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Sample records for tissue cyclic gmp

  1. Specificity of the Cyclic GMP-Binding Activity and of a Cyclic GMP-Dependent Cyclic GMP Phosphodiesterase in Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Walsum, Hans van; Meer, Rob C. van der; Bulgakov, Roman; Konijn, Theo M.

    1982-01-01

    The nucleotide specificity of the cyclic GMP-binding activity in a homogenate of Dictyostelium discoideum was determined by competition of cyclic GMP derivatives with [8-3H] cyclic GMP for the binding sites. The results indicate that cyclic GMP is bound to the binding proteins by hydrogen bonds at

  2. [Effects of cytosolic bacteria on cyclic GMP-AMP synthase expression in human gingival tissues and periodontal ligament cells].

    Science.gov (United States)

    Xiaojun, Yang; Yongmei, Tan; Zhihui, Tian; Ting, Zhou; Wanghong, Zhao; Jin, Hou

    2017-04-01

    This work aims to determine the effect of cytosolic bacteria on the expression of cyclic GMP-AMP synthase (cGAS) in human periodontal ligament cells (hPDLCs) and gingival tissues. The ability of Porphyromonas gingivalis (P. gingivalis) to invade hPDLCs was detected using laser scanning confocal microscope assay at a multiplicity of infection of 10. P. gingivalis-infected cells were sorted by fluorescence-activated cell sorting (FACS). Then, quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect cGAS expression in infected cells. Finally, the location and expression of cGAS in inflammatory and normal gingival tissues were investigated by immunohistochemistry. P. gingivalis actively invaded hPDLCs. Moreover, cGAS expression significantly increased in P. gingivalis-infected cells. Although cGAS was expressed in the epithelial and subepithelial cells of both inflamed and normal gingival tissues, cGAS expression significantly increased in inflamed gingival tissues. Cytosolic bacteria can upregulate cGAS expression in infected cells. These data suggest that cGAS may act as pattern-recognition receptors and participate in recognizing cytosolic nucleic acid pathogen-associated molecular patterns.
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  3. Guanylin peptides: cyclic GMP signaling mechanisms

    Directory of Open Access Journals (Sweden)

    Forte L.R.

    1999-01-01

    Full Text Available Guanylate cyclases (GC serve in two different signaling pathways involving cytosolic and membrane enzymes. Membrane GCs are receptors for guanylin and atriopeptin peptides, two families of cGMP-regulating peptides. Three subclasses of guanylin peptides contain one intramolecular disulfide (lymphoguanylin, two disulfides (guanylin and uroguanylin and three disulfides (E. coli stable toxin, ST. The peptides activate membrane receptor-GCs and regulate intestinal Cl- and HCO3- secretion via cGMP in target enterocytes. Uroguanylin and ST also elicit diuretic and natriuretic responses in the kidney. GC-C is an intestinal receptor-GC for guanylin and uroguanylin, but GC-C may not be involved in renal cGMP pathways. A novel receptor-GC expressed in the opossum kidney (OK-GC has been identified by molecular cloning. OK-GC cDNAs encode receptor-GCs in renal tubules that are activated by guanylins. Lymphoguanylin is highly expressed in the kidney and heart where it may influence cGMP pathways. Guanylin and uroguanylin are highly expressed in intestinal mucosa to regulate intestinal salt and water transport via paracrine actions on GC-C. Uroguanylin and guanylin are also secreted from intestinal mucosa into plasma where uroguanylin serves as an intestinal natriuretic hormone to influence body Na+ homeostasis by endocrine mechanisms. Thus, guanylin peptides control salt and water transport in the kidney and intestine mediated by cGMP via membrane receptors with intrinsic guanylate cyclase activity.

  4. Cyclic GMP-AMP Displays Mucosal Adjuvant Activity in Mice

    OpenAIRE

    Škrnjug, Ivana; Guzmán, Carlos Alberto; Ruecker, Christine

    2014-01-01

    The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP) after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice....

  5. Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles.

    Science.gov (United States)

    Shuhaibar, Leia C; Egbert, Jeremy R; Norris, Rachael P; Lampe, Paul D; Nikolaev, Viacheslav O; Thunemann, Martin; Wen, Lai; Feil, Robert; Jaffe, Laurinda A

    2015-04-28

    Meiosis in mammalian oocytes is paused until luteinizing hormone (LH) activates receptors in the mural granulosa cells of the ovarian follicle. Prior work has established the central role of cyclic GMP (cGMP) from the granulosa cells in maintaining meiotic arrest, but it is not clear how binding of LH to receptors that are located up to 10 cell layers away from the oocyte lowers oocyte cGMP and restarts meiosis. Here, by visualizing intercellular trafficking of cGMP in real-time in live follicles from mice expressing a FRET sensor, we show that diffusion of cGMP through gap junctions is responsible not only for maintaining meiotic arrest, but also for rapid transmission of the signal that reinitiates meiosis from the follicle surface to the oocyte. Before LH exposure, the cGMP concentration throughout the follicle is at a uniformly high level of ∼2-4 μM. Then, within 1 min of LH application, cGMP begins to decrease in the peripheral granulosa cells. As a consequence, cGMP from the oocyte diffuses into the sink provided by the large granulosa cell volume, such that by 20 min the cGMP concentration in the follicle is uniformly low, ∼100 nM. The decrease in cGMP in the oocyte relieves the inhibition of the meiotic cell cycle. This direct demonstration that a physiological signal initiated by a stimulus in one region of an intact tissue can travel across many layers of cells via cyclic nucleotide diffusion through gap junctions could provide a general mechanism for diverse cellular processes.

  6. Cyclic GMP alters Ca exchange in vascular smooth muscle

    International Nuclear Information System (INIS)

    Magliola, L.; Bailey, B.; Jones, A.W.

    1986-01-01

    Contraction and 42 K efflux from vascular smooth muscle stimulated either by norepinephrine (NE) or by K-depolarization is dependent on an increase in cytosolic Ca concentration. The purpose of this study was to determine if cyclic GMP (cGMP) inhibited these processes and if inhibition was secondary to the action of cGMP on Ca movements. Basal cGMP content of rat aorta was 1.2 fmol/mg wet wt. Sodium nitroprusside (NP) increased cGMP ∼2-fold at 1 nM and ∼750-fold at 1 μM with no effect on cAMP levels. A 5 min pretreatment with NP (1 μM) completely prevented tension development induced by 3 μM NE. The same concentration of NP also inhibited NE-stimulated 42 K and 45 Ca efflux > 90 and > 80%, respectively. Removal of NP in the continued presence of NE (3 μM) caused recovery of the 42 K efflux response to ∼75% of control with a half-time of ∼2.5 min. NP (1 μM) also caused a rapid relaxation of aorta contracted with 3 μM NE and a loss of the 42 K efflux response with half-times of 2-3 min. In contrast, 100 μM NP produced only a 50% inhibition of contraction induced by high K (55 mM). Also, NP (1 μM) inhibited K-stimulated 42 K efflux only ∼25%. These results demonstrate both a concentration- and a time-dependent relationship between increases in cGMP induced by NP and decreases in NE-stimulated contraction, 42 K and 45 Ca effluxes. They also indicate that the sensitivity of NE-induced contraction and 42 K efflux to NP is greater than that induced by high K. These studies suggest that cGMP modulates the control sites for Ca exchange in the plasma membrane and sarcoplasmic reticulum

  7. Cyclic GMP-AMP displays mucosal adjuvant activity in mice.

    Directory of Open Access Journals (Sweden)

    Ivana Škrnjug

    Full Text Available The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity--a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines.

  8. Cyclic GMP-AMP displays mucosal adjuvant activity in mice.

    Science.gov (United States)

    Škrnjug, Ivana; Guzmán, Carlos Alberto; Rueckert, Christine; Ruecker, Christine

    2014-01-01

    The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP) after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity--a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines.

  9. Cellular signaling with nitric oxide and cyclic GMP

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    F. Murad

    1999-11-01

    Full Text Available During the past two decades, nitric oxide signaling has been one of the most rapidly growing areas in biology. This simple free radical gas can regulate an ever growing list of biological processes. In most instances nitric oxide mediates its biological effects by activating guanylyl cyclase and increasing cyclic GMP synthesis. However, the identification of effects of nitric oxide that are independent of cyclic GMP is also growing at a rapid rate. The effects of nitric oxide can mediate important physiological regulatory events in cell regulation, cell-cell communication and signaling. Nitric oxide can function as an intracellular messenger, neurotransmitter and hormone. However, as with any messenger molecule, there can be too much or too little of the substance and pathological events ensue. Methods to regulate either nitric oxide formation, metabolism or function have been used therapeutically for more than a century as with nitroglycerin therapy. Current and future research should permit the development of an expanded therapeutic armamentarium for the physician to manage effectively a number of important disorders. These expectations have undoubtedly fueled the vast research interests in this simple molecule.

  10. Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis.

    Science.gov (United States)

    Shaw, Catherine A; Webb, David J; Rossi, Adriano G; Megson, Ian L

    2009-05-07

    Nitric oxide (NO) can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-). In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDMvarphi), and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP) is able to limit apoptosis in this cell type. Characterisation of the NO-related species generated by (Z)-1- [2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-, chloride (GEA-3162) was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR) spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMvarphi. Resultant MDMvarphi were treated for 24 h with DETA/NO (100 - 1000 muM) or GEA-3162 (10 - 300 muM) in the presence or absence of BAY 41-2272 (1 muM), isobutylmethylxanthine (IBMX; 1 muM), 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 muM) or 8-bromo-cGMP (1 mM). Apoptosis in MDMvarphi was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining. Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO) had no effect on cell viability, but ONOO- (GEA-3162) caused a concentration-dependent induction of apoptosis in MDMvarphi. Preconditioning of MDMvarphi with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX), or the NO-independent stimulator of soluble guanylate cyclase, BAY 41-2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner. These results

  11. Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis

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    Rossi Adriano G

    2009-05-01

    Full Text Available Abstract Background Nitric oxide (NO can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-. In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDMϕ, and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP is able to limit apoptosis in this cell type. Methods Characterisation of the NO-related species generated by (Z-1- [2-(2-aminoethyl-N-(2-ammonioethylamino]diazen-1-ium-1,2-diolate (DETA/NO and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl-, chloride (GEA-3162 was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMϕ. Resultant MDMϕ were treated for 24 h with DETA/NO (100 – 1000 μM or GEA-3162 (10 – 300 μM in the presence or absence of BAY 41–2272 (1 μM, isobutylmethylxanthine (IBMX; 1 μM, 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 μM or 8-bromo-cGMP (1 mM. Apoptosis in MDMϕ was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining. Results Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO had no effect on cell viability, but ONOO- (GEA-3162 caused a concentration-dependent induction of apoptosis in MDMϕ. Preconditioning of MDMϕ with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX, or the NO-independent stimulator of soluble guanylate cyclase, BAY 41–2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner

  12. The Cyclic AMP-Vfr Signaling Pathway in Pseudomonas aeruginosa Is Inhibited by Cyclic Di-GMP

    DEFF Research Database (Denmark)

    Almblad, Henrik; Harrison, Joe J; Rybtke, Morten

    2015-01-01

    infection give rise to rugose small colony variants (RSCVs), which are hyper-biofilm-forming mutants that commonly possess mutations that increase production of the biofilm-promoting secondary messenger cyclic di-GMP (c-di-GMP). We show that RSCVs display a decreased production of acute virulence factors...... as a direct result of elevated c-di-GMP content. Overproduction of c-di-GMP causes a decrease in the transcription of virulence factor genes that are regulated by the global virulence regulator Vfr. The low level of Vfr-dependent transcription is caused by a low level of its coactivator, cyclic AMP (c......AMP), which is decreased in response to a high level of c-di-GMP. Mutations that cause reversion of the RSCV phenotype concomitantly reactivate Vfr-cAMP signaling. Attempts to uncover the mechanism underlying the observed c-di-GMP-mediated lowering of cAMP content provided evidence that it is not caused...

  13. Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence.

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    Shi-qi An

    2014-10-01

    Full Text Available Bis-(3',5' cyclic di-guanylate (cyclic di-GMP is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc. This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K(d∼2 µM. Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.

  14. Stimulation of cyclic GMP efflux in human melanocytes by hypergravity generated by centrifugal acceleration

    NARCIS (Netherlands)

    Ivanova, Krassimira; Zadeh, Nahid Hamidi; Block, Ingrid; Das, Pranab K.; Gerzer, Rupert

    2004-01-01

    Gravity alteration (micro- and hypergravity) is known to influence cell functions. As guanosine 3',5'-cyclic monophosphate (cGMP) plays an important role in human melanocyte functions and different guanylyl cyclase isoforms are responsible for cGMP synthesis in human non-metastatic and metastatic

  15. Optogenetic Manipulation of Cyclic Di-GMP (c-di-GMP) Levels Reveals the Role of c-di-GMP in Regulating Aerotaxis Receptor Activity in Azospirillum brasilense.

    Science.gov (United States)

    O'Neal, Lindsey; Ryu, Min-Hyung; Gomelsky, Mark; Alexandre, Gladys

    2017-09-15

    Bacterial chemotaxis receptors provide the sensory inputs that inform the direction of navigation in changing environments. Recently, we described the bacterial second messenger cyclic di-GMP (c-di-GMP) as a novel regulator of a subclass of chemotaxis receptors. In Azospirillum brasilense , c-di-GMP binds to a chemotaxis receptor, Tlp1, and modulates its signaling function during aerotaxis. Here, we further characterize the role of c-di-GMP in aerotaxis using a novel dichromatic optogenetic system engineered for manipulating intracellular c-di-GMP levels in real time. This system comprises a red/near-infrared-light-regulated diguanylate cyclase and a blue-light-regulated c-di-GMP phosphodiesterase. It allows the generation of transient changes in intracellular c-di-GMP concentrations within seconds of irradiation with appropriate light, which is compatible with the time scale of chemotaxis signaling. We provide experimental evidence that binding of c-di-GMP to the Tlp1 receptor activates its signaling function during aerotaxis, which supports the role of transient changes in c-di-GMP levels as a means of adjusting the response of A. brasilense to oxygen gradients. We also show that intracellular c-di-GMP levels in A. brasilense change with carbon metabolism. Our data support a model whereby c-di-GMP functions to imprint chemotaxis receptors with a record of recent metabolic experience, to adjust their contribution to the signaling output, thus allowing the cells to continually fine-tune chemotaxis sensory perception to their metabolic state. IMPORTANCE Motile bacteria use chemotaxis to change swimming direction in response to changes in environmental conditions. Chemotaxis receptors sense environmental signals and relay sensory information to the chemotaxis machinery, which ultimately controls the swimming pattern of cells. In bacteria studied to date, differential methylation has been known as a mechanism to control the activity of chemotaxis receptors and

  16. Evidence for a Messenger Function of Cyclic GMP During Phosphodiesterase Induction in Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Pasveer, Frank J.; Meer, Rob C. van der; Heijden, Paul R. van der; Walsum, Hans van; Konijn, Theo M.

    1982-01-01

    Chemotactic stimulation of vegetative or aggregative Dictyostelium discoideum cells induced a transient elevation of cyclic GMP levels. The addition of chemoattractants to postvegetative cells by pulsing induced phosphodiesterase activity. The following lines of evidence suggest a messenger function

  17. Occurrence of Cyclic di-GMP-Modulating Output Domains in Cyanobacteria: an Illuminating Perspective

    Science.gov (United States)

    Agostoni, Marco; Koestler, Benjamin J.; Waters, Christopher M.; Williams, Barry L.; Montgomery, Beronda L.

    2013-01-01

    ABSTRACT Microorganisms use a variety of metabolites to respond to external stimuli, including second messengers that amplify primary signals and elicit biochemical changes in a cell. Levels of the second messenger cyclic dimeric GMP (c-di-GMP) are regulated by a variety of environmental stimuli and play a critical role in regulating cellular processes such as biofilm formation and cellular motility. Cyclic di-GMP signaling systems have been largely characterized in pathogenic bacteria; however, proteins that can impact the synthesis or degradation of c-di-GMP are prominent in cyanobacterial species and yet remain largely underexplored. In cyanobacteria, many putative c-di-GMP synthesis or degradation domains are found in genes that also harbor light-responsive signal input domains, suggesting that light is an important signal for altering c-di-GMP homeostasis. Indeed, c-di-GMP-associated domains are often the second most common output domain in photoreceptors—outnumbered only by a histidine kinase output domain. Cyanobacteria differ from other bacteria regarding the number and types of photoreceptor domains associated with c-di-GMP domains. Due to the widespread distribution of c-di-GMP domains in cyanobacteria, we investigated the evolutionary origin of a subset of genes. Phylogenetic analyses showed that c-di-GMP signaling systems were present early in cyanobacteria and c-di-GMP genes were both vertically and horizontally inherited during their evolution. Finally, we compared intracellular levels of c-di-GMP in two cyanobacterial species under different light qualities, confirming that light is an important factor for regulating this second messenger in vivo. PMID:23943760

  18. A cyclic GMP signalling module that regulates gliding motility in a malaria parasite.

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    Robert W Moon

    2009-09-01

    Full Text Available The ookinete is a motile stage in the malaria life cycle which forms in the mosquito blood meal from the zygote. Ookinetes use an acto-myosin motor to glide towards and penetrate the midgut wall to establish infection in the vector. The regulation of gliding motility is poorly understood. Through genetic interaction studies we here describe a signalling module that identifies guanosine 3', 5'-cyclic monophosphate (cGMP as an important second messenger regulating ookinete differentiation and motility. In ookinetes lacking the cyclic nucleotide degrading phosphodiesterase delta (PDEdelta, unregulated signalling through cGMP results in rounding up of the normally banana-shaped cells. This phenotype is suppressed in a double mutant additionally lacking guanylyl cyclase beta (GCbeta, showing that in ookinetes GCbeta is an important source for cGMP, and that PDEdelta is the relevant cGMP degrading enzyme. Inhibition of the cGMP-dependent protein kinase, PKG, blocks gliding, whereas enhanced signalling through cGMP restores normal gliding speed in a mutant lacking calcium dependent protein kinase 3, suggesting at least a partial overlap between calcium and cGMP dependent pathways. These data demonstrate an important function for signalling through cGMP, and most likely PKG, in dynamically regulating ookinete gliding during the transmission of malaria to the mosquito.

  19. Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High Affinity Ligand for STING

    OpenAIRE

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J.

    2013-01-01

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2′-OH of GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and 5′-phosphate of GMP. This mo...

  20. The cyclic-di-GMP signaling pathway in the Lyme disease spirochete, Borrelia burgdorferi

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    Elizabeth A. Novak

    2014-05-01

    Full Text Available In nature, the Lyme disease spirochete Borrelia burgdorferi cycles between the unrelated environments of the Ixodes tick vector and mammalian host. In order to survive transmission between hosts, B. burgdorferi must be able to not only detect changes in its environment, but also rapidly and appropriately respond to these changes. One manner in which this obligate parasite regulates and adapts to its changing environment is through cyclic-di-GMP (c-di-GMP signaling. c-di-GMP has been shown to be instrumental in orchestrating the adaptation of B. burgdorferi to the tick environment. B. burgdorferi possesses only one set of c-di-GMP-metabolizing genes (one diguanylate cyclase and two distinct phosphodiesterases and one c-di-GMP-binding PilZ-domain protein designated as PlzA. While studies in the realm of c-di-GMP signaling in B. burgdorferi have exploded in the last few years, there are still many more questions than answers. Elucidation of the importance of c-di-GMP signaling to B. burgdorferi may lead to the identification of mechanisms that are critical for the survival of B. burgdorferi in the tick phase of the enzootic cycle as well as potentially delineate a role (if any c-di-GMP may play in the transmission and virulence of B. burgdorferi during the enzootic cycle, thereby enabling the development of effective drugs for the prevention and/or treatment of Lyme disease.

  1. The participation of elevated levels of cyclic GMP in the recovery from radiation-induced mitotic delay

    International Nuclear Information System (INIS)

    Daniel, J.W.; Oleinick, N.L.

    1984-01-01

    The levels of cyclic AMP and cyclic GMP have been measured in Physarum plasmodia before and after treatment with gamma-radiation, 2 mM caffeine, or combinations of the two agents compared to the length of the radiation-induced mitotic delay. Caffeine alone produces a rapid transient elevation of cyclic AMP and a slower delayed elevation of cyclic GMP. Irradiation elicits an immediate transient increase in cyclic AMP and a later cyclic GMP increase which accompanies or precedes the delayed mitosis. A composite pattern is produced by combinations of radiation and caffeine, a distinctive feature of which is an elevated level of cyclic GMP near the time of the radiation-delayed and caffeine-promoted mitosis. With pretreatment by caffeine, the least radiation-induced mitotic delay occurs when plasmodia are irradiated during the caffeine-elicited increase in cyclic GMP. The plasmodium becomes refractory to the reduction of mitotic delay by caffeine at approximately the time it becomes refractory to the further elevation of cyclic GMP by caffeine. The data support a role for cyclic AMP in the onset of and for cyclic GMP in the recovery from mitotic delay induced by ionizing radiation. (author)

  2. Atrial natriuretic peptide receptor heterogeneity and effects on cyclic GMP accumulation

    International Nuclear Information System (INIS)

    Leitman, D.C.

    1988-01-01

    The effects of atrial natriuretic peptide (ANP), oxytocin (OT) and vasopressin (AVP) on guanylate cyclase activity and cyclic GMP accumulation were examined, since these hormones appear to be intimately associated with blood pressure and intravascular volume homeostasis. ANP was found to increase cyclic GMP accumulation in ten cell culture systems, which were derived from blood vessels, adrenal cortex, kidney, lung, testes and mammary gland. ANP receptors were characterized in intact cultured cells using 125 I-ANP 8-33 . Specific 125 I-ANP binding was saturable and of high affinity. Scratchard analysis of the binding data for all cell types exhibited a straight line, indicating that these cells possessed a single class of binding sites. Despite the presence of linear Scatchard plots, these studies demonstrated that cultured cells possess two functionally and physically distinct ANP-binding sites. Most of the ANP-binding sites in cultured cells have a molecular size of 66,000 daltons under reducing conditions. The identification of cultured cell types in which hormones (ANP and oxytocin) regulate guanylate cyclase activity and increase cyclic GMP synthesis will provide valuable systems to determine the mechanisms of hormone-receptor coupling to guanylate cyclase and the cellular processes regulated by cyclic GMP

  3. Molecular cloning and functional characterization of porcine cyclic GMP-AMP synthase.

    Science.gov (United States)

    Wang, Jiang; Chu, Beibei; Du, Lili; Han, Yingqian; Zhang, Xuemei; Fan, Shuangshuang; Wang, Yueying; Yang, Guoyu

    2015-06-01

    Cyclic GMP-AMP synthase (cGAS), which belongs to the nucleotidyltransferase family, recognizes cytosolic DNA and induces the type I interferon (IFN) pathway through the synthesis of the second messenger cGAMP. In this study, porcine cGAS (p-cGAS) was identified and its tissue distribution, subcellular localization, and functions in innate immunity were characterized. The coding sequence of p-cGAS is 1494 bp long, encodes 497 amino acids, and is most similar (74%) to Bos taurus cGAS. p-cGAS mRNA is abundant in the spleen, duodenum, jejunum, and ileum. The subcellular distribution of p-cGAS is not only in the cytosol, but also on the endoplasmic reticulum (ER) membrane. The overexpression of wild-type p-cGAS in porcine kidney epithelial cells, but not its catalytically inactive mutants, induced IFN-β expression, which was dependent on STING and IRF3. However, the downregulation of p-cGAS by RNA interference markedly reduced IFN-β expression after pseudorabies virus (PRV) infection or poly(dA:dT) transfection. These results demonstrate that p-cGAS is an important DNA sensor, required for IFN-β activation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Cyclic GMP-AMP Synthase is an Innate Immune Sensor of HIV and Other Retroviruses

    OpenAIRE

    Gao, Daxing; Wu, Jiaxi; Wu, You-Tong; Du, Fenghe; Aroh, Chukwuemika; Yan, Nan; Sun, Lijun; Chen, Zhijian J.

    2013-01-01

    Retroviruses, including HIV, can activate innate immune responses, but the host sensors for retroviruses are largely unknown. Here we show that HIV infection activates cyclic-GMP-AMP (cGAMP) synthase (cGAS) to produce cGAMP, which binds to and activates the adaptor protein STING to induce type-I interferons and other cytokines. Inhibitors of HIV reverse transcriptase, but not integrase, abrogated interferon-β induction by the virus, suggesting that the reverse transcribed HIV DNA triggers the...

  5. Cyclic GMP-AMP Synthase is Activated by Double-stranded DNA-Induced Oligomerization

    OpenAIRE

    Li, Xin; Shu, Chang; Yi, Guanghui; Chaton, Catherine T.; Shelton, Catherine L.; Diao, Jiasheng; Zuo, Xiaobing; Kao, C Cheng; Herr, Andrew B.; Li, Pingwei

    2013-01-01

    Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor mediating innate antimicrobial immunity. It catalyzes the synthesis of a noncanonical cyclic dinucleotide 2′,5′ cGAMP that binds to STING and mediates the activation of TBK1 and IRF-3. Activated IRF-3 translocates to the nucleus and initiates the transcription of the IFN-β gene. The structure of mouse cGAS bound to an 18 bp dsDNA revealed that cGAS interacts with dsDNA through two binding sites, forming a 2:2 complex. Enzyme assays and ...

  6. A cyclic GMP-dependent calcium-activated chloride current in smooth-muscle cells from rat mesenteric resistance arteries

    DEFF Research Database (Denmark)

    Matchkov, Vladimir; Aalkjær, Christian; Nilsson, Holger

    2004-01-01

    We have previously demonstrated the presence of a cyclic GMP (cGMP)-dependent calcium-activated inward current in vascular smooth-muscle cells, and suggested this to be of importance in synchronizing smooth-muscle contraction. Here we demonstrate the characteristics of this current. Using......M) in the pipette solution. The current was found to be a calcium-activated chloride current with an absolute requirement for cyclic GMP (EC50 6.4 microM). The current could be activated by the constitutively active subunit of PKG. Current activation was blocked by the protein kinase G antagonist Rp-8-Br-PET-cGMP...... differed from those of the calcium-activated chloride current in pulmonary myocytes, which was cGMP-independent, exhibited a high sensitivity to inhibition by niflumic acid, was unaffected by zinc ions, and showed outward current rectification as has previously been reported for this current. Under...

  7. CRP-Cyclic AMP Regulates the Expression of Type 3 Fimbriae via Cyclic di-GMP in Klebsiella pneumoniae.

    Directory of Open Access Journals (Sweden)

    Ching-Ting Lin

    Full Text Available Klebsiella pneumoniae is the predominant pathogen isolated from liver abscesses of diabetic patients in Asian countries. However, the effects of elevated blood glucose levels on the virulence of this pathogen remain largely unknown. Type 3 fimbriae, encoded by the mrkABCDF genes, are important virulence factors in K. pneumoniae pathogenesis. In this study, the effects of exogenous glucose and the intracellular cyclic AMP (cAMP signaling pathway on type 3 fimbriae expression regulation were investigated. The production of MrkA, the major subunit of type 3 fimbriae, was increased in glucose-rich medium, whereas cAMP supplementation reversed the effect. MrkA production was markedly increased by cyaA or crp deletion, but slightly decreased by cpdA deletion. In addition, the mRNA levels of mrkABCDF genes and the activity of PmrkA were increased in Δcrp strain, as well as the mRNA levels of mrkHIJ genes that encode cyclic di-GMP (c-di-GMP-related regulatory proteins that influence type 3 fimbriae expression. Moreover, the activities of PmrkHI and PmrkJ were decreased in ΔlacZΔcrp strain. These results indicate that CRP-cAMP down-regulates mrkABCDF and mrkHIJ at the transcriptional level. Further deletion of mrkH or mrkI in Δcrp strain diminished the production of MrkA, indicating that MrkH and MrkI are required for the CRP regulation of type 3 fimbriae expression. Furthermore, the high activity of PmrkHI in the ΔlacZΔcrp strain was diminished in ΔlacZΔcrpΔmrkHI, but increased in the ΔlacZΔcrpΔmrkJ strain. Deletion of crp increased the intracellular c-di-GMP concentration and reduced the phosphodiesterase activity. Moreover, we found that the mRNA levels of multiple genes related to c-di-GMP metabolism were altered in Δcrp strain. These indicate that CRP regulates type 3 fimbriae expression indirectly via the c-di-GMP signaling pathway. In conclusion, we found evidence of a coordinated regulation of type 3 fimbriae expression by the CRP

  8. Opposing actions of dibutyryl cyclic AMP and GMP on temperature in conscious guinea-pigs

    Science.gov (United States)

    Kandasamy, S. B.; Williaes, B. A.

    1983-01-01

    It is shown that the intracerebroventricular administration of dibutyryl cyclic AMP (Db-cAMP) induced hyperthermia in guinea pigs which was not mediated through prostaglandins or norepinephrine since a prostaglandin synthesis inhibitor and an alpha-adrenergic receptor blocking agent did not antagonize the hyperthermia. However, the hyperthermic response to Db-cAMP was attenuated by the central administration of a beta-adrenergic receptor antagonist, which indicates that cAMP may be involved, through beta-adrenergic receptors, in the central regulation of heat production and conservation. The central administration of Db-cGMP produced hypothermia which was not mediated via histamine H1 or H2 receptors and serotonin. The antagonism of hypothermia induced by Db-cGMP and acetylcholine + physostigmine by central administration of a cholinergic muscarine receptor antagonist and not by a cholinergic nicotinic receptor antagonist suggests that cholinoceptive neurons and endogenous cGMP may regulate heat loss through cholinergic muscarine receptors. It is concluded that these results indicate a regulatory role in thermoregulation provided by a balance between opposing actions of cAMP and cGMP in guinea pigs.

  9. The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response.

    Science.gov (United States)

    Khan, Mike; Harms, Jerome S; Marim, Fernanda M; Armon, Leah; Hall, Cherisse L; Liu, Yi-Ping; Banai, Menachem; Oliveira, Sergio C; Splitter, Gary A; Smith, Judith A

    2016-12-01

    Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host-Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a ΔbpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the ΔbpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase ΔcgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, ΔbpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  10. Cyclic di-GMP is essential for the survival of the lyme disease spirochete in ticks.

    Directory of Open Access Journals (Sweden)

    Ming He

    2011-06-01

    Full Text Available Cyclic dimeric GMP (c-di-GMP is a bacterial second messenger that modulates many biological processes. Although its role in bacterial pathogenesis during mammalian infection has been documented, the role of c-di-GMP in a pathogen's life cycle within a vector host is less understood. The enzootic cycle of the Lyme disease pathogen Borrelia burgdorferi involves both a mammalian host and an Ixodes tick vector. The B. burgdorferi genome encodes a single copy of the diguanylate cyclase gene (rrp1, which is responsible for c-di-GMP synthesis. To determine the role of c-di-GMP in the life cycle of B. burgdorferi, an Rrp1-deficient B. burgdorferi strain was generated. The rrp1 mutant remains infectious in the mammalian host but cannot survive in the tick vector. Microarray analyses revealed that expression of a four-gene operon involved in glycerol transport and metabolism, bb0240-bb0243, was significantly downregulated by abrogation of Rrp1. In vitro, the rrp1 mutant is impaired in growth in the media containing glycerol as the carbon source (BSK-glycerol. To determine the contribution of the glycerol metabolic pathway to the rrp1 mutant phenotype, a glp mutant, in which the entire bb0240-bb0243 operon is not expressed, was generated. Similar to the rrp1 mutant, the glp mutant has a growth defect in BSK-glycerol medium. In vivo, the glp mutant is also infectious in mice but has reduced survival in ticks. Constitutive expression of the bb0240-bb0243 operon in the rrp1 mutant fully rescues the growth defect in BSK-glycerol medium and partially restores survival of the rrp1 mutant in ticks. Thus, c-di-GMP appears to govern a catabolic switch in B. burgdorferi and plays a vital role in the tick part of the spirochetal enzootic cycle. This work provides the first evidence that c-di-GMP is essential for a pathogen's survival in its vector host.

  11. Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases.

    Science.gov (United States)

    Gao, Daxing; Li, Tuo; Li, Xiao-Dong; Chen, Xiang; Li, Quan-Zhen; Wight-Carter, Mary; Chen, Zhijian J

    2015-10-20

    TREX1 is an exonuclease that digests DNA in the cytoplasm. Loss-of-function mutations of TREX1 are linked to Aicardi-Goutieres Syndrome (AGS) and systemic lupus erythematosus (SLE) in humans. Trex1(-/-) mice exhibit autoimmune and inflammatory phenotypes that are associated with elevated expression of interferon (IFN)-induced genes (ISGs). Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates the IFN pathway. Upon binding to DNA, cGAS is activated to catalyze the synthesis of cGAMP, which functions as a second messenger that binds and activates the adaptor protein STING to induce IFNs and other cytokines. Here we show that genetic ablation of cGas in Trex1(-/-) mice eliminated all detectable pathological and molecular phenotypes, including ISG induction, autoantibody production, aberrant T-cell activation, and lethality. Even deletion of just one allele of cGas largely rescued the phenotypes of Trex1(-/-) mice. Similarly, deletion of cGas in mice lacking DNaseII, a lysosomal enzyme that digests DNA, rescued the lethal autoimmune phenotypes of the DNaseII(-/-) mice. Through quantitative mass spectrometry, we found that cGAMP accumulated in mouse tissues deficient in Trex1 or DNaseII and that this accumulation was dependent on cGAS. These results demonstrate that cGAS activation causes the autoimmune diseases in Trex1(-/-) and DNaseII(-/-) mice and suggest that inhibition of cGAS may lead to prevention and treatment of some human autoimmune diseases caused by self-DNA.

  12. Cyclic GMP-AMP as an Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA.

    Science.gov (United States)

    Kato, Kazuki; Omura, Hiroki; Ishitani, Ryuichiro; Nureki, Osamu

    2017-06-20

    The innate immune system functions as the first line of defense against invading bacteria and viruses. In this context, the cGAS/STING [cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase/STING] signaling axis perceives the nonself DNA associated with bacterial and viral infections, as well as the leakage of self DNA by cellular dysfunction and stresses, to elicit the host's immune responses. In this pathway, the noncanonical cyclic dinucleotide 2',3'-cyclic GMP-AMP (2',3'-cGAMP) functions as a second messenger for signal transduction: 2',3'-cGAMP is produced by the enzyme cGAS upon its recognition of double-stranded DNA, and then the 2',3'-cGAMP is recognized by the receptor STING to induce the phosphorylation of downstream factors, including TBK1 (TANK binding kinase 1) and IRF3 (interferon regulatory factor 3). Numerous crystal structures of the components of this cGAS/STING signaling axis have been reported and these clarify the structural basis for their signal transduction mechanisms. In this review, we summarize recent progress made in the structural dissection of this signaling pathway and indicate possible directions of forthcoming research.

  13. Role of cyclic di-GMP in Xylella fastidiosa biofilm formation, plant virulence, and insect transmission.

    Science.gov (United States)

    Chatterjee, Subhadeep; Killiny, Nabil; Almeida, Rodrigo P P; Lindow, Steven E

    2010-10-01

    Xylella fastidiosa must coordinately regulate a variety of traits contributing to biofilm formation, host plant and vector colonization, and transmission between plants. Traits such as production of extracellular polysaccharides (EPS), adhesins, extracellular enzymes, and pili are expressed in a cell-density-dependent fashion mediated by a cell-to-cell signaling system involving a fatty acid diffusible signaling factor (DSF). The expression of gene PD0279 (which has a GGDEF domain) is downregulated in the presence of DSF and may be involved in intracellular signaling by modulating the levels of cyclic di-GMP. PD0279, designated cyclic di-GMP synthase A (cgsA), is required for biofilm formation, plant virulence, and vector transmission. cgsA mutants exhibited a hyperadhesive phenotype in vitro and overexpressed gumJ, hxfA, hxfB, xadA, and fimA, which promote attachment of cells to surfaces and, hence, biofilm formation. The mutants were greatly reduced in virulence to grape albeit still transmissible by insect vectors, although at a reduced level compared with transmission rates of the wild-type strain, despite the fact that similar numbers of cells of the cgsA mutant were acquired by the insects from infected plants. High levels of EPS were measured in cgsA mutants compared with wild-type strains, and scanning electron microscopy analysis also revealed a thicker amorphous layer surrounding the mutants. Overexpression of cgsA in a cgsA-complemented mutant conferred the opposite phenotypes in vitro. These results suggest that decreases of cyclic di-GMP result from the accumulation of DSF as cell density increases, leading to a phenotypic transition from a planktonic state capable of colonizing host plants to an adhesive state that is insect transmissible.

  14. In Vivo Biochemistry: Single-Cell Dynamics of Cyclic Di-GMP in Escherichia coli in Response to Zinc Overload.

    Science.gov (United States)

    Yeo, Jongchan; Dippel, Andrew B; Wang, Xin C; Hammond, Ming C

    2018-01-09

    Intracellular signaling enzymes drive critical changes in cellular physiology and gene expression, but their endogenous activities in vivo remain highly challenging to study in real time and for individual cells. Here we show that flow cytometry can be performed in complex media to monitor single-cell population distributions and dynamics of cyclic di-GMP signaling, which controls the bacterial colonization program. These in vivo biochemistry experiments are enabled by our second-generation RNA-based fluorescent (RBF) biosensors, which exhibit high fluorescence turn-on in response to cyclic di-GMP. Specifically, we demonstrate that intracellular levels of cyclic di-GMP in Escherichia coli are repressed with excess zinc, but not with other divalent metals. Furthermore, in both flow cytometry and fluorescence microscopy setups, we monitor the dynamic increase in cellular cyclic di-GMP levels upon zinc depletion and show that this response is due to de-repression of the endogenous diguanylate cyclase DgcZ. In the presence of zinc, cells exhibit enhanced cell motility and increased sensitivity to antibiotics due to inhibited biofilm formation. Taken together, these results showcase the application of RBF biosensors in visualizing single-cell dynamic changes in cyclic di-GMP signaling in direct response to environmental cues such as zinc and highlight our ability to assess whether observed phenotypes are related to specific signaling enzymes and pathways.

  15. Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High Affinity Ligand for STING

    Science.gov (United States)

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J.

    2013-01-01

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2′-OH of GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and 5′-phosphate of GMP. This molecule, termed 2′3′-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2′3′-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. PMID:23747010

  16. The crucial role of cyclic GMP in the eclosion hormone mediated signal transduction in the silkworm metamorphoses.

    Science.gov (United States)

    Shibanaka, Y; Hayashi, H; Okada, N; Fujita, N

    1991-10-31

    The signal transduction of the peptide, eclosion hormone, in the silkworm Bombyx mori appears to be mediated via the second messenger cyclic GMP throughout their life cycle. Injection of 8-bromo-cGMP induced the ecdysis behavior in pharate adults with similar latency to eclosion hormone-induced ecdysis; the moulting occurred 50-70 min after the injection. The potency of 8Br-cGMP was 10(2) fold higher than that of cGMP and the efficacy was increased by the co-injection of the phosphodiesterase inhibitor IBMX. On the other hand, in the silkworm pupal ecdysis the eclosion hormone and also 8Br-cGMP induced the moulting behavior in a dose-dependent manner. The adult development of the ability to respond to 8Br-cGMP took place concomitantly with the response to the eclosion hormone. Both the developmental time courses were shifted by a shift of light and dark cycles. Accordingly, the sensitivities to the peptide and cyclic nucleotide developed correspondently under the light and dark circadian rhythm. Thus throughout the silkworm life cycle, eclosion hormone is effective to trigger the ecdysis behavior and cGMP plays a crucial role as the second messenger in the eclosion hormone-mediated signal transduction.

  17. Structural Basis for the Catalytic Mechanism of DncV, Bacterial Homolog of Cyclic GMP-AMP Synthase.

    Science.gov (United States)

    Kato, Kazuki; Ishii, Ryohei; Hirano, Seiichi; Ishitani, Ryuichiro; Nureki, Osamu

    2015-05-05

    Cyclic dinucleotides (CDNs) play key roles as second messengers and signaling molecules in bacteria and metazoans. The newly identified dinucleotide cyclase in Vibrio cholerae (DncV) produces three different CDNs containing two 3'-5' phosphodiester bonds, and its predominant product is cyclic GMP-AMP, whereas mammalian cyclic GMP-AMP synthase (cGAS) produces only cyclic GMP-AMP containing mixed 2'-5' phosphodiester bonds. We report the crystal structures of V. cholerae and Escherichia coli DncV in complex with various nucleotides in the pre-reaction states. The high-resolution structures revealed that DncV preferably recognizes ATP and GTP as acceptor and donor nucleotides, respectively, in the first nucleotidyl transfer reaction. Considering the recently reported intermediate structures, our pre-reaction state structures provide the precise mechanism of 3'-5' linked cyclic AMP-GMP production in bacteria. A comparison with cGAS in the pre-reaction states suggests that the orientation of the acceptor nucleotide primarily determines the distinct linkage specificities between DncV and cGAS. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Multiple Degradation Pathways of Chemoattractant Mediated Cyclic GMP Accumulation in Dictyostelium

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Lookeren Campagne, Michiel M. van; Kesbeke, Fanja

    1983-01-01

    Chemoattractants induce a transient accumulation of cGMP levels in Dictyostelium. Intracellular cGMP levels reach a peak at 10 s and prestimulated cGMP levels are recovered at about 30 s. Intracellular and extracellular cGMP levels were detected simultaneously after stimulation of D. lacteum cells

  19. Reactive oxygen species drive evolution of pro-biofilm variants in pathogens by modulating cyclic-di-GMP levels

    DEFF Research Database (Denmark)

    Chua, Song Lin; Ding, Yichen; Liu, Yang

    2016-01-01

    . Comparative genomic analysis of the RSCVs revealed that mutations in the wspF gene, which encodes for a repressor of WspR diguanylate cyclase (DGC), were responsible for increased intracellular cyclic-di-GMP content and production of Psl exopolysaccharide. Psl provides the first line of defence against ROS...

  20. The enzymatic preparation of [α-32P]nucleoside triphosphates, cyclic [32P]AMP, and cyclic [32P]GMP

    International Nuclear Information System (INIS)

    Walseth, T.F.; Johnson, R.A.

    1979-01-01

    A method has been developed for the enzymatic preparation of α- 32 P-labelled ribo- and deoxyribonucleoside triphosphates, cyclic [ 32 P]AMP, and cyclic [ 32 P]GMP of high specific radioactivity and in high yield from 32 Psub(i). The method also enables the preparation of [γ- 32 P]ATP, [γ- 32 P]GTP, [γ- 32 P]ITP, and [γ- 32 P]-dATP of very high specific activity and in high yield. (Auth.)

  1. Insulin alters the target size of the peripheral cyclic AMP phosphodiesterase but not the integral cyclic GMP-stimulated cyclic AMP phosphodiesterase in liver plasma membranes

    International Nuclear Information System (INIS)

    Wallace, A.V.; Martin, B.R.; Houslay, M.D.

    1990-01-01

    Radiation inactivation of the two high affinity cyclic AMP phosphodiesterases (PDE) found in liver plasma membranes afforded an estimation of their molecular target sizes in situ. The activity of the peripheral plasma membrane PDE decayed as a single exponential with a target size corresponding to a monomer of circa 54 kDa. The integral, cyclic GMP-stimulated PDE decayed as a dimer of circa 125 kDa. Preincubation of plasma membranes with insulin (10nM), prior to irradiation, caused the target size of only the peripheral plasma membrane PDE to increase. We suggest that insulin addition causes the peripheral plasma membrane PDE to alter its coupling to an integral plasma membrane protein with a target size of circa 90 kDa

  2. Cyclic GMP-AMP synthase is activated by double-stranded DNA-induced oligomerization.

    Science.gov (United States)

    Li, Xin; Shu, Chang; Yi, Guanghui; Chaton, Catherine T; Shelton, Catherine L; Diao, Jiasheng; Zuo, Xiaobing; Kao, C Cheng; Herr, Andrew B; Li, Pingwei

    2013-12-12

    Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor mediating innate antimicrobial immunity. It catalyzes the synthesis of a noncanonical cyclic dinucleotide, 2',5' cGAMP, that binds to STING and mediates the activation of TBK1 and IRF-3. Activated IRF-3 translocates to the nucleus and initiates the transcription of the IFN-β gene. The structure of mouse cGAS bound to an 18 bp dsDNA revealed that cGAS interacts with dsDNA through two binding sites, forming a 2:2 complex. Enzyme assays and IFN-β reporter assays of cGAS mutants demonstrated that interactions at both DNA binding sites are essential for cGAS activation. Mutagenesis and DNA binding studies showed that the two sites bind dsDNA cooperatively and that site B plays a critical role in DNA binding. The structure of mouse cGAS bound to dsDNA and 2',5' cGAMP provided insight into the catalytic mechanism of cGAS. These results demonstrated that cGAS is activated by dsDNA-induced oligomerization. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. High levels of cyclic-di-GMP in plant-associated Pseudomonas correlate with evasion of plant immunity.

    Science.gov (United States)

    Pfeilmeier, Sebastian; Saur, Isabel Marie-Luise; Rathjen, John Paul; Zipfel, Cyril; Malone, Jacob George

    2016-05-01

    The plant innate immune system employs plasma membrane-localized receptors that specifically perceive pathogen/microbe-associated molecular patterns (PAMPs/MAMPs). This induces a defence response called pattern-triggered immunity (PTI) to fend off pathogen attack. Commensal bacteria are also exposed to potential immune recognition and must employ strategies to evade and/or suppress PTI to successfully colonize the plant. During plant infection, the flagellum has an ambiguous role, acting as both a virulence factor and also as a potent immunogen as a result of the recognition of its main building block, flagellin, by the plant pattern recognition receptors (PRRs), including FLAGELLIN SENSING2 (FLS2). Therefore, strict control of flagella synthesis is especially important for plant-associated bacteria. Here, we show that cyclic-di-GMP [bis-(3'-5')-cyclic di-guanosine monophosphate], a central regulator of bacterial lifestyle, is involved in the evasion of PTI. Elevated cyclic-di-GMP levels in the pathogen Pseudomonas syringae pv. tomato (Pto) DC3000, the opportunist P. aeruginosa PAO1 and the commensal P. protegens Pf-5 inhibit flagellin synthesis and help the bacteria to evade FLS2-mediated signalling in Nicotiana benthamiana and Arabidopsis thaliana. Despite this, high cellular cyclic-di-GMP concentrations were shown to drastically reduce the virulence of Pto DC3000 during plant infection. We propose that this is a result of reduced flagellar motility and/or additional pleiotropic effects of cyclic-di-GMP signalling on bacterial behaviour. © 2015 THE AUTHORS MOLECULAR PLANT PATHOLOGY PUBLISHED BY BRITISH SOCIETY FOR PLANT PATHOLOGY AND JOHN WILEY & SONS LTD.

  4. Inhibition of excitatory synaptic transmission in the trigeminal motor nucleus by the nitric oxide-cyclic GMP signaling pathway.

    Science.gov (United States)

    Pose, Inés; Silveira, Valentina; Morales, Francisco R

    2011-06-01

    Nitric oxide (NO) and cyclic GMP (cGMP) suppressed glutamatergic synaptic transmission to trigeminal motoneurons in brain stem slices of neonatal rats. Histological studies showed guanylate cyclase (GC) containing fibers in the trigeminal motor pool. Glutamatergic excitatory postsynaptic currents (EPSCs) were recorded from neonatal trigeminal motoneurons in response to stimulation of the supratrigeminal nucleus (SuV). The NO donors DETA/NONOate (DETA/NO), at a concentration which released 275.1 nM of NO, and Spermine/NONOate (Sper/NO) reduced the amplitude of the EPSC to 52.7±0.6% and 60.1±10.8% of control values, respectively. These actions were not blocked by the GC inhibitors, ODQ or NS-2028. However, in the presence of YC-1 or BAY41-2272, modulators of GC that act as NO sensitizers, lower and otherwise ineffective concentrations of DETA/NO induced a reduction of the EPSC to 60.6±5.2%. Moreover, NO effects were mimicked by 8BrcGMP and by Zaprinast, an inhibitor of Phosphodiesterase 5. Glutamatergic currents evoked by exogenous glutamate were not reduced by DETA/NO nor 8BrcGMP. Paired-pulse facilitation was increased by NO donors. Under "minimal stimulation" conditions NO donors and cGMP increased the failure rate of evoked EPSCs. Protein kinase inhibitors antagonized cGMP effects. The results suggest that NO, through the synthesis of cGMP, presynaptically inhibits glutamatergic synaptic transmission on trigeminal motoneurons. We propose that NO has complex actions on motor pools; specific studies are needed to elucidate their physiological significance in the behaving animal. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Expression of Cyclic GMP-AMP Synthase in Patients With Systemic Lupus Erythematosus.

    Science.gov (United States)

    An, Jie; Durcan, Laura; Karr, Reynold M; Briggs, Tracy A; Rice, Gillian I; Teal, Thomas H; Woodward, Joshua J; Elkon, Keith B

    2017-04-01

    Type I interferon (IFN) is implicated in the pathogenesis of systemic lupus erythematosus (SLE) and interferonopathies such as Aicardi-Goutières syndrome. A recently discovered DNA-activated type I IFN pathway, cyclic GMP-AMP synthase (cGAS), has been linked to Aicardi-Goutières syndrome and mouse models of lupus. The aim of this study was to determine whether the cGAS pathway contributes to type I IFN production in patients with SLE. SLE disease activity was measured by the Safety of Estrogens in Lupus Erythematosus National Assessment version of the Systemic Lupus Erythematosus Disease Activity Index. Expression of messenger RNA for cGAS and IFN-stimulated genes (ISGs) was determined by quantitative polymerase chain reaction analysis. Cyclic GMP-AMP (cGAMP) levels were examined by multiple reaction monitoring with ultra-performance liquid chromatography tandem mass spectrometry. Expression of cGAS in peripheral blood mononuclear cells (PBMCs) was significantly higher in SLE patients than in normal controls (n = 51 and n = 20 respectively; P < 0.01). There was a positive correlation between cGAS expression and the IFN score (P < 0.001). The expression of cGAS in PBMCs showed a dose response to type I IFN stimulation in vitro, consistent with it being an ISG. Targeted measurement of cGAMP by tandem mass spectrometry detected cGAMP in 15% of the SLE patients (7 of 48) but none of the normal (0 of 19) or rheumatoid arthritis (0 of 22) controls. Disease activity was higher in SLE patients with cGAMP versus those without cGAMP. Increased cGAS expression and cGAMP in a proportion of SLE patients indicates that the cGAS pathway should be considered as a contributor to type I IFN production. Whereas higher cGAS expression may be a consequence of exposure to type I IFN, detection of cGAMP in patients with increased disease activity indicates potential involvement of this pathway in disease expression. © 2016, American College of Rheumatology.

  6. Topoisomerase 1 Inhibition Promotes Cyclic GMP-AMP Synthase-Dependent Antiviral Responses

    Directory of Open Access Journals (Sweden)

    Geneviève Pèépin

    2017-10-01

    Full Text Available Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1 by low-dose camptothecin (CPT can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40 large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development.

  7. Maintenance of cyclic GMP-AMP homeostasis by ENPP1 is involved in pseudorabies virus infection.

    Science.gov (United States)

    Wang, Jiang; Lu, Shao-Fang; Wan, Bo; Ming, Sheng-Li; Li, Guo-Li; Su, Bing-Qian; Liu, Jiao-Yang; Wei, Yu-Shuang; Yang, Guo-Yu; Chu, Bei-Bei

    2018-03-01

    In a previous study, we demonstrated that porcine cyclic GMP-AMP (cGAMP) synthase (cGAS) catalyzes cGAMP production and is an important DNA sensor for the pseudorabies virus (PRV)-induced activation of interferon β (IFN-β). Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) has recently been identified as the hydrolase of cGAMP in rodents, but its role in porcine cells is not clear. Our recent study demonstrated that porcine ENPP1 is responsible for the homeostasis of cGAMP and is critical for PRV infection. Porcine ENPP1 mRNA is predominantly expressed in muscle. PRV infection was enhanced by ENPP1 overexpression and attenuated by silencing of ENPP1. During PRV infection, the activation of IFN-β and NF-κB was reduced in ENPP1 overexpressed cells and promoted in ENPP1 knockdown cells. Investigation of the molecular mechanisms of ENPP1 during PRV infection showed that ENPP1 hydrolyzed cGAMP in PRV-infected or cGAMP-transfected cells and inhibited IRF3 phosphorylation, reducing IFN-β secretion. These results, combined with those for porcine cGAS, demonstrate that ENPP1 acts coordinately with cGAS to maintain the reservoir of cGAMP and participates in PRV infection. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. Physiological and Molecular Effects of the Cyclic Nucleotides cAMP and cGMP on Arabidopsis thaliana

    KAUST Repository

    Herrera, Natalia M.

    2012-12-01

    The cyclic nucleotide monophosphates (CNs), cAMP and cGMP, are second messengers that participate in the regulation of development, metabolism and adaptive responses. In plants, CNs are associated with the control of pathogen responses, pollen tube orientation, abiotic stress response, membrane transport regulation, stomatal movement and light perception. In this study, we hypothesize that cAMP and cGMP promote changes in the transcription level of genes related to photosynthesis, high light and membrane transport in Arabidopsis thaliana leaves and, that these changes at the molecular level can have functional biological consequences. For this reason we tested if CNs modulate the photosynthetic rate, responses to high light and root ion transport. Real time quantitative PCR was used to assess transcription levels of selected genes and infrared gas analyzers coupled to fluorescence sensors were used to measure the photosynthetic parameters. We present evidence that both cAMP and cGMP modulate foliar mRNA levels early after stimulation. The two CNs trigger different responses indicating that the signals have specificity. A comparison of proteomic and transcriptional changes suggest that both transcriptional and post-transcriptional mechanisms are modulated by CNs. cGMP up-regulates the mRNA levels of components of the photosynthesis and carbon metabolism. However, neither cAMP nor cGMP trigger differences in the rate of carbon assimilation, maximum efficiency of the photosystem II (PSII), or PSII operating efficiency. It was also demonstrated that CN regulate the expression of its own targets, the cyclic nucleotide gated channels - CNGC. Further studies are needed to identify the components of the signaling transduction pathway that mediate cellular changes and their respective regulatory and/or signaling roles.

  9. Topoisomerase 1 Inhibition Promotes Cyclic GMP-AMP Synthase-Dependent Antiviral Responses.

    Science.gov (United States)

    Pépin, Geneviève; Nejad, Charlotte; Ferrand, Jonathan; Thomas, Belinda J; Stunden, H James; Sanij, Elaine; Foo, Chwan-Hong; Stewart, Cameron R; Cain, Jason E; Bardin, Philip G; Williams, Bryan R G; Gantier, Michael P

    2017-10-03

    Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1) by low-dose camptothecin (CPT) can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS) detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40) large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development. IMPORTANCE Recent studies suggest that low-dose DNA-damaging compounds traditionally used in cancer therapy can have opposite effects on antiviral responses, either suppressing (with the example of CPT) or potentiating (with the example of doxorubicin) them. Our work demonstrates that the minor DNA damage promoted by low-dose CPT can also trigger strong antiviral responses, dependent on the presence of viral oncogenes. Taken together, these results call for caution in the therapeutic use of low-dose chemotherapy agents to modulate antiviral responses in humans. Copyright © 2017 Pépin et al.

  10. 3',5'-Cyclic diguanylic acid (c-di-GMP) inhibits basal and growth factor-stimulated human colon cancer cell proliferation

    International Nuclear Information System (INIS)

    Karaolis, David K.R.; Cheng, Kunrong; Lipsky, Michael; Elnabawi, Ahmed; Catalano, Jennifer; Hyodo, Mamoru; Hayakawa, Yoshihiro; Raufman, Jean-Pierre

    2005-01-01

    The novel cyclic dinucleotide, 3',5'-cyclic diguanylic acid, cGpGp (c-di-GMP), is a naturally occurring small molecule that regulates important signaling mechanisms in prokaryotes. Recently, we showed that c-di-GMP has 'drug-like' properties and that c-di-GMP treatment might be a useful antimicrobial approach to attenuate the virulence and pathogenesis of Staphylococcus aureus and prevent or treat infection. In the present communication, we report that c-di-GMP (≤50 μM) has striking properties regarding inhibition of cancer cell proliferation in vitro. c-di-GMP inhibits both basal and growth factor (acetylcholine and epidermal growth factor)-induced cell proliferation of human colon cancer (H508) cells. Toxicity studies revealed that exposure of normal rat kidney cells and human neuroblastoma cells to c-di-GMP at biologically relevant doses showed no lethal cytotoxicity. Cyclic dinucleotides, such as c-di-GMP, represent an attractive and novel 'drug-platform technology' that can be used not only to develop new antimicrobial agents, but also to develop novel therapeutic agents to prevent or treat cancer

  11. The DNA sensor, cyclic GMP-AMP synthase, is essential for induction of IFN-β during Chlamydia trachomatis infection.

    Science.gov (United States)

    Zhang, Yugen; Yeruva, Laxmi; Marinov, Anthony; Prantner, Daniel; Wyrick, Priscilla B; Lupashin, Vladimir; Nagarajan, Uma M

    2014-09-01

    IFN-β has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFN-β expression during chlamydial infection. We determined that three-prime repair exonuclease-1, a host 3' to 5' exonuclease, reduced IFN-β expression significantly during chlamydial infection using small interfering RNA and gene knockout fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cyclic GMP-AMP synthase (cGAS) has been shown to bind cytosolic DNA to generate cyclic GMP-AMP, which binds to the signaling adaptor stimulator of IFN genes (STING) to induce IFN-β expression. We determined that cGAS is required for IFN-β expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFN-β, coculture of cells depleted for either STING or cGAS rescued IFN-β expression. These data demonstrate that cyclic GMP-AMP produced in infected cGAS(+)STING(-) cells can migrate into adjacent cells via gap junctions to function in trans in cGAS(-)STING(+) cells. Furthermore, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is most likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated DNA sensing directs IFN-β expression during Chlamydia trachomatis infection and suggest that effectors from infected cells can directly upregulate IFN-β expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis. Copyright © 2014 by The American Association of Immunologists, Inc.

  12. Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases

    DEFF Research Database (Denmark)

    Issinger, O G; Beier, H; Speichermann, N

    1980-01-01

    Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were...... by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins...... from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates...

  13. Cyclic GMP-AMP Synthase Is Required for Cell Proliferation and Inflammatory Responses in Rheumatoid Arthritis Synoviocytes

    OpenAIRE

    Wang, Yan; Su, Guo-Hua; Zhang, Fang; Chu, Jing-Xue; Wang, Yun-Shan

    2015-01-01

    Rheumatoid arthritis (RA) is characterized by inflammatory cell infiltration, fibroblast-like synoviocytes (FLS) invasive proliferation, and joint destruction. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces immune activation. In this study, we examined whether cGAS plays a role in RA FLS. In this study, cGAS was overexpressed in RA-FLS compared with OA FLS. TNFα stimulation induced cGAS expression in RA FLS. Overexpression of cGAS promoted the proliferation and knockdow...

  14. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING.

    Science.gov (United States)

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J

    2013-07-25

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2'-OH of GMP and 5'-phosphate of AMP, and the other between 3'-OH of AMP and 5'-phosphate of GMP. This molecule, termed 2'3'-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2'3'-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Differential Contribution of the Guanylyl Cyclase-Cyclic GMP-Protein Kinase G Pathway to the Proliferation of Neural Stem Cells Stimulated by Nitric Oxide

    Directory of Open Access Journals (Sweden)

    Bruno P. Carreira

    2012-02-01

    Full Text Available Nitric oxide (NO is an important inflammatory mediator involved in the initial boost in the proliferation of neural stem cells following brain injury. However, the mechanisms underlying the proliferative effect of NO are still unclear. The aim of this work was to investigate whether cyclic GMP (cGMP and the cGMP-dependent kinase (PKG are involved in the proliferative effect triggered by NO in neural stem cells. For this purpose, cultures of neural stem cells isolated from the mouse subventricular zone (SVZ were used. We observed that long-term exposure to the NO donor (24 h, NOC-18, increased the proliferation of SVZ cells in a cGMP-dependent manner, since the guanylate cyclase inhibitor, ODQ, prevented cell proliferation. Similarly to NOC-18, the cGMP analogue, 8-Br-cGMP, also increased cell proliferation. Interestingly, shorter exposures to NO (6 h increased cell proliferation in a cGMP-independent manner via the ERK/MAP kinase pathway. The selective inhibitor of PKG, KT5823, prevented the proliferative effect induced by NO at 24 h but not at 6 h. In conclusion, the proliferative effect of NO is initially mediated by the ERK/MAPK pathway, and at later stages by the GC/cGMP/PKG pathway. Thus, our work shows that NO induces neural stem cell proliferation by targeting these two pathways in a biphasic manner.

  16. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.

    Science.gov (United States)

    Sun, Lijun; Wu, Jiaxi; Du, Fenghe; Chen, Xiang; Chen, Zhijian J

    2013-02-15

    The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers host immune responses such as the production of type I interferons. Cytosolic DNA induces interferons through the production of cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced interferon-β in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and interferon-β induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.

  17. Mutation of the cyclic di-GMP phosphodiesterase gene in Burkholderia lata SK875 attenuates virulence and enhances biofilm formation.

    Science.gov (United States)

    Jung, Hae-In; Kim, Yun-Jung; Lee, Yun-Jung; Lee, Hee-Soo; Lee, Jung-Kee; Kim, Soo-Ki

    2017-10-01

    Burkholderia sp. is a gram-negative bacterium that commonly exists in the environment, and can cause diseases in plants, animals, and humans. Here, a transposon mutant library of a Burkholderia lata isolate from a pig with swine respiratory disease in Korea was screened for strains showing attenuated virulence in Caenorhabditis elegans. One such mutant was obtained, and the Tn5 insertion junction was mapped to rpfR, a gene encoding a cyclic di-GMP phosphodiesterase that functions as a receptor. Mutation of rpfR caused a reduction in growth on CPG agar and swimming motility as well as a rough colony morphology on Congo red agar. TLC analysis showed reduced AHL secretion, which was in agreement with the results from plate-based and bioluminescence assays. The mutant strain produced significantly more biofilm detected by crystal violet staining than the parent strain. SEM of the mutant strain clearly showed that the overproduced biofilm contained a filamentous structure. These results suggest that the cyclic di-GMP phosphodiesterase RpfR plays an important role in quorum sensing modulation of the bacterial virulence and biofilm formation.

  18. Nicorandil directly and cyclic GMP-dependently opens K+ channels in human bypass grafts

    Directory of Open Access Journals (Sweden)

    Marija Marinko

    2015-06-01

    Full Text Available As we previously demonstrated the role of different K+ channels in the action of nicorandil on human saphenous vein (HSV and human internal mammary artery (HIMA, this study aimed to analyse the contribution of the cGMP pathway in nicorandil-induced vasorelaxation and to determine the involvement of cGMP in the K+ channel-activating effect of nicorandil. An inhibitor of soluble guanylate cyclase (GC, ODQ, significantly inhibited nicorandil-induced relaxation, while ODQ plus glibenclamide, a selective ATP-sensitive K+ (KATP channel inhibitor, produced a further inhibition of both vessels. In HSV, ODQ in combination with 4-aminopyridine, a blocker of voltage-gated K+ (KV channels, did not modify the concentration-response to nicorandil compared with ODQ, whereas in HIMA, ODQ plus iberiotoxin, a selective blocker of large-conductance Ca2+-activated K+ (BKCa channels, produced greater inhibition than ODQ alone. We showed that the cGMP pathway plays a significant role in the vasorelaxant effect of nicorandil on HSV and HIMA. It seems that nicorandil directly opens KATP channels in both vessels and BKCa channels in HIMA, although it is possible that stimulation of GC contributes to KATP channels activation in HIMA. Contrary, the activation of KV channels in HSV is probably due to GC activation and increased levels of cGMP.

  19. Cyclic GMP-mediated memory enhancement in the object recognition test by inhibitors of phosphodiesterase-2 in mice.

    Science.gov (United States)

    Lueptow, Lindsay M; Zhan, Chang-Guo; O'Donnell, James M

    2016-02-01

    Cyclic nucleotide phosphodiesterase-2 (PDE2) is a potential therapeutic target for the treatment of cognitive dysfunction. Using the object recognition test (ORT), this study assessed the effects of two PDE2 inhibitors, Bay 60-7550 and ND7001, on learning and memory, and examined underlying mechanisms. To assess the role of PDE2 inhibition on phases of memory, Bay 60-7550 (3 mg/kg) was administered: 30 min prior to training; 0, 1, or 3 h after training; or 30 min prior to recall testing. To assess cyclic nucleotide involvement in PDE2 inhibitor-enhanced memory consolidation, either the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg; intraperitoneal (IP)), soluble guanylyl cyclase inhibitor 1H-[-1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 20 mg/kg; IP), protein kinase G inhibitor KT5823 (2.5 μg; intracerebroventricular (ICV)), or protein kinase A inhibitor H89 (1 μg; ICV) was administered 30 min prior to the PDE2 inhibitor Bay 60-7550 (3 mg/kg) or ND7001 (3 mg/kg). Changes in the phosphorylation of 3'5'-cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) at Ser-133 and vasodilator-stimulated phosphoprotein (VASP) at Ser-239 were determined to confirm activation of cAMP and 3'5'-cyclic guanosine monophosphate (cGMP) signaling. Bay 60-7550 (3 mg/kg) enhanced memory of mice in the ORT when given 30 min prior to training, immediately after training, or 30 min prior to recall. Inhibitors of the cGMP pathway blocked the memory-enhancing effects of both Bay 60-7550 (3 mg/kg) and ND7001 (3 mg/kg) on early consolidation processes. Bay 60-7550 (3 mg/kg) enhanced phosphorylation of CREB and VASP, both targets of cGMP-dependent protein kinase (PKG). These results confirm a potential of PDE2, or components of its signaling pathway, as a therapeutic target for drug discovery focused on restoring memory function.

  20. Clearance of Pseudomonas aeruginosa Foreign-Body Biofilm Infections through Reduction of the Cyclic Di-GMP Level in the Bacteria

    DEFF Research Database (Denmark)

    Christensen, Louise D.; van Gennip, Maria; Rybtke, Morten Theil

    2013-01-01

    Opportunistic pathogenic bacteria can engage in biofilm-based infections that evade immune responses and develop into chronic conditions. Because conventional antimicrobials cannot efficiently eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections....... It has recently been established that the secondary messenger cyclic diguanosine monophosphate (c-di-GMP) functions as a positive regulator of biofilm formation in several different bacteria. In the present study we investigated whether manipulation of the c-di-GMP level in bacteria potentially can...... be used for biofilm control in vivo. We constructed a Pseudomonas aeruginosa strain in which a reduction in the c-di-GMP level can be achieved via induction of the Escherichia coli YhjH c-di-GMP phosphodiesterase. Initial experiments showed that induction of yhjH expression led to dispersal...

  1. Nitric oxide increases cyclic GMP levels, AMP-activated protein kinase (AMPK)alpha1-specific activity and glucose transport in human skeletal muscle

    DEFF Research Database (Denmark)

    Deshmukh, A S; Long, Y C; de Castro Barbosa, T

    2010-01-01

    -nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) would increase intracellular cyclic GMP (cGMP) levels and promote glucose transport. METHODS: Skeletal muscle strips were prepared from vastus lateralis muscle biopsies obtained from seven healthy men. Muscle strips were incubated in the absence or presence...... of 5 mmol/l spermine NONOate or 120 nmol/l insulin. The L6 muscle cells were treated with spermine NONOate (20 micromol/l) and incubated in the absence or presence of insulin (120 nmol/l). The direct effect of spermine NONOate and insulin on glucose transport, cGMP levels and signal transduction...... was determined. RESULTS: In human skeletal muscle, spermine NONOate increased glucose transport 2.4-fold (p GMP levels (80-fold, p

  2. Cyclic GMP-AMP Synthase is a Cytosolic DNA Sensor that Activates the Type-I Interferon Pathway

    Science.gov (United States)

    Sun, Lijun; Wu, Jiaxi; Du, Fenghe; Chen, Xiang; Chen, Zhijian J.

