenylate cyclase inhibitor SQ-22536 (10(-4) M). Galanin did not affect inositol triphosphate release, which, in contrast, was raised by angiotensin-II (10(-8) M). SQ-22536 and the protein kinase (PK)A inhibitor H-89 (10(-5) M) abolished the cortisol response to 10(-8) M galanin, while the phospholipase C inhibitor U-73122 and the PKC inhibitor calphostin-C were ineffective. Preincubation with pertussis toxin (Ptx) (0.5 microg/ml) partially inhibited the cortisol response to galanin. We conclude that galanin stimulates cortisol secretion from human inner adrenocortical cells, acting through GAL-R1 coupled to the adenylate cyclase/PKA-dependent signaling cascade via a Ptx-sensitive Galpha protein. PMID:17982695

  1. A mutation in TRPC6 channels abolishes their activation by hypoosmotic stretch but does not affect activation by diacylglycerol or G protein signaling cascades.

    PubMed

    Wilson, Cory; Dryer, Stuart E

    2014-05-01

    Canonical transient receptor potential-6 (TRPC6) channels have been implicated in the pathogenesis of kidney disease and in the regulation of vascular smooth muscle tone, podocyte function, and a variety of processes in other cell types. The question of whether their gating is intrinsically mechanosensitive has been controversial. In this study we have examined activation of two alleles of TRPC6 transiently expressed in CHO-K1 cells: the wild-type human TRPC6 channel, and TRPC6-N143S, an allele originally identified in a family with autosomal dominant familial focal and segmental glomerulosclerosis (FSGS). We observed that both channel variants carried robust cationic currents that could be evoked by application of membrane-permeable analogs of diacylglycerol (DAG) or by the P2Y receptor agonist ATP. The amplitudes and characteristics of currents evoked by the DAG analog or ATP were indistinguishable in cells expressing the two TRPC6 alleles. By contrast, hypoosmotic stretch evoked robust currents in wild-type TRPC6 channels but had no discernible effect on currents in cells expressing TRPC6-N143S, indicating that the mutant form lacks mechanosensitivity. Coexpression of TRPC6-N143S with wild-type TRPC6 or TRPC3 channels did not alter stretch-evoked responses compared with when TRPC3 channels were expressed by themselves, indicating that TRPC6-N143S does not function as a dominant-negative. These data indicate that mechanical activation and activation evoked by DAG or ATP occur through fundamentally distinct biophysical mechanisms, and they provide support for the hypothesis that protein complexes containing wild-type TRPC6 subunits can be intrinsically mechanosensitive. PMID:24598806

  2. S-allyl cysteine protects against 6-hydroxydopamine-induced neurotoxicity in the rat striatum: involvement of Nrf2 transcription factor activation and modulation of signaling kinase cascades.

    PubMed

    Tobón-Velasco, Julio César; Vázquez-Victorio, Genaro; Macías-Silva, Marina; Cuevas, Elvis; Ali, Syed F; Maldonado, Perla D; González-Trujano, María Eva; Cuadrado, Antonio; Pedraza-Chaverrí, José; Santamaría, Abel

    2012-09-01

    Pharmacological activation at the basal ganglia of the transcription factor Nrf2, guardian of redox homeostasis, holds a strong promise for the slow progression of Parkinson's disease (PD). However, a potent Nrf2 activator in the brain still must be found. In this study, we have investigated the potential use of the antioxidant compound S-allyl cysteine (SAC) in the activation of Nrf2 in 6-hydoxydopamine (6-OHDA)-intoxicated rats. In the rat striatum, SAC by itself promoted the Nrf2 dissociation of Keap-1, its nuclear translocation, the subsequent association with small MafK protein, and further binding of the Nrf2/MafK complex to ARE sequence, as well as the up-regulation of Nrf2-dependent genes encoding the antioxidant enzymes HO-1, NQO-1, GR, and SOD-1. In vivo and in vitro experiments to identify signaling pathways activated by SAC pointed to Akt as the most likely kinase participating in Nrf2 activation by SAC. In PC12 cells, SAC stimulated the activation of Akt and ERK1/2 and inhibited JNK1/2/3 activation. In the rat striatum, the SAC-induced activation of Nrf2 is likely to contribute to inhibit the toxic effects of 6-OHDA evidenced by phase 2 antioxidant enzymes up-regulation, glutathione recovery, and attenuation of reactive oxygen species (ROS), nitric oxide (NO), and lipid peroxides formation. These early protective effects correlated with the long-term preservation of the cellular redox status, the striatal dopamine (DA) and tyrosine hydroxylase (TH) levels, and the improvement of motor skills. Therefore, this study indicates that, in addition to direct scavenging actions, the activation of Nrf2 by SAC might confer neuroprotective responses through the modulation of kinase signaling pathways in rodent models of PD, and suggests that this antioxidant molecule may have a therapeutic value in this human pathology. PMID:22781654

  3. Evolution of Vertebrate Phototransduction: Cascade Activation

    PubMed Central

    Lamb, Trevor D.; Patel, Hardip; Chuah, Aaron; Natoli, Riccardo C.; Davies, Wayne I. L.; Hart, Nathan S.; Collin, Shaun P.; Hunt, David M.

    2016-01-01

    We applied high-throughput sequencing to eye tissue from several species of basal vertebrates (a hagfish, two species of lamprey, and five species of gnathostome fish), and we analyzed the mRNA sequences for the proteins underlying activation of the phototransduction cascade. The molecular phylogenies that we constructed from these sequences are consistent with the 2R WGD model of two rounds of whole genome duplication. Our analysis suggests that agnathans retain an additional representative (that has been lost in gnathostomes) in each of the gene families we studied; the evidence is strong for the G-protein α subunit (GNAT) and the cGMP phosphodiesterase (PDE6), and indicative for the cyclic nucleotide-gated channels (CNGA and CNGB). Two of the species (the hagfish Eptatretus cirrhatus and the lamprey Mordacia mordax) possess only a single class of photoreceptor, simplifying deductions about the composition of cascade protein isoforms utilized in their photoreceptors. For the other lamprey, Geotria australis, analysis of the ratios of transcript levels in downstream and upstream migrant animals permits tentative conclusions to be drawn about the isoforms used in four of the five spectral classes of photoreceptor. Overall, our results suggest that agnathan rod-like photoreceptors utilize the same GNAT1 as gnathostomes, together with a homodimeric PDE6 that may be agnathan-specific, whereas agnathan cone-like photoreceptors utilize a GNAT that may be agnathan-specific, together with the same PDE6C as gnathostomes. These findings help elucidate the evolution of the vertebrate phototransduction cascade from an ancestral chordate phototransduction cascade that existed prior to the vertebrate radiation. PMID:27189541

  4. Evolution of Vertebrate Phototransduction: Cascade Activation.

    PubMed

    Lamb, Trevor D; Patel, Hardip; Chuah, Aaron; Natoli, Riccardo C; Davies, Wayne I L; Hart, Nathan S; Collin, Shaun P; Hunt, David M

    2016-08-01

    We applied high-throughput sequencing to eye tissue from several species of basal vertebrates (a hagfish, two species of lamprey, and five species of gnathostome fish), and we analyzed the mRNA sequences for the proteins underlying activation of the phototransduction cascade. The molecular phylogenies that we constructed from these sequences are consistent with the 2R WGD model of two rounds of whole genome duplication. Our analysis suggests that agnathans retain an additional representative (that has been lost in gnathostomes) in each of the gene families we studied; the evidence is strong for the G-protein α subunit (GNAT) and the cGMP phosphodiesterase (PDE6), and indicative for the cyclic nucleotide-gated channels (CNGA and CNGB). Two of the species (the hagfish Eptatretus cirrhatus and the lamprey Mordacia mordax) possess only a single class of photoreceptor, simplifying deductions about the composition of cascade protein isoforms utilized in their photoreceptors. For the other lamprey, Geotria australis, analysis of the ratios of transcript levels in downstream and upstream migrant animals permits tentative conclusions to be drawn about the isoforms used in four of the five spectral classes of photoreceptor. Overall, our results suggest that agnathan rod-like photoreceptors utilize the same GNAT1 as gnathostomes, together with a homodimeric PDE6 that may be agnathan-specific, whereas agnathan cone-like photoreceptors utilize a GNAT that may be agnathan-specific, together with the same PDE6C as gnathostomes. These findings help elucidate the evolution of the vertebrate phototransduction cascade from an ancestral chordate phototransduction cascade that existed prior to the vertebrate radiation. PMID:27189541

  5. The JAK/STAT signaling cascade in gastric carcinoma (Review).

    PubMed

    Khanna, Puja; Chua, Pei Jou; Bay, Boon Huat; Baeg, Gyeong Hun

    2015-11-01

    Gastric carcinoma remains one of the most prevalent forms of cancer worldwide, despite the decline in incidence rates, increased awareness of the disease and advancement in treatment strategies. Helicobacter pylori infection, dietary factors, lifestyle influences and various genetic aberrations have been shown to contribute to the development and progression of gastric cancer. Recent studies on the genomic landscape of gastric adenocarcinoma have identified several key signaling molecules, including epidermal growth factor receptor family (ErbB) members, vascular endothelial growth factor receptor family (VEGFR) members and PI3K/Akt/mTOR pathway components, that have been implicated in the molecular pathogenesis of gastric cancers. However, clinical trials with compounds that target these molecules have failed to show a significant improvement in overall survival rates when supplemented with conventional therapies. Therefore, it is essential to identify effective prognostic and/or diagnostic biomarkers and develop molecular targeted therapies. The JAK/STAT cascade is a principal signal transduction pathway in cytokine and growth factor signaling, regulating various cellular processes such as cell proliferation, differentiation, migration and survival. Numerous in vivo and in vitro studies have shown that dysregulated JAK/STAT signaling is a driving force in the pathogenesis of various solid cancers as well as hematopoietic malignancies. Hence, a large number of preclinical and clinical studies of drugs targeting this pathway are currently underway. Notably, aberrant JAK/STAT signaling has also been implicated in gastric cancers. In this review, we focus on the ongoing research on the JAK/STAT cascade in gastric carcinoma and discuss the therapeutic potential of targeting JAK/STAT signaling for the treatment of gastric cancer. PMID:26398764

  6. Gene regulatory cascade of senescence-associated NAC transcription factors activated by ETHYLENE-INSENSITIVE2-mediated leaf senescence signalling in Arabidopsis

    PubMed Central

    Kim, Hyo Jung; Hong, Sung Hyun; Kim, You Wang; Lee, Il Hwan; Jun, Ji Hyung; Phee, Bong-Kwan; Rupak, Timilsina; Jeong, Hana; Lee, Yeonmi; Hong, Byoung Seok; Nam, Hong Gil; Woo, Hye Ryun; Lim, Pyung Ok

    2014-01-01

    Leaf senescence is a finely tuned and genetically programmed degeneration process, which is critical to maximize plant fitness by remobilizing nutrients from senescing leaves to newly developing organs. Leaf senescence is a complex process that is driven by extensive reprogramming of global gene expression in a highly coordinated manner. Understanding how gene regulatory networks involved in controlling leaf senescence are organized and operated is essential to decipher the mechanisms of leaf senescence. It was previously reported that the trifurcate feed-forward pathway involving EIN2, ORE1, and miR164 in Arabidopsis regulates age-dependent leaf senescence and cell death. Here, new components of this pathway have been identified, which enhances knowledge of the gene regulatory networks governing leaf senescence. Comparative gene expression analysis revealed six senescence-associated NAC transcription factors (TFs) (ANAC019, AtNAP, ANAC047, ANAC055, ORS1, and ORE1) as candidate downstream components of ETHYLENE-INSENSITIVE2 (EIN2). EIN3, a downstream signalling molecule of EIN2, directly bound the ORE1 and AtNAP promoters and induced their transcription. This suggests that EIN3 positively regulates leaf senescence by activating ORE1 and AtNAP, previously reported as key regulators of leaf senescence. Genetic and gene expression analyses in the ore1 atnap double mutant revealed that ORE1 and AtNAP act in distinct and overlapping signalling pathways. Transient transactivation assays further demonstrated that ORE1 and AtNAP could activate common as well as differential NAC TF targets. Collectively, the data provide insight into an EIN2-mediated senescence signalling pathway that coordinates global gene expression during leaf senescence via a gene regulatory network involving EIN3 and senescence-associated NAC TFs. PMID:24659488

  7. 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

  8. Membrane localization of scaffold proteins promotes graded signaling in the yeast MAP kinase cascade

    PubMed Central

    Takahashi, Satoe; Pryciak, Peter M.

    2008-01-01

    Summary Background Signaling through mitogen-activated protein kinase (MAPK) cascade pathways can show various input-output behaviors, including either switch-like or graded responses to increasing levels of stimulus. Prior studies suggest that switch-like behavior is promoted by positive feedback loops and nonprocessive phosphorylation reactions, but it is unclear whether graded signaling is a default behavior or if it must be enforced by separate mechanisms. Scaffold proteins have been hypothesized to promote graded behavior. Results Here, we experimentally probe the determinants of graded signaling in the yeast mating MAPK pathway. We find that graded behavior is robust, as it resists perturbation by loss of several negative feedback regulators. However, the pathway becomes switch-like when activated by a crosstalk stimulus that bypasses multiple upstream components. To dissect the contributing factors, we developed a method for gradually varying the signal input at different pathway steps in vivo. Input at the beginning of the kinase cascade produced a sharp, threshold-like response. Surprisingly, the scaffold protein Ste5 increased this threshold behavior when limited to the cytosol. However, signaling remained graded whenever Ste5 was allowed to function at the plasma membrane. Conclusions The results suggest that the MAPK cascade module is inherently ultrasensitive, but is converted to a graded system by the pathway-specific activation mechanism. Scaffold-mediated assembly of signaling complexes at the plasma membrane allows faithful propagation of weak signals, which consequently reduces pathway ultrasensitivity. These properties help shape the input-output properties of the system to fit the physiological context. PMID:18722124

  9. 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

  10. Oncoprotein HBXIP Modulates Abnormal Lipid Metabolism and Growth of Breast Cancer Cells by Activating the LXRs/SREBP-1c/FAS Signaling Cascade.

    PubMed

    Zhao, Yu; Li, Hang; Zhang, Yingyi; Li, Leilei; Fang, Runping; Li, Yinghui; Liu, Qian; Zhang, Weiying; Qiu, Liyan; Liu, Fabao; Zhang, Xiaodong; Ye, Lihong

    2016-08-15

    Abnormal lipid metabolism is a hallmark of tumorigenesis. Accumulating evidence demonstrates that fatty acid synthase (FAS, FASN) is a metabolic oncogene that supports the growth and survival of tumor cells and is highly expressed in many cancers. Here, we report that the oncoprotein, hepatitis B X-interacting protein (HBXIP, LAMTOR5) contributes to abnormal lipid metabolism. We show that high expression of HBXIP in 236 breast cancer patients was significantly associated with decreased overall survival and progression-free survival. Interestingly, the expression of HBXIP was positively related to that of FAS in clinical breast cancer tissues, and HBXIP overexpression in breast cancer cells resulted in FAS upregulation. Mechanistically, HBXIP upregulated SREBP-1c (SREBF1), which activates the transcription of FAS, by directly interacting with and coactivating nuclear receptor (NR) liver X receptors (LXR). Physiologically, LXRs are activated via a coactivator containing NR motif in a ligand-dependent manner. However, in breast cancer cells, HBXIP containing the corepressor/nuclear receptor motif with special flanking sequence could coactivate LXRs independent of ligand. Moreover, overexpressed SREBP-1c was able to activate the transcription of HBXIP, forming a positive-feedback loop. Functionally, HBXIP enhanced lipogenesis, resulting in the growth of breast cancer cells in vitro and in vivo Thus, we conclude that the oncoprotein HBXIP contributes to the abnormal lipid metabolism in breast cancer through LXRs/SREBP-1c/FAS signaling, providing new insights into the mechanisms by which cancer cells reprogram lipid metabolism in their favor. Cancer Res; 76(16); 4696-707. ©2016 AACR. PMID:26980761

  11. Safrole induces cell death in human tongue squamous cancer SCC-4 cells through mitochondria-dependent caspase activation cascade apoptotic signaling pathways.

    PubMed

    Yu, Fu-Shun; Huang, An-Cheng; Yang, Jai-Sing; Yu, Chun-Shu; Lu, Chi-Cheng; Chiang, Jo-Hua; Chiu, Chang-Fang; Chung, Jing-Gung

    2012-07-01

    Safrole is one of important food-borne phytotoxin that exhibits in many natural products such as oil of sassafras and spices such as anise, basil, nutmeg, and pepper. This study was performed to elucidate safrole-induced apoptosis in human tongue squamous carcinoma SCC-4 cells. The effect of safrole on apoptosis was measured by flow cytometry and DAPI staining and its regulatory molecules were studied by Western blotting analysis. Safrole-induced apoptosis was accompanied with up-regulation of the protein expression of Bax and Bid and down-regulation of the protein levels of Bcl-2 (up-regulation of the ratio of Bax/Bcl-2), resulting in cytochrome c release, promoted Apaf-1 level and sequential activation of caspase-9 and caspase-3 in a time-dependent manner. We also used real-time PCR to show safrole promoted the mRNA expressions of caspase-3, -8, and -9 in SCC-4 cells. These findings indicate that safrole has a cytotoxic effect in human tongue squamous carcinoma SCC-4 cells by inducing apoptosis. The induction of apoptosis of SCC-4 cells by safrole is involved in mitochondria- and caspase-dependent signal pathways. PMID:21591240

  12. Paper-based microreactor array for rapid screening of cell signaling cascades.

    PubMed

    Huang, Chia-Hao; Lei, Kin Fong; Tsang, Ngan-Ming

    2016-08-01

    Investigation of cell signaling pathways is important for the study of pathogenesis of cancer. However, the related operations used in these studies are time consuming and labor intensive. Thus, the development of effective therapeutic strategies may be hampered. In this work, gel-free cell culture and subsequent immunoassay has been successfully integrated and conducted in a paper-based microreactor array. Study of the activation level of different kinases of cells stimulated by different conditions, i.e., IL-6 stimulation, starvation, and hypoxia, was demonstrated. Moreover, rapid screening of cell signaling cascades after the stimulations of HGF, doxorubicin, and UVB irradiation was respectively conducted to simultaneously screen 40 kinases and transcription factors. Activation of multi-signaling pathways could be identified and the correlation between signaling pathways was discussed to provide further information to investigate the entire signaling network. The present technique integrates most of the tedious operations using a single paper substrate, reduces sample and reagent consumption, and shortens the time required by the entire process. Therefore, it provides a first-tier rapid screening tool for the study of complicated signaling cascades. It is expected that the technique can be developed for routine protocol in conventional biological research laboratories. PMID:27377153

  13. Bombyx mori prothoracicostatic peptide receptor is allosterically activated via a Gα(i/o)-protein-biased signalling cascade by Drosophila sex peptide.

    PubMed

    He, Xiaobai; Zang, Jiashu; Yang, Huipeng; Huang, Haishan; Shi, Ying; Zhu, Chenggang; Zhou, Naiming

    2015-03-01

    In insects, molting and metamorphosis are strictly regulated by ecdysteroids. Ecdysteroid synthesis is positively or negatively controlled by several neuropeptides. The prothoracicostatic peptide (PTSP) BmPTSP (Bombyx mori prothoracicostatic peptide), isolated from the larval brain of B. mori, has been demonstrated to inhibit ecdysteroid synthesis in the prothoracic glands (PGs) [Hua et al. (1999) J. Biol. Chem. 274, 31169-31173]. More recently, the newly recognized B. mori receptor for Drosophila melanogaster sex peptide (DmSP) has been identified as a receptor for BmPTSP. However, details on the signalling pathways and physiological functions of this receptor have remained elusive. In the present paper, we report the functional characterization of the BmPTSP receptor (BmPTSPR)/sex peptide (SP) receptor (SPR) using both mammalian and insect cells. Synthetic DmSP shows the potential to inhibit forskolin (FSK) or adipokinetic hormone (AKH)-induced cAMP-response element (CRE)-driven luciferase (Luc) activity in a manner comparable with synthetic BmPTSP1. However, DmSP displayed a much lower activity in triggering Ca²⁺ mobilization and internalization than did BmPTSP1. Additionally, 6-carboxy-fluorescein fluorophore (FAM)-labelled DmSP and BmPTSP3 were found to bind specifically to BmPTSPR/SPR. The binding of FAM-DmSP was displaced by unlabelled DmSP, but not by unlabelled BmPTSP1 and, vice versa, the binding of FAM-BmPTSP3 was blocked by unlabelled BmPTSP3, but not by unlabelled DmSP. Moreover, internalization assays demonstrated that BmPTSP1, but not DmSP, evoked recruitment of the Bombyx non-visual arrestin, Kurtz, to the activated BmPTSPR/SPR in the plasma membrane. This was followed by induction of internalization. This suggests that BmPTSP1 is probably an endogenous ligand specific for BmPTSPR/SPR. We therefore designate this receptor BmPTSPR. In contrast, DmSP is an allosteric agonist that is biased towards Gα(i/o)-dependent cAMP production and away from Ca

  14. Neuronal Apoptosis Induced by Selective Inhibition of Rac GTPase versus Global Suppression of Rho Family GTPases Is Mediated by Alterations in Distinct Mitogen-activated Protein Kinase Signaling Cascades*

    PubMed Central

    Stankiewicz, Trisha R.; Ramaswami, Sai Anandi; Bouchard, Ron J.; Aktories, Klaus; Linseman, Daniel A.

    2015-01-01

    Rho family GTPases play integral roles in neuronal differentiation and survival. We have shown previously that Clostridium difficile toxin B (ToxB), an inhibitor of RhoA, Rac1, and Cdc42, induces apoptosis of cerebellar granule neurons (CGNs). In this study, we compared the effects of ToxB to a selective inhibitor of the Rac-specific guanine nucleotide exchange factors Tiam1 and Trio (NSC23766). In a manner similar to ToxB, selective inhibition of Rac induces CGN apoptosis associated with enhanced caspase-3 activation and reduced phosphorylation of the Rac effector p21-activated kinase. In contrast to ToxB, caspase inhibitors do not protect CGNs from targeted inhibition of Rac. Also dissimilar to ToxB, selective inhibition of Rac does not inhibit MEK1/2/ERK1/2 or activate JNK/c-Jun. Instead, targeted inhibition of Rac suppresses distinct MEK5/ERK5, p90Rsk, and Akt-dependent signaling cascades known to regulate the localization and expression of the Bcl-2 homology 3 domain-only protein Bad. Adenoviral expression of a constitutively active mutant of MEK5 is sufficient to attenuate neuronal cell death induced by selective inhibition of Rac with NSC23766 but not apoptosis induced by global inhibition of Rho GTPases with ToxB. Collectively, these data demonstrate that global suppression of Rho family GTPases with ToxB causes a loss of MEK1/2/ERK1/2 signaling and activation of JNK/c-Jun, resulting in diminished degradation and enhanced transcription of the Bcl-2 homology 3 domain-only protein Bim. In contrast, selective inhibition of Rac induces CGN apoptosis by repressing unique MEK5/ERK5, p90Rsk, and Akt-dependent prosurvival pathways, ultimately leading to enhanced expression, dephosphorylation, and mitochondrial localization of proapoptotic Bad. PMID:25666619

  15. Neuronal apoptosis induced by selective inhibition of Rac GTPase versus global suppression of Rho family GTPases is mediated by alterations in distinct mitogen-activated protein kinase signaling cascades.

    PubMed

    Stankiewicz, Trisha R; Ramaswami, Sai Anandi; Bouchard, Ron J; Aktories, Klaus; Linseman, Daniel A

    2015-04-10

    Rho family GTPases play integral roles in neuronal differentiation and survival. We have shown previously that Clostridium difficile toxin B (ToxB), an inhibitor of RhoA, Rac1, and Cdc42, induces apoptosis of cerebellar granule neurons (CGNs). In this study, we compared the effects of ToxB to a selective inhibitor of the Rac-specific guanine nucleotide exchange factors Tiam1 and Trio (NSC23766). In a manner similar to ToxB, selective inhibition of Rac induces CGN apoptosis associated with enhanced caspase-3 activation and reduced phosphorylation of the Rac effector p21-activated kinase. In contrast to ToxB, caspase inhibitors do not protect CGNs from targeted inhibition of Rac. Also dissimilar to ToxB, selective inhibition of Rac does not inhibit MEK1/2/ERK1/2 or activate JNK/c-Jun. Instead, targeted inhibition of Rac suppresses distinct MEK5/ERK5, p90Rsk, and Akt-dependent signaling cascades known to regulate the localization and expression of the Bcl-2 homology 3 domain-only protein Bad. Adenoviral expression of a constitutively active mutant of MEK5 is sufficient to attenuate neuronal cell death induced by selective inhibition of Rac with NSC23766 but not apoptosis induced by global inhibition of Rho GTPases with ToxB. Collectively, these data demonstrate that global suppression of Rho family GTPases with ToxB causes a loss of MEK1/2/ERK1/2 signaling and activation of JNK/c-Jun, resulting in diminished degradation and enhanced transcription of the Bcl-2 homology 3 domain-only protein Bim. In contrast, selective inhibition of Rac induces CGN apoptosis by repressing unique MEK5/ERK5, p90Rsk, and Akt-dependent prosurvival pathways, ultimately leading to enhanced expression, dephosphorylation, and mitochondrial localization of proapoptotic Bad. PMID:25666619

  16. An Nfic-hedgehog signaling cascade regulates tooth root development.

    PubMed

    Liu, Yang; Feng, Jifan; Li, Jingyuan; Zhao, Hu; Ho, Thach-Vu; Chai, Yang

    2015-10-01

    Coordination between the Hertwig's epithelial root sheath (HERS) and apical papilla (AP) is crucial for proper tooth root development. The hedgehog (Hh) signaling pathway and Nfic are both involved in tooth root development; however, their relationship has yet to be elucidated. Here, we establish a timecourse of mouse molar root development by histological staining of sections, and we demonstrate that Hh signaling is active before and during root development in the AP and HERS using Gli1 reporter mice. The proper pattern of Hh signaling activity in the AP is crucial for the proliferation of dental mesenchymal cells, because either inhibition with Hh inhibitors or constitutive activation of Hh signaling activity in transgenic mice leads to decreased proliferation in the AP and shorter roots. Moreover, Hh activity is elevated in Nfic(-/-) mice, a root defect model, whereas RNA sequencing and in situ hybridization show that the Hh attenuator Hhip is downregulated. ChIP and RNAscope analyses suggest that Nfic binds to the promoter region of Hhip. Treatment of Nfic(-/-) mice with Hh inhibitor partially restores cell proliferation, AP growth and root development. Taken together, our results demonstrate that an Nfic-Hhip-Hh signaling pathway is crucial for apical papilla growth and proper root formation. This discovery provides insight into the molecular mechanisms regulating tooth root development. PMID:26293299

  17. MEK1-ERKs signal cascade is required for the replication of Enterovirus 71 (EV71).

    PubMed

    Wang, Bo; Zhang, Hao; Zhu, Meng; Luo, Zhijun; Peng, Yihong

    2012-01-01

    The role of the MEK1-ERK signaling cascade in the replication cycle of Enterovirus 71 (EV71), the primary cause of hand, foot and mouth disease (HFMD), has been analyzed. In vitro infection with EV71 induced a biphasic activation of ERK. The two phases of activation appeared to be triggered by different mechanisms, with the first phase being activated by the binding of viral particles to the membrane receptor of host cells and the second probably being in response to the production of new virus particles. Inhibition of ERK activation by U0126 was found to severely impair virus production. A similar reduction in EV71 replication was also observed when MEK1 expression was subject to knockdown using specific siRNAs. By contrast knockdown of MEK2 expression showed that it was dispensable for virus replication cycle, despite both MEK isoforms being activated and translocated to the nucleus equally well in response to virus infection. Overall, this study suggests distinct functions of the two isoforms of MEK in EV71 replication cycle, with an essential role for MEK1 in stimulating the ERK signaling cascade to promote virus replication. Taken together with our previous work on herpes simplex virus type 2 (HSV2) this study highlights MEK1 as a potential broad antiviral molecular target. PMID:22101247

  18. FGF19 promotes epithelial-mesenchymal transition in hepatocellular carcinoma cells by modulating the GSK3β/β- catenin signaling cascade via FGFR4 activation

    PubMed Central

    Zhao, Huakan; Lv, Fenglin; Liang, Guizhao; Huang, Xiaobin; Wu, Gang; Zhang, Wenfa; Yu, Le; Shi, Lei; Teng, Yong

    2016-01-01

    Compelling evidence suggests that the epithelial-mesenchymal transition (EMT) correlates with aggressiveness of tumors and poor survival. FGF19 has been shown to be involved in EMT in cholangiocarcinoma and colorectal cancer, however, molecular mechanisms underlying FGF19-induced EMT process in hepatocellular carcinoma (HCC) remain largely unknown. Here, we show the expression of FGF19 is significantly elevated and negatively associated with the expression of E-cadherin in HCC tissues and cell lines. Ectopic FGF19 expression promotes EMT and invasion in epithelial-like HCC cells through repression of E-cadherin expression, whereas FGF19 knockdown enhances E-cadherin expression and hence diminishes EMT traits in mesenchymal-like HCC cells, suggesting FGF19 exerts its tumor progressing functions as an EMT inducer. Interestingly, depletion of FGF19 cannot abrogate EMT traits in the presence of GSK3β inhibitors. Furthermore, FGF19-induced EMT can be markedly attenuated when FGFR4 is knocked out. These observations clearly indicate that FGFR4/GSK3β/β-catenin axis may play a pivotal role in FGF19-induced EMT in HCC cells. As FGF19 and its specific receptor FGFR4 are frequently amplified in HCC cells, selective targeting this signaling node may lend insights into a potential effective therapeutic approach for blocking metastasis of HCC. PMID:26498355

  19. Search for acoustic signals from high energy cascades

    NASA Technical Reports Server (NTRS)

    Bell, R.; Bowen, T.

    1985-01-01

    High energy cosmic ray secondaries can be detected by means of the cascades they produce when they pass through matter. When the charged particles of these cascades ionize the matter they are traveling through, the heat produced and resulting thermal expansion causes a thermoacoustic wave. These sound waves travel at about one hundred-thousandth the speed of light, and should allow an array of acoustic transducers to resolve structure in the cascade to about 1 cm without high speed electronics or segmentation of the detector.

  20. Dioscin inhibits osteoclast differentiation and bone resorption though down-regulating the Akt signaling cascades

    SciTech Connect

    Qu, Xinhua; Zhai, Zanjing; Liu, Xuqiang; Li, Haowei; Ouyang, Zhengxiao; Wu, Chuanlong; Liu, Guangwang; Fan, Qiming; Tang, Tingting; Qin, An; Dai, Kerong

    2014-01-10

    Highlights: •A natural-derived compound, dioscin, suppresses osteoclast formation and bone resorption. •Dioscin inhibits osteolytic bone loss in vivo. •Dioscin impairs the Akt signaling cascades pathways during osteoclastogenesis. •Dioscin have therapeutic value in treating osteoclast-related diseases. -- Abstract: Bone resorption is the unique function of osteoclasts (OCs) and is critical for both bone homeostasis and pathologic bone diseases including osteoporosis, rheumatoid arthritis and tumor bone metastasis. Thus, searching for natural compounds that may suppress osteoclast formation and/or function is promising for the treatment of osteoclast-related diseases. In this study, we for the first time demonstrated that dioscin suppressed RANKL-mediated osteoclast differentiation and bone resorption in vitro in a dose-dependent manner. The suppressive effect of dioscin is supported by the reduced expression of osteoclast-specific markers. Further molecular analysis revealed that dioscin abrogated AKT phosphorylation, which subsequently impaired RANKL-induced nuclear factor-kappaB (NF-κB) signaling pathway and inhibited NFATc1 transcriptional activity. Moreover, in vivo studies further verified the bone protection activity of dioscin in osteolytic animal model. Together our data demonstrate that dioscin suppressed RANKL-induced osteoclast formation and function through Akt signaling cascades. Therefore, dioscin is a potential natural agent for the treatment of osteoclast-related diseases.

  1. Spatio-temporal dynamcis of a cell signal cascade with negative feedback

    NASA Astrophysics Data System (ADS)

    Maya Bernal, Jose Luis; Ramirez-Santiago, Guillermo

    2014-03-01

    We studied the spatio-temporal dynamics of a system of reactio-diffusion equations that models a cell signal transduction pathway with six cycles and negative feedback. The basic cycle consists of the phosphorylation-dephosphorylation of two antagonic proteins. We found two regimes of saturation of the enzimatic reaction in the kinetic parameters space and determined the conditions for the signal propagation in the steady state. The trajectories for which transduction occurs are defined in terms of the ratio of the enzimatic activities. We found that in spite of the negative feedback the cell signal cascade behaves as an amplifier and produces phosphoprotein concentration gradients within the cell. This model behaves also as a noise filter and as a switch. Supported by DGAPA-UNAM Contract IN118410-3.

  2. Signaling gradients in cascades of two-state reaction-diffusion systems.

    PubMed

    Berezhkovskii, Alexander M; Coppey, Mathieu; Shvartsman, Stanislav Y

    2009-01-27

    Biological networks frequently use cascades, generally defined as chain-like arrangements of similar modules. Spatially lumped cascades can serve as noise filters, time-delay, or thresholding elements. The operation and functional capabilities of spatially distributed cascades are much less understood. Motivated by studies of pattern formation in the early Drosophila embryo, we analyze cascades of 2-state reaction-diffusion systems. At each stage within such as a cascade, a diffusible particle is reversibly bound by immobile traps and can be annihilated in both mobile and immobile states. When trapped, these particles drive the next stage by converting mobile particles of a different type from a passive to active form. The cascade initiated by injection of mobile particles into the first stage. We derive analytical expressions for the steady-state concentration profiles of mobile and immobile particles and analyze how the output of a cascade is controlled by properties of the constituent stages. PMID:19147842

  3. Protein-protein interactions in plant mitogen-activated protein kinase cascades.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2016-02-01

    Mitogen-activated protein kinases (MAPKs) form tightly controlled signaling cascades that play essential roles in plant growth, development, and defense. However, the molecular mechanisms underlying MAPK cascades are still elusive, due largely to our poor understanding of how they relay the signals. Extensive effort has been devoted to characterization of MAPK-substrate interactions to illustrate phosphorylation-based signaling. The diverse MAPK substrates identified also shed light on how spatiotemporal-specific protein-protein interactions function in distinct MAPK cascade-mediated biological processes. This review surveys various technologies used for characterizing MAPK-substrate interactions and presents case studies of MPK4 and MPK6, highlighting the multiple functions of MAPKs. Mass spectrometry-based approaches in identifying MAPK-interacting proteins are emphasized due to their increasing utility and effectiveness. The potential for using MAPKs and their substrates in enhancing plant stress tolerance is also discussed. PMID:26646897

  4. Newly Constructed Network Models of Different WNT Signaling Cascades Applied to Breast Cancer Expression Data

    PubMed Central

    Kramer, Frank; Pukrop, Tobias; Beißbarth, Tim; Bleckmann, Annalen

    2015-01-01

    Introduction WNT signaling is a complex process comprising multiple pathways: the canonical β-catenin-dependent pathway and several alternative non-canonical pathways that act in a β-catenin-independent manner. Representing these intricate signaling mechanisms through bioinformatic approaches is challenging. Nevertheless, a simplified but reliable bioinformatic WNT pathway model is needed, which can be further utilized to decipher specific WNT activation states within e.g. high-throughput data. Results In order to build such a model, we collected, parsed, and curated available WNT signaling knowledge from different pathway databases. The data were assembled to construct computationally suitable models of different WNT signaling cascades in the form of directed signaling graphs. This resulted in four networks representing canonical WNT signaling, non-canonical WNT signaling, the inhibition of canonical WNT signaling and the regulation of WNT signaling pathways, respectively. Furthermore, these networks were integrated with microarray and RNA sequencing data to gain deeper insight into the underlying biology of gene expression differences between MCF-7 and MDA-MB-231 breast cancer cell lines, representing weakly and highly invasive breast carcinomas, respectively. Differential genes up-regulated in the MDA-MB-231 compared to the MCF-7 cell line were found to display enrichment in the gene set originating from the non-canonical network. Moreover, we identified and validated differentially regulated modules representing canonical and non-canonical WNT pathway components specific for the aggressive basal-like breast cancer subtype. Conclusions In conclusion, we demonstrated that these newly constructed WNT networks reliably reflect distinct WNT signaling processes. Using transcriptomic data, we shaped these networks into comprehensive modules of the genes implicated in the aggressive basal-like breast cancer subtype and demonstrated that non-canonical WNT signaling is

  5. The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells

    PubMed Central

    Melillo, Rosa Marina; Castellone, Maria Domenica; Guarino, Valentina; De Falco, Valentina; Cirafici, Anna Maria; Salvatore, Giuliana; Caiazzo, Fiorina; Basolo, Fulvio; Giannini, Riccardo; Kruhoffer, Mogens; Orntoft, Torben; Fusco, Alfredo; Santoro, Massimo

    2005-01-01

    In papillary thyroid carcinomas (PTCs), rearrangements of the RET receptor (RET/PTC) and activating mutations in the BRAF or RAS oncogenes are mutually exclusive. Here we show that the 3 proteins function along a linear oncogenic signaling cascade in which RET/PTC induces RAS-dependent BRAF activation and RAS- and BRAF-dependent ERK activation. Adoptive activation of the RET/PTC-RAS-BRAF axis induced cell proliferation and Matrigel invasion of thyroid follicular cells. Gene expression profiling revealed that the 3 oncogenes activate a common transcriptional program in thyroid cells that includes upregulation of the CXCL1 and CXCL10 chemokines, which in turn stimulate proliferation and invasion. Thus, motile and mitogenic properties are intrinsic to transformed thyroid cells and are governed by an epistatic oncogenic signaling cascade. PMID:15761501

  6. Integration of C/N-nutrient and multiple environmental signals into the ABA signaling cascade

    PubMed Central

    Lu, Yu; Yamaguchi, Junji; Sato, Takeo

    2015-01-01

    Due to their immobility, plants have developed sophisticated mechanisms to robustly monitor and appropriately respond to dynamic changes in nutrient availability. Carbon (C) and nitrogen (N) are especially important in regulating plant metabolism and development, thereby affecting crop productivity. In addition to their independent utilization, the ratio of C to N metabolites in the cell, referred to as the “C/N balance”, is important for the regulation of plant growth, although molecular mechanisms mediating C/N signaling remain unclear. Recently ABI1, a protein phosphatase type 2C (PP2C), was shown to be a regulator of C/N response in Arabidopsis plants. ABI1 functions as a negative regulator of abscisic acid (ABA) signal transduction. ABA is versatile phytohormone that regulates multiple aspects of plant growth and adaptation to environmental stress. This review highlights the regulation of the C/N response mediated by a non-canonical ABA signaling pathway that is independent of ABA biosynthesis, as well as recent findings on the direct crosstalk between multiple cellular signals and the ABA signaling cascade. PMID:26786013

  7. Regulation of MAP kinase signaling cascade by microRNAs in Oryza sativa

    PubMed Central

    Raghuram, Badmi; Sheikh, Arsheed Hussain; Sinha, Alok Krishna

    2014-01-01

    Mitogen activated protein kinase (MAPK) pathway is one of the most conserved signaling cascade in plants regulating a plethora of cellular processes including normal growth and development, abiotic and biotic stress responses. The perception of external cues triggers the phosphorylation of three tier MAPKKK-MAPKK-MAPK cascade which finally modifies a downstream substrate thereby regulating the cellular processes. Whereas, the transcription regulation by MAPKs, mediated through their substrates is well studied in plants, the transcription and post-transcriptional regulation of the MAPK genes are poorly understood. Previous studies from the animals systems suggested the miRNAs regulate the post-transcriptional regulation of MAPK transcripts. Here we attempt to unravel the post-transcriptional regulation of MAPKs by miRNAs in model crop plant Oryza sativa. Using in silico tools, we predict the miRNAs for 98 out of 99 MAPK transcripts. The predicted miRNAs were validated for the biological relevance of their function. The inverse correlation between relative transcript levels between the MAPKs and their predicted miRNAs validated the in silico prediction. Taken together, this report demonstrates the significance of miRNAs in regulation of the MAPK pathway in plants with a new direction to study the plant signaling molecules. PMID:25482813

  8. Tight Interconnection and Multi-Level Control of Arabidopsis MYB44 in MAPK Cascade Signalling

    PubMed Central

    2013-01-01

    Abiotic stress poses a huge, ever-increasing problem to plants and agriculture. The dissection of signalling pathways mediating stress tolerance is a prerequisite to develop more resistant plant species. Mitogen-activated protein kinase (MAPK) cascades are universal signalling modules. In Arabidopsis, the MAPK MPK3 and its upstream regulator MAPK kinase MKK4 initiate the adaptation response to numerous abiotic and biotic stresses. Yet, molecular steps directly linked with MKK4 – MPK3 activation are largely unknown. Starting with a yeast-two-hybrid screen for interacting partners of MKK4, we identified a transcription factor, MYB44. MYB44 is controlled at multiple levels by and strongly inter-connected with MAPK signalling. As we had shown earlier, stress-induced expression of the MYB44 gene is regulated by a MPK3-targeted bZIP transcription factor VIP1. At the protein level, MYB44 interacts with MPK3 in vivo. MYB44 is phosphorylated by MPK3 in vitro at a single residue, Ser145. Although replacement of Ser145 by a non-phosphorylatable (S145A) or phosphomimetic (S145D) residue did not alter MYB44 subcellular localisation, dimerization behaviour nor DNA-binding characteristics, abiotic stress tolerance tests in stable transgenic Arabidopsis plants clearly related S145 phosphorylation to MYB44 function: Compared to Arabidopsis wild type plants, MYB44 overexpressing lines exhibit an enhanced tolerance to osmotic stress and are slightly more sensitive to abscisic acid. Interestingly, overexpression of the S145A variant revealed that impaired phosphorylation does not render the MYB44 protein non-functional. Instead, S145A lines are highly sensitive to abiotic stress, and thereby remarkably similar to mpk3-deficient plants. Its in vivo interaction with the nuclear sub-pools of both MPK3 and MKK4 renders MYB44 the first plant transcription factor to have a second function as putative MAPK cascade scaffolding protein. PMID:23437396

  9. Prostate Cancer Stem Cells: Viewing Signaling Cascades at a Finer Resolution.

    PubMed

    Lin, Xiukun; Farooqi, Ammad Ahmad; Qureshi, Muhammad Zahid; Romero, Mirna Azalea; Tabassum, Sobia; Ismail, Muhammad

    2016-06-01

    It is becoming characteristically more understandable that within tumor cells, there lies a sub-population of tumor cells with "stem cell" like properties and remarkable ability of self-renewal. Many features of these self-renewing cells are comparable with normal stem cells and are termed as "cancer stem cells". Accumulating experimentally verified data has started to scratch the surface of spatio-temporally dysregulated intracellular signaling cascades in the biology of prostate cancer stem cells. We partition this multicomponent review into how different signaling cascades operate in cancer stem cells and how bioactive ingredients isolated from natural sources may modulate signaling network. PMID:26846602

  10. Hydrogen sulphide in cardiovascular system: A cascade from interaction between sulphur atoms and signalling molecules.

    PubMed

    Wang, Ming-Jie; Cai, Wen-Jie; Zhu, Yi-Chun

    2016-05-15

    As a gasotransmitter, hydrogen sulphide exerts its extensive physiological and pathophysiological effects in mammals. The interaction between sulphur atoms and signalling molecules forms a cascade that modulates cellular functions and homeostasis. In this review, we focus on the signalling mechanism underlying the effect of hydrogen sulphide in the cardiovascular system and metabolism as well as the biological relevance to human diseases. PMID:27071836

  11. Cilostazol Induces PGI2 Production via Activation of the Downstream Epac-1/Rap1 Signaling Cascade to Increase Intracellular Calcium by PLCε and to Activate p44/42 MAPK in Human Aortic Endothelial Cells

    PubMed Central

    Hashimoto, Ayako

    2015-01-01

    Background Cilostazol, a selective phosphodiesterase 3 (PDE3) inhibitor, is known as an anti-platelet drug and acts directly on platelets. Cilostazol has been shown to exhibit vascular protection in ischemic diseases. Although vascular endothelium-derived prostaglandin I2 (PGI2) plays an important role in vascular protection, it is unknown whether cilostazol directly stimulates PGI2 synthesis in endothelial cells. Here, we elucidate the mechanism of cilostazol-induced PGI2 stimulation in endothelial cells. Methods and Results Human aortic endothelial cells (HAECs) were stimulated with cilostazol and PGI2 accumulation in the culture media was measured. Cilostazol increased PGI2 synthesis via the arachidonic acid pathway. Cilostazol-induced intracellular calcium also promoted PGI2 synthesis via the inositol 1,4,5-trisphosphate receptor. Using RNAi, silencing of PDE3B abolished the induction effect of cilostazol on PGI2 synthesis and intracellular cAMP accumulation. Inhibition of the exchange protein, which was directly activated by cyclic AMP 1 (Epac-1) and its downstream signal the Ras-like small GTPase (Rap-1), abolished cilostazol-induced PGI2 synthesis, but this did not take place via protein kinase A (PKA). Inhibition of downstream signaling, such as mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) γ, and phospholipase C (PLC) ε, suppressed cilostazol-induced PGI2 synthesis. Conclusions The PDE3/Epac-1/Rap-1 signaling pathway plays an important role in cilostazol-induced PGI2 synthesis. Namely, stimulation of HAECs with cilostazol induces intracellular calcium elevation via the Rap-1/PLCε/IP3 pathway, along with MAPK activation via direct activation by Epac-1/Rap-1 and indirect activation by Epac-1/Rap-1/PI3Kγ, resulting in synergistically induced PGI2 synthesis. PMID:26181635

  12. Stochastic signaling in biochemical cascades and genetic systems in genetically engineered living cells.

    PubMed

    Daniel, Ramiz; Almog, Ronen; Shacham-Diamand, Yosi

    2010-04-01

    Living cells, either prokaryote or eukaryote, can be integrated within whole-cell biochips (WCBCs) for various applications. We investigate WCBCs where information is extracted from the cells via a cascade of biochemical reactions that involve gene expression. The overall biological signal is weak due to small sample volume, low intrinsic cell response, and extrinsic signal loss mechanisms. The low signal-to-noise ratio problem is aggravated during initial detection stages and limits the minimum detectable signal or, alternatively, the minimum detection time. Taking into account the stochastic nature of biochemical process, we find that the signal is accompanied by relatively large noise disturbances. In this work, we use genetically engineered microbe sensors as a model to study the biochips output signal stochastic behavior. In our model, the microbes are designed to express detectable reporter proteins under external induction. We present analytical approximated expressions and numerical simulations evaluating the fluctuations of the synthesized reporter proteins population based on a set of equations modeling a cascade of biochemical and genetic reactions. We assume that the reporter proteins decay more slowly than messenger RNA molecules. We calculate the relation between the noise of the input signal (extrinsic noise) and biochemical reaction statistics (intrinsic noise). We discuss in further details two cases: (1) a cascade with large decay rates of all biochemical reactions compared to the protein decay rate. We show that in this case, the noise amplitude has a positive linear correlation with the number of stages in the cascade. (2) A cascade which includes a stable enzymatic-binding reaction with slow decay rate. We show that in this case, the noise strongly depends on the protein decay rate. Finally, a general observation is presented stating that the noise in whole-cell biochip sensors is determined mainly by the first reactions in the genetic system

  13. Stochastic signaling in biochemical cascades and genetic systems in genetically engineered living cells

    NASA Astrophysics Data System (ADS)

    Daniel, Ramiz; Almog, Ronen; Shacham-Diamand, Yosi

    2010-04-01

    Living cells, either prokaryote or eukaryote, can be integrated within whole-cell biochips (WCBCs) for various applications. We investigate WCBCs where information is extracted from the cells via a cascade of biochemical reactions that involve gene expression. The overall biological signal is weak due to small sample volume, low intrinsic cell response, and extrinsic signal loss mechanisms. The low signal-to-noise ratio problem is aggravated during initial detection stages and limits the minimum detectable signal or, alternatively, the minimum detection time. Taking into account the stochastic nature of biochemical process, we find that the signal is accompanied by relatively large noise disturbances. In this work, we use genetically engineered microbe sensors as a model to study the biochips output signal stochastic behavior. In our model, the microbes are designed to express detectable reporter proteins under external induction. We present analytical approximated expressions and numerical simulations evaluating the fluctuations of the synthesized reporter proteins population based on a set of equations modeling a cascade of biochemical and genetic reactions. We assume that the reporter proteins decay more slowly than messenger RNA molecules. We calculate the relation between the noise of the input signal (extrinsic noise) and biochemical reaction statistics (intrinsic noise). We discuss in further details two cases: (1) a cascade with large decay rates of all biochemical reactions compared to the protein decay rate. We show that in this case, the noise amplitude has a positive linear correlation with the number of stages in the cascade. (2) A cascade which includes a stable enzymatic-binding reaction with slow decay rate. We show that in this case, the noise strongly depends on the protein decay rate. Finally, a general observation is presented stating that the noise in whole-cell biochip sensors is determined mainly by the first reactions in the genetic system

  14. Engineering modular and tunable genetic amplifiers for scaling transcriptional signals in cascaded gene networks.

    PubMed

    Wang, Baojun; Barahona, Mauricio; Buck, Martin

    2014-08-01

    Synthetic biology aims to control and reprogram signal processing pathways within living cells so as to realize repurposed, beneficial applications. Here we report the design and construction of a set of modular and gain-tunable genetic amplifiers in Escherichia coli capable of amplifying a transcriptional signal with wide tunable-gain control in cascaded gene networks. The devices are engineered using orthogonal genetic components (hrpRS, hrpV and PhrpL) from the hrp (hypersensitive response and pathogenicity) gene regulatory network in Pseudomonas syringae. The amplifiers can linearly scale up to 21-fold the transcriptional input with a large output dynamic range, yet not introducing significant time delay or significant noise during signal amplification. The set of genetic amplifiers achieves different gains and input dynamic ranges by varying the expression levels of the underlying ligand-free activator proteins in the device. As their electronic counterparts, these engineered transcriptional amplifiers can act as fundamental building blocks in the design of biological systems by predictably and dynamically modulating transcriptional signal flows to implement advanced intra- and extra-cellular control functions. PMID:25030903

  15. Compartmentalization of NO signaling cascade in skeletal muscles

    SciTech Connect

    Buchwalow, Igor B. . E-mail: buchwalo@uni-muenster.de; Minin, Evgeny A.; Samoilova, Vera E.; Boecker, Werner; Wellner, Maren; Schmitz, Wilhelm; Neumann, Joachim

    2005-05-06

    Skeletal muscle functions regulated by NO are now firmly established. However, the literature on the compartmentalization of NO signaling in myocytes is highly controversial. To address this issue, we examined localization of enzymes engaged in L-arginine-NO-cGMP signaling in the rat quadriceps muscle. Employing immunocytochemical labeling complemented with tyramide signal amplification and electron microscopy, we found NO synthase expressed not only in the sarcolemma, but also along contractile fibers, in the sarcoplasmic reticulum and mitochondria. The expression pattern of NO synthase in myocytes showed striking parallels with the enzymes engaged in L-arginine-NO-cGMP signaling (arginase, phosphodiesterase, and soluble guanylyl cyclase). Our findings are indicative of an autocrine fashion of NO signaling in skeletal muscles at both cellular and subcellular levels, and challenge the notion that the NO generation is restricted to the sarcolemma.

  16. A Gibberellin-Mediated DELLA-NAC Signaling Cascade Regulates Cellulose Synthesis in Rice[OPEN

    PubMed Central

    Huang, Debao; Wang, Shaogan; Zhang, Baocai; Shang-Guan, Keke; Shi, Yanyun; Zhang, Dongmei; Liu, Xiangling; Wu, Kun; Xu, Zuopeng; Fu, Xiangdong; Zhou, Yihua

    2015-01-01

    Cellulose, which can be converted into numerous industrial products, has important impacts on the global economy. It has long been known that cellulose synthesis in plants is tightly regulated by various phytohormones. However, the underlying mechanism of cellulose synthesis regulation remains elusive. Here, we show that in rice (Oryza sativa), gibberellin (GA) signals promote cellulose synthesis by relieving the interaction between SLENDER RICE1 (SLR1), a DELLA repressor of GA signaling, and NACs, the top-layer transcription factors for secondary wall formation. Mutations in GA-related genes and physiological treatments altered the transcription of CELLULOSE SYNTHASE genes (CESAs) and the cellulose level. Multiple experiments demonstrated that transcription factors NAC29/31 and MYB61 are CESA regulators in rice; NAC29/31 directly regulates MYB61, which in turn activates CESA expression. This hierarchical regulation pathway is blocked by SLR1-NAC29/31 interactions. Based on the results of anatomical analysis and GA content examination in developing rice internodes, this signaling cascade was found to be modulated by varied endogenous GA levels and to be required for internode development. Genetic and gene expression analyses were further performed in Arabidopsis thaliana GA-related mutants. Altogether, our findings reveal a conserved mechanism by which GA regulates secondary wall cellulose synthesis in land plants and provide a strategy for manipulating cellulose production and plant growth. PMID:26002868

  17. Angiotensin II-triggered kinase signaling cascade in the central nervous system.

    PubMed

    Bali, Anjana; Jaggi, Amteshwar Singh

    2016-04-01

    Recent studies have projected the renin-angiotensin system as a central component of the physiological and pathological processes of assorted neurological disorders. Its primary effector hormone, angiotensin II (Ang II), not only mediates the physiological effects of vasoconstriction and blood pressure regulation in cardiovascular disease but is also implicated in a much wider range of neuronal activities and diseases, including Alzheimer's disease, neuronal injury, and cognitive disorders. Ang II produces different actions by acting on its two subtypes of receptors (AT1 and AT2); however, the well-known physiological actions of Ang II are mainly mediated through AT1 receptors. Moreover, recent studies also suggest the important functional role of AT2 receptor in the brain. Ang II acts on AT1 receptors and conducts its functions via MAP kinases (ERK1/2, JNK, and p38MAPK), glycogen synthase kinase, Rho/ROCK kinase, receptor tyrosine kinases (PDGF and EGFR), and nonreceptor tyrosine kinases (Src, Pyk2, and JAK/STAT). AT1R-mediated NADPH oxidase activation also leads to the generation of reactive oxygen species, widely implicated in neuroinflammation. These signaling cascades lead to glutamate excitotoxicity, apoptosis, cerebral infarction, astrocyte proliferation, nociception, neuroinflammation, and progression of other neurological disorders. The present review focuses on the Ang II-triggered signal transduction pathways in central nervous system. PMID:26574890

  18. Dioscin inhibits osteoclast differentiation and bone resorption though down-regulating the Akt signaling cascades.

    PubMed

    Qu, Xinhua; Zhai, Zanjing; Liu, Xuqiang; Li, Haowei; Ouyang, Zhengxiao; Wu, Chuanlong; Liu, Guangwang; Fan, Qiming; Tang, Tingting; Qin, An; Dai, Kerong

    2014-01-10

    Bone resorption is the unique function of osteoclasts (OCs) and is critical for both bone homeostasis and pathologic bone diseases including osteoporosis, rheumatoid arthritis and tumor bone metastasis. Thus, searching for natural compounds that may suppress osteoclast formation and/or function is promising for the treatment of osteoclast-related diseases. In this study, we for the first time demonstrated that dioscin suppressed RANKL-mediated osteoclast differentiation and bone resorption in vitro in a dose-dependent manner. The suppressive effect of dioscin is supported by the reduced expression of osteoclast-specific markers. Further molecular analysis revealed that dioscin abrogated AKT phosphorylation, which subsequently impaired RANKL-induced nuclear factor-kappaB (NF-κB) signaling pathway and inhibited NFATc1 transcriptional activity. Moreover, in vivo studies further verified the bone protection activity of dioscin in osteolytic animal model. Together our data demonstrate that dioscin suppressed RANKL-induced osteoclast formation and function through Akt signaling cascades. Therefore, dioscin is a potential natural agent for the treatment of osteoclast-related diseases. PMID:24333429

  19. Bacterial factors exploit eukaryotic Rho GTPase signaling cascades to promote invasion and proliferation within their host

    PubMed Central

    Popoff, Michel R

    2014-01-01

    Actin cytoskeleton is a main target of many bacterial pathogens. Among the multiple regulation steps of the actin cytoskeleton, bacterial factors interact preferentially with RhoGTPases. Pathogens secrete either toxins which diffuse in the surrounding environment, or directly inject virulence factors into target cells. Bacterial toxins, which interfere with RhoGTPases, and to some extent with RasGTPases, catalyze a covalent modification (ADPribosylation, glucosylation, deamidation, adenylation, proteolysis) blocking these molecules in their active or inactive state, resulting in alteration of epithelial and/or endothelial barriers, which contributes to dissemination of bacteria in the host. Injected bacterial virulence factors preferentially manipulate the RhoGTPase signaling cascade by mimicry of eukaryotic regulatory proteins leading to local actin cytoskeleton rearrangement, which mediates bacterial entry into host cells or in contrast escape to phagocytosis and immune defense. Invasive bacteria can also manipulate RhoGTPase signaling through recognition and stimulation of cell surface receptor(s). Changes in RhoGTPase activation state is sensed by the innate immunity pathways and allows the host cell to adapt an appropriate defense response. PMID:25203748

  20. 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

  1. Function of RSKS-1-AAK-2-DAF-16 signaling cascade in enhancing toxicity of multi-walled carbon nanotubes can be suppressed by mir-259 activation in Caenorhabditis elegans

    PubMed Central

    Zhuang, Ziheng; Li, Min; Liu, Hui; Luo, Libo; Gu, Weidong; Wu, Qiuli; Wang, Dayong

    2016-01-01

    Caenorhabditis elegans is an important non-mammalian alternative assay model for toxicological study. Previous study has indicated that exposure to multi-walled carbon nanotubes (MWCNTs) dysregulated the transcriptional expression of mir-259. In this study, we examined the molecular basis for mir-259 in regulating MWCNTs toxicity in nematodes. Mutation of mir-259 induced a susceptible property to MWCNTs toxicity, and MWCNTs exposure induced a significant increase in mir-259::GFP in pharyngeal/intestinal valve and reproductive tract, implying that mir-259 might mediate a protection mechanisms for nematodes against MWCNTs toxicity. RSKS-1, a putative ribosomal protein S6 kinase, acted as the target for mir-259 in regulating MWCNTs toxicity, and mutation of rsks-1 suppressed the susceptible property of mir-259 mutant to MWCNTs toxicity. Moreover, mir-259 functioned in pharynx-intestinal valve and RSKS-1 functioned in pharynx to regulate MWCNTs toxicity. Furthermore, RSKS-1 regulated MWCNTs toxicity by suppressing the function of AAK-2-DAF-16 signaling cascade. Our results will strengthen our understanding the microRNAs mediated protection mechanisms for animals against the toxicity from certain nanomaterials. PMID:27573184

  2. Function of RSKS-1-AAK-2-DAF-16 signaling cascade in enhancing toxicity of multi-walled carbon nanotubes can be suppressed by mir-259 activation in Caenorhabditis elegans.

