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Sample records for require pka activity

  1. The effects of caffeine on sleep in Drosophila require PKA activity, but not the adenosine receptor.

    Science.gov (United States)

    Wu, Mark N; Ho, Karen; Crocker, Amanda; Yue, Zhifeng; Koh, Kyunghee; Sehgal, Amita

    2009-09-02

    Caffeine is one of the most widely consumed stimulants in the world and has been proposed to promote wakefulness by antagonizing function of the adenosine A2A receptor. Here, we show that chronic administration of caffeine reduces and fragments sleep in Drosophila and also lengthens circadian period. To identify the mechanisms underlying these effects of caffeine, we first generated mutants of the only known adenosine receptor in flies (dAdoR), which by sequence is most similar to the mammalian A2A receptor. Mutants lacking dAdoR have normal amounts of baseline sleep, as well as normal homeostatic responses to sleep deprivation. Surprisingly, these mutants respond normally to caffeine. On the other hand, the effects of caffeine on sleep and circadian rhythms are mimicked by a potent phosphodiesterase inhibitor, IBMX (3-isobutyl-1-methylxanthine). Using in vivo fluorescence resonance energy transfer imaging, we find that caffeine induces widespread increase in cAMP levels throughout the brain. Finally, the effects of caffeine on sleep are blocked in flies that have reduced neuronal PKA activity. We suggest that chronic administration of caffeine promotes wakefulness in Drosophila, at least in part, by inhibiting cAMP phosphodiesterase activity.

  2. Different Phases of Long-Term Memory Require Distinct Temporal Patterns of PKA Activity after Single-Trial Classical Conditioning

    Science.gov (United States)

    Michel, Maximilian; Kemenes, Ildiko; Muller, Uli; Kemenes, Gyorgy

    2008-01-01

    The cAMP-dependent protein kinase (PKA) is known to play a critical role in both transcription-independent short-term or intermediate-term memory and transcription-dependent long-term memory (LTM). Although distinct phases of LTM already have been demonstrated in some systems, it is not known whether these phases require distinct temporal patterns…

  3. Stimulation of fibroblast proliferation by neokyotorphin requires Ca influx and activation of PKA, CaMK II and MAPK/ERK.

    Science.gov (United States)

    Sazonova, Olga V; Blishchenko, Elena Yu; Tolmazova, Anna G; Khachin, Dmitry P; Leontiev, Konstantin V; Karelin, Andrey A; Ivanov, Vadim T

    2007-01-01

    Neokyotorphin [TSKYR, hemoglobin alpha-chain fragment (137-141)] has previously been shown to enhance fibroblast proliferation, its effect depending on cell density and serum level. Here we show the dependence of the effect of neokyotorphin on cell type and its correlation with the effect of protein kinase A (PKA) activator 8-Br-cAMP, but not the PKC activator 4beta-phorbol 12-myristate, 13-acetate (PMA). In L929 fibroblasts, the proliferative effect of neokyotorphin was suppressed by the Ca2+ L-type channel inhibitors verapamil or nifedipine, the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, kinase inhibitors H-89 (PKA), KN-62 (Ca2+/calmodulin-dependent kinase II) and PD98059 (mitogen-activated protein kinase). The proliferative effect of 8-Br-cAMP was also suppressed by KN-62 and PD98059. PKC suppression (downregulation with PMA or inhibition with bisindolylmaleimide XI) did not affect neokyotorphin action. The results obtained point to a cAMP-like action for neokyotorphin.

  4. PKA and PKC Are Required for Long-Term but Not Short-Term in Vivo Operant Memory in "Aplysia"

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    Michel, Maximilian; Green, Charity L.; Lyons, Lisa C.

    2011-01-01

    We investigated the involvement of PKA and PKC signaling in a negatively reinforced operant learning paradigm in "Aplysia", learning that food is inedible (LFI). In vivo injection of PKA or PKC inhibitors blocked long-term LFI memory formation. Moreover, a persistent phase of PKA activity, although not PKC activity, was necessary for long-term…

  5. PKA and PKC Are Required for Long-Term but Not Short-Term in Vivo Operant Memory in "Aplysia"

    Science.gov (United States)

    Michel, Maximilian; Green, Charity L.; Lyons, Lisa C.

    2011-01-01

    We investigated the involvement of PKA and PKC signaling in a negatively reinforced operant learning paradigm in "Aplysia", learning that food is inedible (LFI). In vivo injection of PKA or PKC inhibitors blocked long-term LFI memory formation. Moreover, a persistent phase of PKA activity, although not PKC activity, was necessary for long-term…

  6. Hippocampal cAMP/PKA/CREB is required for neuroprotective effect of acupuncture.

    Science.gov (United States)

    Li, Qian-Qian; Shi, Guang-Xia; Yang, Jing-Wen; Li, Zhao-Xin; Zhang, Zhen-Hua; He, Tian; Wang, Jing; Liu, Li-Ying; Liu, Cun-Zhi

    2015-02-01

    Acupuncture has beneficial effects in vascular dementia (VaD) patients. The underlying mechanism, however, remains unknown. The present study was designed to investigate whether the cAMP/PKA/CREB cascade is involved in the mechanism of acupuncture in cerebral multi-infarction rats. In this study, cerebral multi-infarction was modeled in adult Wistar rats by homologous blood clot emboli. After a two-week acupuncture treatment at Zusanli (ST36), hippocampal-dependent memory was tested by employing a radial arm maze test. The hippocampus was isolated for analyses of cAMP concentration, phosphodiesterase (PDE) activity and CREB/pCREB and ERK/pERK expressions. The Morris water maze (MWM) task and CREB phosphorylation were evaluated in the presence of PKA-selective peptide inhibitor (H89). The radial arm maze test results demonstrated that acupuncture treatment at ST36 reversed hippocampal-dependent memory in impaired animals. Compared to those of the impaired group, cAMP concentration, PKA activity and pCREB and pERK expressions were increased following acupuncture therapy. Finally, the blockade of PKA reversed the increase in CREB phosphorylation and the improvement in recognitive function induced by acupuncture treatment. These results suggest that acupuncture could improve hippocampus function by modulating the cAMP/PKA/CREB signaling pathway, which represents a molecular mechanism of acupuncture for recognitive function in cerebral multi-infarction rats.

  7. Involvement of PKA, PKC, and Ca2+ in LPS-activated expression of the chicken lysozyme gene.

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    Regenhard, P; Goethe, R; Phi-van, L

    2001-04-01

    The lysozyme gene is activated in myelomonocytic HD11 cells in response to LPS. In this study, we described the involvement of LPS-activated signal transduction pathways in activation of the lysozyme gene. Pre-treatment of HD 11 cells with H-89, H-7, TMB-8, or KN-93 resulted in inhibition of the LPS-enhanced lysozyme expression, suggesting that PKA, PKC, and Ca2+-dependent protein kinases participate in the LPS activation. CaMKII seems to be required for the processing of lysozyme transcripts. TPA and calcium ionophore A23187, when separately added to HD11 cells, stimulated the lysozyme expression effectively, and forskolin was ineffective. It is interesting that simultaneous treatment of cells with forskolin and calcium ionophore A23187 resulted in a potentiated increase in lysozyme mRNA expression, indicating a synergistic cooperation of PKA and Ca2+. This synergistic effect of PKA and Ca2+ was observed on the expression of a stably integrated CAT construct, controlled by the lysozyme promoter and the -6.1-kb enhancer containing binding sites for C/EBP and NF-kappaB/Rel. Therefore, we discussed the role of C/EBPbeta(NF-M), CREB, and NF-kappaB/Rel as possible targets for phosphorylation mediated by PKA, PKC, and Ca2+.

  8. Evidence for an Elevated Aspartate pKa in the Active Site of Human Aromatase*

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    Di Nardo, Giovanna; Breitner, Maximilian; Bandino, Andrea; Ghosh, Debashis; Jennings, Gareth K.; Hackett, John C.; Gilardi, Gianfranco

    2015-01-01

    Aromatase (CYP19A1), the enzyme that converts androgens to estrogens, is of significant mechanistic and therapeutic interest. Crystal structures and computational studies of this enzyme shed light on the critical role of Asp309 in substrate binding and catalysis. These studies predicted an elevated pKa for Asp309 and proposed that protonation of this residue was required for function. In this study, UV-visible absorption, circular dichroism, resonance Raman spectroscopy, and enzyme kinetics were used to study the impact of pH on aromatase structure and androstenedione binding. Spectroscopic studies demonstrate that androstenedione binding is pH-dependent, whereas, in contrast, the D309N mutant retains its ability to bind to androstenedione across the entire pH range studied. Neither pH nor mutation perturbed the secondary structure or heme environment. The origin of the observed pH dependence was further narrowed to the protonation equilibria of Asp309 with a parallel set of spectroscopic studies using exemestane and anastrozole. Because exemestane interacts with Asp309 based on its co-crystal structure with the enzyme, its binding is pH-dependent. Aromatase binding to anastrozole is pH-independent, consistent with the hypothesis that this ligand exploits a distinct set of interactions in the active site. In summary, we assign the apparent pKa of 8.2 observed for androstenedione binding to the side chain of Asp309. To our knowledge, this work represents the first experimental assignment of a pKa value to a residue in a cytochrome P450. This value is in agreement with theoretical calculations (7.7–8.1) despite the reliance of the computational methods on the conformational snapshots provided by crystal structures. PMID:25425647

  9. Protein implicated in nonsyndromic mental retardation regulates protein kinase A (PKA) activity

    KAUST Repository

    Al-Tawashi, Azza

    2012-02-28

    Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacitytoformdendritesandsynapsesinculture. Atthebiochemical level,CC2D1Atransduces signals to the cyclic adenosine 3?,5?-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation. 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. The Stapled AKAP Disruptor Peptide STAD-2 Displays Antimalarial Activity through a PKA-Independent Mechanism.

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    Briana R Flaherty

    Full Text Available Drug resistance poses a significant threat to ongoing malaria control efforts. Coupled with lack of a malaria vaccine, there is an urgent need for the development of new antimalarials with novel mechanisms of action and low susceptibility to parasite drug resistance. Protein Kinase A (PKA has been implicated as a critical regulator of pathogenesis in malaria. Therefore, we sought to investigate the effects of disrupted PKA signaling as a possible strategy for inhibition of parasite replication. Host PKA activity is partly regulated by a class of proteins called A Kinase Anchoring Proteins (AKAPs, and interaction between HsPKA and AKAP can be inhibited by the stapled peptide Stapled AKAP Disruptor 2 (STAD-2. STAD-2 was tested for permeability to and activity against Plasmodium falciparum blood stage parasites in vitro. The compound was selectively permeable only to infected red blood cells (iRBC and demonstrated rapid antiplasmodial activity, possibly via iRBC lysis (IC50 ≈ 1 μM. STAD-2 localized within the parasite almost immediately post-treatment but showed no evidence of direct association with PKA, indicating that STAD-2 acts via a PKA-independent mechanism. Furosemide-insensitive parasite permeability pathways in the iRBC were largely responsible for uptake of STAD-2. Further, peptide import was highly specific to STAD-2 as evidenced by low permeability of control stapled peptides. Selective uptake and antiplasmodial activity of STAD-2 provides important groundwork for the development of stapled peptides as potential antimalarials. Such peptides may also offer an alternative strategy for studying protein-protein interactions critical to parasite development and pathogenesis.

  11. Activation of the cAMP-PKA pathway Antagonizes Metformin Suppression of Hepatic Glucose Production.

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    He, Ling; Chang, Evan; Peng, Jinghua; An, Hongying; McMillin, Sara M; Radovick, Sally; Stratakis, Constantine A; Wondisford, Fredric E

    2016-05-13

    Metformin is the most commonly prescribed oral anti-diabetic agent worldwide. Surprisingly, about 35% of diabetic patients either lack or have a delayed response to metformin treatment, and many patients become less responsive to metformin over time. It remains unknown how metformin resistance or insensitivity occurs. Recently, we found that therapeutic metformin concentrations suppressed glucose production in primary hepatocytes through AMPK; activation of the cAMP-PKA pathway negatively regulates AMPK activity by phosphorylating AMPKα subunit at Ser-485, which in turn reduces AMPK activity. In this study, we find that metformin failed to suppress glucose production in primary hepatocytes with constitutively activated PKA and did not improve hyperglycemia in mice with hyperglucagonemia. Expression of the AMPKα1(S485A) mutant, which is unable to be phosphorylated by PKA, increased both AMPKα activation and the suppression of glucose production in primary hepatocytes treated with metformin. Intriguingly, salicylate/aspirin prevents the phosphorylation of AMPKα at Ser-485, blocks cAMP-PKA negative regulation of AMPK, and improves metformin resistance. We propose that aspirin/salicylate may augment metformin's hepatic action to suppress glucose production.

  12. PKA and Epac activation mediates cAMP-induced vasorelaxation by increasing endothelial NO production.

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    García-Morales, Verónica; Cuíñas, Andrea; Elíes, Jacobo; Campos-Toimil, Manuel

    2014-03-01

    Vascular relaxation induced by 3',5'-cyclic adenosine monophosphate (cAMP) is both endothelium-dependent and endothelium-independent, although the underlying signaling pathways are not fully understood. Aiming to uncover potential mechanisms, we performed contraction-relaxation experiments on endothelium-denuded and intact rat aorta rings and measured NO levels in isolated human endothelial cells using single cell fluorescence imaging. The vasorelaxant effect of forskolin, an adenylyl cyclase activator, was decreased after selective inhibitor of protein kinase A (PKA), a cAMP-activated kinase, or L-NAME, an endothelial nitric oxide synthase (eNOS) inhibitor, only in intact aortic rings. Both selective activation of PKA with 6-Bnz-cAMP and exchange protein directly activated by cAMP (Epac) with 8-pCPT-2'-O-Me-cAMP significantly relaxed phenylephrine-induced contractions. The vasorelaxant effect of the Epac activator, but not that of the PKA activator, was reduced by endothelium removal. Forskolin, dibutyryl cAMP (a cAMP analogue), 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP increased NO levels in endothelial cells and the forskolin effect was significantly inhibited by inactivation of both Epac and PKA, and eNOS inhibition. Our results indicate that the endothelium-dependent component of forskolin/cAMP-induced vasorelaxation is partially mediated by an increase in endothelial NO release due to an enhanced eNOS activity through PKA and Epac activation in endothelial cells.

  13. Modulation of an active-site cysteine pKa allows PDI to act as a catalyst of both disulfide bond formation and isomerization.

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    Karala, Anna-Riikka; Lappi, Anna-Kaisa; Ruddock, Lloyd W

    2010-03-05

    Protein disulfide isomerase (PDI) plays a central role in disulfide bond formation in the endoplasmic reticulum. It is implicated both in disulfide bond formation and in disulfide bond reduction and isomerization. To be an efficient catalyst of all three reactions requires complex mechanisms. These include mechanisms to modulate the pK(a) values of the active-site cysteines of PDI. Here, we examined the role of arginine 120 in modulating the pK(a) values of these cysteines. We find that arginine 120 plays a significant role in modulating the pK(a) of the C-terminal active-site cysteine in the a domain of PDI and plays a role in determining the reactivity of the N-terminal active-site cysteine but not via direct modulation of its pK(a). Mutation of arginine 120 and the corresponding residue, arginine 461, in the a' domain severely reduces the ability of PDI to catalyze disulfide bond formation and reduction but enhances the ability to catalyze disulfide bond isomerization due to the formation of more stable PDI-substrate mixed disulfides. These results suggest that the modulation of pK(a) of the C-terminal active cysteine by the movement of the side chain of these arginine residues into the active-site locales has evolved to allow PDI to efficiently catalyze both oxidation and isomerization reactions.

  14. Ethanol Activation of PKA Mediates Single-Minded 2 Expression in Neuronal Cells.

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    Wang, Xiaolan; Yang, Zhihua; Sun, Yinan; Zhou, Hanjing; Chu, Guangpin; Zhang, Jing; Meng, Xianfang

    2015-12-01

    Prenatal ethanol exposure can cause extensive apoptotic neurodegeneration throughout the developing central nervous system (CNS), which results in cognitive deficits and memory decline. However, the underlying mechanisms need further study. Single-minded 2 (Sim2), a transcriptional repressor, is reportedly involved in diseases that impair learning and memory, such as Down syndrome (DS) and Alzheimer's disease. It is still unknown whether Sim2 is involved in regulating ethanol-mediated neuronal injury that might ultimately lead to neuronal dysfunction and subsequent learning and memory deficits. To study the effects of ethanol on Sim2 expression and neuronal injury, we used animal models and cell culture experiments. Our results indicated that in SH-SY5Y cells, ethanol exposure increased Sim2 expression and levels of cleaved caspase 3, which is a marker for cells undergoing apoptosis. Silencing Sim2 expression attenuated caspase 3 activation and cellular apoptosis. We also found that protein kinase A (PKA) activation induced Sim2 expression, as did ethanol. Inhibiting the PKA signaling pathway with H-89 decreased Sim2 expression and cleavage of caspase 3 that was induced by ethanol in vivo and in vitro. We further found that PKA regulated Sim2 expression at the transcriptional level. These results demonstrate that ethanol leads to increased Sim2 expression via the PKA pathway, ultimately resulting in apoptotic cell death.

  15. The spatio-temporal dynamics of PKA activity profile during mitosis and its correlation to chromosome segregation.

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    Vandame, Pauline; Spriet, Corentin; Trinel, Dave; Gelaude, Armance; Caillau, Katia; Bompard, Coralie; Biondi, Emanuele; Bodart, Jean-François

    2014-01-01

    The cyclic adenosine monophosphate dependent kinase protein (PKA) controls a variety of cellular processes including cell cycle regulation. Here, we took advantages of genetically encoded FRET-based biosensors, using an AKAR-derived biosensor to characterize PKA activity during mitosis in living HeLa cells using a single-cell approach. We measured PKA activity changes during mitosis. HeLa cells exhibit a substantial increase during mitosis, which ends with telophase. An AKAREV T>A inactive form of the biosensor and H89 inhibitor were used to ascertain for the specificity of the PKA activity measured. On a spatial point of view, high levels of activity near to chromosomal plate during metaphase and anaphase were detected. By using the PKA inhibitor H89, we assessed the role of PKA in the maintenance of a proper division phenotype. While this treatment in our hands did not impaired cell cycle progression in a drastic manner, inhibition of PKA leads to a dramatic increase in chromososme misalignement on the spindle during metaphase that could result in aneuploidies. Our study emphasizes the insights that can be gained with genetically encoded FRET-based biosensors, which enable to overcome the shortcomings of classical methologies and unveil in vivo PKA spatiotemporal profiles in HeLa cells.

  16. In Vivo FRET Imaging of Tumor Endothelial Cells Highlights a Role of Low PKA Activity in Vascular Hyperpermeability.

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    Yamauchi, Fumio; Kamioka, Yuji; Yano, Tetsuya; Matsuda, Michiyuki

    2016-09-15

    Vascular hyperpermeability is a pathological hallmark of cancer. Previous in vitro studies have elucidated roles of various signaling molecules in vascular hyperpermeability; however, the activities of such signaling molecules have not been examined in live tumor tissues for technical reasons. Here, by in vivo two-photon excitation microscopy with transgenic mice expressing biosensors based on Förster resonance energy transfer, we examined the activity of protein kinase A (PKA), which maintains endothelial barrier function. The level of PKA activity was significantly lower in the intratumoral endothelial cells than the subcutaneous endothelial cells. PKA activation with a cAMP analogue alleviated the tumor vascular hyperpermeability, suggesting that the low PKA activity in the endothelial cells may be responsible for the tumor-tissue hyperpermeability. Because the vascular endothelial growth factor (VEGF) receptor is a canonical inducer of vascular hyperpermeability and a molecular target of anticancer drugs, we examined the causality between VEGF receptor activity and the PKA activity. Motesanib, a kinase inhibitor for VEGF receptor, activated tumor endothelial PKA and reduced the vascular permeability in the tumor. Conversely, subcutaneous injection of VEGF decreased endothelial PKA activity and induced hyperpermeability of subcutaneous blood vessels. Notably, in cultured human umbilical vascular endothelial cells, VEGF activated PKA rather than decreasing its activity, highlighting the remarkable difference between its actions in vitro and in vivo These data suggested that the VEGF receptor signaling pathway increases vascular permeability, at least in part, by reducing endothelial PKA activity in the live tumor tissue. Cancer Res; 76(18); 5266-76. ©2016 AACR.

  17. Liberated PKA Catalytic Subunits Associate with the Membrane via Myristoylation to Preferentially Phosphorylate Membrane Substrates.

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    Tillo, Shane E; Xiong, Wei-Hong; Takahashi, Maho; Miao, Sheng; Andrade, Adriana L; Fortin, Dale A; Yang, Guang; Qin, Maozhen; Smoody, Barbara F; Stork, Philip J S; Zhong, Haining

    2017-04-18

    Protein kinase A (PKA) has diverse functions in neurons. At rest, the subcellular localization of PKA is controlled by A-kinase anchoring proteins (AKAPs). However, the dynamics of PKA upon activation remain poorly understood. Here, we report that elevation of cyclic AMP (cAMP) in neuronal dendrites causes a significant percentage of the PKA catalytic subunit (PKA-C) molecules to be released from the regulatory subunit (PKA-R). Liberated PKA-C becomes associated with the membrane via N-terminal myristoylation. This membrane association does not require the interaction between PKA-R and AKAPs. It slows the mobility of PKA-C and enriches kinase activity on the membrane. Membrane-residing PKA substrates are preferentially phosphorylated compared to cytosolic substrates. Finally, the myristoylation of PKA-C is critical for normal synaptic function and plasticity. We propose that activation-dependent association of PKA-C renders the membrane a unique PKA-signaling compartment. Constrained mobility of PKA-C may synergize with AKAP anchoring to determine specific PKA function in neurons. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Acute mechanical stretch promotes eNOS activation in venous endothelial cells mainly via PKA and Akt pathways.

    Directory of Open Access Journals (Sweden)

    Zhenqian Hu

    Full Text Available In the vasculature, physiological levels of nitric oxide (NO protect against various stressors, including mechanical stretch. While endothelial NO production in response to various stimuli has been studied extensively, the precise mechanism underlying stretch-induced NO production in venous endothelial cells remains incompletely understood. Using a model of continuous cellular stretch, we found that stretch promoted phosphorylation of endothelial NO synthase (eNOS at Ser¹¹⁷⁷, Ser⁶³³ and Ser⁶¹⁵ and NO production in human umbilical vein endothelial cells. Although stretch activated the kinases AMPKα, PKA, Akt, and ERK1/2, stretch-induced eNOS activation was only inhibited by kinase-specific inhibitors of PKA and PI3K/Akt, but not of AMPKα and Erk1/2. Similar results were obtained with knockdown by shRNAs targeting the PKA and Akt genes. Furthermore, inhibition of PKA preferentially attenuated eNOS activation in the early phase, while inhibition of the PI3K/Akt pathway reduced eNOS activation in the late phase, suggesting that the PKA and PI3K/Akt pathways play distinct roles in a time-dependent manner. Finally, we investigated the role of these pathways in stretch-induced endothelial exocytosis and leukocyte adhesion. Interestingly, we found that inhibition of the PI3K/Akt pathway increased stretch-induced Weibel-Palade body exocytosis and leukocyte adhesion, while inhibition of the PKA pathway had the opposite effects, suggesting that the exocytosis-promoting effect of PKA overwhelms the inhibitory effect of PKA-mediated NO production. Taken together, the results suggest that PKA and Akt are important regulators of eNOS activation in venous endothelial cells under mechanical stretch, while playing different roles in the regulation of stretch-induced endothelial exocytosis and leukocyte adhesion.

  19. Downregulation of transient K+ channels in dendrites of hippocampal CA1 pyramidal neurons by activation of PKA and PKC.

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    Hoffman, D A; Johnston, D

    1998-05-15

    We have reported recently a high density of transient A-type K+ channels located in the distal dendrites of CA1 hippocampal pyramidal neurons and shown that these channels shape EPSPs, limit the back-propagation of action potentials, and prevent dendritic action potential initiation (). Because of the importance of these channels in dendritic signal propagation, their modulation by protein kinases would be of significant interest. We investigated the effects of activators of cAMP-dependent protein kinase (PKA) and the Ca2+-dependent phospholipid-sensitive protein kinase (PKC) on K+ channels in cell-attached patches from the distal dendrites of hippocampal CA1 pyramidal neurons. Inclusion of the membrane-permeant PKA activators 8-bromo-cAMP (8-br-cAMP) or forskolin in the dendritic patch pipette resulted in a depolarizing shift in the activation curve for the transient channels of approximately 15 mV. Activation of PKC by either of two phorbol esters also resulted in a 15 mV depolarizing shift of the activation curve. Neither PKA nor PKC activation affected the sustained or slowly inactivating component of the total outward current. This downregulation of transient K+ channels in the distal dendrites may be responsible for some of the frequently reported increases in cell excitability found after PKA and PKC activation. In support of this hypothesis, we found that activation of either PKA or PKC significantly increased the amplitude of back-propagating action potentials in distal dendrites.

  20. Activation of PKA leads to mesenchymal-to-epithelial transition and loss of tumor-initiating ability.

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    Pattabiraman, Diwakar R; Bierie, Brian; Kober, Katharina Isabelle; Thiru, Prathapan; Krall, Jordan A; Zill, Christina; Reinhardt, Ferenc; Tam, Wai Leong; Weinberg, Robert A

    2016-03-04

    The epithelial-to-mesenchymal transition enables carcinoma cells to acquire malignancy-associated traits and the properties of tumor-initiating cells (TICs). TICs have emerged in recent years as important targets for cancer therapy, owing to their ability to drive clinical relapse and enable metastasis. Here, we propose a strategy to eliminate mesenchymal TICs by inducing their conversion to more epithelial counterparts that have lost tumor-initiating ability. We report that increases in intracellular levels of the second messenger, adenosine 3',5'-monophosphate, and the subsequent activation of protein kinase A (PKA) induce a mesenchymal-to-epithelial transition (MET) in mesenchymal human mammary epithelial cells. PKA activation triggers epigenetic reprogramming of TICs by the histone demethylase PHF2, which promotes their differentiation and loss of tumor-initiating ability. This study provides proof-of-principle for inducing an MET as differentiation therapy for TICs and uncovers a role for PKA in enforcing and maintaining the epithelial state.

  1. Evaluation of Thiol Raman Activities and pKa Values Using Internally Referenced Raman-Based pH Titration.

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    Suwandaratne, Nuwanthi; Hu, Juan; Siriwardana, Kumudu; Gadogbe, Manuel; Zhang, Dongmao

    2016-04-05

    Thiols, including organothiol and thiol-containing biomolecules, are among the most important classes of chemicals that are used broadly in organic synthesis, biological chemistry, and nanosciences. Thiol pKa values are key indicators of thiol reactivity and functionality. Reported herein is an internally referenced Raman-based pH titration method that enables reliable quantification of thiol pKa values for both mono- and dithiols in water. The degree of thiol ionization is monitored directly using the peak intensity of the S-H stretching feature in the 2600 cm(-1) region relative to an internal reference peak as a function of the titration solution's pH. The thiol pKa values and Raman activity relative to its internal reference were then determined by curve fitting the experimental data with equations derived on the basis of the Henderson-Hasselbalch equation. Using this Raman titration method, we determined for the first time the first and second thiol pKa values for 1,2-benzenedithiol in water. This Raman-based method is convenient to implement, and its underlying theory is easy to follow. It should therefore have broad application for thiol pKa determinations and verification.

  2. Sustained increase of PKA activity in the postcommissural putamen of dyskinetic monkeys.

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    Azkona, Garikoitz; Marcilla, Irene; López de Maturana, Rakel; Sousa, Amaya; Pérez-Navarro, Esther; Luquin, Maria-Rosario; Sanchez-Pernaute, Rosario

    2014-12-01

    Levodopa-induced dyskinesias (LID) are a frequent complication of Parkinson's disease pharmacotherapy that causes significant disability and narrows the therapeutic window. Pharmacological management of LID is challenging partly because the precise molecular mechanisms are not completely understood. Here, our aim was to determine molecular changes that could unveil targetable mechanisms underlying this drug complication. We examined the expression and downstream activity of dopamine receptors (DR) in the striatum of 1-methyl-4-phenyl-1,2,3,6 tetrahydropiridine (MPTP)-lesioned monkeys with and without L-DOPA treatment. Four monkeys were made dyskinetic and other four received a shorter course of L-DOPA and did not develop LID. Our results show that L-DOPA treatment induces an increase in DRD2 and DRD3 expression in the postcommissural putamen, but only DRD3 is correlated with the severity of LID. Dyskinetic monkeys show a hyperactivation of the canonical DRD1-signaling pathway, measured by an increased phosphorylation of protein kinase A (PKA) and its substrates, particularly DARPP32. In contrast, activation of the DRD2-signaling pathway, visible in the levels of Akt phosphorylated on Thr308 and GSK3β on Ser9, is associated with L-DOPA treatment, independently of the presence of dyskinesias. Our data clearly demonstrate that dyskinetic monkeys present a dysregulation of the DRD3 receptor and the DRD1 pathway with a sustained increase of PKA activity in the postcommissural putamen. Importantly, we found that all signaling changes related to long-term L-DOPA administration are exquisitely restricted to the postcommissural putamen, which may be related to the recurrent failure of pharmacological approaches.

  3. Presynaptic α7 nicotinic acetylcholine receptors enhance hippocampal mossy fiber glutamatergic transmission via PKA activation.

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    Cheng, Qing; Yakel, Jerrel L

    2014-01-01

    Nicotinic acetylcholine receptors (nAChRs) are expressed widely in the CNS, and mediate both synaptic and perisynaptic activities of endogenous cholinergic inputs and pharmacological actions of exogenous compounds (e.g., nicotine and choline). Behavioral studies indicate that nicotine improves such cognitive functions as learning and memory. However, the mechanism of nicotine's action on cognitive function remains elusive. We performed patch-clamp recordings from hippocampal CA3 pyramidal neurons to determine the effect of nicotine on mossy fiber glutamatergic synaptic transmission. We found that nicotine in combination with NS1738, an α7 nAChR-positive allosteric modulator, strongly potentiated the amplitude of evoked EPSCs (eEPSCs), and reduced the EPSC paired-pulse ratio. The action of nicotine and NS1738 was mimicked by PNU-282987 (an α7 nAChR agonist), and was absent in α7 nAChR knock-out mice. These data indicate that activation of α7 nAChRs was both necessary and sufficient to enhance the amplitude of eEPSCs. BAPTA applied postsynaptically failed to block the action of nicotine and NS1738, suggesting again a presynaptic action of the α7 nAChRs. We also observed α7 nAChR-mediated calcium rises at mossy fiber giant terminals, indicating the presence of functional α7 nAChRs at presynaptic terminals. Furthermore, the addition of PNU-282987 enhanced action potential-dependent calcium transient at these terminals. Last, the potentiating effect of PNU-282987 on eEPSCs was abolished by inhibition of protein kinase A (PKA). Our findings indicate that activation of α7 nAChRs at presynaptic sites, via a mechanism involving PKA, plays a critical role in enhancing synaptic efficiency of hippocampal mossy fiber transmission.

  4. Application of variable anti-connectivity index to active sites. Modelling pK(a) values of aliphatic monocarboxylic acids.

    Science.gov (United States)

    Sčavničar, A; Balaban, A T; Pompe, M

    2013-01-01

    A partial distance-weighted variable anti-connectivity topological index was introduced for modelling pK(a) values of 31 aliphatic carboxylic acids and haloalkyl-carboxylic acids. The partial distance-weighted variable anti-connectivity index showed superior modelling capabilities compared with the index calculated from the complete graph, because it is capable of accounting correctly for the intramolecular interactions of unconnected vertices to specific bond strengths (active site), thereby improving the RMS(CV) error by about 30% (0.221 pK(a) units).

  5. Improved long-term memory via enhancing cGMP-PKG signaling requires cAMP-PKA signaling.

    Science.gov (United States)

    Bollen, Eva; Puzzo, Daniela; Rutten, Kris; Privitera, Lucia; De Vry, Jochen; Vanmierlo, Tim; Kenis, Gunter; Palmeri, Agostino; D'Hooge, Rudi; Balschun, Detlef; Steinbusch, Harry M W; Blokland, Arjan; Prickaerts, Jos

    2014-10-01

    Memory consolidation is defined by the stabilization of a memory trace after acquisition, and consists of numerous molecular cascades that mediate synaptic plasticity. Commonly, a distinction is made between an early and a late consolidation phase, in which early refers to the first hours in which labile synaptic changes occur, whereas late consolidation relates to stable and long-lasting synaptic changes induced by de novo protein synthesis. How these phases are linked at a molecular level is not yet clear. Here we studied the interaction of the cyclic nucleotide-mediated pathways during the different phases of memory consolidation in rodents. In addition, the same pathways were studied in a model of neuronal plasticity, long-term potentiation (LTP). We demonstrated that cGMP/protein kinase G (PKG) signaling mediates early memory consolidation as well as early-phase LTP, whereas cAMP/protein kinase A (PKA) signaling mediates late consolidation and late-phase-like LTP. In addition, we show for the first time that early-phase cGMP/PKG signaling requires late-phase cAMP/PKA-signaling in both LTP and long-term memory formation.

  6. Feedback regulation between autophagy and PKA

    Science.gov (United States)

    Torres-Quiroz, Francisco; Filteau, Marie; Landry, Christian R

    2015-01-01

    Protein kinase A (PKA) controls diverse cellular processes and homeostasis in eukaryotic cells. Many processes and substrates of PKA have been described and among them are direct regulators of autophagy. The mechanisms of PKA regulation and how they relate to autophagy remain to be fully understood. We constructed a reporter of PKA activity in yeast to identify genes affecting PKA regulation. The assay systematically measures relative protein-protein interactions between the regulatory and catalytic subunits of the PKA complex in a systematic set of genetic backgrounds. The candidate PKA regulators we identified span multiple processes and molecular functions (autophagy, methionine biosynthesis, TORC signaling, protein acetylation, and DNA repair), which themselves include processes regulated by PKA. These observations suggest the presence of many feedback loops acting through this key regulator. Many of the candidate regulators include genes involved in autophagy, suggesting that not only does PKA regulate autophagy but that autophagy also sends signals back to PKA. PMID:26046386

  7. Feedback regulation between autophagy and PKA.

    Science.gov (United States)

    Torres-Quiroz, Francisco; Filteau, Marie; Landry, Christian R

    2015-01-01

    Protein kinase A (PKA) controls diverse cellular processes and homeostasis in eukaryotic cells. Many processes and substrates of PKA have been described and among them are direct regulators of autophagy. The mechanisms of PKA regulation and how they relate to autophagy remain to be fully understood. We constructed a reporter of PKA activity in yeast to identify genes affecting PKA regulation. The assay systematically measures relative protein-protein interactions between the regulatory and catalytic subunits of the PKA complex in a systematic set of genetic backgrounds. The candidate PKA regulators we identified span multiple processes and molecular functions (autophagy, methionine biosynthesis, TORC signaling, protein acetylation, and DNA repair), which themselves include processes regulated by PKA. These observations suggest the presence of many feedback loops acting through this key regulator. Many of the candidate regulators include genes involved in autophagy, suggesting that not only does PKA regulate autophagy but that autophagy also sends signals back to PKA.

  8. Angiotensin II counteracts the effects of cAMP/PKA on NHE3 activity and phosphorylation in proximal tubule cells.

    Science.gov (United States)

    Crajoinas, Renato O; Polidoro, Juliano Z; Carneiro de Morais, Carla P A; Castelo-Branco, Regiane C; Girardi, Adriana C C

    2016-11-01

    Binding of angiotensin II (ANG II) to the AT1 receptor (AT1R) in the proximal tubule stimulates Na(+)/H(+) exchanger isoform 3 (NHE3) activity through multiple signaling pathways. However, the effects of ANG II/AT1R-induced inihibitory G protein (Gi) activation and subsequent decrease in cAMP accumulation on NHE3 regulation are not well established. We therefore tested the hypothesis that ANG II reduces cAMP/PKA-mediated phosphorylation of NHE3 on serine 552 and, in doing so, stimulates NHE3 activity. Under basal conditions, ANG II stimulated NHE3 activity but did not affect PKA-mediated NHE3 phosphorylation at serine 552 in opossum kidney (OKP) cells. However, in the presence of the cAMP-elevating agent forskolin (FSK), ANG II blocked FSK-induced NHE3 inhibition, reduced intracellular cAMP concentrations, lowered PKA activity, and prevented the FSK-mediated increase in NHE3 serine 552 phosphorylation. All effects of ANG II were blocked by pretreating OKP cells with the AT1R antagonist losartan, highlighting the contribution of the AT1R/Gi pathway in ANG II-mediated NHE3 upregulation under cAMP-elevating conditions. Accordingly, Gi inhibition by pertussis toxin treatment decreased NHE3 activity both in vitro and in vivo and, more importantly, prevented the stimulatory effect of ANG II on NHE3 activity in rat proximal tubules. Collectively, our results suggest that ANG II counteracts the effects of cAMP/PKA on NHE3 phosphorylation and inhibition by activating the AT1R/Gi pathway. Moreover, these findings support the notion that NHE3 dephosphorylation at serine 552 may represent a key event in the regulation of renal proximal tubule sodium handling by ANG II in the presence of natriuretic hormones that promote cAMP accumulation and transporter phosphorylation.

  9. Age-related deficits in synaptic plasticity rescued by activating PKA or PKC in sensory neurons of Aplysia californica

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    Andrew T Kempsell

    2015-09-01

    Full Text Available Brain aging is associated with declines in synaptic function that contribute to memory loss, including reduced postsynaptic response to neurotransmitters and decreased neuronal excitability. To understand how aging affects memory in a simple neural circuit, we studied neuronal proxies of memory for sensitization in mature versus advanced age Aplysia. Glutamate- (L-Glu- evoked excitatory currents were facilitated by the neuromodulator serotonin (5-HT in sensory neurons (SN isolated from mature but not aged animals. Activation of PKA and PKC signaling rescued facilitation of L-Glu currents in aged SN. Similarly, PKA and PKC activators restored increased excitability in aged tail SN. These results suggest that altered synaptic plasticity during aging involves defects in second messenger systems

  10. cAMP-Induced Histones H3 Dephosphorylation Is Independent of PKA and MAP Kinase Activations and Correlates With mTOR Inactivation.

    Science.gov (United States)

    Rodriguez, Pedro; Rojas, Juan

    2016-03-01

    cAMP is a second messenger well documented to be involved in the phosphorylation of PKA, MAP kinase, and histone H3 (H3). Early, we reported that cAMP also induced H3 dephosphorylation in a variety of proliferating cell lines. Herein, it is shown that cAMP elicits a biphasic H3 dephosphorylation independent of PKA activation in cycling cells. H89, a potent inhibitor of PKA catalytic sub-unite, could not abolish this effect. Additionally, H89 induces a rapid and biphasic H3 serine 10 dephosphorylation, while a decline in the basal phosphorylation of CREB/ATF-1 is observed. Rp-cAMPS, an analog of cAMP and specific inhibitor of PKA, is unable to suppress cAMP-mediated H3 dephosphorylation, whereas Rp-cAMPS effectively blocks CREB/ATF-1 hyper-phosphorylation by cAMP and its inducers. Interestingly, cAMP exerts a rapid and profound H3 dephosphorylation at much lower concentration (50-fold lower, 0.125 mM) than the concentration required for maximal CREB/ATF-1 phosphorylation (5 mM). Much higher cAMP concentration is required to fully induce CREB/ATF-1 gain in phosphate (5 mM), which correlates with the inhibition of H3 dephosphorylation. Also, the dephosphorylation of H3 does not overlap at onset of MAP kinase phosphorylation pathways, p38 and ERK. Surprisingly, rapamycin (an mTOR inhibitor), cAMP, and its natural inducer isoproterenol, elicit identical dephosphorylation kinetics on both S6K1 ribosomal kinase (a downstream mTOR target) and H3. Finally, cAMP-induced H3 dephosphorylation is PP1/2-dependent. The results suggest that a pathway, requiring much lower cAMP concentration to that required for CREB/ATF-1 hyper-phosphorylation, is responsible for histone H3 dephosphorylation and may be linked to mTOR down regulation.

  11. Activation of the cAMP/PKA pathway induces UT-A1 urea transporter monoubiquitination and targets it for lysosomal degradation.

    Science.gov (United States)

    Su, Hua; Chen, Minguang; Sands, Jeff M; Chen, Guangping

    2013-12-15

    Regulation of urea transporter UT-A1 in the kidney is important for the urinary concentrating mechanism. We previously reported that activation of the cAMP/PKA pathway by forskolin (FSK) leads to UT-A1 ubiquitination, endocytosis, and degradation. In this study, we discovered that FSK-induced UT-A1 ubiquitination is monoubiquitination as judged by immunoblotting with specific ubiquitin antibodies to the different linkages of the ubiquitin chain. UT-A1 monoubiquitination induced by FSK was processed mainly on the cell plasma membrane. Monoubiquitination facilitates UT-A1 endocytosis, and internalized UT-A1 is accumulated in the early endosome. Inhibition of ubiquitination by E1 ubiquitin-activating enzyme inhibitor PYR-41 significantly reduced FSK-induced UT-A1 endocytosis and degradation. Interestingly, FSK-stimulated UT-A1 degradation occurs through a lysosomal protein degradation system. We further found that the PKA phosphorylation sites of UT-A1 at Ser486 and Ser499 are required for FSK-induced UT-A1 monoubiquitination. The physiological significance was confirmed using rat kidney inner medullary collecting duct suspensions, which showed that vasopressin treatment promotes UT-A1 ubiquitination. We conclude that unlike under basal conditions in which UT-A1 is subject to polyubiquitination and proteasome-mediated protein degradation, activation of UT-A1 by FSK induces UT-A1 monoubiquitination and protein lysosomal degradation.

  12. MARK/Par1 Kinase Is Activated Downstream of NMDA Receptors through a PKA-Dependent Mechanism.

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    Laura P Bernard

    Full Text Available The Par1 kinases, also known as microtubule affinity-regulating kinases (MARKs, are important for the establishment of cell polarity from worms to mammals. Dysregulation of these kinases has been implicated in autism, Alzheimer's disease and cancer. Despite their important function in health and disease, it has been unclear how the activity of MARK/Par1 is regulated by signals from cell surface receptors. Here we show that MARK/Par1 is activated downstream of NMDA receptors in primary hippocampal neurons. Further, we show that this activation is dependent on protein kinase A (PKA, through the phosphorylation of Ser431 of Par4/LKB1, the major upstream kinase of MARK/Par1. Together, our data reveal a novel mechanism by which MARK/Par1 is activated at the neuronal synapse.

  13. Melanocortin-induced PKA activation inhibits AMPK activity via ERK-1/2 and LKB-1 in hypothalamic GT1-7 cells.

    Science.gov (United States)

    Damm, Ellen; Buech, Thomas R H; Gudermann, Thomas; Breit, Andreas

    2012-04-01

    α-Melanocyte-stimulating hormone (α-MSH)-induced activation of the melanocortin-4 receptor in hypothalamic neurons increases energy expenditure and inhibits food intake. Active hypothalamic AMP-activated protein kinase (AMPK) has recently been reported to enhance food intake, and in vivo experiments suggested that intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of AMPK activity. However, it is not clear whether α-MSH affects AMPK via direct intracellular signaling cascades or if the release of paracrine factors is involved. Here, we used a murine, hypothalamic cell line (GT1-7 cells) and monitored AMPK phosphorylation at Thr(172), which has been suggested to increase AMPK activity. We found that α-MSH dephosphorylated AMPK at Thr(172) and consequently decreased phosphorylation of the established AMPK substrate acetyl-coenzyme A-carboxylase at Ser(79). Inhibitory effects of α-MSH on AMPK were blocked by specific inhibitors of protein kinase A (PKA) or ERK-1/2, pointing to an important role of both kinases in this process. Because α-MSH-induced activation of ERK-1/2 was blunted by PKA inhibitors, we propose that ERK-1/2 serves as a link between PKA and AMPK in GT1-7 cells. Furthermore, down-regulation of liver kinase B-1, but not inhibition of calcium-calmodulin-dependent kinase kinase-β or TGFβ-activated kinase-1 decreased basal phosphorylation of AMPK and its dephosphorylation induced by α-MSH. Thus, we propose that α-MSH inhibits AMPK activity via a linear pathway, including PKA, ERK-1/2, and liver kinase B-1 in GT1-7 cells. Given the importance of the melanocortin system in the formation of adipositas, detailed knowledge about this pathway might help to develop drugs targeting obesity.

  14. Regulation of Hxt3 and Hxt7 turnover converges on the Vid30 complex and requires inactivation of the Ras/cAMP/PKA pathway in Saccharomyces cerevisiae.

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    Chris Snowdon

    Full Text Available Eukaryotic cells adjust their intracellular protein complement as a mechanism to adapt to changing environmental signals. In Saccharomyces cerevisiae the hexose transporters Hxt3 and Hxt7 are expressed and function on the plasma membrane in high and low glucose abundance, respectively. By contrast, Hxt3 is endocytosed and degraded in the vacuole when cells are starved of glucose and Hxt7 in response to rapamycin treatment or when nitrogen is limiting. Yeast uses several signaling pathways, including the TORC1 and Ras/cAMP/Protein Kinase A (PKA pathways, to adapt to nutrient changes in the environment. The multi-protein Vid30 complex (Vid30c, an E3 ubiquitin ligase required for the degradation of FBPase, assists in this adaptation process in a mechanism that is poorly understood. Here we show the endocytosis and the subsequent degradation of both Hxt3 and Hxt7, in response to different nutrient signals, is dependent on components of the Vid30c. Additionally, we define the signaling events required for the turnover of Hxt3 and Hxt7 by showing that Hxt3 turnover requires Ras2 and PKA inactivation, whereas Hxt7 turnover requires TORC1 and Ras2 inactivation. Further investigation led us to identify Rim15, a kinase that is inhibited by both the TORC1 and Ras/cAMP/PKA pathways, as a key downstream effector in signaling both turnover events. Finally, we show that the turnover of both Hxt3 and Hxt7 is dependent on the essential E3 ubiquitin ligase, Rsp5, indicating that the role of the Vid30c might be indirect of Hxt ubiquitylation.

  15. Reduced lipolysis response to adipose afferent reflex involved in impaired activation of adrenoceptor-cAMP-PKA-hormone sensitive lipase pathway in obesity

    Science.gov (United States)

    Ding, Lei; Zhang, Feng; Zhao, Ming-Xia; Ren, Xing-Sheng; Chen, Qi; Li, Yue-Hua; Kang, Yu-Ming; Zhu, Guo-Qing

    2016-01-01

    Chemical stimulation of white adipose tissue (WAT) causes adipose afferent reflex (AAR) and sympathetic activation. This study is to investigate the effects of AAR on lipolysis and the mechanisms of attenuated lipolysis response to enhanced AAR in obesity. Obesity was caused by high-fat diet for 12 weeks in rats. AAR was induced by injection of capsaicin into inguinal WAT or electrical stimulation of epididymal WAT afferent nerve. AAR caused sympathetic activation, which was enhanced in obesity rats. AAR increased cAMP levels and PKA activity, promoted hormone sensitive lipase (HSL) and perilipin phosphorylation, and increased lipolysis in WAT, which were attenuated in obesity rats. PKA activity, cAMP, perilipin and β-adrenoceptor levels were reduced, while HSL was upregulated in adipocytes from obesity rats. In primary adipocytes, isoproterenol increased cAMP levels and PKA activity, promoted HSL and perilipin phosphorylation, and increased lipolysis, which were attenuated in obesity rats. The attenuated effects of isoproterenol in adipocytes from obesity rats were prevented by a cAMP analogue dbcAMP. The results indicate that reduced lipolysis response to enhanced AAR in obesity is attributed to the impaired activation of β-adrenoceptor-cAMP-PKA-HSL pathway. Increased cAMP level in adipocytes rectifies the attenuated lipolysis in obesity. PMID:27694818

  16. Reduced lipolysis response to adipose afferent reflex involved in impaired activation of adrenoceptor-cAMP-PKA-hormone sensitive lipase pathway in obesity.

    Science.gov (United States)

    Ding, Lei; Zhang, Feng; Zhao, Ming-Xia; Ren, Xing-Sheng; Chen, Qi; Li, Yue-Hua; Kang, Yu-Ming; Zhu, Guo-Qing

    2016-10-03

    Chemical stimulation of white adipose tissue (WAT) causes adipose afferent reflex (AAR) and sympathetic activation. This study is to investigate the effects of AAR on lipolysis and the mechanisms of attenuated lipolysis response to enhanced AAR in obesity. Obesity was caused by high-fat diet for 12 weeks in rats. AAR was induced by injection of capsaicin into inguinal WAT or electrical stimulation of epididymal WAT afferent nerve. AAR caused sympathetic activation, which was enhanced in obesity rats. AAR increased cAMP levels and PKA activity, promoted hormone sensitive lipase (HSL) and perilipin phosphorylation, and increased lipolysis in WAT, which were attenuated in obesity rats. PKA activity, cAMP, perilipin and β-adrenoceptor levels were reduced, while HSL was upregulated in adipocytes from obesity rats. In primary adipocytes, isoproterenol increased cAMP levels and PKA activity, promoted HSL and perilipin phosphorylation, and increased lipolysis, which were attenuated in obesity rats. The attenuated effects of isoproterenol in adipocytes from obesity rats were prevented by a cAMP analogue dbcAMP. The results indicate that reduced lipolysis response to enhanced AAR in obesity is attributed to the impaired activation of β-adrenoceptor-cAMP-PKA-HSL pathway. Increased cAMP level in adipocytes rectifies the attenuated lipolysis in obesity.

  17. Superior Long-Term Synaptic Memory Induced by Combining Dual Pharmacological Activation of PKA and ERK with an Enhanced Training Protocol

    Science.gov (United States)

    Liu, Rong-Yu; Neveu, Curtis; Smolen, Paul; Cleary, Leonard J.; Byrne, John H.

    2017-01-01

    Developing treatment strategies to enhance memory is an important goal of neuroscience research. Activation of multiple biochemical signaling cascades, such as the protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) pathways, is necessary to induce long-term synaptic facilitation (LTF), a correlate of long-term memory (LTM).…

  18. Identification of Ftr1 and Zrt1 as iron and zinc micronutrient transceptors for activation of the PKA pathway in Saccharomyces cerevisiae

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    Joep Schothort

    2017-03-01

    Full Text Available Multiple types of nutrient transceptors, membrane proteins that combine a transporter and receptor function, have now been established in a variety of organisms. However, so far all established transceptors utilize one of the macronutrients, glucose, amino acids, ammonium, nitrate, phosphate or sulfate, as substrate. This is also true for the Saccharomyces cerevisiae transceptors mediating activation of the PKA pathway upon re-addition of a macronutrient to glucose-repressed cells starved for that nutrient, re-establishing a fermentable growth medium. We now show that the yeast high-affinity iron transporter Ftr1 and high-affinity zinc transporter Zrt1 function as transceptors for the micronutrients iron and zinc. We show that replenishment of iron to iron-starved cells or zinc to zinc-starved cells triggers within 1-2 minutes a rapid surge in trehalase activity, a well-established PKA target. The activation with iron is dependent on Ftr1 and with zinc on Zrt1, and we show that it is independent of intracellular iron and zinc levels. Similar to the transceptors for macronutrients, Ftr1 and Zrt1 are strongly induced upon iron and zinc starvation, respectively, and they are rapidly downregulated by substrate-induced endocytosis. Our results suggest that transceptor-mediated signaling to the PKA pathway may occur in all cases where glucose-repressed yeast cells have been starved first for an essential nutrient, causing arrest of growth and low activity of the PKA pathway, and subsequently replenished with the lacking nutrient to re-establish a fermentable growth medium. The broadness of the phenomenon also makes it likely that nutrient transceptors use a common mechanism for signaling to the PKA pathway.

  19. PKA distributions: Contributions from transmutation products and from radioactive decay

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    M.R. Gilbert

    2016-12-01

    Full Text Available The neutrons generated in fusion plasmas interact with materials via nuclear reactions. The resulting transmutations and atomic displacements have life-limiting consequences for fusion reactor components. A detailed understanding of the production, evolution and material consequences of the damage created by cascades of atomic displacements requires, as a vital primary input, a complete description of the energy-spectrum of initial (prompt atomic displacement events (the primary knock on atoms or PKAs produced by direct neutron nuclear interactions. There is also the possibility that the radionuclides produced under transmutation will create further PKAs as they decay, and so the rate of these must also be quantified. This paper presents the latest results from the analysis of PKA spectra under neutron irradiation, focussing particularly on the variation in PKA distributions due to changes in composition under transmutation, but also on the PKA contributions from radioactive decay of materials that become activated under irradiation.

  20. Toxicity of Cry1A toxins from Bacillus thuringiensis to CF1 cells does not involve activation of adenylate cyclase/PKA signaling pathway.

    Science.gov (United States)

    Portugal, Leivi; Muñóz-Garay, Carlos; Martínez de Castro, Diana L; Soberón, Mario; Bravo, Alejandra

    2017-01-01

    Bacillus thuringiensis (Bt) bacteria produce Cry toxins that are able to kill insect pests. Different models explaining the mode of action of these toxins have been proposed. The pore formation model proposes that the toxin creates pores in the membrane of the larval midgut cells after interaction with different receptors such as cadherin, aminopeptidase N and alkaline phosphatase and that this pore formation activity is responsible for the toxicity of these proteins. The alternative model proposes that interaction with cadherin receptor triggers an intracellular cascade response involving protein G, adenylate cyclase (AC) and protein kinase A (PKA). In addition, it was shown that Cry toxins induce a defense response in the larvae involving the activation of mitogen-activated kinases such as MAPK p38 in different insect orders. Here we analyzed the mechanism of action of Cry1Ab and Cry1Ac toxins and a collection of mutants from these toxins in the insect cell line CF1 from Choristoneura fumiferana, that is naturally sensitive to these toxins. Our results show that both toxins induced permeability of K(+) ions into the cells. The initial response after intoxication with Cry1Ab and Cry1Ac toxins involves the activation of a defense response that involves the phosphorylation of MAPK p38. Analysis of activation of PKA and AC activities indicated that the signal transduction involving PKA, AC and cAMP was not activated during Cry1Ab or Cry1Ac intoxication. In contrast we show that Cry1Ab and Cry1Ac activate apoptosis. These data indicate that Cry toxins can induce an apoptotic death response not related with AC/PKA activation. Since Cry1Ab and Cry1Ac toxins affected K(+) ion permeability into the cells, and that mutant toxins affected in pore formation are not toxic to CF1, we propose that pore formation activity of the toxins is responsible of triggering cell death response in CF1cells.

  1. Regulation of SGLT expression and localization through Epac/PKA-dependent caveolin-1 and F-actin activation in renal proximal tubule cells.

    Science.gov (United States)

    Lee, Yu Jin; Kim, Mi Ok; Ryu, Jung Min; Han, Ho Jae

    2012-04-01

    This study demonstrated that exchange proteins directly activated by cAMP (Epac) and protein kinase A (PKA) by 8-bromo (8-Br)-adenosine 3',5'-cyclic monophosphate (cAMP) stimulated [(14)C]-α-methyl-D-glucopyranoside (α-MG) uptake through increased sodium-glucose cotransporters (SGLTs) expression and translocation to lipid rafts in renal proximal tubule cells (PTCs). In PTCs, SGLTs were colocalized with lipid raft caveolin-1 (cav-1), disrupted by methyl-β-cyclodextrin (MβCD). Selective activators of Epac or PKA, 8-Br-cAMP, and forskolin stimulated expressions of SGLTs and α-MG uptake in PTCs. In addition, 8-Br-cAMP-induced PKA and Epac activation increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB), which were involved in expressions of SGLTs. Furthermore, 8-Br-cAMP stimulated SGLTs translocation to lipid rafts via filamentous actin (F-actin) organization, which was blocked by cytochalasin D. In addition, cav-1 and SGLTs stimulated by 8-Br-cAMP were detected in lipid rafts, which were blocked by cytochalasin D. Furthermore, 8-Br-cAMP-induced SGLTs translocation and α-MG uptake were attenuated by inhibition of cav-1 activation with cav-1 small interfering RNA (siRNA) and inhibition of F-actin organization with TRIO and F-actin binding protein (TRIOBP). In conclusion, 8-Br-cAMP stimulated α-MG uptake via Epac and PKA-dependent SGLTs expression and trafficking through cav-1 and F-actin in PTCs.

  2. Ganoderma atrum polysaccharide evokes antitumor activity via cAMP-PKA mediated apoptotic pathway and down-regulation of Ca(2+)/PKC signal pathway.

    Science.gov (United States)

    Zhang, Shenshen; Nie, Shaoping; Huang, Danfei; Huang, Jianqin; Feng, Yanling; Xie, Mingyong

    2014-06-01

    Ganoderma atrum polysaccharide (PSG-1) has been commonly suggested as a candidate for prevention and therapy of cancer. We investigated the antitumor effect and the underlying molecular mechanisms of PSG-1. The results showed that PSG-1 inhibited tumor growth and resulted in tumor cell apoptosis in vivo. Here, the data revealed that PSG-1 caused a markedly increase in cAMP and PKA activities, rather than cGMP and PKC. Moreover, the treatment of PSG-1 induced a dramatic increase in the protein level of PKA. In contrast, the expression of PKC and intracellular [Ca(2+)]i were inhibited. Our study also revealed that treatment with PSG-1 increased the spleen and thymus weights, lymphocyte proliferation and macrophage phagocytic activity in tumor-bearing mice. Taken together, we conclude that PSG-1 could inhibit the tumor growth, possibly in part by enhancing the induction of apoptosis through cAMP-PKA signaling pathway and down-regulation of Ca(2+)/PKC signal pathway, activating host immune function in S180-bearing mice.

  3. Tonic 5nM DA stabilizes neuronal output by enabling bidirectional activity-dependent regulation of the hyperpolarization activated current via PKA and calcineurin.

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    Wulf-Dieter C Krenz

    Full Text Available Volume transmission results in phasic and tonic modulatory signals. The actions of tonic dopamine (DA at type 1 DA receptors (D1Rs are largely undefined. Here we show that tonic 5nM DA acts at D1Rs to stabilize neuronal output over minutes by enabling activity-dependent regulation of the hyperpolarization activated current (I h. In the presence but not absence of 5nM DA, I h maximal conductance (G max was adjusted according to changes in slow wave activity in order to maintain spike timing. Our study on the lateral pyloric neuron (LP, which undergoes rhythmic oscillations in membrane potential with depolarized plateaus, demonstrated that incremental, bi-directional changes in plateau duration produced corresponding alterations in LP I hG max when preparations were superfused with saline containing 5nM DA. However, when preparations were superfused with saline alone there was no linear correlation between LP I hGmax and duty cycle. Thus, tonic nM DA modulated the capacity for activity to modulate LP I h G max; this exemplifies metamodulation (modulation of modulation. Pretreatment with the Ca2+-chelator, BAPTA, or the specific PKA inhibitor, PKI, prevented all changes in LP I h in 5nM DA. Calcineurin inhibitors blocked activity-dependent changes enabled by DA and revealed a PKA-mediated, activity-independent enhancement of LP I hG max. These data suggested that tonic 5nM DA produced two simultaneous, PKA-dependent effects: a direct increase in LP I h G max and a priming event that permitted calcineurin regulation of LP I h. The latter produced graded reductions in LP I hG max with increasing duty cycles. We also demonstrated that this metamodulation preserved the timing of LP's first spike when network output was perturbed with bath-applied 4AP. In sum, 5nM DA permits slow wave activity to provide feedback that maintains spike timing, suggesting that one function of low-level, tonic modulation is to stabilize specific features of a dynamic output.

  4. Tonic 5nM DA stabilizes neuronal output by enabling bidirectional activity-dependent regulation of the hyperpolarization activated current via PKA and calcineurin.

    Science.gov (United States)

    Krenz, Wulf-Dieter C; Rodgers, Edmund W; Baro, Deborah J

    2015-01-01

    Volume transmission results in phasic and tonic modulatory signals. The actions of tonic dopamine (DA) at type 1 DA receptors (D1Rs) are largely undefined. Here we show that tonic 5nM DA acts at D1Rs to stabilize neuronal output over minutes by enabling activity-dependent regulation of the hyperpolarization activated current (I h). In the presence but not absence of 5nM DA, I h maximal conductance (G max) was adjusted according to changes in slow wave activity in order to maintain spike timing. Our study on the lateral pyloric neuron (LP), which undergoes rhythmic oscillations in membrane potential with depolarized plateaus, demonstrated that incremental, bi-directional changes in plateau duration produced corresponding alterations in LP I hG max when preparations were superfused with saline containing 5nM DA. However, when preparations were superfused with saline alone there was no linear correlation between LP I hGmax and duty cycle. Thus, tonic nM DA modulated the capacity for activity to modulate LP I h G max; this exemplifies metamodulation (modulation of modulation). Pretreatment with the Ca2+-chelator, BAPTA, or the specific PKA inhibitor, PKI, prevented all changes in LP I h in 5nM DA. Calcineurin inhibitors blocked activity-dependent changes enabled by DA and revealed a PKA-mediated, activity-independent enhancement of LP I hG max. These data suggested that tonic 5nM DA produced two simultaneous, PKA-dependent effects: a direct increase in LP I h G max and a priming event that permitted calcineurin regulation of LP I h. The latter produced graded reductions in LP I hG max with increasing duty cycles. We also demonstrated that this metamodulation preserved the timing of LP's first spike when network output was perturbed with bath-applied 4AP. In sum, 5nM DA permits slow wave activity to provide feedback that maintains spike timing, suggesting that one function of low-level, tonic modulation is to stabilize specific features of a dynamic output.

  5. PKA spectrum file

    Energy Technology Data Exchange (ETDEWEB)

    Kawai, M. [Toshiba Corp., Kawasaki, Kanagawa (Japan). Nuclear Engineering Lab.

    1997-03-01

    In the Japanese Nuclear Data Committee, the PKA/KERMA file containing PKA spectra, KERMA factors and DPA cross sections in the energy range between 10{sup -5} eV and 50 MeV is being prepared from the evaluated nuclear data. The processing code ESPERANT was developed to calculate quantities of PKA, KERMA and DPA from evaluated nuclear data for medium and heavy elements by using the effective single particle emission approximation (ESPEA). For light elements, the PKA spectra are evaluated by the SCINFUL/DDX and EXIFON codes, simultaneously with other neutron cross sections. The DPA cross sections due to charged particle emitted from light elements are evaluated for high neutron energy above 20 MeV. (author)

  6. PKA activity is essential for relieving the suppression of hyphal growth and appressorium formation by MoSfl1 in Magnaporthe oryzae.

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    Yang Li

    2017-08-01

    Full Text Available In the rice blast fungus Magnaporthe oryzae, the cAMP-PKA pathway regulates surface recognition, appressorium turgor generation, and invasive growth. However, deletion of CPKA failed to block appressorium formation and responses to exogenous cAMP. In this study, we generated and characterized the cpk2 and cpkA cpk2 mutants and spontaneous suppressors of cpkA cpk2 in M. oryzae. Our results demonstrate that CPKA and CPK2 have specific and overlapping functions, and PKA activity is essential for appressorium formation and plant infection. Unlike the single mutants, the cpkA cpk2 mutant was significantly reduced in growth and rarely produced conidia. It failed to form appressoria although the intracellular cAMP level and phosphorylation of Pmk1 MAP kinase were increased. The double mutant also was defective in plant penetration and Mps1 activation. Interestingly, it often produced fast-growing spontaneous suppressors that formed appressoria but were still non-pathogenic. Two suppressor strains of cpkA cpk2 had deletion and insertion mutations in the MoSFL1 transcription factor gene. Deletion of MoSFL1 or its C-terminal 93-aa (MoSFL1ΔCT was confirmed to suppress the defects of cpkA cpk2 in hyphal growth but not appressorium formation or pathogenesis. We also isolated 30 spontaneous suppressors of the cpkA cpk2 mutant in Fusarium graminearum and identified mutations in 29 of them in FgSFL1. Affinity purification and co-IP assays showed that this C-terminal region of MoSfl1 was essential for its interaction with the conserved Cyc8-Tup1 transcriptional co-repressor, which was reduced by cAMP treatment. Furthermore, the S211D mutation at the conserved PKA-phosphorylation site in MoSFL1 partially suppressed the defects of cpkA cpk2. Overall, our results indicate that PKA activity is essential for appressorium formation and proper activation of Pmk1 or Mps1 in M. oryzae, and phosphorylation of MoSfl1 by PKA relieves its interaction with the Cyc8-Tup1 co

  7. Pycnogenol supplementation promotes lipolysis via activation of cAMP-dependent PKA in ob/ob mice and primary-cultured adipocytes.

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    Ho, Jin-Nyoung; Kim, Ok-Kyung; Nam, Da-Eun; Jun, Woojin; Lee, Jeongmin

    2014-01-01

    This study investigated the PKA-dependent inhibitory effect of pycnogenol (Pyc) on lipolysis using ob/ob mice and primary mouse adipocytes. Supplementation of Pyc at 30 mg/kg significantly reduced body weight gain and visceral fat mass. The serum and hepatic triglyceride (TG) and total cholesterol (TC) levels were reduced by Pyc supplementation, and high density lipoprotein (HDL)-cholesterol level significantly increased. In addition, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) mRNA levels increased with Pyc supplementation in adipose tissue of ob/ob mice. The treatment of primary cultured adipocytes with Pyc at 100 μg/mL significantly increased glycerol release, cAMP level by reduction of phosphodiestersae-3B (PDE3B), and HSL levels, but decreased protein levels of perilipin A and fatty acid synthetase (FAS). The PKA inhibitor (H89) clearly blocked the cellular levels of perilipin A and HSL, suggesting that Pyc promotes lipolysis of adipocytes through activation of cAMP-dependent PKA, resulting in induction of HSL and reduction of perilipin A. Therefore, this study may elucidate the possible mechanism of Pyc, which is a candidate for weight loss through stimulation of lipolysis.

  8. Transcriptional activation of peroxisome proliferator-activated receptor-{gamma} requires activation of both protein kinase A and Akt during adipocyte differentiation

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    Kim, Sang-pil [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of); Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of); Chung, Sung Woon [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of); Hong, Ki Whan; Kim, Chi Dae [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of); Bae, Sun Sik, E-mail: sunsik@pusan.ac.kr [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of)

    2010-08-13

    Research highlights: {yields} Elevated cAMP activates both PKA and Epac. {yields} PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. {yields} Akt modulates PPAR-{gamma} transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-{gamma} is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-{gamma}. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-{gamma} was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-{gamma} transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-{gamma} transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-{gamma}, suggesting post-translational activation of PPAR-{gamma} might be critical step for adipogenic gene expression.

  9. Ribosomal protein S6 phosphorylation is controlled by TOR and modulated by PKA in Candida albicans.

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    Chowdhury, Tahmeena; Köhler, Julia R

    2015-10-01

    TOR and PKA signaling pathways control eukaryotic cell growth and proliferation. TOR activity in model fungi, such as Saccharomyces cerevisiae, responds principally to nutrients, e.g., nitrogen and phosphate sources, which are incorporated into the growing cell mass; PKA signaling responds to the availability of the cells' major energy source, glucose. In the fungal commensal and pathogen, Candida albicans, little is known of how these pathways interact. Here, the signal from phosphorylated ribosomal protein S6 (P-S6) was defined as a surrogate marker for TOR-dependent anabolic activity in C. albicans. Nutritional, pharmacologic and genetic modulation of TOR activity elicited corresponding changes in P-S6 levels. The P-S6 signal corresponded to translational activity of a GFP reporter protein. Contributions of four PKA pathway components to anabolic activation were then examined. In high glucose concentrations, only Tpk2 was required to upregulate P-S6 to physiologic levels, whereas all four tested components were required to downregulate P-S6 in low glucose. TOR was epistatic to PKA components with respect to P-S6. In many host niches inhabited by C. albicans, glucose is scarce, with protein being available as a nitrogen source. We speculate that PKA may modulate TOR-dependent cell growth to a rate sustainable by available energy sources, when monomers of anabolic processes, such as amino acids, are abundant.

  10. Caffeine induces beneficial changes in PKA signaling and JNK and ERK activities in the striatum and cortex of Alzheimer's transgenic mice.

    Science.gov (United States)

    Zeitlin, Ross; Patel, Sagar; Burgess, Sarah; Arendash, Gary W; Echeverria, Valentina

    2011-10-12

    Caffeine intake has been associated with a lower incidence of Alzheimer's disease (AD) in humans. In AD mouse models, caffeine significantly decreases senile plaques and amyloid beta (Aβ) levels while also protecting against or reversing cognitive impairment. To understand the mechanism(s) underlying the protective effects of caffeine against AD pathology, we investigated the effects of a two-week treatment with caffeine (3mg/day) in transgenic (APPswe) mice and non-transgenic (NT) mice on signaling factors involved in neuronal plasticity and survival. We evaluated cAMP-dependent protein kinase A (PKA), phospho-cyclic AMP response-element binding protein (phospho-CREB), and the pro-apoptotic protein kinases extracellular signal-regulated kinase 1/2 (phospho-ERK) and phospho-c-Jun N-terminal kinase 1 (phospho-JNK) in the striatum and frontal cortex of caffeine-treated mice. In the striatum, APPswe control mice exhibited a significant decrease in phospho-CREB, as well as significant increases in phospho-JNK and phospho-ERK in comparison to NT mice. Caffeine treatment stimulated PKA activity, increased phospho-CREB levels, and decreased phospho-JNK and phospho-ERK expression in the striatum of APPswe mice, all of which are thought to be beneficial changes for brain function. Even caffeine-treated NT mice exhibited some of these changes in striatum. In the frontal cortex, caffeine did not significantly increase phospho-CREB and PKA activity, but significantly reduced phospho-JNK and phospho-ERK expression in both APPswe and NT mice. These results suggest that caffeine shifts the balance between neurodegeneration and neuronal survival toward the stimulation of pro-survival cascades and inhibition of pro-apoptotic pathways in the striatum and/or cortex, which may contribute to its beneficial effects against AD.

  11. Acidosis Mediates the Switching of Gs-PKA and Gi-PKCε Dependence in Prolonged Hyperalgesia Induced by Inflammation.

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    Huang, Wei-Yu; Dai, Shih-Ping; Chang, Yan-Ching; Sun, Wei-Hsin

    2015-01-01

    Chronic inflammatory pain, when not effectively treated, is a costly health problem and has a harmful effect on all aspects of health-related quality of life. Previous studies suggested that in male Sprague Dawley rats, prostaglandin E2 (PGE2)-induced short-term hyperalgesia depends on protein kinase A (PKA) activity, whereas long-lasting hyperalgesia induced by PGE2 with carrageenan pre-injection, requires protein kinase Cε (PKCε). However, the mechanism underlying the kinase switch with short- to long-term hyperalgesia remains unclear. In this study, we used the inflammatory agents carrageenan or complete Freund's adjuvant (CFA) to induce long-term hyperalgesia, and examined PKA and PKCε dependence and switching time. Hyperalgesia induced by both agents depended on PKA/PKCε and Gs/Gi-proteins, and the switching time from PKA to PKCε and from Gs to Gi was about 3 to 4 h after inflammation induction. Among the single inflammatory mediators tested, PGE2 and 5-HT induced transient hyperalgesia, which depended on PKA and PKCε, respectively. Only acidic solution-induced hyperalgesia required Gs-PKA and Gi-PKCε, and the switch time for kinase dependency matched inflammatory hyperalgesia, in approximately 2 to 4 h. Thus, acidosis in inflamed tissues may be a decisive factor to regulate switching of PKA and PKCε dependence via proton-sensing G-protein-coupled receptors.

  12. Acidosis Mediates the Switching of Gs-PKA and Gi-PKCε Dependence in Prolonged Hyperalgesia Induced by Inflammation.

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    Wei-Yu Huang

    Full Text Available Chronic inflammatory pain, when not effectively treated, is a costly health problem and has a harmful effect on all aspects of health-related quality of life. Previous studies suggested that in male Sprague Dawley rats, prostaglandin E2 (PGE2-induced short-term hyperalgesia depends on protein kinase A (PKA activity, whereas long-lasting hyperalgesia induced by PGE2 with carrageenan pre-injection, requires protein kinase Cε (PKCε. However, the mechanism underlying the kinase switch with short- to long-term hyperalgesia remains unclear. In this study, we used the inflammatory agents carrageenan or complete Freund's adjuvant (CFA to induce long-term hyperalgesia, and examined PKA and PKCε dependence and switching time. Hyperalgesia induced by both agents depended on PKA/PKCε and Gs/Gi-proteins, and the switching time from PKA to PKCε and from Gs to Gi was about 3 to 4 h after inflammation induction. Among the single inflammatory mediators tested, PGE2 and 5-HT induced transient hyperalgesia, which depended on PKA and PKCε, respectively. Only acidic solution-induced hyperalgesia required Gs-PKA and Gi-PKCε, and the switch time for kinase dependency matched inflammatory hyperalgesia, in approximately 2 to 4 h. Thus, acidosis in inflamed tissues may be a decisive factor to regulate switching of PKA and PKCε dependence via proton-sensing G-protein-coupled receptors.

  13. G protein-coupled receptor Gpr4 senses amino acids and activates the cAMP-PKA pathway in Cryptococcus neoformans.

    Science.gov (United States)

    Xue, Chaoyang; Bahn, Yong-Sun; Cox, Gary M; Heitman, Joseph

    2006-02-01

    The Galpha protein Gpa1 governs the cAMP-PKA signaling pathway and plays a central role in virulence and differentiation in the human fungal pathogen Cryptococcus neoformans, but the signals and receptors that trigger this pathway were unknown. We identified seven putative proteins that share identity with known G protein-coupled receptors (GPCRs). One protein, Gpr4, shares limited sequence identity with the Dictyostelium discoideum cAMP receptor cAR1 and the Aspergillus nidulans GPCR protein GprH and also shares structural similarity with the Saccharomyces cerevisiae receptor Gpr1. gpr4 mutants exhibited reduced capsule production and mating defects, similar to gpa1 mutants, and exogenous cAMP suppressed both gpr4 mutant phenotypes. Epistasis analysis provides further evidence that Gpr4 functions upstream of the Galpha subunit Gpa1. Gpr4-Gpr4 homomeric interactions were observed in the yeast two-hybrid assay, and Gpr4 was shown to physically interact with Gpa1 in the split-ubiquitin system. A Gpr4::DsRED fusion protein was localized to the plasma membrane and methionine was found to trigger receptor internalization. The analysis of intracellular cAMP levels showed that gpr4 mutants still respond to glucose but not to certain amino acids, such as methionine. Amino acids might serve as ligands for Gpr4 and could contribute to engage the cAMP-PKA pathway. Activation of the cAMP-PKA pathway by glucose and amino acids represents a nutrient coincidence detection system shared in other pathogenic fungi.

  14. Effect of encapsulation in the anion receptor pocket of sub-domain IIA of human serum albumin on the modulation of pKa of warfarin and structurally similar acidic guests: a possible implication on biological activity.