    2013-01-01

    The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers the host immune responses such as the production of type-I interferons (IFN). Cytosolic DNA induces IFN through the production of cyclic-GMP-AMP (cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced IFNβ in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and IFNβ induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP. PMID:23258413

  3. Cyclic [G(2',5')pA(3',5')p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase.

    Science.gov (United States)

    Gao, Pu; Ascano, Manuel; Wu, Yang; Barchet, Winfried; Gaffney, Barbara L; Zillinger, Thomas; Serganov, Artem A; Liu, Yizhou; Jones, Roger A; Hartmann, Gunther; Tuschl, Thomas; Patel, Dinshaw J

    2013-05-23

    Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2',5')pA and A(3',5')pG phosphodiester linkages, designated c[G(2',5')pA(3',5')p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, resulting in formation of a catalytically competent and accessible nucleotide-binding pocket for generation of c[G(2',5')pA(3',5')p]. We demonstrate that cyclization occurs in a stepwise manner through initial generation of 5'-pppG(2',5')pA prior to cyclization to c[G(2',5')pA(3',5')p], with the latter positioned precisely in the catalytic pocket. Mutants of cGAS dsDNA-binding or catalytic pocket residues exhibit reduced or abrogated activity. Our studies have identified c[G(2',5')pA(3',5')p] as a founding member of a family of metazoan 2',5'-containing cyclic heterodinucleotide second messengers distinct from bacterial 3',5' cyclic dinucleotides. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. DNA-Mediated Cyclic GMP-AMP Synthase-Dependent and -Independent Regulation of Innate Immune Responses.

    Science.gov (United States)

    Motani, Kou; Ito, Shinji; Nagata, Shigekazu

    2015-05-15

    Cytoplasmic DNA activates cyclic GMP-AMP synthase (cGAS) to produce cyclic 2'-5'3'-5'GMP-AMP dinucleotide (2'5 'cGAMP). The binding of 2'5'cGAMP to an adaptor protein, stimulator of IFN genes (STING), activates a transcription factor, IFN regulatory factor 3, leading to the induction of IFN and chemokine gene expression. In this study, we found that the 2'5'cGAMP-dependent STING activation induced highly upregulated CXCL10 gene expression. Formation of a distinct STING dimer, which was detected by native PAGE, was induced by 2'5'cGAMP, but not 3'-5'3'-5'cGAMP. Analysis of DNase II(-/-) mice, which constitutively produce IFN-β and CXCL10, showed the accumulation of 2'5'cGAMP in their fetal livers and spleens, suggesting that the undigested DNA accumulating in DNase II(-/-) cells may have leaked from the lysosomes into the cytoplasm. The DNase II(-/-) mouse embryonic fibroblasts produced 2'5'cGAMP in a cGAS-dependent manner during apoptotic cell engulfment. However, cGAS deficiency did not impair the STING-dependent upregulation of CXCL10 in DNase II(-/-) mouse embryonic fibroblasts that was induced by apoptotic cell engulfment or DNA lipofection. These results suggest the involvement of a cGAS-independent additional DNA sensor(s) that induces the STING-dependent activation of innate immunity. Copyright © 2015 by The American Association of Immunologists, Inc.

  5. Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3′-5′)-cyclic-GMP in virulence

    Science.gov (United States)

    Kulesekara, Hemantha; Lee, Vincent; Brencic, Anja; Liberati, Nicole; Urbach, Jonathan; Miyata, Sachiko; Lee, Daniel G.; Neely, Alice N.; Hyodo, Mamoru; Hayakawa, Yoshihiro; Ausubel, Frederick M.; Lory, Stephen

    2006-01-01

    The opportunistic pathogen Pseudomonas aeruginosa is responsible for systemic infections in immunocompromised individuals and chronic respiratory disease in patients with cystic fibrosis. Cyclic nucleotides are known to play a variety of roles in the regulation of virulence-related factors in pathogenic bacteria. A set of P. aeruginosa genes, encoding proteins that contain putative domains characteristic of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) that are responsible for the maintenance of cellular levels of the second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) was identified in the annotated genomes of P. aeruginosa strains PAO1 and PA14. Although the majority of these genes are components of the P. aeruginosa core genome, several are located on presumptive horizontally acquired genomic islands. A comprehensive analysis of P. aeruginosa genes encoding the enzymes of c-di-GMP metabolism (DGC- and PDE-encoding genes) was carried out to analyze the function of c-di-GMP in two disease-related phenomena, cytotoxicity and biofilm formation. Analysis of the phenotypes of DGC and PDE mutants and overexpressing clones revealed that certain virulence-associated traits are controlled by multiple DGCs and PDEs through alterations in c-di-GMP levels. A set of mutants in selected DGC- and PDE-encoding genes exhibited attenuated virulence in a mouse infection model. Given that insertions in different DGC and PDE genes result in distinct phenotypes, it seems likely that the formation or degradation of c-di-GMP by these enzymes is in highly localized and intimately linked to particular targets of c-di-GMP action. PMID:16477007

  6. YfiBNR mediates cyclic di-GMP dependent small colony variant formation and persistence in Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Jacob G Malone

    2010-03-01

    Full Text Available During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs, auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes Yfi

  7. [Aging reduces contents of endogenous CO, cAMP and cGMP in rat penile tissues].

    Science.gov (United States)

    Qin, Wen-Bo; Wang, Shu-Qiu; Li, Ming; Kang, Yu-Ming; Gui, Shi-Liang; Chi, Bao-Jin

    2009-02-01

    To explore the relationship of aging with the changes of endogenous carbon monoxide (CO), cGMP and cAMP contents in the penile tissues of rats. Twenty-four male rats were equally divided into an 8-month, a 16-month and a 24-month group, and their penile erection was detected by injecting apomorphine, their penile cavernous body harvested, and the contents of CO, cAPM and cGMP detected by improved dual wavelength spectrophotometry. The contents of CO, cAPM and cGMP were reduced with the increase of age, with statistically significant differences between the three age groups (P < 0.01). Aging significantly decreased the contents of CO, cAMP and cGMP in the penile tissues of the rats, which suggests that aging might play an important role in erectile dysfunction.

  8. Cyclic GMP-AMP Synthase Is Required for Cell Proliferation and Inflammatory Responses in Rheumatoid Arthritis Synoviocytes

    Directory of Open Access Journals (Sweden)

    Yan Wang

    2015-01-01

    Full Text Available Rheumatoid arthritis (RA is characterized by inflammatory cell infiltration, fibroblast-like synoviocytes (FLS invasive proliferation, and joint destruction. Cyclic GMP-AMP synthase (cGAS is a cytosolic DNA sensor that induces immune activation. In this study, we examined whether cGAS plays a role in RA FLS. In this study, cGAS was overexpressed in RA-FLS compared with OA FLS. TNFα stimulation induced cGAS expression in RA FLS. Overexpression of cGAS promoted the proliferation and knockdown of cGAS inhibited the proliferation of RA FLS. cGAS overexpression enhanced the production of proinflammatory cytokines and matrix metalloproteinases (MMPs as well as AKT and ERK phosphorylation in TNFα-stimulated FLS. In contrast, cGAS silencing inhibited production of proinflammatory cytokines and matrix metalloproteinases (MMPs as well as AKT and ERK phosphorylation in TNFα-stimulated FLS. These results suggest that cGAS activates the AKT and ERK pathways to promote the inflammatory response of RA FLS, and the development of strategies targeting cGAS may have therapeutic potential for human RA.

  9. The catalytic mechanism of cyclic GMP-AMP synthase (cGAS) and implications for innate immunity and inhibition.

    Science.gov (United States)

    Hall, Justin; Ralph, Erik C; Shanker, Suman; Wang, Hong; Byrnes, Laura J; Horst, Reto; Wong, Jimson; Brault, Amy; Dumlao, Darren; Smith, James F; Dakin, Leslie A; Schmitt, Daniel C; Trujillo, John; Vincent, Fabien; Griffor, Matt; Aulabaugh, Ann E

    2017-12-01

    Cyclic GMP-AMP synthase (cGAS) is activated by ds-DNA binding to produce the secondary messenger 2',3'-cGAMP. cGAS is an important control point in the innate immune response; dysregulation of the cGAS pathway is linked to autoimmune diseases while targeted stimulation may be of benefit in immunoncology. We report here the structure of cGAS with dinucleotides and small molecule inhibitors, and kinetic studies of the cGAS mechanism. Our structural work supports the understanding of how ds-DNA activates cGAS, suggesting a site for small molecule binders that may cause cGAS activation at physiological ATP concentrations, and an apparent hotspot for inhibitor binding. Mechanistic studies of cGAS provide the first kinetic constants for 2',3'-cGAMP formation, and interestingly, describe a catalytic mechanism where 2',3'-cGAMP may be a minor product of cGAS compared with linear nucleotides. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

  10. Cyclic GMP-AMP Synthase Is Required for Cell Proliferation and Inflammatory Responses in Rheumatoid Arthritis Synoviocytes.

    Science.gov (United States)

    Wang, Yan; Su, Guo-Hua; Zhang, Fang; Chu, Jing-Xue; Wang, Yun-Shan

    2015-01-01

    Rheumatoid arthritis (RA) is characterized by inflammatory cell infiltration, fibroblast-like synoviocytes (FLS) invasive proliferation, and joint destruction. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces immune activation. In this study, we examined whether cGAS plays a role in RA FLS. In this study, cGAS was overexpressed in RA-FLS compared with OA FLS. TNFα stimulation induced cGAS expression in RA FLS. Overexpression of cGAS promoted the proliferation and knockdown of cGAS inhibited the proliferation of RA FLS. cGAS overexpression enhanced the production of proinflammatory cytokines and matrix metalloproteinases (MMPs) as well as AKT and ERK phosphorylation in TNFα-stimulated FLS. In contrast, cGAS silencing inhibited production of proinflammatory cytokines and matrix metalloproteinases (MMPs) as well as AKT and ERK phosphorylation in TNFα-stimulated FLS. These results suggest that cGAS activates the AKT and ERK pathways to promote the inflammatory response of RA FLS, and the development of strategies targeting cGAS may have therapeutic potential for human RA.

  11. Cutting edge: Antimalarial drugs inhibit IFN-β production through blockade of cyclic GMP-AMP synthase-DNA interaction.

    Science.gov (United States)

    An, Jie; Woodward, Joshua J; Sasaki, Tomikazu; Minie, Mark; Elkon, Keith B

    2015-05-01

    Type I IFN is strongly implicated in the pathogenesis of systemic autoimmune diseases, such as lupus, and rare monogenic IFNopathies, including Aicardi-Goutières syndrome. Recently, a new DNA-activated pathway involving the enzyme cyclic GMP-AMP synthase (cGAS) was described and potentially linked to Aicardi-Goutières syndrome. To identify drugs that could potentially inhibit cGAS activity, we performed in silico screening of drug libraries. By computational analysis, we identified several antimalarial drugs (AMDs) that were predicted to interact with the cGAS/dsDNA complex. Our studies validated that several AMDs were effective inhibitors of IFN-β production and that they functioned by inhibiting dsDNA stimulation of cGAS. Because AMDs have been widely used in human diseases and have an excellent safety profile, our findings suggest new therapeutic strategies for the treatment of severe debilitating diseases associated with type I IFNs due to cGAS activation. Copyright © 2015 by The American Association of Immunologists, Inc.

  12. Luteinizing hormone signaling phosphorylates and activates the cyclic GMP phosphodiesterase PDE5 in mouse ovarian follicles, contributing an additional component to the hormonally induced decrease in cyclic GMP that reinitiates meiosis.

    Science.gov (United States)

    Egbert, Jeremy R; Yee, Siu-Pok; Jaffe, Laurinda A

    2018-03-01

    Prior to birth, oocytes within mammalian ovarian follicles initiate meiosis, but then arrest in prophase until puberty, when with each reproductive cycle, one or more follicles are stimulated by luteinizing hormone (LH) to resume meiosis in preparation for fertilization. Within preovulatory follicles, granulosa cells produce high levels of cGMP, which diffuses into the oocyte to maintain meiotic arrest. LH signaling restarts meiosis by rapidly lowering the levels of cGMP in the follicle and oocyte. Part of this decrease is mediated by the dephosphorylation and inactivation the NPR2 guanylyl cyclase in response to LH, but the mechanism for the remainder of the cGMP decrease is unknown. At least one cGMP phosphodiesterase, PDE5, is activated by LH signaling, which would contribute to lowering cGMP. PDE5 exhibits increased cGMP-hydrolytic activity when phosphorylated on serine 92, and we recently demonstrated that LH signaling phosphorylates PDE5 on this serine and increases its activity in rat follicles. To test the extent to which this mechanism contributes to the cGMP decrease that restarts meiosis, we generated a mouse line in which serine 92 was mutated to alanine (Pde5-S92A), such that it cannot be phosphorylated. Here we show that PDE5 phosphorylation is required for the LH-induced increase in cGMP-hydrolytic activity, but that this increase has only a modest effect on the LH-induced cGMP decrease in mouse follicles, and does not affect the timing of meiotic resumption. Though we show that the activation of PDE5 is among the mechanisms contributing to the cGMP decrease, these results suggest that another cGMP phosphodiesterase is also activated by LH signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses.

    Science.gov (United States)

    Gao, Daxing; Wu, Jiaxi; Wu, You-Tong; Du, Fenghe; Aroh, Chukwuemika; Yan, Nan; Sun, Lijun; Chen, Zhijian J

    2013-08-23

    Retroviruses, including HIV, can activate innate immune responses, but the host sensors for retroviruses are largely unknown. Here we show that HIV infection activates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) to produce cGAMP, which binds to and activates the adaptor protein STING to induce type I interferons and other cytokines. Inhibitors of HIV reverse transcriptase, but not integrase, abrogated interferon-β induction by the virus, suggesting that the reverse-transcribed HIV DNA triggers the innate immune response. Knockout or knockdown of cGAS in mouse or human cell lines blocked cytokine induction by HIV, murine leukemia virus, and simian immunodeficiency virus. These results indicate that cGAS is an innate immune sensor of HIV and other retroviruses.

  14. Cyclic GMP-AMP Synthase Is an Innate Immune DNA Sensor for Mycobacterium tuberculosis.

    Science.gov (United States)

    Collins, Angela C; Cai, Haocheng; Li, Tuo; Franco, Luis H; Li, Xiao-Dong; Nair, Vidhya R; Scharn, Caitlyn R; Stamm, Chelsea E; Levine, Beth; Chen, Zhijian J; Shiloh, Michael U

    2015-06-10

    Activation of the DNA-dependent cytosolic surveillance pathway in response to Mycobacterium tuberculosis infection stimulates ubiquitin-dependent autophagy and inflammatory cytokine production, and plays an important role in host defense against M. tuberculosis. However, the identity of the host sensor for M. tuberculosis DNA is unknown. Here we show that M. tuberculosis activated cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) in macrophages to produce cGAMP, a second messenger that activates the adaptor protein stimulator of interferon genes (STING) to induce type I interferons and other cytokines. cGAS localized with M. tuberculosis in mouse and human cells and in human tuberculosis lesions. Knockdown or knockout of cGAS in human or mouse macrophages blocked cytokine production and induction of autophagy. Mice deficient in cGAS were more susceptible to lethality caused by infection with M. tuberculosis. These results demonstrate that cGAS is a vital innate immune sensor of M. tuberculosis infection. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    Řlepokura, Katarzyna Anna

    2016-06-01

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

  16. Three Antagonistic Cyclic di-GMP-Catabolizing Enzymes Promote Differential Dot/Icm Effector Delivery and Intracellular Survival at the Early Steps of Legionella pneumophila Infection

    Science.gov (United States)

    Allombert, Julie; Lazzaroni, Jean-Claude; Baïlo, Nathalie; Gilbert, Christophe; Charpentier, Xavier; Doublet, Patricia

    2014-01-01

    Legionella pneumophila is an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages, causing an acute pneumonia in humans. The second messenger cyclic di-GMP (c-di-GMP) has been shown to play key roles in the regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of the L. pneumophila Lens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118), and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities, which are all required for the survival and intracellular replication of L. pneumophila. Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of the Legionella-containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated actions of several type 4 secretion system Dot/Icm effectors proteins, which exhibit differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged, others appeared over- and undertranslocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in the trafficking and biogenesis of the LCV. Our results suggest that L. pneumophila uses cyclic di-GMP signaling to fine-tune effector delivery and ensure effective evasion of the host degradative pathways and establishment of a replicative vacuole. PMID:24379287

  17. Type I interferon induction by Neisseria gonorrhoeae: Dual requirement of cyclic GMP-AMP synthase and Toll-like receptor 4

    OpenAIRE

    Andrade, Warrison A.; Agarwal, Sarika; Mo, Shunyan; Shaffer, Scott A.; Dillard, Joseph P.; Schmidt, Tobias; Hornung, Veit; Fitzgerald, Katherine A.; Kurt-Jones, Evelyn A.; Golenbock, Douglas T.

    2016-01-01

    The innate immune system is the first line of defense against Neisseria gonorrhoeae (GC). Exposure of cells to GC lipooligosaccharides induces a strong immune response, leading to type I interferon (IFN) production via TLR4/MD-2. In addition to living freely in the extracellular space, GC can invade the cytoplasm to evade detection and elimination. Double-stranded DNA introduced into the cytosol binds and activates the enzyme cyclic-GMP-AMP synthase (cGAS), which produces 2′3′-cGAMP and trigg...

  18. The cyclic-di-GMP diguanylate cyclase CdgA has a role in biofilm formation and exopolysaccharide production in Azospirillum brasilense.

    Science.gov (United States)

    Ramírez-Mata, Alberto; López-Lara, Lilia I; Xiqui-Vázquez, Ma Luisa; Jijón-Moreno, Saúl; Romero-Osorio, Angelica; Baca, Beatriz E

    2016-04-01

    In bacteria, proteins containing GGDEF domains are involved in production of the second messenger c-di-GMP. Here we report that the cdgA gene encoding diguanylate cyclase A (CdgA) is involved in biofilm formation and exopolysaccharide (EPS) production in Azospirillum brasilense Sp7. Biofilm quantification using crystal violet staining revealed that inactivation of cdgA decreased biofilm formation. In addition, confocal laser scanning microscopy analysis of green-fluorescent protein-labeled bacteria showed that, during static growth, the biofilms had differential levels of development: bacteria harboring a cdgA mutation exhibited biofilms with considerably reduced thickness compared with those of the wild-type Sp7 strain. Moreover, DNA-specific staining and treatment with DNase I, and epifluorescence studies demonstrated that extracellular DNA and EPS are components of the biofilm matrix in Azospirillum. After expression and purification of the CdgA protein, diguanylate cyclase activity was detected. The enzymatic activity of CdgA-producing cyclic c-di-GMP was determined using GTP as a substrate and flavin adenine dinucleotide (FAD(+)) and Mg(2)(+) as cofactors. Together, our results revealed that A. brasilense possesses a functional c-di-GMP biosynthesis pathway. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  19. Role of the nitric oxide/cyclic GMP/Ca2+ signaling pathway in the pyrogenic effect of interleukin-1beta.

    Science.gov (United States)

    Palmi, Mitri; Meini, Antonella

    2002-04-01

    Interleukin-1beta (IL-1beta) has a wide spectrum of inflammatory, metabolic, haemopoietic, and immunological properties. Because it produces fever when injected into animals and humans, it is considered an endogenous pyrogen. There is evidence to suggest that Ca2+ plays a critical role in the central mechanisms of thermoregulation, and in the intracellular signaling pathways controlling fever induced by IL-1beta and other pyrogens. Data from different labs indicate that Ca2+ and Na+ determine the temperature set point in the posterior hypothalamus (PH) of various mammals and that changes in Ca2+ and PGE2 concentrations in the cerebrospinal fluid (CSF) of these animals are associated with IL-1beta-induced fever. Antipyretic drugs such as acetylsalicylic acid, dexamethasone, and lipocortin 5-(204-212) peptide counteract IL-1beta-induced fever and abolish changes in Ca2+ and PGE2 concentrations in CSF. In vitro studies have established that activation of the nitric oxide (NO)/cyclic GMP (cGMP) pathway is part of the signaling cascade transducing Ca2+ mobilization in response to IL-1beta and that the ryanodine (RY)- and inositol-(1,4,5)-trisphosphate (IP3)-sensitive pools are the main source of the mobilized Ca2+. It is concluded that the NO/cGMP/Ca2+ pathway is part of the signaling cascade subserving some of the multiple functions of IL-1beta.

  20. Necrotic enteritis locus 1 diguanylate cyclase and phosphodiesterase (cyclic-di-GMP) gene mutation attenuates virulence in an avian necrotic enteritis isolate of Clostridium perfringens.

    Science.gov (United States)

    Parreira, Valeria R; Ojha, Shivani; Lepp, Dion; Mehdizadeh Gohari, Iman; Zhou, Hongzhuan; Susta, Leonardo; Gong, Jianhua; Prescott, John F

    2017-09-01

    Necrotic enteritis (NE) caused by netB-positive strains of Clostridium perfringens is an important disease of intensively-reared broiler chickens. It is widely controlled by antibiotic use, but this practice that has come under increasing scrutiny and alternative approaches are required. As part of the search for alternative approaches over the last decade, advances have been made in understanding its pathogenesis but much remains to be understood and applied to the control of NE. The objective of this work was to assess the effect on virulence of mutation of the cyclic-di-GMP signaling genes present on the large pathogenicity locus (NELoc-1) in the tcp-encoding conjugative virulence plasmid, pNetB. For this purpose, the diguanylate cyclase (dgc) and phosphodiesterase (pde) genes were individually insertionally inactivated and the two mutants were subsequently complemented with their respective genes. Southern blotting showed that a single gene insertion was present. Mutation of either gene resulted in almost total attenuation of the mutants to cause NE in experimentally-infected broiler chickens, which was fully restored in each case by complementation of the respective mutated gene. Production of NetB-associated cytotoxicity for Leghorn male hepatoma (LMH) cells was unaffected in mutants. We conclude that the cyclic-di-GMP signaling system is important in controlling virulence in a NE C. perfringens strain and might be a target for control of the disease. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Cyclic [G(2′,5′)pA(3′,5′)p] Is the Metazoan Second Messenger Produced by DNA-Activated Cyclic GMP-AMP Synthase

    OpenAIRE

    Gao, Pu; Ascano, Manuel; Wu, Yang; Barchet, Winfried; Gaffney, Barbara L.; Zillinger, Thomas; Serganov, Artem A.; Liu, Yizhou; Jones, Roger A.; Hartmann, Gunther; Tuschl, Thomas; Patel, Dinshaw J.

    2013-01-01

    Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2′,5′)pA and A(3′,5′)pG phosphodiester linkages, designated c[G(2′,5′) pA(3′,5′)p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, res...

  2. Role of cyclic GMP in cells with the properties of smooth muscle cultured from the rat myometrium

    International Nuclear Information System (INIS)

    Krall, J.F.; Morin, A.

    1986-01-01

    Cells growing in culture with previously described properties of rat uterine smooth muscle accumulated 45 Ca 2+ from the medium. Ca 2+ uptake by these cells was stimulated by the addition to the medium of 8-bromo-cGMP but not by 8-bromo-cAMP. Ca 2+ uptake was also stimulated by carbachol and by the nitro-vasodilator nitroprusside. Although cholinergic agonists have been shown previously to stimulate contraction but not cGMP synthesis in the rat myometrium, both carbachol and nitroprusside stimulated cGMP production by the cultured cells. These results suggested the cells had cholinergic receptor-medicated functions that reflected some neurotransmitter-sensitive properties of uterine smooth muscle in situ. When determined by a specific radioligand binding assay, subcellular fractions of the cultured cells bound muscarinic cholinergic agonists and antagonists with affinities expected of the muscarinic receptor. The cells were also sensitive to the β-adrenergic catecholamine agonist isoproterenol, which stimulated cAMP production but not Ca 2+ uptake. Carbachol failed to inhibit isoproterenol-dependent cAMP production, which is an important property of the cholinergic receptor in uterine smooth muscle in situ. These results suggest some but not all acetylcholine-sensitive properties of uterine smooth muscle may be retained in cell culture

  3. Cyclic Di-GMP Binding by an Assembly ATPase (PilB2) and Control of Type IV Pilin Polymerization in the Gram-Positive Pathogen Clostridium perfringens.

    Science.gov (United States)

    Hendrick, William A; Orr, Mona W; Murray, Samantha R; Lee, Vincent T; Melville, Stephen B

    2017-05-15

    The Gram-positive pathogen Clostridium perfringens possesses type IV pili (TFP), which are extracellular fibers that are polymerized from a pool of pilin monomers in the cytoplasmic membrane. Two proteins that are essential for pilus functions are an assembly ATPase (PilB) and an inner membrane core protein (PilC). Two homologues each of PilB and PilC are present in C. perfringens , called PilB1/PilB2 and PilC1/PilC2, respectively, along with four pilin proteins, PilA1 to PilA4. The gene encoding PilA2, which is considered the major pilin based on previous studies, is immediately downstream of the pilB2 and pilC2 genes. Purified PilB2 had ATPase activity, bound zinc, formed hexamers even in the absence of ATP, and bound the second messenger molecule cyclic di-GMP (c-di-GMP). Circular dichroism spectroscopy of purified PilC2 indicated that it retained its predicted degree of alpha-helical secondary structure. Even though no direct interactions between PilB2 and PilC2 could be detected in vivo or in vitro even in the presence of c-di-GMP, high levels of expression of a diguanylate cyclase from C. perfringens (CPE1788) stimulated polymerization of PilA2 in a PilB2- and PilC2-dependent manner. These results suggest that PilB2 activity is controlled by c-di-GMP levels in vivo but that PilB2-PilC2 interactions are either transitory or of low affinity, in contrast to results reported previously from in vivo studies of the PilB1/PilC1 pair in which PilC1 was needed for polar localization of PilB1. This is the first biochemical characterization of a c-di-GMP-dependent assembly ATPase from a Gram-positive bacterium. IMPORTANCE Type IV pili (TFP) are protein fibers involved in important bacterial functions, including motility, adherence to surfaces and host cells, and natural transformation. All clostridia whose genomes have been sequenced show evidence of the presence of TFP. The genetically tractable species Clostridium perfringens was used to study proteins involved in

  4. Phenotype overlap in Xylella fastidiosa is controlled by the cyclic di-GMP phosphodiesterase Eal in response to antibiotic exposure and diffusible signal factor-mediated cell-cell signaling.

    Science.gov (United States)

    de Souza, Alessandra A; Ionescu, Michael; Baccari, Clelia; da Silva, Aline M; Lindow, Steven E

    2013-06-01

    Eal is an EAL domain protein in Xylella fastidiosa homologous to one involved in resistance to tobramycin in Pseudomonas aeruginosa. EAL and HD-GYP domain proteins are implicated in the hydrolysis of the secondary messenger bis-(3'-5')-cyclic dimeric GMP (cyclic di-GMP). Cell density-dependent communication mediated by a Diffusible Signal Factor (DSF) also modulates cyclic di-GMP levels in X. fastidiosa, thereby controlling the expression of virulence genes and genes involved in insect transmission. The possible linkage of Eal to both extrinsic factors such as antibiotics and intrinsic factors such as quorum sensing, and whether both affect virulence, was thus addressed. Expression of eal was induced by subinhibitory concentrations of tobramycin, and an eal deletion mutant was more susceptible to this antibiotic than the wild-type strain and exhibited phenotypes similar to those of an rpfF deletion mutant blocked in DSF production, such as hypermotility, reduced biofilm formation, and hypervirulence to grape. Consistent with that, the rpfF mutant was more susceptible than the wild-type strain to tobramycin. Therefore, we propose that cell-cell communication and antibiotic stress can apparently lead to similar modulations of cyclic di-GMP in X. fastidiosa, resulting in similar phenotypes. However, the effect of cell density is dominant compared to that of antibiotic stress, since eal is suppressed by RpfF, which may prevent inappropriate behavioral changes in response to antibiotic stress when DSF accumulates.

  5. Cyclic AMP system in muscle tissue during prolonged hypokinesia

    Science.gov (United States)

    Antipenko, Y. A.; Bubeyev, Y. A.; Korovkin, B. F.; Mikhaleva, N. P.

    1980-01-01

    Components of the cyclic Adenosine-cyclic-35-monophosphate (AMP) system in the muscle tissue of white rats were studied during 70-75 days of hypokinesia, created by placing the animals in small booths which restricted their movements, and during the readaptation period. In the initial period, cyclic AMP levels and the activities of phosphodiesterase and adenylate cyclase in muscle tissue were increased. The values for these indices were roughly equal for controls and experimental animals during the adaptation period, but on the 70th day of the experiment cAMP levels dropped, phosphodiesterase activity increased, and the stimulative effect of epinephrine on the activity of adenylate cyclase decreased. The indices under study normalized during the readaptation period.

  6. Cyclic Loading of Growing Tissue in a Bioreactor: Mathematical Model and Asymptotic Analysis

    KAUST Repository

    Pohlmeyer, J. V.; Cummings, L. J.

    2013-01-01

    A simplified 2D mathematical model for tissue growth within a cyclically-loaded tissue engineering scaffold is presented and analyzed. Such cyclic loading has the potential to improve yield and functionality of tissue such as bone and cartilage when

  7. The phytosulfokine (PSK) receptor is capable of guanylate cyclase activity and enabling cyclic GMP-dependent signaling in plants

    KAUST Repository

    Kwezi, Lusisizwe; Ruzvidzo, Oziniel; Wheeler, Janet I.; Govender, Kershini; Iacuone, Sylvana; Thompson, Philip E.; Gehring, Christoph A; Irving, Helen R.

    2011-01-01

    Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase domain and hypothesized that the GC works in conjunction with the kinase in downstream PSK signaling. We expressed the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate Km of 7.5 μM and Vmax of 1800 nmol min-1 mg-1 of protein. This same recombinant protein also has GC activity in vitro that is dependent on the presence of either Mg2+ or Mn2+. Overexpression of the full-length AtPSKR1 receptor in Arabidopsis leaf protoplasts raised the endogenous basal cGMP levels over 20-fold, indicating that the receptor has GC activity in vivo. In addition, PSK-α itself, but not the non-sulfated backbone, induces rapid increases in cGMP levels in protoplasts. Together these results indicate that the PSKR1 contains dual GC and kinase catalytic activities that operate in vivo and that this receptor constitutes a novel class of enzymes with overlapping catalytic domains. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. The phytosulfokine (PSK) receptor is capable of guanylate cyclase activity and enabling cyclic GMP-dependent signaling in plants

    KAUST Repository

    Kwezi, Lusisizwe

    2011-04-19

    Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase domain and hypothesized that the GC works in conjunction with the kinase in downstream PSK signaling. We expressed the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate Km of 7.5 μM and Vmax of 1800 nmol min-1 mg-1 of protein. This same recombinant protein also has GC activity in vitro that is dependent on the presence of either Mg2+ or Mn2+. Overexpression of the full-length AtPSKR1 receptor in Arabidopsis leaf protoplasts raised the endogenous basal cGMP levels over 20-fold, indicating that the receptor has GC activity in vivo. In addition, PSK-α itself, but not the non-sulfated backbone, induces rapid increases in cGMP levels in protoplasts. Together these results indicate that the PSKR1 contains dual GC and kinase catalytic activities that operate in vivo and that this receptor constitutes a novel class of enzymes with overlapping catalytic domains. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Effects of Biotin Supplementation in the Diet on Adipose Tissue cGMP Concentrations, AMPK Activation, Lipolysis, and Serum-Free Fatty Acid Levels.

    Science.gov (United States)

    Boone-Villa, Daniel; Aguilera-Méndez, Asdrubal; Miranda-Cervantes, Adriana; Fernandez-Mejia, Cristina

    2015-10-01

    Several studies have shown that pharmacological concentrations of biotin decrease hyperlipidemia. The molecular mechanisms by which pharmacological concentrations of biotin modify lipid metabolism are largely unknown. Adipose tissue plays a central role in lipid homeostasis. In the present study, we analyzed the effects of biotin supplementation in adipose tissue on signaling pathways and critical proteins that regulate lipid metabolism, as well as on lipolysis. In addition, we assessed serum fatty acid concentrations. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet (control: 1.76 mg biotin/kg; supplemented: 97.7 mg biotin/kg diet) over 8 weeks postweaning. Compared with the control group, biotin-supplemented mice showed an increase in the levels of adipose guanosine 3',5'-cyclic monophosphate (cGMP) (control: 30.3±3.27 pmol/g wet tissue; supplemented: 49.5±3.44 pmol/g wet tissue) and of phosphorylated forms of adenosine 5'-monophosphate-activated protein kinase (AMPK; 65.2%±1.06%), acetyl-coenzyme A (CoA), carboxylase-1 (196%±68%), and acetyl-CoA carboxylase-2 (78.1%±18%). Serum fatty acid concentrations were decreased (control: 1.12±0.04 mM; supplemented: 0.91±0.03 mM), and no change in lipolysis was found (control: 0.29±0.05 μmol/mL; supplemented: 0.33±0.08 μmol/mL). In conclusion, 8 weeks of dietary biotin supplementation increased adipose tissue cGMP content and protein expression of the active form of AMPK and of the inactive forms of acetyl-CoA carboxylase-1 and acetyl-CoA carboxylase-2. Serum fatty acid levels fell, and no change in lipolysis was observed. These findings provide insight into the effects of biotin supplementation on adipose tissue and support its use in the treatment of dyslipidemia.

  10. Phosphodiesterase-9 (PDE9) inhibition with BAY 73-6691 increases corpus cavernosum relaxations mediated by nitric oxide-cyclic GMP pathway in mice.

    Science.gov (United States)

    da Silva, F H; Pereira, M N; Franco-Penteado, C F; De Nucci, G; Antunes, E; Claudino, M A

    2013-01-01

    Phosphodiesterase-9 (PDE9) specifically hydrolyzes cyclic GMP, and was detected in human corpus cavernosum. However, no previous studies explored the selective PDE9 inhibition with BAY 73-6691 in corpus cavernosum relaxations. Therefore, this study aimed to characterize the PDE9 mRNA expression in mice corpus cavernosum, and investigate the effects of BAY 73-6691 in endothelium-dependent and -independent relaxations, along with the nitrergic corpus cavernosum relaxations. Male mice received daily gavage of BAY 73-6691 (or dimethylsulfoxide) at 3 mg kg(-1) per day for 21 days. Relaxant responses to acetylcholine (ACh), nitric oxide (NO) (as acidified sodium nitrite; NaNO2 solution), sildenafil and electrical-field stimulation (EFS) were obtained in corpus cavernosum in control and BAY 73-6691-treated mice. BAY 73-6691 was also added in vitro 30 min before construction of concentration-responses and frequency curves. PDE9A and PDE5 mRNA expression was detected in the mice corpus cavernosum in a similar manner. In vitro addition of BAY 73-6691 neither itself relaxed mice corpus cavernosum nor changed the NaNO2, sildenafil and EFS-induced relaxations. However, in mice treated chronically with BAY 73-6691, the potency (pEC50) values for ACh, NaNO2 and sildenafil were significantly greater compared with control group. The maximal responses (Emax) to NaNO2 and sildenafil were also significantly greater in BAY 73-6691-treated mice. BAY 73-6691 treatment also significantly increased the magnitude and duration of the nitrergic corpus cavernosum relaxations (8-32 Hz). In conclusion, murine corpus cavernosum expresses PDE9 mRNA. Prolonged PDE9 inhibition with BAY 73-6691 amplifies the NO-cGMP-mediated cavernosal responses, and may be of therapeutic value for erectile dysfunction.

  11. Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection

    Directory of Open Access Journals (Sweden)

    Benjamin A. Diner

    2016-11-01

    Full Text Available The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS. Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1 and human cytomegalovirus (HCMV infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses.

  12. Type I Interferon Induction by Neisseria gonorrhoeae: Dual Requirement of Cyclic GMP-AMP Synthase and Toll-like Receptor 4.

    Science.gov (United States)

    Andrade, Warrison A; Agarwal, Sarika; Mo, Shunyan; Shaffer, Scott A; Dillard, Joseph P; Schmidt, Tobias; Hornung, Veit; Fitzgerald, Katherine A; Kurt-Jones, Evelyn A; Golenbock, Douglas T

    2016-06-14

    The innate immune system is the first line of defense against Neisseria gonorrhoeae (GC). Exposure of cells to GC lipooligosaccharides induces a strong immune response, leading to type I interferon (IFN) production via TLR4/MD-2. In addition to living freely in the extracellular space, GC can invade the cytoplasm to evade detection and elimination. Double-stranded DNA introduced into the cytosol binds and activates the enzyme cyclic-GMP-AMP synthase (cGAS), which produces 2'3'-cGAMP and triggers STING/TBK-1/IRF3 activation, resulting in type I IFN expression. Here, we reveal a cytosolic response to GC DNA that also contributes to type I IFN induction. We demonstrate that complete IFN-β induction by live GC depends on both cGAS and TLR4. Type I IFN is detrimental to the host, and dysregulation of iron homeostasis genes may explain lower bacteria survival in cGAS(-/-) and TLR4(-/-) cells. Collectively, these observations reveal cooperation between TLRs and cGAS in immunity to GC infection. Copyright © 2016. Published by Elsevier Inc.

  13. Cyclic GMP-AMP Synthase Is the Cytosolic Sensor of Plasmodium falciparum Genomic DNA and Activates Type I IFN in Malaria.

    Science.gov (United States)

    Gallego-Marin, Carolina; Schrum, Jacob E; Andrade, Warrison A; Shaffer, Scott A; Giraldo, Lina F; Lasso, Alvaro M; Kurt-Jones, Evelyn A; Fitzgerald, Katherine A; Golenbock, Douglas T

    2018-01-15

    Innate immune receptors have a key role in the sensing of malaria and initiating immune responses. As a consequence of infection, systemic inflammation emerges and is directly related to signs and symptoms during acute disease. We have previously reported that plasmodial DNA is the primary driver of systemic inflammation in malaria, both within the phagolysosome and in the cytosol of effector cells. In this article, we demonstrate that Plasmodium falciparum genomic DNA delivered to the cytosol of human monocytes binds and activates cyclic GMP-AMP synthase (cGAS). Activated cGAS synthesizes 2'3'-cGAMP, which we subsequently can detect using liquid chromatography-tandem mass spectrometry. 2'3'-cGAMP acts as a second messenger for STING activation and triggers TBK1/IRF3 activation, resulting in type I IFN production in human cells. This induction of type I IFN was independent of IFI16. Access of DNA to the cytosolic compartment is mediated by hemozoin, because incubation of purified malaria pigment with DNase abrogated IFN-β induction. Collectively, these observations implicate cGAS as an important cytosolic sensor of P. falciparum genomic DNA and reveal the role of the cGAS/STING pathway in the induction of type I IFN in response to malaria parasites. Copyright © 2018 by The American Association of Immunologists, Inc.

  14. Type I Interferon Induction by Neisseria gonorrhoeae: Dual Requirement of Cyclic GMP-AMP Synthase and Toll-like Receptor 4

    Directory of Open Access Journals (Sweden)

    Warrison A. Andrade

    2016-06-01

    Full Text Available The innate immune system is the first line of defense against Neisseria gonorrhoeae (GC. Exposure of cells to GC lipooligosaccharides induces a strong immune response, leading to type I interferon (IFN production via TLR4/MD-2. In addition to living freely in the extracellular space, GC can invade the cytoplasm to evade detection and elimination. Double-stranded DNA introduced into the cytosol binds and activates the enzyme cyclic-GMP-AMP synthase (cGAS, which produces 2′3′-cGAMP and triggers STING/TBK-1/IRF3 activation, resulting in type I IFN expression. Here, we reveal a cytosolic response to GC DNA that also contributes to type I IFN induction. We demonstrate that complete IFN-β induction by live GC depends on both cGAS and TLR4. Type I IFN is detrimental to the host, and dysregulation of iron homeostasis genes may explain lower bacteria survival in cGAS−/− and TLR4−/− cells. Collectively, these observations reveal cooperation between TLRs and cGAS in immunity to GC infection.

  15. Evaluation of Tissue Homogenization to Support the Generation of GMP-Compliant Mesenchymal Stromal Cells from the Umbilical Cord

    Directory of Open Access Journals (Sweden)

    Ryan J. Emnett

    2016-01-01

    Full Text Available Recent studies have demonstrated that the umbilical cord (UC is an excellent source of mesenchymal stromal cells (MSCs. However, current protocols for extracting and culturing UC-MSCs do not meet current good manufacturing practice (cGMP standards, in part due to the use of xenogeneic reagents. To support the development of a cGMP-compliant method, we have examined an enzyme-free isolation method utilizing tissue homogenization (t-H followed by culture in human platelet lysate (PL supplemented media. The yield and viability of cells after t-H were comparable to those obtained after collagenase digestion (Col-D. Importantly, kinetic analysis of cultured cells showed logarithmic growth over 10 tested passages, although the rate of cell division was lower for t-H as compared to Col-D. This slower growth of t-H-derived cells was also reflected in their longer population doubling time. Interestingly, there was no difference in the expression of mesenchymal markers and trilineage differentiation potential of cells generated using either method. Finally, t-H-derived cells had greater clonogenic potential compared to Col-D/FBS but not Col-D/PL and were able to maintain CFU-F capacity through P7. This bench scale study demonstrates the possibility of generating therapeutic doses of good quality UC-MSCs within a reasonable length of time using t-H and PL.

  16. Cyclic [G(2′,5′)pA(3′,5′)p] Is the Metazoan Second Messenger Produced by DNA-Activated Cyclic GMP-AMP Synthase

    Science.gov (United States)

    Gao, Pu; Ascano, Manuel; Wu, Yang; Barchet, Winfried; Gaffney, Barbara L.; Zillinger, Thomas; Serganov, Artem A.; Liu, Yizhou; Jones, Roger A.; Hartmann, Gunther; Tuschl, Thomas; Patel, Dinshaw J.

    2015-01-01

    SUMMARY Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2′,5′)pA and A(3′,5′)pG phosphodiester linkages, designated c[G(2′,5′) pA(3′,5′)p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, resulting in formation of a catalytically competent and accessible nucleotide-binding pocket for generation of c[G(2′,5′)pA(3′,5′)p]. We demonstrate that cyclization occurs in a stepwise manner through initial generation of 5′-pppG(2′,5′)pA prior to cyclization to c[G(2′,5′)pA(3′,5′)p], with the latter positioned precisely in the catalytic pocket. Mutants of cGAS dsDNA-binding or catalytic pocket residues exhibit reduced or abrogated activity. Our studies have identified c[G(2′,5′)pA(3′,5′)p] as a founding member of a family of metazoan 2′,5′-containing cyclic heterodinucleotide second messengers distinct from bacterial 3′,5′ cyclic dinucleotides. PMID:23647843

  17. Radiorestoring activity of few nucleotides on normal tissues of Jerusalem Artichoke after an irradiation with γ rays of 60Co

    International Nuclear Information System (INIS)

    Jonard, Robert; Bayonove, Jacqueline; Riedel, Michel.

    1978-01-01

    The nucleotides tested: adenosine triphosphate (ATP) and cyclic adenosine 3',5'-monophosphate (3',5'-cAMP), guanosine triphosphate (GTP) and cyclic guanosine 3',5'-monophosphate (3',5'-cGMP), are able to restore proliferation to irradiated (γ irradiation, 3,000 rad) Jesusalem Artichoke tissue. The 3',5'-cGMP shows the greater radiorestoring activity [fr

  18. Hypercapnic vasodilatation in isolated rat basilar arteries is exerted via low pH and does not involve nitric oxide synthase stimulation or cyclic GMP production

    DEFF Research Database (Denmark)

    You, J P; Wang, Qian; Zhang, W

    1994-01-01

    this relaxation by 54% and 70%, respectively. The effect of L-NOARG was completely reversed by L-arginine. Blockade of nerve excitation with tetrodotoxin (TTX) had no affect on the 15% CO2 elicited vasodilatation. Measurements of cGMP in vessel segments showed no significant increase in cGMP content in response...... to hypercapnia. L-NOARG and MB, but not TTX, significantly reduced the basal cGMP content in cerebral vessels. Adding 1.5% halothane to the incubation medium did not result in a significant increase in cGMP content. Lowering the pH by cumulative application of 0.12 M HCl resulted in relaxation identical...... elicits vasodilatation of isolated rat basilar arteries by a mechanism independent of nitric oxide synthase (NOS) activity. The markedly reduced basal cGMP levels in cerebral vessels by L-NOARG and MB suggest that there exists a basal NO formation in the cerebral vessel wall....

  19. A multi-angular mass spectrometric view at cyclic nucleotide signaling proteins : Structure/function and protein interactions of cAMP- and cGMP-dependent protein kinase

    NARCIS (Netherlands)

    Scholten, A.

    2006-01-01

    The primary focus of this thesis is the two kinases PKA and PKG, cAMP and cGMP dependent protein kinase respectively. PKA and PKG are studied both at structure/function level as well as at the level of interaction with other proteins in tissue. Our primary methods are all based on mass spectrometry.

  20. Cyclic GMP-dependent protein kinase II is necessary for macrophage M1 polarization and phagocytosis via toll-like receptor 2.

    Science.gov (United States)

    Liao, Wei-Ting; You, Huey-Ling; Li, Changgui; Chang, Jan-Gowth; Chang, Shun-Jen; Chen, Chung-Jen

    2015-05-01

    Cyclic GMP-dependent protein kinase II (cGKII; PRKG2) phosphorylates a variety of biological targets and has been identified as a gout-susceptible gene. However, the regulatory role of cGKII in triggering gout disease has yet to be clarified. Thus, we plan to explore the specific function of cGKII in macrophages related to gout disease. By using cGKII gene knockdown method, we detected macrophage M1/M2 polarization, phagocytosis, and their responses to stimulation by monosodium urate (MSU). cGKII was highly expressed in M1 phenotype, but not in M2, and cGKII knockdown significantly inhibited macrophage M1 polarization by decreasing M1 chemokine markers (CXCL10 and CCL2) and downregulating phagocytosis function. We further identified that cGKII-associated phagocytosis was mediated by upregulating toll-like receptor 2 (TLR2) expression, but not by TLR4. Mimicking gout condition by MSU treatments, we found that MSU alone induced cGKII and TLR2 expression with increased M1 polarization markers and phagocytosis activity. It means that cGKII knockdown significantly inhibited this MSU-induced cGKII-TLR2-phagocytosis axis. Our study showed that cGKII plays a key role in M1 polarization, especially in TLR2-mediated phagocytosis under MSU exposure. The findings provide evidence for the possible role of cGKII as an inflammation exciter in gout disease. Gout-susceptible gene cGKII is necessary for macrophage M1 polarization. cGKII regulates M1 phagocytosis function via TLR2. Monosodium urate treatments increase cGKII expression and related function. This study reveals the role of cGKII in enhancing gouty inflammatory responses.

  1. Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection.

    Science.gov (United States)

    Diner, Benjamin A; Lum, Krystal K; Toettcher, Jared E; Cristea, Ileana M

    2016-11-15

    The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses. How mammalian cells detect and respond to DNA viruses that replicate in the nucleus is poorly understood. Here, we decipher the distinct functions of two viral DNA sensors, IFI16 and cGAS, during active immune signaling upon infection with two herpesviruses, herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV). We show that IFI16

  2. Development and validation of an LC-MS/MS method for quantification of cyclic guanosine 3',5'-monophosphate (cGMP) in clinical applications: a comparison with a EIA method.

    Science.gov (United States)

    Zhang, Yanhua; Dufield, Dawn; Klover, Jon; Li, Wenlin; Szekely-Klepser, Gabriella; Lepsy, Christopher; Sadagopan, Nalini

    2009-02-15

    An LC-MS/MS method was developed and validated to quantify endogenous cyclic guanosine 3',5'-monophosphate (cGMP) in human plasma. The LC-MS/MS and competitive enzyme immunoassay (EIA) assays were compared. cGMP concentrations of 20 human plasma samples were measured by both methods. For the MS-based assay, plasma samples were subjected to a simple protein precipitation procedure by acetonitrile prior to analysis by electrospray ionization LC-MS/MS. De-protonated analytes generated in negative ionization mode were monitored through multiple reaction monitoring (MRM). A stable isotope-labeled internal standard, (13)C(10),(15)N(5)-cGMP, which was biosynthesized in-house, was used in the LC-MS/MS method. The competitive EIA was validated using a commercially available cGMP fluorescence assay kit. The intra-assay accuracy and precision for MS-based assay for cGMP were 6-10.1% CV and -3.6% to 7.3% relative error (RE), respectively, while inter-assay precision and accuracy were 5.6-8.1% CV and -2.1% to 6.3% RE, respectively. The intra-assay accuracy and precision for EIA were 17.9-27.1% CV and -4.9% to 24.5% RE, respectively, while inter-assay precision and accuracy were 15.1-39.5% CV and -30.8% to 4.37% RE, respectively. Near the lower limits of detection, there was little correlation between the cGMP concentration values in human plasma generated by these two methods (R(2)=0.197, P=0.05). Overall, the MS-based assay offered better selectivity, recovery, precision and accuracy over a linear range of 0.5-20ng/mL. The LC-MS/MS method provides an effective tool for the quantitation of cGMP to support clinical mechanistic studies of curative pharmaceuticals.

  3. Nitric oxide-soluble guanylyl cyclase-cyclic GMP signaling in the striatum: New targets for the treatment of Parkinson's disease?

    Directory of Open Access Journals (Sweden)

    Anthony R West

    2011-06-01

    Full Text Available Striatal nitric oxide (NO-producing interneurons play an important role in the regulation of corticostriatal synaptic transmission and motor behavior. Striatal NO synthesis is driven by concurrent activation of NMDA and dopamine (DA D1 receptors. NO diffuses into the dendrites of medium-sized spiny neurons (MSNs which contain high levels of NO receptors called soluble guanylyl cyclases (sGC. NO-mediated activation of sGC leads to the synthesis of the second messenger cGMP. In the intact striatum, transient elevations in intracellular cGMP primarily act to increase neuronal excitability and to facilitate glutamatergic corticostriatal transmission. NO-cGMP signaling also functionally opposes the inhibitory effects of DA D2 receptor activation on corticostriatal transmission. Not surprisingly, abnormal striatal NO-sGC-cGMP signaling becomes apparent following striatal DA depletion, an alteration thought to contribute to pathophysiological changes observed in basal ganglia circuits in Parkinson’s disease (PD. Here, we discuss recent developments in the field which have shed light on the role of NO-sGC-cGMP signaling pathways in basal ganglia dysfunction and motor symptoms associated with PD and L-DOPA-induced dyskinesias.

  4. Isosorbide 5 mononitrate administration increases nitric oxide blood levels and reduces proteinuria in IgA glomerulonephritis patients with abnormal urinary endothelin/cyclic GMP ratio.

    Science.gov (United States)

    Roccatello, D; Mengozzi, G; Ferro, M; Cesano, G; Polloni, R; Mosso, R; Bonetti, G; Inconis, T; Paradisi, L; Sena, L M

    1995-09-01

    An endothelin urinary hyperexcretion, which is not counterbalanced by an adequate increase in cGMP biosynthesis, was previously detected in some patients with IgA Nephropathy (IgAN). Since this imbalance might potentiate local ET1-mediated hemodynamics effects, 9 IgAN patients with an increased (> or = 0.1) urinary ET1/cGMP ratio (group 1) and 5 IgAN patients with comparable renal function and reduced ET1/cGMP ratio (group 2) were given standard doses of isosorbide 5 mononitrate (as a nitric oxide source). Blood nitric oxide (NO) levels, as detected by electron paramagnetic resonance, significantly increased after isosorbide administration (p effective renal plasma flow (p counterbalancing effects of nitric oxide on endothelin-mediated mesangial contraction.

  5. Biophysical Techniques for Detection of cAMP and cGMP in Living Cells

    Directory of Open Access Journals (Sweden)

    Viacheslav O. Nikolaev

    2013-04-01

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

  6. Effects of fenspiride on human bronchial cyclic nucleotide phosphodiesterase isoenzymes: functional and biochemical study.

    Science.gov (United States)

    Cortijo, J; Naline, E; Ortiz, J L; Berto, L; Girard, V; Malbezin, M; Advenier, C; Morcillo, E J

    1998-01-02

    We have investigated the role of human bronchial cyclic nucleotide phosphodiesterases in the effects of fenspiride, a drug endowed with bronchodilator and anti-inflammatory properties. Functional studies on human isolated bronchi showed that fenspiride (10(-6)-3 x 10(-3) M, 30 min) induced a shift to the left of the concentration-response curves for isoprenaline and sodium nitroprusside with -logEC50 values of 4.1+/-0.1 (n = 7) and 3.5+/-0.2 (n = 8), respectively. Biochemical studies were carried out on three human bronchi in which separation of cyclic nucleotide phosphodiesterase isoenzymes was performed by ion exchange chromatography followed by determination of phosphodiesterase activity with a radioisotopic method. Phosphodiesterase 4 (cyclic AMP-specific) and phosphodiesterase 5 (cyclic GMP-specific) were the major phosphodiesterase isoforms present in the human bronchial tissue. The presence of phosphodiesterase 1 (Ca2+/calmodulin-stimulated), phosphodiesterase 2 (cyclic GMP-stimulated) and, in two cases, phosphodiesterase 3 (cyclic GMP-inhibited) was also identified. Fenspiride inhibited phosphodiesterase 4 and phosphodiesterase 3 activities with -logIC50 values of 4.16+/-0.09 and 3.44+/-0.12, respectively. Phosphodiesterase 5 activity was also inhibited with a -logIC50 value of approximately 3.8. Fenspiride (fenspiride is an effective inhibitor of both cyclic AMP and cyclic GMP hydrolytic activity in human bronchial tissues and this action may contribute to its airway effects.

  7. Cyclic Loading of Growing Tissue in a Bioreactor: Mathematical Model and Asymptotic Analysis

    KAUST Repository

    Pohlmeyer, J. V.

    2013-10-24

    A simplified 2D mathematical model for tissue growth within a cyclically-loaded tissue engineering scaffold is presented and analyzed. Such cyclic loading has the potential to improve yield and functionality of tissue such as bone and cartilage when grown on a scaffold within a perfusion bioreactor. The cyclic compression affects the flow of the perfused nutrient, leading to flow properties that are inherently unsteady, though periodic, on a timescale short compared with that of tissue proliferation. A two-timescale analysis based on these well-separated timescales is exploited to derive a closed model for the tissue growth on the long timescale of proliferation. Some sample numerical results are given for the final model, and discussed. © 2013 Society for Mathematical Biology.

  8. The Cyclic Di-GMP Phosphodiesterase Gene Rv1357c/BCG1419c Affects BCG Pellicle Production and In Vivo Maintenance.

    Science.gov (United States)

    Flores-Valdez, Mario Alberto; Aceves-Sánchez, Michel de Jesús; Pedroza-Roldán, César; Vega-Domínguez, Perla Jazmín; Prado-Montes de Oca, Ernesto; Bravo-Madrigal, Jorge; Laval, Françoise; Daffé, Mamadou; Koestler, Ben; Waters, Christopher M

    2015-02-01

    Bacteria living in a surface-attached community that contains a heterogeneous population, coated with an extracellular matrix, and showing drug tolerance (biofilms) are often linked to chronic infections. In mycobacteria, the pellicle mode of growth has been equated to an in vitro biofilm and meets several of the criteria mentioned above, while tuberculosis infection presents a chronic (latent) phase of infection. As mycobacteria lack most genes required to control biofilm production by other microorganisms, we deleted or expressed from the hsp60 strong promoter the only known c-di-GMP phosphodiesterase (PDE) gene in Mycobacterium bovis BCG. We found changes in pellicle production, cellular protein profiles, lipid production, resistance to nitrosative stress and maintenance in lungs and spleens of immunocompetent BALB/mice. Our results show that pellicle production and capacity to remain within the host are linked in BCG. © 2015 International Union of Biochemistry and Molecular Biology.

  9. Mechanical Stimulation of Adipose-Derived Stem Cells for Functional Tissue Engineering of the Musculoskeletal System via Cyclic Hydrostatic Pressure, Simulated Microgravity, and Cyclic Tensile Strain.

    Science.gov (United States)

    Nordberg, Rachel C; Bodle, Josie C; Loboa, Elizabeth G

    2018-01-01

    It is critical that human adipose stem cell (hASC) tissue-engineering therapies possess appropriate mechanical properties in order to restore function of the load bearing tissues of the musculoskeletal system. In an effort to elucidate the hASC response to mechanical stimulation and develop mechanically robust tissue engineered constructs, recent research has utilized a variety of mechanical loading paradigms including cyclic tensile strain, cyclic hydrostatic pressure, and mechanical unloading in simulated microgravity. This chapter describes methods for applying these mechanical stimuli to hASC to direct differentiation for functional tissue engineering of the musculoskeletal system.

  10. 3' : 5'-Cyclic AMP-dependent 3'

    NARCIS (Netherlands)

    Mato, José M.; Krens, Frans A.; Haastert, Peter J.M. van; Konijn, Theo M.

    1977-01-01

    Suspensions of 3':5'-cyclic AMP (cAMP)-sensitive cells of Dictyostelium discoideum responded to a cAMP pulse with increased 3':5'-cyclic GMP (cGMP) levels. Under the assay conditions used (2 × 10^8 cells per ml in 10 mM phosphate buffer, pH 6.0) cAMP (5 × 10-8 M final concentration) increased cGMP

  11. Reduced intracellular c-di-GMP content increases expression of quorum sensing-regulated genes in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Chua, Song Lin; Liu, Yang; Li, Yingying

    2017-01-01

    Cyclic-di-GMP (c-di-GMP) is an intracellular secondary messenger which controls the biofilm life cycle in many bacterial species. High intracellular c-di-GMP content enhances biofilm formation via the reduction of motility and production of biofilm matrix, while low c-di-GMP content in biofilm...... cells leads to increased motility and biofilm dispersal. While the effect of high c-di-GMP levels on bacterial lifestyles is well studied, the physiology of cells at low c-di-GMP levels remains unclear. Here, we showed that Pseudomonas aeruginosa cells with high and low intracellular c-di-GMP contents...... possessed distinct transcriptome profiles. There were 535 genes being upregulated and 432 genes downregulated in cells with low c-di-GMP, as compared to cells with high c-di-GMP. Interestingly, both rhl and pqs quorum-sensing (QS) operons were expressed at higher levels in cells with low intracellular c-di-GMP...

  12. Detection of Cyclic Dinucleotides by STING.

    Science.gov (United States)

    Du, Xiao-Xia; Su, Xiao-Dong

    2017-01-01

    STING (stimulator of interferon genes) is an essential signaling adaptor protein mediating cytosolic DNA-induced innate immunity for both microbial invasion and self-DNA leakage. STING is also a direct receptor for cytosolic cyclic dinucleotides (CDNs), including the microbial secondary messengers c-di-GMP (3',3'-cyclic di-GMP), 3',3'cGAMP (3',3'-cyclic GMP-AMP), and mammalian endogenous 2',3'cGAMP (2',3'-cyclic GMP-AMP) synthesized by cGAS (cyclic GMP-AMP synthase). Upon CDN binding, STING undergoes a conformational change to enable signal transduction by phosphorylation and finally to active IRF3 (Interferon regulatory factor 3) for type I interferon production. Here, we describe some experimental procedures such as Isothermal Titration Calorimetry and luciferase reporter assays to study the CDNs binding and activity by STING proteins.

  13. C-di-GMP regulates antimicrobial peptide resistance in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Chua, Song Lin; Tan, Sean Yang-Yi; Rybtke, Morten Theil

    2013-01-01

    Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is an intracellular second messenger which controls the life styles of many bacteria. A high intracellular level of c-di-GMP induces a biofilm lifestyle, whereas a low intracellular level of c-di-GMP stimulates dispersal of biofilms and promotes...... a planktonic lifestyle. Here, we used expression of different reporters to show that planktonic cells (PCells), biofilm cells (BCells) and cells dispersed from biofilms (DCells) had distinct intracellular c-di-GMP levels. Proteomics analysis showed that the low intracellular c-di-GMP level of DCells induced...... the expression of proteins required for the virulence and development of antimicrobial peptide resistance in P. aeruginosa. In accordance, P. aeruginosa cells with low c-di-GMP levels were found to be more resistant to colistin than P. aeruginosa cells with high c-di-GMP levels. This contradicts the current...

  14. Glass transition temperature of dried lens tissue pretreated with trehalose, maltose, or cyclic tetrasaccharide.

    Science.gov (United States)

    Kawata, Tetsuhiro; Matsuo, Toshihiko; Uchida, Tetsuya

    2014-01-01

    Glass transition temperature is a main indicator for amorphous polymers and biological macromolecules as materials, and would be a key for understanding the role of trehalose in protecting proteins and cells against desiccation. In this study, we measured the glass transition temperature by differential scanning calorimetry of dried lens tissues as a model of a whole biological tissue to know the effect of pretreatment by trehalose and other sugars. Isolated porcine lenses were incubated with saline, 100 or 1000 mM concentration of trehalose, maltose, or cyclic tetrasaccharide dissolved in saline at room temperature for 150 minutes. The solutions were removed and all samples were dried at room temperature in a desiccator until no weight change. The dried tissues were ground into powder and placed in a measuring pan for differential scanning calorimetry. The glass transition temperature of the dried lens tissues, as a mean and standard deviation, was 63.0 ± 6.4°C (n = 3) with saline pretreatment; 53.0 ± 0.8°C and 56.3 ± 2.7°C (n = 3), respectively, with 100 and 1000 mM trehalose pretreatment; 56.0 ± 1.6°C and 55.8 ± 1.1°C (n = 3), respectively, with 100 and 1000 mM maltose pretreatment; 60.0 ± 8.8°C and 59.2 ± 6.3°C (n = 3), respectively, with 100 and 1000 mM cyclic tetrasaccharide pretreatment. The glass transition temperature appeared lower, although not significantly, with trehalose and maltose pretreatments than with saline and cyclic tetrasaccharide pretreatments (P > 0.05, Kruskal-Wallis test). The glass transition temperature of the dried lens tissues with trehalose pretreatment appeared more noticeable on the thermogram, compared with other pretreatments. The glass transition temperature was measured for the first time in the dried lens tissues as an example of a whole biological tissue and might provide a basis for tissue preservation in the dried condition.

  15. Effects of oxytocin and methacholine on cyclic nucleotide levels of rabbit myometrium.

    Science.gov (United States)

    Schlageter, N; Janis, R A; Gualtieri, R T; Hechter, O

    1980-03-01

    The effects of oxytocin and methacholine on cyclic nucleotide levels in estrogen-primed rabbit myometrium were studied in the presence and absence of 1-methyl-3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor. In the absence of MIX, methacholine increased guanosine 3',5'-cyclic monophosphate (cGMP) levels at a time when contraction was decreasing, but had no influence on adenosine 3',5'-cyclic monophosphate (cAMP) levels. In contrast, oxytocin did not elevate cGMP, but rapidly decreased cAMP levels. MIX (1 mM) increased both cAMP and cGMP levels. Oxytocin or methacholine further increased cGMP, indicating activation of guanylate cyclase. Oxytocin- but not methacholine-induced stimulation of guanylate cyclase was abolished in Ca2+-free solution. Oxytocin increased cAMP over the levels produced by MIX alone, whereas methacholine decreased cAMP below the MIX control values; these effects were insensitive to indomethacin. Tissue levels of cGMP and cAMP did not directly correlate with isometric tension. The results also indicate that both oxytocin and methacholine stimulate guanylate cyclase but have opposing effects on adenylate cyclase of rabbit myometrium.

  16. The resistance of cortical bone tissue to failure under cyclic loading is reduced with alendronate.

    Science.gov (United States)

    Bajaj, Devendra; Geissler, Joseph R; Allen, Matthew R; Burr, David B; Fritton, J C

    2014-07-01

    Bisphosphonates are the most prescribed preventative treatment for osteoporosis. However, their long-term use has recently been associated with atypical fractures of cortical bone in patients who present with low-energy induced breaks of unclear pathophysiology. The effects of bisphosphonates on the mechanical properties of cortical bone have been exclusively studied under simple, monotonic, quasi-static loading. This study examined the cyclic fatigue properties of bisphosphonate-treated cortical bone at a level in which tissue damage initiates and is accumulated prior to frank fracture in low-energy situations. Physiologically relevant, dynamic, 4-point bending applied to beams (1.5 mm × 0.5 mm × 10 mm) machined from dog rib (n=12/group) demonstrated mechanical failure and micro-architectural features that were dependent on drug dose (3 groups: 0, 0.2, 1.0mg/kg/day; alendronate [ALN] for 3 years) with cortical bone tissue elastic modulus (initial cycles of loading) reduced by 21% (pbone remodeling, such as the size of osteons (-14%; ALN1.0: 10.5±1.8, VEH: 12.2±1.6, ×10(3) μm2; pbone tissue are altered by high-dose ALN treatment and contribute to reduced mechanical properties under cyclic loading conditions. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. BolA Is Required for the Accurate Regulation of c-di-GMP, a Central Player in Biofilm Formation

    OpenAIRE

    Moreira, Ricardo N.; Dressaire, Clémentine; Barahona, Susana; Galego, Lisete; Kaever, Volkhard; Jenal, Urs; Arraiano, Cecília M.

    2017-01-01

    The bacterial second messenger cyclic dimeric GMP (c-di-GMP) is a nearly ubiquitous intracellular signaling molecule involved in the transition from the motile to the sessile/biofilm state in bacteria. C-di-GMP regulates various cellular processes, including biofilm formation, motility, and virulence. BolA is a transcription factor that promotes survival in different stresses and is also involved in biofilm formation. Both BolA and c-di-GMP participate in the regulation of motility mechanisms...

  18. Partial reconstitution of photoreceptor cGMP phosphodiesterase characteristics in cGMP phosphodiesterase-5.

    Science.gov (United States)

    Granovsky, A E; Artemyev, N O

    2001-06-15

    Photoreceptor cGMP phosphodiesterases (PDE6) are uniquely qualified to serve as effector enzymes in the vertebrate visual transduction cascade. In the dark-adapted photoreceptors, the activity of PDE6 is blocked via tight association with the inhibitory gamma-subunits (Pgamma). The Pgamma block is removed in the light-activated PDE6 by the visual G protein, transducin. Transducin-activated PDE6 exhibits an exceptionally high catalytic rate of cGMP hydrolysis ensuring high signal amplification. To identify the structural determinants for the inhibitory interaction with Pgamma and the remarkable cGMP hydrolytic ability, we sought to reproduce the PDE6 characteristics by mutagenesis of PDE5, a related cyclic GMP-specific, cGMP-binding PDE. PDE5 is insensitive to Pgamma and has a more than 100-fold lower k(cat) for cGMP hydrolysis. Our mutational analysis of chimeric PDE5/PDE6alpha' enzymes revealed that the inhibitory interaction of cone PDE6 catalytic subunits (PDE6alpha') with Pgamma is mediated primarily by three hydrophobic residues at the entry to the catalytic pocket, Met(758), Phe(777), and Phe(781). The maximal catalytic rate of PDE5 was enhanced by at least 10-fold with substitutions of PDE6alpha'-specific glycine residues for the corresponding PDE5 alanine residues, Ala(608) and Ala(612). The Gly residues are adjacent to the highly conserved metal binding motif His-Asn-X-X-His, which is essential for cGMP hydrolysis. Our results suggest that the unique Gly residues allow the PDE6 metal binding site to adopt a more favorable conformation for cGMP hydrolysis.

  19. Short bursts of cyclic mechanical compression modulate tissue formation in a 3D hybrid scaffold.

    Science.gov (United States)

    Brunelli, M; Perrault, C M; Lacroix, D

    2017-07-01

    Among the cues affecting cells behaviour, mechanical stimuli are known to have a key role in tissue formation and mineralization of bone cells. While soft scaffolds are better at mimicking the extracellular environment, they cannot withstand the high loads required to be efficient substitutes for bone in vivo. We propose a 3D hybrid scaffold combining the load-bearing capabilities of polycaprolactone (PCL) and the ECM-like chemistry of collagen gel to support the dynamic mechanical differentiation of human embryonic mesodermal progenitor cells (hES-MPs). In this study, hES-MPs were cultured in vitro and a BOSE Bioreactor was employed to induce cells differentiation by mechanical stimulation. From day 6, samples were compressed by applying a 5% strain ramp followed by peak-to-peak 1% strain sinewaves at 1Hz for 15min. Three different conditions were tested: unloaded (U), loaded from day 6 to day 10 (L1) and loaded as L1 and from day 16 to day 20 (L2). Cell viability, DNA content and osteocalcin expression were tested. Samples were further stained with 1% osmium tetroxide in order to investigate tissue growth and mineral deposition by micro-computed tomography (µCT). Tissue growth involved volumes either inside or outside samples at day 21 for L1, suggesting cyclic stimulation is a trigger for delayed proliferative response of cells. Cyclic load also had a role in the mineralization process preventing mineral deposition when applied at the early stage of culture. Conversely, cyclic load during the late stage of culture on pre-compressed samples induced mineral formation. This study shows that short bursts of compression applied at different stages of culture have contrasting effects on the ability of hES-MPs to induce tissue formation and mineral deposition. The results pave the way for a new approach using mechanical stimulation in the development of engineered in vitro tissue as replacement for large bone fractures. Copyright © 2017 Elsevier Ltd. All rights

  20. Effects of hypokinesia on cyclic nucleotides and hormonal regulation ...

    African Journals Online (AJOL)

    PTH), calcitonin (CT), cyclic nucleotides (cAMP, cGMP) and calcium in the blood of rats, while in urine - phosphate, calcium and cyclic nucleotides. Design: Laboratory based experiment. Setting: Laboratory in the Department of Biochemistry, ...