    PubMed

    Zhuang, Ziheng; Li, Min; Liu, Hui; Luo, Libo; Gu, Weidong; Wu, Qiuli; Wang, Dayong

    2016-01-01

    Caenorhabditis elegans is an important non-mammalian alternative assay model for toxicological study. Previous study has indicated that exposure to multi-walled carbon nanotubes (MWCNTs) dysregulated the transcriptional expression of mir-259. In this study, we examined the molecular basis for mir-259 in regulating MWCNTs toxicity in nematodes. Mutation of mir-259 induced a susceptible property to MWCNTs toxicity, and MWCNTs exposure induced a significant increase in mir-259::GFP in pharyngeal/intestinal valve and reproductive tract, implying that mir-259 might mediate a protection mechanisms for nematodes against MWCNTs toxicity. RSKS-1, a putative ribosomal protein S6 kinase, acted as the target for mir-259 in regulating MWCNTs toxicity, and mutation of rsks-1 suppressed the susceptible property of mir-259 mutant to MWCNTs toxicity. Moreover, mir-259 functioned in pharynx-intestinal valve and RSKS-1 functioned in pharynx to regulate MWCNTs toxicity. Furthermore, RSKS-1 regulated MWCNTs toxicity by suppressing the function of AAK-2-DAF-16 signaling cascade. Our results will strengthen our understanding the microRNAs mediated protection mechanisms for animals against the toxicity from certain nanomaterials. PMID:27573184

  3. Text mining for metabolic pathways, signaling cascades, and protein networks.

    PubMed

    Hoffmann, Robert; Krallinger, Martin; Andres, Eduardo; Tamames, Javier; Blaschke, Christian; Valencia, Alfonso

    2005-05-10

    The complexity of the information stored in databases and publications on metabolic and signaling pathways, the high throughput of experimental data, and the growing number of publications make it imperative to provide systems to help the researcher navigate through these interrelated information resources. Text-mining methods have started to play a key role in the creation and maintenance of links between the information stored in biological databases and its original sources in the literature. These links will be extremely useful for database updating and curation, especially if a number of technical problems can be solved satisfactorily, including the identification of protein and gene names (entities in general) and the characterization of their types of interactions. The first generation of openly accessible text-mining systems, such as iHOP (Information Hyperlinked over Proteins), provides additional functions to facilitate the reconstruction of protein interaction networks, combine database and text information, and support the scientist in the formulation of novel hypotheses. The next challenge is the generation of comprehensive information regarding the general function of signaling pathways and protein interaction networks. PMID:15886388

  4. Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling.

    PubMed Central

    Denhardt, D T

    1996-01-01

    The features of three distinct protein phosphorylation cascades in mammalian cells are becoming clear. These signalling pathways link receptor-mediated events at the cell surface or intracellular perturbations such as DNA damage to changes in cytoskeletal structure, vesicle transport and altered transcription factor activity. The best known pathway, the Ras-->Raf-->MEK-->ERK cascade [where ERK is extracellular-signal-regulated kinase and MEK is mitogen-activated protein (MAP) kinase/ERK kinase], is typically stimulated strongly by mitogens and growth factors. The other two pathways, stimulated primarily by assorted cytokines, hormones and various forms of stress, predominantly utilize p21 proteins of the Rho family (Rho, Rac and CDC42), although Ras can also participate. Diagnostic of each pathway is the MAP kinase component, which is phosphorylated by a unique dual-specificity kinase on both tyrosine and threonine in one of three motifs (Thr-Glu-Tyr, Thr-Phe-Tyr or Thr-Gly-Tyr), depending upon the pathway. In addition to activating one or more protein phosphorylation cascades, the initiating stimulus may also mobilize a variety of other signalling molecules (e.g. protein kinase C isoforms, phospholipid kinases, G-protein alpha and beta gamma subunits, phospholipases, intracellular Ca2+). These various signals impact to a greater or lesser extent on multiple downstream effectors. Important concepts are that signal transmission often entails the targeted relocation of specific proteins in the cell, and the reversible formation of protein complexes by means of regulated protein phosphorylation. The signalling circuits may be completed by the phosphorylation of upstream effectors by downstream kinases, resulting in a modulation of the signal. Signalling is terminated and the components returned to the ground state largely by dephosphorylation. There is an indeterminant amount of cross-talk among the pathways, and many of the proteins in the pathways belong to families

  5. L-Lactate protects neurons against excitotoxicity: implication of an ATP-mediated signaling cascade.

    PubMed

    Jourdain, P; Allaman, I; Rothenfusser, K; Fiumelli, H; Marquet, P; Magistretti, P J

    2016-01-01

    Converging experimental data indicate a neuroprotective action of L-Lactate. Using Digital Holographic Microscopy, we observe that transient application of glutamate (100 μM; 2 min) elicits a NMDA-dependent death in 65% of mouse cortical neurons in culture. In the presence of L-Lactate (or Pyruvate), the percentage of neuronal death decreases to 32%. UK5099, a blocker of the Mitochondrial Pyruvate Carrier, fully prevents L-Lactate-mediated neuroprotection. In addition, L-Lactate-induced neuroprotection is not only inhibited by probenicid and carbenoxolone, two blockers of ATP channel pannexins, but also abolished by apyrase, an enzyme degrading ATP, suggesting that ATP produced by the Lactate/Pyruvate pathway is released to act on purinergic receptors in an autocrine/paracrine manner. Finally, pharmacological approaches support the involvement of the P2Y receptors associated to the PI3-kinase pathway, leading to activation of KATP channels. This set of results indicates that L-Lactate acts as a signalling molecule for neuroprotection against excitotoxicity through coordinated cellular pathways involving ATP production, release and activation of a P2Y/KATP cascade. PMID:26893204

  6. p38 MAPK and PI3K/AKT Signalling Cascades inParkinson’s Disease

    PubMed Central

    Jha, Saurabh Kumar; Jha, Niraj Kumar; Kar, Rohan; Ambasta, Rashmi K; Kumar, Pravir

    2015-01-01

    Parkinson's disease (PD) is a chronic neurodegenerative condition which has the second largest incidence rate among all other neurodegenerative disorders barring Alzheimer's disease (AD). Currently there is no cure and researchers continue to probe the therapeutic prospect in cell cultures and animal models of PD. Out of the several factors contributing to PD prognosis, the role of p38 MAPK (Mitogen activated protein-kinase) and PI3K/AKT signalling module in PD brains is crucial because the impaired balance between the pro- apoptotic and anti-apoptotic pathways trigger unwanted phenotypes such as microglia activation, neuroinflammation, oxidative stress and apoptosis. These factors continue challenging the brain homeostasis in initial stages thereby essentially assisting the dopaminergic (DA) neurons towards progressive degeneration in PD. Neurotherapeutics against PD shall then be targeted against the misregulated accomplices of the p38 and PI3K/AKT cascades. In this review, we have outlined many such established mechanisms involving the p38 MAPK and PI3K/AKT pathways which can offer therapeutic windows for the rectification of aberrant DA neuronal dynamics in PD brains. PMID:26261796

  7. L-Lactate protects neurons against excitotoxicity: implication of an ATP-mediated signaling cascade

    PubMed Central

    Jourdain, P.; Allaman, I.; Rothenfusser, K.; Fiumelli, H.; Marquet, P.; Magistretti, P. J.

    2016-01-01

    Converging experimental data indicate a neuroprotective action of L-Lactate. Using Digital Holographic Microscopy, we observe that transient application of glutamate (100 μM; 2 min) elicits a NMDA-dependent death in 65% of mouse cortical neurons in culture. In the presence of L-Lactate (or Pyruvate), the percentage of neuronal death decreases to 32%. UK5099, a blocker of the Mitochondrial Pyruvate Carrier, fully prevents L-Lactate-mediated neuroprotection. In addition, L-Lactate-induced neuroprotection is not only inhibited by probenicid and carbenoxolone, two blockers of ATP channel pannexins, but also abolished by apyrase, an enzyme degrading ATP, suggesting that ATP produced by the Lactate/Pyruvate pathway is released to act on purinergic receptors in an autocrine/paracrine manner. Finally, pharmacological approaches support the involvement of the P2Y receptors associated to the PI3-kinase pathway, leading to activation of KATP channels. This set of results indicates that L-Lactate acts as a signalling molecule for neuroprotection against excitotoxicity through coordinated cellular pathways involving ATP production, release and activation of a P2Y/KATP cascade. PMID:26893204

  8. Sensitive detection of Escherichia coli O157:H7 based on cascade signal amplification in ELISA.

    PubMed

    Shan, Shan; Liu, Daofeng; Guo, Qi; Wu, Songsong; Chen, Rui; Luo, Kai; Hu, Liming; Xiong, Yonghua; Lai, Weihua

    2016-09-01

    In this study, cascade signal amplification in ELISA involving double-antibody sandwich ELISA and indirectly competitive ELISA was established to sensitively detect Escherichia coli O157:H7. In the double-antibody sandwich ELISA, a complex was formed comprising anti-E. coli O157:H7 polyclonal antibody, E. coli O157:H7, biotinylated anti-E. coli O157:H7 monoclonal antibody, streptavidin, and biotinylated β-lactamase. Penicillin solution was then added into the ELISA well and hydrolyzed by β-lactamase. Afterward, the penicillin solution was transferred to indirectly competitive ELISA. The concentration of penicillin can be sensitively detected in indirectly competitive ELISA. In the cascade signal amplification system, increasing the amount of added E. coli O157:H7 resulted in more β-lactamase and less penicillin. The detection sensitivity of E. coli O157:H7, which was 20cfu/mL with the cascade signal amplification in ELISA, was 1,000-fold higher than that of traditional ELISA. Furthermore, the novel method can be used to detect E. coli O157:H7 in milk (2cfu/g). Therefore, this new signaling strategy will facilitate analyses of highly sensitive foodborne pathogens. PMID:27394946

  9. Non-redundant and complementary functions of adaptor proteins TRAF2 and TRAF3 in a ubiquitination cascade that activates NIK-dependent alternative NF-κB signaling

    PubMed Central

    Vallabhapurapu, Sivakumar; Matsuzawa, Atsushi; Zhang, WeiZhou; Tseng, Ping-Hui; Keats, Jonathan J.; Wang, Haopeng; Vignali, Dario A. A.; Bergsagel, P. Leif; Karin, Michael

    2009-01-01

    The adaptor and signaling proteins TRAF2, TRAF3 and cIAP1 and cIAP2 were suggested to inhibit alternative nuclear factor kappa B (NF-κB) signaling in resting cells by targeting NF-κB inducing kinase (NIK) to ubiquitin-dependent degradation, thus preventing processing of the NF-κB2 precursor protein p100 to release p52. However, the respective functions of TRAF2 and TRAF3 in NIK degradation and activation of alternative NF-κB signaling has remained elusive. We now show that CD40 or BAFF receptor activation resulted in TRAF3 degradation in a cIAP1-cIAP2- and TRAF2- dependent way due to enhanced cIAP1, cIAP2 TRAF3-directed ubiquitin ligase activity. Receptor-induced activation of cIAP1 and cIAP2 correlated with their K63-linked ubiquitination by TRAF2. Degradation of TRAF3 prevented association of NIK with the cIAP1-cIAP2-TRAF2 ubiquitin ligase complex, which resulted in NIK stabilization and NF-κB2-p100 processing. Constitutive activation of this pathway causes perinatal lethality and lymphoid defects. PMID:18997792

  10. Human Neuropeptide S Receptor Is Activated via a Gαq Protein-biased Signaling Cascade by a Human Neuropeptide S Analog Lacking the C-terminal 10 Residues.

    PubMed

    Liao, Yuan; Lu, Bin; Ma, Qiang; Wu, Gang; Lai, Xiangru; Zang, Jiashu; Shi, Ying; Liu, Dongxiang; Han, Feng; Zhou, Naiming

    2016-04-01

    Human neuropeptide S (NPS) and its cognate receptor regulate important biological functions in the brain and have emerged as a future therapeutic target for treatment of a variety of neurological and psychiatric diseases. The human NPS (hNPS) receptor has been shown to dually couple to Gαs- and Gαq-dependent signaling pathways. The human NPS analog hNPS-(1-10), lacking 10 residues from the C terminus, has been shown to stimulate Ca(2+)mobilization in a manner comparable with full-length hNPSin vitrobut seems to fail to induce biological activityin vivo Here, results derived from a number of cell-based functional assays, including intracellular cAMP-response element (CRE)-driven luciferase activity, Ca(2+)mobilization, and ERK1/2 phosphorylation, show that hNPS-(1-10) preferentially activates Gαq-dependent Ca(2+)mobilization while exhibiting less activity in triggering Gαs-dependent CRE-driven luciferase activity. We further demonstrate that both Gαq- and Gαs-coupled signaling pathways contribute to full-length hNPS-mediated activation of ERK1/2, whereas hNPS-(1-10)-promoted ERK1/2 activation is completely inhibited by the Gαqinhibitor UBO-QIC but not by the PKA inhibitor H89. Moreover, the results of Ala-scanning mutagenesis of hNPS-(1-13) indicated that residues Lys(11)and Lys(12)are structurally crucial for the hNPS receptor to couple to Gαs-dependent signaling. In conclusion, our findings demonstrate that hNPS-(1-10) is a biased agonist favoring Gαq-dependent signaling. It may represent a valuable chemical probe for further investigation of the therapeutic potential of human NPS receptor-directed signalingin vivo. PMID:26865629

  11. Electrochemical immunoassay for thyroxine detection using cascade catalysis as signal amplified enhancer and multi-functionalized magnetic graphene sphere as signal tag.

    PubMed

    Han, Jing; Zhuo, Ying; Chai, Yaqin; Yu, Yanqing; Liao, Ni; Yuan, Ruo

    2013-08-01

    This paper constructed a reusable electrochemical immunosensor for the detection of thyroxine at an ultralow concentration using cascade catalysis of cytochrome c (Cyt c) and glucose oxidase (GOx) as signal amplified enhancer. It is worth pointing out that numerous Cyt c and GOx were firstly carried onto the double-stranded DNA polymers based on hybridization chain reaction (HCR), and then the amplified responses could be achieved by cascade catalysis of Cyt c and GOx recycling with the help of glucose. Moreover, multi-functionalized magnetic graphene sphere was synthesized and used as signal tag, which not only exhibited good mechanical properties, large surface area and an excellent electron transfer rate of graphene, but also possessed excellent redox activity and desirable magnetic property. With a sandwich-type immunoreaction, the proposed cascade catalysis amplification strategy could greatly enhance the sensitivity for the detection of thyroxine. Under the optimal conditions, the immunosensor showed a wide linear ranged from 0.05pg mL(-1) to 5ng mL(-1) and a low detection limit down to 15fg mL(-1). Importantly, the proposed method offers promise for reproducible and cost-effective analysis of biological samples. PMID:23870405

  12. Orexin-stimulated MAP kinase cascades are activated through multiple G-protein signalling pathways in human H295R adrenocortical cells: diverse roles for orexins A and B.

    PubMed

    Ramanjaneya, Manjunath; Conner, Alex C; Chen, Jing; Kumar, Prashanth; Brown, James E P; Jöhren, Olaf; Lehnert, Hendrik; Stanfield, Peter R; Randeva, Harpal S

    2009-08-01

    Orexins A and B (ORA and ORB) are neuropeptide hormones found throughout the central nervous system and periphery. They are required for a host of physiological processes including mitogen-activated protein kinase (MAPK) regulation, steroidogenesis, appetite control and energy regulation. While some signalling mechanisms have been proposed for individual recombinant orexin receptors in generic mammalian cell types, it is clear that the peripheral effects of orexin are spatially and temporally complex. This study dissects the different G-protein signalling and MAPK pathways activated in a pluripotent human adrenal H295R cell line capable of all the physiological steps involved in steroidogenesis. Both extracellular receptor kinase 1/2 (ERK1/2) and p38 were phosphorylated rapidly with a subsequent decline, in a time- and dose-dependent manner, in response to both ORA and ORB. Conversely, there was little or no direct activation of the ERK5 or JNK pathway. Analysis using signalling and MAPK inhibitors as well as receptor-specific antagonists determined the precise mediators of the orexin response in these cells. Both ERK1/2 and p38 activation were predominantly G(q)- and to a lesser extent G(s)-mediated; p38 activation even had a small G(i)-component. Effects were broadly comparable for both orexin sub-types ORA and ORB and although most of the effects were transmitted through the orexin receptor-1 subtype, we did observe a role for orexin receptor-2-mediated activation of both ERK1/2 and p38. Cortisol secretion also differed in response to ORA and ORB. These data suggest multiple roles for orexin-mediated MAPK activation in an adrenal cell-line, this complexity may help to explain the diverse biological actions of orexins with wide-ranging consequences for our understanding of the mechanisms initiated by these steroidogenic molecules. PMID:19460850

  13. Loss of mesenchymal bone morphogenetic protein signaling leads to development of reactive stroma and initiation of the gastric neoplastic cascade.

    PubMed

    Roy, Sébastien A B; Allaire, Joannie M; Ouellet, Camille; Maloum-Rami, Faiza; Pomerleau, Véronique; Lemieux, Étienne; Babeu, Jean-Philippe; Rousseau, Jasmin; Paquet, Marilène; Garde-Granger, Perrine; Boudreau, François; Perreault, Nathalie

    2016-01-01

    Bmps are morphogens involved in various gastric cellular functions. Studies in genetically-modified mice have shown that Bmp disruption in gastric epithelial and stromal cell compartments leads to the development of tumorigenesis. Our studies have demonstrated that abrogation of gastric epithelial Bmp signaling alone was not sufficient to recapitulate the neoplastic features associated with total gastric loss of Bmp signaling. Thus, epithelial Bmp signaling does not appear to be a key player in gastric tumorigenesis initiation. These observations suggest a greater role for stromal Bmp signaling in gastric polyposis initiation. In order to identify the specific roles played by mesenchymal Bmp signaling in gastric homeostasis, we generated a mouse model with abrogation of Bmp signaling exclusively in the gastro-intestinal mesenchyme (Bmpr1a(ΔMES)). We were able to expose an unsuspected role for Bmp loss of signaling in leading normal gastric mesenchyme to adapt into reactive mesenchyme. An increase in the population of activated-fibroblasts, suggesting mesenchymal transdifferentiation, was observed in mutant stomach. Bmpr1a(ΔMES) stomachs exhibited spontaneous benign polyps with presence of both intestinal metaplasia and spasmolytic-polypeptide-expressing metaplasia as early as 90 days postnatal. These results support the novel concept that loss of mesenchymal Bmp signaling cascade acts as a trigger in gastric polyposis initiation. PMID:27609464

  14. Loss of mesenchymal bone morphogenetic protein signaling leads to development of reactive stroma and initiation of the gastric neoplastic cascade

    PubMed Central

    Roy, Sébastien A. B.; Allaire, Joannie M.; Ouellet, Camille; Maloum-Rami, Faiza; Pomerleau, Véronique; Lemieux, Étienne; Babeu, Jean-Philippe; Rousseau, Jasmin; Paquet, Marilène; Garde-Granger, Perrine; Boudreau, François; Perreault, Nathalie

    2016-01-01

    Bmps are morphogens involved in various gastric cellular functions. Studies in genetically-modified mice have shown that Bmp disruption in gastric epithelial and stromal cell compartments leads to the development of tumorigenesis. Our studies have demonstrated that abrogation of gastric epithelial Bmp signaling alone was not sufficient to recapitulate the neoplastic features associated with total gastric loss of Bmp signaling. Thus, epithelial Bmp signaling does not appear to be a key player in gastric tumorigenesis initiation. These observations suggest a greater role for stromal Bmp signaling in gastric polyposis initiation. In order to identify the specific roles played by mesenchymal Bmp signaling in gastric homeostasis, we generated a mouse model with abrogation of Bmp signaling exclusively in the gastro-intestinal mesenchyme (Bmpr1aΔMES). We were able to expose an unsuspected role for Bmp loss of signaling in leading normal gastric mesenchyme to adapt into reactive mesenchyme. An increase in the population of activated-fibroblasts, suggesting mesenchymal transdifferentiation, was observed in mutant stomach. Bmpr1aΔMES stomachs exhibited spontaneous benign polyps with presence of both intestinal metaplasia and spasmolytic-polypeptide-expressing metaplasia as early as 90 days postnatal. These results support the novel concept that loss of mesenchymal Bmp signaling cascade acts as a trigger in gastric polyposis initiation. PMID:27609464

  15. 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

  16. Transcription factors and cognate signalling cascades in the regulation of autophagy.

    PubMed

    Chandra, Vemika; Bhagyaraj, Ella; Parkesh, Raman; Gupta, Pawan

    2016-05-01

    Autophagy is a process that maintains the equilibrium between biosynthesis and the recycling of cellular constituents; it is critical for avoiding the pathophysiology that results from imbalance in cellular homeostasis. Recent reports indicate the need for the design of high-throughput screening assays to identify targets and small molecules for autophagy modulation. For such screening, however, a better understanding of the regulation of autophagy is essential. In addition to regulation by various signalling cascades, regulation of gene expression by transcription factors is also critical. This review focuses on the various transcription factors as well as the corresponding signalling molecules that act together to translate the stimuli to effector molecules that up- or downregulate autophagy. This review rationalizes the importance of these transcription factors functioning in tandem with cognate signalling molecules and their interfaces as possible therapeutic targets for more specific pharmacological interventions. PMID:25651938

  17. Specific Activation of the G Protein-coupled Receptor BNGR-A21 by the Neuropeptide Corazonin from the Silkworm, Bombyx mori, Dually Couples to the Gq and Gs Signaling Cascades*

    PubMed Central

    Yang, Jingwen; Huang, Haishan; Yang, Huipeng; He, Xiaobai; Jiang, Xue; Shi, Ying; Alatangaole, Damirin; Shi, Liangen; Zhou, Naiming

    2013-01-01

    Corazonin, an undecapeptide neurohormone sharing a highly conserved amino acid sequence across Insecta, plays different physiological roles in the regulation of heart contraction rates, silk spinning rates, the induction of dark color and morphometric phase changes, and ecdysis. Corazonin receptors have been identified in Drosophila melanogaster, Manduca sexta, and Musca domestica. However, detailed information on the signaling and major physiological functions of corazonin and its receptor is largely unknown. In the current study, using both the mammalian cell line HEK293 and insect cell lines BmN and Sf21, we paired the Bombyx corazonin neuropeptide as a specific endogenous ligand for the Bombyx neuropeptide G protein-coupled receptor A21 (BNGR-A21), and we therefore designated this receptor as BmCrzR. Further characterization indicated that synthetic BmCrz demonstrated a high affinity for and activated BmCrzR, resulting in intracellular cAMP accumulation, Ca2+ mobilization, and ERK1/2 phosphorylation via the Gq- and Gs-coupled signaling pathways. The direct interaction of BmCrzR with BmCrz was confirmed by a rhodamine-labeled BmCrz peptide. Moreover, experiments with double-stranded RNA and synthetic peptide injection suggested a possible role of BmCrz/BmCrzR in the regulation of larval growth and spinning rate. Our present results provide the first in-depth information on BmCrzR-mediated signaling for further elucidation of the BmCrz/BmCrzR system in the regulation of fundamental physiological processes. PMID:23457297

  18. Cascade signal amplification strategy for subattomolar protein detection by rolling circle amplification and quantum dots tagging.

    PubMed

    Cheng, Wei; Yan, Feng; Ding, Lin; Ju, Huangxian; Yin, Yibing

    2010-04-15

    A cascade signal amplification strategy was proposed for detection of protein target at ultralow concentration by combining the rolling circle amplification (RCA) technique with oligonucleotide functionalized quantum dots (QDs), multiplex binding of the biotin-strepavidin system, and anodic stripping voltammetric detection. The RCA product containing tandem-repeat sequences could serve as excellent template for periodic assembly of QDs, which presented per protein recognition event to numerous quantum dot tags for electrochemical readout. Both the RCA and the multiplex binding system showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. Using human vascular endothelial growth factor as a model protein, the designed strategy could quantitatively detect protein down to 16 molecules in a 100 microL sample with a linear calibration range from 1 aM to 1 pM and was amenable to quantification of protein target in complex biological matrixes. The proposed cascade signal amplification strategy would become a powerful tool for proteomics research and clinical diagnostics. PMID:20345087

  19. Quantum dots as active material for quantum cascade lasers: comparison to quantum wells

    NASA Astrophysics Data System (ADS)

    Michael, Stephan; Chow, Weng W.; Schneider, Hans Christian

    2016-03-01

    We review a microscopic laser theory for quantum dots as active material for quantum cascade lasers, in which carrier collisions are treated at the level of quantum kinetic equations. The computed characteristics of such a quantum-dot active material are compared to a state-of-the-art quantum-well quantum cascade laser. We find that the current requirement to achieve a comparable gain-length product is reduced compared to that of the quantum-well quantum cascade laser.

  20. On the modified active region design of interband cascade lasers

    SciTech Connect

    Motyka, M.; Ryczko, K.; Dyksik, M.; Sęk, G.; Misiewicz, J.; Weih, R.; Dallner, M.; Kamp, M.; Höfling, S.

    2015-02-28

    Type II InAs/GaInSb quantum wells (QWs) grown on GaSb or InAs substrates and designed to be integrated in the active region of interband cascade lasers (ICLs) emitting in the mid infrared have been investigated. Optical spectroscopy, combined with band structure calculations, has been used to probe their electronic properties. A design with multiple InAs QWs has been compared with the more common double W-shaped QW and it has been demonstrated that it allows red shifting the emission wavelength and enhancing the transition oscillator strength. This can be beneficial for the improvements of the ICLs performances, especially when considering their long-wavelength operation.

  1. Bipolar disorder: involvement of signaling cascades and AMPA receptor trafficking at synapses

    PubMed Central

    DU, JING; QUIROZ, JORGE; YUAN, PEIXIONG; ZARATE, CARLOS; MANJI, HUSSEINI K.

    2009-01-01

    There is increasing evidence that severe mood disorders are associated with impairment of structural plasticity and cellular resilience. Cumulative data demonstrate that mood stabilizers regulate intracellular signaling cascades, including protein kinase C (PKC), PKA, mitogen-activated protein (MAP) kinase, glycogen synthase kinase 3-β (GSK3-β) and intracellular calcium, which are signaling pathways that regulate synaptic plasticity. In this context, it is noteworthy that a growing body of data indicates that the glutamatergic system, has a major role in neuronal plasticity and cellular resilience, might be involved in the pathophysiology and treatment of mood disorders. AMPA glutamate-receptor trafficking is important in synaptic plasticity and might play crucial roles in maintaining critical neuronal circuits associated with mood. Two clinically effective, structurally dissimilar, antimanic agents, lithium and valproate (VPA), down-regulate synaptic expression of AMPA receptor subunit GluR1 in hippocampus in chronically treated rats. This reduction in synaptic GluR1 by lithium and VPA is due to attenuated phosphorylation of GluR1 at a specific PKA site (residue 845 of GluR1), which is crucial for AMPA receptor insertion. By contrast, imipramine, which can provoke mania, increases synaptic expression of GluR1 in the hippocampus in vivo. Furthermore, there is ample evidence from preclinical and clinical research that the glutamatergic system is involved in the pathophysiology of mood disorders and that many of the somatic treatments used for mood disorders including antidepressants, mood stabilizers, atypical antipsychotic drugs and electroconvulsive therapy have both direct and indirect effects on the glutamatergic system. Given these findings, further research with medications that specifically affect the glutamatergic system is warranted. Recent studies in our lab have shown that riluzole, a FDA approved medicine that regulates the glutamatergic system, shows

  2. Lovastatin causes FaDu hypopharyngeal carcinoma cell death via AMPK-p63-survivin signaling cascade

    PubMed Central

    Yen, Chia-Sheng; Chen, Jung-Chien; Chang, Yi-Fang; Hsu, Ya-Fen; Chiu, Pei-Ting; Shiue, Ching; Chuang, Yu-Fan; Ou, George; Hsu, Ming-Jen

    2016-01-01

    Statins are used widely to lower serum cholesterol and the incidence of cardiovascular diseases. Growing evidence shows that statins also exhibit beneficial effects against cancers. In this study, we investigated the molecular mechanisms involved in lovastatin-induced cell death in Fadu hypopharyngeal carcinoma cells. Lovastatin caused cell cycle arrest and apoptosis in FaDu cells. Lovastatin increased p21cip/Waf1 level while the survivin level was decreased in the presence of lovastatin. Survivin siRNA reduced cell viability and induced cell apoptosis in FaDu cells. Lovastatin induced phosphorylation of AMP-activated protein kinase (AMPK), p38 mitogen-activated protein kinase (MAPK) and transcription factor p63. Lovastatin also caused p63 acetylation and increased p63 binding to survivin promoter region in FaDu cells. AMPK-p38MAPK signaling blockade abrogated lovastatin-induced p63 phosphorylation. Lovastatin’s enhancing effect on p63 acetylation was reduced in HDAC3- or HDAC4- transfected cells. Moreover, transfection of cells with AMPK dominant negative mutant (AMPK-DN), HDAC3, HDAC4 or p63 siRNA significantly reduced lovastatin’s effects on p21cip/Waf1 and survivin. Furthermore, lovastatin inhibited subcutaneous FaDu xenografts growth in vivo. Taken together, lovastatin may activate AMPK-p38MAPK-p63-survivin cascade to cause FaDu cell death. This study establishes, at least in part, the signaling cascade by which lovastatin induces hypopharyngeal carcinoma cell death. PMID:27122225

  3. The role of mitogen-activated protein kinase in insulin and insulin-like growth factor I (IGF-I) signaling cascades for progesterone and IGF-binding protein-1 production in human granulosa cells.

    PubMed

    Seto-Young, Donna; Zajac, Jacek; Liu, Hung-Ching; Rosenwaks, Zev; Poretsky, Leonid

    2003-07-01

    Insulin and IGF-I participate in the regulation of ovulation, steroidogenesis, and IGF-binding protein (IGFBP) production in the ovary. Insulin and IGF-I actions in the ovary are closely related. For example, insulin may amplify IGF-I action in the ovary by up-regulating type I IGF receptors and inhibiting IGFBP-1 production, thus increasing the bioavailability of IGF-I. It is hypothesized that ovarian effects of insulin in insulin-resistant states are mediated via an insulin action pathway(s) distinct from those involved in glucose transport. We previously reported that insulin-induced stimulation of progesterone and inhibition of IGFBP-1 production in the human ovary are mediated by signaling pathways that are independent of phosphatidylinositol 3-kinase, the enzyme whose activation is crucial for glucose transport. We now examined whether activation of MAPK is necessary to mediate insulin-induced or IGF-I-induced stimulation of progesterone or inhibition of IGFBP-1 production in human granulosa cells. Human granulosa cells were obtained during in vitro fertilization. Cells (0.5-1 x 10(5)) were incubated for 24 h in the presence of 0, 10, 10(2), or 10(3) ng/ml insulin or 0, 0.5, 1, 2.5, or 5 ng/ml IGF-I and in the presence or absence of 1 micro M PD98059, a specific inhibitor of ERK1/2 MAPK. The progesterone concentration in the tissue culture medium was measured by RIA (Pantex, Santa Monica, CA), and the IGFBP-1 concentration was measured by immunoradiometric assay (DSL-7800, Diagnostic Systems Laboratories, Inc., Webster, TX). MAPK activity was assessed using the MAPK IP-Kinase assay kit (Upstate Biotechnology, Inc., Lake Placid, NY). ANOVA was used to compare mean values of progesterone or IGFBP-1 concentrations. MAPK was stimulated by insulin up to 350% of the baseline value. Progesterone production in human granulosa cells was stimulated by insulin in a dose-related manner to 123% of the control value (P < 0.001), and IGFBP-1 production was inhibited to 25

  4. A CaMK cascade activates CRE-mediated transcription in neurons of Caenorhabditis elegans

    PubMed Central

    Kimura, Yoshishige; Corcoran, Ethan E.; Eto, Koh; Gengyo-Ando, Keiko; Muramatsu, Masa-aki; Kobayashi, Ryoji; Freedman, Jonathan H.; Mitani, Shohei; Hagiwara, Masatoshi; Means, Anthony R.; Tokumitsu, Hiroshi

    2002-01-01

    Calcium (Ca2+) signals regulate a diverse set of cellular responses, from proliferation to muscular contraction and neuro-endocrine secretion. The ubiquitous Ca2+ sensor, calmodulin (CaM), translates changes in local intracellular Ca2+ concentrations into changes in enzyme activities. Among its targets, the Ca2+/CaM-dependent protein kinases I and IV (CaMKs) are capable of transducing intraneuronal signals, and these kinases are implicated in neuronal gene regulation that mediates synaptic plasticity in mammals. Recently, the cyclic AMP response element binding protein (CREB) has been proposed as a target for a CaMK cascade involving not only CaMKI or CaMKIV, but also an upstream kinase kinase that is also CaM regulated (CaMKK). Here, we report that all components of this pathway are coexpressed in head neurons of Caenorhabditis elegans. Utilizing a transgenic approach to visualize CREB-dependent transcription in vivo, we show that this CaMK cascade regulates CRE-mediated transcription in a subset of head neurons in living nematodes. PMID:12231504

  5. Abrogation of STAT3 signaling cascade by zerumbone inhibits proliferation and induces apoptosis in renal cell carcinoma xenograft mouse model.

    PubMed

    Shanmugam, Muthu K; Rajendran, Peramaiyan; Li, Feng; Kim, Chulwon; Sikka, Sakshi; Siveen, Kodappully Sivaraman; Kumar, Alan Prem; Ahn, Kwang Seok; Sethi, Gautam

    2015-10-01

    Persistent activation of signal transducer and activator of transcription 3 (STAT3) is one of the characteristic features of renal cell carcinoma (RCC) and often linked to its deregulated proliferation, survival, and angiogenesis. In the present report, we investigated whether zerumbone, a sesquiterpene, exerts its anticancer effect through modulation of STAT3 activation pathway. The pharmacological effect of zerumbone on STAT3 activation, associated protein kinases and phosphatase, and apoptosis was investigated using both RCC cell lines and xenograft mouse model. We observed that zerumbone suppressed STAT3 activation in a dose- and time-dependent manner in RCC cells. The suppression was mediated through the inhibition of activation of upstream kinases c-Src, Janus-activated kinase 1, and Janus-activated kinase 2. Pervanadate treatment reversed zerumbone-induced downregulation of STAT3, suggesting the involvement of a tyrosine phosphatase. Indeed, we found that zerumbone induced the expression of tyrosine phosphatase SHP-1 that correlated with its ability to inhibit STAT3 activation. Interestingly, deletion of SHP-1 gene by siRNA abolished the ability of zerumbone to inhibit STAT3 activation. The inhibition of STAT3 activation by zerumbone also caused the suppression of the gene products involved in proliferation, survival, and angiogenesis. Finally, when administered i.p., zerumbone inhibited STAT3 activation in tumor tissues and the growth of human RCC xenograft tumors in athymic nu/nu mice without any side effects. Overall, our results suggest for the first time that zerumbone is a novel blocker of STAT3 signaling cascade and thus has an enormous potential for the treatment of RCC and other solid tumors. PMID:24797723

  6. FUS3 phosphorylates multiple components of the mating signal transduction cascade: evidence for STE12 and FAR1.

    PubMed Central

    Elion, E A; Satterberg, B; Kranz, J E

    1993-01-01

    The mitogen-activated protein (MAP) kinase homologue FUS3 mediates both transcription and G1 arrest in a pheromone-induced signal transduction cascade in Saccharomyces cerevisiae. We report an in vitro kinase assay for FUS3 and its use in identifying candidate substrates. The assay requires catalytically active FUS3 and pheromone induction. STE7, a MAP kinase kinase homologue, is needed for maximal activity. At least seven proteins that specifically associate with FUS3 are phosphorylated in the assay. Many of these substrates are physiologically relevant and are affected by in vivo levels of numerous signal transduction components. One substrate is likely to be the transcription factor STE12. A second is likely to be FAR1, a protein required for G1 arrest. FAR1 was isolated as a multicopy suppressor of a nonarresting fus3 mutant and interacts with FUS3 in a two hybrid system. Consistent with this FAR1 is a good substrate in vitro and generates a FUS3-associated substrate of expected size. These data support a model in which FUS3 mediates transcription and G1 arrest by direct activation of STE12 and FAR1 and phosphorylates many other proteins involved in the response to pheromone. Images PMID:8334305

  7. Photoinduced cytotoxicity of a photochromic diarylethene via caspase cascade activation.

    PubMed

    Okuda, Jun-ya; Tanaka, Yukimi; Kodama, Ryuhei; Sumaru, Kimio; Morishita, Kana; Kanamori, Toshiyuki; Yamazoe, Seiji; Hyodo, Kengo; Yamazaki, Shohei; Miyatake, Tomohiro; Yokojima, Satoshi; Nakamura, Sinichiro; Uchida, Kingo

    2015-07-11

    The photo-generated closed-ring isomer of bis(5-methyl-2-phenylthiazoyl)perfluorocyclopentene shows cytotoxicity to Madin-Darby canine kidney (MDCK) cells through a caspase cascade and induces apoptosis of cells. PMID:26063471

  8. Mitogen-Activated Protein Kinase Cascade MKK7-MPK6 Plays Important Roles in Plant Development and Regulates Shoot Branching by Phosphorylating PIN1 in Arabidopsis.

    PubMed

    Jia, Weiyan; Li, Baohua; Li, Shujia; Liang, Yan; Wu, Xiaowei; Ma, Mei; Wang, Jiyao; Gao, Jin; Cai, Yueyue; Zhang, Yuanya; Wang, Yingchun; Li, Jiayang; Wang, Yonghong

    2016-09-01

    Emerging evidences exhibit that mitogen-activated protein kinase (MAPK/MPK) signaling pathways are connected with many aspects of plant development. The complexity of MAPK cascades raises challenges not only to identify the MAPK module in planta but also to define the specific role of an individual module. So far, our knowledge of MAPK signaling has been largely restricted to a small subset of MAPK cascades. Our previous study has characterized an Arabidopsis bushy and dwarf1 (bud1) mutant, in which the MAP Kinase Kinase 7 (MKK7) was constitutively activated, resulting in multiple phenotypic alterations. In this study, we found that MPK3 and MPK6 are the substrates for phosphorylation by MKK7 in planta. Genetic analysis showed that MKK7-MPK6 cascade is specifically responsible for the regulation of shoot branching, hypocotyl gravitropism, filament elongation, and lateral root formation, while MKK7-MPK3 cascade is mainly involved in leaf morphology. We further demonstrated that the MKK7-MPK6 cascade controls shoot branching by phosphorylating Ser 337 on PIN1, which affects the basal localization of PIN1 in xylem parenchyma cells and polar auxin transport in the primary stem. Our results not only specify the functions of the MKK7-MPK6 cascade but also reveal a novel mechanism for PIN1 phosphorylation, establishing a molecular link between the MAPK cascade and auxin-regulated plant development. PMID:27618482

  9. The regulatory cascade that activates the Hrp regulon in Erwinia herbicola pv. gypsophilae.

    PubMed

    Nizan-Koren, R; Manulis, S; Mor, H; Iraki, N M; Barash, I

    2003-03-01

    The pathogenicity of Erwinia herbicola pv. gypsophilae (Ehg) is dependent on a plasmid (pPATH(Ehg)) that harbors the hrp gene cluster and additional virulence genes. The hrp regulatory cascade of Ehg comprises an hrpXY operon encoding a two-component system; hrpS encoding a transcriptional factor of the NtrC family and hrpL encoding an alternative sigma factor. Results obtained suggest the following signal transduction model for activating the Hrp regulon: phosphorylated HrpY activates hrpS, HrpS activates hrpL, and HrpL activates genes containing "hrp box" promoter. This model was supported by studies on the effects of mutations in the regulatory genes on pathogenicity and complementation analysis. Nonpolar mutations in hrpX did not affect virulence or transcription of downstream genes. Site-directed mutagenesis of the conserved aspartate 57 in HrpY suggested that its phosphorylation is crucial for activating the hrp regulatory cascade. Studies on the effects of mutations in the hrp regulatory genes on transcriptional activity of downstream genes or of their isolated promoters in planta showed dependency of hrpS expression on active HrpY, of hrpL expression on active HrpS, and of hrpN or hrpJ expression on active HrpL. These results were also partially supported by overexpression of regulatory genes under in vitro conditions. The hrpXY is constitutively expressed with high basal levels under repressive conditions, in contrast to hrpS and hrpL, which exhibit low basal expression levels and are environmentally regulated. PMID:12650456

  10. Optical Properties of Active Regions in Terahertz Quantum Cascade Lasers

    NASA Astrophysics Data System (ADS)

    Dyksik, M.; Motyka, M.; Rudno-Rudziński, W.; Sęk, G.; Misiewicz, J.; Pucicki, D.; Kosiel, K.; Sankowska, I.; Kubacka-Traczyk, J.; Bugajski, M.

    2016-07-01

    In this work, AlGaAs/GaAs superlattice, with layers' sequence and compositions imitating the active and injector regions of a quantum cascade laser designed for emission in the terahertz spectral range, was investigated. Three independent absorption-like optical spectroscopy techniques were employed in order to study the band structure of the minibands formed within the conduction band. Photoreflectance measurements provided information about interband transitions in the investigated system. Common transmission spectra revealed, in the target range of intraband transitions, mainly a number of lines associated with the phonon-related processes, including two-phonon absorption. In contrast, differential transmittance realized by means of Fourier-transform spectroscopy was utilized to probe the confined states of the conduction band. The obtained energy separation between the second and third confined electron levels, expected to be predominantly contributing to the lasing, was found to be ~9 meV. The optical spectroscopy measurements were supported by numerical calculations performed in the effective mass approximation and XRD measurements for layers' width verification. The calculated energy spacings are in a good agreement with the experimental values.

  11. Signaling cascades and the importance of moonlight in coral broadcast mass spawning.

    PubMed

    Kaniewska, Paulina; Alon, Shahar; Karako-Lampert, Sarit; Hoegh-Guldberg, Ove; Levy, Oren

    2015-01-01

    Many reef-building corals participate in a mass-spawning event that occurs yearly on the Great Barrier Reef. This coral reproductive event is one of earth's most prominent examples of synchronised behavior, and coral reproductive success is vital to the persistence of coral reef ecosystems. Although several environmental cues have been implicated in the timing of mass spawning, the specific sensory cues that function together with endogenous clock mechanisms to ensure accurate timing of gamete release are largely unknown. Here, we show that moonlight is an important external stimulus for mass spawning synchrony and describe the potential mechanisms underlying the ability of corals to detect environmental triggers for the signaling cascades that ultimately result in gamete release. Our study increases the understanding of reproductive chronobiology in corals and strongly supports the hypothesis that coral gamete release is achieved by a complex array of potential neurohormones and light-sensing molecules. PMID:26668113

  12. 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.

  13. Light-Dependent Translocation of Arrestin in Rod Photoreceptors is Signaled Through a Phospholipase C Cascade and Requires ATP

    PubMed Central

    Orisme, Wilda; Li, Jian; Goldmann, Tobias; Bolch, Susan; Wolfrum, Uwe; Smith, W. Clay

    2009-01-01

    Partitioning of cellular components is a critical mechanism by which cells can regulate their activity. In rod photoreceptors, light induces a large-scale translocation of arrestin from the inner segments to the outer segments. The purpose of this project is to elucidate the signaling pathway necessary to initiate arrestin translocation to the outer segments and the mechanism for arrestin translocation. Mouse retinal organotypic cultures and eyes from transgenic Xenopus tadpoles expressing a fusion of GFP and rod arrestin were treated with both activators and inhibitors of proteins in the phosphoinositide pathway. Confocal microscopy was used to image the effects of the pharmacological agents on arrestin translocation in rod photoreceptors. Retinas were also depleted of ATP using potassium cyanide to assess the requirement for ATP in arrestin translocation. In this study, we demonstrate that components of the G-protein-linked phospholipase C (PLC) pathway play a role in initiating arrestin translocation. Our results show that arrestin translocation can be stimulated by activators of PLC and protein kinase C (PKC), and by cholera toxin in the absence of light. Arrestin translocation to the outer segments is significantly reduced by inhibitors of PLC and PKC. Importantly, we find that treatment with potassium cyanide inhibits arrestin translocation in response to light. Collectively, our results suggest that arrestin translocation is initiated by a G-protein-coupled cascade through PLC and PKC signaling. Furthermore, our results demonstrate that at least the initiation of arrestin translocation requires energy input. PMID:19887106

  14. Protopanaxatirol type ginsenoside Re promotes cyclic growth of hair follicles via inhibiting transforming growth factor β signaling cascades.

    PubMed

    Li, Zheng; Ryu, Seung-Wook; Lee, Jungsul; Choi, Kyungsun; Kim, Sunchang; Choi, Chulhee

    2016-02-19

    Ginsenosides, the major bio-active ingredients included in Panax ginseng, have been known for the hair growth activity and used to treat patients who suffer from hair loss; however, the detailed mechanisms of this action are still largely unknown. This study was conducted to investigate the molecular and cellular mechanisms responsible for hair growth promoting effect of ginsenoside Re (GRe) in vitro and in vivo. Different doses of minoxidil and GRe were administered topically to the back regions of nude mice for up to 45 days, and hair shaft length and hair cycles were determined for hair promoting activities. Topical treatment of GRe significantly increased the hair shaft length and hair existent time, which was comparable to the action of minoxidil. We also demonstrated that GRe stimulated hair shaft elongation in the ex vivo cultures of vibrissa hair follicles isolated from C57BL/6 mouse. Systemic transcriptome analysis by next generation sequencing demonstrated that TGF-β-pathway related genes were selectively down-regulated by treatment of GRe in vivo, and the same treatment suppressed TGF-β-induced phosphorylation of ERK in HeLa cells. The results clearly indicated that GRe is the effective constituent in the ginseng on hair promotion via selective inhibition of the hair growth phase transition related signaling pathways, TGF-β signaling cascades. PMID:26820528

  15. FIA functions as an early signal component of abscisic acid signal cascade in Vicia faba guard cells.

    PubMed

    Sugiyama, Yusuke; Uraji, Misugi; Watanabe-Sugimoto, Megumi; Okuma, Eiji; Munemasa, Shintaro; Shimoishi, Yasuaki; Nakamura, Yoshimasa; Mori, Izumi C; Iwai, Sumio; Murata, Yoshiyuki

    2012-02-01

    An abscisic acid (ABA)-insensitive Vicia faba mutant, fia (fava bean impaired in ABA-induced stomatal closure) had previously been isolated. In this study, it was investigated how FIA functions in ABA signalling in guard cells of Vicia faba. Unlike ABA, methyl jasmonate (MeJA), H(2)O(2), and nitric oxide (NO) induced stomatal closure in the fia mutant. ABA did not induce production of either reactive oxygen species or NO in the mutant. Moreover, ABA did not suppress inward-rectifying K(+) (K(in)) currents or activate ABA-activated protein kinase (AAPK) in mutant guard cells. These results suggest that FIA functions as an early signal component upstream of AAPK activation in ABA signalling but does not function in MeJA signalling in guard cells of Vicia faba. PMID:22131163

  16. A Co-Opted Hormonal Cascade Activates Dormant Adventitious Root Primordia upon Flooding in Solanum dulcamara.

    PubMed

    Dawood, Thikra; Yang, Xinping; Visser, Eric J W; Te Beek, Tim A H; Kensche, Philip R; Cristescu, Simona M; Lee, Sangseok; Floková, Kristýna; Nguyen, Duy; Mariani, Celestina; Rieu, Ivo

    2016-04-01

    Soil flooding is a common stress factor affecting plants. To sustain root function in the hypoxic environment, flooding-tolerant plants may form new, aerenchymatous adventitious roots (ARs), originating from preformed, dormant primordia on the stem. We investigated the signaling pathway behind AR primordium reactivation in the dicot species Solanum dulcamara Transcriptome analysis indicated that flooding imposes a state of quiescence on the stem tissue, while increasing cellular activity in the AR primordia. Flooding led to ethylene accumulation in the lower stem region and subsequently to a drop in abscisic acid (ABA) level in both stem and AR primordia tissue. Whereas ABA treatment prevented activation of AR primordia by flooding, inhibition of ABA synthesis was sufficient to activate them in absence of flooding. Together, this reveals that there is a highly tissue-specific response to reduced ABA levels. The central role for ABA in the response differentiates the pathway identified here from the AR emergence pathway known from rice (Oryza sativa). Flooding and ethylene treatment also induced expression of the polar auxin transporter PIN2, and silencing of this gene or chemical inhibition of auxin transport inhibited primordium activation, even though ABA levels were reduced. Auxin treatment, however, was not sufficient for AR emergence, indicating that the auxin pathway acts in parallel with the requirement for ABA reduction. In conclusion, adaptation of S. dulcamara to wet habitats involved co-option of a hormonal signaling cascade well known to regulate shoot growth responses, to direct a root developmental program upon soil flooding. PMID:26850278

  17. Modeling self-sustained activity cascades in socio-technical networks

    NASA Astrophysics Data System (ADS)

    Piedrahita, P.; Borge-Holthoefer, J.; Moreno, Y.; Arenas, A.

    2013-11-01

    The ability to understand and eventually predict the emergence of information and activation cascades in social networks is core to complex socio-technical systems research. However, the complexity of social interactions makes this a challenging enterprise. Previous works on cascade models assume that the emergence of this collective phenomenon is related to the activity observed in the local neighborhood of individuals, but do not consider what determines the willingness to spread information in a time-varying process. Here we present a mechanistic model that accounts for the temporal evolution of the individual state in a simplified setup. We model the activity of the individuals as a complex network of interacting integrate-and-fire oscillators. The model reproduces the statistical characteristics of the cascades in real systems, and provides a framework to study the time evolution of cascades in a state-dependent activity scenario.

  18. Ascochlorin, an isoprenoid antibiotic inhibits growth and invasion of hepatocellular carcinoma by targeting STAT3 signaling cascade through the induction of PIAS3.

    PubMed

    Dai, Xiaoyun; Ahn, Kwang Seok; Kim, Chulwon; Siveen, Kodappully Sivaraman; Ong, Tina H; Shanmugam, Muthu K; Li, Feng; Shi, Jizhong; Kumar, Alan Prem; Wang, Ling Zhi; Goh, Boon Cher; Magae, Junji; Hui, Kam M; Sethi, Gautam

    2015-04-01

    Deregulated activation of oncogenic transcription factors such as signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in proliferation and survival of hepatocellular carcinoma (HCC). Thus, agents which can inhibit STAT3 activation may have an enormous potential for treatment of HCC patients. Hence, in the present report, we investigated the effect of ascochlorin (ASC), an isoprenoid antibiotic on STAT3 activation cascade in various HCC cell lines and orthotopic mouse model. We observed that ASC could substantially inhibit both constitutive and IL-6/EGF inducible STAT3 activation as well as reduce its DNA binding ability. ASC increased the expression of protein inhibitor of activated STAT3 (PIAS3) which could bind to STAT3 DNA binding domain and thereby down-regulate STAT3 activation. Deletion of PIAS3 gene by siRNA abolished the ability of ASC to inhibit STAT3 activation and induce apoptosis in HCC cells. ASC also modulated the expression of diverse STAT3-regulated oncogenic gene products. Finally, when administered intraperitoneally, ASC also inhibited tumor growth in an orthotopic HCC mouse model and reduced STAT3 activation in tumor tissues. Overall our results indicate that ASC mediates its anti-tumor effects predominantly through the suppression of STAT3 signaling cascade, and can form the basis of novel therapy for HCC patients. PMID:25624051

  19. Participation of signaling cascades in the regulation of erythropoiesis under conditions of cytostatic treatment.

    PubMed

    Dygai, A M; Zhdanov, V V; Miroshnichenko, L A; Udut, E V; Zyuz'kov, G N; Simanina, E V; Chaikovskii, A V; Stavrova, L A; Trofimova, E S; Burmina, Ya V

    2015-01-01

    We studied the role of signaling pathways in the regulation of erythropoiesis against the background of myelosuppression caused by administration of 5-fluorouracil. The important role of cyclic AMP in the maturation of erythroid progenitors after cytostatic treatment was demonstrated. The secretory activity of myelokaryocytes during the period of erythroid hemopoiesis recovery is mainly regulated via the p38 MAPK signaling pathway; non-erythropoietin factors are involved in the formation of erythropoietic activity of adherent cells of the microenvironment. PMID:25578863

  20. Orphan Receptor GPR179 Forms Macromolecular Complexes With Components of Metabotropic Signaling Cascade in Retina ON-Bipolar Neurons

    PubMed Central

    Orlandi, Cesare; Cao, Yan; Martemyanov, Kirill A.