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    Datta, Shubhashis; Halder, Mintu

    2014-01-05

    Supramolecular and bio-supramolecular host assisted pKa shift of biologically relevant acidic guests, warfarin and coumarin 343, has been monitored using both steady-state and time resolved fluorescence spectroscopy. The anion receptors present in sub-domain IIA of human serum albumin (HSA) stabilize the anionic form of the guest and thereby shift pKa towards acidic range. On the other hand, the preferential binding of the neutral form of guests in the non-polar hydrophobic cavity of β-cyclodextrin results in up-shifted pKa. This shifting of pKa of drugs like warfarin, etc., whose therapeutic activity depends on the position of the acid-base equilibrium in human system, is of great importance in pharmacokinetics. The release of the active form of such drugs from macrocyclic carrier and subsequent distribution through the carrier protein should depend on the modulation of the overall pKa window brought about by the encapsulation in these hosts. Present work also suggests that properly optimized encapsulation in appropriate receptor pocket can enhance the bioavailability of drugs. This work also opens up the possibility to use HSA as encapsulator, instead of traditional cyclodextrins or other polymeric hosts, since such system may overcome toxicity as well as biocompatibility issues.

  15. Parathyroid Hormone Activates Phospholipase C (PLC)-Independent Protein Kinase C Signaling Pathway via Protein Kinase A (PKA)-Dependent Mechanism: A New Defined Signaling Route Would Induce Alternative Consideration to Previous Conceptions.

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    Tong, Guojun; Meng, Yue; Hao, Song; Hu, Shaoyu; He, Youhua; Yan, Wenjuan; Yang, Dehong

    2017-04-20

    BACKGROUND Parathyroid hormone (PTH) is an effective anti-osteoporosis agent, after binding to its receptor PTHR1, several signaling pathways, including cAMP/protein kinase A (PKA) and phospholipase C (PLC)/protein kinase C (PKC), are initiated through G proteins; with the cAMP/PKA pathway as the major pathway. Earlier studies have reported that PTHR1 might also activate PKC via a PLC-independent mechanism, but this pathway remains unclear. MATERIAL AND METHODS In HEK293 cells, cAMP accumulation was measured with ELISA and PKC was measured with fluorescence resonance energy transfer (FRET) analysis using CKAR plasmid. In MC3T3-E1 cells, real-time PCR was performed to examine gene expressions. Then assays for cell apoptosis, cell differentiation, alkaline phosphatase activity, and mineralization were performed. RESULTS The FRET analysis found that PTH(1-34), [G1,R19]PTH(1-34) (GR(1-34), and [G1,R19]PTH(1-28) (GR(1-28) were all activated by PKC. The PKC activation ability of GR(1-28) was blocked by cAMP inhibitor (Rp-cAMP) and rescued with the addition of active PKA-α and PKA-β. The PKC activation ability of GR(1-34) was partially inhibited by Rp-cAMP. In MC3T3-E1 cells, gene expressions of ALP, CITED1, NR4a2, and OSX that was regulated by GR(1-28) were significantly changed by the pan-PKC inhibitor Go6983. After pretreatment with Rp-cAMP, the gene expressions of ALP, CITED1, and OPG were differentially regulated by GR(1-28) or GR(1-34), and the difference was blunted by Go6983. PTH(1-34), GR(1-28), and GR(1-34) significantly decreased early apoptosis and augmented osteoblastic differentiation in accordance with the activities of PKA and PKC. CONCLUSIONS PLC-independent PKC activation induced by PTH could be divided into two potential mechanisms: one was PKA-dependent and associated with PTH(1-28); the other was PKA-independent and associated with PTH(29-34). We also found that PTH could activate PLC-independent PKC via PKA-dependent mechanisms.

  16. The yeast transcription factor Crz1 is activated by light in a Ca2+/calcineurin-dependent and PKA-independent manner.

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    Kristofer Bodvard

    Full Text Available Light in the visible range can be stressful to non-photosynthetic organisms. The yeast Saccharomyces cerevisiae has earlier been reported to respond to blue light via activation of the stress-regulated transcription factor Msn2p. Environmental changes also induce activation of calcineurin, a Ca(2+/calmodulin dependent phosphatase, which in turn controls gene transcription by dephosphorylating the transcription factor Crz1p. We investigated the connection between cellular stress caused by blue light and Ca(2+ signalling in yeast by monitoring the nuclear localization dynamics of Crz1p, Msn2p and Msn4p. The three proteins exhibit distinctly different stress responses in relation to light exposure. Msn2p, and to a lesser degree Msn4p, oscillate rapidly between the nucleus and the cytoplasm in an apparently stochastic fashion. Crz1p, in contrast, displays a rapid and permanent nuclear localization induced by illumination, which triggers Crz1p-dependent transcription of its target gene CMK2. Moreover, increased extracellular Ca(2+ levels stimulates the light-induced responses of all three transcription factors, e.g. Crz1p localizes much quicker to the nucleus and a larger fraction of cells exhibits permanent Msn2p nuclear localization at higher Ca(2+ concentration. Studies in mutants lacking Ca(2+ transporters indicate that influx of extracellular Ca(2+ is crucial for the initial stages of light-induced Crz1p nuclear localization, while mobilization of intracellular Ca(2+ stores appears necessary for a sustained response. Importantly, we found that Crz1p nuclear localization is dependent on calcineurin and the carrier protein Nmd5p, while not being affected by increased protein kinase A activity (PKA, which strongly inhibits light-induced nuclear localization of Msn2/4p. We conclude that the two central signalling pathways, cAMP-PKA-Msn2/4 and Ca(2+-calcineurin-Crz1, are both activated by blue light illumination.

  17. Caffeine stimulates locomotor activity in the mammalian spinal cord via adenosine A1 receptor-dopamine D1 receptor interaction and PKA-dependent mechanisms.

    Science.gov (United States)

    Acevedo, JeanMarie; Santana-Almansa, Alexandra; Matos-Vergara, Nikol; Marrero-Cordero, Luis René; Cabezas-Bou, Ernesto; Díaz-Ríos, Manuel

    2016-02-01

    Caffeine is a potent psychostimulant that can have significant and widely variable effects on the activity of multiple neuronal pathways. The most pronounced caffeine-induced behavioral effect seen in rodents is to increase locomotor activity which has been linked to a dose-dependent inhibition of A1 and A(2A) receptors. The effects of caffeine at the level of the lumbar spinal central pattern generator (CPG) network for hindlimb locomotion are lacking. We assessed the effects of caffeine to the locomotor function of the spinal CPG network via extracellular ventral root recordings using the isolated neonatal mouse spinal cord preparation. Addition of caffeine and of an A1 receptor antagonist significantly decreased the cycle period accelerating the ongoing locomotor rhythm, while decreasing burst duration reversibly in most preparations suggesting the role of A1 receptors as the primary target of caffeine. Caffeine and an A1 receptor antagonist failed to stimulate ongoing locomotor activity in the absence of dopamine or in the presence of a D1 receptor antagonist supporting A1/D1 receptor-dependent mechanism of action. The use of caffeine or an A1 receptor blocker failed to stimulate an ongoing locomotor rhythm in the presence of a blocker of the cAMP-dependent protein kinase (PKA) supporting the need of this intracellular pathway for the modulatory effects of caffeine to occur. These results support a stimulant effect of caffeine on the lumbar spinal network controlling hindlimb locomotion through the inhibition of A1 receptors and subsequent activation of D1 receptors via a PKA-dependent intracellular mechanism.

  18. Tau-driven 26S proteasome impairment and cognitive dysfunction can be prevented early in disease by activating cAMP-PKA signaling.

    Science.gov (United States)

    Myeku, Natura; Clelland, Catherine L; Emrani, Sheina; Kukushkin, Nikolay V; Yu, Wai Haung; Goldberg, Alfred L; Duff, Karen E

    2016-01-01

    The ubiquitin proteasome system (UPS) degrades misfolded proteins including those implicated in neurodegenerative diseases. We investigated the effects of tau accumulation on proteasome function in a mouse model of tauopathy and in a cross to a UPS reporter mouse (line Ub-G76V-GFP). Accumulation of insoluble tau was associated with a decrease in the peptidase activity of brain 26S proteasomes, higher levels of ubiquitinated proteins and undegraded Ub-G76V-GFP. 26S proteasomes from mice with tauopathy were physically associated with tau and were less active in hydrolyzing ubiquitinated proteins, small peptides and ATP. 26S proteasomes from normal mice incubated with recombinant oligomers or fibrils also showed lower hydrolyzing capacity in the same assays, implicating tau as a proteotoxin. Administration of an agent that activates cAMP-protein kinase A (PKA) signaling led to attenuation of proteasome dysfunction, probably through proteasome subunit phosphorylation. In vivo, this led to lower levels of aggregated tau and improvements in cognitive performance.

  19. Opposing roles of PKA and EPAC in the cAMP-dependent regulation of schwann cell proliferation and differentiation [corrected].

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    Ketty Bacallao

    Full Text Available In Schwann cells (SCs, cyclic adenosine monophosphate (cAMP not only induces differentiation into a myelinating SC-related phenotype, but also synergistically enhances the mitogenic action of growth factors such as neuregulin. To better understand the molecular mechanism by which cAMP exerts these apparently contradictory functions, we investigated the role of the two main effectors of cAMP, protein kinase A (PKA and the exchange protein activated by cAMP (EPAC, on the proliferation and differentiation of both isolated and axon-related SCs. For these studies, a variety of PKA and EPAC agonists and antagonists were used, including pathway-selective analogs of cAMP and pharmacological inhibitors. Our studies indicated that the activity of PKA rather than EPAC was required for the adjuvant effect of cAMP on S-phase entry, whereas the activity of EPAC rather than PKA was required for SC differentiation and myelin formation. Even though selective EPAC activation had an overall anti-proliferative effect in SCs, it failed to drive the expression of Krox-20, a master regulator of myelination, and that of myelin-specific proteins and lipids, suggesting that EPAC activation was insufficient to drive a full differentiating response. Interestingly, inhibition of EPAC activity resulted in a drastic impairment of SC differentiation and myelin formation but not Krox-20 expression, which indicates an independent mechanism of Krox-20 regulation in response to cAMP. In conclusion, our data supports the idea that the outcome of cAMP signaling in SCs depends on the particular set of effectors activated. Whereas the mitogenic action of cAMP relies exclusively on PKA activity, the differentiating action of cAMP requires a PKA-independent (non-canonical cAMP-specific pathway that is partially transduced by EPAC.

  20. Opposing roles of PKA and EPAC in the cAMP-dependent regulation of schwann cell proliferation and differentiation [corrected].

    Science.gov (United States)

    Bacallao, Ketty; Monje, Paula V

    2013-01-01

    In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) not only induces differentiation into a myelinating SC-related phenotype, but also synergistically enhances the mitogenic action of growth factors such as neuregulin. To better understand the molecular mechanism by which cAMP exerts these apparently contradictory functions, we investigated the role of the two main effectors of cAMP, protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC), on the proliferation and differentiation of both isolated and axon-related SCs. For these studies, a variety of PKA and EPAC agonists and antagonists were used, including pathway-selective analogs of cAMP and pharmacological inhibitors. Our studies indicated that the activity of PKA rather than EPAC was required for the adjuvant effect of cAMP on S-phase entry, whereas the activity of EPAC rather than PKA was required for SC differentiation and myelin formation. Even though selective EPAC activation had an overall anti-proliferative effect in SCs, it failed to drive the expression of Krox-20, a master regulator of myelination, and that of myelin-specific proteins and lipids, suggesting that EPAC activation was insufficient to drive a full differentiating response. Interestingly, inhibition of EPAC activity resulted in a drastic impairment of SC differentiation and myelin formation but not Krox-20 expression, which indicates an independent mechanism of Krox-20 regulation in response to cAMP. In conclusion, our data supports the idea that the outcome of cAMP signaling in SCs depends on the particular set of effectors activated. Whereas the mitogenic action of cAMP relies exclusively on PKA activity, the differentiating action of cAMP requires a PKA-independent (non-canonical) cAMP-specific pathway that is partially transduced by EPAC.

  1. Inhibitory phosphorylation of GSK-3β by AKT, PKA, and PI3K contributes to high NaCl-induced activation of the transcription factor NFAT5 (TonEBP/OREBP).

    Science.gov (United States)

    Zhou, Xiaoming; Wang, Hong; Burg, Maurice B; Ferraris, Joan D

    2013-04-01

    High NaCl activates the transcription factor nuclear factor of activated T cells 5 (NFAT5), leading to increased transcription of osmoprotective target genes. Kinases PKA, PI3K, AKT1, and p38α were known to contribute to the high NaCl-induced increase of NFAT5 activity. We now identify another kinase, GSK-3β. siRNA-mediated knock-down of GSK-3β increases NFAT5 transcriptional and transactivating activities without affecting high NaCl-induced nuclear localization of NFAT5 or NFAT5 protein expression. High NaCl increases phosphorylation of GSK-3β-S9, which inhibits GSK-3β. In GSK-3β-null mouse embryonic fibroblasts transfection of GSK-3β, in which serine 9 is mutated to alanine, so that it cannot be inhibited by phosphorylation at that site, inhibits high NaCl-induced NFAT5 transcriptional activity more than transfection of wild-type GSK-3β. High NaCl-induced phosphorylation of GSK-3β-S9 depends on PKA, PI3K, and AKT, but not p38α. Overexpression of PKA catalytic subunit α or of catalytically active AKT1 reduces inhibition of NFAT5 by GSK-3β, but overexpression of p38α together with its catalytically active upstream kinase, MKK6, does not. Thus, GSK-3β normally inhibits NFAT5 by suppressing its transactivating activity. When activated by high NaCl, PKA, PI3K, and AKT1, but not p38α, increase phosphorylation of GSK-3β-S9, which reduces the inhibitory effect of GSK-3β on NFAT5, and thus contributes to activation of NFAT5.

  2. Regulation of Gβγi-dependent PLC-β3 activity in smooth muscle: inhibitory phosphorylation of PLC-β3 by PKA and PKG and stimulatory phosphorylation of Gαi-GTPase-activating protein RGS2 by PKG.

    Science.gov (United States)

    Nalli, Ancy D; Kumar, Divya P; Al-Shboul, Othman; Mahavadi, Sunila; Kuemmerle, John F; Grider, John R; Murthy, Karnam S

    2014-11-01

    In gastrointestinal smooth muscle, agonists that bind to Gi-coupled receptors activate preferentially PLC-β3 via Gβγ to stimulate phosphoinositide (PI) hydrolysis and generate inositol 1,4,5-trisphosphate (IP3) leading to IP3-dependent Ca(2+) release and muscle contraction. In the present study, we identified the mechanism of inhibition of PLC-β3-dependent PI hydrolysis by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). Cyclopentyl adenosine (CPA), an adenosine A1 receptor agonist, caused an increase in PI hydrolysis in a concentration-dependent fashion; stimulation was blocked by expression of the carboxyl-terminal sequence of GRK2(495-689), a Gβγ-scavenging peptide, or Gαi minigene but not Gαq minigene. Isoproterenol and S-nitrosoglutathione (GSNO) induced phosphorylation of PLC-β3 and inhibited CPA-induced PI hydrolysis, Ca(2+) release, and muscle contraction. The effect of isoproterenol on all three responses was inhibited by PKA inhibitor, myristoylated PKI, or AKAP inhibitor, Ht-31, whereas the effect of GSNO was selectively inhibited by PKG inhibitor, Rp-cGMPS. GSNO, but not isoproterenol, also phosphorylated Gαi-GTPase-activating protein, RGS2, and enhanced association of Gαi3-GTP and RGS2. The effect of GSNO on PI hydrolysis was partly reversed in cells (i) expressing constitutively active GTPase-resistant Gαi mutant (Q204L), (ii) phosphorylation-site-deficient RGS2 mutant (S46A/S64A), or (iii) siRNA for RGS2. We conclude that PKA and PKG inhibit Gβγi-dependent PLC-β3 activity by direct phosphorylation of PLC-β3. PKG, but not PKA, also inhibits PI hydrolysis indirectly by a mechanism involving phosphorylation of RGS2 and its association with Gαi-GTP. This allows RGS2 to accelerate Gαi-GTPase activity, enhance Gαβγi trimer formation, and inhibit Gβγi-dependent PLC-β3 activity.

  3. At immature mossy-fiber-CA3 synapses, correlated presynaptic and postsynaptic activity persistently enhances GABA release and network excitability via BDNF and cAMP-dependent PKA.

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    Sivakumaran, Sudhir; Mohajerani, Majid H; Cherubini, Enrico

    2009-02-25

    In the adult rat hippocampus, the axons of granule cells in the dentate gyrus, the mossy fibers (MF), form excitatory glutamatergic synapses with CA3 principal cells. In neonates, MF release into their targets mainly GABA, which at this developmental stage is depolarizing. Here we tested the hypothesis that, at immature MF-CA3 synapses, correlated presynaptic [single fiber-evoked GABA(A)-mediated postsynaptic potentials (GPSPs)] and postsynaptic activity (back propagating action potentials) may exert a critical control on synaptic efficacy. This form of plasticity, called spike-timing-dependent plasticity (STDP), is a Hebbian type form of learning extensively studied at the level of glutamatergic synapses. Depending on the relative timing, pairing postsynaptic spiking and single MF-GPSPs induced bidirectional changes in synaptic efficacy. In case of positive pairing, spike-timing-dependent-long-term potentiation (STD-LTP) was associated with a persistent increase in GPSP slope and in the probability of cell firing. The transduction pathway involved a rise of calcium in the postsynaptic cell and the combined activity of cAMP-dependent PKA (protein kinase A) and brain-derived neurotrophic factor (BDNF). Retrograde signaling via BDNF and presynaptic TrkB receptors led to a persistent increase in GABA release. In "presynaptically" silent neurons, the enhanced probability of GABA release induced by the pairing protocol, unsilenced these synapses. Shifting E(GABA) from the depolarizing to the hyperpolarizing direction with bumetanide failed to modify synaptic strength. Thus, STD-LTP of GPSPs provides a reliable way to convey information from granule cells to the CA3 associative network at a time when glutamatergic synapses are still poorly developed.

  4. Direct Raman measurement of an elevated base pKa in the active site of a small ribozyme in a precatalytic conformation.

    Science.gov (United States)

    Guo, Man; Spitale, Robert C; Volpini, Rosaria; Krucinska, Jolanta; Cristalli, Gloria; Carey, Paul R; Wedekind, Joseph E

    2009-09-16

    Catalytic RNA molecules can achieve rate acceleration by shifting base pK(a) values toward neutrality. Prior evidence has suggested that base A38 of the hairpin ribozyme plays an important role in phosphoryl transfer, possibly functioning as a general acid, or by orienting a specific water molecule for proton transfer. To address the role of A38, we used Raman spectroscopy to measure directly the pK(a) of the N1-imino moiety in the context of hairpin ribozyme crystals representative of a "precatalytic" conformation. The results revealed that the pK(a) of A38 is shifted to 5.46 +/- 0.05 relative to 3.68 +/- 0.06 derived from a reference solution of the nucleotide AMP. The elevated pK(a) correlates well with the first titration point of the macroscopic pH-rate profile of the hairpin ribozyme in solution and strongly supports A38 as a general acid catalyst in bond scission. The results confirm that A38 is protonated before the transition state, which would promote phosphorane development. Overall, the results establish a cogent structure-function paradigm that expands our understanding of how RNA structure can enhance nucleobase reactivity to catalyze biological reactions.

  5. PKA-chromatin association at stress responsive target genes from Saccharomyces cerevisiae.

    Science.gov (United States)

    Baccarini, Leticia; Martínez-Montañés, Fernando; Rossi, Silvia; Proft, Markus; Portela, Paula

    2015-11-01

    Gene expression regulation by intracellular stimulus-activated protein kinases is essential for cell adaptation to environmental changes. There are three PKA catalytic subunits in Saccharomyces cerevisiae: Tpk1, Tpk2, and Tpk3 and one regulatory subunit: Bcy1. Previously, it has been demonstrated that Tpk1 and Tpk2 are associated with coding regions and promoters of target genes in a carbon source and oxidative stress dependent manner. Here we studied five genes, ALD6, SED1, HSP42, RPS29B, and RPL1B whose expression is regulated by saline stress. We found that PKA catalytic and regulatory subunits are associated with both coding regions and promoters of the analyzed genes in a stress dependent manner. Tpk1 and Tpk2 recruitment was completely abolished in catalytic inactive mutants. BCY1 deletion changed the binding kinetic to chromatin of each Tpk isoform and this strain displayed a deregulated gene expression in response to osmotic stress. In addition, yeast mutants with high PKA activity exhibit sustained association to target genes of chromatin-remodeling complexes such as Snf2-catalytic subunit of the SWI/SNF complex and Arp8-component of INO80 complex, leading to upregulation of gene expression during osmotic stress. Tpk1 accumulation in the nucleus was stimulated upon osmotic stress, while the nuclear localization of Tpk2 and Bcy1 showed no change. We found that each PKA subunit is transported into the nucleus by a different β-karyopherin pathway. Moreover, β-karyopherin mutant strains abolished the chromatin association of Tpk1 or Tpk2, suggesting that nuclear localization of PKA catalytic subunits is required for its association to target genes and properly gene expression.

  6. G Protein-coupled Receptor Gpr4 Senses Amino Acids and Activates the cAMP-PKA Pathway in Cryptococcus neoformansD⃞

    OpenAIRE

    Xue, Chaoyang; Bahn, Yong-Sun; Cox, Gary M.; Heitman, Joseph

    2006-01-01

    The Gα protein Gpa1 governs the cAMP-PKA signaling pathway and plays a central role in virulence and differentiation in the human fungal pathogen Cryptococcus neoformans, but the signals and receptors that trigger this pathway were unknown. We identified seven putative proteins that share identity with known G protein-coupled receptors (GPCRs). One protein, Gpr4, shares limited sequence identity with the Dictyostelium discoideum cAMP receptor cAR1 and the Aspergillus nidulans GPCR protein Gpr...

  7. 慢性束缚应激对大鼠体质量、血清皮质酮和前额叶皮质cAMP-PKA-CREB-BDNF通路的影响%Effect of chronic restraint stress on body weight,serum corticosterone concentration and cAMP-PKA-CREB-BDNF signaling activity in prefrontal cortex in rats

    Institute of Scientific and Technical Information of China (English)

    孟珊珊; 刘洪文

    2011-01-01

    目的 探讨慢性束缚应激对大鼠体质量、血清皮质酮水平和前额叶皮质cAMP-PKA-CREB-BDNF通路的影响.方法 将30只雄性SD大鼠随机分为对照组、慢性束缚应激1周组和慢性束缚应激2周组,每组各10只.比较应激前后大鼠体质量和血清皮质酮水平的变化,放免法观察应激前后大鼠前额叶皮质环磷腺苷(cAMP)含量,试剂盒测定cAMP依赖蛋白激酶(protein kinase,PKA)活性和脑源性神经营养因子(brain derived neurophic factor,BDNF)表达水平,用蛋白免疫印迹技术观察cAMP反应元件结合蛋白质(p-CREB)水平变化.结果 慢性束缚应激组大鼠在应激1周和应激2周的体质量增长[分别为(35±11)g和(23±8)g]均低于对照组[分别为(59±16)g和(68±14)g](P<0.05).束缚应激1周组和束缚应激2周组大鼠血清皮质醇浓度分别为(356.78±112.21)ng/ml、(558.49±98.32)ng/ml,均高于对照组(198.21±101.35)ng/ml(P<0.05,P<0.01).束缚应激1周组和束缚应激2周组大鼠前额叶皮质的cAMP含量、PKA活性、p-CREB和BDNF表达水平低于对照组(P<0.01).结论 慢性束缚应激抑制大鼠体质量的增长,引起血清皮质酮水平增高和前额叶皮质cAMP-PKA-CREB-BDNF信号通路被抑制.%To observe effect of chronic restraint stress on body weight,scrum corticostcronc concentration and cAMP-PKA-CREB-BDNF signaling activity in prcfrontai cortex in the rat. Methods Thirty male SD rats were randomly divided into control group (n = 10), 1 week and 2 weeks (n= 10) restraint stress groups. Before and after restraint stress, body weight and scrum corticostcronc concentration were examined, radio-immunity method Pep TagR detecting system, and western blotting were used to assess cAMP contcrt. PKA activity and the level of p-CREDB in prcfrontai cortex. Results The body weight gains on the first (35 + ll)g and second stressed weeks (23 I: 8)g were significantly lower in restraint stress groups than in controls group (59 ± 16)g and (68

  8. Cysteine pKa Depression by a Protonated Glutamic Acid in Human DJ-1

    Science.gov (United States)

    Witt, Anna C.; Lakshminarasimhan, Mahadevan; Remington, Benjamin C.; Hasim, Sahar; Pozharski, Edwin; Wilson, Mark A.

    2009-01-01

    Human DJ-1, a disease-associated protein that protects cells from oxidative stress, contains an oxidation-sensitive cysteine (C106) that is essential for its cytoprotective activity. The origin of C106 reactivity is obscure, due in part to the absence of an experimentally determined pKa value for this residue. We have used atomic resolution X-ray crystallography and UV spectroscopy to show that C106 has a depressed pKa of 5.4±0.1 and that the C106 thiolate accepts a hydrogen bond from a protonated glutamic acid sidechain (E18). X-ray crystal structures and cysteine pKa analysis of several site-directed substitutions at residue 18 demonstrate that the protonated carboxylic acid sidechain of E18 is required to maximally stabilize the C106 thiolate. A nearby arginine residue (R48) participates in a guanidinium stacking interaction with R28 from the other monomer in the DJ-1 dimer and elevates the pKa of C106 by binding an anion that electrostatically suppresses thiol ionization. Our results show that the ionizable residues (E18, R48 and R28) surrounding C106 affect its pKa in a way that is contrary to expectations based on the typical ionization behavior of glutamic acid and arginine. Lastly, a search of the Protein Data Bank (PDB) produces several candidate hydrogen bonded aspartic/glutamic acid-cysteine interactions, which we propose are particularly common in the DJ-1 superfamily. PMID:18570440

  9. Activation of G protein-coupled estrogen receptor 1 induces coronary artery relaxation via Epac/Rap1-mediated inhibition of RhoA/Rho kinase pathway in parallel with PKA

    Science.gov (United States)

    Yu, Xuan; Zhang, Qiao; Zhao, Yan; Schwarz, Benjamin J.; Stallone, John N.; Heaps, Cristine L.; Han, Guichun

    2017-01-01

    Previously, we reported that cAMP/PKA signaling is involved in GPER-mediated coronary relaxation by activating MLCP via inhibition of RhoA pathway. In the current study, we tested the hypothesis that activation of GPER induces coronary artery relaxation via inhibition of RhoA/Rho kinase pathway by cAMP downstream targets, exchange proteins directly activated by cAMP (Epac) as well as PKA. Our results show that Epac inhibitors, brefeldin A (BFA, 50 μM), or ESI-09 (20 μM), or CE3F4 (100 μM), all partially inhibited porcine coronary artery relaxation response to the selective GPER agonist, G-1 (0.3–3 μM); while concurrent administration of BFA and PKI (5 μM), a PKA inhibitor, almost completely blocked the relaxation effect of G-1. The Epac specific agonist, 8-CPT-2Me-cAMP (007, 1–100 μM), induced a concentration-dependent relaxation response. Furthermore, the activity of Ras-related protein 1 (Rap1) was up regulated by G-1 (1 μM) treatment of porcine coronary artery smooth muscle cells (CASMCs). Phosphorylation of vasodilator-stimulated phosphoprotein (p-VASP) was elevated by G-1 (1 μM) treatment, but not by 007 (50 μM); and the effect of G-1 on p-VASP was blocked by PKI, but not by ESI-09, an Epac antagonist. RhoA activity was similarly down regulated by G-1 and 007, whereas ESI-09 restored most of the reduced RhoA activity by G-1 treatment. Furthermore, G-1 decreased PGF2α-induced p-MYPT1, which was partially reversed with either ESI-09 or PKI; whereas, concurrent administration of ESI-09 and PKI totally prevented the inhibitory effect of G-1. The inhibitory effects of G-1 on p- MLC levels in CASMCs were mostly restored by either ESI-09 or PKI. These results demonstrate that activation of GPER induces coronary artery relaxation via concurrent inhibition of RhoA/Rho kinase by Epac/Rap1 and PKA. GPER could be a potential drug target for preventing and treating cardiovascular diseases. PMID:28278256

  10. The cAMP-PKA Signaling Pathway Regulates Pathogenicity, Hyphal Growth, Appressorial Formation, Conidiation, and Stress Tolerance in Colletotrichum higginsianum

    Directory of Open Access Journals (Sweden)

    Wenjun Zhu

    2017-07-01

    Full Text Available Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. Understanding the mechanisms of the cruciferous plant–C. higginsianum interactions will be important in facilitating efficient control of anthracnose diseases. The cAMP-PKA signaling pathway plays important roles in diverse physiological processes of multiple pathogens. C. higginsianum contains two genes, ChPKA1 and ChPKA2, that encode the catalytic subunits of cyclic AMP (cAMP-dependent protein kinase A (PKA. To analyze the role of cAMP signaling pathway in pathogenicity and development in C. higginsianum, we characterized ChPKA1 and ChPKA2 genes, and adenylate cyclase (ChAC gene. The ChPKA1 and ChAC deletion mutants were unable to cause disease and significantly reduced in hyphal growth, tolerance to cell wall inhibitors, conidiation, and appressorial formation with abnormal germ tubes, but they had an increased tolerance to elevated temperatures and exogenous H2O2. In contrast, the ChPKA2 mutant had no detectable alteration of phenotypes, suggesting that ChPKA1 contributes mainly to PKA activities in C. higginsianum. Moreover, we failed to generate ΔChPKA1ChPKA2 double mutant, indicating that deletion of both PKA catalytic subunits is lethal in C. higginsianum and the two catalytic subunits possibly have overlapping functions. These results indicated that ChPKA1 is the major PKA catalytic subunit in cAMP-PKA signaling pathway and plays significant roles in hyphal growth, pathogenicity, appressorial formation, conidiation, and stress tolerance in C. higginsianum.

  11. Nutrient Control of Yeast Gametogenesis Is Mediated by TORC1, PKA and Energy Availability.

    Directory of Open Access Journals (Sweden)

    Hilla Weidberg

    2016-06-01

    Full Text Available Cell fate choices are tightly controlled by the interplay between intrinsic and extrinsic signals, and gene regulatory networks. In Saccharomyces cerevisiae, the decision to enter into gametogenesis or sporulation is dictated by mating type and nutrient availability. These signals regulate the expression of the master regulator of gametogenesis, IME1. Here we describe how nutrients control IME1 expression. We find that protein kinase A (PKA and target of rapamycin complex I (TORC1 signalling mediate nutrient regulation of IME1 expression. Inhibiting both pathways is sufficient to induce IME1 expression and complete sporulation in nutrient-rich conditions. Our ability to induce sporulation under nutrient rich conditions allowed us to show that respiration and fermentation are interchangeable energy sources for IME1 transcription. Furthermore, we find that TORC1 can both promote and inhibit gametogenesis. Down-regulation of TORC1 is required to activate IME1. However, complete inactivation of TORC1 inhibits IME1 induction, indicating that an intermediate level of TORC1 signalling is required for entry into sporulation. Finally, we show that the transcriptional repressor Tup1 binds and represses the IME1 promoter when nutrients are ample, but is released from the IME1 promoter when both PKA and TORC1 are inhibited. Collectively our data demonstrate that nutrient control of entry into sporulation is mediated by a combination of energy availability, TORC1 and PKA activities that converge on the IME1 promoter.

  12. Nutrient Control of Yeast Gametogenesis Is Mediated by TORC1, PKA and Energy Availability.

    Directory of Open Access Journals (Sweden)

    Hilla Weidberg

    2016-06-01

    Full Text Available Cell fate choices are tightly controlled by the interplay between intrinsic and extrinsic signals, and gene regulatory networks. In Saccharomyces cerevisiae, the decision to enter into gametogenesis or sporulation is dictated by mating type and nutrient availability. These signals regulate the expression of the master regulator of gametogenesis, IME1. Here we describe how nutrients control IME1 expression. We find that protein kinase A (PKA and target of rapamycin complex I (TORC1 signalling mediate nutrient regulation of IME1 expression. Inhibiting both pathways is sufficient to induce IME1 expression and complete sporulation in nutrient-rich conditions. Our ability to induce sporulation under nutrient rich conditions allowed us to show that respiration and fermentation are interchangeable energy sources for IME1 transcription. Furthermore, we find that TORC1 can both promote and inhibit gametogenesis. Down-regulation of TORC1 is required to activate IME1. However, complete inactivation of TORC1 inhibits IME1 induction, indicating that an intermediate level of TORC1 signalling is required for entry into sporulation. Finally, we show that the transcriptional repressor Tup1 binds and represses the IME1 promoter when nutrients are ample, but is released from the IME1 promoter when both PKA and TORC1 are inhibited. Collectively our data demonstrate that nutrient control of entry into sporulation is mediated by a combination of energy availability, TORC1 and PKA activities that converge on the IME1 promoter.

  13. PKA-mediated phosphorylation of EPEC-Tir at serine residues 434 and 463

    Science.gov (United States)

    Kenny, Brendan; Gerhard, Ralf; Tegtmeyer, Nicole; Brandt, Sabine

    2010-01-01

    Type-III or type-IV secretion systems of many Gram-negative bacterial pathogens inject effector proteins into host cells that modulate cellular functions in their favour. A preferred target of these effectors is the actin-cytoskeleton as shown by studies using the gastric pathogens Helicobacter pylori (H. pylori) and enteropathogenic Escherichia coli (EPEC). We recently developed a co-infection approach to study effector protein function and molecular mechanisms by which they highjack cellular signalling cascades. This is exemplified by our observation that EPEC profoundly blocks H. pylori-induced epithelial cell scattering and elongation, a disease-related event requiring the activity of small Rho GTPase Rac1. While this suppressive effect is dependent on the effector protein Tir and the outer-membrane protein Intimin, it unexpectedly revealed evidence for Tir-signalling independent of phosphorylation of Tir at tyrosine residues 454 and 474. Instead, our studies revealed a previously unidentified function for protein kinase A (PKA)-mediated phosphorylation of Tir at serine residues 434 and 463. We demonstrated that EPEC infection activates PKA for Tir phosphorylation. Activated PKA then phosphorylates Rac1 at its serine residue 71 associated with reduced GTP-load and inhibited cell elongation. Phosphorylation of Rho GTPases such as Rac1 might be an interesting novel strategy in microbial pathogenesis. PMID:21326916

  14. Caffeine inhibits the activation of hepatic stellate cells induced by acetaldehyde via adenosine A2A receptor mediated by the cAMP/PKA/SRC/ERK1/2/P38 MAPK signal pathway.

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    He Wang

    Full Text Available Hepatic stellate cell (HSC activation is an essential event during alcoholic liver fibrosis. Evidence suggests that adenosine aggravates liver fibrosis via the adenosine A2A receptor (A2AR. Caffeine, which is being widely consumed during daily life, inhibits the action of adenosine. In this study, we attempted to validate the hypothesis that caffeine influences acetaldehyde-induced HSC activation by acting on A2AR. Acetaldehyde at 50, 100, 200, and 400 μM significantly increased HSC-T6 cells proliferation, and cell proliferation reached a maximum at 48 h after exposure to 200 μM acetaldehyde. Caffeine and the A2AR antagonist ZM241385 decreased the cell viability and inhibited the expression of procollagen type I and type III in acetaldehyde-induced HSC-T6 cells. In addition, the inhibitory effect of caffeine on the expression of procollagen type I was regulated by A2AR-mediated signal pathway involving cAMP, PKA, SRC, and ERK1/2. Interestingly, caffeine's inhibitory effect on the expression of procollagen type III may depend upon the A2AR-mediated P38 MAPK-dependent pathway.Caffeine significantly inhibited acetaldehyde-induced HSC-T6 cells activation by distinct A2AR mediated signal pathway via inhibition of cAMP-PKA-SRC-ERK1/2 for procollagen type I and via P38 MAPK for procollagen type III.

  15. Cyclophilin D deficiency rescues Aβ-impaired PKA/CREB signaling and alleviates synaptic degeneration.

    Science.gov (United States)

    Du, Heng; Guo, Lan; Wu, Xiaoping; Sosunov, Alexander A; McKhann, Guy M; Chen, John Xi; Yan, Shirley ShiDu

    2014-12-01

    The coexistence of neuronal mitochondrial pathology and synaptic dysfunction is an early pathological feature of Alzheimer's disease (AD). Cyclophilin D (CypD), an integral part of mitochondrial permeability transition pore (mPTP), is involved in amyloid beta (Aβ)-instigated mitochondrial dysfunction. Blockade of CypD prevents Aβ-induced mitochondrial malfunction and the consequent cognitive impairments. Here, we showed the elimination of reactive oxygen species (ROS) by antioxidants probucol or superoxide dismutase (SOD)/catalase blocks Aβ-mediated inactivation of protein kinase A (PKA)/cAMP regulatory-element-binding (CREB) signal transduction pathway and loss of synapse, suggesting the detrimental effects of oxidative stress on neuronal PKA/CREB activity. Notably, neurons lacking CypD significantly attenuate Aβ-induced ROS. Consequently, CypD-deficient neurons are resistant to Aβ-disrupted PKA/CREB signaling by increased PKA activity, phosphorylation of PKA catalytic subunit (PKA C), and CREB. In parallel, lack of CypD protects neurons from Aβ-induced loss of synapses and synaptic dysfunction. Furthermore, compared to the mAPP mice, CypD-deficient mAPP mice reveal less inactivation of PKA-CREB activity and increased synaptic density, attenuate abnormalities in dendritic spine maturation, and improve spontaneous synaptic activity. These findings provide new insights into a mechanism in the crosstalk between the CypD-dependent mitochondrial oxidative stress and signaling cascade, leading to synaptic injury, functioning through the PKA/CREB signal transduction pathway.

  16. Cyclic adenosine 3'-5'-monophosphate (cAMP) exerts proliferative and anti-proliferative effects in pituitary cells of different types by activating both cAMP-dependent protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac).

    Science.gov (United States)

    Vitali, E; Peverelli, E; Giardino, E; Locatelli, M; Lasio, G B; Beck-Peccoz, P; Spada, A; Lania, A G; Mantovani, G

    2014-03-05

    In the pituitary the activation of cyclic adenosine 3'-5'-monophosphate (cAMP) dependent pathways generates proliferative signals in somatotrophs, whereas in pituitary cells of other lineages its effect remains uncertain. Moreover, the specific role of the two main cAMP effectors, protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac), has not been defined. Aim of this study was to investigate the effect of cAMP on pituitary adenomatous cells proliferation and to identify PKA and Epac differential involvement. We found that cAMP increased DNA synthesis and cyclin D1 expression in somatotropinomas, whereas it reduced both parameters in prolactinomas and nonfunctioning adenomas, these effects being replicated in corresponding cell lines. Moreover, the divergent cAMP effects were mimicked by Epac and PKA analogs, which activated Rap1 and CREB, respectively. In conclusion, we demonstrated that cAMP exerted opposite effects on different pituitary cell types proliferation, these effects being mediated by both Epac and PKA.

  17. Reorganized PKA-AKAP associations in the failing human heart.

    Science.gov (United States)

    Aye, Thin-Thin; Soni, Siddarth; van Veen, Toon A B; van der Heyden, Marcel A G; Cappadona, Salvatore; Varro, Andras; de Weger, Roel A; de Jonge, Nicolaas; Vos, Marc A; Heck, Albert J R; Scholten, Arjen

    2012-02-01

    Here we reveal that the characterization of large-scale re-arrangements of signaling scaffolds induced by heart failure can serve as a novel concept to identify more specific therapeutic targets. In the mammalian heart, the cAMP pathway, with the cAMP-dependent protein kinase (PKA) in a central role, acts directly downstream of adrenergic receptors to mediate cardiac contractility and rhythm. Heart failure, characterized by severe alterations in adrenergic stimulation is, amongst other interventions, often treated with β-blockers. Contrasting results, however, have shown both beneficial and detrimental effects of decreased cAMP levels in failing hearts. We hypothesize that the origin of this behavior lies in the complex spatiotemporal organization of the regulatory subunit of PKA (PKA-R), which associates tightly with various A-kinase anchoring proteins (AKAPs) to specifically localize PKA's activity. Using chemical proteomics directly applied to human patient and control heart tissue we demonstrate that the association profile of PKA-R with several AKAPs is severely altered in the failing heart, for instance effecting the interaction between PKA and the novel AKAP SPHKAP was 6-fold upregulated upon failing heart conditions. Also a significant increase in captured cGMP-dependent protein kinase (PKG) and phosphodiesterase 2 (PDE2) was observed. The observed altered profiles can already explain many aspects of the aberrant cAMP-response in the failing human heart, validating that this dataset may provide a resource for several novel, more specific, treatment options. This article is part of a Special Issue entitled "Local Signaling in Myocytes".

  18. PKA controls calcium influx into motor neurons during a rhythmic behavior.

    Directory of Open Access Journals (Sweden)

    Han Wang

    Full Text Available Cyclic adenosine monophosphate (cAMP has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels.

  19. PKA controls calcium influx into motor neurons during a rhythmic behavior.

    Directory of Open Access Journals (Sweden)

    Han Wang

    Full Text Available Cyclic adenosine monophosphate (cAMP has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels.

  20. Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling.

    Science.gov (United States)

    Bachmann, Verena A; Mayrhofer, Johanna E; Ilouz, Ronit; Tschaikner, Philipp; Raffeiner, Philipp; Röck, Ruth; Courcelles, Mathieu; Apelt, Federico; Lu, Tsan-Wen; Baillie, George S; Thibault, Pierre; Aanstad, Pia; Stelzl, Ulrich; Taylor, Susan S; Stefan, Eduard

    2016-07-12

    Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.

  1. Biphasic Erk1/2 activation sequentially involving Gs and Gi signaling is required in beta3-adrenergic receptor-induced primary smooth muscle cell proliferation.

    Science.gov (United States)

    Hadi, Tarik; Barrichon, Marina; Mourtialon, Pascal; Wendremaire, Maeva; Garrido, Carmen; Sagot, Paul; Bardou, Marc; Lirussi, Frédéric

    2013-05-01

    The beta3 adrenergic receptor (B3-AR) reportedly induces cell proliferation, but the signaling pathways that were proposed, involving either Gs or Gi coupling, remain controversial. To further investigate the role of G protein coupling in B3-AR induced proliferation, we stimulated primary human myometrial smooth muscle cells with SAR150640 (B3-AR agonist) in the absence or presence of variable G-protein inhibitors. Specific B3-AR stimulation led to an Erk1/2 induced proliferation. We observed that the proliferative effects of B3-AR require two Erk1/2 activation peaks (the first after 3min, the second at 8h). Erk1/2 activation at 3min was mimicked by forskolin (adenylyl-cyclase activator), and was resistant to pertussis toxin (Gi inhibitor), suggesting a Gs protein signaling. This first signaling also required the downstream Gs signaling effectors PKA and Src. However, Erk1/2 activation at 8h turned out to be pertussis toxin-dependent, and PKA-independent, indicating a Gi signaling pathway in which Src and PI3K were required. The pharmacological inhibition of both the Gs and Gi pathway abolished B3-AR-induced proliferation. Altogether, these data indicate that B3-AR-induced proliferation depends on the biphasic activation of Erk1/2 sequentially induced by the Gs/PKA/Src and Gi/Src/PI3K signaling pathways. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. PKA regulatory subunit expression in tooth development.

    Science.gov (United States)

    de Sousa, Sílvia Ferreira; Kawasaki, Katsushige; Kawasaki, Maiko; Volponi, Ana Angelova; Gomez, Ricardo Santiago; Gomes, Carolina Cavaliéri; Sharpe, Paul T; Ohazama, Atsushi

    2014-05-01

    Protein kinase A (PKA) plays critical roles in many biological processes including cell proliferation, cell differentiation, cellular metabolism and gene regulation. Mutation in PKA regulatory subunit, PRKAR1A has previously been identified in odontogenic myxomas, but it is unclear whether PKA is involved in tooth development. The aim of the present study was to assess the expression of alpha isoforms of PKA regulatory subunit (Prkar1a and Prkar2a) in mouse and human odontogenesis by in situ hybridization. PRKAR1A and PRKAR2A mRNA transcription was further confirmed in a human deciduous germ by qRT-PCR. Mouse Prkar1a and human PRKAR2A exhibited a dynamic spatio-temporal expression in tooth development, whereas neither human PRKAR1A nor mouse Prkar2a showed their expression in odontogenesis. These isoforms thus showed different expression pattern between human and mouse tooth germs. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Ionization constants pKa of cardiolipin.

    Directory of Open Access Journals (Sweden)

    Gerd Olofsson

    Full Text Available Cardiolipin is a phospholipid found in the inner mitochondrial membrane and in bacteria, and it is associated with many physiological functions. Cardiolipin has a dimeric structure consisting of two phosphatidyl residues connected by a glycerol bridge and four acyl chains, and therefore it can carry two negative charges. The pKa values of the phosphate groups have previously been reported to differ widely with pKa1 = 2.8 and pKa2 = 7.5-9.5. Still, there are several examples of experimental observations from cardiolipin-containing systems that do not fit with this dissociation behavior. Therefore, we have carried out pH-titration and titration calorimetric experiments on two synthetic cardiolipins, 1,1',2,2'-tetradecanoyl cardiolipin, CL (C14:0, and 1,1',2,2'-tetraoctadecenoyl cardiolipin, CL (C18:1. Our results show that both behave as strong dibasic acids with pKa1 about the same as the first pKa of phosphoric acid, 2.15, and pKa2 about one unit larger. The characterization of the acidic properties of cardiolipin is crucial for the understanding of the molecular organization in self-assembled systems that contain cardiolipin, and for their biological function.

  4. Interaction of PACAP with Sonic hedgehog reveals complex regulation of the hedgehog pathway by PKA.

    Science.gov (United States)

    Niewiadomski, Pawel; Zhujiang, Annie; Youssef, Mary; Waschek, James A

    2013-11-01

    Sonic hedgehog (Shh) signaling is essential for proliferation of cerebellar granule cell progenitors (cGCPs) and its aberrant activation causes a cerebellar cancer medulloblastoma. Pituitary adenylate cyclase activating polypeptide (PACAP) inhibits Shh-driven proliferation of cGCPs and acts as tumor suppressor in murine medulloblastoma. We show that PACAP blocks canonical Shh signaling by a mechanism that involves activation of protein kinase A (PKA) and inhibition of the translocation of the Shh-dependent transcription factor Gli2 into the primary cilium. PKA is shown to play an essential role in inhibiting gene transcription in the absence of Shh, but global PKA activity levels are found to be a poor predictor of the degree of Shh pathway activation. We propose that the core Shh pathway regulates a small compartmentalized pool of PKA in the vicinity of primary cilia. GPCRs that affect global PKA activity levels, such as the PACAP receptor, cooperate with the canonical Shh signal to regulate Gli protein phosphorylation by PKA. This interaction serves to fine-tune the transcriptional and physiological function of the Shh pathway.

  5. Cardiomyocytes with disrupted CFTR function require CaMKII and Ca(2+)-activated Cl(-) channel activity to maintain contraction rate.

    Science.gov (United States)

    Sellers, Zachary M; De Arcangelis, Vania; Xiang, Yang; Best, Philip M

    2010-07-01

    The physiological role of the cystic fibrosis transmembrane conductance regulator (CFTR) in cardiomyocytes remains unclear. Using spontaneously beating neonatal ventricular cardiomyocytes from wild-type (WT) or CFTR knockout (KO) mice, we examined the role of CFTR in the modulation of cardiomyocyte contraction rate. Contraction rates of spontaneously beating myocytes were captured by video imaging. Real-time changes in intracellular ([Ca(2+)](i)) and protein kinase A (PKA) activity were measured by fura-2 and fluorescence resonance energy transfer, respectively. Acute inhibition of CFTR in WT cardiomyocytes using the CFTR inhibitor CFTR(inh)-172 transiently inhibited the contraction rate. By contrast, cardiomyocytes from CFTR KO mice displayed normal contraction rates. Further investigation revealed that acute inhibition of CFTR activity in WT cardiomyocytes activated L-type Ca(2+) channels, leading to a transient increase of [Ca(2+)](i) and inhibition of PKA activity. Additionally, we found that contraction rate normalization following acute CFTR inhibition in WT cardiomyocytes or chronic deletion in cardiomyocytes from CFTR KO mice requires the activation of Ca(2+)/calmodulin-dependent kinase II (CaMKII) and Ca(2+)-activated Cl(-) channels (CaCC) because simultaneous addition of myristoylated-autocamtide-2-related inhibitory peptide or niflumic acid and CFTR(inh)-172 to WT cardiomyocytes or treatment of cardiomyoctes from CFTR KO mice with these agents caused sustained attenuation of contraction rates. Our results demonstrate that regulation of cardiomyocyte contraction involves CFTR. They also reveal that activation of CaMKII and CaCC compensates for loss of CFTR function. Increased dependence on CaMKII upon loss of CFTR function might leave cystic fibrosis patients at increased risk of heart dysfunction and disease.

  6. Cardiomyocytes with disrupted CFTR function require CaMKII and Ca2+-activated Cl− channel activity to maintain contraction rate

    Science.gov (United States)

    Sellers, Zachary M; De Arcangelis, Vania; Xiang, Yang; Best, Philip M

    2010-01-01

    The physiological role of the cystic fibrosis transmembrane conductance regulator (CFTR) in cardiomyocytes remains unclear. Using spontaneously beating neonatal ventricular cardiomyocytes from wild-type (WT) or CFTR knockout (KO) mice, we examined the role of CFTR in the modulation of cardiomyocyte contraction rate. Contraction rates of spontaneously beating myocytes were captured by video imaging. Real-time changes in intracellular ([Ca2+]i) and protein kinase A (PKA) activity were measured by fura-2 and fluorescence resonance energy transfer, respectively. Acute inhibition of CFTR in WT cardiomyocytes using the CFTR inhibitor CFTRinh-172 transiently inhibited the contraction rate. By contrast, cardiomyocytes from CFTR KO mice displayed normal contraction rates. Further investigation revealed that acute inhibition of CFTR activity in WT cardiomyoctyes activated L-type Ca2+ channels, leading to a transient increase of [Ca2+]i and inhibition of PKA activity. Additionally, we found that contraction rate normalization following acute CFTR inhibition in WT cardiomyocytes or chronic deletion in cardiomyocytes from CFTR KO mice requires the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and Ca2+-activated Cl− channels (CaCC) because simultaneous addition of myristoylated-autocamtide-2-related inhibitory peptide or niflumic acid and CFTRinh-172 to WT cardiomyocytes or treatment of cardiomyoctes from CFTR KO mice with these agents caused sustained attenuation of contraction rates. Our results demonstrate that regulation of cardiomyocyte contraction involves CFTR. They also reveal that activation of CaMKII and CaCC compensates for loss of CFTR function. Increased dependence on CaMKII upon loss of CFTR function might leave cystic fibrosis patients at increased risk of heart dysfunction and disease. PMID:20442264

  7. Chemical tools selectively target components of the PKA system

    Directory of Open Access Journals (Sweden)

    Drewianka Stephan

    2009-02-01

    Full Text Available Abstract Background In the eukaryotic cell the cAMP-dependent protein kinase (PKA is a key enzyme in signal transduction and represents the main target of the second messenger cAMP. Here we describe the design, synthesis and characterisation of specifically tailored cAMP analogs which can be utilised as a tool for affinity enrichment and purification as well as for proteomics based analyses of cAMP binding proteins. Results Two sets of chemical binders were developed based on the phosphorothioate derivatives of cAMP, Sp-cAMPS and Rp-cAMPS acting as cAMP-agonists and -antagonists, respectively. These compounds were tested via direct surface plasmon resonance (SPR analyses for their binding properties to PKA R-subunits and holoenzyme. Furthermore, these analogs were used in an affinity purification approach to analyse their binding and elution properties for the enrichment and improvement of cAMP binding proteins exemplified by the PKA R-subunits. As determined by SPR, all tested Sp-analogs provide valuable tools for affinity chromatography. However, Sp-8-AEA-cAMPS displayed (i superior enrichment properties while maintaining low unspecific binding to other proteins in crude cell lysates, (ii allowing mild elution conditions and (iii providing the capability to efficiently purify all four isoforms of active PKA R-subunit in milligram quantities within 8 h. In a chemical proteomics approach both sets of binders, Rp- and Sp-cAMPS derivatives, can be employed. Whereas Sp-8-AEA-cAMPS preferentially binds free R-subunit, Rp-AHDAA-cAMPS, displaying antagonist properties, not only binds to the free PKA R-subunits but also to the intact PKA holoenzyme both from recombinant and endogenous sources. Conclusion In summary, all tested cAMP analogs were useful for their respective application as an affinity reagent which can enhance purification of cAMP binding proteins. Sp-8-AEA-cAMPS was considered the most efficient analog since Sp-8-AHA-cAMPS and Sp-2-AHA

  8. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Seon Sook; Lee, Eun Hye [Department of Molecular Bioscience, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Lee, Kooyeon [Department of Bio-Health Technology, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Jo, Su-Hyun, E-mail: suhyunjo@kangwon.ac.kr [Department of Physiology, BK21 Plus Graduate Program, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Seo, Su Ryeon, E-mail: suryeonseo@kangwon.ac.kr [Department of Molecular Bioscience, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of)

    2015-04-17

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression.

  9. PGE2 promotes angiogenesis through EP4 and PKA Cγ pathway

    Science.gov (United States)

    Zhang, Yushan

    2011-01-01

    Inflammation is increasingly recognized as a critical mediator of angiogenesis, and unregulated angiogenic response is involved in human diseases, including cancer. Proinflammatory prostaglandin E2 (PGE2) is secreted by many cell types and plays important roles in the process of angiogenesis via activation of cognate EP1-4 receptors. Here, we provide evidence that PGE2 promotes the in vitro tube formation of human microvascular endothelial cells, ex vivo vessel outgrowth of aortic rings, and actual in vivo angiogenesis. Use of EP subtype-selective agonists and antagonists suggested EP4 mediates the prostaglandin-induced tube formation, and this conclusion was substantiated with small interfering RNA to specifically knockdown the EP4 expression. EP4 couples to Gαs, leading to activation of protein kinase A (PKA). Inhibition of PKA activity or knockdown of PKA catalytic subunit γ with RNAi attenuates the PGE2-induced tube formation. Further, knocking down the expression of Rap1A, HSPB6, or endothelial NO synthase, which serve as PKA-activatable substrates, inhibits the tube formation, whereas knockdown of RhoA or glycogen synthase kinase 3β that are inactivated after phosphorylation by PKA increases the tube formation. These results support the existence of EP4-to-PKA angiogenic signal and provide rationale for use of selective EP4 signal inhibitors as a probable strategy to control pathologic angiogenesis. PMID:21926356

  10. Protein kinase A increases type-2 inositol 1,4,5-trisphosphate receptor activity by phosphorylation of serine 937.

    Science.gov (United States)

    Betzenhauser, Matthew J; Fike, Jenna L; Wagner, Larry E; Yule, David I

    2009-09-11

    Protein kinase A (PKA) phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) represents a mechanism for shaping intracellular Ca(2+) signals following a concomitant elevation in cAMP. Activation of PKA results in enhanced Ca(2+) release in cells that express predominantly InsP(3)R2. PKA is known to phosphorylate InsP(3)R2, but the molecular determinants of this effect are not known. We have expressed mouse InsP(3)R2 in DT40-3KO cells that are devoid of endogenous InsP(3)R and examined the effects of PKA phosphorylation on this isoform in unambiguous isolation. Activation of PKA increased Ca(2+) signals and augmented the single channel open probability of InsP(3)R2. A PKA phosphorylation site unique to the InsP(3)R2 was identified at Ser(937). The enhancing effects of PKA activation on this isoform required the phosphorylation of Ser(937), since replacing this residue with alanine eliminated the positive effects of PKA activation. These results provide a mechanism responsible for the enhanced Ca(2+) signaling following PKA activation in cells that express predominantly InsP(3)R2.

  11. NMR determination of lysine pKa values in the Pol lambda lyase domain: mechanistic implications.

    Science.gov (United States)

    Gao, Guanghua; DeRose, Eugene F; Kirby, Thomas W; London, Robert E

    2006-02-14

    The base excision repair (BER) process requires removal of an abasic deoxyribose-5-phosphate group, a catalytic activity that has been demonstrated for the N-terminal 8 kDa domain of DNA polymerase beta (Pol beta), and for the homologous domain of DNA polymerase lambda (Pol lambda). Previous studies have demonstrated that this activity results from formation of a Schiff base adduct of the abasic deoxyribose C-1' with a lysine residue (K312 in the case of Pol lambda), followed by a beta-elimination reaction. To better understand the underlying chemistry, we have determined pKa values for the lysine residues in the Pol lambda lyase domain labeled with [epsilon-13C]lysine. At neutral pH, the H(epsilon) protons on 3 of the 10 lysine residues in this domain, K287, K291, and K312, exhibit chemical shift inequivalence that results from immobilization of the lysyl side chains. For K287 and K291, this results from the K287-E261 and K291-E298 salt bridge interactions, while for K312, immobilization apparently results from steric and hydrogen-bonding interactions that constrain the position of the lysine side chain. The pKa value of K312 is depressed to 9.58, a value indicating that at physiological pH K312 will exist predominantly in the protonated form. Titration of the domain with hairpin DNA containing a 5'-tetrahydrofuran terminus to model the abasic site produced shifts of the labeled lysine resonances that were in fast exchange but appeared to be complete at a stoichiometry of approximately 1:1.3, consistent with a dissociation constant of approximately 1 microM. The epsilon-proton shifts of K273 were the most sensitive to the addition of the DNA, apparently due to changes in the relative orientation between K273 and W274 in the DNA complex. The average pKa values increased by 0.55, consistent with the formation of some DNA-lysine salt bridges and with the general pH increase expected to result from a reduction in the net positive charge of the complex. A general

  12. Inhibition of the cAMP/PKA/CREB Pathway Contributes to the Analgesic Effects of Electroacupuncture in the Anterior Cingulate Cortex in a Rat Pain Memory Model

    Directory of Open Access Journals (Sweden)

    Xiao-Mei Shao

    2016-01-01

    Full Text Available Pain memory is considered as endopathic factor underlying stubborn chronic pain. Our previous study demonstrated that electroacupuncture (EA can alleviate retrieval of pain memory. This study was designed to observe the different effects between EA and indomethacin (a kind of nonsteroid anti-inflammatory drugs, NSAIDs in a rat pain memory model. To explore the critical role of protein kinase A (PKA in pain memory, a PKA inhibitor was microinjected into anterior cingulate cortex (ACC in model rats. We further investigated the roles of the cyclic adenosine monophosphate (cAMP, PKA, cAMP response element-binding protein (CREB, and cAMP/PKA/CREB pathway in pain memory to explore the potential molecular mechanism. The results showed that EA alleviates the retrieval of pain memory while indomethacin failed. Intra-ACC microinjection of a PKA inhibitor blocked the occurrence of pain memory. EA reduced the activation of cAMP, PKA, and CREB and the coexpression levels of cAMP/PKA and PKA/CREB in the ACC of pain memory model rats, but indomethacin failed. The present findings identified a critical role of PKA in ACC in retrieval of pain memory. We propose that the proper mechanism of EA on pain memory is possibly due to the partial inhibition of cAMP/PKA/CREB signaling pathway by EA.

  13. pK(a) prediction from "Quantum Chemical Topology" descriptors.

    Science.gov (United States)

    Harding, A P; Wedge, D C; Popelier, P L A

    2009-08-01

    Knowing the pK(a) of a compound gives insight into many properties relevant to many industries, in particular the pharmaceutical industry during drug development processes. In light of this, we have used the theory of Quantum Chemical Topology (QCT), to provide ab initio descriptors that are able to accurately predict pK(a) values for 228 carboxylic acids. This Quantum Topological Molecular Similarity (QTMS) study involved the comparison of 5 increasingly more expensive levels of theory to conclude that HF/6-31G(d) and B3LYP/6-311+G(2d,p) provided an accurate representation of the compounds studies. We created global and subset models for the carboxylic acids using Partial Least Square (PLS), Support Vector Machines (SVM), and Radial Basis Function Neural Networks (RBFNN). The models were extensively validated using 4-, 7-, and 10-fold cross-validation, with the validation sets selected based on systematic and random sampling. HF/6-31G(d) in conjunction with SVM provided the best statistics when taking into account the large increase in CPU time required to optimize the geometries at the B3LYP/6-311+G(2d,p) level. The SVM models provided an average q(2) value of 0.886 and an RMSE value of 0.293 for all the carboxylic acids, a q(2) of 0.825 and RMSE of 0.378 for the ortho-substituted acids, a q(2) of 0.923 and RMSE of 0.112 for the para- and meta-substituted acids, and a q(2) of 0.906 and RMSE of 0.268 for the aliphatic acids. Our method compares favorably to ACD/Laboratories, VCCLAB, SPARC, and ChemAxon's pK(a) prediction software based of the RMSE calculated by the leave-one-out method.

  14. Sensory deprivation unmasks a PKA-dependent synaptic plasticity mechanism that operates in parallel with CaMKII.

    Science.gov (United States)

    Hardingham, Neil; Wright, Nick; Dachtler, James; Fox, Kevin

    2008-12-10

    Calcium/calmodulin kinase II (CaMKII) is required for LTP and experience-dependent potentiation in the barrel cortex. Here, we find that whisker deprivation increases LTP in the layer IV to II/III pathway and that PKA antagonists block the additional LTP. No LTP was seen in undeprived CaMKII-T286A mice, but whisker deprivation again unmasked PKA-sensitive LTP. Infusion of a PKA agonist potentiated EPSPs in deprived wild-types and deprived CaMKII-T286A point mutants but not in undeprived animals of either genotype. The PKA-dependent potentiation mechanism was not present in GluR1 knockouts. Infusion of a PKA antagonist caused depression of EPSPs in undeprived but not deprived cortex. LTD was occluded by whisker deprivation and blocked by PKA manipulation, but not blocked by cannabinoid antagonists. NMDA receptor currents were unaffected by sensory deprivation. These results suggest that sensory deprivation causes synaptic depression by reversing a PKA-dependent process that may act via GluR1.

  15. PKA regulatory subunit 1A inactivating mutation induces serotonin signaling in primary pigmented nodular adrenal disease.

    Science.gov (United States)

    Bram, Zakariae; Louiset, Estelle; Ragazzon, Bruno; Renouf, Sylvie; Wils, Julien; Duparc, Céline; Boutelet, Isabelle; Rizk-Rabin, Marthe; Libé, Rossella; Young, Jacques; Carson, Dennis; Vantyghem, Marie-Christine; Szarek, Eva; Martinez, Antoine; Stratakis, Constantine A; Bertherat, Jérôme; Lefebvre, Hervé

    2016-09-22

    Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent hypercortisolism. The disease is primarily caused by germline mutations of the protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene, which induces constitutive activation of PKA in adrenocortical cells. Hypercortisolism is thought to result from PKA hyperactivity, but PPNAD tissues exhibit features of neuroendocrine differentiation, which may lead to stimulation of steroidogenesis by abnormally expressed neurotransmitters. We hypothesized that serotonin (5-HT) may participate in the pathophysiology of PPNAD-associated hypercortisolism. We show that PPNAD tissues overexpress the 5-HT synthesizing enzyme tryptophan hydroxylase type 2 (Tph2) and the serotonin receptors types 4, 6, and 7, leading to formation of an illicit stimulatory serotonergic loop whose pharmacological inhibition in vitro decreases cortisol production. In the human PPNAD cell line CAR47, the PKA inhibitor H-89 decreases 5-HT4 and 5-HT7 receptor expression. Moreover, in the human adrenocortical cell line H295R, inhibition of PRKAR1A expression increases the expression of Tph2 and 5-HT4/6/7 receptors, an effect that is blocked by H-89. These findings show that the serotonergic process observed in PPNAD tissues results from PKA activation by PRKAR1A mutations. They also suggest that Tph inhibitors may represent efficient treatments of hypercortisolism in patients with PPNAD.

  16. PKA regulatory subunit 1A inactivating mutation induces serotonin signaling in primary pigmented nodular adrenal disease

    Science.gov (United States)

    Bram, Zakariae; Louiset, Estelle; Renouf, Sylvie; Duparc, Céline; Boutelet, Isabelle; Rizk-Rabin, Marthe; Libé, Rossella; Young, Jacques; Carson, Dennis; Vantyghem, Marie-Christine; Szarek, Eva; Martinez, Antoine; Stratakis, Constantine A.; Bertherat, Jérôme

    2016-01-01

    Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent hypercortisolism. The disease is primarily caused by germline mutations of the protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene, which induces constitutive activation of PKA in adrenocortical cells. Hypercortisolism is thought to result from PKA hyperactivity, but PPNAD tissues exhibit features of neuroendocrine differentiation, which may lead to stimulation of steroidogenesis by abnormally expressed neurotransmitters. We hypothesized that serotonin (5-HT) may participate in the pathophysiology of PPNAD-associated hypercortisolism. We show that PPNAD tissues overexpress the 5-HT synthesizing enzyme tryptophan hydroxylase type 2 (Tph2) and the serotonin receptors types 4, 6, and 7, leading to formation of an illicit stimulatory serotonergic loop whose pharmacological inhibition in vitro decreases cortisol production. In the human PPNAD cell line CAR47, the PKA inhibitor H-89 decreases 5-HT4 and 5-HT7 receptor expression. Moreover, in the human adrenocortical cell line H295R, inhibition of PRKAR1A expression increases the expression of Tph2 and 5-HT4/6/7 receptors, an effect that is blocked by H-89. These findings show that the serotonergic process observed in PPNAD tissues results from PKA activation by PRKAR1A mutations. They also suggest that Tph inhibitors may represent efficient treatments of hypercortisolism in patients with PPNAD. PMID:27699247

  17. PKA-mediated phosphorylation of EPEC-Tir at serine residues 434 and 463: A novel pathway in regulating Rac1 GTPase function.

    Science.gov (United States)

    Backert, Steffen; Kenny, Brendan; Gerhard, Ralf; Tegtmeyer, Nicole; Brandt, Sabine

    2010-03-01

    Type-III or type-IV secretion systems of many Gram-negative bacterial pathogens inject effector proteins into host cells that modulate cellular functions in their favour. A preferred target of these effectors is the actin-cytoskeleton as shown by studies using the gastric pathogens Helicobacter pylori (H. pylori) and enteropathogenic Escherichia coli (EPEC). We recently developed a co-infection approach to study effector protein function and molecular mechanisms by which they highjack cellular signalling cascades. This is exemplified by our observation that EPEC profoundly blocks H. pylori-induced epithelial cell scattering and elongation, a disease-related event requiring the activity of small Rho GTPase Rac1. While this suppressive effect is dependent on the effector protein Tir and the outer-membrane protein Intimin, it unexpectedly revealed evidence for Tir-signalling independent of phosphorylation of Tir at tyrosine residues 454 and 474. Instead, our studies revealed a previously unidentified function for protein kinase A (PKA)-mediated phosphorylation of Tir at serine residues 434 and 463. We demonstrated that EPEC infection activates PKA for Tir phosphorylation. Activated PKA then phosphorylates Rac1 at its serine residue 71 associated with reduced GTP-load and inhibited cell elongation. Phosphorylation of Rho GTPases such as Rac1 might be an interesting novel strategy in microbial pathogenesis.

  18. Reduced ATGL-mediated lipolysis attenuates β-adrenergic-induced AMPK signaling, but not the induction of PKA-targeted genes, in adipocytes and adipose tissue.

    Science.gov (United States)

    MacPherson, Rebecca E K; Dragos, Steven M; Ramos, Sofhia; Sutton, Charles; Frendo-Cumbo, Scott; Castellani, Laura; Watt, Matthew J; Perry, Christopher G R; Mutch, David M; Wright, David C

    2016-08-01

    5'-AMP-activated protein kinase (AMPK) is activated as a consequence of lipolysis and has been shown to play a role in regulation of adipose tissue mitochondrial content. Conversely, the inhibition of lipolysis has been reported to potentiate the induction of protein kinase A (PKA)-targeted genes involved in the regulation of oxidative metabolism. The purpose of the current study was to address these apparent discrepancies and to more fully examine the relationship between lipolysis, AMPK, and the β-adrenergic-mediated regulation of gene expression. In 3T3-L1 adipocytes, the adipose tissue triglyceride lipase (ATGL) inhibitor ATGListatin attenuated the Thr(172) phosphorylation of AMPK by a β3-adrenergic agonist (CL 316,243) independent of changes in PKA signaling. Similarly, CL 316,243-induced increases in the Thr(172) phosphorylation of AMPK were reduced in adipose tissue from whole body ATGL-deficient mice. Despite reductions in the activation of AMPK, the induction of PKA-targeted genes was intact or, in some cases, increased. Similarly, markers of mitochondrial content and respiration were increased in adipose tissue from ATGL knockout mice independent of changes in the Thr(172) phosphorylation of AMPK. Taken together, our data provide evidence that AMPK is not required for the regulation of adipose tissue oxidative capacity in conditions of reduced fatty acid release. Copyright © 2016 the American Physiological Society.

  19. Estrogenic flavonoids: structural requirements for biological activity.

    Science.gov (United States)

    Miksicek, R J

    1995-01-01

    A systematic survey of polycyclic phenols has been performed to identify members of this chemical group with estrogenic activity. Twelve compounds were found to be able to stimulate the transcriptional activity of the human estrogen receptor expressed in cultured cells by transient transfection. These natural estrogens belong to several distinct, but chemically related classes including chalcones, flavanones, flavones, flavonols, and isoflavones. Selected examples of estrogenic flavonoids were further analyzed to determine their biological potencies and their relative affinities for binding to the estrogen receptor. These data are interpreted with respect to the molecular structure of polycyclic phenols required for hormonal activity as nonsteroidal estrogens.

  20. Expression of a dominant negative PKA mutation in the kidney elicits a diabetes insipidus phenotype.

    Science.gov (United States)

    Gilbert, Merle L; Yang, Linghai; Su, Thomas; McKnight, G Stanley

    2015-03-15

    PKA plays a critical role in water excretion through regulation of the production and action of the antidiuretic hormone arginine vasopressin (AVP). The AVP prohormone is produced in the hypothalamus, where its transcription is regulated by cAMP. Once released into the circulation, AVP stimulates antidiuresis through activation of vasopressin 2 receptors in renal principal cells. Vasopressin 2 receptor activation increases cAMP and activates PKA, which, in turn, phosphorylates aquaporin (AQP)2, triggering apical membrane accumulation, increased collecting duct permeability, and water reabsorption. We used single-minded homolog 1 (Sim1)-Cre recombinase-mediated expression of a dominant negative PKA regulatory subunit (RIαB) to disrupt kinase activity in vivo and assess the role of PKA in fluid homeostasis. RIαB expression gave rise to marked polydipsia and polyuria; however, neither hypothalamic Avp mRNA expression nor urinary AVP levels were attenuated, indicating a primary physiological effect on the kidney. RIαB mice displayed a marked deficit in urinary concentrating ability and greatly reduced levels of AQP2 and phospho-AQP2. Dehydration induced Aqp2 mRNA in the kidney of both control and RIαB-expressing mice, but AQP2 protein levels were still reduced in RIαB-expressing mutants, and mice were unable to fully concentrate their urine and conserve water. We conclude that partial PKA inhibition in the kidney leads to posttranslational effects that reduce AQP2 protein levels and interfere with apical membrane localization. These findings demonstrate a distinct physiological role for PKA signaling in both short- and long-term regulation of AQP2 and characterize a novel mouse model of diabetes insipidus.

  1. Studies of mice with cyclic AMP-dependent protein kinase (PKA) defects reveal the critical role of PKA's catalytic subunits in anxiety

    Science.gov (United States)

    Briassoulis, George; Keil, Margaret F.; Naved, Bilal; Liu, Sophie; Starost, Matthew F.; Nesterova, Maria; Gokarn, Nirmal; Batistatos, Anna; Wu, T. John; Stratakis, Constantine A.

    2016-01-01

    Cyclic adenosine mono-phosphate-dependent protein kinase (PKA) is critically involved in the regulation of behavioral responses. Previous studies showed that PKA's main regulatory subunit, R1α, is involved in anxiety-like behaviors. The purpose of this study was to determine how the catalytic subunit, Cα, might affect R1α's function and determine its effects on anxiety-related behaviors. The marble bury (MB) and elevated plus maze (EPM) tests were used to assess anxiety-like behavior and the hotplate test to assess nociception in wild type (WT) mouse, a Prkar1a heterozygote (Prkar1a+/-) mouse with haploinsufficiency for the regulatory subunit (R1α), a Prkaca heterozygote (Prkaca+/-) mouse with haploinsufficiency for the catalytic subunit (Cα), and a double heterozygote mouse (Prkar1a+/-/Prkaca+/-) with haploinsufficiency for both R1α and Cα. We then examined specific brain nuclei involved in anxiety. Results of MB test showed a genotype effect, with increased anxiety-like behavior in Prkar1a+/- and Prkar1a+/-/Prkaca+/- compared to WT mice. In the EPM, Prkar1a+/- spent significantly less time in the open arms, while Prkaca+/- and Prkar1a+/-/Prkaca+/- mice displayed less exploratory behavior compared to WT mice. The loss of one Prkar1a allele was associated with a significant increase in PKA activity in the basolateral (BLA) and central (CeA) amygdala and ventromedial hypothalamus (VMH) in both Prkar1a+/- and Prkar1a+/- /Prkaca+/- mice. Alterations of PKA activity induced by haploinsufficiency of its main regulatory or most important catalytic subunits result in anxiety-like behaviors. The BLA, CeA, and VMH are implicated in mediating these PKA effects in brain. PMID:26992826

  2. Receptor-mediated Ca2+ and PKC signaling triggers the loss of cortical PKA compartmentalization through the redistribution of gravin.

    Science.gov (United States)

    Schott, Micah B; Grove, Bryon

    2013-11-01

    A-Kinase Anchoring Proteins (AKAPs) direct the flow of cellular information by positioning multiprotein signaling complexes into proximity with effector proteins. However, certain AKAPs are not stationary but can undergo spatiotemporal redistribution in response to stimuli. Gravin, a 300kD AKAP that intersects with a diverse signaling array, is localized to the plasma membrane but has been shown to translocate to the cytosol following the elevation of intracellular calcium ([Ca(2+)]i). Despite the potential for gravin redistribution to impact multiple signaling pathways, the dynamics of this event remain poorly understood. In this study, quantitative microscopy of cells expressing gravin-EGFP revealed that Ca(2+) elevation caused the complete translocation of gravin from the cell cortex to the cytosol in as little as 60s of treatment with ionomycin or thapsigargin. In addition, receptor mediated signaling was also shown to cause gravin redistribution following ATP treatment, and this event required both [Ca(2+)]i elevation and PKC activation. To understand the mechanism for Ca(2+) mediated gravin dynamics, deletion of calmodulin-binding domains revealed that a fourth putative calmodulin binding domain called CB4 (a.a. 670-694) is critical for targeting gravin to the cell cortex despite its location downstream of gravin's membrane-targeting domains, which include an N-terminal myristoylation site and three polybasic domains. Finally, confocal microscopy of cells co-transfected with gravin-EYFP and PKA RII-ECFP revealed that gravin redistribution mediated by ionomycin, thapsigargin, and ATP each triggered the gravin-dependent loss of PKA localized at the cell cortex. Our results support the hypothesis that gravin redistribution regulates cross-talk between PKA-dependent signaling and receptor-mediated events involving Ca(2+) and PKC. © 2013.