  1. Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation

    KAUST Repository

    Kim, Jeong Joo

    2016-04-09

    Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG. Kim et al. obtain the first crystal structure of the PKG I R domain bound with cGMP representing its activated state. It reveals a symmetric R dimer where cGMP molecules provide dimeric contacts. This R-R interaction prevents the high-affinity inhibitory interaction between R-C domain and sustains activation. © 2016 Elsevier Ltd.

  2. Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation

    KAUST Repository

    Kim, Jeong  Joo; Lorenz, Robin; Arold, Stefan T.; Reger, Albert  S.; Sankaran, Banumathi; Casteel, Darren  E.; Herberg, Friedrich  W.; Kim, Choel

    2016-01-01

    Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG. Kim et al. obtain the first crystal structure of the PKG I R domain bound with cGMP representing its activated state. It reveals a symmetric R dimer where cGMP molecules provide dimeric contacts. This R-R interaction prevents the high-affinity inhibitory interaction between R-C domain and sustains activation. © 2016 Elsevier Ltd.

  3. In vivo cyclic loading as a potent stimulatory signal for bone formation inside tissue engineering scaffold

    Directory of Open Access Journals (Sweden)

    A Roshan-Ghias

    2010-02-01

    Full Text Available In clinical situations, bone defects are often located at load bearing sites. Tissue engineering scaffolds are future bone substitutes and hence they will be subjected to mechanical stimulation. The goal of this study was to test if cyclic loading can be used as stimulatory signal for bone formation in a bone scaffold. Poly(L-lactic acid (PLA/ 5% beta-tricalcium phosphate (beta-TCP scaffolds were implanted in both distal femoral epiphyses of eight rats. Right knees were stimulated (10N, 4Hz, 5 min five times, every two days, starting from the third day after surgery while left knees served as control. Finite element study of the in vivo model showed that the strain applied to the scaffold is similar to physiological strains. Using micro-computed tomography (CT, all knees were scanned five times after the surgery and the related bone parameters of the newly formed bone were quantified. Statistical modeling was used to estimate the evolution of these parameters as a function of time and loading. The results showed that mechanical stimulation had two effects on bone volume (BV: an initial decrease in BV at week 2, and a long-term increase in the rate of bone formation by 28%. At week 13, the BV was then significantly higher in the loaded scaffolds.

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

    Directory of Open Access Journals (Sweden)

    Martin eThunemann

    2014-10-01

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

  5. Improvements in the automated radioimmunoassay for cAMP or cGMP

    International Nuclear Information System (INIS)

    Brooker, G.

    1988-01-01

    The work others in developing antibodies and the original radioimmunoassay for cyclic nucleotides provides the basis for these sensitive assays. The acetylation radioimmunoassay for cyclic nucleotides has enabled the measurement of cyclic AMP and cyclic GMP in very small biological samples. This is because accurate determinations can be made in samples containing less than 1 fmol of cyclic AMP or cyclic GMP. The Gamma-Flo automated radioimmunoassay system has been adapted to these assays such that cyclic nucleotides can be automatically measured at a rate of about 60 samples/hr. The Gamma-Flo instrument provides high-precision assays and eliminates human intervention in all steps of the radioimmunoassay. The automated assay has been in continuous operation in our laboratory over the last 10 years and this chapter summarizes the methodology and delineates improvements which have occurred over that time frame. Details for the preparation of the radioligands apply also to the manual acetylated radioimmunoassay for cyclic nucleotides

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

    KAUST Repository

    Turek, Ilona

    2016-03-05

    The functional homologues of vertebrate natriuretic peptides (NPs), the plant natriuretic peptides (PNPs), are a novel class of peptidic hormones that signal via guanosine 3′,5′-cyclic monophosphate (cGMP) and systemically affect plant salt and water balance and responses to biotrophic plant pathogens. Although there is increasing understanding of the complex roles of PNPs in plant responses at the systems level, little is known about the underlying signaling mechanisms. Here we report isolation and identification of a novel Leucine-Rich Repeat (LRR) protein that directly interacts with A. thaliana PNP, AtPNP-A. In vitro binding studies revealed that the Arabidopsis AtPNP-A binds specifically to the LRR protein, termed AtPNP-R1, and the active region of AtPNP-A is sufficient for the interaction to occur. Importantly, the cytosolic part of the AtPNP-R1, much like in some vertebrate NP receptors, harbors a catalytic center diagnostic for guanylyl cyclases and the recombinant AtPNP-R1 is capable of catalyzing the conversion of guanosine triphosphate to cGMP. In addition, we show that AtPNP-A causes rapid increases of cGMP levels in wild type (WT) leaf tissue while this response is significantly reduced in the atpnp-r1 mutants. AtPNP-A also causes cGMP-dependent net water uptake into WT protoplasts, and hence volume increases, whereas responses of the protoplasts from the receptor mutant are impaired. Taken together, our results suggest that the identified LRR protein is an AtPNP-A receptor essential for the PNP-dependent regulation of ion and water homeostasis in plants and that PNP- and vertebrate NP-receptors and their signaling mechanisms share surprising similarities. © 2016 Springer Science+Business Media Dordrecht

  7. cGMP Signaling in the Cardiovascular System—The Role of Compartmentation and Its Live Cell Imaging

    Science.gov (United States)

    Bork, Nadja I.; Nikolaev, Viacheslav O.

    2018-01-01

    The ubiquitous second messenger 3′,5′-cyclic guanosine monophosphate (cGMP) regulates multiple physiologic processes in the cardiovascular system. Its intracellular effects are mediated by stringently controlled subcellular microdomains. In this review, we will illustrate the current techniques available for real-time cGMP measurements with a specific focus on live cell imaging methods. We will also discuss currently accepted and emerging mechanisms of cGMP compartmentation in the cardiovascular system. PMID:29534460

  8. Cyclic deformation-induced solute transport in tissue scaffolds with computer designed, interconnected, pore networks: experiments and simulations.

    Science.gov (United States)

    Den Buijs, Jorn Op; Dragomir-Daescu, Dan; Ritman, Erik L

    2009-08-01

    Nutrient supply and waste removal in porous tissue engineering scaffolds decrease from the periphery to the center, leading to limited depth of ingrowth of new tissue into the scaffold. However, as many tissues experience cyclic physiological strains, this may provide a mechanism to enhance solute transport in vivo before vascularization of the scaffold. The hypothesis of this study was that pore cross-sectional geometry and interconnectivity are of major importance for the effectiveness of cyclic deformation-induced solute transport. Transparent elastic polyurethane scaffolds, with computer-programmed design of pore networks in the form of interconnected channels, were fabricated using a 3D printing and injection molding technique. The scaffold pores were loaded with a colored tracer for optical contrast, cyclically compressed with deformations of 10 and 15% of the original undeformed height at 1.0 Hz. Digital imaging was used to quantify the spatial distribution of the tracer concentration within the pores. Numerical simulations of a fluid-structure interaction model of deformation-induced solute transport were compared to the experimental data. The results of experiments and modeling agreed well and showed that pore interconnectivity heavily influences deformation-induced solute transport. Pore cross-sectional geometry appears to be of less relative importance in interconnected pore networks. Validated computer models of solute transport can be used to design optimal scaffold pore geometries that will enhance the convective transport of nutrients inside the scaffold and the removal of waste, thus improving the cell survivability deep inside the scaffold.

  9. Biomimetic fetal rotation bioreactor for engineering bone tissues-Effect of cyclic strains on upregulation of osteogenic gene expression.

    Science.gov (United States)

    Ravichandran, Akhilandeshwari; Wen, Feng; Lim, Jing; Chong, Mark Seow Khoon; Chan, Jerry K Y; Teoh, Swee-Hin

    2018-04-01

    Cells respond to physiological mechanical stresses especially during early fetal development. Adopting a biomimetic approach, it is necessary to develop bioreactor systems to explore the effects of physiologically relevant mechanical strains and shear stresses for functional tissue growth and development. This study introduces a multimodal bioreactor system that allows application of cyclic compressive strains on premature bone grafts that are cultured under biaxial rotation (chamber rotation about 2 axes) conditions for bone tissue engineering. The bioreactor is integrated with sensors for dissolved oxygen levels and pH that allow real-time, non-invasive monitoring of the culture parameters. Mesenchymal stem cells-seeded polycaprolactone-β-tricalcium phosphate scaffolds were cultured in this bioreactor over 2 weeks in 4 different modes-static, cyclic compression, biaxial rotation, and multimodal (combination of cyclic compression and biaxial rotation). The multimodal culture resulted in 1.8-fold higher cellular proliferation in comparison with the static controls within the first week. Two weeks of culture in the multimodal bioreactor utilizing the combined effects of optimal fluid flow conditions and cyclic compression led to the upregulation of osteogenic genes alkaline phosphatase (3.2-fold), osteonectin (2.4-fold), osteocalcin (10-fold), and collagen type 1 α1 (2-fold) in comparison with static cultures. We report for the first time, the independent and combined effects of mechanical stimulation and biaxial rotation for bone tissue engineering using a bioreactor platform with non-invasive sensing modalities. The demonstrated results show leaning towards the futuristic vision of using a physiologically relevant bioreactor system for generation of autologous bone grafts for clinical implantation. Copyright © 2018 John Wiley & Sons, Ltd.

  10. Cyclic hexapeptide-conjugated nanoparticles enhance curcumin delivery to glioma tumor cells and tissue

    Directory of Open Access Journals (Sweden)

    Zhang X

    2017-08-01

    Full Text Available Xuemei Zhang,1–3 Xuejuan Li,1,4 Hongchen Hua,1 Aiping Wang,1 Wanhui Liu,1–3 Youxin Li,1–3 Fenghua Fu,1–3 Yanan Shi,5 Kaoxiang Sun1 1School of Pharmacy, Yantai University, Yantai, Shandong Province, People’s Republic of China; 2State Key Laboratory of Long-acting and Targeting Drug Delivery System, Yantai, Shandong Province, People’s Republic of China; 3Luye Pharmaceutical Co., Ltd., Shandong Province, People’s Republic of China; 4National Engineering and Technology Research Center of Chirality Pharmaceutical, Lunan Pharmaceutical Group Co., Ltd., Shandong Province, People’s Republic of China; 5School of Pharmacy, Binzhou Medical University, Shandong Province, People’s Republic of China Abstract: Glioma has one of the highest mortality rates among primary brain tumors. The clinical treatment for glioma is very difficult due to its infiltration and specific growth locations. To achieve improved drug delivery to a brain tumor, we report the preparation and in vitro and in vivo evaluation of curcumin nanoparticles (Cur-NPs. The cyclic hexapeptide c(RGDf(N-meVK-C (cHP has increased affinity for cells that overexpress integrins and was designed to target Cur-NPs to tumors. Functional polyethyleneglycol-modified poly(D,L-lactide-co-glycolide (PEG-PLGA conjugated to cHP was synthesized, and targeted Cur-NPs were prepared using a self-assembly nanoprecipitation process. The physicochemical properties and the in vitro cytotoxicity, accuracy, and penetration capabilities of Cur-NPs targeting cells with high levels of integrin expression were investigated. The in vivo targeting and penetration capabilities of the NPs were also evaluated against glioma in rats using in vivo imaging equipment. The results showed that the in vitro cytotoxicity of the targeted cHP-modified curcumin nanoparticles (cHP/Cur-NPs was higher than that of either free curcumin or non-targeted Cur-NPs due to the superior ability of the cHP/Cur-NPs to target tumor cells

  11. [Effect of twirling-reinforcing-reducing needling manipulations on contents of serum acetylcholine and arterial NOS and cGMP in stress-induced hypertension rats].

    Science.gov (United States)

    Liu, Wei; Zhu, Ling-Qun; Chen, Si-Si; Lu, Shu-Chao; Tang, Jie; Liu, Qing-Guo

    2015-04-01

    To observe the effect of twirling-reinforcing or reducing needling manipulations on plasma acetylcholine (Ach) content and expression of nitric oxide synthetase (NOS) and cyclic guanosine monophosphate (cGMP) in thoracic artery tissue in stress-induced hypertension rats. A total of 60 male rats were randomly divided into blank control, model, acupuncture (no-needle-manipulation) , twirling-reinforcing needling and twirling-reducing needling groups (n = 12 in each group). The stress hypertension model was established by giving the animals with noise and electric shock stimulation (paw), twice a day for 15 days. Acupuncture stimulation was applied to bilateral "Taichong" (LR 3) for 1 min, followed by retaining the needles for 20 min. The treatment was conducted once daily for 7 days. Systolic blood pressure of the rat's tail was detected with non-invasive method and plasma Ach, and NOS and cGMP contents in the thoracic artery tissue were measured using ELISA method. Compared with the control group, the systolic blood pressure was significantly higher in the model group after 15 days' stress stimulation (P arterial NOS and cGMP were markedly down-regulated (P arterial cGMP content was found in the no-needle-manipulation group (P > 0.05). The effect of the twirling-reducing needling was superior to that of no-needle-manipulation and twirling-reinforcing needling in lowering blood pressure and raising plasma Ach content (P hypertensive effect in stress hypertension rats, which may be associated with its effects in raising blood Ach, and arterial NOS and cGMP levels.

  12. The role of cGMP signalling in regulating life cycle progression of Plasmodium.

    OpenAIRE

    Hopp, CS; Bowyer, PW; Baker, DA

    2012-01-01

    The 3′-5′-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is the main mediator of cGMP signalling in the malaria parasite. This article reviews the role of PKG in Plasmodium falciparum during gametogenesis and blood stage schizont rupture, as well as the role of the Plasmodium berghei orthologue in ookinete differentiation and motility, and liver stage schizont development. The current views on potential effector proteins downstream of PKG and the mechanisms that may regu...

  13. Effects of cyclic compression on the mechanical properties and calcification process of immature chick bone tissue in culture.

    Science.gov (United States)

    Maeda, Eijiro; Nakagaki, Masashi; Ichikawa, Katsuhisa; Nagayama, Kazuaki; Matsumoto, Takeo

    2017-06-01

    Contribution of mechanical loading to tissue growth during both the development and post-natal maturation is of a particular interest, as its understanding would be important to strategies in bone tissue engineering and regenerative medicine. The present study has been performed to investigate how immature bone responds to mechanical loading using an ex vivo culture system. A slice of the tibia, with the thickness of 3 mm, was obtained from 0-day-old chick. For the ex vivo culture experiment in conjunction with cyclic compressive loading, we developed a custom-made, bioreactor system where both the load and the deformation applied to the specimen was recorded. Cyclic compression, with an amplitude of 0.3 N corresponding to 1 to 2% compressive strain, was applied to immature bone specimen during a 3-day culture period at an overall loading rate 3-4 cycles/min, in the presence of β-glycerol phosphate and dexamethasone in culture medium. The stress-strain relationship was obtained at the beginning and the end of the culture experiment. In addition, analyses for alkaline phosphate release, cell viability and tissue calcification were also performed. It was exhibited that elastic moduli of bone slices were significantly elevated at the end of the 3-day culture in the presence of cyclic compression, which was a similar phenomenon to significant elevation of the elastic moduli of bone tissue by the maturation from 0-day old to 3-day old. By contrast, no significant changes in the moduli were observed in the absence of cyclic compression or in deactivated, cell-free samples. The increases in the moduli were coincided with the increase in calcified area in the bone samples. It was confirmed that immature bone can respond to compressive loading in vitro and demonstrate the growth of bone matrix, similar to natural, in vivo maturation. The elevation of the elastic moduli was attributable to the increased calcified area and the realignment of collagen fibers parallel to

  14. Effects of cyclic compression on the mechanical properties and calcification process of immature chick bone tissue in culture

    Directory of Open Access Journals (Sweden)

    Eijiro Maeda

    2017-06-01

    Full Text Available Contribution of mechanical loading to tissue growth during both the development and post-natal maturation is of a particular interest, as its understanding would be important to strategies in bone tissue engineering and regenerative medicine. The present study has been performed to investigate how immature bone responds to mechanical loading using an ex vivo culture system. A slice of the tibia, with the thickness of 3 mm, was obtained from 0-day-old chick. For the ex vivo culture experiment in conjunction with cyclic compressive loading, we developed a custom-made, bioreactor system where both the load and the deformation applied to the specimen was recorded. Cyclic compression, with an amplitude of 0.3 N corresponding to 1 to 2% compressive strain, was applied to immature bone specimen during a 3-day culture period at an overall loading rate 3–4 cycles/min, in the presence of β-glycerol phosphate and dexamethasone in culture medium. The stress-strain relationship was obtained at the beginning and the end of the culture experiment. In addition, analyses for alkaline phosphate release, cell viability and tissue calcification were also performed. It was exhibited that elastic moduli of bone slices were significantly elevated at the end of the 3-day culture in the presence of cyclic compression, which was a similar phenomenon to significant elevation of the elastic moduli of bone tissue by the maturation from 0-day old to 3-day old. By contrast, no significant changes in the moduli were observed in the absence of cyclic compression or in deactivated, cell-free samples. The increases in the moduli were coincided with the increase in calcified area in the bone samples. It was confirmed that immature bone can respond to compressive loading in vitro and demonstrate the growth of bone matrix, similar to natural, in vivo maturation. The elevation of the elastic moduli was attributable to the increased calcified area and the realignment of collagen

  15. Cyclic nucleotides and radioresistnace

    International Nuclear Information System (INIS)

    Kulinskij, V.I.; Mikheeva, G.A.; Zel'manovich, B.M.

    1982-01-01

    The addition of glucose to meat-peptone broth does not change the radiosensitizing effect (RSE) of cAMP at the logarithmic phase (LP) and the radioprotective effect (RPE) at the stationary phase (SP), but sensitization, characteristic of cGMP, disappears in SP and turns into RPE in LP. Introduction of glucose into the broth for 20 min eliminates all the effects of both cyclic nucleotides in the cya + strain while cya - mutant exhibits RSE. RSE of both cyclic nucleotides is only manifested on minimal media. These data brought confirmation of the dependence of the influence of cyclic media. These data brought confirmation of the dependence of the influence of cyclic nucleotides on radioresistance upon the metabolic status of the cell [ru

  16. Identification and Characterization of c-di-GMP Metabolic Enzymes of Leptospira interrogans and c-di-GMP Fluctuations After Thermal Shift and Infection

    Directory of Open Access Journals (Sweden)

    Guohui Xiao

    2018-04-01

    Full Text Available Leptospirosis is a widespread zoonotic disease caused by pathogenic Leptospira species. The most common species, Leptospira interrogans, can transfer from contaminated soil or water to the human body. It is able to survive these changing environments through sensing and responding to the changes of environmental cues. Cyclic di-GMP (c-di-GMP is a special secondary messenger in bacteria, which can respond to the environment and regulate diverse bacterial behaviors. The c-di-GMP levels in bacterial cells are regulated by diguanylatecyclases (DGC and phosphodiesterases (PDE, which are responsible for synthesizing or hydrolyzing c-di-GMP, respectively. In this study, distribution and phylogenetics of c-di-GMP metabolic genes among 15 leptospiral species were systematically analyzed. Bioinformatics analysis revealed that leptospiral species contain a multitude of c-di-GMP metabolic genes. C-di-GMP metabolic genes in L. interrogans strain Lai 56601 were further analyzed and the results showed that these genes have very diverse expression patterns. Most of the putative DGCs and PDEs possess enzymatic activities, as determined by riboswitch-based dual-fluorescence reporters in vivo or HPLC in vitro. Furtherer analysis of subdomains from GGDEF-containing proteins revealed that the ability to synthesize c-di-GMP was lost when the GAF domain from LA1483 and PAS domain from LA2932 were deleted, while deletion of the REC domain from LA2528 did not affect its ability to synthesize c-di-GMP. Furthermore, high temperatures generally resulted in low c-di-GMP concentrations in L. interrogans and most of the c-di-GMP metabolic genes exhibited differential temperature regulation. Also, infection of murine J774A.1 cells resulted in reduced c-di-GMP levels, while no significant change of c-di-GMP metabolic genes on transcriptional levels were observed during the infection of J774A.1 cells. Taken together, these results provide a basic platform for future studies of c-di-GMP

  17. c-di-GMP is an Effective Immunomodulator and Vaccine Adjuvant Against Pneumococcal Infection

    Science.gov (United States)

    Ogunniyi, Abiodun D.; Paton, James C.; Kirby, Alun C.; McCullers, Jonathan A.; Cook, Jan; Hyodo, Mamoru; Hayakawa, Yoshihiro; Karaolis, David K. R.

    2009-01-01

    Cyclic diguanylate (c-di-GMP) is a unique bacterial intracellular signaling molecule capable of stimulating enhanced protective innate immunity against various bacterial infections. The effects of intranasal pretreatment with c-di-GMP, or intraperitoneal coadministration of c-di-GMP with the pneumolysin toxoid (PdB) or PspA before pneumococcal challenge, was investigated in mice. We found that c-di-GMP had no significant direct short-term effect on the growth rate of S. pneumoniae either in vitro or in vivo. However, intranasal pretreatment of mice with c-di-GMP resulted in significant decrease in bacterial load in lungs and blood after serotypes 2 and 3 challenge, and significant decrease in lung titers after serotype 4 challenge. Potential cellular mediators of these enhanced protective responses were identified in lungs and draining lymph nodes. Intraperitoneal coadministration of c-di-GMP with PdB or PspA before challenge resulted in significantly higher antigen-specific antibody titers and increased survival of mice, compared to that obtained with alum adjuvant. These findings demonstrate that local or systemic c-di-GMP administration stimulates innate and adaptive immunity against invasive pneumococcal disease. We propose that c-di-GMP can be used as an effective broad spectrum immunomodulator and vaccine adjuvant to prevent infectious diseases. PMID:18640167

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

    Science.gov (United States)

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

    2012-05-01

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

  19. The role of cGMP signalling in regulating life cycle progression of Plasmodium.

    Science.gov (United States)

    Hopp, Christine S; Bowyer, Paul W; Baker, David A

    2012-08-01

    The 3'-5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is the main mediator of cGMP signalling in the malaria parasite. This article reviews the role of PKG in Plasmodium falciparum during gametogenesis and blood stage schizont rupture, as well as the role of the Plasmodium berghei orthologue in ookinete differentiation and motility, and liver stage schizont development. The current views on potential effector proteins downstream of PKG and the mechanisms that may regulate cyclic nucleotide levels are presented. Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  20. The search for mutations in the gene for the beta subunit of the cGMP phosphodiesterase (PDEB) in patients with autosomal recessive retinitis pigmentosa

    DEFF Research Database (Denmark)

    Riess, O; Noerremoelle, A; Weber, B

    1992-01-01

    The finding of a mutation in the beta subunit of the cyclic GMP (cGMP) phosphodiesterase gene causing retinal degeneration in mice (the Pdeb gene) prompted a search for disease-causing mutations in the human phosphodiesterase gene (PDEB gene) in patients with retinitis pigmentosa. All 22 exons...

  1. Allometric relationships to liver tissue concentrations of cyclic volatile methyl siloxanes in Atlantic cod

    International Nuclear Information System (INIS)

    Warner, Nicholas A.; Nøst, Therese H.; Andrade, Hector; Christensen, Guttorm

    2014-01-01

    Spatial distribution and relationship of allometric measurements (length, weight and age) to liver concentrations of cyclic volatile methyl siloxanes (cVMS) including octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5) and dodecamethylcyclosiloxane (D6) in Atlantic cod (Gadus morhua) collected near the community of Tromsø in Northern Norway were assessed. These congeners were benchmarked against known persistent polychlorinated biphenyls (PCBs 153 and 180) to assess accumulation behavior of cVMS. D5 was the dominate cVMS detected in all fish livers with lipid normalized concentrations up to 10 times or greater than those observed for PCB 153 and 180. D4 and D6 concentration were negatively correlated with fish length and weight, indicating a greater elimination capacity compared to uptake processes with increasing fish size for these chemicals. These results indicate relationships between allometric measurements and cVMS concentrations may account for concentration variations observed within fish and should be assessed in future studies evaluating cVMS bioaccumulation potential. - Highlights: • cVMS spatial distribution investigated within cod surrounding an Arctic community. • Highest cVMS concentrations detected in biota collected near human settlements. • Cod liver concentrations of D5 were higher compared to PCBs. • D4 and D6 liver concentrations were negatively correlated with fish length/weight. - Liver concentrations of cVMS congeners decreased with increasing fish length and weight in Atlantic cod collected near emission sources of cVMS

  2. cGMP and NHR signaling co-regulate expression of insulin-like peptides and developmental activation of infective larvae in Strongyloides stercoralis.

    Directory of Open Access Journals (Sweden)

    Jonathan D Stoltzfus

    2014-07-01

    Full Text Available The infectious form of the parasitic nematode Strongyloides stercoralis is a developmentally arrested third-stage larva (L3i, which is morphologically similar to the developmentally arrested dauer larva in the free-living nematode Caenorhabditis elegans. We hypothesize that the molecular pathways regulating C. elegans dauer development also control L3i arrest and activation in S. stercoralis. This study aimed to determine the factors that regulate L3i activation, with a focus on G protein-coupled receptor-mediated regulation of cyclic guanosine monophosphate (cGMP pathway signaling, including its modulation of the insulin/IGF-1-like signaling (IIS pathway. We found that application of the membrane-permeable cGMP analog 8-bromo-cGMP potently activated development of S. stercoralis L3i, as measured by resumption of feeding, with 85.1 ± 2.2% of L3i feeding in 200 µM 8-bromo-cGMP in comparison to 0.6 ± 0.3% in the buffer diluent. Utilizing RNAseq, we examined L3i stimulated with DMEM, 8-bromo-cGMP, or the DAF-12 nuclear hormone receptor (NHR ligand Δ7-dafachronic acid (DA--a signaling pathway downstream of IIS in C. elegans. L3i stimulated with 8-bromo-cGMP up-regulated transcripts of the putative agonistic insulin-like peptide (ILP -encoding genes Ss-ilp-1 (20-fold and Ss-ilp-6 (11-fold in comparison to controls without stimulation. Surprisingly, we found that Δ7-DA similarly modulated transcript levels of ILP-encoding genes. Using the phosphatidylinositol-4,5-bisphosphate 3-kinase inhibitor LY294002, we demonstrated that 400 nM Δ7-DA-mediated activation (93.3 ± 1.1% L3i feeding can be blocked using this IIS inhibitor at 100 µM (7.6 ± 1.6% L3i feeding. To determine the tissues where promoters of ILP-encoding genes are active, we expressed promoter::egfp reporter constructs in transgenic S. stercoralis post-free-living larvae. Ss-ilp-1 and Ss-ilp-6 promoters are active in the hypodermis and neurons and the Ss-ilp-7 promoter is active in the

  3. The role of cGMP hydrolysing phosphodiesterases 1 and 5 in cerebral artery dilatation

    DEFF Research Database (Denmark)

    Kruuse, Christina; Rybalkin, S D; Khurana, T S

    2001-01-01

    The aim was to investigate the presence and activity of cGMP hydrolysing phosphodiesterases in guinea pig basilar arteries and the effect of selective and non-selective phosphodiesterase inhibitors on cerebral artery dilatation involving the nitric oxide (NO)-guanosine cyclic 3'5-monophosphate (cGMP...... a close relation to the nitric oxide-cGMP pathway. The responses to zaprinast and dipyridamole, however, were not only moderately affected, but also restored by sodium nitroprusside (0.1 microM) pretreatment. At high concentrations, the dilatory effects of zaprinast and dipyridamole were partly caused...... by cGMP-independent mechanisms. Targeting the phosphodiesterases present in cerebral arteries, with selective inhibitors or activators of phosphodiesterase, may be a possible new way of treating cerebrovascular disease....

  4. Study on the change of cyclic nucleotide in mice with yang vacuity disease

    International Nuclear Information System (INIS)

    Zhu Xinhua; Shen Ling; Wang Shuguang

    2002-01-01

    To study the relation between Yang Vacuity disease happening, development and cyclic nucleotide response, and prove curative effects of some assisting Yang drug, the plasma cAMP, cGMP and cAMP/cGMP levels were detected by radioimmunoassay in the Yang Vacuity group and curing group. Results: showed: (1) Yang Vacuity group: the symptoms were clear, death rate was high, the plasma cAMP and cAMP/cGMP increased obviously, it suggests that cyclic nucleotide was imbalance. (2) Curing group: the symptoms of Yang Vacuity disease were improved obviously, death rate dropped, cAMP declined, cGMP increased, while cAMP/cGMP reached the normal level, it showed that cyclic nucleotide of the body had altered greatly. (3) It is a reference target for Yang Vacuity. (4) Assisting yang drug (Sini Decoction) had a close relation with correcting imbalance of cyclic nucleotide

  5. Molecular properties of mammalian proteins that interact with cGMP: protein kinases, cation channels, phosphodiesterases, and multi-drug anion transporters.

    Science.gov (United States)

    Francis, Sharron H; Blount, Mitsi A; Zoraghi, Roya; Corbin, Jackie D

    2005-09-01

    Cyclic GMP is a critical second messenger signaling molecule in many mammalian cell types. It is synthesized by a family of guanylyl cyclases that is activated in response to stimuli from hormones such as natriuretic peptides, members of the guanylin family, and chemical stimuli including nitric oxide and carbon monoxide. The resulting elevation of cGMP modulates myriad physiological processes. Three major groups of cellular proteins bind cGMP specifically at allosteric sites; interaction of cGMP with these sites modulates the activities and functions of other domains within these protein groups to bring about physiological effects. These proteins include the cyclic nucleotide (cN)-dependent protein kinases, cN-gated cation channels, and cGMP-binding phosphodiesterases (PDE). Cyclic GMP also interacts with the catalytic sites of many cN PDEs and with some members of the multi-drug anion transporter family (MRPs) which can extrude nucleotides from cells. The allosteric cN-binding sites in the kinases and the cN-gated channels are evolutionarily and biochemically related, whereas the allosteric cGMP-binding sites in PDEs (also known as GAF domains), the catalytic sites of PDEs , and the ligand-binding sites in the MRPs are evolutionarily and biochemically distinct from each other and from those in the kinase and channel families. The sites that interact with cGMP within each of these groups of proteins have unique properties that provide for cGMP binding. Within a given cell, cGMP can potentially interact with members of all these groups of proteins if they are present. The relative abundance and affinities of these various cGMP-binding sites in conjunction with their subcellular compartmentation, proximity to cyclases and PDEs, and post-translational modification contribute importantly in determining the impact of these respective proteins to cGMP signaling within a particular cell.

  6. Critical Role of Nitric Oxide-cGMP Cascade in the Formation of cAMP-Dependent Long-Term Memory

    Science.gov (United States)

    Aonuma, Hitoshi; Mizunami, Makoto; Matsumoto, Yukihisa; Unoki, Sae

    2006-01-01

    Cyclic AMP pathway plays an essential role in formation of long-term memory (LTM). In some species, the nitric oxide (NO)-cyclic GMP pathway has been found to act in parallel and complementary to the cAMP pathway for LTM formation. Here we describe a new role of the NO-cGMP pathway, namely, stimulation of the cAMP pathway to induce LTM. We have…

  7. Plasma levels of cAMP, cGMP and CGRP in sildenafil-induced headache

    DEFF Research Database (Denmark)

    Kruuse, Christina Rostrup; Frandsen, E; Schifter, S

    2004-01-01

    Sildenafil, a selective inhibitor of the cyclic guanosine monophosphate (cGMP) degrading phosphodiestrase 5 (PDE5), induced migraine without aura in 10 of 12 migraine patients and in healthy subjects it induced significantly more headache than placebo. The aim of the present study was to determine...... whether the pain-inducing effects of sildenafil would be reflected in plasma levels of important signalling molecules in migraine: cGMP, cyclic adenosine monophosphate (cAMP) and calcitonin gene-related peptide (CGRP). Ten healthy subjects (four women, six men) and 12 patients (12 women) suffering from...... migraine without aura were included in two separate double-blind, placebo-controlled, cross-over studies in which placebo or sildenafil 100 mg was administered orally. Plasma levels of CGRP, cAMP and cGMP were determined in blood from the antecubital vein. Despite the ability of sildenafil to induce...

  8. The Potential of GMP-Compliant Platelet Lysate to Induce a Permissive State for Cardiovascular Transdifferentiation in Human Mediastinal Adipose Tissue-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Camilla Siciliano

    2015-01-01

    Full Text Available Human adipose tissue-derived mesenchymal stem cells (ADMSCs are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL, a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs.

  9. The Potential of GMP-Compliant Platelet Lysate to Induce a Permissive State for Cardiovascular Transdifferentiation in Human Mediastinal Adipose Tissue-Derived Mesenchymal Stem Cells

    Science.gov (United States)

    Bordin, Antonella; Ponti, Donatella; Iudicone, Paola; Rendina, Erino Angelo; Calogero, Antonella; Pierelli, Luca; Ibrahim, Mohsen; De Falco, Elena

    2015-01-01

    Human adipose tissue-derived mesenchymal stem cells (ADMSCs) are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL), a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs. PMID:26495284

  10. Structural and functional characteristics of cGMP-dependent methionine oxidation in Arabidopsis thaliana proteins

    KAUST Repository

    Marondedze, Claudius; Turek, Ilona; Parrott, Brian Jonathan; Thomas, Ludivine; Jankovic, Boris R.; Lilley, Kathryn S; Gehring, Christoph A

    2013-01-01

    molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by c

  11. Antidepressant-like properties of sildenafil in a genetic rat model of depression: Role of cholinergic cGMP-interactions

    DEFF Research Database (Denmark)

    Liebenberg, Nico; Brink, Christiaan; Brand, Linda

    2008-01-01

    Background: The N-methyl-D-aspartate (NMDA)/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway has been implicated in the neurobiology of depression. Recently we suggested a possible complex interaction between the cholinergic and NO-cGMP pathways in the antidepressant-like response....... Conclusions: Using a genetic animal model of depression, we have confirmed the antidepressant-like property of sildenafil following “unmasking” by concomitant block of muscarinic receptors. These findings hint at a novel interaction between the cGMP and cholinergic systems in depression, and suggest...

  12. Cyclic Nucleotide Signalling in Kidney Fibrosis

    Directory of Open Access Journals (Sweden)

    Elisabeth Schinner

    2015-01-01

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

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

    Science.gov (United States)

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

    2002-11-01

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

  14. Implementation of good manufacturing practices (GMP) on human blood irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Boghi, Claudio; Napolitano, Celia M.; Ferreira, Danilo C.; Rela, Paulo Roberto [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)]. E-mails: cboghi@uol.com.br; cmnapoli@ipen.br; dancarde@ig.com.br; prela@ipen.br; Zarate, Herman S. [Comission Chilena de Energia Nuclear, Santiago (Chile)]. E-mail: hzarate@cchen.cl

    2007-07-01

    The irradiation of human blood is used to avoid the TA-GVHD (transfusion-associated graft-versus-host-disease), a rare but devastating adverse effect of leukocytes present in blood components for a immuno-competent transfusion recipients. Usually this irradiation practice is performed to a physical elimination of lymphocytes. The implementation of the GMP will assure that the properly dose in a range of 25 Gy to 50 Gy will be delivered to the blood in the bag collected in a blood tissue bank. The studies to establish the GMP were developed under the guidelines of the standard ISO 11137 - Sterilization of health care products - Requirements for validation and routine control - Radiation sterilization. In this work, two dosimetric systems were used for dose mapping during the studies of irradiator qualification, loading pattern, irradiation process validation and auditing. The CaSO{sub 4}: Dy dosimeter presented difficulties concerning to uncertainty on dose measurement, stability, trace ability and calibration system. The PMMA and gafchromic dosimetric systems have shown a better performance and were adopted on establishment of GMP procedures. The irradiation tests have been done using a Gammacell 220 Irradiator. The developed GMP can be adapted for different types of gamma irradiators, allowing to set up a quality assurance program for blood irradiation. (author)

  15. Implementation of good manufacturing practices (GMP) on human blood irradiation

    International Nuclear Information System (INIS)

    Boghi, Claudio; Napolitano, Celia M.; Ferreira, Danilo C.; Rela, Paulo Roberto; Zarate, Herman S.

    2007-01-01

    The irradiation of human blood is used to avoid the TA-GVHD (transfusion-associated graft-versus-host-disease), a rare but devastating adverse effect of leukocytes present in blood components for a immuno-competent transfusion recipients. Usually this irradiation practice is performed to a physical elimination of lymphocytes. The implementation of the GMP will assure that the properly dose in a range of 25 Gy to 50 Gy will be delivered to the blood in the bag collected in a blood tissue bank. The studies to establish the GMP were developed under the guidelines of the standard ISO 11137 - Sterilization of health care products - Requirements for validation and routine control - Radiation sterilization. In this work, two dosimetric systems were used for dose mapping during the studies of irradiator qualification, loading pattern, irradiation process validation and auditing. The CaSO 4 : Dy dosimeter presented difficulties concerning to uncertainty on dose measurement, stability, trace ability and calibration system. The PMMA and gafchromic dosimetric systems have shown a better performance and were adopted on establishment of GMP procedures. The irradiation tests have been done using a Gammacell 220 Irradiator. The developed GMP can be adapted for different types of gamma irradiators, allowing to set up a quality assurance program for blood irradiation. (author)

  16. Good manufacturing practices (GMP utilized on human blood irradiation process

    Directory of Open Access Journals (Sweden)

    Cláudio Boghi

    2008-01-01

    Full Text Available Irradiation of human blood is used to avoid the TA-GVHD (transfusion-associated graft-versus-host-disease, a rare but devastating adverse effect of leukocytes present in blood components for immunocompetent transfusion recipients. Usually this irradiation practice is performed to a physical elimination of lymphocytes. The implementation of the GMP will assure that the properly dose in a range of 25Gy to 50Gy will be delivered to the blood in the bag collected in a blood tissue bank. The studies to establish the GMP were developed under the guidelines of the standard ISO 11137 - Sterilization of health care products - Requirements for validation and routine control - Radiation sterilization. In this work, two dosimetric systems were used for dose mapping during the studies of irradiator qualification, loading pattern, irradiation process validation and auditing. The CaSO4: Dy dosimeter presented difficulties concerning to uncertainty on dose measurement, stability, trace ability and calibration system. The PMMA and gafchromic dosimetric systems have shown a better performance and were adopted on establishment of GMP procedures. The irradiation tests have been done using a Gammacell 220 Irradiator. The developed GMP can be adapted for different types of gamma irradiators, allowing to set up a quality assurance program for blood irradiation.

  17. Amyloid-β Peptide Is Needed for cGMP-Induced Long-Term Potentiation and Memory.

    Science.gov (United States)

    Palmeri, Agostino; Ricciarelli, Roberta; Gulisano, Walter; Rivera, Daniela; Rebosio, Claudia; Calcagno, Elisa; Tropea, Maria Rosaria; Conti, Silvia; Das, Utpal; Roy, Subhojit; Pronzato, Maria Adelaide; Arancio, Ottavio; Fedele, Ernesto; Puzzo, Daniela

    2017-07-19

    High levels of amyloid-β peptide (Aβ) have been related to Alzheimer's disease pathogenesis. However, in the healthy brain, low physiologically relevant concentrations of Aβ are necessary for long-term potentiation (LTP) and memory. Because cGMP plays a key role in these processes, here we investigated whether the cyclic nucleotide cGMP influences Aβ levels and function during LTP and memory. We demonstrate that the increase of cGMP levels by the phosphodiesterase-5 inhibitors sildenafil and vardenafil induces a parallel release of Aβ due to a change in the approximation of amyloid precursor protein (APP) and the β-site APP cleaving enzyme 1. Moreover, electrophysiological and behavioral studies performed on animals of both sexes showed that blocking Aβ function, by using anti-murine Aβ antibodies or APP knock-out mice, prevents the cGMP-dependent enhancement of LTP and memory. Our data suggest that cGMP positively regulates Aβ levels in the healthy brain which, in turn, boosts synaptic plasticity and memory. SIGNIFICANCE STATEMENT Amyloid-β (Aβ) is a key pathogenetic factor in Alzheimer's disease. However, low concentrations of endogenous Aβ, mimicking levels of the peptide in the healthy brain, enhance hippocampal long-term potentiation (LTP) and memory. Because the second messenger cGMP exerts a central role in LTP mechanisms, here we studied whether cGMP affects Aβ levels and function during LTP. We show that cGMP enhances Aβ production by increasing the APP/BACE-1 convergence in endolysosomal compartments. Moreover, the cGMP-induced enhancement of LTP and memory was disrupted by blockade of Aβ, suggesting that the physiological effect of the cyclic nucleotide on LTP and memory is dependent upon Aβ. Copyright © 2017 the authors 0270-6474/17/376926-12$15.00/0.

  18. The Role of Cyclic Nucleotide Signaling Pathways in Cancer: Targets for Prevention and Treatment

    Energy Technology Data Exchange (ETDEWEB)

    Fajardo, Alexandra M.; Piazza, Gary A. [Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Ave, Suite 3029, Mobile, AL 36604 (United States); Tinsley, Heather N., E-mail: htinsley@montevallo.edu [Department of Biology, Chemistry, and Mathematics, University of Montevallo, Station 6480, Montevallo, AL 35115 (United States)

    2014-02-26

    For more than four decades, the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) have been recognized as important signaling molecules within cells. Under normal physiological conditions, cyclic nucleotides regulate a myriad of biological processes such as cell growth and adhesion, energy homeostasis, neuronal signaling, and muscle relaxation. In addition, altered cyclic nucleotide signaling has been observed in a number of pathophysiological conditions, including cancer. While the distinct molecular alterations responsible for these effects vary depending on the specific cancer type, several studies have demonstrated that activation of cyclic nucleotide signaling through one of three mechanisms—induction of cyclic nucleotide synthesis, inhibition of cyclic nucleotide degradation, or activation of cyclic nucleotide receptors—is sufficient to inhibit proliferation and activate apoptosis in many types of cancer cells. These findings suggest that targeting cyclic nucleotide signaling can provide a strategy for the discovery of novel agents for the prevention and/or treatment of selected cancers.

  19. The arabidopsis cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara Elizabeth

    2016-05-11

    Background Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals. Methods An affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling. Results A total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment. Conclusions We propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.

  20. Chemotaxis to cyclic AMP and folic acid is mediated by different G proteins in Dictyostelium discoideum

    NARCIS (Netherlands)

    Kesbeke, Fanja; Haastert, Peter J.M. van; Wit, René J.W. de; Snaar-Jagalska, B. Ewa

    1990-01-01

    Mutant Frigid A (fgdA) of Dictyostelium discoideum is defective in a functional Gα2 subunit of a G protein and is characterized by a complete blockade of the cyclic AMP-mediated sensory transduction steps, including cyclic AMP relay, chemotaxis and the cyclic GMP response. Folic acid-mediated

  1. Single amino acids in the carboxyl terminal domain of aquaporin-1 contribute to cGMP-dependent ion channel activation

    Directory of Open Access Journals (Sweden)

    Yool Andrea J

    2003-10-01

    Full Text Available Abstract Background Aquaporin-1 (AQP1 functions as an osmotic water channel and a gated cation channel. Activation of the AQP1 ion conductance by intracellular cGMP was hypothesized to involve the carboxyl (C- terminus, based on amino acid sequence alignments with cyclic-nucleotide-gated channels and cGMP-selective phosphodiesterases. Results Voltage clamp analyses of human AQP1 channels expressed in Xenopus oocytes demonstrated that the nitric oxide donor, sodium nitroprusside (SNP; 3–14 mM activated the ionic conductance response in a dose-dependent manner. Block of soluble guanylate cyclase prevented the response. Enzyme immunoassays confirmed a linear dose-dependent relationship between SNP and the resulting intracellular cGMP levels (up to 1700 fmol cGMP /oocyte at 14 mM SNP. Results here are the first to show that the efficacy of ion channel activation is decreased by mutations of AQP1 at conserved residues in the C-terminal domain (aspartate D237 and lysine K243. Conclusions These data support the idea that the limited amino acid sequence similarities found between three diverse classes of cGMP-binding proteins are significant to the function of AQP1 as a cGMP-gated ion channel, and provide direct evidence for the involvement of the AQP1 C-terminal domain in cGMP-mediated ion channel activation.

  2. Acute stress-induced antinociception is cGMP-dependent but heme oxygenase-independent

    International Nuclear Information System (INIS)

    Carvalho-Costa, P.G.; Branco, L.G.S.; Leite-Panissi, C.R.A.

    2014-01-01

    Endogenous carbon monoxide (CO), which is produced by the enzyme heme oxygenase (HO), participates as a neuromodulator in physiological processes such as thermoregulation and nociception by stimulating the formation of 3′,5′-cyclic guanosine monophosphate (cGMP). In particular, the acute physical restraint-induced fever of rats can be blocked by inhibiting the enzyme HO. A previous study reported that the HO-CO-cGMP pathway plays a key phasic antinociceptive role in modulating noninflammatory acute pain. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in antinociception induced by acute stress in male Wistar rats (250-300 g; n=8/group) using the analgesia index (AI) in the tail flick test. The results showed that antinociception induced by acute stress was not dependent on the HO-CO-cGMP pathway, as neither treatment with the HO inhibitor ZnDBPG nor heme-lysinate altered the AI. However, antinociception was dependent on cGMP activity because pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) blocked the increase in the AI induced by acute stress

  3. Acute stress-induced antinociception is cGMP-dependent but heme oxygenase-independent

    Energy Technology Data Exchange (ETDEWEB)

    Carvalho-Costa, P.G. [Programa de Graduação em Psicobiologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Branco, L.G.S. [Departamento de Morfologia, Fisiologia e Patologia Básica, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Leite-Panissi, C.R.A. [Programa de Graduação em Psicobiologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Departamento de Morfologia, Fisiologia e Patologia Básica, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2014-09-19

    Endogenous carbon monoxide (CO), which is produced by the enzyme heme oxygenase (HO), participates as a neuromodulator in physiological processes such as thermoregulation and nociception by stimulating the formation of 3′,5′-cyclic guanosine monophosphate (cGMP). In particular, the acute physical restraint-induced fever of rats can be blocked by inhibiting the enzyme HO. A previous study reported that the HO-CO-cGMP pathway plays a key phasic antinociceptive role in modulating noninflammatory acute pain. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in antinociception induced by acute stress in male Wistar rats (250-300 g; n=8/group) using the analgesia index (AI) in the tail flick test. The results showed that antinociception induced by acute stress was not dependent on the HO-CO-cGMP pathway, as neither treatment with the HO inhibitor ZnDBPG nor heme-lysinate altered the AI. However, antinociception was dependent on cGMP activity because pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) blocked the increase in the AI induced by acute stress.

  4. The CRP/FNR family protein Bcam1349 is a c-di-GMP effector that regulates biofilm formation in the respiratory pathogen Burkholderia cenocepacia

    DEFF Research Database (Denmark)

    Fazli, Mustafa; O'Connell, Aileen; Nilsson, Martin

    2011-01-01

    Burkholderia cenocepacia is an opportunistic respiratory pathogen that can cause severe infections in immune-compromised individuals and is associated with poor prognosis for patients suffering from cystic fibrosis. The second messenger cyclic diguanosine monophosphate (c-di-GMP) has been shown...... to control a wide range of functions in bacteria, but little is known about these regulatory mechanisms in B. cenocepacia. Here we investigated the role that c-di-GMP plays in the regulation of biofilm formation and virulence in B. cenocepacia. Elevated intracellular levels of c-di-GMP promoted wrinkly...... colony, pellicle and biofilm formation in B. cenocepacia. A screen for transposon mutants unable to respond to elevated levels of c-di-GMP led to the identification of the mutant bcam1349 that did not display increased biofilm and pellicle formation with excessive c-di-GMP levels, and displayed a biofilm...

  5. Immunohistochemical distribution of cAMP- and cGMP-phosphodiesterase (PDE) isoenzymes in the human prostate

    NARCIS (Netherlands)

    Uckert, Stefan; Oelke, Matthias; Stief, Christian G.; Andersson, K.-E.; Jonas, Udo; Hedlund, Petter

    2006-01-01

    With the introduction of sildenafil citrate (Viagra), the concept of phosphodiesterase (PDE) inhibition has gained tremendous interest in the field of urology. Cyclic nucleotide second messengers cGMP and cAMP have been assumed to be involved in the control of the normal function of the prostate.

  6. A potent series targeting the malarial cGMP-dependent protein kinase clears infection and blocks transmission

    NARCIS (Netherlands)

    Baker, D.A.; Stewart, L.B.; Large, J.M.; Bowyer, P.W.; Ansell, K.H.; Jimenez-Diaz, M.B.; Bakkouri, M. El; Birchall, K.; Dechering, K.J.; Bouloc, N.S.; Coombs, P.J.; Whalley, D.; Harding, D.J.; Smiljanic-Hurley, E.; Wheldon, M.C.; Walker, E.M.; Dessens, J.T.; Lafuente, M.J.; Sanz, L.M.; Gamo, F.J.; Ferrer, S.B.; Hui, R.; Bousema, T.; Angulo-Barturen, I.; Merritt, A.T.; Croft, S.L.; Gutteridge, W.E.; Kettleborough, C.A.; Osborne, S.A.

    2017-01-01

    To combat drug resistance, new chemical entities are urgently required for use in next generation anti-malarial combinations. We report here the results of a medicinal chemistry programme focused on an imidazopyridine series targeting the Plasmodium falciparum cyclic GMP-dependent protein kinase

  7. In vitro and in vivo generation and characterization of Pseudomonas aeruginosa biofilm-dispersed cells via c-di-GMP manipulation

    DEFF Research Database (Denmark)

    Chua, Song Lin; Hultqvist, Louise D; Yuan, Mingjun

    2015-01-01

    Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a global secondary bacterial messenger that controls the formation of drug-resistant multicellular biofilms. Lowering the intracellular c-di-GMP content can disperse biofilms, and it is proposed as a biofilm eradication strategy...... biofilms by reducing the intracellular c-di-GMP content through modulation of phosphodiesterases (PDEs). Unlike conventional protocols that demonstrate biofilm dispersal by biomass quantification, our protocols enable physiological characterization of the dispersed cells. Biomarkers of dispersed cells...

  8. ASEAN GMP and pharmaceutical industries in Indonesia.

    Science.gov (United States)

    Soesilo, S; Sitorus, U

    1995-01-01

    Indonesia was appointed by the ASEAN Technical Cooperation in Pharmaceutical as a focal point and to coordinate the development of practical guidelines for the implementation of GMP. The ASEAN GMP Guidelines were endorsed by the ASEAN Technical Cooperation in Pharmaceutical in 1988, which among others required separation of Beta-Lactam dedicated facilities and three degrees of cleanliness for production areas. As it was realised that drug manufacturers in developing countries need more detailed guidelines to be able to implement the GMP, an Operational Manual for GMP was also prepared for providing examples of SOPs lay-outs, documentation etc. It was agreed by the technical cooperation group to leave the implementation of GMP to each member country. However, the ASEAN Manual for Inspection of GMP was drafted and endorsed by the group and training of ASEAN Drug Inspectors was organized to support the implementation. The ASEAN GMP is being implemented in Indonesia through a five-year, stepwise implementation plan, starting in 1989.

  9. Cyclic guanosine monophosphate in the regulation of the cell function

    Directory of Open Access Journals (Sweden)

    Małgorzata Zbrojkiewicz

    2016-12-01

    Full Text Available Intracellular concentration of cGMP depends on the activity of guanylate cyclase, responsible for its synthesis, on the activity of cyclic nucleotide degrading enzymes - phosphodiesterases (PDEs. There are two forms of guanylate cyclase: the membrane-bound cyclase and the soluble form. The physiological activators of the membrane guanylate cyclase are natriuretic peptides (NPs, and of the cytosolic guanylate cyclase - nitric oxide (NO and carbon monoxide (CO. Intracellular cGMP signaling pathways arise from its direct effect on the activity of G protein kinases, phosphodiesterases and cyclic nucleotide dependent cation channels. It has been shown in recent years that cGMP can also affect other signal pathways in cell signaling activity involving Wnt proteins and sex hormones. The increased interest in the research on the role of cGMP, resulted also in the discovery of its role in the regulation of phototransduction in the eye, neurotransmission, calcium homeostasis, platelet aggregation, heartbeat, bone remodeling, lipid metabolism and the activity of the cation channels. Better understanding of the mechanisms of action of cGMP in the regulation of cell function can create new opportunities for the cGMP affecting drugs use in the pharmacotherapy.

  10. C-di-GMP regulates Pseudomonas aeruginosa stress response to tellurite during both planktonic and biofilm modes of growth

    DEFF Research Database (Denmark)

    Chua, Song Lin; Sivakumar, Krishnakumar; Rybtke, Morten Levin

    2015-01-01

    tellurite (TeO3(2-)) exposure induced the intracellular content of the secondary messenger cyclic di-GMP (c-di-GMP) of Pseudomonas aeruginosa. Two diguanylate cyclases (DGCs), SadC and SiaD, were responsible for the increased intracellular content of c-di-GMP. Enhanced c-di-GMP levels by TeO3(2-) further...... increased P. aeruginosa biofilm formation and resistance to TeO3(2-). P. aeruginosa ΔsadCΔsiaD and PAO1/p(lac)-yhjH mutants with low intracellular c-di-GMP content were more sensitive to TeO3(2-) exposure and had low relative fitness compared to the wild-type PAO1 planktonic and biofilm cultures exposed...... to TeO3(2-). Our study provided evidence that c-di-GMP level can play an important role in mediating stress response in microbial communities during both planktonic and biofilm modes of growth....

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

    International Nuclear Information System (INIS)

    Forte, L.R.; Krause, W.J.; Freeman, R.H.

    1988-01-01

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

  12. CSF concentrations of cAMP and cGMP are lower in patients with Creutzfeldt-Jakob disease but not Parkinson's disease and amyotrophic lateral sclerosis.

    Directory of Open Access Journals (Sweden)

    Patrick Oeckl

    Full Text Available BACKGROUND: The cyclic nucleotides cyclic adenosine-3',5'-monophosphate (cAMP and cyclic guanosine-3',5'-monophosphate (cGMP are important second messengers and are potential biomarkers for Parkinson's disease (PD, amyotrophic lateral sclerosis (ALS and Creutzfeldt-Jakob disease (CJD. METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigated by liquid chromatography/tandem mass spectrometry (LC-MS/MS the cerebrospinal fluid (CSF concentrations of cAMP and cGMP of 82 patients and evaluated their diagnostic potency as biomarkers. For comparison with a well-accepted biomarker, we measured tau concentrations in CSF of CJD and control patients. CJD patients (n = 15 had lower cAMP (-70% and cGMP (-55% concentrations in CSF compared with controls (n = 11. There was no difference in PD, PD dementia (PDD and ALS cases. Receiver operating characteristic (ROC curve analyses confirmed cAMP and cGMP as valuable diagnostic markers for CJD indicated by the area under the curve (AUC of 0.86 (cAMP and 0.85 (cGMP. We calculated a sensitivity of 100% and specificity of 64% for cAMP and a sensitivity of 67% and specificity of 100% for cGMP. The combination of both nucleotides increased the sensitivity to 80% and specificity to 91% for the term cAMPxcGMP (AUC 0.92 and to 93% and 100% for the ratio tau/cAMP (AUC 0.99. CONCLUSIONS/SIGNIFICANCE: We conclude that the CSF determination of cAMP and cGMP may easily be included in the diagnosis of CJD and could be helpful in monitoring disease progression as well as in therapy control.

  13. Micro pore arrays in free standing cyclic olefin copolymer membranes: fabrication and surface functionalization strategies for in-vitro barrier tissue models

    Science.gov (United States)

    Gel, M.; Kandasamy, S.; Cartledge, K.; Be, C. L.; Haylock, D.

    2013-12-01

    In recent years there has been growing interest in micro engineered in-vitro models of tissues and organs. These models are designed to mimic the in-vivo like physiological conditions with a goal to study human physiology in an organ-specific context or to develop in-vitro disease models. One of the challenges in the development of these models is the formation of barrier tissues in which the permeability is controlled locally by the tissues cultured at the interface. In-vitro models of barrier tissues are typically created by generating a monolayer of cells grown on thin porous membranes. This paper reports a robust preparation method for free standing porous cyclic olefin copolymer (COC) membranes. We also demonstrate that gelatin coated membranes facilitate formation of highly confluent monolayer of HUVECs. Membranes with thickness in the range of 2-3 um incorporating micro pores with diameter approximately 20 um were fabricated and integrated with microfluidic channels. The performance of the device was demonstrated with a model system mimicking the endothelial barrier in bone marrow sinusoids.

  14. Transient Kinetics of a cGMP-dependent cGMP-specific Phosphodiesterase from Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Lookeren Campagne, Michiel M. van

    1984-01-01

    Chemotactic stimulation of Dictyostelium discoideum cells induces a fast transient increase of cGMP levels which reach a peak at 10 s. Prestimulation levels are recovered in ~30 s, which is achieved mainly by the action of a guanosine 3',5'-monophosphate cGMP-specific phosphodiesterase. This enzyme

  15. Involvement of cyclic nucleotides in locust flight muscle metabolism

    NARCIS (Netherlands)

    Worm, R.A.A.

    1980-01-01

    1. Flight had no significant effect on the levels of c-AMP of c-GMP in the flight muscles of Locusta migratoria. 2. Injections of 0.01 or 0.1 corpus cardiacum equivalents into the abdominal cavity did not elicit any effect on cyclic nucleotide levels either. 3. Injection of A23187 resulted in

  16. cGMP-Phosphodiesterase Inhibition Prevents Hypoxia-Induced Cell Death Activation in Porcine Retinal Explants.

    Directory of Open Access Journals (Sweden)

    Lorena Olivares-González

    Full Text Available Retinal hypoxia and oxidative stress are involved in several retinal degenerations including diabetic retinopathy, glaucoma, central retinal artery occlusion, or retinopathy of prematurity. The second messenger cyclic guanosine monophosphate (cGMP has been reported to be protective for neuronal cells under several pathological conditions including ischemia/hypoxia. The purpose of this study was to evaluate whether the accumulation of cGMP through the pharmacological inhibition of phosphodiesterase (PDE with Zaprinast prevented retinal degeneration induced by mild hypoxia in cultures of porcine retina. Exposure to mild hypoxia (5% O2 for 24h reduced cGMP content and induced retinal degeneration by caspase dependent and independent (PARP activation mechanisms. Hypoxia also produced a redox imbalance reducing antioxidant response (superoxide dismutase and catalase activities and increasing superoxide free radical release. Zaprinast reduced mild hypoxia-induced cell death through inhibition of caspase-3 or PARP activation depending on the cell layer. PDE inhibition also ameliorated the effects of mild hypoxia on antioxidant response and the release of superoxide radical in the photoreceptor layer. The use of a PKG inhibitor, KT5823, suggested that cGMP-PKG pathway is involved in cell survival and antioxidant response. The inhibition of PDE, therefore, could be useful for reducing retinal degeneration under hypoxic/ischemic conditions.

  17. 3,7-Bis(2-hydroxyethyl)icaritin, a potent inhibitor of phosphodiesterase-5, prevents monocrotaline-induced pulmonary arterial hypertension via NO/cGMP activation in rats.

    Science.gov (United States)

    Lan, Tao-Hua; Chen, Xiao-Ling; Wu, Yun-Shan; Qiu, Hui-Liang; Li, Jun-Zhe; Ruan, Xin-Min; Xu, Dan-Ping; Lin, Dong-Qun

    2018-06-15

    Pulmonary arterial hypertension (PAH) is a chronic progressive disease which leads to elevated pulmonary arterial pressure and right heart failure. 3,7-Bis(2-hydroxyethyl)icaritin (ICT), an icariin derivatives, was reported to have potent inhibitory activity on phosphodiesterase type 5 (PDE5) which plays a crucial role in the pathogenesis of PAH. The present study was designed to investigate the effects of ICT on monocrotaline (MCT)-induced PAH rat model and reveal the underlying mechanism. MCT-induced PAH rat models were established with intragastric administration of ICT (10, 20, 40 mg/kg/d), Icariin (ICA) (40 mg/kg/d) and Sildenafil (25 mg/kg/d). The mean pulmonary arterial pressure (mPAP) and right ventricle hypertrophy index (RVHI) were measured. Pulmonary artery remodeling was assessed by H&E staining. Blood and lung tissue were collected to evaluate the level of endothelin 1 (ET-1), nitric oxide (NO), and cyclic guanosine monophosphate (cGMP). The expressions endothelial nitric oxide synthase (eNOS) and PDE5A in lung tissues were determined by Western blot analysis. The results showed that ICT reduced RVHI and mPAP, and reversed lung vascular remodeling in rats with MCT-induced PAH. ICT also reversed MCT-induced ET-1 elevation, NO and cGMP reduction in serum or lung tissue. Moreover, ICT administration significantly induced eNOS activation and PDE5A inhibition. ICT with lower dose had better effects than ICA. In summary, ICT is more effective in preventing MCT-induced PAH in rats via NO/cGMP activation compared with ICA. These findings demonstrate a novel mechanism of the action of ICT that may have value in prevention of PAH. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Potentiation of cGMP signaling increases oxygen delivery and oxidative metabolism in contracting skeletal muscle of older but not young humans

    DEFF Research Database (Denmark)

    Nyberg, Michael Permin; Piil, Peter Bergmann; Egelund, Jon

    2015-01-01

    regulation remain unresolved. Cyclic guanosine monophosphate (cGMP) is one of the main second messengers that mediate smooth muscle vasodilation and alterations in cGMP signaling could, therefore, be one mechanism by which skeletal muscle perfusion is impaired with advancing age. The current study aimed...... to evaluate the effect of inhibiting the main enzyme involved in cGMP degradation, phosphodiesterase 5 (PDE5), on blood flow and O2 delivery in contracting skeletal muscle of young and older humans. A group of young (23 ± 1 years) and a group of older (72 ± 2 years) male human subjects performed submaximal...... in the older subjects correlated with the increase in leg O2 uptake (r (2) = 0.843). These findings suggest an insufficient O2 delivery to the contracting skeletal muscle of aged individuals and that reduced cGMP availability is a novel mechanism underlying impaired skeletal muscle perfusion with advancing age....

  19. The exopolysaccharide gene cluster Bcam1330-Bcam1341 is involved in Burkholderia cenocepacia biofilm formation, and its expression is regulated by c-di-GMP and Bcam1349

    DEFF Research Database (Denmark)

    Fazli, Mustafa; McCarthy, Yvonne; Givskov, Michael

    2013-01-01

    In Burkholderia cenocepacia, the second messenger cyclic diguanosine monophosphate (c-di-GMP) has previously been shown to positively regulate biofilm formation and the expression of cellulose and type-I fimbriae genes through binding to the transcriptional regulator Bcam1349. Here, we provide...... evidence that cellulose and type-I fimbriae are not involved in B. cenocepacia biofilm formation in flow chambers, and we identify a novel Bcam1349/c-di-GMP-regulated exopolysaccharide gene cluster which is essential for B. cenocepacia biofilm formation. Overproduction of Bcam1349 in trans promotes wrinkly...... matrix exopolysaccharide and to be essential for flow-chamber biofilm formation. We demonstrate that Bcam1349 binds to the promoter region of genes in the Bcam1330-Bcam1341 cluster and that this binding is enhanced by the presence of c-di-GMP. Furthermore, we demonstrate that overproduction of both c-di-GMP...

  20. Molecular Analysis of Sensory Axon Branching Unraveled a cGMP-Dependent Signaling Cascade

    Directory of Open Access Journals (Sweden)

    Alexandre Dumoulin

    2018-04-01

    Full Text Available Axonal branching is a key process in the establishment of circuit connectivity within the nervous system. Molecular-genetic studies have shown that a specific form of axonal branching—the bifurcation of sensory neurons at the transition zone between the peripheral and the central nervous system—is regulated by a cyclic guanosine monophosphate (cGMP-dependent signaling cascade which is composed of C-type natriuretic peptide (CNP, the receptor guanylyl cyclase Npr2, and cGMP-dependent protein kinase Iα (cGKIα. In the absence of any one of these components, neurons in dorsal root ganglia (DRG and cranial sensory ganglia no longer bifurcate, and instead turn in either an ascending or a descending direction. In contrast, collateral axonal branch formation which represents a second type of axonal branch formation is not affected by inactivation of CNP, Npr2, or cGKI. Whereas axon bifurcation was lost in mouse mutants deficient for components of CNP-induced cGMP formation; the absence of the cGMP-degrading enzyme phosphodiesterase 2A had no effect on axon bifurcation. Adult mice that lack sensory axon bifurcation due to the conditional inactivation of Npr2-mediated cGMP signaling in DRG neurons demonstrated an altered shape of sensory axon terminal fields in the spinal cord, indicating that elaborate compensatory mechanisms reorganize neuronal circuits in the absence of bifurcation. On a functional level, these mice showed impaired heat sensation and nociception induced by chemical irritants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are normal. These data point to a critical role of axon bifurcation for the processing of acute pain perception.

  1. Molecular Analysis of Sensory Axon Branching Unraveled a cGMP-Dependent Signaling Cascade.

    Science.gov (United States)

    Dumoulin, Alexandre; Ter-Avetisyan, Gohar; Schmidt, Hannes; Rathjen, Fritz G

    2018-04-24

    Axonal branching is a key process in the establishment of circuit connectivity within the nervous system. Molecular-genetic studies have shown that a specific form of axonal branching—the bifurcation of sensory neurons at the transition zone between the peripheral and the central nervous system—is regulated by a cyclic guanosine monophosphate (cGMP)-dependent signaling cascade which is composed of C-type natriuretic peptide (CNP), the receptor guanylyl cyclase Npr2, and cGMP-dependent protein kinase Iα (cGKIα). In the absence of any one of these components, neurons in dorsal root ganglia (DRG) and cranial sensory ganglia no longer bifurcate, and instead turn in either an ascending or a descending direction. In contrast, collateral axonal branch formation which represents a second type of axonal branch formation is not affected by inactivation of CNP, Npr2, or cGKI. Whereas axon bifurcation was lost in mouse mutants deficient for components of CNP-induced cGMP formation; the absence of the cGMP-degrading enzyme phosphodiesterase 2A had no effect on axon bifurcation. Adult mice that lack sensory axon bifurcation due to the conditional inactivation of Npr2-mediated cGMP signaling in DRG neurons demonstrated an altered shape of sensory axon terminal fields in the spinal cord, indicating that elaborate compensatory mechanisms reorganize neuronal circuits in the absence of bifurcation. On a functional level, these mice showed impaired heat sensation and nociception induced by chemical irritants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are normal. These data point to a critical role of axon bifurcation for the processing of acute pain perception.

  2. Differential regulation of c-di-GMP metabolic enzymes by environmental signals modulates biofilm formation in Yersinia pestis

    Directory of Open Access Journals (Sweden)

    Gai-Xian eRen

    2016-06-01

    Full Text Available Cyclic diguanylate (c-di-GMP is essential for Yersinia pestis biofilm formation, which is important for flea-borne blockage-dependent plague transmission. Two diguanylate cyclases (DGCs, HmsT and HmsD and one phosphodiesterase (PDE, HmsP are responsible for the synthesis and degradation of c-di-GMP in Y. pestis. Here, we systematically analyzed the effect of various environmental signals on regulation of the biofilm phenotype, the c-di-GMP levels, and expression of HmsT, HmsD and HmsP in Y. pestis. Biofilm formation was higher in the presence of nonlethal high concentration of CaCl2, MgCl2, CuSO4, sucrose, sodium dodecyl sulfonate, or dithiothreitol, and was lower in the presence of FeCl2 or NaCl. In addition, we found that HmsD plays a major role in biofilm formation in acidic or redox environments. These environmental signals differentially regulated expression of HmsT, HmsP and HmsD, resulting in changes in the intracellular levels of c-di-GMP in Y. pestis. Our results suggest that bacteria can sense various environmental signals, and differentially regulates their DGCs and PDEs to coordinately regulate and adapt metabolism of c-di-GMP and biofilm formation to changing environments.