    2013-01-01

    Purpose. In the mammalian retina, synaptic transmission between light-excited rod photoreceptors and downstream ON-bipolar neurons is indispensable for dim vision, and disruption of this process leads to congenital stationary night blindness in human patients. The ON-bipolar neurons use the metabotropic signaling cascade, initiated by the mGluR6 receptor, to generate depolarizing responses to light-induced changes in neurotransmitter glutamate release from the photoreceptor axonal terminals. Evidence for the identity of the components involved in transducing these signals is growing rapidly. Recently, the orphan receptor, GPR179, a member of the G protein-coupled receptor (GPCR) superfamily, has been shown to be indispensable for the synaptic responses of ON-bipolar cells. In our study, we investigated the interaction of GPR179 with principle components of the signal transduction cascade. Methods. We used immunoprecipitation and proximity ligation assays in transfected cells and native retinas to characterize the protein–protein interactions involving GPR179. The influence of cascade components on GPR179 localization was examined through immunohistochemical staining of the retinas from genetic mouse models. Results. We demonstrated that, in mouse retinas, GPR179 forms physical complexes with the main components of the metabotropic cascade, recruiting mGluR6, TRPM1, and the RGS proteins. Elimination of mGluR6 or RGS proteins, but not TRPM1, detrimentally affects postsynaptic targeting or GPR179 expression. Conclusions. These observations suggest that the mGluR6 signaling cascade is scaffolded as a macromolecular complex in which the interactions between the components ensure the optimal spatiotemporal characteristics of signal transduction. PMID:24114537

  1. 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.

  2. Cigarette smoke exposure triggers the autophagic cascade via activation of the AMPK pathway in mice.

    PubMed

    Furlong, Hayley C; Stämpfli, Martin R; Gannon, Anne M; Foster, Warren G

    2015-10-01

    We previously demonstrated that cigarette smoke (CS) exposure decreases primordial follicle counts and induces autophagy in ovarian granulosa cells in preference to apoptosis. Therefore, the objective of this study was to investigate molecular targets underlying smoke-induced activation of the reparative autophagy pathway in the ovary. Briefly, ovarian homogenates were prepared from adult female mice exposed to mainstream CS twice daily for 8 wk, using a whole-body exposure system. A gene array revealed that CS exposure induced a greater than 2-fold significant increase in the expression of proautophagic genes Cdkn1b, Map1lc3a, Bad, and Sqstm1/p62. A significant increase in Prkaa2, Pik3c3, and Maplc31b expression, as well as a significant decrease in Akt1 and Mtor expression, was detected by quantitative PCR. The 5'-AMP-activated protein kinase catalytic subunit (AMPK) alpha1 + alpha2 and ATG7 protein expression was significantly increased, whereas AKT1, mTOR, CDKN1B/p27, and CXCR4 proteins were significantly decreased in CS exposed versus control ovaries. Up-regulation of AMPK alpha1 + alpha2, a known initiator of autophagic signaling, and ATG7 further suggests activation of the autophagy cascade. Two prosurvival factors, AKT and mTOR, were decreased in expression, an outcome that favors induction of the autophagy pathway, whereas decreased levels of CDKN1B is suggestive of cell cycle dysregulation. In summary, our data suggest that CS exposure induces ovarian follicle loss through induction of the autophagic cascade via the AMPK pathway together with inhibition of antiautophagic markers AKT and mTOR. We further postulate that toxicant-induced dysregulation of reparative autophagy is a novel pathway central to impaired follicle development and subfertility. PMID:26377221

  3. Human melanopsin forms a pigment maximally sensitive to blue light (λmax ≈ 479 nm) supporting activation of G(q/11) and G(i/o) signalling cascades.

    PubMed

    Bailes, Helena J; Lucas, Robert J

    2013-05-22

    A subset of mammalian retinal ganglion cells expresses an opsin photopigment (melanopsin, Opn4) and is intrinsically photosensitive. The human retina contains melanopsin, but the literature lacks a direct investigation of its spectral sensitivity or G-protein selectivity. Here, we address this deficit by studying physiological responses driven by human melanopsin under heterologous expression in HEK293 cells. Luminescent reporters for common second messenger systems revealed that light induces a high amplitude increase in intracellular calcium and a modest reduction in cAMP in cells expressing human melanopsin, implying that this pigment is able to drive responses via both Gq and Gi/o class G-proteins. Melanopsins from mouse and amphioxus had a similar profile of G-protein coupling in HEK293 cells, but chicken Opn4m and Opn4x pigments exhibited some Gs activity in addition to a strong Gq/11 response. An action spectrum for the calcium response in cells expressing human melanopsin had the predicted form for an opsin : vitamin A1 pigment and peaked at 479 nm. The G-protein selectivity and spectral sensitivity of human melanopsin is similar to that previously described for rodents, supporting the utility of such laboratory animals for developing methods of manipulating this system using light or pharmacological agents. PMID:23554393

  4. Signaling cascades and the importance of moonlight in coral broadcast mass spawning

    PubMed Central

    Kaniewska, Paulina; Alon, Shahar; Karako-Lampert, Sarit; Hoegh-Guldberg, Ove; Levy, Oren

    2015-01-01

    Many reef-building corals participate in a mass-spawning event that occurs yearly on the Great Barrier Reef. This coral reproductive event is one of earth's most prominent examples of synchronised behavior, and coral reproductive success is vital to the persistence of coral reef ecosystems. Although several environmental cues have been implicated in the timing of mass spawning, the specific sensory cues that function together with endogenous clock mechanisms to ensure accurate timing of gamete release are largely unknown. Here, we show that moonlight is an important external stimulus for mass spawning synchrony and describe the potential mechanisms underlying the ability of corals to detect environmental triggers for the signaling cascades that ultimately result in gamete release. Our study increases the understanding of reproductive chronobiology in corals and strongly supports the hypothesis that coral gamete release is achieved by a complex array of potential neurohormones and light-sensing molecules. DOI: http://dx.doi.org/10.7554/eLife.09991.001 PMID:26668113

  5. 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

  6. Ostrinia furnacalis serpin-3 regulates melanization cascade by inhibiting a prophenoloxidase-activating protease.

    PubMed

    Chu, Yuan; Zhou, Fan; Liu, Yang; Hong, Fang; Wang, Guirong; An, Chunju

    2015-06-01

    Serine protease cascade-mediated prophenolxidase activation is a prominent innate immune response in insect defense against the invading pathogens. Serpins regulate this reaction to avoid excessive activation. However, the function of serpins in most insect species, especially in some non-model agriculture insect pests, is largely unknown. We here cloned a full-length cDNA for a serpin, named as serpin-3, from Asian corn borer, Ostrinia furnacalis (Guenée). The open reading frame of serpin-3 encodes 462-amino acid residue protein with a 19-residue signal peptide. It contains a reactive center loop strikingly similar to the proteolytic activation site in prophenoloxidase. Sequence comparison indicates that O. furnacalis serpin-3 is an apparent ortholog of Manduca sexta serpin-3, a defined negative regulator of melanization reaction. Serpin-3 mRNA and protein levels significantly increase after a bacterial or fungal injection. Recombinant serpin-3 efficiently blocks prophenoloxidase activation in larval plasma in a concentration-dependent manner. It forms SDS-stable complexes with serine protease 13 (SP13), and prevents SP13 from cleaving prophenoloxidase. Injection of recombinant serpin-3 into larvae results in decreased fungi-induced melanin synthesis and reduced the expression of attacin, cecropin, gloverin, and peptidoglycan recognition protein-1 genes in the fat body. Altogether, serpin-3 plays important roles in the regulation of prophenoloxidase activation and antimicrobial peptide production in O. furnacalis larvae. PMID:25818483

  7. Caspase-10 triggers Bid cleavage and caspase cascade activation in FasL-induced apoptosis.

    PubMed

    Milhas, Delphine; Cuvillier, Olivier; Therville, Nicole; Clavé, Patricia; Thomsen, Mogens; Levade, Thierry; Benoist, Hervé; Ségui, Bruno

    2005-05-20

    In contrast to caspase-8, controversy exists as to the ability of caspase-10 to mediate apoptosis in response to FasL. Herein, we have shown activation of caspase-10, -3, and -7 as well as B cell lymphoma-2-interacting domain (Bid) cleavage and cytochrome c release in caspase-8-deficient Jurkat (I9-2) cells treated with FasL. Apoptosis was clearly induced as illustrated by nuclear and DNA fragmentation. These events were inhibited by benzyloxycarbonyl-VAD-fluoromethyl ketone, a broad spectrum caspase inhibitor, indicating that caspases were functionally and actively involved. Benzyloxycarbonyl-AEVD-fluoromethyl ketone, a caspase-10 inhibitor, had a comparable effect. FasL-induced cell death was not completely abolished by caspase inhibitors in agreement with the existence of a cytotoxic caspase-independent pathway. In subpopulations of I9-2 cells displaying distinct caspase-10 expression levels, cell sensitivity to FasL correlated with caspase-10 expression. A robust caspase activation, Bid cleavage, and DNA fragmentation were observed in cells with high caspase-10 levels but not in those with low levels. In vitro, caspase-10, as well as caspase-8, could cleave Bid to generate active truncated Bid (p15). Altogether, our data strongly suggest that caspase-10 can serve as an initiator caspase in Fas signaling leading to Bid processing, caspase cascade activation, and apoptosis. PMID:15772077

  8. Teaching an Old Hormone New Tricks: Cytosolic Ca2+ Elevation Involvement in Plant Brassinosteroid Signal Transduction Cascades1[W][OPEN

    PubMed Central

    Zhao, Yichen; Qi, Zhi; Berkowitz, Gerald A.

    2013-01-01

    Brassinosteroids (BRs) are hormones that control many aspects of plant growth and development, acting at the cell level to promote division and expansion. BR regulation of plant and plant cell function occurs through altered expression of many genes. Transcriptional reprogramming downstream from cell perception of this hormone is currently known to be mediated by a phosphorylation/dephosphorylation (“phosphorelay”) cascade that alters the stability of two master transcription regulators. Here, we provide evidence that BR perception by their receptor also causes an elevation in cytosolic Ca2+, initiating a Ca2+ signaling cascade in Arabidopsis (Arabidopsis thaliana) cell cytosol. BR-dependent increases in the expression of some genes (INDOLE-3-ACETIC ACID-INDUCIBLE1 and PHYTOCHROME B ACTIVATION-TAGGED SUPPRESSOR1) were impaired in wild-type plants by a Ca2+ channel blocker and also in the defense-no-death (dnd1) mutant, which lacks a functional cyclic GMP-activated cell membrane Ca2+-conducting channel. Alternatively, mutations that impair the BR phosphorelay cascade did not much affect the BR-dependent expression of these genes. Similar effects of the Ca2+ channel blocker and dnd1 mutation were observed on a BR plant growth phenotype, deetiolation of the seedling hypocotyl. Further evidence presented in this report suggests that a BR-dependent elevation in cyclic GMP may be involved in the Ca2+ signaling cascade initiated by this hormone. The work presented here leads to a new model of the molecular steps that mediate some of the cell responses to this plant hormone. PMID:23852441

  9. Mitogen-activated protein kinase cascade required for regulation of development and secondary metabolism in Neurospora crassa.

    PubMed

    Park, Gyungsoon; Pan, Songqin; Borkovich, Katherine A

    2008-12-01

    Mitogen-activated protein kinase (MAPK) signaling cascades are composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In this study, we characterize components of a MAPK cascade in Neurospora crassa (mik-1, MAPKKK; mek-1, MAPKK; and mak-1, MAPK) homologous to that controlling cell wall integrity in Saccharomyces cerevisiae. Growth of basal hyphae is significantly reduced in mik-1, mek-1, and mak-1 deletion mutants on solid medium. All three mutants formed short aerial hyphae and the formation of asexual macroconidia was reduced in Deltamik-1 mutants and almost abolished in Deltamek-1 and Deltamak-1 strains. In contrast, the normally rare asexual spores, arthroconidia, were abundant in cultures of the three mutants. Deltamik-1, Deltamek-1, and Deltamak-1 mutants were unable to form protoperithecia or perithecia when used as females in a sexual cross. The MAK-1 MAPK was not phosphorylated in Deltamik-1 and Deltamek-1 mutants, consistent with the involvement of MIK-1, MEK-1, and MAK-1 in the same signaling cascade. Interestingly, we observed increased levels of mRNA and protein for tyrosinase in the mutants under nitrogen starvation, a condition favoring sexual differentiation. Tyrosinase is an enzyme that catalyzes production of the secondary metabolite l-DOPA melanin. These results implicate the MAK-1 pathway in regulation of development and secondary metabolism in filamentous fungi. PMID:18849472

  10. Hypothesis for thermal activation of the caspase cascade in apoptotic cell death at elevated temperatures

    NASA Astrophysics Data System (ADS)

    Pearce, John A.

    2013-02-01

    Apoptosis is an especially important process affecting disease states from HIV-AIDS to auto-immune disease to cancer. A cascade of initiator and executioner capsase functional proteins is the hallmark of apoptosis. When activated the various caspases activate other caspases or cleave structural proteins of the cytoskeleton, resulting in "blebbing" of the plasma membrane forming apoptotic bodies that completely enclose the disassembled cellular components. Containment of the cytosolic components within the apoptotic bodies differentiates apoptosis from necroptosis and necrosis, both of which release fragmented cytosol and other cellular constituents into the intracellular space. Biochemical models of caspase activation reveal the extensive feedback loops characteristic of apoptosis. They clearly explain the failure of Arrhenius models to give accurate predictions of cell survival curves in hyperthermic heating protocols. Nevertheless, each of the individual reaction velocities can reasonably be assumed to follow Arrhenius kinetics. If so, the thermal sensitivity of the reaction velocity to temperature elevation is: ∂k/∂T = Ea [k/RT2]. Particular reaction steps described by higher activation energies, Ea, are likely more thermally-sensitive than lower energy reactions and may initiate apoptosis in the absence of other stress signals. Additionally, while the classical irreversible Arrhenius formulation fails to accurately represent many cell survival and/or dye uptake curves - those that display an early stage shoulder region - an expanded reversible model of the law of mass action equation seems to prove effective and is directly based on a firm theoretical thermodynamic foundation.

  11. Identification of signalling cascades involved in red blood cell shrinkage and vesiculation

    PubMed Central

    Kostova, Elena B.; Beuger, Boukje M.; Klei, Thomas R.L.; Halonen, Pasi; Lieftink, Cor; Beijersbergen, Roderick; van den Berg, Timo K.; van Bruggen, Robin

    2015-01-01

    Even though red blood cell (RBC) vesiculation is a well-documented phenomenon, notably in the context of RBC aging and blood transfusion, the exact signalling pathways and kinases involved in this process remain largely unknown. We have established a screening method for RBC vesicle shedding using the Ca2+ ionophore ionomycin which is a rapid and efficient method to promote vesiculation. In order to identify novel pathways stimulating vesiculation in RBC, we screened two libraries: the Library of Pharmacologically Active Compounds (LOPAC) and the Selleckchem Kinase Inhibitor Library for their effects on RBC from healthy donors. We investigated compounds triggering vesiculation and compounds inhibiting vesiculation induced by ionomycin. We identified 12 LOPAC compounds, nine kinase inhibitors and one kinase activator which induced RBC shrinkage and vesiculation. Thus, we discovered several novel pathways involved in vesiculation including G protein-coupled receptor (GPCR) signalling, the phosphoinositide 3-kinase (PI3K)–Akt (protein kinase B) pathway, the Jak–STAT (Janus kinase–signal transducer and activator of transcription) pathway and the Raf–MEK (mitogen-activated protein kinase kinase)–ERK (extracellular signal-regulated kinase) pathway. Moreover, we demonstrated a link between casein kinase 2 (CK2) and RBC shrinkage via regulation of the Gardos channel activity. In addition, our data showed that inhibition of several kinases with unknown functions in mature RBC, including Alk (anaplastic lymphoma kinase) kinase and vascular endothelial growth factor receptor 2 (VEGFR-2), induced RBC shrinkage and vesiculation. PMID:25757360

  12. Vehicular Exhaust Particles Promote Allergic Airway Inflammation via an Aryl Hydrocarbon Receptor-Notch Signaling Cascade

    PubMed Central

    Xia, Mingcan; Viera-Hutchins, Loida; Garcia-Lloret, Maria; Rivas, Magali Noval; Wise, Petra; MGhee, Sean A.; Chatila, Zena K.; Daher, Nancy; Sioutas, Constantinos; Chatila, Talal A.

    2015-01-01

    Background Traffic-related particulate matter (PM) has been linked to heightened incidence of asthma and allergic diseases. However, molecular mechanisms by which PM exposure promote allergic diseases remain elusive. Objective We sought to determine the expression, function and regulation of pathways involved in the promotion by PM of allergic airway inflammation. Methods We employed gene expression transcriptional profiling, in vitro culture assays, and vivo murine models of allergic airway inflammation. Results We identified genes of the Notch pathway, most notably Jagged 1 (Jag1), as targets of PM induction in human monocytes and murine dendritic cells (DCs). PM, especially ultrafine particles (UFP), upregulated T helper cytokine, IgE production and allergic airway inflammation in mice in a Jag1 and Notch-dependent manner especially in the context of the pro-asthmatic IL-4 receptor allele Il4raR576. PM-induced Jag1 expression was mediated by the aryl hydrocarbon receptor (AhR), which bound to and activated AhR response elements in the Jag1 promoter. Pharmacological antagonism of AhR or its lineage-specific deletion in CD11c+ cells abrogated the augmentation of airway inflammation by PM. Conclusion PM activate an AhR-Jag1-Notch cascade to promote allergic airway inflammation in concert with pro-asthmatic alleles. PMID:25825216

  13. Radiation induced nuclear factor kappa-B signaling cascade study in mammalian cells by improved detection systems

    NASA Astrophysics Data System (ADS)

    Chishti, Arif Ali; Baumstark-Khan, Christa; Hellweg, Christine; Reitz, Guenther

    To enable long-term human space flight cellular radiation response to densely ionizing radiation needs to be better understood for developing appropriate countermeasures to mitigate acute effects and late radiation risks for the astronaut. The biological effectiveness of accelerated heavy ions with high linear energy transfer (LET) for effecting DNA damage response pathways as a gateway to cell death or survival is of major concern, not only for tumor radiotherapy but also for new regimes of space missions. Ionizing radiation modulates several signaling pathways resulting in transcription factor activation. NF-kappaB is one of the important transcription factors that respond to changes in the environment of a mammalian cell and plays a key role in many biological processes relevant to radiation response, such as apoptosis, inflammation and carcinogenesis. From medical and biological point of view it is important to understand radiation induced NF-kappaB signaling cascade. For studying NF-kappaB signaling, green fluorescent proteins EGFP and d2EGFP were used previously (Advances in Space Research, 36: 1673-1679, 2005). The current study aims to improve reporter assays by the use of a destabilized variant of red fluorescent protein tdTomato (DD-tdTomato) which gives high fluorescence signals and a better signal/noise ratio for NF-kappaB activation. The reporter system HEK-pNFkappaB-DD-tdTomato-C8 is a dual reporter system which can provide both discrete and cumulative signals after exposure to ionizing radiation (X-rays, heavy ions). In the presence of Shield-1, the fluorescent protein DD-tdTomato is not degraded but accumulated inside the cell which helps to quantify the fold induction of NF-kappaB-dependent gene expression. The minimum dose required to activate NF-kappaB is 6 Gy but accumulated signals data shows that NF-kappaB is activated after 3 Gy in the presence of Shield-1. Average dose and number of heavy ions’ hits per nucleus necessary to double the NF

  14. Neonatal Dexamethasone Treatment Leads to Alterations in Cell Signaling Cascades Controlling Hepatic and Cardiac Function in Adulthood

    PubMed Central

    Adigun, Abayomi A.; Wrench, Nicola; Seidler, Frederic J.; Slotkin, Theodore A.

    2009-01-01

    Increasing evidence indicates that early-life glucocorticoid exposure, either involving stress or the therapy of preterm labor, contributes to metabolic and cardiovascular disorders in adulthood. We investigated cellular mechanisms underlying these effects by administering dexamethasone (DEX) to neonatal rats on postnatal (PN) days 1–3 or 7–9, using doses spanning the threshold for somatic growth impairment: 0.05, 0.2 and 0.8 mg/kg. In adulthood, we assessed the effects on hepatic and cardiac cell function mediated through the adenylyl cyclase (AC) signaling cascade, which controls neuronal and hormonal inputs that regulate hepatic glucose metabolism and cardiac contractility. Treatment on PN1-3 produced heterologous sensitization of hepatic signaling, with upregulation of AC itself leading to parallel increases in the responses to β-adrenergic or glucagon receptor stimulation, or to activation of G-proteins by fluoride. The effects were seen at the lowest dose but increasing DEX past the point of somatic growth impairment led to loss of the effect in females. Nonmonotonic effects were also present in the heart, where males showed AC sensitization at the lowest dose, with decreasing effects as the dose was raised; females showed progressive deficits of cardiac AC activity. Shifting the exposure to PN7-9 still elicited AC sensitization but with a greater offsetting contribution at the higher doses. Our findings show that, in contrast to growth restriction, the glucocorticoids associated with stress or the therapy of preterm labor are more sensitive and more important contributors to the cellular abnormalities underlying subsequent metabolic and cardiovascular dysfunction. PMID:19853034

  15. A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

    PubMed

    Mulder, Monique P C; Witting, Katharina; Berlin, Ilana; Pruneda, Jonathan N; Wu, Kuen-Phon; Chang, Jer-Gung; Merkx, Remco; Bialas, Johanna; Groettrup, Marcus; Vertegaal, Alfred C O; Schulman, Brenda A; Komander, David; Neefjes, Jacques; El Oualid, Farid; Ovaa, Huib

    2016-07-01

    Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades. PMID:27182664

  16. Differences in the control of basal L-type Ca(2+) current by the cyclic AMP signaling cascade in frog, rat, and human cardiac myocytes.

    PubMed

    Treinys, Rimantas; Bogdelis, Andrius; Rimkutė, Lina; Jurevičius, Jonas; Skeberdis, Vytenis Arvydas

    2016-07-01

    β-adrenergic receptors (β-ARs) mediate the positive inotropic effects of catecholamines by cAMP-dependent phosphorylation of the L-type Ca(2+) channels (LTCCs), which provide Ca(2+) for the initiation and regulation of cell contraction. The overall effect of cAMP-modulating agents on cardiac calcium current (I Ca,L) and contraction depends on the basal activity of LTCCs which, in turn, depends on the basal activities of key enzymes involved in the cAMP signaling cascade. Our current work is a comparative study demonstrating the differences in the basal activities of β-ARs, adenylyl cyclase, phosphodiesterases, phosphatases, and LTCCs in the frog and rat ventricular and human atrial myocytes. The main conclusion is that the basal I Ca,L, and consequently the contractile function of the heart, is secured from unnecessary elevation of its activity and energy consumption at the several "checking-points" of cAMP-dependent signaling cascade and the loading of these "checking-points" may vary in different species and tissues. PMID:26676115

  17. Effects of acute and chronic treatment elicited by lamotrigine on behavior, energy metabolism, neurotrophins and signaling cascades in rats.

    PubMed

    Abelaira, Helena M; Réus, Gislaine Z; Ribeiro, Karine F; Zappellini, Giovanni; Ferreira, Gabriela K; Gomes, Lara M; Carvalho-Silva, Milena; Luciano, Thais F; Marques, Scherolin O; Streck, Emilio L; Souza, Cláudio T; Quevedo, João

    2011-12-01

    The present study was aimed to investigate the behavioral and molecular effects of lamotrigine. To this aim, Wistar rats were treated with lamotrigine (10 and 20 mg/kg) or imipramine (30 mg/kg) acutely and chronically. The behavior was assessed using forced swimming test. Brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), Proteina Kinase B (PKB, AKT), glycogen synthase kinase 3 (GSK-3) and B-cell lymphoma 2 (Bcl-2) levels, citrate synthase, creatine kinase and mitochondrial chain (I, II, II-III and IV) activities were assessed in the brain. The results showed that both treatments reduced the immobility time. The BDNF were increased in the prefrontal after acute treatment with lamotrigine (20 mg/kg), and the BDNF and NGF were increased in the prefrontal after chronic treatment with lamotrigine in all doses. The AKT increased and Bcl-2 and GSK-3 decreased after both treatments in all brain areas. The citrate synthase and creatine kinase increased in the amygdala after acute treatment with imipramine. Chronic treatment with imipramine and lamotrigine (10 mg/kg) increased the creatine kinase in the hippocampus. The complex I was reduced and the complex II, II-III and IV were increased, but related with treatment and brain area. In conclusion, lamotrigine exerted antidepressant-like, which can be attributed to its effects on pathways related to depression, such as neurotrophins, metabolism energy and signaling cascade. PMID:22044672

  18. Opioid-receptor (OR) signaling cascades in rat cerebral cortex and model cell lines: the role of plasma membrane structure.

    PubMed

    Ujčíková, H; Brejchová, J; Vošahlíková, M; Kagan, D; Dlouhá, K; Sýkora, J; Merta, L; Drastichová, Z; Novotný, J; Ostašov, P; Roubalová, L; Parenti, M; Hof, M; Svoboda, P

    2014-01-01

    . Validity of this conclusion was supported by the analysis of an immediate PM environment of cholesterol molecules in living delta-OR-G(i)1alpha-HEK293 cells by fluorescent probes 22- and 25-NBD-cholesterol. The alteration of plasma membrane structure by cholesterol depletion made the membrane more hydrated. Understanding of the positive and negative feedback regulatory loops among different OR-initiated signaling cascades (micro-, delta-, and kappa-OR) is crucial for understanding of the long-term mechanisms of drug addiction as the decrease in functional activity of micro-OR may be compensated by increase of delta-OR and/or kappa-OR signaling. PMID:24564656

  19. 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

  20. The mTOR signalling cascade: paving new roads to cure neurological disease.

    PubMed

    Crino, Peter B

    2016-07-01

    Defining the multiple roles of the mechanistic (formerly 'mammalian') target of rapamycin (mTOR) signalling pathway in neurological diseases has been an exciting and rapidly evolving story of bench-to-bedside translational research that has spanned gene mutation discovery, functional experimental validation of mutations, pharmacological pathway manipulation, and clinical trials. Alterations in the dual contributions of mTOR - regulation of cell growth and proliferation, as well as autophagy and cell death - have been found in developmental brain malformations, epilepsy, autism and intellectual disability, hypoxic-ischaemic and traumatic brain injuries, brain tumours, and neurodegenerative disorders. mTOR integrates a variety of cues, such as growth factor levels, oxygen levels, and nutrient and energy availability, to regulate protein synthesis and cell growth. In line with the positioning of mTOR as a pivotal cell signalling node, altered mTOR activation has been associated with a group of phenotypically diverse neurological disorders. To understand how altered mTOR signalling leads to such divergent phenotypes, we need insight into the differential effects of enhanced or diminished mTOR activation, the developmental context of these changes, and the cell type affected by altered signalling. A particularly exciting feature of the tale of mTOR discovery is that pharmacological mTOR inhibitors have shown clinical benefits in some neurological disorders, such as tuberous sclerosis complex, and are being considered for clinical trials in epilepsy, autism, dementia, traumatic brain injury, and stroke. PMID:27340022

  1. Redox metabolites signal polymicrobial biofilm development via the NapA oxidative stress cascade in Aspergillus

    PubMed Central

    Zheng, He; Kim, Jaekuk; Liew, Mathew; Yan, John K.; Herrera, Oscar; Bok, JinWoo; Kelleher, Neil L.; Keller, Nancy P.; Wang, Yun

    2014-01-01

    Summary Background Filamentous fungi and bacteria form mixed-species biofilms in nature and diverse clinical contexts. They secrete a wealth of redox-active small molecule secondary metabolites, which are traditionally viewed as toxins that inhibit growth of competing microbes. Results Here we report that these “toxins” can act as interspecies signals, affecting filamentous fungal development via oxidative stress regulation. Specifically, in co-culture biofilms, Pseudomonas aeruginosa phenazine-derived metabolites differentially modulated Aspergillus fumigatus development, shifting from weak vegetative growth to induced asexual sporulation (conidiation) along a decreasing phenazine gradient. The A. fumigatus morphological shift correlated with the production of phenazine radicals and concomitant reactive oxygen species (ROS) production generated by phenazine redox cycling. Phenazine conidiation signaling was conserved in the genetic model A. nidulans, and mediated by NapA, a homolog of AP-1-like bZIP transcription factor, which is essential for the response to oxidative stress in humans, yeast, and filamentous fungi. Expression profiling showed phenazine treatment induced a NapA-dependent response of the global oxidative stress metabolome including the thioredoxin, glutathione and NADPH-oxidase systems. Conidiation induction in A. nidulans by another microbial redox-active secondary metabolite, gliotoxin, also required NapA. Conclusions This work highlights that microbial redox metabolites are key signals for sporulation in filamentous fungi, which are communicated through an evolutionarily conserved eukaryotic stress response pathway. It provides a foundation for interspecies signaling in environmental and clinical biofilms involving bacteria and filamentous fungi. PMID:25532893

  2. 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

  3. Characterization of photomorphogenic responses and signaling cascades controlled by phytochrome-A expressed in different tissues.

    PubMed

    Kirchenbauer, Daniel; Viczián, András; Ádám, Éva; Hegedűs, Zoltán; Klose, Cornelia; Leppert, Michael; Hiltbrunner, Andreas; Kircher, Stefan; Schäfer, Eberhard; Nagy, Ferenc

    2016-07-01

    The photoreceptor phytochrome A acts as a light-dependent molecular switch and regulates responses initiated by very low fluences of light (VLFR) and high fluences (HIR) of far-red light. PhyA is expressed ubiquitously, but how phyA signaling is orchestrated to regulate photomorphogenesis is poorly understood. To address this issue, we generated transgenic Arabidopsis thaliana phyA-201 mutant lines expressing the biologically active phyA-YFP photoreceptor in different tissues, and analyzed the expression of several reporter genes, including ProHY5:HY5-GFP and Pro35S:CFP-PIF1, and various FR-HIR-dependent physiological responses. We show that phyA action in one tissue is critical and sufficient to regulate flowering time and root growth; control of cotyledon and hypocotyl growth requires simultaneous phyA activity in different tissues; and changes detected in the expression of reporters are not restricted to phyA-containing cells. We conclude that FR-HIR-controlled morphogenesis in Arabidopsis is mediated partly by tissue-specific and partly by intercellular signaling initiated by phyA. Intercellular signaling is critical for many FR-HIR induced responses, yet it appears that phyA modulates the abundance and activity of key regulatory transcription factors in a tissue-autonomous fashion. PMID:27027866

  4. Sending Mixed Signals: The Expanding Role of Molecular Cascade Mutations in Malformations of Cortical Development and Epilepsy

    PubMed Central

    Iffland, Philip H.; Crino, Peter B.

    2016-01-01

    Advances in gene sequencing techniques have led to a dramatic increase in the number of signaling cascade and cytoskeletal assembly mutations associated with malformations of cortical development and epilepsy. At the forefront of this research are novel mutations found in regulators of the PI3K/AKT/mTOR cascade and tubulin-associated malformations of cortical development. However, there is limited understanding of the consequences of these newly discovered germline and somatic mutations on cellular function or how these changes in cell biology may lead to areas—large or small—of malformed cortex and recurrent spontaneous seizures. We summarize and discuss what is currently known in this field in an effort to shine light on vast gaps in our knowledge of relatively common causes of cortical malformations. PMID:27330441

  5. Ultraspecific electrochemical DNA biosensor by coupling spontaneous cascade DNA branch migration and dual-signaling sensing strategy.

    PubMed

    Wang, Ting; Zhou, Lili; Bai, Shulian; Zhang, Zhang; Li, Junlong; Jing, Xiaoying; Xie, Guoming

    2016-04-15

    Using spontaneous cascade DNA branch migration and dual-signaling sensing strategy, we developed a novel universal electrochemical biosensor for the highly specific and sensitive detection of nucleic acids. A target strand (Ts) competitively hybridized with a ferrocene (Fc)-labeled signal probe (Fc-S1), which was blocked by a protector strand (Ps), after strand displacement to form the Ts/Fc-S1 duplex. A methylene blue (MB)-modified signal probe (MB-S2) was immobilized on the Au electrode surface by hybridizing with a thiolated capture probe (Cp). Then, the obtained reactants (Ts/Fc-S1 and MB-S2/Cp) suffered spontaneous DNA branch migration and produced two hybridization products (Fc-S1/Cp and MB-S2/Ts). These reactions led to the dissociation of MB molecules and the collection of Fc molecules. The detection mechanism of this DNA biosensor involved distance variation between the redox tags and the Au electrode, which was associated with target-induced cascade DNA branch migration. Moreover, we rationally designed the cascade DNA branch migration to occur spontaneously with ΔG° ≈ 0, at which slight thermodynamic changes caused by base mismatch exerted a disproportionately large effect on the hybridization yield. This "signal-on/off" sensing system exhibited a remarkable analytical performance and an ultrahigh discrimination capability even against a single-base mismatch. The maximum discrimination factor (DF) of base mutations or alterations can reach 17.9. Therefore, our electrochemical biosensor might hold a great potential for further applications in biomedical research and early clinical diagnosis. PMID:26657589

  6. Distinct Signaling Cascades Elicited by Different Formyl Peptide Receptor 2 (FPR2) Agonists

    PubMed Central

    Cattaneo, Fabio; Parisi, Melania; Ammendola, Rosario

    2013-01-01

    The formyl peptide receptor 2 (FPR2) is a remarkably versatile transmembrane protein belonging to the G-protein coupled receptor (GPCR) family. FPR2 is activated by an array of ligands, which include structurally unrelated lipids and peptide/proteins agonists, resulting in different intracellular responses in a ligand-specific fashion. In addition to the anti-inflammatory lipid, lipoxin A4, several other endogenous agonists also bind FPR2, including serum amyloid A, glucocorticoid-induced annexin 1, urokinase and its receptor, suggesting that the activation of FPR2 may result in potent pro- or anti-inflammatory responses. Other endogenous ligands, also present in biological samples, include resolvins, amyloidogenic proteins, such as beta amyloid (Aβ)-42 and prion protein (Prp)106–126, the neuroprotective peptide, humanin, antibacterial peptides, annexin 1-derived peptides, chemokine variants, the neuropeptides, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP)-27, and mitochondrial peptides. Upon activation, intracellular domains of FPR2 mediate signaling to G-proteins, which trigger several agonist-dependent signal transduction pathways, including activation of phospholipase C (PLC), protein kinase C (PKC) isoforms, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway, the mitogen-activated protein kinase (MAPK) pathway, p38MAPK, as well as the phosphorylation of cytosolic tyrosine kinases, tyrosine kinase receptor transactivation, phosphorylation and nuclear translocation of regulatory transcriptional factors, release of calcium and production of oxidants. FPR2 is an attractive therapeutic target, because of its involvement in a range of normal physiological processes and pathological diseases. Here, we review and discuss the most significant findings on the intracellular pathways and on the cross-communication between FPR2 and tyrosine kinase receptors triggered by different FPR2 agonists. PMID

  7. Melanopsin-Expressing Amphioxus Photoreceptors Transduce Light via a Phospholipase C Signaling Cascade

    PubMed Central

    Angueyra, Juan Manuel; Pulido, Camila; Malagón, Gerardo; Nasi, Enrico; Gomez, Maria del Pilar

    2012-01-01

    Melanopsin, the receptor molecule that underlies light sensitivity in mammalian ‘circadian’ receptors, is homologous to invertebrate rhodopsins and has been proposed to operate via a similar signaling pathway. Its downstream effectors, however, remain elusive. Melanopsin also expresses in two distinct light-sensitive cell types in the neural tube of amphioxus. This organism is the most basal extant chordate and can help outline the evolutionary history of different photoreceptor lineages and their transduction mechanisms; moreover, isolated amphioxus photoreceptors offer unique advantages, because they are unambiguously identifiable and amenable to single-cell physiological assays. In the present study whole-cell patch clamp recording, pharmacological manipulations, and immunodetection were utilized to investigate light transduction in amphioxus photoreceptors. A Gq was identified and selectively localized to the photosensitive microvillar membrane, while the pivotal role of phospholipase C was established pharmacologically. The photocurrent was profoundly depressed by IP3 receptor antagonists, highlighting the importance of IP3 receptors in light signaling. By contrast, surrogates of diacylglycerol (DAG), as well as poly-unsaturated fatty acids failed to activate a membrane conductance or to alter the light response. The results strengthen the notion that calcium released from the ER via IP3-sensitive channels may fulfill a key role in conveying - directly or indirectly - the melanopsin-initiated light signal to the photoconductance; moreover, they challenge the dogma that microvillar photoreceptors and phoshoinositide-based light transduction are a prerogative of invertebrate eyes. PMID:22235344

  8. Delta Opioid activation of the Mitogen-activated protein kinase cascade does not require transphosphorylation of Receptor Tyrosine Kinases

    PubMed Central

    Kramer, H Kenneth; Onoprishvili, Irma; Andria, Matthew L; Hanna, Kayane; Sheinkman, Karina; Haddad, Lisa B; Simon, Eric J

    2002-01-01

    Background In this study, we investigated the mechanism(s) by which delta opioids induce their potent activation of extracellular signal-regulated protein kinases (ERKs) in different cell lines expressing the cloned δ-opioid receptor (δ-OR). While it has been known for some time that OR stimulation leads to the phosphorylation of both ERK isoforms, the exact progression of events has remained elusive. Results Our results indicate that the transphosphorylation of an endogenous epidermal growth factor receptor (EGFR) in the human embryonic kidney (HEK-293) cell line does not occur when co-expressed δ-ORs are stimulated by the δ-opioid agonist, D-Ser-Leu-enkephalin-Thr (DSLET). Moreover, neither pre-incubation of cultures with the selective EGFR antagonist, AG1478, nor down-regulation of the EGFR to a point where EGF could no longer activate ERKs had an inhibitory effect on ERK activation by DSLET. These results appear to rule out any structural or catalytic role for the EGFR in the δ-opioid-mediated MAPK cascade. To confirm these results, we used C6 glioma cells, a cell line devoid of the EGFR. In δ-OR-expressing C6 glioma cells, opioids produce a robust phosphorylation of ERK 1 and 2, whereas EGF has no stimulatory effect. Furthermore, antagonists to the RTKs that are endogenously expressed in C6 glioma cells (insulin receptor (IR) and platelet-derived growth factor receptor (PDGFR)) were unable to reduce opioid-mediated ERK activation. Conclusion Taken together, these data suggest that the transactivation of resident RTKs does not appear to be required for OR-mediated ERK phosphorylation and that the tyrosine-phosphorylated δ-OR, itself, is likely to act as its own signalling scaffold. PMID:11897012

  9. Moringa oleifera Lam.: Protease activity against blood coagulation cascade

    PubMed Central

    Satish, A; Sairam, Sudha; Ahmed, Faiyaz; Urooj, Asna

    2012-01-01

    Background: The present study evaluated the protease activity of aqueous extracts of Moringa oleifera (Moringaceae) leaf (MOL) and root (MOR). Materials and Methods: Protease activity was assayed using casein, human plasma clot and human fibrinogen as substrates. Results: Caseinolytic activity of MOL was significantly higher (P ≤ 0.05) than that of MOR. Similar observations were found in case of human plasma clot hydrolyzing activity, wherein MOL caused significantly higher (P ≤ 0.05) plasma clot hydrolysis than MOR. Zymographic techniques were used to detect proteolytic enzymes following electrophoretic separation in gels. Further, both the extracts exhibited significant procoagulant activity as reflected by a significant decrease (P ≤ 0.05) in recalcification time, accompanied by fibrinogenolytic and fibrinolytic activities; clotting time was decreased from 180 ± 10 sec to 119 ± 8 sec and 143 ± 10 sec by MOL and MOR, respectively, at a concentration of 2.5 mg/mL. Fibrinogenolytic (human fibrinogen) and fibrinolytic activity (human plasma clot) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), plate method and colorimetric method. Zymographic profile indicated that both the extracts exerted their procoagulant activity by selectively hydrolyzing Aα and Bβ subunits of fibrinogen to form fibrin clot, thereby exhibiting fibrinogenolytic activity. However, prolonged incubation resulted in degradation of the formed fibrin clot, suggesting fibrinolytic like activity. Conclusions: These findings support the traditional usage of M. oleifera extracts for wound healing. PMID:22224061

  10. Differential control of Yorkie activity by LKB1/AMPK and the Hippo/Warts cascade in the central nervous system

    PubMed Central

    Gailite, Ieva; Aerne, Birgit L.; Tapon, Nicolas

    2015-01-01

    The Hippo (Hpo) pathway is a highly conserved tumor suppressor network that restricts developmental tissue growth and regulates stem cell proliferation and differentiation. At the heart of the Hpo pathway is the progrowth transcriptional coactivator Yorkie [Yki–Yes-activated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) in mammals]. Yki activity is restricted through phosphorylation by the Hpo/Warts core kinase cascade, but increasing evidence indicates that core kinase-independent modes of regulation also play an important role. Here, we examine Yki regulation in the Drosophila larval central nervous system and uncover a Hpo/Warts-independent function for the tumor suppressor kinase liver kinase B1 (LKB1) and its downstream effector, the energy sensor AMP-activated protein kinase (AMPK), in repressing Yki activity in the central brain/ventral nerve cord. Although the Hpo/Warts core cascade restrains Yki in the optic lobe, it is dispensable for Yki target gene repression in the late larval central brain/ventral nerve cord. Thus, we demonstrate a dramatically different wiring of Hpo signaling in neighboring cell populations of distinct developmental origins in the central nervous system. PMID:26324895

  11. Erlotinib inhibits T-cell-mediated immune response via down-regulation of the c-Raf/ERK cascade and Akt signaling pathway

    SciTech Connect

    Luo Qiong; Gu Yanhong; Zheng Wei; Wu Xingxin; Gong Fangyuan; Gu Liyun; Sun Yang; Xu Qiang

    2011-03-01

    Erlotinib is a potent inhibitor of epidermal growth factor receptor tyrosine kinase and has been demonstrated to treat advanced or metastatic non-small cell lung cancer to prolong survival after failure of first-line or second-line chemotherapy. However, little is known about its effects on immune system. In the present study, we aimed to investigate the immunosuppressive activity of erlotinib on T lymphocytes both in vitro and in vivo, and further explore its potential molecular mechanism. Erlotinib exerted a significant inhibition on the T cell proliferation and activation induced by concanavalin A, anti-CD3 plus anti-CD28, staphylococcal enterotoxin B or phorbol myristate acetate respectively in a concentration-dependent manner and it also inhibited the secretion of the proinflammatory cytokines such as IL-2 and IFN-{gamma} of activated T cells. Further study showed that erlotinib caused G0/G1 arrest and suppressed the phosphorylations of c-Raf, ERK and Akt in activated T cells. Moreover, erlotinib significantly ameliorated picryl chloride-induced ear contact dermatitis in a dose-dependent manner in vivo. In summary, these findings suggest that erlotinib may cause the impairment of T-cell-mediated immune response both in vitro and in vivo through inhibiting T cell proliferation and activation, which is closely associated with its potent down-regulation of the c-Raf/ERK cascade and Akt signaling pathway. - Graphical abstract: Erlotinib may cause the impairment of T-cell-mediated immune response both in vitro and in vivo through inhibiting T cell proliferation and activation, which is closely associated with its potent down-regulation of the c-Raf/ERK cascade and Akt signaling pathway. Display Omitted

  12. 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

  13. The extracellular signal-regulated kinase mitogen-activated protein kinase/ribosomal S6 protein kinase 1 cascade phosphorylates cAMP response element-binding protein to induce MUC5B gene expression via D-prostanoid receptor signaling.

    PubMed

    Choi, Yeon Ho; Lee, Sang-Nam; Aoyagi, Hiroki; Yamasaki, Yasundo; Yoo, Jung-Yoon; Park, Boryung; Shin, Dong Min; Yoon, Ho-Geun; Yoon, Joo-Heon

    2011-09-30

    Mucus hypersecretion is a prominent feature of respiratory diseases, and MUC5B is a major airway mucin. Mucin gene expression can be affected by inflammatory mediators, including prostaglandin (PG) D(2,) an inflammatory mediator synthesized by hematopoietic PGD synthase (H-PGDS). PGD(2) binds to either D-prostanoid receptor (DP1) or chemoattractant receptor homologous molecule expressed on T-helper type 2 cells (CRTH2). We investigated the mechanisms by which PGD(2) induces MUC5B gene expression in airway epithelial cells. Western blot analysis showed that H-PGDS was highly expressed in nasal polyps. Similar results were obtained for PGD(2) expression. In addition, we could clearly detect the expressions of both H-PGDS and DP1 in nasal epithelial cells but not CRTH2. We demonstrated that PGD(2) increased MUC5B gene expression in normal human nasal epithelial cells as well as in NCI-H292 cells in vitro. S5751, a DP1 antagonist, inhibited PGD(2)-induced MUC5B expression, whereas a CRTH2 antagonist (OC0459) did not. These data suggest that PGD(2) induced MUC5B expression via DP1. Pretreatment with extracellular signal-regulated kinase (ERK) inhibitor (PD98059) blocked both PGD(2)-induced ERK mitogen-activated protein kinase (MAPK) activation and MUC5B expression. Proximity ligation assays showed direct interaction between RSK1 and cAMP response element-binding protein (CREB). Stimulation with PGD(2) caused an increase in intracellular cAMP levels, whereas intracellular Ca(2+) did not have such an effect. PGD(2)-induced MUC5B mRNA levels were regulated by CREB via direct interaction with two cAMP-response element sites (-921/-914 and -900/-893). Finally, we demonstrated that PGD(2) can induce MUC5B overproduction via ERK MAPK/RSK1/CREB signaling and that DP1 receptor may have suppressive effects in controlling MUC5B overproduction in the airway. PMID:21832046

  14. A Signaling Cascade from miR444 to RDR1 in Rice Antiviral RNA Silencing Pathway.

    PubMed

    Wang, Huacai; Jiao, Xiaoming; Kong, Xiaoyu; Hamera, Sadia; Wu, Yao; Chen, Xiaoying; Fang, Rongxiang; Yan, Yongsheng

    2016-04-01

    Plant RNA-DEPENDENT RNA POLYMERASE1 (RDR1) is a key component of the antiviral RNA-silencing pathway, contributing to the biogenesis of virus-derived small interfering RNAs. This enzyme also is responsible for producing virus-activated endogenous small interfering RNAs to stimulate the broad-spectrum antiviral activity through silencing host genes. The expression of RDR1 orthologs in various plants is usually induced by virus infection. However, the molecular mechanisms of activation of RDR1 expression in response to virus infection remain unknown. Here, we show that a monocot-specific microRNA, miR444, is a key factor in relaying the antiviral signaling from virus infection to OsRDR1 expression. The expression of miR444 is enhanced by infection with Rice stripe virus (RSV), and overexpression of miR444 improves rice (Oryza sativa) resistance against RSV infection accompanied by the up-regulation of OsRDR1 expression. We further show that three miR444 targets, the MIKC(C)-type MADS box proteins OsMADS23, OsMADS27a, and OsMADS57, form homodimers and heterodimers between them to repress the expression of OsRDR1 by directly binding to the CArG motifs of its promoter. Consequently, an increased level of miR444 diminishes the repressive roles of OsMADS23, OsMADS27a, and OsMADS57 on OsRDR1 transcription, thus activating the OsRDR1-dependent antiviral RNA-silencing pathway. We also show that overexpression of miR444-resistant OsMADS57 reduced OsRDR1 expression and rice resistance against RSV infection, and knockout of OsRDR1 reduced rice resistance against RSV infection. In conclusion, our results reveal a molecular cascade in the rice antiviral pathway in which miR444 and its MADS box targets directly control OsRDR1 transcription. PMID:26858364

  15. Corticotropin-Releasing Factor Mediates Pain-Induced Anxiety through the ERK1/2 Signaling Cascade in Locus Coeruleus Neurons

    PubMed Central

    Borges, Gisela Patrícia; Micó, Juan Antonio; Neto, Fani Lourença

    2015-01-01

    Background: The corticotropin-releasing factor is a stress-related neuropeptide that modulates locus coeruleus activity. As locus coeruleus has been involved in pain and stress-related patologies, we tested whether the pain-induced anxiety is a result of the corticotropin-releasing factor released in the locus coeruleus. Methods: Complete Freund’s adjuvant-induced monoarthritis was used as inflammatory chronic pain model. α-Helical corticotropin-releasing factor receptor antagonist was microinjected into the contralateral locus coeruleus of 4-week-old monoarthritic animals. The nociceptive and anxiety-like behaviors, as well as phosphorylated extracellular signal-regulated kinases 1/2 and corticotropin-releasing factor receptors expression, were quantified in the paraventricular nucleus and locus coeruleus. Results: Monoarthritic rats manifested anxiety and increased phosphorylated extracellular signal-regulated kinases 1/2 levels in the locus coeruleus and paraventricular nucleus, although the expression of corticotropin-releasing factor receptors was unaltered. α-Helical corticotropin-releasing factor antagonist administration reversed both the anxiogenic-like behavior and the phosphorylated extracellular signal-regulated kinases 1/2 levels in the locus coeruleus. Conclusions: Pain-induced anxiety is mediated by corticotropin-releasing factor neurotransmission in the locus coeruleus through extracellular signal-regulated kinases 1/2 signaling cascade. PMID:25716783

  16. Cascading Activation across Levels of Representation in Children's Lexical Processing

    ERIC Educational Resources Information Center

    Huang, Yi Ting; Snedeker, Jesse

    2011-01-01

    Recent work in adult psycholinguistics has demonstrated that activation of semantic representations begins long before phonological processing is complete. This incremental propagation of information across multiple levels of analysis is a hallmark of adult language processing but how does this ability develop? In two experiments, we elicit…

  17. A signal amplification strategy using the cascade catalysis of gold nanoclusters and glucose dehydrogenase for ultrasensitive detection of thrombin.

    PubMed

    Han, Jing; Zhuo, Ying; Chai, Yaqin; Gui, Guofeng; Zhao, Min; Zhu, Qiang; Yuan, Ruo

    2013-12-15

    This work reports a novel signal amplification strategy for ultrasensitive detection of thrombin by cascade catalysis of gold nanoclusters (AuNCs) and glucose dehydrogenase (GDH). Herein, the AuNCs prepared by using polyamidoamine dendrimer as template were constructed not only as nanocarriers for anchoring the large amounts of secondary thrombin aptamers but also as nanocatalysts to catalyze the oxidation of NADH efficiently. Moreover, a large amount of GDH was loaded through the immobilization technology of DNA hybridization and a large amount of toluidine blue (Tb) was intercalated into the DNA grooves via electrostatic interaction. Significantly, the electrochemical signal was greatly enhanced based on cascade catalysis: firstly, GDH catalyzed the oxidation of glucose to gluconolactone with the concomitant generation of NADH in the presence of NAD(+). Then, AuNCs as nanocatalysts could effectively catalyze NADH to produce NAD(+) with the help of Tb as redox probe. Under the optimal conditions, the proposed aptasensor exhibits a linear range of 1.0×10(-14)-5×10(-9) M with a low detection limit of 3.3×10(-15) M for thrombin detection and shows high sensitivity and good specificity. PMID:23850783

  18. AMPK-SKP2-CARM1 signalling cascade in transcriptional regulation of autophagy.

    PubMed

    Shin, Hi-Jai R; Kim, Hyunkyung; Oh, Sungryong; Lee, Jun-Gi; Kee, Minjung; Ko, Hyun-Jeong; Kweon, Mi-Na; Won, Kyoung-Jae; Baek, Sung Hee

    2016-06-23

    Autophagy is a highly conserved self-digestion process, which is essential for maintaining homeostasis and viability in response to nutrient starvation. Although the components of autophagy in the cytoplasm have been well studied, the molecular basis for the transcriptional and epigenetic regulation of autophagy is poorly understood. Here we identify co-activator-associated arginine methyltransferase 1 (CARM1) as a crucial component of autophagy in mammals. Notably, CARM1 stability is regulated by the SKP2-containing SCF (SKP1-cullin1-F-box protein) E3 ubiquitin ligase in the nucleus, but not in the cytoplasm, under nutrient-rich conditions. Furthermore, we show that nutrient starvation results in AMP-activated protein kinase (AMPK)-dependent phosphorylation of FOXO3a in the nucleus, which in turn transcriptionally represses SKP2. This repression leads to increased levels of CARM1 protein and subsequent increases in histone H3 Arg17 dimethylation. Genome-wide analyses reveal that CARM1 exerts transcriptional co-activator function on autophagy-related and lysosomal genes through transcription factor EB (TFEB). Our findings demonstrate that CARM1-dependent histone arginine methylation is a crucial nuclear event in autophagy, and identify a new signalling axis of AMPK-SKP2-CARM1 in the regulation of autophagy induction after nutrient starvation. PMID:27309807

  19. Gadd45 Proteins as Critical Signal Transducers Linking NF-κB to MAPK Cascades

    PubMed Central

    Yang, Z.; Song, L.; Huang, C.

    2013-01-01

    The growth arrest and DNA damage-inducible 45 (Gadd45) proteins are a group of critical signal transducers that are involved in regulations of many cellular functions. Accumulated data indicate that all three Gadd45 proteins (i.e., Gadd45α, Gadd45β, and Gadd45γ) play essential roles in connecting an upstream sensor module, the transcription Nuclear Factor-κB (NF-κB), to a transcriptional regulating module, mitogen-activated protein kinase (MAPK). This NF-κB-Gadd45(s)-MAPK pathway responds to various kinds of extracellular stimuli and regulates such cell activities as growth arrest, differentiation, cell survival, and apoptosis. Defects in this pathway can also be related to oncogenesis. In the first part of this review, the functions of Gadd45 proteins, and briefly NF-κB and MAPK, are summarized. In the second part, the mechanisms by which Gadd45 proteins are regulated by NF-κB, and how they affect MAPK activation, are reviewed. PMID:20025601

  20. Premature senescence involving p53 and p16 is activated in response to constitutive MEK/MAPK mitogenic signaling

    PubMed Central

    Lin, Athena W.; Barradas, Marta; Stone, James C.; van Aelst, Linda; Serrano, Manuel; Lowe, Scott W.

    1998-01-01

    Oncogenic Ras transforms immortal rodent cells to a tumorigenic state, in part, by constitutively transmitting mitogenic signals through the mitogen-activated protein kinase (MAPK) cascade. In primary cells, Ras is initially mitogenic but eventually induces premature senescence involving the p53 and p16INK4a tumor suppressors. Constitutive activation of MEK (a component of the MAPK cascade) induces both p53 and p16, and is required for Ras-induced senescence of normal human fibroblasts. Furthermore, activated MEK permanently arrests primary murine fibroblasts but forces uncontrolled mitogenesis and transformation in cells lacking either p53 or INK4a. The precisely opposite response of normal and immortalized cells to constitutive activation of the MAPK cascade implies that premature senescence acts as a fail-safe mechanism to limit the transforming potential of excessive Ras mitogenic signaling. Consequently, constitutive MAPK signaling activates p53 and p16 as tumor suppressors. PMID:9765203

  1. Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation

    PubMed Central

    Huo, Yanwu; Nam, Ki Hyun; Ding, Fang; Lee, Heejin; Wu, Lijie; Xiao, Yibei; Farchione, F. Daniel; Zhou, Sharleen; Rajashankar, Raj; Kurinov, Igor; Zhang, Rongguang; Ke, Ailong

    2014-01-01

    CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids using an RNA-mediated interference mechanism. Interference in Type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Cas3 bound to ss-DNA substrate and show that it is an obligated 3′-to-5′ ss-DNase preferentially accepting substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ss-DNA cleavage. ATP coordination and conformational flexibility are revealed for the SF2-type helicase moiety. Cas3 is specifically guided towards Cascade-bound target DNA with a correct PAM sequence, through physical interactions to both the non-target substrate strand and the CasA protein. The cascade of recognition events ensures a well-controlled DNA targeting and degradation of alien DNA by Cascade and Cas3. PMID:25132177

  2. Active Wavelength Control of an External Cavity Quantum Cascade Laser

    PubMed Central

    Tsai, Tracy; Wysocki, Gerard

    2012-01-01

    We present an active wavelength control system for grating-based external cavity lasers that increases the accuracy of predicting the lasing wavelength based on the grating equation and significantly improves scan-to-scan wavelength/frequency repeatability. The ultimate 3σ precision of a frequency scan is determined by the scan-to-scan repeatability of 0.042 cm−1. Since this control method can be applied to any external cavity laser with little to no modification, such a precision provides an excellent opportunity for spectroscopic applications that target molecular absorption lines at standard atmospheric conditions. PMID:23483850

  3. Hypochlorite-Modified Albumin Upregulates ICAM-1 Expression via a MAPK-NF-κB Signaling Cascade: Protective Effects of Apocynin.