  3. Active cAMP-dependent protein kinase incorporated within highly purified HIV-1 particles is required for viral infectivity and interacts with viral capsid protein.

    Science.gov (United States)

    Cartier, Christine; Hemonnot, Bénédicte; Gay, Bernard; Bardy, Martine; Sanchiz, Céline; Devaux, Christian; Briant, Laurence

    2003-09-12

    Host cell components, including protein kinases such as ERK-2/mitogen-activated protein kinase, incorporated within human immunodeficiency virus type 1 (HIV-1) virions play a pivotal role in the ability of HIV to infect and replicate in permissive cells. The present work provides evidence that the catalytic subunit of cAMP-dependent protein kinase (C-PKA) is packaged within HIV-1 virions as demonstrated using purified subtilisin-digested viral particles. Virus-associated C-PKA was shown to be enzymatically active and able to phosphorylate synthetic substrate in vitro. Suppression of virion-associated C-PKA activity by specific synthetic inhibitor had no apparent effect on viral precursor maturation and virus assembly. However, virus-associated C-PKA activity was demonstrated to regulate HIV-1 infectivity as assessed by single round infection assays performed by using viruses produced from cells expressing an inactive form of C-PKA. In addition, virus-associated C-PKA was found to co-precipitate with and to phosphorylate the CAp24gag protein. Altogether our results indicate that virus-associated C-PKA regulates HIV-1 infectivity, possibly by catalyzing phosphorylation of the viral CAp24gag protein.

  4. cAMP/PKA Signaling Inhibits Osteogenic Differentiation and Bone Formation in Rodent Models

    NARCIS (Netherlands)

    Siddappa, Ramakrishnaiah; Mulder, Winfried; Steeghs, Ilse; Klundert, van de Christine; Fernandes, Hugo; Liu, Jun; Arends, Roel; Blitterswijk, van Clemens; Boer, de Jan

    2009-01-01

    We previously demonstrated that cAMP-mediated protein kinase A (PKA) activation induces in vitro osteogenesis and in vivo bone formation by human mesenchymal stem cells (hMSCs). To analyze the species-specific response of this phenomenon and to translate our findings into a clinical trial, suitable

  5. Environmental enrichment modifies the PKA-dependence of hippocampal LTP and improves hippocampus-dependent memory.

    Science.gov (United States)

    Duffy, S N; Craddock, K J; Abel, T; Nguyen, P V

    2001-01-01

    cAMP-dependent protein kinase (PKA) is critical for the expression of some forms of long-term potentiation (LTP) in area CA1 of the mouse hippocampus and for hippocampus-dependent memory. Exposure to spatially enriched environments can modify LTP and improve behavioral memory in rodents, but the molecular bases for the enhanced memory performance seen in enriched animals are undefined. We tested the hypothesis that exposure to a spatially enriched environment may alter the PKA dependence of hippocampal LTP. Hippocampal slices from enriched mice showed enhanced LTP following a single burst of 100-Hz stimulation in the Schaffer collateral pathway of area CA1. In slices from nonenriched mice, this single-burst form of LTP was less robust and was unaffected by Rp-cAMPS, an inhibitor of PKA. In contrast, the enhanced LTP in enriched mice was attenuated by Rp-cAMPS. Enriched slices expressed greater forskolin-induced, cAMP-dependent synaptic facilitation than did slices from nonenriched mice. Enriched mice showed improved memory for contextual fear conditioning, whereas memory for cued fear conditioning was unaffected following enrichment. Our data indicate that exposure of mice to spatial enrichment alters the PKA dependence of LTP and enhances one type of hippocampus-dependent memory. Environmental enrichment can transform the pharmacological profile of hippocampal LTP, possibly by altering the threshold for activity-dependent recruitment of the cAMP-PKA signaling pathway following electrical and chemical stimulation. We suggest that experience-dependent plasticity of the PKA dependence of hippocampal LTP may be important for regulating the efficacy of hippocampus-based memory.

  6. Compartmentalization Role of A-Kinase Anchoring Proteins (AKAPs in Mediating Protein Kinase A (PKA Signaling and Cardiomyocyte Hypertrophy

    Directory of Open Access Journals (Sweden)

    Abeer Rababa'h

    2014-12-01

    Full Text Available The Beta-adrenergic receptors (β-ARs stimulation enhances contractility through protein kinase-A (PKA substrate phosphorylation. This PKA signaling is conferred in part by PKA binding to A-kinase anchoring proteins (AKAPs. AKAPs coordinate multi-protein signaling networks that are targeted to specific intracellular locations, resulting in the localization of enzyme activity and transmitting intracellular actions of neurotransmitters and hormones to its target substrates. In particular, mAKAP (muscle-selective AKAP has been shown to be present on the nuclear envelope of cardiomyocytes with various proteins including: PKA-regulatory subunit (RIIα, phosphodiesterase-4D3, protein phosphatase-2A, and ryanodine receptor (RyR2. Therefore, through the coordination of spatial-temporal signaling of proteins and enzymes, mAKAP controls cyclic-adenosine monophosphate (cAMP levels very tightly and functions as a regulator of PKA-mediated substrate phosphorylation leading to changes in calcium availability and myofilament calcium sensitivity. The goal of this review is to elucidate the critical compartmentalization role of mAKAP in mediating PKA signaling and regulating cardiomyocyte hypertrophy by acting as a scaffolding protein. Based on our literature search and studying the structure–function relationship between AKAP scaffolding protein and its binding partners, we propose possible explanations for the mechanism by which mAKAP promotes cardiac hypertrophy.

  7. Piezoelectric Power Requirements for Active Vibration Control

    Science.gov (United States)

    Brennan, Matthew C.; McGowan, Anna-Maria Rivas

    1997-01-01

    This paper presents a method for predicting the power consumption of piezoelectric actuators utilized for active vibration control. Analytical developments and experimental tests show that the maximum power required to control a structure using surface-bonded piezoelectric actuators is independent of the dynamics between the piezoelectric actuator and the host structure. The results demonstrate that for a perfectly-controlled system, the power consumption is a function of the quantity and type of piezoelectric actuators and the voltage and frequency of the control law output signal. Furthermore, as control effectiveness decreases, the power consumption of the piezoelectric actuators decreases. In addition, experimental results revealed a non-linear behavior in the material properties of piezoelectric actuators. The material non- linearity displayed a significant increase in capacitance with an increase in excitation voltage. Tests show that if the non-linearity of the capacitance was accounted for, a conservative estimate of the power can easily be determined.

  8. Calculated pKa Variations Expose Dynamic Allosteric Communication Networks.

    Science.gov (United States)

    Lang, Eric J M; Heyes, Logan C; Jameson, Geoffrey B; Parker, Emily J

    2016-02-17

    Allosteric regulation of protein function, the process by which binding of an effector molecule provokes a functional response from a distal site, is critical for metabolic pathways. Yet, the way the allosteric signal is communicated remains elusive, especially in dynamic, entropically driven regulation mechanisms for which no major conformational changes are observed. To identify these dynamic allosteric communication networks, we have developed an approach that monitors the pKa variations of ionizable residues over the course of molecular dynamics simulations performed in the presence and absence of an allosteric regulator. As the pKa of ionizable residues depends on their environment, it represents a simple metric to monitor changes in several complex factors induced by binding an allosteric effector. These factors include Coulombic interactions, hydrogen bonding, and solvation, as well as backbone motions and side chain fluctuations. The predictions that can be made with this method concerning the roles of ionizable residues for allosteric communication can then be easily tested experimentally by changing the working pH of the protein or performing single point mutations. To demonstrate the method's validity, we have applied this approach to the subtle dynamic regulation mechanism observed for Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first enzyme of aromatic biosynthesis. We were able to identify key communication pathways linking the allosteric binding site to the active site of the enzyme and to validate these findings experimentally by reestablishing the catalytic activity of allosterically inhibited enzyme via modulation of the working pH, without compromising the binding affinity of the allosteric regulator.

  9. Constitutive Activation of the G-Protein Subunit G[alpha]s within Forebrain Neurons Causes PKA-Dependent Alterations in Fear Conditioning and Cortical "Arc" mRNA Expression

    Science.gov (United States)

    Kelly, Michele P.; Cheung, York-Fong; Favilla, Christopher; Siegel, Steven J.; Kanes, Stephen J.; Houslay, Miles D.; Abel, Ted

    2008-01-01

    Memory formation requires cAMP signaling; thus, this cascade has been of great interest in the search for cognitive enhancers. Given that medications are administered long-term, we determined the effects of chronically increasing cAMP synthesis in the brain by expressing a constitutively active isoform of the G-protein subunit G[alpha]s…

  10. Mechanisms Underlying the Antidepressant Response of Acupuncture via PKA/CREB Signaling Pathway

    National Research Council Canada - National Science Library

    Huili Jiang; Xuhui Zhang; Yu Wang; Huimin Zhang; Jing Li; Xinjing Yang; Bingcong Zhao; Chuntao Zhang; Miao Yu; Mingmin Xu; Qiuyun Yu; Xingchen Liang; Xiang Li; Peng Shi; Tuya Bao

    2017-01-01

    .... The objective was to identify the mechanisms underlying the antidepressant response of acupuncture through PKA signaling pathway in depression rats by employing the PKA signaling pathway inhibitor...

  11. PKA Phosphorylation of NCLX Reverses Mitochondrial Calcium Overload and Depolarization, Promoting Survival of PINK1-Deficient Dopaminergic Neurons

    Directory of Open Access Journals (Sweden)

    Marko Kostic

    2015-10-01

    Full Text Available Mitochondrial Ca2+ overload is a critical, preceding event in neuronal damage encountered during neurodegenerative and ischemic insults. We found that loss of PTEN-induced putative kinase 1 (PINK1 function, implicated in Parkinson disease, inhibits the mitochondrial Na+/Ca2+ exchanger (NCLX, leading to impaired mitochondrial Ca2+ extrusion. NCLX activity was, however, fully rescued by activation of the protein kinase A (PKA pathway. We further show that PKA rescues NCLX activity by phosphorylating serine 258, a putative regulatory NCLX site. Remarkably, a constitutively active phosphomimetic mutant of NCLX (NCLXS258D prevents mitochondrial Ca2+ overload and mitochondrial depolarization in PINK1 knockout neurons, thereby enhancing neuronal survival. Our results identify an mitochondrial Ca2+ transport regulatory pathway that protects against mitochondrial Ca2+ overload. Because mitochondrial Ca2+ dyshomeostasis is a prominent feature of multiple disorders, the link between NCLX and PKA may offer a therapeutic target.

  12. Novel cytochrome P450, cyp6a17, is required for temperature preference behavior in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jongkyun Kang

    Full Text Available Perception of temperature is an important brain function for organisms to survive. Evidence suggests that temperature preference behavior (TPB in Drosophila melanogaster, one of poikilothermal animals, is regulated by cAMP-dependent protein kinase (PKA signaling in mushroom bodies of the brain. However, downstream targets for the PKA signaling in this behavior have not been identified. From a genome-wide search for the genes regulated by PKA activity in the mushroom bodies, we identified the cyp6a17 Cytochrome P450 gene as a new target for PKA. Our detailed analysis of mutants by genetic, molecular and behavioral assays shows that cyp6a17 is essential for temperature preference behavior. cyp6a17 expression is enriched in the mushroom bodies of the adult brain. Tissue-specific knockdown and rescue experiments demonstrate that cyp6a17 is required in the mushroom bodies for normal temperature preference behavior. This is the first study, to our knowledge, to show PKA-dependent expression of a cytochrome P450 gene in the mushroom bodies and its role as a key factor for temperature preference behavior. Taken together, this study reveals a new PKA-Cytochrome P450 pathway that regulates the temperature preference behavior.

  13. Altered Regulation of Protein Kinase A Activity in the Medial Prefrontal Cortex of Normal and Brain-Injured Animals Actively Engaged in a Working Memory Task

    OpenAIRE

    Kobori, Nobuhide; Moore, Anthony N.; Pramod K Dash

    2015-01-01

    Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signaling is required for short- and long-term memory. In contrast, enhanced PKA activity has been shown to impair working memory, a prefrontal cortex (PFC)-dependent, transient form of memory critical for cognition and goal-directed behaviors. Working memory can be impaired after traumatic brain injury (TBI) in the absence of overt damage to the PFC. The cellular and molecular mechanisms that contribute to this deficit ar...

  14. Advanced Extravehicular Activity Pressure Garment Requirements Development

    Science.gov (United States)

    Ross, Amy

    2014-01-01

    The NASA Johnson Space Center advanced pressure garment technology development team is addressing requirements development for exploration missions. Lessons learned from the Z-2 high fidelity prototype development have reiterated that clear low-level requirements and verification methods reduce risk to the government, improve efficiency in pressure garment design efforts, and enable the government to be a smart buyer. The expectation is to provide requirements at the specification level that are validated so that their impact on pressure garment design is understood. Additionally, the team will provide defined verification protocols for the requirements. However, in reviewing exploration space suit high level requirements there are several gaps in the team's ability to define and verify related lower level requirements. This paper addresses the efforts in requirement areas such as mobility/fit/comfort and environmental protection (dust, radiation, plasma, secondary impacts) to determine the by what method the requirements can be defined and use of those methods for verification. Gaps exist at various stages. In some cases component level work is underway, but no system level effort has begun, in other cases no effort has been initiated to close the gap. Status of ongoing efforts and potential approaches to open gaps are discussed.

  15. The kinetics of acylation and deacylation of penicillin acylase from Escherichia coli ATCC 11105: evidence for lowered pKa values of groups near the catalytic centre.

    Science.gov (United States)

    Morillas, M; Goble, M L; Virden, R

    1999-01-01

    Penicillin G acylase catalysed the hydrolysis of 4-nitrophenyl acetate with a kcat of 0.8 s-1 and a Km of 10 microM at pH 7.5 and 20 degreesC. Results from stopped-flow experiments fitted a dissociation constant of 0.16 mM for the Michaelis complex, formation of an acetyl enzyme with a rate constant of 32 s-1 and a subsequent deacylation step with a rate constant of 0.81 s-1. Non-linear Van't Hoff and Arrhenius plots for these parameters, measured at pH 7.5, may be partly explained by a conformational transition affecting catalytic groups, but a linear Arrhenius plot for the ratio of the rate constant for acylation relative to KS was consistent with energy-compensation between the binding of the substrate and catalysis of the formation of the transition state. At 20 degreesC, the pH-dependence of kcat was similar to that of kcat/Km, indicating that formation of the acyl-enzyme did not affect the pKa values (6.5 and 9.0) of an acidic and basic group in the active enzyme. The heats of ionization deduced from values of pKa for kcat, which measures the rate of deacylation, are consistent with alpha-amino and guanidinium groups whose pKa values are decreased in a non-polar environment. It is proposed that, for catalytic activity, the alpha-amino group of the catalytic SerB1 and the guanidinium group of ArgB263 are required in neutral and protonated states respectively. PMID:9931321

  16. Glutamate Counteracts Dopamine/PKA Signaling via Dephosphorylation of DARPP-32 Ser-97 and Alteration of Its Cytonuclear Distribution.

    Science.gov (United States)

    Nishi, Akinori; Matamales, Miriam; Musante, Veronica; Valjent, Emmanuel; Kuroiwa, Mahomi; Kitahara, Yosuke; Rebholz, Heike; Greengard, Paul; Girault, Jean-Antoine; Nairn, Angus C

    2017-01-27

    The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1 receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca(2+)-regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Cav1.2 channel current block by the PKA inhibitor H-89 in rat tail artery myocytes via a PKA-independent mechanism: Electrophysiological, functional, and molecular docking studies.

    Science.gov (United States)

    Fusi, Fabio; Trezza, Alfonso; Spiga, Ottavia; Sgaragli, Giampietro; Bova, Sergio

    2017-09-15

    To characterize the role of cAMP-dependent protein kinase (PKA) in regulating vascular Ca(2+) current through Cav1.2 channels [ICa1.2], we have documented a marked capacity of the isoquinoline H-89, widely used as a PKA inhibitor, to reduce current amplitude. We hypothesized that the ICa1.2 inhibitory activity of H-89 was mediated by mechanisms unrelated to PKA inhibition. To support this, an in-depth analysis of H-89 vascular effects on both ICa1.2 and contractility was undertaken by performing whole-cell patch-clamp recordings and functional experiments in rat tail main artery single myocytes and rings, respectively. H-89 inhibited ICa1.2 with a pIC50 (M) value of about 5.5, even under conditions where PKA activity was either abolished by both the PKA antagonists KT5720 and protein kinase inhibitor fragment 6-22 amide or enhanced by the PKA stimulators 6-Bnz-cAMP and 8-Br-cAMP. Inhibition of ICa1.2 by H-89 appeared almost irreversible upon washout, was charge carrier- and voltage-dependent, and antagonised by the Cav1.2 channel agonist (S)-(-)-Bay K 8644. H-89 did not alter both potency and efficacy of verapamil, did not affect current kinetics or voltage-dependent activation, while shifting to the left the 50% voltage of inactivation in a concentration-dependent manner. H-89 docked at the α1C subunit in a pocket region close to that of (S)-(-)-Bay K 8644 docking, forming a hydrogen bond with the same, key amino acid residue Tyr-1489. Finally, both high K(+)- and (S)-(-)-Bay K 8644-induced contractions of rings were fully reverted by H-89. In conclusion, these results indicate that H-89 inhibited vascular ICa1.2 and, consequently, the contractile function through a PKA-independent mechanism. Therefore, caution is recommended when interpreting experiments where H-89 is used to inhibit vascular smooth muscle PKA. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. L-carnitine contributes to enhancement of neurogenesis from mesenchymal stem cells through Wnt/β-catenin and PKA pathway.

    Science.gov (United States)

    Fathi, Ezzatollah; Farahzadi, Raheleh; Charoudeh, Hojjatollah Nozad

    2017-03-01

    The identification of factors capable of enhancing neurogenesis has great potential for cellular therapies in neurodegenerative diseases. Multiple studies have shown the neuroprotective effects of L-carnitine (LC). This study determined whether neuronal differentiation of rat adipose tissue-derived mesenchymal stem cells (ADSCs) can be activated by LC. In this study, protein kinase A (PKA) and Wnt/β-catenin pathways were detected to show if this activation was due to these pathways. The expression of LC-induced neurogenesis markers in ADSCs was characterized using real-time PCR. ELISA was conducted to assess the expression of cyclic adenosine monophosphate (cAMP) and PKA. The expression of β-catenin, reduced dickkopf1 (DKK1), low-density lipoprotein receptor-related protein 5 (LRP5), Wnt1, and Wnt3a genes as Wnt/β-catenin signaling members were used to detect the Wnt/β-catenin pathway. It was observed that LC could promote neurogenesis in ADSCs as well as expression of some neurogenic markers. Moreover, LC causes to increase the cAMP levels and PKA activity. Treatment of ADSCs with H-89 (dihydrochloride hydrate) as PKA inhibitor significantly inhibited the promotion of neurogenic markers, indicating that the PKA signaling pathway could be involved in neurogenesis induction. Analyses of real-time PCR data showed that the mRNA expressions of β-catenin, DKK1, LRP5c-myc, Wnt1, and Wnt3a were increased in the presence of LC. Therefore, the present study showed that LC promotes ADSCs neurogenesis and the LC-induced neurogenic markers could be due to both the PKA and Wnt/β-catenin signaling pathway. Impact statement Neural tissue has long been believed as incapable of regeneration and the identification of cell types and factors capable of neuronal differentiation has generated intense interest. Mesenchymal stem cells (MSCs) are considered as potential targets for stem cell-based therapy. L-carnitin (LC) as an antioxidant may have neuroprotective effects in

  19. Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM

    Science.gov (United States)

    Demeautis, Claire; Sipieter, François; Roul, Julien; Chapuis, Catherine; Padilla-Parra, Sergi; Riquet, Franck B.; Tramier, Marc

    2017-01-01

    Monitoring of different signalling enzymes in a single assay using multiplex biosensing provides a multidimensional workspace to elucidate biological processes, signalling pathway crosstalk, and determine precise sequence of events at the single living cell level. In this study, we interrogate the complexity in cAMP/PKA-MAPK/ERK1&2 crosstalk by using multi-parameter biosensing experiments to correlate biochemical activities simultaneously in time and space. Using a single excitation wavelength dual colour FLIM method we are able to detect fluorescence lifetime images of two donors to simultaneously measure PKA and ERK1&2 kinase activities in the same cellular localization by using FRET biosensors. To this end, we excite two FRET donors mTFP1 and LSSmOrange with a 440 nm wavelength and we alleviate spectral bleed-through associated limitations with the very dim-fluorescent acceptor ShadowG for mTFP1 and the red-shifted mKate2 for LSSmOrange. The simultaneous recording of PKA and ERK1&2 kinase activities reveals concomitant EGF-mediated activations of both kinases in HeLa cells. Under these conditions the subsequent Forskolin-induced cAMP release reverses the transient increase of EGF-mediated ERK1&2 kinase activity while reinforcing PKA activation. Here we propose a validated methodology for multiparametric kinase biosensing in living cells using FRET-FLIM. PMID:28106114

  20. Measurement of pKa values of newly synthesized heteroarylaminoethanols by CZE.

    Science.gov (United States)

    Lisková, Anna; Slampová, Andrea

    2007-04-01

    Heteroarylaminoethanol derivates are drugs which affect sympathetic nervous system and are used for medications of hypertension. In solutions they behave like weak bases and their pK(a) values represent important information on their potential biological uptake, pharmacological activity and in vivo biodisponsibility. This article brings the measurement of pK(a) values of the series of seven new important heteroarylaminoethanols, compounds with potential vasodilating, beta-adrenolytic and antioxidant activity, by capillary zone electrophoresis (CZE) with diode-array detection. It has been shown that capillary zone electrophoresis measurements of pK(a) can be easily performed with very small quantities of studied substances, and, due to CZE separation power, the purity of samples is not of key importance. Moreover, the CZE method is fast and reliable, providing that suitable operational conditions are selected. The method is based on the measurement of the effective mobility curves within a suitable pH range and related regression analysis where pK(BH)(+) and electrophoretic mobility of BH(+) are explicitly involved. The selection of sufficient operational buffers is of key importance for accurate and reproducible results, and, this article brings step by step the consideration procedure involved in this process. Further, this paper brings principles of least square regression analysis of non-linear function corresponding to exact explicit formula for mobility curve of monovalent weak base.

  1. Requirements engineering for human activity systems

    CSIR Research Space (South Africa)

    Erasmus, J

    2014-10-01

    Full Text Available . Office of the Chief Engineer, NASA, 2007. —. Systems Engineering Handbook. Washington, D.C.: National Aeronautics and Space Administration, 2007. Office of the Deputy Under Secretary of Defense for Acquisition and Technology, Systems and Software... and special tools necessary to perform the verification (NASA 2007). This information will also allow for proper planning and scheduling of the verification activities to be performed throughout the development and realisation of the product. Measures...

  2. Transducin-like enhancer of split-6 (TLE6) is a substrate of protein kinase A activity during mouse oocyte maturation.

    Science.gov (United States)

    Duncan, Francesca E; Padilla-Banks, Elizabeth; Bernhardt, Miranda L; Ord, Teri S; Jefferson, Wendy N; Moss, Stuart B; Williams, Carmen J

    2014-03-01

    Fully grown oocytes in the ovary are arrested at prophase of meiosis I because of high levels of intraoocyte cAMP that maintain increased levels of cAMP-dependent protein kinase (PKA) activity. Following the luteinizing hormone surge at the time of ovulation, cAMP levels drop, resulting in a reduction in PKA activity that triggers meiotic resumption. Although much is known about the molecular mechanisms of how PKA activity fluctuations initiate the oocyte's reentry into meiosis, significantly less is known about the requirement for PKA activity in the oocyte after exit from the prophase I arrest. Here we show that although PKA activity decreases in the oocyte upon meiotic resumption, it increases throughout meiotic progression from the time of germinal vesicle breakdown (GVBD) until the metaphase II (MII) arrest. Blocking this meiotic maturation-associated increase in PKA activity using the pharmacological inhibitor H89 resulted in altered kinetics of GVBD, defects in chromatin and spindle dynamics, and decreased ability of oocytes to reach MII. These effects appear to be largely PKA specific because inhibitors targeting other kinases did not have the same outcomes. To determine potential proteins that may require PKA phosphorylation during meiosis, we separated oocyte protein extracts on an SDS-PAGE gel, extracted regions of the gel that had corresponding immune reactivity towards an anti-PKA substrate antibody, and performed mass spectrometry and microsequencing. Using this approach, we identified transducin-like enhancer of split-6 (TLE6)-a maternal effect gene that is part of the subcortical maternal complex-as a putative PKA substrate. TLE6 localized to the oocyte cortex throughout meiosis in a manner that is spatially and temporally consistent with the localization of critical PKA subunits. Moreover, we demonstrated that TLE6 becomes phosphorylated in a narrow window following meiotic resumption, and H89 treatment can completely block this phosphorylation

  3. Different roles of PKC and PKA in effect of interferon-γ on proliferation and collagen synthesis of fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Xuan-fen ZHANG; Shu-zhong GUO; Kai-hua LU; Hui-yuan LI; Xiang-dong LI; Lin-xi ZHANG; Li YANG

    2004-01-01

    AIM: To study the signal roles of protein kinase C (PKC) and protein kinase A (PKA) in the influence of interferony (IFN-γ) on proliferation and collagen synthesis of fibroblasts derived from hypertrophic scar (HS-FB) and normal skin (NS-FB). METHODS: HS-FB and NS-FB were cultured and passaged in Dulbecco's modified Eagle's medium (DMEM). Activity of PKC and PKA were assayed by transferring phosphorus (32p) into substrate after treatment with IFN-γ 1000 kU/L at 10, 30, 60, and 120 min. Cell proliferation was determined with MTT assay.The collagen synthesis was measured with [3H]proline incorporation and Type III pre-collagen was determined with radioimmunoassay. RESULTS: After exposure to IFN-γ 1000 kU/L for 30 min, PKC activity of HS-FB and respectively (P<0.05). After exposure to IFN-y 1000 kU/L for 60 and 120 min, PKA activities of HS-FB increased The PKA activities of NS-FB also increased from 0.52+0.03 nmol.min-1.g-1of control to 0.68±0.03 and 0.89±0.05 nmol.min-1.g-1, respectively (P<0.05). The proliferation and collagen synthesis were enhanced by PKC activator (containing phosphatidylserine, diacylglycerol and Ca2+) and PKA inhibitor [H7250 μmol/L, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine], and inhibited by PKC inhibior (GF109 250 μmol/L) and PKA activator (cAMP 25 rmol/L)(P<0.01). GF109 abrogated increased proliferation and collagen synthesis by IFN-y but it did not affect the inhibitory effects of IFN-γ. At 120 min H7 reversed the inhibitory functions of IFN-γ. CONCLUSION: IFN-γ transiently increased proliferation and collagen synthesis of HS-FB and NS-FB by activation of PKC and subsequently inhibited proliferation and collagen synthesis by activation of PKA.

  4. AKAP3 synthesis is mediated by RNA binding proteins and PKA signaling during mouse spermiogenesis.

    Science.gov (United States)

    Xu, Kaibiao; Yang, Lele; Zhao, Danyun; Wu, Yaoyao; Qi, Huayu

    2014-06-01

    Mammalian spermatogenesis is regulated by coordinated gene expression in a spatiotemporal manner. The spatiotemporal regulation of major sperm proteins plays important roles during normal development of the male gamete, of which the underlying molecular mechanisms are poorly understood. A-kinase anchoring protein 3 (AKAP3) is one of the major components of the fibrous sheath of the sperm tail that is formed during spermiogenesis. In the present study, we analyzed the expression of sperm-specific Akap3 and the potential regulatory factors of its protein synthesis during mouse spermiogenesis. Results showed that the transcription of Akap3 precedes its protein synthesis by about 2 wk. Nascent AKAP3 was found to form protein complex with PKA and RNA binding proteins (RBPs), including PIWIL1, PABPC1, and NONO, as revealed by coimmunoprecipitation and protein mass spectrometry. RNA electrophoretic gel mobility shift assay showed that these RBPs bind sperm-specific mRNAs, of which proteins are synthesized during the elongating stage of spermiogenesis. Biochemical and cell biological experiments demonstrated that PIWIL1, PABPC1, and NONO interact with each other and colocalize in spermatids' RNA granule, the chromatoid body. In addition, NONO was found in extracytoplasmic granules in round spermatids, whereas PIWIL1 and PABPC1 were diffusely localized in cytoplasm of elongating spermatids, indicating their participation at different steps of mRNA metabolism during spermatogenesis. Interestingly, type I PKA subunits colocalize with PIWIL1 and PABPC1 in the cytoplasm of elongating spermatids and cosediment with the RBPs in polysomal fractions on sucrose gradients. Further biochemical analyses revealed that activation of PKA positively regulates AKAP3 protein synthesis without changing its mRNA level in elongating spermatids. Taken together, these results indicate that PKA signaling directly participates in the regulation of protein translation in postmeiotic male germ cells

  5. Molecular characterisation of cAMP-dependent protein kinase (PK-A) catalytic subunit isoforms in the male tick, Amblyomma hebraeum.

    Science.gov (United States)

    Tabish, Mohammad; Clegg, Roger A; Turner, Philip C; Jonczy, Jan; Rees, Huw H; Fisher, Michael J

    2006-12-01

    The cAMP-dependent protein kinase (protein kinase A, PK-A) plays a central role in the regulation of diverse aspects of cellular activity. Specifically, PK-A appears to play a key controlling role in the maturation of spermatids. Using a PCR-based approach, with degenerate primers from the highly conserved regions of the PK-A catalytic (C) subunit in combination with 5' and 3' RACE, we have cloned three cDNAs for the PK-A C-subunit of the male tick, Amblyomma hebraeum. The three cDNAs have open reading frames of 1059, 1275 and 1404bp which encode proteins of 40.6, 48.2 and 52.5kDa, respectively. These transcripts appear to arise from 5' alternative splicing of RNA derived from a single gene for the PK-A C-subunit. One isoform (AH-PK-A C1), in common with PK-A C-subunits from a range of species, contains a consensus sequence for N-myristoylation. RT-PCR and Western blot experiments suggest that the three splice variants are expressed ubiquitously; however, expression of the myristoylatable AH-PK-A C1 isoform is predominant in all investigated tissues (accessory gland, midgut, Malpighian tubules, salivary gland, testis and immature spermatids). There is no evidence for a sperm-specific PK-A C-subunit (Cs) in tick sperm; however, tyrosine protein phosphorylation, previously shown to be modulated by PK-A activity during mammalian sperm maturation, was observed in tick sperm.

  6. Epac and PKA: a tale of two intracellular cAMP receptors

    Institute of Scientific and Technical Information of China (English)

    Xiaodong Cheng; Zhenyu Ji; Tamara Tsalkova; Fang Mei

    2008-01-01

    cAMP-mediated signaling pathways regulate a multitude of important biological processes under both physiological and pathological conditions,including diabetes,heart failure and cancer.In eukaryotic cells,the effects of cAMP are mediated by two ubiquitously expressed intracellular cAMP receptors,the classic protein kinase A (PKA)/cAMP-dependent protein kinase and the recently discovered exchange protein directly activated by cAMP(Epac)/cAMP-regulated guanine nucleotide exchange factors.Like PKA,Epac contains an evolutionally conserved cAMP binding domain that acts as a molecular switch for sensing intracellular second messenger cAMP levels to control diverse biological functions.The existence of two families of cAMP effectors provides a mechanism for a more precise and integrated control of the cAMP signaling pathways in a spatial and temporal manner.Depending upon the specific cellular environments as well as their relative abundance,distrbution and localization,Epac and PKA may act independently,converge synergistically or oppose each other in regulating a specific cellular function.

  7. GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling.

    Directory of Open Access Journals (Sweden)

    Margarita Marroquin-Guzman

    2015-04-01

    Full Text Available Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development.

  8. GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling.

    Science.gov (United States)

    Marroquin-Guzman, Margarita; Wilson, Richard A

    2015-04-01

    Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR) signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development.

  9. Predicting pKa for proteins using COSMO-RS

    DEFF Research Database (Denmark)

    Andersson, Martin Peter; Jensen, Jan Halborg; Stipp, Susan Louise Svane

    2013-01-01

    We have used the COSMO-RS implicit solvation method to calculate the equilibrium constants, pKa, for deprotonation of the acidic residues of the ovomucoid inhibitor protein, OMTKY3. The root mean square error for comparison with experimental data is only 0.5 pH units and the maximum error 0.8 p......H units. The results show that the accuracy of pKa prediction using COSMO-RS is as good for large biomolecules as it is for smaller inorganic and organic acids and that the method compares very well to previous pKa predictions of the OMTKY3 protein using Quantum Mechanics/Molecular Mechanics. Our approach...

  10. cAMP effects in neuroendocrine tumors: The role of Epac and PKA in cell proliferation and adhesion.

    Science.gov (United States)

    Vitali, E; Cambiaghi, V; Spada, A; Tresoldi, A; Zerbi, A; Peverelli, E; Carnaghi, C; Mantovani, G; Lania, A G

    2015-12-10

    cAMP effects have been initially attributed to protein kinase A (PKA) activation. Subsequently, two exchange proteins directly activated by cAMP (Epac1/2) have been identified as cAMP targets. Aim of this study was to investigate cAMP effects in pancreatic-NET (P-NET) and bronchial carcinoids and in corresponding cell lines (QGP-1 and H727) on cell proliferation and adhesion and to determine PKA and Epac role in mediating these effects. We found that cAMP increased cyclin D1 expression in P-NET and QGP-1 cells, whereas it had opposite effects on bronchial carcinoids and H727 cells and it promoted cell adhesion in QGP-1 and H727 cells. These effects are mimicked by Epac and PKA specific analogs, activating the small GTPase Rap1. In conclusion, we demonstrated that cAMP exerted divergent effects on proliferation and promoted cell adhesion of different neuroendocrine cell types, these effects being mediated by both Epac and PKA and involving the same effector GTPase Rap1.

  11. PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Halayko Andrew J

    2009-09-01

    Full Text Available Abstract Background Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8. IL-8 production is in part regulated via activation of Gq-and Gs-coupled receptors. Here we study the role of the cyclic AMP (cAMP effectors protein kinase A (PKA and exchange proteins directly activated by cAMP (Epac1 and Epac2 in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response. Methods IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors Clostridium difficile toxin B-1470 (GTPases, U0126 (extracellular signal-regulated kinases ERK1/2 and Rp-8-CPT-cAMPS (PKA. The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used. Results The β2-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-O-Me-cAMPS mimicked the effects of 8-pCPT-2'-O-Me-cAMP, whereas the negative control 8-pCPT-2'-O-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP induced GTP

  12. 27 CFR 40.526 - Minimum manufacturing and activity requirements.

    Science.gov (United States)

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 2 2010-04-01 2010-04-01 false Minimum manufacturing and activity requirements. 40.526 Section 40.526 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX... Manufacturers of Processed Tobacco § 40.526 Minimum manufacturing and activity requirements. A permit...

  13. 27 CFR 40.256 - Minimum manufacturing and activity requirements.

    Science.gov (United States)

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 2 2010-04-01 2010-04-01 false Minimum manufacturing and... Provisions Relating to Operations § 40.256 Minimum manufacturing and activity requirements. The minimum manufacturing and activity requirement prescribed in § 40.61(b) of this part is a continuing condition of...

  14. PKA-induced phosphorylation of ERα at serine 305 and high PAK1 levels is associated with sensitivity to tamoxifen in ER-positive breast cancer.

    Science.gov (United States)

    Kok, Marleen; Zwart, Wilbert; Holm, Caroline; Fles, Renske; Hauptmann, Michael; Van't Veer, Laura J; Wessels, Lodewyk F A; Neefjes, Jacques; Stål, Olle; Linn, Sabine C; Landberg, Göran; Michalides, Rob

    2011-01-01

    Phosphorylation of estrogen receptor α at serine 305 (ERαS305-P) by protein kinase A (PKA) or p21-activated kinase 1 (PAK1) has experimentally been associated with tamoxifen sensitivity. Here, we investigated the clinical application of this knowledge to predict tamoxifen resistance in ER-positive breast cancer patients. Using immunohistochemistry, a score including PAK1 and co-expression of PKA and ERαS305-P (PKA/ERαS305-P) was developed on a training set consisting of 103 patients treated with tamoxifen for metastatic disease, and validated on 231 patients randomized between adjuvant tamoxifen or no treatment. In the training set, PAK1 levels were associated with tumor progression after tamoxifen (HR 1.57, 95% CI 0.99-2.48), as was co-expression of PKA and ERαS305-P (HR 2.00, 95% CI 1.14-3.52). In the validation set, a significant tamoxifen benefit was found among the 73% patients negative for PAK1 and PKA/ERαS305-P (HR 0.54, 95% CI 0.34-0.87), while others (27%) were likely to have no benefit from tamoxifen (HR 0.88, 95% 0.42-1.82). The test for interaction showed a significant difference in recurrence-free survival between groups defined by PAK1 and PKA/ERαS305-P (P = 0.037). Elevated PAK1 and PKA/ERαS305-P appeared to influence tamoxifen sensitivity. Both PAK1 and PKA/ERαS305-P levels were associated with sensitivity to tamoxifen in breast tumors and the combination of these variables should be considered in predicting tamoxifen benefit.

  15. Parathyroid hormone induces adipocyte lipolysis via PKA-mediated phosphorylation of hormone-sensitive lipase.

    Science.gov (United States)

    Larsson, Sara; Jones, Helena A; Göransson, Olga; Degerman, Eva; Holm, Cecilia

    2016-03-01

    Parathyroid hormone (PTH) is secreted from the parathyroid glands in response to low plasma calcium levels. Besides its classical actions on bone and kidney, PTH may have other important effects, including metabolic effects, as suggested for instance by increased prevalence of insulin resistance and type 2 diabetes in patients with primary hyperparathyroidism. Moreover, secondary hyperparathyroidism may contribute to the metabolic derangements that characterize states of vitamin D deficiency. PTH has been shown to induce adipose tissue lipolysis, but the details of the lipolytic action of PTH have not been described. Here we used primary mouse adipocytes to show that intact PTH (1-84) as well as the N-terminal fragment (1-37) acutely stimulated lipolysis in a dose-dependent manner, whereas the C-terminal fragment (38-84) was without lipolytic effect. The lipolytic action of PTH was paralleled by phosphorylation of known protein kinase A (PKA) substrates, i.e. hormone-sensitive lipase (HSL) and perilipin. The phosphorylation of HSL in response to PTH occurred at the known PKA sites S563 and S660, but not at the non-PKA site S565. PTH-induced lipolysis, as well as phosphorylation of HSL at S563 and S660, was blocked by both the PKA-inhibitor H89 and the adenylate cyclase inhibitor MDL-12330A, whereas inhibitors of extracellular-regulated kinase (ERK), protein kinase B (PKB), AMP-activated protein kinase (AMPK) and Ca(2+)/calmodulin-dependent protein kinase (CaMK) had little or no effect. Inhibition of phosphodiesterase 4 (PDE4) strongly potentiated the lipolytic action of PTH, whereas inhibition of PDE3 had no effect. Our results show that the lipolytic action of PTH is mediated by the PKA signaling pathway with no or minor contribution of other signaling pathways and, furthermore, that the lipolytic action of PTH is limited by simultaneous activation of PDE4. Knowledge of the signaling pathways involved in the lipolytic action of PTH is important for our

  16. Improvement of Two-Way Active Avoidance Memory Requires Protein Kinase A Activation and Brain-Derived Neurotrophic Factor Expression in the Dorsal Hippocampus

    OpenAIRE

    Datta, Subimal; Siwek, Donald F.; Huang, Max P.

    2009-01-01

    Previous studies have shown that two-way active avoidance (TWAA) memory processing involves a functional interaction between the pontine wave (P wave) generator and the CA3 region of the dorsal hippocampus (DH-CA3). The present experiments examined whether the interaction between P wave generator activity and the DH-CA3 involves the intracellular protein kinase A (PKA) signaling system. In the first series of experiments, rats were subjected to a session of TWAA training followed immediately ...

  17. Assigning the pKa's of Polyprotic Acids.

    Science.gov (United States)

    Bodner, George M.

    1986-01-01

    Discusses (1) polyproptic acids for which the difference between K-a's is large; (2) the Henderson-Hasselbach equation; (3) polyprotic acids for which the difference between K-a's is small; (4) analysis of microscopic dissociation constants for cysteine; and (5) analysis of pK-a data. (JN)

  18. A Proteomics Investigation of Anchored PKA-RI Signaling

    NARCIS (Netherlands)

    Kovanich, D.

    2013-01-01

    Compartmentalization of kinases and phosphatases plays an important role in the specificity of second messenger mediated signaling events. Localization of the cAMP-dependent protein kinase is mediated by interaction of its regulatory subunit (PKA-R) with the versatile family of A-kinase anchoring pr

  19. High Througput pKa Prediction Using Semi Empirical Methods

    CERN Document Server

    Jensen, Jan H

    2015-01-01

    A large proportion of organic molecules relevant to medicine and biotechnology contain one or more ionizable groups, which means that fundamental physical and chemical properties (e.g. the charge of the molecule) depend on the pH of the surroundings via the corresponding pKa values of the molecules. As drug- and material design increasingly is being done through high throughput screens, fast - yet accurate - computational pKa prediction methods are becoming crucial to the design process. Current empirical pKa predictors are increasingly found to fail because they are being applied to parts of chemical space for which experimental parameterization data is lacking. We propose to develop a pKa predictor that, due its quantum mechanical foundation, is more generally applicable but still fast enough to be used in high throughput screening. The method has the potential to impact virtually any biotechnological design process involving organic molecules as we will demonstrate for metabolic engineering and organic bat...

  20. Panax red ginseng extract regulates energy expenditures by modulating PKA dependent lipid mobilization in adipose tissue.

    Science.gov (United States)

    Cho, Hae-Mi; Kang, Young-Ho; Yoo, Hanju; Yoon, Seung-Yong; Kang, Sang-Wook; Chang, Eun-Ju; Song, Youngsup

    2014-05-16

    Regulation of balance between lipid accumulation and energy consumption is a critical step for the maintenance of energy homeostasis. Here, we show that Panax red ginseng extract treatments increased energy expenditures and prevented mice from diet induced obesity. Panax red ginseng extracts strongly activated Hormone Specific Lipase (HSL) via Protein Kinase A (PKA). Since activation of HSL induces lipolysis in WAT and fatty acid oxidation in brown adipose tissue (BAT), these results suggest that Panax red ginseng extracts reduce HFD induced obesity by regulating lipid mobilization. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation

    Science.gov (United States)

    Ferretti, Anabela C.; Tonucci, Facundo M.; Hidalgo, Florencia; Almada, Evangelina; Larocca, Maria C.; Favre, Cristián

    2016-01-01

    The signaling pathways that govern survival response in hepatic cancer cells subjected to nutritional restriction have not been clarified yet. In this study we showed that liver cancer cells undergoing glucose deprivation both arrested in G0/G1 and died mainly by apoptosis. Treatment with the AMPK activator AICAR phenocopied the effect of glucose deprivation on cell survival, whereas AMPK silencing in HepG2/C3A, HuH-7 or SK-Hep-1 cells blocked the cell cycle arrest and the increase in apoptotic death induced by glucose starvation. Both AMPK and PKA were promptly activated after glucose withdrawal. PKA signaling had a dual role during glucose starvation: whereas it elicited an early decreased in cell viability, it later improved this parameter. We detected AMPK phosphorylation (AMPKα(Ser173)) by PKA, which was increased in glucose starved cells and was associated with diminution of AMPK activation. To better explore this inhibitory effect, we constructed a hepatocarcinoma derived cell line which stably expressed an AMPK mutant lacking that PKA phosphorylation site: AMPKα1(S173C). Expression of this mutant significantly decreased viability in cells undergoing glucose starvation. Furthermore, after 36 h of glucose deprivation, the index of AMPKα1(S173C) apoptotic cells doubled the apoptotic index observed in control cells. Two main remarks arise: 1. AMPK is the central signaling kinase in the scenario of cell cycle arrest and death induced by glucose starvation in hepatic cancer cells; 2. PKA phosphorylation of Ser173 comes out as a strong control point that limits the antitumor effects of AMPK in this situation. PMID:26894973

  2. 12 CFR 980.3 - New business activity notice requirement.

    Science.gov (United States)

    2010-01-01

    ... undertaking a new business activity, except as provided in § 980.4(b), a Bank shall submit to the Finance... 12 Banks and Banking 7 2010-01-01 2010-01-01 false New business activity notice requirement. 980.3 Section 980.3 Banks and Banking FEDERAL HOUSING FINANCE BOARD NEW FEDERAL HOME LOAN BANK ACTIVITIES...

  3. Activation of a Non-cAMP/PKA Signaling Pathway Downstream of the PTH/PTHrP Receptor Is Essential for a Sustained Hypophosphatemic Response to PTH Infusion in Male Mice

    Science.gov (United States)

    Song, Lige; Liu, Minlin; Segawa, Hiroko; Miyamoto, Ken-Ichi; Bringhurst, F. Richard; Kronenberg, Henry M.; Jüppner, Harald

    2013-01-01

    PTH increases urinary Pi excretion by reducing expression of two renal cotransporters [NaPi-IIa (Npt2a) and NaPi-IIc (Npt2c)]. In contrast to acute transporter regulation that is cAMP/protein kinase A dependent, long-term effects require phospholipase C (PLC) signaling by the PTH/PTHrP receptor (PPR). To determine whether the latter pathway regulates Pi through Npt2a and/or Npt2c, wild-type mice (Wt) and animals expressing a mutant PPR incapable of PLC activation (DD) were tested in the absence of one (Npt2a−/− or Npt2c−/−) or both phosphate transporters (2a/2c-dko). PTH infusion for 8 days caused a rapid and persistent decrease in serum Pi in Wt mice, whereas serum Pi in DD mice fell only transiently for the first 2 days. Consistent with these findings, fractional Pi excretion index was increased initially in both animals, but this increase persisted only when the PPR Wt was present. The hypophosphatemic response to PTH infusion was impaired only slightly in PPR Wt/Npt2c−/− or DD/Npt2c−/− mice. Despite lower baselines, PTH infusion in PPR Wt/Npt2a−/− mice decreased serum Pi further, an effect that was attenuated in DD/Npt2a−/− mice. Continuous PTH had no effect on serum Pi in 2a/2c-dko mice. PTH administration increased serum 1,25 dihydroxyvitamin D3 levels in Wt and DD mice and increased levels above the elevated baseline with ablation of either but not of both transporters. Continuous PTH elevated serum fibroblast growth factor 23 and blood Ca2+ equivalently in all groups of mice. Our data indicate that PLC signaling at the PPR contributes to the long-term effect of PTH on Pi homeostasis but not to the regulation of 1,25 dihydroxyvitamin D3, fibroblast growth factor 23, or blood Ca2+. PMID:23515284

  4. Improvement of two-way active avoidance memory requires protein kinase a activation and brain-derived neurotrophic factor expression in the dorsal hippocampus.

    Science.gov (United States)

    Datta, Subimal; Siwek, Donald F; Huang, Max P

    2009-07-01

    Previous studies have shown that two-way active avoidance (TWAA) memory processing involves a functional interaction between the pontine wave (P wave) generator and the CA3 region of the dorsal hippocampus (DH-CA3). The present experiments examined whether the interaction between P wave generator activity and the DH-CA3 involves the intracellular protein kinase A (PKA) signaling system. In the first series of experiments, rats were subjected to a session of TWAA training followed immediately by bilateral microinjection of either the PKA activation inhibitor (KT-5720) or vehicle control into the DH-CA3 and tested for TWAA memory 24 h later. The results indicated that immediate KT-5720 infusion impaired improvement of TWAA performance. Additional experiments showed that KT-5720 infusion also blocked TWAA training-induced BDNF expression in the DH-CA3. Together, these findings suggest that the PKA activation and BDNF expression in the DH-CA3 is essential for the improvement of TWAA memory.

  5. Validation of Power Requirement Model for Active Loudspeakers

    DEFF Research Database (Denmark)

    Schneider, Henrik; Madsen, Anders Normann; Bjerregaard, Ruben

    2015-01-01

    The actual power requirement of an active loudspeaker during playback of music has not received much attention in the literature. This is probably because no single and simple solution exists and because a complete system knowledge from input voltage to output sound pressure level is required....... There are however many advantages that could be harvested from such knowledge like size, cost and efficiency improvements. In this paper a recently proposed power requirement model for active loudspeakers is experimentally validated and the model is expanded to include the closed and vented type enclosures...

  6. cAMP/PKA regulates osteogenesis, adipogenesis and ratio of RANKL/OPG mRNA expression in mesenchymal stem cells by suppressing leptin.

    Directory of Open Access Journals (Sweden)

    Der-Chih Yang

    Full Text Available BACKGROUND: Mesenchymal stem cells (MSCs are a pluripotent cell type that can differentiate into adipocytes, osteoblasts and other cells. The reciprocal relationship between adipogenesis and osteogenesis was previously demonstrated; however, the mechanisms remain largely unknown. METHODS AND FINDINGS: We report that activation of PKA by 3-isobutyl-1 methyl xanthine (IBMX and forskolin enhances adipogenesis, the gene expression of PPARgamma2 and LPL, and downregulates the gene expression of Runx2 and osteopontin, markers of osteogenesis. PKA activation also decreases the ratio of Receptor Activator of the NF-kappaB Ligand to Osteoprotegerin (RANKL/OPG gene expression - the key factors of osteoclastogenesis. All these effects are mediated by the cAMP/PKA/CREB pathway by suppressing leptin, and may contribute to PKA stimulators-induced in vivo bone loss in developing zebrafish. CONCLUSIONS: Using MSCs, the center of a newly proposed bone metabolic unit, we identified cAMP/PKA signaling, one of the many signaling pathways that regulate bone homeostasis via controlling cyto-differentiation of MSCs and altering RANKL/OPG gene expression.

  7. UML activity diagrams in requirements specification of logic controllers

    Science.gov (United States)

    Grobelna, Iwona; Grobelny, Michał

    2015-12-01

    Logic controller specification can be prepared using various techniques. One of them is the wide understandable and user-friendly UML language and its activity diagrams. Using formal methods during the design phase increases the assurance that implemented system meets the project requirements. In the approach we use the model checking technique to formally verify a specification against user-defined behavioral requirements. The properties are usually defined as temporal logic formulas. In the paper we propose to use UML activity diagrams in requirements definition and then to formalize them as temporal logic formulas. As a result, UML activity diagrams can be used both for logic controller specification and for requirements definition, what simplifies the specification and verification process.

  8. Mutations of PKA cyclic nucleotide-binding domains reveal novel aspects of cyclic nucleotide selectivity.

    Science.gov (United States)

    Lorenz, Robin; Moon, Eui-Whan; Kim, Jeong Joo; Schmidt, Sven H; Sankaran, Banumathi; Pavlidis, Ioannis V; Kim, Choel; Herberg, Friedrich W

    2017-07-06

    Cyclic AMP and cyclic GMP are ubiquitous second messengers that regulate the activity of effector proteins in all forms of life. The main effector proteins, the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), are preferentially activated by cAMP and cGMP, respectively. However, the molecular basis of this cyclic nucleotide selectivity is still not fully understood. Analysis of isolated cyclic nucleotide-binding (CNB) domains of PKA regulatory subunit type Iα (RIα) reveals that the C-terminal CNB-B has a higher cAMP affinity and selectivity than the N-terminal CNB-A. Here, we show that introducing cGMP-specific residues using site-directed mutagenesis reduces the selectivity of CNB-B, while the combination of two mutations (G316R/A336T) results in a cGMP-selective binding domain. Furthermore, introducing the corresponding mutations (T192R/A212T) into the PKA RIα CNB-A turns this domain into a highly cGMP-selective domain, underlining the importance of these contacts for achieving cGMP specificity. Binding data with the generic purine nucleotide 3',5'-cyclic inosine monophosphate (cIMP) reveal that introduced arginine residues interact with the position 6 oxygen of the nucleobase. Co-crystal structures of an isolated CNB-B G316R/A336T double mutant with either cAMP or cGMP reveal that the introduced threonine and arginine residues maintain their conserved contacts as seen in PKG I CNB-B. These results improve our understanding of cyclic nucleotide binding and the molecular basis of cyclic nucleotide specificity. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  9. Molecular evolution of a-kinase anchoring protein (AKAP-7: implications in comparative PKA compartmentalization

    Directory of Open Access Journals (Sweden)

    Johnson Keven R

    2012-07-01

    Full Text Available Abstract Background A-Kinase Anchoring Proteins (AKAPs are molecular scaffolding proteins mediating the assembly of multi-protein complexes containing cAMP-dependent protein kinase A (PKA, directing the kinase in discrete subcellular locations. Splice variants from the AKAP7 gene (AKAP15/18 are vital components of neuronal and cardiac phosphatase complexes, ion channels, cardiac Ca2+ handling and renal water transport. Results Shown in evolutionary analyses, the formation of the AKAP7-RI/RII binding domain (required for AKAP/PKA-R interaction corresponds to vertebrate-specific gene duplication events in the PKA-RI/RII subunits. Species analyses of AKAP7 splice variants shows the ancestral AKAP7 splice variant is AKAP7α, while the ancestral long form AKAP7 splice variant is AKAP7γ. Multi-species AKAP7 gene alignments, show the recent formation of AKAP7δ occurs with the loss of native AKAP7γ in rats and basal primates. AKAP7 gene alignments and two dimensional Western analyses indicate that AKAP7γ is produced from an internal translation-start site that is present in the AKAP7δ cDNA of mice and humans but absent in rats. Immunofluorescence analysis of AKAP7 protein localization in both rat and mouse heart suggests AKAP7γ replaces AKAP7δ at the cardiac sarcoplasmic reticulum in species other than rat. DNA sequencing identified Human AKAP7δ insertion-deletions (indels that promote the production of AKAP7γ instead of AKAP7δ. Conclusions This AKAP7 molecular evolution study shows that these vital scaffolding proteins developed in ancestral vertebrates and that independent mutations in the AKAP7 genes of rodents and early primates has resulted in the recent formation of AKAP7δ, a splice variant of likely lesser importance in humans than currently described.

  10. Determination of pK(a) of felodipine using UV-Visible spectroscopy.

    Science.gov (United States)

    Pandey, M M; Jaipal, A; Kumar, A; Malik, R; Charde, S Y

    2013-11-01

    In the present study, for the first time, experimental pKa value of felodipine is reported. Dissociation constant, pKa, is one of the very important physicochemical properties of drugs. It is of paramount significance from the perspective of pharmaceutical analysis and dosage form design. The method used for the pKa determination of felodipine was essentially a UV-Visible spectrophotometric method. The spectrophotometric method for the pKa determination was opted by acknowledging the established fact that spectrophotometric determination of pKa produces most precise values. The pKa of felodipine was found to be 5.07. Furthermore, the ruggedness of the determined value is also validated in this study in order to produce exact pKa of the felodipine. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Intrinsic, pro-apoptotic effects of IGFBP-3 on breast cancer cells are reversible: Involvement of PKA, Rho and ceramide.

    Directory of Open Access Journals (Sweden)

    Claire M Perks

    2011-05-01

    Full Text Available We established previously that IGFBP-3 could exert positive or negative effects on cell function depending upon the extracellular matrix composition and by interacting with integrin signalling. To elicit its pro-apoptotic effects IGFBP-3 bound to caveolin-1 and the beta 1 integrin receptor and increased their association culminating in MAPK activation. Disruption of these complexes or blocking the beta 1 integrin receptor reversed these intrinsic actions of IGFBP-3. In this study we have examined the signalling pathway between integrin receptor binding and MAPK activation that mediates the intrinsic, pro-apoptotic actions of IGFBP-3. We found on inhibiting protein kinase A(PKA, Rho associated kinase (ROCK and ceramide, the accentuating effects of IGFBP-3 on apoptotic triggers were reversed, such that IGFBP-3 then conferred cell survival. We established that IGFBP-3 activated Rho, the upstream regulator of ROCK and that beta1 integrin and PKA were upstream of Rho activation, whereas the involvement of ceramide was downstream. The beta 1 integrin, PKA, Rho and ceramide were all upstream of MAPK activation. These data highlight key components involved in the pro-apoptotic effects of IGFBP-3 and that inhibiting them leads to a reversal in the action of IGFBP-3.

  12. The octadecaneuropeptide ODN protects astrocytes against hydrogen peroxide-induced apoptosis via a PKA/MAPK-dependent mechanism.

    Directory of Open Access Journals (Sweden)

    Yosra Hamdi

    Full Text Available Astrocytes synthesize and release endozepines, a family of regulatory peptides, including the octadecaneuropeptide (ODN an endogenous ligand of both central-type benzodiazepine (CBR and metabotropic receptors. We have recently shown that ODN exerts a protective effect against hydrogen peroxide (H(2O(2-induced oxidative stress in astrocytes. The purpose of the present study was to determine the type of receptor and the transduction pathways involved in the protective effect of ODN in cultured rat astrocytes. We have first observed a protective activity of ODN at very low concentrations that was abrogated by the metabotropic ODN receptor antagonist cyclo(1-8[DLeu(5]OP, but not by the CBR antagonist flumazenil. We have also found that the metabotropic ODN receptor is positively coupled to adenylyl cyclase in astrocytes and that the glioprotective action of ODN upon H(2O(2-induced astrocyte death is PKA- and MEK-dependent, but PLC/PKC-independent. Downstream of PKA, ODN induced ERK phosphorylation, which in turn activated the expression of the anti-apoptotic gene Bcl-2 and blocked the stimulation by H(2O(2 of the pro-apoptotic gene Bax. The effect of ODN on the Bax/Bcl-2 balance contributed to abolish the deleterious action of H(2O(2 on mitochondrial membrane integrity and caspase-3 activation. Finally, the inhibitory effect of ODN on caspase-3 activity was shown to be PKA and MEK-dependent. In conclusion, the present results demonstrate that the potent glioprotective action of ODN against oxidative stress involves the metabotropic ODN receptor coupled to the PKA/ERK-kinase pathway to inhibit caspase-3 activation.

  13. The Golgi apparatus is a functionally distinct Ca2+ store regulated by the PKA and Epac branches of the β1-adrenergic signaling pathway.

    Science.gov (United States)

    Yang, Zhaokang; Kirton, Hannah M; MacDougall, David A; Boyle, John P; Deuchars, James; Frater, Brenda; Ponnambalam, Sreenivasan; Hardy, Matthew E; White, Edward; Calaghan, Sarah C; Peers, Chris; Steele, Derek S

    2015-10-13

    Ca(2+) release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. We found that the Golgi apparatus was the source of prolonged Ca(2+) release events that originated near the nuclei of primary cardiomyocytes. Golgi Ca(2+) release was unaffected by depletion of sarcoplasmic reticulum Ca(2+), and disruption of the Golgi apparatus abolished Golgi Ca(2+) release without affecting sarcoplasmic reticulum function, suggesting functional and spatial independence of Golgi and sarcoplasmic reticulum Ca(2+) stores. β1-Adrenoceptor stimulation triggers the production of the second messenger cAMP, which activates the Epac family of Rap guanine nucleotide exchange factors and the kinase PKA (protein kinase A). Phosphodiesterases (PDEs), including those in the PDE3 and PDE4 families, degrade cAMP. Activation of β1-adrenoceptors stimulated Golgi Ca(2+) release, an effect that required activation of Epac, PKA, and the kinase CaMKII. Inhibition of PDE3s or PDE4s potentiated β1-adrenergic-induced Golgi Ca(2+) release, which is consistent with compartmentalization of cAMP signaling near the Golgi apparatus. Interventions that stimulated Golgi Ca(2+) release appeared to increase the trafficking of vascular endothelial growth factor receptor-1 (VEGFR-1) from the Golgi apparatus to the surface membrane of cardiomyocytes. In cardiomyocytes from rats with heart failure, decreases in the abundance of PDE3s and PDE4s were associated with increased Golgi Ca(2+) release events. These data suggest that the Golgi apparatus is a focal point for β1-adrenergic-stimulated Ca(2+) signaling and that the Golgi Ca(2+) store functions independently from the sarcoplasmic reticulum and the global Ca(2+) transients that trigger contraction in cardiomyocytes.

  14. Modeling and predicting pKa values of mono-hydroxylated polychlorinated biphenyls (HO-PCBs) and polybrominated diphenyl ethers (HO-PBDEs) by local molecular descriptors.

    Science.gov (United States)

    Yu, Haiying; Wondrousch, Dominik; Yuan, Quan; Lin, Hongjun; Chen, Jianrong; Hong, Huachang; Schüürmann, Gerrit

    2015-11-01

    Hydroxylated polychlorinated biphenyls (HO-PCBs) and polybrominated diphenyl ethers (HO-PBDEs) are attracting considerable concerns because of their multiple endocrine-disrupting effects and wide existence in environment and organisms. The hydroxyl groups enable these chemicals to be ionizable, and dissociation constant, pKa, becomes an important parameter for investigating their environmental behavior and biological activities. In this study, a new pKa prediction model was developed using local molecular descriptors. The dataset contains 21 experimental pKa values of HO-PCBs and HO-PBDEs. The optimized geometries by ab initio HF/6-31G(∗∗) algorithm were used to calculate the site-specific molecular readiness to accept or donate electron charges. The developed model obtained good statistical performance, which significantly outperformed commercial software ACD and SPARC. Mechanism analysis indicates that pKa values increase with the charge-limited donor energy EQocc on hydroxyl oxygen atom and decrease with the energy-limited acceptor charge QEvac on hydroxyl hydrogen atom. The regression model was also applied to calculate pKa values for all 837 mono-hydroxylated PCBs and PBDEs in each class, aiming to provide basic data for the ecological risk assessment of these chemicals.

  15. Determining Partition Coefficient (Log P), Distribution Coefficient (Log D) and Ionization Constant (pKa) in Early Drug Discovery.

    Science.gov (United States)

    Bharate, Sonali S; Kumar, Vikas; Vishwakarma, Ram A

    2016-01-01

    An early prediction of physicochemical properties is highly desirable during drug discovery to find out a viable lead candidate. Although there are several methods available to determine partition coefficient (log P), distribution coefficient (log D) and ionization constant (pKa), none of them involves simple and fixed, miniaturized protocols for diverse set of compounds. Therefore, it is necessary to establish simple, uniform and medium-throughput protocols requiring small sample quantities for the determination of these physicochemical properties. Log P and log D were determined by shake flask method, wherein, the compound was partitioned between presaturated noctanol and water phase (water/PBS pH 7.4) and the concentration of compound in each phase was determined by HPLC. The pKa determination made use of UV spectrophotometric analysis in a 96-well microtiter plate containing a series of aqueous buffers ranging from pH 1.0 to 13.0. The medium-throughput miniaturized protocols described herein, for determination of log P, log D and pKa, are straightforward to set up and require very small quantities of sample (< 5 mg for all three properties). All established protocols were validated using diverse set of compounds.

  16. Ionization behavior of amino lipids for siRNA delivery: determination of ionization constants, SAR, and the impact of lipid pKa on cationic lipid-biomembrane interactions.

    Science.gov (United States)

    Zhang, Jingtao; Fan, Haihong; Levorse, Dorothy A; Crocker, Louis S

    2011-03-01

    Ionizable amino lipids are being pursued as an important class of materials for delivering small interfering RNA (siRNA) therapeutics, and research is being conducted to elucidate the structure-activity relationships (SAR) of these lipids. The pK(a) of cationic lipid headgroups is one of the critical physiochemical properties of interest due to the strong impact of lipid ionization on the assembly and performance of these lipids. This research focused on developing approaches that permit the rapid determination of the relevant pK(a) of the ionizable amino lipids. Two distinct approaches were investigated: (1) potentiometric titration of amino lipids dissolved in neutral surfactant micelles; and (2) pH-dependent partitioning of a fluorescent dye to cationic liposomes formulated from amino lipids. Using the approaches developed here, the pK(a) values of cationic lipids with distinct headgroups were measured and found to be significantly lower than calculated values. It was also found that lipid-lipid interaction has a strong impact on the pK(a) values of lipids. Lysis of model biomembranes by cationic lipids was used to evaluate the impact of lipid pK(a) on the interaction between cationic lipids and cell membranes. It was found that cationic lipid-biomembrane interaction depends strongly on lipid pK(a) and solution pH, and this interaction is much stronger when amino lipids are highly charged. The presence of an optimal pK(a) range of ionizable amino lipids for siRNA delivery was suggested based on these results. The pK(a) methods reported here can be used to support the SAR screen of cationic lipids for siRNA delivery, and the information revealed through studying the impact of pK(a) on the interaction between cationic lipids and cell membranes will contribute significantly to the design of more efficient siRNA delivery vehicles.

  17. Requirements Specication for Ampliers and Power Supplies in Active Loudspeakers

    DEFF Research Database (Denmark)

    Schneider, Henrik; Jensen, Lasse Crone; Petersen, Lars Press

    2014-01-01

    This work aims to provide designers with a method to develop a requirements specication for power supplies and ampliers in active loudspeakers. The motivation is to avoid over-sizing and unnecessary cost. A realistic estimation of the power supplied during playback of audio in a given loudspeaker...

  18. Review: Bilirubin pKa studies; new models and theories indicate high pKa values in water, dimethylformamide and DMSO

    Directory of Open Access Journals (Sweden)

    Ostrow J

    2010-03-01

    Full Text Available Abstract Background Correct aqueous pKa values of unconjugated bilirubin (UCB, a poorly-soluble, unstable substance, are essential for understanding its functions. Our prior solvent partition studies, of unlabeled and [14C] UCB, indicated pKa values above 8.0. These high values were attributed to effects of internal H-bonding in UCB. Many earlier and subsequent studies have reported lower pKa values, some even below 5.0, which are often used to describe the behavior of UCB. We here review 18 published studies that assessed aqueous pKa values of UCB, critically evaluating their methodologies in relation to essential preconditions for valid pKa measurements (short-duration experiments with purified UCB below saturation and accounting for self-association of UCB. Results These re-assessments identified major deficiencies that invalidate the results of all but our partition studies. New theoretical modeling of UCB titrations shows remarkable, unexpected effects of self-association, yielding falsely low pKa estimates, and provides some rationalization of the titration anomalies. The titration behavior reported for a soluble thioether conjugate of UCB at high aqueous concentrations is shown to be highly anomalous. Theoretical re-interpretations of data in DMSO and dimethylformamide show that those indirectly-derived aqueous pKa values are unacceptable, and indicate new, high average pKa values for UCB in non-aqueous media (>11 in DMSO and, probably, >10 in dimethylformamide. Conclusions No reliable aqueous pKa values of UCB are available for comparison with our partition-derived results. A companion paper shows that only the high pKa values can explain the pH-dependence of UCB binding to phospholipids, cyclodextrins, and alkyl-glycoside and bile salt micelles.

  19. Glucagon stimulates hepatic FGF21 secretion through a PKA- and EPAC-dependent posttranscriptional mechanism.

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    Holly A Cyphert

    Full Text Available Previous studies have shown that whole body deletion of the glucagon receptor suppresses the ability of starvation to increase hepatic fibroblast growth factor 21 (FGF21 expression and plasma FGF21 concentration. Here, we investigate the mechanism by which glucagon receptor activation increases hepatic FGF21 production. Incubating primary rat hepatocyte cultures with glucagon, dibutyryl cAMP or forskolin stimulated a 3-4-fold increase in FGF21 secretion. The effect of these agents on FGF21 secretion was not associated with an increase in FGF21 mRNA abundance. Glucagon induction of FGF21 secretion was additive with the stimulatory effect of a PPARα activator (GW7647 on FGF21 secretion. Inhibition of protein kinase A (PKA and downstream components of the PKA pathway [i.e. AMP-activated protein kinase and p38 MAPK] suppressed glucagon activation of FGF21 secretion. Incubating hepatocytes with an exchange protein directly activated by cAMP (EPAC-selective cAMP analog [i.e. 8-(4-chlorophenylthio-2'-O-methyladenosine-3', 5'-cyclic monophosphate (cpTOME], stimulated a 3.9-fold increase FGF21 secretion, whereas inhibition of the EPAC effector, Rap1, suppressed glucagon activation of FGF21 secretion. Treatment of hepatocytes with insulin also increased FGF21 secretion. In contrast to glucagon, insulin activation of FGF21 secretion was associated with an increase in FGF21 mRNA abundance. Glucagon synergistically interacted with insulin to stimulate a further increase in FGF21 secretion and FGF21 mRNA abundance. These results demonstrate that glucagon increases hepatic FGF21 secretion via a posttranscriptional mechanism and provide evidence that both the PKA branch and EPAC branch of the cAMP pathway play a role in mediating this effect. These results also identify a novel synergistic interaction between glucagon and insulin in the regulation of FGF21 secretion and FGF21 mRNA abundance. We propose that this insulin/glucagon synergism plays a role in

  20. Setting an upper limit on the myoglobin iron(IV)hydroxide pK(a): insight into axial ligand tuning in heme protein catalysis.

    Science.gov (United States)

    Yosca, Timothy H; Behan, Rachel K; Krest, Courtney M; Onderko, Elizabeth L; Langston, Matthew C; Green, Michael T

    2014-06-25

    To provide insight into the iron(IV)hydroxide pK(a) of histidine ligated heme proteins, we have probed the active site of myoglobin compound II over the pH range of 3.9-9.5, using EXAFS, Mössbauer, and resonance Raman spectroscopies. We find no indication of ferryl protonation over this pH range, allowing us to set an upper limit of 2.7 on the iron(IV)hydroxide pK(a) in myoglobin. Together with the recent determination of an iron(IV)hydroxide pK(a) ∼ 12 in the thiolate-ligated heme enzyme cytochrome P450, this result provides insight into Nature's ability to tune catalytic function through its choice of axial ligand.

  1. Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex

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    Shinobu eNomura

    2014-08-01

    Full Text Available Noradrenergic fibers innervate the entire cerebral cortex, whereas the cortical distribution ofdopaminergic fibers is more restricted. However, the relative functional impact ofnoradrenalin and dopamine receptors in various cortical regions is largely unknown. Using aspecific genetic label, we first confirmed that noradrenergic fibers innervate the entire cortexwhereas dopaminergic fibers were present in all layers of restricted medial and lateral areasbut only in deep layers of other areas. Imaging of a genetically-encoded sensor revealed thatnoradrenalin and dopamine widely activate PKA in cortical pyramidal neurons of frontal,parietal and occipital regions with scarce dopaminergic fibers. Responses to noradrenalin hadhigher amplitude, velocity and occurred at more than 10 fold lower dose than those elicited bydopamine, whose amplitude and velocity increased along the antero-posterior axis. Thepharmacology of these responses was consistent with the involvement of Gs-coupled beta1adrenergic and D1/D5 dopaminergic receptors, but the inhibition of both noradrenalin anddopamine responses by beta adrenergic antagonists was suggestive of the existence of beta1-D1/D5 heteromeric receptors. Responses also involved Gi-coupled alpha2 adrenergic and D2-like dopaminergic receptors that markedly reduced their amplitude and velocity andcontributed to their cell-to-cell heterogeneity. Our results reveal that noradrenalin anddopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex withmoderate regional and laminar differences. These receptors can thus mediate widespreadeffects of both catecholamines, which are reportedly co-released by cortical noradrenergicfibers beyond the territory of dopaminergic fibers.

  2. PKA phosphorylation redirects ERα to promoters of a unique gene set to induce tamoxifen resistance.

    Science.gov (United States)

    de Leeuw, R; Flach, K; Bentin Toaldo, C; Alexi, X; Canisius, S; Neefjes, J; Michalides, R; Zwart, W

    2013-07-25

    Protein kinase A (PKA)-induced estrogen receptor alpha (ERα) phosphorylation at serine residue 305 (ERαS305-P) can induce tamoxifen (TAM) resistance in breast cancer. How this phospho-modification affects ERα specificity and translates into TAM resistance is unclear. Here, we show that S305-P modification of ERα reprograms the receptor, redirecting it to new transcriptional start sites, thus modulating the transcriptome. By altering the chromatin-binding pattern, Ser305 phosphorylation of ERα translates into a 26-gene expression classifier that identifies breast cancer patients with a poor disease outcome after TAM treatment. MYC-target genes and networks were significantly enriched in this gene classifier that includes a number of selective targets for ERαS305-P. The enhanced expression of MYC increased cell proliferation in the presence of TAM. We demonstrate that activation of the PKA signaling pathway alters the transcriptome by redirecting ERα to new transcriptional start sites, resulting in altered transcription and TAM resistance.

  3. pKa and solubility of drugs in water, ethanol, and 1-octanol.

    Science.gov (United States)

    Domańska, Urszula; Pobudkowska, Aneta; Pelczarska, Aleksandra; Gierycz, Paweł

    2009-07-02

    Dissociation constants and corresponding pK(a) values of five drugs were obtained with the Bates-Schwarzenbach method using a Perkin-Elmer Lambda 35 UV/vis spectrophotometer at temperature 298.15 K in the buffer solutions. Atropine, promethazine hydrochloride, ibuprofen, flurbiprofen, and meclofenamic acid sodium salt exhibited pK(a) values of 10.3, 6.47, 5.38, 4.50, and 4.39, respectively. The equilibrium mole fraction solubilities of six drugs were measured in a range of temperatures from 240 to 340 K in three important solvents for drugs: water, ethanol, and 1-octanol using the dynamic method. The basic thermal properties of pure drugs, i.e., melting and glass-transition temperatures, as well as the enthalpy of melting and the molar heat capacity at glass transition (at constant pressure) have been measured with the differential scanning microcalorimetry technique (DSC). Molar volumes have been calculated with the Barton group contribution method. The experimental solubility data have been correlated by means of three commonly known G(E) equations: the Wilson, NRTL, and UNIQUAC, with the assumption that the systems studied here have revealed simple eutectic mixtures. As a measure of goodness of correlation, the root-mean-square deviations of temperature have been used. The activity coefficients of the drugs in saturated solutions for each correlated binary mixture were calculated from the experimental data.

  4. Time required for motor activity in lucid dreams.

    Science.gov (United States)

    Erlacher, Daniel; Schredl, Michael

    2004-12-01

    The present study investigated the relationship between the time required for specific tasks (counting and performing squats) in lucid dreams and in the waking state. Five proficient lucid dreamers (26-34 yr. old, M=29.8, SD=3.0; one woman and four men) participated. Analysis showed that the time needed for counting in a lucid dream is comparable to the time needed for counting in wakefulness, but motor activities required more time in lucid dreams than in the waking state.