  3. The effect of cyclic hydrostatic pressure on the functional development of cartilaginous tissues engineered using bone marrow derived mesenchymal stem cells.

    Science.gov (United States)

    Meyer, E G; Buckley, C T; Steward, A J; Kelly, D J

    2011-10-01

    Mechanical signals can play a key role in regulating the chondrogenic differentiation of mesenchymal stem cells (MSCs). The objective of this study was to determine if the long-term application of cyclic hydrostatic pressure could be used to improve the functional properties of cartilaginous tissues engineered using bone marrow derived MSCs. MSCs were isolated from the femora of two porcine donors, expanded separately under identical conditions, and then suspended in cylindrical agarose hydrogels. Constructs from both donors were maintained in a chemically defined media supplemented with TGF-β3 for 42 days. TGF-β3 was removed from a subset of constructs from day 21 to 42. Loaded groups were subjected to 10 MPa of cyclic hydrostatic pressurisation at 1 Hz for one hour/day, five days/week. Loading consisted either of continuous hydrostatic pressure (CHP) initiated at day 0, or delayed hydrostatic pressure (DHP) initiated at day 21. Free swelling (FS) constructs were cultured in parallel as controls. Constructs were assessed at days 0, 21 and 42. MSCs isolated from both donors were morphologically similar, demonstrated comparable colony forming unit-fibroblast (CFU-F) numbers, and accumulated near identical levels of collagen and GAG following 42 days of free swelling culture. Somewhat unexpectedly the two donors displayed a differential response to hydrostatic pressure. For one donor the application of CHP resulted in increased collagen and GAG accumulation by day 42, resulting in an increased dynamic modulus compared to FS controls. In contrast, CHP had no effect on matrix accumulation for the other donor. The application of DHP had no effect on either matrix accumulation or construct mechanical properties for both donors. Variability in the response to hydrostatic pressure was also observed for three further donors. In conclusion, this study demonstrates that the application of long-term hydrostatic pressure can be used to improve the functional properties of

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

    Science.gov (United States)

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

    2015-09-08

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

  5. Gibberellic acid and cGMP-dependent transcriptional regulation in arabidopsis thaliana

    KAUST Repository

    Bastian, René

    2010-03-01

    An ever increasing amount of transcriptomic data and analysis tools provide novel insight into complex responses of biological systems. Given these resources we have undertaken to review aspects of transcriptional regulation in response to the plant hormone gibberellic acid (GA) and its second messenger guanosine 3\\',5\\'-cyclic monophosphate (cGMP) in Arabidopsis thaliana, both wild type and selected mutants. Evidence suggests enrichment of GA-responsive (GARE) elements in promoters of genes that are transcriptionally upregulated in response to cGMP but downregulated in a GA insensitive mutant (ga1-3). In contrast, in the genes upregulated in the mutant, no enrichment in the GARE is observed suggesting that GARE motifs are diagnostic for GA-induced and cGMP-dependent transcriptional upregulation. Further, we review how expression studies of GA-dependent transcription factors and transcriptional networks based on common promoter signatures derived from ab initio analyses can contribute to our understanding of plant responses at the systems level. © 2010 Landes Bioscience.

  6. Complex regulatory network encompassing the Csr, c-di-GMP and motility systems of Salmonella Typhimurium.

    Science.gov (United States)

    Jonas, Kristina; Edwards, Adrianne N; Ahmad, Irfan; Romeo, Tony; Römling, Ute; Melefors, Ojar

    2010-02-01

    Bacterial survival depends on the ability to switch between sessile and motile lifestyles in response to changing environmental conditions. In many species, this switch is governed by (3'-5')-cyclic-diguanosine monophosphate (c-di-GMP), a signalling molecule, which is metabolized by proteins containing GGDEF and/or EAL domains. Salmonella Typhimurium contains 20 such proteins. Here, we show that the RNA-binding protein CsrA regulates the expression of eight genes encoding GGDEF, GGDEF-EAL and EAL domain proteins. CsrA bound directly to the mRNA leaders of five of these genes, suggesting that it may regulate these genes post-transcriptionally. The c-di-GMP-specific phosphodiesterase STM3611, which reciprocally controls flagella function and production of biofilm matrix components, was regulated by CsrA binding to the mRNA, but was also indirectly regulated by CsrA through the FlhDC/FliA flagella cascade and STM1344. STM1344 is an unconventional (c-di-GMP-inactive) EAL domain protein, recently identified as a negative regulator of flagella gene expression. Here, we demonstrate that CsrA directly downregulates expression of STM1344, which in turn regulates STM3611 through fliA and thus reciprocally controls motility and biofilm factors. Altogether, our data reveal that the concerted and complex regulation of several genes encoding GGDEF/EAL domain proteins allows CsrA to control the motility-sessility switch in S. Typhimurium at multiple levels.

  7. Microbiological criteria for good manufacturing practice (GMP)

    Energy Technology Data Exchange (ETDEWEB)

    Farkas, J [Inst. of Preservation and Livestock Products Technology, Univ. of Horticulture and Food Industry, Budapest (Hungary); Zukal, E [Inst. of Preservation and Livestock Products Technology, Univ. of Horticulture and Food Industry, Budapest (Hungary)

    1992-01-01

    Good manufacturing practice (GMP) consist of an effective manufacturing operation and an effective application of food control. GMP is best supported by the Hazard Analysis Critical Control Point system (HACCP) of the preventive quality assurance, which requires that food irradiation as any food processing technology should be used only with foods of an acceptable quality and adequate handling and storage procedures should precede and follow the processing. The paper concentrates on the first element of the HACCP system for an irradiation plant: the incoming product control, i.e. whether GMP of foods to be irradiated can be assessed by establishing microbiological criteria for their previous good manufacturing practice. In this regard, it summarizes considerations and findings of a ''Consultation on Microbiological Criteria for Foods to be Further Processed Including by Irradiation'' held in 1989 by the International Consultative Group on Food irradiation at the Headquarters of the World Health Organization, Geneva. Difficulties in establishing reference values and defining good manufacturing practices will be pointed out. (orig.)

  8. Microbiological criteria for good manufacturing practice (GMP)

    Energy Technology Data Exchange (ETDEWEB)

    Farkas, J. (Inst. of Preservation and Livestock Products Technology, Univ. of Horticulture and Food Industry, Budapest (Hungary)); Zukal, E. (Inst. of Preservation and Livestock Products Technology, Univ. of Horticulture and Food Industry, Budapest (Hungary))

    1992-01-01

    Good manufacturing practice (GMP) consist of an effective manufacturing operation and an effective application of food control. GMP is best supported by the Hazard Analysis Critical Control Point system (HACCP) of the preventive quality assurance, which requires that food irradiation as any food processing technology should be used only with foods of an acceptable quality and adequate handling and storage procedures should precede and follow the processing. The paper concentrates on the first element of the HACCP system for an irradiation plant: the incoming product control, i.e. whether GMP of foods to be irradiated can be assessed by establishing microbiological criteria for their previous good manufacturing practice. In this regard, it summarizes considerations and findings of a ''Consultation on Microbiological Criteria for Foods to be Further Processed Including by Irradiation'' held in 1989 by the International Consultative Group on Food irradiation at the Headquarters of the World Health Organization, Geneva. Difficulties in establishing reference values and defining good manufacturing practices will be pointed out. (orig.)

  9. Microbiological criteria for good manufacturing practice (GMP)

    International Nuclear Information System (INIS)

    Farkas, J.; Zukal, E.

    1992-01-01

    Good manufacturing practice (GMP) consist of an effective manufacturing operation and an effective application of food control. GMP is best supported by the Hazard Analysis Critical Control Point system (HACCP) of the preventive quality assurance, which requires that food irradiation as any food processing technology should be used only with foods of an acceptable quality and adequate handling and storage procedures should precede and follow the processing. The paper concentrates on the first element of the HACCP system for an irradiation plant: the incoming product control, i.e. whether GMP of foods to be irradiated can be assessed by establishing microbiological criteria for their previous good manufacturing practice. In this regard, it summarizes considerations and findings of a ''Consultation on Microbiological Criteria for Foods to be Further Processed Including by Irradiation'' held in 1989 by the International Consultative Group on Food irradiation at the Headquarters of the World Health Organization, Geneva. Difficulties in establishing reference values and defining good manufacturing practices will be pointed out. (orig.) [de

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

    Science.gov (United States)

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

    2014-01-01

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

  11. About ATMPs, SOPs and GMP: The Hurdles to Produce Novel Skin Grafts for Clinical Use.

    Science.gov (United States)

    Hartmann-Fritsch, Fabienne; Marino, Daniela; Reichmann, Ernst

    2016-09-01

    The treatment of severe full-thickness skin defects represents a significant and common clinical problem worldwide. A bio-engineered autologous skin substitute would significantly reduce the problems observed with today's gold standard. Within 15 years of research, the Tissue Biology Research Unit of the University Children's Hospital Zurich has developed autologous tissue-engineered skin grafts based on collagen type I hydrogels. Those products are considered as advanced therapy medicinal products (ATMPs) and are routinely produced for clinical trials in a clean room facility following the guidelines for good manufacturing practice (GMP). This article focuses on hurdles observed for the translation of ATMPs from research into the GMP environment and clinical application. Personalized medicine in the field of rare diseases has great potential. However, ATMPs are mainly developed and promoted by academia, hospitals, and small companies, which face many obstacles such as high financial burdens.

  12. The Oxidative Stress Agent Hypochlorite Stimulates c-di-GMP Synthesis and Biofilm Formation in Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Nikola Strempel

    2017-11-01

    Full Text Available The opportunistic human pathogen Pseudomonas aeruginosa is able to survive under a variety of often harmful environmental conditions due to a multitude of intrinsic and adaptive resistance mechanisms, including biofilm formation as one important survival strategy. Here, we investigated the adaptation of P. aeruginosa PAO1 to hypochlorite (HClO, a phagocyte-derived host defense compound and frequently used disinfectant. In static biofilm assays, we observed a significant enhancement in initial cell attachment in the presence of sublethal HClO concentrations. Subsequent LC-MS analyses revealed a strong increase in cyclic-di-GMP (c-di-GMP levels suggesting a key role of this second messenger in HClO-induced biofilm development. Using DNA microarrays, we identified a 26-fold upregulation of ORF PA3177 coding for a putative diguanylate cyclase (DGC, which catalyzes the synthesis of the second messenger c-di-GMP – an important regulator of bacterial motility, sessility and persistence. This DGC PA3177 was further characterized in more detail demonstrating its impact on P. aeruginosa motility and biofilm formation. In addition, cell culture assays attested a role for PA3177 in the response of P. aeruginosa to human phagocytes. Using a subset of different mutants, we were able to show that both Pel and Psl exopolysaccharides are effectors in the PA3177-dependent c-di-GMP network.

  13. Magnesium Lithospermate B, an Active Extract of Salvia miltiorrhiza, Mediates sGC/cGMP/PKG Translocation in Experimental Vasospasm

    Directory of Open Access Journals (Sweden)

    Chih-Zen Chang

    2014-01-01

    Full Text Available Background. Soluble guanylyl cyclases (sGCs and Ras homolog gene family, member A (rhoA/Ras homolog gene family kinase(rho-kinase plays a role in vascular smooth muscle relaxation in subarachnoid hemorrhage (SAH. It is of interest to examine the effect of MLB on rhoA/ROCK and sGC/cGMP/PKG expression. Methods. A rodent SAH model was employed. Tissue samples were for sGCα1, sGCβ1, PKG, rhoA, ROCK (Western blot, and cGMP (ELISA measurement. Results. MLB morphologically improved convolution of the internal elastic lamina, distortion of endothelial wall, and necrosis of the smooth muscle in the SAH rats. Expressed cGMP, sGCα1, sGCβ1, and PKG in the SAH groups were reduced (P<0.01, and MLB precondition significantly induced cGMP, sGCα1, sGCβ1, and PKG. L-NAME reversed the vasodilation effect of MLB, reduced the bioexpression of PKG and cGMP (P<0.01, and tends to reduce sGCα1 level and induce rhoA, ROCK level in MLB precondition + SAH groups. Conclusion. These results demonstrate that sGC/cGMP/PKG and NO/ET pathways play pivotal roles in SAH-induced vasospasm. Through activating sGC/cGMP/PKG pathway and partially by inactivating rho-kinase in a NO-dependent mechanism, MLB shows promise to be an effective strategy for the treatment of this disease entity.

  14. BolA Is Required for the Accurate Regulation of c-di-GMP, a Central Player in Biofilm Formation.

    Science.gov (United States)

    Moreira, Ricardo N; Dressaire, Clémentine; Barahona, Susana; Galego, Lisete; Kaever, Volkhard; Jenal, Urs; Arraiano, Cecília M

    2017-09-19

    The bacterial second messenger cyclic dimeric GMP (c-di-GMP) is a nearly ubiquitous intracellular signaling molecule involved in the transition from the motile to the sessile/biofilm state in bacteria. C-di-GMP regulates various cellular processes, including biofilm formation, motility, and virulence. BolA is a transcription factor that promotes survival in different stresses and is also involved in biofilm formation. Both BolA and c-di-GMP participate in the regulation of motility mechanisms leading to similar phenotypes. Here, we establish the importance of the balance between these two factors for accurate regulation of the transition between the planktonic and sessile lifestyles. This balance is achieved by negative-feedback regulation of BolA and c-di-GMP. BolA not only contributes directly to the motility of bacteria but also regulates the expression of diguanylate cyclases and phosphodiesterases. This expression modulation influences the synthesis and degradation of c-di-GMP, while this signaling metabolite has a negative influence in bolA mRNA transcription. Finally, we present evidence of the dominant role of BolA in biofilm, showing that, even in the presence of elevated c-di-GMP levels, biofilm formation is reduced in the absence of BolA. C-di-GMP is one of the most important bacterial second messengers involved in several cellular processes, including virulence, cell cycle regulation, biofilm formation, and flagellar synthesis. In this study, we unravelled a direct connection between the bolA morphogene and the c-di-GMP signaling molecule. We show the important cross-talk that occurs between these two molecular regulators during the transition between the motile/planktonic and adhesive/sessile lifestyles in Escherichia coli This work provides important clues that can be helpful in the development of new strategies, and the results can be applied to other organisms with relevance for human health. IMPORTANCE Bacterial cells have evolved several

  15. Crystallization and preliminary X-ray analysis of the flagellar motor `brake' molecule YcgR with c-di-GMP from Escherichia coli.

    Science.gov (United States)

    Hou, Yanjie; Li, De Feng; Wang, Da Cheng

    2013-06-01

    In Escherichia coli and Salmonella enterica, bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a ubiquitous bacterial second-messenger molecule that participates in many cellular processes, can regulate flagellar motor speed and reduce cell swimming velocity by binding to the PilZ-containing protein YcgR. Here, the crystallization and preliminary X-ray crystallographic analysis of YcgR with c-di-GMP are reported. The crystals diffracted to 2.3 Å resolution and belonged to space group R3:H, with unit-cell parameters a = b = 93.96, c = 109.61 Å. The asymmetric unit appeared to contain one subunit with a Matthews coefficient of 3.21 Å(3) Da(-1). The results reported here provide a sound basis for solving the crystal structure of YcgR with c-di-GMP and revealing its structure-function relationship based on the three-dimensional structure.

  16. Activation of a cGMP-sensitive calcium-dependent chloride channel may cause transition from calcium waves to whole-cell oscillations in smooth muscle cells

    DEFF Research Database (Denmark)

    Jacobsen, Jens Christian; Aalkjær, Christian; Nilsson, Holger

    2007-01-01

    waves sweeping through the cytoplasm when the SR is stimulated to release calcium. A rise in cyclic guanosine monophosphate (cGMP) leads to the experimentally observed transition from waves to whole-cell calcium oscillations. At the same time membrane potential starts to oscillate and the frequency...... approximately doubles. In this transition, the simulated results point to a key role for a recently discovered cGMP-sensitive calcium-dependent chloride channel. This channel depolarizes the membrane in response to calcium released from the SR. In turn, depolarization causes uniform opening of L-type calcium...... onset of oscillations in membrane potential within the individual cell may underlie sudden intercellular synchronization and the appearance of vasomotion. Key words: Vasomotion, Chloride channel, cGMP, Mathematical model, Calcium waves....

  17. Effect of ionizing radiation on calcium and cyclic nucleotides metabolism in rats of different age

    International Nuclear Information System (INIS)

    Efimova, N.I.; Libenson, S.V.

    1982-01-01

    Some features of mechanism of calcium homeostasis and cyclic nucleotide exchange breakage in case of acute radiation injury of rats of various age were studied. It is established that calcium level in blood in nonpuberal animals, calcium and cAMP excretion with urine are minimal and reach maximum at puberal age. cGMP excretion with urine and concentrational levels of cAMP and cGMP in blood do not change with age. It is shown that calcium excretion with urine decreases adaptively in conditions of acute radiation injury in rats of all age groups. Maximal shifts in cAMP/cGMP ratio were noted in nonpuberal animals, whereas maximal adaptive-compensatory abilities in the regulation system of calcium homeostasis and cyclic nucleotides are typical to adolescent puberal animals

  18. Cyclic mononucleotides modulate potassium and calcium flux responses to H2O2 in Arabidopsis roots

    KAUST Repository

    Ordoñez, Natalia Maria

    2014-02-13

    Cyclic mononucleotides are messengers in plant stress responses. Here we show that hydrogen peroxide (H2O2) induces rapid net K+-efflux and Ca2+-influx in Arabidopsis roots. Pre-treatment with either 10 μM cAMP or cGMP for 1 or 24 h does significantly reduce net K+-leakage and Ca2+-influx, and in the case of the K+-fluxes, the cell permeant cyclic mononucleotides are more effective. We also examined the effect of 10 μM of the cell permeant 8-Br-cGMP on the Arabidopsis microsomal proteome and noted a specific increase in proteins with a role in stress responses and ion transport, suggesting that cGMP is sufficient to directly and/or indirectly induce complex adaptive changes to cellular stresses induced by H2O2. © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  19. Quality Risk Management: Putting GMP Controls First.

    Science.gov (United States)

    O'Donnell, Kevin; Greene, Anne; Zwitkovits, Michael; Calnan, Nuala

    2012-01-01

    This paper presents a practical way in which current approaches to quality risk management (QRM) may be improved, such that they better support qualification, validation programs, and change control proposals at manufacturing sites. The paper is focused on the treatment of good manufacturing practice (GMP) controls during QRM exercises. It specifically addresses why it is important to evaluate and classify such controls in terms of how they affect the severity, probability of occurrence, and detection ratings that may be assigned to potential failure modes or negative events. It also presents a QRM process that is designed to directly link the outputs of risk assessments and risk control activities with qualification and validation protocols in the GMP environment. This paper concerns the need for improvement in the use of risk-based principles and tools when working to ensure that the manufacturing processes used to produce medicines, and their related equipment, are appropriate. Manufacturing processes need to be validated (or proven) to demonstrate that they can produce a medicine of the required quality. The items of equipment used in such processes need to be qualified, in order to prove that they are fit for their intended use. Quality risk management (QRM) tools can be used to support such qualification and validation activities, but their use should be science-based and subject to as little subjectivity and uncertainty as possible. When changes are proposed to manufacturing processes, equipment, or related activities, they also need careful evaluation to ensure that any risks present are managed effectively. This paper presents a practical approach to how QRM may be improved so that it better supports qualification, validation programs, and change control proposals in a more scientific way. This improved approach is based on the treatment of what are called good manufacturing process (GMP) controls during those QRM exercises. A GMP control can be considered

  20. Specific GMP guidelines for radiopharmaceutical products

    International Nuclear Information System (INIS)

    2000-01-01

    These guidelines are intended to complement those provided in ''Good manufacturing practices for pharmaceutical products'', as well as the GMP for sterile pharmaceutical products. The regulatory procedures necessary to control radiopharmaceutical products are in large part determined by the sources of products and methods of manufacture. Manufacturing procedures within the scope of these guidelines include: preparation of radiopharmaceuticals in hospital radiopharmacies, preparation of radiopharmaceuticals in centralized radiopharmacies, production of radiopharmaceuticals in nuclear centres, institutes or industrial manufacturers, preparation and production of radiopharmaceuticals in Positron Emission Tomography (PET) centres

  1. Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression

    International Nuclear Information System (INIS)

    Nygaard, Gyrid; Herfindal, Lars; Kopperud, Reidun; Aragay, Anna M.; Holmsen, Holm; Døskeland, Stein Ove; Kleppe, Rune; Selheim, Frode

    2014-01-01

    Highlights: • We investigated the impact of cyclic nucleotide analogues on platelet activation. • Different time dependence were found for inhibition of platelet activation. • Additive effect was found using PKA- and PKG-activating analogues. • Our results may explain some of the discrepancies reported for cNMP signalling. - Abstract: In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation

  2. Cyclic mononucleotides modulate potassium and calcium flux responses to H2O2 in Arabidopsis roots

    KAUST Repository

    Ordoñ ez, Natalia Maria; Marondedze, Claudius; Thomas, Ludivine; Pasqualini, Stefania; Shabala, Lana; Shabala, Sergey; Gehring, Christoph A

    2014-01-01

    Cyclic mononucleotides are messengers in plant stress responses. Here we show that hydrogen peroxide (H2O2) induces rapid net K+-efflux and Ca2+-influx in Arabidopsis roots. Pre-treatment with either 10 μM cAMP or cGMP for 1 or 24 h does

  3. Decreased levels of guanosine 3', 5'-monophosphate (cGMP) in cerebrospinal fluid (CSF) are associated with cognitive decline and amyloid pathology in Alzheimer's disease.

    Science.gov (United States)

    Ugarte, Ana; Gil-Bea, Francisco; García-Barroso, Carolina; Cedazo-Minguez, Ángel; Ramírez, M Javier; Franco, Rafael; García-Osta, Ana; Oyarzabal, Julen; Cuadrado-Tejedor, Mar

    2015-06-01

    Levels of the cyclic nucleotides guanosine 3', 5'-monophosphate (cGMP) or adenosine 3', 5'-monophosphate (cAMP) that play important roles in memory processes are not characterized in Alzheimer's disease (AD). The aim of this study was to analyse the levels of these nucleotides in cerebrospinal fluid (CSF) samples from patients diagnosed with clinical and prodromal stages of AD and study the expression level of the enzymes that hydrolyzed them [phosphodiesterases (PDEs)] in the brain of AD patients vs. For cGMP and cAMP CSF analysis, the cohort (n = 79) included cognitively normal participants (subjective cognitive impairment), individuals with stable mild cognitive impairment or AD converters (sMCI and cMCI), and mild AD patients. A high throughput liquid chromatography-tandem mass spectrometry method was used. Interactions between CSF cGMP or cAMP with mini-mental state examination (MMSE) score, CSF Aβ(1-42) and CSF p-tau were analysed. For PDE4, 5, 9 and 10 expression analysis, brains of AD patients vs. controls (n = 7 and n = 8) were used. cGMP, and not cAMP levels, were significantly lower in the CSF of patients diagnosed with mild AD when compared with nondemented controls. CSF levels of cGMP showed a significant association with MMSE-diagnosed clinical dementia and with CSF biomarker Aβ42 in AD patients. Significant increase in PDE5 expression was detected in temporal cortex of AD patients compared with that of age-matched healthy control subjects. No changes in the expression of others PDEs were detected. These results support the potential involvement of cGMP in the pathological and clinical development of AD. The cGMP reduction in early stages of AD might participate in the aggravation of amyloid pathology and cognitive decline. © 2014 British Neuropathological Society.

  4. Cyclic multiverses

    Science.gov (United States)

    Marosek, Konrad; Dąbrowski, Mariusz P.; Balcerzak, Adam

    2016-09-01

    Using the idea of regularization of singularities due to the variability of the fundamental constants in cosmology we study the cyclic universe models. We find two models of oscillating and non-singular mass density and pressure (`non-singular' bounce) regularized by varying gravitational constant G despite the scale factor evolution is oscillating and having sharp turning points (`singular' bounce). Both violating (big-bang) and non-violating (phantom) null energy condition models appear. Then, we extend this idea on to the multiverse containing cyclic individual universes with either growing or decreasing entropy though leaving the net entropy constant. In order to get an insight into the key idea, we consider the doubleverse with the same geometrical evolution of the two `parallel' universes with their physical evolution [physical coupling constants c(t) and G(t)] being different. An interesting point is that there is a possibility to exchange the universes at the point of maximum expansion - the fact which was already noticed in quantum cosmology. Similar scenario is also possible within the framework of Brans-Dicke theory where varying G(t) is replaced by the dynamical Brans-Dicke field φ(t) though these theories are slightly different.

  5. Genetic Dissection of the Regulatory Network Associated with High C-di-GMP Levels in Pseudomonas putida KT2440

    Directory of Open Access Journals (Sweden)

    María Isabel Ramos-González

    2016-07-01

    Full Text Available Most bacteria grow in nature forming multicellular structures named biofilms. The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP is a key player in the regulation of the transition from planktonic to sessile lifestyles and this regulation is crucial in the development of biofilms. In Pseudomonas putida KT2440, Rup4959, a multidomain response regulator with diguanylate cyclase activity, when overexpressed causes an increment in the intracellular levels of c-di-GMP that gives rise to a pleiotropic phenotype consisting of increased biofilm formation and crinkly colony morphology. In a broad genomic screen we have isolated mutant derivatives that lose the crinkly morphology, designed as cfc (crinkle free colony. A total of nineteen different genes have been identified as being related with the emergence of the cfc phenotype either because the expression or functionality of Rup4959 is compromised, or due to a lack of transduction of the c-di-GMP signal to downstream elements involved in the acquisition of the phenotype. Discernment between these possibilities was investigated by using a c-di-GMP biosensor and by HPLC-MS quantification of the second messenger. Interestingly five of the identified genes encode proteins with AAA+ ATPase domain. Among the bacterial determinants found in this screen are the global transcriptional regulators GacA, AlgU and FleQ and two enzymes involved in the arginine biosynthesis pathway. We present evidences that this pathway seems to be an important element to both the availability of the free pool of the second messenger c-di-GMP and to its further transduction as a signal for biosynthesis of biopolimers. In addition we have identified an uncharacterized hybrid sensor histidine kinase whose phosphoaceptor conserved histidine residue has been shown in this work to be required for in vivo activation of the orphan response regulator Rup4959, which suggests these two elements constitute a two

  6. Involvement of NO-cGMP pathway in anti-hyperalgesic effect of PDE5 inhibitor tadalafil in experimental hyperalgesia.

    Science.gov (United States)

    Otari, K V; Upasani, C D

    2015-08-01

    The association of elevated level of cyclic guanosine monophosphate (cGMP) with inhibition of hyperalgesia and involvement of nitric oxide (NO)-cGMP pathway in the modulation of pain perception was previously reported. Phosphodiesterases 5 (PDE5) inhibitors, sildenafil and tadalafil (TAD) used in erectile dysfunction, are known to act via the NO-cGMP pathway. TAD exerts its action by increasing the levels of intracellular cGMP. Hence, the present study investigated the effect of TAD 5, 10, or 20 mg/kg, per os (p.o.) or L-NAME 20 mg/kg, intraperitoneally (i.p.) and TAD (20 mg/kg, p.o.) in carrageenan- and diabetes-induced hyperalgesia in rats using hot plate test at 55 ± 2 °C. In carrageenan- and diabetes-induced hyperalgesia, TAD (10 and 20 mg/kg, p.o.) significantly increased paw withdrawal latencies (PWLs) as compared to the control group. L-NAME significantly decreased PWLs as compared to the normal group and aggravated the hyperalgesia. Moreover, significant difference in PWLs of L-NAME and TAD 20 was evident. Co-administration of L-NAME (20 mg/kg) with TAD (20 mg/kg) showed significant difference in PWLs as compared to the TAD (20 mg/kg), indicating L-NAME reversed and antagonized TAD-induced anti-hyperalgesia. This suggested an important role of NO-cGMP pathway in TAD-induced anti-hyperalgesic effect.

  7. Regulation of Burkholderia cenocepacia biofilm formation by RpoN and the c-di-GMP effector BerB

    DEFF Research Database (Denmark)

    Fazli, Mustafa; Rybtke, Morten Levin; Steiner, Elisabeth

    2017-01-01

    Knowledge about the molecular mechanisms that are involved in the regulation of biofilm formation is essential for the development of biofilm-control measures. It is well established that the nucleotide second messenger cyclic diguanosine monophosphate (c-di-GMP) is a positive regulator of biofilm...... formation in many bacteria, but more knowledge about c-di-GMP effectors is needed. We provide evidence that c-di-GMP, the alternative sigma factor RpoN (σ54), and the enhancer-binding protein BerB play a role in biofilm formation of Burkholderia cenocepacia by regulating the production of a biofilm......-stabilizing exopolysaccharide. Our findings suggest that BerB binds c-di-GMP, and activates RpoN-dependent transcription of the berA gene coding for a c-di-GMP-responsive transcriptional regulator. An increased level of the BerA protein in turn induces the production of biofilm-stabilizing exopolysaccharide in response to high...

  8. The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation.

    Directory of Open Access Journals (Sweden)

    Kátia R L Schwarz

    Full Text Available This study aimed to determine the influence of cyclic guanosine 3'5'-monophosphate (cGMP and cGMP-dependent kinase (PKG during in vitro maturation (IVM on lipolysis-related parameters in bovine cumulus-oocyte complexes (COCs, and on embryo development and cryosurvival. COCs were matured with cGMP/PKG modulators and assessed for metaphase II rates (MII, cGMP levels, lipid content in oocytes (OO, transcript abundance for genes involved in lipolysis (ATGL and lipid droplets (PLIN2 in cumulus cells (CC and OO, and presence of phosphorylated (active hormone sensitive lipase (HSLser563 in OO. Embryo development, lipid contents and survival to vitrification were also assessed. Phosphodiesterase 5 inhibition (PDE5; cGMP-hydrolyzing enzyme with 10-5M sildenafil (SDF during 24 h IVM increased cGMP in COCs (56.9 vs 9.5 fMol/COC in untreated controls, p<0.05 and did not affect on maturation rate (84.3±6.4% MII. Fetal calf serum (FCS in IVM medium decreased cGMP in COCs compared to bovine serum albumin (BSA + SDF (19.6 vs 66.5 fMol/COC, respectively, p<0.05. FCS increased lipid content in OO (40.1 FI, p<0.05 compared to BSA (34.6 FI, while SDF decreased (29.8 and 29.6 FI, with BSA or FCS, respectively p<0.05. PKG inhibitor (KT5823 reversed this effect (38.9 FI, p<0.05. ATGL and PLIN2 transcripts were detected in CC and OO, but were affected by cGMP and PKG only in CC. HSLser563 was detected in OO matured with or without modulators. Reduced lipid content in embryos were observed only when SDF was added during IVM and IVC (27.6 FI compared to its use in either or none of the culture periods (34.2 FI, p<0.05. Survival to vitrification was unaffected by SDF. In conclusion, cGMP and PKG are involved in lipolysis in OO and possibly in CC and embryos; serum negatively affects this pathway, contributing to lipid accumulation, and cGMP modulation may reduce lipid contents in oocytes and embryos, but without improving embryo cryotolerance.

  9. Biofilm formation and antibiotic production in Ruegeria mobilis are influenced by intracellular concentrations of cyclic dimeric guanosinmonophosphate

    DEFF Research Database (Denmark)

    D'Alvise, Paul; Magdenoska, Olivera; Melchiorsen, Jette

    2014-01-01

    species Ruegeria mobilis are associated with intracellular concentrations of the signal compound cyclic dimeric guanosinmonophosphate (c-di-GMP), which in bacteria regulates transitions between motile and sessile life stages. Genes for diguanylate cyclases and phosphodiesterases, which are involved in c-di-GMP...... signalling, were found in the genome of R. mobilis strain F1926. Ion pair chromatography-tandem mass spectrometry revealed 20-fold higher c-di-GMP concentrations per cell in biofilm-containing cultures than in planktonic cells. An introduced diguanylate cyclase gene increased c-di-GMP and enhanced biofilm...... formation and production of the potent antibiotic tropodithietic acid (TDA). An introduced phosphodiesterase gene decreased c-di-GMP and reduced biofilm formation and TDA production. tdaC, a key gene for TDA biosynthesis, was expressed only in attached or biofilm-forming cells, and expression was induced...

  10. Supplementary Material for: The arabidopsis cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara; Meier, Stuart; Gehring, Christoph A

    2016-01-01

    Abstract Background Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals. Methods An affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling. Results A total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment. Conclusions We propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.

  11. Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens

    International Nuclear Information System (INIS)

    Amikam, D.; Benziman, M.

    1989-01-01

    The occurrence of the novel regulatory nucleotide bis(3',5')-cyclic diguanylic acid (c-di-GMP) and its relation to cellulose biogenesis in the plant pathogen Agrobacterium tumefaciens was studied. c-di-GMP was detected in acid extracts of 32 P-labeled cells grown in various media, and an enzyme responsible for its formation from GTP was found to be present in cell-free preparations. Cellulose synthesis in vivo was quantitatively assessed with [ 14 C]glucose as a tracer. The organism produced cellulose during growth in the absence of plant cells, and this capacity was retained in resting cells. Synthesis of a cellulosic product from UDP-glucose in vitro with membrane preparations was markedly stimulated by c-di-GMP and its precursor GTP and was further enhanced by Ca2+. The calcium effect was attributed to inhibition of a c-di-GMP-degrading enzyme shown to be present in the cellulose synthase-containing membranes

  12. Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy

    Directory of Open Access Journals (Sweden)

    Stylianos Michalakis

    2018-03-01

    Full Text Available The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP or cyclic adenosine monophosphate (cAMP. Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca2+ concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN channels and voltage-gated potassium channels (KCN. In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application.

  13. Synthesis and degradation of cyclic nucleotides in brain after a high dose of ionizing radiation

    International Nuclear Information System (INIS)

    Hunt, W.A.; Dalton, T.K.

    1981-01-01

    Previous data from our laboratory have indicated that a high dose of ionizing radiation can deplete the cyclic nucleotides guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) on several areas of the rat brain. cGMP is more sensitive to radiation than cAMP and does not recover for at least 24 h after irradiation. The response of cAMP is transient and recovery occurs within 4 h. The purpose of the present paper is to determine whether alternations in the activity of the synthetic and degradative enzymes that regulate cyclic nucleotide levels could account for the observed effects. Guanylate and adenylate cyclase and cGMP and cAMP phosphodiesterase activities were determined 10 min after irradiation with 10,000 rad of high-energy electrons. No alteration was detected under these experimental conditions. The data suggest that the reduction in cyclic nucleotides is not a direct effect on their metabolic enzymes and is probably secondary to some as yet-undefined action of radiation on the brain

  14. Spatiotemporal and functional characterisation of the Plasmodium falciparum cGMP-dependent protein kinase.

    Directory of Open Access Journals (Sweden)

    Christine S Hopp

    Full Text Available Signalling by 3'-5'-cyclic guanosine monophosphate (cGMP exists in virtually all eukaryotes. In the apicomplexan parasite Plasmodium, the cGMP-dependent protein kinase (PKG has previously been reported to play a critical role in four key stages of the life cycle. The Plasmodium falciparum isoform (PfPKG is essential for the initiation of gametogenesis and for blood stage schizont rupture and work on the orthologue from the rodent malaria parasite P. berghei (PbPKG has shown additional roles in ookinete differentiation and motility as well as liver stage schizont development. In the present study, PfPKG expression and subcellular location in asexual blood stages was investigated using transgenic epitope-tagged PfPKG-expressing P. falciparum parasites. In Western blotting experiments and immunofluorescence analysis (IFA, maximal PfPKG expression was detected at the late schizont stage. While IFA suggested a cytosolic location, a degree of overlap with markers of the endoplasmic reticulum (ER was found and subcellular fractionation showed some association with the peripheral membrane fraction. This broad localisation is consistent with the notion that PfPKG, as with the mammalian orthologue, has numerous cellular substrates. This idea is further supported by the global protein phosphorylation pattern of schizonts which was substantially changed following PfPKG inhibition, suggesting a complex role for PfPKG during schizogony.

  15. Investigation of the role of the NO-cGMP pathway on YC-1 and DEA/NO effects on thoracic aorta smooth muscle responses in a rat preeclampsia model.

    Science.gov (United States)

    Turgut, Nergiz Hacer; Temiz, Tijen Kaya; Turgut, Bülent; Karadas, Baris; Parlak, Mesut; Bagcivan, Ihsan

    2013-10-01

    The present study was designed to investigate the effects of YC-1, a nitric oxide (NO)-independent soluble guanylate cyclase (sGC) activator, and DEA/NO, a NO donor, on smooth muscle responses in the preeclampsia model with suramin-treated rats and on the levels of cyclic guanosine monophosphate (cGMP) of thoracic aorta rings isolated from term-pregnant rats. Rats of 2 groups, control group and suramin group, were given intraperitoneal injection of saline or suramin, respectively. Suramin injection caused increased blood pressure, protein in urine, and fetal growth retardation. Thoracic aorta rings were exposed to contractile and relaxant agents. KCl contraction and papaverine relaxation responses were similar. Relaxation responses of YC-1 and DEA/NO decreased in suramin group. In both groups in the presence of ODQ, a sGC inhibitor, the relaxation responses of YC-1 and DEA/NO decreased. The cGMP content was determined by radioimmunoassay technique. The content of cGMP in the suramin group decreased. In the presence of YC-1 and DEA/NO in both groups, cGMP content increased, but in ODQ-added groups, there was a significant decrease. We conclude that in preeclampsia, the decrease of relaxation responses and the decrease of cGMP content could be due to the reduction in stimulation of sGC and the decrease in cGMP levels.

  16. Implementation of Good Maufacturing Practices (GMP) in the Kitchen Hospital

    OpenAIRE

    Sari, Fitria Novita

    2016-01-01

    Abstract: Food safety is one of the important thing in public health improvement in Indonesia. Hospitals are required to keep food safety for patients by conducting the principle Good Manufacturing Practices (GMP). The purpose of this research to -identify the application of GMP in Installation Nutrition Hospital. Design of this study was using descriptive research in observational method with cross sectional design. Variables the treatment were the physical building, utility, equipment, stor...

  17. Ethanol extract of seeds of Oenothera odorata induces vasorelaxation via endothelium-dependent NO-cGMP signaling through activation of Akt-eNOS-sGC pathway.

    Science.gov (United States)

    Kim, Hye Yoom; Oh, Hyuncheol; Li, Xiang; Cho, Kyung Woo; Kang, Dae Gill; Lee, Ho Sub

    2011-01-27

    The vasorelaxant effect of ethanol extract of seeds of Oenothera odorata (Onagraceae) (one species of evening primroses) (ESOO) and its mechanisms involved were defined. Changes in vascular tension, guanosine 3',5'-cyclic monophosphate (cGMP) levels, and Akt expression were measured in carotid arterial rings from rats. Seeds of Oenothera odorata were extracted with ethanol (94%) and the extract was filtered, concentrated and stored at -70°C. ESOO relaxed endothelium-intact, but not endothelium-denuded, carotid arterial rings in a concentration-dependent manner. Similarly, ESOO increased cGMP levels of the carotid arterial rings. Pretreatment of endothelium-intact arterial rings with L-NAME, an inhibitor of nitric oxide synthase (NOS), or ODQ, an inhibitor of soluble guanylyl cyclase (sGC), blocked the ESOO-induced vasorelaxation and increase in cGMP levels. Nominally Ca(2+)-free but not L-typed Ca(2+) channel inhibition attenuated the ESOO-induced vasorelaxation. Thapsigargin, Gd(3+), and 2-aminoethyl diphenylborinate, modulators of store-operated Ca(2+) entry (SOCE), significantly attenuated the ESOO-induced vasorelaxation and increase in cGMP levels. Further, wortmannin, an inhibitor of Akt, attenuated the ESOO-induced vasorelaxation and increases in cGMP levels and phosphorylated Akt2 expression. K(+) channel blockade with TEA, 4-aminopyridine, and glibenclamide attenuated the ESOO-induced vascular relaxation. Taken together, the present study demonstrates that ESOO relaxes vascular smooth muscle via endothelium-dependent NO-cGMP signaling through activation of the Akt-eNOS-sGC pathway. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  18. The effect of ultraviolet light on the cyclic nucleotide system of human fibroblasts

    International Nuclear Information System (INIS)

    Fertel, R.H.; Tejwani, G.A.; Albrightson, C.R.; Hart, R.W.

    1981-01-01

    The concentrations of cyclic AMP and cyclic GMP in in human skin fibroblasts in culture were determined after exposing the cells to varying fluences of UV (254 nm) light. The cyclic nucleotide concentrations of cells irradiated in the log phase of growth were unchanged relative to controls. In contrast, there was a rise in the concentration of cyclic AMP in cells irradiated after they reached confluency. The increase in concentration was observed as early as 30 min after irradiation, reached a maximum of about 200% of control at 4 to 6 h after exposure, and returned to control values by 24 h after irradiation. The effect was proportional to a UV fluence from 5 to 20 J/m 2 , and was blocked by the addition of the UV absorbing agent para-aminobenzoic acid. In contrast, the results indicated that UV light had no effect on the concentration of cyclic GMP in human fibroblast cell cultures. Because of the importance of cyclic nucleotides in the regulation of cellular function, it is reasonable to hypothesize that changes in cyclic AMP induced by UV light may effect the extranuclear functions of irradiated cells. (author)

  19. Beneficial effects of combined benazepril-amlodipine on cardiac nitric oxide, cGMP, and TNF-alpha production after cardiac ischemia.

    Science.gov (United States)

    Siragy, Helmy M; Xue, Chun; Webb, Randy L

    2006-05-01

    The aim of this study was to determine if myocardial inflammation is increased after myocardial ischemia and whether angiotensin-converting enzyme inhibitors, calcium channel blockers, or diuretics decrease mediators of inflammation in rats with induced myocardial ischemia. Changes in cardiac interstitial fluid (CIF) levels of nitric oxide metabolites (NOX), cyclic guanosine 3',5'-monophosphate (cGMP), angiotensin II (Ang II), and tumor necrosis factor-alpha (TNF-alpha) were monitored with/without oral administration of benazepril, amlodipine, combined benazepril-amlodipine, or hydrochlorothiazide. Using a microdialysis technique, levels of several mediators of inflammation were measured after sham operation or 30-minute occlusion of the left anterior descending coronary artery. Compared with sham animals, levels of CIF NOX and cGMP were decreased in animals with ischemia (P Benazepril or amlodipine significantly increased NOX levels (P benazepril significantly increased cGMP (P benazepril-amlodipine further increased CIF NOX and cGMP (P benazepril alone, or combined benazepril-amlodipine significantly reduced TNF-alpha (P benazepril-amlodipine may be beneficial for managing cardiac ischemia.

  20. Analysis of proton wires in the enzyme active site suggests a mechanism of c-di-GMP hydrolysis by the EAL domain phosphodiesterases.

    Science.gov (United States)

    Grigorenko, Bella L; Knyazeva, Marina A; Nemukhin, Alexander V

    2016-11-01

    We report for the first time a hydrolysis mechanism of the cyclic dimeric guanosine monophosphate (c-di-GMP) by the EAL domain phosphodiesterases as revealed by molecular simulations. A model system for the enzyme-substrate complex was prepared on the base of the crystal structure of the EAL domain from the BlrP1 protein complexed with c-di-GMP. The nucleophilic hydroxide generated from the bridging water molecule appeared in a favorable position for attack on the phosphorus atom of c-di-GMP. The most difficult task was to find a pathway for a proton transfer to the O3' atom of c-di-GMP to promote the O3'P bond cleavage. We show that the hydrogen bond network extended over the chain of water molecules in the enzyme active site and the Glu359 and Asp303 side chains provides the relevant proton wires. The suggested mechanism is consistent with the structural, mutagenesis, and kinetic experimental studies on the EAL domain phosphodiesterases. Proteins 2016; 84:1670-1680. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. Ribozyme Activity of RNA Nonenzymatically Polymerized from 3 ',5 '-Cyclic GMP

    Czech Academy of Sciences Publication Activity Database

    Pino, S.; Costanzo, G.; Giorgi, A.; Šponer, Jiří; Šponer, Judit E.; Di Mauro, E.

    2013-01-01

    Roč. 15, č. 12 (2013), s. 5362-5383 ISSN 1099-4300 R&D Projects: GA ČR(CZ) GAP208/10/2302; GA MŠk(CZ) ED1.1.00/02.0068 Institutional support: RVO:68081707 Keywords : RNA polymerization * RNA origin * ribozymes Subject RIV: BO - Biophysics Impact factor: 1.564, year: 2013

  2. Topoisomerase 1 Inhibition Promotes Cyclic GMP-AMP Synthase-Dependent Antiviral Responses

    OpenAIRE

    Pépin, Geneviève; Nejad, Charlotte; Ferrand, Jonathan; Thomas, Belinda J.; Stunden, H. James; Sanij, Elaine; Foo, Chwan-Hong; Stewart, Cameron R.; Cain, Jason E.; Bardin, Philip G.; Williams, Bryan R. G.; Gantier, Michael P.

    2017-01-01

    ABSTRACT Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1) by low-dose camptothecin (CPT) can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through c...

  3. Fluorescence-Based Reporter for Gauging Cyclic Di-GMP Levels in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Rybtke, Morten T.; Borlee, Bradley R.; Murakami, Keiji

    2012-01-01

    The increased tolerance toward the host immune system and antibiotics displayed by biofilm-forming Pseudomonas aeruginosa and other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the dev...

  4. Host Immune Response to Bacterial Cyclic Diguanylic Acid (c-di-GMP)

    Science.gov (United States)

    2009-07-01

    charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work...thymidine incorporation by an automatic beta counter. Tests were per- formed in triplicates, and results were expressed as the mean cpm . Flow cytometry...thymidine was added and allowed to incubate for an additional 18 h. T cell proliferation was measured by [3H]thymidine uptake ( cpm ). , p 0.01, as

  5. The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling

    KAUST Repository

    Wheeler, Janet I.

    2017-05-08

    The brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) is a member of the leucine rich repeat receptor like kinase family. The intracellular kinase domain of BRI1 is an active kinase and also encapsulates a guanylate cyclase catalytic centre. Using liquid chromatography tandem mass spectrometry, we confirmed that the recombinant cytoplasmic domain of BRI1 generates pmol amounts of cGMP per μg protein with a preference for magnesium over manganese as a co-factor. Importantly, a functional BRI1 kinase is essential for optimal cGMP generation. Therefore, the guanylate cyclase activity of BRI1 is modulated by the kinase while cGMP, the product of the guanylate cyclase, in turn inhibits BRI1 kinase activity. Furthermore, we show using Arabidopsis root cell cultures that cGMP rapidly potentiates phosphorylation of the downstream substrate BRASSINOSTEROID SIGNALING KINASE 1 (BSK1). Taken together, our results suggest that cGMP acts as a modulator that enhances downstream signaling while dampening signal generation from the receptor. This article is protected by copyright. All rights reserved.

  6. The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling

    KAUST Repository

    Wheeler, Janet I.; Wong, Aloysius Tze; Marondedze, Claudius; Groen, Arnoud J.; Kwezi, Lusisizwe; Freihat, Lubna; Vyas, Jignesh; Raji, Misjudeen; Irving, Helen R.; Gehring, Christoph A

    2017-01-01

    The brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) is a member of the leucine rich repeat receptor like kinase family. The intracellular kinase domain of BRI1 is an active kinase and also encapsulates a guanylate cyclase catalytic centre. Using liquid chromatography tandem mass spectrometry, we confirmed that the recombinant cytoplasmic domain of BRI1 generates pmol amounts of cGMP per μg protein with a preference for magnesium over manganese as a co-factor. Importantly, a functional BRI1 kinase is essential for optimal cGMP generation. Therefore, the guanylate cyclase activity of BRI1 is modulated by the kinase while cGMP, the product of the guanylate cyclase, in turn inhibits BRI1 kinase activity. Furthermore, we show using Arabidopsis root cell cultures that cGMP rapidly potentiates phosphorylation of the downstream substrate BRASSINOSTEROID SIGNALING KINASE 1 (BSK1). Taken together, our results suggest that cGMP acts as a modulator that enhances downstream signaling while dampening signal generation from the receptor. This article is protected by copyright. All rights reserved.

  7. Tissue

    Directory of Open Access Journals (Sweden)

    David Morrissey

    2012-01-01

    Full Text Available Purpose. In vivo gene therapy directed at tissues of mesenchymal origin could potentially augment healing. We aimed to assess the duration and magnitude of transene expression in vivo in mice and ex vivo in human tissues. Methods. Using bioluminescence imaging, plasmid and adenoviral vector-based transgene expression in murine quadriceps in vivo was examined. Temporal control was assessed using a doxycycline-inducible system. An ex vivo model was developed and optimised using murine tissue, and applied in ex vivo human tissue. Results. In vivo plasmid-based transgene expression did not silence in murine muscle, unlike in liver. Although maximum luciferase expression was higher in muscle with adenoviral delivery compared with plasmid, expression reduced over time. The inducible promoter cassette successfully regulated gene expression with maximum levels a factor of 11 greater than baseline. Expression was re-induced to a similar level on a temporal basis. Luciferase expression was readily detected ex vivo in human muscle and tendon. Conclusions. Plasmid constructs resulted in long-term in vivo gene expression in skeletal muscle, in a controllable fashion utilising an inducible promoter in combination with oral agents. Successful plasmid gene transfection in human ex vivo mesenchymal tissue was demonstrated for the first time.

  8. The GDP-switched GAF domain of DcpA modulates the concerted synthesis/hydrolysis of c-di-GMP in Mycobacterium smegmatis.

    Science.gov (United States)

    Chen, Hui-Jie; Li, Na; Luo, Ye; Jiang, Yong-Liang; Zhou, Cong-Zhao; Chen, Yuxing; Li, Qiong

    2018-04-09

    The second messenger c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate] plays a key role in bacterial growth, survival and pathogenesis, and thus its intracellular homeostasis should be finely maintained. Mycobacterium smegmatis encodes a GAF (mammalian c G MP-regulated phosphodiesterases, Anabaena a denylyl cyclases and Escherichia coli transcription activator F hlA) domain containing bifunctional enzyme DcpA ( d iguanylate c yclase and p hosphodiesterase A ) that catalyzes the synthesis and hydrolysis of c-di-GMP . Here, we found that M. smegmatis DcpA catalyzes the hydrolysis of c-di-GMP at a higher velocity, compared with synthetic activity, resulting in a sum reaction from the ultimate substrate GTP to the final product pGpG [5'-phosphoguanylyl-(3'-5')-guanosine]. Fusion with the N-terminal GAF domain enables the GGDEF (Gly-Gly-Asp-Glu-Phe) domain of DcpA to dimerize and accordingly gain synthetic activity. Screening of putative metabolites revealed that GDP is the ligand of the GAF domain. Binding of GDP to the GAF domain down-regulates synthetic activity, but up-regulates hydrolytic activity, which, in consequence, might enable a timely response to the transient accumulation of c-di-GMP at the stationary phase or under stresses. Combined with the crystal structure of the EAL (Glu-Ala-Leu) domain and the small-angle X-ray scattering data, we propose a putative regulatory model of the GAF domain finely tuned by the intracellular GTP/GDP ratio. These findings help us to better understand the concerted control of the synthesis and hydrolysis of c-di-GMP in M. smegmatis in various microenvironments. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  9. Structural and functional characteristics of cGMP-dependent methionine oxidation in Arabidopsis thaliana proteins

    KAUST Repository

    Marondedze, Claudius

    2013-01-05

    Background: Increasing structural and biochemical evidence suggests that post-translational methionine oxidation of proteins is not just a result of cellular damage but may provide the cell with information on the cellular oxidative status. In addition, oxidation of methionine residues in key regulatory proteins, such as calmodulin, does influence cellular homeostasis. Previous findings also indicate that oxidation of methionine residues in signaling molecules may have a role in stress responses since these specific structural modifications can in turn change biological activities of proteins. Findings. Here we use tandem mass spectrometry-based proteomics to show that treatment of Arabidopsis thaliana cells with a non-oxidative signaling molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by cGMP-dependent methionine oxidation is functionally enriched for stress response proteins. Furthermore, we also noted distinct signatures in the frequency of amino acids flanking oxidised and un-oxidised methionine residues on both the C- and N-terminus. Conclusions: Given both a structural and functional bias in methionine oxidation events in response to a signaling molecule, we propose that these are indicative of a specific role of such post-translational modifications in the direct or indirect regulation of cellular responses. The mechanisms that determine the specificity of the modifications remain to be elucidated. 2013 Marondedze et al.; licensee BioMed Central Ltd.

  10. Short-term dehydroepiandrosterone treatment increases platelet cGMP production in elderly male subjects.

    Science.gov (United States)

    Martina, Valentino; Benso, Andrea; Gigliardi, Valentina Ramella; Masha, Andi; Origlia, Carla; Granata, Riccarda; Ghigo, Ezio

    2006-03-01

    Several clinical and population-based studies suggest that dehydroepiandrosterone (DHEA) and its sulphate (DHEA-S) play a protective role against atherosclerosis and coronary artery disease in human. However, the mechanisms underlying this action are still unknown. It has recently been suggested that DHEA-S could delay atheroma formation through an increase in nitric oxide (NO) production. Twenty-four aged male subjects [age (mean +/- SEM): 65.4 +/- 0.7 year; range: 58.2-67.6 years] underwent a blinded placebo controlled study receiving DHEA (50 mg p.o. daily at bedtime) or placebo for 2 months. Platelet cyclic guanosine-monophosphate (cGMP) concentration (as marker of NO production) and serum levels of DHEA-S, DHEA, IGF-I, insulin, glucose, oestradiol (E(2)), testosterone, plasminogen activator inhibitor (PAI)-1 antigen (PAI-1 Ag), homocysteine and lipid profile were evaluated before and after the 2-month treatment with DHEA or placebo. At the baseline, all variables in the two groups were overlapping. All parameters were unchanged after treatment with placebo. Conversely, treatment with DHEA (a) increased (P < 0.001 vs. baseline) platelet cGMP (111.9 +/- 7.1 vs. 50.1 +/- 4.1 fmol/10(6) plts), DHEA-S (13.6 +/- 0.8 vs. 3.0 +/- 0.3 micromol/l), DHEA (23.6 +/- 1.7 vs. 15.3 +/- 1.4 nmol/l), testosterone (23.6 +/- 1.0 vs. 17.7 +/- 1.0 nmol/l) and E(2) (72.0 +/- 5.0 vs. 60.0 +/- 4.0 pmol/l); and (b) decreased (P < 0.05 vs. baseline) PAI-1 Ag (27.4 +/- 3.8 vs. 21.5 +/- 2.5 ng/ml) and low-density lipoprotein (LDL) cholesterol (3.4 +/- 0.2 vs. 3.0 +/- 0.2 mmol/l). IGF-I, insulin, glucose, triglycerides, total cholesterol, HDL cholesterol, HDL2 cholesterol, HDL3 cholesterol, apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB) and homocysteine levels were not modified by DHEA treatment. This study shows that short-term treatment with DHEA increased platelet cGMP production, a marker of NO production, in healthy elderly subjects. This effect is coupled with a decrease in PAI-1

  11. cGMP signalling : different ways to create a pathway

    NARCIS (Netherlands)

    Roelofs, Jeroen; Smith, Janet L.; Haastert, Peter J.M. van

    Recently, a novel cGMP signalling cascade was uncovered in Dictyostelium, a eukaryote that diverged from the lineage leading to metazoa after plants and before yeast. In both Dictyostelium and metazoa, the ancient cAMP-binding (cNB) motif of bacterial CAP has been modified and assembled with other

  12. Procedures development and methodology of control for application of good manufacture practices (GMP) on human blood irradiation

    International Nuclear Information System (INIS)

    Boghi, Claudio

    2008-01-01

    The irradiation of human blood is used to avoid the TA-GVHD (transfusion-associated graft-versus-host-disease), a rare but devastating adverse effect of leukocytes present in blood components for a immunocompetent transfusion recipients. Usually this irradiation practice is performed to a physical elimination of leukocytes. The implementation of the procedures will assure that the properly dose in a range of 25 Gy to 50 Gy will be delivered to the blood in the bag collected in a blood tissue bank. The studies of the procedures in order to establish a GMP (Good Manufacturing Practices) were developed under the guidelines of the standard ISO 11137 - Sterilization of health care products - Requirements for validation and routine control - Radiation sterilization. In this work, two dosimetric systems were used for dose mapping during the studies of irradiator qualification, loading pattern, irradiation process validation and auditing. The CaS0 4 :Dy dosimeter presented difficulties concerning to uncertainty on dose measurement, stability, traceability and calibration. The PMMA and Gafchromic dosimetric systems have shown a better performance and were adopted on studies of irradiators qualification that are necessary to implementation of GMP. The irradiation tests have been done in a Gammacell 220 irradiator. The developed procedures can be adapted for different kinds of gamma irradiators, allowing implanting a quality assurance program and a GMP for blood irradiation. (author)

  13. Brain-natriuretic peptide and cyclic guanosine monophosphate as biomarkers of myxomatous mitral valve disease in dogs

    DEFF Research Database (Denmark)

    Moesgaard, Sophia Gry; Falk, Bo Torkel; Teerlink, Tom

    2011-01-01

    Elevations in the plasma concentrations of natriuretic peptides correlate with increased severity of myxomatous mitral valve disease (MMVD) in dogs. This study correlates the severity of MMVD with the plasma concentrations of the biomarkers N-terminal fragment of the pro-brain-natriuretic peptide...... (NT-proBNP) and its second messenger, cyclic guanosine monophosphate (cGMP). Furthermore, the l-arginine:asymmetric dimethylarginine (ADMA) ratio was measured as an index of nitric oxide availability. The study included 75 dogs sub-divided into five groups based on severity of MMVD as assessed...... by clinical examination and echocardiography. Plasma NT-proBNP and cGMP concentrations increased with increasing valve dysfunction and were significantly elevated in dogs with heart failure. The cGMP:NT-proBNP ratio decreased significantly in dogs with heart failure, suggesting the development of natriuretic...

  14. A conjugate of decyltriphenylphosphonium with plastoquinone can carry cyclic adenosine monophosphate, but not cyclic guanosine monophosphate, across artificial and natural membranes.

    Science.gov (United States)

    Firsov, Alexander M; Rybalkina, Irina G; Kotova, Elena A; Rokitskaya, Tatyana I; Tashlitsky, Vadim N; Korshunova, Galina A; Rybalkin, Sergei D; Antonenko, Yuri N

    2018-02-01

    The present study demonstrated for the first time the interaction between adenosine 3',5'-cyclic monophosphate (cAMP), one of the most important signaling compounds in living organisms, and the mitochondria-targeted antioxidant plastoquinonyl-decyltriphenylphosphonium (SkQ1). The data obtained on model liquid membranes and human platelets revealed the ability of SkQ1 to selectively transport cAMP, but not guanosine 3',5'-cyclic monophosphate (cGMP), across both artificial and natural membranes. In particular, SkQ1 elicited translocation of cAMP from the source to the receiving phase of a Pressman-type cell, while showing low activity with cGMP. Importantly, only conjugate with plastoquinone, but not dodecyl-triphenylphosphonium, was effective in carrying cAMP. In human platelets, SkQ1 also appeared to serve as a carrier of cAMP, but not cGMP, from outside to inside the cell, as measured by phosphorylation of the vasodilator stimulated phosphoprotein. The SkQ1-induced transfer of cAMP across the plasma membrane found here can be tentatively suggested to interfere with cAMP signaling pathways in living cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Hfq-dependent, co-ordinate control of cyclic diguanylate synthesis and catabolism in the plague pathogen Yersinia pestis.

    Science.gov (United States)

    Bellows, Lauren E; Koestler, Benjamin J; Karaba, Sara M; Waters, Christopher M; Lathem, Wyndham W

    2012-11-01

    Yersinia pestis, the cause of the disease plague, forms biofilms to enhance flea-to-mammal transmission. Biofilm formation is dependent on exopolysaccharide synthesis and is controlled by the intracellular levels of the second messenger molecule cyclic diguanylate (c-di-GMP), but the mechanisms by which Y. pestis regulates c-di-GMP synthesis and turnover are not fully understood. Here we show that the small RNA chaperone Hfq contributes to the regulation of c-di-GMP levels and biofilm formation by modulating the abundance of both the c-di-GMP phosphodiesterase HmsP and the diguanylate cyclase HmsT. To do so, Hfq co-ordinately promotes hmsP mRNA accumulation while simultaneously decreasing the stability of the hmsT transcript. Hfq-dependent regulation of HmsP occurs at the transcriptional level while the regulation of HmsT is post-transcriptional and is localized to the 5' untranslated region/proximal coding sequence of the hmsT transcript. Decoupling HmsP from Hfq-based regulation is sufficient to overcome the effects of Δhfq on c-di-GMP and biofilm formation. We propose that Y. pestis utilizes Hfq to link c-di-GMP levels to environmental conditions and that the disregulation of c-di-GMP turnover in the absence of Hfq may contribute to the severe attenuation of Y. pestis lacking this RNA chaperone in animal models of plague. © 2012 Blackwell Publishing Ltd.

  16. Design of GMP compliance radiopharmaceutical production facility in MINT

    International Nuclear Information System (INIS)

    Anwar Abd Rahman; Shaharum Ramli; M Rizal Mamat Ibrahim; Rosli Darmawan; Yusof Azuddin Ali; Jusnan Hashim

    2005-01-01

    In 1985, MINT built the only radiopharmaceutical production facility in Malaysia. The facility was designed based on IAEA (International Atomic Energy Agency) standard guidelines which provide radiation safety to the staff and the surrounding environment from radioactive contamination. Since 1999, BPFK (Biro Pengawalan Farmaseutikal Kebangsaan) has used the guidelines from Pharmaceutical Inspection Convention Scheme (PICS) to meet the requirements of the Good Manufacturing Practice (GMP) for Pharmaceutical Products. In the guidelines, the pharmaceutical production facility shall be designed based on clean room environment. In order to design a radiopharmaceutical production facility, it is important to combine the concept of radiation safety and clean room to ensure that both requirements from GMP and IAEA are met. The design requirement is necessary to set up a complete radiopharmaceutical production facility, which is safe, has high production quality and complies with the Malaysian and International standards. (Author)

  17. Specificity of Good Manufacturing Practice (GMP) for Biomedical Cell Products.

    Science.gov (United States)

    Tulina, M A; Pyatigorskaya, N V

    2018-03-01

    The article describes special aspects of Good Manufacturing Practice (GMP) for biomedical cell products (BMCP) that imply high standards of aseptics throughout the entire productio process, strict requirements to donors and to the procedure of biomaterial isolation, guaranty of tracing BMCP products, defining processing procedures which allow to identify BMCP as minimally manipulated; continuous quality control and automation of the control process at all stages of manufacturing, which will ensure product release simultaneously with completion of technological operations.

  18. Cooperative DNA binding of heterologous proteins: Evidence for contact between the cyclic AMP receptor protein and RNA polymerase

    International Nuclear Information System (INIS)

    Ren, Y.L.; Garges, S.; Adhya, S.; Krakow, J.S.

    1988-01-01

    Four cAMP-independent receptor protein mutants (designated CRP* mutants) isolated previously are able to activate in vivo gene transcription in the absence of cAMP and their activity can be enhanced by cAMP or cGMP. One of the four mutant proteins, CRP*598 (Arg-142 to His, Ala-144 to Thr), has been characterized with regard to its conformational properties and ability to bind to and support abortive initiation from the lac promoter. Binding of wild-type CRP to its site on the lac promoter and activation of abortive initiation by RNA polymerase on this promoter are effected by cAMP but not by cGMP. CRP*598 can activate lacP + -directed abortive initiation in the presence of cAMP and less efficiently in the presence of cGMP or in the absence of cyclic nucleotide. DNase I protection (footprinting) indicates that cAMP-CRP* binds to its site on the lac promoter whereas unliganded CRP* and cGMP-CRP* form a stable complex with the [ 32 P]lacP + fragment only in the presence of RNA polymerase, showing cooperative binding of two heterologous proteins. This cooperative binding provides strong evidence for a contact between CRP and RNA polymerase for activation of transcription. Although cGMP binds to CRP, it cannot replace cAMP in effecting the requisite conformational transition necessary for site-specific promoter binding

  19. Effect of radioprotectant WR 2721 on cyclic nucleotides, prostaglandins, and lysosomes

    International Nuclear Information System (INIS)

    Trocha, P.J.; Catravas, G.N.

    1983-01-01

    Within 1 hr after ip injection of the radioprotectant WR 2721 into rats, splenic cGMP levels dropped and remained suppressed for 6 hr before returning to normal. However, if rats were exposed to ionizing radiation 30-40 min after WR 2721 treatment, they had higher cGMP levels at 3 hr postirradiation than the nonirradiUted, drug-treated controls, but the cGMP content was still found to be lower than that of the irradiated nondrug-treated controls. Radiation exposure of animals pretreated with WR 2721 also resulted in higher liver and spleen levels of cAMP and additional elevations in spleen prostaglandin content, compared with irradiated controls at 3-6 hr after radiation treatment. The secondary fluctuations of lysosomal enzyme activities, prostaglandin content, and cyclic nucleotide levels were also altered in irradiated rats pretreated with WR 2721 when compared with irradiated controls. Liver and spleen lysosomal β-glucuronidase activities, spleen cAMP and cGMP levels, and spleen prostaglandin concentrations were closer to physiological levels at 3 days postirradiation in rats given WR 2721 before the radiation treatment

  20. Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling

    KAUST Repository

    Gehring, Christoph A.

    2017-10-04

    The cyclic nucleotide monophosphates (cNMPs), and notably 3′,5′-cyclic guanosine monophosphate (cGMP) and 3′,5′-cyclic adenosine monophosphate (cAMP) are now accepted as key signaling molecules in many processes in plants including growth and differentiation, photosynthesis, and biotic and abiotic defense. At the single molecule level, we are now beginning to understand how cNMPs modify specific target molecules such as cyclic nucleotide-gated channels, while at the systems level, a recent study of the Arabidopsis cNMP interactome has identified novel target molecules with specific cNMP-binding domains. A major advance came with the discovery and characterization of a steadily increasing number of guanylate cyclases (GCs) and adenylate cyclases (ACs). Several of the GCs are receptor kinases and include the brassinosteroid receptor, the phytosulfokine receptor, the Pep receptor, the plant natriuretic peptide receptor as well as a nitric oxide sensor. We foresee that in the near future many more molecular mechanisms and biological roles of GCs and ACs and their catalytic products will be discovered and further establish cNMPs as a key component of plant responses to the environment.

  1. Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling

    KAUST Repository

    Gehring, Christoph A; Turek, Ilona S.

    2017-01-01

    The cyclic nucleotide monophosphates (cNMPs), and notably 3′,5′-cyclic guanosine monophosphate (cGMP) and 3′,5′-cyclic adenosine monophosphate (cAMP) are now accepted as key signaling molecules in many processes in plants including growth and differentiation, photosynthesis, and biotic and abiotic defense. At the single molecule level, we are now beginning to understand how cNMPs modify specific target molecules such as cyclic nucleotide-gated channels, while at the systems level, a recent study of the Arabidopsis cNMP interactome has identified novel target molecules with specific cNMP-binding domains. A major advance came with the discovery and characterization of a steadily increasing number of guanylate cyclases (GCs) and adenylate cyclases (ACs). Several of the GCs are receptor kinases and include the brassinosteroid receptor, the phytosulfokine receptor, the Pep receptor, the plant natriuretic peptide receptor as well as a nitric oxide sensor. We foresee that in the near future many more molecular mechanisms and biological roles of GCs and ACs and their catalytic products will be discovered and further establish cNMPs as a key component of plant responses to the environment.

  2. The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling

    CSIR Research Space (South Africa)

    Wheeler, J

    2017-06-01

    Full Text Available ) with the ll PickUp Injection mode using the loading pump at 15 ll min�1 flow rate for 3 min. Samples were then loaded on a RSLC, 75 lm 9 500 mm, nanoVi- per, C18, 2 lm, 100 �A column (Acclaim, PepMap) retrofitted to an EASY-spray source with a flow rate of 300... receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling Janet I. Wheeler1,2,†, Aloysius Wong3,4, Claudius Marondedze3,5, Arnoud J. Groen5, Lusisizwe Kwezi1,6, Lubna Freihat1, Jignesh Vyas1, Misjudeen A. Raji7, Helen R. Irving1...