    PubMed

    Tang, Dong-dong; Niu, Hong-xin; Peng, Fen-fen; Long, Hai-bo; Liu, Zong-rui; Zhao, Hao; Chen, Yi-hua

    2016-01-01

    Hypochlorite-modified albumin (HOCl-alb) has been linked to endothelial dysfunction, which plays an important role in the development of hypertension, diabetes, and chronic kidney disease. However, whether HOCl-alb induces endothelial dysfunction via vascular inflammation and whether a signaling pathway is involved are unknown and have not been investigated. HOCl-alb was found to upregulate ICAM-1 expression in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent manner. HOCl-alb time-dependently phosphorylated ERK1/2 and p38(MAPK). HOCl-alb also activated NF-κB. ICAM-1 expression was dose-dependently inhibited by U0126 (a specific inhibitor of MEK1/2, a signal upstream from ERK1/2), SB203580 (a specific inhibitor of p38(MAPK)), and SN50 (a specific inhibitor of NF-κB). U0126 and SB203580 both counteracted the activation of NF-κB, whereas the phosphorylation of ERK1/2 and p38(MAPK) was not blocked by SN50. ERK1/2 phosphorylation was blocked by U0126 but not by SB203580, and p38(MAPK) activity was reduced by SB203580 but not by U0126. Apocynin, a specific NADPH oxidase (NOX) inhibitor, inhibited ICAM-1 expression and the activity of ERK1/2, p38(MAPK), and NF-κB. These results indicate that HOCl-alb-induced ICAM-1 expression is caused by the activation of a redox-sensitive intracellular signal cascade involving ERK1/2 and p38(MAPK), culminating in the activation of NF-κB and involving NOXs among the upstream signals. PMID:26881015

  4. Hypochlorite-Modified Albumin Upregulates ICAM-1 Expression via a MAPK–NF-κB Signaling Cascade: Protective Effects of Apocynin

    PubMed Central

    Tang, Dong-dong; Niu, Hong-xin; Peng, Fen-fen; Long, Hai-bo; Liu, Zong-rui; Zhao, Hao; Chen, Yi-hua

    2016-01-01

    Hypochlorite-modified albumin (HOCl-alb) has been linked to endothelial dysfunction, which plays an important role in the development of hypertension, diabetes, and chronic kidney disease. However, whether HOCl-alb induces endothelial dysfunction via vascular inflammation and whether a signaling pathway is involved are unknown and have not been investigated. HOCl-alb was found to upregulate ICAM-1 expression in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent manner. HOCl-alb time-dependently phosphorylated ERK1/2 and p38MAPK. HOCl-alb also activated NF-κB. ICAM-1 expression was dose-dependently inhibited by U0126 (a specific inhibitor of MEK1/2, a signal upstream from ERK1/2), SB203580 (a specific inhibitor of p38MAPK), and SN50 (a specific inhibitor of NF-κB). U0126 and SB203580 both counteracted the activation of NF-κB, whereas the phosphorylation of ERK1/2 and p38MAPK was not blocked by SN50. ERK1/2 phosphorylation was blocked by U0126 but not by SB203580, and p38MAPK activity was reduced by SB203580 but not by U0126. Apocynin, a specific NADPH oxidase (NOX) inhibitor, inhibited ICAM-1 expression and the activity of ERK1/2, p38MAPK, and NF-κB. These results indicate that HOCl-alb-induced ICAM-1 expression is caused by the activation of a redox-sensitive intracellular signal cascade involving ERK1/2 and p38MAPK, culminating in the activation of NF-κB and involving NOXs among the upstream signals. PMID:26881015

  5. A Cascade of Wnt, Eda, and Shh Signaling Is Essential for Touch Dome Merkel Cell Development

    PubMed Central

    Thoresen, Daniel T.; Miao, Lingling; Williams, Jonathan S.; Wang, Chaochen; Atit, Radhika P.; Wong, Sunny Y.

    2016-01-01

    The Sonic hedgehog (Shh) signaling pathway regulates developmental, homeostatic, and repair processes throughout the body. In the skin, touch domes develop in tandem with primary hair follicles and contain sensory Merkel cells. The developmental signaling requirements for touch dome specification are largely unknown. We found dermal Wnt signaling and subsequent epidermal Eda/Edar signaling promoted Merkel cell morphogenesis by inducing Shh expression in early follicles. Lineage-specific gene deletions revealed intraepithelial Shh signaling was necessary for Merkel cell specification. Additionally, a Shh signaling agonist was sufficient to rescue Merkel cell differentiation in Edar-deficient skin. Moreover, Merkel cells formed in Fgf20 mutant skin where primary hair formation was defective but Shh production was preserved. Although developmentally associated with hair follicles, fate mapping demonstrated Merkel cells primarily originated outside the hair follicle lineage. These findings suggest that touch dome development requires Wnt-dependent mesenchymal signals to establish reciprocal signaling within the developing ectoderm, including Eda signaling to primary hair placodes and ultimately Shh signaling from primary follicles to extrafollicular Merkel cell progenitors. Shh signaling often demonstrates pleiotropic effects within a structure over time. In postnatal skin, Shh is known to regulate the self-renewal, but not the differentiation, of touch dome stem cells. Our findings relate the varied effects of Shh in the touch dome to the ligand source, with locally produced Shh acting as a morphogen essential for lineage specification during development and neural Shh regulating postnatal touch dome stem cell maintenance. PMID:27414798

  6. A Cascade of Wnt, Eda, and Shh Signaling Is Essential for Touch Dome Merkel Cell Development.

    PubMed

    Xiao, Ying; Thoresen, Daniel T; Miao, Lingling; Williams, Jonathan S; Wang, Chaochen; Atit, Radhika P; Wong, Sunny Y; Brownell, Isaac

    2016-07-01

    The Sonic hedgehog (Shh) signaling pathway regulates developmental, homeostatic, and repair processes throughout the body. In the skin, touch domes develop in tandem with primary hair follicles and contain sensory Merkel cells. The developmental signaling requirements for touch dome specification are largely unknown. We found dermal Wnt signaling and subsequent epidermal Eda/Edar signaling promoted Merkel cell morphogenesis by inducing Shh expression in early follicles. Lineage-specific gene deletions revealed intraepithelial Shh signaling was necessary for Merkel cell specification. Additionally, a Shh signaling agonist was sufficient to rescue Merkel cell differentiation in Edar-deficient skin. Moreover, Merkel cells formed in Fgf20 mutant skin where primary hair formation was defective but Shh production was preserved. Although developmentally associated with hair follicles, fate mapping demonstrated Merkel cells primarily originated outside the hair follicle lineage. These findings suggest that touch dome development requires Wnt-dependent mesenchymal signals to establish reciprocal signaling within the developing ectoderm, including Eda signaling to primary hair placodes and ultimately Shh signaling from primary follicles to extrafollicular Merkel cell progenitors. Shh signaling often demonstrates pleiotropic effects within a structure over time. In postnatal skin, Shh is known to regulate the self-renewal, but not the differentiation, of touch dome stem cells. Our findings relate the varied effects of Shh in the touch dome to the ligand source, with locally produced Shh acting as a morphogen essential for lineage specification during development and neural Shh regulating postnatal touch dome stem cell maintenance. PMID:27414798

  7. 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

  8. Digital data set of volcano hazards for active Cascade Volcanos, Washington

    USGS Publications Warehouse

    Schilling, Steve P.

    1996-01-01

    Scientists at the Cascade Volcano Observatory have completed hazard assessments for the five active volcanos in Washington. The five studies included Mount Adams (Scott and others, 1995), Mount Baker (Gardner and others, 1995), Glacier Peak (Waitt and others, 1995), Mount Rainier (Hoblitt and others, 1995) and Mount St. Helens (Wolfe and Pierson, 1995). Twenty Geographic Information System (GIS) data sets have been created that represent the hazard information from the assessments. The twenty data sets have individual Open File part numbers and titles

  9. The EAL domain protein YciR acts as a trigger enzyme in a c-di-GMP signalling cascade in E. coli biofilm control

    PubMed Central

    Lindenberg, Sandra; Klauck, Gisela; Pesavento, Christina; Klauck, Eberhard; Hengge, Regine

    2013-01-01

    C-di-GMP—which is produced by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases (PDEs)—is a ubiquitous second messenger in bacterial biofilm formation. In Escherichia coli, several DGCs (YegE, YdaM) and PDEs (YhjH, YciR) and the MerR-like transcription factor MlrA regulate the transcription of csgD, which encodes a biofilm regulator essential for producing amyloid curli fibres of the biofilm matrix. Here, we demonstrate that this system operates as a signalling cascade, in which c-di-GMP controlled by the DGC/PDE pair YegE/YhjH (module I) regulates the activity of the YdaM/YciR pair (module II). Via multiple direct interactions, the two module II proteins form a signalling complex with MlrA. YciR acts as a connector between modules I and II and functions as a trigger enzyme: its direct inhibition of the DGC YdaM is relieved when it binds and degrades c-di-GMP generated by module I. As a consequence, YdaM then generates c-di-GMP and—by direct and specific interaction—activates MlrA to stimulate csgD transcription. Trigger enzymes may represent a general principle in local c-di-GMP signalling. PMID:23708798

  10. Regulatory Cross-Talks and Cascades in Rice Hormone Biosynthesis Pathways Contribute to Stress Signaling

    PubMed Central

    Deb, Arindam; Grewal, Rumdeep K.; Kundu, Sudip

    2016-01-01

    Crosstalk among different hormone signaling pathways play an important role in modulating plant response to both biotic and abiotic stress. Hormone activity is controlled by its bio-availability, which is again influenced by its biosynthesis. Thus, independent hormone biosynthesis pathways must be regulated and co-ordinated to mount an integrated response. One of the possibilities is to use cis-regulatory elements to orchestrate expression of hormone biosynthesis genes. Analysis of CREs, associated with differentially expressed hormone biosynthesis related genes in rice leaf under Magnaporthe oryzae attack and drought stress enabled us to obtain insights about cross-talk among hormone biosynthesis pathways at the transcriptional level. We identified some master transcription regulators that co-ordinate different hormone biosynthesis pathways under stress. We found that Abscisic acid and Brassinosteroid regulate Cytokinin conjugation; conversely Brassinosteroid biosynthesis is affected by both Abscisic acid and Cytokinin. Jasmonic acid and Ethylene biosynthesis may be modulated by Abscisic acid through DREB transcription factors. Jasmonic acid or Salicylic acid biosynthesis pathways are co-regulated but they are unlikely to influence each others production directly. Thus, multiple hormones may modulate hormone biosynthesis pathways through a complex regulatory network, where biosynthesis of one hormone is affected by several other contributing hormones. PMID:27617021

  11. 17β-estradiol regulates the differentiation of cementoblasts via Notch signaling cascade.

    PubMed

    Liao, Jing; Zhou, Zeyuan; Huang, Li; Li, Yuyu; Li, Jingtao; Zou, Shujuan

    2016-08-12

    Estrogen has been well recognized as a key factor in the homeostasis of bone and periodontal tissue, but the way it regulates the activities of cementoblasts, the cell population maintaining cementum has not been fully understood. In this study, we examined the expression of estrogen receptor in OCCM-30 cells and the effect of 17β-estradiol (E2) on the proliferation and differentiation of OCCM-30 cells. We found that both estrogen receptor α and β were expressed in OCCM-30 cells. E2 exerted no significant influence on the proliferation of OCCM-30 cells, but inhibited the transcription and translation of BSP and Runx2 in the early phase of osteogenic induction except the BSP mRNA. Afterwards in the late phase of osteogenic induction, E2 enhanced the transcription and translation of BSP and Runx2 and promoted the calcium deposition. In addition, the expression level of Notch1, NICD and Hey1 mRNAs responded to exogenous E2 in a pattern similar to that of the osteoblastic markers. DAPT could attenuate the effect of E2 on the expression of osteoblastic markers. These findings indicated that E2 might regulate the differentiation of cementoblasts via Notch signaling. PMID:27289020

  12. Regulatory Cross-Talks and Cascades in Rice Hormone Biosynthesis Pathways Contribute to Stress Signaling.

    PubMed

    Deb, Arindam; Grewal, Rumdeep K; Kundu, Sudip

    2016-01-01

    Crosstalk among different hormone signaling pathways play an important role in modulating plant response to both biotic and abiotic stress. Hormone activity is controlled by its bio-availability, which is again influenced by its biosynthesis. Thus, independent hormone biosynthesis pathways must be regulated and co-ordinated to mount an integrated response. One of the possibilities is to use cis-regulatory elements to orchestrate expression of hormone biosynthesis genes. Analysis of CREs, associated with differentially expressed hormone biosynthesis related genes in rice leaf under Magnaporthe oryzae attack and drought stress enabled us to obtain insights about cross-talk among hormone biosynthesis pathways at the transcriptional level. We identified some master transcription regulators that co-ordinate different hormone biosynthesis pathways under stress. We found that Abscisic acid and Brassinosteroid regulate Cytokinin conjugation; conversely Brassinosteroid biosynthesis is affected by both Abscisic acid and Cytokinin. Jasmonic acid and Ethylene biosynthesis may be modulated by Abscisic acid through DREB transcription factors. Jasmonic acid or Salicylic acid biosynthesis pathways are co-regulated but they are unlikely to influence each others production directly. Thus, multiple hormones may modulate hormone biosynthesis pathways through a complex regulatory network, where biosynthesis of one hormone is affected by several other contributing hormones. PMID:27617021

  13. MAPK signaling cascades mediate distinct glucocorticoid resistance mechanisms in pediatric leukemia

    PubMed Central

    Jones, Courtney L.; Gearheart, Christy M.; Fosmire, Susan; Delgado-Martin, Cristina; Evensen, Nikki A.; Bride, Karen; Waanders, Angela J.; Pais, Faye; Wang, Jinhua; Bhatla, Teena; Bitterman, Danielle S.; de Rijk, Simone R.; Bourgeois, Wallace; Dandekar, Smita; Park, Eugene; Burleson, Tamara M.; Madhusoodhan, Pillai Pallavi; Teachey, David T.; Raetz, Elizabeth A.; Hermiston, Michelle L.; Müschen, Markus; Loh, Mignon L.; Hunger, Stephen P.; Zhang, Jinghui; Garabedian, Michael J.; Porter, Christopher C.

    2015-01-01

    The outcome for pediatric acute lymphoblastic leukemia (ALL) patients who relapse is dismal. A hallmark of relapsed disease is acquired resistance to multiple chemotherapeutic agents, particularly glucocorticoids. In this study, we performed a genome-scale short hairpin RNA screen to identify mediators of prednisolone sensitivity in ALL cell lines. The incorporation of these data with an integrated analysis of relapse-specific genetic and epigenetic changes allowed us to identify the mitogen-activated protein kinase (MAPK) pathway as a mediator of prednisolone resistance in pediatric ALL. We show that knockdown of the specific MAPK pathway members MEK2 and MEK4 increased sensitivity to prednisolone through distinct mechanisms. MEK4 knockdown increased sensitivity specifically to prednisolone by increasing the levels of the glucocorticoid receptor. MEK2 knockdown increased sensitivity to all chemotherapy agents tested by increasing the levels of p53. Furthermore, we demonstrate that inhibition of MEK1/2 with trametinib increased sensitivity of ALL cells and primary samples to chemotherapy in vitro and in vivo. To confirm a role for MAPK signaling in patients with relapsed ALL, we measured the activation of the MEK1/2 target ERK in matched diagnosis-relapse primary samples and observed increased phosphorylated ERK levels at relapse. Furthermore, relapse samples have an enhanced response to MEK inhibition compared to matched diagnosis samples in xenograft models. Together, our data indicate that inhibition of the MAPK pathway increases chemosensitivity to glucocorticoids and possibly other agents and that the MAPK pathway is an attractive target for prevention and/or treatment of relapsed disease. PMID:26324703

  14. MAPK signaling cascades mediate distinct glucocorticoid resistance mechanisms in pediatric leukemia.

    PubMed

    Jones, Courtney L; Gearheart, Christy M; Fosmire, Susan; Delgado-Martin, Cristina; Evensen, Nikki A; Bride, Karen; Waanders, Angela J; Pais, Faye; Wang, Jinhua; Bhatla, Teena; Bitterman, Danielle S; de Rijk, Simone R; Bourgeois, Wallace; Dandekar, Smita; Park, Eugene; Burleson, Tamara M; Madhusoodhan, Pillai Pallavi; Teachey, David T; Raetz, Elizabeth A; Hermiston, Michelle L; Müschen, Markus; Loh, Mignon L; Hunger, Stephen P; Zhang, Jinghui; Garabedian, Michael J; Porter, Christopher C; Carroll, William L

    2015-11-01

    The outcome for pediatric acute lymphoblastic leukemia (ALL) patients who relapse is dismal. A hallmark of relapsed disease is acquired resistance to multiple chemotherapeutic agents, particularly glucocorticoids. In this study, we performed a genome-scale short hairpin RNA screen to identify mediators of prednisolone sensitivity in ALL cell lines. The incorporation of these data with an integrated analysis of relapse-specific genetic and epigenetic changes allowed us to identify the mitogen-activated protein kinase (MAPK) pathway as a mediator of prednisolone resistance in pediatric ALL. We show that knockdown of the specific MAPK pathway members MEK2 and MEK4 increased sensitivity to prednisolone through distinct mechanisms. MEK4 knockdown increased sensitivity specifically to prednisolone by increasing the levels of the glucocorticoid receptor. MEK2 knockdown increased sensitivity to all chemotherapy agents tested by increasing the levels of p53. Furthermore, we demonstrate that inhibition of MEK1/2 with trametinib increased sensitivity of ALL cells and primary samples to chemotherapy in vitro and in vivo. To confirm a role for MAPK signaling in patients with relapsed ALL, we measured the activation of the MEK1/2 target ERK in matched diagnosis-relapse primary samples and observed increased phosphorylated ERK levels at relapse. Furthermore, relapse samples have an enhanced response to MEK inhibition compared to matched diagnosis samples in xenograft models. Together, our data indicate that inhibition of the MAPK pathway increases chemosensitivity to glucocorticoids and possibly other agents and that the MAPK pathway is an attractive target for prevention and/or treatment of relapsed disease. PMID:26324703

  15. An electrochemical biosensor for sensitive detection of microRNA-155: combining target recycling with cascade catalysis for signal amplification.

    PubMed

    Wu, Xiaoyan; Chai, Yaqin; Zhang, Pu; Yuan, Ruo

    2015-01-14

    In this work, a new electrochemical biosensor based on catalyzed hairpin assembly target recycling and cascade electrocatalysis (cytochrome c (Cyt c) and alcohol oxidase (AOx)) for signal amplification was constructed for highly sensitive detection of microRNA (miRNA). It is worth pointing out that target recycling was achieved only based on strand displacement process without the help of nuclease. Moreover, porous TiO2 nanosphere was synthesized, which could offer more surface area for Pt nanoparticles (PtNPs) enwrapping and enhance the amount of immobilized DNA strand 1 (S1) and Cyt c accordingly. With the mimicking sandwich-type reaction, the cascade catalysis amplification strategy was carried out by AOx catalyzing ethanol to acetaldehyde with the concomitant formation of high concentration of H2O2, which was further electrocatalyzed by PtNPs and Cyt c. This newly designed biosensor provided a sensitive detection of miRNA-155 from 0.8 fM to 1 nM with a relatively low detection limit of 0.35 fM. PMID:25495913

  16. Anti-complementary constituents of Houttuynia cordata and their targets in complement activation cascade.

    PubMed

    Jiang, Yun; Lu, Yan; Zhang, Yun-Yi; Chen, Dao-Feng

    2014-01-01

    Activity-guided fractionation for complement inhibitors led to the isolation of 23 known compounds from Houttuynia cordata Thunb. Seven flavonoids, two alkaloids, one coumarin and two phenols showed anti-complementary activity. Preliminary inhibitory mechanism of four flavonoids, including quercitrin, afzelin, isoquercitrin and quercetin in the complement activation cascade were examined for the first time. The results indicated that the target components of flavonols are different from those of flavonosides, and the glycoside moieties may be necessary to block C3 and C4 components. PMID:24423008

  17. Active mode locking of quantum cascade lasers in an external ring cavity.

    PubMed

    Revin, D G; Hemingway, M; Wang, Y; Cockburn, J W; Belyanin, A

    2016-01-01

    Stable ultrashort light pulses and frequency combs generated by mode-locked lasers have many important applications including high-resolution spectroscopy, fast chemical detection and identification, studies of ultrafast processes, and laser metrology. While compact mode-locked lasers emitting in the visible and near infrared range have revolutionized photonic technologies, the systems operating in the mid-infrared range where most gases have their strong absorption lines, are bulky and expensive and rely on nonlinear frequency down-conversion. Quantum cascade lasers are the most powerful and versatile compact light sources in the mid-infrared range, yet achieving their mode-locked operation remains a challenge, despite dedicated effort. Here we report the demonstration of active mode locking of an external-cavity quantum cascade laser. The laser operates in the mode-locked regime at room temperature and over the full dynamic range of injection currents. PMID:27147409

  18. Active mode locking of quantum cascade lasers in an external ring cavity

    NASA Astrophysics Data System (ADS)

    Revin, D. G.; Hemingway, M.; Wang, Y.; Cockburn, J. W.; Belyanin, A.

    2016-05-01

    Stable ultrashort light pulses and frequency combs generated by mode-locked lasers have many important applications including high-resolution spectroscopy, fast chemical detection and identification, studies of ultrafast processes, and laser metrology. While compact mode-locked lasers emitting in the visible and near infrared range have revolutionized photonic technologies, the systems operating in the mid-infrared range where most gases have their strong absorption lines, are bulky and expensive and rely on nonlinear frequency down-conversion. Quantum cascade lasers are the most powerful and versatile compact light sources in the mid-infrared range, yet achieving their mode-locked operation remains a challenge, despite dedicated effort. Here we report the demonstration of active mode locking of an external-cavity quantum cascade laser. The laser operates in the mode-locked regime at room temperature and over the full dynamic range of injection currents.

  19. Active mode locking of quantum cascade lasers in an external ring cavity

    PubMed Central

    Revin, D. G.; Hemingway, M.; Wang, Y.; Cockburn, J. W.; Belyanin, A.

    2016-01-01

    Stable ultrashort light pulses and frequency combs generated by mode-locked lasers have many important applications including high-resolution spectroscopy, fast chemical detection and identification, studies of ultrafast processes, and laser metrology. While compact mode-locked lasers emitting in the visible and near infrared range have revolutionized photonic technologies, the systems operating in the mid-infrared range where most gases have their strong absorption lines, are bulky and expensive and rely on nonlinear frequency down-conversion. Quantum cascade lasers are the most powerful and versatile compact light sources in the mid-infrared range, yet achieving their mode-locked operation remains a challenge, despite dedicated effort. Here we report the demonstration of active mode locking of an external-cavity quantum cascade laser. The laser operates in the mode-locked regime at room temperature and over the full dynamic range of injection currents. PMID:27147409

  20. The Role of Phospholipase D and MAPK Signaling Cascades in the Adaption of Lichen Microalgae to Desiccation: Changes in Membrane Lipids and Phosphoproteome.

    PubMed

    Gasulla, Francisco; Barreno, Eva; Parages, María L; Cámara, Joaquín; Jiménez, Carlos; Dörmann, Peter; Bartels, Dorothea

    2016-09-01

    Classically, lichen phycobionts are described as poikilohydric organisms able to undergo desiccation due to the constitutive presence of molecular protection mechanisms. However, little is known about the induction of cellular responses in lichen phycobionts during drying. The analysis of the lipid composition of the desiccated lichen microalga Asterochloris erici revealed the unusual accumulation of highly polar lipids (oligogalactolipids and phosphatidylinositol), which prevents the fusion of membranes during stress, but also the active degradation of cone-shaped lipids (monogalactosyldiacylglycerol and phosphatidylethanolamine) to stabilize membranes in desiccated cells. The level of phosphatidic acid increased 7-fold during desiccation, implicating a possible role for phospholipase D (PLD) in the response to osmotic stress. Inhibition of PLD with 1-butanol markedly impaired the recovery of photosynthesis activity in A. erici upon desiccation and salt stress (2 M NaCl). These two hyperosmotic stresses caused the phosphorylation of c-Jun N-terminal kinase (JNK) and p38-like mitogen-activated protein kinase (MAPK) and the dephosphorylation of extracellular signal-regulated kinase (ERK). The incubation with 1-butanol reduced the phosphorylation of JNK-like proteins and increased the dephosphorylation of ERK-like proteins, which indicates an upstream control of MAPK cascades by PLD. The phosphoproteome showed that desiccation caused the phosphorylation of several proteins in A. erici, most of them involved in protein turnover. The results demonstrate that lichen phycobionts possess both constitutive and inducible protective mechanisms to acquire desiccation tolerance. Among others, these responses are controlled by the PLD pathway through the activation of MAPK cascades. PMID:27335354

  1. A Co-Opted Hormonal Cascade Activates Dormant Adventitious Root Primordia upon Flooding in Solanum dulcamara1[OPEN

    PubMed Central

    Dawood, Thikra; Kensche, Philip R.; Cristescu, Simona M.; Mariani, Celestina

    2016-01-01

    Soil flooding is a common stress factor affecting plants. To sustain root function in the hypoxic environment, flooding-tolerant plants may form new, aerenchymatous adventitious roots (ARs), originating from preformed, dormant primordia on the stem. We investigated the signaling pathway behind AR primordium reactivation in the dicot species Solanum dulcamara. Transcriptome analysis indicated that flooding imposes a state of quiescence on the stem tissue, while increasing cellular activity in the AR primordia. Flooding led to ethylene accumulation in the lower stem region and subsequently to a drop in abscisic acid (ABA) level in both stem and AR primordia tissue. Whereas ABA treatment prevented activation of AR primordia by flooding, inhibition of ABA synthesis was sufficient to activate them in absence of flooding. Together, this reveals that there is a highly tissue-specific response to reduced ABA levels. The central role for ABA in the response differentiates the pathway identified here from the AR emergence pathway known from rice (Oryza sativa). Flooding and ethylene treatment also induced expression of the polar auxin transporter PIN2, and silencing of this gene or chemical inhibition of auxin transport inhibited primordium activation, even though ABA levels were reduced. Auxin treatment, however, was not sufficient for AR emergence, indicating that the auxin pathway acts in parallel with the requirement for ABA reduction. In conclusion, adaptation of S. dulcamara to wet habitats involved co-option of a hormonal signaling cascade well known to regulate shoot growth responses, to direct a root developmental program upon soil flooding. PMID:26850278

  2. 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

  3. Antihyperglycemic potentials of a threatened plant, Helonias dioica: antioxidative stress responses and the signaling cascade.

    PubMed

    Chakraborty, Debrup; Samadder, Asmita; Dutta, Suman; Khuda-Bukhsh, Anisur Rahman

    2012-01-01

    Helonias dioica (HD) is a threatened species of herb growing in North America. It is used as a traditional medicine for treating various ailments particularly related to reproductive issues. The root is reported to contain approximately 10% of a saponin (chamaelirin; C(36)H(62)O(18)) apart from certain other fatty acids. As saponins are known to have hypoglycemic effects, we suspected its possible antihyperglycemic potentials. We injected intraperitoneally alloxan (ALX) at the dose of 200 mg/kg body weight (bw) to induce hyperglycemia in mice and tested possible hypoglycemic effects of HD in vivo by deploying two doses (100 and 200 mg/kg bw, respectively). We also tested its effects on the isolated pancreatic islets cells in vitro. We used various standard protocols like reactive oxygen species (ROS) generation and DNA damage, activities of biomarkers like catalase (CAT), superoxide dismutase (SOD), lipid peroxidase (LPO), reduced glutathione (GSH) of the pancreas tissue and glucokinase and glycogen content of the liver of hyperglycemic mice. With a mechanistic approach, we also tracked down the possible signaling pathway involved. We found an elevated level of ROS generation, LPO and overexpression of inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), p38 Map kinase (p38 MAPK), nuclear factor (NF)-κβ, interferon gamma (IFN-γ), cytochrome c, caspase 3, poly [ADP ribose] polymerase (PARP) and cyclo oxygenase 2 (COX2) in ALX-induced diabetic mouse. Treatment of hyperglycemic mice with both the doses of HD showed a significant decrease with respect to all these parameters of study. Thus, our results suggest that HD prevents ALX-induced islet cell damage and possesses antihyperglycemic and antioxidative potentials. PMID:22169161

  4. GPR179 Is Required for High Sensitivity of the mGluR6 Signaling Cascade in Depolarizing Bipolar Cells

    PubMed Central

    Ray, Thomas A.; Heath, Kathryn M.; Hasan, Nazarul; Noel, Jennifer M.; Samuels, Ivy S.; Martemyanov, Kirill A.; Peachey, Neal S.

    2014-01-01

    Parallel visual pathways are initiated at the first retinal synapse by signaling between the rod and cone photoreceptors and two general classes of bipolar cells. For normal function, ON or depolarizing bipolar cells (DBCs) require the G-protein-coupled receptor, mGluR6, an intact G-protein-coupled cascade and the transient receptor potential melastatin 1 (TRPM1) cation channel. In addition, another seven transmembrane protein, GPR179, is required for DBC function and recruits the regulators of G-protein signaling (RGS) proteins, RGS7 and RGS11, to the dendritic tips of the DBCs. Here we use the Gpr179nob5 mouse, which lacks GPR179 and has a no b-wave electroretinogram (ERG) phenotype, to demonstrate that despite the absence of both GPR179 and RGS7/RGS11, a small dark-adapted ERG b-wave remains and can be enhanced with long duration flashes. Consistent with the ERG, the mGluR6-mediated gating of TRPM1 can be evoked pharmacologically in Gpr179nob5 and RGS7−/−/RGS11−/− rod BCs if strong stimulation conditions are used. In contrast, direct gating of TRPM1 by capsaicin in RGS7−/−/RGS11−/− and WT rod BCs is similar, but severely compromised in Gpr179nob5 rod BCs. Noise and standing current analyses indicate that the remaining channels in Gpr179nob5 and RGS7−/−/RGS11−/− rod BCs have a very low open probability. We propose that GPR179 along with RGS7 and RGS11 controls the ability of the mGluR6 cascade to gate TRPM1. In addition to its role in localizing RGS7 and RGS11 to the dendritic tips, GPR179 via a direct interaction with the TRPM1 channel alters its ability to be gated directly by capsaicin. PMID:24790204

  5. The ATM Signaling Cascade Promotes Recombination-Dependent Pachytene Arrest in Mouse Spermatocytes

    PubMed Central

    Lange, Julian; Jasin, Maria; Keeney, Scott; Roig, Ignasi

    2015-01-01

    Most mutations that compromise meiotic recombination or synapsis in mouse spermatocytes result in arrest and apoptosis at the pachytene stage of the first meiotic prophase. Two main mechanisms are thought to trigger arrest: one independent of the double-strand breaks (DSBs) that initiate meiotic recombination, and another activated by persistent recombination intermediates. Mechanisms underlying the recombination-dependent arrest response are not well understood, so we sought to identify factors involved by examining mutants deficient for TRIP13, a conserved AAA+ ATPase required for the completion of meiotic DSB repair. We find that spermatocytes with a hypomorphic Trip13 mutation (Trip13mod/mod) arrest with features characteristic of early pachynema in wild type, namely, fully synapsed chromosomes without incorporation of the histone variant H1t into chromatin. These cells then undergo apoptosis, possibly in response to the arrest or in response to a defect in sex body formation. However, TRIP13-deficient cells that additionally lack the DSB-responsive kinase ATM progress further, reaching an H1t-positive stage (i.e., similar to mid/late pachynema in wild type) despite the presence of unrepaired DSBs. TRIP13-deficient spermatocytes also progress to an H1t-positive stage if ATM activity is attenuated by hypomorphic mutations in Mre11 or Nbs1 or by elimination of the ATM-effector kinase CHK2. These mutant backgrounds nonetheless experience an apoptotic block to further spermatogenic progression, most likely caused by failure to form a sex body. DSB numbers are elevated in Mre11 and Nbs1 hypomorphs but not Chk2 mutants, thus delineating genetic requirements for the ATM-dependent negative feedback loop that regulates DSB numbers. The findings demonstrate for the first time that ATM-dependent signaling enforces the normal pachytene response to persistent recombination intermediates. Our work supports the conclusion that recombination defects trigger spermatocyte arrest

  6. Smad4-Irf6 genetic interaction and TGFβ-mediated IRF6 signaling cascade are crucial for palatal fusion in mice

    PubMed Central

    Iwata, Jun-ichi; Suzuki, Akiko; Pelikan, Richard C.; Ho, Thach-Vu; Sanchez-Lara, Pedro A.; Urata, Mark; Dixon, Michael J.; Chai, Yang

    2013-01-01

    Cleft palate is one of the most common human birth defects and is associated with multiple genetic and environmental risk factors. Although mutations in the genes encoding transforming growth factor beta (TGFβ) signaling molecules and interferon regulatory factor 6 (Irf6) have been identified as genetic risk factors for cleft palate, little is known about the relationship between TGFβ signaling and IRF6 activity during palate formation. Here, we show that TGFβ signaling regulates expression of Irf6 and the fate of the medial edge epithelium (MEE) during palatal fusion in mice. Haploinsufficiency of Irf6 in mice with basal epithelial-specific deletion of the TGFβ signaling mediator Smad4 (Smad4fl/fl;K14-Cre;Irf6+/R84C) results in compromised p21 expression and MEE persistence, similar to observations in Tgfbr2fl/fl;K14-Cre mice, although the secondary palate of Irf6+/R84C and Smad4fl/fl;K14-Cre mice form normally. Furthermore, Smad4fl/fl;K14-Cre;Irf6+/R84C mice show extra digits that are consistent with abnormal toe and nail phenotypes in individuals with Van der Woude and popliteal pterygium syndromes, suggesting that the TGFβ/SMAD4/IRF6 signaling cascade might be a well-conserved mechanism in regulating multiple organogenesis. Strikingly, overexpression of Irf6 rescued p21 expression and MEE degeneration in Tgfbr2fl/fl;K14-Cre mice. Thus, IRF6 and SMAD4 synergistically regulate the fate of the MEE, and TGFβ-mediated Irf6 activity is responsible for MEE degeneration during palatal fusion in mice. PMID:23406900

  7. Rottlerin Inhibits Lonicera japonica-Induced Photokilling in Human Lung Cancer Cells through Cytoskeleton-Related Signaling Cascade

    PubMed Central

    You, Bang-Jau; Wu, Yang-Chang; Bao, Bo-Ying; Wu, Chi-Yu; Yang, Ya-Win; Chang, Yu-Hao; Lee, Hong-Zin

    2011-01-01

    This study demonstrated that many apoptotic signaling pathways, such as Rho family, PKC family, MAP kinase family, and mitochondria-mediated apoptotic pathway, were triggered by Lonicera japonica extracts and irradiation in CH27 cells. Rottlerin, a PKCδ -selective inhibitor, reversed the photoactivated Lonicera japonica extract-induced decrease in PKCδ protein expression and change in cell morphology in this study. In addition, rottlerin inhibited the photoactivated Lonicera japonica-induced decrease in protein expression of Ras, ERK, p38, PKCα, and PKCε, which are the kinases of prosurvival signaling pathway. We also demonstrated that pretreatment with rottlerin prevented actin microfilaments and microtubules from damage during the photoactivated Lonicera japonica-induced CH27 cell death. Furthermore, the promotion of the cytoskeleton-related signaling cascade following rottlerin by upregulation of cytoskeleton-related mediators (p38, HSP27, FAK, paxillin, and tubulin) and molecules of downstream of F-actin (mitochondria-mediated apoptosis pathway) reduces CH27 cell death, indicating that cytoskeleton is the potential target in the photoactivated Lonicera japonicaextract-induced photokilling of CH27 cells. PMID:21331326

  8. Linewidth Enhancement Factor and Amplification of Angle-Modulated Optical Signals in Injection-Locked Quantum Cascade Lasers

    NASA Astrophysics Data System (ADS)

    Chattopadhyay, Taraprasad; Bhattacharyya, Prosenjit

    2016-03-01

    This paper presents a nonlinear analysis of the effect of linewidth enhancement factor (LEF) on the amplification of angle-modulated optical signals in injection-locked mid-infrared (IR) quantum cascade lasers (QCLs). A higher value of LEF tends to conserve the output angle modulation index of the amplified mid-IR signal particularly in the low-modulation frequency region. Further, a higher value of signal injection ratio produces a wider bandwidth of the locked QCL amplifier. The LEF introduces asymmetry in the lockband (LB) of the injection-locked QCL and this asymmetry increases with the increase in the value of LEF. Typically ratio of calculated lower-side LB to upper-side LB for an injection power level of - 20 dB and a LEF of unity is 1.67. The electron relaxation time in the uppermost subband lasing level in a three-level system has a profound effect on the LB asymmetry in a QCL.

  9. Thermal conductivity tensors of the cladding and active layers of interband cascade lasers

    NASA Astrophysics Data System (ADS)

    Zhou, Chuanle; Cui, Boya; Vurgaftman, I.; Canedy, C. L.; Kim, C. S.; Kim, M.; Bewley, W. W.; Merritt, C. D.; Abell, J.; Meyer, J. R.; Grayson, M.

    2014-12-01

    The cross-plane and in-plane thermal conductivities of the W-active stages and InAs/AlSb superlattice optical cladding layer of an interband cascade laser (ICL) were characterized for temperatures ranging from 15 K to 324 K. The in-plane thermal conductivity of the active layer is somewhat larger than the cross-plane value at temperatures above about 30 K, while the thermal conductivity tensor becomes nearly isotropic at the lowest temperatures studied. These results will improve ICL performance simulations and guide the optimization of thermal management.

  10. 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.

  11. 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

  12. Tools used to study how protein complexes are assembled in signaling cascades

    PubMed Central

    Dwane, Susan

    2011-01-01

    Most proteins do not function on their own but as part of large signaling complexes that are arranged in every living cell in response to specific environmental cues. Proteins interact with each other either constitutively or transiently and do so with different affinity. When identifying the role played by a protein inside a cell, it is essential to define its particular cohort of binding partners so that the researcher can predict what signaling pathways the protein is engaged in. Once identified and confirmed, the information might allow the interaction to be manipulated by pharmacological inhibitors to help fight disease. PMID:22002082

  13. Overexpression of miR-223 Tips the Balance of Pro- and Anti-hypertrophic Signaling Cascades toward Physiologic Cardiac Hypertrophy.

    PubMed

    Yang, Liwang; Li, Yutian; Wang, Xiaohong; Mu, Xingjiang; Qin, Dongze; Huang, Wei; Alshahrani, Saeed; Nieman, Michelle; Peng, Jiangtong; Essandoh, Kobina; Peng, Tianqing; Wang, Yigang; Lorenz, John; Soleimani, Manoocher; Zhao, Zhi-Qing; Fan, Guo-Chang

    2016-07-22

    MicroRNAs (miRNAs) have been extensively examined in pathological cardiac hypertrophy. However, few studies focused on profiling the miRNA alterations in physiological hypertrophic hearts. In this study we generated a transgenic mouse model with cardiac-specific overexpression of miR-223. Our results showed that elevation of miR-223 caused physiological cardiac hypertrophy with enhanced cardiac function but no fibrosis. Using the next generation RNA sequencing, we observed that most of dys-regulated genes (e.g. Atf3/5, Egr1/3, Sfrp2, Itgb1, Ndrg4, Akip1, Postn, Rxfp1, and Egln3) in miR-223-transgenic hearts were associated with cell growth, but they were not directly targeted by miR-223. Interestingly, these dys-regulated genes are known to regulate the Akt signaling pathway. We further identified that miR-223 directly interacted with 3'-UTRs of FBXW7 and Acvr2a, two negative regulators of the Akt signaling. However, we also validated that miR-223 directly inhibited the expression of IGF-1R and β1-integrin, two positive regulators of the Akt signaling. Lastly, Western blotting did reveal that Akt was activated in miR-223-overexpressing hearts. Adenovirus-mediated overexpression of miR-223 in neonatal rat cardiomyocytes induced cell hypertrophy, which was blocked by the addition of MK2206, a specific inhibitor of Akt Taken together, these data represent the first piece of work showing that miR-223 tips the balance of promotion and inactivation of Akt signaling cascades toward activation of Akt, a key regulator of physiological cardiac hypertrophy. Thus, our study suggests that the ultimate phenotype outcome of a miRNA may be decided by the secondary net effects of the whole target network rather than by several primary direct targets in an organ/tissue. PMID:27226563

  14. Do signal transduction cascades influence survival in triple-negative breast cancer? A preliminary study

    PubMed Central

    Mumm, Jan-Niclas; Kölbl, Alexandra C; Jeschke, Udo; Andergassen, Ulrich

    2016-01-01

    Background Triple-negative breast cancer (TNBC) is a rather aggressive form of breast cancer, comprised by early metastasis formation and reduced overall survival of the affected patients. Steroid hormone receptors and the human epidermal growth factor receptor 2 are not overexpressed, limiting therapeutic options. Therefore, new treatment options have to be investigated. The aim of our preliminary study was to detect coherences between some molecules of intracellular signal transduction pathways and survival of patients with TNBC, in order to obtain some hints for new therapeutical solutions. Methods Thirty-one paraffin-embedded tumor tissue samples, which were determined to be negative for steroid hormone receptors as well as human epidermal growth factor receptor 2, were immunohistochemically stained for a number of signal transduction molecules from several signaling pathways. β-Catenin, HIF1α, MCL, Notch1, LRP6, XBP1, and FOXP3 were stained with specific antibodies, and their staining was correlated with patient survival by Kaplan–Meier analyses. Results Only two of the investigated molecules have shown correlation with overall survival. Cytoplasmic staining of HIF1α and centro-tumoral lymphocyte FOXP3 staining showed statistically significant correlations with survival. Conclusion The coherence of signal transduction molecules with survival of patients with TNBC is still controversially discussed in the literature. Our study comprises one more mosaic stone in the elucidation of these intracellular processes and their influences on patient outcome. Lots of research still has to be done in this field, but it would be worthwhile as it may offer new therapeutic targets for a group of patients with breast cancer, which is still hard to treat. PMID:27307757

  15. 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

  16. Two cascaded SOAs used as intensity modulators for adaptively modulated optical OFDM signals in optical access networks.

    PubMed

    Hamié, Ali; Hamzé, Mohamad; Taki, Haidar; Makouk, Layaly; Sharaiha, Ammar; Alaeddine, Ali; Al Housseini, Ali; Giacoumidis, Elias; Tang, J M

    2014-06-30

    Detailed theoretical and numerical investigations of the transmission performance of adaptively modulated optical orthogonal frequency division multiplexed (AMOOFDM) signals are undertaken, for the first time, in optical amplification and chromatic dispersion (CD) compensation free single mode fiber (SMF) intensity-modulated and direct-detection (IMDD) systems using two cascaded semiconductor optical amplifiers in a counterpropagating configuration as an intensity modulator (TC-SOA-CC-IM). A theoretical model describing the characteristics of this configuration is developed. Extensive performance comparisons are also made between the TC-SOA-CC and the single SOA intensity modulators. It is shown that, the TC-SOA-CC reaches its strongly saturated region using a lower input optical power much faster than the single SOA resulting in significantly reduced effective carrier lifetime and thus wide TC-SOA-CC bandwidths. It is shown that at low input optical power, we can increase the signal line rate almost 115% which will be more than twice the transmission performance offered by single SOA. In addition, the TC-SOA-CC-IM is capable of supporting signal line rates higher than corresponding to the SOA-IM by using 10dB lower input optical powers. For long transmission distance, the TC-SOA-CC-IM has much stronger CD compensation capability compared to the SOA-IM. In addition the use of TC-SOA-CC-IM is more effective regarding the capability to benefit from the CD compensation for shorter distances starting at 60km SMF, whilst for the SOA-IM starting at 90km. PMID:24977835

  17. Transcriptomics of Actinorhizal Symbioses Reveals Homologs of the Whole Common Symbiotic Signaling Cascade1[W

    PubMed Central

    Hocher, Valérie; Alloisio, Nicole; Auguy, Florence; Fournier, Pascale; Doumas, Patrick; Pujic, Petar; Gherbi, Hassen; Queiroux, Clothilde; Da Silva, Corinne; Wincker, Patrick; Normand, Philippe; Bogusz, Didier

    2011-01-01

    Comparative transcriptomics of two actinorhizal symbiotic plants, Casuarina glauca and Alnus glutinosa, was used to gain insight into their symbiotic programs triggered following contact with the nitrogen-fixing actinobacterium Frankia. Approximately 14,000 unigenes were recovered in roots and 3-week-old nodules of each of the two species. A transcriptomic array was designed to monitor changes in expression levels between roots and nodules, enabling the identification of up- and down-regulated genes as well as root- and nodule-specific genes. The expression levels of several genes emblematic of symbiosis were confirmed by quantitative polymerase chain reaction. As expected, several genes related to carbon and nitrogen exchange, defense against pathogens, or stress resistance were strongly regulated. Furthermore, homolog genes of the common and nodule-specific signaling pathways known in legumes were identified in the two actinorhizal symbiotic plants. The conservation of the host plant signaling pathway is all the more surprising in light of the lack of canonical nod genes in the genomes of its bacterial symbiont, Frankia. The evolutionary pattern emerging from these studies reinforces the hypothesis of a common genetic ancestor of the Fabid (Eurosid I) nodulating clade with a genetic predisposition for nodulation. PMID:21464474

  18. Gigabit radio-over-fiber link for converged baseband and millimeter-wave band signal transmission using cascaded injection-locked Fabry-Pérot laser diodes.

    PubMed

    Hong, Moon-Ki; Won, Yong-Yuk; Han, Sang-Kook

    2009-05-11

    A novel scheme, for both baseband and millimeter-wave band gigabit data transmission in radio-over-fiber system, is proposed and experimentally demonstrated by using cascaded injection-locked Fabry- Pérot laser diodes. It was able to improve suppression ratio of carrier suppressed signal using the cascaded injection-locking. The suppression ratio improvement of the optical carrier suppressed signal of 20 dB was verified. Applying this mechanism, 60-GHz millimeter-wave carrier of enhanced signal quality could be accomplished. Its peak power and phase noise were obtained as -40 dBm and -103.5 dBm/Hz respectively, which was suitable for 60-GHz data transmission. In addition, a successful bidirectional transmission of 1.25-Gbps wired and wireless data was achieved by adopting remodulation technique using a gain-saturated reflective semiconductor optical amplifier for uplink. PMID:19434116

  19. Apoptosis induced in rats by 4-vinylcyclohexene diepoxide is associated with activation of the caspase cascades.

    PubMed

    Hu, X; Christian, P J; Thompson, K E; Sipes, I G; Hoyer, P B

    2001-07-01

    Previous studies have shown that ovotoxicity induced in rats by dosing with 4-vinylcyclohexene diepoxide (VCD) is likely via acceleration of the normal rate of atresia (apoptosis). The present study was designed to investigate the apoptosis-related caspase cascades as a component of this phenomenon in isolated ovarian small follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., on Day 1; a time when ovotoxicity has not been initiated), or dosed daily for 15 days (80 mg/kg, i.p., on Day 15; a time when significant ovotoxicity is underway). Ovaries were collected after the final dose. Small preantral follicles (25-100 microm in diameter) were isolated, cellular fractions were prepared, and cleavage activity or protein expression levels of caspases-3, -8, and -9 were measured. Cytosolic caspase-3 activity was increased in small follicles (P < 0.01) by VCD treatment (Day 1, 2.86 +/- 0.23; Day 15, 3.25 +/- 0.64, VCD/control, n = 3). This activation was not seen in large or antral follicles (not targeted by VCD). Procaspase-3 protein was increased(P < 0.05) by VCD treatment 212% over controls in small ovarian follicles in Day 15, but not Day 1-dosed rats. Immunofluorescence staining intensity was evaluated by confocal microscopy. Caspase-3 protein, located in the cytosolic compartment of oocytes and granulosa cells of preantral follicles in various stages of development, was selectively increased (P < 0.05) in primordial and small primary follicles from Day 15 VCD-dosed rats. Caspase-8 activity was increased in small follicles in Day 15, but not in Day 1-treated rats; whereas caspase-9 activity was increased by VCD on Day 1 in the mitochondrial fraction. Thus, these data provide evidence that accelerated atresia induced in small ovarian follicles in rats by VCD is associated with activation of a caspase-mediated cascade. PMID:11420227

  20. Effects of high-orbit spaceflight on signaling cascades and apoptosis in immune cells from mice flied on board the BION-M1 satellite

    NASA Astrophysics Data System (ADS)

    Novoselova, Elena; Shenkman, Boris; Lunin, Sergey; Parfenyuk, Svetlana; Novoselova, Tatyana; Fesenko, Eugeny

    The study was designed to evaluate immune cell activity in male C57bl mice after a 30-day high-orbit spaceflight (550 km, higher than conventional manned spaceflights) on board the BION-M1 satellite (Roskosmos Program, Russia). For the present study, thymus, spleens and plasma samples were collected from mice 12 h after landing and, additionally, 7 days subsequently. Assessing the activity of NF-kappaB signaling cascade by measuring Rel A (p65) protein phosphorylation in splenic lymphocytes, we showed that the NF-kappaB activity was significantly increased at 12 h after landing. Contrariwise, one week after landing, the NF-kappaB activity was markedly decreased, even below to the control values. Interestingly, after landing there were no significant changes in SAPK/JNK cascade activity in splenic lymphocytes as well as in the expression of transcription factor IRF3 in thymus cells. To assess the apoptosis status in thymus lymphocytes, levels of p53 protein and its phosphorylated form were measured in thymic lymphocytes. It is known that p53 plays an important role in the cellular response to DNA damage, genomic aberrations, and other characteristic of apoptosis. The results showed that the high-orbit spaceflight environment caused some increase in level of p53 protein, but most notably, activated phosphorylated form of p53 protein. Calculated ratio of active and inactive forms of the protein (ph-p53/p53) 12 h after landing increased by more than 2-fold, indicating the apparent induction of apoptosis in thymus cells. Interestingly, 7 days after the landing, this ratio was not restored, but rather increased: the specified ratio was 4 times higher as compared to the ground-based control. We can conclude that response to the prolonged high-orbit spaceflight is not like the classic "stress response", which is usually observed under various stressful factors. It is known that the stress response is surely accompanied by increased SAPK/JNK cascade activity as well as the

  1. Combination of spices and herbal extract restores macrophage foam cell migration and abrogates the athero-inflammatory signalling cascade of atherogenesis.

    PubMed

    Nimgulkar, Chetan; Ghosh, Sudip; Sankar, Anand B; Uday, Kumar P; Surekha, M V; Madhusudhanachary, P; Annapurna, B R; Raghu, P; Bharatraj, Dinesh Kumar

    2015-09-01

    The trapping of lipid-laden macrophages in the arterial intima is a critical but reversible step in atherogenesis. However, information about possible treatments for this condition is lacking. Here, we hypothesized that combining the polyphenol-rich fractions (PHC) of commonly consumed spices (Allium sativum L (Liliaceae), Zingiber officinale R (Zingiberaceae), Curcuma longa L (Zingiberaceae)) and herbs (Terminalia arjuna (R) W & A (Combretaceae) and Cyperus rotundus L (Cyperaceae)) prevents foam cell formation and atherogenesis. Using an in vitro foam cell formation assay, we found that PHC significantly inhibited lipid-laden macrophage foam cell formation compared to the depleted polyphenol fraction of PHC (F-PHC). We further observed that PHC attenuated the LDL and LPS induced CD36, p-FAK and PPAR-γ protein expression in macrophages and increased their migration. NK-κB-DNA interaction, TNF-α, ROS generation, and MMP9 and MMP2 protein expression were suppressed in PHC-treated macrophages. The anti-atherosclerotic activity of PHC was investigated in a high fat- and cholesterol-fed rabbit model. The inhibition of foam cell deposition within the aortic intima and atheroma formation confirmed the atheroprotective activity of PHC. Therefore, we conclude that the armoury of polyphenols in PHC attenuates the CD36 signalling cascade-mediated foam cell formation, enhances the migration of these cells and prevents atherogenesis. PMID:25869517

  2. Cocaine elicits autophagic cytotoxicity via a nitric oxide-GAPDH signaling cascade.

    PubMed

    Guha, Prasun; Harraz, Maged M; Snyder, Solomon H

    2016-02-01

    Cocaine exerts its behavioral stimulant effects by facilitating synaptic actions of neurotransmitters such as dopamine and serotonin. It is also neurotoxic and broadly cytotoxic, leading to overdose deaths. We demonstrate that the cytotoxic actions of cocaine reflect selective enhancement of autophagy, a process that physiologically degrades metabolites and cellular organelles, and that uncontrolled autophagy can also lead to cell death. In brain cultures, cocaine markedly increases levels of LC3-II and depletes p62, both actions characteristic of autophagy. By contrast, cocaine fails to stimulate cell death processes reflecting parthanatos, monitored by cleavage of poly(ADP ribose)polymerase-1 (PARP-1), or necroptosis, assessed by levels of phosphorylated mixed lineage kinase domain-like protein. Pharmacologic inhibition of autophagy protects neurons against cocaine-induced cell death. On the other hand, inhibition of parthanatos, necroptosis, or apoptosis did not change cocaine cytotoxicity. Depletion of ATG5 or beclin-1, major mediators of autophagy, prevents cocaine-induced cell death. By contrast, depleting caspase-3, whose cleavage reflects apoptosis, fails to alter cocaine cytotoxicity, and cocaine does not alter caspase-3 cleavage. Moreover, depleting PARP-1 or RIPK1, key mediators of parthanatos and necroptosis, respectively, did not prevent cocaine-induced cell death. Autophagic actions of cocaine are mediated by the nitric oxide-glyceraldehyde-3-phosphate dehydrogenase signaling pathway. Thus, cocaine-associated autophagy is abolished by depleting GAPDH via shRNA; by the drug CGP3466B, which prevents GAPDH nitrosylation; and by mutating cysteine-150 of GAPDH, its site of nitrosylation. Treatments that selectively influence cocaine-associated autophagy may afford therapeutic benefit. PMID:26787898

  3. Cocaine elicits autophagic cytotoxicity via a nitric oxide-GAPDH signaling cascade

    PubMed Central

    Guha, Prasun; Harraz, Maged M.; Snyder, Solomon H.

    2016-01-01

    Cocaine exerts its behavioral stimulant effects by facilitating synaptic actions of neurotransmitters such as dopamine and serotonin. It is also neurotoxic and broadly cytotoxic, leading to overdose deaths. We demonstrate that the cytotoxic actions of cocaine reflect selective enhancement of autophagy, a process that physiologically degrades metabolites and cellular organelles, and that uncontrolled autophagy can also lead to cell death. In brain cultures, cocaine markedly increases levels of LC3-II and depletes p62, both actions characteristic of autophagy. By contrast, cocaine fails to stimulate cell death processes reflecting parthanatos, monitored by cleavage of poly(ADP ribose)polymerase-1 (PARP-1), or necroptosis, assessed by levels of phosphorylated mixed lineage kinase domain-like protein. Pharmacologic inhibition of autophagy protects neurons against cocaine-induced cell death. On the other hand, inhibition of parthanatos, necroptosis, or apoptosis did not change cocaine cytotoxicity. Depletion of ATG5 or beclin-1, major mediators of autophagy, prevents cocaine-induced cell death. By contrast, depleting caspase-3, whose cleavage reflects apoptosis, fails to alter cocaine cytotoxicity, and cocaine does not alter caspase-3 cleavage. Moreover, depleting PARP-1 or RIPK1, key mediators of parthanatos and necroptosis, respectively, did not prevent cocaine-induced cell death. Autophagic actions of cocaine are mediated by the nitric oxide-glyceraldehyde-3-phosphate dehydrogenase signaling pathway. Thus, cocaine-associated autophagy is abolished by depleting GAPDH via shRNA; by the drug CGP3466B, which prevents GAPDH nitrosylation; and by mutating cysteine-150 of GAPDH, its site of nitrosylation. Treatments that selectively influence cocaine-associated autophagy may afford therapeutic benefit. PMID:26787898

  4. The collagen triple helix repeat containing 1 facilitates hepatitis B virus-associated hepatocellular carcinoma progression by regulating multiple cellular factors and signal cascades.