  5. PKA and ERK, but not PKC, in the amygdala contribute to pain-related synaptic plasticity and behavior

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    Ramsey Cara

    2008-07-01

    Full Text Available Abstract The laterocapsular division of the central nucleus of the amygdala (CeLC has emerged as an important site of pain-related plasticity and pain modulation. Glutamate and neuropeptide receptors in the CeLC contribute to synaptic and behavioral changes in the arthritis pain model, but the intracellular signaling pathways remain to be determined. This study addressed the role of PKA, PKC, and ERK in the CeLC. Adult male Sprague-Dawley rats were used in all experiments. Whole-cell patch-clamp recordings of CeLC neurons were made in brain slices from normal rats and from rats with a kaolin/carrageenan-induced monoarthritis in the knee (6 h postinduction. Membrane-permeable inhibitors of PKA (KT5720, 1 μM; cAMPS-Rp, 10 μM and ERK (U0126, 1 μM activation inhibited synaptic plasticity in slices from arthritic rats but had no effect on normal transmission in control slices. A PKC inhibitor (GF109203x, 1 μM and an inactive structural analogue of U0126 (U0124, 1 μM had no effect. The NMDA receptor-mediated synaptic component was inhibited by KT5720 or U0126; their combined application had additive effects. U0126 did not inhibit synaptic facilitation by forskolin-induced PKA-activation. Administration of KT5720 (100 μM, concentration in microdialysis probe or U0126 (100 μM into the CeLC, but not striatum (placement control, inhibited audible and ultrasonic vocalizations and spinal reflexes of arthritic rats but had no effect in normal animals. GF109203x (100 μM and U0124 (100 μM did not affect pain behavior. The data suggest that in the amygdala PKA and ERK, but not PKC, contribute to pain-related synaptic facilitation and behavior by increasing NMDA receptor function through independent signaling pathways.

  6. Short-Chain Fatty Acids Inhibit Growth Hormone and Prolactin Gene Transcription via cAMP/PKA/CREB Signaling Pathway in Dairy Cow Anterior Pituitary Cells

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    Jian-Fa Wang

    2013-10-01

    Full Text Available Short-chain fatty acids (SCFAs play a key role in altering carbohydrate and lipid metabolism, influence endocrine pancreas activity, and as a precursor of ruminant milk fat. However, the effect and detailed mechanisms by which SCFAs mediate bovine growth hormone (GH and prolactin (PRL gene transcription remain unclear. In this study, we detected the effects of SCFAs (acetate, propionate, and butyrate on the activity of the cAMP/PKA/CREB signaling pathway, GH, PRL, and Pit-1 gene transcription in dairy cow anterior pituitary cells (DCAPCs. The results showed that SCFAs decreased intracellular cAMP levels and a subsequent reduction in PKA activity. Inhibition of PKA activity decreased CREB phosphorylation, thereby inhibiting GH and PRL gene transcription. Furthermore, PTX blocked SCFAs- inhibited cAMP/PKA/CREB signaling pathway. These data showed that the inhibition of GH and PRL gene transcription induced by SCFAs is mediated by Gi activation and that propionate is more potent than acetate and butyrate in inhibiting GH and PRL gene transcription. In conclusion, this study identifies a biochemical mechanism for the regulation of SCFAs on bovine GH and PRL gene transcription in DCAPCs, which may serve as one of the factors that regulate pituitary function in accordance with dietary intake.

  7. Involvement of the Notch Pathway in Terminal Astrocytic Differentiation: Role of PKA

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    Carla Angulo-Rojo

    2013-11-01

    Full Text Available The Notch pathway is a highly conserved signaling system essential for modulating neurogenesis and promoting astrogenesis. Similarly, the cAMP signaling cascade can promote astrocytic commitment in several cell culture models, such as the C6 glioma cell line. These cells have the capacity to differentiate into oligodendrocytes or astrocytes, characteristics that allow their use as a glial progenitor model. In this context, we explore here the plausible involvement of cAMP in Notch-dependent signal transactions. The exposure of C6 cells to a non-hydrolysable cAMP analogue resulted in a sustained augmentation of Notch activity, as detected by nuclear translocation of its intracellular domain portion (NICD and transcriptional activity. The cAMP effect is mediated through the activation of the γ-secretase complex, responsible for Notch cleavage and is sensitive to inhibitors of the cAMP-dependent protein kinase, PKA. As expected, Notch cleavage and nuclear translocation resulted in the up-regulation of the mRNA levels of one of its target genes, the transcription factor Hair and enhancer of split 5. Moreover, the glutamate uptake activity, as well as the expression of astrocytic markers such as glial fibrillary acidic protein, S100β protein and GLAST was also enhanced in cAMP-exposed cells. Our results clearly suggest that during the process of C6 astrocytic differentiation, cAMP activates the PKA/γ-secretase/NICD/RBPJK pathway and Notch1 expression, leading to transcriptional activation of the genes responsible for glial progenitor cell fate decision.

  8. Involvement of the Notch pathway in terminal astrocytic differentiation: role of PKA

    Directory of Open Access Journals (Sweden)

    Carla Angulo‑Rojo

    2013-12-01

    Full Text Available The Notch pathway is a highly conserved signaling system essential for modulating neurogenesis and promoting astrogenesis. Similarly, the cAMP signaling cascade can promote astrocytic commitment in several cell culture models, such as the C6 glioma cell line. These cells have the capacity to differentiate into oligodendrocytes or astrocytes, characteristics that allow their use as a glial progenitor model. In this context, we explore here the plausible involvement of cAMP in Notch-dependent signal transactions. The exposure of C6 cells to a non-hydrolysable cAMP analogue resulted in a sustained augmentation of Notch activity, as detected by nuclear translocation of its intracellular domain portion (NICD and transcriptional activity. The cAMP effect is mediated through the activation of the γ-secretase complex, responsible for Notch cleavage and is sensitive to inhibitors of the cAMP-dependent protein kinase, PKA. As expected, Notch cleavage and nuclear translocation resulted in the up-regulation of the mRNA levels of one of its target genes, the transcription factor Hair and enhancer of split 5. Moreover, the glutamate uptake activity, as well as the expression of astrocytic markers such as glial fibrillary acidic protein, S100β protein and GLAST was also enhanced in cAMP-exposed cells. Our results clearly suggest that during the process of C6 astrocytic differentiation, cAMP activates the PKA/γ-secretase/NICD/RBPJκ pathway and Notch1 expression, leading to transcriptional activation of the genes responsible for glial progenitor cell fate decision.

  9. Mechanisms Underlying the Antidepressant Response of Acupuncture via PKA/CREB Signaling Pathway

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    Huili Jiang

    2017-01-01

    Full Text Available Protein kinase A (PKA/cAMP response element-binding (CREB protein signaling pathway, contributing to impaired neurogenesis parallel to depressive-like behaviors, has been identified as the crucial factor involved in the antidepressant response of acupuncture. However, the molecular mechanisms associated with antidepressant response of acupuncture, neurogenesis, and depressive-like behaviors ameliorating remain unexplored. The objective was to identify the mechanisms underlying the antidepressant response of acupuncture through PKA signaling pathway in depression rats by employing the PKA signaling pathway inhibitor H89 in in vivo experiments. Our results indicated that the expression of hippocampal PKA-α and p-CREB was significantly downregulated by chronic unpredicted mild stress (CUMS procedures. Importantly, acupuncture reversed the downregulation of PKA-α and p-CREB. The expression of PKA-α was upregulated by fluoxetine, but not p-CREB. No significant difference was found between Acu and FLX groups on the expression of PKA-α and p-CREB. Interestingly, H89 inhibited the effects of acupuncture or fluoxetine on upregulating the expression of p-CREB, but not PKA-α. There was no significant difference in expression of CREB among the groups. Conclusively, our findings further support the hypothesis that acupuncture could ameliorate depressive-like behaviors by regulating PKA/CREB signaling pathway, which might be mainly mediated by regulating the phosphorylation level of CREB.

  10. Active Prospective Control Is Required for Effective Sensorimotor Learning

    OpenAIRE

    Snapp-Childs, W; Casserly, E; Mon-Williams, M; Bingham, GP

    2013-01-01

    Passive modeling of movements is often used in movement therapy to overcome disabilities caused by stroke or other disorders (e.g. Developmental Coordination Disorder or Cerebral Palsy). Either a therapist or, recently, a specially designed robot moves or guides the limb passively through the movement to be trained. In contrast, action theory has long suggested that effective skill acquisition requires movements to be actively generated. Is this true? In view of the former, we explicitly test...

  11. Dopamine receptors modulate cytotoxicity of natural killer cells via cAMP-PKA-CREB signaling pathway.

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    Wei Zhao

    Full Text Available Dopamine (DA, a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R with agonist SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R with agonist quinpirole attenuated NK cells. Simultaneously, SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of SKF38393 were blocked by SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA, prevented the SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC, counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The

  12. Autophagy is required for the activation of NFκB.

    Science.gov (United States)

    Criollo, Alfredo; Chereau, Fanny; Malik, Shoaib Ahmad; Niso-Santano, Mireia; Mariño, Guillermo; Galluzzi, Lorenzo; Maiuri, Maria Chiara; Baud, Véronique; Kroemer, Guido

    2012-01-01

    It is well-established that the activation of the inhibitor of NFκB (IκBα) kinase (IKK) complex is required for autophagy induction by multiple stimuli. Here, we show that in autophagy-competent mouse embryonic fibroblasts (MEFs), distinct autophagic triggers, including starvation, mTOR inhibition with rapamycin and p53 inhibition with cyclic pifithrin α lead to the activation of IKK, followed by the phosphorylation-dependent degradation of IκBα and nuclear translocation of NFκB. Remarkably, the NFκB signaling pathway was blocked in MEFs lacking either the essential autophagy genes Atg5 or Atg7. In addition, we found that tumor necrosis factor α (TNFα)-induced NFκB nuclear translocation is abolished in both Atg5- and Atg7-deficient MEFs. Similarly, the depletion of essential autophagy modulators, including ATG5, ATG7, Beclin 1 and VPS34, by RNA interference inhibited TNFα-driven NFκB activation in two human cancer cell lines. In conclusion, it appears that, at least in some instances, autophagy is required for NFκB activation, highlighting an intimate crosstalk between these two stress response signaling pathways.

  13. Identification of a novel TGFβ/PKA signaling transduceome in mediating control of cell survival and metastasis in colon cancer.

    Directory of Open Access Journals (Sweden)

    Sanjib Chowdhury

    Full Text Available BACKGROUND: Understanding drivers for metastasis in human cancer is important for potential development of therapies to treat metastases. The role of loss of TGFβ tumor suppressor activities in the metastatic process is essentially unknown. METHODOLOGY/PRINCIPAL FINDINGS: Utilizing in vitro and in vivo techniques, we have shown that loss of TGFβ tumor suppressor signaling is necessary to allow the last step of the metastatic process - colonization of the metastatic site. This work demonstrates for the first time that TGFβ receptor reconstitution leads to decreased metastatic colonization. Moreover, we have identified a novel TGFβ/PKA tumor suppressor pathway that acts directly on a known cell survival mechanism that responds to stress with the survivin/XIAP dependent inhibition of caspases that effect apoptosis. The linkage between the TGFβ/PKA transduceome signaling and control of metastasis through induction of cell death was shown by TGFβ receptor restoration with reactivation of the TGFβ/PKA pathway in receptor deficient metastatic colon cancer cells leading to control of aberrant cell survival. CONCLUSION/SIGNIFICANCE: This work impacts our understanding of the possible mechanisms that are critical to the growth and maintenance of metastases as well as understanding of a novel TGFβ function as a metastatic suppressor. These results raise the possibility that regeneration of attenuated TGFβ signaling would be an effective target in the treatment of metastasis. Our work indicates the clinical potential for developing anti-metastasis therapy based on inhibition of this very important aberrant cell survival mechanism by the multifaceted TGFβ/PKA transduceome induced pathway. Development of effective treatments for metastatic disease is a pressing need since metastases are the major cause of death in solid tumors.

  14. Liver-specific activities of FGF19 require Klotho beta.

    Science.gov (United States)

    Lin, Benjamin C; Wang, Manping; Blackmore, Craig; Desnoyers, Luc R

    2007-09-14

    Hepatocyte function is regulated by members of the fibroblast growth factor (FGF) family of proteins, but little is known about the specific molecular mechanisms of this endocrine pathway. FGF19 regulates bile acid homeostasis and gall bladder filling; FGF19 binds only to FGF receptor 4 (FGFR4), but its liver-specific activity cannot be explained solely by the distribution of this receptor. Although it has been suggested that Klotho beta (KLB) may have a role in mediating FGF19 activity, we have provided for the first time definitive evidence that KLB is required for FGF19 binding to FGFR4, intracellular signaling, and downstream modulation of gene expression. We have shown that FGFR4 is widely distributed in mouse, whereas KLB distribution is more restricted. Liver was the only organ in which both genes were abundantly expressed. We show that in mice, FGF19 injection triggers liver-specific induction of c-Fos and repression of CYP7A1. The tissue-specific activity of FGF19 supports the unique intersection of KLB and FGFR4 distribution in liver. These studies define KLB as a novel FGFR4 coreceptor required for FGF19 liver specific functions.

  15. Active prospective control is required for effective sensorimotor learning.

    Directory of Open Access Journals (Sweden)

    Winona Snapp-Childs

    Full Text Available Passive modeling of movements is often used in movement therapy to overcome disabilities caused by stroke or other disorders (e.g. Developmental Coordination Disorder or Cerebral Palsy. Either a therapist or, recently, a specially designed robot moves or guides the limb passively through the movement to be trained. In contrast, action theory has long suggested that effective skill acquisition requires movements to be actively generated. Is this true? In view of the former, we explicitly tested the latter. Previously, a method was developed that allows children with Developmental Coordination Disorder to produce effective movements actively, so as to improve manual performance to match that of typically developing children. In the current study, we tested practice using such active movements as compared to practice using passive movement. The passive movement employed, namely haptic tracking, provided a strong test of the comparison, one that showed that the mere inaction of the muscles is not the problem. Instead, lack of prospective control was. The result was no effective learning with passive movement while active practice with prospective control yielded significant improvements in performance.

  16. Active prospective control is required for effective sensorimotor learning.

    Science.gov (United States)

    Snapp-Childs, Winona; Casserly, Elizabeth; Mon-Williams, Mark; Bingham, Geoffrey P

    2013-01-01

    Passive modeling of movements is often used in movement therapy to overcome disabilities caused by stroke or other disorders (e.g. Developmental Coordination Disorder or Cerebral Palsy). Either a therapist or, recently, a specially designed robot moves or guides the limb passively through the movement to be trained. In contrast, action theory has long suggested that effective skill acquisition requires movements to be actively generated. Is this true? In view of the former, we explicitly tested the latter. Previously, a method was developed that allows children with Developmental Coordination Disorder to produce effective movements actively, so as to improve manual performance to match that of typically developing children. In the current study, we tested practice using such active movements as compared to practice using passive movement. The passive movement employed, namely haptic tracking, provided a strong test of the comparison, one that showed that the mere inaction of the muscles is not the problem. Instead, lack of prospective control was. The result was no effective learning with passive movement while active practice with prospective control yielded significant improvements in performance.

  17. Retinoic acid activates two pathways required for meiosis in mice.

    Directory of Open Access Journals (Sweden)

    Jana Koubova

    2014-08-01

    Full Text Available In all sexually reproducing organisms, cells of the germ line must transition from mitosis to meiosis. In mice, retinoic acid (RA, the extrinsic signal for meiotic initiation, activates transcription of Stra8, which is required for meiotic DNA replication and the subsequent processes of meiotic prophase. Here we report that RA also activates transcription of Rec8, which encodes a component of the cohesin complex that accumulates during meiotic S phase, and which is essential for chromosome synapsis and segregation. This RA induction of Rec8 occurs in parallel with the induction of Stra8, and independently of Stra8 function, and it is conserved between the sexes. Further, RA induction of Rec8, like that of Stra8, requires the germ-cell-intrinsic competence factor Dazl. Our findings strengthen the importance of RA and Dazl in the meiotic transition, provide important details about the Stra8 pathway, and open avenues to investigate early meiosis through analysis of Rec8 induction and function.

  18. Retinoic acid activates two pathways required for meiosis in mice.

    Directory of Open Access Journals (Sweden)

    Jana Koubova

    2014-08-01

    Full Text Available In all sexually reproducing organisms, cells of the germ line must transition from mitosis to meiosis. In mice, retinoic acid (RA, the extrinsic signal for meiotic initiation, activates transcription of Stra8, which is required for meiotic DNA replication and the subsequent processes of meiotic prophase. Here we report that RA also activates transcription of Rec8, which encodes a component of the cohesin complex that accumulates during meiotic S phase, and which is essential for chromosome synapsis and segregation. This RA induction of Rec8 occurs in parallel with the induction of Stra8, and independently of Stra8 function, and it is conserved between the sexes. Further, RA induction of Rec8, like that of Stra8, requires the germ-cell-intrinsic competence factor Dazl. Our findings strengthen the importance of RA and Dazl in the meiotic transition, provide important details about the Stra8 pathway, and open avenues to investigate early meiosis through analysis of Rec8 induction and function.

  19. Cysteine 904 is required for maximal insulin degrading enzyme activity and polyanion activation.

    Directory of Open Access Journals (Sweden)

    Eun Suk Song

    Full Text Available Cysteine residues in insulin degrading enzyme have been reported as non-critical for its activity. We found that converting the twelve cysteine residues in rat insulin degrading enzyme (IDE to serines resulted in a cysteine-free form of the enzyme with reduced activity and decreased activation by polyanions. Mutation of each cysteine residue individually revealed cysteine 904 as the key residue required for maximal activity and polyanion activation, although other cysteines affect polyanion binding to a lesser extent. Based on the structure of IDE, Asn 575 was identified as a potential hydrogen bond partner for Cys904 and mutation of this residue also reduced activity and decreased polyanion activation. The oligomerization state of IDE did not correlate with its activity, with the dimer being the predominant form in all the samples examined. These data suggest that there are several conformational states of the dimer that affect activity and polyanion activation.

  20. Schwann cell myelination requires integration of laminin activities.

    Science.gov (United States)

    McKee, Karen K; Yang, Dong-Hua; Patel, Rajesh; Chen, Zu-Lin; Strickland, Sidney; Takagi, Junichi; Sekiguchi, Kiyotoshi; Yurchenco, Peter D

    2012-10-01

    Laminins promote early stages of peripheral nerve myelination by assembling basement membranes (BMs) on Schwann cell surfaces, leading to activation of β1 integrins and other receptors. The BM composition, structural bonds and ligands needed to mediate this process, however, are not well understood. Mice hypomorphic for laminin γ1-subunit expression that assembled endoneurial BMs with reduced component density exhibited an axonal sorting defect with amyelination but normal Schwann cell proliferation, the latter unlike the null. To identify the basis for this, and to dissect participating laminin interactions, LAMC1 gene-inactivated dorsal root ganglia were treated with recombinant laminin-211 and -111 lacking different architecture-forming and receptor-binding activities, to induce myelination. Myelin-wrapping of axons by Schwann cells was found to require higher laminin concentrations than either proliferation or axonal ensheathment. Laminins that were unable to polymerize through deletions that removed critical N-terminal (LN) domains, or that lacked cell-adhesive globular (LG) domains, caused reduced BMs and almost no myelination. Laminins engineered to bind weakly to α6β1 and/or α7β1 integrins through their LG domains, even though they could effectively assemble BMs, decreased myelination. Proliferation depended upon both integrin binding to LG domains and polymerization. Collectively these findings reveal that laminins integrate scaffold-forming and cell-adhesion activities to assemble an endoneurial BM, with myelination and proliferation requiring additional α6β1/α7β1-laminin LG domain interactions, and that a high BM ligand/structural density is needed for efficient myelination.

  1. Hormonal activation of a kinase cascade localized at the mitochondria is required for StAR protein activity.

    Science.gov (United States)

    Poderoso, Cecilia; Maloberti, Paula; Duarte, Alejandra; Neuman, Isabel; Paz, Cristina; Cornejo Maciel, Fabiana; Podesta, Ernesto J

    2009-03-01

    It is known that ERK1/2 and MEK1/2 participate in the regulation of Star gene transcription. However, their role in StAR protein post-transcriptional regulation is not described yet. In this study we analyzed the relationship between the MAPK cascade and StAR protein phosphorylation and function. We have demonstrated that (a) steroidogenesis in MA-10 Leydig cells depends on the specific of ERK1/2 activation at the mitochondria; (b) ERK1/2 phosphorylation is driven by mitochondrial PKA and constitutive MEK1/2 in this organelle; (c) active ERK1/2 interacts with StAR protein, leads to StAR protein phosphorylation at Ser(232) only in the presence of cholesterol; (d) directed mutagenesis of Ser(232) (S232A) inhibited in vitro StAR protein phosphorylation by ERK1; (e) transient transfection of MA-10 cells with StAR S232A cDNA markedly reduced the yield of progesterone production. We show that StAR protein is a substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric complex that regulates cholesterol transport.

  2. Experimental approaches for measuring pKa's in RNA and DNA.

    Science.gov (United States)

    Thaplyal, Pallavi; Bevilacqua, Philip C

    2014-01-01

    RNA and DNA carry out diverse functions in biology including catalysis, splicing, gene regulation, and storage of genetic information. Interest has grown in understanding how nucleic acids perform such sophisticated functions given their limited molecular repertoire. RNA can fold into diverse shapes that often perturb pKa values and allow it to ionize appreciably under biological conditions, thereby extending its molecular diversity. The goal of this chapter is to enable experimental measurement of pKa's in RNA and DNA. A number of experimental methods for measuring pKa values in RNA and DNA have been developed over the last 10 years, including RNA cleavage kinetics; UV-, fluorescence-, and NMR-detected pH titrations; and Raman crystallography. We begin with general considerations for choosing a pKa assay and then describe experimental conditions, advantages, and disadvantages for these assays. Potential pitfalls in measuring a pKa are provided including the presence of apparent pKa's due to a kinetic pKa or coupled acid- and alkali-promoted RNA unfolding, as well as degradation of RNA, precipitation of metal hydroxides and poor baselines. Use of multiple data fitting procedures and the study of appropriate mutants are described as ways to avoid some of these pitfalls. Application of these experimental methods to RNA and DNA will increase the number of available nucleic acid pKa values in the literature, which should deepen insight into biology and provide benchmarks for pKa calculations. Future directions for measuring pKa's in nucleic acids are discussed.

  3. Photosensitization is required for antiretroviral activity of hypericin

    Science.gov (United States)

    Carpenter, Susan; Tossberg, John; Kraus, George A.

    1991-06-01

    In a seminal series of papers, Meruelo and co-workers have described the potent antiretroviral effect of hypericin. Interestingly, hypericin was found to inhibit not only the production of infectious virus from chronically infected cells, but was also shown to directly inhibit reverse transcriptase activity of mature virions. The effect of hypericin on cells chronically infected with equine infectious anemia virus (EIAV), a retrovirus genetically related to HIV, is demonstrated. At concentrations of 10 (mu) g/ml, hypericin reduced production of infectious EIAV by 99.99 without causing obvious cytopathic effects. Interestingly, the results indicated that the antiretroviral activity of hypericin was wholly dependent on the presence of light. No decrease in viral infectivity was observed when hypericin and virus were incubated in the dark. Moreover, it appeared that light was an absolute requirement for the antiviral activity, as even high concentrations of hypericin (10 (mu) g/ml) were unable to reduce infectivity of as few as 100 infectious virions.

  4. Protein kinase A regulation of P2X(4) receptors: requirement for a specific motif in the C-terminus.

    Science.gov (United States)

    Brown, David A; Yule, David I

    2010-02-01

    The P2X purinergic receptor sub-family of ligand-gated ion channels are subject to protein kinase modulation. We have previously demonstrated that P2X(4)R signaling can be positively regulated by increasing intracellular cAMP levels. The molecular mechanism underlying this effect was, however, unknown. The present study initially addressed whether protein kinase A (PKA) activation was required. Subsequently a mutational approach was utilized to determine which region of the receptor was required for this potentiation. In both DT-40 3KO and HEK-293 cells transiently expressing P2X(4)R, forskolin treatment enhanced ATP-mediated signaling. Specific PKA inhibitors prevented the forskolin-induced enhancement of ATP-mediated inward currents in P2X(4)R expressing HEK-293 cells. To define which region of the P2X(4)R was required for the potentiation, mutations were generated in the cytoplasmic C-terminal tail. It was determined that a limited region of the C-terminus, consisting of a non-canonical tyrosine based sorting motif, was required for the effects of PKA. Of note, this region does not harbor any recognizable PKA phosphorylation motifs, and no direct phosphorylation of P2X(4)R was detected, suggesting that PKA phosphorylation of an accessory protein interacts with the endocytosis motif in the C-terminus of the P2X(4)R. In support of this notion, using Total Internal Reflection Fluorescence Microscopy (TIRF)\\ P2X(4)-EGFP was shown to accumulate at/near the plasma membrane following forskolin treatment. In addition, disrupting the endocytosis machinery using a dominant-negative dynamin construct also prevented the PKA-mediated enhancement of ATP-stimulated Ca(2+) signals. Our results are consistent with a novel mechanism of P2XR regulation, whereby PKA activity, without directly phosphorylating P2X(4)R, markedly enhances ATP-stimulated P2X(4)R currents and hence cytosolic Ca(2+) signals. This may occur at least in part, by altering the trafficking of a population of

  5. Phosphorylation of both nucleoplasmin domains is required for activation of its chromatin decondensation activity

    DEFF Research Database (Denmark)

    Bañuelos, Sonia; Omaetxebarria, Miren J; Ramos, Isbaal

    2007-01-01

    Nucleoplasmin (NP) is a histone chaperone involved in nucleosome assembly, chromatin decondensation at fertilization, and apoptosis. To carry out these activities NP has to interact with different types of histones, an interaction that is regulated by phosphorylation. Here we have identified...... were independently or simultaneously "activated" indicates that activation or phosphorylation of both protein domains is required for NP to efficiently extract linker-type histones from chromatin....

  6. Solubility and pKa determination of six structurally related phenothiazines.

    Science.gov (United States)

    Domańska, Urszula; Pelczarska, Aleksandra; Pobudkowska, Aneta

    2011-12-12

    Solubilities of six structurally related phenothiazines, namely chlorpromazine hydrochloride, fluphenazine dihydrochloride, promazine hydrochloride, thioridazine hydrochloride, trifluoperazine dihydrochloride, and triflupromazine hydrochloride at constant pH were measured in the temperature range from 290 K to 350 K in three important drugs solvents: water, ethanol and 1-octanol using the dynamic method and UV-vis method. Dissociation constants and corresponding pK(a) values of drugs were obtained with Bates-Schwarzenbach method at temperature 298.15K in the buffer solutions. Our experimental pK(a) values for chlorpromazine hydrochloride, fluphenazine dihydrochloride, promazine hydrochloride, thioridazine hydrochloride, trifluoperazine dihydrochloride, and triflupromazine hydrochloride are 9.15, 10.01, 9.37, 8.89, 8.97, and 9.03, respectively. The basic thermal properties of pure drugs i.e. melting and solid-solid phase transition as well as glass-transition temperatures, the enthalpy of melting and phase transitions and the molar heat capacity at glass transition (at constant pressure) were measured with differential scanning microcalorimetry (DSC) technique. Molar volumes were calculated with Barton group contribution method. The experimental solubility data were correlated by means of three commonly known G(E) equations: the Wilson, NRTL and UNIQUAC with the assumption that the systems studied here have revealed simple eutectic mixtures. The root-mean-square deviations of temperature were used for the precision of the correlation. The activity coefficients of drugs at saturated solutions in each correlated binary mixture were calculated from the experimental data. These new data will help in all prediction-methods and their precision.

  7. Regulation of fear extinction versus other affective behaviors by discrete cortical scaffolding complexes associated with NR2B and PKA signaling.

    Science.gov (United States)

    Corcoran, K A; Leaderbrand, K; Jovasevic, V; Guedea, A L; Kassam, F; Radulovic, J

    2015-10-13

    In patients suffering from post-traumatic stress disorder (PTSD), fear evoked by trauma-related memories lasts long past the traumatic event and it is often complicated by general anxiety and depressed mood. This poses a treatment challenge, as drugs beneficial for some symptoms might exacerbate others. For example, in preclinical studies, antagonists of the NR2B subunit of N-methyl-d-aspartate receptors and activators of cAMP-dependent protein kinase (PKA) act as potent antidepressants and anxiolytics, but they block fear extinction. Using mice, we attempted to overcome this problem by interfering with individual NR2B and PKA signaling complexes organized by scaffolding proteins. We infused cell-permeable Tat peptides that displaced either NR2B from receptor for activated C kinase 1 (RACK1), or PKA from A-kinase anchor proteins (AKAPs) or microtubule-associated proteins (MAPs). The infusions were targeted to the retrosplenial cortex, an area involved in both fear extinction of remotely acquired memories and in mood regulation. Tat-RACK1 and Tat-AKAP enhanced fear extinction, all peptides reduced anxiety and none affected baseline depression-like behavior. However, disruption of PKA complexes distinctively interfered with the rapid antidepressant actions of the N-methyl-D-aspartate receptors antagonist MK-801 in that Tat-MAP2 blocked, whereas Tat-AKAP completely inverted the effect of MK-801 from antidepressant to depressant. These effects were unrelated to the MK-801-induced changes of brain-derived neurotrophic factor messenger RNA levels. Together, the findings suggest that NR2B-RACK1 complexes specifically contribute to fear extinction, and may provide a target for the treatment of PTSD. AKAP-PKA, on the other hand, appears to modulate fear extinction and antidepressant responses in opposite directions.

  8. Drosophila TRPML is required for TORC1 activation.

    Science.gov (United States)

    Wong, Ching-On; Li, Ruoxia; Montell, Craig; Venkatachalam, Kartik

    2012-09-11

    Loss-of-function mutations in TRPML1 (transient receptor potential mucolipin 1) cause the lysosomal storage disorder, mucolipidosis type IV (MLIV). Here, we report that flies lacking the TRPML1 homolog displayed incomplete autophagy and reduced viability during the pupal period--a phase when animals rely on autophagy for nutrients. We show that TRPML was required for fusion of amphisomes with lysosomes, and its absence led to accumulation of vesicles of significantly larger volume and higher luminal Ca(2+). We also found that trpml(1) mutant cells showed decreased TORC1 (target of rapamycin complex 1) signaling and a concomitant upregulation of autophagy induction. Both of these defects in the mutants were reversed by genetically activating TORC1 or by feeding the larvae a high-protein diet. The high-protein diet also reduced the pupal lethality and the increased volume of acidic vesicles. Conversely, further inhibition of TORC1 activity by rapamycin exacerbated the mutant phenotypes. Finally, TORC1 exerted reciprocal control on TRPML function. A high-protein diet caused cortical localization of TRPML, and this effect was blocked by rapamycin. Our findings delineate the interrelationship between the TRPML and TORC1 pathways and raise the intriguing possibility that a high-protein diet might reduce the severity of MLIV.

  9. Curcumin Protects Neurons from Glutamate-Induced Excitotoxicity by Membrane Anchored AKAP79-PKA Interaction Network

    Directory of Open Access Journals (Sweden)

    Kui Chen

    2015-01-01

    Full Text Available Now stimulation of AMPA receptor as well as its downstream pathways is considered as potential central mediators in antidepressant mechanisms. As a signal integrator which binds to AMPA receptor, A-kinase anchoring protein 79-(AKAP79- PKA complex is regarded as a potential drug target to exert neuroprotective effects. A well-tolerated and multitarget drug curcumin has been confirmed to exert antidepressant-like effects. To explore whether AKAP79-PKA complex is involved in curcumin-mediated antiexcitotoxicity, we detected calcium signaling, subcellular location of AKAP79-PKA complex, phosphorylation of glutamate receptor, and ERK and AKT cascades. In this study, we found that curcumin protected neurons from glutamate insult by reducing Ca2+ influx and blocking the translocation of AKAP79 from cytomembrane to cytoplasm. In parallel, curcumin enhanced the phosphorylation of AMPA receptor and its downstream pathways in PKA-dependent manner. If we pretreated cells with PKA anchoring inhibitor Ht31 to disassociate PKA from AKAP79, no neuroprotective effects were observed. In conclusion, our results show that AKAP79-anchored PKA facilitated the signal relay from AMPA receptor to AKT and ERK cascades, which may be crucial for curcumin-mediated antiexcitotoxicity.

  10. Ginsenosides Rb1 and Rg1 Stimulate Melanogenesis in Human Epidermal Melanocytes via PKA/CREB/MITF Signaling

    Directory of Open Access Journals (Sweden)

    Mao Lin

    2014-01-01

    Full Text Available Reduced or defective melanin skin pigmentation may cause many hypopigmentation disorders and increase the risk of damage to the skin triggered by UV irradiation. Ginsenosides Rb1 and Rg1 have many molecular targets including the cAMP-response element-binding protein (CREB, which is involved in melanogenesis. This study aimed to investigate the effects of ginsenosides Rb1 and Rg1 on melanogenesis in human melanocytes and their related mechanisms. The effects of Rb1 and Rg1 on cell viability, tyrosinase activity, cellular melanin content and protein levels of tyrosinase, microphthalmia-associated transcription factor (MITF, and activation of CREB in melanocytes were assessed. Results showed that Rb1 or Rg1 significantly increased cellular melanin content and tyrosinase activity in a dose-dependent manner. By contrast, the cell viability of melanocytes remained unchanged. After exposure to Rb1 or Rg1, the protein levels of tyrosinase, MITF, and phosphorylated CREB were significantly increased. Furthermore, pretreatment with the selective PKA inhibitor H-89 significantly blocked the Rb1- or Rg1-induced increase of melanin content. These findings indicated that Rb1 and Rg1 increased melanogenesis and tyrosinase activity in human melanocytes, which was associated with activation of PKA/CREB/MITF signaling. The effects and mechanisms of Rb1 or Rg1 on skin pigmentation deserve further study.

  11. Regulatory subunits of PKA define an axis of cellular proliferation/differentiation in ovarian cancer cells

    Directory of Open Access Journals (Sweden)

    Hall John C

    2008-09-01

    Full Text Available Abstract Background The regulatory subunit of cAMP-dependent protein kinase (PKA exists in two isoforms, RI and RII, which distinguish the PKA isozymes, type I (PKA-I and type II (PKA-II. Evidence obtained from a variety of different experimental approaches has shown that the relative levels of type I and type II PKA in cells can play a major role in determining the balance between cell growth and differentiation. In order to characterize the effect of PKA type I and type II regulatory subunits on gene transcription at a global level, the PKA regulatory subunit genes for RIα and RIIβ were stably transfected into cells of the ovarian cancer cell line (OVCAR8. Results RIα transfected cells exhibit hyper-proliferative growth and RIIβ transfected cells revert to a relatively quiescent state. Profiling by microarray revealed equally profound changes in gene expression between RIα, RIIβ, and parental OVCAR cells. Genes specifically up-regulated in RIα cells were highly enriched for pathways involved in cell growth while genes up-regulated in RIIβ cells were enriched for pathways involved in differentiation. A large group of genes (~3600 was regulated along an axis of proliferation/differentiation between RIα, parental, and RIIβ cells. RIα/wt and RIIβ/wt gene regulation was shown by two separate and distinct gene set analytical methods to be strongly cross-correlated with a generic model of cellular differentiation. Conclusion Overexpression of PKA regulatory subunits in an ovarian cancer cell line dramatically influences the cell phenotype. The proliferation phenotype is strongly correlated with recently identified clinical biomarkers predictive of poor prognosis in ovarian cancer suggesting a possible pivotal role for PKA regulation in disease progression.

  12. Effect of methylation on the side-chain pKa value of arginine.

    Science.gov (United States)

    Evich, Marina; Stroeva, Ekaterina; Zheng, Yujun George; Germann, Markus W

    2016-02-01

    Arginine methylation is important in biological systems. Recent studies link the deregulation of protein arginine methyltransferases with certain cancers. To assess the impact of methylation on interaction with other biomolecules, the pKa values of methylated arginine variants were determined using NMR data. The pKa values of monomethylated, symmetrically dimethylated, and asymmetrically dimethylated arginine are similar to the unmodified arginine (14.2 ± 0.4). Although the pKa value has not been significantly affected by methylation, consequences of methylation include changes in charge distribution and steric effects, suggesting alternative mechanisms for recognition. © 2015 The Protein Society.

  13. Phosphorylation of endothelial NOS contributes to simvastatin protection against myocardial no-reflow and infarction in reperfused swine hearts: partially via the PKA signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Xiang-dong LI; Yue-jin YANG; Yong-jian GENG; Jing-lin ZHAO; Hai-tao ZHANG; Yu-tong CHENG; Yi-ling WU

    2012-01-01

    Aim:The cholesterol-lowering drugs statins could enhance the activities of endothelial nitric oxide synthase (eNOS) and protect myocardium during ischemia and reperfusion.The aim of this study was to examine whether protein kinase A (PKA) was involved in statinmediated eNOS phosphorylation and cardioprotection.Methods:6-Month-old Chinese minipigs (20-30 kg) underwent a 1.5-h occlusion and 3-h reperfusion of the left anterior descending coronary artery (LAD).In the sham group,the LAD was encircled by a suture but not occluded.Hemodynamic and cardiac function was monitored using a polygraph.Plasma activity of creatine kinase and the tissue activities of PKA and NOS were measured spectrophotometrically.p-CREB,eNOS and p-eNOS levels were detected using Western blotting.Sizes of the area at risk,the area of no-reflow and the area of necrosis were measured morphologically.Results:Pretreatment of the animals with simvastatin (SIM,2 mg/kg,po) before reperfusion significantly decreased the plasma activity of creatine kinase,an index of myocardial necrosis,and reduced the no-reflow size (from 50.4%±2.4% to 36.1%+2.1%,P<0.01) and the infarct size (from 79.0%±2.7% to 64.1%±4.5%,P<0.01).SIM significantly increased the activities of PKA and constitutive NOS,and increased Ser133 p-CREB protein,Ser1179 p-eNOS,and Ser635 p-eNOS in ischemic myocardium.Intravenous infusion of the PKA inhibitor H-89 (1 μg·kg1·min-1) partially abrogated the SIM-induced cardioprotection and eNOS phosphorylation.In contrast,intravenous infusion of the eNOS inhibitor L-NNA (10 mg·kg1) completely abrogated the SIM-induced cardioprotection and eNOS phosphorylation during ischemia and reperfusion,but did not affect the activity of PKA.Conclusion:Pretreatment with a single dose of SIM 2.5 h before reperfusion attenuates myocardial no-reflow and infarction through increasing eNOS phosphorylation at Ser1179 and Ser635 that was partially mediated via the PKA signaling pathway.

  14. The pKa Cooperative: a collaborative effort to advance structure-based calculations of pKa values and electrostatic effects in proteins.

    Science.gov (United States)

    Nielsen, Jens E; Gunner, M R; García-Moreno, Bertrand E

    2011-12-01

    The pK(a) Cooperative (http://www.pkacoop.org) was organized to advance development of accurate and useful computational methods for structure-based calculation of pK(a) values and electrostatic energies in proteins. The Cooperative brings together laboratories with expertise and interest in theoretical, computational, and experimental studies of protein electrostatics. To improve structure-based energy calculations, it is necessary to better understand the physical character and molecular determinants of electrostatic effects. Thus, the Cooperative intends to foment experimental research into fundamental aspects of proteins that depend on electrostatic interactions. It will maintain a depository for experimental data useful for critical assessment of methods for structure-based electrostatics calculations. To help guide the development of computational methods, the Cooperative will organize blind prediction exercises. As a first step, computational laboratories were invited to reproduce an unpublished set of experimental pK(a) values of acidic and basic residues introduced in the interior of staphylococcal nuclease by site-directed mutagenesis. The pK(a) values of these groups are unique and challenging to simulate owing to the large magnitude of their shifts relative to normal pK(a) values in water. Many computational methods were tested in this first Blind Prediction Challenge and critical assessment exercise. A workshop was organized in the Telluride Science Research Center to objectively assess the performance of many computational methods tested on this one extensive data set. This volume of Proteins: Structure, Function, and Bioinformatics introduces the pK(a) Cooperative, presents reports submitted by participants in the Blind Prediction Challenge, and highlights some of the problems in structure-based calculations identified during this exercise. Copyright © 2011 Wiley-Liss, Inc.

  15. Computational chemical analysis of unconjugated bilirubin anions and insights into pKa values clarification

    Science.gov (United States)

    Vega-Hissi, Esteban G.; Estrada, Mario R.; Lavecchia, Martín J.; Pis Diez, Reinaldo

    2013-01-01

    The pKa, the negative logarithm of the acid dissociation equilibrium constant, of the carboxylic acid groups of unconjugated bilirubin in water is a discussed issue because there are quite different experimental values reported. Using quantum mechanical calculations we have studied the conformational behavior of unconjugated bilirubin species (in gas phase and in solution modeled implicitly and explicitly) to provide evidence that may clarify pKa values because of its pathophysiological relevance. Our results show that rotation of carboxylate group, which is not restricted, settles it in a suitable place to establish stronger interactions that stabilizes the monoanion and the dianion to be properly solvated, demonstrating that the rationalization used to justify the high pKa values of unconjugated bilirubin is inappropriate. Furthermore, low unconjugated bilirubin (UCB) pKa values were estimated from a linear regression analysis.

  16. Role of AC-cAMP-PKA Cascade in Antidepressant Action of Electroacupuncture Treatment in Rats

    Directory of Open Access Journals (Sweden)

    Jian-hua Liu

    2012-01-01

    Full Text Available Adenylyl cyclase (AC-cyclic adenosine monophosphate (cAMP-cAMP-dependent protein kinase A (PKA cascade is considered to be associated with the pathogenesis and treatment of depression. The present study was conducted to explore the role of the cAMP cascade in antidepressant action of electroacupuncture (EA treatment for chronic mild stress (CMS-induced depression model rats. The results showed that EA improved significantly behavior symptoms in depression and dysfunction of AC-cAMP-PKA signal transduction pathway induced by CMS, which was as effective as fluoxetine. Moreover, the antidepressant effects of EA rather than Fluoxetine were completely abolished by H89, a specific PKA inhibitor. Consequently, EA has a significant antidepressant treatment in CMS-induced depression model rats, and AC-cAMP-PKA signal transduction pathway is crucial for it.

  17. First principles pKa calculations on carboxylic acids using the SMD solvation model: effect of thermodynamic cycle, model chemistry, and explicit solvent molecules.

    Science.gov (United States)

    Sutton, Catherine C R; Franks, George V; da Silva, Gabriel

    2012-10-04

    Aqueous pK(a) values are calculated from first principles for a set of carboxylic acids using the SMD solvation model with various model chemistries, thermodynamic cycles, and treatments of explicit solvation. In all, 108 unique theoretical protocols are examined. The direct (D) and water proton exchange (PX) cycles are trialled along with a new approach, termed the semidirect (SD) cycle. The SD thermodynamic cycle offers some improvements over the D and PX schemes, as it bypasses the gas-phase heterolytic bond dissociation calculation required in the conventional D approach while also avoiding an aqueous OH(-) calculation required by the PX method when using water as the reference acid. With all three cycles, the recommended model chemistry employs M05-2X/cc-pVTZ Gibbs energies of solvation with a single discrete water molecule and a high-level composite method for the gas-phase reaction energies. With the SD cycle, these calculations result in a mean unsigned error of less than 1 pK(a) units, with respective mean signed error and maximum unsigned error of less than 0.5 and 2 pK(a) units. Similar results are obtained with the D and PX cycles, and further improvement is required in both the gas and aqueous phase ab initio energy calculations before we can truly discriminate between the thermodynamic cycles investigated here.

  18. Activated Rac1 requires gp130 for Stat3 activation, cell proliferation and migration

    Energy Technology Data Exchange (ETDEWEB)

    Arulanandam, Rozanne; Geletu, Mulu [Departments of Microbiology and Immunology and Pathology and Molecular Medicine, and Queen' s University Cancer Institute, Queen' s University, Botterell Hall, Rm. 713, Kingston, Ontario, Canada K7L 3N6 (Canada); Feracci, Helene [Universite Bordeaux 1, Centre de Recherche Paul Pascal, CNRS UPR 8641, 33600 Pessac (France); Raptis, Leda, E-mail: raptisl@queensu.ca [Departments of Microbiology and Immunology and Pathology and Molecular Medicine, and Queen' s University Cancer Institute, Queen' s University, Botterell Hall, Rm. 713, Kingston, Ontario, Canada K7L 3N6 (Canada)

    2010-03-10

    Rac1 (Rac) is a member of the Rho family of small GTPases which controls cell migration by regulating the organization of actin filaments. Previous results suggested that mutationally activated forms of the Rho GTPases can activate the Signal Transducer and Activator of Transcription-3 (Stat3), but the exact mechanism is a matter of controversy. We recently demonstrated that Stat3 activity of cultured cells increases dramatically following E-cadherin engagement. To better understand this pathway, we now compared Stat3 activity levels in mouse HC11 cells before and after expression of the mutationally activated Rac1 (Rac{sup V12}), at different cell densities. The results revealed for the first time a dramatic increase in protein levels and activity of both the endogenous Rac and Rac{sup V12} with cell density, which was due to inhibition of proteasomal degradation. In addition, Rac{sup V12}-expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that Rac{sup V12} is able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that Rac{sup V12} expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity, indicating that these cytokines may be responsible for the Stat3 activation by Rac{sup V12}. The upregulation of IL6 family cytokines was required for cell migration and proliferation induced by Rac{sup V12}, as shown by gp130 knockdown experiments, thus demonstrating that the gp130/Stat3 axis represents an essential effector of activated Rac for the regulation of key cellular functions.

  19. Environmental Enrichment Modifies the PKA-Dependence of Hippocampal LTP and Improves Hippocampus-Dependent Memory

    OpenAIRE

    Duffy, Steven N.; Craddock, Kenneth J.; Abel, Ted; Nguyen, Peter V.

    2001-01-01

    cAMP-dependent protein kinase (PKA) is critical for the expression of some forms of long-term potentiation (LTP) in area CA1 of the mouse hippocampus and for hippocampus-dependent memory. Exposure to spatially enriched environments can modify LTP and improve behavioral memory in rodents, but the molecular bases for the enhanced memory performance seen in enriched animals are undefined. We tested the hypothesis that exposure to a spatially enriched environment may alter the PKA dependence of h...

  20. TNF-alpha/IFN-gamma-induced iNOS expression increased by prostaglandin E2 in rat primary astrocytes via EP2-evoked cAMP/PKA and intracellular calcium signaling.

    Science.gov (United States)

    Hsiao, Han-Yun; Mak, Oi-Tong; Yang, Chung-Shi; Liu, Yu-Peng; Fang, Kuan-Ming; Tzeng, Shun-Fen

    2007-01-15

    Astrocytes, the most abundant glia in the central nervous system (CNS), produce a large amount of prostaglandin E(2) (PGE(2)) in response to proinflammatory mediators after CNS injury. However, it is unclear whether PGE(2) has a regulatory role in astrocytic activity under the inflamed condition. In the present work, we showed that PGE(2) increased inducible nitric oxide synthase (iNOS) production by tumor necrosis factor-alpha and interferon-gamma (T/I) in astrocytes. Pharmacological and RNA interference approaches further indicated the involvement of the receptor EP2 in PGE(2)-induced iNOS upregulation in T/I-treated astrocytes. Quantitative real-time polymerase chain reaction and gel mobility shift assays also demonstrated that PGE(2) increased iNOS transcription through EP2-induced cAMP/protein kinase A (PKA)-dependent pathway. Consistently, the effect of EP2 was significantly attenuated by the PKA inhibitor KT-5720 and partially suppressed by the inhibitor (SB203580) of p38 mitogen-activated protein kinase (p38MAPK), which serves as one of the downstream components of the PKA-dependent pathway. Interestingly, EP2-mediated PKA signaling appeared to increase intracellular Ca(2+) release through inositol triphosphate (IP3) receptor activation, which might in turn stimulate protein kinase C (PKC) activation to promote iNOS production in T/I-primed astrocytes. By analyzing the expression of astrocytic glial fibrillary acidic protein (GFAP), we found that PGE(2) alone only triggered the EP2-induced cAMP/PKA/p38MAPK signaling pathway in astrocytes. Collectively, PGE(2) may enhance T/I-induced astrocytic activation by augmenting iNOS/NO production through EP2-mediated cross-talk between cAMP/PKA and IP3/Ca(2+) signaling pathways.

  1. Discovery of potent and selective urea-based ROCK inhibitors: Exploring the inhibitor's potency and ROCK2/PKA selectivity by 3D-QSAR, molecular docking and molecular dynamics simulations.

    Science.gov (United States)

    Mei, Ding; Yin, Yan; Wu, Fanhong; Cui, Jiaxing; Zhou, Hong; Sun, Guofeng; Jiang, Yu; Feng, Yangbo

    2015-05-15

    An activity model and a selectivity model from 3D-QSAR studies were established by CoMFA and CoMSIA to explore the SAR. Then docking was used to study the binding modes between ligand and kinases (ROCK2 and PKA), and the molecular docking results were further validated by MD simulations. Computational results suggested that substitution containing positive charge attached to the middle phenyl ring, or electropositive group in urea linker was favored for both activity and ROCK2/PKA selectivity. Finally, three compounds were designed, and biological evaluation demonstrated that these molecular models were effective for guiding the design of potent and selective ROCK inhibitors.

  2. 20 CFR 665.330 - Are the NAFTA-TAA program requirements for rapid response also required activities?

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 3 2010-04-01 2010-04-01 false Are the NAFTA-TAA program requirements for... WORKFORCE INVESTMENT ACT Rapid Response Activities § 665.330 Are the NAFTA-TAA program requirements for... WIA are made available to workers who, under the NAFTA Implementation Act (Public Law 103-182), are...

  3. The Anomalous pKa of Tyr-9 in Glutathione S-Transferase A1-1 Catalyzes Product Release*

    Science.gov (United States)

    Ibarra, Catherine A.; Chowdhury, Pramit; Petrich, Jacob W.; Atkins, William M.

    2007-01-01

    The pKa of the catalytic Tyr-9 in glutathione S-transferase (GST) A1-1 is lowered from 10.3 to ~8.1 in the apoenzyme and ~9.0 with a GSH conjugate bound at the active site. However, a clear functional role for the unusual Tyr-9 pKa has not been elucidated. GSTA1-1 also includes a dynamic C terminus that undergoes a ligand-dependent disorder-to-order transition. Previous studies suggest a functional link between Tyr-9 ionization and C-terminal dynamics. Here we directly probe the role of Tyr-9 ionization in ligand binding and C-terminal conformation. An engineered mutant of rGSTA1-1, W21F/F222W, which contains a single Trp at the C terminus, was used as a fluorescent reporter of pH-dependent C-terminal dynamics. This mutant exhibited a pH-dependent change in Trp-222 emission properties consistent with changes in C-terminal solvation or conformation. The apparent pKa values for the conformational transition were 7.9 ± 0.1 and 9.3 ± 0.1 for the apoenzyme and ligand-bound enzyme, respectively, in excellent agreement with the pKa for Tyr-9 in these states. The Y9F/W21F/F222W mutant, however, exhibited no such pH-dependent changes. Time-resolved fluorescence anisotropy studies revealed a ligand-dependent, Tyr-9-dependent, change in the order parameter of Trp-222. However, no pH dependence was observed. In equilibrium and pre-steady-state ligand binding studies, product conjugate had a decreased equilibrium binding affinity (KD), concomitant with increased binding and dissociation rates, at higher pH values. Furthermore, the recovered pKa values for the pH-dependent microscopic rate constants ranged from 7.7 to 8.4, also in agreement with the pKa of Tyr-9. In contrast, the Y9F/W21F/F222W mutant had no pH-dependent transition in KD or rate constants for ligand binding or dissociation. The combined results indicate that the macroscopic populations of “open” and “closed” states of the C terminus are not determined solely by the ionization state of Tyr-9. However

  4. Advanced Extra-Vehicular Activity Pressure Garment Requirements Development

    Science.gov (United States)

    Ross, Amy; Aitchison, Lindsay; Rhodes, Richard

    2015-01-01

    The NASA Johnson Space Center advanced pressure garment technology development team is addressing requirements development for exploration missions. Lessons learned from the Z-2 high fidelity prototype development have reiterated that clear low-level requirements and verification methods reduce risk to the government, improve efficiency in pressure garment design efforts, and enable the government to be a smart buyer. The expectation is to provide requirements at the specification level that are validated so that their impact on pressure garment design is understood. Additionally, the team will provide defined verification protocols for the requirements. However, in reviewing exploration space suit high level requirements there are several gaps in the team's ability to define and verify related lower level requirements. This paper addresses the efforts in requirement areas such as mobility/fit/comfort and environmental protection (dust, radiation, plasma, secondary impacts) to determine the method by which the requirements can be defined and use of those methods for verification. Gaps exist at various stages. In some cases component level work is underway, but no system level effort has begun; in other cases no effort has been initiated to close the gap. Status of on-going efforts and potential approaches to open gaps are discussed.

  5. Thyroid-stimulating hormone inhibits adipose triglyceride lipase in 3T3-L1 adipocytes through the PKA pathway.

    Directory of Open Access Journals (Sweden)

    Dongqing Jiang

    Full Text Available Thyroid-stimulating hormone (TSH has been shown to play an important role in the regulation of triglyceride (TG metabolism in adipose tissue. Adipose triglyceride lipase (ATGL is a rate-limiting enzyme controlling the hydrolysis of TG. Thus far, it is unclear whether TSH has a direct effect on the expression of ATGL. Because TSH function is mediated through the TSH receptor (TSHR, TSHR knockout mice (Tshr-/- mice (supplemented with thyroxine were used in this study to determine the effects of TSHR deletion on ATGL expression. These effects were verified in 3T3-L1 adipocytes and potential underlying mechanisms were explored. In the Tshr-/- mice, ATGL expression in epididymal adipose tissue was significantly increased compared with that in Tshr+/+ mice. ATGL expression was observed to increase with the differentiation process of 3T3-L1 preadipocytes. In mature 3T3-L1 adipocytes, TSH significantly suppressed ATGL expression at both the protein and mRNA levels in a dose-dependent manner. Forskolin, which is an activator of adenylate cyclase, suppressed the expression of ATGL in 3T3-L1 adipocytes. The inhibitory effects of TSH on ATGL expression were abolished by H89, which is a protein kinase A (PKA inhibitor. These results indicate that TSH has an inhibitory effect on ATGL expression in mature adipocytes. The associated mechanism is related to PKA activation.

  6. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    Science.gov (United States)

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  7. Differential regulation of glucose transport activity in yeast by specific cAMP signatures.

    Science.gov (United States)

    Bermejo, Clara; Haerizadeh, Farzad; Sadoine, Mayuri S C; Chermak, Diane; Frommer, Wolf B

    2013-06-15

    Successful colonization and survival in variable environments require a competitive advantage during the initial growth phase after experiencing nutrient changes. Starved yeast cells anticipate exposure to glucose by activating the Hxt5p (hexose transporter 5) glucose transporter, which provides an advantage during early phases after glucose resupply. cAMP and glucose FRET (fluorescence resonance energy transfer) sensors were used to identify three signalling pathways that co-operate in the anticipatory Hxt5p activity in glucose-starved cells: as expected the Snf1 (sucrose nonfermenting 1) AMP kinase pathway, but, surprisingly, the sugar-dependent G-protein-coupled Gpr1 (G-protein-coupled receptor 1)/cAMP/PKA (protein kinase A) pathway and the Pho85 (phosphate metabolism 85)/Plc (phospholipase C) 6/7 pathway. Gpr1/cAMP/PKA are key elements of a G-protein-coupled sugar response pathway that produces a transient cAMP peak to induce growth-related genes. A novel function of the Gpr1/cAMP/PKA pathway was identified in glucose-starved cells: during starvation the Gpr1/cAMP/PKA pathway is required to maintain Hxt5p activity in the absence of glucose-induced cAMP spiking. During starvation, cAMP levels remain low triggering expression of HXT5, whereas cAMP spiking leads to a shift to the high capacity Hxt isoforms.

  8. Human breast cancer cell-mediated bone collagen degradation requires plasminogen activation and matrix metalloproteinase activity

    Directory of Open Access Journals (Sweden)

    Hill Peter A

    2005-02-01

    Full Text Available Abstract Background Breast cancer cells frequently metastasize to the skeleton and induce extensive bone destruction. Cancer cells produce proteinases, including matrix metalloproteinases (MMPs and the plasminogen activator system (PAS which promote invasion of extracellular matrices, but whether these proteinases degrade bone matrix is unclear. To characterize the role that breast cancer cell proteinases play in bone degradation we compared the effects of three human breast cancer cell lines, MDA-MB-231, ZR-75-1 and MCF-7 with those of a normal breast epithelial cell line, HME. The cell lines were cultured atop radiolabelled matrices of either mineralized or non-mineralized bone or type I collagen, the principal organic constituent of bone. Results The 3 breast cancer cell lines all produced significant degradation of the 3 collagenous extracellular matrices (ECMs whilst the normal breast cell line was without effect. Breast cancer cells displayed an absolute requirement for serum to dissolve collagen. Degradation of collagen was abolished in plasminogen-depleted serum and could be restored by the addition of exogenous plasminogen. Localization of plasmin activity to the cell surface was critical for the degradation process as aprotinin, but not α2 antiplasmin, prevented collagen dissolution. During ECM degradation breast cancer cell lines expressed urokinase-type plasminogen activator (u-PA and uPA receptor, and MMPs-1, -3, -9,-13, and -14. The normal breast epithelial cell line expressed low levels of MMPs-1, and -3, uPA and uPA receptor. Inhibitors of both the PAS (aprotinin and PA inhibitor-1 and MMPs (CT1166 and tisue inhibitor of metalloproteinase blocked collagen degradation, demonstrating the requirement of both plasminogen activation and MMP activity for degradation. The activation of MMP-13 in human breast cancer cells was prevented by plasminogen activator inhibitor-1 but not by tissue inhibitor of metalloproteinase-1, suggesting

  9. Rationalization of the pKa values of alcohols and thiols using atomic charge descriptors and its application to the prediction of amino acid pKa's

    DEFF Research Database (Denmark)

    Ugur, Ilke; Marion, Antoine; Parant, Stéphane

    2014-01-01

    approaches (gas phase or continuum solvent-based approaches), with five distinct atomic charge models (Mulliken, Löwdin, NPA, Merz-Kollman, and CHelpG), and with nine different DFT functionals combined with 16 different basis sets. Moreover, the capability of semiempirical methods (AM1, RM1, PM3, and PM6......) to also predict pKa's of thiols, phenols, and alcohols is analyzed. From our benchmarks, the best combination to reproduce experimental pKa's is to compute NPA atomic charge using the CPCM model at the B3LYP/3-21G and M062X/6-311G levels for alcohols (R(2) = 0.995) and thiols (R(2) = 0.986), respectively...... of the experimental pKa's of phenols, alcohols, and thiols. Our protocol is based on the linear relationship between computed atomic charges of the anionic form of the molecules (being either phenolates, alkoxides, or thiolates) and their respective experimental pKa values. It is tested with different environment...

  10. Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes

    DEFF Research Database (Denmark)

    Petersen, Rasmus Koefoed; Madsen, Lise; Pedersen, Lone Møller

    2008-01-01

    Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for cAMP-dependent ......Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for c......AMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho...

  11. Estrogenic activity of diarylheptanoids from Curcuma comosa Roxb. Requires metabolic activation.

    Science.gov (United States)

    Winuthayanon, Wipawee; Suksen, Kanoknetr; Boonchird, Chuenchit; Chuncharunee, Aporn; Ponglikitmongkol, Mathurose; Suksamrarn, Apichart; Piyachaturawat, Pawinee

    2009-02-11

    Curcuma comosa Roxb. has traditionally been used as a dietary supplement for health promotion in peri- and postmenopausal women in Thailand. We investigated the estrogenic activity of 7 naturally occurring diarylheptanoids from the extracts of C. comosa both in vitro and in vivo. A yeast recombinant system containing human estrogen receptor alpha, coactivator TIF2 and a beta-galactosidase reporter gene was used to determine estrogenic activity of diarylheptanoids metabolically activated with rat liver S9-fraction prior to the assay. The most potent compound was (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol, with a relative potency of 4% compared to 17beta-estradiol. The metabolic activation of diarylheptanoids markedly enhanced their efficiency. The chemical structure required for estrogenic activity of diarylheptanoids was the presence of a keto group at C3 and absence of hydroxyl moiety in ring B. Only diarylheptanoids showing full estrogenic efficiency in vitro were able to elicit uterotrophic activity of in immature ovariectomized rat. This is the first evidence for in vivo estrogenic activity of diarylheptanoids from C. comosa. This novel class of natural phytoestrogens has the potential to be developed for use as dietary supplement in the treatment of menopausal symptoms.

  12. Opposing needling promotes behavior recovery and exerts neuroprotection via the cAMP/PKA/CREB signal transduction pathway in transient MCAO rats

    Science.gov (United States)

    JIANG, YIJING; YANG, SHANLI; TAO, JING; LIN, ZHICHENG; YE, XIAOQIAN; YOU, YONGMEI; PENG, JUN; HONG, ZHENFENG; CHEN, LIDIAN

    2016-01-01

    The aim of the present study was to investigate whether the cyclic adenosine 3′,5′-monophosphate (cAMP)/protein kinase A(PKA)/cAMP-responsive element binding protein (CREB) signal transduction pathway triggered by γ-aminobutyric acid class B (GABAB) receptor activation is involved in neuroprotection against ischemia and behavioral recovery induced by opposing needling (ON). A total of 80 rats were randomly divided into four groups: A sham operation group, an ischemia group, an ON group and an ON group effectively inhibited by the GABAB receptor antagonist, CGP35384 (n=20/group). The behavior of the rats was assessed by their neurological deficit score, whereas the impairment of gait was examined using the CatWalk system. The volume of cerebral infarction was examined upon treatment with 2,3,5-triphenyltetrazolium chloride. The expression levels of CREB, GABAB1 and GABAB2 were examined by western blotting and reverse transcription-quantitative polymerase chain reaction, and the activity of adenylyl cyclase (AC), cAMP and PKA in the serum was detected using an enzyme-linked immunosorbent assay. In the present study, in comparison with other groups, the ON group exhibited a reduced score for the neurological deficit, the stride length and swing speed were improved, and the volume of infarction was reduced. However, these effects were reversed upon administration of CGP35384. Additionally, the expression levels of CREB, GABAB1 and GABAB2 were increased in the ON group. The levels of AC, cAMP and PKA in the serum were also increased in the ON group, whereas the addition of CGP35384 reversed these effects. The results of the present study demonstrated that ON markedly protected the brain against transient cerebral ischemic injury, and this effect was possibly mediated by the activation of the GABAB/cAMP/PKA/CREB signal transduction pathway. These findings implied that ON may be a potential therapeutic method for treating stroke. PMID:26780954

  13. PKA, novel PKC isoforms, and ERK is mediating PACAP auto-regulation via PAC1R in human neuroblastoma NB-1 cells.

    Science.gov (United States)

    Georg, Birgitte; Falktoft, Birgitte; Fahrenkrug, Jan

    2016-12-01

    The neuropeptide PACAP is expressed throughout the central and peripheral nervous system where it modulates diverse physiological functions including neuropeptide gene expression. We here report that in human neuroblastoma NB-1 cells PACAP transiently induces its own expression. Maximal PACAP mRNA expression was found after stimulation with PACAP for 3h. PACAP auto-regulation was found to be mediated by activation of PACAP specific PAC1Rs as PACAP had >100-fold higher efficacy than VIP, and the PAC1R selective agonist Maxadilan potently induced PACAP gene expression. Experiments with pharmacological kinase inhibitors revealed that both PKA and novel but not conventional PKC isozymes were involved in the PACAP auto-regulation. Inhibition of MAPK/ERK kinase (MEK) also impeded the induction, and we found that PKA, novel PKC and ERK acted in parallel and were thus not part of the same pathways. The expression of the transcription factor EGR1 previously ascribed as target of PACAP signalling was found to be transiently induced by PACAP and pharmacological inhibition of either PKC or MEK1/2 abolished PACAP mediated EGR1 induction. In contrast, inhibition of PKA mediated increased PACAP mediated EGR1 induction. Experiments using siRNA against EGR1 to lower the expression did however not affect the PACAP auto-regulation indicating that this immediate early gene product is not part of PACAP auto-regulation in NB-1 cells. We here reveal that in NB-1 neuroblastoma cells, PACAP induces its own expression by activation of PAC1R, and that the signalling is different from the PAC1R signalling mediating induction of VIP in the same cells. PACAP auto-regulation depends on parallel activation of PKA, novel PKC isoforms, and ERK, while EGR1 does not seem to be part of the PACAP auto-regulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Multidisciplinary and Active/Collaborative Approaches in Teaching Requirements Engineering

    Science.gov (United States)

    Rosca, Daniela

    2005-01-01

    The requirements engineering course is a core component of the curriculum for the Master's in Software Engineering programme, at Monmouth University (MU). It covers the process, methods and tools specific to this area, together with the corresponding software quality issues. The need to produce software engineers with strong teamwork and…

  15. Multidisciplinary and Active/Collaborative Approaches in Teaching Requirements Engineering

    Science.gov (United States)

    Rosca, Daniela

    2005-01-01

    The requirements engineering course is a core component of the curriculum for the Master's in Software Engineering programme, at Monmouth University (MU). It covers the process, methods and tools specific to this area, together with the corresponding software quality issues. The need to produce software engineers with strong teamwork and…

  16. 10 CFR 490.507 - Credit activity reporting requirements.

    Science.gov (United States)

    2010-01-01

    ... 490.507 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ALTERNATIVE FUEL TRANSPORTATION PROGRAM... person or fleet applying for allocation of alternative fueled vehicle credits must submit a credit... acquired in model years before the first model year the fleet or covered person is required to...

  17. 20 CFR 655.1302 - Required pre-filing activity.

    Science.gov (United States)

    2010-04-01

    ... Temporary Employment Certification must maintain documentation of its advertising and recruitment efforts as... satisfy all the requirements for newspaper advertisements contained in § 655.103 and comply with the... Secretary. (g) Newspaper advertisements. (1) During the period of time that the job order is...

  18. 13 CFR 107.590 - Licensee's requirement to maintain active operations.

    Science.gov (United States)

    2010-01-01

    ... active operations. 107.590 Section 107.590 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION SMALL BUSINESS INVESTMENT COMPANIES Managing the Operations of a Licensee Requirement to Conduct Active...) Permitted exceptions to activity requirements. You are considered active if your failure to meet...

  19. Computer programs for calculating pKa: a comparative study for 3-(3-(2-nitrophenylprop-2-enoyl-2H-1-benzopyran-2-one

    Directory of Open Access Journals (Sweden)

    SELMA ŠPIRTOVIĆ-HALILOVIĆ

    2010-02-01

    Full Text Available Coumarin-based compounds containing a chalcone moiety exhibit antimicrobial activity. These substances are potential drugs and it is important to determine their pKa values. However, they are almost insoluble in water. The dissociation constant was experimentally determined by potentiometric titration for 3-[3-(2-nitrophenylprop-2-enoyl]-2H-1-benzopyran-2-one because this compound shows good activity and solubility. A number of different computer programs for the calculation of the dissociation constant of chemical compounds have been developed. The pKa value of the target compound was calculated using three different computer programs, i.e., the ACD/pKa, CSpKaPredictor and ADME/ToxWEB programs, which are based on different theoretical approaches. The analysis demonstrated good agreement between the experimentally observed pKa value of 3-[3-(2-nitrophenylprop-2-enoyl]-2H-1-benzopyran-2-one and the value calculated using the computer program CSpKa.

  20. HPRT-deficiency dysregulates cAMP-PKA signaling and phosphodiesterase 10A expression: mechanistic insight and potential target for Lesch-Nyhan Disease?

    Science.gov (United States)

    Guibinga, Ghiabe-Henri; Murray, Fiona; Barron, Nikki

    2013-01-01

    Lesch-Nyhan Disease (LND) is the result of mutations in the X-linked gene encoding the purine metabolic enzyme, hypoxanthine guanine phosphoribosyl transferase (HPRT). LND gives rise to severe neurological anomalies including mental retardation, dystonia, chorea, pyramidal signs and a compulsive and aggressive behavior to self injure. The neurological phenotype in LND has been shown to reflect aberrant dopaminergic signaling in the basal ganglia, however there are little data correlating the defect in purine metabolism to the neural-related abnormalities. In the present studies, we find that HPRT-deficient neuronal cell lines have reduced CREB (cAMP response element-binding protein) expression and intracellular cyclic AMP (cAMP), which correlates with attenuated CREB-dependent transcriptional activity and a reduced phosphorylation of protein kinase A (PKA) substrates such as synapsin (p-syn I). Of interest, we found increased expression of phosphodiesterase 10A (PDE10A) in HPRT-deficient cell lines and that the PDE10 inhibitor papaverine and PDE10A siRNA restored cAMP/PKA signaling. Furthermore, reconstitution of HPRT expression in mutant cells partly increased cAMP signaling synapsin phosphorylation. In conclusion, our data show that HPRT-deficiency alters cAMP/PKA signaling pathway, which is in part due to the increased of PDE10A expression and activity. These findings suggest a mechanistic insight into the possible causes of LND and highlight PDE10A as a possible therapeutic target for this intractable neurological disease.

  1. PKA Inhibitor H89 (N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulfonamide Attenuates Synaptic Dysfunction and Neuronal Cell Death following Ischemic Injury

    Directory of Open Access Journals (Sweden)

    Juhyun Song

    2015-01-01

    Full Text Available The cyclic AMP-dependent protein kinase (PKA, which activates prosurvival signaling proteins, has been implicated in the expression of long-term potentiation and hippocampal long-term memory. It has come to light that H89 commonly known as the PKA inhibitor have diverse roles in the nervous system that are unrelated to its role as a PKA inhibitor. We have investigated the role of H89 in ischemic and reperfusion injury. First, we examined the expression of postsynaptic density protein 95 (PSD95, microtubule-associated protein 2 (MAP2, and synaptophysin in mouse brain after middle cerebral artery occlusion injury. Next, we examined the role of H89 pretreatment on the expression of brain-derived neurotrophic factor (BDNF, PSD95, MAP2, and the apoptosis regulators Bcl2 and cleaved caspase-3 in cultured neuroblastoma cells exposed to hypoxia and reperfusion injury. In addition, we investigated the alteration of AKT activation in H89 pretreated neuroblastoma cells under hypoxia and reperfusion injury. The data suggest that H89 may contribute to brain recovery after ischemic stroke by regulating neuronal death and proteins related to synaptic plasticity.

  2. Ser1928 phosphorylation by PKA stimulates the L-type Ca2+ channel CaV1.2 and vasoconstriction during acute hyperglycemia and diabetes

    Science.gov (United States)

    Nystoriak, Matthew A.; Nieves-Cintrón, Madeline; Patriarchi, Tommaso; Buonarati, Olivia R.; Prada, Maria Paz; Morotti, Stefano; Grandi, Eleonora; Fernandes, Julia Dos Santos; Forbush, Katherine; Hofmann, Franz; Sasse, Kent C.; Scott, John D.; Ward, Sean M.; Hell, Johannes W.; Navedo, Manuel F.

    2017-01-01

    Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type CaV1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in CaV1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the CaV1.2 channel pore-forming subunit (α1C) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in CaV1.2 channel activity were associated with PKA activity, leading to α1C phosphorylation at Ser1928. Compared to arteries from low-fat diet (LFD)–fed mice and nondiabetic patients, arteries from high-fat diet (HFD)–fed mice and from diabetic patients had increased Ser1928 phosphorylation and CaV1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser1928 phosphorylation and CaV1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser1928 phosphorylation, arterial myocytes and arteries from knockin mice expressing a CaV1.2 with Ser1928 mutated to alanine (S1928A) lacked glucose-mediated increases in CaV1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in CaV1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1C phosphorylation at Ser1928 in stimulating CaV1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes. PMID:28119464

  3. Adenovirus vector E4 gene regulates connexin 40 and 43 expression in endothelial cells via PKA and PI3K signal pathways.

    Science.gov (United States)

    Zhang, Fan; Cheng, Joseph; Lam, George; Jin, David K; Vincent, Loïc; Hackett, Neil R; Wang, Shiyang; Young, Lauren M; Hempstead, Barbara; Crystal, Ronald G; Rafii, Shahin

    2005-05-13

    Connexins (Cxs) provide a means for intercellular communication and play important roles in the pathophysiology of vascular cardiac diseases. Infection of endothelial cells (ECs) with first-generation E1/E3-deleted E4+ adenovirus (AdE4+) selectively modulates the survival and angiogenic potential of ECs by as of yet unrecognized mechanisms. We show here that AdE4+ vectors potentiate Cx expression in ECs in vitro and in mouse heart tissue. Infection of ECs with AdE4+, but not AdE4-, resulted in a time- and dose-dependent induction of junctional Cx40 expression and suppression of Cx43 protein and mRNA expression. Treatment of ECs with PKA inhibitor H89 or PI3K inhibitor LY294002 prevented the AdE4+-mediated regulation of Cx40 and Cx43 that was associated with diminished AdE4+-mediated survival of ECs. Moreover, both PKA activity and cAMP-response element (CRE)-binding activity were enhanced by treatment of ECs with AdE4+. However, there is no causal evidence of a cross-talk between the 2 modulatory pathways, PKA and PI3K. Remarkably, Cx40 immunostaining was markedly increased and Cx43 was decreased in the heart tissue of mice treated with intra-tracheal AdE4+. Taken together, these results suggest that AdE4+ may play an important role in the regulation of Cx expression in ECs, and that these effects are mediated by both the PKA/CREB and PI3K signaling pathways.

  4. Cholesterol enhances neuron susceptibility to apoptotic stimuli via cAMP/PKA/CREB-dependent up-regulation of Kv2.1.

    Science.gov (United States)

    Zhou, Meng-Hua; Yang, Guang; Jiao, Song; Hu, Chang-Long; Mei, Yan-Ai

    2012-02-01

    Cholesterol is a major component of membrane lipid rafts. It is more abundant in the brain than in other tissues and plays a critical role in maintaining brain function. We report here that a significant enhancement in apoptosis in rat cerebellar granule neurons (CGNs) was observed upon incubation with 5mM K(+) /serum free (LK-S) medium. Cholesterol enrichment further potentiated CGN apoptosis incubated under LK-S medium. On the contrary, cholesterol depletion using methyl-beta-cyclodextrin protected the CGNs from apoptosis induced by LK-S treatment. Cholesterol enrichment, however, did not induce apoptosis in CGNs that have been incubated with 25mM K(+) /serum medium. Mechanistically, increased I(K) currents and DNA fragmentation were found in CGNs incubated in LK-S, which was further potentiated in the presence of cholesterol. Cholesterol-treated CGNs also exhibited increased cAMP levels and up-regulation of Kv2.1 expression. Increased levels of activated form of PKA and phospho-CREB further supported activation of the cAMP/PKA pathway upon treatment of CGNs with cholesterol-containing LK-S medium. Conversely, inhibition of PKA or small G protein Gs abolished the increase in I(K) current and the potentiation of Kv2.1 expression, leading to reduced susceptibility of CGNs to LK-S and cholesterol-induced apoptosis. Our results demonstrate that the elevation of membrane cholesterol enhances CGN susceptibility to apoptotic stimuli via cAMP/PKA/CREB-dependent up-regulation of Kv2.1. Our data provide new evidence for the role of cholesterol in eliciting neuronal cell death. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  5. KC-46 Workforce Requirements for Depot Maintenance Activation

    Science.gov (United States)

    2014-03-27

    Community College of the Air force CFR: Code of Federal Regulation CLS: Contract Logistic Service CoF: Complex of the Future CSR : Case Study...maintain. Organization H, an organization that provides air freight transportation , overcomes this challenge by mixing seasoned technicians with new...safety and efficiency of flight in this country” (US Department of Transportation , 2013). The safe operation of aircraft requires the strict adherence

  6. A compartmental model of the cAMP/PKA/MAPK pathway in Bio-PEPA

    Directory of Open Access Journals (Sweden)

    Federica Ciocchetta

    2009-11-01

    Full Text Available The vast majority of biochemical systems involve the exchange of information between different compartments, either in the form of transportation or via the intervention of membrane proteins which are able to transmit stimuli between bordering compartments. The correct quantitative handling of compartments is, therefore, extremely important when modelling real biochemical systems. The Bio-PEPA process algebra is equipped with the capability of explicitly defining quantitative information such as compartment volumes and membrane surface areas. Furthermore, the recent development of the Bio-PEPA Eclipse Plug-in allows us to perform a correct stochastic simulation of multi-compartmental models. Here we present a Bio-PEPA compartmental model of the cAMP/PKA/MAPK pathway. We analyse the system using the Bio-PEPA Eclipse Plug-in and we show the correctness of our model by comparison with an existing ODE model. Furthermore, we perform computational experiments in order to investigate certain properties of the pathway. Specifically, we focus on the system response to the inhibition and strengthening of feedback loops and to the variation in the activity of key pathway reactions and we observe how these modifications affect the behaviour of the pathway. These experiments are useful to understand the control and regulatory mechanisms of the system.