  3. Differential Regulation of cGMP Signaling in Human Melanoma Cells at Altered Gravity: Simulated Microgravity Down-Regulates Cancer-Related Gene Expression and Motility

    Science.gov (United States)

    Ivanova, Krassimira; Eiermann, Peter; Tsiockas, Wasiliki; Hemmersbach, Ruth; Gerzer, Rupert

    2018-03-01

    Altered gravity is known to affect cellular function by changes in gene expression and cellular signaling. The intracellular signaling molecule cyclic guanosine-3',5'-monophosphate (cGMP), a product of guanylyl cyclases (GC), e.g., the nitric oxide (NO)-sensitive soluble GC (sGC) or natriuretic peptide-activated GC (GC-A/GC-B), is involved in melanocyte response to environmental stress. NO-sGC-cGMP signaling is operational in human melanocytes and non-metastatic melanoma cells, whereas up-regulated expression of GC-A/GC-B and inducible NO synthase (iNOS) are found in metastatic melanoma cells, the deadliest skin cancer. Here, we investigated the effects of altered gravity on the mRNA expression of NOS isoforms, sGC, GC-A/GC-B and multidrug resistance-associated proteins 4/5 (MRP4/MRP5) as selective cGMP exporters in human melanoma cells with different metastatic potential and pigmentation. A specific centrifuge (DLR, Cologne Germany) was used to generate hypergravity (5 g for 24 h) and a fast-rotating 2-D clinostat (60 rpm) to simulate microgravity values ≤ 0.012 g for 24 h. The results demonstrate that hypergravity up-regulates the endothelial NOS-sGC-MRP4/MRP5 pathway in non-metastatic melanoma cells, but down-regulates it in simulated microgravity when compared to 1 g. Additionally, the suppression of sGC expression and activity has been suggested to correlate inversely to tumor aggressiveness. Finally, hypergravity is ineffective in highly metastatic melanoma cells, whereas simulated microgravity down-regulates predominantly the expression of the cancer-related genes iNOS and GC-A/GC-B (shown additionally on protein levels) as well as motility in comparison to 1 g. The results suggest that future studies in real microgravity can benefit from considering GC-cGMP signaling as possible factor for melanocyte transformation.

  4. cGMP-dependent protein kinase type I is implicated in the regulation of the timing and quality of sleep and wakefulness.

    Directory of Open Access Journals (Sweden)

    Sonja Langmesser

    Full Text Available Many effects of nitric oxide (NO are mediated by the activation of guanylyl cyclases and subsequent production of the second messenger cyclic guanosine-3',5'-monophosphate (cGMP. cGMP activates cGMP-dependent protein kinases (PRKGs, which can therefore be considered downstream effectors of NO signaling. Since NO is thought to be involved in the regulation of both sleep and circadian rhythms, we analyzed these two processes in mice deficient for cGMP-dependent protein kinase type I (PRKG1 in the brain. Prkg1 mutant mice showed a strikingly altered distribution of sleep and wakefulness over the 24 hours of a day as well as reductions in rapid-eye-movement sleep (REMS duration and in non-REM sleep (NREMS consolidation, and their ability to sustain waking episodes was compromised. Furthermore, they displayed a drastic decrease in electroencephalogram (EEG power in the delta frequency range (1-4 Hz under baseline conditions, which could be normalized after sleep deprivation. In line with the re-distribution of sleep and wakefulness, the analysis of wheel-running and drinking activity revealed more rest bouts during the activity phase and a higher percentage of daytime activity in mutant animals. No changes were observed in internal period length and phase-shifting properties of the circadian clock while chi-squared periodogram amplitude was significantly reduced, hinting at a less robust oscillator. These results indicate that PRKG1 might be involved in the stabilization and output strength of the circadian oscillator in mice. Moreover, PRKG1 deficiency results in an aberrant pattern, and consequently a reduced quality, of sleep and wakefulness, possibly due to a decreased wake-promoting output of the circadian system impinging upon sleep.

  5. A study on relations between the levels of GMP-140 and microangiopathy in NIDDM

    International Nuclear Information System (INIS)

    Du Tongxin; Wang Zizheng; Shi Hongzhen

    1995-01-01

    The relations between the level of GMP-140 and microangiopathy in NIDDM for earlier diagnosis or better treatment are investigated, the level of GMP-140 in both platelet and plasma was measured. The level of GMP-140 in both platelet and plasma in 104 cases with NIDDM (55 with and 49 without microagiopathy) and 38 controls were assayed by RIA and also simultaneously with direct platelet count. The level of GMP-140 in both platelet and plasma in NIDDM was remarkably higher than that in controls (P 1 = 0.69, r 2 = 0.75). No differences existed in platelet count between NIDDM and controls. The level of GMP-140 and ophthalmoscopic study had no change after decreasing the concentration of blood glucose (<7.8 mmol/L) and administrating aspirin for 6 months. Microangiopathy in NIDDM had close relation with platelet function and the level of GMP-140

  6. Gauging and visualizing c-di-GMP levels in pseudomonas aeruginosa using fluorescence-based biosensors

    DEFF Research Database (Denmark)

    Rybtke, Morten; Chua, Song Lin; Yam, Joey Kuok Hoong

    2017-01-01

    Recent research has shown that the molecule c-di-GMP is an important second messenger regulating various functions in bacteria. In particular, the implication of c-di-GMP as a positive regulator of adhesion and biofilm formation has gained momentum as a highly relevant research topic, as detailed...... knowledge about the underlying regulatory mechanisms may enable the development of measures to control biofilms in both industrial and medical settings. Accordingly, it is in many cases of interest to measure the c-di-GMP level in bacteria under specific conditions or in specific mutant strains. We have...... developed a collection of fluorescence-based c-di-GMP biosensors capable of gauging the c-di-GMP level in Pseudomonas aeruginosa and closely related bacteria. Here, we describe protocols for the use of these biosensors in gauging and visualizing cellular c-di-GMP levels of P. aeruginosa both in in vitro...

  7. Evaluation of GMP-compliant culture media for in vitro expansion of human bone marrow mesenchymal stromal cells.

    Science.gov (United States)

    Wuchter, Patrick; Vetter, Marcel; Saffrich, Rainer; Diehlmann, Anke; Bieback, Karen; Ho, Anthony D; Horn, Patrick

    2016-06-01

    Mesenchymal stromal cells (MSCs) from human bone marrow serve as a resource for cell-based therapies in regenerative medicine. Clinical applications require standardized protocols according to good manufacturing practice (GMP) guidelines. Donor variability as well as the intrinsic heterogeneity of MSC populations must be taken into consideration. The composition of the culture medium is a key factor in successful MSC expansion. The aim of this study was to comparatively assess the efficiency of xeno-free human platelet lysate (HPL)-based cell expansion with two commercially available media-StemPro MSC SFM CTS (for human ex vivo tissue and cell culture processing applications) and MSCGM (non-GMP-compliant, for research only)-in an academic setting as the first optimization step toward GMP-compliant manufacturing. We report the feasibility of MSC expansion up to the yielded cell number with all three media. MSCs exhibited the typical fibroblastoid morphology, with distinct differences in cell size depending on the medium. The differentiation capacity and characteristic immunophenotype were confirmed for all MSC populations. Proliferation was highest using StemPro MSC SFM CTS, whereas HPL medium was more cost-effective and its composition could be adjusted individually according to the respective needs. In summary, we present a comprehensive evaluation of GMP-compatible culture media for MSC expansion. Both StemPro and HPL medium proved to be suitable for clinical application and allowed sufficient cell proliferation. Specific differences were observed and should be considered according to the intended use. This study provides a detailed cost analysis and tools that may be helpful for the establishment of GMP-compliant MSC expansion. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  8. c-di-GMP Regulates Various Phenotypes and Insecticidal Activity of Gram-Positive Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    Yang Fu

    2018-02-01

    Full Text Available C-di-GMP has been well investigated to play significant roles in the physiology of many Gram-negative bacteria. However, its effect on Gram-positive bacteria is less known. In order to more understand the c-di-GMP functions in Gram-positive bacteria, we have carried out a detailed study on the c-di-GMP-metabolizing enzymes and their physiological functions in Bacillus thuringiensis, a Gram-positive entomopathogenic bacterium that has been applied as an insecticide successfully. We performed a systematic study on the ten putative c-di-GMP-synthesizing enzyme diguanylate cyclases (DGCs and c-di-GMP-degrading enzyme phosphodiesterases (PDEs in B. thuringiensis BMB171, and artificially elevated the intracellular c-di-GMP level in BMB171 by deleting one or more pde genes. We found increasing level of intracellular c-di-GMP exhibits similar activities as those in Gram-negative bacteria, including altered activities in cell motility, biofilm formation, and cell-cell aggregation. Unexpectedly, we additionally found a novel function exhibited by the increasing level of c-di-GMP to promote the insecticidal activity of this bacterium against Helicoverpa armigera. Through whole-genome transcriptome profile analyses, we found that 4.3% of the B. thuringiensis genes were differentially transcribed when c-di-GMP level was increased, and 77.3% of such gene products are involved in some regulatory pathways not reported in other bacteria to date. In summary, our study represents the first comprehensive report on the c-di-GMP-metabolizing enzymes, their effects on phenotypes, and the transcriptome mediated by c-di-GMP in an important Gram-positive bacterium.

  9. Captive solvent [11C]acetate synthesis in GMP conditions

    International Nuclear Information System (INIS)

    Soloviev, Dmitri; Tamburella, Claire

    2006-01-01

    Reliable procedure for the production of 1-[ 11 C]acetate in GMP conditions was developed based on a combination of the captive-solvent Grignard reaction conducted in the sterile catheter followed by the convenient solid-phase extraction purification on a series of ion-exchange cartridges. The described procedure proved to be reliable in more than 30 patient productions. The process provides stable radiochemical yields (65% EOB) of sodium acetate (1-[ 11 C]) of the Ph.Eur. quality (radiochemical purity better than 95%) in a short time (5 min)

  10. Derivation of xeno-free and GMP-grade human embryonic stem cells--platforms for future clinical applications.

    Directory of Open Access Journals (Sweden)

    Shelly E Tannenbaum

    Full Text Available Clinically compliant human embryonic stem cells (hESCs should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs.

  11. Involvement of NO/cGMP pathway in the antidepressant-like effect of gabapentin in mouse forced swimming test.

    Science.gov (United States)

    Ostadhadi, Sattar; Kordjazy, Nastaran; Haj-Mirzaian, Arya; Ameli, Sanaz; Akhlaghipour, Golnoosh; Dehpour, AhmadReza

    2016-04-01

    Based on clinical studies regarding the beneficial effect of gabapentin in depression, we aimed to evaluate the antidepressant-like properties of gabapentin in mice and also the participation of nitric oxide (NO)/cyclic guanosine monophosphate pathway in this effect. The following drugs were used in this study: gabapentin; N(G)-nitro-L-arginine methyl ester (L-NAME), a non-specific NO synthase (NOS) inhibitor; 7-nitroindazole, a specific neuronal NOS inhibitor; aminoguanidine, a specific inducible NOS inhibitor; L-arginine, a NO precursor; and sildenafil, a phosphodiestrase inhibitor. Finally, we studied the behavioral effects through the forced swimming test (FST) and the changes of the hippocampus NO level through nitrite assay. The immobility time was significantly reduced after gabapentin administration. Co-administration of non-effective doses of gabapentin and L-NAME or 7-nitroindazole (7-NI) resulted in antidepressant-like effect in FST, while aminoguanidine did not affect the immobility time of gabapentin-treated mice. Furthermore, the antidepressant-like property of gabapentin was prevented by L-arginine or sildenafil. Also, the hippocampal nitrite level was significantly lower in gabapentin-treated mice relative to saline-injected mice, and co-administration of 7-NI with sub-effective gabapentin caused a significant decrease in hippocampal nitrite levels. Our results indicate that the antidepressant-like effect of gabapentin in the mice FST model is mediated at least in part through nitric oxide/cyclic guanosine monophosphate (cGMP) pathway.

  12. Understanding the link between GMP and dough: from glutenin particles in flour towards developed dough

    NARCIS (Netherlands)

    Don, C.; Lichtendonk, W.J.; Plijter, J.J.; Hamer, R.J.

    2003-01-01

    Clear correlations exist for glutenin macropolymer (GMP) quantity and rheological properties vs. wheat quality and dough rheological properties, but real insight in understanding these links is still missing. The observation that GMP consists of glutenin particles opens up new possibilities to

  13. Understanding the link between GMP and dough: From glutenin particles in flour towards developed dough

    NARCIS (Netherlands)

    Don, C.; Lichtendonk, W.J.; Plijter, J.J.; Hamer, R.J.

    2003-01-01

    Clear correlations exist for glutenin macropolymer (GMP) quantity and rheological properties vs. wheat quality and dough rheological properties, but real insight in understanding these links is still missing. The observation that GMP consists of glutenin particles opens up new possibilities to

  14. Novel Radioligands for Cyclic Nucleotide Phosphodiesterase Imaging with Positron Emission Tomography: An Update on Developments Since 2012

    Directory of Open Access Journals (Sweden)

    Susann Schröder

    2016-05-01

    Full Text Available Cyclic nucleotide phosphodiesterases (PDEs are a class of intracellular enzymes that inactivate the secondary messenger molecules, cyclic adenosine monophosphate (cAMP and cyclic guanosine monophosphate (cGMP. Thus, PDEs regulate the signaling cascades mediated by these cyclic nucleotides and affect fundamental intracellular processes. Pharmacological inhibition of PDE activity is a promising strategy for treatment of several diseases. However, the role of the different PDEs in related pathologies is not completely clarified yet. PDE-specific radioligands enable non-invasive visualization and quantification of these enzymes by positron emission tomography (PET in vivo and provide an important translational tool for elucidation of the relationship between altered expression of PDEs and pathophysiological effects as well as (pre-clinical evaluation of novel PDE inhibitors developed as therapeutics. Herein we present an overview of novel PDE radioligands for PET published since 2012.

  15. Generalized Wideband Cyclic MUSIC

    Directory of Open Access Journals (Sweden)

    Zhang-Meng Liu

    2009-01-01

    Full Text Available The method of Spectral Correlation-Signal Subspace Fitting (SC-SSF fails to separate wideband cyclostationary signals with coherent second-order cyclic statistics (SOCS. Averaged Cyclic MUSIC (ACM method made up for the drawback to some degree via temporally averaging the cyclic cross-correlation of the array output. This paper interprets ACM from another perspective and proposes a new DOA estimation method by generalizing ACM for wideband cyclostationary signals. The proposed method successfully makes up for the aforementioned drawback of SC-SSF and obtains a more satisfying performance than ACM. It is also demonstrated that ACM is a simplified form of the proposed method when only a single spectral frequency is exploited, and the integration of the frequencies within the signal bandwidth helps the new method to outperform ACM.

  16. Purification and characterization of cGMP binding protein-phosphodiesterase from rat lung

    International Nuclear Information System (INIS)

    Francis, S.H.; Walseth, T.F.; Corbin, J.D.

    1986-01-01

    The cGMP binding protein-phosphodiesterase (cG-BPP) with a phosphodiesterase specific activity of 7 μM/min/mg has been purified from rat lung by sequential chromatography on DEAE-cellulose, Blue-Sepharose, zinc chelate affinity adsorbent and HPLC-DEAE. Migration of the major band on SDS-PAGE corresponds to a MW of ∼93,000. Both cGMP phosphodiesterase activity and cGMP binding from the HPLC-DEAE profile correlate with this band. Since the authors previous work has determined the native MW to be ∼177,000, this suggests a dimeric structure comprised of two 93,000 MW subunits for the rat lung cG-BPP. At low cGMP concentrations, cGMP binding is stimulated ∼20-fold by histone and ∼5-fold by 3-isobutyl-1-methylxanthine(IBMX). The purified protein has one component of cGMP dissociation with a rate constant of 0.045/min. Photolysis of the purified protein in the presence of 32 P-cGMP labels the 93,000 MW band and this labeling is increased by IBMX, indicating that the 93,000 MW band is a subunit of the cGMP-BPP. This implies that the enzyme preparation is nearly homogeneous, a conclusion also supported by a minimum [ 3 H]-cGMP binding stoichiometry of 0.5 mol per 93,000 subunit. An additional protein band with a MW of ∼90,000 also occurs in these preparations which exhibits behavior similar to the 93,000 MW protein. N 2 -Hexyl-cGMP inhibits phosphodiesterase activity by competing with cGMP for hydrolysis at the catalytic site but not at the binding site. N 2 -Hexyl cGMP actually increases cGMP binding. This provides the first evidence that cGMP binding is increased by compounds hydrolyzed at the catalytic site. This interaction between the binding and phosphodiesterase sites could be important in the regulation of the functions of these sites in vivo

  17. Radioimmunoassay for platelet activation specific protein GMP-140 on the platelet surface and in plasma

    International Nuclear Information System (INIS)

    Wu Guoxin; Li Jianyong; Ruan Changgeng

    1991-08-01

    Using monoclonal antibody (McAb) SZ-51 which is specific for an alpha-granule membrane protein (GMP-140) on the surface of human activated platelets, the platelet GMP-140 expression in fixed whole blood was measured by direct radioimmunoassay and GMP-140 microparticles in plasma was measured by sandwich method. The GMP-140 molecules per platelet or milliliter (mL) were calculated for the following subjects; acute myocardial infarction; cerebro thrombosis; diabetic mellitus; asthma attack; epidemic hemorrhagic fever etc.. By comparing with the concentration of thromboxane B 2 (TXB 2 ) and von Willebrand factor (vWF) in plasma, it is confirmed that the measurement of GMP-140 molecules is better than that of TXB 2 and vWF. It is a sensitive and specific method for evaluating the platelet activation degree in vivo. The establishment of this method will be useful to diagnosing the thrombotic disorders and studying the pathogenesis of some other diseases

  18. The Role of Aquaporin 1 Activated by cGMP in Myocardial Edema Caused by Cardiopulmonary Bypass in Sheep

    Directory of Open Access Journals (Sweden)

    Fang-bao Ding

    2013-11-01

    Full Text Available Background/Aims: Most cardiac procedures involve the use of cardiopulmonary bypass (CPB, which pumps oxygenated blood to the body while the heart and lungs are isolated. CPB can cause profound alterations V in the homeostasis of physiological fluids, which often results in myocardial edema. In our study, we used sheep CPB model of in vivo and in vitro to assess the relationship between cGMP and AQP1 during CPB. Methods: ODQ, a specific inhibitor of soluble guanylate cyclase (sGC, was used to treat the CPB animals or cardiomyocytes. Left ventricular function of each group was determined by pressure-volume system. Water content of myocardial tissue was assessed by dry-wet weight, and cardiomyocytes water permeability was also calculated. The concentration of cGMP was determined by Radioimmunoassay (RIA. mRNA and protein expression of AQP1 were detected by real-time PCR and western blot, respectively. Results: The relative expression level of AQP1 mRNA and protein at each time point (0, 6, 12, 24 or 48 h after CPB was significantly increased (1.18-fold at 12 h, 1.77-fold at 24 h and 2.18-fold at 48h compared with each sham group, the protein expression of AQP1 also showed a rising trend after CPB. The degree of myocardial edema (75.1% at 12 h, 79.3% at 24 h and 81.0% at 48h increased following the CPB surgery. The mRNA expression level of AQP1 was significantly decreased by 39.7% (pin vitro experiments showed the same changing trends as in vivo. Conclusion: cGMP pathway controls water channels and then affects water intake during CPB through an AQP1-mediated pathway.

  19. Andrographolide inhibits hypoxia-induced hypoxia-inducible factor 1α and endothelin 1 expression through the heme oxygenase 1/CO/cGMP/MKP-5 pathways in EA.hy926 cells.

    Science.gov (United States)

    Lin, Hung-Chih; Su, Shih-Li; Lin, Wan-Chun; Lin, Ai-Hsuan; Yang, Ya-Chen; Lii, Chong-Kuei; Chen, Haw-Wen

    2018-03-01

    Andrographolide is a potent anti-inflammatory agent found in Andrographis paniculata. Endothelin 1 (ET-1) is an endothelium-derived vasoconstrictor with pro-inflammatory properties secreted in response to hypoxia. Mitogen-activated protein kinase phosphatase 5 (MKP-5) is a dual-specificity phosphatase that dephosphorylates threonine and tyrosine residues of MAPKs. We showed previously that hypoxia-induced HIF-1α expression and ET-1 secretion are dependent on p38 MAPK in EA.hy926 cells. Here, we investigate what role MKP-5 plays in andrographolide's inhibition of hypoxia-induced expression of HIF-1α and ET-1. Hypoxic conditions were created using the hypoxia-mimetic agent CoCl 2 . Andrographolide enhanced HO-1 and MKP-5 expression and cellular cGMP content in addition to inhibiting hypoxia-induced ROS generation. Concomitantly, the HO-1 byproduct CO and the cGMP analogue 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) increased MKP-5 expression, and pretreatment with CO and 8-Br-cGMP inhibited hypoxia-induced HIF-1α and ET-1 expression. Transfection of HO-1 siRNA or pretreatment with the HO-1 inhibitor ZnPP-9 or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a specific inhibitor of soluble guanylate cyclase, reduced andrographolide-induced MKP-5 expression. Moreover, silencing MKP-5 or treatment with the phosphatase inhibitor vanadate abrogated andrographolide's suppressing hypoxia-induced p38 MAPK activation and HIF-1α expression. The inhibition of hypoxia-induced HIF-1α and ET-1 expression by andrographolide is likely associated with HO-1/CO/cGMP/MKP-5 pathways, which is involved in inhibiting hypoxia-induced p38 MAPK activation. © 2017 Wiley Periodicals, Inc.

  20. The arabidopsis cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara Elizabeth; Meier, Stuart Kurt; Gehring, Christoph A

    2016-01-01

    Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms

  1. High-Performance Liquid Chromatography (HPLC)-Based Detection and Quantitation of Cellular c-di-GMP.

    Science.gov (United States)

    Petrova, Olga E; Sauer, Karin

    2017-01-01

    The modulation of c-di-GMP levels plays a vital role in the regulation of various processes in a wide array of bacterial species. Thus, investigation of c-di-GMP regulation requires reliable methods for the assessment of c-di-GMP levels and turnover. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis has become a commonly used approach to accomplish these goals. The following describes the extraction and HPLC-based detection and quantification of c-di-GMP from Pseudomonas aeruginosa samples, a procedure that is amenable to modifications for the analysis of c-di-GMP in other bacterial species.

  2. Expansion of Human Tregs from Cryopreserved Umbilical Cord Blood for GMP-Compliant Autologous Adoptive Cell Transfer Therapy

    Directory of Open Access Journals (Sweden)

    Howard R. Seay

    2017-03-01

    Full Text Available Umbilical cord blood is a traditional and convenient source of cells for hematopoietic stem cell transplantation. Thymic regulatory T cells (Tregs are also present in cord blood, and there is growing interest in the use of autologous Tregs to provide a low-risk, fully human leukocyte antigen (HLA-matched cell product for treating autoimmune diseases, such as type 1 diabetes. Here, we describe a good manufacturing practice (GMP-compatible Treg expansion protocol using fluorescence-activated cell sorting, resulting in a mean 2,092-fold expansion of Tregs over a 16-day culture for a median yield of 1.26 × 109 Tregs from single-donor cryopreserved units. The resulting Tregs passed prior clinical trial release criteria for Treg purity and sterility, including additional rigorous assessments of FOXP3 and Helios expression and epigenetic analysis of the FOXP3 Treg-specific demethylated region (TSDR. Compared with expanded adult peripheral blood Tregs, expanded cord blood Tregs remained more naive, as assessed by continued expression of CD45RA, produced reduced IFN-γ following activation, and effectively inhibited responder T cell proliferation. Immunosequencing of the T cell receptor revealed a remarkably diverse receptor repertoire within cord blood Tregs that was maintained following in vitro expansion. These data support the feasibility of generating GMP-compliant Tregs from cord blood for adoptive cell transfer therapies and highlight potential advantages in terms of safety, phenotypic stability, autoantigen specificity, and tissue distribution.

  3. 8-Nitro-cGMP promotes bone growth through expansion of growth plate cartilage.

    Science.gov (United States)

    Hoshino, Marie; Kaneko, Kotaro; Miyamoto, Yoichi; Yoshimura, Kentaro; Suzuki, Dai; Akaike, Takaaki; Sawa, Tomohiro; Ida, Tomoaki; Fujii, Shigemoto; Ihara, Hideshi; Tanaka, Junichi; Tsukuura, Risa; Chikazu, Daichi; Mishima, Kenji; Baba, Kazuyoshi; Kamijo, Ryutaro

    2017-09-01

    In endochondral ossification, growth of bones occurs at their growth plate cartilage. While it is known that nitric oxide (NO) synthases are required for proliferation of chondrocytes in growth plate cartilage and growth of bones, the precise mechanism by which NO facilitates these process has not been clarified yet. C-type natriuretic peptide (CNP) also positively regulate elongation of bones through expansion of the growth plate cartilage. Both NO and CNP are known to use cGMP as the second messenger. Recently, 8-nitro-cGMP was identified as a signaling molecule produced in the presence of NO in various types of cells. Here, we found that 8-nitro-cGMP is produced in proliferating chondrocytes in the growth plates, which was enhanced by CNP, in bones cultured ex vivo. In addition, 8-nitro-cGMP promoted bone growth with expansion of the proliferating zone as well as increase in the number of proliferating cells in the growth plates. 8-Nitro-cGMP also promoted the proliferation of chondrocytes in vitro. On the other hand, 8-bromo-cGMP enhanced the growth of bones with expansion of hypertrophic zone of the growth plates without affecting either the width of proliferating zone or proliferation of chondrocytes. These results indicate that 8-nitro-cGMP formed in growth plate cartilage accelerates chondrocyte proliferation and bone growth as a downstream molecule of NO. Copyright © 2017. Published by Elsevier Inc.

  4. Production of GMP certified herbal products in Malaysian Nuclear Agency

    International Nuclear Information System (INIS)

    Daryl Jesus Arapoc; Bohari Yaacob; Zainah Adam

    2015-01-01

    This paper will discuss on up scaling production of herbal product. A pilot scale production plant were developed in Block 37 and equipped with automated and semi-automated production machines which have the capacity to produce 100 thousand pieces of tablet per hour. In order to ensure the quality of the products, raw material inspections, IPQC and FPQC will be done on each batch. Besides that, certification of GMP will be done concurrently. One of the products that will be launch soon is the Mas Cotek tablet. This product is the result of numerous years of researches that had been done in BTP. This includes formulation and production of the product itself. It is hope that more herbal products can be produce in near future. (author)

  5. Cyclic Voltammograms from First Principles

    DEFF Research Database (Denmark)

    Karlberg, Gustav; Jaramillo, Thomas; Skulason, Egill

    2007-01-01

    Cyclic voltammetry is a fundamental experimental tool for characterizing electrochemical surfaces. Whereas cyclic voltammetry is widely used within the field of electrochemistry, a way to quantitatively and directly relate the cyclic voltammogram to ab initio calculations has been lacking, even f...

  6. The anti-cancerous drug doxorubicin decreases the c-di-GMP content in Pseudomonas aeruginosa but promotes biofilm formation

    DEFF Research Database (Denmark)

    Groizeleau, Julie; Rybtke, Morten; Andersen, Jens Bo

    2016-01-01

    Current antibiotic treatments are insufficient in eradicating bacterial biofilms, which represent the primary cause of chronic bacterial infections. Thus, there is an urgent need for new strategies to eradicate biofilm infections. The second messenger c-di-GMP is a positive regulator of biofilm...... formation in many clinically relevant bacteria. It is hypothesized that drugs lowering the intracellular level of c-di-GMP will force biofilm bacteria into a more treatable planktonic lifestyle. To identify compounds capable of lowering c-di-GMP levels in Pseudomonas aeruginosa, we screened 5000 compounds...... for their potential c-di-GMP-lowering effect using a recently developed c-di-GMP biosensor strain. Our screen identified the anti-cancerous drug doxorubicin as a potent c-di-GMP inhibitor. In addition, the drug decreased the transcription of many biofilm-related genes. However, despite its effect on the c-di-GMP...

  7. The regulatory role of the NO/cGMP signal transduction cascade during larval attachment and metamorphosis of the barnacle Balanus (=Amphibalanus) amphitrite

    KAUST Repository

    Zhang, Y.

    2012-08-01

    The barnacle Balanus amphitrite is among the most dominant fouling species on intertidal rocky shores in tropical and subtropical areas and is thus a target organism in antifouling research. After being released from adults, the swimming nauplius undertakes six molting cycles and then transforms into a cyprid. Using paired antennules, a competent cyprid actively explores and selects a suitable substratum for attachment and metamorphosis (collectively known as settlement). This selection process involves the reception of exogenous signals and subsequent endogenous signal transduction. To investigate the involvement of nitric oxide (NO) and cyclic GMP (cGMP) during larval settlement of B. amphitrite, we examined the effects of an NO donor and an NO scavenger, two nitric oxide synthase (NOS) inhibitors and a soluble guanylyl cyclase (sGC) inhibitor on settling cyprids. We found that the NO donor sodium nitroprusside (SNP) inhibited larval settlement in a dose-dependent manner. In contrast, both the NO scavenger carboxy-PTIO and the NOS inhibitors aminoguanidine hemisulfate (AGH) and S-methylisothiourea sulfate (SMIS) significantly accelerated larval settlement. Suppression of the downstream guanylyl cyclase (GC) activity using a GC-selective inhibitor ODQ could also significantly accelerate larval settlement. Interestingly, the settlement inhibition effects of SNP could be attenuated by ODQ at all concentrations tested. In the developmental expression profiling of NOS and sGC, the lowest expression of both genes was detected in the cyprid stage, a crucial stage for the larval decision to attach and metamorphose. In summary, we concluded that NO regulates larval settlement via mediating downstream cGMP signaling.

  8. The regulatory role of the NO/cGMP signal transduction cascade during larval attachment and metamorphosis of the barnacle Balanus (=Amphibalanus) amphitrite

    KAUST Repository

    Zhang, Y.; He, L.-S.; Zhang, G.; Xu, Y.; Lee, O.-O.; Matsumura, K.; Qian, P.-Y.

    2012-01-01

    The barnacle Balanus amphitrite is among the most dominant fouling species on intertidal rocky shores in tropical and subtropical areas and is thus a target organism in antifouling research. After being released from adults, the swimming nauplius undertakes six molting cycles and then transforms into a cyprid. Using paired antennules, a competent cyprid actively explores and selects a suitable substratum for attachment and metamorphosis (collectively known as settlement). This selection process involves the reception of exogenous signals and subsequent endogenous signal transduction. To investigate the involvement of nitric oxide (NO) and cyclic GMP (cGMP) during larval settlement of B. amphitrite, we examined the effects of an NO donor and an NO scavenger, two nitric oxide synthase (NOS) inhibitors and a soluble guanylyl cyclase (sGC) inhibitor on settling cyprids. We found that the NO donor sodium nitroprusside (SNP) inhibited larval settlement in a dose-dependent manner. In contrast, both the NO scavenger carboxy-PTIO and the NOS inhibitors aminoguanidine hemisulfate (AGH) and S-methylisothiourea sulfate (SMIS) significantly accelerated larval settlement. Suppression of the downstream guanylyl cyclase (GC) activity using a GC-selective inhibitor ODQ could also significantly accelerate larval settlement. Interestingly, the settlement inhibition effects of SNP could be attenuated by ODQ at all concentrations tested. In the developmental expression profiling of NOS and sGC, the lowest expression of both genes was detected in the cyprid stage, a crucial stage for the larval decision to attach and metamorphose. In summary, we concluded that NO regulates larval settlement via mediating downstream cGMP signaling.

  9. Post-translational Analysis of Arabidopsis thaliana Proteins in Response to Cyclic Guanosine Monophosphate Treatment

    KAUST Repository

    Parrott, Brian

    2011-12-12

    The introduction of mass spectrometry techniques to the field of biology has made possible the exploration of the proteome as a whole system as opposed to prior techniques, such as anti-body based assays or yeast two-hybrid studies, which were strictly limited to the study of a few proteins at a time. This practice has allowed for a systems biology approach of exploring the proteome, with the possibility of viewing entire pathways over increments of time. In this study, the effect of treating Arabidopsis thaliana suspension culture cells with 3’,5’-cyclic guanosine monophosphate (cGMP), which is a native second messenger, was examined. Samples were collected at four time points and proteins were extracted and enriched for both oxidation and phosphorylation before analysis via mass spectrometry. Preliminary results suggest a tendency towards an increased number of phosphorylated proteins as a result of cGMP treatment. The data also showed a sharp increase in methionine oxidation in response to the treatment, occurring within the first ten minutes. This finding suggests that cGMP may utilize methionine oxidation as a mechanism of signal transduction. As such, this study corroborates a growing body of evidence supporting the inclusion of methionine oxidation in intracellular signaling pathways.

  10. HOST liner cyclic facilities

    Science.gov (United States)

    Schultz, D.

    1983-01-01

    The HOST Liner Cyclic Program is utilizing two types of test apparatus, rectangular box rigs and a full annular rig. To date two quartz lamp cyclic box rigs have been tested and a third is to begin testing in late October 1983. The box rigs are used to evaluate 5x8 inch rectangular linear samples. A 21 inch diameter outer liner simulator is also being built up for testing beginning in April 1984. All rigs are atmospheric rigs. The first box rig, a three 6-kVA lamp installation, was operated under adverse conditions to determine feasibility of using quartz lamps for cyclic testing. This work was done in December 1981 and looked promising. The second box rig, again using three 6-kVA lamps, was operated to obtain instrumentation durability information and initial data input to a Finite Element Model. This limited test program was conducted in August 1983. Five test plates were run. Instrumentation consisted of strain gages, thermocouples and thermal paint. The strain gages were found to fail at 1200 F as expected though plates were heated to 1700 F. The third box rig, containing four 6-kVA lamps, is in build up for testing to begin in late October 1983. In addition to 33 percent greater power input, this rig has provision for 400 F backside line cooling air and a viewing port suitable for IR camera viewing. The casing is also water cooled for extended durability.

  11. Inotropic responses of the frog ventricle to adenosine triphosphate and related changes in endogenous cyclic nucleotides.

    Science.gov (United States)

    Flitney, F W; Singh, J

    1980-07-01

    1. A study has been made of a well documented but poorly understood response of the isolated frog ventricle to treatment with exogenous adenosine 5' triphosphate (ATP). Measurements of membrane potential, isometric twitch tension and levels of endogenous 3',5'-cyclic nucleotides have been made at various times during the ATP-induced response. 2. ATP elicits a characteristic triphasic response, which comprises an initial, abrupt increase in contractility, rising to a maximum within a few beats (first phase); followed by a period when the twitch amplitude falls, sometimes to below the control level (second phase); and superceded by a more slowly developing and longer-lasting increase in contractile force (third phase). The response is unaffected by atropine, propranolol or phentolamine. However, the prostaglandin synthetase inhibitor indomethacin depresses the first phase and entirely suppresses the third phase. 3. The inotropic effects of ATP are accompanied by changes in the shape of the action potential. These effects are dose-related. The duration of the action potential (D-30mV) and its positive overshoot (O) are increased during all phases of the response, for [ATP]o's up to 10(-5) M. However, at higher [ATP]o's, D-30mV and O ar both reduced during the second phase (but not the first or third phase), when isometric twitch tension is also depressed. The relationship between action potential duration and twitch tension (P) for different [ATP]o's is linear for all three phases of the response, but the slopes of the curves (delta P/delta D) are markedly different, indicating that the sensitivity of the contractile system to membrane depolarization is not constant, but varies continuously throughout the response. 4. ATP has a potent stimulatory effect on the metabolism of endogenous 3',5'-cyclic nucleotides. The time courses of the changes in adenosine 3','5-cyclic monophosphate (3',5'-cyclic AMP) and guanosine 3',5'-cyclic monophosphate (3',5'-cyclic GMP) are

  12. Microgravity changes in heart structure and cyclic-AMP metabolism

    Science.gov (United States)

    Philpott, D. E.; Fine, A.; Kato, K.; Egnor, R.; Cheng, L.

    1985-01-01

    The effects of microgravity on cardiac ultrastructure and cyclic AMP metabolism in tissues of rats flown on Spacelab 3 are reported. Light and electron microscope studies of cell structure, measurements of low and high Km phosphodiesterase activity, cyclic AMP-dependent protein kinase activity, and regulatory subunit compartmentation show significant deviations in flight animals when compared to ground controls. The results indicate that some changes have occurred in cellular responses associated with catecholamine receptor interactions and intracellular signal processing.

  13. cGMP-dependent protein kinase type II knockout mice exhibit working memory impairments, decreased repetitive behavior, and increased anxiety-like traits.

    Science.gov (United States)

    Wincott, Charlotte M; Abera, Sinedu; Vunck, Sarah A; Tirko, Natasha; Choi, Yoon; Titcombe, Roseann F; Antoine, Shannon O; Tukey, David S; DeVito, Loren M; Hofmann, Franz; Hoeffer, Charles A; Ziff, Edward B

    2014-10-01

    Neuronal activity regulates AMPA receptor trafficking, a process that mediates changes in synaptic strength, a key component of learning and memory. This form of plasticity may be induced by stimulation of the NMDA receptor which, among its activities, increases cyclic guanosine monophosphate (cGMP) through the nitric oxide synthase pathway. cGMP-dependent protein kinase type II (cGKII) is ultimately activated via this mechanism and AMPA receptor subunit GluA1 is phosphorylated at serine 845. This phosphorylation contributes to the delivery of GluA1 to the synapse, a step that increases synaptic strength. Previous studies have shown that cGKII-deficient mice display striking spatial learning deficits in the Morris Water Maze compared to wild-type littermates as well as lowered GluA1 phosphorylation in the postsynaptic density of the prefrontal cortex (Serulle et al., 2007; Wincott et al., 2013). In the current study, we show that cGKII knockout mice exhibit impaired working memory as determined using the prefrontal cortex-dependent Radial Arm Maze (RAM). Additionally, we report reduced repetitive behavior in the Marble Burying task (MB), and heightened anxiety-like traits in the Novelty Suppressed Feeding Test (NSFT). These data suggest that cGKII may play a role in the integration of information that conveys both anxiety-provoking stimuli as well as the spatial and environmental cues that facilitate functional memory processes and appropriate behavioral response. Published by Elsevier Inc.

  14. Supercritical fluid chromatography for GMP analysis in support of pharmaceutical development and manufacturing activities.

    Science.gov (United States)

    Hicks, Michael B; Regalado, Erik L; Tan, Feng; Gong, Xiaoyi; Welch, Christopher J

    2016-01-05

    Supercritical fluid chromatography (SFC) has long been a preferred method for enantiopurity analysis in support of pharmaceutical discovery and development, but implementation of the technique in regulated GMP laboratories has been somewhat slow, owing to limitations in instrument sensitivity, reproducibility, accuracy and robustness. In recent years, commercialization of next generation analytical SFC instrumentation has addressed previous shortcomings, making the technique better suited for GMP analysis. In this study we investigate the use of modern SFC for enantiopurity analysis of several pharmaceutical intermediates and compare the results with the conventional HPLC approaches historically used for analysis in a GMP setting. The findings clearly illustrate that modern SFC now exhibits improved precision, reproducibility, accuracy and robustness; also providing superior resolution and peak capacity compared to HPLC. Based on these findings, the use of modern chiral SFC is recommended for GMP studies of stereochemistry in pharmaceutical development and manufacturing. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. KAJIAN PENERAPAN GOOD MANUFACTURING PRACTICE (GMP DI INDUSTRI RAJUNGAN PT.KELOLA MINA LAUT MADURA

    Directory of Open Access Journals (Sweden)

    Bhiaztika Ristyanadi

    2016-11-01

    Full Text Available Good manufacturing practice is the first step implementation of food safety regulation. PT. Kelola Mina Laut is one of chilled sea crab producers in Madura. It has four branches in Madura,those are in Tanjung Bumi, Noreh, Sampang, and Lobuk. The objective of this research is to assess the effectiveness GMP in four branches of PT. Kelola Mina Laut. The research  uses field observation, data analysis and GMP development as the method. Based on GMP analysis, four branches of PT. Kelola Mina Laut appear to have a cummulative score between 337-369, in which Lobuk has the highest score. Therefore, it can be concluded that PT. Kelola Mina Laut has applied most of GMP elements

  16. Physiological and Molecular Effects of the Cyclic Nucleotides cAMP and cGMP on Arabidopsis thaliana

    KAUST Repository

    Herrera, Natalia M.

    2012-01-01

    transport in Arabidopsis thaliana leaves and, that these changes at the molecular level can have functional biological consequences. For this reason we tested if CNs modulate the photosynthetic rate, responses to high light and root ion transport. Real time

  17. Cyclic approximation to stasis

    Directory of Open Access Journals (Sweden)

    Stewart D. Johnson

    2009-06-01

    Full Text Available Neighborhoods of points in $mathbb{R}^n$ where a positive linear combination of $C^1$ vector fields sum to zero contain, generically, cyclic trajectories that switch between the vector fields. Such points are called stasis points, and the approximating switching cycle can be chosen so that the timing of the switches exactly matches the positive linear weighting. In the case of two vector fields, the stasis points form one-dimensional $C^1$ manifolds containing nearby families of two-cycles. The generic case of two flows in $mathbb{R}^3$ can be diffeomorphed to a standard form with cubic curves as trajectories.

  18. Accelerated cyclic corrosion tests

    Directory of Open Access Journals (Sweden)

    Prošek T.

    2016-06-01

    Full Text Available Accelerated corrosion testing is indispensable for material selection, quality control and both initial and residual life time prediction for bare and painted metallic, polymeric, adhesive and other materials in atmospheric exposure conditions. The best known Neutral Salt Spray (NSS test provides unrealistic conditions and poor correlation to exposures in atmosphere. Modern cyclic accelerated corrosion tests include intermittent salt spray, wet and dry phases and eventually other technical phases. They are able to predict the material performance in service more correctly as documented on several examples. The use of NSS should thus be restricted for quality control.

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

    Directory of Open Access Journals (Sweden)

    Justin Schleede

    2015-10-01

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

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

    Science.gov (United States)

    Schleede, Justin; Blair, Seth S

    2015-10-01

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

  1. Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

    Directory of Open Access Journals (Sweden)

    Hou Ssu-Yu

    2010-06-01

    Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP-eNOS/NO-cyclic

  2. A potent series targeting the malarial cGMP-dependent protein kinase clears infection and blocks transmission.

    Science.gov (United States)

    Baker, David A; Stewart, Lindsay B; Large, Jonathan M; Bowyer, Paul W; Ansell, Keith H; Jiménez-Díaz, María B; El Bakkouri, Majida; Birchall, Kristian; Dechering, Koen J; Bouloc, Nathalie S; Coombs, Peter J; Whalley, David; Harding, Denise J; Smiljanic-Hurley, Ela; Wheldon, Mary C; Walker, Eloise M; Dessens, Johannes T; Lafuente, María José; Sanz, Laura M; Gamo, Francisco-Javier; Ferrer, Santiago B; Hui, Raymond; Bousema, Teun; Angulo-Barturén, Iñigo; Merritt, Andy T; Croft, Simon L; Gutteridge, Winston E; Kettleborough, Catherine A; Osborne, Simon A

    2017-09-05

    To combat drug resistance, new chemical entities are urgently required for use in next generation anti-malarial combinations. We report here the results of a medicinal chemistry programme focused on an imidazopyridine series targeting the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG). The most potent compound (ML10) has an IC 50 of 160 pM in a PfPKG kinase assay and inhibits P. falciparum blood stage proliferation in vitro with an EC 50 of 2.1 nM. Oral dosing renders blood stage parasitaemia undetectable in vivo using a P. falciparum SCID mouse model. The series targets both merozoite egress and erythrocyte invasion, but crucially, also blocks transmission of mature P. falciparum gametocytes to Anopheles stephensi mosquitoes. A co-crystal structure of PvPKG bound to ML10, reveals intimate molecular contacts that explain the high levels of potency and selectivity we have measured. The properties of this series warrant consideration for further development to produce an antimalarial drug.Protein kinases are promising drug targets for treatment of malaria. Here, starting with a medicinal chemistry approach, Baker et al. generate an imidazopyridine that selectively targets Plasmodium falciparum PKG, inhibits blood stage parasite growth in vitro and in mice and blocks transmission to mosquitoes.

  3. [Asthma and cyclic neutropenia].

    Science.gov (United States)

    Salazar Cabrera, A N; Berrón Pérez, R; Ortega Martell, J A; Onuma Takane, E

    1996-01-01

    We report a male with history of recurrent infections (recurrent oral aphtous disease [ROAD], middle ear infections and pharyngo amigdalitis) every 3 weeks since he was 7 months old. At the age of 3 years cyclic neutropenia was diagnosed with cyclic fall in the total neutrophil count in blood smear every 21 days and prophylactic antimicrobial therapy was indicated. Episodic events every 3 weeks of acute asthma and allergic rhinitis were detected at the age of 6 years old and specific immunotherapy to Bermuda grass was given during 3 years with markedly improvement in his allergic condition but not in the ROAD. He came back until the age of 16 with episodic acute asthma and ROAD. The total neutrophil count failed to 0 every 21 days and surprisingly the total eosinophil count increased up to 2,000 at the same time, with elevation of serum IgE (412 Ul/mL). Specific immunotherapy to D.pt. and Aller.a. and therapy with timomodulin was indicated. After 3 months we observed clinical improvement in the asthmatic condition and the ROAD disappeared, but the total neutrophil count did not improve. We present this case as a rare association between 2 diseases with probably no etiological relationship but may be physiopatological that could help to understand more the pathogenesis of asthma.

  4. Ekpyrotic and cyclic cosmology

    International Nuclear Information System (INIS)

    Lehners, Jean-Luc

    2008-01-01

    Ekpyrotic and cyclic cosmologies provide theories of the very early and of the very late universe. In these models, the big bang is described as a collision of branes - and thus the big bang is not the beginning of time. Before the big bang, there is an ekpyrotic phase with equation of state w=P/(ρ) >>1 (where P is the average pressure and ρ the average energy density) during which the universe slowly contracts. This phase resolves the standard cosmological puzzles and generates a nearly scale-invariant spectrum of cosmological perturbations containing a significant non-Gaussian component. At the same time it produces small-amplitude gravitational waves with a blue spectrum. The dark energy dominating the present-day cosmological evolution is reinterpreted as a small attractive force between our brane and a parallel one. This force eventually induces a new ekpyrotic phase and a new brane collision, leading to the idea of a cyclic universe. This review discusses the detailed properties of these models, their embedding in M-theory and their viability, with an emphasis on open issues and observational signatures

  5. GMP-compliant isolation and large-scale expansion of bone marrow-derived MSC.

    Directory of Open Access Journals (Sweden)

    Natalie Fekete

    Full Text Available BACKGROUND: Mesenchymal stromal cells (MSC have gained importance in tissue repair, tissue engineering and in immunosupressive therapy during the last years. Due to the limited availability of MSC in the bone marrow, ex vivo amplification prior to clinical application is requisite to obtain therapeutic applicable cell doses. Translation of preclinical into clinical-grade large-scale MSC expansion necessitates precise definition and standardization of all procedural parameters including cell seeding density, culture medium and cultivation devices. While xenogeneic additives such as fetal calf serum are still widely used for cell culture, its use in the clinical context is associated with many risks, such as prion and viral transmission or adverse immunological reactions against xenogeneic components. METHODS AND FINDINGS: We established animal-free expansion protocols using platelet lysate as medium supplement and thereby could confirm its safety and feasibility for large-scale MSC isolation and expansion. Five different GMP-compliant standardized protocols designed for the safe, reliable, efficient and economical isolation and expansion of MSC was performed and MSC obtained were analyzed for differentiation capacity by qPCR and histochemistry. Expression of standard MSC markers as defined by the International Society for Cellular Therapy as well as expression of additional MSC markers and of various chemokine and cytokine receptors was analysed by flow cytometry. Changes of metabolic markers and cytokines in the medium were addressed using the LUMINEX platform. CONCLUSIONS: The five different systems for isolation and expansion of MSC described in this study are all suitable to produce at least 100 millions of MSC, which is commonly regarded as a single clinical dose. Final products are equal according to the minimal criteria for MSC defined by the ISCT. We showed that chemokine and integrin receptors analyzed had the same expression pattern

  6. Ca 2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca 2+ channels

    KAUST Repository

    Qia, Zhi

    2010-11-18

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

  7. Z₂-double cyclic codes

    OpenAIRE

    Borges, J.

    2014-01-01

    A binary linear code C is a Z2-double cyclic code if the set of coordinates can be partitioned into two subsets such that any cyclic shift of the coordinates of both subsets leaves invariant the code. These codes can be identified as submodules of the Z2[x]-module Z2[x]/(x^r − 1) × Z2[x]/(x^s − 1). We determine the structure of Z2-double cyclic codes giving the generator polynomials of these codes. The related polynomial representation of Z2-double cyclic codes and its duals, and the relation...

  8. Manual for Cyclic Triaxial Test

    DEFF Research Database (Denmark)

    Shajarati, Amir; Sørensen, Kris Wessel; Nielsen, Søren Kjær

    This manual describes the different steps that is included in the procedure for conducting a cyclic triaxial test at the geotechnical Laboratory at Aalborg University. Furthermore it contains a chapter concerning some of the background theory for the static triaxial tests. The cyclic/dynamic tria......This manual describes the different steps that is included in the procedure for conducting a cyclic triaxial test at the geotechnical Laboratory at Aalborg University. Furthermore it contains a chapter concerning some of the background theory for the static triaxial tests. The cyclic...

  9. Dynamic Changes of Endothelium Derived Factors and Cyclic Nucleotides in Ascites Broilers and Control of L-arginine

    Institute of Scientific and Technical Information of China (English)

    HAN Bo; WANG Xiao-long

    2003-01-01

    A flock of AA breed chickens were reared in peterstme brood-vait chamber using high energypelleted feed, at 14 days of age, 400 birds were separated into 3 groups randomly as follows: 100 birds wereexposed to normal ambient temperature (20℃) as control group, 150 birds were exposed to low ambient tem-perature (11℃) in order to induce ascites as treatment Ⅰ group, another 100 birds were also exposed to lowambient temperature (11℃) and fed the diets containing 1% L-arginine for ascitic prophylactic treatment astreatment Ⅱ group. The blood samples were collected on 3, 4, 5, 6, 7 wk, respectively, to measure the con-tents of plasma endothelin (ET-1), angiotensin Ⅱ (Ang Ⅱ ) and cyclic adenosine monophosphate (cAMP) andcyclic guanosine monophosphate (cGMP). The results indicated that the contents of cAMP, cGMP, Ang Ⅱand the ratio of cAMP/cGMP in treatment Ⅰ and ascitic broilers were higher than that of correspondent con-trol group(P<0.01, P<0.05), ET-1 of preascitic broilers were higher than that of control group(P<0.05),while there were insignificant differences with later ascitic broilers, the contents of cAMP and cGMP in treat-ment Ⅱ were higher than treatment Ⅰ and control group(P<0.01, P<0.05), whereas, the ratio of cAMP/cGMP and the contents of Ang Ⅱ were gradually decreased than that of control group(P<0.05), the contentsof ET-1 were not changed. By further analysis, the increased plasma Ang Ⅱ at low ambient temperature condi-tion in broilers made endothelium cell secretion of increased ET-1, cAMP, cGMP and decreased NO. So lowtemperature accelarated ascites syndrome in broilers. Supplemented L-arginine can decrease ET-1, cAMP andcAMP/cGMP. It is concluded that cAMP mediated pulmonary hypertension syndrome in broilers.

  10. Regulation of the Na(+)-K(+)-2Cl(-) cotransporter by cGMP/cGMP-dependent protein kinase I after furosemide administration.

    Science.gov (United States)

    Limmer, Franziska; Schinner, Elisabeth; Castrop, Hayo; Vitzthum, Helga; Hofmann, Franz; Schlossmann, Jens

    2015-10-01

    Sodium chloride reabsorption in the thick ascending limb of the loop of Henle is mediated by the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). The loop diuretic furosemide is a potent inhibitor of NKCC2. However, less is known about the mechanism regulating the electrolyte transporter. Considering the well-established effects of nitric oxide on NKCC2 activity, cGMP is likely involved in this regulation. cGMP-dependent protein kinase I (cGKI; PKGI) is a cGMP target protein that phosphorylates different substrates after activation through cGMP. We investigated the potential correlation between the cGMP/cGKI pathway and NKCC2 regulation. We treated wild-type (wt) and cGKIα-rescue mice with furosemide. cGKIα-rescue mice expressed cGKIα only under the control of the smooth muscle-specific transgelin (SM22) promoter in a cGKI deficient background. Furosemide treatment increased the urine excretion of sodium and chloride in cGKIα-rescue mice compared to that in wt mice. We analyzed the phosphorylation of NKCC2 by western blotting and immunostaining using the phosphospecific antibody R5. The administration of furosemide significantly increased the phosphorylated NKCC2 signal in wt but not in cGKIα-rescue mice. NKCC2 activation led to its phosphorylation and membrane translocation. To examine whether cGKI was involved in this process, we analyzed vasodilator-stimulated phosphoprotein, which is phosphorylated by cGKI. Furosemide injection resulted in increased vasodilator-stimulated phosphoprotein phosphorylation in wt mice. We hypothesize that furosemide administration activated cGKI, leading to NKCC2 phosphorylation and membrane translocation. This cGKI-mediated pathway could be a mechanism to compensate for the inhibitory effect of furosemide on NKCC2. © 2015 FEBS.

  11. The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING.

    Science.gov (United States)

    Diner, Elie J; Burdette, Dara L; Wilson, Stephen C; Monroe, Kathryn M; Kellenberger, Colleen A; Hyodo, Mamoru; Hayakawa, Yoshihiro; Hammond, Ming C; Vance, Russell E

    2013-05-30

    The presence of foreign DNA in the cytosol of mammalian cells elicits a potent antiviral interferon response. Recently, cytosolic DNA was proposed to induce the synthesis of cyclic GMP-AMP (cGAMP) upon binding to an enzyme called cGAMP synthase (cGAS). cGAMP activates an interferon response by binding to a downstream receptor called STING. Here, we identify natural variants of human STING (hSTING) that are poorly responsive to cGAMP yet, unexpectedly, are normally responsive to DNA and cGAS signaling. We explain this paradox by demonstrating that the cGAS product is actually a noncanonical cyclic dinucleotide, cyclic [G(2'-5')pA(3'-5')p], which contains a single 2'-5' phosphodiester bond. Cyclic [G(2'-5')pA(3'-5')p] potently activates diverse hSTING receptors and, therefore, may be a useful adjuvant or immunotherapeutic. Our results indicate that hSTING variants have evolved to distinguish conventional (3'-5') cyclic dinucleotides, known to be produced mainly by bacteria, from the noncanonical cyclic dinucleotide produced by mammalian cGAS. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  12. The Innate Immune DNA Sensor cGAS Produces a Noncanonical Cyclic Dinucleotide that Activates Human STING

    Directory of Open Access Journals (Sweden)

    Elie J. Diner

    2013-05-01

    Full Text Available The presence of foreign DNA in the cytosol of mammalian cells elicits a potent antiviral interferon response. Recently, cytosolic DNA was proposed to induce the synthesis of cyclic GMP-AMP (cGAMP upon binding to an enzyme called cGAMP synthase (cGAS. cGAMP activates an interferon response by binding to a downstream receptor called STING. Here, we identify natural variants of human STING (hSTING that are poorly responsive to cGAMP yet, unexpectedly, are normally responsive to DNA and cGAS signaling. We explain this paradox by demonstrating that the cGAS product is actually a noncanonical cyclic dinucleotide, cyclic [G(2′-5′pA(3′-5′p], which contains a single 2′-5′ phosphodiester bond. Cyclic [G(2′-5′pA(3′-5′p] potently activates diverse hSTING receptors and, therefore, may be a useful adjuvant or immunotherapeutic. Our results indicate that hSTING variants have evolved to distinguish conventional (3′-5′ cyclic dinucleotides, known to be produced mainly by bacteria, from the noncanonical cyclic dinucleotide produced by mammalian cGAS.

  13. Studies on Relationship between Serum Nitric Oxide and Plasma Cyclic Guanosine Monophosphate and Prolonged Bleeding after Medical Abortion as well as Prophylaxis and Treatment of Bleeding with Traditional Chinese Medicine

    Institute of Scientific and Technical Information of China (English)

    廖玎玲; 谭布珍; 辛华; 贺晓菊

    1999-01-01

    Objectives To study the relationship between serum nitric oxide(NO and plasma cyclic guanosine monophosphate(cGMP)and prolonged bleeding after medical abortion.Methods A total of 120women having received medical abortions at random were recruited and divided into two groups:the one(Group A,n=60) taking “Gong-Fu Mixture(Uterus-Recovering Mixture)”and the other(Group B,n=60)not taking it after abortion.On d 10,20 and 30 after medical abortion,serum NO and plasma cGMP were tested before and after mifepristone administration and 10 d later by Gresis reac-tion method and radioimmunoassay respectively.Results NO concentration in serum and cGMP concentration in plasma decreased signifi-cantly after taking mifeprlstone given(P<0. 05).Ten days later,the number of thos ewith bleeding discontinuation in the group A was significantly greater than that in the group B(P<0.05).Serum NO level and plasma cGMP level in the group A de-creased more significantly than those in the group B(P<0. 05).Conclusion The slow decrease of serum NO and plasma cGMP is closely related to prolonged bleeding after medical abortion.“Gong-Fu Mixture(uterus-recovering mixture)”is effective in prevention and treatment of prolonged bleeding.

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

    Science.gov (United States)

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

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

  15. The Role of Growth Factors (VEGF, TGF-β1 and Cyclic Guanosine Monophosphate in the Formation of Pulmonary Hypertension in Children with Bronchopulmonary Dysplasia

    Directory of Open Access Journals (Sweden)

    A.S. Senatorova

    2013-10-01

    Full Text Available In 82 children with bronchopulmonary dysplasia (from 1 to 36 months of corrected age we investigated the level of VEGF, TGF-β1 in blood and cyclic guanosine monophosphate (cGMP in sputum. It was revealed that children with bronchopulmonary dysplasia had a significant increase in TGF-β1 (p < 0.05 and cGMP (p < 0.01–0.001, reduced VEGF (p < 0.05, indicating inhibition of angiogenesis, activation of fibrosis factors and endothelium-dependent vasodilation. Reliable direct dependence of activation of TGF-β1 in blood and cGMP in sputum, as well as inverse correlation between VEGF in blood and rLA had been proved, which gave reason to think of pulmonary hypertension as an adverse factor in fibrosis activation and angiogenesis inhibition in children with bronchopulmonary dysplasia. Reduced oxygen saturation and oxygen partial pressure moderately activated cGMP, but did not provide a sufficient reduction of pressure in the pulmonary artery.

  16. Adenosine 3':5'-cyclic monophosphate in higher plants: Isolation and characterization of adenosine 3':5'-cyclic monophosphate from Kalanchoe and Agave.

    Science.gov (United States)

    Ashton, A R; Polya, G M

    1977-01-01

    1.3':5'-Cyclic AMP was extensively purified from Kalanchoe daigremontiana and Agave americana by neutral alumina and anion- and cation-exchange column chromatography. Inclusion of 3':5'-cyclic [8-3H]AMP from the point of tissue extraction permitted calculation of yields. The purification procedure removed contaminating material that was shown to interfere with the 3':5'-cyclic AMP estimation and characterization procedures. 2. The partially purified 3':5'-cyclic AMP was quantified by means of a radiochemical saturation assay using an ox heart 3':5'-cyclic AMP-binding protein and by an assay involving activation of a mammalian protein kinase. 3. The plant 3':5'-cyclic AMP co-migrated with 3':5'-cyclic [8-3H]AMP on cellulose chromatography, poly(ethyleneimine)-cellulose chromatography and silica-gel t.l.c. developed with several solvent systems. 4. The plant 3':5'-cyclic AMP was degraded by ox heart 3':5'-cyclic nucleotide phosphodiesterase at the same rates as authentic 3':5'-cyclic AMP. 1-Methyl-3-isobutylxanthine (1 mM), a specific inhibitor of the 3':5'-cyclic nucleotide phosphodieterase, completely inhibited such degradation. 5. The concentrations of 3':5'-cyclic AMP satisfying the above criteria in Kalanchoe and Agave were 2-6 and 1 pmol/g fresh wt. respectively. Possible bacterial contribution to these analyses was estimated to be less than 0.002pmol/g fresh wt. Evidence for the occurrence of 3':5'-cyclic AMP in plants is discussed. PMID:196595

  17. cGMP-dependent protein kinase I, the circadian clock, sleep and learning

    OpenAIRE

    Feil, Robert; Hölter, Sabine M; Weindl, Karin; Wurst, Wolfgang; Langmesser, Sonja; Gerling, Andrea; Feil, Susanne; Albrecht, Urs

    2009-01-01

    The second messenger cGMP controls cardiovascular and gastrointestinal homeostasis in mammals. However, its physiological relevance in the nervous system is poorly understood.1 Now, we have reported that the cGMP-dependent protein kinase type I (PRKG1) is implicated in the regulation of the timing and quality of sleep and wakefulness.2 Prkg1 mutant mice showed altered distribution of sleep and wakefulness as well as reduction in rapid-eye-movement sleep (REMS) duration and in non-REMS consoli...

  18. PDE1A inhibition elicits cGMP-dependent relaxation of rat mesenteric arteries

    DEFF Research Database (Denmark)

    Khammy, Makhala Michell; Dalsgaard, Thomas; Larsen, Peter Hjorringgaard

    2017-01-01

    (EC50 = 32 nM). Inhibition of NOS with L-NAME, soluble GC with ODQ, or PKG with Rp-8-Br-PET-cGMP all attenuated PDE1 inhibition-induced relaxation, whereas PKA inhibition with H89 had no effect. CONCLUSION AND IMPLICATIONS: Pde1a was the dominant PDE1 isoform present in VSMC and relaxation mediated...... by PDE1A-inhibition was predominantly driven by enhanced cGMP signalling. These results imply that isoform-selective PDE1 inhibitors are powerful investigative tools allowing examination of physiological and pathological roles of PDE1 isoforms....

  19. Prognosis of Cyclic Vomiting Syndrome

    Directory of Open Access Journals (Sweden)

    J. Gordon Millichap

    2016-03-01

    Full Text Available Investigators from Teikyo University School of Medicine, Tokyo, Japan, evaluated the clinical features, prognosis, and prophylaxis of cyclic vomiting syndrome and the relationship between the syndrome and levels of adrenocorticotropic/antidiuretic hormones (ACTH/ADH.

  20. Functionalized linear and cyclic polyolefins

    Energy Technology Data Exchange (ETDEWEB)

    Tuba, Robert; Grubbs, Robert H.

    2018-02-13

    This invention relates to methods and compositions for preparing linear and cyclic polyolefins. More particularly, the invention relates to methods and compositions for preparing functionalized linear and cyclic polyolefins via olefin metathesis reactions. Polymer products produced via the olefin metathesis reactions of the invention may be utilized for a wide range of materials applications. The invention has utility in the fields of polymer and materials chemistry and manufacture.

  1. Cyclic Processing for Context Fusion

    DEFF Research Database (Denmark)

    Kjærgaard, Mikkel Baun

    2007-01-01

    Many machine-learning techniques use feedback information. However, current context fusion systems do not support this because they constrain processing to be structured as acyclic processing. This paper proposes a generalization which enables the use of cyclic processing in context fusion systems....... A solution is proposed to the inherent problem of how to avoid uncontrollable looping during cyclic processing. The solution is based on finding cycles using graph-coloring and breaking cycles using time constraints....

  2. A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

    Science.gov (United States)

    Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

    1996-01-01

    Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

  3. Unraveling the cellular context of cyclic nucleotide signaling proteins by chemical proteomics

    NARCIS (Netherlands)

    Corradini, E.

    2015-01-01

    Understanding the molecular mechanisms which regulate signal transduction is fundamental to the development of therapeutic molecules for the treatment of several diseases. In particular, signaling proteins, such as cyclic nucleotide dependent enzymes are the orchestrators of many tissue functions.

  4. Heat shock factor-1 intertwines insulin/IGF-1, TGF-β and cGMP signaling to control development and aging

    Directory of Open Access Journals (Sweden)

    Barna János

    2012-11-01

    Full Text Available Abstract Background Temperature affects virtually all cellular processes. A quick increase in temperature challenges the cells to undergo a heat shock response to maintain cellular homeostasis. Heat shock factor-1 (HSF-1 functions as a major player in this response as it activates the transcription of genes coding for molecular chaperones (also called heat shock proteins that maintain structural integrity of proteins. However, the mechanisms by which HSF-1 adjusts fundamental cellular processes such as growth, proliferation, differentiation and aging to the ambient temperature remain largely unknown. Results We demonstrate here that in Caenorhabditis elegans HSF-1 represses the expression of daf-7 encoding a TGF-β (transforming growth factor-beta ligand, to induce young larvae to enter the dauer stage, a developmentally arrested, non-feeding, highly stress-resistant, long-lived larval form triggered by crowding and starvation. Under favorable conditions, HSF-1 is inhibited by crowding pheromone-sensitive guanylate cyclase/cGMP (cyclic guanosine monophosphate and systemic nutrient-sensing insulin/IGF-1 (insulin-like growth factor-1 signaling; loss of HSF-1 activity allows DAF-7 to promote reproductive growth. Thus, HSF-1 interconnects the insulin/IGF-1, TGF-β and cGMP neuroendocrine systems to control development and longevity in response to diverse environmental stimuli. Furthermore, HSF-1 upregulates another TGF-β pathway-interacting gene, daf-9/cytochrome P450, thereby fine-tuning the decision between normal growth and dauer formation. Conclusion Together, these results provide mechanistic insight into how temperature, nutrient availability and population density coordinately influence development, lifespan, behavior and stress response through HSF-1.

  5. Candida albicans ethanol stimulates Pseudomonas aeruginosa WspR-controlled biofilm formation as part of a cyclic relationship involving phenazines.

    Directory of Open Access Journals (Sweden)

    Annie I Chen

    2014-10-01

    Full Text Available In chronic infections, pathogens are often in the presence of other microbial species. For example, Pseudomonas aeruginosa is a common and detrimental lung pathogen in individuals with cystic fibrosis (CF and co-infections with Candida albicans are common. Here, we show that P. aeruginosa biofilm formation and phenazine production were strongly influenced by ethanol produced by the fungus C. albicans. Ethanol stimulated phenotypes that are indicative of increased levels of cyclic-di-GMP (c-di-GMP, and levels of c-di-GMP were 2-fold higher in the presence of ethanol. Through a genetic screen, we found that the diguanylate cyclase WspR was required for ethanol stimulation of c-di-GMP. Multiple lines of evidence indicate that ethanol stimulates WspR signaling through its cognate sensor WspA, and promotes WspR-dependent activation of Pel exopolysaccharide production, which contributes to biofilm maturation. We also found that ethanol stimulation of WspR promoted P. aeruginosa colonization of CF airway epithelial cells. P. aeruginosa production of phenazines occurs both in the CF lung and in culture, and phenazines enhance ethanol production by C. albicans. Using a C. albicans adh1/adh1 mutant with decreased ethanol production, we found that fungal ethanol strongly altered the spectrum of P. aeruginosa phenazines in favor of those that are most effective against fungi. Thus, a feedback cycle comprised of ethanol and phenazines drives this polymicrobial interaction, and these relationships may provide insight into why co-infection with both P. aeruginosa and C. albicans has been associated with worse outcomes in cystic fibrosis.

  6. Antidepressant effect of pramipexole in mice forced swimming test: A cross talk between dopamine receptor and NMDA/nitric oxide/cGMP pathway.