    PubMed

    Zhang, Rui; Cao, Yanhua; Bai, Lan; Zhu, Chengliang; Li, Rui; He, Hui; Liu, Yingle; Wu, Kailang; Liu, Fang; Wu, Jianguo

    2015-12-01

    Hepatitis B virus (HBV) infection is one of the major causes of acute and chronic liver diseases, fulminant hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HCC accounts for more than 85% of primary liver cancers and is the seventh most common cancer and the third leading cause of cancer-related deaths. However, the mechanism by which HBV induces HCC is largely unknown. Collagen triple helixes repeat containing 1 (CTHRC1) is a secreted protein and has characteristics of a circulating hormone with potentially broad implications for cell metabolism and physiology. CTHRC1 is associated with human cancers, but its effect on HCC is unknown. Here, we revealed that CTHRC1 expression is highly correlated with HCC progression in HBV-infected patients, and demonstrated that HBV stimulates CTHRC1 expression by activating nuclear factor-kappa B (NF-κB) and cAMP response element binding protein (CREB), through extracellular signal-regulated kinase/c-Jun N-terminal kinase (ERK/c-JNK) pathway. In addition, CTHRC1 activates hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) through regulating phosphoinosmde-3-kinase/protein kinase B/mammalian target of rapamycin (PI-3K/AKT/mTOR) pathway. More interestingly, CTHRC1 enhances colony formation, migration, and invasion of hepatoma cells by regulating p53 and stimulating matrix metalloproteinase-9 (MMP-9) expression. In addition, knock-down of CTHRC1 results in the repression of HBV-associated carcinogenesis in nude mice. Thus, we revealed a novel mechanism by which HBV facilitates HCC development through activating the oncoprotein CTHRC1, which in turn enhances HBV-related HCC progression by stimulates colony formation, migration, and invasion of hepatoma cells through regulating multiple cellular factors and signal cascades. PMID:25263696

  5. A defect in the inflammation-primed macrophage-activation cascade in osteopetrotic rats.

    PubMed

    Yamamoto, N; Lindsay, D D; Naraparaju, V R; Ireland, R A; Popoff, S N

    1994-05-15

    Macrophages were activated by administration of lysophosphatidylcholine (lyso-Pc) or dodecylglycerol (DDG) to wild-type rats but not in osteopetrotic (op) mutant rats. In vitro treatment of wild-type rat peritoneal cells with lyso-Pc or DDG efficiently activated macrophages whereas treatment of op mutant rat peritoneal cells with lyso-Pc or DDG did not activate macrophages. The inflammation-primed macrophage activation cascade in rats requires participation of B lymphocytes and vitamin D binding protein (DBP). Lyso-Pc-inducible beta-galactosidase of wild-type rat B lymphocytes can convert DBP to the macrophage-activating factor (MAF), whereas B lymphocytes of the op mutant rats were shown to be deficient in lyso-Pc-inducible beta-galactosidase. DBP is conserved among mammalian species. Treatment of human DBP (Gc1 protein) with commercial glycosidases yields an extremely high titrated MAF as assayed on mouse and rat macrophages. Because the enzymatically generated MAF (GcMAF) bypasses the role of lymphocytes in macrophage activation, the op mutant rat macrophages were efficiently activated by administration of a small quantity (100 pg/rat) of GcMAF. Likewise, in vitro treatment of op rat peritoneal cells with as little as 40 pg GcMAF/ml activated macrophages. PMID:8176226

  6. 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

  7. Identification of a Novel lincRNA-p21-miR-181b-PTEN Signaling Cascade in Liver Fibrosis.

    PubMed

    Yu, Fujun; Lu, Zhongqiu; Chen, Bicheng; Dong, Peihong; Zheng, Jianjian

    2016-01-01

    Previously, we found that long intergenic noncoding RNA-p21 (lincRNA-p21) inhibits hepatic stellate cell (HSC) activation and liver fibrosis via p21. However, the underlying mechanism of the antifibrotic role of lincRNA-p21 in liver fibrosis remains largely unknown. Here, we found that lincRNA-p21 expression was significantly downregulated during liver fibrosis. In LX-2 cells, the reduction of lincRNA-p21 induced by TGF-β1 was in a dose- and time-dependent manner. lincRNA-p21 expression was reduced in liver tissues from patients with liver cirrhosis when compared with that of healthy controls. Notably, lincRNA-p21 overexpression contributed to the suppression of HSC activation. lincRNA-p21 suppressed HSC proliferation and induced a significant reduction in α-SMA and type I collagen. All these effects induced by lincRNA-p21 were blocked down by the loss of PTEN, suggesting that lincRNA-p21 suppressed HSC activation via PTEN. Further study demonstrated that microRNA-181b (miR-181b) was involved in the effects of lincRNA-p21 on HSC activation. The effects of lincRNA-p21 on PTEN expression and HSC activation were inhibited by miR-181b mimics. We demonstrated that lincRNA-p21 enhanced PTEN expression by competitively binding miR-181b. In conclusion, our results disclose a novel lincRNA-p21-miR-181b-PTEN signaling cascade in liver fibrosis and suggest lincRNA-p21 as a promising molecular target for antifibrosis therapy. PMID:27610008

  8. Identification of a Novel lincRNA-p21-miR-181b-PTEN Signaling Cascade in Liver Fibrosis

    PubMed Central

    Yu, Fujun; Lu, Zhongqiu; Chen, Bicheng

    2016-01-01

    Previously, we found that long intergenic noncoding RNA-p21 (lincRNA-p21) inhibits hepatic stellate cell (HSC) activation and liver fibrosis via p21. However, the underlying mechanism of the antifibrotic role of lincRNA-p21 in liver fibrosis remains largely unknown. Here, we found that lincRNA-p21 expression was significantly downregulated during liver fibrosis. In LX-2 cells, the reduction of lincRNA-p21 induced by TGF-β1 was in a dose- and time-dependent manner. lincRNA-p21 expression was reduced in liver tissues from patients with liver cirrhosis when compared with that of healthy controls. Notably, lincRNA-p21 overexpression contributed to the suppression of HSC activation. lincRNA-p21 suppressed HSC proliferation and induced a significant reduction in α-SMA and type I collagen. All these effects induced by lincRNA-p21 were blocked down by the loss of PTEN, suggesting that lincRNA-p21 suppressed HSC activation via PTEN. Further study demonstrated that microRNA-181b (miR-181b) was involved in the effects of lincRNA-p21 on HSC activation. The effects of lincRNA-p21 on PTEN expression and HSC activation were inhibited by miR-181b mimics. We demonstrated that lincRNA-p21 enhanced PTEN expression by competitively binding miR-181b. In conclusion, our results disclose a novel lincRNA-p21-miR-181b-PTEN signaling cascade in liver fibrosis and suggest lincRNA-p21 as a promising molecular target for antifibrosis therapy. PMID:27610008

  9. Candesartan and glycyrrhizin ameliorate ischemic brain damage through downregulation of the TLR signaling cascade.

    PubMed

    Barakat, Waleed; Safwet, Nancy; El-Maraghy, Nabila N; Zakaria, Mohamed N M

    2014-02-01

    Stroke is the second leading cause of death in industrialized countries and the most frequent cause of permanent disability in adults worldwide. The final outcome of stroke is determined not only by the volume of the ischemic core, but also by the extent of secondary brain damage inflicted to penumbral tissues by brain swelling, impaired microcirculation, and inflammation. The only drug approved for the treatment ischemic stroke is recombinant tissue plasminogen activator (rt-PA). The current study was designed to investigate the protective effects of candesartan (0.15 mg/kg, orally) and glycyrrhizin (30 mg/kg, orally) experimentally-induced ischemic brain damage in C57BL/6 mice (middle cerebral artery occlusion, MCAO) in comparison to the effects of a standard neuroprotective drug (cerebrolysin, 7.5 mg/kg, IP). All drugs were administered 30 min before and 24h after MCAO. Both candesartan and glycyrrhizin ameliorated the deleterious effects of MCAO as indicated by the improvement in the performance of the animals in behaviour tests, reduction in brain infarction, neuronal degeneration, and leukocyte infiltration. In addition, MCAO induced a significant upregulation in the different elements of the TLR pathway including TLR-2 and TLR-4, Myd88, TRIF and IRF-3 and the downstream effectors TNF-α, IL-1β, IL-6 and NF-kB. All these changes were significantly ameliorated by treatment with candesartan and glycyrrhizin. The results of the current study represent a new indication for both candesartan and glycyrrhizin in the management of ischemic stroke with effects comparable to those of the standard neuroprotective drug cerebrolysin. PMID:24378346

  10. The emerging roles of serine protease cascades in the epidermis.

    PubMed

    Ovaere, Petra; Lippens, Saskia; Vandenabeele, Peter; Declercq, Wim

    2009-09-01

    It has become clear in recent years that serine proteases have an important role in epidermal homeostasis, and the signaling cascades are gradually being identified. For example, matriptase, prostasin and furin are implicated in a cascade that could activate ENaC, leading to epidermal barrier formation and hydration, probably in part through their involvement in filaggrin processing. Kallikreins can form a signaling cascade to coordinate corneocyte desquamation. Knowledge is also emerging about how endogenous inhibitors, calcium and pH control these cascades. It is becoming clear that some skin pathologies are associated with deregulated serine protease activity. Therefore, a deeper knowledge of the regulation of these serine protease cascades could form the basis for development of appropriate treatments for skin disorders such as Netherton syndrome. PMID:19726197

  11. Analysis of the multiple roles of gld-1 in germline development: Interactions with the sex determination cascade and the glp-1 signaling pathway

    SciTech Connect

    Francis, R.; Schedl, T.; Maine, E.

    1995-02-01

    The Caenorhabditis elegans gene gld-1 is essential for oocyte development; in gld-1 (null) hermaphrodites, a tumor forms where oogenesis would normally occur. We use genetic epistasis analysis to demonstrate that tumor formation is dependent on the sexual fate of the germline. When the germline sex determination pathway is set in the female mode (terminal fem/fog genes inactive), gld-1 (null) germ cells exit meiotic prophase and proliferate to form a tumor, but when the pathway is et in the male mode, they develop into sperm. We conclude that the gld-1 (null) phenotype is cell-type specific and that gld-1(+) acts at the end of the cascade to direct oogenesis. We also use cell ablation and epistasis analysis to examine the dependence of tumor formation on the glp-1 signaling pathway. Although glp-1 activity promotes tumor growth, it is not essential for tumor formation by gld-1 (null) germ cells. These data also reveal that gld-1(+) plays a nonessential (and sex nonspecific) role in regulating germ cell proliferation before their entry into meiosis. Thus gld-1(+) may negatively regulate proliferation at two distinct points in germ cell development: before entry into meiotic prophase in both sexes (nonessential premeiotic gld-1 function) and during meiotic prophase when the sex determination pathway is set in the female mode (essential meiotic gld-1 function). 46 refs., 9 figs., 4 tabs.

  12. Analysis of the Multiple Roles of Gld-1 in Germline Development: Interactions with the Sex Determination Cascade and the Glp-1 Signaling Pathway

    PubMed Central

    Francis, R.; Maine, E.; Schedl, T.

    1995-01-01

    The Caenorhabditis elegans gene gld-1 is essential for oocyte development; in gld-1 (null) hermaphrodites, a tumor forms where oogenesis would normally occur. We use genetic epistasis analysis to demonstrate that tumor formation is dependent on the sexual fate of the germline. When the germline sex determination pathway is set in the female mode (terminal fem/fog genes inactive), gld-1 (null) germ cells exit meiotic prophase and proliferate to form a tumor, but when the pathway is set in the male mode, they develop into sperm. We conclude that the gld-1 (null) phenotype is cell-type specific and that gld-1 (+) acts at the end of the cascade to direct oogenesis. We also use cell ablation and epistasis analysis to examine the dependence of tumor formation on the glp-1 signaling pathway. Although glp-1 activity promotes tumor growth, it is not essential for tumor formation by gld-1 (null) germ cells. These data also reveal that gld-1 (+) plays a nonessential (and sex nonspecific) role in regulating germ cell proliferation before their entry into meiosis. Thus gld-1 (+) may negatively regulate proliferation at two distinct points in germ cell development: before entry into meiotic prophase in both sexes (nonessential premeiotic gld-1 function) and during meiotic prophase when the sex determination pathway is set in the female mode (essential meiotic gld-1 function). PMID:7713420

  13. Calcium-Ask1-MKK7-JNK2-c-Src Signaling Cascade Mediates Disruption of Intestinal Epithelial Tight Junctions by Dextran Sulfate Sodium

    PubMed Central

    Samak, Geetha; Chaudhry, Kamaljit K.; Gangwar, Ruchika; Narayanan, Damodaran; Jaggar, Jonathan H.; Rao, RadhaKrishna

    2015-01-01

    Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with the symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca2+ concentration, and depletion of intracellular Ca2+ by BAPTA or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of Ask1 or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased Tyr-phosphorylation of occludin, ZO-1, E-cadherin and β-catenin. SP600125 abrogated DSS-induced Tyr-phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto phosphorylation of c-Src. This study demonstrates that Ca2+-Ask1-MKK7-JNK2-cSrc signaling cascade mediates DSS-induced tight junction disruption and barrier dysfunction. PMID:25377781

  14. Integration of Apoptosis Signal-Regulating Kinase 1-Mediated Stress Signaling with the Akt/Protein Kinase B-IκB Kinase Cascade

    PubMed Central

    Puckett, Mary C.; Goldman, Erinn H.; Cockrell, Lisa M.; Huang, Bei; Kasinski, Andrea L.; Du, Yuhong; Wang, Cun-Yu; Lin, Anning; Ichijo, Hidenori; Khuri, Fadlo

    2013-01-01

    Cellular processes are tightly controlled through well-coordinated signaling networks that respond to conflicting cues, such as reactive oxygen species (ROS), endoplasmic reticulum (ER) stress signals, and survival factors to ensure proper cell function. We report here a direct interaction between inhibitor of κB kinase (IKK) and apoptosis signal-regulating kinase 1 (ASK1), unveiling a critical node at the junction of survival, inflammation, and stress signaling networks. IKK can be activated by growth factor stimulation or tumor necrosis factor alpha engagement. IKK forms a complex with and phosphorylates ASK1 at a sensor site, Ser967, leading to the recruitment of 14-3-3, counteracts stress signal-triggered ASK1 activation, and suppresses ASK1-mediated functions. An inhibitory role of IKK in JNK signaling has been previously reported to depend on NF-κB-mediated gene expression. Our data suggest that IKK has a dual role: a transcription-dependent and a transcription-independent action in controlling the ASK1-JNK axis, coupling IKK to ROS and ER stress response. Direct phosphorylation of ASK1 by IKK also defines a novel IKK phosphorylation motif. Because of the intimate involvement of ASK1 in diverse diseases, the IKK/ASK1 interface offers a promising target for therapeutic development. PMID:23530055

  15. 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

  16. A label-free and cascaded dual-signaling amplified electrochemical aptasensing platform for sensitive prion assay.

    PubMed

    Yu, Peng; Zhang, Xiaohua; Xiong, Erhu; Zhou, Jiawan; Li, Xiaoyu; Chen, Jinhua

    2016-11-15

    Prion proteins, as an important biomarker of prion disease, are responsible for the transmissible spongiform encephalopathies (a group of fatal neurodegenerative diseases). Hence, the sensitive detection of prion protein is very essential for biological studies and medical diagnostics. In this paper, a novel label-free and cascaded dual-signaling amplified electrochemical strategy was developed for sensitive and selective analysis of cellular prion protein (PrP(C)). The recognition elements included double-stranded DNA consisted of PrP(C)-binding aptamer (DNA1) and its partially complementary DNA (DNA2), and ordered mesoporous carbon probe (OMCP) fabricated by sealing the electroactive ferrocenecarboxylic acid (Fc) into its inner pores and then using single-stranded DNA (DNA3) as the gatekeeper. In the presence of PrP(C), DNA1 could bind the target protein and free DNA2. More importantly, DNA2 could hybridize with DNA3 to form a rigid duplex DNA and thus triggered the exonuclease III (Exo III) cleavage process to realize the DNA2 recycling, accompanied by opening more biogates and releasing more Fc. The released Fc could be further used as a competitive guest of β-cyclodextrin (β-CD) to displace the Rhodamine B (RhB) on the electrode. As a result, an amplified oxidation peak current of Fc (RhB) increased (decreased) with the increase of PrP(C) concentration. When "ΔI=ΔIFc+|ΔIRhB|" (ΔIFc and ΔIRhB were the change values of the oxidation peak currents of Fc and RhB, respectively.) was used as the response signal for quantitative determination of PrP(C), the detection limit was 7.6fM (3σ), which was much lower than that of the most reported methods for PrP(C) assay. This strategy provided a simple and sensitive approach for the detection of PrP(C) and has a great potential for bioanalysis, disease diagnostics, and clinical biomedicine applications. PMID:27208480

  17. A RelA-dependent two-tiered regulated proteolysis cascade controls synthesis of a contact-dependent intercellular signal in Myxococcus xanthus.

    PubMed

    Konovalova, Anna; Löbach, Stephanie; Søgaard-Andersen, Lotte

    2012-04-01

    Proteolytic cleavage of precursor proteins to generate intercellular signals is a common mechanism in all cells. In Myxococcus xanthus the contact-dependent intercellular C-signal is a 17 kDa protein (p17) generated by proteolytic cleavage of the 25 kDa csgA protein (p25), and is essential for starvation-induced fruiting body formation. p25 accumulates in the outer membrane and PopC, the protease that cleaves p25, in the cytoplasm of vegetative cells. PopC is secreted in response to starvation, therefore, restricting p25 cleavage to starving cells. We focused on identifying proteins critical for PopC secretion in response to starvation. PopC secretion depends on the (p)ppGpp synthase RelA and the stringent response, and is regulated post-translationally. PopD, which is encoded in an operon with PopC, forms a soluble complex with PopC and inhibits PopC secretion whereas the integral membrane AAA+ protease FtsH(D) is required for PopC secretion. Biochemical and genetic evidence suggest that in response to starvation, RelA is activated and induces the degradation of PopD thereby releasing pre-formed PopC for secretion and that FtsH(D) is important for PopD degradation. Hence, regulated PopC secretion depends on regulated proteolysis. Accordingly, p17 synthesis depends on a proteolytic cascade including FtsH(D) -dependent degradation of PopD and PopC-dependent cleavage of p25. PMID:22404381

  18. Phonological Activation of Category Coordinates during Speech Planning is Observable in Children but Not in Adults: Evidence for Cascaded Processing

    ERIC Educational Resources Information Center

    Jescheniak, Jorg D.; Hahne, Anja; Hoffmann, Stefanie; Wagner, Valentin

    2006-01-01

    There is a long-standing debate in the area of speech production on the question of whether only words selected for articulation are phonologically activated (as maintained by serial-discrete models) or whether this is also true for their semantic competitors (as maintained by forward-cascading and interactive models). Past research has addressed…

  19. Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases.

    PubMed Central

    Juo, P; Kuo, C J; Reynolds, S E; Konz, R F; Raingeaud, J; Davis, R J; Biemann, H P; Blenis, J

    1997-01-01

    The Fas receptor mediates a signalling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. In this study Fas activated the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70S6k. Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3 (glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1 beta-converting enzyme) family proteases to induce apoptosis from studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK. In this study, crmA antagonized, and YVAD-CMK and Z-VAD-FMK completely inhibited, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide was resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in the current work indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signalling cascades. PMID:8972182

  20. Activation and quenching of the phototransduction cascade in retinal cones as inferred from electrophysiology and mathematical modeling

    PubMed Central

    Astakhova, Luba; Firsov, Michael

    2015-01-01

    Purpose To experimentally identify and quantify factors responsible for the lower sensitivity of retinal cones compared to rods. Methods Electrical responses of frog rods and fish (Carassius) cones to short flashes of light were recorded using the suction pipette technique. A fast solution changer was used to apply a solution that fixed intracellular Ca2+ concentration at the prestimulus level, thereby disabling Ca2+ feedback, to the outer segment (OS). The results were analyzed with a specially designed mathematical model of phototransduction. The model included all basic processes of activation and quenching of the phototransduction cascade but omitted unnecessary mechanistic details of each step. Results Judging from the response versus intensity curves, Carassius cones were two to three orders of magnitude less sensitive than frog rods. There was a large scatter in sensitivity among individual cones, with red-sensitive cones being on average approximately two times less sensitive than green-sensitive ones. The scatter was mostly due to different signal amplification, since the kinetic parameters of the responses among cones were far less variable than sensitivity. We argue that the generally accepted definition of the biochemical amplification in phototransduction cannot be used for comparing amplification in rods and cones, since it depends on an irrelevant factor, that is, the cell’s volume. We also show that the routinely used simplified parabolic curve fitting to an initial phase of the response leads to a few-fold underestimate of the amplification. We suggest a new definition of the amplification that only includes molecular parameters of the cascade activation, and show how it can be derived from experimental data. We found that the mathematical model with unrestrained parameters can yield an excellent fit to experimental responses. However, the fits with wildly different sets of parameters can be virtually indistinguishable, and therefore cannot

  1. 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

  2. The aging brain. Changes in the neuronal insulin/insulin receptor signal transduction cascade trigger late-onset sporadic Alzheimer disease (SAD). A mini-review.

    PubMed

    Hoyer, Siegfried

    2002-07-01

    Aging of the brain has been demonstrated to be the main risk factor for late-onset sporadic AD what is in contrast to early-onset familial AD in which mutations predominate the pathology. Aging of the brain was found to be associated with a multitude of aberrancies from normal in morphological, cellular and molecular terms. Recent findings provide clear evidence that the function of the neuronal insulin/insulin receptor signal transduction cascade is of pivotal significance to maintain normal cerebral blood flow and oxidative energy metabolism, work of the endoplasmatic reticulum/Golgi apparatus and the cell cycle in terminally differentiated neurons no longer in the cell cycle. It has become evident that normal metabolism of both amyloid precursor protein and tau-protein is part of interactive processes controlled by the neuronal I/IR signal transduction cascade. In normal brain aging, the function of this cascade starts to fail compared to normal resulting in adverse effects in CBF/oxidative energy metabolism, work of the endoplasmatic reticulum/Golgi apparatus and cell cycle. The aberrancies may not be drastic, but multifold and permanently existing, inclusive the metabolism of APP and tau-protein. The amount of intraneuronally formed betaA4 may increase, and tau-protein may become hyperphosphorylated. These processes as a whole may increase the vulnerability of the aging brain and may facilitate the generation of late-onset sporadic AD. PMID:12111436

  3. 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

  4. Neuronal MHC Class I Expression Is Regulated by Activity Driven Calcium Signaling

    PubMed Central

    Peng, Yaqin; Liu, Jiane; Miao, Fengqin; Zhang, Jianqiong

    2015-01-01

    MHC class I (MHC-I) molecules are important components of the immune system. Recently MHC-I have been reported to also play important roles in brain development and synaptic plasticity. In this study, we examine the molecular mechanism(s) underlying activity-dependent MHC-I expression using hippocampal neurons. Here we report that neuronal expression level of MHC-I is dynamically regulated during hippocampal development after birth in vivo. Kainic acid (KA) treatment significantly increases the expression of MHC-I in cultured hippocampal neurons in vitro, suggesting that MHC-I expression is regulated by neuronal activity. In addition, KA stimulation decreased the expression of pre- and post-synaptic proteins. This down-regulation is prevented by addition of an MHC-I antibody to KA treated neurons. Further studies demonstrate that calcium-dependent protein kinase C (PKC) is important in relaying KA simulation activation signals to up-regulated MHC-I expression. This signaling cascade relies on activation of the MAPK pathway, which leads to increased phosphorylation of CREB and NF-κB p65 while also enhancing the expression of IRF-1. Together, these results suggest that expression of MHC-I in hippocampal neurons is driven by Ca2+ regulated activation of the MAPK signaling transduction cascade. PMID:26263390

  5. Mangiferin Attenuates Diabetic Nephropathy by Inhibiting Oxidative Stress Mediated Signaling Cascade, TNFα Related and Mitochondrial Dependent Apoptotic Pathways in Streptozotocin-Induced Diabetic Rats

    PubMed Central

    Pal, Pabitra Bikash; Sinha, Krishnendu; Sil, Parames C.

    2014-01-01

    Oxidative stress plays a crucial role in the progression of diabetic nephropathy in hyperglycemic conditions. It has already been reported that mangiferin, a natural C-glucosyl xanthone and polyhydroxy polyphenol compound protects kidneys from diabetic nephropathy. However, little is known about the mechanism of its beneficial action in this pathophysiology. The present study, therefore, examines the detailed mechanism of the beneficial action of mangiferin on STZ-induced diabetic nephropathy in Wister rats as the working model. A significant increase in plasma glucose level, kidney to body weight ratio, glomerular hypertrophy and hydropic changes as well as enhanced nephrotoxicity related markers (BUN, plasma creatinine, uric acid and urinary albumin) were observed in the experimental animals. Furthermore, increased oxidative stress related parameters, increased ROS production and decreased the intracellular antioxidant defenses were detected in the kidney. Studies on the oxidative stress mediated signaling cascades in diabetic nephropathy demonstrated that PKC isoforms (PKCα, PKCβ and PKCε), MAPKs (p38, JNK and ERK1/2), transcription factor (NF-κB) and TGF-β1 pathways were involved in this pathophysiology. Besides, TNFα was released in this hyperglycemic condition, which in turn activated caspase 8, cleaved Bid to tBid and finally the mitochorndia-dependent apoptotic pathway. In addition, oxidative stress also disturbed the proapoptotic-antiapoptotic (Bax and Bcl-2) balance and activated mitochorndia-dependent apoptosis via caspase 9, caspase 3 and PARP cleavage. Mangiferin treatment, post to hyperglycemia, successfully inhibited all of these changes and protected the cells from apoptotic death. PMID:25233093

  6. Effects of fluorotelomer alcohol 8:2 FTOH on steroidogenesis in H295R cells: Targeting the cAMP signalling cascade

    SciTech Connect

    Liu Chunsheng; Zhang Xiaowei; Chang Hong; Jones, Paul; Wiseman, Steve; Naile, Jonathan; Hecker, Markus; Giesy, John P.; Zhou Bingsheng

    2010-09-15

    Previous studies have demonstrated that perfluorinated chemicals (PFCs) can affect reproduction by disruption of steroidogenesis in experimental animals. However, the underlying mechanism(s) of this disruption remain unknown. Here we investigated the effects and mechanisms of action of 1H, 1H, 2H, 2H-perfluoro-decan-1-ol (8:2 FTOH) on steroidogenesis using a human adrenocortical carcinoma cell line (H295R) as a model. H295R cells were exposed to 0, 7.4, 22.2 or 66.6 {mu}M 8:2 FTOH for 24 h and productions of progesterone, 17{alpha}-OH-progesterone, androstenedione, testosterone, deoxycorticosterone, corticosterone and cortisol were quantified by HPLC-MS/MS. With the exception of progesterone, 8:2 FTOH treatment significantly decreased production of all hormones in the high dose group. Exposure to 8:2 FTOH significantly down-regulated cAMP-dependent mRNA expression and protein abundance of several key steroidogenic enzymes, including StAR, CYP11A, CYP11B1, CYP11B2, CYP17 and CYP21. Furthermore, a dose-dependent decrease of cellular cAMP levels was observed in H295R cells exposed to 8:2 FTOH. The observed responses are consistent with reduced cellular cAMP levels. Exposure to 8:2 FTOH resulted in significantly less basal (+ GTP) and isoproterenol-stimulated adenylate cyclase activities, but affected neither total cellular ATP level nor basal (-GTP) or NaF-stimulated adenylate cyclase activities, suggesting that inhibition of steroidogenesis may be due to an alteration in membrane properties. Metabolites of 8:2 FTOH were not detected by HPLC-MS/MS, suggesting that 8:2 FTOH was not metabolized by H295R cells. Overall, the results show that 8:2 FTOH may inhibit steroidogenesis by disrupting the cAMP signalling cascade.

  7. Imperatorin exhibits anticancer activities in human colon cancer cells via the caspase cascade.

    PubMed

    Zheng, Yi Mei; Lu, Amy Xiaoxu; Shen, James Zheng; Kwok, Amy Ho Yan; Ho, Wing Shing

    2016-04-01

    Despite advances in medical treatments for colon cancer, it remains one of the leading causes of cancer-related mortality among men. Thus, more efficacious treatment strategies for colon cancer are needed. Imperatorin is one of the major ingredients present in the root of Angelica dahurica, and has been used in herbal formulations for the treatment of hypertension and cardiovascular diseases. However, the medical properties of imperatorin remain unclear. In the present study, the anti‑proliferative activities of imperatorin were investigated in the HT‑29 colon cancer cell line. The results showed that imperatorin significantly inhibited HT‑29 colon cancer cell growth with an IC50 value of 78 µM. Imperatorin induced the apoptosis of colon cancer cells through upregulation of p53 and the caspase cascade. Our findings revealed that imperatorin induced cell cycle arrest in the G1 phase. The apoptotic index showed a steady increment when the imperatorin concentration was increased. The results suggest that imperatorin exerts considerable anti‑proliferative activities in HT‑29 colon cancer cells and highlight the potential of imperatorin as an anticancer agent for colon cancer. PMID:26794238

  8. Ascorbic acid suppresses endotoxemia and NF-κB signaling cascade in alcoholic liver fibrosis in guinea pigs: A mechanistic approach

    SciTech Connect

    Abhilash, P.A.; Harikrishnan, R.; Indira, M.

    2014-01-15

    Alcohol consumption increases the small intestinal bacterial overgrowth (SIBO) and intestinal permeability of endotoxin. The endotoxin mediated inflammatory signaling plays a major role in alcoholic liver fibrosis. We evaluated the effect of ascorbic acid (AA), silymarin and alcohol abstention on the alcohol induced endotoxemia and NF-κB activation cascade pathway in guinea pigs (Cavia porcellus). Guinea pigs were administered ethanol at a daily dose of 4 g/kg b.wt for 90 days. After 90 days, ethanol administration was stopped. The ethanol treated animals were divided into abstention, silymarin (250 mg/kg b.wt) and AA (250 mg/kg b.wt) supplemented groups and maintained for 30 days. The SIBO, intestinal permeability and endotoxin were significantly increased in the ethanol group. The mRNA expressions of intestinal proteins claudin, occludin and zona occludens-1 were significantly decreased in ethanol group. The mRNA levels of inflammatory receptors, activity of IKKβ and the protein expressions of phospho-IκBα, NF-κB, TNF-α, TGF-β{sub 1} and IL-6 were also altered in ethanol group. The expressions of fibrosis markers α-SMA, α{sub 1} (I) collagen and sirius red staining in the liver revealed the induction of fibrosis. But the supplementation of AA could induce greater reduction of ethanol induced SIBO, intestinal barrier defects, NF-κB activation and liver fibrosis than silymarin. The possible mechanism may be the inhibitory effect of AA on SIBO, intestinal barrier defect and IKKβ, which decreased the activation of NF-κB and synthesis of cytokines. This might have led to suppression of HSCs activation and liver fibrosis. - Highlights: • Alcohol increases intestinal bacterial overgrowth and permeability of endotoxin. • Endotoxin mediated inflammation plays a major role in alcoholic liver fibrosis. • Ascorbic acid reduces endotoxemia, NF-κB activation and proinflammatory cytokines. • AA's action is by inhibition of SIBO, IKKβ and alteration of

  9. 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

  10. Measurement of constitutive MAPK and PI3K/AKT signaling activity in human cancer cell lines

    PubMed Central

    Paraiso, Kim H.T.; Van Der Kooi, Kaisa; Messina, Jane L.; Smalley, Keiran S. M.

    2014-01-01

    The growth and survival of cancer cells is often driven by constitutive activity in the mitogen activated protein kinase (MAPK) and phospho-inositide 3-kinase (PI3K)/AKT signaling pathways. Activity in these signal transduction cascades is known to contribute to the uncontrolled growth and resistance to apoptosis that characterizes tumor progression. There is now a great deal of interest in therapeutically targeting these pathways in cancer using small molecule inhibitors. In this chapter we describe methods to measure constitutive MAPK and AKT activity in melanoma cell lines, with a focus upon Western blotting, phospho-flow cytometry and immunofluorescence staining techniques. PMID:21036250

  11. Insulin-degrading enzyme as a downstream target of insulin receptor signaling cascade: implications for Alzheimer's disease intervention.

    PubMed

    Zhao, Lixia; Teter, Bruce; Morihara, Takashi; Lim, Giselle P; Ambegaokar, Surendra S; Ubeda, Oliver J; Frautschy, Sally A; Cole, Greg M

    2004-12-01

    Insulin-degrading enzyme (IDE) is one of the proteins that has been demonstrated to play a key role in degrading beta-amyloid (Abeta) monomer in vitro and in vivo, raising the possibility of upregulating IDE as an approach to reduce Abeta. Little is known, however, about the cellular and molecular regulation of IDE protein. Because one of the main functions of IDE is to degrade insulin, we hypothesized that there is a negative feedback mechanism whereby stimulation of insulin receptor-mediated signaling upregulates IDE to prevent chronic activation of the pathway. We show that treatment of primary hippocampal neurons with insulin increased IDE protein levels by approximately 25%. Insulin treatment also led to phosphatidylinositol-3 (PI3) kinase activation evidenced by Akt phosphorylation, which was blocked by PI3 kinase inhibitors, wortmannin and LY 294002. Inhibition of PI3 kinase abolished the IDE upregulation by insulin, indicating a cause-effect relationship between insulin signaling and IDE upregulation. Further support for this link was provided by the findings that deficient insulin signaling (decreased PI3 kinase subunit P85) was correlated with reduced IDE in Alzheimer's disease (AD) brains and in Tg2576 Swedish amyloid precursor protein transgenic mice fed a safflower oil-enriched ("Bad") diet used to accelerate pathogenesis. Consistent with IDE function in the degradation of Abeta monomer, the IDE decrease in the Bad diet-fed Tg2576 mice was associated with increased Abeta monomer levels. These in vitro and in vivo analyses validate the use of enhanced CNS insulin signaling as a potential strategy for AD intervention to correct the IDE defects occurring in AD. PMID:15590928

  12. Neutrophil proteolytic activation cascades: a possible mechanistic link between chronic periodontitis and coronary heart disease.

    PubMed

    Alfakry, Hatem; Malle, Ernst; Koyani, Chintan N; Pussinen, Pirkko J; Sorsa, Timo

    2016-01-01

    Cardiovascular diseases are chronic inflammatory diseases that affect a large segment of society. Coronary heart disease (CHD), the most common cardiovascular disease, progresses over several years and affects millions of people worldwide. Chronic infections may contribute to the systemic inflammation and enhance the risk for CHD. Periodontitis is one of the most common chronic infections that affects up to 50% of the adult population. Under inflammatory conditions the activation of endogenous degradation pathways mediated by immune responses leads to the release of destructive cellular molecules from both resident and immigrant cells. Matrix metalloproteinases (MMPs) and their regulators can activate each other and play an important role in immune response via degrading extracellular matrix components and modulating cytokines and chemokines. The action of MMPs is required for immigrant cell recruitment at the site of inflammation. Stimulated neutrophils represent the major pathogen-fighting immune cells that upregulate expression of several proteinases and oxidative enzymes, which can degrade extracellular matrix components (e.g. MMP-8, MMP-9 and neutrophil elastase). The activity of MMPs is regulated by endogenous inhibitors and/or candidate MMPs (e.g. MMP-7). The balance between MMPs and their inhibitors is thought to mirror the proteolytic burden. Thus, neutrophil-derived biomarkers, including myeloperoxidase, may activate proteolytic destructive cascades that are involved in subsequent immune-pathological events associated with both periodontitis and CHD. Here, we review the existing studies on the contribution of MMPs and their regulators to the infection-related pathology. Also, we discuss the possible proteolytic involvement and role of neutrophil-derived enzymes as an etiological link between chronic periodontitis and CHD. PMID:26608308

  13. 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

  14. Electron-phonon interaction in three-barrier nanosystems as active elements of quantum cascade detectors

    SciTech Connect

    Tkach, N. V. Seti, Ju. A.; Grynyshyn, Yu. B.

    2015-04-15

    The theory of electron tunneling through an open nanostructure as an active element of a quantum cascade detector is developed, which takes into account the interaction of electrons with confined and interface phonons. Using the method of finite-temperature Green’s functions and the electron-phonon Hamiltonian in the representation of second quantization over all system variables, the temperature shifts and electron-level widths are calculated and the contributions of different electron-phonon-interaction mechanisms to renormalization of the spectral parameters are analyzed depending on the geometrical configuration of the nanosystem. Due to weak electron-phonon coupling in a GaAs/Al{sub 0.34}Ga{sub 0.66}As-based resonant tunneling nanostructure, the temperature shift and rf field absorption peak width are not very sensitive to the electron-phonon interaction and result from a decrease in potential barrier heights caused by a difference in the temperature dependences of the well and barrier band gaps.

  15. Brain injury caused by chronic fetal hypoxemia is mediated by inflammatory cascade activation.

    PubMed

    Guo, Rong; Hou, Weijian; Dong, Yafeng; Yu, Zhiyong; Stites, Josh; Weiner, Carl P

    2010-06-01

    The prevalence of cerebral palsy (CP) shows little temporal or geographic variation and is associated with preterm birth, maternal/fetal infection/inflammation, and fetal growth restriction (IUGR), a potential surrogate for chronic fetal hypoxemia (CHX). We previously demonstrated CHX causes a fetal inflammatory response syndrome (FIRS). Herein, we test the hypothesis that CHX may cause fetal brain injury by upregulating inflammatory cytokine cascades, culminating in apoptosis pathway activation. Time-mated guinea pigs were housed in 12% or 10.5% O(2) for the last 21% of gestation. Chronic fetal hypoxemia increased the lactate/pyruvate and decreased the glutathione (GSH)/oxidized glutathione (GSSH) ratios, confirming a shift to a prooxidant state. The end result was a >30% decrease in hippocampal neuron density. Based on a microarray spotted with 113 cytokines and receptors, 22 genes were upregulated by CHX in proportion to the degree of hypoxia; the findings were confirmed by quantitative polymerase chain reaction (PCR). Thus, CHX triggers fetal brain inflammation inversely proportional to its severity characterized by increased apoptosis and neuronal loss. We suggest CHX fetal brain injury is not directly caused by oxygen deprivation but rather is an adaptive response that becomes maladaptive. PMID:20360591

  16. Phospholipase C{gamma}1 stimulates transcriptional activation of the matrix metalloproteinase-3 gene via the protein kinase C/Raf/ERK cascade

    SciTech Connect

    Shin, Soon Young; Choi, Ha Young; Ahn, Bong-Hyun; Son, Sang Wook; Lee, Young Han . E-mail: younghan@hanyang.ac.kr

    2007-02-16

    The phospholipid hydrolase phospholipase C{gamma}1 (PLC{gamma}1) plays a major role in regulation of cell proliferation, development, and cell motility. Overexpression of PLC{gamma}1 is associated with tumor development, and it is overexpressed in some tumors. Matrix metalloproteinase-3 (MMP-3) is a protein involved in tumor invasion and metastasis. Here, we demonstrate that overexpression of PLC{gamma}1 stimulates MMP-3 expression at the transcriptional level via the PKC-mediated Raf/MEK1/ERK signaling cascade. We propose that modulation of PLC{gamma}1 activity might be of value in controlling the activity of MMPs, which are important regulators of invasion and metastasis in malignant tumors.

  17. 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

  18. 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

  19. The ternary complex factor Net contains two distinct elements that mediate different responses to MAP kinase signalling cascades.

    PubMed

    Ducret, C; Maira, S M; Lutz, Y; Wasylyk, B

    2000-10-19

    The ternary complex factors (TCFs), Elk-1, Sap-1a and Net, are key integrators of the transcriptional response to different signalling pathways. Classically, three MAP kinase pathways, involving ERK, JNK, and p38, transduce various extracellular stimuli to the nucleus. Net is a repressor that is converted into an activator by Ras/ERK signalling. Net is also exported from the nucleus in response to stress stimuli transduced through the JNK pathway, leading to relief from repression. Here we show that ERK and p38 bind to the D box and that binding is required for phosphorylation of the adjacent C-terminally located C-domain. The D box as well as the phosphorylation sites in the C-domain (the DC element) are required for transcription activation by Ras. On the other hand, JNK binds to the J box in the middle of the protein, and binding is required for phosphorylation of the adjacent EXport motif. Both the binding and phosphorylation sites (the JEX element) are important for Net export. In conclusion, specific targeting of Net by MAP kinase pathways involves two different docking sites and phosphorylation of two different domains. These two elements, DC and JEX, mediate two distinct functional responses. PMID:11042694

  20. "RED" matters when naming "CAR": The cascading activation of nontarget properties.

    PubMed

    Roux, Sébastien; Bonin, Patrick

    2016-03-01

    Seven experiments tested, whether when naming a colored object (e.g., CAR), its color (e.g., red) is phonologically encoded. In the first experiment, adults had to say aloud the names of colored line drawings of objects that were each displayed among 3 black-and-white line drawings (Experiment 1a) or that were presented alone (Experiment 1b). Naming times were shorter in Experiment 1a, but not in Experiment 1b, when both the color and object names were phonologically related (e.g., blue ball). In Experiment 2a, adults had to name objects having diagnostic colors (e.g., banana, tomato) while hearing distractor words. Compared with unrelated distractors, object naming times were longer when the distractors were phonologically related to the names of the colors, indicating that the names of the colors were activated. In Experiment 2b, this inhibitory effect did not surface when the same pictures were displayed in black and white, indicating that it originates from the perceptual level. In Experiment 3a, we used the same paradigm as in Experiment 2 (a and b) with objects having "plausible," but nondiagnostic, colors (e.g., red CAR). The inhibitory effect of color-related distractors turned out to be reliable but it vanished when regular colored-line drawings were used (Experiment 3b) and when colors and objects were spatially segregated (Experiment 3c). Taken together, the findings strongly suggest that under certain circumstances, an object's properties are phonologically activated during object naming. These findings are accounted for in terms of the general attentional view of cascading of Oppermann, Jescheniak, Schriefers, and Görges (2010). (PsycINFO Database Record PMID:26389629

  1. Oxidant Stress and Signal Transduction in the Nervous System with the PI 3-K, Akt, and mTOR Cascade

    PubMed Central

    Maiese, Kenneth; Chong, Zhao Zhong; Wang, Shaohui; Shang, Yan Chen

    2012-01-01

    Oxidative stress impacts multiple systems of the body and can lead to some of the most devastating consequences in the nervous system especially during aging. Both acute and chronic neurodegenerative disorders such as diabetes mellitus, cerebral ischemia, trauma, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and tuberous sclerosis through programmed cell death pathways of apoptosis and autophagy can be the result of oxidant stress. Novel therapeutic avenues that focus upon the phosphoinositide 3-kinase (PI 3-K), Akt (protein kinase B), and the mammalian target of rapamycin (mTOR) cascade and related pathways offer exciting prospects to address the onset and potential reversal of neurodegenerative disorders. Effective clinical translation of these pathways into robust therapeutic strategies requires intimate knowledge of the complexity of these pathways and the ability of this cascade to influence biological outcome that can vary among disorders of the nervous system. PMID:23203037

  2. 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

  3. 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.

  4. 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

  5. When activity requires breaking up: LEKTI proteolytic activation cascade for specific proteinase inhibition.

    PubMed

    Furio, Laetitia; Hovnanian, Alain

    2011-11-01

    Lymphoepithelial Kazal-type related inhibitor (LEKTI) is a multidomain proteinase inhibitor whose defective expression causes Netherton syndrome (NS). LEKTI is encoded by SPINK5, which is also a susceptibility gene for atopic disease. In this issue, Fortugno et al. report an elegant and thorough study of the LEKTI proteolytic activation process in which they identify the precise nature of the cleavage sites used and the bioactive fragments generated. They propose a proteolytic activation model in human skin and confirm differential inhibition of kallikrein (KLK) 5, 7, and 14 by the major physiological LEKTI fragments. They show that these bioactive fragments inhibit KLK-mediated proteolysis of desmoglein 1 (DSG1) and suggest a fine-tuned inhibition process controlling target serine proteinase (SP) activity. PMID:21997416

  6. Integrated Broadband Quantum Cascade Laser

    NASA Technical Reports Server (NTRS)

    Mansour, Kamjou (Inventor); Soibel, Alexander (Inventor)

    2016-01-01

    A broadband, integrated quantum cascade laser is disclosed, comprising ridge waveguide quantum cascade lasers formed by applying standard semiconductor process techniques to a monolithic structure of alternating layers of claddings and active region layers. The resulting ridge waveguide quantum cascade lasers may be individually controlled by independent voltage potentials, resulting in control of the overall spectrum of the integrated quantum cascade laser source. Other embodiments are described and claimed.

  7. Tyrosine phosphorylation is a mandatory proximal step in radiation-induced activation of the protein kinase C signaling pathway in human B-lymphocyte precursors.

    PubMed Central

    Uckun, F M; Schieven, G L; Tuel-Ahlgren, L M; Dibirdik, I; Myers, D E; Ledbetter, J A; Song, C W

    1993-01-01

    Ionizing radiation triggers a signal in human B-lymphocyte precursors that is intimately linked to an active protein-tyrosine kinase regulatory pathway. We show that in B-lymphocyte precursors, irradiation with gamma-rays leads to (i) stimulation of phosphatidylinositol turnover; (ii) downstream activation by covalent modification of multiple serine-specific protein kinases, including protein kinase C; and (iii) activation of nuclear factor kappa B. All of the radiation-induced signals were effectively prevented by the protein-tyrosine kinase inhibitors genistein and herbimycin A. Thus, tyrosine phosphorylation is an important and perhaps mandatory proximal step in the activation of the protein kinase C signaling cascade in human B-lymphocyte precursors. Our report expands current knowledge of the radiation-induced signaling cascade by clarifying the chronological sequence of biochemical events that follow irradiation. Images PMID:8419931

  8. Neuropeptidergic Signaling and Active Feeding State Inhibit Nociception in Caenorhabditis elegans.

    PubMed

    Ezcurra, Marina; Walker, Denise S; Beets, Isabel; Swoboda, Peter; Schafer, William R

    2016-03-16

    Food availability and nutritional status are important cues affecting behavioral states. Here we report that, in Caenorhabditis elegans, a cascade of dopamine and neuropeptide signaling acts to inhibit nociception in food-poor environments. In the absence of food, animals show decreased sensitivity and increased adaptation to soluble repellents sensed by the polymodal ASH nociceptors. The effects of food on adaptation are affected by dopamine and neuropeptide signaling; dopamine acts via the DOP-1 receptor to decrease adaptation on food, whereas the neuropeptide receptors NPR-1 and NPR-2 act to increase adaptation off food. NPR-1 and NPR-2 function cell autonomously in the ASH neurons to increase adaptation off food, whereas the DOP-1 receptor controls neuropeptide release from interneurons that modulate ASH activity indirectly. These results indicate that feeding state modulates nociception through the interaction of monoamine and neuropeptide signaling pathways. PMID:26985027

  9. Neuropeptidergic Signaling and Active Feeding State Inhibit Nociception in Caenorhabditis elegans

    PubMed Central

    Ezcurra, Marina; Walker, Denise S.; Beets, Isabel; Swoboda, Peter

    2016-01-01

    Food availability and nutritional status are important cues affecting behavioral states. Here we report that, in Caenorhabditis elegans, a cascade of dopamine and neuropeptide signaling acts to inhibit nociception in food-poor environments. In the absence of food, animals show decreased sensitivity and increased adaptation to soluble repellents sensed by the polymodal ASH nociceptors. The effects of food on adaptation are affected by dopamine and neuropeptide signaling; dopamine acts via the DOP-1 receptor to decrease adaptation on food, whereas the neuropeptide receptors NPR-1 and NPR-2 act to increase adaptation off food. NPR-1 and NPR-2 function cell autonomously in the ASH neurons to increase adaptation off food, whereas the DOP-1 receptor controls neuropeptide release from interneurons that modulate ASH activity indirectly. These results indicate that feeding state modulates nociception through the interaction of monoamine and neuropeptide signaling pathways. PMID:26985027

  10. Analysis of signal transduction pathways during anoxia exposure in a marine snail: a role for p38 MAP kinase and downstream signaling cascades.

    PubMed

    Larade, Kevin; Storey, Kenneth B

    2006-01-01

    The responses of members of the three main MAPK families (ERK, JNK/SAPK, p38 MAPK), as well as selected peripheral pathways, were examined in hepatopancreas of the marine periwinkle, Littorina littorea, to determine if anoxia exposure influenced the total protein content or the phosphorylation status of any key components. The content of active phospho-p38 MAPK was 2-fold higher in hepatopancreas from anoxic snails relative to controls. A 1.7-fold increase in the amount of phospho-Hsp27 and a 1.3-fold increase in phospho-CREB correlated well with the changes in p38 MAPK phosphorylation. Activation of these factors via p38 MAPK may be vital to the reorganization of metabolic responses to anoxia in hepatopancreas. No changes in components of the JNK/SAPK and ERK pathways occurred and transcription factors involved in lipid metabolism did not appear to be affected by anoxia. The present analysis of a variety of signaling pathways has implicated the p38 MAPK pathway as a key anoxia-responsive signal transduction pathway in L. littorea. PMID:16326124

  11. Antitumor Activity of Tenacissoside H on Esophageal Cancer through Arresting Cell Cycle and Regulating PI3K/Akt-NF-κB Transduction Cascade

    PubMed Central

    Jia, Yong-sen; Hu, Xue-qin; Gabriella, Hegyi; Qin, Li-juan; Meggyeshazi, Nora

    2015-01-01

    Objective. The purpose of the study was to elucidate the molecular mechanism of tenacissoside H (TDH) inhibiting esophageal carcinoma infiltration and proliferation. Methods. In vitro, EC9706 cells were treated with TDH. Cells proliferation and cell cycle were assayed. PI3K and NF-κB mRNAs expression were determined by real time PCR. In vivo, model of nude mice with tumor was established. Mice were treated with TDH. Inhibition ratio of tumor volume was calculated. PCNA expression was examined. Protein expression in PI3K/Akt-NF-κB signaling pathway was determined. Results. In vitro, TDH significantly inhibited cells proliferation in a time-and-dose-dependent manner. TDH arrested the cell cycle in S phase and significantly inhibited PI3K and NF-κB mRNA expression, compared with blank controlled group (P < 0.05). In vivo, TDH strongly inhibits tumor growth and volume. PCNA expression was significantly decreased after treatment of TDH. TDH downregulated proteins expression in PI3K/Akt-NF-κB transduction cascade (P < 0.05). Conclusion. TDH inhibited esophageal carcinoma infiltration and proliferation both in vitro and in vivo. The anticancer activity has relation to arresting the cell cycle at the S phase, inhibited the PCNA expression of transplanted tumors in nude mice, and regulated the protein expression in the PI3K/Akt-NF-κB transduction cascade. PMID:26495015

  12. Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR{sub 1} activation

    SciTech Connect

    Blanc-Brude, Olivier P. . E-mail: olivier.blanc-brude@larib.inserm.fr; Archer, Fabienne; Leoni, Patricia; Derian, Claudia; Bolsover, Steven; Laurent, Geoffrey J.; Chambers, Rachel C.

    2005-03-10

    Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR{sub 1}). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR{sub 1}-deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR{sub 1}-specific agonists and inhibitors were used to demonstrate that PAR{sub 1} mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR{sub 1} and not PAR{sub 2}. These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis.

  13. 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

  14. Observation of Infrared Free-Induction Decay and Optical Nutation Signals from Nitrous Oxide Using a Current Modulated Quantum Cascade Laser

    SciTech Connect

    Duxbury, Geoffrey; Kelly, James F.; Blake, Thomas A.; Langford, Nigel

    2012-05-07

    Free induction decay (FID), optical nutation and rapid passage induced (RP) signals in nitrous oxide, under both optically thin and optically thick conditions, have been observed using a rapid current pulse modulation, or chirp, applied to the slow current ramp of a quantum cascade (QC) laser. The variation in optical depth was achieved by increasing the pressure of nitrous oxide in a long pathlength multipass absorption cell. This allows the variation of optical depth to be achieved over a range of low gas pressures. Since, even at the highest gas pressure used in the cell, the chirp rate of the QC laser is faster than the collisional reorientation time of the molecules, there is minimal collisional damping, allowing a large macroscopic polarization of the molecular dipoles to develop. This is referred to as rapid passage induced gain. The resultant FID signals are enhanced due to the constructive interference between the field within the gas generated by the slow ramp of the laser (pump), and the fast chirp of the laser (probe) signal generated by pulse modulation of the continuously operating QC laser. The FID signals obtained at large 2 optical depth have not been observed previously in the mid-infrared regions, and unusual oscillatory signals have been observed at the highest gas pressures used.

  15. Observation of infrared free-induction decay and optical nutation signals from nitrous oxide using a current modulated quantum cascade laser.

    PubMed

    Duxbury, Geoffrey; Kelly, James F; Blake, Thomas A; Langford, Nigel

    2012-05-01

    Free induction decay (FID), optical nutation, and rapid passage induced signals in nitrous oxide, under both optically thin and optically thick conditions, have been observed using a rapid current pulse modulation, or chirp, applied to the slow current ramp of a quantum cascade (QC) laser. The variation in optical depth was achieved by increasing the pressure of nitrous oxide in a long path length multipass absorption cell. This allows the variation of optical depth to be achieved over a range of low gas pressures. Since, even at the highest gas pressure used in the cell, the chirp rate of the QC laser is faster than the collisional reorientation time of the molecules, there is minimal collisional damping, allowing a large macroscopic polarization of the molecular dipoles to develop. This is referred to as rapid passage induced polarization. The resultant FID signals are enhanced due to the constructive interference between the field within the gas generated by the slow ramp of the laser (pump), and that of the fast chirp of the laser (probe) signal generated by pulse modulation of the continuously operating QC laser. The FID signals obtained at large optical depth have not been observed previously in the mid-infrared regions, and unusual oscillatory signals have been observed at the highest gas pressures used. PMID:22583240

  16. X-ray irradiation activates K+ channels via H2O2 signaling.

    PubMed

    Gibhardt, Christine S; Roth, Bastian; Schroeder, Indra; Fuck, Sebastian; Becker, Patrick; Jakob, Burkhard; Fournier, Claudia; Moroni, Anna; Thiel, Gerhard

    2015-01-01

    Ionizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca2+-activated-K+-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H2O2 (HyPer) and for the dominant redox-buffer glutathione (Grx1-roGFP2) to monitor with high spatial and temporal resolution, radiation-triggered excursions of H2O2 in A549 and HEK293 cells. The data show that challenging cells with ≥1 Gy X-rays or with UV-A laser micro-irradiation causes a rapid rise of H2O2 in the nucleus and in the cytosol. This rise, which is determined by the rate of H2O2 production and glutathione-buffering, is sufficient for triggering a signaling cascade that involves an elevation of cytosolic Ca2+ and eventually an activation of hIK channels. PMID:26350345

  17. X-ray irradiation activates K+ channels via H2O2 signaling

    PubMed Central

    Gibhardt, Christine S.; Roth, Bastian; Schroeder, Indra; Fuck, Sebastian; Becker, Patrick; Jakob, Burkhard; Fournier, Claudia; Moroni, Anna; Thiel, Gerhard

    2015-01-01

    Ionizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca2+-activated-K+-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H2O2 (HyPer) and for the dominant redox-buffer glutathione (Grx1-roGFP2) to monitor with high spatial and temporal resolution, radiation-triggered excursions of H2O2 in A549 and HEK293 cells. The data show that challenging cells with ≥1 Gy X-rays or with UV-A laser micro-irradiation causes a rapid rise of H2O2 in the nucleus and in the cytosol. This rise, which is determined by the rate of H2O2 production and glutathione-buffering, is sufficient for triggering a signaling cascade that involves an elevation of cytosolic Ca2+ and eventually an activation of hIK channels. PMID:26350345

  18. 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

  19. 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

  20. 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

  1. Active phase-nulling of the self-mixing phase in a terahertz frequency quantum cascade laser.

    PubMed

    Dean, P; Keeley, J; Valavanis, A; Bertling, K; Lim, Y L; Taimre, T; Alhathlool, R; Li, L H; Indjin, D; Rakić, A D; Linfield, E H; Davies, A G

    2015-03-15

    We demonstrate an active phase-nulling scheme for terahertz (THz) frequency quantum cascade lasers (QCLs) under optical feedback, by active electronic feedback control of the emission frequency. Using this scheme, the frequency tuning rate of a THz QCL is characterized, with significantly reduced experimental complexity compared to alternative approaches. Furthermore, we demonstrate real-time displacement sensing of targets, overcoming the resolution limits imposed by quantization in previously implemented fringe-counting methods. Our approach is readily applicable to high-frequency vibrometry and surface profiling of targets, as well as frequency-stabilization schemes for THz QCLs. PMID:25768154

  2. Identification of Trypanocidal Activity for Known Clinical Compounds Using a New Trypanosoma cruzi Hit-Discovery Screening Cascade.

    PubMed

    De Rycker, Manu; Thomas, John; Riley, Jennifer; Brough, Stephen J; Miles, Tim J; Gray, David W

    2016-04-01

    Chagas disease is a significant health problem in Latin America and the available treatments have significant issues in terms of toxicity and efficacy. There is thus an urgent need to develop new treatments either via a repurposing strategy or through the development of new chemical entities. A key first step is the identification of compounds with anti-Trypanosoma cruzi activity from compound libraries. Here we describe a hit discovery screening cascade designed to specifically identify hits that have the appropriate anti-parasitic properties to warrant further development. The cascade consists of a primary imaging-based assay followed by newly developed and appropriately scaled secondary assays to predict the cidality and rate-of-kill of the compounds. Finally, we incorporated a cytochrome P450 CYP51 biochemical assay to remove compounds that owe their phenotypic response to inhibition of this enzyme. We report the use of the cascade in profiling two small libraries containing clinically tested compounds and identify Clemastine, Azelastine, Ifenprodil, Ziprasidone and Clofibrate as molecules having appropriate profiles. Analysis of clinical derived pharmacokinetic and toxicity data indicates that none of these are appropriate for repurposing but they may represent suitable start points for further optimisation for the treatment of Chagas disease. PMID:27082760

  3. Identification of Trypanocidal Activity for Known Clinical Compounds Using a New Trypanosoma cruzi Hit-Discovery Screening Cascade

    PubMed Central

    De Rycker, Manu; Thomas, John; Riley, Jennifer; Brough, Stephen J.; Miles, Tim J.; Gray, David W.

    2016-01-01

    Chagas disease is a significant health problem in Latin America and the available treatments have significant issues in terms of toxicity and efficacy. There is thus an urgent need to develop new treatments either via a repurposing strategy or through the development of new chemical entities. A key first step is the identification of compounds with anti-Trypanosoma cruzi activity from compound libraries. Here we describe a hit discovery screening cascade designed to specifically identify hits that have the appropriate anti-parasitic properties to warrant further development. The cascade consists of a primary imaging-based assay followed by newly developed and appropriately scaled secondary assays to predict the cidality and rate-of-kill of the compounds. Finally, we incorporated a cytochrome P450 CYP51 biochemical assay to remove compounds that owe their phenotypic response to inhibition of this enzyme. We report the use of the cascade in profiling two small libraries containing clinically tested compounds and identify Clemastine, Azelastine, Ifenprodil, Ziprasidone and Clofibrate as molecules having appropriate profiles. Analysis of clinical derived pharmacokinetic and toxicity data indicates that none of these are appropriate for repurposing but they may represent suitable start points for further optimisation for the treatment of Chagas disease. PMID:27082760

  4. Diet-induced obesity elevates colonic TNF-alpha in mice and is accompanied by an activation of Wnt signaling: a mechanism for obesity-associated colorectal cancer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inflammation associated with obesity may play a role in colorectal carcinogenesis, but the underlying mechanism remains unclear. This study investigated whether the Wnt pathway, an intracellular signaling cascade that plays a critical role in colorectal carcinogenesis, is activated by obesity-induce...

  5. Inhibition on JAK-STAT3 Signaling Transduction Cascade Is Taken by Bioactive Peptide Alpha-S2 Casein Protein from Goat Ethawah Breed Milk

    PubMed Central

    Rohmah, Rista Nikmatu; Hardiyanti, Ferlany; Fatchiyah, Fatchiyah

    2015-01-01

    Background: RA is a systemic inflammatory disease that causes developing comorbidity conditions. This condition can cause by overproduction of pro-inflammatory cytokine. In a previous study, we have found bioactive peptide CSN1S2 from Ethawah goat milk for anti-inflammatory for repair the ileum destruction. However, the signaling transduction cascade of bioactive peptides inhibits inflammation still not clear yet. Therefore, we analyzed the signaling transduction cascade via JAK-STAT3 pathway by in vivo and in silico. Methods: The ileum was isolated DNA and amplification with specific primer. The sequence was analyzed using the Sanger sequencing method. Modeling 3D-structure was predicted by SWISS-MODEL and virtual interaction was analyzed by docking system using Pymol and Discovery Studio 4.0 software. Results: This study showed that STAT3 has target gene 480bp. The normal group and normal treating- CSN1S2 of goat milk have similarity from gene bank. Whereas, RA group had transversion mutation that the purine change into pyrimidine even cause frameshift mutation. Interestingly, after treating with the CSN1S2 protein of goat milk shows reverse to the normal acid sequence group. Based on in silico study, from eight peptides, only three peptides of CSN1S2 protein, which carried by PePT1 to enter the small intestine. The fragments are PepT1-41-NMAIHPR-47; PepT1-182-KISQYYQK-189 and PepT1-214-TNAIPYVR-221. We have found just one bioactive peptide of f182-KISQYYQK-189 is able bind to STAT3. The energy binding of f182-KISQYYQK-189 and RA-STAT3 amino acid, it was Σ = -402.43 kJ/mol and the energy binding of f182-KISQYYQK-189 and RAS-STAT3 amino acid is decreasing into Σ = -407.09 kJ/mol. Conclusion: This study suggested that the fragment 182-KISQYYQK-189 peptides from Ethawah goat milk may act as an anti-inflammatory agent via JAK-STAT3 signal transduction cascade at the cellular level. PMID:26483598

  6. Pharmacology of Signaling Induced by Dopamine D1-Like Receptor Activation

    PubMed Central

    Undieh, Ashiwel S.

    2010-01-01

    Dopamine D1-like receptors consisting of D1 and D5 subtypes are intimately implicated in dopaminergic regulation of fundamental neurophysiologic processes such as mood, motivation, cognitive function, and motor activity. Upon stimulation, D1-like receptors initiate signal transduction cascades that are mediated through adenylyl cyclase or phosphoinositide metabolism, with subsequent enhancement of multiple downstream kinase cascades. The latter actions propagate and further amplify the receptor signals, thus predisposing D1-like receptors to multifaceted interactions with various other mediators and receptor systems. The adenylyl cyclase response to dopamine or selective D1-like receptor agonists is reliably associated with the D1 subtype, while emerging evidence indicates that the phosphoinositide responses in native brain tissues may be preferentially mediated through stimulation of the D5 receptor. Besides classic coupling of each receptor subtype to specific G proteins, additional biophysical models are advanced in attempts to account for differential subcellular distribution, heteromolecular oligomerization, and activity-dependent selectivity of the receptors. It is expected that significant advances in understanding of dopamine neurobiology will emerge from current and anticipated studies directed at uncovering the molecular mechanisms of D5 coupling to phosphoinositide signaling, the structural features that might enhance pharmacological selectivity for D5 versus D1 subtypes, the mechanism by which dopamine may modulate phosphoinositide synthesis, the contributions of the various responsive signal mediators to D1 or D5 interactions with D2-like receptors, and the spectrum of dopaminergic functions that may be attributed to each receptor subtype and signaling pathway. PMID:20547182

  7. The TIR-domain-containing adapter inducing interferon-β-dependent signaling cascade plays a crucial role in ischemia–reperfusion-induced retinal injury, whereas the contribution of the myeloid differentiation primary response 88-dependent signaling cascade is not as pivotal

    PubMed Central

    Dvoriantchikova, Galina; Santos, Andrea Rachelle C; Danek, Dagmara; Dvoriantchikova, Xenia; Ivanov, Dmitry

    2014-01-01

    Toll-like receptor 4 (Tlr4) plays an important role in ischemia–reperfusion (IR)-induced retinal inflammation and damage. However, the role of two Tlr4-dependent signaling cascades, myeloid differentiation primary response 88 (Myd88) and TIR-domain-containing adapter inducing interferon-β (Trif), in retinal IR injury is poorly understood. In this study, we investigated the contribution of the Myd88-dependent and Trif-dependent signaling cascades in retinal damage and inflammation triggered by IR, by using Myd88 knockout (Myd88KO) and Trif knockout (TrifKO) mice. Retinal IR injury was induced by unilateral elevation of intraocular pressure for 45 min by direct corneal cannulation. To study IR-induced retinal ganglion cell (RGC) death in vitro, we used an oxygen and glucose deprivation (OGD) model. Our data suggested that Myd88 was present in many retinal layers of sham-operated and ischemic mice, whereas Trif was mainly present in the ganglion cell layer (GCL). The level of Myd88 was increased in the retina after IR. We found that retinas of TrifKO mice had a significantly reduced neurotoxic pro-inflammatory response and significantly increased survival of the GCL neurons after IR. Although Myd88KO mice had relatively low levels of inflammation in ischemic retinas, their levels of IR-induced retinal damage were notably higher than those of TrifKO mice. We also found that Trif-deficient RGCs were more resistant to death induced by OGD than were RGCs isolated from Myd88KO mice. These data suggested that, as compared with the Myd88-dependent signaling cascade, Trif signaling contributes significantly to retinal damage after IR. PMID:24754835

  8. 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

  9. 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

  10. Hypothesis: the pathogenesis of AIDS. Activation of the T- and B-cell cascades.

    PubMed Central

    Evans, A. S.

    1984-01-01

    The hypothesis is presented that a human T-cell leukemia virus (HTLV) or a related agent produces a lytic response of T cells manifested by the acquired immune deficiency syndrome (AIDS) and a proliferative response represented by the adult leukemia/lymphoma (ATL) syndromes. The sequence or cascade of T-cell events following loss of T4 helper cells in AIDS includes reactivation of Epstein-Barr virus and a B-cell cascade of cytomegalovirus, resulting in Kaposi sarcoma in genetically susceptible persons, and of other intracellular agents (CNS viruses, M. avium intracellulari, T. gondii); opportunistic infections also occur. A comparison of AIDS and ATL syndromes is presented and the details of the B-cell cascade are outlined. The usefulness of prospective serological/immunological studies is discussed in an effort to determine the temporal sequence of infection by the candidate agents and their relation to the appearance of T4/T8 reversal and of the clinical features of AIDS. PMID:6093395

  11. 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

  12. A computational model on the modulation of mitogen-activated protein kinase (MAPK) and Akt pathways in heregulin-induced ErbB signalling.

    PubMed Central

    Hatakeyama, Mariko; Kimura, Shuhei; Naka, Takashi; Kawasaki, Takuji; Yumoto, Noriko; Ichikawa, Mio; Kim, Jae-Hoon; Saito, Kazuki; Saeki, Mihoro; Shirouzu, Mikako; Yokoyama, Shigeyuki; Konagaya, Akihiko

    2003-01-01

    ErbB tyrosine kinase receptors mediate mitogenic signal cascade by binding a variety of ligands and recruiting the different cassettes of adaptor proteins. In the present study, we examined heregulin (HRG)-induced signal transduction of ErbB4 receptor and found that the phosphatidylinositol 3'-kinase (PI3K)-Akt pathway negatively regulated the extracellular signal-regulated kinase (ERK) cascade by phosphorylating Raf-1 on Ser(259). As the time-course kinetics of Akt and ERK activities seemed to be transient and complex, we constructed a mathematical simulation model for HRG-induced ErbB4 receptor signalling to explain the dynamics of the regulation mechanism in this signal transduction cascade. The model reflected well the experimental results observed in HRG-induced ErbB4 cells and in other modes of growth hormone-induced cell signalling that involve Raf-Akt cross-talk. The model suggested that HRG signalling is regulated by protein phosphatase 2A as well as Raf-Akt cross-talk, and protein phosphatase 2A modulates the kinase activity in both the PI3K-Akt and MAPK (mitogen-activated protein kinase) pathways. PMID:12691603

  13. Synthesis of imidazoles via cascade reaction of nitroallylic acetates with amidines and studies on their trypanocidal activity.

    PubMed

    Kumar, Tarun; Verma, Deepti; Menna-Barreto, Rubem F S; Valença, Wagner O; da Silva Júnior, Eufrânio N; Namboothiri, Irishi N N

    2015-02-21

    A one-pot, two step synthesis of highly substituted imidazoles has been carried out in good to excellent yields for the first time via a cascade intermolecular aza-SN2'-intramolecular aza-Michael addition involving a variety of Morita-Baylis-Hillman acetates of nitroalkenes and amidines in the presence of DABCO at room temperature. The synthetic and biological utility of the products has been demonstrated. In particular, some of the imidazoles exhibited potent activity against T. cruzi, the etiological agent of Chagas disease. PMID:25573664

  14. Matriptase activation connects tissue factor-dependent coagulation initiation to epithelial proteolysis and signaling.

    PubMed

    Le Gall, Sylvain M; Szabo, Roman; Lee, Melody; Kirchhofer, Daniel; Craik, Charles S; Bugge, Thomas H; Camerer, Eric

    2016-06-23

    The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF-expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2) and pro-urokinase. An activation pathway-selective PAR2 mutant resistant to direct cleavage by TF:FVIIa and FXa was activated by these proteases when cells co-expressed pro-matriptase, and matriptase transactivation was necessary for efficient cleavage and activation of wild-type PAR2 by physiological concentrations of TF:FVIIa and FXa. The coagulation initiation complex induced rapid and prolonged enhancement of the barrier function of epithelial monolayers that was dependent on matriptase transactivation and PAR2 signaling. These observations suggest that the coagulation cascade engages matriptase to help coordinate epithelial defense and repair programs after injury or infection, and that matriptase may contribute to TF-driven pathogenesis in cancer and inflammation. PMID:27114461

  15. A cascade signal amplification strategy for surface enhanced Raman spectroscopy detection of thrombin based on DNAzyme assistant DNA recycling and rolling circle amplification.

    PubMed

    Gao, Fenglei; Du, Lili; Tang, Daoquan; Lu, Yao; Zhang, Yanzhuo; Zhang, Lixian

    2015-04-15

    A sensitive protocol for surface enhanced Raman spectroscopy (SERS) detection of thrombin is designed with R6G-Ag NPs as a signal tag by combining DNAzyme assistant DNA recycling and rolling circle amplification (RCA). Molecular beacon (MB) as recognition probe immobilizes on the glass slides and performs the amplification procedure. After thrombin-induced structure-switching DNA hairpins of probe 1, the DNAzyme is liberated from the caged structure, which hybridizes with the MB for cleavage of the MB in the presence of cofactor Zn(2+) and initiates the DNA recycling process, leading to the cleavage of a large number of MB and the generation of numerous primers for triggering RCA reaction. The long amplified RCA product which contained hundreds of tandem-repeat sequences, which can bind with oligonucleotide functionalized Ag NPs reporters. The attached signal tags can be easily read out by SERS. Because of the cascade signal amplification, these newly designed protocols provides a sensitive SERS detection of thrombin down to the femolar level (2.3fM) with a linear range of 5 orders of magnitude (from 10(-14) to 10(-9)M) and have high selectivity toward its target protein. The proposed method is expected to be a good clinical tool for the diagnosis of a thrombotic disease. PMID:25497982

  16. Sub-nanometrically resolved chemical mappings of quantum-cascade laser active regions

    NASA Astrophysics Data System (ADS)

    Pantzas, Konstantinos; Beaudoin, Grégoire; Patriarche, Gilles; Largeau, Ludovic; Mauguin, Olivia; Pegolotti, Giulia; Vasanelli, Angela; Calvar, Ariane; Amanti, Maria; Sirtori, Carlo; Sagnes, Isabelle

    2016-05-01

    A procedure that produces sub-nanometrically resolved chemical mappings of MOCVD-grown InGaAs/InAlAs/InP quantum cascade lasers is presented. The chemical mappings reveal that, although the structure is lattice-matched to InP, the InAlAs barriers do not attain the nominal aluminum content—48%—and are, in fact, InGaAlAs quaternaries. This information is used to adjust the aluminum precursor flow and fine-tune the composition of the barriers, resulting in a significant improvement of the fabricated lasers.

  17. 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

  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. Dopamine D₁-D₂ receptor heteromer regulates signaling cascades involved in addiction: potential relevance to adolescent drug susceptibility.

    PubMed

    Perreault, Melissa L; O'Dowd, Brian F; George, Susan R

    2014-01-01

    Adolescence is a developmental period that has been associated with heightened sensitivity to psychostimulant-induced reward, thus placing adolescents at increased risk to develop drug addiction. Although alterations in dopamine-induced synaptic plasticity are perhaps the most critical factor in mediating addiction processes, developmental differences in the cell signaling mechanisms that contribute to synaptic plasticity, and their contribution to adolescent reward sensitivity, has been grossly understudied. The most abundant dopamine receptors, the D1 and D2 receptors, as well as the dopamine D1-D2 receptor heteromer, exhibit age-dependent and brain region-specific changes in their expression and function and are responsible for regulating cell signaling pathways known to significantly contribute to the neurobiological mechanisms underlying addiction. The D1-D2 receptor heteromer, for instance, has been associated with calcium calmodulin kinase IIα, brain-derived neurotrophic factor and glycogen synthase kinase 3 (GSK-3) signaling, three proteins highly implicated in the regulation of glutamate transmission and synaptic plasticity and which regulate addiction to amphetamine, opioids and cocaine. Therefore, in this review the importance of these signaling proteins as potential mediators of addiction susceptibility in adolescence will be highlighted, and the therapeutic potential of the D1-D2 receptor heteromer in addiction will be discussed. It is the overall goal of this review to draw attention to the research gap in dopamine-induced cell signaling in the adolescent brain--knowledge that would provide much-needed insights into adolescent addiction vulnerability. PMID:24820626

  20. A novel pathway spatiotemporally activates Rac1 and redox signaling in response to fluid shear stress

    PubMed Central

    Liu, Yunhao; Collins, Caitlin; Kiosses, William B.; Murray, Ann M.; Joshi, Monika; Shepherd, Tyson R.; Fuentes, Ernesto J.

    2013-01-01

    Hemodynamic forces regulate embryonic organ development, hematopoiesis, vascular remodeling, and atherogenesis. The mechanosensory stimulus of blood flow initiates a complex network of intracellular pathways, including activation of Rac1 GTPase, establishment of endothelial cell (EC) polarity, and redox signaling. The activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can be modulated by the GTP/GDP state of Rac1; however, the molecular mechanisms of Rac1 activation by flow are poorly understood. Here, we identify a novel polarity complex that directs localized Rac1 activation required for downstream reactive oxygen species (ROS) production. Vav2 is required for Rac1 GTP loading, whereas, surprisingly, Tiam1 functions as an adaptor in a VE-cadherin–p67phox–Par3 polarity complex that directs localized activation of Rac1. Furthermore, loss of Tiam1 led to the disruption of redox signaling both in vitro and in vivo. Our results describe a novel molecular cascade that regulates redox signaling by the coordinated regulation of Rac1 and by linking components of the polarity complex to the NADPH oxidase. PMID:23733346

  1. Stress-activated signaling responses leading to apoptosis following photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Oleinick, Nancy L.; He, Jin; Xue, Liang-yan; Separovic, Duska

    1998-05-01

    Photodynamic treatment with the phthalocyanine Pc 4, a mitochondrially localizing photosensitizer, is an efficient inducer of cell death by apoptosis, a cell suicide pathway that can be triggered by physiological stimuli as well as by various types of cellular damage. Upon exposure of the dye- loaded cells to red light, several stress signalling pathways are rapidly activated. In murine L5178Y-R lymphoblasts, caspase activation and other hallmarks of the final phase of apoptosis are observed within a few minutes post-PDT. In Chinese hamster CHO-K1 cells, the first signs of apoptosis are not observed for 1 - 2 hours. The possible involvement of three parallel mitogen-activated protein kinase (MAPK) signalling pathways has been investigated. The extracellular- regulated kinases (ERK-1 and ERK-2), that are thought to promote cell growth, are not appreciably altered by PDT. However, PDT causes marked activation of the stress-activated protein kinase (SAPK) cascade in both cell types and of the p38/HOG-type kinase in CHO cells. Both of these latter pathways have been demonstrated to be associated with apoptosis. A specific inhibitor of the ERK pathway did not alter PDT-induced apoptosis; however, an inhibitor of the p38 pathway partially blocked PDT-induced apoptosis. Blockage of the SAPK pathway is being pursued by a genetic approach. It appears that the SAPK and p38 pathways may participate in signaling apoptosis in response to PDT with Pc 4.

  2. Cascaded active silicon microresonator array cross-connect circuits for WDM networks-on-chip

    NASA Astrophysics Data System (ADS)

    Poon, Andrew W.; Xu, Fang; Luo, Xianshu

    2008-02-01

    We propose a design of an optical switch on a silicon chip comprising a 5 × 5 array of cascaded waveguide-crossing-coupled microring resonator-based switches for photonic networks-on-chip applications. We adopt our recently demonstrated design of multimode-interference (MMI)-based wire waveguide crossings, instead of conventional plain waveguide crossings, for the merits of low loss and low crosstalk. The microring resonator is integrated with a lateral p-i-n diode for carrier-injection-based GHz-speed on-off switching. All 25 microring resonators are assumed to be identical within a relatively wide resonance line width. The optical circuit switch can employ a single wavelength channel or multiple wavelength channels that are spaced by the microring resonator free spectral range. We analyze the potential performance of the proposed photonic network in terms of (i) light path cross-connections loss budget, and (ii) DC on-off power consumption for establishing a light path. As a proof-of-concept, our initial experiments on cascaded passive silicon MMI-crossing-coupled microring resonators demonstrate 3.6-Gbit/s non-return-to-zero data transmissions at on- and off-resonance wavelengths.

  3. 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

  4. Terahertz generation in mid-infrared quantum cascade lasers with a dual-upper-state active region

    SciTech Connect

    Fujita, Kazuue Hitaka, Masahiro; Ito, Akio; Edamura, Tadataka; Yamanishi, Masamichi; Jung, Seungyong; Belkin, Mikhail A.

    2015-06-22

    We report the performance of room temperature terahertz sources based on intracavity difference-frequency generation in mid-infrared quantum cascade lasers with a dual-upper-state (DAU) active region. DAU active region design is theoretically expected to produce larger optical nonlinearity for terahertz difference-frequency generation, compared to the active region designs of the bound-to-continuum type used previously. Fabricated buried heterostructure devices with a two-section buried distributed feedback grating and the waveguide designed for Cherenkov difference-frequency phase-matching scheme operate in two single-mode mid-infrared wavelengths at 10.7 μm and 9.7 μm and produce terahertz output at 2.9 THz with mid-infrared to terahertz conversion efficiency of 0.8 mW/W{sup 2} at room temperature.

  5. 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

  6. 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

  7. Phonetic basis of phonemic paraphasias in aphasia: Evidence for cascading activation.

    PubMed

    Kurowski, Kathleen; Blumstein, Sheila E

    2016-02-01

    Phonemic paraphasias are a common presenting symptom in aphasia and are thought to reflect a deficit in which selecting an incorrect phonemic segment results in the clear-cut substitution of one phonemic segment for another. The current study re-examines the basis of these paraphasias. Seven left hemisphere-damaged aphasics with a range of left hemisphere lesions and clinical diagnoses including Broca's, Conduction, and Wernicke's aphasia, were asked to produce syllable-initial voiced and voiceless fricative consonants, [z] and [s], in CV syllables followed by one of five vowels [i e a o u] in isolation and in a carrier phrase. Acoustic analyses were conducted focusing on two acoustic parameters signaling voicing in fricative consonants: duration and amplitude properties of the fricative noise. Results show that for all participants, regardless of clinical diagnosis or lesion site, phonemic paraphasias leave an acoustic trace of the original target in the error production. These findings challenge the view that phonemic paraphasias arise from a mis-selection of phonemic units followed by its correct implementation, as traditionally proposed. Rather, they appear to derive from a common mechanism with speech errors reflecting the co-activation of a target and competitor resulting in speech output that has some phonetic properties of both segments. PMID:26808838

  8. Jasmonate-based wound signal transduction requires activation of WIPK, a tobacco mitogen-activated protein kinase.

    PubMed Central

    Seo, S; Sano, H; Ohashi, Y

    1999-01-01

    A gene encoding a tobacco mitogen-activated protein kinase (WIPK) is transcriptionally activated in response to wounding. Transgenic tobacco plants, in which expression of endogenous wipk was suppressed, did not accumulate jasmonic acid or its methyl ester when wounded, suggesting that WIPK is involved in jasmonate-mediated wound signal transduction. Here, we demonstrate that activation of WIPK is required for triggering the jasmonate-mediated signal transduction cascade that occurs when wild-type tobacco plants are wounded. We also show that when plants are wounded, WIPK is rapidly and transiently activated, whereas the quantity of WIPK protein is maintained at a constant level. A transgenic tobacco plant in which the wipk gene was constitutively expressed at a high level showed constitutive enzymatic activation of WIPK and exhibited three- to fourfold higher levels of jasmonate than did its wild-type counterpart. This plant also showed constitutive accumulation of jasmonate-inducible proteinase inhibitor II transcripts. These results show that WIPK is activated in response to wounding, which subsequently causes an increase in jasmonate synthesis. PMID:9927645

  9. Redox Signaling as a Therapeutic Target to Inhibit Myofibroblast Activation in Degenerative Fibrotic Disease

    PubMed Central

    Berger, Peter; Zenzmaier, Christoph

    2014-01-01

    Degenerative fibrotic diseases encompass numerous systemic and organ-specific disorders. Despite their associated significant morbidity and mortality, there is currently no effective antifibrotic treatment. Fibrosis is characterized by the development and persistence of myofibroblasts, whose unregulated deposition of extracellular matrix components disrupts signaling cascades and normal tissue architecture leading to organ failure and death. The profibrotic cytokine transforming growth factor beta (TGFβ) is considered the foremost inducer of fibrosis, driving myofibroblast differentiation in diverse tissues. This review summarizes recent in vitro and in vivo data demonstrating that TGFβ-induced myofibroblast differentiation is driven by a prooxidant shift in redox homeostasis. Elevated NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) supported by concomitant decreases in nitric oxide (NO) signaling and reactive oxygen species scavengers are central factors in the molecular pathogenesis of fibrosis in numerous tissues and organs. Moreover, complex interplay between NOX4-derived H2O2 and NO signaling regulates myofibroblast differentiation. Restoring redox homeostasis via antioxidants or NOX4 inactivation as well as by enhancing NO signaling via activation of soluble guanylyl cyclases or inhibition of phosphodiesterases can inhibit and reverse myofibroblast differentiation. Thus, dysregulated redox signaling represents a potential therapeutic target for the treatment of wide variety of different degenerative fibrotic disorders. PMID:24701562

  10. 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

  11. Magmatic activity beneath the quiescent Three Sisters volcanic center, central Oregon Cascade Range, USA

    USGS Publications Warehouse

    Wicks, Charles W., Jr.; Dzurisin, Daniel; Ingebritsen, Steven E.; Thatcher, Wayne R.; Lu, Zhong; Iverson, Justin

    2002-01-01

    Images from satellite interferometric synthetic aperture radar (InSAR) reveal uplift of a broad ???10 km by 20 km area in the Three Sisters volcanic center of the central Oregon Cascade Range, ???130 km south of Mt. St. Helens. The last eruption in the volcanic center occurred ???1500 years ago. Multiple satellite images from 1992 through 2000 indicate that most if not all of ???100 mm of observed uplift occurred between September 1998 and October 2000. Geochemical (water chemistry) anomalies, first noted during 1990, coincide with the area of uplift and suggest the existence of a crustal magma reservoir prior to the uplift. We interpret the uplift as inflation caused by an ongoing episode of magma intrusion at a depth of ???6.5 km.

  12. Stratigraphic development and hydrothermal activity in the central western Cascade Range, Oregon

    SciTech Connect

    Cummings, M.L.; Bull, M.K. ); Pollock, J.M. ); Thompson, G.D. )

    1990-11-10

    Two volcanic sequences bounded by erosional unconformities compose the stratigraphy of the North Santiam mining district, Western Cascade Range, Oregon. Diorite, grandodiorite, and leucocratic quartz porphyry dikes, stocks, and sills intrude the breccias, flows, and tuffs of a volcanic center in the older Sardine Formation. Tourmaline-bearing breccia pipes are associated with the porphyritic granodiorite intrusions. An erosional unconformity separates the Sardine Formation from the overlying Elk Lake formation. The alteration patterns in the two formations are consistent with the development of hydrothermal systems during the eruption of each formation. However, the development of the two hydrothermal systems is separated by a period of erosion of the older volcanic pile. Early formation of mineralization that resembles porphyry copper deposits occurred within the Sardine Formation, and later, after eruption of the Elk Lake formation, epithermal veins and alteration developed along faults, fractures, and the margins of dikes in the Sardine Formation.

  13. Induction of matrix metalloproteinase activation cascades based on membrane-type 1 matrix metalloproteinase: associated activation of gelatinase A, gelatinase B and collagenase 3.

    PubMed Central

    Cowell, S; Knäuper, V; Stewart, M L; D'Ortho, M P; Stanton, H; Hembry, R M; López-Otín, C; Reynolds, J J; Murphy, G

    1998-01-01

    SW1353 chondrosarcoma cells cultured in the presence of interleukin-1, concanavalin A or PMA secreted procollagenase 3 (matrix metalloproteinase-13). The enzyme was detected in the culture medium by Western blotting using a specific polyclonal antibody raised against recombinant human procollagenase 3. Oncostatin M enhanced the interleukin-1-induced production of procollagenase 3, whereas interleukin-4 decreased procollagenase 3 synthesis. The enzyme was latent except when the cells had been treated with concanavalin A, when a processed form of 48 kDa, which corresponds to the active form, was found in the culture medium and collagenolytic activity was detected by degradation of 14C-labelled type I collagen. The concanavalin A-induced activation of procollagenase 3 coincided with the processing of progelatinase A (matrix metalloproteinase-2) by the cells, as measured by gelatin zymography. In addition, progelatinase B (matrix metalloproteinase-9) was activated when gelatinase A and collagenase 3 were in their active forms. Concanavalin A treatment of SW1353 cells increased the amount of membrane-type-1 matrix metalloproteinase protein in the cell membranes, suggesting that this membrane-bound enzyme participates in an activation cascade involving collagenase 3 and the gelatinases. This cascade was effectively inhibited by tissue inhibitors of metalloproteinases-2 and -3. Tissue inhibitor of metalloproteinases-1, which is a much weaker inhibitor of membrane-type 1 matrix metalloproteinase than tissue inhibitors of metalloproteinases-2 and -3 [Will, Atkinson, Butler, Smith and Murphy (1996) J. Biol. Chem. 271, 17119-17123], was a weaker inhibitor of the activation cascade. PMID:9531484

  14. Broadly continuously tunable slot waveguide quantum cascade lasers based on a continuum-to-continuum active region design

    SciTech Connect

    Meng, Bo; Zeng, Yong Quan; Liang, Guozhen; Hu, Xiao Nan; Rodriguez, Etienne; Wang, Qi Jie

    2015-09-14

    We report our progress in the development of broadly tunable single-mode slot waveguide quantum cascade lasers based on a continuum-to-continuum active region design. The electroluminescence spectrum of the continuum-to-continuum active region design has a full width at half maximum of 440 cm{sup −1} at center wavelength ∼10 μm at room temperature (300 K). Devices using the optimized slot waveguide structure and the continuum-to-continuum design can be tuned continuously with a lasing emission over 42 cm{sup −1}, from 9.74 to 10.16 μm, at room temperature by using only current tuning scheme, together with a side mode suppression ratio of above 15 dB within the whole tuning range.

  15. 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

  16. 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.

  17. Inhibition of host extracellular signal-regulated kinase (ERK) activation decreases new world alphavirus multiplication in infected cells

    SciTech Connect

    Voss, Kelsey; Amaya, Moushimi; Mueller, Claudius; Roberts, Brian; Kehn-Hall, Kylene; Bailey, Charles; Petricoin, Emanuel; Narayanan, Aarthi

    2014-11-15

    New World alphaviruses belonging to the family Togaviridae are classified as emerging infectious agents and Category B select agents. Our study is focused on the role of the host extracellular signal-regulated kinase (ERK) in the infectious process of New World alphaviruses. Infection of human cells by Venezuelan equine encephalitis virus (VEEV) results in the activation of the ERK-signaling cascade. Inhibition of ERK1/2 by the small molecule inhibitor Ag-126 results in inhibition of viral multiplication. Ag-126-mediated inhibition of VEEV was due to potential effects on early and late stages of the infectious process. While expression of viral proteins was down-regulated in Ag-126 treated cells, we did not observe any influence of Ag-126 on the nuclear distribution of capsid. Finally, Ag-126 exerted a broad-spectrum inhibitory effect on New World alphavirus multiplication, thus indicating that the host kinase, ERK, is a broad-spectrum candidate for development of novel therapeutics against New World alphaviruses. - Highlights: • VEEV infection activated multiple components of the ERK signaling cascade. • Inhibition of ERK activation using Ag-126 inhibited VEEV multiplication. • Activation of ERK by Ceramide C6 increased infectious titers of TC-83. • Ag-126 inhibited virulent strains of all New World alphaviruses. • Ag-126 treatment increased percent survival of infected cells.

  18. Cascade DNA logic device programmed ratiometric DNA analysis and logic devices based on a fluorescent dual-signal probe of a G-quadruplex DNAzyme.

    PubMed

    Fan, Daoqing; Zhu, Jinbo; Zhai, Qingfeng; Wang, Erkang; Dong, Shaojun

    2016-03-01

    Herein, two fluorescence sensitive substrates of G-quadruplex/hemin DNAzyme with inverse responses (Scopoletin and Amplex Red) were simultaneously used in one homogeneous system to construct a cascade advanced DNA logic device for the first time (a functional logic device (a three input based DNA calliper) cascade with an advanced non-arithmetic logic gate (1 to 2 decoder)). This cascade logic device was applied to label-free ratiometric target DNA detection and length measurement. PMID:26882417

  19. Potentiation of acid-sensing ion channel activity by peripheral group I metabotropic glutamate receptor signaling.

    PubMed

    Gan, Xiong; Wu, Jing; Ren, Cuixia; Qiu, Chun-Yu; Li, Yan-Kun; Hu, Wang-Ping

    2016-05-01

    Glutamate activates peripheral group I metabotropic glutamate receptors (mGluRs) and contributes to inflammatory pain. However, it is still not clear the mechanisms are involved in group I mGluR-mediated peripheral sensitization. Herein, we report that group I mGluRs signaling sensitizes acid-sensing ion channels (ASICs) in dorsal root ganglion (DRG) neurons and contributes to acidosis-evoked pain. DHPG, a selective group I mGluR agonist, can potentiate the functional activity of ASICs, which mediated the proton-induced events. DHPG concentration-dependently increased proton-gated currents in DRG neurons. It shifted the proton concentration-response curve upwards, with a 47.3±7.0% increase of the maximal current response to proton. Group I mGluRs, especially mGluR5, mediated the potentiation of DHPG via an intracellular cascade. DHPG potentiation of proton-gated currents disappeared after inhibition of intracellular Gq/11 proteins, PLCβ, PKC or PICK1 signaling. Moreover, DHPG enhanced proton-evoked membrane excitability of rat DRG neurons and increased the amplitude of the depolarization and the number of spikes induced by acid stimuli. Finally, peripherally administration of DHPG dose-dependently exacerbated nociceptive responses to intraplantar injection of acetic acid in rats. Potentiation of ASIC activity by group I mGluR signaling in rat DRG neurons revealed a novel peripheral mechanism underlying group I mGluRs involvement in hyperalgesia. PMID:26946972

  20. Single molecule analysis of B cell receptor motion during signaling activation

    NASA Astrophysics Data System (ADS)

    Rey Suarez, Ivan; Koo, Peter; Mochrie, Simon; Song, Wenxia; Upadhyaya, Arpita

    B cells are an essential part of the adaptive immune system. They patrol the body looking for signs of infection in the form of antigen on the surface of antigen presenting cells. The binding of the B cell receptor (BCR) to antigen induces signaling cascades that lead to B cell activation and eventual production of high affinity antibodies. During activation, BCR organize into signaling microclusters, which are platforms for signal amplification. The physical processes underlying receptor movement and aggregation are not well understood. Here we study the dynamics of single BCRs on activated murine primary B cells using TIRF imaging and single particle tracking. The tracks obtained are analyzed using perturbation expectation-maximization (pEM) a systems-level analysis that allows the identification of different short-time diffusive states from a set of single particle tracks. We identified five different diffusive states on wild type cells, which correspond to different molecular states of the BCR. By using actin polymerization inhibitors and mutant cells lacking important actin regulators we were able to identify the BCR molecule configuration associated with each diffusive state.

  1. Cardiovascular Protective Effect of Metformin and Telmisartan: Reduction of PARP1 Activity via the AMPK-PARP1 Cascade

    PubMed Central

    Shang, Fenqing; Zhang, Jiao; Li, Zhao; Zhang, Jin; Yin, Yanjun; Wang, Yaqiong; Marin, Traci L.; Gongol, Brendan; Xiao, Han; Zhang, You-yi; Chen, Zhen; Shyy, John Y-J; Lei, Ting

    2016-01-01

    Hyperglycemia and hypertension impair endothelial function in part through oxidative stress-activated poly (ADP-ribose) polymerase 1 (PARP1). Biguanides and angiotensin II receptor blockers (ARBs) such as metformin and telmisartan have a vascular protective effect. We used cultured vascular endothelial cells (ECs), diabetic and hypertensive rodent models, and AMPKα2-knockout mice to investigate whether metformin and telmisartan have a beneficial effect on the endothelium via AMP-activated protein kinase (AMPK) phosphorylation of PARP1 and thus inhibition of PARP1 activity. The results showed that metformin and telmisartan, but not glipizide and metoprolol, activated AMPK, which phosphorylated PARP1 Ser-177 in cultured ECs and the vascular wall of rodent models. Experiments using phosphorylated/de-phosphorylated PARP1 mutants show that AMPK phosphorylation of PARP1 leads to decreased PARP1 activity and attenuated protein poly(ADP-ribosyl)ation (PARylation), but increased endothelial nitric oxide synthase (eNOS) activity and silent mating type information regulation 2 homolog 1 (SIRT1) expression. Taken together, the data presented here suggest biguanides and ARBs have a beneficial effect on the vasculature by the cascade of AMPK phosphorylation of PARP1 to inhibit PARP1 activity and protein PARylation in ECs, thereby mitigating endothelial dysfunction. PMID:26986624

  2. Cardiovascular Protective Effect of Metformin and Telmisartan: Reduction of PARP1 Activity via the AMPK-PARP1 Cascade.

    PubMed

    Shang, Fenqing; Zhang, Jiao; Li, Zhao; Zhang, Jin; Yin, Yanjun; Wang, Yaqiong; Marin, Traci L; Gongol, Brendan; Xiao, Han; Zhang, You-Yi; Chen, Zhen; Shyy, John Y-J; Lei, Ting

    2016-01-01

    Hyperglycemia and hypertension impair endothelial function in part through oxidative stress-activated poly (ADP-ribose) polymerase 1 (PARP1). Biguanides and angiotensin II receptor blockers (ARBs) such as metformin and telmisartan have a vascular protective effect. We used cultured vascular endothelial cells (ECs), diabetic and hypertensive rodent models, and AMPKα2-knockout mice to investigate whether metformin and telmisartan have a beneficial effect on the endothelium via AMP-activated protein kinase (AMPK) phosphorylation of PARP1 and thus inhibition of PARP1 activity. The results showed that metformin and telmisartan, but not glipizide and metoprolol, activated AMPK, which phosphorylated PARP1 Ser-177 in cultured ECs and the vascular wall of rodent models. Experiments using phosphorylated/de-phosphorylated PARP1 mutants show that AMPK phosphorylation of PARP1 leads to decreased PARP1 activity and attenuated protein poly(ADP-ribosyl)ation (PARylation), but increased endothelial nitric oxide synthase (eNOS) activity and silent mating type information regulation 2 homolog 1 (SIRT1) expression. Taken together, the data presented here suggest biguanides and ARBs have a beneficial effect on the vasculature by the cascade of AMPK phosphorylation of PARP1 to inhibit PARP1 activity and protein PARylation in ECs, thereby mitigating endothelial dysfunction. PMID:26986624

  3. Spinal Toll-like receptor signaling and nociceptive processing: Regulatory balance between TIRAP and TRIF cascades mediated by TNF and IFNβ

    PubMed Central

    Stokes, Jennifer A.; Corr, Maripat; Yaksh, Tony L.

    2013-01-01

    Toll-like Receptors (TLRs) play a pivotal role in inflammatory processes and individual TLRs have been investigated in nociception. Here, we examine overlapping and diverging roles of spinal TLRs and their associated adaptor proteins in nociceptive processing. Intrathecal (IT) TLR2, TLR3, or TLR4 ligands (-L) evoked persistent (7 day) tactile allodynia (TA) that was abolished in respective TLR deficient mice. Using Tnf−/− mice, we found that IT TLR2 and TLR4 TA was TNF-dependent, while TLR3 was TNF-independent. In toll-interleukin 1 receptor (TIR) domain containing adaptor protein (Tirap−/−) mice (downstream to TLR2 and TLR4), allodynia after IT TLR2-L and TLR4-L was abolished. Unexpectedly, in TIR-domain-containing adapter-inducing interferon-β (Triflps2) mice (downstream of TLR3 and TLR4), TLR3-L allodynia was abrogated, but intrathecal TLR4-L produced a persistent increase (>21 days) in TA. Consistent with a role for interferon (IFN)β (downstream to TRIF) in regulating recovery after IT TLR4-L, prolonged allodynia was noted in Ifnar1−/− mice. Further, IT IFNβ given to Triflps2 mice reduced TLR4 allodynia. Hence, spinal TIRAP and TRIF cascades differentially lead to robust TA by TNF dependent and independent pathways, while activation of TRIF modulated processing through type I IFN receptors. Based on these results, we believe that processes leading to the activation of these spinal TLRs initiate TNF-dependent and -independent cascades, which contribute to the associated persistent pain state. In addition, TRIF pathways are able to modulate the TNF-dependent pain state through IFNβ. PMID:23489833

  4. Irisin promotes osteoblast proliferation and differentiation via activating the MAP kinase signaling pathways.

    PubMed

    Qiao, Xiaoyong; Yong Qiao, Xiao; Nie, Ying; Ma, Yaxian; Xian Ma, Ya; Chen, Yan; Cheng, Ran; Yin, Weiyao; Yao Yinrg, Wei; Hu, Ying; Xu, Wenming; Ming Xu, Wen; Xu, Liangzhi; Zhi Xu, Liang

    2016-01-01

    Physical exercise is able to improve skeletal health. However, the mechanisms are poorly known. Irisin, a novel exercise-induced myokine, secreted by skeletal muscle in response to exercise, have been shown to mediate beneficial effects of exercise in many disorders. In the current study, we demonstrated that irisin promotes osteoblast proliferation, and increases the expression of osteoblastic transcription regulators, such as Runt-related transcription factor-2, osterix/sp7; and osteoblast differentiation markers, including alkaline phosphatase, collagen type 1 alpha-1, osteocalcin, and osteopontin in vitro. Irisin also increase ALP activity and calcium deposition in cultured osteoblast. These osteogenic effects were mediated by activating the p38 mitogen-activated protein kinase (p-p38 MAPK) and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK by SB023580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runx2 expression and ALP activity. Together our observation suggest that irisin directly targets osteoblast, promoting osteoblast proliferation and differentiation via activating P38/ERK MAP kinase signaling cascades in vitro. Whether irisin can be utilized as the therapeutic agents for osteopenia and osteoporosis is worth to be further pursued. PMID:26738434

  5. Irisin promotes osteoblast proliferation and differentiation via activating the MAP kinase signaling pathways

    PubMed Central

    Yong Qiao, Xiao; Nie, Ying; Xian Ma, Ya; Chen, Yan; Cheng, Ran; Yao Yinrg, Wei; Hu, Ying; Ming Xu, Wen; Zhi Xu, Liang

    2016-01-01

    Physical exercise is able to improve skeletal health. However, the mechanisms are poorly known. Irisin, a novel exercise-induced myokine, secreted by skeletal muscle in response to exercise, have been shown to mediate beneficial effects of exercise in many disorders. In the current study, we demonstrated that irisin promotes osteoblast proliferation, and increases the expression of osteoblastic transcription regulators, such as Runt-related transcription factor-2, osterix/sp7; and osteoblast differentiation markers, including alkaline phosphatase, collagen type 1 alpha-1, osteocalcin, and osteopontin in vitro. Irisin also increase ALP activity and calcium deposition in cultured osteoblast. These osteogenic effects were mediated by activating the p38 mitogen-activated protein kinase (p-p38 MAPK) and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK by SB023580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runx2 expression and ALP activity. Together our observation suggest that irisin directly targets osteoblast, promoting osteoblast proliferation and differentiation via activating P38/ERK MAP kinase signaling cascades in vitro. Whether irisin can be utilized as the therapeutic agents for osteopenia and osteoporosis is worth to be further pursued. PMID:26738434

  6. Reconfigurable optical inter-channel interference mitigation for spectrally overlapped QPSK signals using nonlinear wave mixing in cascaded PPLN waveguides.

    PubMed

    Cao, Yinwen; Ziyadi, Morteza; Mohajerin-Ariaei, Amirhossein; Almaiman, Ahmed; Liao, Peicheng; Bao, Changjing; Alishahi, Fatemeh; Falahpour, Ahmad; Shamee, Bishara; Yang, Jeng-Yuan; Akasaka, Youichi; Sekiya, Motoyoshi; Tur, Moshe; Langrock, Carsten; Fejer, Martin; Touch, Joseph; Willner, Alan E

    2016-07-15

    A reconfigurable all-optical inter-channel interference (ICI) mitigation method is proposed for an overlapped channel system that avoids the need for multi-channel detection and channel spacing estimation. The system exhibits a 0.5-dB implementation penalty compared to a single-channel baseline system. Experiments using a dual-carrier QPSK overlapped system with both 20G-baud and 25G-baud under different channel spacing conditions evaluate the performance of the method. Improved signal constellation and receiver sensitivity demonstrate the effectiveness of this approach. This results in over 4-dB OSNR of benefit when the system Q-factor reaches a forward error correction (FEC) threshold of 8.5 dB under less-than-baudrate channel spacing conditions. PMID:27420503

  7. Monolithically, widely tunable quantum cascade lasers based on a heterogeneous active region design

    NASA Astrophysics Data System (ADS)

    Zhou, Wenjia; Bandyopadhyay, Neelanjan; Wu, Donghai; McClintock, Ryan; Razeghi, Manijeh

    2016-06-01

    Quantum cascade lasers (QCLs) have become important laser sources for accessing the mid-infrared (mid-IR) spectral range, achieving watt-level continuous wave operation in a compact package at room temperature. However, up to now, wavelength tuning, which is desirable for most applications, has relied on external cavity feedback or exhibited a limited monolithic tuning range. Here we demonstrate a widely tunable QCL source over the 6.2 to 9.1 μm wavelength range with a single emitting aperture by integrating an eight-laser sampled grating distributed feedback laser array with an on-chip beam combiner. The laser gain medium is based on a five-core heterogeneous QCL wafer. A compact tunable laser system was built to drive the individual lasers within the array and produce any desired wavelength within the available spectral range. A rapid, broadband spectral measurement (520 cm‑1) of methane using the tunable laser source shows excellent agreement to a measurement made using a standard low-speed infrared spectrometer. This monolithic, widely tunable laser technology is compact, with no moving parts, and will open new opportunities for MIR spectroscopy and chemical sensing.

  8. Monolithically, widely tunable quantum cascade lasers based on a heterogeneous active region design

    PubMed Central

    Zhou, Wenjia; Bandyopadhyay, Neelanjan; Wu, Donghai; McClintock, Ryan; Razeghi, Manijeh

    2016-01-01

    Quantum cascade lasers (QCLs) have become important laser sources for accessing the mid-infrared (mid-IR) spectral range, achieving watt-level continuous wave operation in a compact package at room temperature. However, up to now, wavelength tuning, which is desirable for most applications, has relied on external cavity feedback or exhibited a limited monolithic tuning range. Here we demonstrate a widely tunable QCL source over the 6.2 to 9.1 μm wavelength range with a single emitting aperture by integrating an eight-laser sampled grating distributed feedback laser array with an on-chip beam combiner. The laser gain medium is based on a five-core heterogeneous QCL wafer. A compact tunable laser system was built to drive the individual lasers within the array and produce any desired wavelength within the available spectral range. A rapid, broadband spectral measurement (520 cm−1) of methane using the tunable laser source shows excellent agreement to a measurement made using a standard low-speed infrared spectrometer. This monolithic, widely tunable laser technology is compact, with no moving parts, and will open new opportunities for MIR spectroscopy and chemical sensing. PMID:27270634

  9. RBM4a-regulated splicing cascade modulates the differentiation and metabolic activities of brown adipocytes

    PubMed Central

    Lin, Jung-Chun; Lu, Yi-Han; Liu, Yun-Ru; Lin, Ying-Ju

    2016-01-01

    RNA-binding motif protein 4a (RBM4a) reportedly reprograms splicing profiles of the insulin receptor (IR) and myocyte enhancer factor 2C (MEF2C) genes, facilitating the differentiation of brown adipocytes. Using an RNA-sequencing analysis, we first compared the gene expressing profiles between wild-type and RBM4a−/− brown adipocytes. The ablation of RBM4a led to increases in the PTBP1, PTBP2 (nPTB), and Nova1 proteins, whereas elevated RBM4a reduced the expression of PTBP1 and PTBP2 proteins in brown adipocytes through an alternative splicing-coupled nonsense-mediated decay mechanism. Subsequently, RBM4a indirectly shortened the half-life of the Nova1 transcript which was comparatively stable in the presence of PTBP2. RBM4a diminished the influence of PTBP2 in adipogenic development by reprogramming the splicing profiles of the FGFR2 and PKM genes. These results constitute a mechanistic understanding of the RBM4a-modulated splicing cascade during the brown adipogenesis. PMID:26857472

  10. Monolithically, widely tunable quantum cascade lasers based on a heterogeneous active region design.

    PubMed

    Zhou, Wenjia; Bandyopadhyay, Neelanjan; Wu, Donghai; McClintock, Ryan; Razeghi, Manijeh

    2016-01-01

    Quantum cascade lasers (QCLs) have become important laser sources for accessing the mid-infrared (mid-IR) spectral range, achieving watt-level continuous wave operation in a compact package at room temperature. However, up to now, wavelength tuning, which is desirable for most applications, has relied on external cavity feedback or exhibited a limited monolithic tuning range. Here we demonstrate a widely tunable QCL source over the 6.2 to 9.1 μm wavelength range with a single emitting aperture by integrating an eight-laser sampled grating distributed feedback laser array with an on-chip beam combiner. The laser gain medium is based on a five-core heterogeneous QCL wafer. A compact tunable laser system was built to drive the individual lasers within the array and produce any desired wavelength within the available spectral range. A rapid, broadband spectral measurement (520 cm(-1)) of methane using the tunable laser source shows excellent agreement to a measurement made using a standard low-speed infrared spectrometer. This monolithic, widely tunable laser technology is compact, with no moving parts, and will open new opportunities for MIR spectroscopy and chemical sensing. PMID:27270634

  11. Opposite cross-talk by oleate and palmitate on insulin signaling in hepatocytes through macrophage activation.

    PubMed

    Pardo, Virginia; González-Rodríguez, Águeda; Guijas, Carlos; Balsinde, Jesús; Valverde, Ángela M

    2015-05-01

    Chronic low grade inflammation in adipose tissue during obesity is associated with an impairment of the insulin signaling cascade. In this study, we have evaluated the impact of palmitate or oleate overload of macrophage/Kupffer cells in triggering stress-mediated signaling pathways, in lipoapoptosis, and in the cross-talk with insulin signaling in hepatocytes. RAW 264.7 macrophages or Kupffer cells were stimulated with oleate or palmitate, and levels of M1/M2 polarization markers and the lipidomic profile of eicosanoids were analyzed. Whereas proinflammatory cytokines and total eicosanoids were elevated in macrophages/Kupffer cells stimulated with palmitate, enhanced arginase 1 and lower leukotriene B4 (LTB4) levels were detected in macrophages stimulated with oleate. When hepatocytes were pretreated with conditioned medium (CM) from RAW 264.7 or Kupffer cells loaded with palmitate (CM-P), phosphorylation of stress kinases and endoplasmic reticulum stress signaling was increased, insulin signaling was impaired, and lipoapoptosis was detected. Conversely, enhanced insulin receptor-mediated signaling and reduced levels of the phosphatases protein tyrosine phosphatase 1B (PTP1B) and phosphatase and tensin homolog (PTEN) were found in hepatocytes treated with CM from macrophages stimulated with oleate (CM-O). Supplementation of CM-O with LTB4 suppressed insulin sensitization and increased PTP1B and PTEN. Furthermore, LTB4 decreased insulin receptor tyrosine phosphorylation in hepatocytes, activated the NFκB pathway, and up-regulated PTP1B and PTEN, these effects being mediated by LTB4 receptor BTL1. In conclusion, oleate and palmitate elicit an opposite cross-talk between macrophages/Kupffer cells and hepatocytes. Whereas CM-P interferes at the early steps of insulin signaling, CM-O increases insulin sensitization, possibly by reducing LTB4. PMID:25792746

  12. Magnetic Nanoparticles as Mediators of Ligand-Free Activation of EGFR Signaling

    PubMed Central

    Fritsch, Cornelia; Klaver, Arjen; Kanger, Johannes S.; Jovin, Thomas M.; Arndt-Jovin, Donna J.

    2013-01-01

    Background Magnetic nanoparticles (NPs) are of particular interest in biomedical research, and have been exploited for molecular separation, gene/drug delivery, magnetic resonance imaging, and hyperthermic cancer therapy. In the case of cultured cells, magnetic manipulation of NPs provides the means for studying processes induced by mechanotransduction or by local clustering of targeted macromolecules, e.g. cell surface receptors. The latter are normally activated by binding of their natural ligands mediating key signaling pathways such as those associated with the epidermal growth factor (EGFR). However, it has been reported that EGFR may be dimerized and activated even in the absence of ligands. The present study assessed whether receptor clustering induced by physical means alone suffices for activating EGFR in quiescent cells. Methodology/Principal Findings The EGFR on A431 cells was specifically targeted by superparamagnetic iron oxide NPs (SPIONs) carrying either a ligand-blocking monoclonal anti-EGFR antibody or a streptavidin molecule for targeting a chimeric EGFR incorporating a biotinylated amino-terminal acyl carrier peptide moiety. Application of a magnetic field led to SPION magnetization and clustering, resulting in activation of the EGFR, a process manifested by auto and transphosphorylation and downstream signaling. The magnetically-induced early signaling events were similar to those inherent to the ligand dependent EGFR pathways. Magnetization studies indicated that the NPs exerted magnetic dipolar forces in the sub-piconewton range with clustering dependent on Brownian motion of the receptor-SPION complex and magnetic field strength. Conclusions/Significance We demonstrate that EGFR on the cell surface that have their ligand binding-pocket blocked by an antibody are still capable of transphosphorylation and initiation of signaling cascades if they are clustered by SPIONs either attached locally or targeted to another site of the receptor

  13. Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19)

    PubMed Central

    Sangphech, Naunpun; Osborne, Barbara A.; Palaga, Tanapat

    2014-01-01

    Macrophages play critical roles in innate immune defense by sensing microbes using pattern-recognition receptors. Lipopolysaccharide (LPS) stimulates macrophages via TLR, which leads to activation of downstream signaling cascades. In this study, we investigated the roles of a conserved signaling pathway, Notch signaling, in regulating the downstream signaling cascades of the LPS/TLR4 pathways in macrophages. Using a phosphoproteomic approach and a gamma-secretase inhibitor (GSI) to suppress the processing and activation of Notch signaling, we identified regulator of G protein signaling 19 (RGS19) as a target protein whose phosphorylation was affected by GSI treatment. RGS19 is a guanosine triphosphatase (GTPase)-activating protein that functions to negatively regulate G protein-coupled receptors via Gαi/Gαq-linked signaling. Stimulation of RAW264.7 cells with LPS increased the level of the phosphorylated form of RGS19, while LPS stimulation in the presence of GSI decreased its level. GSI treatment did not alter the mRNA level of rgs19. Treatment with GSI or silencing of rgs19 in macrophages impaired the phosphorylation of Akt Thr308 upon LPS stimulation. Furthermore, targeted deletion of a DNA-binding protein and binding partner of the Notch receptor, RBP-Jκ/CSL, in macrophages resulted in delayed and decreased Akt phosphorylation. Because the PI3K/Akt pathway regulates cell survival in various cell types, the cell cycle and cell death were assayed upon GSI treatment, phosphatidylinositol 3 kinase (PI3K) inhibitor treatment or silencing of rgs19. GSI treatment resulted in decreased cell populations in the G1 and S phases, while it increased the cell population of cell death. Similarly, silencing of rgs19 resulted in a decreased cell population in the G1 phase and an increased cell population in the subG1 phase. Inhibition of Akt phosphorylation by PI3K inhibitor in LPS-stimulated macrophages increased cell population in G1 phase, suggesting a possible cell cycle

  14. Activation of gene expression by metal-responsive signal transduction pathways.

    PubMed Central

    Adams, Timothy K; Saydam, Nurten; Steiner, Florian; Schaffner, Walter; Freedman, Jonathan H

    2002-01-01

    Metallothioneins are small, cysteine-rich, metal-binding proteins that play important roles in maintaining intracellular metal homeostasis and in transition metal detoxification. MTF-1 (metal transcription factor-1) plays a central role in regulating the metal-inducible, transcriptional activation of metallothionein. Here we report that the phosphorylation of MTF-1 plays a critical role in the activation of MTF-1/metal-responsive element-mediated transcription. Inhibitor studies indicate that signal transduction cascades, including those mediated by protein kinase C, tyrosine kinase, and casein kinase II, are essential for zinc- and cadmium-inducible transcription. In addition, calcium signaling is also involved in regulating transcription. In contrast, cAMP-dependent protein kinase may not be directly involved in the metal response. Contrary to what has been reported for other transcription factors, the inhibition of transcriptional activation does not impair the binding of MTF-1 to DNA, suggesting that phosphorylation is not regulating DNA binding. Elevated phosphorylation of MTF-1 is observed under conditions of protein kinase C inhibition, suggesting that dephosphorylation of this transcription factor mediates its activation. PMID:12426137

  15. Modeling and analysis of early events in T-lymphocyte antigen-activated intracellular-signaling pathways

    NASA Astrophysics Data System (ADS)

    Zheng, Yanan; Balakrishnan, Venkataramanan; Buzzard, Greg; Geahlen, Robert; Harrison, Marietta; Rundell, Ann

    2005-12-01

    The T-cell antigen-activated signaling pathway is a highly regulated intracellular biochemical system that is crucial for initiating an appropriate adaptive immune response. To improve the understanding of the complex regulatory mechanisms controlling the early events in T-cell signaling, a detailed mathematical model was developed that utilizes ordinary differential equations to describe chemical reactions of the signaling pathway. The model parameter values were constrained by experimental data on the activation of a specific signaling intermediate and indicated an initial rapid cascade of phosphorylation events followed by a comparatively slow signal downregulation. Nonlinear analysis of the model suggested that thresholding and bistability occur as a result of the embedded positive and negative feedback loops within the model. These nonlinear system properties may enhance the T-cell receptor specificity and provide sub-threshold noise filtering with switch-like behavior to ensure proper cell response. Additional analysis using a reduced second-order model led to further understanding of the observed system behavior. Moreover, the interactions between the positive and negative feedback loops enabled the model to exhibit, among a variety of other feasible dynamics, a sustained oscillation that corresponds to a stable limit cycle in the two-dimensional phase plane. Quantitative analysis in this paper has helped identify potential regulatory mechanisms in the early T-cell signaling events. This integrated approach provides a framework to quantify and discover the ensemble of interconnected T-cell antigen-activated signaling pathways from limited experimental data.

  16. 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

  17. Signal-dependent Hydrolysis of Phosphatidylinositol 4,5-Bisphosphate without Activation of Phospholipase C

    PubMed Central

    Lev, Shaya; Katz, Ben; Tzarfaty, Vered; Minke, Baruch

    2012-01-01

    In Drosophila, a phospholipase C (PLC)-mediated signaling cascade, couples photo-excitation of rhodopsin to the opening of the transient receptor potential (TRP) and TRP-like (TRPL) channels. A lipid product of PLC, diacylglycerol (DAG), and its metabolites, polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, how can one separate between the increase in putative second messengers, change in pH, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) depletion when exploring the TRPL gating mechanism? To answer this question we co-expressed the TRPL channels together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method that reduced plasma membrane-associated PI(4,5)P2 in HEK cells within seconds without activating PLC. Upon the addition of a dimerizing drug, PI(4,5)P2 was selectively hydrolyzed in the cell membrane without producing DAG, inositol trisphosphate, or calcium signals. We show that PI(4,5)P2 is not an inhibitor of TRPL channel activation. PI(4,5)P2 hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, whereas PUFA did activate the channels. Moreover, a reduction in PI(4,5)P2 levels or inhibition of DAG lipase during PLC activity suppressed the PLC-activated TRPL current. This suggests that PI(4,5)P2 is a crucial substrate for PLC-mediated activation of the channels, whereas PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating. PMID:22065576

  18. Garcinol, a polyisoprenylated benzophenone modulates multiple proinflammatory signaling cascades leading to the suppression of growth and survival of head and neck carcinoma.

    PubMed

    Li, Feng; Shanmugam, Muthu K; Chen, Luxi; Chatterjee, Snehajyoti; Basha, Jeelan; Kumar, Alan Prem; Kundu, Tapas K; Sethi, Gautam

    2013-08-01

    Constitutive activation of proinflammatory transcription factors such as STAT3 and NF-κB plays a pivotal role in the proliferation and survival of squamous cell carcinoma of the head and neck (HNSCC). Thus, the agents that can modulate deregulated STAT3 and NF-κB activation have a great potential both for the prevention and treatment of HNSCC. In the present report, we investigated the potential effects of garcinol, an active component of Garcinia indica on various inflammatory mediators involved in HNSCC progression using cell lines and xenograft mouse model. We found that garcinol inhibited constitutively activated STAT3 in HNSCC cells in a time- and dose-dependent manner, which correlated with the suppression of the upstream kinases (c-Src, JAK1, and JAK2) in HNSCC cells. Also, we noticed that the generation of reactive oxygen species is involved in STAT3 inhibitory effect of garcinol. Furthermore, garcinol exhibited an inhibitory effect on the constitutive NF-κB activation, mediated through the suppression of TGF-β-activated kinase 1 (TAK1) and inhibitor of IκB kinase (IKK) activation in HNSCC cells. Garcinol also downregulated the expression of various gene products involved in proliferation, survival, and angiogenesis that led to the reduction of cell viability and induction of apoptosis in HNSCC cells. When administered intraperitoneally, garcinol inhibited the growth of human HNSCC xenograft tumors in male athymic nu/nu mice. Overall, our results suggest for the first time that garcinol mediates its antitumor effects in HNSCC cells and mouse model through the suppression of multiple proinflammatory cascades. PMID:23803415

  19. 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

  20. 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

  1. 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

  2. Cerium oxide nanoparticles promote neurogenesis and abrogate hypoxia-induced memory impairment through AMPK–PKC–CBP signaling cascade

    PubMed Central

    Arya, Aditya; Gangwar, Anamika; Singh, Sushil Kumar; Roy, Manas; Das, Mainak; Sethy, Niroj Kumar; Bhargava, Kalpana

    2016-01-01

    Structural and functional integrity of the brain is adversely affected by reduced oxygen saturation, especially during chronic hypoxia exposure and often encountered by altitude travelers or dwellers. Hypoxia-induced generation of reactive nitrogen and oxygen species reportedly affects the cortex and hippocampus regions of the brain, promoting memory impairment and cognitive dysfunction. Cerium oxide nanoparticles (CNPs), also known as nanoceria, switch between +3 and +4 oxidation states and reportedly scavenge superoxide anions, hydrogen peroxide, and peroxynitrite in vivo. In the present study, we evaluated the neuroprotective as well as the cognition-enhancing activities of nanoceria during hypobaric hypoxia. Using polyethylene glycol-coated 3 nm nanoceria (PEG-CNPs), we have demonstrated efficient localization of PEG-CNPs in rodent brain. This resulted in significant reduction of oxidative stress and associated damage during hypoxia exposure. Morris water maze-based memory function tests revealed that PEG-CNPs ameliorated hypoxia-induced memory impairment. Using microscopic, flow cytometric, and histological studies, we also provide evidences that PEG-CNPs augmented hippocampus neuronal survival and promoted neurogenesis. Molecular studies revealed that PEG-CNPs promoted neurogenesis through the 5′-adenine monophosphate-activated protein kinase–protein kinase C–cyclic adenosine monophosphate response element-binding protein binding (AMPK-PKC-CBP) protein pathway. Our present study results suggest that nanoceria can be translated as promising therapeutic molecules for neurodegenerative diseases. PMID:27069362

  3. Stimulation by extracellular ATP and UTP of the stress-activated protein kinase cascade in rat renal mesangial cells

    PubMed Central

    Huwiler, Andrea; van Rossum, Gerda; Wartmann, Markus; Pfeilschifter, Josef

    1997-01-01

    Extracellular adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP) have been shown to activate a nucleotide receptor (P2U receptor) in rat mesangial cells that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases C and D, respectively. This is followed by an increased activity of the mitogen-activated protein kinase cascade and cell proliferation. Here we show that ATP and UTP potently stimulate the stress-activated protein kinase pathway and phosphorylation of the transcription factor c-Jun.Both nucleotides stimulated a rapid (within 5 min) and concentration-dependent activation of stress-activated protein kinases as measured by the phosphorylation of c-Jun in a solid phase kinase assay.When added at 100 μM the rank order of potency of a series of nucleotide analogues for stimulation of c-Jun phosphorylation was UTP>ATP=UDP=ATPγS>2-methylthio-ATP>βγ-imido-ATP= ADP>AMP=UMP=adenosine=uridine. Activation of stress-activated protein kinase activity by ATP and UTP was dose-dependently attenuated by suramin.Down-regulation of protein kinase C-α, -δ and -ε isoenzymes by 24 h treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate did not inhibit ATP- and UTP-induced activation of c-Jun phosphorylation. Furthermore, the specific protein kinase C inhibitors, CGP 41251 and Ro 31-8220, did not inhibit nucleotide-stimulated c-Jun phosphorylation, suggesting that protein kinase C is not involved in ATP- and UTP-triggered stress-activated protein kinase activation.Pretreatment of the cells with pertussis toxin or the tyrosine kinase inhibitor, genistein, strongly attenuated ATP- and UTP-induced c-Jun phosphorylation. Furthermore, N-acetyl-cysteine completely blocked the activation of stress-activated protein kinase in response to extracellular nucleotide stimulation.In summary, these results suggest that ATP and UTP trigger the activation of the stress-activated protein kinase module in mesangial cells by a

  4. Cinchona alkaloid squaramide catalyzed enantioselective hydrazination/cyclization cascade reaction of α-isocyanoacetates and azodicarboxylates: synthesis of optically active 1,2,4-triazolines.

    PubMed

    Zhao, Mei-Xin; Bi, Hong-Lei; Zhou, Hao; Yang, Hui; Shi, Min

    2013-09-20

    An efficient enantioselective hydrazination/cyclization cascade reaction of α-substituted isocyanoacetates to azodicarboxylates catalyzed by Cinchona alkaloid derived squaramide catalysts has been investigated, affording the optically active 1,2,4-triazolines in excellent yields (up to 99%) and good to excellent enantioselectivities (up to 97% ee) under mild conditions. PMID:23984761

  5. Activation of the Amyloid Cascade in Apolipoprotein E4 Transgenic Mice Induces Lysosomal Activation and Neurodegeneration Resulting in Marked Cognitive Deficits

    PubMed Central

    Belinson, Haim; Lev, Dimitri; Masliah, Eliezer

    2008-01-01

    The allele E4 of apolipoprotein E (apoE4), the most prevalent genetic risk factor for Alzheimer's disease, is associated histopathologically with elevated levels of brain amyloid. This led to the suggestion that the pathological effects of apoE4 are mediated by cross-talk interactions with amyloid β peptide (Aβ), which accentuate the pathological effects of the amyloid cascade. The mechanisms underlying the Aβ-mediated pathological effects of apoE4 are unknown. We have shown recently that inhibition of the Aβ-degrading enzyme neprilysin in brains of wild-type apoE3 and apoE4 mice results in rapid and similar elevations in their total brain Aβ levels. However, the nucleation and aggregation of Aβ in these mice were markedly affected by the apoE genotype and were specifically enhanced in the apoE4 mice. We presently used the neprilysin inhibition paradigm to analyze the neuropathological and cognitive effects that are induced by apoE4 after activation of the amyloid cascade. This revealed that apoE4 stimulates isoform specifically the degeneration of hippocampal CA1 neurons and of entorhinal and septal neurons, which is accompanied by the accumulation of intracellular Aβ and apoE and with lysosomal activation. Furthermore, these neuropathological effects are associated isoform specifically with the occurrence of pronounced cognitive deficits in the ApoE4 mice. These findings provide the first in vivo evidence regarding the cellular mechanisms underlying the pathological cross talk between apoE4 and Aβ, as well as a novel model system of neurodegeneration in vivo that is uniquely suitable for studying the early stages of the amyloid cascade and the effects thereon of apoE4. PMID:18448646

  6. A hot-spot-active magnetic graphene oxide substrate for microRNA detection based on cascaded chemiluminescence resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Bi, Sai; Chen, Min; Jia, Xiaoqiang; Dong, Ying

    2015-02-01

    Herein, a cascaded chemiluminescence resonance energy transfer (C-CRET) process was demonstrated from horseradish peroxidase (HRP)-mimicking DNAzyme-catalyzed luminol-H2O2 to fluorescein and further to graphene oxide (GO) when HRP-mimicking DNAzyme/fluorescein was in close proximity to the GO surface. The proposed C-CRET system was successfully implemented to construct three modes of C-CRET hot-spot-active substrates (modes I, II and III) by covalently immobilizing HRP-mimicking DNAzyme/fluorescein-labeled hairpin DNAs (hot-spot-generation probes) on magnetic GO (MGO), resulting in a signal ``off'' state due to the quenching of the luminol/H2O2/HRP-mimicking DNAzyme/fluorescein CRET system by GO. Upon the introduction of microRNA-122 (miRNA-122), the targets (mode I) or the new triggers that were generated through a strand displacement reaction (SDR) initiated by miRNA-122 (modes II and III) hybridized with the loop domains of hairpin probes on MGO to form double-stranded (modes I and II) or triplex-stem structures (mode III), causing an ``open'' configuration of the hairpin probe and a CRET signal ``on'' state, thus achieving sensitive and selective detection of miRNA-122. More importantly, the substrate exhibited excellent controllability, reversibility and reproducibility through SDR and magnetic separation (modes II and III), especially sequence-independence for hairpin probes in mode III, holding great potential for the development of a versatile platform for optical biosensing.Herein, a cascaded chemiluminescence resonance energy transfer (C-CRET) process was demonstrated from horseradish peroxidase (HRP)-mimicking DNAzyme-catalyzed luminol-H2O2 to fluorescein and further to graphene oxide (GO) when HRP-mimicking DNAzyme/fluorescein was in close proximity to the GO surface. The proposed C-CRET system was successfully implemented to construct three modes of C-CRET hot-spot-active substrates (modes I, II and III) by covalently immobilizing HRP-mimicking DNAzyme

  7. 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

  8. A hot-spot-active magnetic graphene oxide substrate for microRNA detection based on cascaded chemiluminescence resonance energy transfer.

    PubMed

    Bi, Sai; Chen, Min; Jia, Xiaoqiang; Dong, Ying

    2015-02-28

    Herein, a cascaded chemiluminescence resonance energy transfer (C-CRET) process was demonstrated from horseradish peroxidase (HRP)-mimicking DNAzyme-catalyzed luminol-H2O2 to fluorescein and further to graphene oxide (GO) when HRP-mimicking DNAzyme/fluorescein was in close proximity to the GO surface. The proposed C-CRET system was successfully implemented to construct three modes of C-CRET hot-spot-active substrates (modes I, II and III) by covalently immobilizing HRP-mimicking DNAzyme/fluorescein-labeled hairpin DNAs (hot-spot-generation probes) on magnetic GO (MGO), resulting in a signal "off" state due to the quenching of the luminol/H2O2/HRP-mimicking DNAzyme/fluorescein CRET system by GO. Upon the introduction of microRNA-122 (miRNA-122), the targets (mode I) or the new triggers that were generated through a strand displacement reaction (SDR) initiated by miRNA-122 (modes II and III) hybridized with the loop domains of hairpin probes on MGO to form double-stranded (modes I and II) or triplex-stem structures (mode III), causing an "open" configuration of the hairpin probe and a CRET signal "on" state, thus achieving sensitive and selective detection of miRNA-122. More importantly, the substrate exhibited excellent controllability, reversibility and reproducibility through SDR and magnetic separation (modes II and III), especially sequence-independence for hairpin probes in mode III, holding great potential for the development of a versatile platform for optical biosensing. PMID:25644330

  9. 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

  10. Prolactin-Stimulated Activation of ERK1/2 Mitogen-Activated Protein Kinases is Controlled by PI3-Kinase/Rac/PAK Signaling Pathway in Breast Cancer Cells

    PubMed Central

    Aksamitiene, Edita; Achanta, Sirisha; Kolch, Walter; Kholodenko, Boris N.; Hoek, Jan B.; Kiyatkin, Anatoly

    2011-01-01

    There is strong evidence that deregulation of prolactin (PRL) signaling contributes to pathogenesis and chemoresistance of breast cancer. Therefore, understanding cross-talk between distinct signal transduction pathways triggered by activation of the prolactin receptor (PRL-R), is essential for elucidating the pathogenesis of metastatic breast cancer. In this study, we applied a sequential inhibitory analysis of various signaling intermediates to examine the hierarchy of protein interactions within the PRL signaling network and to evaluate the relative contributions of multiple signaling branches downstream of PRL-R to the activation of the extracellular signal-regulated kinases ERK1 and ERK2 in T47D and MCF-7 human breast cancer cells. Quantitative measurements of the phosphorylation/activation patterns of proteins showed that PRL simultaneously activated Src family kinases (SFKs) and the JAK/STAT, phosphoinositide-3 (PI3)-kinase/Akt and MAPK signaling pathways. The specific blockade or siRNA-mediated suppression of SFK/FAK, JAK2/STAT5, PI3-kinase/PDK1/Akt, Rac/PAK or Ras regulatory circuits revealed that (1) the PI3-kinase/Akt pathway is required for activation of the MAPK/ERK signaling cascade upon PRL stimulation; (2) PI3-kinase-mediated activation of the c-Raf-MEK1/2-ERK1/2 cascade occurs independent of signaling dowstream of STATs, Akt and PKC, but requires JAK2, SFKs and FAK activities; (3) activated PRL-R mainly utilizes the PI3-kinase-dependent Rac/PAK pathway rather than the canonical Shc/Grb2/SOS/Ras route to initiate and sustain ERK1/2 signaling. By interconnecting diverse signaling pathways PLR may enhance proliferation, survival, migration and invasiveness of breast cancer cells. PMID:21726627

  11. Prolactin-stimulated activation of ERK1/2 mitogen-activated protein kinases is controlled by PI3-kinase/Rac/PAK signaling pathway in breast cancer cells.

    PubMed

    Aksamitiene, Edita; Achanta, Sirisha; Kolch, Walter; Kholodenko, Boris N; Hoek, Jan B; Kiyatkin, Anatoly

    2011-11-01

    There is strong evidence that deregulation of prolactin (PRL) signaling contributes to pathogenesis and chemoresistance of breast cancer. Therefore, understanding cross-talk between distinct signal transduction pathways triggered by activation of the prolactin receptor (PRL-R), is essential for elucidating the pathogenesis of metastatic breast cancer. In this study, we applied a sequential inhibitory analysis of various signaling intermediates to examine the hierarchy of protein interactions within the PRL signaling network and to evaluate the relative contributions of multiple signaling branches downstream of PRL-R to the activation of the extracellular signal-regulated kinases ERK1 and ERK2 in T47D and MCF-7 human breast cancer cells. Quantitative measurements of the phosphorylation/activation patterns of proteins showed that PRL simultaneously activated Src family kinases (SFKs) and the JAK/STAT, phosphoinositide-3 (PI3)-kinase/Akt and MAPK signaling pathways. The specific blockade or siRNA-mediated suppression of SFK/FAK, JAK2/STAT5, PI3-kinase/PDK1/Akt, Rac/PAK or Ras regulatory circuits revealed that (1) the PI3-kinase/Akt pathway is required for activation of the MAPK/ERK signaling cascade upon PRL stimulation; (2) PI3-kinase-mediated activation of the c-Raf-MEK1/2-ERK1/2 cascade occurs independent of signaling dowstream of STATs, Akt and PKC, but requires JAK2, SFKs and FAK activities; (3) activated PRL-R mainly utilizes the PI3-kinase-dependent Rac/PAK pathway rather than the canonical Shc/Grb2/SOS/Ras route to initiate and sustain ERK1/2 signaling. By interconnecting diverse signaling pathways PLR may enhance proliferation, survival, migration and invasiveness of breast cancer cells. PMID:21726627

  12. A new genetic model of activity-induced Ras signaling dependent pre-synaptic plasticity in Drosophila

    PubMed Central

    Freeman, Amanda; Bowers, Mallory; Mortimer, Alysia Vrailas; Timmerman, Christina; Roux, Stephanie; Ramaswami, Mani; Sanyal, Subhabrata

    2010-01-01

    Techniques to induce activity-dependent neuronal plasticity in vivo allow the underlying signaling pathways to be studied in their biological context. Here, we demonstrate activity-induced plasticity at neuromuscular synapses of Drosophila double mutant for comatose (an NSF mutant) and Kum (a SERCA mutant), and present an analysis of the underlying signaling pathways. comt; Kum (CK) double mutants exhibit increased locomotor activity under normal culture conditions, concomitant with a larger neuromuscular junction synapse and stably elevated evoked transmitter release. The observed enhancements of synaptic size and transmitter release in CK mutants are completely abrogated by: a) reduced activity of motor neurons; b) attenuation of the Ras/ERK signaling cascade; or c) inhibition of the transcription factors Fos and CREB. all of which restrict synaptic properties to near wild type levels. Together, these results document neural activity-dependent plasticity of motor synapses in CK animals that requires Ras/ERK signaling and normal transcriptional activity of Fos and CREB. Further, novel in vivo reporters of neuronal Ras activation and Fos transcription also confirm increased signaling through a Ras/AP-1 pathway in motor neurons of CK animals, consistent with results from our genetic experiments. Thus, this study: a) provides a robust system in which to study activity-induced synaptic plasticity in vivo; b) establishes a causal link between neural activity, Ras signaling, transcriptional regulation and pre-synaptic plasticity in glutamatergic motor neurons of Drosophila larvae; and c) presents novel, genetically encoded reporters for Ras and AP-1 dependent signaling pathways in Drosophila. PMID:20193670

  13. Optical filtering enabled by cascaded parametric amplification.

    PubMed

    McKinstrie, C J; Dailey, J M; Agarwal, A; Toliver, P

    2016-06-27

    A cascaded parametric amplifier consists of a first parametric amplifier, which amplifies an input signal and generates an idler, which is a copy of the signal, a signal processor, which controls the phases of the signal and idler, and a second parametric amplifier, which combines the signal and idler in a phase-sensitive manner. In this paper, cascaded parametric amplification is modeled and the conditions required to maximize the constructive-destructive extinction ratio are determined. The results show that a cascaded parametric amplifier can be operated as a filter: A desired signal-idler pair is amplified, whereas undesired signal-idler pairs are deamplified. For the desired signal and idler, the noise figures of the filtering process (input signal-to-noise ratio divided by the output ratios) are only slightly higher than those of the copying process: Signal-processing functionality can be achieved with only a minor degradation in signal quality. PMID:27410581

  14. Simultaneous EEG-fMRI Reveals a Temporal Cascade of Task-Related and Default-Mode Activations During a Simple Target Detection Task

    PubMed Central

    Walz, Jennifer M.; Goldman, Robin I.; Carapezza, Michael; Muraskin, Jordan; Brown, Truman R.; Sajda, Paul

    2013-01-01

    Focused attention continuously and inevitably fluctuates, and to completely understand the mechanisms responsible for these modulations it is necessary to localize the brain regions involved. During a simple visual oddball task, neural responses measured by electroencephalography (EEG) modulate primarily with attention, but source localization of the correlates is a challenge. In this study we use single-trial analysis of simultaneously-acquired scalp EEG and functional magnetic resonance image (fMRI) data to investigate the blood oxygen level dependent (BOLD) correlates of modulations in task-related attention, and we unravel the temporal cascade of these transient activations. We hypothesize that activity in brain regions associated with various task-related cognitive processes modulates with attention, and that their involvements occur transiently in a specific order. We analyze the fMRI BOLD signal by first regressing out the variance linked to observed stimulus and behavioral events. We then correlate the residual variance with the trial-to-trial variation of EEG discriminating components for identical stimuli, estimated at a sequence of times during a trial. Post-stimulus and early in the trial, we find activations in right-lateralized frontal regions and lateral occipital cortex, areas that are often linked to task-dependent processes, such as attentional orienting, and decision certainty. After the behavioral response we see correlates in areas often associated with the default-mode network and introspective processing, including precuneus, angular gyri, and posterior cingulate cortex. Our results demonstrate that during simple tasks both task-dependent and default-mode networks are transiently engaged, with a distinct temporal ordering and millisecond timescale. PMID:23962956

  15. Shared signaling networks active in B cells isolated from genetically distinct mouse models of lupus

    PubMed Central

    Wu, Tianfu; Qin, Xiangmei; Kurepa, Zoran; Kumar, Kirthi Raman; Liu, Kui; Kanta, Hasna; Zhou, Xin J.; Satterthwaite, Anne B.; Davis, Laurie S.; Mohan, Chandra

    2007-01-01

    Though B cells play key roles in lupus pathogenesis, the molecular circuitry and its dysregulation in these cells as disease evolves remain poorly understood. To address this, a comprehensive scan of multiple signaling axes using multiplexed Western blotting was undertaken in several different murine lupus strains. PI3K/AKT/mTOR (mTOR, mammalian target of rapamycin), MEK1/Erk1/2, p38, NF-κB, multiple Bcl-2 family members, and cell-cycle molecules were observed to be hyperexpressed in lupus B cells in an age-dependent and lupus susceptibility gene–dose–dependent manner. Therapeutic targeting of the AKT/mTOR axis using a rapamycin (sirolimus) derivative ameliorated the serological, cellular, and pathological phenotypes associated with lupus. Surprisingly, the targeting of this axis was associated with the crippling of several other signaling axes. These studies reveal that lupus pathogenesis is contingent upon the activation of an elaborate network of signaling cascades that is shared among genetically distinct mouse models and raise hope that targeting pivotal nodes in these networks may offer therapeutic benefit. PMID:17641780

  16. Biological Activities of Reactive Oxygen and Nitrogen Species: Oxidative Stress versus Signal Transduction

    PubMed Central

    Weidinger, Adelheid; Kozlov, Andrey V.

    2015-01-01

    In the past, reactive oxygen and nitrogen species (RONS) were shown to cause oxidative damage to biomolecules, contributing to the development of a variety of diseases. However, recent evidence has suggested that intracellular RONS are an important component of intracellular signaling cascades. The aim of this review was to consolidate old and new ideas on the chemical, physiological and pathological role of RONS for a better understanding of their properties and specific activities. Critical consideration of the literature reveals that deleterious effects do not appear if only one primary species (superoxide radical, nitric oxide) is present in a biological system, even at high concentrations. The prerequisite of deleterious effects is the formation of highly reactive secondary species (hydroxyl radical, peroxynitrite), emerging exclusively upon reaction with another primary species or a transition metal. The secondary species are toxic, not well controlled, causing irreversible damage to all classes of biomolecules. In contrast, primary RONS are well controlled (superoxide dismutase, catalase), and their reactions with biomolecules are reversible, making them ideal for physiological/pathophysiological intracellular signaling. We assume that whether RONS have a signal transducing or damaging effect is primarily defined by their quality, being primary or secondary RONS, and only secondly by their quantity. PMID:25884116

  17. Pathological axonal death through a MAPK cascade that triggers a local energy deficit

    PubMed Central

    Yang, Jing; Wu, Zhuhao; Renier, Nicolas; Simon, David J.; Uryu, Kunihiro; Park, David S.; Greer, Peter A.; Tournier, Cathy; Davis, Roger J.; Tessier-Lavigne, Marc

    2015-01-01

    Summary Axonal death disrupts functional connectivity of neural circuits and is a critical feature of many neurodegenerative disorders. Pathological axon degeneration often occurs independently of known programmed death pathways, but the underlying molecular mechanisms remain largely unknown. Using traumatic injury as a model, we systematically investigate mitogen-activated protein kinase (MAPK) families, and delineate a MAPK cascade that represents the early degenerative response to axonal injury. The adaptor protein Sarm1 is required for activation of this MAPK cascade, and this Sarm1-MAPK pathway disrupts axonal energy homeostasis, leading to ATP depletion before physical breakdown of damaged axons. The protective cytoNmnat1/Wlds protein inhibits activation of this MAPK cascade. Further, MKK4, a key component in the Sarm1-MAPK pathway, is antagonized by AKT signaling, which modulates the degenerative response by limiting activation of downstream JNK signaling. Our results reveal a regulatory mechanism that integrates distinct signals to instruct pathological axon degeneration. PMID:25594179

  18. Single-cell mass cytometry of TCR signaling: amplification of small initial differences results in low ERK activation in NOD mice.

    PubMed

    Mingueneau, Michael; Krishnaswamy, Smita; Spitzer, Matthew H; Bendall, Sean C; Stone, Erica L; Hedrick, Stephen M; Pe'er, Dana; Mathis, Diane; Nolan, Garry P; Benoist, Christophe

    2014-11-18

    Signaling from the T-cell receptor (TCR) conditions T-cell differentiation and activation, requiring exquisite sensitivity and discrimination. Using mass cytometry, a high-dimensional technique that can probe multiple signaling nodes at the single-cell level, we interrogate TCR signaling dynamics in control C57BL/6 and autoimmunity-prone nonobese diabetic (NOD) mice, which show ineffective ERK activation after TCR triggering. By quantitating signals at multiple steps along the signaling cascade and parsing the phosphorylation level of each node as a function of its predecessors, we show that a small impairment in initial pCD3ζ activation resonates farther down the signaling cascade and results in larger defects in activation of the ERK1/2-S6 and IκBα modules. This nonlinear property of TCR signaling networks, which magnifies small initial differences during signal propagation, also applies in cells from B6 mice activated at different levels of intensity. Impairment in pCD3ζ and pSLP76 is not a feedback consequence of a primary deficiency in ERK activation because no proximal signaling defect was observed in Erk2 KO T cells. These defects, which were manifest at all stages of T-cell differentiation from early thymic pre-T cells to memory T cells, may condition the imbalanced immunoregulation and tolerance in NOD T cells. More generally, this amplification of small initial differences in signal intensity may explain how T cells discriminate between closely related ligands and adopt strongly delineated cell fates. PMID:25362052

  19. Single-cell mass cytometry of TCR signaling: Amplification of small initial differences results in low ERK activation in NOD mice

    PubMed Central

    Mingueneau, Michael; Krishnaswamy, Smita; Spitzer, Matthew H.; Bendall, Sean C.; Stone, Erica L.; Hedrick, Stephen M.; Pe'er, Dana; Mathis, Diane; Nolan, Garry P.; Benoist, Christophe

    2014-01-01

    Signaling from the T-cell receptor (TCR) conditions T-cell differentiation and activation, requiring exquisite sensitivity and discrimination. Using mass cytometry, a high-dimensional technique that can probe multiple signaling nodes at the single-cell level, we interrogate TCR signaling dynamics in control C57BL/6 and autoimmunity-prone nonobese diabetic (NOD) mice, which show ineffective ERK activation after TCR triggering. By quantitating signals at multiple steps along the signaling cascade and parsing the phosphorylation level of each node as a function of its predecessors, we show that a small impairment in initial pCD3ζ activation resonates farther down the signaling cascade and results in larger defects in activation of the ERK1/2–S6 and IκBα modules. This nonlinear property of TCR signaling networks, which magnifies small initial differences during signal propagation, also applies in cells from B6 mice activated at different levels of intensity. Impairment in pCD3ζ and pSLP76 is not a feedback consequence of a primary deficiency in ERK activation because no proximal signaling defect was observed in Erk2 KO T cells. These defects, which were manifest at all stages of T-cell differentiation from early thymic pre-T cells to memory T cells, may condition the imbalanced immunoregulation and tolerance in NOD T cells. More generally, this amplification of small initial differences in signal intensity may explain how T cells discriminate between closely related ligands and adopt strongly delineated cell fates. PMID:25362052

  20. Prolonged agonist stimulation does not alter the protein composition of membrane domains in spite of dramatic changes induced in a specific signaling cascade.

    PubMed

    Matousek, Petr; Novotny, Jirí; Rudajev, Vladimír; Svoboda, Petr

    2005-01-01

    Protein composition of membrane domains prepared by three different procedures (mechanical homogenization, alkaline treatment with 1 M Na2CO3 [pH 11.0], or extraction with nonionic detergent Triton X-100), and isolated from the bulk of plasma membranes by flotation on equilibrium sucrose density gradients, was analyzed by two-dimensional (2D) electrophoresis and compared in preparations from control (quiescent) and agonist-stimulated human embryonic kidney cells (HEK)293 or S49 cells. HEK293 cells (clone e2m11) stably expressing high levels of thyrotropin-releasing hormone receptor and G11alpha protein were stimulated by thyrotropin-releasing hormone and S49 lymphoma cells by the beta-adrenergic receptor agonist isoprenaline. Whereas sustained exposure (16 h) of both cell lines to the appropriate hormones led to substantial cellular redistribution and downregulation of the cognate G proteins (G(q)alpha/G11alpha and G(s)alpha, respectively), the distribution and levels of nonstimulated G(i) proteins remained unchanged. The 2D electrophoretic analysis of membrane domains distinguished approx 150-170 major proteins in these structures and none of these proteins was significantly altered by prolonged agonist stimulation. Furthermore, specific immunochemical determination of a number of plasma membrane markers, including transmembrane and glycosyl-phosphatidylinositol-anchored peripheral proteins, confirmed that their detergent-extractability/solubility was not influenced by hormone treatment. Collectively, our present data indicate that sustained hormone stimulation of target cells does not alter the basic protein composition of membrane domain/raft compartments of the plasma membrane in spite of marked changes proceeding in a given signaling cascade. PMID:15673926

  1. Two adaptor molecules of MyD88 and TRAF6 in Apostichopus japonicus Toll signaling cascade: molecular cloning and expression analysis.

    PubMed

    Lu, Yali; Li, Chenghua; Zhang, Peng; Shao, Yina; Su, Xiurong; Li, Ye; Li, Taiwu

    2013-12-01

    Myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6) are two key adaptor molecules in Toll-like receptor signal transduction that triggers downstream cascades involved in innate immunity. Here we reported the isolation and characterization the full-length cDNAs of MyD88 and TRAF6 from sea cucumber Apostichopus japonicus (denoted as AjMyD88 and AjTRAF6, respectively). Both of two factors shared a remarkable high degree of structural conservation with their mammalian and Drosophila orthologs, such as a typical death domain (DD) and a conservative Toll/IL-1R (TIR) domain for the deduced amino acid of AjMyD88, a zinc finger of RING-type, two zinc fingers of TRAF-type, a coiled-coil region, and a MATH domain for that of AjTRAF6. Constitutive expression patterns were also observed in the two genes with different expression levels. AjMyD88 mRNA transcripts were higher expressed in intestine and respiratory trees, and AjTRAF6 were abundant in coelomocytes and tentacle. During Vibrio splendidus challenge experiment, the expression levels of two genes were increased significantly with larger amplitude and longer duration in AjTRAF6. The peak expression levels were detected at 6 h for AjMyD88 with 1.80-fold increase, and at 24 h for AjTRAF6 with 2.73-fold increase compared with that in the control group. All these results suggested that AjMyD88 and AjTRAF6 might be involved into immune response toward V. splendidus challenge. PMID:23886491

  2. Proteolytic activation cascade of the Netherton syndrome-defective protein, LEKTI, in the epidermis: implications for skin homeostasis.

    PubMed

    Fortugno, Paola; Bresciani, Alberto; Paolini, Chantal; Pazzagli, Chiara; El Hachem, May; D'Alessio, Marina; Zambruno, Giovanna

    2011-11-01

    Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is the defective protein of the ichthyosiform condition Netherton syndrome (NS). Strongly expressed in the most differentiated epidermal layers, LEKTI is a serine protease inhibitor synthesized as three different high-molecular-weight precursors, which are rapidly processed into shorter fragments and secreted extracellularly. LEKTI polypeptides interact with several proteases to regulate skin barrier homeostasis as well as inflammatory and/or immunoallergic responses. Here, by combining antibody mapping, N-terminal sequencing, and site-specific mutagenesis, we defined the amino-acid sequence of most of the LEKTI polypeptides physiologically generated in human epidermis. We also identified three processing intermediates not described so far. Hence, a proteolytic cascade model for LEKTI activation is proposed. We then pinpointed the most effective fragments against the desquamation-related kallikreins (KLKs) and we proved that LEKTI is involved in stratum corneum shedding as some of its polypeptides inhibit the KLK-mediated proteolysis of desmoglein-1. Finally, we quantified the individual LEKTI fragments in the uppermost epidermis, showing that the ratios between LEKTI polypeptides and active KLK5 are compatible with a fine-tuned inhibition. These findings are relevant both to the understanding of skin homeostasis regulation and to the design of novel therapeutic strategies for NS. PMID:21697885

  3. Fine Time Course Expression Analysis Identifies Cascades of Activation and Repression and Maps a Putative Regulator of Mammalian Sex Determination

    PubMed Central

    Looger, Loren L.; Ohler, Uwe; Capel, Blanche

    2013-01-01

    In vertebrates, primary sex determination refers to the decision within a bipotential organ precursor to differentiate as a testis or ovary. Bifurcation of organ fate begins between embryonic day (E) 11.0–E12.0 in mice and likely involves a dynamic transcription network that is poorly understood. To elucidate the first steps of sexual fate specification, we profiled the XX and XY gonad transcriptomes at fine granularity during this period and resolved cascades of gene activation and repression. C57BL/6J (B6) XY gonads showed a consistent ∼5-hour delay in the activation of most male pathway genes and repression of female pathway genes relative to 129S1/SvImJ, which likely explains the sensitivity of the B6 strain to male-to-female sex reversal. Using this fine time course data, we predicted novel regulatory genes underlying expression QTLs (eQTLs) mapped in a previous study. To test predictions, we developed an in vitro gonad primary cell assay and optimized a lentivirus-based shRNA delivery method to silence candidate genes and quantify effects on putative targets. We provide strong evidence that Lmo4 (Lim-domain only 4) is a novel regulator of sex determination upstream of SF1 (Nr5a1), Sox9, Fgf9, and Col9a3. This approach can be readily applied to identify regulatory interactions in other systems. PMID:23874228

  4. Soma influences GSC progeny differentiation via the cell adhesion-mediated steroid-let-7-Wingless signaling cascade that regulates chromatin dynamics

    PubMed Central

    König, Annekatrin; Shcherbata, Halyna R.

    2015-01-01

    ABSTRACT It is known that signaling from the germline stem cell niche is required to maintain germline stem cell identity in Drosophila. However, it is not clear whether the germline stem-cell daughters differentiate by default (because they are physically distant from the niche) or whether additional signaling is necessary to initiate the differentiation program. Previously, we showed that ecdysteroid signaling cell non-autonomously regulates early germline differentiation via its soma-specific co-activator and co-repressor, Taiman and Abrupt. Now, we demonstrate that this regulation is modulated by the miRNA let-7, which acts in a positive feedback loop to confer ecdysone signaling robustness via targeting its repressor, the transcription factor Abrupt. This feedback loop adjusts ecdysteroid signaling in response to some stressful alterations in the external and internal conditions, which include temperature stress and aging, but not nutritional deprivation. Upon let-7 deficit, escort cells fail to properly differentiate: their shape, division, and cell adhesive characteristics are perturbed. These cells have confused cellular identity and form columnar-like rather than squamous epithelium and fail to send protrusions in between differentiating germline cysts, affecting soma-germline communication. Particularly, levels of the homophilic cell adhesion protein Cadherin, which recruits Wg signaling transducer β-catenin, are increased in mutant escort cells and, correspondingly, in the adjacent germline cells. Readjustment of heterotypic (soma-germline) cell adhesion modulates Wg signaling intensity in the germline, which in turn regulates histone modifications that promote expression of the genes necessary to trigger early germline differentiation. Thus, our data first show the intrinsic role for Wg signaling in the germline and support a model where the soma influences the tempo of germline differentiation in response to external conditions. PMID:25661868

  5. A Cell-Autonomous Molecular Cascade Initiated by AMP-Activated Protein Kinase Represses Steroidogenesis

    PubMed Central

    Abdou, Houssein S.; Bergeron, Francis

    2014-01-01

    Steroid hormones regulate essential physiological processes, and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by luteinizing hormone (LH) via its receptor leading to increased cyclic AMP (cAMP) production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Steroidogenesis then passively decreases with the degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP-to-AMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis, including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutively steroidogenic R2C cells. We have also determined that maximum AMPK activation following stimulation of steroidogenesis in MA-10 Leydig cells occurs when steroid hormone production has reached a plateau. Our data identify AMPK as a molecular rheostat that actively represses steroid hormone biosynthesis to preserve cellular energy homeostasis and prevent excess steroid production. PMID:25225331

  6. Activation of toll-like receptor signaling pathways leading to nitric oxide-mediated antiviral responses.

    PubMed

    Abdul-Cader, Mohamed Sarjoon; Amarasinghe, Aruna; Abdul-Careem, Mohamed Faizal

    2016-08-01

    Toll-like receptors (TLRs), well-characterized pattern-recognizing receptors of the innate arm of the immune system, are vital in detecting pathogen-associated molecular patterns (PAMPs). The TLR-PAMP interaction initiates an intracellular signaling cascade, predominantly culminating in upregulation of antiviral components, including inducible nitric oxide synthase (iNOS). After activation, various TLR pathways can promote iNOS production via the myeloid differentiation primary response-88 (MyD-88) adapter protein. Subsequently, iNOS facilitates production of nitric oxide (NO), a highly reactive and potent antiviral molecule that can inhibit replication of RNA and DNA viruses. Furthermore, NO can diffuse freely across cell membranes and elicit antiviral mechanisms in various ways, including direct and indirect damage to viral genomes. This review emphasizes current knowledge of NO-mediated antiviral responses elicited after activation of TLR signaling pathways. PMID:27233799

  7. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    SciTech Connect

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald

    2015-03-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

  8. Spinal 5-HT3 receptors mediate descending facilitation and contribute to behavioral hypersensitivity via a reciprocal neuron-glial signaling cascade

    PubMed Central

    2014-01-01

    Background It has been recently recognized that the descending serotonin (5-HT) system from the rostral ventromedial medulla (RVM) in the brainstem and the 5-HT3 receptor subtype in the spinal dorsal horn are involved in enhanced descending pain facilitation after tissue and nerve injury. However, the mechanisms underlying the activation of the 5-HT3 receptor and its contribution to facilitation of pain remain unclear. Results In the present study, activation of spinal 5-HT3 receptors by intrathecal injection of a selective 5-HT3 receptor agonist SR 57227 induced spinal glial hyperactivity, neuronal hyperexcitability and pain hypersensitivity in rats. We found that there was neuron-to-microglia signaling via the chemokine fractalkine, microglia to astrocyte signaling via cytokine IL-18, astrocyte to neuronal signaling by IL-1β, and enhanced activation of NMDA receptors in the spinal dorsal horn. Glial hyperactivation in spinal dorsal horn after hindpaw inflammation was also attenuated by molecular depletion of the descending 5-HT system by intra-RVM Tph-2 shRNA interference. Conclusions These findings offer new insights into the cellular and molecular mechanisms at the spinal level responsible for descending 5-HT-mediated pain facilitation during the development of persistent pain after tissue and nerve injury. New pain therapies should focus on prime targets of descending facilitation-induced glial involvement, and in particular the blocking of intercellular signaling transduction between neurons and glia. PMID:24913307

  9. 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

  10. Dietary and behavioral interventions protect against age related activation of caspase cascades in the canine brain.

    PubMed

    Snigdha, Shikha; Berchtold, Nicole; Astarita, Giuseppe; Saing, Tommy; Piomelli, Daniele; Cotman, Carl W

    2011-01-01

    Lifestyle interventions such as diet, exercise, and cognitive training represent a quietly emerging revolution in the modern approach to counteracting age-related declines in brain health. Previous studies in our laboratory have shown that long-term dietary supplementation with antioxidants and mitochondrial cofactors (AOX) or behavioral enrichment with social, cognitive, and exercise components (ENR), can effectively improve cognitive performance and reduce brain pathology of aged canines, including oxidative damage and Aβ accumulation. In this study, we build on and extend our previous findings by investigating if the interventions reduce caspase activation and ceramide accumulation in the aged frontal cortex, since caspase activation and ceramide accumulation are common convergence points for oxidative damage and Aβ, among other factors associated with the aged and AD brain. Aged beagles were placed into one of four treatment groups: CON--control environment/control diet, AOX--control environment/antioxidant diet, ENR--enriched environment/control diet, AOX/ENR--enriched environment/antioxidant diet for 2.8 years. Following behavioral testing, brains were removed and frontal cortices were analyzed to monitor levels of active caspase 3, active caspase 9 and their respective cleavage products such as tau and semaphorin7a, and ceramides. Our results show that levels of activated caspase-3 were reduced by ENR and AOX interventions with the largest reduction occurring with combined AOX/ENR group. Further, reductions in caspase-3 correlated with reduced errors in a reversal learning task, which depends on frontal cortex function. In addition, animals treated with an AOX arm showed reduced numbers of cells expressing active caspase 9 or its cleavage product semaphorin 7A, while ENR (but not AOX) reduced ceramide levels. Overall, these data demonstrate that lifestyle interventions curtail activation of pro-degenerative pathways to improve cellular health and are the

  11. Activation of TAK1 by Chemotactic and Growth Factors, and Its Impact on Human Neutrophil Signaling and Functional Responses.

    PubMed

    Sylvain-Prévost, Stéphanie; Ear, Thornin; Simard, François A; Fortin, Carl F; Dubois, Claire M; Flamand, Nicolas; McDonald, Patrick P

    2015-12-01

    The MAP3 kinase, TAK1, is known to act upstream of IKK and MAPK cascades in several cell types, and is typically activated in response to cytokines (e.g., TNF, IL-1) and TLR ligands. In this article, we report that in human neutrophils, TAK1 can also be activated by different classes of inflammatory stimuli, namely, chemoattractants and growth factors. After stimulation with such agents, TAK1 becomes rapidly and transiently activated. Blocking TAK1 kinase activity with a highly selective inhibitor (5z-7-oxozeaenol) attenuated the inducible phosphorylation of ERK occurring in response to these stimuli but had little or no effect on that of p38 MAPK or PI3K. Inhibition of TAK1 also impaired MEKK3 (but not MEKK1) activation by fMLF. Moreover, both TAK1 and the MEK/ERK module were found to influence inflammatory cytokine expression and release in fMLF- and GM-CSF-activated neutrophils, whereas the PI3K pathway influenced this response independently of TAK1. Besides cytokine production, other responses were found to be under TAK1 control in neutrophils stimulated with chemoattractants and/or GM-CSF, namely, delayed apoptosis and leukotriene biosynthesis. Our data further emphasize the central role of TAK1 in controlling signaling cascades and functional responses in primary neutrophils, making it a promising target for therapeutic intervention in view of the foremost role of neutrophils in several chronic inflammatory conditions. PMID:26491199

  12. Activation of the MAP Kinase Cascade by Exogenous Calcium-Sensing Receptor

    SciTech Connect

    Hobson, Susan A.; Wright, Jay W.; Lee, Fred; Mcneil, Scott; Bilderback, Tim R.; Rodland, Karin D.

    2003-02-01

    In Rat-1 fibroblasts and ovarian surface epithelial cells, extracellular calcium induces a proliferative response which appears to be mediated by the G-protein coupled Calcium-sensing Receptor (CaR), as expression of the non-functional CaR-R795W mutant inhibits both thymidine incorporation and activation of the extracellular-regulated kinase (ERK) in response to calcium. In this report we utilized CaR-transfected HEK293 cells to demonstrate that functional CaR is necessary and sufficient for calcium-induced ERK activation. CaR-dependent ERK activation was blocked by co-expression of the Ras dominant-negative mutant, Ras N17, and by exposure to the phosphatidyl inositol 3' kinase inhibitors wortmannin and LY294002. In contrast to Rat-1 fibroblasts, CaR-mediated in vitro kinase activity of ERK2 was unaffected by tyrosine kinase inhibitor herbimycin in CaR-transfected HEK293 cells. These results suggest that usage of distinct pathways downstream of the CaR varies in a cell-type specific manner, suggesting a potential mechanism by which activation of the CaR could couple to distinct calcium-dependent responses.

  13. Suppression of NF-κB signaling by andrographolide with a novel mechanism in human platelets: regulatory roles of the p38 MAPK-hydroxyl radical-ERK2 cascade.

    PubMed

    Lu, Wan J; Lin, Kuan H; Hsu, Ming J; Chou, Duen S; Hsiao, George; Sheu, Joen R

    2012-10-01

    Andrographolide, a novel nuclear factor-κB (NF-κB) inhibitor, is isolated from leaves of Andrographis paniculata. Platelet activation is relevant to a variety of coronary heart diseases. Our recent studies revealed that andrographolide possesses potent antiplatelet activity by activating the endothelial nitric oxide synthase (eNOS)-NO-cyclic GMP pathway. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of andrographolide in NF-κB-mediated events in platelets. In this study, NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were time-dependently activated by collagen in human platelets, and these signaling events were attenuated by andrographolide (35 and 75 μM). ODQ and KT5823, respective inhibitors of guanylate cyclase and cyclic GMP-dependent kinase (PKG), strongly reversed andrographolide-mediated inhibition of platelet aggregation, relative [Ca(2+)]i mobilization, and IKKβ, and p65 phosphorylation. In addition, SB203580 (an inhibitor of p38 MAPK), but not PD98059 (an inhibitor of ERKs), markedly abolished IKKβ and p65 phosphorylation. SB203580, NAC (a free-radical scavenger), and BAY11-7082 (an inhibitor of NF-κB) all diminished ERK2 phosphorylation, whereas PD98059, BAY11-7082, and NAC had no effects on p38 MAPK phosphorylation. Furthermore, SB203580, but not BAY11-7082 or PD98059, reduced collagen-induced hydroxyl radical ((·)HO) formation. KT5823 also markedly reversed andrographolide-mediated inhibition of p38 MAPK and ERK2 phosphorylation, and hydroxyl radical formation in platelets. In conclusion, this study demonstrated that andrographolide may involve an increase in cyclic GMP/PKG, followed by inhibition of the p38 MAPK/(·)HO-NF-κB-ERK2 cascade in activated platelets. Therefore, andrographolide may have a high therapeutic potential

  14. 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

  15. Activation of the prophenoloxidase cascade and initiation of nodule formation in locusts by bacterial lipopolysaccharides.

    PubMed

    Ratcliffe, N A; Brookman, J L; Rowley, A F

    1991-01-01

    The activation of the prophenoloxidase (proPO) system of the locusts, Schistocerca gregaria and Locusta migratoria, by several bacterial lipopolysaccharides (LPS) is described. Activation of proPO by LPS occurred only in the presence of whole blood homogenates and not with hemocyte lysate preparations alone. Levels of phenoloxidase generated by the different LPSs in vitro were also correlated with numbers of nodules formed in vivo by injection of these LPSs. This further strengthens the evidence for the involvement of proPO activation in the insect cellular defenses. Finally, the wisdom in using anticoagulants in order to stabilize fragile hemocytes in studies on the proPO system is discussed. PMID:1904828

  16. Role of a mitogen-activated protein kinase cascade in ion flux-mediated turgor regulation in fungi.

    PubMed

    Lew, Roger R; Levina, Natalia N; Shabala, Lana; Anderca, Marinela I; Shabala, Sergey N

    2006-03-01

    Fungi normally maintain a high internal hydrostatic pressure (turgor) of about 500 kPa. In response to hyperosmotic shock, there are immediate electrical changes: a transient depolarization (1 to 2 min) followed by a sustained hyperpolarization (5 to 10 min) prior to turgor recovery (10 to 60 min). Using ion-selective vibrating probes, we established that the transient depolarization is due to Ca(2+) influx and the sustained hyperpolarization is due to H(+) efflux by activation of the plasma membrane H(+)-ATPase. Protein synthesis is not required for H(+)-ATPase activation. Net K(+) and Cl(-) uptake occurs at the same time as turgor recovery. The magnitude of the ion uptake is more than sufficient to account for the osmotic gradients required for turgor to return to its original level. Two osmotic mutants, os-1 and os-2, homologs of a two-component histidine kinase sensor and the yeast high osmotic glycerol mitogen-activated protein (MAP) kinase, respectively, have lower turgor than the wild type and do not exhibit the sustained hyperpolarization after hyperosmotic treatment. The os-1 mutant does not exhibit all of the wild-type turgor-adaptive ion fluxes (Cl(-) uptake increases, but net K(+) flux barely changes and net H(+) efflux declines) (os-2 was not examined). Both os mutants are able to regulate turgor but at a lower level than the wild type. Our results demonstrate that a MAP kinase cascade regulates ion transport, activation of the H(+)-ATPase, and net K(+) and Cl(-) uptake during turgor regulation. Other pathways regulating turgor must also exist. PMID:16524903

  17. Role of a Mitogen-Activated Protein Kinase Cascade in Ion Flux-Mediated Turgor Regulation in Fungi

    PubMed Central

    Lew, Roger R.; Levina, Natalia N.; Shabala, Lana; Anderca, Marinela I.; Shabala, Sergey N.

    2006-01-01

    Fungi normally maintain a high internal hydrostatic pressure (turgor) of about 500 kPa. In response to hyperosmotic shock, there are immediate electrical changes: a transient depolarization (1 to 2 min) followed by a sustained hyperpolarization (5 to 10 min) prior to turgor recovery (10 to 60 min). Using ion-selective vibrating probes, we established that the transient depolarization is due to Ca2+ influx and the sustained hyperpolarization is due to H+ efflux by activation of the plasma membrane H+-ATPase. Protein synthesis is not required for H+-ATPase activation. Net K+ and Cl− uptake occurs at the same time as turgor recovery. The magnitude of the ion uptake is more than sufficient to account for the osmotic gradients required for turgor to return to its original level. Two osmotic mutants, os-1 and os-2, homologs of a two-component histidine kinase sensor and the yeast high osmotic glycerol mitogen-activated protein (MAP) kinase, respectively, have lower turgor than the wild type and do not exhibit the sustained hyperpolarization after hyperosmotic treatment. The os-1 mutant does not exhibit all of the wild-type turgor-adaptive ion fluxes (Cl− uptake increases, but net K+ flux barely changes and net H+ efflux declines) (os-2 was not examined). Both os mutants are able to regulate turgor but at a lower level than the wild type. Our results demonstrate that a MAP kinase cascade regulates ion transport, activation of the H+-ATPase, and net K+ and Cl− uptake during turgor regulation. Other pathways regulating turgor must also exist. PMID:16524903

  18. Phonological Activation of Ignored Pictures: Further Evidence for a Cascade Model of Lexical Access

    ERIC Educational Resources Information Center

    Navarrete, Eduardo; Costa, Albert

    2005-01-01

    Four experiments are reported exploring whether distractor pictures activate their phonological properties in the course of speech production. In Experiment 1, participants were presented with two pictures and were asked to name one while ignoring the other. Distractor pictures were phonologically related, semantically related or unrelated to the…

  19. 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

  20. Hybrid coordination-network-engineering for bridging cascaded channels to activate long persistent phosphorescence in the second biological window

    PubMed Central

    Qin, Xixi; Li, Yang; Zhang, Ruili; Ren, Jinjun; Gecevicius, Mindaugas; Wu, Yiling; Sharafudeen, Kaniyarakkal; Dong, Guoping; Zhou, Shifeng; Ma, Zhijun; Qiu, Jianrong

    2016-01-01

    We present a novel “Top-down” strategy to design the long phosphorescent phosphors in the second biological transparency window via energy transfer. Inherence in this approach to material design involves an ingenious engineering for hybridizing the coordination networks of hosts, tailoring the topochemical configuration of dopants, and bridging a cascaded tunnel for transferring the persistent energy from traps, to sensitizers and then to acceptors. Another significance of this endeavour is to highlight a rational scheme for functionally important hosts and dopants, Cr/Nd co-doped Zn1−xCaxGa2O4 solid solutions. Such solid-solution is employed as an optimized host to take advantage of its characteristic trap site level to establish an electron reservoir and network parameters for the precipitation of activators Nd3+ and Cr3+. The results reveal that the strategy employed here has the great potential, as well as opens new opportunities for future new-wavelength, NIR phosphorescent phosphors fabrication with many potential multifunctional bio-imaging applications. PMID:26843129

  1. Hybrid coordination-network-engineering for bridging cascaded channels to activate long persistent phosphorescence in the second biological window

    NASA Astrophysics Data System (ADS)

    Qin, Xixi; Li, Yang; Zhang, Ruili; Ren, Jinjun; Gecevicius, Mindaugas; Wu, Yiling; Sharafudeen, Kaniyarakkal; Dong, Guoping; Zhou, Shifeng; Ma, Zhijun; Qiu, Jianrong

    2016-02-01

    We present a novel “Top-down” strategy to design the long phosphorescent phosphors in the second biological transparency window via energy transfer. Inherence in this approach to material design involves an ingenious engineering for hybridizing the coordination networks of hosts, tailoring the topochemical configuration of dopants, and bridging a cascaded tunnel for transferring the persistent energy from traps, to sensitizers and then to acceptors. Another significance of this endeavour is to highlight a rational scheme for functionally important hosts and dopants, Cr/Nd co-doped Zn1-xCaxGa2O4 solid solutions. Such solid-solution is employed as an optimized host to take advantage of its characteristic trap site level to establish an electron reservoir and network parameters for the precipitation of activators Nd3+ and Cr3+. The results reveal that the strategy employed here has the great potential, as well as opens new opportunities for future new-wavelength, NIR phosphorescent phosphors fabrication with many potential multifunctional bio-imaging applications.

  2. Hybrid coordination-network-engineering for bridging cascaded channels to activate long persistent phosphorescence in the second biological window.

    PubMed

    Qin, Xixi; Li, Yang; Zhang, Ruili; Ren, Jinjun; Gecevicius, Mindaugas; Wu, Yiling; Sharafudeen, Kaniyarakkal; Dong, Guoping; Zhou, Shifeng; Ma, Zhijun; Qiu, Jianrong

    2016-01-01

    We present a novel "Top-down" strategy to design the long phosphorescent phosphors in the second biological transparency window via energy transfer. Inherence in this approach to material design involves an ingenious engineering for hybridizing the coordination networks of hosts, tailoring the topochemical configuration of dopants, and bridging a cascaded tunnel for transferring the persistent energy from traps, to sensitizers and then to acceptors. Another significance of this endeavour is to highlight a rational scheme for functionally important hosts and dopants, Cr/Nd co-doped Zn1-xCaxGa2O4 solid solutions. Such solid-solution is employed as an optimized host to take advantage of its characteristic trap site level to establish an electron reservoir and network parameters for the precipitation of activators Nd(3+) and Cr(3+). The results reveal that the strategy employed here has the great potential, as well as opens new opportunities for future new-wavelength, NIR phosphorescent phosphors fabrication with many potential multifunctional bio-imaging applications. PMID:26843129

  3. A Palladium(II)-Catalyzed C-H Activation Cascade Sequence for Polyheterocycle Formation.

    PubMed

    Cooper, Stephen P; Booker-Milburn, Kevin I

    2015-05-26

    Polyheterocycles are found in many natural products and are useful moieties in functional materials and drug design. As part of a program towards the synthesis of Stemona alkaloids, a novel palladium(II)-catalyzed C-H activation strategy for the construction of such systems has been developed. Starting from simple 1,3-dienyl-substituted heterocycles, a large range of polycyclic systems containing pyrrole, indole, furan and thiophene moieties can be synthesized in a single step. PMID:25872492

  4. Methamphetamine-induced neurotoxicity and microglial activation are not mediated by fractalkine receptor signaling

    PubMed Central

    Thomas, David M.; Francescutti-Verbeem, Dina M.; Kuhn, Donald M.

    2009-01-01

    Methamphetamine (METH) damages dopamine (DA) nerve endings by a process that has been linked to microglial activation but the signaling pathways that mediate this response have not yet been delineated. Cardona et al. [Nat. Neurosci. 9 (2006), 917] recently identified the microglial-specific fractalkine receptor (CX3CR1) as an important mediator of MPTP-induced neurodegeneration of DA neurons. Because the CNS damage caused by METH and MPTP is highly selective for the DA neuronal system in mouse models of neurotoxicity, we hypothesized that the CX3CR1 plays a role in METH-induced neurotoxicity and microglial activation. Mice in which the CX3CR1 gene has been deleted and replaced with a cDNA encoding enhanced green fluorescent protein (eGFP) were treated with METH and examined for striatal neurotoxicity. METH depleted DA, caused microglial activation, and increased body temperature in CX3CR1 knockout mice to the same extent and over the same time course seen in wild-type controls. The effects of METH in CX3CR1 knockout mice were not gender-dependent and did not extend beyond the striatum. Striatal microglia expressing eGFP constitutively show morphological changes after METH that are characteristic of activation. This response was restricted to the striatum and contrasted sharply with unresponsive eGFP-microglia in surrounding brain areas that are not damaged by METH. We conclude from these studies that CX3CR1 signaling does not modulate METH neurotoxicity or microglial activation. Furthermore, it appears that striatal-resident microglia respond to METH with an activation cascade and then return to a surveying state without undergoing apoptosis or migration. PMID:18410508

  5. Phosphoproteomic analysis of induced resistance reveals activation of signal transduction processes by beneficial and pathogenic interaction in grapevine.

    PubMed

    Perazzolli, Michele; Palmieri, Maria Cristina; Matafora, Vittoria; Bachi, Angela; Pertot, Ilaria

    2016-05-20

    Protein phosphorylation regulates several key processes of the plant immune system. Protein kinases and phosphatases are pivotal regulators of defense mechanisms elicited by resistance inducers. However, the phosphorylation cascades that trigger the induced resistance mechanisms in plants have not yet been deeply investigated. The beneficial fungus Trichoderma harzianum T39 (T39) induces resistance against grapevine downy mildew (Plasmopara viticola), but its efficacy could be further improved by a better understanding of the cellular regulations involved. We investigated quantitative changes in the grapevine phosphoproteome during T39-induced resistance to get an overview of regulatory mechanisms of downy mildew resistance. Immunodetection experiments revealed activation of the 45 and 49kDa kinases by T39 treatment both before and after pathogen inoculation, and the phosphoproteomic analysis identified 103 phosphopeptides that were significantly affected by the phosphorylation cascades during T39-induced resistance. Peptides affected by T39 treatment showed comparable phosphorylation levels after P. viticola inoculation, indicating activation of the microbial recognition machinery before pathogen infection. Phosphorylation profiles of proteins related to photosynthetic processes and protein ubiquitination indicated a partial overlap of cellular responses in T39-treated and control plants. However, phosphorylation changes of proteins involved in response to stimuli, signal transduction, hormone signaling, gene expression regulation, and RNA metabolism were exclusively elicited by P. viticola inoculation in T39-treated plants. These results highlighted the relevance of phosphorylation changes during T39-induced resistance and identified key regulator candidates of the grapevine defense against downy mildew. PMID:27010348

  6. 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.

  7. Leucocyte expression of genes implicated in the plasminogen activation cascade is modulated by yoghurt peptides.

    PubMed

    Theodorou, Georgios; Politis, Ioannis

    2016-08-01

    The urokinase-plasminogen activator (u-PA), its receptor (u-PAR) and the inhibitors of u-PA (PAI-1 and PAI-2) provide a multi-molecular system in leucocytes that exerts pleiotropic functions influencing the development of inflammatory and immune responses. The objective of the present study was to examine the ability of water soluble extracts (WSE) obtained from traditional Greek yoghurt made from bovine or ovine milk to modulate the expression of u-PA, u-PAR, PAI-1 and PAI-2 in ovine monocytes and neutrophils. WSE were obtained from 8 commercial traditional type Greek yoghurts made from ovine or bovine milk. WSE upregulated the expression of all 4 u-PA related genes in monocytes but the upregulation was much higher in the PAI-1 (10-fold) than in u-PA and u-PAR (3-4 fold) thus, shifting the system towards inhibition. In line with this observation, WSE reduced total and membrane-bound u-PA activity in monocytes. In neutrophils, WSE caused small (50-60%) but significant (P < 0·05) reductions in expression of u-PAR and PAI-2 but had no effect on expression of u-PA, PAI-1 and on total cell-associated and membrane-bound u-PA activity. WSE from yoghurts made from bovine or ovine milk were essentially equally effective in affecting the u-PA system except for the u-PAR gene in ovine neutrophils that was affected (reduced) by the ovine and not the bovine WSE. In conclusion, peptides present in WSE modulated the expression of u-PA related genes but the effect was much more prominent in monocytes than in neutrophils. PMID:27600972

  8. Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis.

    PubMed

    Krithika, Ramakrishnan; Srivastava, Prabhakar Lal; Rani, Bajaj; Kolet, Swati P; Chopade, Manojkumar; Soniya, Mantri; Thulasiram, Hirekodathakallu V

    2015-01-01

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)(+) dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP(+) yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis. PMID:25651761

  9. Characterization of 10-Hydroxygeraniol Dehydrogenase from Catharanthus roseus Reveals Cascaded Enzymatic Activity in Iridoid Biosynthesis

    PubMed Central

    Krithika, Ramakrishnan; Srivastava, Prabhakar Lal; Rani, Bajaj; Kolet, Swati P.; Chopade, Manojkumar; Soniya, Mantri; Thulasiram, Hirekodathakallu V.

    2015-01-01

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)+ dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP+ yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis. PMID:25651761

  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. Oncolytic Activity of a Recombinant Measles Virus, Blind to Signaling Lymphocyte Activation Molecule, Against Colorectal Cancer Cells

    PubMed Central

    Amagai, Yosuke; Fujiyuki, Tomoko; Yoneda, Misako; Shoji, Koichiro; Furukawa, Yoichi; Sato, Hiroki; Kai, Chieko

    2016-01-01

    Oncolytic virotherapy is a distinctive antitumor therapy based on the cancer-cell-specific infectivity and killing activity of viruses, which exert a considerable antitumor effect with only a few treatments. Because colorectal cancer cells often acquire resistance to the molecular-targeted therapies and alternative treatments are called for, in this study, we evaluated the oncolytic activity against colorectal cancer cells of a recombinant measles virus (rMV-SLAMblind), which is blind to signaling lymphocytic activation molecule (SLAM) and infects target cells via nectin-4/poliovirus receptor-related 4 protein. We examined 10 cell lines including 8 cell lines that were resistant to epidermal-growth-factor-receptor (EGFR) targeted therapy. rMV-SLAMblind infected and lysed the nectin-4-positive cell lines dependently on nectin-4 expression, in spite of mutation in EGFR cascade. Tumour progression in xenograft models was also abrogated by the virus, and the infection of cancer cells in vivo by the virus was demonstrated with both flow cytometry and a histological analysis. Therefore, rMV-SLAMblind is considered a novel therapeutic agent for colorectal cancers, including those resistant to molecular-targeted therapies. PMID:27090874

  12. Oncolytic Activity of a Recombinant Measles Virus, Blind to Signaling Lymphocyte Activation Molecule, Against Colorectal Cancer Cells.

    PubMed

    Amagai, Yosuke; Fujiyuki, Tomoko; Yoneda, Misako; Shoji, Koichiro; Furukawa, Yoichi; Sato, Hiroki; Kai, Chieko

    2016-01-01

    Oncolytic virotherapy is a distinctive antitumor therapy based on the cancer-cell-specific infectivity and killing activity of viruses, which exert a considerable antitumor effect with only a few treatments. Because colorectal cancer cells often acquire resistance to the molecular-targeted therapies and alternative treatments are called for, in this study, we evaluated the oncolytic activity against colorectal cancer cells of a recombinant measles virus (rMV-SLAMblind), which is blind to signaling lymphocytic activation molecule (SLAM) and infects target cells via nectin-4/poliovirus receptor-related 4 protein. We examined 10 cell lines including 8 cell lines that were resistant to epidermal-growth-factor-receptor (EGFR) targeted therapy. rMV-SLAMblind infected and lysed the nectin-4-positive cell lines dependently on nectin-4 expression, in spite of mutation in EGFR cascade. Tumour progression in xenograft models was also abrogated by the virus, and the infection of cancer cells in vivo by the virus was demonstrated with both flow cytometry and a histological analysis. Therefore, rMV-SLAMblind is considered a novel therapeutic agent for colorectal cancers, including those resistant to molecular-targeted therapies. PMID:27090874

  13. The timing of protein kinase activation events in the cascade that regulates mitotic progression in Tradescantia stamen hair cells.

    PubMed Central

    Wolniak, S M; Larsen, P M

    1995-01-01

    peptide appeared to act as a competitive inhibitor to reveal inflection points when protein kinase activation was occurring or when endogenous substrate levels approached levels of the peptide. The timing of these inflection points coincides with the changes in protein kinase activities during prometaphase and metaphase, as indicated by our perfusions of cells with the broad spectrum kinase inhibitors. Collectively, our results suggest that the cascade that culminates in anaphase is complex and involves several successive protein kinase activation steps punctuated by the activation of one or more protein phosphatases in mid-metaphase. PMID:7539650

  14. 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

  15. Protein corona changes mediated by surface modification of amorphous silica nanoparticles suppress acute toxicity and activation of intrinsic coagulation cascade in mice

    NASA Astrophysics Data System (ADS)

    Yoshida, Tokuyuki; Yoshioka, Yasuo; Morishita, Yuki; Aoyama, Michihiko; Tochigi, Saeko; Hirai, Toshiro; Tanaka, Kota; Nagano, Kazuya; Kamada, Haruhiko; Tsunoda, Shin-ichi; Nabeshi, Hiromi; Yoshikawa, Tomoaki; Higashisaka, Kazuma; Tsutsumi, Yasuo

    2015-06-01

    Recently, nanomaterial-mediated biological effects have been shown to be governed by the interaction of nanomaterials with some kinds of proteins in biological fluids, and the physical characteristics of the nanomaterials determine the extent and type of their interactions with proteins. Here, we examined the relationships between the surface properties of amorphous silica nanoparticles with diameters of 70 nm (nSP70), their interactions with some proteins in biological fluids, and their toxicity in mice after intravenous administration. The surface modification of nSP70 with amino groups (nSP70-N) prevented acute lethality and abnormal activation of the coagulation cascade found in the nSP70-treated group of mice. Since our previous study showed that coagulation factor XII played a role in the nSP70-mediated abnormal activation of the coagulation cascade, we examined the interaction of nSP70 and nSP70-N with coagulation factor XII. Coagulation factor XII bonded to the surface of nSP70 to a greater extent than that observed for nSP70-N, and consequently more activation of coagulation factor XII was observed for nSP70 than for nSP70-N. Collectively, our results suggest that controlling the interaction of nSP70 with blood coagulation factor XII by modifying the surface properties would help to inhibit the nSP70-mediated abnormal activation of the blood coagulation cascade.

  16. Activation of PPAR-γ and PTEN Cascade Participates in Lovastatin-mediated Accelerated Differentiation of Oligodendrocyte Progenitor Cells

    PubMed Central

    Paintlia, Ajaib S; Paintlia, Manjeet K; Singh, Avtar K; Singh, Inderjit

    2010-01-01

    Previously, we and others documented that statins including—lovastatin (LOV) promote the differentiation of oligodendrocyte progenitor cells (OPCs) and remyelination in experimental autoimmune encephalomyelitis (EAE), an multiple sclerosis (MS) model. Conversely, some recent studies demonstrated that statins negatively influence oligodendrocyte (OL) differentiation in vitro and remyelination in a cuprizone-CNS demyelinating model. Therefore, herein, we first investigated the cause of impaired differentiation of OLs by statins in vitro settings. Our observations indicated that the depletion of cholesterol was detrimental to LOV treated OPCs under cholesterol/serum-deprived culture conditions similar to that were used in conflicting studies. However, the depletion of geranylgeranyl-pp under normal cholesterol homeostasis conditions enhanced the phenotypic commitment and differentiation of LOV-treated OPCs ascribed to inhibition of RhoA-Rho kinase. Interestingly, this effect of LOV was associated with increased activation and expression of both PPAR-γ and PTEN in OPCs as confirmed by various pharmacological and molecular based approaches. Furthermore, PTEN was involved in an inhibition of OPCs proliferation via PI3K-Akt inhibition and induction of cell cycle arrest at G1 phase, but without affecting their cell survival. These effects of LOV on OPCs in vitro were absent in the CNS of normal rats chronically treated with LOV concentrations used in EAE indicating that PPAR-γ induction in normal brain may be tightly regulated — providing evidences that statins are therapeutically safe for humans. Collectively, these data provide initial evidence that statin-mediated activation of the PPAR-γ — PTEN cascade participates in OL differentiation, thus suggesting new therapeutic-interventions for MS or related CNS-demyelinating diseases. PMID:20578043

  17. 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

  18. 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

  19. The Prosurvival IKK-Related Kinase IKKε Integrates LPS and IL17A Signaling Cascades to Promote Wnt-Dependent Tumor Development in the Intestine.

    PubMed

    Göktuna, Serkan Ismail; Shostak, Kateryna; Chau, Tieu-Lan; Heukamp, Lukas C; Hennuy, Benoit; Duong, Hong-Quan; Ladang, Aurélie; Close, Pierre; Klevernic, Iva; Olivier, Fabrice; Florin, Alexandra; Ehx, Grégory; Baron, Frédéric; Vandereyken, Maud; Rahmouni, Souad; Vereecke, Lars; van Loo, Geert; Büttner, Reinhard; Greten, Florian R; Chariot, Alain

    2016-05-01

    Constitutive Wnt signaling promotes intestinal cell proliferation, but signals from the tumor microenvironment are also required to support cancer development. The role that signaling proteins play to establish a tumor microenvironment has not been extensively studied. Therefore, we assessed the role of the proinflammatory Ikk-related kinase Ikkε in Wnt-driven tumor development. We found that Ikkε was activated in intestinal tumors forming upon loss of the tumor suppressor Apc Genetic ablation of Ikkε in β-catenin-driven models of intestinal cancer reduced tumor incidence and consequently extended survival. Mechanistically, we attributed the tumor-promoting effects of Ikkε to limited TNF-dependent apoptosis in transformed intestinal epithelial cells. In addition, Ikkε was also required for lipopolysaccharide (LPS) and IL17A-induced activation of Akt, Mek1/2, Erk1/2, and Msk1. Accordingly, genes encoding pro-inflammatory cytokines, chemokines, and anti-microbial peptides were downregulated in Ikkε-deficient tissues, subsequently affecting the recruitment of tumor-associated macrophages and IL17A synthesis. Further studies revealed that IL17A synergized with commensal bacteria to trigger Ikkε phosphorylation in transformed intestinal epithelial cells, establishing a positive feedback loop to support tumor development. Therefore, TNF, LPS, and IL17A-dependent signaling pathways converge on Ikkε to promote cell survival and to establish an inflammatory tumor microenvironment in the intestine upon constitutive Wnt activation. Cancer Res; 76(9); 2587-99. ©2016 AACR. PMID:26980769

  20. 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

  1. 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

  2. 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

  3. Interleukin 6 Is Required for Pancreatic Cancer Progression by Promoting MAPK Signaling Activation and Oxidative Stress Resistance

    PubMed Central

    Zhang, Yaqing; Yan, Wei; Collins, Meredith A.; Bednar, Filip; Rakshit, Sabita; Zetter, Bruce R.; Stanger, Ben Z.; Chung, Ivy; Rhim, Andrew D.; di Magliano, Marina Pasca

    2013-01-01

    Pancreatic cancer, one of the deadliest human malignancies, is almost invariably associated with the presence of an oncogenic form of Kras. Mice expressing oncogenic Kras in the pancreas recapitulate the step-wise progression of the human disease. The inflammatory cytokine interleukin 6 (IL6) is often expressed by multiple cell types within the tumor microenvironment. Here, we show that IL6 is required for the maintenance and progression of pancreatic cancer precursor lesions. In fact, the lack of IL6 completely ablates cancer progression even in presence of oncogenic Kras. Mechanistically, we show that IL6 synergizes with oncogenic Kras to activate the reactive oxygen species (ROS) detoxification program downstream of the MAPK/ERK signaling cascade. In addition, IL6 regulates the inflammatory microenvironment of pancreatic cancer throughout its progression, providing several signals that are essential for carcinogenesis. Thus, IL6 emerges as a key player at all stages of pancreatic carcinogenesis, and a potential therapeutic target. PMID:24097820

  4. Overexpression of Amyloid- β Protein Precursor Induces Mitochondrial Oxidative Stress and Activates the Intrinsic Apoptotic Cascade

    PubMed Central

    Bartley, Matthew G.; Marquardt, Kristin; Kirchhof, Danielle; Wilkins, Heather M.; Patterson, David; Linseman, Daniel A.

    2015-01-01

    Alzheimer's disease (AD) is a debilitating cognitive disorder which is characterized pathologically by amyloid-β plaques and neurofibrillary tangles. Aberrant processing of amyloid beta protein precursor (AβPP) into amyloid-β fragments underlies the formation of senile plaques. Moreover, amyloid-β fragments, particularly Aβ42, exert direct toxic effects within neurons including the induction of mitochondrial oxidative stress (MOS). Interestingly, individuals with Down Syndrome (DS) frequently develop early onset AD as a major co-morbid phenotype. One hypothesis for AD associated with DS involves the overexpression of wild type (WT) AβPP protein, due to its location on chromosome 21. However, the mechanism by which the overexpression of WT AβPP might trigger MOS and induce cell death is presently unclear. Here we show that transient overexpression of DsRed2-tagged AβPP (WT) in CHO cells induces the activation of caspase-3 and nuclear fragmentation indicative of apoptosis. AβPP localizes to the mitochondrial fraction of transfected CHO cells and its overexpression causes glutathione (GSH)-sensitive opening of the mitochondrial permeability transition pore (mPTP) and cytochrome c release. MOS and intrinsic apoptosis induced by AβPP were significantly inhibited by the co-expression of Bcl-2 or treatment with either GSH or Boc, a pan-caspase inhibitor. Furthermore, the mPTP inhibitor, cyclosporin A, also significantly protected CHO cells from apoptosis induced by AβPP overexpression. Finally, co-treatment with a β-secretase inhibitor did not significantly protect CHO cells from AβPP overexpression and Aβ42 levels were undetectable in transfected CHO cells. Therefore, the mechanism of AβPP induced MOS and apoptosis is independent of the production of Aβ42. However, a γ-secretase inhibitor showed significant protection of the CHO cells against AβPP overexpression. Thus, suggesting a possible role of the AβPP intracellular domain in this apoptotic

  5. 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

  6. 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

  7. The HrpX/HrpY two-component system activates hrpS expression, the first step in the regulatory cascade controlling the Hrp regulon in Pantoea stewartii subsp. stewartii.

    PubMed

    Merighi, Massimo; Majerczak, Doris R; Stover, Elizabeth H; Coplin, David L

    2003-03-01

    A regulatory cascade activating hrp/hrc type III secretion and effector genes was delineated in Pantoea stewartii subsp. stewartii, a bacterial pathogen of corn. Four hrp regulatory genes were characterized: hrpX and hrpY encode the sensor kinase and response regulator, respectively, of a two-component signal transduction system; hrpS encodes an NtrC-like transcriptional enhancer; and hrpL encodes an alternative sigma factor. Epistasis analysis, expression studies using gene fusions, and genetic reconstruction of each step in Escherichia coli were used to delineate the following pathway: HrpY activates hrpS and also positively autoregulates the hrpXY operon. In turn, HrpS is required for full activation of the sigma54-dependent hrpL promoter. Finally, HrpL controls expression of all known hrp and wts genes. In vitro, hrpS and all downstream hrp genes were regulated by pH and salt concentration. Mutants with in-frame deletions in hrpX were still partially virulent on corn but were unable to sense the chemical or metabolic signals that induce hrp genes in vitro. Site-directed mutagenesis of HrpY indicated that aspartate 57 is the probable phosphorylation site and that it is needed for activity. These findings suggest that both HrpX and an alternate mechanism are involved in the activation of HrpY in planta. PMID:12650455

  8. Step-taper active-region quantum cascade lasers for carrier-leakage suppression and high internal differential efficiency

    NASA Astrophysics Data System (ADS)

    Kirch, J. D.; Chang, C.-C.; Boyle, C.; Mawst, L. J.; Lindberg, D.; Earles, T.; Botez, D.

    2016-03-01

    By stepwise tapering both the barrier heights and quantum-well depths in the active regions of 8.7 μm- and 8.4 μm-emitting quantum cascade lasers (QCLs) virtually complete carrier-leakage suppression is achieved, as evidenced by high values for both the threshold-current characteristic temperature coefficient T0 (283 K and 242 K) and the slope-efficiency characteristic temperature coefficient T1 (561 K and 279 K), over the 20-60 °C heatsink-temperature range, for low- and high-doped devices, respectively. Such high values are obtained while the threshold-current density is kept relatively low for 35-period, low- and high-doped devices: 1.58 kA/cm2 and 1.88 kA/cm2, respectively. In addition, due to resonant extraction from the lower laser level, high differential-transition-efficiency values (89-90%) are obtained. In turn, the slope-efficiency for 3 mm-long, 35-period high-reflectivity (HR)-coated devices are: 1.15-1.23 W/A; that is, 30- 40 % higher than for same-geometry and similar-doping conventional 8-9 μm-emitting QCLs. As a result of both efficient carrier-leakage suppression as well as fast and efficient carrier extraction, the values for the internal differential efficiency are found to be ≍ 86%, by comparison to typical values in the 58-67 % range for conventional QCLs emitting in the 7-11 μm wavelength range.

  9. Enhanced hydrogen production from waste activated sludge by cascade utilization of organic matter in microbial electrolysis cells.

    PubMed

    Lu, Lu; Xing, Defeng; Liu, Bingfeng; Ren, Nanqi

    2012-03-15

    Fermentative hydrogen production from waste activated sludge (WAS) has low H2 yield because WAS contains limited amounts of carbohydrate suitable for use by hydrogen-producing bacteria. Here, augmentation of hydrogen production from WAS by microbial electrolysis cells (MECs) was implemented. H2 yields of 3.89±0.39 mg-H2/g-DS (5.67±0.61 mg-H2/g-VSS) from raw WAS and 6.78±0.94 mg-H2/g-DS (15.08±1.41 mg-H2/g-VSS) from alkaline-pretreated WAS were obtained in the two-chamber MECs (TMECs). This was several times higher than yields obtained previously by fermentation. Single-chamber MECs (SMECs) with low internal resistance showed a H2 production rate that 13 times that of TMECs with similar H2 yield when alkaline-pretreated WAS was used. However, methanogenesis was detected after several batch cycles. A yield balance calculation revealed that carbohydrates were not the only substrates for electrohydrogenesis. Protein and its acidification products, such as volatile fatty acids are also responsible for a portion of H2 generation in MEC. Characterization of WAS in TMECs by three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy with parallel factor analysis indicated that electrohydrogenesis reacted on the extracellular polymeric substances and intracellular substances of WAS. Cascade utilization of organic matter in MECs increased hydrogen production from WAS. MECs showed high hydrogen yield from WAS, fewer H2 sinks, and insensitivity to temperature. Optimizing MEC configurations and operation conditions and improving the pretreatment processes of WAS are necessary before practical application can take place on a large scale. PMID:22197264

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. A novel role of sesamol in inhibiting NF-κB-mediated signaling in platelet activation

    PubMed Central

    2011-01-01

    Background Platelet activation is relevant to a variety of coronary heart diseases. Our previous studies revealed that sesamol possesses potent antiplatelet activity through increasing cyclic AMP formation. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of sesamol in NF-κB-mediated platelet function. Methods Platelet aggregation, Fura 2-AM fluorescence, and immunoblotting analysis were used in this study. Results NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were markedly activated by collagen (1 μg/ml) in washed human platelets, and these signaling events were attenuated by sesamol (2.5~25 μM). Furthermore, SQ22536 and ODQ, inhibitors of adenylate cyclase and guanylate cyclase, respectively, strongly reversed the sesamol (25 μM)-mediated inhibitory effects of IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation stimulated by collagen. The protein kinase A (PKA) inhibitor, H89, also reversed sesamol-mediated inhibition of IκBα degradation. Moreover, BAY11-7082, an NF-κB inhibitor, abolished IκBα degradation, phospholipase C (PLC)γ2 phosphorylation, protein kinase C (PKC) activation, [Ca2+]i mobilization, and platelet aggregation stimulated by collagen. Preincubation of platelets with the inhibitors, SQ22536 and H89, both strongly reversed sesamol-mediated inhibition of platelet aggregation and [Ca2+]i mobilization. Conclusions Sesamol activates cAMP-PKA signaling, followed by inhibition of the NF-κB-PLC-PKC cascade, thereby leading to inhibition of [Ca2+]i mobilization and platelet aggregation. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our data demonstrating that inhibition of NF-κB interferes with platelet function may have a great impact when

  19. Synthesis of 1,2,3,4-tetrasubstituted naphthalenes through a cascade reaction triggered by silyl acetal activation.

    PubMed

    Yanai, Hikaru; Ishii, Nobuyuki; Matsumoto, Takashi

    2016-06-28

    1,2,3,4-Tetrasubstituted naphthalenes bearing four different substituents were synthesized in a regioselective manner through a fluoride-induced cascade reaction of lactol silyl ethers, which could be easily prepared from 4-alkynylisocoumarins and ketene silyl acetal. PMID:27254271

  20. 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

  1. 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

  2. The regulatory role of the NO/cGMP signal transduction cascade during larval attachment and metamorphosis of the barnacle Balanus (=Amphibalanus) amphitrite.

    PubMed

    Zhang, Yu; He, Li-Sheng; Zhang, Gen; Xu, Ying; Lee, On-On; Matsumura, Kiyotaka; Qian, Pei-Yuan

    2012-11-01

    The barnacle Balanus amphitrite is among the most dominant fouling species on intertidal rocky shores in tropical and subtropical areas and is thus a target organism in antifouling research. After being released from adults, the swimming nauplius undertakes six molting cycles and then transforms into a cyprid. Using paired antennules, a competent cyprid actively explores and selects a suitable substratum for attachment and metamorphosis (collectively known as settlement). This selection process involves the reception of exogenous signals and subsequent endogenous signal transduction. To investigate the involvement of nitric oxide (NO) and cyclic GMP (cGMP) during larval settlement of B. amphitrite, we examined the effects of an NO donor and an NO scavenger, two nitric oxide synthase (NOS) inhibitors and a soluble guanylyl cyclase (sGC) inhibitor on settling cyprids. We found that the NO donor sodium nitroprusside (SNP) inhibited larval settlement in a dose-dependent manner. In contrast, both the NO scavenger carboxy-PTIO and the NOS inhibitors aminoguanidine hemisulfate (AGH) and S-methylisothiourea sulfate (SMIS) significantly accelerated larval settlement. Suppression of the downstream guanylyl cyclase (GC) activity using a GC-selective inhibitor ODQ could also significantly accelerate larval settlement. Interestingly, the settlement inhibition effects of SNP could be attenuated by ODQ at all concentrations tested. In the developmental expression profiling of NOS and sGC, the lowest expression of both genes was detected in the cyprid stage, a crucial stage for the larval decision to attach and metamorphose. In summary, we concluded that NO regulates larval settlement via mediating downstream cGMP signaling. PMID:22855617

  3. Screening of UV-B-induced genes from apple peels by SSH: possible involvement of MdCOP1-mediated signaling cascade genes in anthocyanin accumulation.

    PubMed

    Peng, Ting; Saito, Takanori; Honda, Chikako; Ban, Yusuke; Kondo, Satoru; Liu, Ji-Hong; Hatsuyama, Yoshimichi; Moriguchi, Takaya

    2013-07-01

    Suppression subtractive hybridization (SSH) was employed to identify candidate genes involved in red coloration in apple peel with the ultraviolet (UV)-B-treated 'Mutsu'. After reverse Northern blotting verification, nearly 80 clones were successfully sequenced. Large portions of the expressed sequence tags (ESTs) are well characterized anthocyanin biosynthesis-related genes, such as chalcone synthase (11A5), flavonol synthase (12F3), anthocyanidin synthase (11H5) and UDP-glycosyl transferase (14A12) whose presence proved the success of SSH. Eight ESTs were selected for quantitative real-time polymerase chain reaction analysis and their expressions were all elevated in 'Induction', further confirming the reliability of the SSH library. One EST, 11F4 (CONSTITUTIVE PHOTOMORPHOGENIC 1: COP1) with putative function in light signal relay was further analyzed in 'Mutsu' and 'Tsugaru', along with MdHY5 (ELONGATED HYPOCOTYL 5: the downstream target of COP1), MdMYB22 (a possible flavonol-specific activator under the regulation of HY5, belonging to the SG7/PRODUCTION OF FLAVONOL GLYCOSIDES family) and MdMYBA. Results showed that MdCOP1, MdHY5, MdMYB22 and MdMYBA were all UV-B inducible genes and anthocyanin accumulation occurred after their increased expressions. Moreover, their expressions and anthocyanin content were enhanced under UV-B plus 17°C treatment. The presence of G box, a known consensus binding site of HY5, in the MdMYBA promoter region implicated that it could be regulated by MdHY5, which was verified by the result of the yeast one-hybrid analysis. Our data suggested that UV-B irradiation would induce the utmost upstream light signaling factor, MdCOP1, which activates MdHY5 signaling by binding to the promoter regions of MdMYBs, and finally leads to the red coloration of apple peels. PMID:23171407

  4. Sequential signaling cascade of IL-6 and PGC-1α is involved in high glucose-induced podocyte loss and growth arrest

    SciTech Connect

    Kim, Dong Il; Park, Soo Hyun

    2013-06-14

    Highlights: •The pathophysiological role of IL-6 in high glucose-induced podocyte loss. •The novel role of PGC-1α in the development of diabetic nephropathy. •Signaling of IL-6 and PGC-1α in high glucose-induced dysfunction of podocyte. -- Abstract: Podocyte loss, which is mediated by podocyte apoptosis, is implicated in the onset of diabetic nephropathy. In this study, we investigated the involvement of interleukin (IL)-6 in high glucose-induced apoptosis of rat podocytes. We also examined the pathophysiological role of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) in this system. High glucose treatment induced not only podocyte apoptosis but also podocyte growth arrest. High glucose treatment also increased IL-6 secretion and activated IL-6 signaling. The high glucose-induced podocyte apoptosis was blocked by IL-6 neutralizing antibody. IL-6 treatment or overexpression induced podocyte apoptosis and growth arrest, and IL-6 siRNA transfection blocked high glucose-induced podocyte apoptosis and growth arrest. Furthermore, high glucose or IL-6 treatment increased PGC-1α expression, and PGC-1α overexpression also induced podocyte apoptosis and growth arrest. PGC-1α siRNA transfection blocked high glucose-induced podocyte apoptosis and growth arrest. Collectively, these findings showed that high glucose promoted apoptosis and cell growth arrest in podocytes via IL-6 signaling. In addition, PGC-1α is involved in podocyte apoptosis and cell growth arrest. Therefore, blocking IL-6 and its downstream mediators such as IL6Rα, gp130 and PGC-1α may attenuate the progression of diabetic nephropathy.

  5. The mitogen-activated protein kinase (MAPK) cascade controls phosphatase and tensin homolog (PTEN) expression through multiple mechanisms.

    PubMed

    Ciuffreda, Ludovica; Di Sanza, Cristina; Cesta Incani, Ursula; Eramo, Adriana; Desideri, Marianna; Biagioni, Francesca; Passeri, Daniela; Falcone, Italia; Sette, Giovanni; Bergamo, Paola; Anichini, Andrea; Sabapathy, Kanaga; McCubrey, James A; Ricciardi, Maria Rosaria; Tafuri, Agostino; Blandino, Giovanni; Orlandi, Augusto; De Maria, Ruggero; Cognetti, Francesco; Del Bufalo, Donatella; Milella, Michele

    2012-06-01

    The mitogen-activated protein kinase (MAPK) and PI3K pathways are regulated by extensive crosstalk, occurring at different levels. In tumors, transactivation of the alternate pathway is a frequent "escape" mechanism, suggesting that combined inhibition of both pathways may achieve synergistic antitumor activity. Here we show that, in the M14 melanoma model, simultaneous inhibition of both MEK and mammalian target of rapamycin (mTOR) achieves synergistic effects at suboptimal concentrations, but becomes frankly antagonistic in the presence of relatively high concentrations of MEK inhibitors. This observation led to the identification of a novel crosstalk mechanism, by which either pharmacologic or genetic inhibition of constitutive MEK signaling restores phosphatase and tensin homolog (PTEN) expression, both in vitro and in vivo, and inhibits downstream signaling through AKT and mTOR, thus bypassing the need for double pathway blockade. This appears to be a general regulatory mechanism and is mediated by multiple mechanisms, such as MAPK-dependent c-Jun and miR-25 regulation. Finally, PTEN upregulation appears to be a major effector of MEK inhibitors' antitumor activity, as cancer cells in which PTEN is inactivated are consistently more resistant to the growth inhibitory and anti-angiogenic effects of MEK blockade. PMID:22215152

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. Endothelin signaling activates Mef2c expression in the neural crest through a MEF2C-dependent positive-feedback transcriptional pathway.

    PubMed

    Hu, Jianxin; Verzi, Michael P; Robinson, Ashley S; Tang, Paul Ling-Fung; Hua, Lisa L; Xu, Shan-Mei; Kwok, Pui-Yan; Black, Brian L

    2015-08-15

    Endothelin signaling is essential for neural crest development, and dysregulated Endothelin signaling is associated with several neural crest-related disorders, including Waardenburg and other syndromes. However, despite the crucial roles of this pathway in neural crest development and disease, the transcriptional effectors directly activated by Endothelin signaling during neural crest development remain incompletely elucidated. Here, we establish that the MADS box transcription factor MEF2C is an immediate downstream transcriptional target and effector of Endothelin signaling in the neural crest. We show that Endothelin signaling activates Mef2c expression in the neural crest through a conserved enhancer in the Mef2c locus and that CRISPR-mediated deletion of this Mef2c neural crest enhancer from the mouse genome abolishes Endothelin induction of Mef2c expression. Moreover, we demonstrate that Endothelin signaling activates neural crest expression of Mef2c by de-repressing MEF2C activity through a Calmodulin-CamKII-histone deacetylase signaling cascade. Thus, these findings identify a MEF2C-dependent, positive-feedback mechanism for Endothelin induction and establish MEF2C as an immediate transcriptional effector and target of Endothelin signaling in the neural crest. PMID:26160899

  12. Sustained activation of fibroblast transforming growth factor-beta/Smad signaling in a murine model of scleroderma.

    PubMed

    Takagawa, Shinsuke; Lakos, Gabriella; Mori, Yasuji; Yamamoto, Toshiyuki; Nishioka, Kiyoshi; Varga, John

    2003-07-01

    Transforming growth factor-beta is responsible for triggering a cascade of events leading to fibrosis in scleroderma. The Smads are intracellular signal transducers recently shown to mediate fibroblast activation and other profibrotic responses elicited by transforming growth factor-betain vitro. To understand better the involvement of Smads in the pathogenesis of fibrosis, we examined Smad expression and activation in situ in a murine model of scleroderma. Bleomycin injections induced striking dermal infiltration with macrophages by 3 d, and progressive fibrosis by 2 wk. Infiltrating macrophages and resident fibroblasts expressed Smad3, the positive mediator for transforming growth factor-beta responses. Importantly, in bleomycin-injected skin, fibroblasts showed predominantly nuclear localization of Smad3 and intense staining for phospho-Smad2/3. Furthermore, phosphorylated Smad2/3 in fibroblasts was detected even after the resolution of inflammation. Expression of Smad7, the endogenous inhibitor of transforming growth factor-beta/Smad signaling, was strongly induced in dermal cells by transforming growth factor-beta, but not by bleomycin injections. Collectively, these results indicate that bleomycin-induced murine scleroderma is associated with rapid and sustained induction of transforming growth factor-beta/Smad signaling in resident dermal fibroblasts. Despite apparent activation of the intracellular transforming growth factor-beta signaling pathway in the lesional dermis, the expression of transforming growth factor-beta-inducible Smad7 was not upregulated. In light of the critical function of Smad7 as an endogenous inhibitor of Smad signaling that restricts the duration and magnitude of transforming growth factor-beta responses, and as a mediator of apoptosis, relative Smad7 deficiency observed in the present studies may account for sustained activation of transforming growth factor-beta/Smad signaling in lesional tissues. These findings raise the

  13. Membrane estradiol signaling in the brain

    PubMed Central

    Micevych, Paul; Dominguez, Reymundo

    2009-01-01

    While the physiology of membrane-initiated estradiol signaling in the nervous system has remained elusive, a great deal of progress has been made toward understanding the activation of cell signaling. Membrane-initiated estradiol signaling activates G proteins and their downstream cascades, but the identity of membrane receptors and the proximal signaling mechanism(s) have been more difficult to elucidate. Mounting evidence suggests that classical intracellular estrogen receptor-α (ERα) and ERβ are trafficked to the membrane to mediate estradiol cell signaling. Moreover, an interaction of membrane ERα and ERβ with metabotropic glutamate receptors has been identified that explains the pleomorphic actions of membrane-initiated estradiol signaling. This review focuses on the mechanism of actions initiated by membrane estradiol receptors and discusses the role of scaffold proteins and signaling cascades involved in the regulation of nociception, sexual receptivity and the synthesis of neuroprogesterone, an important component in the central nervous system signaling. PMID:19416735

  14. 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

  15. 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

  16. Stimulation of urokinase-type plasminogen activator receptor expression by PMA requires JNK1-dependent and -independent signaling modules.

    PubMed

    Gum, R; Juarez, J; Allgayer, H; Mazar, A; Wang, Y; Boyd, D

    1998-07-16

    The urokinase-type plasminogen activator receptor (u-PAR) has been implicated in tumor progression, and previous studies have shown that the expression of this gene is strongly up-regulated by PMA. Although the signaling mechanism by which PMA modulates u-PAR expression is not known, the effect of this phorbol ester on the expression of other genes has been ascribed to activation of the c-Raf-1-ERK signaling pathway. However, in the current study we examined an alternate possibility that the inductive effect of PMA on u-PAR expression also required a JNK1-dependent signaling cascade usually associated with stress-inducing stimuli. PMA treatment of the u-PAR-deficient OVCAR-3 ovarian cancer cells, which contain low JNK activities, resulted in a rapid (5 min) increase in JNK activity. Maximal JNK activity (12-fold induction) occurred after 30 min; this preceding the earliest detected rise in u-PAR protein (2 h). Dose-response studies with PMA also indicated that the increased JNK activity was tightly correlated with elevated u-PAR protein levels. The stimulation of u-PAR promoter activity by PMA required an intact upstream AP-1 motif (-184) and in PMA-treated cells this motif was bound with c-Jun as indicated from mobility shift assays. PMA up-regulated the c-Jun trans acting activity as indicated by the higher activity of a GAL4-regulated luciferase reporter in phorbol-ester-treated cells co-transfected with an expression vector encoding the c-Jun transactivation domain fused to the GAL4 DNA-binding domain. The ability of PMA to stimulate u-PAR promoter activity was effectively titrated out by the co-expression of either a kinase-defective JNK1 or a dominant negative MEKK1 the latter being an upstream activator of JNK1. Conversely, u-PAR promoter activity was stimulated by the co-expression of a constitutively active MEKK1 and this induction was antagonized by the inclusion of the kinase-defective JNK1 plasmid. We also determined the biological significance of the

  17. Farnesol activates the intrinsic pathway of apoptosis and the ATF4-ATF3-CHOP cascade of ER stress in human T lymphoblastic leukemia Molt4 cells.

    PubMed

    Joo, Joung Hyuck; Ueda, Eiichiro; Bortner, Carl D; Yang, Xiao-Ping; Liao, Grace; Jetten, Anton M

    2015-10-01

    In this study, we demonstrate that treatment of T lymphoblastic leukemic Molt4 cells with farnesol activates the apoptosome via the intrinsic pathway of apoptosis. This induction was associated with changes in the level of intracellular potassium and calcium, the dissipation of the mitochondrial and plasma membrane potential, release of cytochrome c, activation of several caspases, and PARP cleavage. The induction of apoptosis by farnesol was inhibited by the addition of the pan-caspase inhibitor Z-VAD-fmk and by the exogenous expression of the anti-apoptotic protein Bcl2. Analysis of the gene expression profiles by microarray analysis revealed that farnesol increased the expression of several genes related to the unfolded protein response (UPR), including CHOP and CHAC1. This induction was associated with the activation of the PERK-eIF2α-ATF3/4 cascade, but not the XBP-1 branch of the UPR. Although farnesol induced activation of the ERK1/2, p38, and JNK pathways, inhibition of these MAPKs had little effect on farnesol-induced apoptosis or the induction of UPR-related genes. Our data indicate that the induction of apoptosis in leukemic cells by farnesol is mediated through a pathway that involves activation of the apoptosome via the intrinsic pathway and induction of the PERK-eIF2α-ATF3/4 cascade in a manner that is independent of the farnesol-induced activation of MAPKs. PMID:26275811

  18. Anti-Breast Cancer Potential of Quercetin via the Akt/AMPK/Mammalian Target of Rapamycin (mTOR) Signaling Cascade

    PubMed Central

    Rivera Rivera, Amilcar; Castillo-Pichardo, Linette; Gerena, Yamil; Dharmawardhane, Suranganie

    2016-01-01

    The Akt/adenosine monophosphate protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway has emerged as a critical signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth. Thus, dysregulation of this pathway contributes to the development of metabolic disorders such as obesity, type 2diabetes, and cancer. We previously reported that a combination of grape polyphenols (resveratrol, quercetin and catechin: RQC), at equimolar concentrations, reduces breast cancer (BC) growth and metastasis in nude mice, and inhibits Akt and mTOR activities and activates AMPK, an endogenous inhibitor of mTOR, in metastatic BC cells. The objective of the present study was to determine the contribution of individual polyphenols to the effect of combined RQC on mTOR signaling. Metastatic BC cells were treated with RQC individually or in combination, at various concentrations, and the activities (phosphorylation) of AMPK, Akt, and the mTOR downstream effectors, p70S6 kinase (p70S6K) and 4E binding protein (4EBP1), were determined by Western blot. Results show that quercetin was the most effective compound for Akt/mTOR inhibition. Treatment with quercetin at 15μM had a similar effect as the RQC combination in the inhibition of BC cell proliferation, apoptosis, and migration. However, cell cycle analysis showed that the RQC treatment arrested BC cells in the G1 phase, while quercetin arrested the cell cycle in G2/M. In vivo experiments, using SCID mice with implanted tumors from metastatic BC cells, demonstrated that administration of quercetin at 15mg/kg body weight resulted in a ~70% reduction in tumor growth. In conclusion, quercetin appears to be a viable grape polyphenol for future development as an anti BC therapeutic. PMID:27285995

  19. Anti-Breast Cancer Potential of Quercetin via the Akt/AMPK/Mammalian Target of Rapamycin (mTOR) Signaling Cascade.

    PubMed

    Rivera Rivera, Amilcar; Castillo-Pichardo, Linette; Gerena, Yamil; Dharmawardhane, Suranganie

    2016-01-01

    The Akt/adenosine monophosphate protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway has emerged as a critical signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth. Thus, dysregulation of this pathway contributes to the development of metabolic disorders such as obesity, type 2diabetes, and cancer. We previously reported that a combination of grape polyphenols (resveratrol, quercetin and catechin: RQC), at equimolar concentrations, reduces breast cancer (BC) growth and metastasis in nude mice, and inhibits Akt and mTOR activities and activates AMPK, an endogenous inhibitor of mTOR, in metastatic BC cells. The objective of the present study was to determine the contribution of individual polyphenols to the effect of combined RQC on mTOR signaling. Metastatic BC cells were treated with RQC individually or in combination, at various concentrations, and the activities (phosphorylation) of AMPK, Akt, and the mTOR downstream effectors, p70S6 kinase (p70S6K) and 4E binding protein (4EBP1), were determined by Western blot. Results show that quercetin was the most effective compound for Akt/mTOR inhibition. Treatment with quercetin at 15μM had a similar effect as the RQC combination in the inhibition of BC cell proliferation, apoptosis, and migration. However, cell cycle analysis showed that the RQC treatment arrested BC cells in the G1 phase, while quercetin arrested the cell cycle in G2/M. In vivo experiments, using SCID mice with implanted tumors from metastatic BC cells, demonstrated that administration of quercetin at 15mg/kg body weight resulted in a ~70% reduction in tumor growth. In conclusion, quercetin appears to be a viable grape polyphenol for future development as an anti BC therapeutic. PMID:27285995

  20. 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

  1. 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

  2. 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

  3. Wnt5a can both activate and repress Wnt/β-catenin signaling during mouse embryonic development

    PubMed Central

    van Amerongen, Renée; Fuerer, Christophe; Mizutani, Makiko; Nusse, Roel

    2012-01-01

    Embryonic development is controlled by a small set of signal transduction pathways, with vastly different phenotypic outcomes depending on the time and place of their recruitment. How the same molecular machinery can elicit such specific and distinct responses, remains one of the outstanding questions in developmental biology. Part of the answer may lie in the high inherent genetic complexity of these signaling cascades, as observed for the Wnt-pathway. The mammalian genome encodes multiple Wnt proteins and receptors, each of which show dynamic and tightly controlled expression patterns in the embryo. Yet how these components interact in the context of the whole organism remains unknown. Here we report the generation of a novel, inducible transgenic mouse model that allows spatiotemporal control over the expression of Wnt5a, a protein implicated in many developmental processes and multiple Wnt-signaling responses. We show that ectopic Wnt5a expression from E10.5 onwards results in a variety of developmental defects, including loss of hair follicles and reduced bone formation in the skull. Moreover, we find that Wnt5a can have dual signaling activities during mouse embryonic development. Specifically, Wnt5a is capable of both inducing and repressing β-catenin/TCF signaling in vivo, depending on the time and site of expression and the receptors expressed by receiving cells. These experiments show for the first time that a single mammalian Wnt protein can have multiple signaling activities in vivo, thereby furthering our understanding of how signaling specificity is achieved in a complex developmental context. PMID:22771246

  4. Oxidative Stress–Induced JNK1/2 Activation Triggers Proapoptotic Signaling and Apoptosis That Leads to Diabetic Embryopathy

    PubMed Central

    Li, Xuezheng; Weng, Hongbo; Xu, Cheng; Reece, E. Albert; Yang, Peixin

    2012-01-01

    Oxidative stress and apoptosis are implicated in the pathogenesis of diabetic embryopathy. The proapoptotic c-Jun NH2-terminal kinases (JNK)1/2 activation is associated with diabetic embryopathy. We sought to determine whether 1) hyperglycemia-induced oxidative stress is responsible for the activation of JNK1/2 signaling, 2) JNK1 contributes to the teratogenicity of hyperglycemia, and 3) both JNK1 and JNK2 activation cause activation of downstream transcription factors, caspase activation, and apoptosis, resulting in neural tube defects (NTDs). Wild-type (WT) embryos from nondiabetic WT dams and WT, superoxide dismutase (SOD)1–overexpressing, jnk1+/−, jnk1−/−, and jnk2−/− embryos exposed to maternal hyperglycemia were used to assess JNK1/2 activation, NTDs, activation of transcription factors downstream of JNK1/2, caspase cascade, and apoptosis. SOD1 overexpression abolished diabetes-induced activation of JNK1/2 and their downstream effectors: phosphorylation of c-Jun, activating transcription factor 2, and E twenty-six–like transcription factor 1 and dephosphorylation of forkhead box class O3a. jnk1−/− embryos had significantly lower incidences of NTDs than those of WT or jnk1+/− embryos. Either jnk1 or jnk2 gene deletion blocked diabetes-induced activation of JNK1/2 signaling, caspases 3 and 8, and apoptosis in Sox1+ neural progenitors of the developing neural tube. Our results show that JNK1 and JNK2 are equally involved in diabetic embryopathy and that the oxidative stress–JNK1/2–caspase pathway mediates the proapoptotic signals and the teratogenicity of maternal diabetes. PMID:22688338

  5. Hyperactive RAS/PI3-K/MAPK Signaling Cascade in Migration and Adhesion of Nf1 Haploinsufficient Mesenchymal Stem/Progenitor Cells.

    PubMed

    Zhou, Yuan; He, Yongzheng; Sharma, Richa; Xing, Wen; Estwick, Selina A; Wu, Xiaohua; Rhodes, Steven D; Xu, Mingjiang; Yang, Feng-Chun

    2015-01-01

    Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in the NF1 tumor suppressor gene, which affect approximately 1 out of 3000 individuals. Patients with NF1 suffer from a range of malignant and nonmalignant manifestations such as plexiform neurofibromas and skeletal abnormalities. We previously demonstrated that Nf1 haploinsufficiency in mesenchymal stem/progenitor cells (MSPCs) results in impaired osteoblastic differentiation, which may be associated with the skeletal manifestations in NF1 patients. Here we sought to further ascertain the role of Nf1 in modulating the migration and adhesion of MSPCs of the Nf1 haploinsufficient (Nf1(+/-)) mice. Nf1(+/-) MSPCs demonstrated increased nuclear-cytoplasmic ratio, increased migration, and increased actin polymerization as compared to wild-type (WT) MSPCs. Additionally, Nf1(+/-) MSPCs were noted to have significantly enhanced cell adhesion to fibronectin with selective affinity for CH271 with an overexpression of its complimentary receptor, CD49e. Nf1(+/-) MSPCs also showed hyperactivation of phosphoinositide 3-kinase (PI3-K) and mitogen activated