  7. Involvement of PKA and HO-1 signaling in anti-inflammatory effects of surfactin in BV-2 microglial cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Sun Young; Kim, Ji-Hee [Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Keumjeong-gu, Busan 609-735 (Korea, Republic of); Lee, Sang Joon [Department of Microbiology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Keumjeong-gu, Busan 609-735 (Korea, Republic of); Kim, YoungHee, E-mail: yheekim@pusan.ac.kr [Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Keumjeong-gu, Busan 609-735 (Korea, Republic of)

    2013-04-01

    Surfactin, one of the most powerful biosurfactants, is a bacterial cyclic lipopeptide. Here, we investigated the anti-neuroinflammatory properties of surfactin in lipoteichoic acid (LTA)-stimulated BV-2 microglial cells. Surfactin significantly inhibited excessive production of the pro-inflammatory mediators TNF-α, IL-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1), prostaglandin E{sub 2} (PGE{sub 2}), nitric oxide (NO) and reactive oxygen species (ROS), and suppressed the expression of matrix metalloproteinase-9 (MMP-9), inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). Subsequent mechanistic studies revealed that surfactin inhibited LTA-induced nuclear factor-kappaB (NF-κB) and signal transducer and activator of transcription-1 (STAT-1) activation. However, surfactin increases the phosphorylation of the STAT-3, a component of the homeostatic mechanism causing anti-inflammatory events. We also demonstrated that surfactin induces heme oxygenase-1 (HO-1) expression and nuclear factor-regulated factor-2 (Nrf-2) activation, and that the anti-inflammatory effects of surfactin are abrogated by small interfering RNA-mediated knock-down of HO-1 or Nrf-2. Interestingly, we found that surfactin increased the level of cAMP and induced phosphorylation of cAMP responsive element binding protein (CREB) in microglial cells. Furthermore, treatment with the protein kinase A (PKA) inhibitor, H-89, blocked HO-1 induction by surfactin and abolished surfactin's suppressive effects on ROS and NO production. These results indicate that HO-1 and its upstream effector, PKA, play a pivotal role in the anti-neuroinflammatory response of surfactin in LTA-stimulated microglia. Therefore, surfactin might have therapeutic potential for neuroprotective agents to treat inflammatory and neurodegenerative diseases. - Highlights: ► Surfactin inhibits proinflammatory mediator synthesis in LTA-activated BV-2 cells. ► Surfactin suppresses NF-κB and STAT-1, but potentiates

  8. Progress in the prediction of pKa values in proteins

    Energy Technology Data Exchange (ETDEWEB)

    Alexov, Emil; Mehler, Ernest L.; Baker, Nathan A.; Baptista, Antonio; Huang, Yong; Milletti, Francesca; Nielsen, Jens E.; Farrell, Damien; Carstensen, Tommy; Olsson, Mats H.; Shen, Jana K.; Warwicker, Jim; Williams, Sarah; Word, J Michael

    2011-12-15

    The pKa-cooperative aims to provide a forum for experimental and theoretical researchers interested in protein pKa values and protein electrostatics in general. The first round of the pKa -cooperative, which challenged computational labs to carry out blind predictions against pKas experimentally determined in the laboratory of Bertrand Garcia-Moreno, was completed and results discussed at the Telluride meeting (July 6-10, 2009). This paper serves as an introduction to the reports submitted by the blind prediction participants that will be published in a special issue of PROTEINS: Structure, Function and Bioinformatics. Here we briefly outline existing approaches for pKa calculations, emphasizing methods that were used by the participants in calculating the blind pKa values in the first round of the cooperative. We then point out some of the difficulties encountered by the participating groups in making their blind predictions, and finally try to provide some insights for future developments aimed at improving the accuracy of pKa calculations.

  9. PTPIP51: A New Interaction Partner of the Insulin Receptor and PKA in Adipose Tissue

    Directory of Open Access Journals (Sweden)

    M. A. Bobrich

    2013-01-01

    Full Text Available Aims. Our previous experiments revealed an association of PTPIP51 (protein tyrosine phosphatase interacting protein 51 with the insulin signalling pathway through PTP1B and 14-3-3beta. We aimed to clarify the role of PTPIP51 in adipocyte metabolism. Methods. Four groups of ten C57Bl/6 mice each were used. Two groups were fed a standard diet; two groups were fed a high-fat diet. Two groups (one high-fat diet and one standard diet were submitted to endurance training, while the remaining two groups served as untrained control groups. After ten weeks, we measured glucose tolerance of the mice. Adipose tissue samples were analyzed by immunofluorescence and Duolink proximity ligation assay to quantify interactions of PTPIP51 with either insulin receptor (IR or PKA. Results. PTPIP51 and the IR and PTPIP51 and PKA, respectively, were colocalized in all groups. Standard diet animals that were submitted to endurance training showed low PTPIP51-IR and PTPIP51-PKA interactions. The interaction levels of both the IR and PKA differed between the feeding and training groups. Conclusion. PTPIP51 might serve as a linking protein in adipocyte metabolism by connecting the IR-triggered lipogenesis with the PKA-dependent lipolysis. PTPIP51 interacts with both proteins, therefore being a potential gateway for the cooperation of both pathways.

  10. Assembly of a Notch transcriptional activation complex requires multimerization.

    Science.gov (United States)

    Vasquez-Del Carpio, Rodrigo; Kaplan, Fred M; Weaver, Kelly L; VanWye, Jeffrey D; Alves-Guerra, Marie-Clotilde; Robbins, David J; Capobianco, Anthony J

    2011-04-01

    Notch transmembrane receptors direct essential cellular processes, such as proliferation and differentiation, through direct cell-to-cell interactions. Inappropriate release of the intracellular domain of Notch (N(ICD)) from the plasma membrane results in the accumulation of deregulated nuclear N(ICD) that has been linked to human cancers, notably T-cell acute lymphoblastic leukemia (T-ALL). Nuclear N(ICD) forms a transcriptional activation complex by interacting with the coactivator protein Mastermind-like 1 and the DNA binding protein CSL (for CBF-1/Suppressor of Hairless/Lag-1) to regulate target gene expression. Although it is well understood that N(ICD) forms a transcriptional activation complex, little is known about how the complex is assembled. In this study, we demonstrate that N(ICD) multimerizes and that these multimers function as precursors for the stepwise assembly of the Notch activation complex. Importantly, we demonstrate that the assembly is mediated by N(ICD) multimers interacting with Skip and Mastermind. These interactions form a preactivation complex that is then resolved by CSL to form the Notch transcriptional activation complex on DNA.

  11. 10 CFR 810.8 - Activities requiring specific authorization.

    Science.gov (United States)

    2010-01-01

    ...-nuclear-weapon states that do not have full-scope IAEA safeguards agreements in force. Afghanistan Albania...-Bissau* Haiti* India* Iran Iraq* Israel* Kazakhstan Kenya* Korea, People's Democratic Republic of* Kuwait... technology for an activity in any foreign country. (c) Engaging in or providing assistance or training in any...

  12. 33 CFR 322.3 - Activities requiring permits.

    Science.gov (United States)

    2010-07-01

    ... States that are part of the civil works activities of the Corps of Engineers, unless covered by a... separate regulations. Agreement for construction or engineering services performed for other agencies by... history and/or provisions of the Act should clearly demonstrate that Congress was approving the...

  13. Tapered whiskers are required for active tactile sensation

    Science.gov (United States)

    Hires, Samuel Andrew; Pammer, Lorenz; Svoboda, Karel; Golomb, David

    2013-01-01

    Many mammals forage and burrow in dark constrained spaces. Touch through facial whiskers is important during these activities, but the close quarters makes whisker deployment challenging. The diverse shapes of facial whiskers reflect distinct ecological niches. Rodent whiskers are conical, often with a remarkably linear taper. Here we use theoretical and experimental methods to analyze interactions of mouse whiskers with objects. When pushed into objects, conical whiskers suddenly slip at a critical angle. In contrast, cylindrical whiskers do not slip for biologically plausible movements. Conical whiskers sweep across objects and textures in characteristic sequences of brief sticks and slips, which provide information about the tactile world. In contrast, cylindrical whiskers stick and remain stuck, even when sweeping across fine textures. Thus the conical whisker structure is adaptive for sensor mobility in constrained environments and in feature extraction during active haptic exploration of objects and surfaces. DOI: http://dx.doi.org/10.7554/eLife.01350.001 PMID:24252879

  14. Local arginase 1 activity is required for cutaneous wound healing.

    Science.gov (United States)

    Campbell, Laura; Saville, Charis R; Murray, Peter J; Cruickshank, Sheena M; Hardman, Matthew J

    2013-10-01

    Chronic nonhealing wounds in the elderly population are associated with a prolonged and excessive inflammatory response, which is widely hypothesized to impede healing. Previous studies have linked alterations in local L-arginine metabolism, principally mediated by the enzymes arginase (Arg) and inducible nitric oxide synthase (iNOS), to pathological wound healing. Over subsequent years, interest in Arg/iNOS has focused on the classical versus alternatively activated (M1/M2) macrophage paradigm. Although the role of iNOS during healing has been studied, Arg contribution to healing remains unclear. Here, we report that Arg is dynamically regulated during acute wound healing. Pharmacological inhibition of local Arg activity directly perturbed healing, as did Tie2-cre-mediated deletion of Arg1, revealing the importance of Arg1 during healing. Inhibition or depletion of Arg did not alter alternatively activated macrophage numbers but instead was associated with increased inflammation, including increased influx of iNOS(+) cells and defects in matrix deposition. Finally, we reveal that in preclinical murine models reduced Arg expression directly correlates with delayed healing, and as such may represent an important future therapeutic target.

  15. Active diver thermal protection requirements for cold water diving.

    Science.gov (United States)

    Lippitt, M W; Nuckols, M L

    1983-07-01

    An analysis of the supplemental heating requirements for military divers, both surface-tended and free-swimming, is presented. Specific categories of diver heat loss, including respiratory losses and suit convective losses, are characterized over a range of water temperatures, depths, and breathing gas mixtures. The need for a 1-kW diver heater is identified to accommodate deep dives where the limitation of a surface-supplied hot water source and a long hot water umbilical pose an unacceptable burden. A 0.5-kW heater is shown to be satisfactory to extend the performance of existing closed circuit-breathing apparatuses for shallow water operations in water temperatures as low as 40 degrees F. Substantial benefits in heat savings are shown through the use of passive regenerative breath heaters and alternative suit inflation gases for drysuit use.

  16. 蛋白激酶A与疼痛调控%PKA and Pain Regulation

    Institute of Scientific and Technical Information of China (English)

    朱红妮; 赵小霞; 孙志宏; 白占涛

    2015-01-01

    蛋白激酶A( PKA)是G蛋白偶联受体所介导细胞信号通路中重要的信号转导因子,广泛参与了伤害性信息的调控和痛觉过敏的形成.本文聚焦PKA的结构特点、分布、功能及其对疼痛的调控机制,以充实基于PKA信号通路的疼痛机制.%Protein kinase A was an important signal transduction factor participating in cell signaling pathways which was mediated by G protein coupled receptor,and involvement in the regulation of nociceptive information and the formation of hyperalgesia widely. In this paper,the structure characteristics,distribution,functions of PKA and its pain regulation were reviewed so as to enrich pain mechanism based on PKA signaling pathway.

  17. Slicing-independent RISC activation requires the argonaute PAZ domain.

    Science.gov (United States)

    Gu, Shuo; Jin, Lan; Huang, Yong; Zhang, Feijie; Kay, Mark A

    2012-08-21

    Small RNAs regulate genetic networks through a ribonucleoprotein complex called the RNA-induced silencing complex (RISC), which, in mammals, contains at its center one of four Argonaute proteins (Ago1-Ago4). A key regulatory event in the RNA interference (RNAi) and microRNA (miRNA) pathways is Ago loading, wherein double-stranded small-RNA duplexes are incorporated into RISC (pre-RISC) and then become single-stranded (mature RISC), a process that is not well understood. The Agos contain an evolutionarily conserved PAZ (Piwi/Argonaute/Zwille) domain whose primary function is to bind the 3' end of small RNAs. We created multiple PAZ-domain-disrupted mutant Ago proteins and studied their biochemical properties and biological functionality in cells. We found that the PAZ domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer-substrate duplex RNAs, PAZ-disrupted Agos bound duplex small interfering RNAs, but were unable to unwind or eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved PAZ domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA- and RNAi-based therapeutics.

  18. pKa values in proteins determined by electrostatics applied to molecular dynamics trajectories.

    Science.gov (United States)

    Meyer, Tim; Knapp, Ernst-Walter

    2015-06-09

    For a benchmark set of 194 measured pKa values in 13 proteins, electrostatic energy computations are performed in which pKa values are computed by solving the Poisson-Boltzmann equation. In contrast to the previous approach of Karlsberg(+) (KB(+)) that essentially used protein crystal structures with variations in their side chain conformations, the present approach (KB2(+)MD) uses protein conformations from four molecular dynamics (MD) simulations of 10 ns each. These MD simulations are performed with different specific but fixed protonation patterns, selected to sample the conformational space for the different protonation patterns faithfully. The root-mean-square deviation between computed and measured pKa values (pKa RMSD) is shown to be reduced from 1.17 pH units using KB(+) to 0.96 pH units using KB2(+)MD. The pKa RMSD can be further reduced to 0.79 pH units, if each conformation is energy-minimized with a dielectric constant of εmin = 4 prior to calculating the electrostatic energy. The electrostatic energy expressions upon which the computations are based have been reformulated such that they do not involve terms that mix protein and solvent environment contributions and no thermodynamic cycle is needed. As a consequence, conformations of the titratable residues can be treated independently in the protein and solvent environments. In addition, the energy terms used here avoid the so-called intrinsic pKa and can therefore be interpreted without reference to arbitrary protonation states and conformations.

  19. P-glycoprotein ATPase activity requires lipids to activate a switch at the first transmission interface.

    Science.gov (United States)

    Loo, Tip W; Clarke, David M

    2016-04-01

    P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump. A common feature of ABC proteins is that they are organized into two wings. Each wing contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates and ATP bind at the interface between the TMDs and NBDs, respectively. Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. P-gps crystallized in the presence of detergent show an open structure. Human P-gp is inactive in detergent but basal ATPase activity is restored upon addition of lipids. The lipids might cause closure of the wings to bring the NBDs close together to allow ATP hydrolysis. We show however, that cross-linking the wings together did not activate ATPase activity when lipids were absent suggesting that lipids may induce other structural changes required for ATPase activity. We then tested the effect of lipids on disulfide cross-linking of mutants at the first transmission interface between intracellular loop 4 (TMD2) and NBD1. Mutants L443C/S909C and L443C/R905C but not G471C/S909C and V472C/S909C were cross-linked with oxidant when in membranes. The mutants were then purified and cross-linked with or without lipids. Mutants G471C/S909C and V472C/S909C cross-linked only in the absence of lipids whereas mutants L443C/S909C and L443C/R905C were cross-linked only in the presence of lipids. The results suggest that lipids activate a switch at the first transmission interface and that the structure of P-gp is different in detergents and lipids.

  20. Histamine derived from probiotic Lactobacillus reuteri suppresses TNF via modulation of PKA and ERK signaling.

    Directory of Open Access Journals (Sweden)

    Carissa M Thomas

    Full Text Available Beneficial microbes and probiotic species, such as Lactobacillus reuteri, produce biologically active compounds that can modulate host mucosal immunity. Previously, immunomodulatory factors secreted by L. reuteri ATCC PTA 6475 were unknown. A combined metabolomics and bacterial genetics strategy was utilized to identify small compound(s produced by L. reuteri that were TNF-inhibitory. Hydrophilic interaction liquid chromatography-high performance liquid chromatography (HILIC-HPLC separation isolated TNF-inhibitory compounds, and HILIC-HPLC fraction composition was determined by NMR and mass spectrometry analyses. Histamine was identified and quantified in TNF-inhibitory HILIC-HPLC fractions. Histamine is produced from L-histidine via histidine decarboxylase by some fermentative bacteria including lactobacilli. Targeted mutagenesis of each gene present in the histidine decarboxylase gene cluster in L. reuteri 6475 demonstrated the involvement of histidine decarboxylase pyruvoyl type A (hdcA, histidine/histamine antiporter (hdcP, and hdcB in production of the TNF-inhibitory factor. The mechanism of TNF inhibition by L. reuteri-derived histamine was investigated using Toll-like receptor 2 (TLR2-activated human monocytoid cells. Bacterial histamine suppressed TNF production via activation of the H(2 receptor. Histamine from L. reuteri 6475 stimulated increased levels of cAMP, which inhibited downstream MEK/ERK MAPK signaling via protein kinase A (PKA and resulted in suppression of TNF production by transcriptional regulation. In summary, a component of the gut microbiome, L. reuteri, is able to convert a dietary component, L-histidine, into an immunoregulatory signal, histamine, which suppresses pro-inflammatory TNF production. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases.

  1. Activation of STING requires palmitoylation at the Golgi.

    Science.gov (United States)

    Mukai, Kojiro; Konno, Hiroyasu; Akiba, Tatsuya; Uemura, Takefumi; Waguri, Satoshi; Kobayashi, Toshihide; Barber, Glen N; Arai, Hiroyuki; Taguchi, Tomohiko

    2016-06-21

    Stimulator of interferon genes (STING) is essential for the type I interferon response against DNA pathogens. In response to the presence of DNA and/or cyclic dinucleotides, STING translocates from the endoplasmic reticulum to perinuclear compartments. However, the role of this subcellular translocation remains poorly defined. Here we show that palmitoylation of STING at the Golgi is essential for activation of STING. Treatment with palmitoylation inhibitor 2-bromopalmitate (2-BP) suppresses palmitoylation of STING and abolishes the type I interferon response. Mutation of two membrane-proximal Cys residues (Cys88/91) suppresses palmitoylation, and this STING mutant cannot induce STING-dependent host defense genes. STING variants that constitutively induce the type I interferon response were found in patients with autoimmune diseases. The response elicited by these STING variants is effectively inhibited by 2-BP or an introduction of Cys88/91Ser mutation. Our results may lead to new treatments for cytosolic DNA-triggered autoinflammatory diseases.

  2. Gearbox design for uncertain load requirements using active robust optimization

    Science.gov (United States)

    Salomon, Shaul; Avigad, Gideon; Purshouse, Robin C.; Fleming, Peter J.

    2016-04-01

    Design and optimization of gear transmissions have been intensively studied, but surprisingly the robustness of the resulting optimal design to uncertain loads has never been considered. Active Robust (AR) optimization is a methodology to design products that attain robustness to uncertain or changing environmental conditions through adaptation. In this study the AR methodology is utilized to optimize the number of transmissions, as well as their gearing ratios, for an uncertain load demand. The problem is formulated as a bi-objective optimization problem where the objectives are to satisfy the load demand in the most energy efficient manner and to minimize production cost. The results show that this approach can find a set of robust designs, revealing a trade-off between energy efficiency and production cost. This can serve as a useful decision-making tool for the gearbox design process, as well as for other applications.

  3. Controlling the pKa of the bacteriorhodopsin Schiff base by use of artificial retinal analogues.

    OpenAIRE

    1986-01-01

    Artificial bacteriorhodopsin pigments based on synthetic retinal analogues carrying an electron-withdrawing CF3 substituent group were prepared. The effects of CF3 on the spectra, photocycles, and Schiff base pKa values of the pigments were analyzed. A reduction of 5 units in the pKa of the Schiff base is observed when the CF3 substituent is located at the C-13 polyene position, in the vicinity of the protonated Schiff base nitrogen. The results lead to the unambiguous characterization of the...

  4. Is the prediction of pKa values by constant-pH molecular dynamics being hindered by inherited problems?

    Science.gov (United States)

    Machuqueiro, Miguel; Baptista, António M

    2011-12-01

    In this study, we investigate two factors that can hinder the performance of constant-pH molecular dynamics methods in predicting protein pK(a) values, using hen egg white lysozyme as a test system. The first factor is related to the molecular definition and pK(a) value of model compounds in the Poisson-Boltzmann framework. We address this by defining the model compound as a molecular fragment with an associated pK(a) value that is calibrated against experimental data, which results in a decrease of 0.12 units in pK(a) errors. The second addressed factor is the possibility that detrimental structural distortions are being introduced in the simulations by the underlying molecular mechanics force field. This issue is investigated by analyzing how the gradual structural rearrangements affect the predicted pK(a) values. The two GROMOS force fields studied here (43A1 and 53A6) yield good pK(a) predictions, although a time-dependent performance is observed: 43A1 performs better after a few nanoseconds of structural reorganization (pK(a) errors of ~0.45), while 53A6 gives the best prediction right at the first nanosecond (pK(a) errors of 0.42). These results suggest that the good performance of constant-pH molecular dynamics methods could be further improved if these force field limitations were overcome. Copyright © 2011 Wiley-Liss, Inc.

  5. Inhibitory Effect of Dried Pomegranate Concentration Powder on Melanogenesis in B16F10 Melanoma Cells; Involvement of p38 and PKA Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Su Jin Kang

    2015-10-01

    Full Text Available Plants rich in antioxidant substances may be useful for preventing skin aging. Pomegranates, containing flavonoids and other polyphenolic compounds, are widely consumed due to their beneficial properties. We examined the underlying mechanisms of dried pomegranate concentrate powder (PCP on melanin synthesis in B16F10 melanoma cells. The antioxidant effects of PCP were determined by measuring free radical scavenging capacity and transcript levels of antioxidant enzymes. To explore the inhibitory effects of PCP on melanin synthesis, we measured tyrosinase activity and melanin content in α-melanocyte stimulating hormone (α-MSH-stimulated B16F10 cells. In addition, the levels of tyrosinase-related protein-1 (TRP-1, TRP-2, tyrosinase, and microphthalmia-associated transcription factor (MITF expression were determined by Western blotting. Changes in the phosphorylation status of protein kinase A (PKA, cAMP response element-binding protein (CREB, mitogen-activated protein kinases (MAPKs, phosphatidylinositol 3-kinase (PI3K, serine/threonine kinase Akt, and glycogen kinase 3β (GSK3β were also examined. The free radical scavenging activity of PCP increased in a dose-dependent manner. In PCP-treated B16F10 cells, transcript levels of glutathione peroxidase-1 (GPx-1 were increased compared with α-MSH-stimulated cells. In addition, PCP led to the down-regulation of phospho-p38, phospho-PKA, phospho-CREB, phospho-GSK3β, MITF, and TRP-1 compared with α-MSH-stimulated B16F10 cells. We believe this effect may be associated with PCP activity, which leads to the inhibition of melanin production and tyrosinase activity. These results suggest that PCP decreases tyrosinase activity and melanin production via inactivation of the p38 and PKA signaling pathways, and subsequently decreases phosphorylation of CREB, MITF, and melanogenic enzymes. These observations provided new insights on the molecular mechanisms of the skin-whitening property of PCP.

  6. 77 FR 6815 - Agency Information Collection Activities: Country of Origin Marking Requirements for Containers...

    Science.gov (United States)

    2012-02-09

    ... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Country of Origin... requirement concerning Country of Origin Marking Requirements for Containers or Holders. This request for...: Title: Country of Origin Marking Requirements for Containers or Holders. OMB Number: 1651-0057....

  7. 20 CFR 404.1311 - Ninety-day active service requirement for World War II veterans.

    Science.gov (United States)

    2010-04-01

    ... World War II veterans. 404.1311 Section 404.1311 Employees' Benefits SOCIAL SECURITY ADMINISTRATION... Uniformed Services World War II Veterans § 404.1311 Ninety-day active service requirement for World War II veterans. (a) The 90 days of active service required for World War II veterans do not have to...

  8. 30 CFR 280.30 - What activities will not require environmental analysis?

    Science.gov (United States)

    2010-07-01

    ... Obligations Under This Part Environmental Issues § 280.30 What activities will not require environmental... 30 Mineral Resources 2 2010-07-01 2010-07-01 false What activities will not require environmental... environmental impact and will normally be categorically excluded from additional environmental analysis. The...

  9. Design requirements document for project W-520, immobilized low-activity waste disposal

    Energy Technology Data Exchange (ETDEWEB)

    Ashworth, S.C.

    1998-08-06

    This design requirements document (DRD) identifies the functions that must be performed to accept, handle, and dispose of the immobilized low-activity waste (ILAW) produced by the Tank Waste Remediation System (TWRS) private treatment contractors and close the facility. It identifies the requirements that are associated with those functions and that must be met. The functional and performance requirements in this document provide the basis for the conceptual design of the Tank Waste Remediation System Immobilized Low-Activity Waste disposal facility project (W-520) and provides traceability from the program-level requirements to the project design activity.

  10. Acid-base crystalline complexes and the pK(a) rule

    NARCIS (Netherlands)

    Cruz-Cabeza, A.J.

    2012-01-01

    Differences in the predicted aqueous pKa values (ΔpKa) have been calculated for 6465 crystalline complexes containing ionised and non-ionised acid-base pairs in the Cambridge Structural Database. A linear relationship between ΔpKa and the probability of proton transfer between acid-base pairs has be

  11. p85 regulatory subunit of PI3K mediates cAMP-PKA and estrogens biological effects on growth and survival.

    Science.gov (United States)

    Cosentino, C; Di Domenico, M; Porcellini, A; Cuozzo, C; De Gregorio, G; Santillo, M R; Agnese, S; Di Stasio, R; Feliciello, A; Migliaccio, A; Avvedimento, E V

    2007-03-29

    Cyclic adenosine 3'5' monophosphate (cAMP) and protein kinase A (PKA) cooperate with phosphatidylinositol 3' kinase (PI3K) signals in the control of growth and survival. To determine the molecular mechanism(s) involved, we identified and mutagenized a specific serine (residue 83) in p85alpha(PI3K), which is phosphorylated in vivo and in vitro by PKA. Expression of p85alpha(PI3K) mutants (alanine or aspartic substitutions) significantly altered the biological responses of the cells to cAMP. cAMP protection from anoikis was reduced in cells expressing the alanine version p85alpha(PI3K). These cells did not arrest in G1 in the presence of cAMP, whereas cells expressing the aspartic mutant p85D accumulated in G1 even in the absence of cAMP. S phase was still efficiently inhibited by cAMP in cells expressing both mutants. The binding of PI3K to Ras p21 was greatly reduced in cells expressing p85A in the presence or absence of cAMP. Conversely, expression of the aspartic mutant stimulated robustly the binding of PI3K to p21 Ras in the presence of cAMP. Mutation in the Ser 83 inhibited cAMP, but not PDGF stimulation of PI3K. Conversely, the p85D aspartic mutant amplified cAMP stimulation of PI3K activity. Phosphorylation of Ser 83 by cAMP-PKA in p85alpha(PI3K) was also necessary for estrogen signaling as expression of p85A or p85D mutants inhibited or amplified, respectively, the binding of estrogen receptor to p85alpha and AKT phosphorylation induced by estrogens. The data presented indicate that: (1) phosphorylation of Ser 83 in p85alpha(PI3K) is critical for cAMP-PKA induced G1 arrest and survival in mouse 3T3 fibroblasts; (2) this site is necessary for amplification of estrogen signals by cAMP-PKA and related receptors. Finally, these data suggest a general mechanism of PI3K regulation by cAMP, operating in various cell types and under different conditions.

  12. Prediction of the pKa's of aqueous metal ion +2 complexes.

    Science.gov (United States)

    Jackson, Virgil E; Felmy, Andrew R; Dixon, David A

    2015-03-26

    Aqueous metal ions play an important role in many areas of chemistry. The acidities of [Be(H2O)4](2+), [M(H2O)6](2+), M = Mg(2+), Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), and Hg(2+), and [M(H2O)n](2+), M = Ca(2+) and Sr(2+), n = 7 and 8, complexes have been predicted using density functional theory, second-order Møller-Plesset perturbation theory (MP2), and coupled cluster CCSD(T) theory in the gas phase. pKa's in aqueous solution were predicted by using self-consistent reaction field (SCRF) calculations with different solvation models. The most common binding motif of the majority of the metal +2 complexes is coordination number (CN) 6, with each hexaaquo cluster having reasonably high symmetry for the best arrangement of the water molecules in the first solvation shell. Be(2+) is tetracoordinated, but a second solvation shell of 8 waters is needed to predict the pKa. The Ca(2+) and Sr(2+) aquo clusters have a coordination number of 7 or 8 as found in terms of the energy of the reaction M(H2O)7(2+) + H2O → M(H2O)8(2+) and the pKa values. The calculated geometries are in reasonable agreement with experiment. The SCRF calculations with the conductor-like screening model (COSMO), and the conductor polarized continuum model (CPCM) using COSMO-RS radii, consistently agree best with experiment at the MP2/aug-cc-pVDZ and CCSD(T)/aug-cc-pVDZ levels of theory. The CCSD(T) level provides the most accurate pKa's, and the MP2 level also provides reliable predictions. Our predictions were used to elucidate the properties of metal +2 ion complexes. The pKa predictions provide confirmation of the size of the first solvation shell sizes. The calculations show that it is still difficult to predict pKa's using this cluster/implicit solvent approach to better than 1 pKa unit.

  13. Enhancement of osteogenic differentiation of rat adipose tissue-derived mesenchymal stem cells by zinc sulphate under electromagnetic field via the PKA, ERK1/2 and Wnt/β-catenin signaling pathways

    Science.gov (United States)

    Fathi, Ezzatollah; Farahzadi, Raheleh

    2017-01-01

    Zinc ion as an essential trace element and electromagnetic fields (EMFs) has been reported to be involved in the regulation of bone metabolism. The aim of this study was to elucidate the effects of zinc sulphate (ZnSO4) on the osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (ADSCs) in the presence of EMF as a strategy in osteoporosis therapy. Alkaline phophatase (ALP) activity measurement, calcium assay and expression of several osteoblastic marker genes were examined to assess the effect of ZnSO4 on the osteogenic differentiation of ADSCs under EMF. The expression of cAMP and PKA was evaluated by ELISA. The expression of β-catenin, Wnt1, Wnt3a, low-density lipoprotein receptor-related protein 5 (LRP5) and reduced dickkopf1 (DKK1) genes were used to detect the Wnt/β-catenin pathway. It was found that ZnSO4, in the presence of EMF, resulted in an increase in the expression of osteogenic genes, ALP activity and calcium levels. EMF, in the presence of ZnSO4, increased the cAMP level and protein kinase A (PKA) activity. Treatment of ADSCs with (MAPK)/ERK kinase 1/2 inhibitor, or PKA inhibitor, significantly inhibited the promotion of osteogenic markers, indicating that the induction of osteogenesis was dependent on the ERK and PKA signaling pathways. Real-time PCR analysis showed that ZnSO4, in the presence of EMF, increased the mRNA expressions of β-catenin, Wnt1, Wnt3a, LRP5 and DKK1. In this study, it was shown that 0.432 μg/ml ZnSO4, in the presence of 50 Hz, 20 mT EMF, induced the osteogenic differentiation of ADSCs via PKA, ERK1/2 and Wnt/β-catenin signaling pathways. PMID:28339498

  14. Reproducing basic pKa values for turkey ovomucoid third domain using a polarizable force field.

    Science.gov (United States)

    Click, Timothy H; Kaminski, George A

    2009-06-04

    We have extended our previous studies of calculating acidity constants for the acidic residues found in the turkey ovomucoid third domain protein (OMTKY3) by determining the relative pKa values for the basic residues (Lys13, Arg21, Lys29, Lys34, His52, and Lys55). A polarizable force field (PFF) was employed. The values of the pKa were found by direct comparison of energies of solvated protonated and deprotonated forms of the protein. Poisson-Boltzmann (PBF) and surface generalized Born (SGB) continuum solvation models represent the hydration, and a nonpolarizable fixed-charge OPLS-AA force field was used for comparison. Our results indicate that (i) the pKa values of the basic residues can be found in close agreement with the experimental values when a PFF is used in conjunction with the PBF solvation model, (ii) it is sufficient to take into the account only the residues which are in close proximity (hydrogen bonded) to the residue in question, and (iii) the PBF solvation model is superior to the SGB solvation model for these pKa calculations. The average error with the PBF/PFF model is only 0.7 pH unit, compared with 2.2 and 6.1 units for the PBF/OPLS and SGB/OPLS, respectively. The maximum deviation of the PBF/PFF results from the experimental values is 1.7 pH units compared with 6.0 pH units for the PBF/OPLS. Moreover, the best results were obtained while using an advanced nonpolar energy calculation scheme. The overall conclusion is that this methodology and force field are suitable for the accurate assessment of pKa shifts for both acidic and basic protein residues.

  15. Electrostatic pKa computations in proteins: role of internal cavities.

    Science.gov (United States)

    Meyer, Tim; Kieseritzky, Gernot; Knapp, Ernst-Walter

    2011-12-01

    The solvent accessible surface area (SASA) algorithm is conventionally used to characterize protein surfaces in electrostatic energy computations of proteins. Unfortunately, it often fails to find narrow cavities inside a protein. As a consequence pK(a) computations based on this algorithm perform badly. In this study a new cavity-algorithm is introduced, which solves this problem and provides improved pK(a) values. The procedure is applied to 20 pK(a) values of titratable groups introduced as point mutations in SNase variants, where crystal structures are available. The computations of these pK(a)s are particular challenging, since they are placed in a rather hydrophobic environment. For nine mutants, where the titratable residue is in contact with a large cavity, the RMSD(pKa) between computed and measured pK(a) values is 2.04, which is a considerable improvement as compared to the original results obtained with Karlsberg(+) (http://agknapp.chemie.fu-berlin.de/karlsberg/) that yielded an RMSD(pKa) of 8.8. However, for 11 titratable residues the agreement with experiments remains poor (RMSD(pKa) = 6.01). Considering 15 pK(a)s of SNase, which are in a more conventional less hydrophobic protein environment, the RMSD(pKa) is 2.1 using the SASA-algorithm and 1.7 using the new cavity-algorithm. The agreement is reasonable but less good than what one would expect from the general performance of Karlsberg(+) indicating that SNase belongs to the more difficult proteins with respect to pK(a) computations. We discuss the possible reasons for the remaining discrepancies between computed and measured pK(a)s.

  16. Peroxisome Proliferator-Activated Receptor γ Activity is Required for Appropriate Cardiomyocyte Differentiation

    Directory of Open Access Journals (Sweden)

    Maryam Peymani

    2016-07-01

    Full Text Available Objective Peroxisome proliferator-activated receptor γ (PPARγ is a member of the PPAR nuclear receptor superfamily. Although PPARγ acts as a master transcription factor in adipocyte differentiation, it is also associated with a variety of cell functions including carbohydrate and lipid metabolism, glucose homeostasis, cell proliferation and cell differentiation. This study aimed to assess the expression level of PPARγ in order to address its role in cardiac cell differentiation of mouse embryonic stem cells (mESCs. Materials and Methods In this an intervening study, mESCs were subjected to cardiac differentiation. Total RNA was extracted from the cells and quantitative real time polymerase chain reaction (qPCR was carried out to estimate level of gene expression. Furthermore, the requirement of PPARγ in cardiac differentiation of mESCs, during cardiac progenitor cells (CPCs formation, was examined by applying the respective agonist and antagonist. Results The obtained data revealed an elevation in the expression level of PPARγ during spontaneous formation of CPCs and cardiomyocytes. Our results indicated that during CPC formation, PPARγ inactivation via treatment with GW9662 (GW reduced expression of CPC and cardiac markers. Conclusion We conclude that PPARγ modulation has an effective role on cardiac differentiation of mESCs at the early stage of cardiomyogenesis.

  17. Intrinsically disordered inhibitor of glutamine synthetase is a functional protein with random-coil-like pKa values.

    Science.gov (United States)

    Cozza, Concetta; Neira, José L; Florencio, Francisco J; Muro-Pastor, M Isabel; Rizzuti, Bruno

    2017-06-01

    The sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria allows the incorporation of ammonium into carbon skeletons. In the cyanobacterium Synechocystis sp. PCC 6803, the activity of GS is modulated by the interaction with proteins, which include a 65-residue-long intrinsically disordered protein (IDP), the inactivating factor IF7. This interaction is regulated by the presence of charged residues in both IF7 and GS. To understand how charged amino acids can affect the binding of an IDP with its target and to provide clues on electrostatic interactions in disordered states of proteins, we measured the pKa values of all IF7 acidic groups (Glu32, Glu36, Glu38, Asp40, Asp58, and Ser65, the backbone C-terminus) at 100 mM NaCl concentration, by using NMR spectroscopy. We also obtained solution structures of IF7 through molecular dynamics simulation, validated them on the basis of previous experiments, and used them to obtain theoretical estimates of the pKa values. Titration values for the two Asp and three Glu residues of IF7 were similar to those reported for random-coil models, suggesting the lack of electrostatic interactions around these residues. Furthermore, our results suggest the presence of helical structure at the N-terminus of the protein and of conformational changes at acidic pH values. The overall experimental and in silico findings suggest that local interactions and conformational equilibria do not play a role in determining the electrostatic features of the acidic residues of IF7. © 2017 The Protein Society.

  18. Efg1 directly regulates ACE2 expression to mediate cross talk between the cAMP/PKA and RAM pathways during Candida albicans morphogenesis.

    Science.gov (United States)

    Saputo, Sarah; Kumar, Anuj; Krysan, Damian J

    2014-09-01

    The cyclic AMP/protein kinase A (cAMP/PKA) and regulation of Ace2 and morphogenesis (RAM) pathways are important regulators of the yeast-to-hypha transition in Candida albicans that interact genetically during this process. To further understand this interaction, we have characterized the expression of ACE2 during morphogenesis. In normoxic, planktonic conditions, ACE2 expression is very low in stationary-phase cells at both the mRNA and protein levels. Upon shifting to Spider medium, ACE2/Ace2p levels increase. Although Ace2 is not absolutely required for hypha formation, ace2Δ/Δ mutants show delayed hypha formation in Spider medium (but not others) and morphological changes to the hyphal tip and lateral yeast. We also show that Efg1 directly binds the promoter of Ace2 in stationary phase, and ACE2 levels are increased in strains lacking Efg1 and the protein kinase A proteins Tpk1 and Tpk2, indicating that the PKA pathway directly regulates ACE2 expression. ACE2 expression is positively regulated by Tec1 and Brg1, which bind the promoters of ACE2 in hyphal cells but not in the yeast phase. Under embedded conditions, Efg1 is dispensable for filamentation and Ace2 is required. We have found that ACE2 expression is much higher in embedded cells than in planktonic cells, providing a potential rationale for this observation. Taken together, our observations indicate that the PKA pathway directly regulates the RAM pathway under specific conditions and are consistent with a model where the two pathways carry out similar functions that depend on the specific environmental context.

  19. EFFECT OF PHORBOL ESTER ON cAMP-DEPENDENT PROTEIN KINASE ACTIVITY IN CARDIOMYOCYTES

    Institute of Scientific and Technical Information of China (English)

    周文华; 肖殿模; 郑超强; 王小鲁; 张俊保

    1995-01-01

    Cardiomyocytes isolated from neonatal rats were treated with phorbol-12-myristate-13-acetate(PMA) ranging from 10-11 to 10-7mol/L for 20 min,causing cytosol protein kinase A (PKA) activity to decrease while particulate PKA activity increase in a concentration-dependent manner.The change of PKA activity induced by PMA was abolished completely by pretreatment of polymyxin B or depletion of protein kinase C (PKC).Type Ⅱ PKA activity in particulate fraction was enhanced remarkably,while that of type I PKA was not altered when the cells were treated with 100 nmol/L PMA.The results suggested that subcellular distribution and activity of PKA in cardiomyocytes may be regulated by PKC.

  20. Bacillus bombysepticus α-Toxin Binding to G Protein-Coupled Receptor Kinase 2 Regulates cAMP/PKA Signaling Pathway to Induce Host Death.

    Directory of Open Access Journals (Sweden)

    Ping Lin

    2016-03-01

    Full Text Available Bacterial pathogens and their toxins target host receptors, leading to aberrant behavior or host death by changing signaling events through subversion of host intracellular cAMP level. This is an efficient and widespread mechanism of microbial pathogenesis. Previous studies describe toxins that increase cAMP in host cells, resulting in death through G protein-coupled receptor (GPCR signaling pathways by influencing adenylyl cyclase or G protein activity. G protein-coupled receptor kinase 2 (GRK2 has a central role in regulation of GPCR desensitization. However, little information is available about the pathogenic mechanisms of toxins associated with GRK2. Here, we reported a new bacterial toxin-Bacillus bombysepticus (Bb α-toxin that was lethal to host. We showed that Bb α-toxin interacted with BmGRK2. The data demonstrated that Bb α-toxin directly bound to BmGRK2 to promote death by affecting GPCR signaling pathways. This mechanism involved stimulation of Gαs, increase level of cAMP and activation of protein kinase A (PKA. Activated cAMP/PKA signal transduction altered downstream effectors that affected homeostasis and fundamental biological processes, disturbing the structural and functional integrity of cells, resulting in death. Preventing cAMP/PKA signaling transduction by inhibitions (NF449 or H-89 substantially reduced the pathogenicity of Bb α-toxin. The discovery of a toxin-induced host death specifically linked to GRK2 mediated signaling pathway suggested a new model for bacterial toxin action. Characterization of host genes whose expression and function are regulated by Bb α-toxin and GRK2 will offer a deeper understanding of the pathogenesis of infectious diseases caused by pathogens that elevate cAMP.

  1. Determination of Histidine pKa Values in the Propeptides of Furin and Proprotein Convertase 1/3 Using Histidine Hydrogen-Deuterium Exchange Mass Spectrometry.

    Science.gov (United States)

    Elferich, Johannes; Williamson, Danielle M; David, Larry L; Shinde, Ujwal

    2015-08-04

    Propeptides of proprotein convertases regulate activation of their protease domains by sensing the organellar pH within the secretory pathway. Earlier experimental work highlighted the importance of a conserved histidine residue within the propeptide of a widely studied member, furin. A subsequent evolutionary analysis found an increase in histidine content within propeptides of secreted eukaryotic proteases compared with their prokaryotic orthologs. However, furin activates in the trans-golgi network at a pH of 6.5 while a paralog, proprotein convertase 1/3, activates in secretory vesicles at a pH of 5.5. It is unclear how a conserved histidine can mediate activation at two different pH values. In this manuscript, we measured the pKa values of histidines within the propeptides of furin and proprotein convertase 1/3 using a histidine hydrogen-deuterium exchange mass spectrometry approach. The high density of histidine residues combined with an abundance of basic residues provided challenges for generation of peptide ions with unique histidine residues, which were overcome by employing ETD fragmentation. During this analysis, we found slow hydrogen-deuterium exchange in residues other than histidine at basic pH. Finally, we demonstrate that the pKa of the conserved histidine in proprotein convertase 1/3 is acid-shifted compared with furin and is consistent with its lower pH of activation.

  2. Predicting the pKa and stability of organic acids and bases at an oil-water interface.

    Science.gov (United States)

    Andersson, M P; Olsson, M H M; Stipp, S L S

    2014-06-10

    We have used density functional theory and the implicit solvent model, COSMO-RS, to investigate how the acidity constant, pKa, of organic acids and bases adsorbed at the organic compound-aqueous solution interface changes, compared to its value in the aqueous phase. The pKa determine the surface charge density of the molecules that accumulate at the fluid-fluid interface. We have estimated the pKa by comparing the stability of the protonated and unprotonated forms of a series of molecules in the bulk aqueous solution and at an interface where parts of each molecule reside in the hydrophobic phase and the rest remains in the hydrophilic phase. We found that the pKa for acids is shifted by ∼1 pH unit to higher values compared to the bulk water pKa, whereas they are shifted to lower values by a similar amount for bases. Because this pKa shift is similar in magnitude for each of the molecules studied, we propose that the pKa for molecules at a water-organic compound interface can easily be predicted by adding a small shift to the aqueous pKa. This shift is general and correlates with the functional group. We also found that the relative composition of molecules at the fluid-fluid interface is not the same as in the bulk. For example, species such as carboxylic acids are enriched at the interface, where they can dominate surface properties, even when they are a modest component in the bulk fluid. For high surface concentrations of carboxylic acid groups at an interface, such as a self-assembled monolayer, we have demonstrated that the pKa depends on the degree of deprotonation through direct hydrogen bonding between protonated and deprotonated acidic headgroups.

  3. 77 FR 27787 - Agency Information Collection Activities: Customs Modernization Act Recordkeeping Requirements

    Science.gov (United States)

    2012-05-11

    ... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Customs Modernization... review and approval in accordance with the Paperwork Reduction Act: Customs Modernization Act Record... techniques or other forms of information. Title: Customs Modernization Act Recordkeeping Requirements....

  4. HPRT-deficiency dysregulates cAMP-PKA signaling and phosphodiesterase 10A expression: mechanistic insight and potential target for Lesch-Nyhan Disease?

    Directory of Open Access Journals (Sweden)

    Ghiabe-Henri Guibinga

    Full Text Available Lesch-Nyhan Disease (LND is the result of mutations in the X-linked gene encoding the purine metabolic enzyme, hypoxanthine guanine phosphoribosyl transferase (HPRT. LND gives rise to severe neurological anomalies including mental retardation, dystonia, chorea, pyramidal signs and a compulsive and aggressive behavior to self injure. The neurological phenotype in LND has been shown to reflect aberrant dopaminergic signaling in the basal ganglia, however there are little data correlating the defect in purine metabolism to the neural-related abnormalities. In the present studies, we find that HPRT-deficient neuronal cell lines have reduced CREB (cAMP response element-binding protein expression and intracellular cyclic AMP (cAMP, which correlates with attenuated CREB-dependent transcriptional activity and a reduced phosphorylation of protein kinase A (PKA substrates such as synapsin (p-syn I. Of interest, we found increased expression of phosphodiesterase 10A (PDE10A in HPRT-deficient cell lines and that the PDE10 inhibitor papaverine and PDE10A siRNA restored cAMP/PKA signaling. Furthermore, reconstitution of HPRT expression in mutant cells partly increased cAMP signaling synapsin phosphorylation. In conclusion, our data show that HPRT-deficiency alters cAMP/PKA signaling pathway, which is in part due to the increased of PDE10A expression and activity. These findings suggest a mechanistic insight into the possible causes of LND and highlight PDE10A as a possible therapeutic target for this intractable neurological disease.

  5. Involvement of PKA-dependent upregulation of nNOS-CGRP in adrenomedullin-initiated mechanistic pathway underlying CFA-induced response in rats.

    Science.gov (United States)

    Wang, Dongmei; Ruan, Liqin; Hong, Yanguo; Chabot, Jean-Guy; Quirion, Rémi

    2013-01-01

    We have previously shown that intrathecal administration of the adrenomedullin (AM) receptor antagonist AM(22-52) produces a long-lasting anti-hyperalgesia effect. This study examined the hypothesis that AM recruits other pronociceptive mediators in complete Freund's adjuvant (CFA)-induced inflammation. Injection of CFA in the hindpaw of rat produced an increase in the expression of nNOS in dorsal root ganglion (DRG) and the spinal dorsal horn. An intrathecal administration of AM(22-52), but not the CGRP antagonist BIBN4096BS, abolished the CFA-induced increase of nNOS. Moreover, AM-induced increase of CGRP was inhibited by the nNOS inhibitors L-NAME and 7-nitroindazole in cultured ganglion explants. Addition of AM to ganglion cultures induced an increase in nNOS protein, which was attenuated by the PKA inhibitor H-89. Treatment with AM also concentration-dependently increased cAMP content and pPKA protein level, but not its non-phosphorylated form, in cultured ganglia. In addition, nNOS was shown to be co-localized with the AM receptor components calcitonin receptor-like receptor and receptor activity-modifying protein 2- and 3 in DRG neurons. The present study suggests that the enhanced activity of nitric oxide (NO) mediates the biological action of AM at the spinal level and that AM recruits NO-CGRP via cAMP/PKA signaling in a mechanistic pathway underlying CFA-induced hyperalgesia. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Interferon regulatory factor 1 is required for mouse Gbp gene activation by gamma interferon.

    OpenAIRE

    1995-01-01

    Full-scale transcriptional activation of the mouse Gbp genes by gamma interferon (IFN-gamma) requires protein synthesis in embryonic fibroblasts. Although the Gbp-1 and Gbp-2 promoters contain binding sites for transcription factors Stat1 and IFN regulatory factor 1 (IRF-1), deletion analysis revealed that the Stat1 binding site is dispensable for IFN-gamma inducibility of Gbp promoter constructs in transfected fibroblasts. However, activation of the mouse Gbp promoter by IFN-gamma requires t...

  7. Modulation of signaling through GPCR-cAMP-PKA pathways by PDE4 depends on stimulus intensity: Possible implications for the pathogenesis of acrodysostosis without hormone resistance.

    Science.gov (United States)

    Motte, Emmanuelle; Le Stunff, Catherine; Briet, Claire; Dumaz, Nicolas; Silve, Caroline

    2017-02-15

    In acrodysostosis without hormone resistance, a disease caused by phosphodiesterase (PDE)-4D mutations, increased PDE activity leads to bone developmental defects but with normal renal responses to PTH. To identify potential mechanisms for these disparate responses, we compared the effect of PDE activity on hormone signaling through the GPCR-Gsα-cAMP-PKA pathway in cells from two lineages, HEK-293 cells stably overexpressing PTH1R (HEKpthr) and human dermal fibroblasts, including studies evaluating cAMP levels using an Epac-based BRET-sensor for cAMP (CAMYEL). For ligand-induced responses inducing strong cAMP accumulation, the inhibition of PDE4 activity resulted in relatively small further increases. In contrast, when ligand-induced cAMP accumulation was of lesser intensity, the inhibition of PDE4 had a more pronounced effect. Similar results were obtained evaluating downstream events (cellular CREB phosphorylation and CRE-luciferase activity). Thus, the ability of PDE4 to modulate signaling through GPCR-cAMP-PKA pathways can depend on the cell type and stimulus intensity.

  8. 42 CFR 424.516 - Additional provider and supplier requirements for enrolling and maintaining active enrollment...

    Science.gov (United States)

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Additional provider and supplier requirements for... Billing Privileges § 424.516 Additional provider and supplier requirements for enrolling and maintaining... active enrollment status for a provider or supplier when that provider or supplier certifies that...

  9. 76 FR 69204 - Anti-Money Laundering Program and Suspicious Activity Reporting Requirements for Housing...

    Science.gov (United States)

    2011-11-08

    ... Financial Crimes Enforcement Network 31 CFR Parts 1010 and 1030 RIN 1506-AB14 Anti-Money Laundering Program... requiring them to establish anti-money laundering programs and report suspicious activities pursuant to the Bank Secrecy Act. The proposal to require these organizations to establish anti-money laundering...

  10. 77 FR 8148 - Anti-Money Laundering Program and Suspicious Activity Report Filing Requirements for Residential...

    Science.gov (United States)

    2012-02-14

    ... Financial Crimes Enforcement Network 31 CFR Parts 1010 and 1029 RIN 1506-AB02 Anti-Money Laundering Program... requiring them to establish anti-money laundering programs and report suspicious activities under the Bank... impose anti-money laundering (``AML'') program requirements on financial institutions.\\3\\ The authority...

  11. 10 CFR 73.50 - Requirements for physical protection of licensed activities.

    Science.gov (United States)

    2010-01-01

    ... loss of primary power. (f) Testing and maintenance. Each licensee shall test and maintain intrusion... activities. 73.50 Section 73.50 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) PHYSICAL PROTECTION OF PLANTS AND MATERIALS Physical Protection Requirements at Fixed Sites § 73.50 Requirements for...

  12. Cytokine-induced activation of Mixed Lineage Kinase 3 requires TRAF2 and TRAF6

    OpenAIRE

    Korchnak, Amanda C.; Zhan, Yu; Aguilar, Michael T.; Chadee, Deborah N.

    2009-01-01

    Mixed Lineage Kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates multiple mitogen activated protein kinase (MAPK) pathways in response to growth factors, stresses and the pro-inflammatory cytokine, tumor necrosis factor (TNF). MLK3 is required for optimal activation of stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling by TNF, however, the mechanism by which MLK3 is recruited and activated by the TNF receptor remains poorly und...

  13. Prediction of pKa values using the PM6 semiempirical method

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    Jimmy C. Kromann

    2016-08-01

    Full Text Available The PM6 semiempirical method and the dispersion and hydrogen bond-corrected PM6-D3H+ method are used together with the SMD and COSMO continuum solvation models to predict pKa values of pyridines, alcohols, phenols, benzoic acids, carboxylic acids, and phenols using isodesmic reactions and compared to published ab initio results. The pKa values of pyridines, alcohols, phenols, and benzoic acids considered in this study can generally be predicted with PM6 and ab initio methods to within the same overall accuracy, with average mean absolute differences (MADs of 0.6–0.7 pH units. For carboxylic acids, the accuracy (0.7–1.0 pH units is also comparable to ab initio results if a single outlier is removed. For primary, secondary, and tertiary amines the accuracy is, respectively, similar (0.5–0.6, slightly worse (0.5–1.0, and worse (1.0–2.5, provided that di- and tri-ethylamine are used as reference molecules for secondary and tertiary amines. When applied to a drug-like molecule where an empirical pKa predictor exhibits a large (4.9 pH unit error, we find that the errors for PM6-based predictions are roughly the same in magnitude but opposite in sign. As a result, most of the PM6-based methods predict the correct protonation state at physiological pH, while the empirical predictor does not. The computational cost is around 2–5 min per conformer per core processor, making PM6-based pKa prediction computationally efficient enough to be used for high-throughput screening using on the order of 100 core processors.

  14. Adult active transport in the Netherlands: an analysis of its contribution to physical activity requirements.

    Directory of Open Access Journals (Sweden)

    Elliot Fishman

    Full Text Available Modern, urban lifestyles have engineered physical activity out of everyday life and this presents a major threat to human health. The Netherlands is a world leader in active travel, particularly cycling, but little research has sought to quantify the cumulative amount of physical activity through everyday walking and cycling.Using data collected as part of the Dutch National Travel Survey (2010 - 2012, this paper determines the degree to which Dutch walking and cycling contributes to meeting minimum level of physical activity of 150 minutes of moderate intensity aerobic activity throughout the week. The sample includes 74,465 individuals who recorded at least some travel on the day surveyed. As physical activity benefits are cumulative, all walking and cycling trips are analysed, including those to and from public transport. These trips are then converted into an established measure of physical activity intensity, known as metabolic equivalents of tasks. Multivariate Tobit regression models were performed on a range of socio-demographic, transport resources, urban form and meteorological characteristics.The results reveal that Dutch men and women participate in 24 and 28 minutes of daily physical activity through walking and cycling, which is 41% and 55% more than the minimum recommended level. It should be noted however that some 57% of the entire sample failed to record any walking or cycling, and an investigation of this particular group serves as an important topic of future research. Active transport was positively related with age, income, bicycle ownership, urban density and air temperature. Car ownership had a strong negative relationship with physically active travel.The results of this analysis demonstrate the significance of active transport to counter the emerging issue of sedentary lifestyle disease. The Dutch experience provides other countries with a highly relevant case study in the creation of environments and cultures that

  15. Computing pK(A) values of hexa-aqua transition metal complexes.

    Science.gov (United States)

    Galstyan, Gegham; Knapp, Ernst-Walter

    2015-01-15

    Aqueous pKA values for 15 hexa-aqua transition metal complexes were computed using a combination of quantum chemical and electrostatic methods. Two different structure models were considered optimizing the isolated complexes in vacuum or in presence of explicit solvent using a QM/MM approach. They yield very good agreement with experimentally measured pKA values with an overall root mean square deviation of about 1 pH unit, excluding a single but different outlier for each of the two structure models. These outliers are hexa-aqua Cr(III) for the vacuum and hexa-aqua Mn(III) for the QM/MM structure model. Reasons leading to the deviations of the outlier complexes are partially explained. Compared to previous approaches from the same lab the precision of the method was systematically improved as discussed in this study. The refined methods to obtain the appropriate geometries of the complexes, developed in this work, may allow also the computation of accurate pKA values for multicore transition metal complexes in different oxidation states.

  16. Computing pKa Values with a Mixing Hamiltonian Quantum Mechanical/Molecular Mechanical Approach.

    Science.gov (United States)

    Liu, Yang; Fan, Xiaoli; Jin, Yingdi; Hu, Xiangqian; Hu, Hao

    2013-09-10

    Accurate computation of the pKa value of a compound in solution is important but challenging. Here, a new mixing quantum mechanical/molecular mechanical (QM/MM) Hamiltonian method is developed to simulate the free-energy change associated with the protonation/deprotonation processes in solution. The mixing Hamiltonian method is designed for efficient quantum mechanical free-energy simulations by alchemically varying the nuclear potential, i.e., the nuclear charge of the transforming nucleus. In pKa calculation, the charge on the proton is varied in fraction between 0 and 1, corresponding to the fully deprotonated and protonated states, respectively. Inspired by the mixing potential QM/MM free energy simulation method developed previously [H. Hu and W. T. Yang, J. Chem. Phys. 2005, 123, 041102], this method succeeds many advantages of a large class of λ-coupled free-energy simulation methods and the linear combination of atomic potential approach. Theory and technique details of this method, along with the calculation results of the pKa of methanol and methanethiol molecules in aqueous solution, are reported. The results show satisfactory agreement with the experimental data.

  17. A TRPV2–PKA Signaling Module for Transduction of Physical Stimuli in Mast Cells

    Science.gov (United States)

    Stokes, Alexander J.; Shimoda, Lori M.N.; Koblan-Huberson, Murielle; Adra, Chaker N.; Turner, Helen

    2004-01-01

    Cutaneous mast cell responses to physical (thermal, mechanical, or osmotic) stimuli underlie the pathology of physical urticarias. In vitro experiments suggest that mast cells respond directly to these stimuli, implying that a signaling mechanism couples functional responses to physical inputs in mast cells. We asked whether transient receptor potential (vanilloid) (TRPV) cation channels were present and functionally coupled to signaling pathways in mast cells, since expression of this channel subfamily confers sensitivity to thermal, osmotic, and pressure inputs. Transcripts for a range of TRPVs were detected in mast cells, and we report the expression, surface localization, and oligomerization of TRPV2 protein subunits in these cells. We describe the functional coupling of TRPV2 protein to calcium fluxes and proinflammatory degranulation events in mast cells. In addition, we describe a novel protein kinase A (PKA)–dependent signaling module, containing PKA and a putative A kinase adapter protein, Acyl CoA binding domain protein (ACBD)3, that interacts with TRPV2 in mast cells. We propose that regulated phosphorylation by PKA may be a common pathway for TRPV modulation. PMID:15249591

  18. Genetic and functional studies implicate synaptic overgrowth and ring gland cAMP/PKA signaling defects in the Drosophila melanogaster neurofibromatosis-1 growth deficiency.

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    James A Walker

    2013-11-01

    Full Text Available Neurofibromatosis type 1 (NF1, a genetic disease that affects 1 in 3,000, is caused by loss of a large evolutionary conserved protein that serves as a GTPase Activating Protein (GAP for Ras. Among Drosophila melanogaster Nf1 (dNf1 null mutant phenotypes, learning/memory deficits and reduced overall growth resemble human NF1 symptoms. These and other dNf1 defects are relatively insensitive to manipulations that reduce Ras signaling strength but are suppressed by increasing signaling through the 3'-5' cyclic adenosine monophosphate (cAMP dependent Protein Kinase A (PKA pathway, or phenocopied by inhibiting this pathway. However, whether dNf1 affects cAMP/PKA signaling directly or indirectly remains controversial. To shed light on this issue we screened 486 1(st and 2(nd chromosome deficiencies that uncover >80% of annotated genes for dominant modifiers of the dNf1 pupal size defect, identifying responsible genes in crosses with mutant alleles or by tissue-specific RNA interference (RNAi knockdown. Validating the screen, identified suppressors include the previously implicated dAlk tyrosine kinase, its activating ligand jelly belly (jeb, two other genes involved in Ras/ERK signal transduction and several involved in cAMP/PKA signaling. Novel modifiers that implicate synaptic defects in the dNf1 growth deficiency include the intersectin-related synaptic scaffold protein Dap160 and the cholecystokinin receptor-related CCKLR-17D1 drosulfakinin receptor. Providing mechanistic clues, we show that dAlk, jeb and CCKLR-17D1 are among mutants that also suppress a recently identified dNf1 neuromuscular junction (NMJ overgrowth phenotype and that manipulations that increase cAMP/PKA signaling in adipokinetic hormone (AKH-producing cells at the base of the neuroendocrine ring gland restore the dNf1 growth deficiency. Finally, supporting our previous contention that ALK might be a therapeutic target in NF1, we report that human ALK is expressed in cells that

  19. A protein kinase A (PKA)/β-catenin pathway sustains the BMP2/DLX3-induced osteogenic differentiation in dental follicle cells (DFCs).

    Science.gov (United States)

    Viale-Bouroncle, S; Klingelhöffer, C; Ettl, T; Reichert, T E; Morsczeck, C

    2015-03-01

    The directed expression of osteogenic transcription factors via a balanced activation of signaling pathways is an important prerequisite for the development of mineralized tissues. A positive-feedback loop of the BMP2-dependent SMAD signaling pathway and the DLX3 transcription factor (BMP2/DLX3 pathway) directs the osteogenic differentiation of periodontal precursor cells from the dental follicle (DFCs). However, little is known how this BMP2/DLX3 pathway interacts with other crucial signaling pathways such as the WNT/β-catenin signaling pathway. This study investigated the interaction between the BMP2/DLX3 pathway and the WNT pathway during the osteogenic differentiation of DFCs. BMP2 induced the WNT/β-catenin pathway in DFCs and phosphorylates β-catenin via protein kinase A (PKA). Moreover, only BMP2 facilitated the binding of LEF1/SMAD4/β-catenin complex to the DLX3 promoter, while an inducer of the canonical WNT pathway, WNT3A, act as an inhibitor. Although WNT3A inhibits the osteogenic differentiation of DFCs the expression of β-catenin was crucial for both the expression of DLX3 and for the osteogenic differentiation. In conclusion, while the activation of the canonical WNT pathway inhibits the osteogenic differentiation of DFCs, β-catenin sustains the BMP2/DLX3-mediated osteogenic differentiation via the activation of PKA.

  20. PKA and cAMP/CNG Channels Independently Regulate the Cholinergic Ca2+-Response of Drosophila Mushroom Body Neurons1,2,3

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    Pavot, Pierre; Carbognin, Elena

    2015-01-01

    Abstract The mushroom bodies (MBs), one of the main structures in the adult insect brain, play a critical role in olfactory learning and memory. Though historical genes such as dunce and rutabaga, which regulate the level of cAMP, were identified more than 30 years ago, their in vivo effects on cellular and physiological mechanisms and particularly on the Ca2+-responses still remain largely unknown. In this work, performed in Drosophila, we took advantage of in vivo bioluminescence imaging, which allowed real-time monitoring of the entire MBs (both the calyx/cell-bodies and the lobes) simultaneously. We imaged neuronal Ca2+-activity continuously, over a long time period, and characterized the nicotine-evoked Ca2+-response. Using both genetics and pharmacological approaches to interfere with different components of the cAMP signaling pathway, we first show that the Ca2+-response is proportional to the levels of cAMP. Second, we reveal that an acute change in cAMP levels is sufficient to trigger a Ca2+-response. Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has PKA-independent effects through the cyclic nucleotide-gated Ca2+-channel (CNG). Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects in the calyx/cell-bodies and in the lobes, suggesting a differential and regionalized regulation of AC. Our results provide insights into the complex Ca2+-response in the MBs, leading to the conclusion that cAMP modulates the Ca2+-responses through both PKA-dependent and -independent mechanisms, the latter through CNG-channels. PMID:26464971

  1. Phenothiazines solution complexity - Determination of pKa and solubility-pH profiles exhibiting sub-micellar aggregation at 25 and 37°C.

    Science.gov (United States)

    Pobudkowska, Aneta; Ràfols, Clara; Subirats, Xavier; Bosch, Elisabeth; Avdeef, Alex

    2016-10-10

    The ionization constants (pKa) and the pH-dependent solubility (log S-pH) of six phenothiazine derivatives (promazine hydrochloride, chlorpromazine hydrochloride, triflupromazine hydrochloride, fluphenazine dihydrochloride, perphenazine free base, and trifluoperazine dihydrochloride) were determined at 25 and 37°C. The pKa values of these low-soluble surface active molecules were determined by the cosolvent method (n-propanol/water at 37°C and methanol/water at 25°C). The log S-pH profiles were measured at 24h incubation time in 0.15M phosphate buffers. The log S-pH "shape-template" method, which critically depends on accurate pKa values (determined independently of solubility data), was used to propose speciation models, which were subsequently refined by rigorous mass-action weighted regression procedure described recently. Differential scanning calorimetry (DSC), UV-visible spectrophotometry, potentiometric, and high performance liquid chromatography (HPLC) measurements were used to characterize the compounds. The intrinsic solubility (S0) values of the three least-soluble drugs (chlorpromazine·HCl, triflupromazine·HCl, and trifluoperazine·2HCl) at 25°C were 0.5, 1.1, and 2.7μg/mL (resp.). These values increased to 5.5, 9.2, and 8.7μg/mL (resp.) at the physiological temperature. The enthalpies of solution for the latter compounds were exceptionally high positive (endothermic) values (99-152kJ·mol(-1)). Cationic sub-micellar aggregates were evident (from the distortions in the log S-pH profiles) for chlorpromazine, fluphenazine, perphenazine, and trifluoperazine at 25°C. The effects persisted at 37°C for chlorpromazine and trifluoperazine. The solids in suspension were apparently amorphous in cases where the drugs were introduced as the chloride salts.

  2. Determination of Surface pKa of Pure Mercaptoacetic Acid and 2- Mercaptobenzothiazole Mixed Monolayers by Impedance Titration

    Institute of Scientific and Technical Information of China (English)

    Guang Han LU; Chuan Yin LIU; Hong Yan ZHAO; Wei LIU; Li Ping JIANG; Ling Yan JIANG

    2004-01-01

    Interfacial proton transfer reactions of pure mercaptoacetic acid (MA) and 2-mercaptobenzothiazole (Mbz) mixed self-assembled monolayers (SAMs) have been studied using a.c. impedance titration method. The charge-transfer resistance (Rct) is measured with the monolayer composition and the ionic strength of pH solution. The surface pKa can be obtained by the plots of Rct and pH, the reasons of shifts of surface pKa are also explained.

  3. pkaPS: prediction of protein kinase A phosphorylation sites with the simplified kinase-substrate binding model

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    Schneider Georg

    2007-01-01

    Full Text Available Abstract Background Protein kinase A (cAMP-dependent kinase, PKA is a serine/threonine kinase, for which ca. 150 substrate proteins are known. Based on a refinement of the recognition motif using the available experimental data, we wished to apply the simplified substrate protein binding model for accurate prediction of PKA phosphorylation sites, an approach that was previously successful for the prediction of lipid posttranslational modifications and of the PTS1 peroxisomal translocation signal. Results Approximately 20 sequence positions flanking the phosphorylated residue on both sides have been found to be restricted in their sequence variability (region -18...+23 with the site at position 0. The conserved physical pattern can be rationalized in terms of a qualitative binding model with the catalytic cleft of the protein kinase A. Positions -6...+4 surrounding the phosphorylation site are influenced by direct interaction with the kinase in a varying degree. This sequence stretch is embedded in an intrinsically disordered region composed preferentially of hydrophilic residues with flexible backbone and small side chain. This knowledge has been incorporated into a simplified analytical model of productive binding of substrate proteins with PKA. Conclusion The scoring function of the pkaPS predictor can confidently discriminate PKA phosphorylation sites from serines/threonines with non-permissive sequence environments (sensitivity of ~96% at a specificity of ~94%. The tool "pkaPS" has been applied on the whole human proteome. Among new predicted PKA targets, there are entirely uncharacterized protein groups as well as apparently well-known families such as those of the ribosomal proteins L21e, L22 and L6. Availability The supplementary data as well as the prediction tool as WWW server are available at http://mendel.imp.univie.ac.at/sat/pkaPS. Reviewers Erik van Nimwegen (Biozentrum, University of Basel, Switzerland, Sandor Pongor (International

  4. Penetapan Harga PKA Dari Ketoprofen Bpfi Dan Beberapa Sediaan Tablet Ketoprofen Yang Terdapat Dipasaran Dengan Metode Spektrofotometri Ultraviolet Secara Isobestik

    OpenAIRE

    Rina Astuti

    2010-01-01

    Ketoprofen yang terdapat dipasaran diproduksi oleh beberapa industri farmasi dan masing-masing industri memiliki formulasi yang berbeda, dengan perbedaan formulasi ini tentu akan mempengaruhi harga pKa dari molekul obat yang akan menentukan absorpsi dan distribusi obat dalam jaringan-jaringan tubuh. Harga pKa yang diperoleh dari suatu senyawa tergantung dari metode yang digunakan, diantaranya perhitungan secara ? Hammett (Siswandono & Soekardjo, 1998), secara Spektrofotometri Ultraviolet m...

  5. Calcium signaling and the MAPK cascade are required for sperm activation in Caenorhabditis elegans.

    Science.gov (United States)

    Liu, Zhiyu; Wang, Bin; He, Ruijun; Zhao, Yanmei; Miao, Long

    2014-02-01

    In nematode, sperm activation (or spermiogenesis), a process in which the symmetric and non-motile spermatids transform into polarized and crawling spermatozoa, is critical for sperm cells to acquire fertilizing competence. SPE-8 dependent and SPE-8 independent pathways function redundantly during sperm activation in both males and hermaphrodites of Caenorhabditis elegans. However, the downstream signaling for both pathways remains unclear. Here we show that calcium signaling and the MAPK cascade are required for both SPE-8 dependent and SPE-8 independent sperm activation, implying that both pathways share common downstream signaling components during sperm activation. We demonstrate that activation of the MAPK cascade is sufficient to activate spermatids derived from either wild-type or spe-8 group mutant males and that activation of the MAPK cascade bypasses the requirement of calcium signal to induce sperm activation, indicating that the MAPK cascade functions downstream of or parallel with the calcium signaling during sperm activation. Interestingly, the persistent activation of MAPK in activated spermatozoa inhibits Major Sperm Protein (MSP)-based cytoskeleton dynamics. We demonstrate that MAPK plays dual roles in promoting pseudopod extension during sperm activation but also blocking the MSP-based, amoeboid motility of the spermatozoa. Thus, though nematode sperm are crawling cells, morphologically distinct from flagellated sperm, and the molecular machinery for motility of amoeboid and flagellated sperm is different, both types of sperm might utilize conserved signaling pathways to modulate sperm maturation.

  6. Functional dissection of the alphavirus capsid protease: sequence requirements for activity

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    Pützer Brigitte M

    2010-11-01

    Full Text Available Abstract Background The alphavirus capsid is multifunctional and plays a key role in the viral life cycle. The nucleocapsid domain is released by the self-cleavage activity of the serine protease domain within the capsid. All alphaviruses analyzed to date show this autocatalytic cleavage. Here we have analyzed the sequence requirements for the cleavage activity of Chikungunya virus capsid protease of genus alphavirus. Results Amongst alphaviruses, the C-terminal amino acid tryptophan (W261 is conserved and found to be important for the cleavage. Mutating tryptophan to alanine (W261A completely inactivated the protease. Other amino acids near W261 were not having any effect on the activity of this protease. However, serine protease inhibitor AEBSF did not inhibit the activity. Through error-prone PCR we found that isoleucine 227 is important for the effective activity. The loss of activity was analyzed further by molecular modelling and comparison of WT and mutant structures. It was found that lysine introduced at position 227 is spatially very close to the catalytic triad and may disrupt electrostatic interactions in the catalytic site and thus inactivate the enzyme. We are also examining other sequence requirements for this protease activity. Conclusions We analyzed various amino acid sequence requirements for the activity of ChikV capsid protease and found that amino acids outside the catalytic triads are important for the activity.

  7. Comparison of Predicted pKa Values for Some Amino-Acids, Dipeptides and Tripeptides, Using COSMO-RS, ChemAxon and ACD/Labs Methods Comparaison des valeurs de pKa de quelques acides aminés, dipeptides et tripeptides, prédites en utilisant les méthodes COSMO-RS, ChemAxon et ACD/Labs

    Directory of Open Access Journals (Sweden)

    Toure O.

    2013-05-01

    Full Text Available Liquid-phase pKa values play a key role in food science. Chemical properties of molecules depend largely on whether they are ionized or not. Most organic molecules are capable of gaining and/or losing a proton in aqueous solutions. Proton transfer most. frequently occurs between water and any ionizable atom of the organic molecule. The molecule’s response to profanation or deprotonation depends significantly on the site that was disturbed by proton transfer. Partial charge distribution in the molecule also varies with protonation of the acidlbase active sites. Then it can he used to determine the pKa of a molecule. First, we use the COSMO-RS method, a combination of the quantum chemical dielectric continuum solvation model COSMO with a statistical thermodynamics treatment fin- more Realistic Solvation (RS simulations, for the direct prediction of pKa constants of about 50 molecules (amino-acids, dipeptides and tripeptides. Then, we compare our results with experimental data and the pKa values predicted using two other methods. We used respectively the ChemAxon method using a program based on the calculation of partial charge of atoms in the molecule and the ACD/Labs method that enables to calculate single pKa values. for all possible dissociation centers when the rest of the molecule is considered neutral, using an internal database containing chemical structures and their experimental pKa values. The averaged Root Mean Square Error (RMSE of the predicted pKa values for each method compared to experimental results were respectively 0.596 for COSMO-RS, 0.445 for ChemAxon and 0.490 for ACD/Labs. While ACDILabs and ChemAxon are parameterized using a large set ofexperimental data (including several of the studied molecules, the COSMO- RS method was used in a fully predictive way. Regarding these results, COSMO-RS appears as a promising method to predict the pKa values of molecules of interest in food science with scarce available pKa values such

  8. T-cell receptor-induced JNK activation requires proteolytic inactivation of CYLD by MALT1.

    Science.gov (United States)

    Staal, Jens; Driege, Yasmine; Bekaert, Tine; Demeyer, Annelies; Muyllaert, David; Van Damme, Petra; Gevaert, Kris; Beyaert, Rudi

    2011-05-04

    The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is central to lymphocyte activation and lymphomagenesis. MALT1 mediates antigen receptor signalling to NF-κB by acting as a scaffold protein. Furthermore, MALT1 has proteolytic activity that contributes to optimal NF-κB activation by cleaving the NF-κB inhibitor A20. Whether MALT1 protease activity is involved in other signalling pathways, and the identity of the relevant substrates, is unknown. Here, we show that T-cell receptors (TCR) activation, as well as overexpression of the oncogenic API2-MALT1 fusion protein, results in proteolytic inactivation of CYLD by MALT1, which is specifically required for c-jun N-terminal kinase (JNK) activation and the inducible expression of a subset of genes. These results indicate a novel role for MALT1 proteolytic activity in TCR-induced JNK activation and reveal CYLD cleavage as the underlying mechanism.