    Science.gov (United States)

    Ostadhadi, Sattar; Imran Khan, Muhammad; Norouzi-Javidan, Abbas; Dehpour, Ahmad-Reza

    2016-07-01

    Pramipexole is a dopamine D2 receptor agonist indicated for treating Parkinson disorder. This study was aimed to investigate the effect of pramipexole in forced swimming test (FST) in mice and the possible involvement of activation of D2 receptors and inhibition of N-methyl-d-aspartate (NMDA) receptors and nitric oxide-cyclic guanosine monophosphate (NO-cGMP) on this effect. Intraperitoneal administration of pramipexole (1-3mg/kg) reduced the immobility time in the FST similar to fluoxetine (20mg/kg, i.p.). This effect of pramipexole (1mg/kg, i.p.) was ceased when mice were pretreated with haloperidol (0.15mg/kg, i.p,) and sulpiride (5mg/kg, i.p) as D2 receptor antagonists, NMDA (75mg/kg,i.p.), l-arginine (750mg/kg, i.p., a substrate for nitric oxide synthase) or sildenafil (5mg/kg, i.p., a phosphodiesterase 5 inhibitor). The administration of MK-801 (0.05mg/kg, i.p., a NMDA receptor antagonist) l-NG-Nitro arginine methyl ester (l-NAME, 10mg/kg, i.p., a non-specific nitric oxide synthase (NOS) inhibitor), 7-nitroindazole (30mg/kg, i.p., a neuronal NOS inhibitor) and methylene blue (10mg/kg, i.p.), an inhibitor of both NOS and soluble guanylyl cyclase (sGC) in combination with the sub-effective dose of pramipexole (0.3mg/kg, i.p.) reduced the immobility. Altogether, our data suggest that the antidepressant-like effect of pramipexole is dependent on the activation of D2 receptor and inhibition of either NMDA receptors and/or NO-cGMP synthesis. These results contribute to the understanding of the mechanisms underlying the antidepressant-like effect of pramipexole and reinforce the role of D2 receptors, NMDA receptors and l-arginine-NO-GMP pathway in the antidepressant mechanism of this agent. Copyright © 2016. Published by Elsevier Masson SAS.

  7. Systemic induction of NO-, redox- and cGMP signalling in the pumpkin extrafascicular phloem upon local leaf wounding

    Directory of Open Access Journals (Sweden)

    Frank eGaupels

    2016-02-01

    Full Text Available Cucurbits developed the unique extrafascicular phloem (EFP as a defensive structure against herbivorous animals. Mechanical leaf injury was previously shown to induce a systemic wound response in the EFP of pumpkin (Cucurbita maxima. Here, we demonstrate that the phloem antioxidant system and protein modifications by NO are strongly regulated during this process. Activities of the central antioxidant enzymes dehydroascorbate reductase, glutathione reductase and ascorbate reductase were rapidly down-regulated at 30 min with a second minimum at 24 h after wounding. As a consequence levels of total ascorbate and glutathione also decreased with similar bi-phasic kinetics. These results hint towards a wound-induced shift in the redox status of the EFP. Nitric oxide (NO is another important player in stress-induced redox signalling in plants. Therefore, we analysed NO-dependent protein modifications in the EFP. Six to 48 h after leaf damage total S-nitrosothiol content and protein S-nitrosylation were clearly reduced, which was contrasted by a pronounced increase in protein tyrosine nitration. Collectively, these findings suggest that NO-dependent S-nitrosylation turned into peroxynitrite-mediated protein nitration upon a stress-induced redox shift probably involving the accumulation of reactive oxygen species within the EFP. Using the biotin switch assay and anti-nitrotyrosine antibodies we identified 9 candidate S-nitrosylated and 6 candidate tyrosine-nitrated phloem proteins. The wound-responsive Phloem Protein 16-1 (PP16-1 and Cyclophilin 18 (CYP18 as well as the 26.5 kD isoform of Phloem Protein 2 (PP2 were amenable to both NO modifications and could represent important redox-sensors within the cucurbit EFP. We also found that leaf injury triggered the systemic accumulation of cyclic guanosine monophosphate (cGMP in the EFP and discuss the possible function of this second messenger in systemic NO and redox signalling within the EFP.

  8. A novel crosstalk between Alk7 and cGMP signaling differentially regulates brown adipocyte function

    Directory of Open Access Journals (Sweden)

    Aileen Balkow

    2015-08-01

    Conclusions: We found a so far unknown crosstalk between cGMP and Alk7 signaling pathways. Tight regulation of Alk7 is required for efficient differentiation of brown adipocytes. Alk7 has differential effects on adipogenic differentiation and the development of the thermogenic program in brown adipocytes.

  9. HMW-GS affect the properties of glutenin particles in GMP and thus flour quality

    NARCIS (Netherlands)

    Don, C.; Mann, G.; Bekes, F.; Hamer, R.J.

    2006-01-01

    Using a unique set of deletion lines, (Olympic×Gabo, varying in high molecular weight glutenin subunit (HMW-GS) composition, but with the same genetic background) it was shown that the presence of glutenin particles in glutenin macropolymer (GMP) is directly related to the presence of certain

  10. The added value of Good Manufacturing Practices (GMP) in the production of radiopharmaceuticals

    NARCIS (Netherlands)

    Gerrits, Edwin; Woerdenbag, Herman; Luurtsema, Geert; de Hooge, Marjolijn; Boersma, Hendrikus

    2017-01-01

    Manufacturers of medicinal products including radiopharmaceuticals have to follow regulations from their governmental organizations as well as professional societies to ensure built-in quality combined with patient safety issues. This chapter is a concise review of Good Manufacturing Practices (GMP)

  11. GMP and AMP as methyl radical traps in the reaction with pentaamminemethylcobalt(III)

    DEFF Research Database (Denmark)

    Kofod, Pauli

    2004-01-01

    as the PBN/13??H3 adduct (PBN = phenyl-N-tert-butylnitrone). When the purine nucleotide was used in large excess, the efficiency of the trapping by the C8 atom was determined by integration of the 13C NMR signals to be 20-25% for GMP and 15-20% for AMP, respectively, at 37??C. ?? 2004 Elsevier Inc. All...

  12. Pro-survival Effects of 17β-Estradiol on Osteocytes Are Mediated by Nitric Oxide/cGMP via Differential Actions of cGMP-dependent Protein Kinases I and II*

    Science.gov (United States)

    Marathe, Nisha; Rangaswami, Hema; Zhuang, Shunhui; Boss, Gerry R.; Pilz, Renate B.

    2012-01-01

    Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser136 and Ser155; these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD. PMID:22117068

  13. Changes of platelet GMP-140 in diabetic nephropathy and its multi-factor regression analysis

    International Nuclear Information System (INIS)

    Wang Zizheng; Du Tongxin; Wang Shukui

    2001-01-01

    The relation of platelet GMP-140 and its related factors with diabetic nephropathy was studied. 144 patients of diabetic mellitus without nephropathy (group without DN, mean suffering duration of 25.5 +- 18.6 months); 80 with diabetic nephropathy (group DN, mean suffering duration of 58.7 +- 31.6 months) and 50 normal controls were chosen in the research. Platelet GMP-140, plasma α 1 -MG, β 2 -MG, and 24 hour urine albumin (ALB), IgG, α 1 -MG, β 2 -MG were detected by RIA, while HBA 1 C via chromatographic separation and FBG, PBG, Ch, TG, HDL, FG via biochemical methods. All the data had been processed with software on computer with t-test and linear regression, and multi-factor analysis were done also. The levels of platelet GMP-140, FG, DBP, TG, HBA 1 C and PBG in group DN were significantly higher than those of group without DN and normal control (P 0.05), while they were higher than those of normal controls. Multi-factor analysis of platelet GMP-140 with TG, DBP and HBA 1 C were performed in 80 patients with DN (P 1 C are the independent factors enhancing the activation of platelets. The disturbance of lipid metabolism in type II diabetic mellitus may also enhance the activation of platelets. Elevation of blood pressure may accelerate the initiation and deterioration of DN in which change of platelet GMP-140 is an independent factor. Elevation of HBA 1 C and blood glucose are related closely to the diabetic nephropathy

  14. Cohesive Properties of the Caulobacter crescentus Holdfast Adhesin Are Regulated by a Novel c-di-GMP Effector Protein

    Directory of Open Access Journals (Sweden)

    Kathrin S. Sprecher

    2017-03-01

    Full Text Available When encountering surfaces, many bacteria produce adhesins to facilitate their initial attachment and to irreversibly glue themselves to the solid substrate. A central molecule regulating the processes of this motile-sessile transition is the second messenger c-di-GMP, which stimulates the production of a variety of exopolysaccharide adhesins in different bacterial model organisms. In Caulobacter crescentus, c-di-GMP regulates the synthesis of the polar holdfast adhesin during the cell cycle, yet the molecular and cellular details of this control are currently unknown. Here we identify HfsK, a member of a versatile N-acetyltransferase family, as a novel c-di-GMP effector involved in holdfast biogenesis. Cells lacking HfsK form highly malleable holdfast structures with reduced adhesive strength that cannot support surface colonization. We present indirect evidence that HfsK modifies the polysaccharide component of holdfast to buttress its cohesive properties. HfsK is a soluble protein but associates with the cell membrane during most of the cell cycle. Coincident with peak c-di-GMP levels during the C. crescentus cell cycle, HfsK relocalizes to the cytosol in a c-di-GMP-dependent manner. Our results indicate that this c-di-GMP-mediated dynamic positioning controls HfsK activity, leading to its inactivation at high c-di-GMP levels. A short C-terminal extension is essential for the membrane association, c-di-GMP binding, and activity of HfsK. We propose a model in which c-di-GMP binding leads to the dispersal and inactivation of HfsK as part of holdfast biogenesis progression.

  15. Cyclic nucleotide dependent dephosphorylation of regulator of G-protein signaling 18 in human platelets.

    LENUS (Irish Health Repository)

    Gegenbauer, Kristina

    2013-11-01

    Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets.

  16. On Improvements of Cyclic MUSIC

    Directory of Open Access Journals (Sweden)

    H. Howard Fan

    2005-01-01

    Full Text Available Many man-made signals encountered in communications exhibit cyclostationarity. By exploiting cyclostationarity, cyclic MUSIC has been shown to be able to separate signals with different cycle frequencies, thus, to be able to perform signal selective direction of-arrival (DOA estimation. However, as will be shown in this paper, the DOA estimation of cyclic MUSIC is actually biased. We show in this paper that by properly choosing the frequency for evaluating the steering vector, the bias of DOA estimation can be substantially reduced and the performance can be improved. Furthermore, we propose another algorithm exploiting cyclic conjugate correlation to further improve the performance of DOA estimation. Simulation results show the effectiveness of both of our methods.

  17. High-throughput screening for compounds that modulate the cellular c-di-GMP level in bacteria

    DEFF Research Database (Denmark)

    Groizeleau, Julie; Andersen, Jens Bo; Givskov, Michael

    2017-01-01

    . The secondary messenger c-di-GMP is a positive regulator of biofilm formation in many clinically relevant bacteria, and it is assumed that drugs that lower the intracellular level of c-di-GMP will force biofilm bacteria into a more treatable planktonic lifestyle. We describe a protocol for high......-throughput screening of chemical libraries for compounds that lower the c-di-GMP level in bacteria, and potentially can serve as lead compounds in the development of novel biofilm dismantling drugs....

  18. REM sleep deprivation induces endothelial dysfunction and hypertension in middle-aged rats: Roles of the eNOS/NO/cGMP pathway and supplementation with L-arginine.

    Science.gov (United States)

    Jiang, Jiaye; Gan, Zhongyuan; Li, Yuan; Zhao, Wenqi; Li, Hanqing; Zheng, Jian-Pu; Ke, Yan

    2017-01-01

    Sleep loss can induce or aggravate the development of cardiovascular and cerebrovascular diseases. However, the molecular mechanism underlying this phenomenon is poorly understood. The present study was designed to investigate the effects of REM sleep deprivation on blood pressure in rats and the underlying mechanisms of these effects. After Sprague-Dawley rats were subjected to REM sleep deprivation for 5 days, their blood pressures and endothelial function were measured. In addition, one group of rats was given continuous access to L-arginine supplementation (2% in distilled water) for the 5 days before and the 5 days of REM sleep deprivation to reverse sleep deprivation-induced pathological changes. The results showed that REM sleep deprivation decreased body weight, increased blood pressure, and impaired endothelial function of the aortas in middle-aged rats but not young rats. Moreover, nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) concentrations as well as endothelial NO synthase (eNOS) phosphorylation in the aorta were decreased by REM sleep deprivation. Supplementation with L-arginine could protect against REM sleep deprivation-induced hypertension, endothelial dysfunction, and damage to the eNOS/NO/cGMP signaling pathway. The results of the present study suggested that REM sleep deprivation caused endothelial dysfunction and hypertension in middle-aged rats via the eNOS/NO/cGMP pathway and that these pathological changes could be inhibited via L-arginine supplementation. The present study provides a new strategy to inhibit the signaling pathways involved in insomnia-induced or insomnia-enhanced cardiovascular diseases.

  19. Procedures development and methodology of control for application of good manufacture practices (GMP) on human blood irradiation; Desenvolvimento de procedimentos e metodologia de controle para aplicacao de boas praticas de fabricacao (BPF) na irradiacao de sangue humano

    Energy Technology Data Exchange (ETDEWEB)

    Boghi, Claudio

    2008-07-01

    The irradiation of human blood is used to avoid the TA-GVHD (transfusion-associated graft-versus-host-disease), a rare but devastating adverse effect of leukocytes present in blood components for a immunocompetent transfusion recipients. Usually this irradiation practice is performed to a physical elimination of leukocytes. The implementation of the procedures will assure that the properly dose in a range of 25 Gy to 50 Gy will be delivered to the blood in the bag collected in a blood tissue bank. The studies of the procedures in order to establish a GMP (Good Manufacturing Practices) were developed under the guidelines of the standard ISO 11137 - Sterilization of health care products - Requirements for validation and routine control - Radiation sterilization. In this work, two dosimetric systems were used for dose mapping during the studies of irradiator qualification, loading pattern, irradiation process validation and auditing. The CaS0{sub 4}:Dy dosimeter presented difficulties concerning to uncertainty on dose measurement, stability, traceability and calibration. The PMMA and Gafchromic dosimetric systems have shown a better performance and were adopted on studies of irradiators qualification that are necessary to implementation of GMP. The irradiation tests have been done in a Gammacell 220 irradiator. The developed procedures can be adapted for different kinds of gamma irradiators, allowing implanting a quality assurance program and a GMP for blood irradiation. (author)

  20. Design of a cyclic multiverse

    Energy Technology Data Exchange (ETDEWEB)

    Piao Yunsong, E-mail: yspiao@gucas.ac.c [College of Physical Sciences, Graduate School of Chinese Academy of Sciences, Beijing 100049 (China)

    2010-08-09

    Recently, it has been noticed that the amplification of the amplitude of curvature perturbation cycle by cycle can lead to a cyclic multiverse scenario, in which the number of universes increases cycle by cycle. However, this amplification will also inevitably induce either the ultimate end of corresponding cycle, or the resulting spectrum of perturbations inside corresponding universe is not scale invariant, which baffles the existence of observable universes. In this Letter, we propose a design of a cyclic multiverse, in which the observable universe can emerges naturally. The significance of a long period of dark energy before the turnaround of each cycle for this implementing is shown.

  1. Design of a cyclic multiverse

    International Nuclear Information System (INIS)

    Piao Yunsong

    2010-01-01

    Recently, it has been noticed that the amplification of the amplitude of curvature perturbation cycle by cycle can lead to a cyclic multiverse scenario, in which the number of universes increases cycle by cycle. However, this amplification will also inevitably induce either the ultimate end of corresponding cycle, or the resulting spectrum of perturbations inside corresponding universe is not scale invariant, which baffles the existence of observable universes. In this Letter, we propose a design of a cyclic multiverse, in which the observable universe can emerges naturally. The significance of a long period of dark energy before the turnaround of each cycle for this implementing is shown.

  2. Nature of a solar cyclicity

    International Nuclear Information System (INIS)

    Romanchuk, P.R.

    1981-01-01

    The paper contains a critical review of works on studying a cyclic character of solar activity. An introduction of cyclic curves with a frequency spectrum is established to be insolvent. The Wolf, Newcomb and Waldmeier approach seems to be useful. Some evidence is given in favour of the author's conception of solar activity ciclicity of a tide nature. It is accounted for a continuous double and single effect of planets, a resonant character of this effect due to which a 10-year period of Jupiter and Saturn is transformed into an 11-year cycle of activity [ru

  3. Similar expression patterns of bestrophin-4 and cGMP dependent Ca2+-activated chloride channel activity in the vasculature

    DEFF Research Database (Denmark)

    Bouzinova, Elena V.; Larsen, Per; Matchkov, Vladimir

    2008-01-01

    (abstract by Matchkov et. al) that siRNA mediated downregulation of bestrophin-4 is associated with the disappearance of a recently demonstrated2 cGMP-dependent Ca2+-activated Cl- current in vascular smooth muscle cells (SMCs). Here we study the distribution of bestrophin-4-and cGMP dependent Cl- channel...... expressed epitope) Western blot detected a ~65 kDa band in cell lysates from rat mesenteric small arteries and aorta, which was not seen in pulmonary arteries and when preincubated with the immunizing peptide. The distribution of bestrophin-4 mRNA and protein has a pattern similar to the cGMP-dependent Cl......- current in SMCs of different origins. Immunohistochemistry identified bestrophin-4 both in endothelial and SMCs of the vascular tree in the brain, heart, kidney and mesentery, but not in the lungs. We suggest that bestrophin-4 is important for the cGMP dependent, Ca2+ activated Cl- conductance in many...

  4. Effects of the NO/soluble guanylate cyclase/cGMP system on the functions of human platelets.

    Science.gov (United States)

    Makhoul, Stephanie; Walter, Elena; Pagel, Oliver; Walter, Ulrich; Sickmann, Albert; Gambaryan, Stepan; Smolenski, Albert; Zahedi, René P; Jurk, Kerstin

    2018-06-01

    Platelets are circulating sentinels of vascular integrity and are activated, inhibited, or modulated by multiple hormones, vasoactive substances or drugs. Endothelium- or drug-derived NO strongly inhibits platelet activation via activation of the soluble guanylate cyclase (sGC) and cGMP elevation, often in synergy with cAMP-elevation by prostacyclin. However, the molecular mechanisms and diversity of cGMP effects in platelets are poorly understood and sometimes controversial. Recently, we established the quantitative human platelet proteome, the iloprost/prostacyclin/cAMP/protein kinase A (PKA)-regulated phosphoproteome, and the interactions of the ADP- and iloprost/prostacyclin-affected phosphoproteome. We also showed that the sGC stimulator riociguat is in vitro a highly specific inhibitor, via cGMP, of various functions of human platelets. Here, we review the regulatory role of the cGMP/protein kinase G (PKG) system in human platelet function, and our current approaches to establish and analyze the phosphoproteome after selective stimulation of the sGC/cGMP pathway by NO donors and riociguat. Present data indicate an extensive and diverse NO/riociguat/cGMP phosphoproteome, which has to be compared with the cAMP phosphoproteome. In particular, sGC/cGMP-regulated phosphorylation of many membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca 2+ regulation, suggests that the sGC/cGMP system targets multiple signaling networks rather than a limited number of PKG substrate proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Good Manufacturing Practices (GMP) manufacturing of advanced therapy medicinal products: a novel tailored model for optimizing performance and estimating costs.

    Science.gov (United States)

    Abou-El-Enein, Mohamed; Römhild, Andy; Kaiser, Daniel; Beier, Carola; Bauer, Gerhard; Volk, Hans-Dieter; Reinke, Petra

    2013-03-01

    Advanced therapy medicinal products (ATMP) have gained considerable attention in academia due to their therapeutic potential. Good Manufacturing Practice (GMP) principles ensure the quality and sterility of manufacturing these products. We developed a model for estimating the manufacturing costs of cell therapy products and optimizing the performance of academic GMP-facilities. The "Clean-Room Technology Assessment Technique" (CTAT) was tested prospectively in the GMP facility of BCRT, Berlin, Germany, then retrospectively in the GMP facility of the University of California-Davis, California, USA. CTAT is a two-level model: level one identifies operational (core) processes and measures their fixed costs; level two identifies production (supporting) processes and measures their variable costs. The model comprises several tools to measure and optimize performance of these processes. Manufacturing costs were itemized using adjusted micro-costing system. CTAT identified GMP activities with strong correlation to the manufacturing process of cell-based products. Building best practice standards allowed for performance improvement and elimination of human errors. The model also demonstrated the unidirectional dependencies that may exist among the core GMP activities. When compared to traditional business models, the CTAT assessment resulted in a more accurate allocation of annual expenses. The estimated expenses were used to set a fee structure for both GMP facilities. A mathematical equation was also developed to provide the final product cost. CTAT can be a useful tool in estimating accurate costs for the ATMPs manufactured in an optimized GMP process. These estimates are useful when analyzing the cost-effectiveness of these novel interventions. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  6. Potential coupling effects of ammonia-oxidizing and anaerobic ammonium-oxidizing bacteria on completely autotrophic nitrogen removal over nitrite biofilm formation induced by the second messenger cyclic diguanylate.

    Science.gov (United States)

    Wang, Chao; Liu, Sitong; Xu, Xiaochen; Zhao, Chuanqi; Yang, Fenglin; Wang, Dong

    2017-05-01

    The objective of this study was to investigate the influence of extracellular polymeric substance (EPS) on the coupling effects between ammonia-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing (anammox) bacteria for the completely autotrophic nitrogen removal over nitrite (CANON) biofilm formation in a moving bed biofilm reactor (MBBR). Analysis of the quantity of EPS and cyclic diguanylate (c-di-GMP) confirmed that the contents of polysaccharides and c-di-GMP were correlated in the AOB sludge, anammox sludge, and CANON biofilm. The anammox sludge secreted more EPS (especially polysaccharides) than AOB with a markedly higher c-di-GMP content, which could be used by the bacteria to regulate the synthesis of exopolysaccharides that are ultimately used as a fixation matrix, for the adhesion of biomass. Indeed, increased intracellular c-di-GMP concentrations in the anammox sludge enhanced the regulation of polysaccharides to promote the adhesion of AOB and formation of the CANON biofilm. Overall, the results of this study provide new comprehensive information regarding the coupling effects of AOB and anammox bacteria for the nitrogen removal process.

  7. IL-4 induces cAMP and cGMP in human monocytic cells

    Directory of Open Access Journals (Sweden)

    B. Dugas

    1995-01-01

    Full Text Available Human monocytes, preincubated with IFN-γ respond to IL-4 by a cGMP increase through activation of an inducible NO synthase. Here, IL-4 was found to induce an accumulation of cGMP (1 – 3 min and cAMP (20 – 25 min in unstimulated monocytes. This was impaired with NOS inhibitors, but also with EGTA and calcium/calmodulin inhibitors. These results suggest that: (1 IL-4 may stimulate different NOS isoforms in resting and IFN-γ activated monocytes, and (2 cAMP accumulation may be partially dependent on the NO pathway. By RT-PCR, a type III constitutive NOS mRNA was detected in U937 monocytic cells. IL-4 also increased the [Ca2+]i in these cells. Different NOS may thus be expressed in monocytic cells depending on their differentiation and the signals they receive.

  8. cGMP signaling as a target for the prevention and treatment of breast cancer.

    Science.gov (United States)

    Windham, Perrin F; Tinsley, Heather N

    2015-04-01

    One in eight women in the United States will be diagnosed with invasive breast cancer in her lifetime. Advances in therapeutic strategies, diagnosis, and improved awareness have resulted in a significant reduction in breast cancer related mortality. However, there is a continued need for more effective and less toxic drugs for both the prevention and the treatment of breast cancer in order to see a continued decline in the morbidity and mortality associated with this disease. Recent studies suggest that the cGMP signaling pathway may be aberrantly regulated in breast cancer. As such, this pathway may serve as a source of novel targets for future breast cancer drug discovery efforts. This review provides an overview of cGMP signaling in normal physiology and in breast cancer as well as current strategies being investigated for targeting this pathway in breast cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Deformation mechanisms in cyclic creep and fatigue

    International Nuclear Information System (INIS)

    Laird, C.

    1979-01-01

    Service conditions in which static and cyclic loading occur in conjunction are numerous. It is argued that an understanding of cyclic creep and cyclic deformation are necessary both for design and for understanding creep-fatigue fracture. Accordingly a brief, and selective, review of cyclic creep and cyclic deformation at both low and high strain amplitudes is provided. Cyclic loading in conjunction with static loading can lead to creep retardation if cyclic hardening occurs, or creep acceleration if softening occurs. Low strain amplitude cyclic deformation is understood in terms of dislocation loop patch and persistent slip band behavior, high strain deformation in terms of dislocation cell-shuttling models. While interesting advances in these fields have been made in the last few years, the deformation mechanisms are generally poorly understood

  10. Sequencing Cyclic Peptides by Multistage Mass Spectrometry

    Science.gov (United States)

    Mohimani, Hosein; Yang, Yu-Liang; Liu, Wei-Ting; Hsieh, Pei-Wen; Dorrestein, Pieter C.; Pevzner, Pavel A.

    2012-01-01

    Some of the most effective antibiotics (e.g., Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. While hundreds of biomedically important cyclic peptides have been sequenced, the computational techniques for sequencing cyclic peptides are still in their infancy. Previous methods for sequencing peptide antibiotics and other cyclic peptides are based on Nuclear Magnetic Resonance spectroscopy, and require large amount (miligrams) of purified materials that, for most compounds, are not possible to obtain. Recently, development of mass spectrometry based methods has provided some hope for accurate sequencing of cyclic peptides using picograms of materials. In this paper we develop a method for sequencing of cyclic peptides by multistage mass spectrometry, and show its advantages over single stage mass spectrometry. The method is tested on known and new cyclic peptides from Bacillus brevis, Dianthus superbus and Streptomyces griseus, as well as a new family of cyclic peptides produced by marine bacteria. PMID:21751357

  11. Cyclic peptide therapeutics: past, present and future.

    Science.gov (United States)

    Zorzi, Alessandro; Deyle, Kaycie; Heinis, Christian

    2017-06-01

    Cyclic peptides combine several favorable properties such as good binding affinity, target selectivity and low toxicity that make them an attractive modality for the development of therapeutics. Over 40 cyclic peptide drugs are currently in clinical use and around one new cyclic peptide drug enters the market every year on average. The vast majority of clinically approved cyclic peptides are derived from natural products, such as antimicrobials or human peptide hormones. New powerful techniques based on rational design and in vitro evolution have enabled the de novo development of cyclic peptide ligands to targets for which nature does not offer solutions. A look at the cyclic peptides currently under clinical evaluation shows that several have been developed using such techniques. This new source for cyclic peptide ligands introduces a freshness to the field, and it is likely that de novo developed cyclic peptides will be in clinical use in the near future. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Exogenous Hydrogen Peroxide Contributes to Heme Oxygenase-1 Delaying Programmed Cell Death in Isolated Aleurone Layers of Rice Subjected to Drought Stress in a cGMP-Dependent Manner.

    Science.gov (United States)

    Wang, Guanghui; Xiao, Yu; Deng, Xiaojiang; Zhang, Heting; Li, Tingge; Chen, Huiping

    2018-01-01

    Hydrogen peroxide (H 2 O 2 ) is a reactive oxygen species (ROS) that plays a dual role in plant cells. Here, we discovered that drought (20% polyethylene glycol-6000, PEG)-triggered decreases of HO-1 transcript expression and HO activity. However, exogenous H 2 O 2 contributed toward the increase in HO-1 gene expression and activity of the enzyme under drought stress. Meanwhile, the HO-1 inducer hematin could mimic the effects of the H 2 O 2 scavengers ascorbic acid (AsA) and dimethylthiourea (DMTU) and the H 2 O 2 synthesis inhibitor diphenyleneiodonium (DPI) for scavenging or diminishing drought-induced endogenous H 2 O 2 . Conversely, the zinc protoporphyrin IX (ZnPPIX), an HO-1-specific inhibitor, reversed the effects of hematin. We further analyzed the endogenous H 2 O 2 levels and HO-1 transcript expression levels of aleurone layers treated with AsA, DMTU, and DPI in the presence of exogenous H 2 O 2 under drought stress, respectively. The results showed that in aleurone layers subjected to drought stress, when the endogenous H 2 O 2 level was inhibited, the effect of exogenous H 2 O 2 on the induction of HO-1 was enhanced. Furthermore, exogenous H 2 O 2 -activated HO-1 effectively enhanced amylase activity. Application of 8-bromoguanosine 3',5'-cyclic guanosine monophosphate (8-Br-cGMP) (the membrane permeable cGMP analog) promoted the effect of exogenous H 2 O 2 -delayed PCD of aleurone layers in response to drought stress. More importantly, HO-1 delayed the programmed cell death (PCD) of aleurone layers by cooperating with nitric oxide (NO), and the delayed effect of NO on PCD was achieved via mediation by cGMP under drought stress. In short, in rice aleurone layers, exogenous H 2 O 2 (as a signaling molecule) triggered HO-1 and delayed PCD via cGMP which possibly induced amylase activity under drought stress. In contrast, as a toxic by-product of cellular metabolism, the drought-generated H 2 O 2 promoted cell death.

  13. Exogenous Hydrogen Peroxide Contributes to Heme Oxygenase-1 Delaying Programmed Cell Death in Isolated Aleurone Layers of Rice Subjected to Drought Stress in a cGMP-Dependent Manner

    Science.gov (United States)

    Wang, Guanghui; Xiao, Yu; Deng, Xiaojiang; Zhang, Heting; Li, Tingge; Chen, Huiping

    2018-01-01

    Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) that plays a dual role in plant cells. Here, we discovered that drought (20% polyethylene glycol-6000, PEG)-triggered decreases of HO-1 transcript expression and HO activity. However, exogenous H2O2 contributed toward the increase in HO-1 gene expression and activity of the enzyme under drought stress. Meanwhile, the HO-1 inducer hematin could mimic the effects of the H2O2 scavengers ascorbic acid (AsA) and dimethylthiourea (DMTU) and the H2O2 synthesis inhibitor diphenyleneiodonium (DPI) for scavenging or diminishing drought-induced endogenous H2O2. Conversely, the zinc protoporphyrin IX (ZnPPIX), an HO-1-specific inhibitor, reversed the effects of hematin. We further analyzed the endogenous H2O2 levels and HO-1 transcript expression levels of aleurone layers treated with AsA, DMTU, and DPI in the presence of exogenous H2O2 under drought stress, respectively. The results showed that in aleurone layers subjected to drought stress, when the endogenous H2O2 level was inhibited, the effect of exogenous H2O2 on the induction of HO-1 was enhanced. Furthermore, exogenous H2O2-activated HO-1 effectively enhanced amylase activity. Application of 8-bromoguanosine 3′,5′-cyclic guanosine monophosphate (8-Br-cGMP) (the membrane permeable cGMP analog) promoted the effect of exogenous H2O2-delayed PCD of aleurone layers in response to drought stress. More importantly, HO-1 delayed the programmed cell death (PCD) of aleurone layers by cooperating with nitric oxide (NO), and the delayed effect of NO on PCD was achieved via mediation by cGMP under drought stress. In short, in rice aleurone layers, exogenous H2O2 (as a signaling molecule) triggered HO-1 and delayed PCD via cGMP which possibly induced amylase activity under drought stress. In contrast, as a toxic by-product of cellular metabolism, the drought-generated H2O2 promoted cell death. PMID:29449858

  14. Simulation of an Aspheric Glass Lens Forming Behavior in Progressive GMP Process

    International Nuclear Information System (INIS)

    Chang, Sung Ho; Lee, Young Min; Kang, Jeong Jin; Hong, Seok Kwan; Shin, Gwang Ho; Heo, Young Moo; Jung, Tae Sung

    2007-01-01

    Recently, GMP(Glass Molding Press) process is mainly used to produce aspheric glass lenses. Because glass lens is heated at high temperature above Tg (Transformation Temperature) for forming the glass, the quality of aspheric glass lens is deteriorated by residual stresses which are generated in a aspheric glass lens after forming. In this study, as a fundamental study to develop the mold for progressive GMP process, we conducted a aspheric glass lens forming simulation. Prior to a aspheric glass lens forming simulation, compression and thermal conductivity tests were carried out to obtain mechanical and thermal properties of K-PBK40 which is newly developed material for precision molding, and flow characteristics of K-PBK40 were obtained at high temperature. Then, using the flow characteristics obtained, compression simulation was carried out and compared with the experimental result for the purpose of verifying the obtained flow characteristics. Finally, a glass lens press simulation in progressive GMP process was carried out and we could forecast the shape of deformed glass lenses and residual stresses contribution in the structure of deformed glass lenses after forming

  15. A proposed impact assessment method for genetically modified plants (AS-GMP Method)

    International Nuclear Information System (INIS)

    Jesus-Hitzschky, Katia Regina Evaristo de; Silveira, Jose Maria F.J. da

    2009-01-01

    An essential step in the development of products based on biotechnology is an assessment of their potential economic impacts and safety, including an evaluation of the potential impact of transgenic crops and practices related to their cultivation on the environment and human or animal health. The purpose of this paper is to provide an assessment method to evaluate the impact of biotechnologies that uses quantifiable parameters and allows a comparative analysis between conventional technology and technologies using GMOs. This paper introduces a method to perform an impact analysis associated with the commercial release and use of genetically modified plants, the Assessment System GMP Method. The assessment is performed through indicators that are arranged according to their dimension criterion likewise: environmental, economic, social, capability and institutional approach. To perform an accurate evaluation of the GMP specific indicators related to genetic modification are grouped in common fields: genetic insert features, GM plant features, gene flow, food/feed field, introduction of the GMP, unexpected occurrences and specific indicators. The novelty is the possibility to include specific parameters to the biotechnology under assessment. In this case by case analysis the factors of moderation and the indexes are parameterized to perform an available assessment.

  16. Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase.

    Directory of Open Access Journals (Sweden)

    Louisa McRobert

    2008-06-01

    Full Text Available Malaria parasite transmission requires differentiation of male and female gametocytes into gametes within a mosquito following a blood meal. A mosquito-derived molecule, xanthurenic acid (XA, can trigger gametogenesis, but the signalling events controlling this process in the human malaria parasite Plasmodium falciparum remain unknown. A role for cGMP was revealed by our observation that zaprinast (an inhibitor of phosphodiesterases that hydrolyse cGMP stimulates gametogenesis in the absence of XA. Using cGMP-dependent protein kinase (PKG inhibitors in conjunction with transgenic parasites expressing an inhibitor-insensitive mutant PKG enzyme, we demonstrate that PKG is essential for XA- and zaprinast-induced gametogenesis. Furthermore, we show that intracellular calcium (Ca2+ is required for differentiation and acts downstream of or in parallel with PKG activation. This work defines a key role for PKG in gametogenesis, elucidates the hierarchy of signalling events governing this process in P. falciparum, and demonstrates the feasibility of selective inhibition of a crucial regulator of the malaria parasite life cycle.

  17. Good manufacturing practice (GMP) compliance in the biologics sector: plasma fractionation.

    Science.gov (United States)

    Ways, J P; Preston, M S; Baker, D; Huxsoll, J; Bablak, J

    1999-12-01

    The U.S. blood supply is the safest it has ever been. Due to blood safety and the introduction of viral inactivation/clearance technologies, protein therapies derived from human blood have also in recent years had a history of product safety. Nevertheless, since 1995, the plasma-fractionation industry has experienced increased compliance-related actions by the Food and Drug Administration (FDA), as shown by a substantive increase in the number of FDA 483 inspectional observations, FDA warning letters and other FDA regulatory action. An evaluation of these trends shows that they reflect the implementation by the FDA of increased inspectional interest in the plasma-fractionation industry and an evolution of inspectional practices and standards of current good manufacturing practice (cGMP). Plasma fractionators have responded to FDA actions by carefully evaluating and addressing each inspectional observation, assessing impact to product and taking appropriate actions, including corrective actions to prevent future occurrence. They have made major investments in facilities, quality systems, personnel and training to meet the evolving standards of cGMP and in an effort to implement these standards systemically. Through industry associations, manufacturers have further enhanced product safety by adopting additional voluntary standards for plasma to prevent the entry of potentially unsuitable plasma into the production process. The industry remains committed to application of cGMP and to working with the FDA in further evolution of these standards while striving to assure a continued supply of safe, pure and effective plasma-derived therapies.

  18. [The Contribution of GMP-grade Hospital Preparation to Translational Research].

    Science.gov (United States)

    Yonezawa, Atsushi; Kajiwara, Moto; Minami, Ikuko; Omura, Tomohiro; Nakagawa, Shunsaku; Matsubara, Kazuo

    2015-01-01

    Translational research is important for applying the outcomes of basic research studies to practical medical treatments. In exploratory early-phase clinical trials for an innovative therapy, researchers should generally manufacture investigational agents by themselves. To provide investigational agents with safety and high quality in clinical studies, appropriate production management and quality control are essential. In the Department of Pharmacy of Kyoto University Hospital, a manufacturing facility for sterile drugs was established, independent of existing manufacturing facilities. Manuals on production management and quality control were developed according to Good Manufacturing Practices (GMP) for Investigational New Drugs (INDs). Advanced clinical research has been carried out using investigational agents manufactured in our facility. These achievements contribute to both the safety of patients and the reliability of clinical studies. In addition, we are able to do licensing-out of our technique for the manufacture of investigational drugs. In this symposium, we will introduce our GMP grade manufacturing facility for sterile drugs and discuss the role of GMP grade hospital preparation in translational research.

  19. Monopod bucket foundations under cyclic lateral loading

    DEFF Research Database (Denmark)

    Foglia, Aligi; Ibsen, Lars Bo

    on bucket foundations under lateral cyclic loading. The test setup is described in detail and a comprehensive experimental campaign is presented. The foundation is subjected to cyclic overturning moment, cyclic horizontal loading and constant vertical loading, acting on the same plane for thousands...

  20. 40 CFR 721.2120 - Cyclic amide.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Cyclic amide. 721.2120 Section 721... Cyclic amide. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as a cyclic amide (PMN P-92-131) is subject to reporting under this section for the...

  1. Identification, activity and disulfide connectivity of C-di-GMP regulating proteins in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Kajal Gupta

    2010-11-01

    Full Text Available C-di-GMP, a bacterial second messenger plays a key role in survival and adaptation of bacteria under different environmental conditions. The level of c-di-GMP is regulated by two opposing activities, namely diguanylate cyclase (DGC and phosphodiesterase (PDE-A exhibited by GGDEF and EAL domain, respectively in the same protein. Previously, we reported a bifunctional GGDEF-EAL domain protein, MSDGC-1 from Mycobacterium smegmatis showing both these activities (Kumar and Chatterji, 2008. In this current report, we have identified and characterized the homologous protein from Mycobacterium tuberculosis (Rv 1354c named as MtbDGC. MtbDGC is also a bifunctional protein, which can synthesize and degrade c-di-GMP in vitro. Further we expressed Mtbdgc in M. smegmatis and it was able to complement the MSDGC-1 knock out strain by restoring the long term survival of M. smegmatis. Another protein Rv 1357c, named as MtbPDE, is an EAL domain protein and degrades c-di-GMP to pGpG in vitro. Rv1354c and 1357c have seven cysteine amino acids in their sequence, distributed along the full length of the protein. Disulfide bonds play an important role in stabilizing protein structure and regulating protein function. By proteolytic digestion and mass spectrometric analysis of MtbDGC, connectivity between cysteine pairs Cys94-Cys584, Cys2-Cys479 and Cys429-Cys614 was determined, whereas the third cysteine (Cys406 from N terminal was found to be free in MtbDGC protein, which was further confirmed by alkylation with iodoacetamide labeling. Bioinformatics modeling investigations also supported the pattern of disulfide connectivity obtained by Mass spectrometric analysis. Cys406 was mutated to serine by site directed mutagenesis and the mutant MtbC406S was not found to be active and was not able to synthesize or degrade c-di-GMP. The disulfide connectivity established here would help further in understanding the structure - function relationship in MtbDGC.

  2. GMP-140 binds to a glycoprotein receptor on human neutrophils: Evidence for a lectin-like interaction

    International Nuclear Information System (INIS)

    Moore, K.L.; Varki, A.; McEver, R.P.

    1991-01-01

    GMP-140 is a rapidly inducible receptor for neutrophils and monocytes expressed on activated platelets and endothelial cells. It is a member of the selectin family of lectin-like cell surface molecules that mediate leukocyte adhesion. We used a radioligand binding assay to characterize the interaction of purified GMP-140 with human neutrophils. Unstimulated neutrophils rapidly bound [125I]GMP-140 at 4 degrees C, reaching equilibrium in 10-15 min. Binding was Ca2+ dependent, reversible, and saturable at 3-6 nM free GMP-140 with half-maximal binding at approximately 1.5 nM. Receptor density and apparent affinity were not altered when neutrophils were stimulated with 4 beta-phorbol 12-myristate 13-acetate. Treatment of neutrophils with proteases abolished specific binding of [125I]GMP-140. Binding was also diminished when neutrophils were treated with neuraminidase from Vibrio cholerae, which cleaves alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids, or from Newcastle disease virus, which cleaves only alpha 2-3- and alpha 2-8-linked sialic acids. Binding was not inhibited by an mAb to the abundant myeloid oligosaccharide, Lex (CD15), or by the neoglycoproteins Lex-BSA and sialyl-Lex-BSA. We conclude that neutrophils constitutively express a glycoprotein receptor for GMP-140, which contains sialic acid residues that are essential for function. These findings support the concept that GMP-140 interacts with leukocytes by a lectin-like mechanism

  3. A generalized allosteric mechanism for cis-regulated cyclic nucleotide binding domains.

    Directory of Open Access Journals (Sweden)

    Alexandr P Kornev

    2008-04-01

    Full Text Available Cyclic nucleotides (cAMP and cGMP regulate multiple intracellular processes and are thus of a great general interest for molecular and structural biologists. To study the allosteric mechanism of different cyclic nucleotide binding (CNB domains, we compared cAMP-bound and cAMP-free structures (PKA, Epac, and two ionic channels using a new bioinformatics method: local spatial pattern alignment. Our analysis highlights four major conserved structural motifs: 1 the phosphate binding cassette (PBC, which binds the cAMP ribose-phosphate, 2 the "hinge," a flexible helix, which contacts the PBC, 3 the beta(2,3 loop, which provides precise positioning of an invariant arginine from the PBC, and 4 a conserved structural element consisting of an N-terminal helix, an eight residue loop and the A-helix (N3A-motif. The PBC and the hinge were included in the previously reported allosteric model, whereas the definition of the beta(2,3 loop and the N3A-motif as conserved elements is novel. The N3A-motif is found in all cis-regulated CNB domains, and we present a model for an allosteric mechanism in these domains. Catabolite gene activator protein (CAP represents a trans-regulated CNB domain family: it does not contain the N3A-motif, and its long range allosteric interactions are substantially different from the cis-regulated CNB domains.

  4. On charge-3 cyclic monopoles

    International Nuclear Information System (INIS)

    Braden, H W; D'Avanzo, Antonella; Enolski, V Z

    2011-01-01

    We determine the spectral curve of charge-3 BPS su(2) monopoles with C 3 cyclic symmetry. The symmetry means that the genus 4 spectral curve covers a (Toda) spectral curve of genus 2. A well adapted homology basis is presented enabling the theta functions and monopole data of the genus 4 curve to be given in terms of genus 2 data. The Richelot correspondence, a generalization of the arithmetic mean, is used to solve for this genus 2 curve. Results of other approaches are compared

  5. On numerically pluricanonical cyclic coverings

    International Nuclear Information System (INIS)

    Kulikov, V S; Kharlamov, V M

    2014-01-01

    We investigate some properties of cyclic coverings f:Y→X (where X is a complex surface of general type) branched along smooth curves B⊂X that are numerically equivalent to a multiple of the canonical class of X. Our main results concern coverings of surfaces of general type with p g =0 and Miyaoka-Yau surfaces. In particular, such coverings provide new examples of multi-component moduli spaces of surfaces with given Chern numbers and new examples of surfaces that are not deformation equivalent to their complex conjugates

  6. Cyclic graphs and Apery's theorem

    International Nuclear Information System (INIS)

    Sorokin, V N

    2002-01-01

    This is a survey of results about the behaviour of Hermite-Pade approximants for graphs of Markov functions, and a survey of interpolation problems leading to Apery's result about the irrationality of the value ζ(3) of the Riemann zeta function. The first example is given of a cyclic graph for which the Hermite-Pade problem leads to Apery's theorem. Explicit formulae for solutions are obtained, namely, Rodrigues' formulae and integral representations. The asymptotic behaviour of the approximants is studied, and recurrence formulae are found

  7. A system for cyclical voltametry

    International Nuclear Information System (INIS)

    Silva, R.P. da; Chierice, G.O.

    1974-01-01

    The constrution of a system composed by two instruments, voltametric circuit and potenciostate is depicted. Both instruments junction joined so that the voltametric circuit works as a triangular pulse generator, capable of operating with independent ascendant and descendant slope change, with unique pulse of continuous regime. The circuit of the potenciostate is composed of an amplifier with high entrance impedance and capable of supplying relatively high currents at the exit. The equipment was tested to study the aqueous Pb 2+ system in mercury electrode. this system depicted for the cyclical-voltometry technique set in use at I.E.A., Sao Paulo (Brazil), has very good linearity

  8. Effect of ginger, Paullinia cupana, muira puama and l- citrulline, singly or in combination, on modulation of the inducible nitric oxide- NO-cGMP pathway in rat penile smooth muscle cells.

    Science.gov (United States)

    Ferrini, Monica G; Garcia, Eduardo; Abraham, Andrea; Artaza, Jorge N; Nguyen, Sabine; Rajfer, Jacob

    2018-06-01

    COMP-4 is a natural compound-based dietary supplement consisting of the combination of ginger, Paullinia cupana, muira puama and l-citrulline, which when given long-term has been shown in the aged rat to a) upregulate iNOS in the penile smooth muscle cells (SMC), b) reverse the corporal SMC apoptosis and fibrosis associated with corporal veno-occlusive dysfunction (CVOD), and c) improve resulting erectile function. To elucidate the mechanism of how COMP-4 and its individual components modulate the iNOS-cGMP pathway, an in vitro study was conducted using a rat corporal primary SMC culture to determine its effect on NOS, soluble guanylate cyclase (sGC), cGMP and the phosphodiesterase 5 enzyme (PDE5). Primary SMC cultures using the explant technique were initiated by cutting small pieces of corporal tissue from 8 week old Sprague-Dawley rats. The SMC were grown in Dulbecco media with 20% fetal calf serum. The SMC were then incubated with or without COMP-4 (0.69 mg/ml) or its ingredients alone (ginger: 0.225 mg/ml; muira puama, Paullinia cupana and l-citrulline each at 0.9 mg/ml) for up to 24 h mRNA and protein were extracted and used for the determination of NOS, sGC and PDE5 content. cGMP content was determined by ELISA. L-NIL (4 μM) was used as an inhibitor of iNOS activity. Compared to the control values, COMP-4 upregulated expression of cGMP by 85%, induced a 42 fold increase in sGC as well as a 15 fold increase in both iNOS protein and mRNA content while it decreased both PDE5 mRNA and protein content each by about 50%. L-NIL completely inhibited the effect of COMP-4 on cGMP production. When compared with each of the individual four components of COMP-4, it appears that COMP-4 itself had the most profound effect in modulating each one the specific steps within the iNOS-cGMP pathway. This in vitro study demonstrates that COMP-4 is capable of activating the endogenous cellular iNOS-cGMP pathway within the CSM cells, which is theorized to be responsible

  9. Thrombin has biphasic effects on the nitric oxide-cGMP pathway in endothelial cells and contributes to experimental pulmonary hypertension.

    Directory of Open Access Journals (Sweden)

    Katrin F Nickel

    Full Text Available BACKGROUND: A potential role for coagulation factors in pulmonary arterial hypertension has been recently described, but the mechanism of action is currently not known. Here, we investigated the interactions between thrombin and the nitric oxide-cGMP pathway in pulmonary endothelial cells and experimental pulmonary hypertension. PRINCIPAL FINDINGS: Chronic treatment with the selective thrombin inhibitor melagatran (0.9 mg/kg daily via implanted minipumps reduced right ventricular hypertrophy in the rat monocrotaline model of experimental pulmonary hypertension. In vitro, thrombin was found to have biphasic effects on key regulators of the nitric oxide-cGMP pathway in endothelial cells (HUVECs. Acute thrombin stimulation led to increased expression of the cGMP-elevating factors endothelial nitric oxide synthase (eNOS and soluble guanylate cyclase (sGC subunits, leading to increased cGMP levels. By contrast, prolonged exposition of pulmonary endothelial cells to thrombin revealed a characteristic pattern of differential expression of the key regulators of the nitric oxide-cGMP pathway, in which specifically the factors contributing to cGMP elevation (eNOS and sGC were reduced and the cGMP-hydrolyzing PDE5 was elevated (qPCR and Western blot. In line with the differential expression of key regulators of the nitric oxide-cGMP pathway, a reduction of cGMP by prolonged thrombin stimulation was found. The effects of prolonged thrombin exposure were confirmed in endothelial cells of pulmonary origin (HPAECs and HPMECs. Similar effects could be induced by activation of protease-activated receptor-1 (PAR-1. CONCLUSION: These findings suggest a link between thrombin generation and cGMP depletion in lung endothelial cells through negative regulation of the nitric oxide-cGMP pathway, possibly mediated via PAR-1, which could be of relevance in pulmonary arterial hypertension.

  10. Cyclic AMP in rat pancreatic islets

    International Nuclear Information System (INIS)

    Grill, V.; Borglund, E.; Cerasi, E.; Uppsala Univ.

    1977-01-01

    The incorporation of [ 3 H]adenine into cyclic AMP was studied in rat pancreatic islets under varying conditions of labeling. Prolonging the exposure to [ 3 H]adenine progressively augmented the islet cyclic [ 3 H]AMP level. Islets labeled for different periods of time and subsequently incubated (without adenine) in the presence of D-glucose or cholera toxin showed stimulations of intra-islet cyclic [ 3 H]AMP that were proportionate to the levels of radioactive nucleotide present under non-stimulatory conditions. Labeling the islets in a high glucose concentration (27.7 mM) did not modify the nucleotide responses to glucose or cholera toxin. The specific activity of cyclic [ 3 H]AMP, determined by simultaneous assay of cyclic [ 3 H]AMP and total cyclic AMP, was not influenced by glucose or cholera toxin. Glucose had no effect on the specific activity of labeled ATP

  11. A Scientific Calculator for Exact Real Number Computation Based on LRT, GMP and FC++

    Directory of Open Access Journals (Sweden)

    J. A. Hernández

    2012-03-01

    Full Text Available Language for Redundant Test (LRT is a programming language for exact real number computation. Its lazy evaluation mechanism (also called call-by-need and its infinite list requirement, make the language appropriate to be implemented in a functional programming language such as Haskell. However, a direction translation of the operational semantics of LRT into Haskell as well as the algorithms to implement basic operations (addition subtraction, multiplication, division and trigonometric functions (sin, cosine, tangent, etc. makes the resulting scientific calculator time consuming and so inefficient. In this paper, we present an alternative implementation of the scientific calculator using FC++ and GMP. FC++ is a functional C++ library while GMP is a GNU multiple presicion library. We show that a direct translation of LRT in FC++ results in a faster scientific calculator than the one presented in Haskell.El lenguaje de verificación redundante (LRT, por sus siglas en inglés es un lenguaje de programación para el cómputo con números reales exactos. Su método de evaluación lazy (o mejor conocido como llamada por necesidad y el manejo de listas infinitas requerido, hace que el lenguaje sea apropiado para su implementación en un lenguaje funcional como Haskell. Sin embargo, la implementación directa de la semántica operacional de LRT en Haskell así como los algoritmos para funciones básicas (suma, resta, multiplicación y división y funciones trigonométricas (seno, coseno, tangente, etc hace que la calculadora científica resultante sea ineficiente. En este artículo, presentamos una implementación alternativa de la calculadora científica usando FC++ y GMP. FC++ es una librería que utiliza el paradigma Funcional en C++ mientras que GMP es una librería GNU de múltiple precisión. En el artículo mostramos que la implementación directa de LRT en FC++ resulta en una librería más eficiente que la implementada en Haskell.

  12. GMP reverses the facilitatory effect of glutamate on inhibitory avoidance task in rats.

    Science.gov (United States)

    Rubin, M A; Jurach, A; da Costa Júnior, E M; Lima, T T; Jiménez-Bernal, R E; Begnini, J; Souza, D O; de Mello, C F

    1996-09-02

    Previous studies have demonstrated that post-training intrahippocampal glutamate administration improves inhibitory avoidance task performance in rats. Antagonism of the agonist actions of glutamate by guanine nucleotides has been shown at the molecular and behavioural level. In the present investigation we demonstrate that intrahippocampal co-administration of GMP (guanosine 5'-monophosphate) reverses the facilitatory effect of glutamate on the inhibitory avoidance learning paradigm and inhibits [3H]glutamate binding in hippocampal synaptic plasma membranes. These results suggest that guanine nucleotides may modulate glutamate actions.

  13. Plasma-focused cyclic accelerators

    International Nuclear Information System (INIS)

    Mondelli, A.A.; Chernin, D.P.

    1985-01-01

    The use of ambient plasma to neutralize the transverse forces of an intense particle beam has been known for many years. Most recently, the so-called ion-focused regime (IFR) for beam propagation has been used as a means of focusing intense electron beams in linear accelerators and suggested for injecting an electron beam across magnetic field lines into a high-current cyclic accelerator. One technique for generating the required background plasma for IFR propagation is to use a laser to ionize ambient gas in the accelerator chamber. For cyclic accelerators a technique is required for carrying the plasma channel and the beam around a bend. Multiple laser-generated channels with dipole magnetic fields to switch the beam from one channel to the next have been tested at Sandia. This paper discusses an alternative means of plasma production for IFR, viz. by using rf breakdown. For this approach the accelerator chamber acts as a waveguide. With a suitable driving frequency, a waveguide mode can be driven which has its peak field intensity on the axis with negligible fields at the chamber walls. The plasma production and hence the beam propagation is thereby isolated from the walls. This technique is not limited to toroidal accelerators. It may be applied to any accelerator or recirculator geometry as well as for beam steering and for injection or extraction of beams in closed accelerator configurations

  14. Cyclic Vomiting Syndrome in Children

    Directory of Open Access Journals (Sweden)

    T.V. Sorokman

    2016-08-01

    Full Text Available Introduction. Cyclic vomiting syndrome (CVS — is a fairly common disease of unknown etiology that affects children of all age groups and sometimes adult population and refers to the functional disorders of the gastrointestinal tract. Objective: to evaluate the effectiveness of the usage of Rehydron Optim for oral rehydration therapy in children. Materials and methods. The treatment of 40 children aged 3 to 11 years with CVS (15 persons and primary acetonemic syndrome (25 persons in the period of acetonemic crisis, including 15 boys and 25 girls, was analyzed. All children were observed in the outpatient department of the Regional children’s hospital of Chernivtsi. Diagnosis was established based on anamnesis, clinical and laboratory data. Patients underwent required clinico-biological tests and instrumental examinations. The dynamics of the following syndromes was investigated: pain, vomiting, dehydration and intoxication. Rehydration therapy in all cases was oral with the usage of Rehydron Optim. Results of the study and their discussion. A cyclical vomiting was observed in children with primary acetonemic syndrome with satisfactory condition in attack-free period. Migraine-like headaches prevailed in 36 patients (80 %, and the age of these patients was older than 7 years. Same children had episodes of paroxysmal autonomic failure. Almost all surveyed children had in their family history the risk factors for CVS development. All children had positive dynamics of the main basic clinical manifestations on the background of oral rehydration therapy using Rehydron Optim. Within the 1st day of oral rehydration therapy with Rehydron Optim in children, we have noted a significant decrease in the incidence of lethargy, vomiting, spastic abdominal pain, smell of acetone in the exhaled air (p < 0.05. In children with the I degree of dehydration, clinical signs of dehydration were not seen before the treatment, and children with the II degree had an

  15. [Cyclic Cushing's Syndrome - rare or rarely recognized].

    Science.gov (United States)

    Kiałka, Marta; Doroszewska, Katarzyna; Mrozińska, Sandra; Milewicz, Tomasz; Stochmal, Ewa

    2015-01-01

    Cyclic Cushing's syndrome is a type of Cushing's disease which is characterized by alternating periods of increasing and decreasing levels of cortisol in the blood. The diagnostic criteria for cyclic Cushing's syndrome are at least three periods of hypercortisolism alternating with at least two episodes of normal levels of serum cortisol concentration. The epidemiology, signs, symptoms, pathogenesis and treatment of cyclic Cushing's syndrome have been discussed.

  16. Changes in calmodulin concentration and cyclic 3',5'-nucleotide phosphodiesterase activity in skeletal muscle of hyper- and hypothyroid rats.

    Science.gov (United States)

    Mano, T; Iwase, K; Yoshimochi, I; Sawai, Y; Oda, N; Nishida, Y; Mokuno, T; Kotake, M; Nakai, A; Hayakawa, N

    1995-08-01

    Hyper- and hypothyroid states occasionally induce skeletal muscle dysfunction i.e. periodic paralysis and thyroid myopathy. The etiology of these diseases remains unclear, but several findings suggest that the catecholamine-beta-receptor-cAMP system or other messenger systems are disturbed in these diseases. In this context, we evaluated changes in the cyclic 3',5'-nucleotide metabolic enzyme, cyclic 3',5'-nucleotide phosphodiesterase (PDE) and calmodulin concentrations in skeletal muscles of hyper- and hypothyroid rats. Activities of cyclic AMP-PDE were low in skeletal muscle both from hyper- and hypothyroid rats, and calmodulin concentration was high in hyperthyroid and low in hypothyroid rats, as compared with normal rats. DE-52 column chromatographic analysis showed that the cGMP hydrolytic activity in peak I and the cAMP hydrolytic activity in peak II were decreased in hypothyroid rats, whereas cAMP hydrolytic activity in peak III was unchanged. The cAMP hydrolytic activity in peak III was decreased in hyperthyroid rats, but the activities in peaks I and II were unchanged. These findings indicate that cAMP and calmodulin may have some role in skeletal muscle function in the hyperthyroid state, and that cAMP and calmodulin-dependent metabolism may be suppressed in the hypothyroid state.

  17. Studies on the production of endogenous pyrogen by rabbit monocytes: the role of calcium and cyclic nucleotides.

    Science.gov (United States)

    Sigal, S L; Duff, G W; Atkins, E

    1985-01-01

    Rabbit monocytes stimulated with endotoxin produced endogenous pyrogen, even under conditions of high or low extracellular calcium concentrations. Maximal production occurred when the concentration was in the near-physiological range. Prolonged incubation of cells with a calcium chelator prevented subsequent activation with endotoxin, an effect which was rapidly reversible by re-addition of calcium but not other cations. Addition of small amounts of lanthanum, which acts as a calcium channel blocker, prevented the restoration of pyrogen production, indicating that entry of the added calcium into the monocyte was required. Incorporation of a calcium ionophore into the cell membrane did not stimulate pyrogen production, and no measurable influx or efflux of calcium occurred during stimulation with endotoxin. These observations suggest that a slowly exchangeable calcium pool is necessary for the production of endogenous pyrogen, but that a rise in intracellular calcium is not by itself a necessary or sufficient stimulus. This stands in contrast to other biological systems in which Ca2+ directly couples stimulus and hormone secretion. Incubation of cells with agents shown to increase cyclic 3',5' AMP or cyclic 3',5' GMP levels in monocytes similarly did not stimulate pyrogen production or modulate its production by endotoxin stimulation. Thus, cyclic nucleotides also did not play a detectable role as intracellular messengers in this system. Future work is required to define more clearly the mechanism for the production of endogenous pyrogen, given its marked effects on the immune system through lymphocyte activation and temperature regulation.

  18. A concise discussion of the regulatory role of cGMP kinase I in cardiac physiology and pathology.

    Science.gov (United States)

    Hofmann, Franz

    2018-06-22

    The underlying cause of cardiac hypertrophy, fibrosis, and heart failure has been investigated in great detail using different mouse models. These studies indicated that cGMP and cGMP-dependent protein kinase type I (cGKI) may ameliorate these negative phenotypes in the adult heart. Recently, evidence has been published that cardiac mitochondrial BKCa channels are a target for cGKI and that activation of mitoBKCa channels may cause some of the positive effects of conditioning in ischemia/reperfusion injury. It will be pointed out that most studies could not present convincing evidence that it is the cGMP level and the activity cGKI in specific cardiac cells that reduces hypertrophy or heart failure. However, anti-fibrotic compounds stimulating nitric oxide-sensitive guanylyl cyclase may be an upcoming therapy for abnormal cardiac remodeling.

  19. Exercise training improves blood flow to contracting skeletal muscle of older men via enhanced cGMP signaling

    DEFF Research Database (Denmark)

    Piil, Peter Bergmann; Smith Jørgensen, Tue; Egelund, Jon

    2018-01-01

    Physical activity has the potential to offset age-related impairments in the regulation of blood flow and O2 delivery to the exercising muscles; however, the mechanisms underlying this effect of physical activity remain poorly understood. The present study examined the role of cGMP in training...... a period of aerobic high-intensity exercise training. To determine the role of cGMP signaling, pharmacological inhibition of phosphodiesterase 5 (PDE5) was performed. Before training, inhibition of PDE5 increased (P... group; however, these effects of PDE5 inhibition were not detected after training. These findings suggest a role for enhanced cGMP signaling in the training-induced improvement of regulation of blood flow in contracting skeletal muscle of older men....

  20. Modeling Cyclic Variation of Intracranial Pressure

    National Research Council Canada - National Science Library

    Daley, M

    2001-01-01

    ...) recording during mechanical ventilation are due to cyclic extravascular compressional modulation primarily of the cerebral venous bed, an established isovolumetric model of cerebrospinal fluid...

  1. Behaviour of Cohesionless Soils During Cyclic Loading

    DEFF Research Database (Denmark)

    Shajarati, Amir; Sørensen, Kris Wessel; Nielsen, Søren Kjær

    Offshore wind turbine foundations are typically subjected to cyclic loading from both wind and waves, which can lead to unacceptable deformations in the soil. However, no generally accepted standardised method is currently available, when accounting for cyclic loading during the design of offshore...... wind turbine foundations. Therefore a literature study is performed in order to investigate existing research treating the behaviour of cohesionless soils, when subjected to cyclic loading. The behaviour of a soil subjected to cyclic loading is found to be dependent on; the relative density, mean...

  2. Cyclical subnormal separation in A-groups

    International Nuclear Information System (INIS)

    Makarfi, M.U.

    1995-12-01

    Three main results, concerning A-groups in respect of cyclical subnormal separation as defined in, are presented. It is shown in theorem A that any A-group that is generated by elements of prime order and satisfying the cyclical subnormal separation conditions is metabelian. The two other main results give necessary and sufficient conditions for A-groups, that are split extensions of certain abelian p-groups by a metabelian p'-group, to satisfy the cyclical subnormal separation condition. There is also a result which shows that A-groups with elementary abelian Sylow subgroups are cyclically separated as defined. (author). 7 refs

  3. Aging has the opposite effect on cAMP and cGMP circadian variations in rat Leydig cells.

    Science.gov (United States)

    Baburski, Aleksandar Z; Sokanovic, Srdjan J; Andric, Silvana A; Kostic, Tatjana S

    2017-05-01

    The Leydig cell physiology displays a circadian rhythm driven by a complex interaction of the reproductive axis hormones and circadian system. The final output of this regulatory process is circadian pattern of steroidogenic genes expression and testosterone production. Aging gradually decreases robustness of rhythmic testosterone secretion without change in pattern of LH secretion. Here, we analyzed effect of aging on circadian variation of cAMP and cGMP signaling in Leydig cells. Results showed opposite effect of aging on cAMP and cGMP daily variation. Reduced amplitude of cAMP circadian oscillation was probably associated with changed expression of genes involved in cAMP production (increased circadian pattern of Adcy7, Adcy9, Adcy10 and decreased Adcy3); cAMP degradation (increased Pde4a, decreased Pde8b, canceled rhythm of Pde4d, completely reversed circadian pattern of Pde7b and Pde8a); and circadian expression of protein kinase A subunits (Prkac/PRKAC and Prkar2a). Aging stimulates expression of genes responsible for cGMP production (Nos2, Gucy1a3 and Gucy1b3/GUCYB3) and degradation (Pde5a, Pde6a and Pde6h) but the overall net effect is elevation of cGMP circadian oscillations in Leydig cells. In addition, the expression of cGMP-dependent kinase, Prkg1/PRKG1 is up-regulated. It seems that aging potentiate cGMP- and reduce cAMP-signaling in Leydig cells. Since both signaling pathways affect testosterone production and clockwork in the cells, further insights into these signaling pathways will help to unravel disorders linked to the circadian timing system, aging and reproduction.

  4. cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING.

    Science.gov (United States)

    Ablasser, Andrea; Goldeck, Marion; Cavlar, Taner; Deimling, Tobias; Witte, Gregor; Röhl, Ingo; Hopfner, Karl-Peter; Ludwig, Janos; Hornung, Veit

    2013-06-20

    Detection of cytoplasmic DNA represents one of the most fundamental mechanisms of the innate immune system to sense the presence of microbial pathogens. Moreover, erroneous detection of endogenous DNA by the same sensing mechanisms has an important pathophysiological role in certain sterile inflammatory conditions. The endoplasmic-reticulum-resident protein STING is critically required for the initiation of type I interferon signalling upon detection of cytosolic DNA of both exogenous and endogenous origin. Next to its pivotal role in DNA sensing, STING also serves as a direct receptor for the detection of cyclic dinucleotides, which function as second messenger molecules in bacteria. DNA recognition, however, is triggered in an indirect fashion that depends on a recently characterized cytoplasmic nucleotidyl transferase, termed cGAMP synthase (cGAS), which upon interaction with DNA synthesizes a dinucleotide molecule that in turn binds to and activates STING. We here show in vivo and in vitro that the cGAS-catalysed reaction product is distinct from previously characterized cyclic dinucleotides. Using a combinatorial approach based on mass spectrometry, enzymatic digestion, NMR analysis and chemical synthesis we demonstrate that cGAS produces a cyclic GMP-AMP dinucleotide, which comprises a 2'-5' and a 3'-5' phosphodiester linkage >Gp(2'-5')Ap(3'-5')>. We found that the presence of this 2'-5' linkage was required to exert potent activation of human STING. Moreover, we show that cGAS first catalyses the synthesis of a linear 2'-5'-linked dinucleotide, which is then subject to cGAS-dependent cyclization in a second step through a 3'-5' phosphodiester linkage. This 13-membered ring structure defines a novel class of second messenger molecules, extending the family of 2'-5'-linked antiviral biomolecules.

  5. cGMP and nitric oxide modulate thrombin-induced endothelial permeability : Regulation via different pathways in human aortic and umbilical vein endothelial cells

    NARCIS (Netherlands)

    Draijer, R.; Atsma, D.E.; Laarse, A. van der; Hinsbergh, V.W.M. van

    1995-01-01

    Previous studies have demonstrated that cGMP and cAMP reduce the endothelial permeability for fluids and macromolecules when the endothelial permeability is increased by thrombin. In this study, we have investigated the mechanism by which cGMP improves the endothelial barrier function and examined

  6. Interactions in the aqueous phase and adsorption at the air-water interface of caseinoglycomacropeptide (GMP) and beta-lactoglobulin mixed systems.

    Science.gov (United States)

    Martinez, María J; Sánchez, Cecilio Carrera; Patino, Juan M Rodríguez; Pilosof, Ana M R

    2009-01-01

    The aim of this work was to study the interactions and adsorption of caseinoglycomacropeptide (GMP) and GMP:beta-lactoglobulin (beta-lg) mixed system in the aqueous phase and at the air-water interface. The existence of associative interactions between GMP and beta-lg in the aqueous phase was investigated by dynamic light scattering, differential scanning calorimetry (DSC), fluorometry and native PAGE-electrophoresis. The surface pressure isotherm and the static and dynamic surface pressure were determined by tensiometry and surface dilatational properties. The results showed that GMP presented higher surface activity than beta-lg at a concentration of 4%wt but beta-lg showed higher film forming ability. In the mixed systems beta-lg dominated the static and dynamic surface pressure and the rheological properties of interfacial films suggesting that beta-lg hinders GMP adsorption because, in simple competition, GMP should dominate because of its higher surface activity. The surface predominance of beta-lg can be attributed to binding of GMP to beta-lg in the aqueous phase that prevents GMP adsorption on its own.

  7. cGMP-dependent protein kinase I, the circadian clock, sleep and learning.

    Science.gov (United States)

    Feil, Robert; Hölter, Sabine M; Weindl, Karin; Wurst, Wolfgang; Langmesser, Sonja; Gerling, Andrea; Feil, Susanne; Albrecht, Urs

    2009-07-01

    The second messenger cGMP controls cardiovascular and gastrointestinal homeostasis in mammals. However, its physiological relevance in the nervous system is poorly understood.1 Now, we have reported that the cGMP-dependent protein kinase type I (PRKG1) is implicated in the regulation of the timing and quality of sleep and wakefulness.2Prkg1 mutant mice showed altered distribution of sleep and wakefulness as well as reduction in rapid-eye-movement sleep (REMS) duration and in non-REMS consolidation. Furthermore, the ability to sustain waking episodes was compromised. These observations were also reflected in wheel-running and drinking activity. A decrease in electroencephalogram power in the delta frequency range (1-4 Hz) under baseline conditions was observed, which was normalized after sleep deprivation. Together with the finding that circadian clock amplitude is reduced in Prkg1 mutants these results indicate a decrease of the wake-promoting output of the circadian system affecting sleep. Because quality of sleep might affect learning we tested Prkg1 mutants in several learning tasks and find normal spatial learning but impaired object recognition memory in these animals. Our findings indicate that Prkg1 impinges on circadian rhythms, sleep and distinct aspects of learning.

  8. Developing a tool for the preparation of GMP audit of pharmaceutical contract manufacturer.

    Science.gov (United States)

    Linna, Anu; Korhonen, Mirka; Mannermaa, Jukka-Pekka; Airaksinen, Marja; Juppo, Anne Mari

    2008-06-01

    Outsourcing is rapidly growing in the pharmaceutical industry. When the manufacturing activities are outsourced, control of the product's quality has to be maintained. One way to confirm contract manufacturer's GMP (Good Manufacturing Practice) compliance is auditing. Audits can be supported for instance by using GMP questionnaires. The objective of this study was to develop a tool for the audit preparation of pharmaceutical contract manufacturers and to validate its contents by using Delphi method. At this phase of the study the tool was developed for non-sterile finished product contract manufacturers. A modified Delphi method was used with expert panel consisting of 14 experts from pharmaceutical industry, authorities and university. The content validity of the developed tool was assessed by a Delphi questionnaire round. The response rate in Delphi questionnaire round was 86%. The tool consisted of 103 quality items, from which 90 (87%) achieved the pre-defined agreement rate level (75%). Thirteen quality items which did not achieve the pre-defined agreement rate were excluded from the tool. The expert panel suggested only minor changes to the tool. The results show that the content validity of the developed audit preparation tool was good.

  9. Phenotypic stability and plasticity in GMP-derived cells as determined by their underlying regulatory network.

    Science.gov (United States)

    Ramírez, Carlos; Mendoza, Luis

    2018-04-01

    Blood cell formation has been recognized as a suitable system to study celular differentiation mainly because of its experimental accessibility, and because it shows characteristics such as hierarchical and gradual bifurcated patterns of commitment, which are present in several developmental processes. Although hematopoiesis has been extensively studied and there is a wealth of molecular and cellular data about it, it is not clear how the underlying molecular regulatory networks define or restrict cellular differentiation processes. Here, we infer the molecular regulatory network that controls the differentiation of a blood cell subpopulation derived from the granulocyte-monocyte precursor (GMP), comprising monocytes, neutrophils, eosinophils, basophils and mast cells. We integrate published qualitative experimental data into a model to describe temporal expression patterns observed in GMP-derived cells. The model is implemented as a Boolean network, and its dynamical behavior is studied. Steady states of the network can be clearly identified with the expression profiles of monocytes, mast cells, neutrophils, basophils, and eosinophils, under wild-type and mutant backgrounds. All scripts are publicly available at https://github.com/caramirezal/RegulatoryNetworkGMPModel. lmendoza@biomedicas.unam.mx. Supplementary data are available at Bioinformatics online.

  10. Experiences of using the GMP audit preparation tool in pharmaceutical contract manufacturer audits.

    Science.gov (United States)

    Linna, Anu; Korhonen, Mirka; Airaksinen, Marja; Juppo, Anne Mari

    2010-06-01

    Use of external contractors is nowadays inevitable in the pharmaceutical industry. Therefore the amount of current good manufacturing practice audits has been increasing. During the audit, a large amount of items should be covered in a limited amount of time. Consequently, pharmaceutical companies should have systematic and effective ways to manage and prepare for the audits. This study is a continuation to the earlier study, where a tool for the preparation of cGMP audit was developed and its content was validated. The objective of this study was to evaluate the usefulness of the developed tool in audit preparation and during the actual cGMP audit. Three qualitative research methods were used in this study (observation, interviews, and opinion survey). First, the validity of the information given through the tool was examined by comparing the responses to the actual conditions observed during the contract manufacturer audits (n = 15). Additionally the opinions of the contract manufacturers of the tool were gathered (n = 10) and the auditors were interviewed (n = 2). The developed tool was proven to be useful in audit preparation phase from both the auditor's and the contract manufacturers' point of view. Furthermore, using the tool can also save some time when performing the audit. The results show that using the tool can give significant support in audit preparation phase and also during the actual audit.

  11. Plasma-focused cyclic accelerators

    International Nuclear Information System (INIS)

    Mondelli, A.A.; Chernin, D.P.

    1985-01-01

    The use of ambient plasma to neutralize the transverse forces of an intense particle beam has been known for many years. Most recently, the so-called ion-focused regime (IFR) for beam propagation has been used as a means of focusing intense electron beams in linear accelerators and suggested for injecting an electron beam across magnetic field lines into a high-current cyclic accelerator. One technique for generating the required background plasma for IFR propagation is to use a laser to ionize ambient gas in the accelerator chamber. This paper discusses an alternative means of plasma production for IFR, viz. by using RF breakdown. For this approach the accelerator chamber acts as a waveguide. This technique is not limited to toroidal accelerators. It may be applied to any accelerator or recirculator geometry as well as for beam steering and for injection or extraction of beams in closed accelerator configurations

  12. Bestrophin-3 (vitelliform macular dystrophy 2-like 3 protein) is essential for the cGMP-dependent calcium-activated chloride conductance in vascular smooth muscle cells

    DEFF Research Database (Denmark)

    Matchkov, Vladimir; Larsen, Per; Bouzinova, Elena V.

    2008-01-01

    have recently characterized a cGMP-dependent Ca(2+)-activated Cl(-) current with unique characteristics in smooth muscle cells. This novel current has been shown to coexist with a "classic" (cGMP-independent) Ca(2+)-activated Cl(-) current and to have characteristics distinct from those previously...... known for Ca(2+)-activated Cl(-) currents. Here, we suggest that a bestrophin, a product of the Best gene family, is responsible for the cGMP-dependent Ca(2+)-activated Cl(-) current based on similarities between the membrane currents produced by heterologous expressions of bestrophins and the cGMP......-dependent Ca(2+)-activated Cl(-) current. This is supported by similarities in the distribution pattern of the cGMP-dependent Ca(2+)-activated Cl(-) current and bestrophin-3 (the product of Best-3 gene) expression in different smooth muscle. Furthermore, downregulation of Best-3 gene expression with small...

  13. THE ANTI-FIBROTIC ACTIONS OF RELAXIN ARE MEDIATED THROUGH A NO-sGC-cGMP-DEPENDENT PATHWAY IN RENAL MYOFIBROBLASTS IN VITRO AND ENHANCED BY THE NO DONOR, DIETHYLAMINE NONOATE

    Directory of Open Access Journals (Sweden)

    Chao eWang

    2016-03-01

    Full Text Available INTRODUCTION: The anti-fibrotic hormone, relaxin, has been inferred to disrupt TGF-beta1/Smad2 phosphorylation (pSmad2 signal transduction and promote collagen-degrading gelatinase activity via a nitric oxide (NO-dependent pathway. Here, we determined the extent to which NO, soluble guanylate cyclase (sGC and cyclic guanosine monophosphate (cGMP were directly involved in the anti-fibrotic actions of relaxin using a selective NO scavenger and sGC inhibitor, and comparing and combining relaxin’s effects with that of an NO donor. METHODS AND RESULTS: Primary renal cortical myofibroblasts isolated from injured rat kidneys were treated with human recombinant relaxin (RLX; 16.8nM, the NO donor, diethylamine NONOate (DEA/NO; 0.5-5uM or the combined effects of RLX (16.8nM and DEA/NO (5uM over 72 hours. The effects of RLX (16.8nM and DEA/NO (5uM were also evaluated in the presence of the NO scavenger, hydroxocobalamin (HXC; 100uM or sGC inhibitor, ODQ (5uM over 72 hours. Furthermore, the effects of RLX (30nM, DEA/NO (5uM and RLX (30nM+DEA/NO (5uM on cGMP levels were directly measured, in the presence or absence of ODQ (5uM. Changes in matrix metalloproteinase (MMP-2, MMP-9 (cell media, pSmad2 and α-smooth muscle actin (α-SMA; a measure myofibroblast differentiation (cell layer were assessed by gelatin zymography and Western blotting, respectively. At the highest concentration tested, both RLX and DEA/NO promoted MMP-2 and MMP-9 levels by 25-33%, while inhibiting pSmad2 and α-SMA expression by up to 50% (all p<0.05 vs untreated and vehicle-treated cells. However, 5uM of DEA/NO was required to produce the effects seen with 16.8nM of RLX over 72 hours. The anti-fibrotic effects of RLX or DEA/NO alone were completely abrogated by HXC and ODQ (both p<0.01 vs RLX alone or DEA/NO alone, but were significantly enhanced when added in combination (all p<0.05 vs RLX alone. Additionally, the direct cGMP-promoting effects of RLX, DEA/NO and RLX+DEA/NO (which all

  14. Assay of cyclic nucleotide phosphodiesterase using radiolabeled and fluorescent substrates

    International Nuclear Information System (INIS)

    Kincaid, R.L.; Manganiello, V.C.

    1988-01-01

    There are four major classes of phosphodiesterase with different specificities for cAMP and cGMP and different allosteric regulators. Type I phosphodiesterase is activated by calmodulin plus Ca/sup 2+/ and has a higher affinity for cGMP than cAMP. Type II phosphodiesterase likewise has a higher affinity for cGMP than cAMP, but the activity toward one substrate is markedly stimulated by low (micromolar) concentrations of the other nucleotide. Type III phosphodiesterase has a higher affinity for cAMP than cGMP; its activity is increased in responsive cells by certain hormones, e.g., insulin, isoproterenol. Type IV phosphodiesterase is the cGMP-specific enzyme, which also has an allosteric binding site for cGMP. An example of this class of enzyme is the one from retinal rod outer segments, which is activated by light via rhodopsin and the guanine nucleotide-binding protein transducin. There appears to be little structural relatedness among these enzymes based on immunologic analysis, consistent with the possibility that divergent forms evolved from an ancestral enzyme. Determination of the amount of a specific form of phosphodiesterase in crude material is often difficult. Modification of assay conditions by judicious choice of substrate and/or inhibitor concentrations may selectively favor (or reduce) the activity of a particular form; in many instances, however, some fractionation of enzymes may be necessary. This is discussed more fully in the final section of this chapter

  15. Cyclic completion of the anamorphic universe

    Science.gov (United States)

    Ijjas, Anna

    2018-04-01

    Cyclic models of the universe have the advantage of avoiding initial conditions problems related to postulating any sort of beginning in time. To date, the best known viable examples of cyclic models have been ekpyrotic. In this paper, we show that the recently proposed anamorphic scenario can also be made cyclic. The key to the cyclic completion is a classically stable, non-singular bounce. Remarkably, even though the bounce construction was originally developed to connect a period of contraction with a period of expansion both described by Einstein gravity, we show here that it can naturally be modified to connect an ordinary contracting phase described by Einstein gravity with a phase of anamorphic smoothing. The paper will present the basic principles and steps in constructing cyclic anamorphic models.

  16. Cyclic sciatica caused by infiltrative endometriosis: MRI findings

    Energy Technology Data Exchange (ETDEWEB)

    Yekeler, Ensar; Kumbasar, Basak; Tunaci, Atadan; Barman, Ahmet; Tunaci, Mehtap [Department of Radiology, Istanbul Faculty of Medicine, Istanbul University, 34390, Capa, Istanbul (Turkey); Bengisu, Ergin [Department of Obstetrics and Gynecology, Istanbul Faculty of Medicine, Istanbul University, Istanbul (Turkey); Yavuz, Ekrem [Department of Pathology, Istanbul Faculty of Medicine, Istanbul University, 34390, Capa, Istanbul (Turkey)

    2004-03-01

    Endometriosis, an important gynecological disorder of reproductive women, affects most commonly the ovaries and less frequently the gastrointestinal tract, chest, urinary tract, and soft tissues. Endometriosis classically appears on MRI as a mass with a large cystic component and variable signal intensities on T1- and T2-weighted images due to the presence of variable degradation of hemorrhagic products. Endometriosis in an atypical location, an infiltrative appearance and without cystic-hemorrhagic components has rarely been described. We report on a 33-year-old woman with cyclic sciatica due to histologically documented infiltrative endometriosis involving the area of the left sciatic notch. (orig.)

  17. Cyclic sciatica caused by infiltrative endometriosis: MRI findings

    International Nuclear Information System (INIS)

    Yekeler, Ensar; Kumbasar, Basak; Tunaci, Atadan; Barman, Ahmet; Tunaci, Mehtap; Bengisu, Ergin; Yavuz, Ekrem

    2004-01-01

    Endometriosis, an important gynecological disorder of reproductive women, affects most commonly the ovaries and less frequently the gastrointestinal tract, chest, urinary tract, and soft tissues. Endometriosis classically appears on MRI as a mass with a large cystic component and variable signal intensities on T1- and T2-weighted images due to the presence of variable degradation of hemorrhagic products. Endometriosis in an atypical location, an infiltrative appearance and without cystic-hemorrhagic components has rarely been described. We report on a 33-year-old woman with cyclic sciatica due to histologically documented infiltrative endometriosis involving the area of the left sciatic notch. (orig.)

  18. Hyperactivity and memory/learning deficits evoked by developmental exposure to nicotine and/or ethanol are mitigated by cAMP and cGMP signaling cascades activation.

    Science.gov (United States)

    Abreu-Villaça, Yael; Carvalho-Graça, Anna C; Skinner, Gabriela; Lotufo, Bruna M; Duarte-Pinheiro, Vitor H S; Ribeiro-Carvalho, Anderson; Manhães, Alex C; Filgueiras, Claudio C

    2018-04-10

    Pregnant smoking women are frequently episodic drinkers. Here, we investigated whether ethanol exposure restricted to the brain growth spurt period when combined with chronic developmental exposure to nicotine aggravates memory/learning deficits and hyperactivity, and associated cAMP and cGMP signaling disruption. To further investigate the role of these signaling cascades, we verified whether vinpocetine (a phosphodiesterase inhibitor) ameliorates the neurochemical and behavioral outcomes. Swiss mice had free access to nicotine (NIC, 50 μg/ml) or water to drink during gestation and until the 8th postnatal day (PN8). Ethanol (ETOH, 5 g/kg, i.p.) or saline were injected in the pups every other day from PN2 to PN8. At PN30, animals either received vinpocetine (20 mg/kg, i.p.) or vehicle before being tested in the step-down passive avoidance or open field. Memory/learning was impaired in NIC, ETOH and NIC + ETOH mice, and vinpocetine mitigated ETOH- and NIC + ETOH-induced deficits. Locomotor hyperactivity identified in ETOH and NIC + ETOH mice was ameliorated by vinpocetine. While cyclic nucleotides levels in cerebral cortex and hippocampus were reduced by NIC, ETOH and NIC + ETOH, this outcome was more consistent in the latter group. As observed for behavior, vinpocetine normalized NIC + ETOH nucleotides levels. pCREB levels were also increased in response to vinpocetine, with stronger effects in the NIC + ETOH group. Exposure to both drugs of abuse worsens behavioral and neurochemical disruption. These findings and the amelioration of deleterious effects by vinpocetine support the idea that cAMP and cGMP signaling contribute to nicotine- and ethanol-induced hyperactivity and memory/learning deficits. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Competitive protein binding assay

    International Nuclear Information System (INIS)

    Kaneko, Toshio; Oka, Hiroshi

    1975-01-01

    The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

  20. The DNA sensor, cyclic GMP-AMP synthase (cGAS) is essential for induction of IFN beta during Chlamydia trachomatis infection1

    Science.gov (United States)

    Zhang, Yugen; Yeruva, Laxmi; Marinov, Anthony; Prantner, Daniel; Wyrick, Priscilla; Lupashin, Vladimir; Nagarajan, Uma M.

    2014-01-01

    IFNβ has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFNβ expression during chlamydial infection. We determined that TREX-1, a host 3’to 5’ exonuclease, reduced IFNβ expression significantly during chlamydial infection using siRNA and gene knock out fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cGAS has been shown to bind cytosolic DNA to generate cGAMP, which binds to the signaling adaptor STING to induce IFNβ expression. We determined that cGAS is required for IFNβ expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFNβ, co-culture of cells depleted for either STING or cGAS rescued IFNβ expression. These data demonstrate that cGAMP produced in infected cGAS+STING− cells can migrate into adjacent cells via gap junctions to function in trans in cGAS−STING+ cells. Further, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated-DNA sensing directs IFNβ expression during C.trachomatis infection and suggests that effectors from infected cells can directly upregulate IFNβ expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis. PMID:25070851

  1. Cyclic di-GMP regulation of the bvg-repressed genes and the orphan response regulator RisA in Bordetella pertussis

    Science.gov (United States)

    Expression of Bordetella pertussis virulence factors is activated by the BvgAS two-component system. Under modulating growth conditions BvgAS indirectly represses another set of genes through the action of BvgR, a bvg-activated protein. BvgR blocks activation of the response regulator RisA which is ...

  2. Application of halophilic nuclease H of Micrococcus varians subsp. halophilus to commercial production of flavoring agent 5'-GMP.

    Science.gov (United States)

    Kamekura, M; Hamakawa, T; Onishi, H

    1982-01-01

    RNA was degraded at 60 degrees C for 24 h by halophilic nuclease H in supernatants from broth cultures of Micrococcus varians subsp. halophilus containing 12% NaCl. Since contaminating 5'-nucleotidase exhibited almost no activity under these conditions, the 5'-GMP formed could be recovered from the reaction mixture, and the yield was 805 mg from 5 g of RNA. PMID:6184020

  3. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    Science.gov (United States)

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  4. Nuclear cGMP-dependent kinase regulates gene expression via activity-dependent recruitment of a conserved histone deacetylase complex.

    Directory of Open Access Journals (Sweden)

    Yan Hao

    2011-05-01

    Full Text Available Elevation of the second messenger cGMP by nitric oxide (NO activates the cGMP-dependent protein kinase PKG, which is key in regulating cardiovascular, intestinal, and neuronal functions in mammals. The NO-cGMP-PKG signaling pathway is also a major therapeutic target for cardiovascular and male reproductive diseases. Despite widespread effects of PKG activation, few molecular targets of PKG are known. We study how EGL-4, the Caenorhabditis elegans PKG ortholog, modulates foraging behavior and egg-laying and seeks the downstream effectors of EGL-4 activity. Using a combination of unbiased forward genetic screen and proteomic analysis, we have identified a conserved SAEG-1/SAEG-2/HDA-2 histone deacetylase complex that is specifically recruited by activated nuclear EGL-4. Gene expression profiling by microarrays revealed >40 genes that are sensitive to EGL-4 activity in a SAEG-1-dependent manner. We present evidence that EGL-4 controls egg laying via one of these genes, Y45F10C.2, which encodes a novel protein that is expressed exclusively in the uterine epithelium. Our results indicate that, in addition to cytoplasmic functions, active EGL-4/PKG acts in the nucleus via a conserved Class I histone deacetylase complex to regulate gene expression pertinent to behavioral and physiological responses to cGMP. We also identify transcriptional targets of EGL-4 that carry out discrete components of the physiological response.

  5. The effects of test environment and cyclic stretching on the failure properties of human patellar tendons

    International Nuclear Information System (INIS)

    Haut, R.C.; Powlison, A.C.

    1990-01-01

    There is a need to document the mechanical properties of patellar tendon allografts used for reconstructive surgery of the damaged anterior cruciate ligament, especially the effects of irradiation sterilization. The purpose of this study was to investigate the influences of in vitro test environment and low-level cyclic stretching prior to failure tests on nonirradiated and irradiated human graft tissues. Bilateral patellar tendons were split and each half processed accordingly. Some graft tissues were stretched cyclically at 2.5 mm deformation before failure. Experiments were performed in a 37 degrees C saline bath or with tissues moistened with a drip of the same. The irradiated grafts relaxed less and generated less slack length in the drip environment than the nonirradiated controls. Cyclic stretching did not alter failure characteristics of either graft tissue. While no significant differences in the tensile responses or failure characteristics were noted for irradiated and nonirradiated grafts in the drip, in the bath environment the nonirradiated tissues had greater strength and modulus. This resulted in there being a significant difference between irradiated and nonirradiated tissue responses in a heated saline bath environment. These experimental results exemplify the need to control in vitro test environments in the evaluation of various sterilization and preservation protocols for soft tissue allografts

  6. Cyclic characteristics of earthquake time histories

    International Nuclear Information System (INIS)

    Hall, J.R. Jr; Shukla, D.K.; Kissenpfennig, J.F.

    1977-01-01

    From an engineering standpoint, an earthquake record may be characterized by a number of parameters, one of which is its 'cyclic characteristics'. The cyclic characteristics are most significant in fatigue analysis of structures and liquefaction analysis of soils where, in addition to the peak motion, cyclic buildup is significant. Whereas duration peak amplitude and response spectra for earthquakes have been studied extensively, the cyclic characteristics of earthquake records have not received an equivalent attention. Present procedures to define the cyclic characteristics are generally based upon counting the number of peaks at various amplitude ranges on a record. This paper presents a computer approach which describes a time history by an amplitude envelope and a phase curve. Using Fast Fourier Transform Techniques, an earthquake time history is represented as a projection along the x-axis of a rotating vector-the length the vector is given by the amplitude spectra-and the angle between the vector and x-axis is given by the phase curve. Thus one cycle is completed when the vector makes a full rotation. Based upon Miner's cumulative damage concept, the computer code automatically combines the cycles of various amplitudes to obtain the equivalent number of cycles of a given amplitude. To illustrate the overall results, the cyclic characteristics of several real and synthetic earthquake time histories have been studied and are presented in the paper, with the conclusion that this procedure provides a physical interpretation of the cyclic characteristics of earthquakes. (Auth.)

  7. Identification of the gamma subunit-interacting residues on photoreceptor cGMP phosphodiesterase, PDE6alpha '.

    Science.gov (United States)

    Granovsky, A E; Artemyev, N O

    2000-12-29

    Photoreceptor cGMP phosphodiesterase (PDE6) is the effector enzyme in the G protein-mediated visual transduction cascade. In the dark, the activity of PDE6 is shut off by the inhibitory gamma subunit (Pgamma). Chimeric proteins between cone PDE6alpha' and cGMP-binding and cGMP-specific PDE (PDE5) have been constructed and expressed in Sf9 cells to study the mechanism of inhibition of PDE6 catalytic activity by Pgamma. Substitution of the segment PDE5-(773-820) by the corresponding PDE6alpha'-(737-784) sequence in the wild-type PDE5 or in a PDE5/PDE6alpha' chimera containing the catalytic domain of PDE5 results in chimeric enzymes capable of inhibitory interaction with Pgamma. The catalytic properties of the chimeric PDEs remained similar to those of PDE5. Ala-scanning mutational analysis of the Pgamma-binding region, PDE6alpha'-(750-760), revealed PDE6alpha' residues essential for the interaction. The M758A mutation markedly impaired and the Q752A mutation moderately impaired the inhibition of chimeric PDE by Pgamma. The analysis of the catalytic properties of mutant PDEs and a model of the PDE6 catalytic domain suggest that residues Met(758) and Gln(752) directly bind Pgamma. A model of the PDE6 catalytic site shows that PDE6alpha'-(750-760) forms a loop at the entrance to the cGMP-binding pocket. Binding of Pgamma to Met(758) would effectively block access of cGMP to the catalytic cavity, providing a structural basis for the mechanism of PDE6 inhibition.

  8. cGMP-phosphodiesterase inhibition enhances photic responses and synchronization of the biological circadian clock in rodents.

    Directory of Open Access Journals (Sweden)

    Santiago A Plano

    Full Text Available The master circadian clock in mammals is located in the hypothalamic suprachiasmatic nuclei (SCN and is synchronized by several environmental stimuli, mainly the light-dark (LD cycle. Light pulses in the late subjective night induce phase advances in locomotor circadian rhythms and the expression of clock genes (such as Per1-2. The mechanism responsible for light-induced phase advances involves the activation of guanylyl cyclase (GC, cGMP and its related protein kinase (PKG. Pharmacological manipulation of cGMP by phosphodiesterase (PDE inhibition (e.g., sildenafil increases low-intensity light-induced circadian responses, which could reflect the ability of the cGMP-dependent pathway to directly affect the photic sensitivity of the master circadian clock within the SCN. Indeed, sildenafil is also able to increase the phase-shifting effect of saturating (1200 lux light pulses leading to phase advances of about 9 hours, as well as in C57 a mouse strain that shows reduced phase advances. In addition, sildenafil was effective in both male and female hamsters, as well as after oral administration. Other PDE inhibitors (such as vardenafil and tadalafil also increased light-induced phase advances of locomotor activity rhythms and accelerated reentrainment after a phase advance in the LD cycle. Pharmacological inhibition of the main downstream target of cGMP, PKG, blocked light-induced expression of Per1. Our results indicate that the cGMP-dependent pathway can directly modulate the light-induced expression of clock-genes within the SCN and the magnitude of light-induced phase advances of overt rhythms, and provide promising tools to design treatments for human circadian disruptions.

  9. GMP production of [18F]FDOPA and issues concerning its quality analyses as in USP 'Fluorodopa F 18 Injection'

    International Nuclear Information System (INIS)

    Kao, C.K.; Hsu Wenlin; Xie Hengli; Lin Mingchi; Lan Wenchun; Chao Haoyu

    2011-01-01

    Lately, 6-[ 18 F]fluoro-L-DOPA (FDOPA) has found increase in its clinical demand for whole-body positron emission tomography (PET) scans, and two key issues in fulfilling this demand are the difficulties in producing FDOPA under the recently imposed PET drug good manufacturing practice (GMP) regulations and in providing it in the quality meeting the terms of major compendia. This paper describes the approaches for the GMP production of FDOPA and for the product testing to meet the standard of United States Pharmacopeia (USP) 'Fluorodopa F 18 Injection.' FDOPA was produced by the carrier-added electrophilic aromatic substitution reaction in the facility complying Pharmaceutical Inspection Cooperation Scheme clean room standard. The special aseptic handling technique was applied to minimize the bioburden. The product quality control followed all testing items and procedures, including three different settings of high performance liquid chromatography (HPLC). The process yielded FDOPA average 2.60±0.26 GBq (N=22) in every batch. All qualities of the product were within the specifications described in the USP 'Fluorodopa F 18 Injection.' The entire production was audited by the government authority and certified to comply with the latest PET drug GMP regulation. Our efforts in producing FDOPA following all aspects of GMP requirements have resulted in a product with the USP quality and certified as GMP complied. The routine production yields enough doses for three to four whole-body scans in each batch. The issues discussed in the report provide good reference for producers planning in routine production for PET drugs that are not commonly produced or with complicated compendial quality control tests. (author)

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

    Science.gov (United States)

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

    2015-04-08

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

  11. The effects of irradiation on the cytosol glucocorticoid receptor and concentrations of corticosterone and cyclic nucleotides in the rat liver

    International Nuclear Information System (INIS)

    Teshima, Teruki; Mori, Masaki; Honke, Yoshifumi

    1983-01-01

    The effects of irradiation on both the cytosol glucocorticoid receptor and concentrations of corticosterone and cyclic nucleotides in the rat liver were investigated. The liver concentrations of corticosterone and cyclic nucleotides were measured by radioimmunoassay before and after the irradiation of 1,000 rad/l fraction. The glucocorticoid receptor in the liver cytosol was determined by the measurement of the cytosol binding to 3 H-dexamethasone. The cytosol and nuclear corticosterone levels reached a peak 1 day after the irradiation of the rat liver and declined to the control levels after 2 days. The increase in corticosterone levels may be due to the direct stimulation of the right adrenal gland and/ or the stress induced by the irradiation. The binding capacity of the glucocorticoid receptor in rat liver cytosol decreased to the minimum 1 day after the irradiation, and the recovery occurred at 4 days. The Kd value of the glucocorticoid receptor remained unchanged from 1 hour until 4 days but was high at 4 and 7 days. The distinctly increased levels of cyclic GMP in the rat liver were found from 1 hour through 7 days after the irradiation, while cyclic AMP did not change. The inversed relationship between the cytosol glucocorticoid receptor and corticosterone levels in cytosol and the nuclei indicates that the receptor-bound corticosterone in cytosol can be transferred to a nucleus and remain there in the presence of appropriate amounts of corticosterone in cytosol, after which the receptor is released from the nucleus into cytosol. The high Kd values observed 4 -- 7 days after the irradiation may be either due to the direct effect of irradiation or to the replenishment of the receptor with a low affinity. (author)

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

    Science.gov (United States)

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

    2013-01-01

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

  13. Relation of intracellular cyclic AMP to the shape of mammalian cell survival curves

    International Nuclear Information System (INIS)

    Lehnert, S.

    1975-01-01

    Results of experiments with V79 cells growing in tissue culture indicate that the reproductive survival of cells following irradiation is influenced by the level of intracellular 3', 5'-cyclic adenosine monophosphate (cyclic AMP) at the time of irradiation. Cells containing high levels of cyclic AMP induced by treatments with drugs show a characteristic survival curve in which the extent of the shoulder is increased so that the survival after low doses is enhanced. The exponential slope or D 0 , however, is decreased so that at high doses the survival of cells containing high levels of cyclic AMP may be less than that of controls. Naturally occurring changes in radiosensitivity such as those observed as cells pass through the division cycle, may also be related to parallel changes in cyclic AMP concentration occurring during the cycle. Injection of mice with compounds producing elevated cyclic AMP prior to whole-body irradiation increases survival at seven days post-irradiation. The shape of the survival curve for intestinal stem cells in these mice differs from that of the control in having an increased extrapolation number; no change in D 0 is observed in this in vivo situation. (author)

  14. Cyclic voltammetry and reduction mechanistic studies of ...

    African Journals Online (AJOL)

    styrylpyrylium perchlorates have been evaluated using cyclic voltammetry, in comparison to their non-methylated derivatives values. The reduction peak of all studied compounds remained chemically irreversible. The presence of the ...

  15. A cyclically actuated electrolytic drug delivery device

    KAUST Repository

    Yi, Ying; Buttner, Ulrich; Foulds, Ian G.

    2015-01-01

    This work, focusing on an implantable drug delivery system, presents the first prototype electrolytic pump that combines a catalytic reformer and a cyclically actuated mode. These features improve the release performance and extend the lifetime

  16. Introduction of a cyclic-fermentation method

    Energy Technology Data Exchange (ETDEWEB)

    Makarova, C P

    1958-01-01

    Equipment is described, consisting of 8 kettles, which permits a cyclic fermentation process and continuous ethanol production; 100% yields of ethanol are obtained, based on the starch content in grain.

  17. Results on Cyclic Signal Processing Systems

    National Research Council Canada - National Science Library

    Vaidyanathan, P

    1998-01-01

    .... A number of related problems such as the paraunitary interpolation problem and the cyclic paraunitary factorizability problem can be understood in a unified way by using the realization matrix...

  18. Cyclical Variability of Prominences, CMEs and Flares

    Indian Academy of Sciences (India)

    tribpo

    For many years, qualitative studies were made about the cyclical ... plan to review the more recent research concerning all these topics. Key words. ... are distributed in three narrow zones, which show different types of time-latitude behaviour.

  19. Anodic selective functionalization of cyclic amine derivatives

    OpenAIRE

    Onomura, Osamu

    2012-01-01

    Anodic reactions are desirable methods from the viewpoint of Green Chemistry, since no toxic oxidants are necessary for the oxidation of organic molecules. This review introduces usefulness of anodic oxidation and successive reaction for selective functionalization of cyclic amine derivatives.

  20. Macromolecular Networks Containing Fluorinated Cyclic Moieties

    Science.gov (United States)

    2015-12-12

    Briefing Charts 3. DATES COVERED (From - To) 17 Nov 2015 – 12 Dec 2015 4. TITLE AND SUBTITLE Macromolecular Networks Containing Fluorinated Cyclic... FLUORINATED CYCLIC MOIETIES 12 December 2015 Andrew J. Guenthner,1 Scott T. Iacono,2 Cynthia A. Corley,2 Christopher M. Sahagun,3 Kevin R. Lamison,4...Reinforcements Good Flame, Smoke, & Toxicity Characteristics Low Water Uptake with Near Zero Coefficient of Hygroscopic Expansion ∆ DISTRIBUTION A

  1. The Cyclicality of New Product Introductions

    OpenAIRE

    Kostas Axarloglou

    2003-01-01

    This study analyzes empirically the cyclical nature of the timing of new product introductions in U.S. manufacturing. New product introductions vary more in nonseasonal frequencies than in seasonal frequencies. However, the seasons alone account for only a small part of their total variability with demand factors being much more important. Demand fluctuations account for 35%80% and 17%43%, respectively, of the seasonal and cyclical variability of new product introductions in various industrie...

  2. The calculation of dissipated work, elastoplastic cyclic stress and cyclic strain in a structure

    International Nuclear Information System (INIS)

    Wang Xucheng; Xie Yihuan.

    1986-01-01

    With the development of the reactor technique, there is being an increasing interest in the calculation of elastoplastic response of a structure to its complex loading. This paper introduces a constitutive relation of a material for discribing unloading property, and uses it in an analysis of a real structure under a cyclic loading. The results, which include cyclic stress, cyclic strain and dissipated work, are meaningful in the researches of the structure behavior under complex loading and of the structural safety

  3. Inhibition of Cyclic Adenosine Monophosphate-Specific Phosphodiesterase by Various Food Plant-Derived Phytotherapeutic Agents.

    Science.gov (United States)

    Röhrig, Teresa; Pacjuk, Olga; Hernández-Huguet, Silvia; Körner, Johanna; Scherer, Katharina; Richling, Elke

    2017-11-04

    Background: Phosphodiesterases (PDEs) play a major role in the regulation of cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-mediated pathways. Their inhibitors exhibit anti-inflammatory, vasodilatory and antithrombotic effects. Therefore, consumption of foods with PDE-inhibiting potential may possess beneficial influence on the risk of cardiovascular diseases. Methods: Four plant extracts ( Arbutus unedo , Camellia sinensis , Cynara scolymus , Zingiber officinale ) with promising ingredient profiles and physiological effects were tested for their ability to inhibit cAMP-specific PDE in vitro in a radioactive assay. Results: Strawberry tree fruit ( Arbutus unedo ) and tea ( Camellia sinensis ) extracts did not inhibit PDE markedly. Alternatively, artichoke ( Cynara scolymus ) extract had a significant inhibitory influence on PDE activity (IC 50 = 0.9 ± 0.1 mg/mL) as well as its flavone luteolin (IC 50 = 41 ± 10 μM) and 3,4-dicaffeoylquinic acid (IC 50 > 1.0 mM). Additionally, the ginger ( Zingiber officinale ) extract and one of its constituents, [6]-gingerol, significantly inhibited PDE (IC 50 = 1.7 ± 0.2 mg/mL and IC 50 > 1.7 mM, respectively). Crude fractionation of ginger extract showed that substances responsible for PDE inhibition were in the lipoid fraction (IC 50 = 455 ± 19 μg/mL). Conclusions: A PDE-inhibitory effect was shown for artichoke and ginger extract. Whether PDE inhibition in vivo can be achieved through ingestion of artichoke or ginger extracts leading to physiological effects concerning cardiovascular health should be addressed in future research.

  4. Inhibition of Cyclic Adenosine Monophosphate-Specific Phosphodiesterase by Various Food Plant-Derived Phytotherapeutic Agents

    Directory of Open Access Journals (Sweden)

    Teresa Röhrig

    2017-11-01

    Full Text Available Background: Phosphodiesterases (PDEs play a major role in the regulation of cyclic adenosine monophosphate (cAMP- and cyclic guanosine monophosphate (cGMP-mediated pathways. Their inhibitors exhibit anti-inflammatory, vasodilatory and antithrombotic effects. Therefore, consumption of foods with PDE-inhibiting potential may possess beneficial influence on the risk of cardiovascular diseases. Methods: Four plant extracts (Arbutus unedo, Camellia sinensis, Cynara scolymus, Zingiber officinale with promising ingredient profiles and physiological effects were tested for their ability to inhibit cAMP-specific PDE in vitro in a radioactive assay. Results: Strawberry tree fruit (Arbutus unedo and tea (Camellia sinensis extracts did not inhibit PDE markedly. Alternatively, artichoke (Cynara scolymus extract had a significant inhibitory influence on PDE activity (IC50 = 0.9 ± 0.1 mg/mL as well as its flavone luteolin (IC50 = 41 ± 10 μM and 3,4-dicaffeoylquinic acid (IC50 > 1.0 mM. Additionally, the ginger (Zingiber officinale extract and one of its constituents, [6]-gingerol, significantly inhibited PDE (IC50 = 1.7 ± 0.2 mg/mL and IC50 > 1.7 mM, respectively. Crude fractionation of ginger extract showed that substances responsible for PDE inhibition were in the lipoid fraction (IC50 = 455 ± 19 μg/mL. Conclusions: A PDE-inhibitory effect was shown for artichoke and ginger extract. Whether PDE inhibition in vivo can be achieved through ingestion of artichoke or ginger extracts leading to physiological effects concerning cardiovascular health should be addressed in future research.

  5. A bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis.

    Science.gov (United States)

    Dey, Bappaditya; Dey, Ruchi Jain; Cheung, Laurene S; Pokkali, Supriya; Guo, Haidan; Lee, Jong-Hee; Bishai, William R

    2015-04-01

    Detection of cyclic-di-adenosine monophosphate (c-di-AMP), a bacterial second messenger, by the host cytoplasmic surveillance pathway (CSP) is known to elicit type I interferon (IFN) responses, which are crucial to antimicrobial defense. However, the mechanisms and role of c-di-AMP signaling in Mycobacterium tuberculosis virulence remain unclear. Here we show that resistance to tuberculosis requires CSP-mediated detection of c-di-AMP produced by M. tuberculosis and that levels of c-di-AMP modulate the fate of infection. We found that a di-adenylate cyclase (disA or dacA)-overexpressing M. tuberculosis strain that secretes excess c-di-AMP activates the interferon regulatory factor (IRF) pathway with enhanced levels of IFN-β, elicits increased macrophage autophagy, and exhibits substantial virulence attenuation in mice. We show that c-di-AMP-mediated IFN-β induction during M. tuberculosis infection requires stimulator of interferon genes (STING)-signaling. We observed that c-di-AMP induction of IFN-β is independent of the cytosolic nucleic acid receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), but cGAS nevertheless contributes substantially to the overall IFN-β response to M. tuberculosis infection. In sum, our results reveal c-di-AMP to be a key mycobacterial pathogen-associated molecular pattern (PAMP) driving host type I IFN responses and autophagy. These findings suggest that modulating the levels of this small molecule may lead to novel immunotherapeutic strategies against tuberculosis.

  6. Effect of cyclic training model on terminal structure of rabbit Achilles tendon: an experimental study

    OpenAIRE

    Chang-lin HUANG; Wang GAO; Tao HUANG; Zhen-hai GUO

    2012-01-01

    Objective  To observe the effect of cyclic training on histomorphology of the terminal structure of rabbit Achilles tendon, and explore its preventive effect on training-based enthesiopathy. Methods  Seventy-two Japanese white rabbits were randomly assigned to four groups: control group, jumping group, running group and cyclic training group, 18 for each. Three rabbits of each group were sacrificed at the 2nd, 3rd, 4th, 6th, 8th and 10th week. The terminal insertion tissues of bilateral Achil...

  7. Full GMP-Compliant Validation of Bone Marrow-Derived Human CD133+ Cells as Advanced Therapy Medicinal Product for Refractory Ischemic Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Daniela Belotti

    2015-01-01

    Full Text Available According to the European Medicine Agency (EMA regulatory frameworks, Advanced Therapy Medicinal Products (ATMP represent a new category of drugs in which the active ingredient consists of cells, genes, or tissues. ATMP-CD133 has been widely investigated in controlled clinical trials for cardiovascular diseases, making CD133+ cells one of the most well characterized cell-derived drugs in this field. To ensure high quality and safety standards for clinical use, the manufacturing process must be accomplished in certified facilities following standard operative procedures (SOPs. In the present work, we report the fully compliant GMP-grade production of ATMP-CD133 which aims to address the treatment of chronic refractory ischemic heart failure. Starting from bone marrow (BM, ATMP-CD133 manufacturing output yielded a median of 6.66 × 106 of CD133+ cells (range 2.85 × 106–30.84 × 106, with a viability ranged between 96,03% and 99,97% (median 99,87% and a median purity of CD133+ cells of 90,60% (range 81,40%–96,20%. Based on these results we defined our final release criteria for ATMP-CD133: purity ≥ 70%, viability ≥ 80%, cellularity between 1 and 12 × 106 cells, sterile, and endotoxin-free. The abovementioned criteria are currently applied in our Phase I clinical trial (RECARDIO Trial.

  8. Full GMP-compliant validation of bone marrow-derived human CD133(+) cells as advanced therapy medicinal product for refractory ischemic cardiomyopathy.

    Science.gov (United States)

    Belotti, Daniela; Gaipa, Giuseppe; Bassetti, Beatrice; Cabiati, Benedetta; Spaltro, Gabriella; Biagi, Ettore; Parma, Matteo; Biondi, Andrea; Cavallotti, Laura; Gambini, Elisa; Pompilio, Giulio

    2015-01-01

    According to the European Medicine Agency (EMA) regulatory frameworks, Advanced Therapy Medicinal Products (ATMP) represent a new category of drugs in which the active ingredient consists of cells, genes, or tissues. ATMP-CD133 has been widely investigated in controlled clinical trials for cardiovascular diseases, making CD133(+) cells one of the most well characterized cell-derived drugs in this field. To ensure high quality and safety standards for clinical use, the manufacturing process must be accomplished in certified facilities following standard operative procedures (SOPs). In the present work, we report the fully compliant GMP-grade production of ATMP-CD133 which aims to address the treatment of chronic refractory ischemic heart failure. Starting from bone marrow (BM), ATMP-CD133 manufacturing output yielded a median of 6.66 × 10(6) of CD133(+) cells (range 2.85 × 10(6)-30.84 × 10(6)), with a viability ranged between 96,03% and 99,97% (median 99,87%) and a median purity of CD133(+) cells of 90,60% (range 81,40%-96,20%). Based on these results we defined our final release criteria for ATMP-CD133: purity ≥ 70%, viability ≥ 80%, cellularity between 1 and 12 × 10(6) cells, sterile, and endotoxin-free. The abovementioned criteria are currently applied in our Phase I clinical trial (RECARDIO Trial).

  9. Full GMP-Compliant Validation of Bone Marrow-Derived Human CD133+ Cells as Advanced Therapy Medicinal Product for Refractory Ischemic Cardiomyopathy

    Science.gov (United States)

    Belotti, Daniela; Gaipa, Giuseppe; Bassetti, Beatrice; Cabiati, Benedetta; Spaltro, Gabriella; Biagi, Ettore; Parma, Matteo; Biondi, Andrea; Cavallotti, Laura; Gambini, Elisa; Pompilio, Giulio

    2015-01-01

    According to the European Medicine Agency (EMA) regulatory frameworks, Advanced Therapy Medicinal Products (ATMP) represent a new category of drugs in which the active ingredient consists of cells, genes, or tissues. ATMP-CD133 has been widely investigated in controlled clinical trials for cardiovascular diseases, making CD133+ cells one of the most well characterized cell-derived drugs in this field. To ensure high quality and safety standards for clinical use, the manufacturing process must be accomplished in certified facilities following standard operative procedures (SOPs). In the present work, we report the fully compliant GMP-grade production of ATMP-CD133 which aims to address the treatment of chronic refractory ischemic heart failure. Starting from bone marrow (BM), ATMP-CD133 manufacturing output yielded a median of 6.66 × 106 of CD133+ cells (range 2.85 × 106–30.84 × 106), with a viability ranged between 96,03% and 99,97% (median 99,87%) and a median purity of CD133+ cells of 90,60% (range 81,40%–96,20%). Based on these results we defined our final release criteria for ATMP-CD133: purity ≥ 70%, viability ≥ 80%, cellularity between 1 and 12 × 106 cells, sterile, and endotoxin-free. The abovementioned criteria are currently applied in our Phase I clinical trial (RECARDIO Trial). PMID:26495296

  10. 21 CFR 862.1230 - Cyclic AMP test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cyclic AMP test system. 862.1230 Section 862.1230....1230 Cyclic AMP test system. (a) Identification. A cyclic AMP test system is a device intended to measure the level of adenosine 3′, 5′-monophosphate (cyclic AMP) in plasma, urine, and other body fluids...

  11. Construction and expression of a functional monoclonal antibody SZ-51 specific for GMP-140 chimeric fab fragment in Escherichia coli

    International Nuclear Information System (INIS)

    Gu Jianming; Zhang Xiaomin; Xia Lijun; Wan Haiying; Liu Yue; Li Peixia; Ruan Changgeng

    1996-04-01

    The variable region cDNAs of a monoclonal antibody SZ-51 specific for α-granule membrane protein (GMP-140) on the surface of activated human platelets were spliced with the constant region cDNA of the heavy chain CH1 and light chain k of human Ig G by means of the gene recombination techniques. The above recombinant gene was amplified by the polymerase chain reaction (PCR). The expression vector of phage plasmid pHEN1 SZ-51 Fab/Hu was constructed. The pHEN1-51 Fab/Hu was introduced into non-suppressor E. coli HB2151. The amount of expression of SZ-51 chimeric Fab/Hu measured by quantitative ELISA was about 500 μg/L. Western blot demonstrated that the SZ-51 chimeric Fab fragment could specifically bind to GMP-140. (2 figs.)

  12. The effects of nitric oxide-cGMP pathway stimulation on dopamine in the medial preoptic area and copulation in DHT-treated castrated male rats.

    Science.gov (United States)

    Sato, Satoru M; Wersinger, Scott R; Hull, Elaine M

    2007-08-01

    Dopamine (DA) in the medial preoptic area (MPOA) provides important facilitative influence on male rat copulation. We have shown that the nitric oxide-cGMP (NO-cGMP) pathway modulates MPOA DA levels and copulation. We have also shown that systemic estradiol (E(2)) maintains neuronal NO synthase (nNOS) immunoreactivity in the MPOA of castrates, as well as relatively normal DA levels. This effect of E(2) on nNOS probably accounts for at least some of the previously demonstrated behavioral facilitation by intra-MPOA E(2) administration in castrates. Therefore, we hypothesized that stimulation of the MPOA NO-cGMP pathway in dihydrotestosterone (DHT)-treated castrates should restore DA levels and copulatory behaviors. Reverse-dialysis of a NO donor, sodium nitroprusside (SNP), increased extracellular DA in the MPOA of DHT-treated castrates and restored the ability to copulate to ejaculation in half of the animals. A cGMP analog, 8-Br-cGMP, also increased extracellular DA, though not as robustly, but did not restore copulatory ability. The effectiveness of the NO donor in restoring copulation and MPOA DA levels is consistent with our hypothesis. However, the lack of behavioral effects of 8-Br-cGMP, despite its increase in MPOA DA, suggests that NO may have additional mediators in the MPOA in the regulation of copulation. Furthermore, the suboptimal copulation seen in the NO donor-treated animals suggests the importance of extra-MPOA systems in the regulation of copulation.

  13. Clinical application of combined detection of serum hs-CRP, GMP-140 and cTnI in patients with coronary heart diseases

    International Nuclear Information System (INIS)

    Qin Jibao; Wu Zhaozeng

    2010-01-01

    Objective: To explore the clinical significance of changes of serum hs-CRP, GMP-140 and cTnI levels in patients with coronary heart diseases. Methods: Serum GMP-140 (with RIA), cTnI (with ELISA) and hs-CRP (with immuno turbidity method) levels were determined in 91 patients with coronary heart diseases (42 SAP, 34UAP, 15AMI) and 35 controls. Results: Serum hs-CRP, GMP-140, cTnI levels in patients with coronary heart diseases were significantly higher than those in controls (P <0.01). Among the patients with of coronary heart diseases, the magnitude of changes of the levels of serum hs-CRP, GMP-140 and cTnI levels in AMI and UAP groups were significantly larger than those in SAP group (P < 0.05). Serum hs-CRP levels were positively correlated with serum GMP-140 and cTnI levels (r = 0.6214, 0.6023, P < 0.01). Conclusion: Serum hs-CRP, GMP140 and cTnI levels were closely related to the diseases process of coronary heart diseases and were of great clinical importance for assessment of the disease and outcome prediction. (authors)

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

    Directory of Open Access Journals (Sweden)

    Marina ePolito

    2013-11-01

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

  15. Cyclic transformation of orbital angular momentum modes

    International Nuclear Information System (INIS)

    Schlederer, Florian; Krenn, Mario; Fickler, Robert; Malik, Mehul; Zeilinger, Anton

    2016-01-01

    The spatial modes of photons are one realization of a QuDit, a quantum system that is described in a D-dimensional Hilbert space. In order to perform quantum information tasks with QuDits, a general class of D-dimensional unitary transformations is needed. Among these, cyclic transformations are an important special case required in many high-dimensional quantum communication protocols. In this paper, we experimentally demonstrate a cyclic transformation in the high-dimensional space of photonic orbital angular momentum (OAM). Using simple linear optical components, we show a successful four-fold cyclic transformation of OAM modes. Interestingly, our experimental setup was found by a computer algorithm. In addition to the four-cyclic transformation, the algorithm also found extensions to higher-dimensional cycles in a hybrid space of OAM and polarization. Besides being useful for quantum cryptography with QuDits, cyclic transformations are key for the experimental production of high-dimensional maximally entangled Bell-states. (paper)

  16. On the equivalence of cyclic and quasi-cyclic codes over finite fields

    Directory of Open Access Journals (Sweden)

    Kenza Guenda

    2017-07-01

    Full Text Available This paper studies the equivalence problem for cyclic codes of length $p^r$ and quasi-cyclic codes of length $p^rl$. In particular, we generalize the results of Huffman, Job, and Pless (J. Combin. Theory. A, 62, 183--215, 1993, who considered the special case $p^2$. This is achieved by explicitly giving the permutations by which two cyclic codes of prime power length are equivalent. This allows us to obtain an algorithm which solves the problem of equivalency for cyclic codes of length $p^r$ in polynomial time. Further, we characterize the set by which two quasi-cyclic codes of length $p^rl$ can be equivalent, and prove that the affine group is one of its subsets.

  17. Islet isolation and GMP, ISO 9001:2000: what do we need--a 3-year experience.

    Science.gov (United States)

    Hengster, P; Hermann, M; Pirkebner, D; Draxl, A; Margreiter, R

    2005-10-01

    Pancreatic islet cell isolation and transplantation has been performed for many years at several institutions. Although all institutions aim to produce high-quality islets, applied standards widely deviate from standards in the pharmaceutical industry. The legal situation within the European Union has changed requirements for setting up and running such a laboratory. The process is now clearly defined as a production of a pharmaceutical and therefore must be licensed by federal authorities. Analysis of workload for establishing an islet isolation program that fulfil GMP and ISO 9001 criteria including an estimation of costs and the impact of such a system on the isolation process. The definition of quality parameters and documentation is a central issue of all islet isolation laboratories. Therefore, GMP and ISO 9001:2000 do not add additional work per se. On the other hand, clear guidelines, a clear policy, working place descriptions, forms, checklists, and, particularly standard operating procedures, are instrumental for smooth functioning within the department. Collection of data such as errors, improvement measures, and preventive measures reduces subsequent costs. A clear definition of responsibilities minimizes organizational problems. Steering of inspection devices prevents bias errors and validating the processes clearly points out incorrect assumptions. Documentation helps to prove the correctness of the production at any time and is of use also for scientific evaluations. We strongly feel that GMP criteria are mandatory and together with an ISO 9001:2000 quality management system offers significant advantages for the process of islet isolation and a continuous improvement process.

  18. Increasing plasma fibrinogen, but unchanged levels of intraplatelet cyclic nucleotides, plasma endothelin-1, factor VII, and neopterin during cholesterol lowering with fluvastatin.

    Science.gov (United States)

    Gottsäter, A; Anwaar, I; Lind, P; Mattiasson, I; Lindgärde, F

    1999-04-01

    Lipid-lowering statin treatment reduces cardiovascular morbidity and mortality and improves endothelial function in patients with hypercholesterolemia. The aim of the present study was to evaluate plasma levels of fibrinogen, factor VII, and the macrophage-derived inflammatory mediator neopterin during lipid lowering. In addition, the endothelial production of platelet antiaggregatory and vasodilatory factors such as nitric oxide and prostacyclin, and vasoconstrictive factors such as endothelin-1, was assessed. Plasma fibrinogen, factor VII, endothelin-1, and the neopterin and intraplatelet nitric oxide and prostacyclin mediators cyclic 3'-5'guanosine monophosphate (cGMP) and cyclic 3'-5'adenosine monophosphate (cAMP) were measured before and 6 months after the institution of treatment with fluvastatin in 17 patients (eight men and nine women, median age 60 years) with vascular disease and previously untreated hypercholesterolemia. After 6 months, a decrease of 1.62 mmol/l [1.26-2.18 (19%); P factor VII [from 1.14 IE/ml (0.58-1.38) to 1.22 IE/ml (0.96-1.46); NS], or plasma neopterin [from 8.6 nmol/l (7.1-11.5) to 8.7 nmol/l (7.9-11.3); NS]. In conclusion, during cholesterol-lowering treatment with fluvastatin, plasma levels of fibrinogen increased whereas intraplatelet cyclic nucleotide levels and plasma endothelin-1, factor VII and neopterin levels were unchanged.

  19. HOST liner cyclic facilities: Facility description

    Science.gov (United States)

    Schultz, D.

    1982-01-01

    A quartz lamp box, a quartz lamp annular rig, and a low pressure liner cyclic can rig planned for liner cyclic tests are described. Special test instrumentation includes an IR-TV camera system for measuring liner cold side temperatures, thin film thermocouples for measuring liner hot side temperatures, and laser and high temperature strain gages for obtaining local strain measurements. A plate temperature of 2,000 F was obtained in an initial test of an apparatus with three quartz lamps. Lamp life, however, appeared to be limited for the standard commercial quartz lamps available. The design of vitiated and nonvitiated preheaters required for the quartz lamp annular rig and the cyclic can test rigs is underway.

  20. Cyclic cellular automata in 3D

    International Nuclear Information System (INIS)

    Reiter, Clifford A.

    2011-01-01

    Highlights: → We explore the self-organization of cyclic cellular automata in 3D. → Von Neumann, Moore and two types of intermediate neighborhoods are investigated. → Random neighborhoods self organize through phases into complex nested structures. → Demons are seen to have many alternatives in 3D. - Abstract: Cyclic cellular automata in two dimensions have long been intriguing because they self organize into spirals and that behavior can be analyzed. The form for the patterns that develop is highly dependent upon the form of the neighborhood. We extend this work to three dimensional cyclic cellular automata and observe self organization dependent upon the neighborhood type. This includes neighborhood types intermediate between Von Neumann and Moore neighborhoods. We also observe that the patterns include nested shells with the appropriate forms but that the nesting is far more complex than the spirals that occur in two dimensions.

  1. Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca²⁺ signals at key decision points in the life cycle of malaria parasites.

    Directory of Open Access Journals (Sweden)

    Mathieu Brochet

    2014-03-01

    Full Text Available Many critical events in the Plasmodium life cycle rely on the controlled release of Ca²⁺ from intracellular stores to activate stage-specific Ca²⁺-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca²⁺ required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca²⁺ signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca²⁺ effectors, PKG emerges as a unifying factor to control multiple cellular Ca²⁺ signals essential for malaria parasite development and transmission.

  2. cGMP-Dependent Protein Kinase Inhibitors in Health and Disease

    Directory of Open Access Journals (Sweden)

    Jens Schlossmann

    2013-02-01

    Full Text Available cGMP-dependent protein kinases (PKG exhibit diverse physiological functions in the mammalian system e.g., in vascular and gastrointestinal smooth muscles, in platelets, in kidney, in bone growth, nociception and in the central nervous system. Furthermore, PKG were found in insects and in the malaria parasite Plasmodium falciparum. Two different genes of PKG exist: a the PKG-I gene that is expressed as cytosolic PKG-Iα or PKG-Iβ isoform, and b the PKG-II gene, which expresses the membrane associated PKG-II protein. The enzyme kinetics, the localization and the substrates of these PKG enzymes differ utilizing different physiological functions. Various inhibitors of PKG were developed directed against diverse functional regions of the kinase. These inhibitors of PKG have been used to analyse the specific functions of these enzymes. The review article will summarize these different inhibitors regarding their specificity and their present applications in vitro and in vivo. Furthermore, it will be discussed that the distinct inhibition of the PKG enzymes could be used as a valuable pharmacological target e.g., in the treatment of cardiovascular diseases, diarrhea, cancer or malaria.

  3. GMP-grade platelet lysate enhances proliferation and migration of tenon fibroblasts.

    Science.gov (United States)

    Carducci, Augusto; Scafetta, Gaia; Siciliano, Camilla; Carnevale, Roberto; Rosa, Paolo; Coccia, Andrea; Mangino, Giorgio; Bordin, Antonella; Vingolo, Enzo Maria; Pierelli, Luca; Lendaro, Eugenio; Ragona, Giuseppe; Frati, Giacomo; De Falco, Elena

    2016-01-01

    Tenon's fibroblasts (TFs), widely employed as in vitro model for many ophthalmological studies, are routinely cultured with FBS. Platelet Lysate (PL), a hemoderivate enriched with growth factors and cytokines has been largely tested in several clinical applications and as substitute of FBS in culture. Here, we investigate whether PL can exert biological effects on TF populations similarly to other cell types. Results show that PL significantly enhances cell proliferation and migration vs. FBS, without influencing cell size/granularity. Upregulation of EGF, VEGF, KDR, MMP2-9, FAK mRNA levels also occurs and phosphorylation of AKT but not of ERK1/2 is significantly enhanced. The inhibition of the PI3kinase/AKT pathway with the specific inhibitor wortmannin, decreases PL-induced cell migration but not proliferation. Condition supernatants containing PL show increased bioavailability of Nitric Oxide and reduced levels of 8-Iso-PGF2-alpha, correlating with cell proliferation and migration. Pro-angiogenic/inflammatory soluble factors (GRO, Angiogenin, EGF, I-309, PARC) are exclusively or greater expressed in media containing PL than FBS. GMP-grade PL preparations positively influence in vitro biological effects of TFs representing a suitable and safer alternative to FBS.

  4. Microneedle characterisation: the need for universal acceptance criteria and GMP specifications when moving towards commercialisation.

    Science.gov (United States)

    Lutton, Rebecca E M; Moore, Jessica; Larrañeta, Eneko; Ligett, Stephen; Woolfson, A David; Donnelly, Ryan F

    2015-08-01

    With interest in microneedles as a novel drug transdermal delivery system increasing rapidly since the late 1990s (Margetts and Sawyer Contin Educ Anaesthesia Crit Care Pain. 7(5):171-76, 2007), a diverse range of microneedle systems have been fabricated with varying designs and dimensions. However, there are still very few commercially available microneedle products. One major issue regarding microneedle manufacture on an industrial scale is the lack of specific quality standards for this novel dosage form in the context of Good Manufacturing Practice (GMP). A range of mechanical characterisation tests and microneedle insertion analysis techniques are used by researchers working on microneedle systems to assess the safety and performance profiles of their various designs. The lack of standardised tests and equipment used to demonstrate microneedle mechanical properties and insertion capability makes it difficult to directly compare the in use performance of candidate systems. This review highlights the mechanical tests and insertion analytical techniques used by various groups to characterise microneedles. This in turn exposes the urgent need for consistency across the range of microneedle systems in order to promote innovation and the successful commercialisation of microneedle products.

  5. An ant-plant mutualism through the lens of cGMP-dependent kinase genes.

    Science.gov (United States)

    Malé, Pierre-Jean G; Turner, Kyle M; Doha, Manjima; Anreiter, Ina; Allen, Aaron M; Sokolowski, Marla B; Frederickson, Megan E

    2017-09-13

    In plant-animal mutualisms, how an animal forages often determines how much benefit its plant partner receives. In many animals, foraging behaviour changes in response to foraging gene expression or activation of the cGMP-dependent protein kinase (PKG) that foraging encodes. Here, we show that this highly conserved molecular mechanism affects the outcome of a plant-animal mutualism. We studied the two PKG genes of Allomerus octoarticulatus, an Amazonian ant that defends the ant-plant Cordia nodosa against herbivores. Some ant colonies are better 'bodyguards' than others. Working in the field in Peru, we found that colonies fed with a PKG activator recruited more workers to attack herbivores than control colonies. This resulted in less herbivore damage. PKG gene expression in ant workers correlated with whether an ant colony discovered an herbivore and how much damage herbivores inflicted on leaves in a complex way; natural variation in expression levels of the two genes had significant interaction effects on ant behaviour and herbivory. Our results suggest a molecular basis for ant protection of plants in this mutualism. © 2017 The Author(s).

  6. Holographic entanglement entropy and cyclic cosmology

    Science.gov (United States)

    Frampton, Paul H.

    2018-06-01

    We discuss a cyclic cosmology in which the visible universe, or introverse, is all that is accessible to an observer while the extroverse represents the total spacetime originating from the time when the dark energy began to dominate. It is argued that entanglement entropy of the introverse is the more appropriate quantity to render infinitely cyclic, rather than the entropy of the total universe. Since vanishing entanglement entropy implies disconnected spacetimes, at the turnaround when the introverse entropy is zero the disconnected extroverse can be jettisoned with impunity.

  7. Entire cyclic cohomology and modular theory

    International Nuclear Information System (INIS)

    Stoytchev, O.Ts.

    1992-04-01

    We display a close relationship between C* and W*-dynamical systems with KMS states on them and entire cyclic cohomology theory. We construct a character form which assigns to each such system (A, α, R) an even entire cyclic cocycle of the subalgebra A of differentiable (with respect to the given automorphism group) elements of A. We argue that the most interesting case is the von Neumann algebra one, where the automorphism group is determined uniquely by the faithful normal state on the algebra (the modular group) and where the character may provide important information about the algebra. (author). 11 refs

  8. Effects of cyclic stretch on proliferation of mesenchymal stem cells and their differentiation to smooth muscle cells

    International Nuclear Information System (INIS)

    Ghazanfari, Samane; Tafazzoli-Shadpour, Mohammad; Shokrgozar, Mohammad Ali

    2009-01-01

    Bone marrow mesenchymal stem cells (MSCs) are capable of differentiating into a variety of cell types such as vascular smooth muscle cells (SMCs). In this study, we investigated influence of cyclic stretch on proliferation of hMSCs for different loading conditions, alignment of actin filaments, and consequent differentiation to SMCs. Isolated cells from bone marrow were exposed to cyclic stretch utilizing a customized device. Cell proliferation was examined by MTT assay, alignment of actin fibers by a designed image processing code, and cell differentiation by fluorescence staining. Results indicated promoted proliferation of hMSCs by cyclic strain, enhanced by elevated strain amplitude and number of cycles. Such loading regulated smooth muscle α-actin, and reoriented actin fibers. Cyclic stretch led to differentiation of hMSCs to SMCs without addition of growth factor. It was concluded that applying appropriate loading treatment on hMSCs could enhance proliferation capability, and produce functional SMCs for engineered tissues.

  9. Adrenomedullin stimulates cyclic AMP production in the airway epithelial cells of guinea-pigs and in the human epithelial cell line

    Directory of Open Access Journals (Sweden)

    Takashi Kawaguchi

    1999-01-01

    Full Text Available This study was designed to examine the effects of adrenomedullin (AM on airway epithelial cells. Primary cultures of guinea-pig tracheal epithelial cells and the human bronchiolar epithelial cell line NCI-H441 were used. Intracellular cyclic adenosine monophosphate (cAMP, cyclic guanosine monophosphate (cGMP, prostaglandin E2 (PGE2, and stable end-products of nitric oxide were assayed. Adrenomedullin (10−6 mol/L stimulated cAMP production in guinea-pig epithelial cells. Indomethacin (10−5 mol/L significantly decreased the basal level of intracellular cAMP in guinea-pig epithelial cells, but not in NCI-H441 cells. However, AM did not stimulate production of PGE2, a major product that can increase cAMP formation. In the case of NCI-H441 cells, AM (10−8 – 10−6 mol/L did not significantly affect intracellular cGMP levels or nitrite content in conditioned medium. Adrenomedullin and calcitonin gene-related peptide (CGRP each stimulated cAMP production in NCI-H441 cells, but AM-stimulated cAMP production was antagonized by the CGRP fragment CGRP8–37. These findings suggest that AM stimulates cAMP production and functionally competes with CGRP for binding sites in airway epithelial cells, at least in human epithelial cells, but that it does not stimulate the release of PGE2 and nitric oxide. Though cyclooxygenase products contribute to some extent to cAMP formation in guinea-pigs, AM independently stimulates intracellular cAMP formation in airway epithelial cells.

  10. Diguanylate cyclase null mutant reveals that C-Di-GMP pathway regulates the motility and adherence of the extremophile bacterium Acidithiobacillus caldus.

    Directory of Open Access Journals (Sweden)

    Matías Castro

    Full Text Available An understanding of biofilm formation is relevant to the design of biological strategies to improve the efficiency of the bioleaching process and to prevent environmental damages caused by acid mine/rock drainage. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in different biomining bacteria. In many bacteria, the intracellular levels of c-di-GMP molecules regulate the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development, through different kinds of effectors. Thus, we recently started a study of the c-di-GMP pathway in several biomining bacteria including Acidithiobacillus caldus. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs and degraded by phosphodiesterases (PDEs. We previously reported the existence of intermediates involved in c-di-GMP pathway from different Acidithiobacillus species. Here, we report our work related to At. caldus ATCC 51756. We identified several putative-ORFs encoding DGC and PDE and effector proteins. By using total RNA extracted from At. caldus cells and RT-PCR, we demonstrated that these genes are expressed. We also demonstrated the presence of c-di-GMP by mass spectrometry and showed that genes for several of the DGC enzymes were functional by heterologous genetic complementation in Salmonella enterica serovar Typhimurium mutants. Moreover, we developed a DGC defective mutant strain (Δc1319 that strongly indicated that the c-di-GMP pathway regulates the swarming motility and adherence to sulfur surfaces by At. caldus. Together, our results revealed that At. caldus possesses a functional c-di-GMP pathway which could be significant for ores colonization during the bioleaching process.

  11. Equibiaxial cyclic stretch stimulates fibroblasts to rapidly remodel fibrin.

    Science.gov (United States)

    Balestrini, Jenna Leigh; Billiar, Kristen Lawrence

    2006-01-01

    Understanding the effects of the mechanical environment on wound healing is critical for developing more effective treatments to reduce scar formation and contracture. The aim of this study was to investigate the effects of dynamic mechanical stretch on cell-mediated early wound remodeling independent of matrix alignment which obscures more subtle remodeling mechanisms. Cyclic equibiaxial stretch (16% stretch at 0.2 Hz) was applied to fibroblast-populated fibrin gel in vitro wound models for eight days. Compaction, density, tensile strength, and collagen content were quantified as functional measures of remodeling. Stretched samples were approximately ten times stronger, eight-fold more dense, and eight times thinner than statically cultured samples. These changes were accompanied by a 15% increase in net collagen but no significant differences in cell number or viability. When collagen crosslinking was inhibited in stretched samples, the extensibility increased and the strength decreased. The apparent weakening was due to a reduction in compaction rather than a decrease in ability of the tissue to withstand tensile forces. Interestingly, inhibiting collagen crosslinking had no measurable effects on the statically cultured samples. These results indicate that amplified cell-mediated compaction and even a slight addition in collagen content play substantial roles in mechanically induced wound strengthening. These findings increase our understanding of how mechanical forces guide the healing response in skin, and the methods employed in this study may also prove valuable tools for investigating stretch-induced remodeling of other planar connective tissues and for creating mechanically robust engineered tissues.

  12. Cyclic Plastic Deformation and Welding Simulation

    NARCIS (Netherlands)

    Ten Horn, C.H.L.J.