  9. Meeting Resident Scholarly Activity Requirements Through a Longitudinal Quality Improvement Curriculum.

    Science.gov (United States)

    Simasek, Madeline; Ballard, Stephanie L; Phelps, Phillip; Pingul-Ravano, Rowena; Kolb, N Randall; Finkelstein, Alan; Weaver-Agostoni, Jacqueline; Takedai, Teiichi

    2015-03-01

    Quality improvement (QI) skills are learned during residency, yet there are few reports of the scholarly activity outcomes of a QI curriculum in a primary care program. We examined whether scholarly activity can result from a longitudinal, experiential QI curriculum that involves residents, clinic staff, and faculty. The University of Pittsburgh Medical Center Shadyside Family Medicine Residency implemented a required longitudinal outpatient practice improvement rotation (LOPIR) curriculum in 2005. The rotation format includes weekly multidisciplinary work group meetings alternating with resident presentations delivered to the entire program. Residents present the results of a literature review and provide 2 interim project updates to the residency. A completed individual project is required for residency graduation, with project results presented at Residency Research Day. Scholarly activity outcomes of the curriculum were analyzed using descriptive statistics. As of 2014, 60 residents completed 3 years of the LOPIR curriculum. All residents satisfied the 2014 Accreditation Council for Graduate Medical Education (ACGME) scholarly activity and QI requirements with a literature review presentation in postgraduate year 2, and the presentation of a completed QI project at Residency Research Day. Residents have delivered 83 local presentations, 13 state/regional presentations, and 2 national presentations. Residents received 7 awards for QI posters, as well as 3 grants totaling $21,639. The educational program required no additional curriculum time, few resources, and was acceptable to residents, faculty, and staff. LOPIR is an effective way to meet and exceed the 2014 ACGME scholarly activity requirements for family medicine residents.

  10. Limited Ca2+ and PKA-pathway dependent neurogenic differentiation of human adult mesenchymal stem cells as compared to fetal neuronal stem cells.

    Science.gov (United States)

    Lepski, Guilherme; Jannes, Cinthia Elim; Maciaczyk, Jaroslaw; Papazoglou, Anna; Mehlhorn, Alexander T; Kaiser, Stefan; Teixeira, Manoel Jacobsen; Marie, Suely K N; Bischofberger, Josef; Nikkhah, Guido

    2010-01-15

    The ability of mesenchymal stem cells to generate functional neurons in culture is still a matter of controversy. In order to assess this issue, we performed a functional comparison between neuronal differentiation of human MSCs and fetal-derived neural stem cells (NSCs) based on morphological, immunocytochemical, and electrophysiological criteria. Furthermore, possible biochemical mechanisms involved in this process were presented. NF200 immunostaining was used to quantify the yield of differentiated cells after exposure to cAMP. The addition of a PKA inhibitor and Ca(2+) blockers to the differentiation medium significantly reduced the yield of differentiated cells. Activation of CREB was also observed on MSCs during maturation. Na(+)-, K(+)-, and Ca(2+)-voltage-dependent currents were recorded from MSCs-derived cells. In contrast, significantly larger Na(+) currents, firing activity, and spontaneous synaptic currents were recorded from NSCs. Our results indicate that the initial neuronal differentiation of MSCs is induced by cAMP and seems to be dependent upon Ca(2+) and the PKA pathway. However, compared to fetal neural stem cells, adult mesenchymal counterparts are limited in their neurogenic potential. Despite the similar yield of neuronal cells, NSCs achieved a more mature functional state. Description of the underlying mechanisms that govern MSCs' differentiation toward a stable neuronal phenotype and their limitations provides a unique opportunity to enhance our understanding of stem cell plasticity.

  11. Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine Cβ2

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    Ørstavik Sigurd

    2006-08-01

    Full Text Available Abstract Background Two main genes encoding the catalytic subunits Cα and Cβ of cyclic AMP dependent protein kinase (PKA have been identified in all vertebrates examined. The murine, bovine and human Cβ genes encode several splice variants, including the splice variant Cβ2. In mouse Cβ2 has a relative molecular mass of 38 kDa and is only expressed in the brain. In human and bovine Cβ2 has a relative molecular mass of 47 kDa and is mainly expressed in lymphoid tissues. Results We identified a novel 47 kDa splice variant encoded by the mouse Cβ gene that is highly expressed in lymphoid cells. Cloning, expression, and production of a sequence-specific antiserum and characterization of PKA catalytic subunit activities demonstrated the 47 kDa protein to be a catalytically active murine homologue of human and bovine Cβ2. Based on the present results and the existence of a human brain-specifically expressed Cβ splice variant designated Cβ4 that is identical to the former mouse Cβ2 splice variant, the mouse splice variant has now been renamed mouse Cβ4. Conclusion Murine lymphoid tissues express a protein that is a homologue of human and bovine Cβ2. The murine Cβ gene encodes the splice variants Cβ1, Cβ2, Cβ3 and Cβ4, as is the case with the human Cβ gene.

  12. HIF-1α activation by intermittent hypoxia requires NADPH oxidase stimulation by xanthine oxidase.

    Science.gov (United States)

    Nanduri, Jayasri; Vaddi, Damodara Reddy; Khan, Shakil A; Wang, Ning; Makarenko, Vladislav; Semenza, Gregg L; Prabhakar, Nanduri R

    2015-01-01

    Hypoxia-inducible factor 1 (HIF-1) mediates many of the systemic and cellular responses to intermittent hypoxia (IH), which is an experimental model that simulates O2 saturation profiles occurring with recurrent apnea. IH-evoked HIF-1α synthesis and stability are due to increased reactive oxygen species (ROS) generated by NADPH oxidases, especially Nox2. However, the mechanisms by which IH activates Nox2 are not known. We recently reported that IH activates xanthine oxidase (XO) and the resulting increase in ROS elevates intracellular calcium levels. Since Nox2 activation requires increased intracellular calcium levels, we hypothesized XO-mediated calcium signaling contributes to Nox activation by IH. We tested this possibility in rat pheochromocytoma PC12 cells subjected to IH consisting alternating cycles of hypoxia (1.5% O2 for 30 sec) and normoxia (21% O2 for 5 min). Kinetic analysis revealed that IH-induced XO preceded Nox activation. Inhibition of XO activity either by allopurinol or by siRNA prevented IH-induced Nox activation, translocation of the cytosolic subunits p47phox and p67phox to the plasma membrane and their interaction with gp91phox. ROS generated by XO also contribute to IH-evoked Nox activation via calcium-dependent protein kinase C stimulation. More importantly, silencing XO blocked IH-induced upregulation of HIF-1α demonstrating that HIF-1α activation by IH requires Nox2 activation by XO.

  13. Direct interaction of the molecular scaffolds POSH and JIP is required for apoptotic activation of JNKs.

    Science.gov (United States)

    Kukekov, Nickolay V; Xu, Zhiheng; Greene, Lloyd A

    2006-06-02

    A sequential pathway (the JNK pathway) that includes activation of Rac1/Cdc42, mixed lineage kinases, MAP kinase kinases 4 and 7, and JNKs plays a required role in many paradigms of apoptotic cell death. However, the means by which this pathway is assembled and directed toward apoptotic death has been unclear. Here, we report that propagation of the apoptotic JNK pathway requires the cooperative interaction of two molecular scaffolds, POSH and JIPs. POSH (plenty of SH3s) is a multidomain GTP-Rac1-interacting protein that binds and promotes activation of mixed lineage kinases. JIPs are reported to bind MAP kinase kinases 4/7 and JNKs. We find that POSH and JIPs directly associate with one another to form a multiprotein complex, PJAC (POSH-JIP apoptotic complex), that includes all of the known kinase components of the pathway. Our observations indicate that this complex is required for JNK activation and cell death in response to apoptotic stimuli.

  14. Conserved mycobacterial lipoglycoproteins activate TLR2 but also require glycosylation for MHC class II-restricted T cell activation.

    Science.gov (United States)

    Sieling, Peter A; Hill, Preston J; Dobos, Karen M; Brookman, Kerry; Kuhlman, Andrew M; Fabri, Mario; Krutzik, Stephan R; Rea, Thomas H; Heaslip, Darragh G; Belisle, John T; Modlin, Robert L

    2008-05-01

    CD4(+) T cell clones derived from a leprosy lesion and patient blood were used to monitor the isolation and identification of an Ag associated with the self-limited form of the disease. Biochemical purification and genetic analysis identified the T cell Ag as a conserved mycobacterial lipoglycoprotein LprG. LprG-mediated activation of CD4(+) T cells required specific MHC class II restriction molecules and intracellular processing. Although LprG activated TLR2, this alone was not sufficient to stimulate or inhibit T cell activation. A striking finding was that the carbohydrate moieties of LprG were required for optimal T cell activation, because recombinant LprG produced in Escherichia coli, or recombinant LprG produced in Mycobacterium smegmatis and digested by alpha-mannosidase, did not activate T cells. This study demonstrates that the universe of bacterial T cell Ags includes lipoglycoproteins, which act as TLR2 ligands but also require glycosylation for MHC class II-restricted T cell activation in vivo.

  15. Requirements, design and pilot study of a physical activity activation system using virtual communities

    NARCIS (Netherlands)

    Elloumi, Lamia; Meijerink, Margot; van Beijnum, Bernhard J.F.; Hermens, Hermanus J.; Fred, Ana; Gamboa, Hugo

    2017-01-01

    Increasing researches support the importance of physical activity in maintaining health and preventing from diseases. Nowadays, researchers from different disciplines and health organizations are putting a lot of effort on interventions and strategies to increase the motivation of people in being

  16. The lipid moiety of brincidofovir is required for in vitro antiviral activity against Ebola virus.

    Science.gov (United States)

    McMullan, Laura K; Flint, Mike; Dyall, Julie; Albariño, César; Olinger, Gene G; Foster, Scott; Sethna, Phiroze; Hensley, Lisa E; Nichol, Stuart T; Lanier, E Randall; Spiropoulou, Christina F

    2016-01-01

    Brincidofovir (BCV) is the 3-hexadecyloxy-1-propanol (HDP) lipid conjugate of the acyclic nucleoside phosphonate cidofovir (CDV). BCV has established broad-spectrum activity against double-stranded DNA (dsDNA) viruses; however, its activity against RNA viruses has been less thoroughly evaluated. Here, we report that BCV inhibited infection of Ebola virus in multiple human cell lines. Unlike the mechanism of action for BCV against cytomegalovirus and other dsDNA viruses, phosphorylation of CDV to the diphosphate form appeared unnecessary. Instead, antiviral activity required the lipid moiety and in vitro activity against EBOV was observed for several HDP-nucleotide conjugates.

  17. Substrate Shuttling Between Active Sites of Uroporphyrinogen Decarboxylase in Not Required to Generate Coproporphyrinogen

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, J.; Warby, C; Whitby, F; Kushner, J; Hill, C

    2009-01-01

    Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.

  18. 30 CFR 285.617 - What activities require a revision to my SAP, and when will MMS approve the revision?

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 2 2010-07-01 2010-07-01 false What activities require a revision to my SAP... OUTER CONTINENTAL SHELF Plans and Information Requirements Activities Under An Approved Sap § 285.617 What activities require a revision to my SAP, and when will MMS approve the revision? (a) You must...

  19. Electrostatic Energetics of Bacillus subtilis Ribonuclease P Protein Determined by Nuclear Magnetic Resonance-Based Histidine pKa Measurements.

    Science.gov (United States)

    Mosley, Pamela L; Daniels, Kyle G; Oas, Terrence G

    2015-09-08

    The pKa values of ionizable groups in proteins report the free energy of site-specific proton binding and provide a direct means of studying pH-dependent stability. We measured histidine pKa values (H3, H22, and H105) in the unfolded (U), intermediate (I), and sulfate-bound folded (F) states of RNase P protein, using an efficient and accurate nuclear magnetic resonance-monitored titration approach that utilizes internal reference compounds and a parametric fitting method. The three histidines in the sulfate-bound folded protein have pKa values depressed by 0.21 ± 0.01, 0.49 ± 0.01, and 1.00 ± 0.01 units, respectively, relative to that of the model compound N-acetyl-l-histidine methylamide. In the unliganded and unfolded protein, the pKa values are depressed relative to that of the model compound by 0.73 ± 0.02, 0.45 ± 0.02, and 0.68 ± 0.02 units, respectively. Above pH 5.5, H22 displays a separate resonance, which we have assigned to I, whose apparent pKa value is depressed by 1.03 ± 0.25 units, which is ∼0.5 units more than in either U or F. The depressed pKa values we observe are consistent with repulsive interactions between protonated histidine side chains and the net positive charge of the protein. However, the pKa differences between F and U are small for all three histidines, and they have little ionic strength dependence in F. Taken together, these observations suggest that unfavorable electrostatics alone do not account for the fact that RNase P protein is intrinsically unfolded in the absence of ligand. Multiple factors encoded in the P protein sequence account for its IUP property, which may play an important role in its function.

  20. Toward accurate prediction of pKa values for internal protein residues: the importance of conformational relaxation and desolvation energy.

    Science.gov (United States)

    Wallace, Jason A; Wang, Yuhang; Shi, Chuanyin; Pastoor, Kevin J; Nguyen, Bao-Linh; Xia, Kai; Shen, Jana K

    2011-12-01

    Proton uptake or release controls many important biological processes, such as energy transduction, virus replication, and catalysis. Accurate pK(a) prediction informs about proton pathways, thereby revealing detailed acid-base mechanisms. Physics-based methods in the framework of molecular dynamics simulations not only offer pK(a) predictions but also inform about the physical origins of pK(a) shifts and provide details of ionization-induced conformational relaxation and large-scale transitions. One such method is the recently developed continuous constant pH molecular dynamics (CPHMD) method, which has been shown to be an accurate and robust pK(a) prediction tool for naturally occurring titratable residues. To further examine the accuracy and limitations of CPHMD, we blindly predicted the pK(a) values for 87 titratable residues introduced in various hydrophobic regions of staphylococcal nuclease and variants. The predictions gave a root-mean-square deviation of 1.69 pK units from experiment, and there were only two pK(a)'s with errors greater than 3.5 pK units. Analysis of the conformational fluctuation of titrating side-chains in the context of the errors of calculated pK(a) values indicate that explicit treatment of conformational flexibility and the associated dielectric relaxation gives CPHMD a distinct advantage. Analysis of the sources of errors suggests that more accurate pK(a) predictions can be obtained for the most deeply buried residues by improving the accuracy in calculating desolvation energies. Furthermore, it is found that the generalized Born implicit-solvent model underlying the current CPHMD implementation slightly distorts the local conformational environment such that the inclusion of an explicit-solvent representation may offer improvement of accuracy.

  1. Novel zinc(II)phthalocyanines bearing azo-containing schiff base: Determination of pKa values, absorption, emission, enzyme inhibition and photochemical properties

    Science.gov (United States)

    Kantar, Cihan; Mavi, Vildan; Baltaş, Nimet; İslamoğlu, Fatih; Şaşmaz, Selami

    2016-10-01

    Azo-containing schiff bases are well known and there are many studies about their various properties in literature. However, phthalocyanines bearing azo-containing schiff bases, their spectral, analytical and biological properties are unknown. Therefore, new zinc (II) phthalocyanines bearing azo-containing schiff base were synthesized and investigated to determine pKa values, absorption, emission, enzyme inhibition and photochemical properties. Emission spectra were reported and large Stokes shift values were determined for all compounds, indicating that all molecules exhibit excited state intramolecular proton transfer. These phthalocyanines were the first examples of phthalocyanine showing excited state intramolecular proton transfer. Singlet oxygen quantum yields of zinc (II) phthalocyanines were determined. pKa values and indicator properties of all compounds were investigated by potentiometry. All compounds were assayed for inhibitory activity against bovine milk xanthine oxidase and acetylcholinesterase enzyme in vitro. Compound 2 showed the high inhibitory effect against xanthine oxidase (IC50 = 0.24 ± 0.01 μM). However, phthalocyanine compounds did not show enzyme inhibitor behavior.

  2. Defect-Related Luminescent Hydroxyapatite-Enhanced Osteogenic Differentiation of Bone Mesenchymal Stem Cells Via an ATP-Induced cAMP/PKA Pathway.

    Science.gov (United States)

    Wang, Chao; Liu, Dandan; Zhang, Cuimiao; Sun, Jiadong; Feng, Weipei; Liang, Xing-Jie; Wang, Shuxiang; Zhang, Jinchao

    2016-05-11

    Novel defect-related hydroxyapatite (DHAP), which combines the advantages of HAP and defect-related luminescence, has the potential application in tissue engineering and biomedical area, because of its excellent capability of monitoring the osteogenic differentiation and material biodegradation. Although the extracellular mechanism of DHAP minerals and PO4(3-) functioning in osteogenic differentiation has been widely studied, the intracellular molecular mechanism through which PO4(3-) mediates osteogenesis of bone mesenchymal stem cells (BMSCs) is not clear. We examined a previously unknown molecular mechanism through which PO4(3-) promoted osteogenesis of BMSCs with an emphasis on adenosine-triphosphate (ATP)-induced cAMP/PKA pathway. Our studies showed that DHAP could be uptaken into lysosome, in which PO4(3-) was released from DHAP, because of the acid environment of lysosome. The released PO4(3-) interacted with ADP to form ATP, and then degraded into adenosine, an ATP metabolite, which interacted with A2b adenosine receptor to activate the cAMP/PKA pathway, resulting in the high expression of osteogenesis-related genes, such as Runx2, BMP-2, and OCN. These findings first revealed the function of ATP-metabolism in bone physiological homeostasis, which may be developed to cure bone metabolic diseases.

  3. Interleukin-1 beta converting enzyme requires oligomerization for activity of processed forms in vivo.

    Science.gov (United States)

    Gu, Y; Wu, J; Faucheu, C; Lalanne, J L; Diu, A; Livingston, D J; Su, M S

    1995-05-01

    Interleukin-1 beta converting enzyme (ICE) is composed of 10' (p10) and 20 kDa (p20) subunits, which are derived from a common 45 kDa precursor. Recent crystallographic studies have shown that ICE exists as a tetramer (p20/p10)2 in the crystal lattice. We provide evidence that the p10 and p20 subunits of ICE associate as oligomers in transfected COS cells. Using intragenic complementation, we show that the activity of a p10/p10 interface mutant defective in autoprocessing can be restored by co-expression with active site ICE mutants. Different active site mutants can also complement each other by oligomerization to form active ICE. These studies indicate that ICE precursor polypeptides may associate in different quaternary structures and that oligomerization is required for autoprocessing. Furthermore, integenic complementation of active site mutants of ICE and an ICE homolog restores autoprocessing activity, suggesting that hetero-oligomerization occurs between ICE homologs.

  4. cAMP/PKA信号通路在肾上腺肿瘤中的作用%Effect of cAMP/PKA Signaling Pathways on Adrenal Tumors

    Institute of Scientific and Technical Information of China (English)

    张科; 刘昌荣

    2016-01-01

    Objective To investigate effect of cyclic adenosine monophosphate/protein kinase a ( cAMP/PKA) signaling pathways on adrenal tumors. Methods Human H295R cells of adrenal tumor cells were randomly divided into control group, group A and B. Control group did not undergo any treatment;group A was incubated with cAMP deriva-tives db-cAMP;group B was incubated with db-cAMP and inhibitor of cAMP/PKA signaling pathway H-89. In three groups, PKA activities were detected, and phosphorylation levels of CREB protein were also detected by Western Blot method;72 h cell proliferation levels were detected by CCK8 assay, and capacities of hormone secretion were detected by glucocorticoid secretion experiment. Results Levels of PKA activity, CREB protein phosphorylation, cell proliferation and glucocorticoid secretion were significantly higher in group A than those in control group and group B (P0. 05). Conclusion The ab-normal activation of cAMP/PKA signaling pathways can promote formation and functional effect of adrenal tumors.%目的:探讨cAMP/PKA信号通路在肾上腺肿瘤中的作用。方法将人肾上腺肿瘤H295R细胞随机分为对照组、实验组A及实验组B,对照组不进行处理,实验组A加入cAMP衍生物db-cAMP,实验组B加入db-cAMP及cAMP/PKA信号通路抑制剂H-89,分别检测细胞PKA活性;应用Western Blot方法检测cAMP/PKA信号通路下游CREB蛋白的磷酸化水平;CCK8实验检测细胞72 h的增殖水平;糖皮质激素分泌实验检测3组H295R的激素分泌能力。结果实验组A的PKA活性水平、CREB蛋白的磷酸化水平、细胞增殖水平、糖皮质激素分泌水平均较对照组和实验组B显著升高(P<0.05);而实验组B与对照组比较差异无统计学意义(P<0.05)。结论 cAMP/PKA信号通路的异常活化能够促进肾上腺肿瘤的形成与功能的发挥。

  5. Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase

    DEFF Research Database (Denmark)

    VanderKuur, J A; Wang, X; Zhang, L

    1994-01-01

    Growth hormone (GH) has recently been shown to activate the GH receptor (GHR)-associated tyrosine kinase JAK2. In the present study, regions of the GHR required for JAK2 association with GHR were identified. GH-dependent JAK2 association with GHR was detected in Chinese hamster ovary (CHO) cells...

  6. 77 FR 13617 - Agency Information Collection Activities: Customs Modernization Act Recordkeeping Requirements

    Science.gov (United States)

    2012-03-07

    ... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Customs Modernization... concerning the Customs Modernization Act Recordkeeping Requirements. This request for comment is being made... American Free Trade Agreement Implementation Act, Title VI, known as the Customs Modernization Act (Mod...

  7. 30 CFR 251.4 - Types of G&G activities that require permits or Notices.

    Science.gov (United States)

    2010-07-01

    ... G&G exploration, including deep stratigraphic tests, for oil, gas, or sulphur resources. If you... 30 Mineral Resources 2 2010-07-01 2010-07-01 false Types of G&G activities that require permits or Notices. 251.4 Section 251.4 Mineral Resources MINERALS MANAGEMENT SERVICE, DEPARTMENT OF THE...

  8. Conformational preferences and pK(a) value of selenocysteine residue.

    Science.gov (United States)

    Byun, Byung Jin; Kang, Young Kee

    2011-05-01

    The conformational preferences of the L-selenocysteine (Sec) dipeptides with selenol and selenolate groups (Ac-Sec-NHMe and Ac-Sec(-) -NHMe, respectively) and the apparent (i.e., macroscopic) pK(a) value of the Sec residue have been studied using the dispersion-corrected density functionals M06-2X and B2PLYP-D with the implicit solvation method in the gas phase and in water. In the gas phase, the backbone-to-backbone and/or side chain-to-backbone hydrogen bonds are found to contribute in stabilizing the most preferred conformations for the Sec and Sec(-) residues, as seen for the Cys and Cys(-) residues. However, the polyproline II-like conformations prevail over the conformations with the backbone-to-backbone hydrogen bonds in water because of the weakened hydrogen bonds by the favorable direct interactions between the backbone CO and HN groups and water molecules. The Sec and Sec(-) residues are found to adopt more various conformations than the Cys and Cys(-) residues in water, although the most preferred conformations of the neutral and/or anionic forms of the two residues are similar each other in the gas phase and in water. Using the statistically weighted free energies of the Sec and Sec(-) dipeptides in the gas phase and their solvation free energies, the pK(a) value of the Sec residue is estimated to be 5.47 at 25°C, which is in good agreement with the experimental value of 5.43 ± 0.02. It is found that the lower pK(a) value of the selenol side chain for the Sec residue by ∼3 units than the thiol side chain for the Cys residue is ascribed to the higher gas-phase acidity of the Sec residue. Copyright © 2011 Wiley Periodicals, Inc.

  9. Aspartate embedding depth affects pHLIP's insertion pKa.

    Science.gov (United States)

    Fendos, Justin; Barrera, Francisco N; Engelman, Donald M

    2013-07-09

    We have used the pHlow insertion peptide (pHLIP) family to study the role of aspartate embedding depth in pH-dependent transmembrane peptide insertion. pHLIP binds to the surface of a lipid bilayer as a largely unstructured monomer at neutral pH. When the pH is lowered, pHLIP inserts spontaneously across the membrane as a spanning α-helix. pHLIP insertion is reversible when the pH is adjusted back to a neutral value. One of the critical events facilitating pHLIP insertion is the protonation of aspartates in the spanning domain of the peptide: the negative side chains of these residues convert to uncharged, polar forms, facilitating insertion by altering the hydrophobicity of the spanning domain. To examine this protonation mechanism further, we created pHLIP sequence variants in which the two spanning aspartates (D14 and D25) were moved up or down in the sequence. We hypothesized that the aspartate depth in the inserted state would directly affect the proton affinity of the acidic side chains, altering the pKa of pH-dependent insertion. To this end, we also mutated the arginine at position 11 to determine whether arginine snorkeling modulates the insertion pKa by affecting the aspartate depth. Our results indicate that both types of mutations change the insertion pKa, supporting the idea that the aspartate depth is a participating parameter in determining the pH dependence. We also show that pHLIP's resistance to aggregation can be altered with our mutations, identifying a new criterion for improving the performance of pHLIP in vivo when targeting acidic disease tissues such as cancer and inflammation.

  10. Factors Required for Activation of Urease as a Virulence Determinant in Cryptococcus neoformans

    Science.gov (United States)

    Singh, Arpita; Panting, Robert J.; Varma, Ashok; Saijo, Tomomi; Waldron, Kevin J.; Jong, Ambrose; Ngamskulrungroj, Popchai; Chang, Yun C.; Rutherford, Julian C.; Kwon-Chung, Kyung J.

    2013-01-01

    ABSTRACT Urease in Cryptococcus neoformans plays an important role in fungal dissemination to the brain and causing meningoencephalitis. Although urea is not required for synthesis of apourease encoded by URE1, the available nitrogen source affected the expression of URE1 as well as the level of the enzyme activity. Activation of the apoenzyme requires three accessory proteins, Ure4, Ure6, and Ure7, which are homologs of the bacterial urease accessory proteins UreD, UreF, and UreG, respectively. A yeast two-hybrid assay showed positive interaction of Ure1 with the three accessory proteins encoded by URE4, URE6, and URE7. Metalloproteomic analysis of cryptococcal lysates using inductively coupled plasma mass spectrometry (ICP-MS) and a biochemical assay of urease activity showed that, as in many other organisms, urease is a metallocentric enzyme that requires nickel transported by Nic1 for its catalytic activity. The Ure7 accessory protein (bacterial UreG homolog) binds nickel likely via its conserved histidine-rich domain and appears to be responsible for the incorporation of Ni2+ into the apourease. Although the cryptococcal genome lacks the bacterial UreE homolog, Ure7 appears to combine the functions of bacterial UreE and UreG, thus making this pathogen more similar to that seen with the plant system. Brain invasion by the ure1, ure7, and nic1 mutant strains that lack urease activity was significantly less effective in a mouse model. This indicated that an activated urease and not the Ure1 protein was responsible for enhancement of brain invasion and that the factors required for urease activation in C. neoformans resemble those of plants more than those of bacteria. PMID:23653445

  11. Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling.

    Science.gov (United States)

    Zahanich, Ihor; Sirenko, Syevda G; Maltseva, Larissa A; Tarasova, Yelena S; Spurgeon, Harold A; Boheler, Kenneth R; Stern, Michael D; Lakatta, Edward G; Maltsev, Victor A

    2011-01-01

    There is an intense interest in differentiating embryonic stem cells to engineer biological pacemakers as an alternative to electronic pacemakers for patients with cardiac pacemaker function deficiency. Embryonic stem cell-derived cardiocytes (ESCs), however, often exhibit dysrhythmic excitations. Using Ca(2+) imaging and patch-clamp techniques, we studied requirements for generation of spontaneous rhythmic action potentials (APs) in late-stage mouse ESCs. Sarcoplasmic reticulum (SR) of ESCs generates spontaneous, rhythmic, wavelet-like Local Ca(2+)Releases (LCRs) (inhibited by ryanodine, tetracaine, or thapsigargin). L-type Ca(2+)current (I(CaL)) induces a global Ca(2+) release (CICR), depleting the Ca(2+) content SR which resets the phases of LCR oscillators. Following a delay, SR then generates a highly synchronized spontaneous Ca(2+)release of multiple LCRs throughout the cell. The LCRs generate an inward Na(+)/Ca(2+)exchanger (NCX) current (absent in Na(+)-free solution) that ignites the next AP. Interfering with SR Ca(2+) cycling (ryanodine, caffeine, thapsigargin, cyclopiazonic acid, BAPTA-AM), NCX (Na(+)-free solution), or I(CaL) (nifedipine) results in dysrhythmic excitations or cessation of automaticity. Inhibition of cAMP/PKA signaling by a specific PKA inhibitor, PKI, decreases SR Ca(2+) loading, substantially reducing both spontaneous LCRs (number, size, and amplitude) and rhythmic AP firing. In contrast, enhancing PKA signaling by cAMP increases the LCRs (number, size, duration) and converts irregularly beating ESCs to rhythmic "pacemaker-like" cells. SR Ca(2+) loading and LCR activity could be also increased with a selective activation of SR Ca(2+) pumping by a phospholamban antibody. We conclude that SR Ca(2+) loading and spontaneous rhythmic LCRs are driven by inherent cAMP/PKA activity. I(CaL) synchronizes multiple LCR oscillators resulting in strong, partially synchronized diastolic Ca(2+) release and NCX current. Rhythmic ESC automaticity can be

  12. Inquiry-Based Laboratory Activity to Investigate Physical Growth Requirements of Microorganisms

    Directory of Open Access Journals (Sweden)

    Michelle Furlong

    2014-08-01

    Full Text Available Standard "cookbook" laboratory activities that are used to teach students the optimal physical growth conditions of microorganisms should be modified so that they more effectively foster student's higher order cognitive skills and attract student interest.  This paper describes a laboratory activity that engages students in an inquiry-based approach to studying the physical growth requirements of microorganisms.  In this activity, students design and implement an experiment to obtain pure cultures of specific microorganisms, with distinct growth properties, that are provided to them in a mixed culture.

  13. The molecular biology of memory: cAMP, PKA, CRE, CREB-1, CREB-2, and CPEB.

    Science.gov (United States)

    Kandel, Eric R

    2012-05-14

    The analysis of the contributions to synaptic plasticity and memory of cAMP, PKA, CRE, CREB-1, CREB-2, and CPEB has recruited the efforts of many laboratories all over the world. These are six key steps in the molecular biological delineation of short-term memory and its conversion to long-term memory for both implicit (procedural) and explicit (declarative) memory. I here first trace the background for the clinical and behavioral studies of implicit memory that made a molecular biology of memory storage possible, and then detail the discovery and early history of these six molecular steps and their roles in explicit memory.

  14. The molecular biology of memory: cAMP, PKA, CRE, CREB-1, CREB-2, and CPEB

    Directory of Open Access Journals (Sweden)

    Kandel Eric R

    2012-05-01

    Full Text Available Abstract The analysis of the contributions to synaptic plasticity and memory of cAMP, PKA, CRE, CREB-1, CREB-2, and CPEB has recruited the efforts of many laboratories all over the world. These are six key steps in the molecular biological delineation of short-term memory and its conversion to long-term memory for both implicit (procedural and explicit (declarative memory. I here first trace the background for the clinical and behavioral studies of implicit memory that made a molecular biology of memory storage possible, and then detail the discovery and early history of these six molecular steps and their roles in explicit memory.

  15. Dkk1 haploinsufficiency requires expression of Bmp2 for bone anabolic activity.

    Science.gov (United States)

    Intini, Giuseppe; Nyman, Jeffry S

    2015-06-01

    Bone fractures remain a serious health burden and prevention and enhanced healing of fractures have been obtained by augmenting either BMP or Wnt signaling. However, whether BMP and Wnt signaling are both required or are self-sufficient for anabolic and fracture healing activities has never been fully elucidated. Mice haploinsufficient for Dkk1 (Dkk1(+/-)) exhibit a high bone mass phenotype due to an up-regulation of canonical Wnt signaling while mice lacking Bmp2 expression in the limbs (Bmp2(c/c);Prx1::cre) succumb to spontaneous fracture and are unable to initiate fracture healing; combined, these mice offer an opportunity to examine the requirement for activated BMP signaling on the anabolic and fracture healing activity of Wnts. When Dkk1(+/-) mice were crossed with Bmp2(c/c);Prx1::cre mice, the offspring bearing both genetic alterations were unable to increase bone mass and heal fractures, indicating that increased canonical Wnt signaling is unable to exploit its activity in absence of Bmp2. Thus, our data suggest that BMP signaling is required for Wnt-mediated anabolic activity and that therapies aimed at preventing fractures and fostering fracture repair may need to target both pathways for maximal efficacy.

  16. Prostaglandins from Cytosolic Phospholipase A2α/Cyclooxygenase-1 Pathway and Mitogen-activated Protein Kinases Regulate Gene Expression in Candida albicans-infected Macrophages.

    Science.gov (United States)

    Yun, Bogeon; Lee, HeeJung; Jayaraja, Sabarirajan; Suram, Saritha; Murphy, Robert C; Leslie, Christina C

    2016-03-25

    In Candida albicans-infected resident peritoneal macrophages, activation of group IVA cytosolic phospholipase A2(cPLA2α) by calcium- and mitogen-activated protein kinases triggers the rapid production of prostaglandins I2 and E2 through cyclooxygenase (COX)-1 and regulates gene expression by increasing cAMP. InC. albicans-infected cPLA2α(-/-)or COX-1(-/-)macrophages, expression ofI l10,Nr4a2, and Ptgs2 was lower, and expression ofTnfα was higher, than in wild type macrophages. Expression was reconstituted with 8-bromo-cAMP, the PKA activator 6-benzoyl-cAMP, and agonists for prostaglandin receptors IP, EP2, and EP4 in infected but not uninfected cPLA2α(-/-)or COX-1(-/-)macrophages. InC. albicans-infected cPLA2α(+/+)macrophages, COX-2 expression was blocked by IP, EP2, and EP4 receptor antagonists, indicating a role for both prostaglandin I2 and E2 Activation of ERKs and p38, but not JNKs, by C. albicansacted synergistically with prostaglandins to induce expression of Il10,Nr4a2, and Ptgs2. Tnfα expression required activation of ERKs and p38 but was suppressed by cAMP. Results using cAMP analogues that activate PKA or Epacs suggested that cAMP regulates gene expression through PKA. However, phosphorylation of cAMP-response element-binding protein (CREB), the cAMP-regulated transcription factor involved inIl10,Nr4a2,Ptgs2, andTnfα expression, was not mediated by cAMP/PKA because it was similar inC. albicans-infected wild type and cPLA2α(-/-)or COX-1(-/-)macrophages. CREB phosphorylation was blocked by p38 inhibitors and induced by the p38 activator anisomycin but not by the PKA activator 6-benzoyl-cAMP. Therefore, MAPK activation inC. albicans-infected macrophages plays a dual role by promoting the cPLA2α/prostaglandin/cAMP/PKA pathway and CREB phosphorylation that coordinately regulate immediate early gene expression.

  17. Anthrax lethal factor cleavage of Nlrp1 is required for activation of the inflammasome.

    Directory of Open Access Journals (Sweden)

    Jonathan L Levinsohn

    Full Text Available NOD-like receptor (NLR proteins (Nlrps are cytosolic sensors responsible for detection of pathogen and danger-associated molecular patterns through unknown mechanisms. Their activation in response to a wide range of intracellular danger signals leads to formation of the inflammasome, caspase-1 activation, rapid programmed cell death (pyroptosis and maturation of IL-1β and IL-18. Anthrax lethal toxin (LT induces the caspase-1-dependent pyroptosis of mouse and rat macrophages isolated from certain inbred rodent strains through activation of the NOD-like receptor (NLR Nlrp1 inflammasome. Here we show that LT cleaves rat Nlrp1 and this cleavage is required for toxin-induced inflammasome activation, IL-1 β release, and macrophage pyroptosis. These results identify both a previously unrecognized mechanism of activation of an NLR and a new, physiologically relevant protein substrate of LT.

  18. The IUPAC aqueous and non-aqueous experimental pKa data repositories of organic acids and bases.

    Science.gov (United States)

    Slater, Anthony Michael

    2014-10-01

    Accurate and well-curated experimental pKa data of organic acids and bases in both aqueous and non-aqueous media are invaluable in many areas of chemical research, including pharmaceutical, agrochemical, specialty chemical and property prediction research. In pharmaceutical research, pKa data are relevant in ligand design, protein binding, absorption, distribution, metabolism, elimination as well as solubility and dissolution rate. The pKa data compilations of the International Union of Pure and Applied Chemistry, originally in book form, have been carefully converted into computer-readable form, with value being added in the process, in the form of ionisation assignments and tautomer enumeration. These compilations offer a broad range of chemistry in both aqueous and non-aqueous media and the experimental conditions and original reference for all pKa determinations are supplied. The statistics for these compilations are presented and the utility of the computer-readable form of these compilations is examined in comparison to other pKa compilations. Finally, information is provided about how to access these databases.

  19. Correlations and predictions of carboxylic acid pKa values using intermolecular structure and properties of hydrogen-bonded complexes.

    Science.gov (United States)

    Tao, Li; Han, Jun; Tao, Fu-Ming

    2008-01-31

    Density functional theory calculations have been preformed on a series of hydrogen-bonded complexes of substituted aliphatic and aromatic carboxylic acids with ammonia. Molecular properties, particularly those related to hydrogen bonding, have been carefully examined for their interdependence as well as dependence on the acidity of the acid. The bond length and stretching frequency of the hydroxyl group and the hydrogen-bond length and energy of the complex are shown to be highly correlated with each other and are linearly correlated with available literature pKa values of the carboxylic acids. The linear correlations resulting from the fit to the available pKa values can be used to predict the pKa values of similar carboxylic acids. The pKa values so predicted using the different molecular properties are highly consistent and in good agreement with the literature values. This study suggests that calculated molecular properties of hydrogen-bonded complexes allow effective and systematic prediction of pKa values for a large range of organic acids using the established linear correlations. This approach is unique in its capability to determine the acidity of a particular functional group or the local acidity within a large molecular system such as a protein.

  20. Accurate pKa calculation of the conjugate acids of alkanolamines, alkaloids and nucleotide bases by quantum chemical methods.

    Science.gov (United States)

    Gangarapu, Satesh; Marcelis, Antonius T M; Zuilhof, Han

    2013-04-02

    The pKa of the conjugate acids of alkanolamines, neurotransmitters, alkaloid drugs and nucleotide bases are calculated with density functional methods (B3LYP, M08-HX and M11-L) and ab initio methods (SCS-MP2, G3). Implicit solvent effects are included with a conductor-like polarizable continuum model (CPCM) and universal solvation models (SMD, SM8). G3, SCS-MP2 and M11-L methods coupled with SMD and SM8 solvation models perform well for alkanolamines with mean unsigned errors below 0.20 pKa units, in all cases. Extending this method to the pKa calculation of 35 nitrogen-containing compounds spanning 12 pKa units showed an excellent correlation between experimental and computational pKa values of these 35 amines with the computationally low-cost SM8/M11-L density functional approach. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Preanalytical requirements for flow cytometric evaluation of platelet activation: choice of anticoagulant.

    Science.gov (United States)

    Mody, M; Lazarus, A H; Semple, J W; Freedman, J

    1999-06-01

    Accurate assessment of in vivo or in vitro platelet activation requires optimal preanalytical conditions to prevent artefactual in vitro activation of the platelets. The choice of anticoagulant is one of the critical preanalytical conditions as anticoagulants exert different effects on the activation of platelets ex vivo. We tested the effectiveness of Diatube-H (also known as CTAD; sodium citrate, theophylline, adenosine and dipyridamole) and citrate vacutainer tubes in preventing artefactual activation of platelets and preserving functional reserve. Platelet surface expression of the CD62P (reflecting alpha granule release), CD63 (reflecting lysosomal release) and modulation of normal platelet membrane glycoproteins CD41a and CD42b, were measured in whole blood and in isolated platelets immediately after collection and at 6, 24 and 48 h after venipuncture. Samples taken into Diatube-H showed less spontaneous platelet activation than did those taken into citrate. To measure in vitro platelet functional reserve, thrombin was added as agonist to blood stored for varying periods up to 48 h. Although Diatube-H suppressed in vitro platelet activation for up to 4 h, in samples kept for 6-24 h before thrombin addition, the inhibitory effect was lost and platelets responded fully to agonist activation. Hence, Diatube-H preserved platelets and allowed for measurement of in vivo platelet activation as well as thrombin-induced in vitro platelet activation after 6-24 h, in both whole blood and isolated platelets.

  2. Gliadins induce TNFalpha production through cAMP-dependent protein kinase A activation in intestinal cells (Caco-2).

    Science.gov (United States)

    Laparra Llopis, José Moisés; Sanz Herranz, Yolanda

    2010-06-01

    Celiac disease is an autoimmune enteropathy caused by a permanent intolerance to gliadins. In this study the effects of two gliadin-derived peptides (PA2, PQPQLPYPQPQLP and PA9, QLQPFPQPQLPY) on TNFalpha production by intestinal epithelial cells (Caco-2) and whether these effects were related to protein kinase A (PKA) and/or -C (PKC) activities have been evaluated. Caco-2 cell cultures were challenged with several sets of gliadin peptides solutions (0.25 mg/mL), with/without different activators of PKA or PKC, bradykinin (Brdkn) and pyrrolidine dithiocarbamate (PDTC). The gliadin-derived peptides assayed represent the two major immunodominant epitopes of the peptide 33-mer of alpha-gliadin (56-88) (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF). Both peptides induced the TNFalpha production triggering the inflammatory cell responses, the PA2 being more effective. The addition of the peptides in the presence of dibutyril cyclic AMP (cAMP), Brdkn or PDTC, inhibited the TNFalpha production. The PKC-activator phorbol 12-myristate 13-diacetate additionally increased the PA2- and PA9-induced TNFalpha production. These results link the gliadin-derived peptides induced TNFalpha production through cAMP-dependent PKA activation, where ion channels controlling calcium influx into cells could play a protective role, and requires NF-kappaB activation.

  3. Technical note: A procedure to estimate glucose requirements of an activated immune system in steers.

    Science.gov (United States)

    Kvidera, S K; Horst, E A; Abuajamieh, M; Mayorga, E J; Sanz Fernandez, M V; Baumgard, L H

    2016-11-01

    Infection and inflammation impede efficient animal productivity. The activated immune system ostensibly requires large amounts of energy and nutrients otherwise destined for synthesis of agriculturally relevant products. Accurately determining the immune system's in vivo energy needs is difficult, but a better understanding may facilitate developing nutritional strategies to maximize productivity. The study objective was to estimate immune system glucose requirements following an i.v. lipopolysaccharide (LPS) challenge. Holstein steers (148 ± 9 kg; = 15) were jugular catheterized bilaterally and assigned to 1 of 3 i.v.

  4. Physiological crosstalk between the AC/PKA and PLC/PKC pathways modulates melatonin-mediated, monochromatic-light-induced proliferation of T-lymphocytes in chickens.

    Science.gov (United States)

    Guo, Qingyun; Wang, Zixu; Dong, Yulan; Cao, Jing; Chen, Yaoxing

    2017-06-28

    Previous study has demonstrated that melatonin plays a critical role in monochromatic-light-induced lymphocyte proliferation in response to T cell mitogen concanavalin A (ConA). However, its intracellular mechanism is still unclear. In this study, we investigate the intracellular signal pathways of melatonin receptor-mediated T-lymphocyte proliferation in the spleens of chicks exposed to different light wavelengths. Results showed that green light enhanced T-lymphocyte proliferation by 2.46-6.83% and increased splenic mRNA and protein expressions of melatonin receptor subtypes (Mel1a, Mel1b and Mel1c) by 16.05-40.43% compared with the white, red and blue light groups. However, pinealectomy resulted in a decrease in T-lymphocyte proliferation and melatonin receptor expression with no statistically significant differences between the different light groups. In vitro experiments showed that the Mel1b selective antagonist 4P-PDOT, the Mel1c selective antagonist prazosin and the mitogen-activated protein kinase kinase-1 (MEK-1) inhibitor PD98059 suppressed both melatonin-induced lymphocyte proliferation in response to ConA and melatonin- and ConA-stimulated extracellular signal-regulated kinase 1/2 (ERK1/2) activity but that the Mel1a/Mel1b non-selective antagonist luzindole did not. In addition, pretreatment with forskolin (FSK, the adenylyl cyclase activator), H89 (the PKA inhibitor), U73122 (the PLC inhibitor) or Go6983 (the broad spectrum PKC inhibitor) markedly attenuated melatonin- and ConA-stimulated T-lymphocyte proliferation and ERK1/2 activity. These results demonstrate that melatonin mediates green-light-induced T-lymphocyte proliferation via the Mel1b and Mel1c receptors by triggering crosstalk between the cAMP/PKA and PLC/PKC signal pathways followed by ERK1/2 activation.

  5. Specific Sirt1 Activator-mediated Improvement in Glucose Homeostasis Requires Sirt1-Independent Activation of AMPK

    Directory of Open Access Journals (Sweden)

    Sung-Jun Park

    2017-04-01

    Full Text Available The specific Sirt1 activator SRT1720 increases mitochondrial function in skeletal muscle, presumably by activating Sirt1. However, Sirt1 gain of function does not increase mitochondrial function, which raises a question about the central role of Sirt1 in SRT1720 action. Moreover, it is believed that the metabolic effects of SRT1720 occur independently of AMP-activated protein kinase (AMPK, an important metabolic regulator that increases mitochondrial function. Here, we show that SRT1720 activates AMPK in a Sirt1-independent manner and SRT1720 activates AMPK by inhibiting a cAMP degrading phosphodiesterase (PDE in a competitive manner. Inhibiting the cAMP effector protein Epac prevents SRT1720 from activating AMPK or Sirt1 in myotubes. Moreover, SRT1720 does not increase mitochondrial function or improve glucose tolerance in AMPKα2 knockout mice. Interestingly, weight loss induced by SRT1720 is not sufficient to improve glucose tolerance. Therefore, contrary to current belief, the metabolic effects produced by SRT1720 require AMPK, which can be activated independently of Sirt1.

  6. Specific Sirt1 Activator-mediated Improvement in Glucose Homeostasis Requires Sirt1-Independent Activation of AMPK.

    Science.gov (United States)

    Park, Sung-Jun; Ahmad, Faiyaz; Um, Jee-Hyun; Brown, Alexandra L; Xu, Xihui; Kang, Hyeog; Ke, Hengming; Feng, Xuesong; Ryall, James; Philp, Andrew; Schenk, Simon; Kim, Myung K; Sartorelli, Vittorio; Chung, Jay H

    2017-04-01

    The specific Sirt1 activator SRT1720 increases mitochondrial function in skeletal muscle, presumably by activating Sirt1. However, Sirt1 gain of function does not increase mitochondrial function, which raises a question about the central role of Sirt1 in SRT1720 action. Moreover, it is believed that the metabolic effects of SRT1720 occur independently of AMP-activated protein kinase (AMPK), an important metabolic regulator that increases mitochondrial function. Here, we show that SRT1720 activates AMPK in a Sirt1-independent manner and SRT1720 activates AMPK by inhibiting a cAMP degrading phosphodiesterase (PDE) in a competitive manner. Inhibiting the cAMP effector protein Epac prevents SRT1720 from activating AMPK or Sirt1 in myotubes. Moreover, SRT1720 does not increase mitochondrial function or improve glucose tolerance in AMPKα2 knockout mice. Interestingly, weight loss induced by SRT1720 is not sufficient to improve glucose tolerance. Therefore, contrary to current belief, the metabolic effects produced by SRT1720 require AMPK, which can be activated independently of Sirt1. Published by Elsevier B.V.

  7. Structure of D-AKAP2:PKA RI Complex: Insights into AKAP Specificity and Selectivity

    Energy Technology Data Exchange (ETDEWEB)

    Sarma, Ganapathy N.; Kinderman, Francis S.; Kim, Choel; von Daake, Sventja; Chen, Lirong; Wang, Bi-Cheng; Taylor, Susan S. (UCSD); (Georgia)

    2010-11-22

    A-kinase anchoring proteins (AKAPs) regulate cyclic AMP-dependent protein kinase (PKA) signaling in space and time. Dual-specific AKAP 2 (D-AKAP2) binds to the dimerization/docking (D/D) domain of both RI and RII regulatory subunits of PKA with high affinity. Here we have determined the structures of the RI{alpha} D/D domain alone and in complex with D-AKAP2. The D/D domain presents an extensive surface for binding through a well-formed N-terminal helix, and this surface restricts the diversity of AKAPs that can interact. The structures also underscore the importance of a redox-sensitive disulfide in affecting AKAP binding. An unexpected shift in the helical register of D-AKAP2 compared to the RII{alpha}:D-AKAP2 complex structure makes the mode of binding to RI{alpha} novel. Finally, the comparison allows us to deduce a molecular explanation for the sequence and spatial determinants of AKAP specificity.

  8. Involvement of PI3K and PKA pathways in mouse tongue epithelial differentiation.

    Science.gov (United States)

    Jung, Jae-Kwang; Jung, Hye-In; Neupane, Sanjiv; Kim, Ki-Rim; Kim, Ji-Youn; Yamamoto, Hitoshi; Cho, Sung-Won; Lee, Youngkyun; Shin, Hong-In; Sohn, Wern-Joo; Kim, Jae-Young

    2017-01-01

    In mice, tongue epithelial differentiation is mainly regulated by the interactions among various signalling molecules including Fgf signalling pathways. However, the subsequent signalling modulations for epithelial maturation, initiated by Fgf signalling, remain to be elucidated. Therefore, we employed an in vitro tongue organ cultivation system along with the applications of various pharmacological inhibitors against the intracellular signalling molecules of Fgf signalling pathways, including H89, LY294002, PD98059, and U0126. Following treatments with LY294002 and H89, inhibitors for PI3K and PKA, respectively, the decreased thickness of the tongue epithelium was observed along with the alteration in cell proliferative and apoptotic patterns. Meanwhile, cultivated tongues treated with MEK inhibitor U0126 or PD98059 showed significantly decreased cell proliferation in the tongue epithelium and the mesenchyme. Based on these results, we suggest that the tongue epithelium is differentiated into multiple epithelial cell layers via the PI3K and PKA pathways in tissue-specific manner during the epithelial-mesenchymal interactions.

  9. Influence of subcascade formation on displacement damage at high PKA energies

    Energy Technology Data Exchange (ETDEWEB)

    Stoller, R.E. [Oak Ridge National Lab., TN (United States); Greenwood, L.R. [Pacific Northwest National Lab., Richland, WA (United States)

    1997-08-01

    The design of first generation fusion reactors will have to be rely on radiation effects data obtained from experiments conducted in fission reactors. Two issues must be addressed to use this data with confidence. The first is differences in the neutron energy spectrum, and the second is differences in nuclear transmutation rates. Differences in the neutron energy spectra are reflected in the energy spectra of the primary knockon atoms (PKA). The issue of PKA energy effects has been addressed through the use of displacement cascade simulations using the method of molecular dynamics (MD). Although MD simulations can provide a detailed picture of the formation and evolution of displacement cascades, they impose a substantial computational burden. However, recent advances in computing equipment permit the simulation of high energy displacement events involving more than one-million atoms; the results presented here encompass MD cascade simulation energies from near the displacement threshold to as high as 40 keV. Two parameters have been extracted from the MD simulations: the number of point defects that remain after the displacement event is completed and the fraction of the surviving interstitials that are contained in clusters. The MD values have been normalized to the number of atomic displacements calculated with the secondary displacement model by Norgett, Robinson, and Torrens (NRT).

  10. 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside confers neuroprotection in cell and animal models of ischemic stroke through calpain1/PKA/CREB-mediated induction of neuronal glucose transporter 3

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Shu; Cheng, Qiong; Li, Lu; Liu, Mei; Yang, Yumin; Ding, Fei, E-mail: dingfei@ntu.edu.cn

    2014-06-15

    Salidroside is proven to be a neuroprotective agent of natural origin, and its analog, 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside (named SalA-4 g), has been synthesized in our lab. In this study, we showed that SalA-4 g promoted neuronal survival and inhibited neuronal apoptosis in primary hippocampal neurons exposed to oxygen and glucose deprivation (OGD) and in rats subjected to ischemia by transient middle cerebral artery occlusion (MCAO), respectively, and that SalA-4 g was more neuroprotective than salidroside. We further found that SalA-4 g elevated glucose uptake in OGD-injured primary hippocampal neurons and increased the expression and recruitment of glucose transporter 3 (GLUT3) in ischemic brain. Signaling analysis revealed that SalA-4 g triggered the phosphorylation of CREB, and increased the expression of PKA RII in primary hippocampal neurons exposed to OGD injury, while inhibition of PKA/CREB by H-89 alleviated the elevation in glucose uptake and GLUT3 expression, and blocked the protective effects of SalA-4 g. Moreover, SalA-4 g was noted to inhibit intracellular Ca{sup 2+} influx and calpain1 activation in OGD-injured primary hippocampal neurons. Our results suggest that SalA-4 g neuroprotection might be mediated by increased glucose uptake and elevated GLUT3 expression through calpain1/PKA/CREB pathway. - Highlights: • A salidroside (Sal) analog (SalA-4 g) is prepared to be more neuroprotective than Sal. • SalA-4 g protected hippocampal neurons from oxygen and glucose deprivation insult. • SalA-4 g reduced ischemic injury after transient middle cerebral artery occlusion in rats. • Neuroprotection of SalA-4 g was mediated by GLUT3 level via calpain/PKA/CREB pathway.

  11. Effect of LIF-withdrawal on acetylcholine synthesis in the embryonic stem cell line CGR8 is not mediated by STAT3, PI3Ks or cAMP/PKA pathways.

    Science.gov (United States)

    Michel-Schmidt, Rosmarie; Kirkpatrick, Charles James; Wessler, Ignaz

    2015-11-01

    Acetylcholine (ACh) acts as a local cellular signaling molecule and is widely expressed in nature, including mammalian cells and embryonic stem cells. The murine embryonic stem cell line CGR8 synthesizes and releases substantial amounts of ACh. Particularly during early differentiation - a period associated with multiple alterations in geno-/phenotype functions - synthesis and release of ACh are increased by 10-fold. In murine stem cells second messengers of the STAT-3, PI3K and cAMP/PKA pathways are involved in maintaining self-renewal and pluripotency. The present experiments were designed to test whether blockers of these signaling pathways enhance ACh cell content in the presence of LIF, i.e. when CGR8 is pluripotent. NSC74859, an inhibitor of STAT-3, affected neither the proliferation rate nor ACh cell content, whereas the more sensitive STAT-3 inhibitor FLLL31 reduced the proliferation rate and increased ACh cell content by about 3-fold. The PI3K inhibitor LY294002 reduced the proliferation rate but did not modify the ACh cell content, whereas the PKA inhibitor H89 produced effects comparable to FLLL31. Interestingly, in control experiments a strong inverse correlation was found between cell density and ACh cell content, which could explain the 3-fold increase in the ACh cell content observed in the presence of FLLL31 and H89. Forskolin, a PKA activator, had no effect. In conclusion, it appears unlikely that the 10-fold increase in ACh cell content induced by LIF removal, i.e. during early differentiation, is mediated by second messengers of the STAT-3, PI3K and cAMP/PKA pathways. However, the PI3K pathway appears to be involved in control of the inverse relation between cell density and ACh cell content, because this correlation was significantly attenuated in the presence of LY294002.

  12. Monoubiquitination and activity of the paracaspase MALT1 requires glutamate 549 in the dimerization interface.

    Directory of Open Access Journals (Sweden)

    Katrin Cabalzar

    Full Text Available The mucosa-associated lymphoid tissue protein-1 (MALT1, also known as paracaspase is a protease whose activity is essential for the activation of lymphocytes and the growth of cells derived from human diffuse large B-cell lymphomas of the activated B-cell subtype (ABC DLBCL. Crystallographic approaches have shown that MALT1 can form dimers via its protease domain, but why dimerization is relevant for the biological activity of MALT1 remains largely unknown. Using a molecular modeling approach, we predicted Glu 549 (E549 to be localized within the MALT1 dimer interface and thus potentially relevant. Experimental mutation of this residue into alanine (E549A led to a complete impairment of MALT1 proteolytic activity. This correlated with an impaired capacity of the mutant to form dimers of the protease domain in vitro, and a reduced capacity to promote NF-κB activation and transcription of the growth-promoting cytokine interleukin-2 in antigen receptor-stimulated lymphocytes. Moreover, this mutant could not rescue the growth of ABC DLBCL cell lines upon MALT1 silencing. Interestingly, the MALT1 mutant E549A was unable to undergo monoubiquitination, which we identified previously as a critical step in MALT1 activation. Collectively, these findings suggest a model in which E549 at the dimerization interface is required for the formation of the enzymatically active, monoubiquitinated form of MALT1.

  13. Monoubiquitination and activity of the paracaspase MALT1 requires glutamate 549 in the dimerization interface.

    Science.gov (United States)

    Cabalzar, Katrin; Pelzer, Christiane; Wolf, Annette; Lenz, Georg; Iwaszkiewicz, Justyna; Zoete, Vincent; Hailfinger, Stephan; Thome, Margot

    2013-01-01

    The mucosa-associated lymphoid tissue protein-1 (MALT1, also known as paracaspase) is a protease whose activity is essential for the activation of lymphocytes and the growth of cells derived from human diffuse large B-cell lymphomas of the activated B-cell subtype (ABC DLBCL). Crystallographic approaches have shown that MALT1 can form dimers via its protease domain, but why dimerization is relevant for the biological activity of MALT1 remains largely unknown. Using a molecular modeling approach, we predicted Glu 549 (E549) to be localized within the MALT1 dimer interface and thus potentially relevant. Experimental mutation of this residue into alanine (E549A) led to a complete impairment of MALT1 proteolytic activity. This correlated with an impaired capacity of the mutant to form dimers of the protease domain in vitro, and a reduced capacity to promote NF-κB activation and transcription of the growth-promoting cytokine interleukin-2 in antigen receptor-stimulated lymphocytes. Moreover, this mutant could not rescue the growth of ABC DLBCL cell lines upon MALT1 silencing. Interestingly, the MALT1 mutant E549A was unable to undergo monoubiquitination, which we identified previously as a critical step in MALT1 activation. Collectively, these findings suggest a model in which E549 at the dimerization interface is required for the formation of the enzymatically active, monoubiquitinated form of MALT1.

  14. Requirement for caspase-8 in NF-kappaB activation by antigen receptor.

    Science.gov (United States)

    Su, Helen; Bidère, Nicolas; Zheng, Lixin; Cubre, Alan; Sakai, Keiko; Dale, Janet; Salmena, Leonardo; Hakem, Razqallah; Straus, Stephen; Lenardo, Michael

    2005-03-04

    Caspase-8, a proapoptotic protease, has an essential role in lymphocyte activation and protective immunity. We show that caspase-8 deficiency (CED) in humans and mice specifically abolishes activation of the transcription factor nuclear factor kappaB (NF-kappaB) after stimulation through antigen receptors, Fc receptors, or Toll-like receptor 4 in T, B, and natural killer cells. Caspase-8 also causes the alphabeta complex of the inhibitor of NF-kappaB kinase (IKK) to associate with the upstream Bcl10-MALT1 (mucosa-associated lymphatic tissue) adapter complex. Recruitment of the IKKalpha, beta complex, its activation, and the nuclear translocation of NF-kappaB require enzyme activity of full-length caspase-8. These findings thus explain the paradoxical association of defective apoptosis and combined immunodeficiency in human CED.

  15. Theoretical pKa prediction of the α-phosphate moiety of uridine 5‧-diphosphate-GlcNAc

    Science.gov (United States)

    Vipperla, Bhavaniprasad; Griffiths, Thomas M.; Wang, Xingyong; Yu, Haibo

    2017-01-01

    The pKa value of the α-phosphate moiety of uridine 5‧-diphosphate-GlcNAc (UDP-GlcNAc) has been successfully calculated using density functional theory methods in conjunction with the Polarizable Continuum Models. Theoretical methods were benchmarked over a dataset comprising of alkyl phosphates. B3LYP/6-31+G(d,p) calculations using SMD solvation model provide excellent agreement with the experimental data. The predicted pKa for UDP-GlcNAc is consistent with most recent NMR studies but much higher than what it has long been thought to be. The importance of this study is evident that the predicted pKa for UDP-GlcNAc supports its potential role as a catalytic base in the substrate-assisted biocatalysis.

  16. Sonic hedgehog-induced histone deacetylase activation is required for cerebellar granule precursor hyperplasia in medulloblastoma.

    Directory of Open Access Journals (Sweden)

    Seung Joon Lee

    Full Text Available Medulloblastoma, the most common pediatric brain tumor, is thought to arise from deregulated proliferation of cerebellar granule precursor (CGP cells. Sonic hedgehog (Shh is the primary mitogen that regulates proliferation of CGP cells during the early stages of postnatal cerebellum development. Aberrant activation of Shh signaling during this time has been associated with hyperplasia of CGP cells and eventually may lead to the development of medulloblastoma. The molecular targets of Shh signaling involved in medulloblastoma formation are still not well-understood. Here, we show that Shh regulates sustained activation of histone deacetylases (HDACs and that this activity is required for continued proliferation of CGP cells. Suppression of HDAC activity not only blocked the Shh-induced CGP proliferation in primary cell cultures, but also ameliorated aberrant CGP proliferation at the external germinal layer (EGL in a medulloblastoma mouse model. Increased levels of mRNA and protein of several HDAC family members were found in medulloblastoma compared to wild type cerebellum suggesting that HDAC activity is required for the survival/progression of tumor cells. The identification of a role of HDACs in the early steps of medulloblastoma formation suggests there may be a therapeutic potential for HDAC inhibitors in this disease.

  17. Balanced Activity of Three Mitotic Motors Is Required for Bipolar Spindle Assembly and Chromosome Segregation

    Directory of Open Access Journals (Sweden)

    Roy G.H.P. van Heesbeen

    2014-08-01

    Full Text Available Bipolar spindle assembly requires force to organize the microtubule network. Here, we show that three motor proteins, namely Eg5, Kif15, and dynein, act together to produce the right force balance in the spindle. Excessive inward force results in monopolar spindle formation, while excessive outward force generation results in unstable spindles with splayed spindle poles. Blocking activity of all three motors prevents bipolar spindle formation, but established bipolar spindles are refractory to loss of all motor activity. Further analysis shows that although these preformed spindles remain bipolar, outward force generation is required to establish sufficient tension on kinetochores and to accomplish successful chromosome segregation. Together, these results show how Eg5, Kif15, and dynein work together to build a bipolar spindle and reveal an important role for antagonistic motors in chromosome segregation.

  18. How Ca2+-permeable AMPA receptors, the kinase PKA, and the phosphatase PP2B are intertwined in synaptic LTP and LTD.

    Science.gov (United States)

    Hell, Johannes W

    2016-04-26

    Both synaptic long-term potentiation (LTP) and long-term depression (LTD) are thought to be critical for memory formation. Dell'Acqua and co-workers now demonstrate that transient postsynaptic incorporation of Ca(2+)-permeable (CP) α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is required for LTD in the exemplary hippocampal CA1 region in 2-week-old mice. Mechanistically, LTD depends on AKAP150-anchored protein kinase A (PKA) to promote the initial functional recruitment of CP-AMPARs during LTD induction and on AKAP150-anchored protein phosphatase 2B (PP2B) to trigger their subsequent removal as part of the lasting depression of synaptic transmission.

  19. Requirement of succinate dehydrogenase activity for symbiotic bacteroid differentiation of Rhizobium meliloti in alfalfa nodules.

    OpenAIRE

    Gardiol, A E; Truchet, G L; Dazzo, F. B.

    1987-01-01

    Transmission electron microscopy was used to study the cellular morphologies of a wild-type Rhizobium meliloti strain (L5-30), a nitrogen fixation-ineffective (Fix-) succinate dehydrogenase mutant (Sdh-) strain, and a Fix+ Sdh+ revertant strain within alfalfa nodules and after free-living growth in a minimal medium containing 27 mM mannitol plus 20 mM succinate. The results showed a requirement of succinate dehydrogenase activity for symbiotic differentiation and maintenance of R. meliloti ba...

  20. Effects of Requiring Physical Fitness in a Lecture-Based College Course: Students' Attitudes toward Physical Activity

    Science.gov (United States)

    Esslinger, Keri A.; Grimes, Amanda R.; Pyle, Elizabeth

    2016-01-01

    In this study, we investigated students' attitudes toward physical activity (PA) when including a required PA component in a university-required personal wellness class. The study included (a) an experimental group of students enrolled in a personal wellness course in which there was a required PA requirement and (b) a control group of students…

  1. Decision Support System Requirements Definition for Human Extravehicular Activity Based on Cognitive Work Analysis.

    Science.gov (United States)

    Miller, Matthew James; McGuire, Kerry M; Feigh, Karen M

    2017-06-01

    The design and adoption of decision support systems within complex work domains is a challenge for cognitive systems engineering (CSE) practitioners, particularly at the onset of project development. This article presents an example of applying CSE techniques to derive design requirements compatible with traditional systems engineering to guide decision support system development. Specifically, it demonstrates the requirements derivation process based on cognitive work analysis for a subset of human spaceflight operations known as extravehicular activity. The results are presented in two phases. First, a work domain analysis revealed a comprehensive set of work functions and constraints that exist in the extravehicular activity work domain. Second, a control task analysis was performed on a subset of the work functions identified by the work domain analysis to articulate the translation of subject matter states of knowledge to high-level decision support system requirements. This work emphasizes an incremental requirements specification process as a critical component of CSE analyses to better situate CSE perspectives within the early phases of traditional systems engineering design.

  2. The Chromatin Remodeler CHD8 Is Required for Activation of Progesterone Receptor-Dependent Enhancers

    Science.gov (United States)

    Giannopoulou, Eugenia G.; Soronellas, Daniel; Vázquez-Chávez, Elena; Vicent, Guillermo P.; Elemento, Olivier; Beato, Miguel; Reyes, José C.

    2015-01-01

    While the importance of gene enhancers in transcriptional regulation is well established, the mechanisms and the protein factors that determine enhancers activity have only recently begun to be unravelled. Recent studies have shown that progesterone receptor (PR) binds regions that display typical features of gene enhancers. Here, we show by ChIP-seq experiments that the chromatin remodeler CHD8 mostly binds promoters under proliferation conditions. However, upon progestin stimulation, CHD8 re-localizes to PR enhancers also enriched in p300 and H3K4me1. Consistently, CHD8 depletion severely impairs progestin-dependent gene regulation. CHD8 binding is PR-dependent but independent of the pioneering factor FOXA1. The SWI/SNF chromatin-remodelling complex is required for PR-dependent gene activation. Interestingly, we show that CHD8 interacts with the SWI/SNF complex and that depletion of BRG1 and BRM, the ATPases of SWI/SNF complex, impairs CHD8 recruitment. We also show that CHD8 is not required for H3K27 acetylation, but contributes to increase accessibility of the enhancer to DNaseI. Furthermore, CHD8 was required for RNAPII recruiting to the enhancers and for transcription of enhancer-derived RNAs (eRNAs). Taken together our data demonstrate that CHD8 is involved in late stages of PR enhancers activation. PMID:25894978

  3. The chromatin Remodeler CHD8 is required for activation of progesterone receptor-dependent enhancers.

    Science.gov (United States)

    Ceballos-Chávez, María; Subtil-Rodríguez, Alicia; Giannopoulou, Eugenia G; Soronellas, Daniel; Vázquez-Chávez, Elena; Vicent, Guillermo P; Elemento, Olivier; Beato, Miguel; Reyes, José C

    2015-04-01

    While the importance of gene enhancers in transcriptional regulation is well established, the mechanisms and the protein factors that determine enhancers activity have only recently begun to be unravelled. Recent studies have shown that progesterone receptor (PR) binds regions that display typical features of gene enhancers. Here, we show by ChIP-seq experiments that the chromatin remodeler CHD8 mostly binds promoters under proliferation conditions. However, upon progestin stimulation, CHD8 re-localizes to PR enhancers also enriched in p300 and H3K4me1. Consistently, CHD8 depletion severely impairs progestin-dependent gene regulation. CHD8 binding is PR-dependent but independent of the pioneering factor FOXA1. The SWI/SNF chromatin-remodelling complex is required for PR-dependent gene activation. Interestingly, we show that CHD8 interacts with the SWI/SNF complex and that depletion of BRG1 and BRM, the ATPases of SWI/SNF complex, impairs CHD8 recruitment. We also show that CHD8 is not required for H3K27 acetylation, but contributes to increase accessibility of the enhancer to DNaseI. Furthermore, CHD8 was required for RNAPII recruiting to the enhancers and for transcription of enhancer-derived RNAs (eRNAs). Taken together our data demonstrate that CHD8 is involved in late stages of PR enhancers activation.

  4. Activation of Notch in lgd mutant cells requires the fusion of late endosomes with the lysosome.

    Science.gov (United States)

    Schneider, Markus; Troost, Tobias; Grawe, Ferdi; Martinez-Arias, Alfonso; Klein, Thomas

    2013-01-15

    The tumour suppressor Lethal (2) giant discs (Lgd) is a regulator of endosomal trafficking of the Notch signalling receptor as well as other transmembrane proteins in Drosophila. The loss of its function results in an uncontrolled ligand-independent activation of the Notch signalling receptor. Here, we investigated the consequences of loss of lgd function and the requirements for the activation of Notch. We show that the activation of Notch in lgd cells is independent of Kuz and dependent on γ-secretase. We found that the lgd cells have a defect that delays degradation of transmembrane proteins, which are residents of the plasma membrane. Furthermore, our results show that the activation of Notch in lgd cells occurs in the lysosome. By contrast, the pathway is activated at an earlier phase in mutants of the gene that encodes the ESCRT-III component Shrub, which is an interaction partner of Lgd. We further show that activation of Notch appears to be a general consequence of loss of lgd function. In addition, electron microscopy of lgd cells revealed that they contain enlarged multi-vesicular bodies. The presented results further elucidate the mechanism of uncontrolled Notch activation upon derailed endocytosis.

  5. Temporal and Spatial Requirement of EMF1 Activity for Arabidopsis Vegetative and Reproductive Development

    Institute of Scientific and Technical Information of China (English)

    Rosario Sánchez; Minjung Y.Kim; Myriam Calonje; Yong-Hwan Moon; Z.Renee Sung

    2009-01-01

    EMBRYONIC FLOWER (EMF) genes are required to maintain vegetative development via repression of flower homeotic genes in Arabidopsis.Removal of EMF gene function caused plants to flower upon germination,producing abnormal and sterile flowers.The pleiotropic effect of emf1 mutation suggests its requirement for gene programs involved in diverse developmental processes.Transgenic plants harboring EMF1 promoter::glucuronidase (GUS) reporter gene were generated to investigate the temporal and spatial expression pattern of EMF1.These plants displayed differential GUS activity in vegetative and flower tissues,consistent with the role of EMF1 in regulating multiple gene programs.EMF1::GUS expression pattern in emf mutants suggests organ-specific auto-regulation.Sense- and antisense (as) EMF1 cDNA were expressed under the control of stage- and tissue-specific promoters in transgenic plants.Characterization of these transgenic plants showed that EMF1 activity is required in meristematic as well as differentiating tissues to rescue emf mutant phenotype.Temporal removal or reduction of EMF1 activity in the embryo or shoot apex of wild-type seedlings was sufficient to cause early flowering and terminal flower formation in adult plants.Such reproductive cell memory is reflected in the flower MADS-box gene activity expressed prior to flowering in these early flowering plants.However,temporal removal of EMF1 activity in flower meristem did not affect flower development.Our results are consistent with EMF1's primary role in repressing flowering in order to allow for vegetative growth.

  6. Copper is required for cobalt-induced transcriptional activity of hypoxia-inducible factor-1.

    Science.gov (United States)

    Qiu, Liying; Ding, Xueqin; Zhang, Zhen; Kang, Y James

    2012-08-01

    Cobalt inhibits prolyl hydroxylases, leading to the accumulation of hypoxia-inducible factor-1α (HIF-1α) and a concomitant increase in the transcriptional activity of HIF-1. Therefore, cobalt has been under development as a drug for activating HIF-1 under some disease conditions. However, it has been shown that ischemic conditions resulted in the loss of copper, and the activation of HIF-1 would not occur unless copper was supplemented. The present study was undertaken to test the hypothesis that copper is also required for the cobalt activation of HIF-1 transcriptional activity. Human umbilical vein endothelial cells subjected to treatment with cobalt chloride (CoCl(2)) at concentrations above 25 μM for 2 h resulted in an accumulation of HIF-1α, which was determined by Western blot analysis, and an increase in the expression of vascular endothelial growth factor (VEGF), which was determined by real-time reverse transcription-polymerase chain reaction analysis for mRNA levels and enzyme-linked immunosorbent assay analysis for protein levels. The copper chelator tetraethylenepentamine at 25 μM did not significantly affect the accumulation of HIF-1α but blocked increases in VEGF mRNA and protein levels, an effect that could be reversed by the addition of 25 μM copper sulfate (CuSO(4)). In addition, gene silencing of the copper chaperone for Cu,Zn-superoxide dismutase blocked VEGF expression with little effect on cobalt-induced HIF-1α accumulation. The present study thus demonstrates that copper was required for cobalt-activated transcriptional activity of HIF-1, although copper did not affect cobalt-induced accumulation of HIF-1α in the cells.

  7. HDAC1 activates FoxO and is both sufficient and required for skeletal muscle atrophy

    Science.gov (United States)

    Beharry, Adam W.; Sandesara, Pooja B.; Roberts, Brandon M.; Ferreira, Leonardo F.; Senf, Sarah M.; Judge, Andrew R.

    2014-01-01

    ABSTRACT The Forkhead box O (FoxO) transcription factors are activated, and necessary for the muscle atrophy, in several pathophysiological conditions, including muscle disuse and cancer cachexia. However, the mechanisms that lead to FoxO activation are not well defined. Recent data from our laboratory and others indicate that the activity of FoxO is repressed under basal conditions via reversible lysine acetylation, which becomes compromised during catabolic conditions. Therefore, we aimed to determine how histone deacetylase (HDAC) proteins contribute to activation of FoxO and induction of the muscle atrophy program. Through the use of various pharmacological inhibitors to block HDAC activity, we demonstrate that class I HDACs are key regulators of FoxO and the muscle-atrophy program during both nutrient deprivation and skeletal muscle disuse. Furthermore, we demonstrate, through the use of wild-type and dominant-negative HDAC1 expression plasmids, that HDAC1 is sufficient to activate FoxO and induce muscle fiber atrophy in vivo and is necessary for the atrophy of muscle fibers that is associated with muscle disuse. The ability of HDAC1 to cause muscle atrophy required its deacetylase activity and was linked to the induction of several atrophy genes by HDAC1, including atrogin-1, which required deacetylation of FoxO3a. Moreover, pharmacological inhibition of class I HDACs during muscle disuse, using MS-275, significantly attenuated both disuse muscle fiber atrophy and contractile dysfunction. Together, these data solidify the importance of class I HDACs in the muscle atrophy program and indicate that class I HDAC inhibitors are feasible countermeasures to impede muscle atrophy and weakness. PMID:24463822

  8. Determination of the minimal amount of Tat activity required for human immunodeficiency virus type 1 replication.

    Science.gov (United States)

    Verhoef, K; Koper, M; Berkhout, B

    1997-10-27

    The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent trans-activator of transcription from the viral LTR promoter. Previous mutagenesis studies have identified domains within Tat responsible for binding to its TAR RNA target and for transcriptional activation. The minimal Tat activation domain is composed of the N-terminal 48 residues, and mutational analyses identified a cluster of critical cysteines. The importance of four highly conserved aromatic amino acids within the activation domain has not been thoroughly investigated. We have systematically substituted these aromatic residues (Y26, F32, F38, Y47) of the HIV-1 LAI Tat protein with other aromatic residues (conservative mutation) or alanine (nonconservative mutation). The activity of the mutant Tat constructs was measured in different cell lines by transfection with a LTR-CAT reporter plasmid. The range of transcriptional activities measured for this set of Tat mutants allowed careful assessment of the level of Tat activity required for optimal viral replication. To test this, the mutant Tat genes were introduced into the pLAI infectious molecular clone and tested for their effect on virus replication in a T-cell line. We found that a twofold reduction in Tat activity already affects viral replication, and no virus replication was measured for Tat mutants with less than 15% activity. This strict correlation between Tat activity and viral replication demonstrates the importance of the Tat function to viral fitness. Interestingly, a less pronounced replication defect was observed in primary cell types. This finding may correlate with the frequent detection of proviruses with Tat-inactivating mutations in clinical samples. Copyright 1997 Academic Press.

  9. Elevated extracellular calcium increases fibroblast growth factor-2 gene and protein expression levels via a cAMP/PKA dependent pathway in cementoblasts.

    Science.gov (United States)

    Kanaya, Sousuke; Nemoto, Eiji; Ebe, Yukari; Somerman, Martha J; Shimauchi, Hidetoshi

    2010-09-01

    Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Elevated levels of extracellular Ca(2+) have been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of extracellular Ca(2+) signaling in cementogenesis has not been examined. Using RT-PCR, we found that elevated levels of extracellular Ca(2+) increase fibroblast growth factor (FGF)-2 gene expression with a peak at 6h. Pretreatment with a protein kinase A (PKA) inhibitor, H89, or an adenylate cyclase inhibitor, MDL-12,330A, inhibited Ca(2+)-stimulated Fgf-2 expression. In contrast, pretreatment with the protein kinase C (PKC) inhibitor GF-109203X or the phospholipase C (PLC) inhibitor U73122 did not affect the expression of Fgf-2 transcripts, suggesting that the increase in Fgf-2 expression was dependent on the PKA but not the PLC/PKC signaling pathway. Treatment with an activator of adenylate cyclase, forskolin, or a cell-permeable analog of cAMP, 8-Br-cAMP, enhanced Ca(2+)-stimulated Fgf-2 expression, but a single treatment with forskolin or 8-Br-cAMP did not, suggesting that cAMP generation is indispensable but not sufficient for Ca(2+)-stimulated FGF2 expression. Next, we examined the cation specificity of the putative receptor and showed that treatment with trivalent/divalent inorganic ions, Ca(2+), Gd(3+), Sr(2+), or Al(3+), caused a dose-dependent increase in Fgf-2 mRNA levels in a cAMP-dependent fashion, whereas Mg(2+) and the organic ions neomycin and spermine had no effect on Fgf-2 gene expression levels. These findings suggest that an extracellular Ca(2+)-sensing mechanism is present in cementoblasts and its activation leads to FGF-2 stimulation in a cAMP/PKA dependent fashion. Understanding the pathway regulating key genes involved in modulating the

  10. Active ADP-ribosylation Factor-1 (ARF1) Is Required for Mitotic Golgi Fragmentation*S

    Science.gov (United States)

    Xiang, Yi; Seemann, Joachim; Bisel, Blaine; Punthambaker, Sukanya; Wang, Yanzhuang

    2012-01-01

    In mammalian cells the Golgi apparatus undergoes an extensive disassembly process at the onset of mitosis that is believed to facilitate equal partitioning of this organelle into the two daughter cells. However, the underlying mechanisms for this fragmentation process are so far unclear. Here we have investigated the role of the ADP-ribosylation factor-1 (ARF1) in this process to determine whether Golgi fragmentation in mitosis is mediated by vesicle budding. ARF1 is a small GTPase that is required for COPI vesicle formation from the Golgi membranes. Treatment of Golgi membranes with mitotic cytosol or with purified coatomer together with wild type ARF1 or its constitutive active form, but not the inactive mutant, converted the Golgi membranes into COPI vesicles. ARF1-depleted mitotic cytosol failed to fragment Golgi membranes. ARF1 is associated with Golgi vesicles generated in vitro and with vesicles in mitotic cells. In addition, microinjection of constitutive active ARF1 did not affect mitotic Golgi fragmentation or cell progression through mitosis. Our results show that ARF1 is active during mitosis and that this activity is required for mitotic Golgi fragmentation. PMID:17562717

  11. Active ADP-ribosylation factor-1 (ARF1) is required for mitotic Golgi fragmentation.

    Science.gov (United States)

    Xiang, Yi; Seemann, Joachim; Bisel, Blaine; Punthambaker, Sukanya; Wang, Yanzhuang

    2007-07-27

    In mammalian cells the Golgi apparatus undergoes an extensive disassembly process at the onset of mitosis that is believed to facilitate equal partitioning of this organelle into the two daughter cells. However, the underlying mechanisms for this fragmentation process are so far unclear. Here we have investigated the role of the ADP-ribosylation factor-1 (ARF1) in this process to determine whether Golgi fragmentation in mitosis is mediated by vesicle budding. ARF1 is a small GTPase that is required for COPI vesicle formation from the Golgi membranes. Treatment of Golgi membranes with mitotic cytosol or with purified coatomer together with wild type ARF1 or its constitutive active form, but not the inactive mutant, converted the Golgi membranes into COPI vesicles. ARF1-depleted mitotic cytosol failed to fragment Golgi membranes. ARF1 is associated with Golgi vesicles generated in vitro and with vesicles in mitotic cells. In addition, microinjection of constitutive active ARF1 did not affect mitotic Golgi fragmentation or cell progression through mitosis. Our results show that ARF1 is active during mitosis and that this activity is required for mitotic Golgi fragmentation.

  12. Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases.

    Science.gov (United States)

    Meunier, Etienne; Dick, Mathias S; Dreier, Roland F; Schürmann, Nura; Kenzelmann Broz, Daniela; Warming, Søren; Roose-Girma, Merone; Bumann, Dirk; Kayagaki, Nobuhiko; Takeda, Kiyoshi; Yamamoto, Masahiro; Broz, Petr

    2014-05-15

    Lipopolysaccharide from Gram-negative bacteria is sensed in the host cell cytoplasm by a non-canonical inflammasome pathway that ultimately results in caspase-11 activation and cell death. In mouse macrophages, activation of this pathway requires the production of type-I interferons, indicating that interferon-induced genes have a critical role in initiating this pathway. Here we report that a cluster of small interferon-inducible GTPases, the so-called guanylate-binding proteins, is required for the full activity of the non-canonical caspase-11 inflammasome during infections with vacuolar Gram-negative bacteria. We show that guanylate-binding proteins are recruited to intracellular bacterial pathogens and are necessary to induce the lysis of the pathogen-containing vacuole. Lysis of the vacuole releases bacteria into the cytosol, thus allowing the detection of their lipopolysaccharide by a yet unknown lipopolysaccharide sensor. Moreover, recognition of the lysed vacuole by the danger sensor galectin-8 initiates the uptake of bacteria into autophagosomes, which results in a reduction of caspase-11 activation. These results indicate that host-mediated lysis of pathogen-containing vacuoles is an essential immune function and is necessary for efficient recognition of pathogens by inflammasome complexes in the cytosol.

  13. Therapeutic requirements in active ulcerative proctitis: A single-centre study.

    Science.gov (United States)

    Calafat, Margalida; Lobatón, Triana; Mañosa, Míriam; Marín, Laura; Caballero, Noemí; Larraín, Macarena; Cabré, Eduard; Domènech, Eugeni

    2017-06-27

    Ulcerative proctitis (UP) presents distinctive clinical characteristics, outcomes and therapeutic approaches as compared to left-sided and extensive ulcerative colitis (UC). To describe the current therapeutic requirements and clinical outcomes in patients with active UP. Retrospective observational study conducted in a referral IBD centre. Patients with UP in follow-up between 1989 and 2014 were included. The clinical characteristics, as well as the different treatments and drug formulations administered to treat flares, were recorded. Out of 687 UC patients, 101 patients (15%) with UP were included. Median follow-up was 8 years (IQR 3-14) and 49% of patients presented disease activity during the study period. Topical mesalazine monotherapy (90%) was the most commonly administered treatment for disease activity (mostly as suppositories), followed by topical steroids (47%) and oral mesalazine (56%) in monotherapy or combination therapy. Only 14% and 16% of patients required oral prednisone and beclomethasone, respectively. In clinical practice, active UP presents mostly favourable outcomes. Mesalazine suppositories are by far the most used treatment for these patients. Copyright © 2017 Elsevier España, S.L.U., AEEH y AEG. All rights reserved.

  14. Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint.

    Science.gov (United States)

    Wu, Juan; Huang, Yu-Fan; Zhou, Xin-Ke; Zhang, Wei; Lian, Yi-Fan; Lv, Xiao-Bin; Gao, Xiu-Rong; Lin, Hui-Kuan; Zeng, Yi-Xin; Huang, Jian-Qing

    2015-01-01

    The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3-ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.

  15. GlnB is specifically required for Azospirillum brasilense NifA activity in Escherichia coli.

    Science.gov (United States)

    Araújo, Luiza M; Monteiro, Rose A; Souza, Emanuel M; Steffens, M Berenice R; Rigo, Liu U; Pedrosa, Fábio O; Chubatsu, Leda S

    2004-01-01

    The Azospirillum brasilense transcription regulator NifA and the nitrogen-status signaling proteins GlnB, GlnZ and GlnK were expressed in Escherichia coli and analyzed for their ability to activate nif gene expression. When expressed separately, none of the proteins were able to activate nifH promoter expression in any tested conditions; in contrast, nifH expression was observed in cells grown in the absence of ammonium and oxygen and when expressing simultaneously NifA and GlnB proteins, but not when expressing NifA and GlnZ or GlnK. Our results show that the GlnB protein is required for transcription activation by Azospirillum brasilense NifA and it cannot be replaced by GlnZ or GlnK.

  16. Exposure to extremely low-frequency electromagnetic fields modulates Na+ currents in rat cerebellar granule cells through increase of AA/PGE2 and EP receptor-mediated cAMP/PKA pathway.

    Directory of Open Access Journals (Sweden)

    Yan-Lin He

    Full Text Available Although the modulation of Ca(2+ channel activity by extremely low-frequency electromagnetic fields (ELF-EMF has been studied previously, few reports have addressed the effects of such fields on the activity of voltage-activated Na(+ channels (Na(v. Here, we investigated the effects of ELF-EMF on Na(v activity in rat cerebellar granule cells (GCs. Our results reveal that exposing cerebellar GCs to ELF-EMF for 10-60 min significantly increased Na(v currents (I(Na by 30-125% in a time- and intensity-dependent manner. The Na(v channel steady-state activation curve, but not the steady-state inactivation curve, was significantly shifted (by 5.2 mV towards hyperpolarization by ELF-EMF stimulation. This phenomenon is similar to the effect of intracellular application of arachidonic acid (AA and prostaglandin E(2 (PGE(2 on I(Na in cerebellar GCs. Increases in intracellular AA, PGE(2 and phosphorylated PKA levels in cerebellar GCs were observed following ELF-EMF exposure. Western blottings indicated that the Na(V 1.2 protein on the cerebellar GCs membrane was increased, the total expression levels of Na(V 1.2 protein were not affected after exposure to ELF-EMF. Cyclooxygenase inhibitors and PGE(2 receptor (EP antagonists were able to eliminate this ELF-EMF-induced increase in phosphorylated PKA and I(Na. In addition, ELF-EMF exposure significantly enhanced the activity of PLA(2 in cerebellar GCs but did not affect COX-1 or COX-2 activity. Together, these data demonstrate for the first time that neuronal I(Na is significantly increased by ELF-EMF exposure via a cPLA2 AA PGE(2 EP receptors PKA signaling pathway.

  17. Exposure to extremely low-frequency electromagnetic fields modulates Na+ currents in rat cerebellar granule cells through increase of AA/PGE2 and EP receptor-mediated cAMP/PKA pathway.

    Science.gov (United States)

    He, Yan-Lin; Liu, Dong-Dong; Fang, Yan-Jia; Zhan, Xiao-Qin; Yao, Jin-Jing; Mei, Yan-Ai

    2013-01-01

    Although the modulation of Ca(2+) channel activity by extremely low-frequency electromagnetic fields (ELF-EMF) has been studied previously, few reports have addressed the effects of such fields on the activity of voltage-activated Na(+) channels (Na(v)). Here, we investigated the effects of ELF-EMF on Na(v) activity in rat cerebellar granule cells (GCs). Our results reveal that exposing cerebellar GCs to ELF-EMF for 10-60 min significantly increased Na(v) currents (I(Na)) by 30-125% in a time- and intensity-dependent manner. The Na(v) channel steady-state activation curve, but not the steady-state inactivation curve, was significantly shifted (by 5.2 mV) towards hyperpolarization by ELF-EMF stimulation. This phenomenon is similar to the effect of intracellular application of arachidonic acid (AA) and prostaglandin E(2) (PGE(2)) on I(Na) in cerebellar GCs. Increases in intracellular AA, PGE(2) and phosphorylated PKA levels in cerebellar GCs were observed following ELF-EMF exposure. Western blottings indicated that the Na(V) 1.2 protein on the cerebellar GCs membrane was increased, the total expression levels of Na(V) 1.2 protein were not affected after exposure to ELF-EMF. Cyclooxygenase inhibitors and PGE(2) receptor (EP) antagonists were able to eliminate this ELF-EMF-induced increase in phosphorylated PKA and I(Na). In addition, ELF-EMF exposure significantly enhanced the activity of PLA(2) in cerebellar GCs but did not affect COX-1 or COX-2 activity. Together, these data demonstrate for the first time that neuronal I(Na) is significantly increased by ELF-EMF exposure via a cPLA2 AA PGE(2) EP receptors PKA signaling pathway.

  18. Fractal patterns of neural activity exist within the suprachiasmatic nucleus and require extrinsic network interactions.

    Science.gov (United States)

    Hu, Kun; Meijer, Johanna H; Shea, Steven A; vanderLeest, Henk Tjebbe; Pittman-Polletta, Benjamin; Houben, Thijs; van Oosterhout, Floor; Deboer, Tom; Scheer, Frank A J L

    2012-01-01

    The mammalian central circadian pacemaker (the suprachiasmatic nucleus, SCN) contains thousands of neurons that are coupled through a complex network of interactions. In addition to the established role of the SCN in generating rhythms of ~24 hours in many physiological functions, the SCN was recently shown to be necessary for normal self-similar/fractal organization of motor activity and heart rate over a wide range of time scales--from minutes to 24 hours. To test whether the neural network within the SCN is sufficient to generate such fractal patterns, we studied multi-unit neural activity of in vivo and in vitro SCNs in rodents. In vivo SCN-neural activity exhibited fractal patterns that are virtually identical in mice and rats and are similar to those in motor activity at time scales from minutes up to 10 hours. In addition, these patterns remained unchanged when the main afferent signal to the SCN, namely light, was removed. However, the fractal patterns of SCN-neural activity are not autonomous within the SCN as these patterns completely broke down in the isolated in vitro SCN despite persistence of circadian rhythmicity. Thus, SCN-neural activity is fractal in the intact organism and these fractal patterns require network interactions between the SCN and extra-SCN nodes. Such a fractal control network could underlie the fractal regulation observed in many physiological functions that involve the SCN, including motor control and heart rate regulation.

  19. Autocatalytic activation of the furin zymogen requires removal of the emerging enzyme's N-terminus from the active site.

    Directory of Open Access Journals (Sweden)

    Katarzyna Gawlik

    Full Text Available Before furin can act on protein substrates, it must go through an ordered process of activation. Similar to many other proteinases, furin is synthesized as a zymogen (profurin which becomes active only after the autocatalytic removal of its auto-inhibitory prodomain. We hypothesized that to activate profurin its prodomain had to be removed and, in addition, the emerging enzyme's N-terminus had to be ejected from the catalytic cleft.We constructed and analyzed the profurin mutants in which the egress of the emerging enzyme's N-terminus from the catalytic cleft was restricted. Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107 downward arrowAsp(108, but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75 downward arrowSer(76 cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108. These correctly processed furin mutants, however, remained self-inhibited by the constrained N-terminal sequence which continuously occupied the S' sub-sites of the catalytic cleft and interfered with the functional activity. Further, using the in vitro cleavage of the purified prodomain and the analyses of colon carcinoma LoVo cells with the reconstituted expression of the wild-type and mutant furins, we demonstrated that a three-step autocatalytic processing including the cleavage of the prodomain at the previously unidentified Arg-Leu-Gln-Arg(89 downward arrowGlu(90 site, is required for the efficient activation of furin.Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms. In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.

  20. Prostaglandin E-2 differentially modulates IL-5 gene expression in activated human T lymphocytes depending on the costimulatory signal

    NARCIS (Netherlands)

    Borger, P; Vellenga, E; Gringhuis, SI; Timmerman, JAB; Lummen, C; Postma, DS; Kauffman, HF

    1998-01-01

    Background: Protein kinase A (PKA) activation is documented to be inhibitory for T helper cell (T-H1)-like cytokines (IL-2, IFN-gamma), whereas T-H2-like cytokines (IL-4, IL-5) are not affected or upregulated. We have recently shown that IL-4 gene expression can be inhibited by PKA activation but de

  1. Direct activation of RhoA by reactive oxygen species requires a redox-sensitive motif.

    Directory of Open Access Journals (Sweden)

    Amir Aghajanian

    Full Text Available BACKGROUND: Rho family GTPases are critical regulators of the cytoskeleton and affect cell migration, cell-cell adhesion, and cell-matrix adhesion. As with all GTPases, their activity is determined by their guanine nucleotide-bound state. Understanding how Rho proteins are activated and inactivated has largely focused on regulatory proteins such as guanine nucleotide exchange factors (GEFs and GTPase activating proteins (GAPs. However, recent in vitro studies have indicated that GTPases may also be directly regulated by redox agents. We hypothesized that this redox-based mechanism occurs in cells and affects cytoskeletal dynamics, and in this report we conclude this is indeed a novel mechanism of regulating the GTPase RhoA. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we show that RhoA can be directly activated by reactive oxygen species (ROS in cells, and that this requires two critical cysteine residues located in a unique redox-sensitive motif within the phosphoryl binding loop. First, we show that ROS can reversibly activate RhoA and induce stress fiber formation, a well characterized readout of RhoA activity. To determine the role of cysteine residues in this mechanism of regulation, we generated cysteine to alanine RhoA mutants. Mutation of these cysteines abolishes ROS-mediated activation and stress fiber formation, indicating that these residues are critical for redox-regulation of RhoA. Importantly, these mutants maintain the ability to be activated by GEFs. CONCLUSIONS/SIGNIFICANCE: Our findings identify a novel mechanism for the regulation of RhoA in cells by ROS, which is independent of classical regulatory proteins. This mechanism of regulation may be particularly relevant in pathological conditions where ROS are generated and the cellular redox-balance altered, such as in asthma and ischemia-reperfusion injury.

  2. Transition of arrestin into the active receptor-binding state requires an extended interdomain hinge.

    Science.gov (United States)

    Vishnivetskiy, Sergey A; Hirsch, Joel A; Velez, Maria-Gabriela; Gurevich, Yulia V; Gurevich, Vsevolod V

    2002-11-15

    Arrestins selectively bind to the phosphorylated activated form of G protein-coupled receptors, thereby blocking further G protein activation. Structurally, arrestins consist of two domains topologically connected by a 12-residue long loop, which we term the "hinge" region. Both domains contain receptor-binding elements. The relative size and shape of arrestin and rhodopsin suggest that dramatic changes in arrestin conformation are required to bring all of its receptor-binding elements in contact with the cytoplasmic surface of the receptor. Here we use the visual arrestin/rhodopsin system to test the hypothesis that the transition of arrestin into its active receptor-binding state involves a movement of the two domains relative to each other that might be limited by the length of the hinge. We have introduced three insertions and 24 deletions in the hinge region and measured the binding of all of these mutants to light-activated phosphorylated (P-Rh*), dark phosphorylated (P-Rh), dark unphosphorylated (Rh), and light-activated unphosphorylated rhodopsin (Rh*). The addition of 1-3 extra residues to the hinge has no effect on arrestin function. In contrast, sequential elimination of 1-8 residues results in a progressive decrease in P-Rh* binding without changing arrestin selectivity for P-Rh*. These results suggest that there is a minimum length of the hinge region necessary for high affinity binding, consistent with the idea that the two domains move relative to each other in the process of arrestin transition into its active receptor-binding state. The same length of the hinge is also necessary for the binding of "constitutively active" arrestin mutants to P-Rh*, dark P-Rh, and Rh*, suggesting that the active (receptor-bound) arrestin conformation is essentially the same in both wild type and mutant forms.

  3. Active source requirements for assay of sludge drums on the BIR WIT system

    Energy Technology Data Exchange (ETDEWEB)

    Roberson, G.P.; Camp, D.C.

    1998-04-27

    The design of the active source for active and passive computed tomography (A&PCT) is critical with respect to accuracy and throughput. The A&PCT active source requirements are highly dependent upon the attenuation properties of the waste matrix within the drum. On of the most highly attenuating waste matrices is sludge. This waste stream will consist of solidified aqueous waste consisting of IDC 001 first stage sludge and IDC 007 wet sludge. Also, the stream consists of solidified organic waste known as code IDC 003 organic setups. We have evaluated the sludge drum data that was previously acquired on the WIT system and have determined that the active source activity must be increased to provide reasonable throughput. The sludge drum that is evaluated here is drum CEPRF11. CEPRF11 is a test drum that was part of the Nondestructive Assay system Capability Evaluation Project (CEP) and contained an actual Rocky Flats waste that is categorized as code 003 solidified organic waste. The full drum was evaluated and found to be somewhat homogenous; therefore, a single slice is arbitrarily chosen to represent the entire drum. Slice number 8 is used and is located approximately at the center of the drum. Figure 1 shows the averaged projections for different energies derived from the active sinogram of slice number 8 from the CEPRF11 drum. This is the average of all the projections of slice 8 taken over 180 degrees with an active integration time of 6 seconds. Figure 2 is also a graph showing the average of all the projections for slice 8; however, the active integration time is 30 seconds.

  4. SHANK-associated RH domain interacting protein (SHARPIN) is required for optimal NLRP3 inflammasome activation

    Science.gov (United States)

    Gurung, Prajwal; Lamkanfi, Mohamed; Kanneganti, Thirumala-Devi

    2015-01-01

    The NLRP3 inflammasome is a multimeric protein complex assembled in response to a wide array of pathogens and danger-associated molecular patterns. Despite the ability of NLRP3 to respond to diverse cues, the mechanisms controlling assembly of this complex are contested. Recently published studies show that HOIL-1, a member of the linear ubiquitin chain assembly complex (LUBAC), contributes to activation of the NLRP3 inflammasome. SHARPIN, along with HOIP and HOIL-1 assembles the LUBAC complex. Herein, we examined whether SHARPIN is required for the activation of NLRP3 inflammasome. Utilizing Sharpincpdm macrophages (deficient in SHARPIN expression), we demonstrate that SHARPIN is required for optimal activation of the NLRP3 inflammasome by both canonical and non-canonical stimuli. Furthermore, Sharpincpdm macrophages had dramatic defects in both NFκB and MAP kinase pathways, suggesting a role in transcriptional priming of the NLRP3 inflammasome. In conclusion, our study identified SHARPIN as a novel regulator of the NLRP3 inflammasome. PMID:25637014

  5. Cereblon expression is required for the antimyeloma activity of lenalidomide and pomalidomide.

    Science.gov (United States)

    Zhu, Yuan Xiao; Braggio, Esteban; Shi, Chang-Xin; Bruins, Laura A; Schmidt, Jessica E; Van Wier, Scott; Chang, Xiu-Bao; Bjorklund, Chad C; Fonseca, Rafael; Bergsagel, P Leif; Orlowski, Robert Z; Stewart, A Keith

    2011-11-03

    The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their antitumor effects remains unclear. We investigated the role of cereblon (CRBN), a primary teratogenic target of thalidomide, in the antimyeloma activity of IMiDs. CRBN depletion is initially cytotoxic to human myeloma cells, but surviving cells with stable CRBN depletion become highly resistant to both lenalidomide and pomalidomide, but not to the unrelated drugs bortezomib, dexamethasone, and melphalan. Acquired deletion of CRBN was found to be the primary genetic event differentiating isogenic MM1.S cell lines cultured to be sensitive or resistant to lenalidomide and pomalidomide. Gene expression changes induced by lenalidomide were dramatically suppressed in the presence of CRBN depletion, further demonstrating that CRBN is required for lenalidomide activity. Downstream targets of CRBN include interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. Patients exposed to, and putatively resistant to, lenalidomide had lower CRBN levels in paired samples before and after therapy. In summary, CRBN is an essential requirement for IMiD activity and a possible biomarker for the clinical assessment of antimyeloma efficacy.

  6. TYK2 kinase activity is required for functional type I interferon responses in vivo.

    Directory of Open Access Journals (Sweden)

    Michaela Prchal-Murphy

    Full Text Available Tyrosine kinase 2 (TYK2 is a member of the Janus kinase (JAK family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2(K923E mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein's stability. An inhibitory function was only observed upon over-expression of TYK2(K923Ein vitro. Tyk2(K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors.

  7. BPTF is required for c-MYC transcriptional activity and in vivo tumorigenesis

    Science.gov (United States)

    Richart, Laia; Carrillo-de Santa Pau, Enrique; Río-Machín, Ana; de Andrés, Mónica P.; Cigudosa, Juan C.; Lobo, Víctor J. Sánchez-Arévalo; Real, Francisco X.

    2016-01-01

    c-MYC oncogene is deregulated in most human tumours. Histone marks associated with transcriptionally active genes define high-affinity c-MYC targets. The mechanisms involved in their recognition by c-MYC are unknown. Here we report that c-MYC interacts with BPTF, a core subunit of the NURF chromatin-remodelling complex. BPTF is required for the activation of the full c-MYC transcriptional programme in fibroblasts. BPTF knockdown leads to decreased c-MYC recruitment to DNA and changes in chromatin accessibility. In Bptf-null MEFs, BPTF is necessary for c-MYC-driven proliferation, G1–S progression and replication stress, but not for c-MYC-driven apoptosis. Bioinformatics analyses unveil that BPTF levels correlate positively with c-MYC-driven transcriptional signatures. In vivo, Bptf inactivation in pre-neoplastic pancreatic acinar cells significantly delays tumour development and extends survival. Our findings uncover BPTF as a crucial c-MYC co-factor required for its biological activity and suggest that the BPTF-c-MYC axis is a potential therapeutic target in cancer. PMID:26729287

  8. Requirement of histone deacetylase activity for the expression of critical photoreceptor genes

    Directory of Open Access Journals (Sweden)

    Cepko Constance L

    2007-06-01

    Full Text Available Abstract Background Histone deacetylases (HDACs play a major role in the regulation of gene transcription, often leading to transcriptional repression, as well as other effects following deacetylation of non-histone proteins. Results To investigate the role of HDACs in the developing mammalian retina, a general inhibitor of HDACs, trichostatin-A (TSA, was used to treat newborn murine retinae in explant cultures. Inhibition of HDAC activity resulted in a reduction in RNA levels for genes that regulate retinal development, as well as cell cycle regulators. Several of the genes encode transcription factors essential for rod photoreceptor development, Otx2, Nrl, and Crx. Using luciferase reporter assays, the promoter activity of both Nrl and Crx was found to be compromised by HDAC inhibition. Furthermore, downregulation of gene expression by HDAC inhibition didn't require de novo protein synthesis, and was associated with hyperacetylation of histones and non-histone proteins. Finally, HDAC inhibition in retinal explant cultures resulted in increased cell death, reduction in proliferation, a complete loss of rod photoreceptors and Müller glial cells, and an increase in bipolar cells. Conclusion HDAC activity is required for the expression of critical pro-rod transcription factors and the development of rod photoreceptor cells.

  9. ARK, the Apaf-1 related killer in Drosophila, requires diverse domains for its apoptotic activity.

    Science.gov (United States)

    Srivastava, M; Scherr, H; Lackey, M; Xu, D; Chen, Z; Lu, J; Bergmann, A

    2007-01-01

    In mammals and Drosophila, apoptotic caspases are under positive control of the CED-4-like proteins Apaf-1 and ARK, respectively. In an EMS-mutagenesis screen, we isolated 33 ark mutants as recessive suppressors of hid-induced apoptosis. The ark mutants are loss-of-function alleles characterized by reduced developmental apoptosis. Using the phenotypic series of these alleles, we identified helical domain I in the nucleotide oligomerization domain as critical for ARK's apoptotic activity. Interestingly, the WD40 region may also have an unanticipated positive requirement for the apoptotic activity of ARK. Considering structural information, we discuss the roles of these domains for assembly and activity of the ARK apoptosome, and propose that the WD40 region is anti-apoptotic in the absence of apoptotic signals, and pro-apoptotic in the presence of such signals. Furthermore, a defined null allele reveals that ark is required for most, but not all apoptosis suggesting the existence of an ARK-independent apoptotic pathway.

  10. Critical evaluation of buffering solutions for pKa determination by capillary electrophoresis.

    Science.gov (United States)

    Fuguet, Elisabet; Reta, Mario; Gibert, Carme; Rosés, Martí; Bosch, Elisabeth; Ràfols, Clara

    2008-07-01

    The performance of the most common and also some other less common CE buffers has been tested for the pKa determination of several types of compounds (pyridine, amines, and phenols). The selected buffers cover a pH ranging from 3.7 to 11.8. Whereas some buffers, like acetic acid/acetate, BisTrisH+/BisTris, TrisH+/Tris, CHES/CHES-, and CAPS/CAPS- can be used with all type of analytes, others like ammonium/ammonia, butylammonium/butylammonia, ethylammonium/ethylammonia, diethylammonium/diethylammonia, and hydrogenphosphate/phosphate are not recommended because they interact with a wide range of compounds. The rest of the tested buffers (dihydrogenphosphate/hydrogenphosphate, MES/MES-, HEPES/HEPES-, and boric acid/borate) can show specific interactions depending on the nature of the analytes, and their use in some applications should be restricted.

  11. Development and validation of a FIA/UV-vis method for pK(a) determination of oxime based acetylcholinesterase reactivators.

    Science.gov (United States)

    Musil, Karel; Florianova, Veronika; Bucek, Pavel; Dohnal, Vlastimil; Kuca, Kamil; Musilek, Kamil

    2016-01-05

    Acetylcholinesterase reactivators (oximes) are compounds used for antidotal treatment in case of organophosphorus poisoning. The dissociation constants (pK(a1)) of ten standard or promising acetylcholinesterase reactivators were determined by ultraviolet absorption spectrometry. Two methods of spectra measurement (UV-vis spectrometry, FIA/UV-vis) were applied and compared. The soft and hard models for calculation of pK(a1) values were performed. The pK(a1) values were recommended in the range 7.00-8.35, where at least 10% of oximate anion is available for organophosphate reactivation. All tested oximes were found to have pK(a1) in this range. The FIA/UV-vis method provided rapid sample throughput, low sample consumption, high sensitivity and precision compared to standard UV-vis method. The hard calculation model was proposed as more accurate for pK(a1) calculation. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Ketamine plus imipramine treatment induces antidepressant-like behavior and increases CREB and BDNF protein levels and PKA and PKC phosphorylation in rat brain.

    Science.gov (United States)

    Réus, Gislaine Z; Stringari, Roberto B; Ribeiro, Karine F; Ferraro, Ana K; Vitto, Marcelo F; Cesconetto, Patrícia; Souza, Claúdio T; Quevedo, João

    2011-08-01

    A growing body of evidence has pointed to the N-methyl-d-aspartate (NMDA) receptor antagonists as a potential therapeutic target for the treatment of major depression. The present study investigated the possibility of synergistic interactions between antidepressant imipramine with the uncompetitive NMDA receptor antagonist ketamine. Wistar rats were acutely treated with ketamine (5 and 10mg/kg) and imipramine (10 and 20mg/kg) and then subjected to forced swimming tests. The cAMP response element bindig (CREB) and brain-derived neurotrophic factor (BDNF) protein levels and protein kinase C (PKC) and protein kinase A (PKA) phosphorylation were assessed in the prefrontal cortex, hippocampus and amygdala by imunoblot. Imipramine at the dose of 10mg/kg and ketamine at the dose of 5mg/kg did not have effect on the immobility time; however, the effect of imipramine (10 and 20mg/kg) was enhanced by both doses of ketamine. Ketamine and imipramine alone or in combination at all doses tested did not modify locomotor activity. Combined treatment with ketamine and imipramine produced stronger increases of CREB and BDNF protein levels in the prefrontal cortex, hippocampus and amygdala, and PKA phosphorylation in the hippocampus and amygdala and PKC phosphorylation in prefrontal cortex. The results described indicate that co-administration of antidepressant imipramine with ketamine may induce a more pronounced antidepressant activity than treatment with each antidepressant alone. This finding may be of particular importance in the case of drug-resistant patients and could suggest a method of obtaining significant antidepressant actions whilst limiting side effects.

  13. Caspase-1 activity is required for UVB-induced apoptosis of human keratinocytes.

    Science.gov (United States)

    Sollberger, Gabriel; Strittmatter, Gerhard E; Grossi, Serena; Garstkiewicz, Martha; Auf dem Keller, Ulrich; French, Lars E; Beer, Hans-Dietmar

    2015-05-01

    Caspase-1 has a crucial role in innate immunity as the protease activates the proinflammatory cytokine prointerleukin(IL)-1β. Furthermore, caspase-1 induces pyroptosis, a lytic form of cell death that supports inflammation. Activation of caspase-1 occurs in multi-protein complexes termed inflammasomes, which assemble upon sensing of stress signals. In the skin and in skin-derived keratinocytes, UVB irradiation induces inflammasome-dependent IL-1 secretion and sunburn. Here we present evidence that caspase-1 and caspase-4 are required for UVB-induced apoptosis. In UVB-irradiated human primary keratinocytes, apoptosis occurs significantly later than inflammasome activation but depends on caspase-1 activity. However, it proceeds independently of inflammasome activation. By a proteomics approach, we identified the antiapoptotic Bap31 as a putative caspase-1 substrate. Caspase-1-dependent apoptosis is possibly a recent process in evolution as it was not detected in mice. These results suggest a protective role of caspase-1 in keratinocytes during UVB-induced skin cancer development through the induction of apoptosis.

  14. Eyes absent tyrosine phosphatase activity is not required for Drosophila development or survival.

    Directory of Open Access Journals (Sweden)

    Meng Jin

    Full Text Available Eyes absent (Eya is an evolutionarily conserved transcriptional coactivator and protein phosphatase that regulates multiple developmental processes throughout the metazoans. Drosophila eya is necessary for survival as well as for the formation of the adult eye. Eya contains a tyrosine phosphatase domain, and mutations altering presumptive active-site residues lead to strongly reduced activities in ectopic eye induction, in vivo genetic rescue using the Gal4-UAS system, and in vitro phosphatase assays. However, these mutations have not been analyzed during normal development with the correct levels, timing, and patterns of endogenous eya expression. To investigate whether the tyrosine phosphatase activity of Eya plays a role in Drosophila survival or normal eye formation, we generated three eya genomic rescue (eyaGR constructs that alter key active-site residues and tested them in vivo. In striking contrast to previous studies, all eyaGR constructs fully restore eye formation as well as viability in an eya null mutant background. We conclude that the tyrosine phosphatase activity of Eya is not required for normal eye development or survival in Drosophila. Our study suggests the need for a re-evaluation of the mechanism of Eya action and underscores the importance of studying genes in their native context.

  15. Carboxyl terminal of rhodopsin kinase is required for the phosphorylation of photo—activated rhodopsin

    Institute of Scientific and Technical Information of China (English)

    YUQINGMING; LANMA; 等

    1998-01-01

    Human rhodopsin kinase (RK) and a carboxyl terminus-truncated mutant RK lacking the last 59 amino acids (RKC) were expressed in human embryonic kidney 293 cells to investigate the role of the carboxyl terminus of RK in recognition and phosphorylation of rhodopsin.RKC,like the wild-type RK,was detected in both plasma membranes and cytosolic fractions.The Cterminal truncated rhodopsin kinase was unable to phosphorylate photo-activated rhodopsin,but possesses kinase activity similar to the wild-type RK in phosphorylation of small peptide substrate.It suggests that the truncation did not disturb the gross structures of RK catalytic domain.Our results also show that RKC failed to translocate to photo-activated rod out segments.Taken together,our study demonstrate the carboxyl terminus of RK is required for phosphorylation of photo-activated rhodopsin and strongly indicate that carboxyl-terminus of RK may be involved in interaction with photo-activated rhodopsin.

  16. Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation.

    Science.gov (United States)

    Van Gorp, Hanne; Saavedra, Pedro H V; de Vasconcelos, Nathalia M; Van Opdenbosch, Nina; Vande Walle, Lieselotte; Matusiak, Magdalena; Prencipe, Giusi; Insalaco, Antonella; Van Hauwermeiren, Filip; Demon, Dieter; Bogaert, Delfien J; Dullaers, Melissa; De Baere, Elfride; Hochepied, Tino; Dehoorne, Joke; Vermaelen, Karim Y; Haerynck, Filomeen; De Benedetti, Fabrizio; Lamkanfi, Mohamed

    2016-12-13

    Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease worldwide. It is caused by mutations in the inflammasome adaptor Pyrin, but how FMF mutations alter signaling in FMF patients is unknown. Herein, we establish Clostridium difficile and its enterotoxin A (TcdA) as Pyrin-activating agents and show that wild-type and FMF Pyrin are differentially controlled by microtubules. Diverse microtubule assembly inhibitors prevented Pyrin-mediated caspase-1 activation and secretion of IL-1β and IL-18 from mouse macrophages and human peripheral blood mononuclear cells (PBMCs). Remarkably, Pyrin inflammasome activation persisted upon microtubule disassembly in PBMCs of FMF patients but not in cells of patients afflicted with other autoinflammatory diseases. We further demonstrate that microtubules control Pyrin activation downstream of Pyrin dephosphorylation and that FMF mutations enable microtubule-independent assembly of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) micrometer-sized perinuclear structures (specks). The discovery that Pyrin mutations remove the obligatory requirement for microtubules in inflammasome activation provides a conceptual framework for understanding FMF and enables immunological screening of FMF mutations.

  17. PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase.

    Science.gov (United States)

    Leenders, Frauke; Möpert, Kristin; Schmiedeknecht, Anett; Santel, Ansgar; Czauderna, Frank; Aleku, Manuela; Penschuck, Silke; Dames, Sibylle; Sternberger, Maria; Röhl, Thomas; Wellmann, Axel; Arnold, Wolfgang; Giese, Klaus; Kaufmann, Jörg; Klippel, Anke

    2004-08-18

    Chronic activation of the phosphoinositide 3-kinase (PI3K)/PTEN signal transduction pathway contributes to metastatic cell growth, but up to now effectors mediating this response are poorly defined. By simulating chronic activation of PI3K signaling experimentally, combined with three-dimensional (3D) culture conditions and gene expression profiling, we aimed to identify novel effectors that contribute to malignant cell growth. Using this approach we identified and validated PKN3, a barely characterized protein kinase C-related molecule, as a novel effector mediating malignant cell growth downstream of activated PI3K. PKN3 is required for invasive prostate cell growth as assessed by 3D cell culture assays and in an orthotopic mouse tumor model by inducible expression of short hairpin RNA (shRNA). We demonstrate that PKN3 is regulated by PI3K at both the expression level and the catalytic activity level. Therefore, PKN3 might represent a preferred target for therapeutic intervention in cancers that lack tumor suppressor PTEN function or depend on chronic activation of PI3K.

  18. RAB8B Is Required for Activity and Caveolar Endocytosis of LRP6

    Directory of Open Access Journals (Sweden)

    Kubilay Demir

    2013-09-01

    Full Text Available Wnt/β-catenin signaling plays an important role in embryonic development and adult tissue homeostasis. When Wnt ligands bind to the receptor complex, LRP5/6 coreceptors are activated by phosphorylation and concomitantly endocytosed. In vertebrates, Wnt ligands induce caveolin-dependent endocytosis of LRP6 to relay signal downstream, whereas antagonists such as Dickkopf promote clathrin-dependent endocytosis, leading to inhibition. However, little is known about how LRP6 is directed to different internalization mechanisms, and how caveolin-dependent endocytosis is mediated. In an RNAi screen, we identified the Rab GTPase RAB8B as being required for Wnt/β-catenin signaling. RAB8B depletion reduces LRP6 activity, β-catenin accumulation, and induction of Wnt target genes, whereas RAB8B overexpression promotes LRP6 activity and internalization and rescues inhibition of caveolar endocytosis. In Xenopus laevis and Danio rerio, RAB8B morphants show lower Wnt activity during embryonic development. Our results implicate RAB8B as an essential evolutionary conserved component of Wnt/β-catenin signaling through regulation of LRP6 activity and endocytosis.

  19. Jack bean (Canavalia ensiformis) urease. Probing acid-base groups of the active site by pH variation.

    Science.gov (United States)

    Krajewska, Barbara; Ciurli, Stefano

    2005-07-01

    A pH-variation study of jack bean (Canavalia ensiformis) urease steady-state kinetic parameters and of the inhibition constant of boric acid, a urease competitive inhibitor, was performed using both noninhibitory organic (MES, HEPES and CHES) and inhibitory inorganic (phosphate) buffers, in an effort to elucidate the functions exercised in the catalysis by the ionizable groups of the enzyme active site. The results obtained are consistent with the requirement for three groups utilized by urease with pK(a)s equal to 5.3+/-0.2, 6.6+/-0.2 and 9.1+/-0.4. Based on the appearance of the ionization step with pK(a)=5.3 in v(max)-pH, K(M)-pH and K(i)-pH profiles, we assigned this group as participating both in the substrate binding and catalytic reaction. As shown by its presence in v(max)-pH and K(M)-pH curves, the obvious role of the group with pK(a)=9.1 is the participation in the catalytic reaction. One function of the group featuring pK(a)=6.6, which was derived from a two-maxima v(max)-pH profile obtained upon increasing phosphate buffer concentration, an effect the first time observed for urease-phosphate systems, is the substrate binding, another possible function being modulation of the active site structure controlled by the ionic strength. It is also possible that the pK(a)=6.6 is a merger of two pK(a)s close in value. The study establishes that regular bell-shaped activity-pH profiles, commonly reported for urease, entail more complex pH-dependent behavior of the urease active site ionizable groups, which could be experimentally derived using species interacting with the enzyme, in addition to changing solution pH and ionic strength.

  20. Active Mirror Predictive and Requirements Verification Software (AMP-ReVS)

    Science.gov (United States)

    Basinger, Scott A.

    2012-01-01

    This software is designed to predict large active mirror performance at various stages in the fabrication lifecycle of the mirror. It was developed for 1-meter class powered mirrors for astronomical purposes, but is extensible to other geometries. The package accepts finite element model (FEM) inputs and laboratory measured data for large optical-quality mirrors with active figure control. It computes phenomenological contributions to the surface figure error using several built-in optimization techniques. These phenomena include stresses induced in the mirror by the manufacturing process and the support structure, the test procedure, high spatial frequency errors introduced by the polishing process, and other process-dependent deleterious effects due to light-weighting of the mirror. Then, depending on the maturity of the mirror, it either predicts the best surface figure error that the mirror will attain, or it verifies that the requirements for the error sources have been met once the best surface figure error has been measured. The unique feature of this software is that it ties together physical phenomenology with wavefront sensing and control techniques and various optimization methods including convex optimization, Kalman filtering, and quadratic programming to both generate predictive models and to do requirements verification. This software combines three distinct disciplines: wavefront control, predictive models based on FEM, and requirements verification using measured data in a robust, reusable code that is applicable to any large optics for ground and space telescopes. The software also includes state-of-the-art wavefront control algorithms that allow closed-loop performance to be computed. It allows for quantitative trade studies to be performed for optical systems engineering, including computing the best surface figure error under various testing and operating conditions. After the mirror manufacturing process and testing have been completed, the

  1. Lin28a protects against diabetic cardiomyopathy via the PKA/ROCK2 pathway.

    Science.gov (United States)

    Sun, Shuhong; Zhang, Mingming; Lin, Jie; Hu, Jianqiang; Zhang, Rongqing; Li, Congye; Wei, Tianlu; Sun, Dongdong; Wei, Jianqin; Wang, Haichang

    2016-01-01

    Lin28a enhances glucose uptake and insulin-sensitivity. However, the role of Lin28a on experimental diabetic cardiomyopathy (DCM) is not well understood. We investigated the potential role and mechanism ofLin28a in diabetes-induced myocardial dysfunction in mice. Diabetes was induced by intraperitoneal (i.p.) injections of Streptozocin (STZ) in mice. Animals were randomized to be treated with lentivirus carrying Lin28a siRNA or Lin28a cDNA. Cardiac function, cardiomyocyte autophagy, apoptosis and mitochondria morphology in diabetic mice were compared between groups. The target proteins of Lin28a were examined by western blot analysis. Lin28a levels were markedly reduced in the cardiac tissue compared to the control mice. Lin28a overexpression significantly improved left ventricular ejection fraction (LVEF), promoted autophagy, decreased myocardial apoptotic index and alleviated mitochondria cristae destruction in diabetic mice. Lin28a knockdown exacerbated diabetic injury as evidenced by decreased LVEF, increased apoptotic index and aggravated mitochondria cristae destruction. Interestingly, pretreatment with a PKA inhibitor, N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt (H89) abolished the beneficial effects of Lin28a overexpression. RhoA-expression and ROCK2-expression were decreased in vivo after Lin28a overexpression, while Lin28a knockdown increased the expression of RhoA and ROCK2 in diabetic mice. Lin28a protects against DCM through PKA/ROCK2 dependent pathway. Lin28a might serve as a potential therapeutic target for the treatment of the patients with DCM. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. PKA, PKC, and AKAP localization in and around the neuromuscular junction

    Directory of Open Access Journals (Sweden)

    Newton Alexandra

    2001-10-01

    Full Text Available Abstract Background One mechanism that directs the action of the second messengers, cAMP and diacylglycerol, is the compartmentalization of protein kinase A (PKA and protein kinase C (PKC. A-kinase anchoring proteins (AKAPs can recruit both enzymes to specific subcellular locations via interactions with the various isoforms of each family of kinases. We found previously that a new class of AKAPs, dual-specific AKAPs, denoted D-AKAP1 and D-AKAP2, bind to RIα in addition to the RII subunits. Results Immunohistochemistry and confocal microscopy were used here to determine that D-AKAP1 colocalizes with RIα at the postsynaptic membrane of the vertebrate neuromuscular junction (NMJ and the adjacent muscle, but not in the presynaptic region. The labeling pattern for RIα and D-AKAP1 overlapped with mitochondrial staining in the muscle fibers, consistent with our previous work showing D-AKAP1 association with mitochondria in cultured cells. The immunoreactivity of D-AKAP2 was distinct from that of D-AKAP1. We also report here that even though the PKA type II subunits (RIIα and RIIβ are localized at the NMJ, their patterns are distinctive and differ from the other R and D-AKAP patterns examined. PKCβ appeared to colocalize with the AKAP, gravin, at the postsynaptic membrane. Conclusions The kinases and AKAPs investigated have distinct patterns of colocalization, which suggest a complex arrangement of signaling micro-environments. Because the labeling patterns for RIα and D-AKAP 1 are similar in the muscle fibers and at the postsynaptic membrane, it may be that this AKAP anchors RIα in these regions. Likewise, gravin may be an anchor of PKCβ at the NMJ.

  3. Polysaccharide Nanoparticles for Efficient siRNA Targeting in Cancer Cells by Supramolecular pKa Shift

    Science.gov (United States)

    Zhang, Ying-Ming; Yang, Yang; Zhang, Yu-Hui; Liu, Yu

    2016-01-01

    Biomacromolecular pKa shifting is considered as one of the most ubiquitous processes in biochemical events, e.g., the enzyme-catalyzed reaction and protein conformational stabilization. In this paper, we report on the construction of biocompatible polysaccharide nanoparticle with targeting ability and lower toxicity by supramolecular pKa shift strategy. This was realized through a ternary assembly constructed by the dual host‒guest interactions of an adamantane-bis(diamine) conjugate (ADA) with cucurbit[6]uril (CB[6]) and a polysaccharide. The potential application of such biocompatible nanostructure was further implemented by the selective transportation of small interfering RNA (siRNA) in a controlled manner. It is demonstrated that the strong encapsulation of the ADA’s diammonium tail by CB[6] not only reduced the cytotoxicity of the nano-scaled vehicle but also dramatically enhanced cation density through an obvious positive macrocycle-induced pKa shift, which eventually facilitated the subsequent siRNA binding. With a targeted polysaccharide shell containing a cyclodextrin‒hyaluronic acid conjugate, macrocycle-incorporated siRNA polyplexes were specifically delivered into malignant human prostate PC-3 cells. The supramolecular polysaccharide nanoparticles, the formation of which was enabled and promoted by the complexation-assisted pKa shift, may be used as a versatile tool for controlled capture and release of biofunctional substrates. PMID:27363811

  4. Polysaccharide Nanoparticles for Efficient siRNA Targeting in Cancer Cells by Supramolecular pKa Shift

    Science.gov (United States)

    Zhang, Ying-Ming; Yang, Yang; Zhang, Yu-Hui; Liu, Yu

    2016-07-01

    Biomacromolecular pKa shifting is considered as one of the most ubiquitous processes in biochemical events, e.g., the enzyme-catalyzed reaction and protein conformational stabilization. In this paper, we report on the construction of biocompatible polysaccharide nanoparticle with targeting ability and lower toxicity by supramolecular pKa shift strategy. This was realized through a ternary assembly constructed by the dual host‒guest interactions of an adamantane-bis(diamine) conjugate (ADA) with cucurbit[6]uril (CB[6]) and a polysaccharide. The potential application of such biocompatible nanostructure was further implemented by the selective transportation of small interfering RNA (siRNA) in a controlled manner. It is demonstrated that the strong encapsulation of the ADA’s diammonium tail by CB[6] not only reduced the cytotoxicity of the nano-scaled vehicle but also dramatically enhanced cation density through an obvious positive macrocycle-induced pKa shift, which eventually facilitated the subsequent siRNA binding. With a targeted polysaccharide shell containing a cyclodextrin‒hyaluronic acid conjugate, macrocycle-incorporated siRNA polyplexes were specifically delivered into malignant human prostate PC-3 cells. The supramolecular polysaccharide nanoparticles, the formation of which was enabled and promoted by the complexation-assisted pKa shift, may be used as a versatile tool for controlled capture and release of biofunctional substrates.

  5. Synergistic effects of metformin with liraglutide against endothelial dysfunction through GLP-1 receptor and PKA signalling pathway

    Science.gov (United States)

    Ke, Jing; Liu, Ye; Yang, Jin; Lu, Ran; Tian, Qing; Hou, Wenfang; Wang, Guang; Wei, Rui; Hong, Tianpei

    2017-01-01

    Metformin or glucagon-like peptide-1 (GLP-1) analogue liraglutide has cardiovascular benefits. However, it is not clear whether their combined treatment have additive or synergistic effects on the vasculature. In this study, human umbilical vein endothelial cells (HUVECs), exposed to palmitic acid (PA) to induce endothelial dysfunction, were incubated with metformin, liraglutide or their combination. High fat diet (HFD)-fed ApoE−/− mice were randomized into control, metformin, liraglutide, and combination treatment groups. Results showed that in PA-treated HUVECs and HFD-fed ApoE−/− mice, combination of metformin and liraglutide at lower dose significantly improved endothelial dysfunction compared with the single treatment. Metformin upregulated GLP-1 receptor (GLP-1R) level and protein kinase A (PKA) phosphorylation. However, PKA inhibition but not GLP-1R blockade eliminated the protective effects of metformin on endothelial function. Furthermore, AMPK inhibitor compound C abolished the metformin-mediated upregulation of GLP-1R level and PKA phosphorylation. In conclusion, combination of metformin and liraglutide has synergistic protective effects on endothelial function. Moreover, metformin stimulates GLP-1R and PKA signalling via AMPK-dependent pathway, which may account for its synergistic protective effects with liraglutide. Our findings provide new insights on the interaction between metformin and GLP-1, and provide important information for designing new GLP-1-based therapy strategies in treating type 2 diabetes. PMID:28145471

  6. Accurate pKa Calculation of the Conjugate Acids of Alkanolamines, Alkaloids and Nucleotide Bases by Quantum Chemical Methods

    NARCIS (Netherlands)

    Gangarapu, S.; Marcelis, A.T.M.; Zuilhof, H.

    2013-01-01

    The pKa of the conjugate acids of alkanolamines, neurotransmitters, alkaloid drugs and nucleotide bases are calculated with density functional methods (B3LYP, M08-HX and M11-L) and ab initio methods (SCS-MP2, G3). Implicit solvent effects are included with a conductor-like polarizable continuum

  7. Involvement of PI3K/Akt/FoxO3a and PKA/CREB Signaling Pathways in the Protective Effect of Fluoxetine Against Corticosterone-Induced Cytotoxicity in PC12 Cells.

    Science.gov (United States)

    Zeng, Bingqing; Li, Yiwen; Niu, Bo; Wang, Xinyi; Cheng, Yufang; Zhou, Zhongzhen; You, Tingting; Liu, Yonggang; Wang, Haitao; Xu, Jiangping

    2016-08-01

    The selective serotonin reuptake inhibitor fluoxetine is neuroprotective in several brain injury models. It is commonly used to treat major depressive disorder and related conditions, but its mechanism of action remains incompletely understood. Activation of the phosphatidylinositol-3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FoxO3a) and protein kinase A/cAMP-response element binding protein (PKA/CREB) signaling pathways has been strongly implicated in the pathogenesis of depression and might be the downstream target of fluoxetine. Here, we used PC12 cells exposed to corticosterone (CORT) to study the neuroprotective effects of fluoxetine and the involvement of the PI3K/Akt/FoxO3a and PKA/CREB signaling pathways. Our results show that CORT reduced PC12 cells viability by 70 %, and that fluoxetine showed a concentration-dependent neuroprotective effect. Neuroprotective effects of fluoxetine were abolished by inhibition of PI3K, Akt, and PKA using LY294002, KRX-0401, and H89, respectively. Treatment of PC12 cells with fluoxetine resulted in increased phosphorylation of Akt, FoxO3a, and CREB. Fluoxetine also dose-dependently rescued the phosphorylation levels of Akt, FoxO3a, and CREB, following administration of CORT (from 99 to 110, 56 to 170, 80 to 170 %, respectively). In addition, inhibition of PKA and PI3K/Akt resulted in decreased levels of p-CREB, p-Akt, and p-FoxO3a in the presence of fluoxetine. Furthermore, fluoxetine reversed CORT-induced upregulation of p53-upregulated modulator of apoptosis (Puma) and Bcl-2-interacting mediator of cell death (Bim) via the PI3K/Akt/FoxO3a signaling pathway. H89 treatment reversed the effect of fluoxetine on the mRNA level of brain-derived neurotrophic factor, which was decreased in the presence of CORT. Our data indicate that fluoxetine elicited neuroprotection toward CORT-induced cell death that involves dual regulation from PI3K/Akt/FoxO3a and PKA/CREB pathways.

  8. Lipopolysaccharide (LPS-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR.

    Directory of Open Access Journals (Sweden)

    Christy E Trussoni

    Full Text Available Cholangiocytes (biliary epithelial cells actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells, or low passage normal human cholangiocytes (NHC, were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (p<0.05 and proliferation (p<0.01. Additionally, cholangiocytes from LPS-treated mouse livers and human primary sclerosing cholangitis (PSC livers exhibited increased phospho-EGFR (p<0.01. Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes.

  9. Lipopolysaccharide (LPS)-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR).

    Science.gov (United States)

    Trussoni, Christy E; Tabibian, James H; Splinter, Patrick L; O'Hara, Steven P

    2015-01-01

    Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (pphospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes.

  10. Pro-osteogenic trophic effects by PKA activation in human mesenchymal stromal cells

    NARCIS (Netherlands)

    Doorn, Joyce; Peppel, van de Jeroen; Leeuwen, van Johannes P.T.M.; Groen, Nathalie; Blitterswijk, van Clemens A.; Boer, de Jan

    2011-01-01

    Human mesenchymal stromal cells (hMSCs) are able to differentiate into a wide variety of cell types, which makes them an interesting source for tissue engineering applications. On the other hand, these cells also secrete a broad panel of growth factors and cytokines that can exert trophic effects on

  11. Mechanisms for luteinizing hormone induction of growth hormone gene transcription in fish model: crosstalk of the cAMP/PKA pathway with MAPK-and PI3K-dependent cascades.

    Science.gov (United States)

    Sun, Caiyun; He, Mulan; Ko, Wendy K W; Wong, Anderson O L

    2014-02-15

    In our previous studies in grass carp pituitary cells, local production of luteinizing hormone (LH) was shown to induce growth hormone (GH) production and gene expression, which constitutes a major component of the "intrapituitary feedback loop" regulating GH secretion and synthesis via autocrine/paracrine interactions between gonadotrophs and somatotrophs in the carp pituitary. To further investigate the signaling mechanisms mediating LH action at the transcriptional level, promoter studies were performed in GH3 cells co-transfected with the expression vector for carp LH receptor and luciferase-expressing reporter constructs with grass carp GH promoter. In this cell model, treatment with human chorionic gonadotropin (hCG) was effective in increasing GH promoter activity and the responsive sequence was mapped to position -616 and -572 of the grass carp GH promoter. GH promoter activation induced by hCG occurred with concurrent rise in cAMP production, CREB phosphorylation, and could be inhibited by inactivation of adenylate cyclase (AC), PKA, MEK1/2, P(38) MAPK, PI3K and mTOR. AC activation, presumably via cAMP production, could mimic hCG-induced CREB phosphorylation and GH promoter activity, and these stimulatory effects were also sensitive to the blockade of PKA-, MAPK- and PI3K- dependent cascades. These results, as a whole, suggest that LH receptor activation in the carp pituitary may trigger GH gene transcription through CREB phosphorylation as a result of the functional crosstalk of the cAMP/PKA pathway with MAPK-and PI3K-dependent cascades.

  12. Haemolysis induced by α-toxin from Staphylococcus aureus requires P2X receptor activation

    DEFF Research Database (Denmark)

    Skals, Marianne Gerberg; Leipziger, Jens Georg; Prætorius, Helle

    2011-01-01

    -forming bacterial toxins. In this context, it is essential to know whether this is specific to HlyA-induced cell damage or if other bacterial pore-forming toxins involve purinergic signals to orchestrate haemolysis. Here, we investigate if the haemolysis produced by α-toxin from Staphylococcus aureus involves P2...... receptor activation. We observed that α-toxin-induced haemolysis is completely blocked by the unselective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid. Moreover, several selective blockers of P2X1 and P2X7 ionotropic receptors abolished haemolysis in murine and equine...... erythrocytes. Inhibitors of pannexin channels partially reduced the α-toxin induced lysis. Thus, we conclude that α-toxin, similar to HlyA from E. coli produces cell damage by specific activation of a purinergic signalling cascade. These data indicate that pore-forming toxins in general require purinergic...

  13. 77 FR 16796 - Lead Requirements for Lead-Based Paint Activities in Target Housing and Child-Occupied Facilities...

    Science.gov (United States)

    2012-03-22

    ... AGENCY 40 CFR Part 745 Lead Requirements for Lead-Based Paint Activities in Target Housing and Child... requirements, training program accreditation requirements, and work practice standards for lead-based paint... the Arkansas lead-based paint program and passed a new statute establishing a State lead-based paint...

  14. Load requirements for maintaining structural integrity of Hanford single-shell tanks during waste feed delivery and retrieval activities

    Energy Technology Data Exchange (ETDEWEB)

    JULYK, L.J.

    1999-09-22

    This document provides structural load requirements and their basis for maintaining the structural integrity of the Hanford Single-Shell Tanks during waste feed delivery and retrieval activities. The requirements are based on a review of previous requirements and their basis documents as well as load histories with particular emphasis on the proposed lead transfer feed tanks for the privatized vitrification plant.

  15. 28 CFR 5.210 - Amount of detail required in information relating to registrant's activities and expenditures.

    Science.gov (United States)

    2010-07-01

    ... § 5.210 Amount of detail required in information relating to registrant's activities and expenditures... 28 Judicial Administration 1 2010-07-01 2010-07-01 false Amount of detail required in information relating to registrant's activities and expenditures. 5.210 Section 5.210 Judicial...

  16. 20 CFR 404.1321 - Ninety-day active service requirement for post-World War II veterans.

    Science.gov (United States)

    2010-04-01

    ... post-World War II veterans. 404.1321 Section 404.1321 Employees' Benefits SOCIAL SECURITY... of the Uniformed Services Post-World War II Veterans § 404.1321 Ninety-day active service requirement for post-World War II veterans. (a) The 90 days of active service required for post-World War...

  17. The exonuclease activity of DNA polymerase γ is required for ligation during mitochondrial DNA replication

    Science.gov (United States)

    Macao, Bertil; Uhler, Jay P.; Siibak, Triinu; Zhu, Xuefeng; Shi, Yonghong; Sheng, Wenwen; Olsson, Monica; Stewart, James B.; Gustafsson, Claes M.; Falkenberg, Maria

    2015-01-01

    Mitochondrial DNA (mtDNA) polymerase γ (POLγ) harbours a 3′–5′ exonuclease proofreading activity. Here we demonstrate that this activity is required for the creation of ligatable ends during mtDNA replication. Exonuclease-deficient POLγ fails to pause on reaching a downstream 5′-end. Instead, the enzyme continues to polymerize into double-stranded DNA, creating an unligatable 5′-flap. Disease-associated mutations can both increase and decrease exonuclease activity and consequently impair DNA ligation. In mice, inactivation of the exonuclease activity causes an increase in mtDNA mutations and premature ageing phenotypes. These mutator mice also contain high levels of truncated, linear fragments of mtDNA. We demonstrate that the formation of these fragments is due to impaired ligation, causing nicks near the origin of heavy-strand DNA replication. In the subsequent round of replication, the nicks lead to double-strand breaks and linear fragment formation. PMID:26095671

  18. Intracellular disassembly and activity of pertussis toxin require interaction with ATP.

    Science.gov (United States)

    Plaut, Roger D; Scanlon, Karen M; Taylor, Michael; Teter, Ken; Carbonetti, Nicholas H

    2016-08-01

    The active subunit (S1) of pertussis toxin (PT), a major virulence factor of Bordetella pertussis, ADP-ribosylates Gi proteins in the mammalian cell cytosol to inhibit GPCR signaling. The intracellular pathway of PT includes endocytosis and retrograde transport to the trans-Golgi network (TGN) and endoplasmic reticulum (ER). Subsequent translocation of S1 to the cytosol is presumably preceded by dissociation from the holotoxin. In vitro, such dissociation is stimulated by interaction of PT with ATP. To investigate the role of this interaction in cellular events, we engineered a form of PT (PTDM) with changes to two amino acids involved in the interaction with ATP. PTDM was reduced in (1) binding to ATP, (2) dissociability by interaction with ATP, (3) in vitro enzymatic activity and (4) cellular ADP-ribosylation activity. In cells treated with PTDM carrying target sequences for organelle-specific modifications, normal transport to the TGN and ER occurred, but N-glycosylation patterns of the S1 and S4 subunits were consistent with an inability of PTDM to dissociate in the ER. These results indicate a requirement for interaction with ATP for PT dissociation in the ER and cellular activity. They also indicate that the retrograde transport route is the cellular intoxication pathway for PT.

  19. Activation of the Erk Pathway Is Required for TGF-β1-Induced EMT In Vitro

    Directory of Open Access Journals (Sweden)

    Lu Xie

    2004-09-01

    Full Text Available Transforming growth factor-β1 (TGF-β1 can be tumorsuppressive through the activation of the Smadmediated signaling pathway. TGF-β1 can also enhance tumor progression by stimulating epithelial-tomesenchymal transition (EMT through additional pathways. EMT is characterized by the acquisition of a fibroblast-like cell morphology, dissolution of tight junctions, disruption of adherence junctions, and formation of actin stress fibers. There is evidence linking the activation of mitogen-activated protein kinase pathways to the induction of TGF-α1-mediated EMT. However, the role of Erk in the induction of TGF-β1-mediated EMT remains unclear. TGF-β1 treatment of normal murine mammary gland (NMuMG epithelial cells resulted in increased gene expression of Ras, Raf, MEK1/2, and Erki/2, as shown by microarray analysis and real-time polymerase chain reaction. Upon 24 and 48 hours of treatment with TGIF-α1, NMuMG and mouse cortical tubule (MCT epithelial cells underwent EMT as shown by changes in cell morphology, delocalization of zonula occludens-1 and E-cadherin from cell-cell junctions, and formation of actin stress fibers. TGF-β1 treatment also resulted in increased levels of phosphorylated Erk and Erk kinase activity. Treatment with an MEK inhibitor, U0126, inhibited increased Erk phosphorylation and kinase activity, and blocked TGF-β1 -induced EMT in both cell lines. These data show that TGF-β1 induces the activation of the Erk signaling pathway, which is required for TGF-β1 -mediated EMT in vitro.

  20. Efficient T-cell priming and activation requires signaling through prostaglandin E2 (EP) receptors.

    Science.gov (United States)

    Sreeramkumar, Vinatha; Hons, Miroslav; Punzón, Carmen; Stein, Jens V; Sancho, David; Fresno, Manuel; Cuesta, Natalia

    2016-01-01

    Understanding the regulation of T-cell responses during inflammation and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. In this regard, prostaglandin E2 (PGE2) is mostly considered a myeloid-derived immunosuppressive molecule. We describe for the first time that T cells secrete PGE2 during T-cell receptor stimulation. In addition, we show that autocrine PGE2 signaling through EP receptors is essential for optimal CD4(+) T-cell activation in vitro and in vivo, and for T helper 1 (Th1) and regulatory T cell differentiation. PGE2 was found to provide additive co-stimulatory signaling through AKT activation. Intravital multiphoton microscopy showed that triggering EP receptors in T cells is also essential for the stability of T cell-dendritic cell (DC) interactions and Th-cell accumulation in draining lymph nodes (LNs) during inflammation. We further demonstrated that blocking EP receptors in T cells during the initial phase of collagen-induced arthritis in mice resulted in a reduction of clinical arthritis. This could be attributable to defective T-cell activation, accompanied by a decline in activated and interferon-γ-producing CD4(+) Th1 cells in draining LNs. In conclusion, we prove that T lymphocytes secret picomolar concentrations of PGE2, which in turn provide additive co-stimulatory signaling, enabling T cells to attain a favorable activation threshold. PGE2 signaling in T cells is also required for maintaining long and stable interactions with DCs within LNs. Blockade of EP receptors in vivo impairs T-cell activation and development of T cell-mediated inflammatory responses. This may have implications in various pathophysiological settings.

  1. Activation of extracellular signal-regulated kinase but not of p38 mitogen-activated protein kinase pathways in lymphocytes requires allosteric activation of SOS.

    Science.gov (United States)

    Jun, Jesse E; Yang, Ming; Chen, Hang; Chakraborty, Arup K; Roose, Jeroen P

    2013-06-01

    Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation.

  2. The information activity of rail passenger information staff: a foundation for information system requirements

    Directory of Open Access Journals (Sweden)

    Martin Rose

    2006-01-01

    Full Text Available Introduction. This paper examines the goal-directed information activity of passenger information staff, working in the dynamic environment of rail network control. The explicit aim is to define a meaningful set of information system requirements. The report shows how dynamic situations may lead us to question a number of established theories of information science. Method. Passenger information officers (PIOs were observed on duty within the rail command and control headquarters. Observation sessions totally eight hours involved the manual recording of sequential information flows and the associated activity of PIOs. A semi-structured management interview was also conducted to provide further insight into the organizational context. Analysis. A viewpoint-oriented analysis technique was used to analyse sequential data captured during observation sessions. Event sequences that represent and explain the viewpoints were identified and elaborated into detailed scenario descriptions. Results. The analysis both supports and contests a number of established theories from information science. Additionally, a range of 'mandatory' and 'desirable' system requirements are derived from the scenario and viewpoint analyses. Conclusion. Dynamic situations have a significant impact on information behaviour which is not always predicted by current theories of information science.

  3. Histone acetyltransferase activity of MOF is required for adult but not early fetal hematopoiesis in mice.

    Science.gov (United States)

    Valerio, Daria G; Xu, Haiming; Eisold, Meghan E; Woolthuis, Carolien M; Pandita, Tej K; Armstrong, Scott A

    2017-01-05

    K(lysine) acetyltransferase 8 (KAT8, also known as MOF) mediates the acetylation of histone H4 at lysine 16 (H4K16ac) and is crucial for murine embryogenesis. Lysine acetyltransferases have been shown to regulate various stages of normal hematopoiesis. However, the function of MOF in hematopoietic stem cell (HSC) development has not yet been elucidated. We set out to study the role of MOF in general hematopoiesis by using a Vav1-cre-induced conditional murine Mof knockout system and found that MOF is critical for hematopoietic cell maintenance and HSC engraftment capacity in adult hematopoiesis. Rescue experiments with a MOF histone acetyltransferase domain mutant illustrated the requirement for MOF acetyltransferase activity in the clonogenic capacity of HSCs and progenitors. In stark contrast, fetal steady-state hematopoiesis at embryonic day (E) 14.5 was not affected by homozygous Mof deletion despite dramatic loss of global H4K16ac. Hematopoietic defects start manifesting in late gestation at E17.5. The discovery that MOF and its H4K16ac activity are required for adult but not early and midgestational hematopoiesis supports the notion that multiple chromatin regulators may be crucial for hematopoiesis at varying stages of development. MOF is therefore a developmental-stage-specific chromatin regulator found to be essential for adult but not early fetal hematopoiesis.

  4. A modal H∞-norm-based performance requirement for damage-tolerant active controller design

    Science.gov (United States)

    Genari, Helói F. G.; Mechbal, Nazih; Coffignal, Gérard; Nóbrega, Eurípedes G. O.

    2017-04-01

    Damage-tolerant active control (DTAC) is a recent research area that encompasses control design methodologies resulting from the application of fault-tolerant control methods to vibration control of structures subject to damage. The possibility of damage occurrence is not usually considered in the active vibration control design requirements. Damage changes the structure dynamics, which may produce unexpected modal behavior of the closed-loop system, usually not anticipated by the controller design approaches. A modal H∞ norm and a respective robust controller design framework were recently introduced, and this method is here extended to face a new DTAC strategy implementation. Considering that damage affects each vibration mode differently, this paper adopts the modal H∞ norm to include damage as a design requirement. The basic idea is to create an appropriate energy distribution over the frequency range of interest and respective vibration modes, guaranteeing robustness, damage tolerance, and adequate overall performance, taking into account that it is common to have previous knowledge of the structure regions where damage may occur during its operational life. For this purpose, a structural health monitoring technique is applied to evaluate modal modifications caused by damage. This information is used to create modal weighing matrices, conducting to the modal H∞ controller design. Finite element models are adopted for a case study structure, including different damage severities, in order to validate the proposed control strategy. Results show the effectiveness of the proposed methodology with respect to damage tolerance.

  5. Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity.

    Science.gov (United States)

    Vielle-Calzada, J P; Thomas, J; Spillane, C; Coluccio, A; Hoeppner, M A; Grossniklaus, U

    1999-11-15

    In higher plants, seed development requires maternal gene activity in the haploid (gametophytic) as well as diploid (sporophytic) tissues of the developing ovule. The Arabidopsis thaliana gene MEDEA (MEA) encodes a SET-domain protein of the Polycomb group that regulates cell proliferation by exerting a gametophytic maternal control during seed development. Seeds derived from female gametocytes (embryo sacs) carrying a mutant mea allele abort and exhibit cell proliferation defects in both the embryo and the endosperm. In this study we show that the mea mutation affects an imprinted gene expressed maternally in cells of the female gametophyte and after fertilization only from maternally inherited MEA alleles. Paternally inherited MEA alleles are transcriptionally silent in both the young embryo and endosperm. Mutations at the decrease in DNA methylation1 (ddm1) locus are able to rescue mea seeds by functionally reactivating paternally inherited MEA alleles during seed development. Rescued seeds are larger than the wild type and exhibit some of the abnormalities found in aborting mea seeds. Our results indicate that the maintenance of the genomic imprint at the mea locus requires zygotic DDM1 activity. Because DDM1 encodes a putative chromatin remodeling factor, chromatin structure is likely to be interrelated with genomic imprinting in Arabidopsis.

  6. Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex.

    Science.gov (United States)

    Wolle, Daniel; Cleard, Fabienne; Aoki, Tsutomu; Deshpande, Girish; Schedl, Paul; Karch, Francois

    2015-11-01

    Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C.

  7. VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation

    Science.gov (United States)

    Lampropoulou, Anastasia; Senatore, Valentina; Brash, James T.; Liyanage, Sidath E.; Raimondi, Claudio; Bainbridge, James W.

    2017-01-01

    The vascular endothelial growth factor (VEGF) isoform VEGF165 stimulates vascular growth and hyperpermeability. Whereas blood vessel growth is essential to sustain organ health, chronic hyperpermeability causes damaging tissue edema. By combining in vivo and tissue culture models, we show here that VEGF165-induced vascular leakage requires both VEGFR2 and NRP1, including the VEGF164-binding site of NRP1 and the NRP1 cytoplasmic domain (NCD), but not the known NCD interactor GIPC1. In the VEGF165-bound receptor complex, the NCD promotes ABL kinase activation, which in turn is required to activate VEGFR2-recruited SRC family kinases (SFKs). These results elucidate the receptor complex and signaling hierarchy of downstream kinases that transduce the permeability response to VEGF165. In a mouse model with choroidal neovascularisation akin to age-related macular degeneration, NCD loss attenuated vessel leakage without affecting neovascularisation. These findings raise the possibility that targeting NRP1 or its NCD interactors may be a useful therapeutic strategy in neovascular disease to reduce VEGF165-induced edema without compromising vessel growth. PMID:28289053

  8. The Demethylase Activity of FTO (Fat Mass and Obesity Associated Protein Is Required for Preadipocyte Differentiation.

    Directory of Open Access Journals (Sweden)

    Meizi Zhang

    Full Text Available FTO (fat mass and obesity associated gene was genetically identified to be associated with body mass index (BMI, presumably through functional regulation of energy homeostasis. However, the cellular and molecular mechanisms by which FTO functions remain largely unknown. Using 3T3-L1 preadipocyte as a model to study the role of FTO in adipogenesis, we demonstrated that FTO is functionally required for 3T3-L1 differentiation. FTO knock-down with siRNA inhibited preadipocyte differentiation, whereas ectopic over-expression of FTO enhanced the process. The demethylase activity of FTO is required for differentiation. Level of N6-methyladenosine (m6A is decreased in cells over-expressing FTO. In contrast, overexpression of R96Q, a FTO missense mutant lack of demethylase activity, had no effect on cellular m6A level and impeded differentiation. Treatment with Rosiglitazone, a PPARγ agonist, could overcome the differentiation inhibition imposed by R96Q mutant, suggesting the effect of FTO is mediated through PPARγ.

  9. Ornithine Decarboxylase Activity Is Required for Prostatic Budding in the Developing Mouse Prostate.

    Directory of Open Access Journals (Sweden)

    Melissa Gamat

    Full Text Available The prostate is a male accessory sex gland that produces secretions in seminal fluid to facilitate fertilization. Prostate secretory function is dependent on androgens, although the mechanism by which androgens exert their effects is still unclear. Polyamines are small cationic molecules that play pivotal roles in DNA transcription, translation and gene regulation. The rate-limiting enzyme in polyamine biosynthesis is ornithine decarboxylase, which is encoded by the gene Odc1. Ornithine decarboxylase mRNA decreases in the prostate upon castration and increases upon administration of androgens. Furthermore, testosterone administered to castrated male mice restores prostate secretory activity, whereas administering testosterone and the ornithine decarboxylase inhibitor D,L-α-difluromethylornithine (DFMO to castrated males does not restore prostate secretory activity, suggesting that polyamines are required for androgens to exert their effects. To date, no one has examined polyamines in prostate development, which is also androgen dependent. In this study, we showed that ornithine decarboxylase protein was expressed in the epithelium of the ventral, dorsolateral and anterior lobes of the adult mouse prostate. Ornithine decarboxylase protein was also expressed in the urogenital sinus (UGS epithelium of the male and female embryo prior to prostate development, and expression continued in prostatic epithelial buds as they emerged from the UGS. Inhibiting ornithine decarboxylase using DFMO in UGS organ culture blocked the induction of prostatic buds by androgens, and significantly decreased expression of key prostate transcription factor, Nkx3.1, by androgens. DFMO also significantly decreased the expression of developmental regulatory gene Notch1. Other genes implicated in prostatic development including Sox9, Wif1 and Srd5a2 were unaffected by DFMO. Together these results indicate that Odc1 and polyamines are required for androgens to exert their

  10. Maintenance of neuronal size gradient in MNTB requires sound-evoked activity

    Science.gov (United States)

    Weatherstone, Jessica H.; Pilati, Nadia; Wang, Yuan; Forsythe, Ian D.; Rubel, Edwin W.; Tempel, Bruce L.

    2016-01-01

    The medial nucleus of the trapezoid body (MNTB) is an important source of inhibition during the computation of sound location. It transmits fast and precisely timed action potentials at high frequencies; this requires an efficient calcium clearance mechanism, in which plasma membrane calcium ATPase 2 (PMCA2) is a key component. Deafwaddler (dfw2J) mutant mice have a null mutation in PMCA2 causing deafness in homozygotes (dfw2J/dfw2J) and high-frequency hearing loss in heterozygotes (+/dfw2J). Despite the deafness phenotype, no significant differences in MNTB volume or cell number were observed in dfw2J homozygous mutants, suggesting that PMCA2 is not required for MNTB neuron survival. The MNTB tonotopic axis encodes high to low sound frequencies across the medial to lateral dimension. We discovered a cell size gradient along this axis: lateral neuronal somata are significantly larger than medially located somata. This size gradient is decreased in +/dfw2J and absent in dfw2J/dfw2J. The lack of acoustically driven input suggests that sound-evoked activity is required for maintenance of the cell size gradient. This hypothesis was corroborated by selective elimination of auditory hair cell activity with either hair cell elimination in Pou4f3 DTR mice or inner ear tetrodotoxin (TTX) treatment. The change in soma size was reversible and recovered within 7 days of TTX treatment, suggesting that regulation of the gradient is dependent on synaptic activity and that these changes are plastic rather than permanent. NEW & NOTEWORTHY Neurons of the medial nucleus of the trapezoid body (MNTB) act as fast-spiking inhibitory interneurons within the auditory brain stem. The MNTB is topographically organized, with low sound frequencies encoded laterally and high frequencies medially. We discovered a cell size gradient along this axis: lateral neurons are larger than medial neurons. The absence of this gradient in deaf mice lacking plasma membrane calcium ATPase 2 suggests an

  11. Regulation of basal and reserve cardiac pacemaker function by interactions of cAMP mediated PKA-dependent Ca2+ cycling with surface membrane channels

    Science.gov (United States)

    Vinogradova, Tatiana M.; Lakatta, Edward G.

    2009-01-01

    Decades of intensive research of primary cardiac pacemaker, the sinoatrial node, have established potential roles of specific membrane channels in the generation of the diastolic depolarization, the major mechanism allowing sinoatrial node cells generate spontaneous beating. During the last three decades, multiple studies made either in the isolated sinoatrial node or sinoatrial node cells have demonstrated a pivotal role of Ca2+ and, specifically Ca2+-release from sarcoplasmic reticulum, for spontaneous beating of cardiac pacemaker. Recently, spontaneous, rhythmic local subsarcolemmal Ca2+ releases from ryanodine receptors during late half of the diastolic depolarization have been implicated as a vital factor in the generation of sinoatrial node cells spontaneous firing. Local Ca2+ releases are driven by a unique combination of high basal cAMP production by adenylyl cyclases, high basal cAMP degradation by phosphodiesterases and a high level of cAMP-mediated PKA-dependent phosphorylation. These local Ca2+ releases activate an inward Na+-Ca2+ exchange current which accelerates the terminal diastolic depolarization rate and, thus, controls the spontaneous pacemaker firing. Both the basal primary pacemaker beating rate and its modulation via β-adrenergic receptor stimulation appear to be critically dependent upon intact RyR function and local subsarcolemmal sarcoplasmic reticulum generated Ca2+ releases. This review aspires to integrate the traditional viewpoint that has emphasized the supremacy of the ensemble of surface membrane ion channels in spontaneous firing of the primary cardiac pacemaker, and these novel perspectives of cAMP-mediated PKA-dependent Ca2+ cycling in regulation of the heart pacemaker clock, both in the basal state and during β-adrenergic receptor stimulation. PMID:19573534

  12. Chronic stress-induced dendritic reorganization and abundance of synaptosomal PKA-dependent CP-AMPA receptor in the basolateral amygdala in a mouse model of depression.

    Science.gov (United States)

    Yi, Eun-Surk; Oh, Seikwan; Lee, Jang-Kyu; Leem, Yea-Hyun

    2017-05-06

    Chronic stress is a precipitating factor for disorders including depression. The basolateral amygdala (BLA) is a critical substrate that interconnects with stress-modulated neural networks to generate emotion- and mood-related behaviors. The current study shows that 3 h per day of restraint stress for 14 days caused mice to exhibit long-term depressive behaviors, manifested by disrupted sociality and despair levels, which were rescued by fluoxetine. These behavioral changes corresponded with morphological and molecular changes in BLA neurons, including chronic stress-elicited increases in arborization, dendritic length, and spine density of BLA principal neurons. At the molecular level, calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (CP-AMPARs) within the synaptosome exhibited an increased GluR1:GluR2 subunit ratio. We also observed increased GluR1 phosphorylation at Ser 845 and enhanced cyclic AMP-dependent protein kinase (PKA) activity in the BLA. These molecular changes reverted to the basal state post-treatment with fluoxetine. The expression of synaptophysin (SYP) and postsynaptic density protein 95 (PSD-95) at BLA neuronal synapses was also enhanced by chronic stress, which was reversed post-treatment. Finally, chronic stress-provoked depressive behavior was overcome by local blockage of CP-AMPARs in the BLA via stereotaxic injection (IEM-1460). Chronic stress-elicited depressive behavior may be due to hypertrophy of BLA neuronal dendrites and increased of PKA-dependent CP-AMPAR levels in BLA neurons. Furthermore, fluoxetine can reverse chronic stress-triggered cytoarchitectural and functional changes of BLA neurons. These findings provide insights into depression-linked structural and functional changes in BLA neurons. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Histone Acetyltransferase Activity of MOF Is Required for MLL-AF9 Leukemogenesis.

    Science.gov (United States)

    Valerio, Daria G; Xu, Haiming; Chen, Chun-Wei; Hoshii, Takayuki; Eisold, Meghan E; Delaney, Christopher; Cusan, Monica; Deshpande, Aniruddha J; Huang, Chun-Hao; Lujambio, Amaia; Zheng, YuJun George; Zuber, Johannes; Pandita, Tej K; Lowe, Scott W; Armstrong, Scott A

    2017-02-15

    Chromatin-based mechanisms offer therapeutic targets in acute myeloid leukemia (AML) that are of great current interest. In this study, we conducted an RNAi-based screen to identify druggable chromatin regulator-based targets in leukemias marked by oncogenic rearrangements of the MLL gene. In this manner, we discovered the H4K16 histone acetyltransferase (HAT) MOF to be important for leukemia cell growth. Conditional deletion of Mof in a mouse model of MLL-AF9-driven leukemogenesis reduced tumor burden and prolonged host survival. RNA sequencing showed an expected downregulation of genes within DNA damage repair pathways that are controlled by MOF, as correlated with a significant increase in yH2AX nuclear foci in Mof-deficient MLL-AF9 tumor cells. In parallel, Mof loss also impaired global H4K16 acetylation in the tumor cell genome. Rescue experiments with catalytically inactive mutants of MOF showed that its enzymatic activity was required to maintain cancer pathogenicity. In support of the role of MOF in sustaining H4K16 acetylation, a small-molecule inhibitor of the HAT component MYST blocked the growth of both murine and human MLL-AF9 leukemia cell lines. Furthermore, Mof inactivation suppressed leukemia development in an NUP98-HOXA9-driven AML model. Taken together, our results establish that the HAT activity of MOF is required to sustain MLL-AF9 leukemia and may be important for multiple AML subtypes. Blocking this activity is sufficient to stimulate DNA damage, offering a rationale to pursue MOF inhibitors as a targeted approach to treat MLL-rearranged leukemias. Cancer Res; 77(7); 1-10. ©2017 AACR.

  14. Epidermal hyperplasia induced by Raf-MAPK signaling requires Stat3 activation.

    Science.gov (United States)

    Tarutani, Masahito; Nakajima, Kimiko; Takaishi, Mikiro; Ohko, Kentaro; Sano, Shigetoshi

    2013-11-01

    Raf is one of the downstream effectors of Ras GTPases, and plays a key role in regulating cell proliferation and differentiation through the activation of MAPK. We have previously demonstrated that temporal induction of Raf in the epidermis of K14-Raf:ER transgenic mice results in epidermal hyperplasia resembling squamous cell carcinoma and psoriasis. It has been demonstrated that epidermal Stat3 activation is required for psoriasis development, since keratinocyte-specific Stat3 activation in a mouse model elicits a psoriasis-like phenotype, which is reversed by inhibition of Stat3 signaling. The aim of this study was whether Stat3 signaling is involved in Raf-MAPK-dependent epidermal hyperplasia. K14-Raf:ER transgenic mice, in which the 4-hydroxytamoxifen (4OHT)-responsive mutant estrogen receptor ligand binding domain-Raf fusion gene is expressed under control of the keratin 14 promoter, were mated with epidermis-specific Stat3 null mice (K5-Cre.Stat3(flox/flox)). K5-Cre.Stat3(flox/flox) mice were used to define the impact of Stat3 deficiency on Raf-induced epidermal hyperplasia. Over-expression of Raf by 4OHT treatment in K14-Raf:ER;K5-Cre.Stat3(flox/flox) mice greatly attenuated the epidermal hyperplasia and dermal cell infiltrates compared with K14-Raf:ER;K5-Cre.Stat3(flox/WT) mice. Also, up-regulation of psoriasis-associated cytokine profiles, including VEGF, was inhibited in the skin from K14-Raf:ER;K5-Cre.Stat3(flox/flox) mice following 4OHT treatment. These results clearly indicate that Raf-MAPK-dependent psoriatic-like epidermal hyperplasia requires Stat3 signaling in keratinocytes. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  15. Simvastatin requires activation in accessory cells to modulate T-cell responses in asthma and COPD.

    Science.gov (United States)

    Knobloch, Jürgen; Yakin, Yakup; Körber, Sandra; Grensemann, Barbara; Bendella, Zeynep; Boyaci, Niyazi; Gallert, Willem-Jakob; Yanik, Sarah Derya; Jungck, David; Koch, Andrea

    2016-10-05

    T-cell-dependent airway and systemic inflammation triggers the progression of chronic obstructive pulmonary disease (COPD) and asthma. Retrospective studies suggest that simvastatin has anti-inflammatory effects in both diseases but it is unclear, which cell types are targeted. We hypothesized that simvastatin modulates T-cell activity. Circulating CD4+ and CD8+ T-cells, either pure, co-cultured with monocytes or alveolar macrophages (AM) or in peripheral blood mononuclear cells (PBMCs), were ex vivo activated towards Th1/Tc1 or Th2/Tc2 and incubated with simvastatin. Markers for Th1/Tc1 (IFNγ) and Th2/Tc2 (IL-5, IL-13) were measured by ELISA; with PBMCs this was done comparative between 11 healthy never-smokers, 11 current smokers without airflow limitation, 14 smokers with COPD and 11 never-smokers with atopic asthma. T-cell activation induced IFNγ, IL-5 and IL-13 in the presence and absence of accessory cells. Simvastatin did not modulate cytokine expression in pure T-cell fractions. β-hydroxy-simvastatin acid (activated simvastatin) suppressed IL-5 and IL-13 in pure Th2- and Tc2-cells. Simvastatin suppressed IL-5 and IL-13 in Th2-cells co-cultivated with monocytes or AM, which was partially reversed by the carboxylesterase inhibitor benzil. Simvastatin suppressed IL-5 production of Th2/Tc2-cells in PBMCs without differences between cohorts and IL-13 stronger in never-smokers and asthma compared to COPD. Simvastatin induced IFNγ in Th1/Tc1-cells in PBMCs of all cohorts except asthmatics. Simvastatin requires activation in accessory cells likely by carboxylesterase to suppress IL-5 and IL-13 in Th2/Tc2-cells. The effects on Il-13 are partially reduced in COPD. Asthma pathogenesis prevents simvastatin-induced IFNγ up-regulation. Simvastatin has anti-inflammatory effects that could be of interest for asthma therapy.

  16. Active materials for automotive adaptive forward lighting Part 1: system requirements vs. material properties

    Science.gov (United States)

    Keefe, Andrew C.; Browne, Alan L.; Johnson, Nancy L.

    2011-04-01

    Adaptive Frontlighting Systems (AFS in GM usage) improve visibility by automatically optimizing the beam pattern to accommodate road, driving and environmental conditions. By moving, modifying, and/or adding light during nighttime, inclement weather, or in sharp turns, the driver is presented with dynamic illumination not possible with static lighting systems The objective of this GM-HRL collaborative research project was to assess the potential of active materials to decrease the cost, mass, and packaging volume of current electric stepper-motor AFS designs. Solid-state active material actuators, if proved suitable for this application, could be less expensive than electric motors and have lower part count, reduced size and weight, and lower acoustic and EMF noise1. This paper documents Part 1 of the collaborative study, assessing technically mature, commercially available active materials for use as actuators. Candidate materials should reduce cost and improve AFS capabilities, such as increased angular velocity on swivel. Additional benefits to AFS resulting from active materials actuators were to be identified as well such as lower part count. In addition, several notional approaches to AFS were documented to illustrate the potential function, which is developed more fully in Part 2. Part 1 was successful in verifying the feasibility of using two active materials for AFS: shape memory alloys, and piezoelectrics. In particular, this demonstration showed that all application requirements including those on actuation speed, force, and cyclic stability to effect manipulation of the filament assembly and/or the reflector could be met by piezoelectrics (as ultrasonic motors) and SMA wire actuators.

  17. HERV-K activation is strictly required to sustain CD133+ melanoma cells with stemness features.

    Science.gov (United States)

    Argaw-Denboba, Ayele; Balestrieri, Emanuela; Serafino, Annalucia; Cipriani, Chiara; Bucci, Ilaria; Sorrentino, Roberta; Sciamanna, Ilaria; Gambacurta, Alessandra; Sinibaldi-Vallebona, Paola; Matteucci, Claudia

    2017-01-26

    Melanoma is a heterogeneous tumor in which phenotype-switching and CD133 marker have been associated with metastasis promotion and chemotherapy resistance. CD133 positive (CD133+) subpopulation has also been suggested as putative cancer stem cell (CSC) of melanoma tumor. Human endogenous retrovirus type K (HERV-K) has been described to be aberrantly activated during melanoma progression and implicated in the etiopathogenesis of disease. Earlier, we reported that stress-induced HERV-K activation promotes cell malignant transformation and reduces the immunogenicity of melanoma cells. Herein, we investigated the correlation between HERV-K and the CD133+ melanoma cells during microenvironmental modifications. TVM-A12 cell line, isolated in our laboratory from a primary human melanoma lesion, and other commercial melanoma cell lines (G-361, WM-115, WM-266-4 and A375) were grown and maintained in the standard and stem cell media. RNA interference, Real-time PCR, flow cytometry analysis, self-renewal and migration/invasion assays were performed to characterize cell behavior and HERV-K expression. Melanoma cells, exposed to stem cell media, undergo phenotype-switching and expansion of CD133+ melanoma cells, concomitantly promoted by HERV-K activation. Notably, the sorted CD133+ subpopulation showed stemness features, characterized by higher self-renewal ability, embryonic genes expression, migration and invasion capacities compared to the parental cell line. RNA interference-mediated downregulation experiments showed that HERV-K has a decisive role to expand and maintain the CD133+ melanoma subpopulation during microenvironmental modifications. Similarly, non nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine were effective to restrain the activation of HERV-K in melanoma cells, to antagonize CD133+ subpopulation expansion and to induce selective high level apoptosis in CD133+ cells. HERV-K activation promotes melanoma cells phenotype

  18. Programmed cell death activated by Rose Bengal in Arabidopsis thaliana cell suspension cultures requires functional chloroplasts.

    Science.gov (United States)

    Gutiérrez, Jorge; González-Pérez, Sergio; García-García, Francisco; Daly, Cara T; Lorenzo, Oscar; Revuelta, José L; McCabe, Paul F; Arellano, Juan B

    2014-07-01

    Light-grown Arabidopsis thaliana cell suspension culture (ACSC) were subjected to mild photooxidative damage with Rose Bengal (RB) with the aim of gaining a better understanding of singlet oxygen-mediated defence responses in plants. Additionally, ACSC were treated with H2O2 at concentrations that induced comparable levels of protein oxidation damage. Under low to medium light conditions, both RB and H2O2 treatments activated transcriptional defence responses and inhibited photosynthetic activity, but they differed in that programmed cell death (PCD) was only observed in cells treated with RB. When dark-grown ACSC were subjected to RB in the light, PCD was suppressed, indicating that the singlet oxygen-mediated signalling pathway in ACSC requires functional chloroplasts. Analysis of up-regulated transcripts in light-grown ACSC, treated with RB in the light, showed that both singlet oxygen-responsive transcripts and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present. A co-regulation analysis proved that ACSC treated with RB exhibited higher correlation with the conditional fluorescence (flu) mutant than with other singlet oxygen-producing mutants or wild-type plants subjected to high light. However, there was no evidence for the up-regulation of EDS1, suggesting that activation of PCD was not associated with the EXECUTER- and EDS1-dependent signalling pathway described in the flu mutant. Indigo Carmine and Methylene Violet, two photosensitizers unable to enter chloroplasts, did not activate transcriptional defence responses in ACSC; however, whether this was due to their location or to their inherently low singlet oxygen quantum efficiencies was not determined.

  19. CAMKII activation is not required for maintenance of learning-induced enhancement of neuronal excitability.

    Directory of Open Access Journals (Sweden)

    Ori Liraz

    Full Text Available Pyramidal neurons in the piriform cortex from olfactory-discrimination trained rats show enhanced intrinsic neuronal excitability that lasts for several days after learning. Such enhanced intrinsic excitability is mediated by long-term reduction in the post-burst after-hyperpolarization (AHP which is generated by repetitive spike firing. AHP reduction is due to decreased conductance of a calcium-dependent potassium current, the sI(AHP. We have previously shown that learning-induced AHP reduction is maintained by persistent protein kinase C (PKC and extracellular regulated kinase (ERK activation. However, the molecular machinery underlying this long-lasting modulation of intrinsic excitability is yet to be fully described. Here we examine whether the CaMKII, which is known to be crucial in learning, memory and synaptic plasticity processes, is instrumental for the maintenance of learning-induced AHP reduction. KN93, that selectively blocks CaMKII autophosphorylation at Thr286, reduced the AHP in neurons from trained and control rat to the same extent. Consequently, the differences in AHP amplitude and neuronal adaptation between neurons from trained rats and controls remained. Accordingly, the level of activated CaMKII was similar in pirifrom cortex samples taken form trained and control rats. Our data show that although CaMKII modulates the amplitude of AHP of pyramidal neurons in the piriform cortex, its activation is not required for maintaining learning-induced enhancement of neuronal excitability.

  20. CAMKII activation is not required for maintenance of learning-induced enhancement of neuronal excitability.

    Science.gov (United States)

    Liraz, Ori; Rosenblum, Kobi; Barkai, Edi

    2009-01-01

    Pyramidal neurons in the piriform cortex from olfactory-discrimination trained rats show enhanced intrinsic neuronal excitability that lasts for several days after learning. Such enhanced intrinsic excitability is mediated by long-term reduction in the post-burst after-hyperpolarization (AHP) which is generated by repetitive spike firing. AHP reduction is due to decreased conductance of a calcium-dependent potassium current, the sI(AHP). We have previously shown that learning-induced AHP reduction is maintained by persistent protein kinase C (PKC) and extracellular regulated kinase (ERK) activation. However, the molecular machinery underlying this long-lasting modulation of intrinsic excitability is yet to be fully described. Here we examine whether the CaMKII, which is known to be crucial in learning, memory and synaptic plasticity processes, is instrumental for the maintenance of learning-induced AHP reduction. KN93, that selectively blocks CaMKII autophosphorylation at Thr286, reduced the AHP in neurons from trained and control rat to the same extent. Consequently, the differences in AHP amplitude and neuronal adaptation between neurons from trained rats and controls remained. Accordingly, the level of activated CaMKII was similar in pirifrom cortex samples taken form trained and control rats. Our data show that although CaMKII modulates the amplitude of AHP of pyramidal neurons in the piriform cortex, its activation is not required for maintaining learning-induced enhancement of neuronal excitability.

  1. The CXXC finger 5 protein is required for DNA damage-induced p53 activation

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    The tumor suppressor p53 is a critical component of the DNA damage response pathway that induces a set of genes responsible for cell cycle arrest,senescence,apoptosis,and DNA repair.The ataxia te-langiectasia mutated protein kinase(ATM) responds to DNA-damage stimuli and signals p53 stabiliza-tion and activation,thereby facilitating transactivation of p53 inducible genes and maintainence of genome integrity.In this study,we identified a CXXC zinc finger domain containing protein termed CF5 as a critical component in the DNA damage signaling pathway.CF5 induces p53 transcriptional activity and apoptosis in cells expressing wild type p53 but not in p53-deficient cells.Knockdown of CF5 in-hibits DNA damage-induced p53 activation as well as cell cycle arrest.Furthermore,CF5 physically interacts with ATM and is required for DNA damage-induced ATM phosphorylation but not its recruitment to chromatin.These findings suggest that CF5 plays a crucial role in ATM-p53 signaling in response to DNA damage.

  2. Human Cells Require Non-stop Ribosome Rescue Activity in Mitochondria.

    Directory of Open Access Journals (Sweden)

    Heather A Feaga

    2016-03-01

    Full Text Available Bacteria use trans-translation and the alternative rescue factors ArfA (P36675 and ArfB (Q9A8Y3 to hydrolyze peptidyl-tRNA on ribosomes that stall near the 3' end of an mRNA during protein synthesis. The eukaryotic protein ICT1 (Q14197 is homologous to ArfB. In vitro ribosome rescue assays of human ICT1 and Caulobacter crescentus ArfB showed that these proteins have the same activity and substrate specificity. Both ArfB and ICT1 hydrolyze peptidyl-tRNA on nonstop ribosomes or ribosomes stalled with ≤6 nucleotides extending past the A site, but are unable to hydrolyze peptidyl-tRNA when the mRNA extends ≥14 nucleotides past the A site. ICT1 provided sufficient ribosome rescue activity to support viability in C. crescentus cells that lacked both trans-translation and ArfB. Likewise, expression of ArfB protected human cells from death when ICT1 was silenced with siRNA. These data indicate that ArfB and ICT1 are functionally interchangeable, and demonstrate that ICT1 is a ribosome rescue factor. Because ICT1 is essential in human cells, these results suggest that ribosome rescue activity in mitochondria is required in humans.

  3. t-Darpp stimulates protein kinase A activity by forming a complex with its RI regulatory subunit.

    Science.gov (United States)

    Theile, Dirk; Geng, Shuhui; Denny, Erin C; Momand, Jamil; Kane, Susan E

    2017-09-01

    t-Darpp is the truncated form of the dopamine- and cAMP-regulated phosphoprotein of 32kDa (Darpp-32) and has been demonstrated to confer resistance to trastuzumab, a Her2-targeted anticancer agent, via sustained signaling through the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt pathway and activation of protein kinase A (PKA). The mechanism of t-Darpp-mediated PKA activation is poorly understood. In the PKA holoenzyme, when the catalytic subunits are bound to regulatory subunits RI or RII, kinase activity is inhibited. We investigated PKA activity and holoenzyme composition in cell lines overexpressing t-Darpp (SK.tDp) or a T39A phosphorylation mutant (SK.tDp(T39A)), as well as an empty vector control cell line (SK.empty). We also evaluated protein-protein interactions between t-Darpp and PKA catalytic (PKAc) or regulatory subunits RI and RII in those cell lines. SK.tDp cells had elevated PKA activity and showed diminished association of RI with PKAc, whereas SK.tDp(T39A) cells did not have these properties. Moreover, wild type t-Darpp associates with RI. Concurrent expression of Darpp-32 reversed t-Darrp's effects on PKA holoenzyme state, consistent with earlier observations that Darpp-32 reverses t-Darpp's activation of PKA. Together, t-Darpp phosphorylation at T39 seems to be crucial for t-Darpp-mediated PKA activation and this activation appears to occur through an association with RI and sequestering of RI away from PKAc. The t-Darpp-RI interaction could be a druggable target to reduce PKA activity in drug-resistant cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Sustained activation of interferon regulatory factor 3 during infection by paramyxoviruses requires MDA5.

    Science.gov (United States)

    Grandvaux, Nathalie; Guan, Xiaochun; Yoboua, Fabrice; Zucchini, Nicolas; Fink, Karin; Doyon, Priscilla; Martin, Lydie; Servant, Marc J; Chartier, Stéfany

    2014-01-01

    Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are the main cytosolic sensors of single-stranded RNA viruses, including paramyxoviruses, and are required to initiate a quick and robust innate antiviral response. Despite different ligand-binding properties, the consensus view is that RIG-I and MDA5 trigger common signal(s) to activate interferon regulatory factor 3 (IRF-3) and NF-κB, and downstream antiviral and proinflammatory cytokine expression. Here, we performed a thorough analysis of the temporal involvement of RIG-I and MDA5 in the regulation of IRF-3 during respiratory syncytial virus (RSV) infection. Based on specific RNA interference-mediated knockdown of RIG-I and MDA5 in A549 cells, we confirmed that RIG-I is critical for the initiation of IRF-3 phosphorylation, dimerization and downstream gene expression. On the other hand, our experiments yielded the first evidence that knockdown of MDA5 leads to early ubiquitination and proteasomal degradation of active IRF-3. Conversely, ectopic expression of MDA5 prolonged RIG-I-induced IRF-3 activation. Altogether, we provide novel mechanistic insight into the temporal involvement of RIG-I and MDA5 in the innate antiviral response. While RIG-I is essential for initial IRF-3 activation, engagement of induced MDA5 is essential to prevent early degradation of IRF-3, thereby sustaining IRF-3-dependent antiviral gene expression. MDA5 plays a similar role during Sendai virus infection suggesting that this model is not restricted to RSV amongst paramyxoviruses.

  5. Protein phosphatases decrease their activity during capacitation: a new requirement for this event.

    Directory of Open Access Journals (Sweden)

    Janetti R Signorelli

    Full Text Available There are few reports on the role of protein phosphatases during capacitation. Here, we report on the role of PP2B, PP1, and PP2A during human sperm capacitation. Motile sperm were resuspended in non-capacitating medium (NCM, Tyrode's medium, albumin- and bicarbonate-free or in reconstituted medium (RCM, NCM plus 2.6% albumin/25 mM bicarbonate. The presence of the phosphatases was evaluated by western blotting and the subcellular localization by indirect immunofluorescence. The function of these phosphatases was analyzed by incubating the sperm with specific inhibitors: okadaic acid, I2, endothall, and deltamethrin. Different aliquots were incubated in the following media: 1 NCM; 2 NCM plus inhibitors; 3 RCM; and 4 RCM plus inhibitors. The percent capacitated sperm and phosphatase activities were evaluated using the chlortetracycline assay and a phosphatase assay kit, respectively. The results confirm the presence of PP2B and PP1 in human sperm. We also report the presence of PP2A, specifically, the catalytic subunit and the regulatory subunits PR65 and B. PP2B and PP2A were present in the tail, neck, and postacrosomal region, and PP1 was present in the postacrosomal region, neck, middle, and principal piece of human sperm. Treatment with phosphatase inhibitors rapidly (≤1 min increased the percent of sperm depicting the pattern B, reaching a maximum of ∼40% that was maintained throughout incubation; after 3 h, the percent of capacitated sperm was similar to that of the control. The enzymatic activity of the phosphatases decreased during capacitation without changes in their expression. The pattern of phosphorylation on threonine residues showed a sharp increase upon treatment with the inhibitors. In conclusion, human sperm express PP1, PP2B, and PP2A, and the activity of these phosphatases decreases during capacitation. This decline in phosphatase activities and the subsequent increase in threonine phosphorylation may be an important

  6. BDNF activates mTOR to regulate GluR1 expression required for memory formation.

    Directory of Open Access Journals (Sweden)

    Leandro Slipczuk

    Full Text Available BACKGROUND: The mammalian target of Rapamycin (mTOR kinase plays a key role in translational control of a subset of mRNAs through regulation of its initiation step. In neurons, mTOR is present at the synaptic region, where it modulates the activity-dependent expression of locally-translated proteins independently of mRNA synthesis. Indeed, mTOR is necessary for different forms of synaptic plasticity and long-term memory (LTM formation. However, little is known about the time course of mTOR activation and the extracellular signals governing this process or the identity of the proteins whose translation is regulated by this kinase, during mnemonic processing. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that consolidation of inhibitory avoidance (IA LTM entails mTOR activation in the dorsal hippocampus at the moment of and 3 h after training and is associated with a rapid and rapamycin-sensitive increase in AMPA receptor GluR1 subunit expression, which was also blocked by intra-hippocampal delivery of GluR1 antisense oligonucleotides (ASO. In addition, we found that pre- or post-training administration of function-blocking anti-BDNF antibodies into dorsal CA1 hampered IA LTM retention, abolished the learning-induced biphasic activation of mTOR and its readout, p70S6K and blocked GluR1 expression, indicating that BDNF is an upstream factor controlling mTOR signaling during fear-memory consolidation. Interestingly, BDNF ASO hindered LTM retention only when given into dorsal CA1 1 h after but not 2 h before training, suggesting that BDNF controls the biphasic requirement of mTOR during LTM consolidation through different mechanisms: an early one involving BDNF already available at the moment of training, and a late one, happening around 3 h post-training that needs de novo synthesis of this neurotrophin. CONCLUSIONS/SIGNIFICANCE: IN CONCLUSION, OUR FINDINGS DEMONSTRATE THAT: 1 mTOR-mediated mRNA translation is required for memory consolidation during

  7. Active control of periodic fan noise in laptops: spectral width requirements in delayed buffer implementation

    Directory of Open Access Journals (Sweden)

    H. A. Cordourier‐Maruri

    2009-08-01

    Full Text Available An active control system intended for the reduction of strictly periodic noise components in computer cooling fans is described,which is based on high‐performance digital sound device architectures found in some personal computers. The systemovercomes causality and synchronization constrains imposed by delayed buffering, as usually found in computer audioprocessing. Performance of the system is demonstrated and evaluated through measurements in a physical implementation ofactive noise control of synthetic tones combined with laptop fan noise, carried out under anechoic and slightly reverberantconditions. Tests on other types of tonal noise sources, like an electrical transformer, were also carried out. However, its widerapplicability to the cancellation of tonal noise has been proved compromised by weak periodicity issues found and reported inthis work. Also, a study of noise spectral width requirements for successful operation is presented.

  8. Critical involvement of postsynaptic protein kinase activation in long-term potentiation at hippocampal mossy fiber synapses on CA3 interneurons.

    Science.gov (United States)

    Galván, Emilio J; Cosgrove, Kathleen E; Mauna, Jocelyn C; Card, J Patrick; Thiels, Edda; Meriney, Stephen D; Barrionuevo, Germán

    2010-02-24

    Hippocampal mossy fiber (MF) synapses on area CA3 lacunosum-moleculare (L-M) interneurons are capable of undergoing a Hebbian form of NMDA receptor (NMDAR)-independent long-term potentiation (LTP) induced by the same type of high-frequency stimulation (HFS) that induces LTP at MF synapses on pyramidal cells. LTP of MF input to L-M interneurons occurs only at synapses containing mostly calcium-impermeable (CI)-AMPA receptors (AMPARs). Here, we demonstrate that HFS-induced LTP at these MF-interneuron synapses requires postsynaptic activation of protein kinase A (PKA) and protein kinase C (PKC). Brief extracellular stimulation of PKA with forskolin (FSK) alone or in combination with 1-Methyl-3-isobutylxanthine (IBMX) induced a long-lasting synaptic enhancement at MF synapses predominantly containing CI-AMPARs. However, the FSK/IBMX-induced potentiation in cells loaded with the specific PKA inhibitor peptide PKI(6-22) failed to be maintained. Consistent with these data, delivery of HFS to MFs synapsing onto L-M interneurons loaded with PKI(6-22) induced posttetanic potentiation (PTP) but not LTP. Hippocampal sections stained for the catalytic subunit of PKA revealed abundant immunoreactivity in interneurons located in strata radiatum and L-M of area CA3. We also found that extracellular activation of PKC with phorbol 12,13-diacetate induced a pharmacological potentiation of the isolated CI-AMPAR component of the MF EPSP. However, HFS delivered to MF synapses on cells loaded with the PKC inhibitor chelerythrine exhibited PTP followed by a significant depression. Together, our data indicate that MF LTP in L-M interneurons at synapses containing primarily CI-AMPARs requires some of the same signaling cascades as does LTP of glutamatergic input to CA3 or CA1 pyramidal cells.

  9. 30 CFR 250.210 - If I conduct ancillary activities, what reporting and data/information retention requirements...

    Science.gov (United States)

    2010-07-01

    ... OPERATIONS IN THE OUTER CONTINENTAL SHELF Plans and Information Ancillary Activities § 250.210 If I conduct ancillary activities, what reporting and data/information retention requirements must I satisfy? (a... data or information obtained or derived from your ancillary activities. When applicable, MMS...

  10. Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex.

    Science.gov (United States)

    Stewart, Emerson V; Nwosu, Christine C; Tong, Zongtian; Roguev, Assen; Cummins, Timothy D; Kim, Dong-Uk; Hayles, Jacqueline; Park, Han-Oh; Hoe, Kwang-Lae; Powell, David W; Krogan, Nevan J; Espenshade, Peter J

    2011-04-22

    Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1-4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2-conjugating enzyme Ubc4, the Dsc1 RING E3 ligase, and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.

  11. A Study on the Requirements for Fast Active Turbine Tip Clearance Control Systems

    Science.gov (United States)

    DeCastro, Jonathan A.; Melcher, Kevin J.

    2004-01-01

    This paper addresses the requirements of a control system for active turbine tip clearance control in a generic commercial turbofan engine through design and analysis. The control objective is to articulate the shroud in the high pressure turbine section in order to maintain a certain clearance set point given several possible engine transient events. The system must also exhibit reasonable robustness to modeling uncertainties and reasonable noise rejection properties. Two actuators were chosen to fulfill such a requirement, both of which possess different levels of technological readiness: electrohydraulic servovalves and piezoelectric stacks. Identification of design constraints, desired actuator parameters, and actuator limitations are addressed in depth; all of which are intimately tied with the hardware and controller design process. Analytical demonstrations of the performance and robustness characteristics of the two axisymmetric LQG clearance control systems are presented. Takeoff simulation results show that both actuators are capable of maintaining the clearance within acceptable bounds and demonstrate robustness to parameter uncertainty. The present model-based control strategy was employed to demonstrate the tradeoff between performance, control effort, and robustness and to implement optimal state estimation in a noisy engine environment with intent to eliminate ad hoc methods for designing reliable control systems.

  12. O pKa de indicadores ácido-base e os efeitos coloidais The pKa of acid-base indicators and the influence of colloidal systems

    Directory of Open Access Journals (Sweden)

    Bruno Alarcon Fernandes Previdello

    2006-06-01

    Full Text Available An experiment is proposed that can be included in undergraduate courses of chemistry. The subject is the acidity of organic compounds, which are employed as pH indicators, particularly in acid-base titrations. The indicators used are methyl orange, bromophenol blue and bromocresol green in aqueous medium. The influence of colloidal systems on the equilibrium is evaluated by the pKa. The colloids employed are surfactants like sodium dodecyl sulfate, cetyl-trimethylammonium bromide and a polymeric non-ionic F127 (pluronics. The effect of stabilization promoted by the system on the acidic or basic structureof the indicator establishes the action mechanism of the colloid on the pKa values.

  13. Inca: a novel p21-activated kinase-associated protein required for cranial neural crest development.

    Science.gov (United States)

    Luo, Ting; Xu, Yanhua; Hoffman, Trevor L; Zhang, Tailin; Schilling, Thomas; Sargent, Thomas D

    2007-04-01

    Inca (induced in neural crest by AP2) is a novel protein discovered in a microarray screen for genes that are upregulated in Xenopus embryos by the transcriptional activator protein Tfap2a. It has no significant similarity to any known protein, but is conserved among vertebrates. In Xenopus, zebrafish and mouse embryos, Inca is expressed predominantly in the premigratory and migrating neural crest (NC). Knockdown experiments in frog and fish using antisense morpholinos reveal essential functions for Inca in a subset of NC cells that form craniofacial cartilage. Cells lacking Inca migrate successfully but fail to condense into skeletal primordia. Overexpression of Inca disrupts cortical actin and prevents formation of actin "purse strings", which are required for wound healing in Xenopus embryos. We show that Inca physically interacts with p21-activated kinase 5 (PAK5), a known regulator of the actin cytoskeleton that is co-expressed with Inca in embryonic ectoderm, including in the NC. These results suggest that Inca and PAK5 cooperate in restructuring cytoskeletal organization and in the regulation of cell adhesion in the early embryo and in NC cells during craniofacial development.

  14. Hes6 is required for the neurogenic activity of neurogenin and NeuroD.

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    Kasumi Murai

    Full Text Available In the embryonic neural plate, a subset of precursor cells with neurogenic potential differentiates into neurons. This process of primary neurogenesis requires both the specification of cells for neural differentiation, regulated by Notch signaling, and the activity of neurogenic transcription factors such as neurogenin and NeuroD which drive the program of neural gene expression. Here we study the role of Hes6, a member of the hairy enhancer of split family of transcription factors, in primary neurogenesis in Xenopus embryos. Hes6 is an atypical Hes gene in that it is not regulated by Notch signaling and promotes neural differentiation in mouse cell culture models. We show that depletion of Xenopus Hes6 (Xhes6 by morpholino antisense oligonucleotides results in a failure of neural differentiation, a phenotype rescued by both wild type Xhes6 and a Xhes6 mutant unable to bind DNA. However, an Xhes6 mutant that lacks the ability to bind Groucho/TLE transcriptional co-regulators is only partly able to rescue the phenotype. Further analysis reveals that Xhes6 is essential for the induction of neurons by both neurogenin and NeuroD, acting via at least two distinct mechanisms, the inhibition of antineurogenic Xhairy proteins and by interaction with Groucho/TLE family proteins. We conclude Xhes6 is essential for neurogenesis in vivo, acting via multiple mechanisms to relieve inhibition of proneural transcription factor activity within the neural plate.

  15. BMP9-Induced Survival Effect in Liver Tumor Cells Requires p38MAPK Activation

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    María García-Álvaro

    2015-08-01

    Full Text Available The study of bone morphogenetic proteins (BMPs role in tumorigenic processes, and specifically in the liver, has gathered importance in the last few years. Previous studies have shown that BMP9 is overexpressed in about 40% of hepatocellular carcinoma (HCC patients. In vitro data have also shown evidence that BMP9 has a pro-tumorigenic action, not only by inducing epithelial to mesenchymal transition (EMT and migration, but also by promoting proliferation and survival in liver cancer cells. However, the precise mechanisms driving these effects have not yet been established. In the present work, we deepened our studies into the intracellular mechanisms implicated in the BMP9 proliferative and pro-survival effect on liver tumor cells. In HepG2 cells, BMP9 induces both Smad and non-Smad signaling cascades, specifically PI3K/AKT and p38MAPK. However, only the p38MAPK pathway contributes to the BMP9 growth-promoting effect on these cells. Using genetic and pharmacological approaches, we demonstrate that p38MAPK activation, although dispensable for the BMP9 proliferative activity, is required for the BMP9 protective effect on serum withdrawal-induced apoptosis. These findings contribute to a better understanding of the signaling pathways involved in the BMP9 pro-tumorigenic role in liver tumor cells.

  16. Neurotrophic effect of citrus 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone: promotion of neurite outgrowth via cAMP/PKA/CREB pathway in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Hui-Chi Lai

    Full Text Available 5-Hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF, a hydroxylated polymethoxyflavone, is found exclusively in the Citrus genus, particularly in the peels of sweet orange. In this research, we report the first investigation of the neurotrophic effects and mechanism of 5-OH-HxMF in PC12 pheochromocytoma cells. We found that 5-OH-HxMF can effectively induce PC12 neurite outgrowth accompanied with the expression of neuronal differentiation marker protein growth-associated protein-43(GAP-43. 5-OH-HxMF caused the enhancement of cyclic AMP response element binding protein (CREB phosphorylation, c-fos gene expression and CRE-mediated transcription, which was inhibited by 2-naphthol AS-E phosphate (KG-501, a specific antagonist for the CREB-CBP complex formation. Moreover, 5-OH-HxMF-induced both CRE transcription activity and neurite outgrowth were inhibited by adenylate cyclase and protein kinase A (PKA inhibitor, but not MEK1/2, protein kinase C (PKC, phosphatidylinositol 3-kinase (PI3K or calcium/calmodulin-dependent protein kinase (CaMK inhibitor. Consistently, 5-OH-HxMF treatment increased the intracellular cAMP level and downstream component, PKA activity. We also found that addition of K252a, a TrKA antagonist, significantly inhibited NGF- but not 5-OH-HxMF-induced neurite outgrowth. These results reveal for the first time that 5-OH-HxMF is an effective neurotrophic agent and its effect is mainly through a cAMP/PKA-dependent, but TrKA-independent, signaling pathway coupling with CRE-mediated gene transcription. A PKC-dependent and CREB-independent pathway was also involved in its neurotrophic action.

  17. Curcumin Ameliorates the Reduction Effect of PGE2 on Fibrillar β-Amyloid Peptide (1-42-Induced Microglial Phagocytosis through the Inhibition of EP2-PKA Signaling in N9 Microglial Cells.

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    Gen-Lin He

    Full Text Available Inflammatory activation of microglia and β amyloid (Aβ deposition are considered to work both independently and synergistically to contribute to the increased risk of Alzheimer's disease (AD. Recent studies indicate that long-term use of phenolic compounds provides protection against AD, primarily due to their anti-inflammatory actions. We previously suggested that phenolic compound curcumin ameliorated phagocytosis possibly through its anti-inflammatory effects rather than direct regulation of phagocytic function in electromagnetic field-exposed N9 microglial cells (N9 cells. Here, we explored the prostaglandin-E2 (PGE2-related signaling pathway that involved in curcumin-mediated phagocytosis in fibrillar β-amyloid peptide (1-42 (fAβ42-stimulated N9 cells. Treatment with fAβ42 increased phagocytosis of fluorescent-labeled latex beads in N9 cells. This increase was attenuated in a dose-dependent manner by endogenous and exogenous PGE2, as well as a selective EP2 or protein kinase A (PKA agonist, but not by an EP4 agonist. We also found that an antagonist of EP2, but not EP4, abolished the reduction effect of PGE2 on fAβ42-induced microglial phagocytosis. Additionally, the increased expression of endogenous PGE2, EP2, and cyclic adenosine monophosphate (AMP, and activation of vasodilator-stimulated phosphoprotein, cyclic AMP responsive element-binding protein, and PKA were depressed by curcumin administration. This reduction led to the amelioration of the phagocytic abilities of PGE2-stimulated N9 cells. Taken together, these data suggested that curcumin restored the attenuating effect of PGE2 on fAβ42-induced microglial phagocytosis via a signaling mechanism involving EP2 and PKA. Moreover, due to its immune modulatory effects, curcumin may be a promising pharmacological candidate for neurodegenerative diseases.

  18. β-Hydroxybutyric Sodium Salt Inhibition of Growth Hormone and Prolactin Secretion via the cAMP/PKA/CREB and AMPK Signaling Pathways in Dairy Cow Anterior Pituitary Cells

    Directory of Open Access Journals (Sweden)

    Shou-Peng Fu

    2015-02-01

    Full Text Available β-hydroxybutyric acid (BHBA regulates the synthesis and secretion of growth hormone (GH and prolactin (PRL, but its mechanism is unknown. In this study, we detected the effects of BHBA on the activities of G protein signaling pathways, AMPK-α activity, GH, and PRL gene transcription, and GH and PRL secretion in dairy cow anterior pituitary cells (DCAPCs. The results showed that BHBA decreased intracellular cAMP levels and a subsequent reduction in protein kinase A (PKA activity. Inhibition of PKA activity reduced cAMP response element-binding protein (CREB phosphorylation, thereby inhibiting GH and PRL transcription and secretion. The effects of BHBA were attenuated by a specific Gαi inhibitor, pertussis toxin (PTX. In addition, intracellular BHBA uptake mediated by monocarboxylate transporter 1 (MCT1 could trigger AMPK signaling and result in the decrease in GH and PRL mRNA translation in DCAPCs cultured under low-glucose and non-glucose condition when compared with the high-glucose group. This study identifies a biochemical mechanism for the regulatory action of BHBA on GH and PRL gene transcription, translation, and secretion in DCAPCs, which may be one of the factors that regulate pituitary function during the transition period in dairy cows.

  19. pK(a) based protonation states and microspecies for protein-ligand docking.

    Science.gov (United States)

    ten Brink, Tim; Exner, Thomas E

    2010-11-01

    In this paper we present our reworked approach to generate ligand protonation states with our structure preparation tool SPORES (Structure PrOtonation and REcognition System). SPORES can be used for the preprocessing of proteins and protein-ligand complexes as e.g. taken from the Protein Data Bank as well as for the setup of 3D ligand databases. It automatically assigns atom and bond types, generates different protonation, tautomeric states as well as different stereoisomers. In the revised version, pKa calculations with the ChemAxon software MARVIN are used either to determine the likeliness of a combinatorial generated protonation state or to determine the titrable atoms used in the combinatorial approach. Additionally, the MARVIN software is used to predict microspecies distributions of ligand molecules. Docking studies were performed with our recently introduced program PLANTS (Protein-Ligand ANT System) on all protomers resulting from the three different selection methods for the well established CCDC/ASTEX clean data set demonstrating the usefulness of especially the latter approach.

  20. Doses monitoring in radiology: calibration of air kerma-area product (PKA meters

    Directory of Open Access Journals (Sweden)

    Ricardo Andrade Terini

    2013-12-01

    Full Text Available Objective The authors have sought to study the calibration of a clinical PKA meter (Diamentor E2 and a calibrator for clinical meters (PDC in the Laboratory of Ionizing Radiation Metrology at Instituto de Energia e Ambiente - Universidade de São Paulo. Materials and Methods Different qualities of both incident and transmitted beams were utilized in conditions similar to a clinical setting, analyzing the influence from the reference dosimeter, from the distance between meters, from the filtration and from the average beam energy. Calibrations were performed directly against a standard 30 cm3 cylindrical chamber or a parallel-plate monitor chamber, and indirectly against the PDC meter. Results The lowest energy dependence was observed for transmitted beams. The cross calibration between the Diamentor E2 and the PDC meters, and the PDC presented the greatest propagation of uncertainties. Conclusion The calibration coefficient of the PDC meter showed to be more stable with voltage, while the Diamentor E2 calibration coefficient was more variable. On the other hand, the PDC meter presented greater uncertainty in readings (5.0% than with the use of the monitor chamber (3.5% as a reference.

  1. Formulation and stability of a soap microemulsion and the apparent pK(A) herein.

    Science.gov (United States)

    Marcus, Julien; Touraud, Didier; Kunz, Werner

    2013-10-01

    The influence of composition, and added salts and polyols on the stability of an oil-in-water microemulsion formulation and on the apparent pKA (apKA) of the used oleate surfactant is investigated. High temperature favours a decrease of the apKA and leads to the formation of more hydrated micelles. The apKA decreases also when the percentage of ethanol increases. Citronellol molecules do not significantly influence the apKA at concentrations between 0% and 2% w/w. By contrast, with increasing limonene concentration, the apKA increases. It was observed that anions of sodium salts destabilize the microemulsion and high temperatures are needed to recover it. By increasing the concentration of NaCl, a slight increase of the apKA is observed, which can be associated with a non-specific, electrostatic (Debye-Hückel) effect. Cations of chloride salts have different effects depending on their ability to exchange with Na(+) near the carboxylate group. Li(+), Na(+) and K(+) have apparently a salting-out effect. Tetramethylammonium chloride and choline chloride have salting-in effects until respectively 0.6 and 0.4 mol kg(-1). The associations of sorbitol or glycerol with ethanol lead to a salting-in effect and to a decrease of the apKA of Na-Oleate.

  2. pKa based protonation states and microspecies for protein-ligand docking

    Science.gov (United States)

    ten Brink, Tim; Exner, Thomas E.

    2010-11-01

    In this paper we present our reworked approach to generate ligand protonation states with our structure preparation tool SPORES (Structure PrOtonation and REcognition System). SPORES can be used for the preprocessing of proteins and protein-ligand complexes as e.g. taken from the Protein Data Bank as well as for the setup of 3D ligand databases. It automatically assigns atom and bond types, generates different protonation, tautomeric states as well as different stereoisomers. In the revised version, pKa calculations with the ChemAxon software MARVIN are used either to determine the likeliness of a combinatorial generated protonation state or to determine the titrable atoms used in the combinatorial approach. Additionally, the MARVIN software is used to predict microspecies distributions of ligand molecules. Docking studies were performed with our recently introduced program PLANTS (Protein-Ligand ANT System) on all protomers resulting from the three different selection methods for the well established CCDC/ASTEX clean data set demonstrating the usefulness of especially the latter approach.

  3. 42 CFR 495.316 - State monitoring and reporting regarding activities required to receive an incentive payment.

    Science.gov (United States)

    2010-10-01

    ...) Provider adoption, implementation, or upgrade of certified EHR technology activities and payments; and (2... upgraded certified EHR technology. (ii) Aggregated data tables representing the provider adoption... STANDARDS FOR THE ELECTRONIC HEALTH RECORD TECHNOLOGY INCENTIVE PROGRAM Requirements Specific to...

  4. Brain-derived neurotrophic factor inhibits neuromuscular junction maturation in a cAMP-PKA-dependent way.

    Science.gov (United States)

    Song, Wei; Jin, Xiwan Albert

    2015-03-30

    The development of neuromuscular junction (NMJ) is initiated by motor axon's contact with the skeletal muscle cell that is followed by synaptic maturation. Previous studies showed that brain-derived neurotrophic factor (BDNF) enhanced motoneurons' survival and growth but significantly inhibited synaptogenesis. Here, we report that chronic application of BDNF resulted in inhibition in the maturation process both physiologically and morphologically. The response to BDNF was mediated by its cognate receptor TrkB as the effects were abolished by Trk receptor inhibitor K252a. Protein kinase A (PKA) inhibitor reversed the effects of BDNF in inhibiting NMJ maturation. These results indicate that BDNF suppresses NMJ maturation through cAMP-PKA signaling pathway. Together with the previous studies, these results suggest that BDNF suppresses NMJ formation and maturation despite its effects in enhancing neuronal survival and growth.

  5. 30 CFR 285.521 - Do my financial assurance requirements change as activities progress on my limited lease or grant?

    Science.gov (United States)

    2010-07-01

    ... Assurance for Limited Leases, Row Grants, and Rue Grants § 285.521 Do my financial assurance requirements... level of your financial assurance as activities progress on your limited lease or grant. We will base... requirement for increased financial assurance levels for the limited lease or grant by increasing the...

  6. pKa predictions with a coupled finite difference Poisson-Boltzmann and Debye-Hückel method.

    Science.gov (United States)

    Warwicker, Jim

    2011-12-01

    Modeling charge interactions is important for understanding many aspects of biological structure and function, and continuum methods such as Finite Difference Poisson-Boltzmann (FDPB) are commonly employed. Calculations of pH-dependence have identified separate populations; surface groups that can be modeled with a simple Debye-Hückel (DH) model, and buried groups, with stronger resultant interactions that are dependent on detailed conformation. This observation led to the development of a combined FDPB and DH method for pK(a) prediction (termed FD/DH). This study reports application of this method to ionizable groups, including engineered buried charges, in staphylococcal nuclease. The data had been made available to interested research groups before publication of mutant structures and/or pK(a) values. Overall, FD/DH calculations perform as intended with low ΔpK(a) values for surface groups (RMSD between predicted and experimental pK(a) values of 0.74), and much larger ΔpK(a) values for the engineered internal groups, with RMSD = 1.64, where mutant structures were known and RMSD = 1.80, where they were modeled. The weaker resultant interactions of the surface groups are determined mostly by charge-charge interactions. For the buried groups, R(2) for correlation between predicted and measured ΔpK(a) values is 0.74, despite the high RMSDs. Charge-charge interactions are much less important, with the R(2) value for buried group ΔpK(a) values increasing to 0.80 when the term describing charge desolvation alone is used. Engineered charge burial delivers a relatively uniform, nonspecific effect, in terms of pK(a) . How the protein environment adapts in atomic detail to deliver this resultant effect is still an open question.

  7. Endogenous 17ß-estradiol is required for activity-dependent long-term potentiation in the striatum: interaction with the dopaminergic system

    Directory of Open Access Journals (Sweden)

    Alessandro eTozzi

    2015-05-01

    Full Text Available 17β-estradiol (E2, a neurosteroid synthesized by P450-aromatase (ARO, modulates various brain functions. We characterized the role of the locally synthesized E2 on striatal long-term synaptic plasticity and explored possible interactions between E2 receptors (ERs and dopamine (DA receptors in the dorsal striatum of adult male rats. Inhibition of E2 synthesis or antagonism of ERs prevented the induction of long-term potentiation (LTP in both medium spiny neurons (MSNs and cholinergic interneurons (ChIs. Activation of a D1-like DA receptor/cAMP/PKA-dependent pathway restored LTP. In MSNs exogenous E2 reversed the effect of ARO inhibition. Also antagonism of M1 muscarinic receptors prevented the D1-like receptor-mediated restoration of LTP confirming a role for ChIs in controlling the E2-mediated LTP of MSNs. A novel striatal interaction, occurring between ERs and D1-like receptors in both MSNs and ChIs, might be critical to regulate basal ganglia physiology and to compensate synaptic alterations in Parkinson's disease.

  8. Arsenic may be involved in fluoride-induced bone toxicity through PTH/PKA/AP1 signaling pathway.

    Science.gov (United States)

    Zeng, Qi-bing; Xu, Yu-yan; Yu, Xian; Yang, Jun; Hong, Feng; Zhang, Ai-hua

    2014-01-01

    Chronic exposure to combined fluoride and arsenic continues to be a major public health problem worldwide, affecting thousands of people. In recent years, more and more researchers began to focus on the interaction between the fluorine and the arsenic. In this study, the selected investigation site was located in China. The study group was selected from people living in fluoride-arsenic polluted areas due to burning coal. The total number of participants was 196; including the fluoride-arsenic anomaly group (130) and the fluoride-arsenic normal group (63). By observing the changes in gene and protein expression of PTH/PKA/AP1 signaling pathway, the results show that fluoride can increase the expression levels of PTH, PKA, and AP1, but arsenic can only affect the expression of AP1; fluoride and arsenic have an interaction on the expression of AP1. Further study found that fluoride and arsenic can affect the mRNA expression level of c-fos gene (AP1 family members), and have an interaction on the expression of c-fos, but not c-jun. The results indicate that PTH/PKA/AP1 signaling pathway may play an important role in bone toxicity of fluoride. Arsenic can affect the expression of c-fos, thereby affecting the expression of transcription factor AP1, indirectly involved in fluoride-induced bone toxicity. Copyright © 2013. Published by Elsevier B.V.

  9. Memory retrieval of inhibitory avoidance requires histamine H1 receptor activation in the hippocampus.

    Science.gov (United States)

    Fabbri, Roberta; Furini, Cristiane Regina Guerino; Passani, Maria Beatrice; Provensi, Gustavo; Baldi, Elisabetta; Bucherelli, Corrado; Izquierdo, Ivan; de Carvalho Myskiw, Jociane; Blandina, Patrizio

    2016-05-10

    Retrieval represents a dynamic process that may require neuromodulatory signaling. Here, we report that the integrity of the brain histaminergic system is necessary for retrieval of inhibitory avoidance (IA) memory, because rats depleted of histamine through lateral ventricle injections of α-fluoromethylhistidine (a-FMHis), a suicide inhibitor of histidine decarboxylase, displayed impaired IA memory when tested 2 d after training. a-FMHis was administered 24 h after training, when IA memory trace was already formed. Infusion of histamine in hippocampal CA1 of brain histamine-depleted rats (hence, amnesic) 10 min before the retention test restored IA memory but was ineffective when given in the basolateral amygdala (BLA) or the ventral medial prefrontal cortex (vmPFC). Intra-CA1 injections of selective H1 and H2 receptor agonists showed that histamine exerted its effect by activating the H1 receptor. Noteworthy, the H1 receptor antagonist pyrilamine disrupted IA memory retrieval in rats, thus strongly supporting an active involvement of endogenous histamine; 90 min after the retention test, c-Fos-positive neurons were significantly fewer in the CA1s of a-FMHis-treated rats that displayed amnesia compared with in the control group. We also found reduced levels of phosphorylated cAMP-responsive element binding protein (pCREB) in the CA1s of a-FMHis-treated animals compared with in controls. Increases in pCREB levels are associated with retrieval of associated memories. Targeting the histaminergic system may modify the retrieval of emotional memory; hence, histaminergic ligands might reduce dysfunctional aversive memories and improve the efficacy of exposure psychotherapies.

  10. Endoplasmic Reticulum Exit of Golgi-resident Defective for SREBP Cleavage (Dsc) E3 Ligase Complex Requires Its Activity.

    Science.gov (United States)

    Raychaudhuri, Sumana; Espenshade, Peter J

    2015-06-01

    Layers of quality control ensure proper protein folding and complex formation prior to exit from the endoplasmic reticulum. The fission yeast Dsc E3 ligase is a Golgi-localized complex required for sterol regulatory element-binding protein (SREBP) transcription factor activation that shows architectural similarity to endoplasmic reticulum-associated degradation E3 ligases. The Dsc E3 ligase consists of five integral membrane proteins (Dsc1-Dsc5) and functionally interacts with the conserved AAA-ATPase Cdc48. Utilizing an in vitro ubiquitination assay, we demonstrated that Dsc1 has ubiquitin E3 ligase activity that requires the E2 ubiquitin-conjugating enzyme Ubc4. Mutations that specifically block Dsc1-Ubc4 interaction prevent SREBP cleavage, indicating that SREBP activation requires Dsc E3 ligase activity. Surprisingly, Golgi localization of the Dsc E3 ligase complex also requires Dsc1 E3 ligase activity. Analysis of Dsc E3 ligase complex formation, glycosylation, and localization indicated that Dsc1 E3 ligase activity is specifically required for endoplasmic reticulum exit of the complex. These results define enzyme activity-dependent sorting as an autoregulatory mechanism for protein trafficking.

  11. Modulation of β-catenin signaling by glucagon receptor activation.

    Directory of Open Access Journals (Sweden)

    Jiyuan Ke

    Full Text Available The glucagon receptor (GCGR is a member of the class B G protein-coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin-mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R and glucagon-like peptide 1 (GLP-1R receptors. Since low-density-lipoprotein receptor-related protein 5 (Lrp5 is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter-mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1 or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations.

  12. Electrostatic contribution of surface charge residues to the stability of a thermophilic protein: benchmarking experimental and predicted pKa values.

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    Chi-Ho Chan

    Full Text Available Optimization of the surface charges is a promising strategy for increasing thermostability of proteins. Electrostatic contribution of ionizable groups to the protein stability can be estimated from the differences between the pKa values in the folded and unfolded states of a protein. Using this pKa-shift approach, we experimentally measured the electrostatic contribution of all aspartate and glutamate residues to the stability of a thermophilic ribosomal protein L30e from Thermococcus celer. The pKa values in the unfolded state were found to be similar to model compound pKas. The pKa values in both the folded and unfolded states obtained at 298 and 333 K were similar, suggesting that electrostatic contribution of ionizable groups to the protein stability were insensitive to temperature changes. The experimental pKa values for the L30e protein in the folded state were used as a benchmark to test the robustness of pKa prediction by various computational methods such as H++, MCCE, MEAD, pKD, PropKa, and UHBD. Although the predicted pKa values were affected by crystal contacts that may alter the side-chain conformation of surface charged residues, most computational methods performed well, with correlation coefficients between experimental and calculated pKa values ranging from 0.49 to 0.91 (p<0.01. The changes in protein stability derived from the experimental pKa-shift approach correlate well (r = 0.81 with those obtained from stability measurements of charge-to-alanine substituted variants of the L30e protein. Our results demonstrate that the knowledge of the pKa values in the folded state provides sufficient rationale for the redesign of protein surface charges leading to improved protein stability.

  13. 77 FR 26824 - Agency Information Collection; Activity Under OMB Review; Reporting Required for International...

    Science.gov (United States)

    2012-05-07

    ...; Reporting Required for International Civil Aviation Organization (ICAO) AGENCY: Research & Innovative... Required for International Civil Aviation Organization (ICAO). Form No.: BTS Form EF. Type of Review... Convention on International Civil Aviation (Treaty), the United States is obligated to provide ICAO...

  14. Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells.

    Science.gov (United States)

    Voloshanenko, Oksana; Erdmann, Gerrit; Dubash, Taronish D; Augustin, Iris; Metzig, Marie; Moffa, Giusi; Hundsrucker, Christian; Kerr, Grainne; Sandmann, Thomas; Anchang, Benedikt; Demir, Kubilay; Boehm, Christina; Leible, Svenja; Ball, Claudia R; Glimm, Hanno; Spang, Rainer; Boutros, Michael

    2013-01-01

    Aberrant regulation of the Wnt/β-catenin pathway has an important role during the onset and progression of colorectal cancer, with over 90% of cases of sporadic colon cancer featuring mutations in APC or β-catenin. However, it has remained a point of controversy whether these mutations are sufficient to activate the pathway or require additional upstream signals. Here we show that colorectal tumours express elevated levels of Wnt3 and Evi/Wls/GPR177. We found that in colon cancer cells, even in the presence of mutations in APC or β-catenin, downstream signalling remains responsive to Wnt ligands and receptor proximal signalling. Furthermore, we demonstrate that truncated APC proteins bind β-catenin and key components of the destruction complex. These results indicate that cells with mutations in APC or β-catenin depend on Wnt ligands and their secretion for a sufficient level of β-catenin signalling, which potentially opens new avenues for therapeutic interventions by targeting Wnt secretion via Evi/Wls.

  15. CARMA1 is required for Akt-mediated NF-kappaB activation in T cells.

    Science.gov (United States)

    Narayan, Preeti; Holt, Brittany; Tosti, Richard; Kane, Lawrence P

    2006-03-01

    Many details of the generic pathway for induction of NF-kappaB have been delineated, but it is still not clear how multiple, diverse receptor systems are able to converge on this evolutionarily conserved family of transcription factors. Recent studies have shown that the CARMA1, Bcl10, and MALT1 proteins are critical for coupling the common elements of the NF-kappaB pathway to the T-cell receptor (TCR) and CD28. We previously demonstrated a role for the serine/threonine kinase Akt in CD28-mediated NF-kappaB induction. Using a CARMA1-deficient T-cell line, we have now found that the CARMA complex is required for induction of NF-kappaB by Akt, in cooperation with protein kinase C activation. Furthermore, using a novel selective inhibitor of Akt, we confirm that Akt plays a modulatory role in NF-kappaB induction by the TCR and CD28. Finally, we provide evidence for a physical and functional interaction between Akt and CARMA and for Akt-dependent phosphorylation of Bcl10. Therefore, in T cells, Akt impinges upon NF-kappaB signaling through at least two separate mechanisms.

  16. The Design of Effective ICT-Supported Learning Activities: Exemplary Models, Changing Requirements, and New Possibilities

    Directory of Open Access Journals (Sweden)

    Cameron Richards

    2005-01-01

    Full Text Available Despite the imperatives of policy and rhetoric about their integration in formal education, Information and Communication Technologies (ICTs are often used as an "add-on" in many classrooms and in many lesson plans. Nevertheless, many teachers find that interesting and well-planned tasks, projects, and resources provide a key to harnessing the educational potential of digital resources, Internet communications and interactive multimedia to engage the interest, interaction, and knowledge construction of young learners. To the extent that such approaches go beyond and transform traditional "transmission" models of teaching and formal lesson planning, this paper investigates the changing requirements and new possibilities represented by the challenge of integrating ICTs in education in a way which at the same time connects more effectively with both the specific contents of the curriculum and the various stages and elements of the learning process. Case studies from teacher education foundation courses provide an exemplary focus of inquiry in order to better link relevant new theories or models of learning with practice, to build upon related learner-centered strategies for integrating ICT resources and tools, and to incorporate interdependent functions of learning as information access, communication, and applied interactions. As one possible strategy in this direction, the concept of an "ICT-supported learning activity" suggests the need for teachers to approach this increasing challenge more as "designers" of effective and integrated learning rather than mere "transmitters" of skills or information through an add-on use of ICTs.

  17. Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs.

    Directory of Open Access Journals (Sweden)

    Yong-Qiang Zhang

    Full Text Available Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H(+-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma(- phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca(2+ and H(+ surges triggered by the antimicrobial agent amiodarone, and impaired Ca(2+ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.

  18. Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1.

    Science.gov (United States)

    Solanes, Paola; Heuzé, Mélina L; Maurin, Mathieu; Bretou, Marine; Lautenschlaeger, Franziska; Maiuri, Paolo; Terriac, Emmanuel; Thoulouze, Maria-Isabel; Launay, Pierre; Piel, Matthieu; Vargas, Pablo; Lennon-Duménil, Ana-Maria

    2015-03-12

    Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP₃ receptors (IP₃Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP₃R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP₃R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP₃R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment.

  19. The Calmodulin-Binding Transcription Activator CAMTA1 Is Required for Long-Term Memory Formation in Mice

    Science.gov (United States)

    Bas-Orth, Carlos; Tan, Yan-Wei; Oliveira, Ana M. M.; Bengtson, C. Peter; Bading, Hilmar

    2016-01-01

    The formation of long-term memory requires signaling from the synapse to the nucleus to mediate neuronal activity-dependent gene transcription. Synapse-to-nucleus communication is initiated by influx of calcium ions through synaptic NMDA receptors and/or L-type voltage-gated calcium channels and involves the activation of transcription factors by…

  20. Measuring work activities and skill requirements of occupations: experiences from a European pilot study with a web-survey

    NARCIS (Netherlands)

    Tijdens, K.G.; de Ruijter, J.; de Ruijter, E.

    2012-01-01

    Purpose - The purpose of this article is to evaluate a method for measuring work activities and skill requirements of 160 occupations in eight countries, used in EurOccupations, an EU-FP6 project. Additionally, it aims to explore how the internet can be used for measuring work activities and skill r

  1. The Calmodulin-Binding Transcription Activator CAMTA1 Is Required for Long-Term Memory Formation in Mice

    Science.gov (United States)

    Bas-Orth, Carlos; Tan, Yan-Wei; Oliveira, Ana M. M.; Bengtson, C. Peter; Bading, Hilmar

    2016-01-01

    The formation of long-term memory requires signaling from the synapse to the nucleus to mediate neuronal activity-dependent gene transcription. Synapse-to-nucleus communication is initiated by influx of calcium ions through synaptic NMDA receptors and/or L-type voltage-gated calcium channels and involves the activation of transcription factors by…

  2. cAMP/PKA Pathways and S56 Phosphorylation Are Involved in AA/PGE2-Induced Increases in rNaV1.4 Current.

    Directory of Open Access Journals (Sweden)

    Hua Gu

    Full Text Available Arachidonic acid (AA and its metabolites are important second messengers for ion channel modulation. The effects of extracellular application of AA and its non-metabolized analogue on muscle rNaV1.4 Na+ current has been studied, but little is known about the effects of intracellular application of AA on this channel isoform. Here, we report that intracellular application of AA significantly augmented the rNaV1.4 current peak without modulating the steady-state activation and inactivation properties of the rNaV1.4 channel. These results differed from the effects of extracellular application of AA on rNaV1.4 current. The effects of intracellular AA were mimicked by prostaglandin E2 but not eicosatetraynoic acid (ETYA, the non-metabolized analogue of AA, and were eliminated by treatment with cyclooxygenase inhibitors, flufenamic acid, or indomethacin. AA/PGE2-induced activation of rNaV1.4 channels was mimicked by a cAMP analogue (db-cAMP and eliminated by a PKA inhibitor, PKAi. Furthermore, inhibition of EP2 and EP4 (PGE2 receptors with AH6809 and AH23848 reduced the intracellular AA/PGE2-induced increase of rNaV1.4 current. Two mutated channels, rNaV1.4S56A and rNaV1.4T21A, were designed to investigate the role of predicted phosphorylation sites in the AA/PGE2-mediated regulation of rNaV1.4 currents. In rNaV1.4S56A, the effects of intracellular db-cAMP, AA, and PGE2 were significantly reduced. The results of the present study suggest that intracellular AA augments rNaV1.4 current by PGE2/EP receptor-mediated activation of the cAMP/PKA pathway, and that the S56 residue on the channel protein is important for this process.

  3. cAMP/PKA Pathways and S56 Phosphorylation Are Involved in AA/PGE2-Induced Increases in rNaV1.4 Current.

    Science.gov (United States)

    Gu, Hua; Fang, Yan-Jia; Liu, Dong-Dong; Chen, Ping; Mei, Yan-Ai

    2015-01-01

    Arachidonic acid (AA) and its metabolites are important second messengers for ion channel modulation. The effects of extracellular application of AA and its non-metabolized analogue on muscle rNaV1.4 Na+ current has been studied, but little is known about the effects of intracellular application of AA on this channel isoform. Here, we report that intracellular application of AA significantly augmented the rNaV1.4 current peak without modulating the steady-state activation and inactivation properties of the rNaV1.4 channel. These results differed from the effects of extracellular application of AA on rNaV1.4 current. The effects of intracellular AA were mimicked by prostaglandin E2 but not eicosatetraynoic acid (ETYA), the non-metabolized analogue of AA, and were eliminated by treatment with cyclooxygenase inhibitors, flufenamic acid, or indomethacin. AA/PGE2-induced activation of rNaV1.4 channels was mimicked by a cAMP analogue (db-cAMP) and eliminated by a PKA inhibitor, PKAi. Furthermore, inhibition of EP2 and EP4 (PGE2 receptors) with AH6809 and AH23848 reduced the intracellular AA/PGE2-induced increase of rNaV1.4 current. Two mutated channels, rNaV1.4S56A and rNaV1.4T21A, were designed to investigate the role of predicted phosphorylation sites in the AA/PGE2-mediated regulation of rNaV1.4 currents. In rNaV1.4S56A, the effects of intracellular db-cAMP, AA, and PGE2 were significantly reduced. The results of the present study suggest that intracellular AA augments rNaV1.4 current by PGE2/EP receptor-mediated activation of the cAMP/PKA pathway, and that the S56 residue on the channel protein is important for this process.

  4. Dissociation constants of weak acids from ab initio molecular dynamics using metadynamics: influence of the inductive effect and hydrogen bonding on pKa values.

    Science.gov (United States)

    Tummanapelli, Anil Kumar; Vasudevan, Sukumaran

    2014-11-26

    The theoretical estimation of the dissociation constant, or pKa, of weak acids continues to be a challenging field. Here, we show that ab initio Car-Parrinello molecular dynamics simulations in conjunction with metadynamics calculations of the free-energy profile of the dissociation reaction provide reasonable estimates of the pKa value. Water molecules, sufficient to complete the three hydration shells surrounding the acid molecule, were included explicitly in the computation procedure. The free-energy profiles exhibit two distinct minima corresponding to the dissociated and neutral states of the acid, and the difference in their values provides the estimate for pKa. We show for a series of organic acids that CPMD simulations in conjunction with metadynamics can provide reasonable estimates of pKa values. The acids investigated were aliphatic carboxylic acids, chlorine-substituted carboxylic acids, cis- and trans-butenedioic acid, and the