    2003-01-01

    One of the concerns of a fitness for purpose analysis is the quantification of the relevant material properties. It is known from experiments that the mechanical properties of a material can change due to a monotonic plastic deformation or a cyclic plastic deformation. For a fitness for purpose

  13. Undrained Cyclic Behaviour of Dense Frederikshavn Sand

    DEFF Research Database (Denmark)

    Nielsen, Søren Kjær; Ibsen, Lars Bo; Sørensen, Kris Wessel

    2013-01-01

    A modified contour diagram is created for the Frederikshavn Sand in the undrained case for a relative density of ID = 80 %. It can be used to estimate the number of cycles to failure for a given combination of pore pressure, average and cyclic load ratio. The diagram is based on a series of undra...

  14. Driving Force Based Design of Cyclic Distillation

    DEFF Research Database (Denmark)

    Nielsen, Rasmus Fjordbak; Huusom, Jakob Kjøbsted; Abildskov, Jens

    2017-01-01

    with mixed phase feeds. A range of binary test cases, benzene toluene, methanol water, and ethanol water, are evaluated. The advantage of the design approach in cyclic distillation is shown to be analogous to the advantages obtained in conventional continuous distillation, including a minimal utility...

  15. Cyclic Cratonic Carbonates and Phanerozoic Calcite Seas.

    Science.gov (United States)

    Wilkinson, Bruce H.

    1982-01-01

    Discusses causes of cyclicity in cratonic carbonate sequences and evidence for and potential significance of postulated primary calcite sediment components in past Paleozoic seas, outlining problems, focusing on models explaining existing data, and identifying background. Future sedimentary geologists will need to address these and related areas…

  16. Hopf Algebroids and Their Cyclic Theory

    NARCIS (Netherlands)

    Kowalzig, N.

    2009-01-01

    The main objective of this thesis is to clarify concepts of generalised symmetries in noncommutative geometry (i.e., the noncommutative analogue of groupoids and Lie algebroids) and their associated (co)homologies. These ideas are incorporated by the notion of Hopf algebroids and Hopf-cyclic

  17. Cyclic viscoelastoplasticity of polypropylene/nanoclay composites

    DEFF Research Database (Denmark)

    Drozdov, A.; Christiansen, Jesper de Claville

    2012-01-01

    Observations are reported on isotactic polypropylene/organically modified nanoclay hybrids with concentrations of filler ranging from 0 to 5 wt.% in cyclic tensile tests with a stress–controlled program (oscillations between various maximum stresses and the zero minimum stress). A pronounced effe...

  18. Breaking antidunes: Cyclic behavior due to hysteresis

    DEFF Research Database (Denmark)

    Deigaard, Rolf

    2006-01-01

    The cyclic behavior of breaking antidunes (growth, breaking of surface wave, obliteration) is investigated by use of a numerical model. The model includes the transition between supercritical and transcritical flow. As the antidune grows the flow becomes transcritical and a hydraulic jump is form...

  19. Inversion of General Cyclic Heptadiagonal Matrices

    Directory of Open Access Journals (Sweden)

    A. A. Karawia

    2013-01-01

    Full Text Available We describe a reliable symbolic computational algorithm for inverting general cyclic heptadiagonal matrices by using parallel computing along with recursion. The computational cost of it is operations. The algorithm is implementable to the Computer Algebra System (CAS such as MAPLE, MATLAB, and MATHEMATICA. Two examples are presented for the sake of illustration.

  20. Cyclic olefin copolymer-silica nanocomposites foams

    Czech Academy of Sciences Publication Activity Database

    Pegoretti, A.; Dorigato, A.; Biani, A.; Šlouf, Miroslav

    2016-01-01

    Roč. 51, č. 8 (2016), s. 3907-3916 ISSN 0022-2461 R&D Projects: GA MŠk(CZ) LO1507 Institutional support: RVO:61389013 Keywords : cyclic olefin copolymer * nanocomposites * silica Subject RIV: CD - Macromolecular Chemistry Impact factor: 2.599, year: 2016

  1. Steady state oxygen reduction and cyclic voltammetry

    DEFF Research Database (Denmark)

    Rossmeisl, Jan; Karlberg, Gustav; Jaramillo, Thomas

    2008-01-01

    The catalytic activity of Pt and Pt3Ni for the oxygen reduction reaction is investigated by applying a Sabatier model based on density functional calculations. We investigate the role of adsorbed OH on the activity, by comparing cyclic voltammetry obtained from theory with previously published ex...

  2. Characterization of the cGMP-dependent protein kinase SmcGK1 of Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    Silke Leutner

    2011-06-01

    Full Text Available Schistosomes are trematode parasites and of worldwide medical importance for humans and animals. Growth and development of these parasites require a specific host environment, but also permanent communication processes between the two genders. Accumulating molecular evidence indicates that the responsible interactions are mediated by signal transduction processes. Conserved signaling molecules were identified, and first approaches made for their characterization. However, no representative of the conserved family of cGMP-dependent protein kinases (cGKs has been described in this parasite yet. Within the Schistosoma mansoni genome data-set we identified cGK homologs, of which one was investigated in more detail in this study. We present the cloning of SmcGK1, whose sequence shows homology to cGKs of higher eukaryotes. SmcGK1 was found to be gender-independently transcribed in adult schistosomes. The occurrence of SmcGK1 sense and antisense transcripts suggests that the expression of this gene is controlled at the post-transcriptional level. In situ hybridization experiments demonstrated a gonad-preferential expression profile in both genders indicating a role of SmcGK1, at least during sexual development of schistosomes. Using a cGK-specific inhibitor to treat adult schistosomes in vitro finally resulted in a multifaceted phenotype including slow motion, oocyte congestion, and reduced egg production.Esquistossomos são parasitas trematodos de importância médica em todo o mundo para o homem e os animais. O crescimento e o desenvolvimento destes parasitas requerem um ambiente específico do hospedeiro, mas também um processo de comunicação permanente entre parasitas dos dois sexos. Evidência molecular tem se acumulado e indica que as interações são mediadas por processos de transdução de sinal. Moléculas sinalizadoras conservadas foram identificadas, e as primeiras abordagens têm sido feitas para sua caracterização. Contudo, não foi

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

    International Nuclear Information System (INIS)

    Crowley, Richard; FitzGerald, Libby Harvey

    2006-01-01

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

  4. GMP scale-up and banking of pluripotent stem cells for cellular therapy applications.

    Science.gov (United States)

    Ausubel, Lara J; Lopez, Patricia M; Couture, Larry A

    2011-01-01

    Human pluripotent stem cells (PSCs), which include human embryonic stem cells (ESCs) as well as induced pluripotent stem cells (iPSCs), represent an important source of cellular therapies in regenerative medicine and the study of early human development. As such, it is becoming increasingly important to develop methods for the large-scale banking of human PSC lines. There are several well-established methods for the propagation of human PSCs. The key to development of a good manufacturing practice (GMP) bank is to determine a manufacturing method that is amenable to large-scale production using materials that are fully documented. We have developed several banks of hESCs using animal feeder cells, animal-based matrices, or animal-free matrices. Protocols for growing hESCs on mouse embryonic fibroblasts (MEFs) are well established and are very helpful for producing research grade banks of cells. As most human ESCs cultured by research laboratories have been exposed to xenogeneic reagents, it is not imperative that all materials used in the production of a master cell bank be animal-free in origin. Nevertheless, as the field develops, it will no doubt become increasingly important to produce a bank of cells for clinical use without xenogeneic reagents, particularly nonhuman feeder cells which might harbor viruses with potential risk to human health or cell product integrity. Thus, even for cell lines previously exposed to xenogeneic reagents, it is important to minimize any subsequent exposure of the cell lines to additional adventitious agents. We have specifically described procedures for the growth of hESCs on Matrigel, an animal-matrix, and CELLstart, an animal-free matrix, and these can be used to produce hESCs as part of a clinical manufacturing process.

  5. Radiological safety and GMP in the bulk batch manufacturing, formulation and dispensing of radiopharmaceuticals

    International Nuclear Information System (INIS)

    Thulasidhasan, A.; Tiwary, Bikash; Kumar, Uma Sheri; Kale, Pooja; Tiwary, Richa; Gaurav, Ananad; Shah, B.K.; Topale, P.D.; Prabhakar, G.; Sachdev, S.S.

    2010-01-01

    Full text: Radiopharmaceutical Program of BRIT is involved in the manufacturing of ready to use radiopharmaceuticals for therapy for the last three decades. Ready to use Radiopharmaceutical products include, 131 I-Sodium iodide solution and capsules for thyroid carcinoma and metastatic lesions, 131 I MIBG injection for diagnosis and therapy of adrenal medullae tumors and their mets, 153 Sm-EDTMP injection and 32 P- Sodium orthophosphate injection for bone pain palliative treatment. BRIT's radiopharmaceutical production facility is a radioisotope laboratory classified as Type-III facility for handling radioisotopes of Group-II, Group-III and Group-IV approved by AERB. This facility meets all the radiological safety requirements as per AERB guidelines. Production of above mentioned radiopharmaceuticals is carried out in Production Plants (PP), β, γ Glove boxes (GB) and Fume hoods (FH). Typical production procedure involves bulk processing, formulation, sterilization and dispensing of doses. Production Plants (PP) are exclusively designed facilities to carry out the production in a radiologically safe manner, at the same time maintaining aseptic conditions required for injectables as per the current GMP. Each production plant has a leak tight Isolator box, made up of high quality SS which has provisions for remote handling devices like, Tongs, dispensing systems, service points for vacuum, gas, compressed air, water and electric power. This Isolator box is shielded from all sides by required amount of lead (2 inch or 4 inch), and has an access port called 'Transport Port Box' with double door transport lock and is equipped with a trolley. Two filter unit systems are fitted at the top of the plant and each unit comprises of activated Charcoal filter and HEPA filter in tandem, this in turn is connected to special exhaust meant for radioactive gases. Similarly, the designs of β, γ Glove boxes (GB) and Fume hoods (FH) also incorporate all radiological safety features

  6. Identification of cyclic nucleotide gated channels using regular expressions

    KAUST Repository

    Zelman, Alice K.; Dawe, Adam Sean; Berkowitz, Gerald A.

    2013-01-01

    Cyclic nucleotide-gated channels (CNGCs) are nonselective cation channels found in plants, animals, and some bacteria. They have a six-transmembrane/one- pore structure, a cytosolic cyclic nucleotide-binding domain, and a cytosolic calmodulin

  7. Rhodium-Catalyzed Dehydrogenative Borylation of Cyclic Alkenes

    Science.gov (United States)

    Kondoh, Azusa; Jamison, Timothy F.

    2010-01-01

    A rhodium-catalyzed dehydrogenative borylation of cyclic alkenes is described. This reaction provides direct access to cyclic 1-alkenylboronic acid pinacol esters, useful intermediates in organic synthesis. Suzuki-Miyaura cross-coupling applications are also presented. PMID:20107646

  8. Cyclic inelastic deformation aspects of fatigue-crack-growth analysis

    Energy Technology Data Exchange (ETDEWEB)

    Leis, B.N.; Zahoor, A.

    1980-01-01

    This paper concentrates on a J-integral analysis of fatigue crack growth. Data on cyclic plasticity are analyzed showing that there are limitations to the usefulness of the deformation theory in applications to cyclic plasticity. 56 refs.

  9. Compressed sensing with cyclic-S Hadamard matrix for terahertz imaging applications

    Science.gov (United States)

    Ermeydan, Esra Şengün; ćankaya, Ilyas

    2018-01-01

    Compressed Sensing (CS) with Cyclic-S Hadamard matrix is proposed for single pixel imaging applications in this study. In single pixel imaging scheme, N = r . c samples should be taken for r×c pixel image where . denotes multiplication. CS is a popular technique claiming that the sparse signals can be reconstructed with samples under Nyquist rate. Therefore to solve the slow data acquisition problem in Terahertz (THz) single pixel imaging, CS is a good candidate. However, changing mask for each measurement is a challenging problem since there is no commercial Spatial Light Modulators (SLM) for THz band yet, therefore circular masks are suggested so that for each measurement one or two column shifting will be enough to change the mask. The CS masks are designed using cyclic-S matrices based on Hadamard transform for 9 × 7 and 15 × 17 pixel images within the framework of this study. The %50 compressed images are reconstructed using total variation based TVAL3 algorithm. Matlab simulations demonstrates that cyclic-S matrices can be used for single pixel imaging based on CS. The circular masks have the advantage to reduce the mechanical SLMs to a single sliding strip, whereas the CS helps to reduce acquisition time and energy since it allows to reconstruct the image from fewer samples.

  10. Free and total GMP (glycomacropeptide contents of milk during bovine lactation Variação dos teores de GMP (glicomacropeptídeo livre e total no leite bovino durante a lactação

    Directory of Open Access Journals (Sweden)

    Andréa Maria Furlanetti

    2003-12-01

    Full Text Available Individual milk samples taken every two weeks from parturition to the end of lactation from 34 animals of three different herds and breeds were analyzed for free-GMP. A milk pool of each herd was analyzed for free and total GMP (released from k-casein by the action of rennin and the data were correlated with sanitary conditions of animal and udder, phase of lactation and milk production. Most udder problems were concentrated near parturition, with few and spaced occurrences of clinical mastitis. The Californian Mastitis Test (CMT results showed oscillations compatible with the phases of lactation period and environmental conditions. The widest variations in free-GMP occurred as a function of lactation period and as a consequence of clinical or subclinical mastitis. Higher levels were observed at the beginning of lactation (5.87mg L-1 of sialic acid, becoming normal with mean values of about 3.30mg L-1 at the end of the second month, and increasing again during the final third of lactation. On average, the same trends were observed for total GMP released by commercial rennet, beginning with slightly high values (35.59mg L-1, becoming normal by the sixth month with values close to 27.15mg L-1, and rising gradually up to the end of lactation, with 58.35mg L-1 of sialic acid. These results prove to be useful for the correct interpretation of tests applied to milk selection with respect to proteolytic status or even to restrain frauds by the addition of whey to milk.Amostras quinzenais, desde o parto até o final do período de lactação, obtidas de 34 vacas de três diferentes raças e propriedades, foram analisadas quanto à presença de GMP livre. Um "pool" das amostras quinzenais de cada rebanho foi analisada tanto para o conteúdo de GMP livre quanto para o GMP total (liberado da k-caseína pela ação da renina, correlacionando-os com as condições sanitárias do animal e do úbere, à fase da lactação e à produção de leite. A maioria

  11. Direct interaction of the inhibitory gamma-subunit of Rod cGMP phosphodiesterase (PDE6) with the PDE6 GAFa domains.

    Science.gov (United States)

    Muradov, Khakim G; Granovsky, Alexey E; Schey, Kevin L; Artemyev, Nikolai O

    2002-03-26

    Retinal rod and cone cGMP phosphodiesterases (PDE6 family) function as the effector enzyme in the vertebrate visual transduction cascade. The activity of PDE6 catalytic subunits is controlled by the Pgamma-subunits. In addition to the inhibition of cGMP hydrolysis at the catalytic sites, Pgamma is known to stimulate a noncatalytic binding of cGMP to the regulatory GAFa-GAFb domains of PDE6. The latter role of Pgamma has been attributed to its polycationic region. To elucidate the structural basis for the regulation of cGMP binding to the GAF domains of PDE6, a photoexcitable peptide probe corresponding to the polycationic region of Pgamma, Pgamma-21-45, was specifically cross-linked to rod PDE6alphabeta. The site of Pgamma-21-45 cross-linking was localized to Met138Gly139 within the PDE6alpha GAFa domain using mass spectrometric analysis. Chimeras between PDE5 and cone PDE6alpha', containing GAFa and/or GAFb domains of PDE6alpha' have been generated to probe a potential role of the GAFb domains in binding to Pgamma. Analysis of the inhibition of the PDE5/PDE6alpha' chimeras by Pgamma supported the role of PDE6 GAFa but not GAFb domains in the interaction with Pgamma. Our results suggest that a direct binding of the polycationic region of Pgamma to the GAFa domains of PDE6 may lead to a stabilization of the noncatalytic cGMP-binding sites.

  12. Cyclic Soft Groups and Their Applications on Groups

    Directory of Open Access Journals (Sweden)

    Hacı Aktaş

    2014-01-01

    Full Text Available In crisp environment the notions of order of group and cyclic group are well known due to many applications. In this paper, we introduce order of the soft groups, power of the soft sets, power of the soft groups, and cyclic soft group on a group. We also investigate the relationship between cyclic soft groups and classical groups.

  13. Participation of the NO/cGMP/K+ATP pathway in the antinociception induced by Walker tumor bearing in rats

    International Nuclear Information System (INIS)

    Barbosa, A.L.R.; Pinheiro, C.A.; Oliveira, G.J.; Torres, J.N.L.; Moraes, M.O.; Ribeiro, R.A.; Vale, M.L.; Souza, M.H.L.P.

    2012-01-01

    Implantation of Walker 256 tumor decreases acute systemic inflammation in rats. Inflammatory hyperalgesia is one of the most important events of acute inflammation. The L-arginine/NO/cGMP/K + ATP pathway has been proposed as the mechanism of peripheral antinociception mediated by several drugs and physical exercise. The objective of this study was to investigate a possible involvement of the NO/cGMP/K + ATP pathway in antinociception induced in Walker 256 tumor-bearing male Wistar rats (180-220 g). The groups consisted of 5-6 animals. Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. Walker tumor (4th and 7th day post-implantation) reduced prostaglandin E 2 - (PGE 2 , 400 ng/paw; 50 µL; intraplantar injection) and carrageenan-induced hypernociception (500 µg/paw; 100 µL; intraplantar injection). Walker tumor-induced analgesia was reversed (99.3% for carrageenan and 77.2% for PGE 2 ) by a selective inhibitor of nitric oxide synthase (L-NAME; 90 mg/kg, ip) and L-arginine (200 mg/kg, ip), which prevented (80% for carrageenan and 65% for PGE 2 ) the effect of L-NAME. Treatment with the soluble guanylyl cyclase inhibitor ODQ (100% for carrageenan and 95% for PGE 2 ; 8 µg/paw) and the ATP-sensitive K + channel (KATP) blocker glibenclamide (87.5% for carrageenan and 100% for PGE 2 ; 160 µg/paw) reversed the antinociceptive effect of tumor bearing in a statistically significant manner (P < 0.05). The present study confirmed an intrinsic peripheral antinociceptive effect of Walker tumor bearing in rats. This antinociceptive effect seemed to be mediated by activation of the NO/cGMP pathway followed by the opening of KATP channels

  14. Human platelet lysate in mesenchymal stromal cell expansion according to a GMP grade protocol: a cell factory experience.

    Science.gov (United States)

    Becherucci, Valentina; Piccini, Luisa; Casamassima, Serena; Bisin, Silvia; Gori, Valentina; Gentile, Francesca; Ceccantini, Riccardo; De Rienzo, Elena; Bindi, Barbara; Pavan, Paola; Cunial, Vanessa; Allegro, Elisa; Ermini, Stefano; Brugnolo, Francesca; Astori, Giuseppe; Bambi, Franco

    2018-05-02

    The use of platelet lysate (PL) for the ex-vivo expansion of mesenchymal stromal/stem cells (MSCs) was initially proposed by Doucet et al. in 2005, as an alternative to animal serum. Moreover, regulatory authorities discourage the use of fetal bovine serum (FBS) or other animal derivatives, to avoid risk of zoonoses and xenogeneic immune reactions. Even if many studies investigated PL composition, there still are some open issues related to its use in ex-vivo MSC expansion, especially according to good manufacturing practice (GMP) grade protocols. As an authorized cell factory, we report our experience using standardized PL produced by Azienda Ospedaliero Universitaria Meyer Transfusion Service for MSC expansion according to a GMP grade clinical protocol. As suggested by other authors, we performed an in-vitro test on MSCs versus MSCs cultured with FBS that still represents the best way to test PL batches. We compared 12 MSC batches cultured with DMEM 5% PL with similar batches cultured with DMEM 10% FBS, focusing on the MSC proliferation rate, MSC surface marker expression, MSC immunomodulatory and differentiation potential, and finally MSC relative telomere length. Results confirmed the literature data as PL increases cell proliferation without affecting the MSC immunophenotype, immunomodulatory potential, differentiation potential and relative telomere length. PL can be considered a safe alternative to FBS for ex-vivo expansion of MSC according to a GMP grade protocol. Our experience confirms the literature data: a large number of MSCs for clinical applications can be obtained by expansion with PL, without affecting the MSC main features. Our experience underlines the benefits of a close collaboration between the PL producers (transfusion service) and the end users (cell factory) in a synergy of skills and experiences that can lead to standardized PL production.

  15. Salvinorin A preserves cerebral pial artery autoregulation after forebrain ischemia via the PI3K/AKT/cGMP pathway

    Directory of Open Access Journals (Sweden)

    H.P. Dong

    2018-03-01

    Full Text Available This study aimed to investigate the protective effect of salvinorin A on the cerebral pial artery after forebrain ischemia and explore related mechanisms. Thirty Sprague-Dawley rats received forebrain ischemia for 10 min. The dilation responses of the cerebral pial artery to hypercapnia and hypotension were assessed in rats before and 1 h after ischemia. The ischemia reperfusion (IR control group received DMSO (1 µL/kg immediately after ischemia. Two different doses of salvinorin A (10 and 20 µg/kg were administered following the onset of reperfusion. The 5th, 6th, and 7th groups received salvinorin A (20 µg/kg and LY294002 (10 µM, L-NAME (10 μM, or norbinaltorphimine (norBIN, 1 μM after ischemia. The levels of cGMP in the cerebrospinal fluid (CSF were also measured. The phosphorylation of AKT (p-AKT was measured in the cerebral cortex by western blot at 24 h post-ischemia. Cell necrosis and apoptosis were examined by hematoxylin-eosin staining (HE and TUNEL staining, respectively. The motor function of the rats was evaluated at 1, 2, and 5 days post-ischemia. The dilation responses of the cerebral pial artery were significantly impaired after ischemia and were preserved by salvinorin A treatment. In addition, salvinorin A significantly increased the levels of cGMP and p-AKT, suppressed cell necrosis and apoptosis of the cerebral cortex and improved the motor function of the rats. These effects were abolished by LY294002, L-NAME, and norBIN. Salvinorin A preserved cerebral pial artery autoregulation in response to hypercapnia and hypotension via the PI3K/AKT/cGMP pathway.

  16. CHARACTERIZATION OF ORG-20241, A COMBINED PHOSPHODIESTERASE IV/III CYCLIC-NUCLEOTIDE PHOSPHODIESTERASE INHIBITOR FOR ASTHMA

    NARCIS (Netherlands)

    NICHOLSON, CD; BRUIN, J; BARRON, E; SPIERS, [No Value; DEBOER, J; VANAMSTERDAM, RGM; ZAAGSMA, J; KELLY, JJ; DENT, G; GIEMBYCZ, MA; BARNES, PJ

    The pharmacological profile of a novel cyclic nucleotide phosphodiesterase (PDE) inhibitor, Org 20241, has been characterized. The compound selectively inhibits PDE IV (plC(50), 5.2-6.1) and PDE III (plC(50), 4.4-4.6) from animal and human tissues. Org 20241 relaxed preparations of bovine trachea

  17. A cyclic symmetry principle in physics

    International Nuclear Information System (INIS)

    Green, H.S.; Adelaide Univ., SA

    1994-01-01

    Many areas of modern physics are illuminated by the application of a symmetry principle, requiring the invariance of the relevant laws of physics under a group of transformations. This paper examines the implications and some of the applications of the principle of cyclic symmetry, especially in the areas of statistical mechanics and quantum mechanics, including quantized field theory. This principle requires invariance under the transformations of a finite group, which may be a Sylow π-group, a group of Lie type, or a symmetric group. The utility of the principle of cyclic invariance is demonstrated in finding solutions of the Yang-Baxter equation that include and generalize known solutions. It is shown that the Sylow π-groups have other uses, in providing a basis for a type of generalized quantum statistics, and in parametrising a new generalization of Lie groups, with associated algebras that include quantized algebras. 31 refs

  18. Strain gradient effects on cyclic plasticity

    DEFF Research Database (Denmark)

    Niordson, Christian Frithiof; Legarth, Brian Nyvang

    2010-01-01

    Size effects on the cyclic shear response are studied numerically using a recent higher order strain gradient visco-plasticity theory accounting for both dissipative and energetic gradient hardening. Numerical investigations of the response under cyclic pure shear and shear of a finite slab between...... rigid platens have been carried out, using the finite element method. It is shown for elastic–perfectly plastic solids how dissipative gradient effects lead to increased yield strength, whereas energetic gradient contributions lead to increased hardening as well as a Bauschinger effect. For linearly...... hardening materials it is quantified how dissipative and energetic gradient effects promote hardening above that of conventional predictions. Usually, increased hardening is attributed to energetic gradient effects, but here it is found that also dissipative gradient effects lead to additional hardening...

  19. Generalized Toeplitz operators and cyclic vectors

    International Nuclear Information System (INIS)

    Gassier, G.; Mahzouli, H.; Zerouali, E.H.

    2003-04-01

    We give in this paper some asymptotic Von Neumann inequalities for power bounded operators in the class C ρ intersection C 1 . and some spacial von Neumann inequalities associated with non zero elements of the point spectrum, when it is non void, of generalized Toeplitz operators. Introducing perturbed kernel, we consider classes C R which extend the classical classes C ρ . We give results about absolute continuity with respect to the Haar measure for operators in class C R intersection C 1 . This allows us to give new results on cyclic vectors for such operators and provides invariant subspaces for their powers. Relationships between cyclic vectors for T and T* involving generalized Toeplitz operators are given and the commutativity of {T}', the commutant of T is discussed. (author)

  20. Cosmic evolution in a cyclic universe

    International Nuclear Information System (INIS)

    Steinhardt, Paul J.; Turok, Neil

    2002-01-01

    Based on concepts drawn from the ekpyrotic scenario and M theory, we elaborate our recent proposal of a cyclic model of the universe. In this model, the universe undergoes an endless sequence of cosmic epochs which begin with the universe expanding from a 'big bang' and end with the universe contracting to a 'big crunch'. Matching from 'big crunch' to 'big bang' is performed according to the prescription recently proposed with Khoury, Ovrut and Seiberg. The expansion part of the cycle includes a period of radiation and matter domination followed by an extended period of cosmic acceleration at low energies. The cosmic acceleration is crucial in establishing the flat and vacuous initial conditions required for ekpyrosis and for removing the entropy, black holes, and other debris produced in the preceding cycle. By restoring the universe to the same vacuum state before each big crunch, the acceleration ensures that the cycle can repeat and that the cyclic solution is an attractor

  1. Separation of isotopes by cyclical processes

    International Nuclear Information System (INIS)

    Hamrin, C.E. Jr.; Weaver, K.

    1976-01-01

    Various isotopes of hydrogen are separated by a cyclic sorption process in which a gas stream containing the isotopes is periodically passed through a high pressure column containing a palladium sorbent. A portion of the product from the high pressure column is passed through a second column at lower pressure to act as a purge. Before the sorbent in the high pressure column becomes saturated, the sequence is reversed with the stream flowing through the former low-pressure column now at high pressure, and a portion of the product purging the former high pressure column now at low pressure. The sequence is continued in cyclic manner with the product being enriched in a particular isotope

  2. Soil Fatigue Due To Cyclically Loaded Foundations

    OpenAIRE

    Pytlik, Robert Stanislaw

    2016-01-01

    Cyclic loading on civil structures can lead to a reduction of strength of the used materials. A literature study showed that, in contrast to steel structures and material engineering, there are no design codes or standards for fatigue of foundations and the surrounding ground masses in terms of shear strength reduction. Scientific efforts to study the fatigue behaviour of geomaterials are mainly focused on strain accumulation, while the reduction of shear strength of geomaterials has not been...

  3. Reaction of cyclic epoxide compounds with triphenylphosphine

    International Nuclear Information System (INIS)

    Kas'yan, L.I.; Stepanova, N.V.; Galafeeva, M.F.; Boldeskul, I.E.; Trachevskii, V.V.; Zefirov, N.S.

    1987-01-01

    Significant differences were found in the reactivity of a series of epoxides of cycloalkenes and methylenecycloalkanes and diepoxides in reaction with triphenylphosphine, depending both on the steric effects of the cyclic fragments and on their strain. The level of the strain can be judged indirectly from the chemical shifts of the 1 H and 13 C nuclei and the spin-spin coupling constants of the C-H bonds in the epoxide ring

  4. Human skin kinetics of cyclic depsipeptide mycotoxins

    OpenAIRE

    Taevernier, Lien; Veryser, Lieselotte; ROCHE, NATHALIE; De Spiegeleer, Bart

    2014-01-01

    Cyclic depsipeptides (CDPs) are an emerging group of naturally occurring bioactive peptides, some of which are already developed as pharmaceutical drugs, e.g. valinomycin. They are produced by bacteria, marine organisms and fungi [1]. Some CDPs are secondary fungal metabolites, which can be very toxic to humans and animals, and are therefore called mycotoxins. Currently, dermal exposure data of CDP mycotoxins is scarce and fragmentary with a lack of understanding about the local skin and syst...

  5. Modelling of cyclical stratigraphy using Markov chains

    Energy Technology Data Exchange (ETDEWEB)

    Kulatilake, P.H.S.W.

    1987-07-01

    State-of-the-art on modelling of cyclical stratigraphy using first-order Markov chains is reviewed. Shortcomings of the presently available procedures are identified. A procedure which eliminates all the identified shortcomings is presented. Required statistical tests to perform this modelling are given in detail. An example (the Oficina formation in eastern Venezuela) is given to illustrate the presented procedure. 12 refs., 3 tabs. 1 fig.

  6. Markup cyclicality, employment adjustment, and financial constraints

    OpenAIRE

    Askildsen, Jan Erik; Nilsen, Øivind Anti

    2001-01-01

    We investigate the existence of markups and their cyclical behaviour. Markup is not directly observed. Instead, it is given as a price-cost relation that is estimated from a dynamic model of the firm. The model incorporates potential costly employment adjustments and takes into consideration that firms may be financially constrained. When considering size of the future labour stock, financially constrained firms may behave as if they have a higher discount factor, which may affect the realise...

  7. Characterization of cyclic peptides containing disulfide bonds

    OpenAIRE

    Johnson, Mindy; Liu, Mingtao; Struble, Elaine; Hettiarachchi, Kanthi

    2015-01-01

    Unlike linear peptides, analysis of cyclic peptides containing disulfide bonds is not straightforward and demands indirect methods to achieve a rigorous proof of structure. Three peptides that belong to this category, p-Cl-Phe-DPDPE, DPDPE, and CTOP, were analyzed and the results are presented in this paper. The great potential of two dimensional NMR and ESI tandem mass spectrometry was harnessed during the course of peptide characterizations. A new RP-HPLC method for the analysis of trifluor...

  8. Visual search of cyclic spatio-temporal events

    Science.gov (United States)

    Gautier, Jacques; Davoine, Paule-Annick; Cunty, Claire

    2018-05-01

    The analysis of spatio-temporal events, and especially of relationships between their different dimensions (space-time-thematic attributes), can be done with geovisualization interfaces. But few geovisualization tools integrate the cyclic dimension of spatio-temporal event series (natural events or social events). Time Coil and Time Wave diagrams represent both the linear time and the cyclic time. By introducing a cyclic temporal scale, these diagrams may highlight the cyclic characteristics of spatio-temporal events. However, the settable cyclic temporal scales are limited to usual durations like days or months. Because of that, these diagrams cannot be used to visualize cyclic events, which reappear with an unusual period, and don't allow to make a visual search of cyclic events. Also, they don't give the possibility to identify the relationships between the cyclic behavior of the events and their spatial features, and more especially to identify localised cyclic events. The lack of possibilities to represent the cyclic time, outside of the temporal diagram of multi-view geovisualization interfaces, limits the analysis of relationships between the cyclic reappearance of events and their other dimensions. In this paper, we propose a method and a geovisualization tool, based on the extension of Time Coil and Time Wave, to provide a visual search of cyclic events, by allowing to set any possible duration to the diagram's cyclic temporal scale. We also propose a symbology approach to push the representation of the cyclic time into the map, in order to improve the analysis of relationships between space and the cyclic behavior of events.

  9. Cyclic dominance in evolutionary games: a review

    Science.gov (United States)

    Szolnoki, Attila; Mobilia, Mauro; Jiang, Luo-Luo; Szczesny, Bartosz; Rucklidge, Alastair M.; Perc, Matjaž

    2014-01-01

    Rock is wrapped by paper, paper is cut by scissors and scissors are crushed by rock. This simple game is popular among children and adults to decide on trivial disputes that have no obvious winner, but cyclic dominance is also at the heart of predator–prey interactions, the mating strategy of side-blotched lizards, the overgrowth of marine sessile organisms and competition in microbial populations. Cyclical interactions also emerge spontaneously in evolutionary games entailing volunteering, reward, punishment, and in fact are common when the competing strategies are three or more, regardless of the particularities of the game. Here, we review recent advances on the rock–paper–scissors (RPS) and related evolutionary games, focusing, in particular, on pattern formation, the impact of mobility and the spontaneous emergence of cyclic dominance. We also review mean-field and zero-dimensional RPS models and the application of the complex Ginzburg–Landau equation, and we highlight the importance and usefulness of statistical physics for the successful study of large-scale ecological systems. Directions for future research, related, for example, to dynamical effects of coevolutionary rules and invasion reversals owing to multi-point interactions, are also outlined. PMID:25232048

  10. Scale factor duality for conformal cyclic cosmologies

    Energy Technology Data Exchange (ETDEWEB)

    Silva, University Camara da; Lima, A.L. Alves; Sotkov, G.M. [Departamento de Física - CCE,Universidade Federal de Espirito Santo, 29075-900, Vitoria ES (Brazil)

    2016-11-16

    The scale factor duality is a symmetry of dilaton gravity which is known to lead to pre-big-bang cosmologies. A conformal time version of the scale factor duality (SFD) was recently implemented as a UV/IR symmetry between decelerated and accelerated phases of the post-big-bang evolution within Einstein gravity coupled to a scalar field. The problem investigated in the present paper concerns the employment of the conformal time SFD methods to the construction of pre-big-bang and cyclic extensions of these models. We demonstrate that each big-bang model gives rise to two qualitatively different pre-big-bang evolutions: a contraction/expansion SFD model and Penrose’s Conformal Cyclic Cosmology (CCC). A few examples of SFD symmetric cyclic universes involving certain gauged Kähler sigma models minimally coupled to Einstein gravity are studied. We also describe the specific SFD features of the thermodynamics and the conditions for validity of the generalized second law in the case of Gauss-Bonnet (GB) extension of these selected CCC models.

  11. Scale factor duality for conformal cyclic cosmologies

    International Nuclear Information System (INIS)

    Silva, University Camara da; Lima, A.L. Alves; Sotkov, G.M.

    2016-01-01

    The scale factor duality is a symmetry of dilaton gravity which is known to lead to pre-big-bang cosmologies. A conformal time version of the scale factor duality (SFD) was recently implemented as a UV/IR symmetry between decelerated and accelerated phases of the post-big-bang evolution within Einstein gravity coupled to a scalar field. The problem investigated in the present paper concerns the employment of the conformal time SFD methods to the construction of pre-big-bang and cyclic extensions of these models. We demonstrate that each big-bang model gives rise to two qualitatively different pre-big-bang evolutions: a contraction/expansion SFD model and Penrose’s Conformal Cyclic Cosmology (CCC). A few examples of SFD symmetric cyclic universes involving certain gauged Kähler sigma models minimally coupled to Einstein gravity are studied. We also describe the specific SFD features of the thermodynamics and the conditions for validity of the generalized second law in the case of Gauss-Bonnet (GB) extension of these selected CCC models.

  12. GMP-compliant automated synthesis of [{sup 18}F]AV-45 (Florbetapir F 18) for imaging {beta}-amyloid plaques in human brain

    Energy Technology Data Exchange (ETDEWEB)

    Yao, C.-H. [Department of Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Taiwan (China); Lin, K.-J. [Department of Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Taiwan (China); Department of Medical Imaging and Radiological Sciences, Chang Gung University, 259 Wen-Hua 1st Road, Kweishan, Taoyuan 333, Taiwan (China); Weng, C.-C. [Department of Medical Imaging and Radiological Sciences, Chang Gung University, 259 Wen-Hua 1st Road, Kweishan, Taoyuan 333, Taiwan (China); Hsiao, I.-T. [Department of Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Taiwan (China); Department of Medical Imaging and Radiological Sciences, Chang Gung University, 259 Wen-Hua 1st Road, Kweishan, Taoyuan 333, Taiwan (China); Ting, Y.-S. [Department of Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Taiwan (China); Yen, T.-C. [Department of Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Taiwan (China); Department of Medical Imaging and Radiological Sciences, Chang Gung University, 259 Wen-Hua 1st Road, Kweishan, Taoyuan 333, Taiwan (China); Jan, T.-R. [Department and Graduate Institute of Veterinary Medicine, National Taiwan University, Taipei, Taiwan (China); Skovronsky, Daniel [Avid Radiopharmaceuticals, Inc., Philadelphia, PA 19104 (United States); Kung, M.-P. [Department of Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Taiwan (China); Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Wey, S.-P., E-mail: spwey@mail.cgu.edu.t [Department of Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Taiwan (China); Department of Medical Imaging and Radiological Sciences, Chang Gung University, 259 Wen-Hua 1st Road, Kweishan, Taoyuan 333, Taiwan (China)

    2010-12-15

    We report herein the Good Manufacturing Practice (GMP)-compliant automated synthesis of {sup 18}F-labeled styrylpyridine, AV-45 (Florbetapir), a novel tracer for positron emission tomography (PET) imaging of {beta}-amyloid (A{beta}) plaques in the brain of Alzheimer's disease patients. [{sup 18}F]AV-45 was prepared in 105 min using a tosylate precursor with Sumitomo modules for radiosynthesis under GMP-compliant conditions. The overall yield was 25.4{+-}7.7% with a final radiochemical purity of 95.3{+-}2.2% (n=19). The specific activity of [{sup 18}F]AV-45 reached as high as 470{+-}135 TBq/mmol (n=19). The present studies show that [{sup 18}F]AV-45 can be manufactured under GMP-compliant conditions and could be widely available for routine clinical use.

  13. Magneto-optical nanoparticles for cyclic magnetomotive photoacoustic imaging

    Science.gov (United States)

    Arnal, Bastien; Yoon, Soon Joon; Li, Junwei; Gao, Xiaohu; O'Donnell, Matthew

    2018-05-01

    Photoacoustic imaging is a highly promising tool to visualize molecular events with deep tissue penetration. Like most other modalities, however, image contrast under in vivo conditions is far from optimal due to background signals from tissue. Using iron oxide-gold core-shell nanoparticles, we previously demonstrated that magnetomotive photoacoustic (mmPA) imaging can dramatically reduce the influence of background signals and produce high-contrast molecular images. Here we report two significant advances toward clinical translation of this technology. First, we introduce a new class of compact, uniform, magneto-optically coupled core-shell nanoparticle, prepared through localized copolymerization of polypyrrole (PPy) on an iron oxide nanoparticle surface. The resulting iron oxide-PPy nanoparticles solve the photo-instability and small-scale synthesis problems previously encountered by the gold coating approach, and extend the large optical absorption coefficient of the particles beyond 1000 nm in wavelength. In parallel, we have developed a new generation of mmPA imaging featuring cyclic magnetic motion and ultrasound speckle tracking, with an image capture frame rate several hundred times faster than the photoacoustic speckle tracking method demonstrated previously. These advances enable robust artifact elimination caused by physiologic motion and first application of the mmPA technology in vivo for sensitive tumor imaging.

  14. Cost benefit of investment on quality in pharmaceutical manufacturing: WHO GMP pre- and post-certification of a Nigerian pharmaceutical manufacturer.

    Science.gov (United States)

    Anyakora, Chimezie; Ekwunife, Obinna; Alozie, Faith; Esuga, Mopa; Ukwuru, Jonathan; Onya, Steve; Nwokike, Jude

    2017-09-18

    Pharmaceutical companies in Africa need to invest in both facilities and quality management systems to achieve good manufacturing practice (GMP) compliance. Compliance to international GMP standards is important to the attainment of World Health Organization (WHO) prequalification. However, most of the local pharmaceutical manufacturing companies may be deterred from investing in quality because of many reasons, ranging from financial constraints to technical capacity. This paper primarily evaluates benefits against the cost of investing in GMP, using a Nigerian pharmaceutical company, Chi Pharmaceuticals Limited, as a case study. This paper also discusses how to drive more local manufacturers to invest in quality to attain GMP compliance; and proffers practical recommendations for local manufacturers who would want to invest in quality to meet ethical and regulatory obligations. The cost benefit of improving the quality of Chi Pharmaceuticals Limited's facilities and system to attain WHO GMP certification for the production of zinc sulfate 20-mg dispersible tablets was calculated by dividing the annual benefits derived from quality improvement interventions by the annual costs of implementing quality improvement interventions, referred to as a benefit-cost ratio (BCR). Cost benefit of obtaining WHO GMP certification for the production of zinc sulfate 20-mg dispersible tablets was 5.3 (95% confidence interval of 5.0-5.5). Investment in quality improvement intervention is cost-beneficial for local manufacturing companies. Governments and regulators in African countries should support pharmaceutical companies striving to invest in quality. Collaboration of local manufacturing companies with global companies will further improve quality. Local pharmaceutical companies should be encouraged to key into development opportunities available for pharmaceutical companies in Africa.

  15. Ca 2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca 2+ channels

    KAUST Repository

    Qia, Zhi; Verma, Rajeev K.; Gehring, Christoph A; Yamaguchi, Yube; Zhao, Yichen; Ryan, Clarence A.; Berkowitz, Gerald A.

    2010-01-01

    receptor- like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2- dependent cytosolic Ca 2+ elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33

  16. Mitochondrial disorder caused Charles Darwin's cyclic vomiting syndrome

    Directory of Open Access Journals (Sweden)

    Finsterer J

    2014-01-01

    Full Text Available Josef Finsterer,1 John Hayman21Krankenanstalt Rudolfstiftng, Vienna, Austria; 2Department of Pathology, University of Melbourne, Victoria, AustraliaBackground: Charles Darwin (CD, “father of modern biology,” suffered from multisystem illness from early adulthood. The most disabling manifestation was cyclic vomiting syndrome (CVS. This study aims at finding the possible cause of CVS in CD.Methods: A literature search using the PubMed database was carried out, and CD's complaints, as reported in his personal writings and those of his relatives, friends, colleagues, biographers, were compared with various manifestations of mitochondrial disorders (MIDs, known to cause CVS, described in the literature.Results: Organ tissues involved in CD's disease were brain, nerves, muscles, vestibular apparatus, heart, gut, and skin. Cerebral manifestations included episodic headache, visual disturbance, episodic memory loss, periodic paralysis, hysterical crying, panic attacks, and episodes of depression. Manifestations of polyneuropathy included numbness, paresthesias, increased sweating, temperature sensitivity, and arterial hypotension. Muscular manifestations included periods of exhaustion, easy fatigability, myalgia, and muscle twitching. Cardiac manifestations included episodes of palpitations and chest pain. Gastrointestinal manifestations were CVS, dental problems, abnormal seasickness, eructation, belching, and flatulence. Dermatological manifestations included painful lips, dermatitis, eczema, and facial edema. Treatments with beneficial effects to his complaints were rest, relaxation, heat, and hydrotherapy.Conclusion: CVS in CD was most likely due to a multisystem, nonsyndromic MID. This diagnosis is based upon the multisystem nature of his disease, the fact that CVS is most frequently the manifestation of a MID, the family history, the variable phenotypic expression between affected family members, the fact that symptoms were triggered by stress

  17. cGMP-dependent protein kinase Iα associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake

    Directory of Open Access Journals (Sweden)

    Steiner Jennifer A

    2009-08-01

    Full Text Available Abstract Background The Na+/Cl--dependent serotonin (5-hydroxytryptamine, 5-HT transporter (SERT is a critical element in neuronal 5-HT signaling, being responsible for the efficient elimination of 5-HT after release. SERTs are not only targets for exogenous addictive and therapeutic agents but also can be modulated by endogenous, receptor-linked signaling pathways. We have shown that neuronal A3 adenosine receptor activation leads to enhanced presynaptic 5-HT transport in vitro and an increased rate of SERT-mediated 5-HT clearance in vivo. SERT stimulation by A3 adenosine receptors derives from an elevation of cGMP and subsequent activation of both cGMP-dependent protein kinase (PKG and p38 mitogen-activated protein kinase. PKG activators such as 8-Br-cGMP are known to lead to transporter phosphorylation, though how this modification supports SERT regulation is unclear. Results In this report, we explore the kinase isoform specificity underlying the rapid stimulation of SERT activity by PKG activators. Using immortalized, rat serotonergic raphe neurons (RN46A previously shown to support 8-Br-cGMP stimulation of SERT surface trafficking, we document expression of PKGI, and to a lower extent, PKGII. Quantitative analysis of staining profiles using permeabilized or nonpermeabilized conditions reveals that SERT colocalizes with PKGI in both intracellular and cell surface domains of RN46A cell bodies, and exhibits a more restricted, intracellular pattern of colocalization in neuritic processes. In the same cells, SERT demonstrates a lack of colocalization with PKGII in either intracellular or surface membranes. In keeping with the ability of the membrane permeant kinase inhibitor DT-2 to block 8-Br-cGMP stimulation of SERT, we found that DT-2 treatment eliminated cGMP-dependent kinase activity in PKGI-immunoreactive extracts resolved by liquid chromatography. Similarly, treatment of SERT-transfected HeLa cells with small interfering RNAs targeting

  18. Fulfillment of GMP standard, halal standard, and applying HACCP for production process of beef floss (Case study: Ksatria enterprise)

    Science.gov (United States)

    A'diat, Arkan Addien Al; Liquiddanu, Eko; Laksono, Pringgo Widyo; Sutopo, Wahyudi; Suletra, I. Wayan

    2018-02-01

    Along with the increasing number of the modern retail business in Indonesia, give an opportunity to small and medium enterprise (SME) to sell its products through the modern retailer. There are some obstacles faced by the SMEs, one of them is about product standard. Product standard that must be owned by SMEs are GMP standard and halal standard. This research was conducted to know the fulfillment by the beef floss enterprise in jagalan in fulfilling the GMP standard and halal. In addition, Hazard Analysis and Critical Control Points (HACCP) system was applied to analyze the process. HACCP which used in this research was based on the seven principles in SNI (Indonesian National Standard) 01-4852-1998. The seven principles included hazard analysis, critical control point (CCP) determination, critical limit establishment, CCP monitor system establishment, corrective action establishment, verification, and also documentation establishment that must be applied in preparing HACCP plan. Based on this case study, it is concluded that there were 5 CCPs : the boiling process, roasting process, frying process, the beef floss draining process, and the packaging process.

  19. Effects of Tang Niao Ning decoction on ET-1, GMP-140 and TXB2 in diabetic rats

    International Nuclear Information System (INIS)

    Song Jumin; Wang Lifen; Liao Han; Wang Wei; Hua Weiguo

    2003-01-01

    To investigate the effects of Tang Niao Ning decoction on fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), endothelin (ET-1), platelet granule membrane protein (GMP-140), thromboxane B 2 (TXB 2 ), 6-keto-prostaglandin F 1α (6-keto-PGF 1α ), the selected female Wistar rats were modeled by streptozotocin (STZ) and high fat diets, and were randomly divided into two groups as Tang Niao Ning and model group, while the normal control group was set up. The Tang Niao Ning group started to supply decoctions after these rats had been fed one week with high fat diets. The rats were killed after six weeks. Above items were examed. The results showed that the model group displayed marked difference when the experiment finished compared with the normal group. The contents of fasting blood glucose and blood lipid were increased, accompanied with the phenomena of hoisting ET-1, enhancing GMP-140, increasing TXB 2 and droping 6-Keto-PGF 1α . After treatment in time with Tang Niao Ning decoction, all kinds of items were adjusted to normal level gradually. It suggested that the treatment with Tang Niao Ning decoction might decrease FBG and blood lipid. In some ways, the decoction could improve the signs of blood stasis. The regulation mechanisms of the decoction was characteristic of wholism through many channels

  20. Statistical damage constitutive model for rocks subjected to cyclic stress and cyclic temperature

    Science.gov (United States)

    Zhou, Shu-Wei; Xia, Cai-Chu; Zhao, Hai-Bin; Mei, Song-Hua; Zhou, Yu

    2017-10-01

    A constitutive model of rocks subjected to cyclic stress-temperature was proposed. Based on statistical damage theory, the damage constitutive model with Weibull distribution was extended. Influence of model parameters on the stress-strain curve for rock reloading after stress-temperature cycling was then discussed. The proposed model was initially validated by rock tests for cyclic stress-temperature and only cyclic stress. Finally, the total damage evolution induced by stress-temperature cycling and reloading after cycling was explored and discussed. The proposed constitutive model is reasonable and applicable, describing well the stress-strain relationship during stress-temperature cycles and providing a good fit to the test results. Elastic modulus in the reference state and the damage induced by cycling affect the shape of reloading stress-strain curve. Total damage induced by cycling and reloading after cycling exhibits three stages: initial slow increase, mid-term accelerated increase, and final slow increase.

  1. Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

    Science.gov (United States)

    Duffy, Fergal J; O'Donovan, Darragh; Devocelle, Marc; Moran, Niamh; O'Connell, David J; Shields, Denis C

    2015-03-23

    Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.

  2. Cyclic AMP-receptor proteins in heart muscle of rats flown on Cosmos 1887

    Science.gov (United States)

    Mednieks, Maija I.; Popova, Irina A.; Grindeland, Richard E.

    1991-01-01

    The cellular compartmentalization of the cyclic AMP-receptor proteins in heart ventricular tissue obtained from rats flown on the Cosmos 1887 is determined. Photoaffinity labeling of soluble and particular cell fractions with a (32P)-8-azido analog of cyclic AMP is followed by electrophoretic separation of the proteins and by autoradiographic identification of the labeled isoforms of cAPK R subunits. It is shown that RII in the particulate subcellular fraction was significantly decreased in heart cells from rats in the flight group when compared to controls. Protein banding patterns in both the cytoplasmic fraction and in a fraction enriched in chromatin-bound proteins exhibited some variability in tissues of individual animals, but showed no changes that could be directly attributed to flight conditions. No significant change was apparent in the distribution of RI or RII cyclic AMP binding in the soluble fractions. It is inferred that the cardiac cell integrity or its protein content is not compromised under flight conditions.

  3. Nitration Study of Cyclic Ladder Polyphenylsilsesquioxane

    Directory of Open Access Journals (Sweden)

    LIANG Jia-xiang

    2017-05-01

    Full Text Available Several nitration reagents including fuming nitric acid, HNO3-H2SO4, KNO3-H2SO4, HNO3-KNO3, CH3COOH-KNO3, (CH3CO2O-HNO3 were used to nitrate cyclic ladder polyphenylsilsesquioxane (CL-PPSQ in different conditions in order to enhance the compatibility of the CL-PPSQ in polymers, the NO2-PPSQ was obtained. FTIR, element analysis, GPC, TGA and 1H NMR were used to characterize the structures of the nitrated products. The results show that the nitrating abilities of the fuming nitric acid, HNO3-H2SO4 and KNO3-H2SO4 are very strong. Many nitro groups can be linked with phenyl groups in CL-PPSQ, but with low molecular mass, fracture occurs in siloxane segment. However, the Mn of the product NO2-PPSQ sharply drops by 50% compared with that of CL-PPSQ, so the nitration reagents can break the cyclic structure of CL-PPSQ. The nitrating reagents of HNO3-KNO3 and CH3COOH-KNO3 have no nitration effects on CL-PPSQ. At last, NO2-CL-PPSQ was prepared using (CH3CO2O-HNO3 because of the moderate nitration process and ability. The cyclic structure of PPSQ is remained, although the number of —NO2 group is not too much. At the same time, the nitration mechanism using different nitration reagents was analyzed. A certain amount of NO2+, which is a kind of activator owning strong nitration ability, can be found in the fuming nitric acid and H2SO4-HNO3(KNO3 systems. As to the (CH3CO2O-HNO3 system, the main activator is CH3COONO2.

  4. Cosmological D-instantons and cyclic universes

    International Nuclear Information System (INIS)

    Bergshoeff, E A; Collinucci, A; Roest, D; Russo, J G; Townsend, P K

    2005-01-01

    For models of gravity coupled to hyperbolic sigma models, such as the metric-scalar sector of IIB supergravity, we show how smooth trajectories in the 'augmented target space' connect FLRW cosmologies to non-extremal D-instantons through a cosmological singularity. In particular, we find closed cyclic universes that undergo an endless sequence of big-bang to big-crunch cycles separated by instanton 'phases'. We also find 'big-bounce' universes in which a collapsing closed universe bounces off its cosmological singularity to become an open expanding universe

  5. Increase of cyclic durability of pressure vessels

    International Nuclear Information System (INIS)

    Vorona, V.A.; Zvezdin, Yu.I.

    1980-01-01

    The durability of multilayer pressure vessels under cyclic loading is compared with single-layer vessels. The relative conditional durability is calculated taking into account the assumption on the consequent destruction of layers and viewing a vessel wall as an indefinite plate. It is established that the durability is mainly determined by the number of layers and to a lesser degree depends on the relative size of the defect for the given layer thickness. The advantage of the multilayer vessels is the possibility of selecting layer materials so that to exclude the effect of agressive corrosion media on the strength [ru

  6. Calcium phosphate/porous silicon biocomposites prepared by cyclic deposition methods: Spin coating vs electrochemical activation

    Energy Technology Data Exchange (ETDEWEB)

    Hernandez-Montelongo, J., E-mail: jacobo.hernandez@uam.es [Departamento de Física Aplicada, Universidad Autónoma de Madrid, 28049 Madrid (Spain); Gallach, D.; Naveas, N.; Torres-Costa, V. [Departamento de Física Aplicada, Universidad Autónoma de Madrid, 28049 Madrid (Spain); Climent-Font, A. [Departamento de Física Aplicada, Universidad Autónoma de Madrid, 28049 Madrid (Spain); Centro de Microanálisis de Materiales (CMAM), Universidad Autónoma de Madrid, Madrid 28049 (Spain); García-Ruiz, J.P. [Departamento de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049 (Spain); Manso-Silvan, M. [Departamento de Física Aplicada, Universidad Autónoma de Madrid, 28049 Madrid (Spain)

    2014-01-01

    Porous silicon (PSi) provides an excellent platform for bioengineering applications due to its biocompatibility, biodegradability, and bioresorbability. However, to promote its application as bone engineering scaffold, deposition of calcium phosphate (CaP) ceramics in its hydroxyapatite (HAP) phase is in progress. In that sense, this work focuses on the synthesis of CaP/PSi composites by means of two different techniques for CaP deposition on PSi: Cyclic Spin Coating (CSC) and Cyclic Electrochemical Activation (CEA). Both techniques CSC and CEA consisted on alternate Ca and P deposition steps on PSi. Each technique produced specific morphologies and CaP phases using the same independent Ca and P stem-solutions at neutral pH and at room temperature. The brushite (BRU) phase was favored with the CSC technique and the hydroxyapatite (HAP) phase was better synthesized using the CEA technique. Analyses by elastic backscattering spectroscopy (EBS) on CaP/PSi structures synthesized by CEA supported that, by controlling the CEA parameters, an HAP coating with the required Ca/P atomic ratio of 1.67 can be promoted. Biocompatibility was evaluated by bone-derived progenitor cells, which grew onto CaP/PSi prepared by CSC technique with a long-shaped actin cytoskeleton. The density of adhered cells was higher on CaP/PSi prepared by CEA, where cells presented a normal morphological appearance and active mitosis. These results can be used for the design and optimization of CaP/PSi composites with enhanced biocompatibility for bone-tissue engineering. - Highlights: • Proposed cyclic methods produce specific morphologies and CaP phases in biocomposites. • The brushite phase is favored in the biocomposite produced by Cyclic Spin Coating. • The hydroxyapatite phase is favored in the biocomposite produced by Cyclic Electrochemical Activation. • The Ca/P atomic ratio of hydroxyapatite was validated by elastic backscattering spectroscopy. • Cells grown showed morphological and

  7. Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds.

    Science.gov (United States)

    Pelaez, Daniel; Huang, Chun-Yuh Charles; Cheung, Herman S

    2009-01-01

    Mechanical loading has long been shown to modulate cartilage-specific extracellular matrix synthesis. With joint motion, cartilage can experience mechanical loading in the form of compressive, tensile or shearing load, and hydrostatic pressure. Recent studies have demonstrated the capacity of unconfined cyclic compression to induce chondrogenic differentiation of human mesenchymal stem cell (hMSC) in agarose culture. However, the use of a nonbiodegradable material such as agarose limits the applicability of these constructs. Of the possible biocompatible materials available for tissue engineering, fibrin is a natural regenerative scaffold, which possesses several desired characteristics including a controllable degradation rate and low immunogenicity. The objective of the present study was to determine the capability of fibrin gels for supporting chondrogenesis of hMSCs under cyclic compression. To optimize the system, three concentrations of fibrin gel (40, 60, and 80 mg/mL) and three different stimulus frequencies (0.1, 0.5, and 1.0 Hz) were used to examine the effects of cyclic compression on viability, proliferation and chondrogenic differentiation of hMSCs. Our results show that cyclic compression (10% strain) at frequencies >0.5 Hz and gel concentration of 40 mg/mL fibrinogen appears to maintain cellular viability within scaffolds. Similarly, variations in gel component concentration and stimulus frequency can be modified such that a significant chondrogenic response can be achieved by hMSC in fibrin constructs after 8 h of compression spread out over 2 days. This study demonstrates the suitability of fibrin gel for supporting the cyclic compression-induced chondrogenesis of mesenchymal stem cells.

  8. Calcium phosphate/porous silicon biocomposites prepared by cyclic deposition methods: Spin coating vs electrochemical activation

    International Nuclear Information System (INIS)

    Hernandez-Montelongo, J.; Gallach, D.; Naveas, N.; Torres-Costa, V.; Climent-Font, A.; García-Ruiz, J.P.; Manso-Silvan, M.

    2014-01-01

    Porous silicon (PSi) provides an excellent platform for bioengineering applications due to its biocompatibility, biodegradability, and bioresorbability. However, to promote its application as bone engineering scaffold, deposition of calcium phosphate (CaP) ceramics in its hydroxyapatite (HAP) phase is in progress. In that sense, this work focuses on the synthesis of CaP/PSi composites by means of two different techniques for CaP deposition on PSi: Cyclic Spin Coating (CSC) and Cyclic Electrochemical Activation (CEA). Both techniques CSC and CEA consisted on alternate Ca and P deposition steps on PSi. Each technique produced specific morphologies and CaP phases using the same independent Ca and P stem-solutions at neutral pH and at room temperature. The brushite (BRU) phase was favored with the CSC technique and the hydroxyapatite (HAP) phase was better synthesized using the CEA technique. Analyses by elastic backscattering spectroscopy (EBS) on CaP/PSi structures synthesized by CEA supported that, by controlling the CEA parameters, an HAP coating with the required Ca/P atomic ratio of 1.67 can be promoted. Biocompatibility was evaluated by bone-derived progenitor cells, which grew onto CaP/PSi prepared by CSC technique with a long-shaped actin cytoskeleton. The density of adhered cells was higher on CaP/PSi prepared by CEA, where cells presented a normal morphological appearance and active mitosis. These results can be used for the design and optimization of CaP/PSi composites with enhanced biocompatibility for bone-tissue engineering. - Highlights: • Proposed cyclic methods produce specific morphologies and CaP phases in biocomposites. • The brushite phase is favored in the biocomposite produced by Cyclic Spin Coating. • The hydroxyapatite phase is favored in the biocomposite produced by Cyclic Electrochemical Activation. • The Ca/P atomic ratio of hydroxyapatite was validated by elastic backscattering spectroscopy. • Cells grown showed morphological and

  9. Dysfunctional Hyperpolarization-Activated Cyclic Nucleotide-gated Ion Channels in Cardiac Diseases

    Directory of Open Access Journals (Sweden)

    Xiaoqi Zhao

    Full Text Available Abstract Hyperpolarization-activated cyclic nucleotide-gated (HCN channels are reverse voltage-dependent, and their activation depends on the hyperpolarization of the membrane and may be directly or indirectly regulated by the cyclic adenosine monophosphate (cAMP or other signal-transduction cascades. The distribution, quantity and activation states of HCN channels differ in tissues throughout the body. Evidence exhibits that HCN channels play critical roles in the generation and conduction of the electrical impulse and the physiopathological process of some cardiac diseases. They may constitute promising drug targets in the treatment of these cardiac diseases. Pharmacological treatment targeting HCN channels is of benefit to these cardiac conditions.

  10. Potential repair of free radical adducts of dGMP and dG by a series of reductants. A pulse radiolytic study

    International Nuclear Information System (INIS)

    O'Neill, P.; Chapman, P.W.

    1985-01-01

    Using the technique of pulse radiolysis, it has been demonstrated that the interaction of hydroxyl-radical adducts of dG and dGMP with a series of reductants with different oxidation potentials at pH 7.0-7.4 proceeds via an electron transfer process (k approx. 1.4-34 x 10 8 dm 3 mol -1 s -1 ). The one-electron oxidation of dGMP (dG) by Br2-anion radicals was shown to result in the formation of a species, the properties of which are similar to those of the OH-radical adduct of dGMP with oxidizing properties based upon both spectral and kinetic information. The nature of the dGMP species produced on interaction with Br2-anion radicals to produce specific base damage. The implications of these findings are presented in terms of potential free radical repair of hydroxyl radical damage and of synergistic effects whereby one reductant may be regenerated at the expense of another reductant. (author)

  11. The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

    Science.gov (United States)

    Incontro, Salvatore; Ciruela, Francisco; Ziff, Edward; Hofmann, Franz; Sánchez-Prieto, José; Torres, Magdalena

    2014-01-01

    Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity. PMID:23545413

  12. Effect on Antibody and T-Cell Responses of Mixing Five GMP-Produced DNA Plasmids and Administration With Plasmid Expressing GM-CSF

    National Research Council Canada - National Science Library

    Sedegah, M; Charoenvit, Y; Aguiar, J; Sacci, J; Hedstrom, R; Kumar, S; Belmonte, A; Lanar, DE; Jones, TR; Abot, E

    2004-01-01

    .... In preparation for a clinical trial, we assessed the immunogenicity of GMP-produced plasmids encoding five Plasmodium falciparum proteins, PfCSP, PfSSP2, PfEXP1, PfLSA1, and PfLSA3, given as a mixture, or alone...

  13. Inhibition of cGMP-specific phosphodiesterase type 5 reduces sodium excretion and arterial blood pressure in patients with NaCl retention and ascites

    DEFF Research Database (Denmark)

    Thiesson, Helle C; Jensen, Boye L; Jespersen, Bente

    2005-01-01

    ANG II, and aldosterone concentrations significantly after 60 min. Plasma cGMP concentration was increased after 120 and 180 min, and urinary sodium excretion and mean arterial blood pressure were decreased significantly at 120 and 180 min. Plasma ANP concentration, GFR, and RBF did not change after...

  14. The Road to Excellence: : A Case Study of the Application of GMP, ISO 9001 and the EFQM Excellence Model in a Nuclear Medicine Department

    NARCIS (Netherlands)

    van Huizen, L; Ahaus, C.T.B. (Kees)

    2017-01-01

    Definitions are introduced to give insight in the field of work of Quality Management in relation to responsibilities in Nuclear Medicine and Molecular Imaging. A relation model visualizes the relationships. When working on the Road to Quality Excellence. The standards as GMP, GCP, ISO 9001 and EFQM

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

    Science.gov (United States)

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

    2013-01-01

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

  16. Tissue engineering

    CERN Document Server

    Fisher, John P; Bronzino, Joseph D

    2007-01-01

    Increasingly viewed as the future of medicine, the field of tissue engineering is still in its infancy. As evidenced in both the scientific and popular press, there exists considerable excitement surrounding the strategy of regenerative medicine. To achieve its highest potential, a series of technological advances must be made. Putting the numerous breakthroughs made in this field into a broad context, Tissue Engineering disseminates current thinking on the development of engineered tissues. Divided into three sections, the book covers the fundamentals of tissue engineering, enabling technologies, and tissue engineering applications. It examines the properties of stem cells, primary cells, growth factors, and extracellular matrix as well as their impact on the development of tissue engineered devices. Contributions focus on those strategies typically incorporated into tissue engineered devices or utilized in their development, including scaffolds, nanocomposites, bioreactors, drug delivery systems, and gene t...

  17. Gold prices: Analyzing its cyclical behavior

    Directory of Open Access Journals (Sweden)

    Martha Gutiérrez

    2013-07-01

    Full Text Available Gold is a commodity that is seen as a safe haven when a financial crisis strikes, but when stock markets are prosperous, these are more attractive investment alternatives, and so the gold cycle goes on and on. The DJIA/GF (Dow Jones Industrial Average and Gold Fix ratio is chosen to establish the evolution of gold prices in relation to the NYSE. This paper has two goals: to prove that the DJIA/GF ratio is strongly cyclical by using Fourier analysis and to set a predictive neural networks model to forecast the behavior of this ratio during 2011-2020. To this end, business cycle events like the Great Depression along with the 1970s crisis, and the 1950s boom along with the world economic recovery of the 1990s are contrasted in light of the mentioned ratio. Gold prices are found to evolve cyclically with a dominant period of 37 years and are mainly affected by energy prices, financial markets and macroeconomic indicators.

  18. Corticosteroid receptors adopt distinct cyclical transcriptional signatures.

    Science.gov (United States)

    Le Billan, Florian; Amazit, Larbi; Bleakley, Kevin; Xue, Qiong-Yao; Pussard, Eric; Lhadj, Christophe; Kolkhof, Peter; Viengchareun, Say; Fagart, Jérôme; Lombès, Marc

    2018-05-07

    Mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) are two closely related hormone-activated transcription factors that regulate major pathophysiologic functions. High homology between these receptors accounts for the crossbinding of their corresponding ligands, MR being activated by both aldosterone and cortisol and GR essentially activated by cortisol. Their coexpression and ability to bind similar DNA motifs highlight the need to investigate their respective contributions to overall corticosteroid signaling. Here, we decipher the transcriptional regulatory mechanisms that underlie selective effects of MRs and GRs on shared genomic targets in a human renal cellular model. Kinetic, serial, and sequential chromatin immunoprecipitation approaches were performed on the period circadian protein 1 ( PER1) target gene, providing evidence that both receptors dynamically and cyclically interact at the same target promoter in a specific and distinct transcriptional signature. During this process, both receptors regulate PER1 gene by binding as homo- or heterodimers to the same promoter region. Our results suggest a novel level of MR-GR target gene regulation, which should be considered for a better and integrated understanding of corticosteroid-related pathophysiology.-Le Billan, F., Amazit, L., Bleakley, K., Xue, Q.-Y., Pussard, E., Lhadj, C., Kolkhof, P., Viengchareun, S., Fagart, J., Lombès, M. Corticosteroid receptors adopt distinct cyclical transcriptional signatures.

  19. A cyclically actuated electrolytic drug delivery device

    KAUST Repository

    Yi, Ying

    2015-01-01

    This work, focusing on an implantable drug delivery system, presents the first prototype electrolytic pump that combines a catalytic reformer and a cyclically actuated mode. These features improve the release performance and extend the lifetime of the device. Using our platinum (Pt)-coated carbon fiber mesh that acts as a catalytic reforming element, the cyclical mode is improved because the faster recombination rate allows for a shorter cycling time for drug delivery. Another feature of our device is that it uses a solid-drug-in-reservoir (SDR) approach, which allows small amounts of a solid drug to be dissolved in human fluid, forming a reproducible drug solution for long-term therapies. We have conducted proof-of-principle drug delivery studies using such an electrolytic pump and solvent blue 38 as the drug substitute. These tests demonstrate power-controlled and pulsatile release profiles of the chemical substance, as well as the feasibility of this device. A drug delivery rate of 11.44 ± 0.56 μg min-1 was achieved by using an input power of 4 mW for multiple pulses, which indicates the stability of our system. © The Royal Society of Chemistry 2015.

  20. MrkH, a novel c-di-GMP-dependent transcriptional activator, controls Klebsiella pneumoniae biofilm formation by regulating type 3 fimbriae expression.

    Directory of Open Access Journals (Sweden)

    Jonathan J Wilksch

    2011-08-01

    Full Text Available Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae