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

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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.

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...

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.

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...

Diguanylate cyclase null mutant reveals that C-Di-GMP pathway regulates the motility and adherence of the extremophile bacterium Acidithiobacillus caldus.

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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.

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

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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...

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

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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...

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

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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.

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

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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

MrkH, a novel c-di-GMP-dependent transcriptional activator, controls Klebsiella pneumoniae biofilm formation by regulating type 3 fimbriae expression.

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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

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

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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.

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.

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

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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

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

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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.

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

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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.

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

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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.

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

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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.

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

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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

Differential control of Yersinia pestis biofilm formation in vitro and in the flea vector by two c-di-GMP diguanylate cyclases.

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Yi-Cheng Sun

2011-04-01

Full Text Available Yersinia pestis forms a biofilm in the foregut of its flea vector that promotes transmission by flea bite. As in many bacteria, biofilm formation in Y. pestis is controlled by intracellular levels of the bacterial second messenger c-di-GMP. Two Y. pestis diguanylate cyclase (DGC enzymes, encoded by hmsT and y3730, and one phosphodiesterase (PDE, encoded by hmsP, have been shown to control biofilm production in vitro via their opposing c-di-GMP synthesis and degradation activities, respectively. In this study, we provide further evidence that hmsT, hmsP, and y3730 are the only three genes involved in c-di-GMP metabolism in Y. pestis and evaluated the two DGCs for their comparative roles in biofilm formation in vitro and in the flea vector. As with HmsT, the DGC activity of Y3730 depended on a catalytic GGDEF domain, but the relative contribution of the two enzymes to the biofilm phenotype was influenced strongly by the environmental niche. Deletion of y3730 had a very minor effect on in vitro biofilm formation, but resulted in greatly reduced biofilm formation in the flea. In contrast, the predominant effect of hmsT was on in vitro biofilm formation. DGC activity was also required for the Hms-independent autoaggregation phenotype of Y. pestis, but was not required for virulence in a mouse model of bubonic plague. Our results confirm that only one PDE (HmsP and two DGCs (HmsT and Y3730 control c-di-GMP levels in Y. pestis, indicate that hmsT and y3730 are regulated post-transcriptionally to differentially control biofilm formation in vitro and in the flea vector, and identify a second c-di-GMP-regulated phenotype in Y. pestis.

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...

Activation and polar sequestration of PopA, a c-di-GMP effector protein involved in Caulobacter crescentus cell cycle control

DEFF Research Database (Denmark)

Ozaki, Shogo; Schalch-Moser, Annina; Zumthor, Ludwig

2014-01-01

that PopA originated through gene duplication from its paralogue response regulator PleD and subsequent co-option as c-di-GMP effector protein. While the C-terminal catalytic domain (GGDEF) of PleD is activated by phosphorylation of the N-terminal receiver domain, functional adaptation has reversed signal......A to the cell pole in response to c-di-GMP binding. In agreement with the divergent activation and targeting mechanisms, distinct markers sequester PleD and PopA to the old cell pole upon S-phase entry. Together these data indicate that PopA adopted a novel role as topology specificity factor to help recruit...

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

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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.

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...

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

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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.

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.

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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.

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

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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.

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.

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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.

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

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

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

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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-c

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.

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

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

Nuclease-resistant c-di-AMP derivatives that differentially recognize RNA and protein receptors

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Meehan, Robert E.; Torgerson, Chad D.; Gaffney, Barbara L.; Jones, Roger A.; Strobel, Scott A.

2016-01-01

The ability of bacteria to sense environmental cues and adapt is essential for their survival. The use of second-messenger signaling molecules to translate these cues into a physiological response is a common mechanism employed by bacteria. The second messenger 3’-5’-cyclic diadenosine monophosphate (c-di-AMP) has been linked to a diverse set of biological processes involved in maintaining cell viability and homeostasis, as well as pathogenicity. A complex network of both protein and RNA receptors inside the cell activate specific pathways and mediate phenotypic outputs in response to c-di-AMP. Structural analysis of these RNA and protein receptors has revealed the different recognition elements employed by these effectors to bind the same small molecule. Herein, using a series of c-di-AMP analogs, we probed the interactions made with a riboswitch and a phosphodiesterase protein to identify the features important for c-di-AMP binding and recognition. We found that the ydaO riboswitch binds c-di-AMP in two discrete sites with near identical affinity and a Hill coefficient of 1.6. The ydaO riboswitch distinguishes between c-di-AMP and structurally related second messengers by discriminating against an amine at the C2 position, more than a carbonyl at the C6 position. We also identified phosphate-modified analogs that bind both the ydaO RNA and GdpP protein with high affinity, while symmetrically-modified ribose analogs exhibited a substantial decrease in ydaO affinity, but retained high affinity for GdpP. These ligand modifications resulted in increased resistance to enzyme-catalyzed hydrolysis by the GdpP enzyme. Together, these data suggest that these c-di-AMP analogs could be useful as chemical tools to specifically target subsections of the second-messenger signaling pathways. PMID:26789423

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…

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

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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

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

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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.

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

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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

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.

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.

Thrombin has biphasic effects on the nitric oxide-cGMP pathway in endothelial cells and contributes to experimental pulmonary hypertension.

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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.

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.

Guanylin peptides: cyclic GMP signaling mechanisms

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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.

cGMP-dependent protein kinase Iα associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake

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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

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

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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.

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

Characterization of the Xylella fastidiosa PD1671 gene encoding degenerate c-di-GMP GGDEF/EAL domains, and its role in the development of Pierce's disease.

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Cursino, Luciana; Athinuwat, Dusit; Patel, Kelly R; Galvani, Cheryl D; Zaini, Paulo A; Li, Yaxin; De La Fuente, Leonardo; Hoch, Harvey C; Burr, Thomas J; Mowery, Patricia

2015-01-01

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce's disease of grapevines. X. fastidiosa is thought to induce disease by colonizing and clogging xylem vessels through the formation of cell aggregates and bacterial biofilms. Here we examine the role in X. fastidiosa virulence of an uncharacterized gene, PD1671, annotated as a two-component response regulator with potential GGDEF and EAL domains. GGDEF domains are found in c-di-GMP diguanylate cyclases while EAL domains are found in phosphodiesterases, and these domains are for c-di-GMP production and turnover, respectively. Functional analysis of the PD1671 gene revealed that it affected multiple X. fastidiosa virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of gum genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility in vitro but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of X. fastidiosa virulence.

Characterization of the Xylella fastidiosa PD1671 gene encoding degenerate c-di-GMP GGDEF/EAL domains, and its role in the development of Pierce's disease.

Directory of Open Access Journals (Sweden)

Luciana Cursino

Full Text Available Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce's disease of grapevines. X. fastidiosa is thought to induce disease by colonizing and clogging xylem vessels through the formation of cell aggregates and bacterial biofilms. Here we examine the role in X. fastidiosa virulence of an uncharacterized gene, PD1671, annotated as a two-component response regulator with potential GGDEF and EAL domains. GGDEF domains are found in c-di-GMP diguanylate cyclases while EAL domains are found in phosphodiesterases, and these domains are for c-di-GMP production and turnover, respectively. Functional analysis of the PD1671 gene revealed that it affected multiple X. fastidiosa virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of gum genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility in vitro but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of X. fastidiosa virulence.

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.

Characterization of the Xylella fastidiosa PD1671 Gene Encoding Degenerate c-di-GMP GGDEF/EAL Domains, and Its Role in the Development of Pierce’s Disease

Science.gov (United States)

Cursino, Luciana; Athinuwat, Dusit; Patel, Kelly R.; Galvani, Cheryl D.; Zaini, Paulo A.; Li, Yaxin; De La Fuente, Leonardo; Hoch, Harvey C.; Burr, Thomas J.; Mowery, Patricia

2015-01-01

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce’s disease of grapevines. X. fastidiosa is thought to induce disease by colonizing and clogging xylem vessels through the formation of cell aggregates and bacterial biofilms. Here we examine the role in X. fastidiosa virulence of an uncharacterized gene, PD1671, annotated as a two-component response regulator with potential GGDEF and EAL domains. GGDEF domains are found in c-di-GMP diguanylate cyclases while EAL domains are found in phosphodiesterases, and these domains are for c-di-GMP production and turnover, respectively. Functional analysis of the PD1671 gene revealed that it affected multiple X. fastidiosa virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of gum genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility in vitro but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of X. fastidiosa virulence. PMID:25811864

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

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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.

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

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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

The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

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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

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...

Aging has the opposite effect on cAMP and cGMP circadian variations in rat Leydig cells.

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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.

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.

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

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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.

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.

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

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.

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.

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

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

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.

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

[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.

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

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

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

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.

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.

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...

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.

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.

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...

Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c.

Science.gov (United States)

Rotcheewaphan, Suwatchareeporn; Belisle, John T; Webb, Kristofor J; Kim, Hee-Jin; Spencer, John S; Borlee, Bradley R

2016-09-01

The second messenger, bis-(3',5')-cyclic dimeric guanosine monophosphate (cyclic di-GMP), is involved in the control of multiple bacterial phenotypes, including those that impact host-pathogen interactions. Bioinformatics analyses predicted that Mycobacterium leprae, an obligate intracellular bacterium and the causative agent of leprosy, encodes three active diguanylate cyclases. In contrast, the related pathogen Mycobacterium tuberculosis encodes only a single diguanylate cyclase. One of the M. leprae unique diguanylate cyclases (ML1419c) was previously shown to be produced early during the course of leprosy. Thus, functional analysis of ML1419c was performed. The gene encoding ML1419c was cloned and expressed in Pseudomonas aeruginosa PAO1 to allow for assessment of cyclic di-GMP production and cyclic di-GMP-mediated phenotypes. Phenotypic studies revealed that ml1419c expression altered colony morphology, motility and biofilm formation of P. aeruginosa PAO1 in a manner consistent with increased cyclic di-GMP production. Direct measurement of cyclic di-GMP levels by liquid chromatography-mass spectrometry confirmed that ml1419c expression increased cyclic di-GMP production in P. aeruginosa PAO1 cultures in comparison to the vector control. The observed phenotypes and increased levels of cyclic di-GMP detected in P. aeruginosa expressing ml1419c could be abrogated by mutation of the active site in ML1419c. These studies demonstrated that ML1419c of M. leprae functions as diguanylate cyclase to synthesize cyclic di-GMP. Thus, this protein was renamed DgcA (Diguanylate cyclase A). These results also demonstrated the ability to use P. aeruginosa as a heterologous host for characterizing the function of proteins involved in the cyclic di-GMP pathway of a pathogen refractory to in vitro growth, M. leprae.

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

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

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.

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.

In Vitro Assessment of Guanylyl Cyclase Activity of Plant Receptor Kinases

KAUST Repository

Raji, Misjudeen; Gehring, Christoph A

2017-01-01

Cyclic nucleotides such as 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) are increasingly recognized as key signaling molecules in plants, and a growing number of plant mononucleotide cyclases, both adenylate cyclases (ACs) and guanylate cyclases (GCs), have been reported. Catalytically active cytosolic GC domains have been shown to be part of many plant receptor kinases and hence directly linked to plant signaling and downstream cellular responses. Here we detail, firstly, methods to identify and express essential functional GC domains of receptor kinases, and secondly, we describe mass spectrometric methods to quantify cGMP generated by recombinant GCs from receptor kinases in vitro.

In Vitro Assessment of Guanylyl Cyclase Activity of Plant Receptor Kinases

KAUST Repository

Raji, Misjudeen

2017-05-31

Cyclic nucleotides such as 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) are increasingly recognized as key signaling molecules in plants, and a growing number of plant mononucleotide cyclases, both adenylate cyclases (ACs) and guanylate cyclases (GCs), have been reported. Catalytically active cytosolic GC domains have been shown to be part of many plant receptor kinases and hence directly linked to plant signaling and downstream cellular responses. Here we detail, firstly, methods to identify and express essential functional GC domains of receptor kinases, and secondly, we describe mass spectrometric methods to quantify cGMP generated by recombinant GCs from receptor kinases in vitro.

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

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.

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.

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.

Extracellular Protein Kinase A Modulates Intracellular Calcium/Calmodulin-Dependent Protein Kinase II, Nitric Oxide Synthase, and the Glutamate-Nitric Oxide-cGMP Pathway in Cerebellum. Differential Effects in Hyperammonemia.

Science.gov (United States)

Cabrera-Pastor, Andrea; Llansola, Marta; Felipo, Vicente

2016-12-21

Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.

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

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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.

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

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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.

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

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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.

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

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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.

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.

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....

Cyclic GMP-AMP displays mucosal adjuvant activity in mice.

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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.

Cyclic GMP-AMP displays mucosal adjuvant activity in mice.

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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.

IL-4 induces cAMP and cGMP in human monocytic cells

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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.

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

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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.

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.

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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.

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

Altered Regulation of the Diguanylate Cyclase YaiC Reduces Production of Type 1 Fimbriae in a Pst Mutant of Uropathogenic Escherichia coli CFT073.

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Crépin, Sébastien; Porcheron, Gaëlle; Houle, Sébastien; Harel, Josée; Dozois, Charles M

2017-12-15

The pst gene cluster encodes the phosphate-specific transport (Pst) system. Inactivation of the Pst system constitutively activates the two-component regulatory system PhoBR and attenuates the virulence of pathogenic bacteria. In uropathogenic Escherichia coli strain CFT073, attenuation by inactivation of pst is predominantly attributed to the decreased expression of type 1 fimbriae. However, the molecular mechanisms connecting the Pst system and type 1 fimbriae are unknown. To address this, a transposon library was constructed in the pst mutant, and clones were tested for a regain in type 1 fimbrial production. Among them, the diguanylate cyclase encoded by yaiC ( adrA in Salmonella ) was identified to connect the Pst system and type 1 fimbrial expression. In the pst mutant, the decreased expression of type 1 fimbriae is connected by the induction of yaiC This is predominantly due to altered expression of the FimBE-like recombinase genes ipuA and ipbA , affecting at the same time the inversion of the fim promoter switch ( fimS ). In the pst mutant, inactivation of yaiC restored fim -dependent adhesion to bladder cells and virulence. Interestingly, the expression of yaiC was activated by PhoB, since transcription of yaiC was linked to the PhoB-dependent phoA-psiF operon. As YaiC is involved in cyclic di-GMP (c-di-GMP) biosynthesis, an increased accumulation of c-di-GMP was observed in the pst mutant. Hence, the results suggest that one mechanism by which deletion of the Pst system reduces the expression of type 1 fimbriae is through PhoBR-mediated activation of yaiC , which in turn increases the accumulation of c-di-GMP, represses the fim operon, and, consequently, attenuates virulence in the mouse urinary tract infection model. IMPORTANCE Urinary tract infections (UTIs) are common bacterial infections in humans. They are mainly caused by uropathogenic Escherichia coli (UPEC). We previously showed that interference with phosphate homeostasis decreases the

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.

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

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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.

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

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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…

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

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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.

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

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

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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.

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...

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

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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.

Detection of Cyclic Dinucleotides by STING.

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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.

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

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.

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

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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.

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

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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.

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.

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....

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

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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.

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...

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.

PPARα autocrine regulation of Ca²⁺-regulated exocytosis in guinea pig antral mucous cells: NO and cGMP accumulation.

Science.gov (United States)

Tanaka, Saori; Sugiyama, Nanae; Takahashi, Yuko; Mantoku, Daiki; Sawabe, Yukinori; Kuwabara, Hiroko; Nakano, Takashi; Shimamoto, Chikao; Matsumura, Hitoshi; Marunaka, Yoshinori; Nakahari, Takashi

2014-12-15

In antral mucous cells, acetylcholine (ACh, 1 μM) activates Ca(2+)-regulated exocytosis, consisting of a peak in exocytotic events that declines rapidly (initial phase) followed by a second slower decline (late phase) lasting during ACh stimulation. GW7647 [a peroxisome proliferation activation receptor α (PPARα) agonist] enhanced the ACh-stimulated initial phase, and GW6471 (a PPARα antagonist) abolished the GW7647-induced enhancement. However, GW6471 produced the delayed, but transient, increase in the ACh-stimulated late phase, and it also decreased the initial phase and produced the delayed increase in the late phase during stimulation with ACh alone. A similar delayed increase in the ACh-stimulated late phase is induced by an inhibitor of the PKG, Rp8BrPETcGMPS, suggesting that GW6471 inhibits cGMP accumulation. An inhibitor of nitric oxide synthase 1 (NOS1), N(5)-[imino(propylamino)methyl]-L-ornithine hydrochloride (N-PLA), also abolished the GW7647-induced-enhancement of ACh-stimulated initial phase but produced the delayed increase in the late phase. However, in the presence of N-PLA, an NO donor or 8BrcGMP enhanced the ACh-stimulated initial phase and abolished the delayed increase in the late phase. Moreover, GW7647 and ACh stimulated NO production and cGMP accumulation in antral mucosae, which was inhibited by GW6471 or N-PLA. Western blotting and immunohistochemistry revealed that NOS1 and PPARα colocalize in antral mucous cells. In conclusion, during ACh stimulation, a PPARα autocrine mechanism, which accumulates NO via NOS1 leading to cGMP accumulation, modulates the Ca(2+)-regulated exocytosis in antral mucous cells. Copyright © 2014 the American Physiological Society.

Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase.

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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.

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.

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.

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.

cGMP-phosphodiesterase inhibition enhances photic responses and synchronization of the biological circadian clock in rodents.

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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.

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

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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.

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)

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

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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

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

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.

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....

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.

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)

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.

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...

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.

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

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.

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

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...

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....

Nitric oxide participates in cold-inhibited Camellia sinensis pollen germination and tube growth partly via cGMP in vitro.

Directory of Open Access Journals (Sweden)

Yu-Hua Wang

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

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.

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.

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.

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.

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.

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

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

Directory of Open Access Journals (Sweden)

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.

cGMP may have trophic effects on beta cell function comparable to those of cAMP, implying a role for high-dose biotin in prevention/treatment of diabetes.

Science.gov (United States)

McCarty, Mark F

2006-01-01

Incretin hormones have trophic effects on beta cell function that can aid prevention and treatment of diabetes. cAMP is the primary mediator of these effects, and has been shown to potentiate glucose-stimulated insulin secretion, promote proper beta cells differentiation by increasing expression of the crucial transcription factor PDX-1, and prevent beta cell apoptosis. cGMP's role in beta cell function has received far less scrutiny, but there is emerging evidence that it may have a trophic impact on beta cell function analogous to that of cAMP. An increase in plasma glucose boosts beta cell production of cGMP, which acts as a feed-forward mediator to enhance glucose-stimulated insulin secretion. cGMP also has an anti-apoptotic effect in beta cells, and there is now indirect evidence that it promotes expression of PDX-1. Supraphysiological concentrations of biotin can directly activate guanylate cyclase, and there is limited evidence that high intakes of this vitamin can be therapeutically beneficial in diabetics and in rodent models of diabetes. Beneficial effects of cGMP on muscle insulin sensitivity and on control of hepatic glucose output may contribute to biotin's utility in diabetes. The fact that nitric oxide/cGMP exert a range of favorable effects on vascular health should further encourage exploration of biotin's preventive and therapeutic potential. If an appropriate high-dose biotin regimen could achieve a modest systemic increase in guanylate cyclase activity, without entailing unacceptable side effects or risks, such a regimen might have considerable potential for promoting vascular health and preventing or managing diabetes.

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

Science.gov (United States)

Hasebe, Masaharu

2016-01-01

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

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.

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.

Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

KAUST Repository

Kwezi, Lusisizwe

2018-01-22

Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

KAUST Repository

Kwezi, Lusisizwe; Wheeler, Janet I; Marondedze, Claudius; Gehring, Christoph A; Irving, Helen R

2018-01-01

Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

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

The Diguanylate Cyclase HsbD Intersects with the HptB Regulatory Cascade to Control Pseudomonas aeruginosa Biofilm and Motility.

Directory of Open Access Journals (Sweden)

Martina Valentini

2016-10-01

Full Text Available The molecular basis of second messenger signaling relies on an array of proteins that synthesize, degrade or bind the molecule to produce coherent functional outputs. Cyclic di-GMP (c-di-GMP has emerged as a eubacterial nucleotide second messenger regulating a plethora of key behaviors, like the transition from planktonic cells to biofilm communities. The striking multiplicity of c-di-GMP control modules and regulated cellular functions raised the question of signaling specificity. Are c-di-GMP signaling routes exclusively dependent on a central hub or can they be locally administrated? In this study, we show an example of how c-di-GMP signaling gains output specificity in Pseudomonas aeruginosa. We observed the occurrence in P. aeruginosa of a c-di-GMP synthase gene, hsbD, in the proximity of the hptB and flagellar genes cluster. We show that the HptB pathway controls biofilm formation and motility by involving both HsbD and the anti-anti-sigma factor HsbA. The rewiring of c-di-GMP signaling into the HptB cascade relies on the original interaction between HsbD and HsbA and on the control of HsbD dynamic localization at the cell poles.

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...

Characterization of a crp* mutant of the E. coli cAMP receptor protein

International Nuclear Information System (INIS)

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

1987-01-01

One of the crp* mutants previously isolated to activate lac promoter in vivo has been characterized with regard to its biochemical properties. CRP*592 shows a more open conformation than CRP as indicated by its sensitivity to proteolytic attack. Dithionitrobenzoic acid mediated intersubunit crosslinking of CRP requires cAMP; this reaction occurs with unliganded CRP*592. Binding of CRP to its site on the lac promoter and activation of abortive initiation is effected by cAMP but not by cGMP. CRP*592 can activate abortive initiation in the presence of cAMP or cGMP and also at a high CRP*592 concentration in the absence of cyclic nucleotide. DNase I footprinting shows that cAMP-CRP* binds to its site on lac P + while unliganded CRP* and cGMP-CRP* form a stable complex with the [ 32 P]lac P + only in the presence of RNA polymerase. While cGMP binds to CRP it cannot replace cAMP in effecting the conformation necessary for site specific promoter binding; the weakly active unliganded CRP*592 can be shifted to a functional conformation by cAMP, cGMP and RNA polymerase

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.

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.

Bacterial nucleotide-based second messengers.

Science.gov (United States)

Pesavento, Christina; Hengge, Regine

2009-04-01

In all domains of life nucleotide-based second messengers transduce signals originating from changes in the environment or in intracellular conditions into appropriate cellular responses. In prokaryotes cyclic di-GMP has emerged as an important and ubiquitous second messenger regulating bacterial life-style transitions relevant for biofilm formation, virulence, and many other bacterial functions. This review describes similarities and differences in the architecture of the cAMP, (p)ppGpp, and c-di-GMP signaling systems and their underlying signaling principles. Moreover, recent advances in c-di-GMP-mediated signaling will be presented and the integration of c-di-GMP signaling with other nucleotide-based signaling systems will be discussed.

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

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.

[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.

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.

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.

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.

CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor

DEFF Research Database (Denmark)

Dietrich, J; Hou, X; Wegener, A M

1994-01-01

-regulation of the TCR. Furthermore, analysis of a series of CD3 gamma truncation mutants indicated that in addition to S126 phosphorylation a motif C-terminal of S126 was required for TCR down-regulation. Point mutation analyses confirmed this observation and demonstrated that a membrane-proximal di-leucine motif (L131......, indicating that the TCR was down-regulated by endocytosis via clathrin coated pits. Based on the present results and previously published observations on intracellular receptor sorting, a general model for intracellular sorting of receptors containing di-leucine- or tyrosine-based motifs is proposed....

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....

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.

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....

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...

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.

HmsC Controls Yersinia pestis Biofilm Formation in Response to Redox Environment

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Gai-Xian Ren

2017-08-01

Full Text Available Yersinia pestis biofilm formation, controlled by intracellular levels of the second messenger molecule cyclic diguanylate (c-di-GMP, is important for blockage-dependent plague transmission from fleas to mammals. HmsCDE is a tripartite signaling system that modulates intracellular c-di-GMP levels to regulate biofilm formation in Y. pestis. Previously, we found that Y. pestis biofilm formation is stimulated in reducing environments in an hmsCDE-dependent manner. However, the mechanism by which HmsCDE senses the redox state remains elusive. Using a dsbA mutant and the addition of Cu2+ to simulate reducing and oxidizing periplasmic environments, we found that HmsC protein levels are decreased and the HmsC-HmsD protein-protein interaction is weakened in a reducing environment. In addition, we revealed that intraprotein disulphide bonds are critical for HmsC since breakage lowers protein stability and diminishes the interaction with HmsD. Our results suggest that HmsC might play a major role in sensing the environmental changes.

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.

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.

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

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...

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.

Membrane-anchored MucR mediates nitrate-dependent regulation of alginate production in Pseudomonas aeruginosa

KAUST Repository

Wang, Yajie; Hay, Iain D.; Rehman, Zahid Ur; Rehm, Bernd H A

2015-01-01

of MucR impaired alginate promoter activity and global c-di-GMP levels, alginate yields were not directly correlated with alginate promoter activity or c-di-GMP levels, suggesting that nitrate and MucR modulate alginate production at a post

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.

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

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.

Multiple diguanylate cyclase-coordinated regulation of pyoverdine synthesis in Pseudomonas aeruginosa

DEFF Research Database (Denmark)

Chen, Yicai; Yuan, Mingjun; Mohanty, Anee

2015-01-01

The nucleotide signalling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays an essential role in regulating microbial virulence and biofilm formation. C-di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. One...

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

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.

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.

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...

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

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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.

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.

Salvinorin A preserves cerebral pial artery autoregulation after forebrain ischemia via the PI3K/AKT/cGMP pathway

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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.

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...

No link of serotonin 2C receptor editing to serotonin transporter genotype

NARCIS (Netherlands)

Lyddon, R.; Cuppen, E.; Haroutunian, V.; Siever, L.J.; Dracheva, S.

2010-01-01

RNA editing is a post-transcriptional process, which has the potential to alter the function of encoded proteins. In particular, serotonin 2C receptor (5-HT2cR) mRNA editing can produce 24 protein isoforms of varying functionality. Rodent studies have shown that 5-HT2cR editing is dynamically

c-MET receptor tyrosine kinase as a molecular target in advanced hepatocellular carcinoma

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Granito A

2015-04-01

Full Text Available Alessandro Granito,1 Elena Guidetti,1 Laura Gramantieri2,3 1Dipartimento di Scienze Mediche e Chirurgiche Università di Bologna, Bologna, Italy; 2Dipartimento dell'Apparato Digerente, Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 3Centro di Ricerca Biomedica Applicata (CRBA, Azienda Ospedaliero-Universitaria Policlinico S Orsola-Malpighi e Università di Bologna, Bologna, Italy Abstract: c-MET is the membrane receptor for hepatocyte growth factor (HGF, also known as scatter factor or tumor cytotoxic factor, a mitogenic growth factor for hepatocytes. HGF is mainly produced by cells of mesenchymal origin and it mainly acts on neighboring epidermal and endothelial cells, regulating epithelial growth and morphogenesis. HGF/MET signaling has been identified among the drivers of tumorigenesis in human cancers. As such, c-MET is a recognized druggable target, and against it, targeted agents are currently under clinical investigation. c-MET overexpression is a common event in a wide range of human malignancies, including gastric, lung, breast, ovary, colon, kidney, thyroid, and liver carcinomas. Despite c-MET overexpression being reported by a large majority of studies, no evidence for a c-MET oncogenic addiction exists in hepatocellular carcinoma (HCC. In particular, c-MET amplification is a rare event, accounting for 4%–5% of cases while no mutation has been identified in c-MET oncogene in HCC. Thus, the selection of patient subgroups more likely to benefit from c-MET inhibition is challenging. Notwithstanding, c-MET overexpression was reported to be associated with increased metastatic potential and poor prognosis in patients with HCC, providing a rationale for its therapeutic inhibition. Here we summarize the role of activated HGF/MET signaling in HCC, its prognostic relevance, and the implications for therapeutic approaches in HCC. Keywords: hepatocellular carcinoma, c-MET, clinical trials

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

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

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.

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

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

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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.

DMPD: Toll-like receptor 3: a link between toll-like receptor, interferon and viruses. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 15031527 Toll-like receptor 3: a link between toll-like receptor, interferon and virus... (.csml) Show Toll-like receptor 3: a link between toll-like receptor, interferon and viruses. PubmedID 1503...1527 Title Toll-like receptor 3: a link between toll-like receptor, interferon and virus

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.

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

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.

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

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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.

New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus.

Science.gov (United States)

Bowman, Lisa; Zeden, Merve S; Schuster, Christopher F; Kaever, Volkhard; Gründling, Angelika

2016-12-30

Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function, and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane-bound Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5'-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared with the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

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.

Tachykinin-Related Peptides Share a G Protein-Coupled Receptor with Ion Transport Peptide-Like in the Silkworm Bombyx mori.

Directory of Open Access Journals (Sweden)

Chiaki Nagai-Okatani

Full Text Available Recently, we identified an orphan Bombyx mori neuropeptide G protein-coupled receptor (BNGR-A24 as an ion transport peptide-like (ITPL receptor. BNGR-A24 belongs to the same clade as BNGR-A32 and -A33, which were recently identified as natalisin receptors. Since these three BNGRs share high similarities with known receptors for tachykinin-related peptides (TRPs, we examined whether these BNGRs can function as physiological receptors for five endogenous B. mori TRPs (TK-1-5. In a heterologous expression system, BNGR-A24 acted as a receptor for all five TRPs. In contrast, BNGR-A32 responded only to TK-5, and BNGR-A33 did not respond to any of the TRPs. These findings are consistent with recent studies on the ligand preferences for B. mori natalisins. Furthermore, we evaluated whether the binding of ITPL and TRPs to BNGR-A24 is competitive by using a Ca2+ imaging assay. Concomitant addition of a TRP receptor antagonist, spantide I, reduced the responses of BNGR-A24 not only to TK-4 but also to ITPL. The results of a binding assay using fluorescent-labeled BNGR-A24 and ligands demonstrated that the binding of ITPL to BNGR-A24 was inhibited by TK-4 as well as by spantide I, and vice versa. In addition, the ITPL-induced increase in cGMP levels of BNGR-A24-expressing BmN cells was suppressed by the addition of excess TK-4 or spantide I. The intracellular levels of cAMP and cGMP, as second messenger candidates of the TRP signaling, were not altered by the five TRPs, suggesting that these peptides act via different signaling pathways from cAMP and cGMP signaling at least in BmN cells. Taken together, the present findings suggest that ITPL and TRPs are endogenous orthosteric ligands of BNGR-A24 that may activate discrete signaling pathways. This receptor, which shares orthosteric ligands, may constitute an important model for studying ligand-biased signaling.

Fine-tuning of T-cell development by the CD3γ di-leucine-based TCR-sorting motif

DEFF Research Database (Denmark)

Lauritsen, Jens Peter Holst; Boding, Lasse; Buus, Terkild B

2015-01-01

The CD3γ di-leucine-based (diL) receptor-sorting motif plays a central role in TCR down-regulation and in clonal expansion of virus-specific T cells. However, the role of the CD3γ diL motif in T-cell development is not known. In this study, we show that protein kinase C-induced TCR down-regulatio......The CD3γ di-leucine-based (diL) receptor-sorting motif plays a central role in TCR down-regulation and in clonal expansion of virus-specific T cells. However, the role of the CD3γ diL motif in T-cell development is not known. In this study, we show that protein kinase C-induced TCR down...

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.

Membrane-anchored MucR mediates nitrate-dependent regulation of alginate production in Pseudomonas aeruginosa

KAUST Repository

Wang, Yajie

2015-04-29

Alginates exhibit unique material properties suitable for medical and industrial applications. However, if produced by Pseudomonas aeruginosa, it is an important virulence factor in infection of cystic fibrosis patients. The alginate biosynthesis machinery is activated by c-di-GMP imparted by the inner membrane protein, MucR. Here, it was shown that MucR impairs alginate production in response to nitrate in P. aeruginosa. Subsequent site-specific mutagenesis of MucR revealed that the second MHYT sensor motif (MHYT II, amino acids 121–124) of MucR sensor domain was involved in nitrate sensing. We also showed that both c-di-GMP synthesizing and degrading active sites of MucR were important for alginate production. Although nitrate and deletion of MucR impaired alginate promoter activity and global c-di-GMP levels, alginate yields were not directly correlated with alginate promoter activity or c-di-GMP levels, suggesting that nitrate and MucR modulate alginate production at a post-translational level through a localized pool of c-di-GMP. Nitrate increased pel promoter activity in the mucR mutant while in the same mutant the psl promoter activity was independent of nitrate. Nitrate and deletion of mucR did not impact on swarming motility but impaired attachment to solid surfaces. Nitrate and deletion of mucR promoted the formation of biofilms with increased thickness, cell density, and survival. Overall, this study provided insight into the functional role of MucR with respect to nitrate-mediated regulation of alginate biosynthesis. © 2015 Springer-Verlag Berlin Heidelberg

IFI16 and cGAS cooperate in the activation of STING during DNA sensing in human keratinocytes.

Science.gov (United States)

Almine, Jessica F; O'Hare, Craig A J; Dunphy, Gillian; Haga, Ismar R; Naik, Rangeetha J; Atrih, Abdelmadjid; Connolly, Dympna J; Taylor, Jordan; Kelsall, Ian R; Bowie, Andrew G; Beard, Philippa M; Unterholzner, Leonie

2017-02-13

Many human cells can sense the presence of exogenous DNA during infection though the cytosolic DNA receptor cyclic GMP-AMP synthase (cGAS), which produces the second messenger cyclic GMP-AMP (cGAMP). Other putative DNA receptors have been described, but whether their functions are redundant, tissue-specific or integrated in the cGAS-cGAMP pathway is unclear. Here we show that interferon-γ inducible protein 16 (IFI16) cooperates with cGAS during DNA sensing in human keratinocytes, as both cGAS and IFI16 are required for the full activation of an innate immune response to exogenous DNA and DNA viruses. IFI16 is also required for the cGAMP-induced activation of STING, and interacts with STING to promote STING phosphorylation and translocation. We propose that the two DNA sensors IFI16 and cGAS cooperate to prevent the spurious activation of the type I interferon response.

Molecular characterization of the di-leucine-based internalization motif of the T cell receptor

DEFF Research Database (Denmark)

Dietrich, J; Hou, X; Wegener, A M

1996-01-01

Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down-regulated following activation of protein kinases. We have recently shown that both phosphorylation of Ser-126 and the presence of the di-leucine sequence Leu-131 and Leu-132 in CD3 gamma are required f...... are important. 2) Recognition of phosphorylated CD3 gamma by molecules involved in receptor internalization. In this process Ser(P)-126, Asp-127, Leu-131, and Leu-132 are important....

Intrinsic nitric oxide regulates the taste response of the sugar receptor cell in the blowfly, Phormia regina.

Science.gov (United States)

Murata, Yoshihiro; Mashiko, Masashi; Ozaki, Mamiko; Amakawa, Taisaku; Nakamura, Tadashi

2004-01-01

The taste organ in insects is a hair-shaped taste sensory unit having four functionally differentiated contact chemoreceptor cells. In the blowfly, Phormia regina, cGMP has been suggested to be a second messenger for the sugar receptor cell. Generally, cGMP is produced by membranous or soluble guanylyl cyclase (sGC), which can be activated by nitric oxide (NO). In the present paper, we electrophysiologically showed that an NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO), an NO donor, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC 7) or an NO synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) specifically affected the response in the sugar receptor cell, but not in other receptor cells. PTIO, when introduced into the receptor cells in a sensillum aided by sodium deoxycholate (DOC, pH 7.2), depressed the response of sugar receptor cells to sucrose but did not affect those of the salt or water receptor cells. NOC 7, given extracellularly, latently induced the response of sugar receptor cells; and L-NAME, when introduced into the receptor cells, depressed the response of sugar receptor cells. The results clearly suggest that NO, which may be produced by intrinsic NOS in sugar receptor cells, participates in the transduction cascade of these cells in blowfly.

Contribution of altered signal transduction associated to glutamate receptors in brain to the neurological alterations of hepatic encephalopathy

Institute of Scientific and Technical Information of China (English)

Vicente Felipo

2006-01-01

Patients with liver disease may present hepatic encephalopathy (HE), a complex neuropsychiatric syndrome covering a wide range of neurological alterations,including cognitive and motor disturbances. HE reduces the quality of life of the patients and is associated with poor prognosis. In the worse cases HE may lead to coma or death.The mechanisms leading to HE which are not well known are being studied using animal models. The neurological alterations in HE are a consequence of impaired cerebral function mainly due to alterations in neurotransmission. We review here some studies indicating that alterations in neurotransmission associated to different types of glutamate receptors are responsible for some of the cognitive and motor alterations present in HE.These studies show that the function of the signal transduction pathway glutamate-nitric oxide-cGMP associated to the NMDA type of glutamate receptors is impaired in brain in vivo in HE animal models as well as in brain of patients died of HE. Activation of NMDA receptors in brain activates this pathway and increases cGMP. In animal models of HE this increase in cGMP induced by activation of NMDA receptors is reduced,which is responsible for the impairment in learning ability in these animal models. Increasing cGMP by pharmacological means restores learning ability in rats with HE and may be a new therapeutic approach to improve cognitive function in patients with HE.However, it is necessary to previously assess the possible secondary effects.Patients with HE may present psychomotor slowing,hypokinesia and bradykinesia. Animal models of HE also show hypolocomotion. It has been shown in rats with HE that hypolocomotion is due to excessive activation of metabotropic glutamate receptors (mGluRs) in substantia nigra pars reticulata. Blocking mGluR1 in this brain area normalizes motor activity in the rats, suggesting that a similar treatment for patients with HE could be useful to treat psychomotor slowing and

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.

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

Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis

Directory of Open Access Journals (Sweden)

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

Composition of cross-linked 125I-follitropin-receptor complexes

Energy Technology Data Exchange (ETDEWEB)

Shin, J.; Ji, T.H.

1985-10-15

Both of the alpha and beta subunits of intact human follitropin (FSH) were radioiodinated with SVI-sodium iodide and chloramine-T and could be resolved on sodium dodecyl sulfate-polyacrylamide gels. Radioiodinated FSH was affinity-cross-linked with a cleavable (nondisulfide) homobifunctional reagent to its membrane receptor on the porcine granulosa cell surface as well as to a Triton X-100-solubilized form of the receptor. Cross-linked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65, 83, and 117 kDa, in addition to the hormone bands. The hormone alpha beta dimer band corresponded to 43 kDa. Formation of the three bands requires the SVI-hormone to bind specifically to the receptor with subsequent cross-linking. Binding was prevented by an excess of the native hormone but not by other hormones. A monofunctional analog of the cross-linking reagent failed to produce the three bands. Reagent concentration-dependent cross-linking revealed that their formation was sequential; smaller complexes formed first and then larger ones. When gels of cross-linked complexes were treated to cleave covalent cross-links and then electrophoresed in a second dimension, 18-, 22-, and 34-kDa components were released, in addition to the alpha and beta subunits of the hormone.

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....

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...

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.

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.

An unusual protein kinase phosphorylates the chemotactic receptor of Dictystelium discoideum

International Nuclear Information System (INIS)

Meier, K.; Klein, C.

1988-01-01

The authors report the cAMP-dependent phosphorylation of the chemotactic receptor of Dictyostelium discoideum in partially purified plasma membranes. The protein kinase responsible for receptor phosphorylation is associated with this fraction and preferentially phosphorylates the ligand-occupied form of the receptor. 8-Azido[ 32 P]cAMP labeling of the cell surface has shown that the cAMP receptor exists in two forms. A 45-kDa protein is predominant on unstimulated cells. cAMP stimulation results in an increased receptor phosphorylation such that the receptor migrates on NaDodSO 4 /PAGE as a 47-kDa protein. Phosphorylation of the chemotactic receptor is not detected in membrane preparations unless cAMP is added to the incubation mixture. Only under those conditions is the phosphorylated 47-kDa form observed. The requirement for cAMP reflects the fact that the kinase involved preferentially uses the ligand-occupied receptor as a substrate. In vitro phosphorylation of the receptor does not involve tyrosine residues. The enzyme does not appear to be a cAMP- or cGMP-dependent protein kinase nor is it sensitive to guanine nucleotides, Ca 2+ /calmodulin, Ca 2+ /phospholipid, or EGTA. Similarities with the β-adrenergic receptor protein kinase are discussed

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

An Alzheimer Disease-linked Rare Mutation Potentiates Netrin Receptor Uncoordinated-5C-induced Signaling That Merges with Amyloid β Precursor Protein Signaling.

Science.gov (United States)

Hashimoto, Yuichi; Toyama, Yuka; Kusakari, Shinya; Nawa, Mikiro; Matsuoka, Masaaki

2016-06-03

A missense mutation (T835M) in the uncoordinated-5C (UNC5C) netrin receptor gene increases the risk of late-onset Alzheimer disease (AD) and also the vulnerability of neurons harboring the mutation to various insults. The molecular mechanisms underlying T835M-UNC5C-induced death remain to be elucidated. In this study, we show that overexpression of wild-type UNC5C causes low-grade death, which is intensified by an AD-linked mutation T835M. An AD-linked survival factor, calmodulin-like skin protein (CLSP), and a natural ligand of UNC5C, netrin1, inhibit this death. T835M-UNC5C-induced neuronal cell death is mediated by an intracellular death-signaling cascade, consisting of death-associated protein kinase 1/protein kinase D/apoptosis signal-regulating kinase 1 (ASK1)/JNK/NADPH oxidase/caspases, which merges at ASK1 with a death-signaling cascade, mediated by amyloid β precursor protein (APP). Notably, netrin1 also binds to APP and partially inhibits the death-signaling cascade, induced by APP. These results may provide new insight into the amyloid β-independent pathomechanism of AD. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases.

Science.gov (United States)

Lieberman, Ori J; Orr, Mona W; Wang, Yan; Lee, Vincent T

2014-01-17

The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.

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

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

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.

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.

C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage

International Nuclear Information System (INIS)

Tzeng, W.-P.; Frey, Teryl K.

2006-01-01

Rubella virus (RUB) replicons are derivatives of the RUB infectious cDNA clone that retain the nonstructural open reading frame (NS-ORF) that encodes the replicase proteins but not the structural protein ORF (SP-ORF) that encodes the virion proteins. RUB defective interfering (DI) RNAs contain deletions within the SP-ORF and thus resemble replicons. DI RNAs often retain the 5' end of the capsid protein (C) gene that has been shown to modulate virus-specific RNA synthesis. However, when replicons either with or without the C gene were passaged serially in the presence of wt RUB as a source of the virion proteins, it was found that neither replicon was maintained and DI RNAs were generated. The majority DI RNA species contained in-frame deletions in the SP-ORF leading to a fusion between the 5' end of the C gene and the 3' end of the E1 glycoprotein gene. DI infectious cDNA clones were constructed and transcripts from these DI infectious cDNA clones were maintained during serial passage with wt RUB. The C-E1 fusion protein encoded by the DI RNAs was synthesized and was required for maintenance of the DI RNA during serial passage. This is the first report of a functional novel gene product resulting from deletion during DI RNA generation. Thus far, the role of the C-E1 fusion protein in maintenance of DI RNAs during serial passage remained elusive as it was found that the fusion protein diminished rather than enhanced DI RNA synthesis and was not incorporated into virus particles

Atrial natriuretic factor receptor guanylate cyclase, ANF-RGC, transduces two independent signals, ANF and Ca2+

Directory of Open Access Journals (Sweden)

Teresa eDuda

2014-03-01

Full Text Available Atrial natriuretic factor receptor guanylate cyclase, ANF-RGC, was the first discovered member of the mammalian membrane guanylate cyclase family. The hallmark feature of the family is that a single protein contains both the site for recognition of the regulatory signal and the ability to transduce it into the production of the second messenger, cyclic GMP. For over two decades, the family has been classified into two subfamilies, the hormone receptor subfamily with ANF-RGC being its paramount member, and the Ca2+ modulated subfamily, which includes the rod outer segment guanylate cyclases, ROS-GC1 and 2, and the olfactory neuroepithelial guanylate cyclase, ONE-GC. ANF-RGC is the receptor and the signal transducer of the most hypotensive hormones, atrial natriuretic factor (ANF and B-type natriuretic peptide (BNP. After binding these hormones at the extracellular domain it, at its intracellular domain, signals activation of the C-terminal catalytic module and accelerates the production of cyclic GMP. Cyclic GMP then serves the second messenger role in biological responses of ANF and BNP such as natriuresis, diuresis, vasorelaxation and anti-proliferation. Very recently another modus operandi for ANF-RGC was revealed. Its crux is that ANF-RGC activity is also regulated by Ca2+. The Ca2+ sensor neurocalcin  mediates this signaling mechanism. Strikingly, the Ca2+ and ANF signaling mechanisms employ separate structural motifs of ANF-RGC in modulating its core catalytic domain in accelerating the production of cyclic GMP. In this review the biochemistry and physiology of these mechanisms with emphasis on cardiovascular regulation will be discussed.

RISET STRATEGI IMPLEMENTASI C-RAN DI TELKOMSEL MELALUI KOLABORASI JARINGAN NG-PON2 DI TELKOM AKSES MENGGUNAKAN PENDEKATAN METODE STRATEGIC SITUATION ANALYSIS

Directory of Open Access Journals (Sweden)

Tri Susanto

2007-07-01

Full Text Available Cloud Radio Access Network (C-RAN adalah teknologi akses radio yang diyakini mampu menekan biaya CAPEX dan OPEX dari mobile operator dan memberikan performansi yang lebih baik dibandingkan Radio Access Network (RAN tradisional. Salah satu faktor kunci sukses implementasi C-RAN adalah penyediaan jaringan fonthaul yang efektif. Tujuan penelitian ini adalah membuat strategi implementasi C-RAN di Telkomsel melalui kolaborasi jaringan NG-PON2 di Telkom Akses. Pada tulisan ini menggunakan metode analisis kualitatif dengan melakukan strategy situation analysis (SSA yaitu melakukan analisa data eksternal maupun internal dan selanjutnya diformulasikan sebagai dasar pembuatan strategi implementasi. Dari penelitian ini dihasilkan usulan strategi implementasi C-RAN di Telkomsel melalui kolaborasi jaringan NG-PON2 di Telkom Akses.

Fully automated synthesis of the M{sub 1} receptor agonist [{sup 11}C]GSK1034702 for clinical use on an Eckert and Ziegler Modular Lab system

Energy Technology Data Exchange (ETDEWEB)

Huiban, Mickael, E-mail: Mickael.x.huiban@gsk.com [GlaxoSmithKline, Clinical Imaging Centre, Imperial College London, Hammersmith Hospital, Du Cane Road, London, W12 0NN (United Kingdom); Pampols-Maso, Sabina; Passchier, Jan [GlaxoSmithKline, Clinical Imaging Centre, Imperial College London, Hammersmith Hospital, Du Cane Road, London, W12 0NN (United Kingdom)

2011-10-15

A fully automated and GMP compatible synthesis has been developed to reliably label the M{sub 1} receptor agonist GSK1034702 with carbon-11. Stille reaction of the trimethylstannyl precursor with [{sup 11}C]methyl iodide afforded [{sup 11}C]GSK1034702 in an estimated 10{+-}3% decay corrected yield. This method utilises the commercially available modular laboratory equipment and provides high purity [{sup 11}C]GSK1034702 in a formulation suitable for human use. - Highlights: > Preparation of [{sup 11}C]GSK1034702 through a Stille cross-coupling reaction. > Provision of the applicability of commercially available modules for the synthesis of non-standard PET tracers. > Defining specification for heavy metals content in final dose product. > Presenting results from validation of manufacturing process.

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.

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.

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.

Natriuretic peptides stimulate the cardiac sodium pump via NPR-C-coupled NOS activation

DEFF Research Database (Denmark)

William, M.; Hamilton, E.J.; Garcia, A.

2008-01-01

Natriuretic peptides (NPs) and their receptors (NPRs) are expressed in the heart, but their effects on myocyte function are poorly understood. Because NPRs are coupled to synthesis of cGMP, an activator of the sarcolemmal Na(+)-K(+) pump, we examined whether atrial natriuretic peptide (ANP) regul...

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

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.

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.

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.

The cGAS-cGAMP-STING pathway connects DNA damage to inflammation, senescence, and cancer.

Science.gov (United States)

Li, Tuo; Chen, Zhijian J

2018-05-07

Detection of microbial DNA is an evolutionarily conserved mechanism that alerts the host immune system to mount a defense response to microbial infections. However, this detection mechanism also poses a challenge to the host as to how to distinguish foreign DNA from abundant self-DNA. Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is a DNA sensor that triggers innate immune responses through production of the second messenger cyclic GMP-AMP (cGAMP), which binds and activates the adaptor protein STING. However, cGAS can be activated by double-stranded DNA irrespective of the sequence, including self-DNA. Although how cGAS is normally kept inactive in cells is still not well understood, recent research has provided strong evidence that genomic DNA damage leads to cGAS activation to stimulate inflammatory responses. This review summarizes recent findings on how genomic instability and DNA damage trigger cGAS activation and how cGAS serves as a link from DNA damage to inflammation, cellular senescence, and cancer. © 2018 Li and Chen.

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.

Changes of muscarinic cholinergic receptors during aging process of primary cultured neutrons

International Nuclear Information System (INIS)

Fan Guohuang; Yi Ningyu; Xia Zongqin

1996-01-01

The dynamic changes of muscarinic receptor density and its reactivity during aging process in primary cultured neutrons were studied. Muscarinic receptor density was measured by 3 H-QNB binding assay, and muscarinic receptor reactivity was assessed by carbachol stimulation of cGMP formation, the latter was measured by RIA. After 2 weeks' incubation of neonatal rat brain cells, the nutrients began to rupture and the cell bodies shrank markedly showing senescent feature. The muscarinic receptor density reached peak at the 12th day in vitro (12 DIV), but the muscarinic receptor reactivity reached peak at 9 DIV and declined significantly at 12 DIV. The results demonstrated that during aging process of primary cultured neutrons, the decline of muscarinic receptor reactivity is likely prior to the decrease of receptor density

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

Cyclic di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus but dispensable for viability in anaerobic conditions.

Science.gov (United States)

Zeden, Merve S; Schuster, Christopher F; Bowman, Lisa; Zhong, Qiyun; Williams, Huw D; Gründling, Angelika

2018-03-02

Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as Staphylococcus aureus However, the exact role of this molecule has not been identified. dacA , the S. aureus gene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that an S. aureus dacA mutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality of dacA were identified in alsT and opuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system in S. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained in hepS, hemB, ctaA, and qoxB, coding proteins required for respiration. Furthermore, we show that dacA is dispensable for growth in anaerobic conditions. Together, these findings reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration in S. aureus . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Endocytosis of GPI-linked membrane folate receptor-alpha.

Science.gov (United States)

Rijnboutt, S; Jansen, G; Posthuma, G; Hynes, J B; Schornagel, J H; Strous, G J

1996-01-01

GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs. We have studied the biosynthetic transport to and internalization of MFR isoform alpha in KB-cells. MFR-alpha was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-alpha was completely soluble in Triton X-100 at 0 degree C. In contrast, only 33% of the 36-38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37 degrees C or in the presence of saponin at 0 degree C. Similar solubilization characteristics were found when MFR-alpha at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinity-analog of folic acid as a ligand. Triton X-100-insoluble membrane domains containing MFR-alpha could be separated from soluble MFR-alpha on sucrose flotation gradients. Only Triton X-100 soluble MFR-alpha was internalized from the plasma membrane. The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100-resistant membrane domains. Internalized MFR-alpha recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0 degree C. Using immunoelectron microscopy, we found MFR-alpha along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles. In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-alpha bypasses caveolae.

Automated preparation of the dopamine D{sub 2/3} receptor agonist ligand [{sup 11}C]-(+)-PHNO for human PET imaging studies

Energy Technology Data Exchange (ETDEWEB)

Plisson, Christophe, E-mail: Christophe.2.plisson@gsk.com [GlaxoSmithKline, Clinical Imaging Centre, Hammersmith Hospital, London W12 0NN (United Kingdom); Huiban, Mickael; Pampols-Maso, Sabina; Singleton, Goerkem; Hill, Samuel P.; Passchier, Jan [GlaxoSmithKline, Clinical Imaging Centre, Hammersmith Hospital, London W12 0NN (United Kingdom)

2012-02-15

Carbon-11 labelled (+)-4-Propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol ([{sup 11}C]-(+)-PHNO) is used as a high-affinity state, dopamine D{sub 2/3} receptor ligand in clinical PET studies. To facilitate its use, robust, rapid, efficient and GMP compliant methods are required for the manufacturing and QC testing processes. Additionally, to allow for full quantification of the resulting signal in the CNS, a reliable method is required to establish the parent plasma concentration over the course of the scan. This paper provides high-quality methods to support clinical application of [{sup 11}C]-(+)-PHNO. - Highlights: Black-Right-Pointing-Pointer Fully automated synthesis of [{sup 11}C]-(+)-PHNO. Black-Right-Pointing-Pointer Rapid multi-step synthesis and QC analysis. Black-Right-Pointing-Pointer Reproducible synthesis process typically yielding more than 3 GBq of [{sup 11}C]-(+)-PHNO. Black-Right-Pointing-Pointer Very low failure rate.

Activation of the ζ receptor 1 suppresses NMDA responses in rat retinal ganglion cells.

Science.gov (United States)

Zhang, X-J; Liu, L-L; Jiang, S-X; Zhong, Y-M; Yang, X-L

2011-03-17

The sigma receptor 1 (σR1) has been shown to modulate the activity of several voltage- and ligand-gated channels. Using patch-clamp techniques in rat retinal slice preparations, we demonstrated that activation of σR1 by SKF10047 (SKF) or PRE-084 suppressed N-methyl-D-aspartate (NMDA) receptor-mediated current responses from both ON and OFF type ganglion cells (GCs), dose-dependently, and the effect could be blocked by the σR1 antagonist BD1047 or the σR antagonist haloperidol. The suppression by SKF of NMDA currents was abolished with pre-incubation of the G protein inhibitor GDP-β-S or the Gi/o activator mastoparan. We further explored the intracellular signaling pathway responsible for the SKF-induced suppression of NMDA responses. Application of either cAMP/the PKA inhibitor Rp-cAMP or cGMP/the PKG inhibitor KT5823 did not change the SKF-induced effect, suggesting the involvement of neither cAMP/PKA nor cGMP/PKG pathway. In contrast, suppression of NMDA responses by SKF was abolished by internal infusion of the phosphatidylinostiol-specific phospholipase C (PLC) inhibitor U73122, but not by the phosphatidylcholine-PLC inhibitor D609. SKF-induced suppression of NMDA responses was dependent on intracellular Ca2+ concentration ([Ca2+]i), as evidenced by the fact that the effect was abolished when [Ca2+]i was buffered with 10 mM BAPTA. The SKF effect was blocked by xestospongin-C/heparin, IP3 receptor antagonists, but unchanged by ryanodine/caffeine, ryanodine receptor modulators. Furthermore, application of protein kinase C inhibitors Bis IV and Gö6976 eliminated the SKF effect. These results suggest that the suppression of NMDA responses of rat retinal GCs caused by the activation of σR1 may be mediated by a distinct [Ca2+]i-dependent PLC-PKC pathway. This effect of SKF could help ameliorate malfunction of GCs caused by excessive stimulation of NMDA receptors under pathological conditions. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights

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

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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.

An ant-plant mutualism through the lens of cGMP-dependent kinase genes.

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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).

Transmission of innate immune signaling by packaging of cGAMP in viral particles.

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Gentili, Matteo; Kowal, Joanna; Tkach, Mercedes; Satoh, Takeshi; Lahaye, Xavier; Conrad, Cécile; Boyron, Marilyn; Lombard, Bérangère; Durand, Sylvère; Kroemer, Guido; Loew, Damarys; Dalod, Marc; Théry, Clotilde; Manel, Nicolas

2015-09-11

Infected cells detect viruses through a variety of receptors that initiate cell-intrinsic innate defense responses. Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is a cytosolic sensor for many DNA viruses and HIV-1. In response to cytosolic viral DNA, cGAS synthesizes the second messenger 2'3'-cyclic GMP-AMP (cGAMP), which activates antiviral signaling pathways. We show that in cells producing virus, cGAS-synthesized cGAMP can be packaged in viral particles and extracellular vesicles. Viral particles efficiently delivered cGAMP to target cells. cGAMP transfer by viral particles to dendritic cells activated innate immunity and antiviral defenses. Finally, we show that cell-free murine cytomegalovirus and Modified Vaccinia Ankara virus contained cGAMP. Thus, transfer of cGAMP by viruses may represent a defense mechanism to propagate immune responses to uninfected target cells. Copyright © 2015, American Association for the Advancement of Science.

Demonstration of a specific C3a receptor on guinea pig platelets

International Nuclear Information System (INIS)

Fukuoka, Y.; Hugli, T.E.

1988-01-01

Guinea pig platelets reportedly contain receptors specific for the anaphylatoxin C3a based on both ligand-binding studies and functional responses. A portion of the human 125I-C3a that binds to guinea pig platelets is competitively displaced by excess unlabeled C3a; however, the majority of ligand uptake was nonspecific. Uptake of 125I-C3a by guinea pig platelets is maximal in 1 min, and stimulation of guinea pig platelets by thrombin, ADP, or the Ca2+ ionophore A23187 showed little influence on binding of the ligand. Scatchard analysis indicated that approximately 1200 binding sites for C3a exist per cell with an estimated Kd of 8 x 10(-10) M. Human C3a des Arg also binds to guinea pig platelets, but Scatchard analysis indicated that no specific binding occurred. Because the ligand-binding studies were complicated by high levels of nonspecific uptake, we attempted to chemically cross-link the C3a molecule to a specific component on the platelet surface. Cross-linkage of 125I-C3a to guinea pig platelets with bis(sulfosuccinimidyl)suberate revealed radioactive complexes at 105,000 and 115,000 m.w. on SDS-PAGE gels by autoradiographic analysis. In the presence of excess unlabeled C3a, complex formation was inhibited. No cross-linkage could be demonstrated between the inactive 125I-C3a des Arg and the putative C3a-R on guinea pig platelets. Human C3a, but not C3a des Arg induces serotonin release and aggregation of the guinea pig platelets. Human C3a was unable to induce either serotonin release or promote aggregation of human platelets. Uptake of human 125I-C3a by human platelets was not saturable, and Scatchard analysis was inconclusive. Attempts to cross-link 125I-C3a to components on the surface of human platelets also failed to reveal a ligand-receptor complex. Therefore, we conclude that guinea pig platelets have specific surface receptors to C3a and that human platelets appear devoid of receptors to the anaphylatoxin

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

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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.

A polycystin-type transient receptor potential (Trp channel that is activated by ATP

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David Traynor

2017-02-01

Full Text Available ATP and ADP are ancient extra-cellular signalling molecules that in Dictyostelium amoebae cause rapid, transient increases in cytosolic calcium due to an influx through the plasma membrane. This response is independent of hetero-trimeric G-proteins, the putative IP3 receptor IplA and all P2X channels. We show, unexpectedly, that it is abolished in mutants of the polycystin-type transient receptor potential channel, TrpP. Responses to the chemoattractants cyclic-AMP and folic acid are unaffected in TrpP mutants. We report that the DIF morphogens, cyclic-di-GMP, GABA, glutamate and adenosine all induce strong cytoplasmic calcium responses, likewise independently of TrpP. Thus, TrpP is dedicated to purinergic signalling. ATP treatment causes cell blebbing within seconds but this does not require TrpP, implicating a separate purinergic receptor. We could detect no effect of ATP on chemotaxis and TrpP mutants grow, chemotax and develop almost normally in standard conditions. No gating ligand is known for the human homologue of TrpP, polycystin-2, which causes polycystic kidney disease. Our results now show that TrpP mediates purinergic signalling in Dictyostelium and is directly or indirectly gated by ATP.

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

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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

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...

AIM2-Like Receptors Positively and Negatively Regulate the Interferon Response Induced by Cytosolic DNA

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Yuki Nakaya

2017-07-01

Full Text Available Cytosolic DNAs derived from retrotransposons serve as pathogen-associated molecular patterns for pattern recognition receptors (PRRs that stimulate the induction of interferons (IFNs and other cytokines, leading to autoimmune disease. Cyclic GMP-AMP synthase is one PRR that senses retrotransposon DNA, activating type I IFN responses through the stimulator of IFN genes (STING. Absent in melanoma 2 (AIM2-like receptors (ALRs have also been implicated in these pathways. Here we show that the mouse ALR IFI205 senses cytosolic retrotransposon DNA independently of cyclic GMP-AMP production. AIM2 antagonizes IFI205-mediated IFN induction activity by sequestering it from STING. We also found that the complement of genes located in the ALR locus in C57BL/6 and AIM2 knockout mice are different and unique, which has implications for interpretation of the sensing of pathogens in different mouse strains. Our data suggest that members of the ALR family are critical to the host IFN response to endogenous DNA.

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

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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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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

Coupling of g proteins to reconstituted monomers and tetramers of the M2 muscarinic receptor.

Science.gov (United States)

Redka, Dar'ya S; Morizumi, Takefumi; Elmslie, Gwendolynne; Paranthaman, Pranavan; Shivnaraine, Rabindra V; Ellis, John; Ernst, Oliver P; Wells, James W

2014-08-29

G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5'-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[(3)H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the "ternary complex model"). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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.)

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

Fur is a repressor of biofilm formation in Yersinia pestis.

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Fengjun Sun

Full Text Available BACKGROUND: Yersinia pestis synthesizes the attached biofilms in the flea proventriculus, which is important for the transmission of this pathogen by fleas. The hmsHFRS operons is responsible for the synthesis of exopolysaccharide (the major component of biofilm matrix, which is activated by the signaling molecule 3', 5'-cyclic diguanylic acid (c-di-GMP synthesized by the only two diguanylate cyclases HmsT, and YPO0449 (located in a putative operonYPO0450-0448. METHODOLOGY/PRINCIPAL FINDINGS: The phenotypic assays indicated that the transcriptional regulator Fur inhibited the Y. pestis biofilm production in vitro and on nematode. Two distinct Fur box-like sequences were predicted within the promoter-proximal region of hmsT, suggesting that hmsT might be a direct Fur target. The subsequent primer extension, LacZ fusion, electrophoretic mobility shift, and DNase I footprinting assays disclosed that Fur specifically bound to the hmsT promoter-proximal region for repressing the hmsT transcription. In contrast, Fur had no regulatory effect on hmsHFRS and YPO0450-0448 at the transcriptional level. The detection of intracellular c-di-GMP levels revealed that Fur inhibited the c-di-GMP production. CONCLUSIONS/SIGNIFICANCE: Y. pestis Fur inhibits the c-di-GMP production through directly repressing the transcription of hmsT, and thus it acts as a repressor of biofilm formation. Since the relevant genetic contents for fur, hmsT, hmsHFRS, and YPO0450-0448 are extremely conserved between Y. pestis and typical Y. pseudotuberculosis, the above regulatory mechanisms can be applied to Y. pseudotuberculosis.

Crystal structure of poly[N,N-diethyl-2-hy-droxy-ethan-1-aminium [μ3-cyanido-κ(3) C:C:N-di-μ-cyanido-κ(4) C:N-dicuprate(I)

Science.gov (United States)

Corfield, Peter W R; Cleary, Emma; Michalski, Joseph F

2016-07-01

In the title compound, {(C6H16NO)[Cu2(CN)3]} n , the cyanide groups link the Cu(I) atoms into an open three-dimensional anionic network, with the mol-ecular formula Cu2(CN)3 (-). One Cu(I) atom is tetra-hedrally bound to four CN groups, and the other Cu(I) atom is bonded to three CN groups in an approximate trigonal-planar coordination. The tetra-hedrally coordinated Cu(I) atoms are linked into centrosymmetric dimers by the C atoms of two end-on bridging CN groups which bring the Cu(I) atoms into close contact at 2.5171 (7) Å. Two of the cyanide groups bonded to the Cu(I) atoms with trigonal-planar surrounding link the dimeric units into columns along the a axis, and the third links the columns together to form the network. The N,N-di-ethyl-ethano-lamine mol-ecules used in the synthesis have become protonated at the N atoms and are situated in cavities in the network, providing charge neutrality, with no covalent inter-actions between the cations and the anionic network.

Development of GMP-1 a molecular chaperone network modulator protecting mitochondrial function and its assessment in fly and mice models of Alzheimer's disease.

Science.gov (United States)

Pavlov, Pavel F; Hutter-Paier, Birgit; Havas, Daniel; Windisch, Manfred; Winblad, Bengt

2018-04-27

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. It has been shown that amyloid beta peptide (Aβ) and amyloid precursor protein (APP) interact with mitochondria contributing to the mitochondrial dysfunction in AD. Prevention of abnormal protein targeting to mitochondria can protect normal mitochondrial function, increase neuronal survival and at the end, ameliorate symptoms of AD and other neurodegenerative disorders. First steps of mitochondrial protein import are coordinated by molecular chaperones Hsp70 and Hsp90 that bind to the newly synthesized mitochondria-destined proteins and deliver them to the protein import receptors on the surface of organelle. Here, we have described the development of a novel compound named GMP-1 that disrupts interactions between Hsp70/Hsp90 molecular chaperones and protein import receptor Tom70. GMP-1 treatment of SH-SY5Y cells results in decrease in mitochondria-associated APP and protects SH-SY5Y cells from toxic effect of Aβ 1-42 exposure. Experiments in drosophila and mice models of AD demonstrated neuroprotective effect of GMP-1 treatment, improvement in memory and behaviour tests as well as restoration of mitochondrial function. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Sigma receptor ligand N,N'-di-(ortho-tolyl)guanidine inhibits release of acetylcholine in the guinea pig ileum.

Science.gov (United States)

Cambell, B G; Keana, J F; Weber, E

1991-11-26

The inhibition of stimulated contractions of the guinea pig ileum longitudinal muscle/myenteric plexus preparation by sigma receptor ligands has been previously described. In this study, the stimulated release of [3H]acetylcholine from cholinergic nerve terminals in this same preparation was monitored in the presence and absence of sigma receptor ligands. N,N'-Di-(orthotolyl)guanidine (DTG) and other compounds selective for the sigma receptor inhibited stimulated [3H]acetylcholine release. These results suggest that their inhibition of stimulated contractions in this preparation was mediated by inhibition of acetylcholine release.

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.

Activation of Phosphatidylinositol-Linked Dopamine Receptors Induces a Facilitation of Glutamate-Mediated Synaptic Transmission in the Lateral Entorhinal Cortex.

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Iulia Glovaci

Full Text Available The lateral entorhinal cortex receives strong inputs from midbrain dopamine neurons that can modulate its sensory and mnemonic function. We have previously demonstrated that 1 µM dopamine facilitates synaptic transmission in layer II entorhinal cortex cells via activation of D1-like receptors, increased cAMP-PKA activity, and a resulting enhancement of AMPA-receptor mediated currents. The present study assessed the contribution of phosphatidylinositol (PI-linked D1 receptors to the dopaminergic facilitation of transmission in layer II of the rat entorhinal cortex, and the involvement of phospholipase C activity and release of calcium from internal stores. Whole-cell patch-clamp recordings of glutamate-mediated evoked excitatory postsynaptic currents were obtained from pyramidal and fan cells. Activation of D1-like receptors using SKF38393, SKF83959, or 1 µM dopamine induced a reversible facilitation of EPSCs which was abolished by loading cells with either the phospholipase C inhibitor U-73122 or the Ca2+ chelator BAPTA. Neither the L-type voltage-gated Ca2+ channel blocker nifedipine, nor the L/N-type channel blocker cilnidipine, blocked the facilitation of synaptic currents. However, the facilitation was blocked by blocking Ca2+ release from internal stores via inositol 1,4,5-trisphosphate (InsP3 receptors or ryanodine receptors. Follow-up studies demonstrated that inhibiting CaMKII activity with KN-93 failed to block the facilitation, but that application of the protein kinase C inhibitor PKC(19-36 completely blocked the dopamine-induced facilitation. Overall, in addition to our previous report indicating a role for the cAMP-PKA pathway in dopamine-induced facilitation of synaptic transmission, we demonstrate here that the dopaminergic facilitation of synaptic responses in layer II entorhinal neurons also relies on a signaling cascade dependent on PI-linked D1 receptors, PLC, release of Ca2+ from internal stores, and PKC activation which is

Reduced ability of C-type natriuretic peptide (CNP) to activate natriuretic peptide receptor B (NPR-B) causes dwarfism in lbab−/− mice

Science.gov (United States)

Yoder, Andrea R.; Kruse, Andrew C.; Earhart, Cathleen A.; Ohlendorf, Douglas H.; Potter, Lincoln R.

2015-01-01

C-type natriuretic peptide (CNP) stimulates endochondrial ossification by activating the transmembrane guanylyl cyclase, natriuretic peptide receptor-B (NPR-B). Recently, a spontaneous autosomal recessive mutation that causes severe dwarfism in mice was identified. The mutant, called long bone abnormality (lbab), contains a single point mutation that converts an arginine to a glycine in a conserved coding region of the CNP gene, but how this mutation affects CNP activity has not been reported. Here, we determined that thirty to greater than one hundred-fold more CNPlbab was required to activate NPR-B as compared to wild-type CNP in whole cell cGMP elevation and membrane guanylyl cyclase assays. The reduced ability of CNPlbab to activate NPR-B was explained, at least in part, by decreased binding since ten-fold more CNPlbab than wild-type CNP was required to compete with [125I][Tyr0]CNP for receptor binding. Molecular modeling suggested that the conserved arginine is critical for binding to an equally conserved acidic pocket in NPR-B. These results indicate that reduced binding to and activation of NPR-B causes dwarfism in lbab−/− mice. PMID:18554750

Structural and functional analyses of DNA-sensing and immune activation by human cGAS.

Science.gov (United States)

Kato, Kazuki; Ishii, Ryohei; Goto, Eiji; Ishitani, Ryuichiro; Tokunaga, Fuminori; Nureki, Osamu

2013-01-01

The detection of cytosolic DNA, derived from pathogens or host cells, by cytosolic receptors is essential for appropriate host immune responses. Cyclic GMP-AMP synthase (cGAS) is a newly identified cytosolic DNA receptor that produces cyclic GMP-AMP, which activates stimulator of interferon genes (STING), resulting in TBK1-IRF3 pathway activation followed by the production of type I interferons. Here we report the crystal structure of human cGAS. The structure revealed that a cluster of lysine and arginine residues forms the positively charged DNA binding surface of human cGAS, which is important for the STING-dependent immune activation. A structural comparison with other previously determined cGASs and our functional analyses suggested that a conserved zinc finger motif and a leucine residue on the DNA binding surface are crucial for the DNA-specific immune response of human cGAS, consistent with previous work. These structural features properly orient the DNA binding to cGAS, which is critical for DNA-induced cGAS activation and STING-dependent immune activation. Furthermore, we showed that the cGAS-induced activation of STING also involves the activation of the NF-κB and IRF3 pathways. Our results indicated that cGAS is a DNA sensor that efficiently activates the host immune system by inducing two distinct pathways.

Structural and functional analyses of DNA-sensing and immune activation by human cGAS.

Directory of Open Access Journals (Sweden)

Kazuki Kato

Full Text Available The detection of cytosolic DNA, derived from pathogens or host cells, by cytosolic receptors is essential for appropriate host immune responses. Cyclic GMP-AMP synthase (cGAS is a newly identified cytosolic DNA receptor that produces cyclic GMP-AMP, which activates stimulator of interferon genes (STING, resulting in TBK1-IRF3 pathway activation followed by the production of type I interferons. Here we report the crystal structure of human cGAS. The structure revealed that a cluster of lysine and arginine residues forms the positively charged DNA binding surface of human cGAS, which is important for the STING-dependent immune activation. A structural comparison with other previously determined cGASs and our functional analyses suggested that a conserved zinc finger motif and a leucine residue on the DNA binding surface are crucial for the DNA-specific immune response of human cGAS, consistent with previous work. These structural features properly orient the DNA binding to cGAS, which is critical for DNA-induced cGAS activation and STING-dependent immune activation. Furthermore, we showed that the cGAS-induced activation of STING also involves the activation of the NF-κB and IRF3 pathways. Our results indicated that cGAS is a DNA sensor that efficiently activates the host immune system by inducing two distinct pathways.

Site-directed cross-linking: establishing the dimeric structure of the aspartate receptor of bacterial chemotaxis

International Nuclear Information System (INIS)

Milligan, D.L.; Koshland, D.E. Jr.

1988-01-01

Cysteine residues introduced at specific locations in the aspartate receptor of Salmonella typhimurium provide anchor points for cross-linking and serve as chemical markers for structural studies of this oligomeric receptor. These markers have been used to measure the rate of subunit exchange between oligomeric receptors and to show that ligand binding inhibits this exchange. The cysteine-containing receptors can be oxidatively cross-linked to completion within the oligomeric receptor, indicating that the receptor has an even number of subunits. Based on this observation, a technique has been developed that can be used to determine the oligomeric structure of proteins under a variety of experimental conditions. The technique involves the measurement of the effect of dilution by cysteineless receptor subunits on cross-linking and reveals that the aspartate receptor is dimeric in detergent solution, in a mixed-micelle system, and in reconstituted membrane vesicles. Binding of aspartate does not change the oligomeric structure of the receptor, indicating that transmembrane signaling occurs within an oligomeric receptor of constant size

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

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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

Example Plant Model for an International Benchmark Study on DI and C PSA

International Nuclear Information System (INIS)

Shin, Sung Min; Park, Jinkyun; Jang, Wondea; Kang, Hyun Gook

2016-01-01

In this context the risk quantification due to these digitalized safety systems became more important. Although there are many challenges to address about this issue, many countries agreed with the necessity of research on reliability quantification of DI and C system. Based on the agreement of several countries, one of internal research association is planning a benchmark study on this issue by sharing an example digitalized plant model and let each participating member develop its own probabilistic safety assessment (PSA) model of digital I and C systems. Although the DI and C systems are being applied to NPPs, of which modeling method to quantify its reliability still ambiguous. Therefore, an internal research association is planning a benchmark study to address this issue by sharing an example digitalized plant model and let each member develop their own PSA model for DI and C systems

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.)

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.

A Bacterial Receptor PcrK Senses the Plant Hormone Cytokinin to Promote Adaptation to Oxidative Stress

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Fang-Fang Wang

2017-12-01

Full Text Available Summary: Recognition of the host plant is a prerequisite for infection by pathogenic bacteria. However, how bacterial cells sense plant-derived stimuli, especially chemicals that function in regulating plant development, remains completely unknown. Here, we have identified a membrane-bound histidine kinase of the phytopathogenic bacterium Xanthomonas campestris, PcrK, as a bacterial receptor that specifically detects the plant cytokinin 2-isopentenyladenine (2iP. 2iP binds to the extracytoplasmic region of PcrK to decrease its autokinase activity. Through a four-step phosphorelay, 2iP stimulation decreased the phosphorylation level of PcrR, the cognate response regulator of PcrK, to activate the phosphodiesterase activity of PcrR in degrading the second messenger 3′,5′-cyclic diguanylic acid. 2iP perception by the PcrK-PcrR remarkably improves bacterial tolerance to oxidative stress by regulating the transcription of 56 genes, including the virulence-associated TonB-dependent receptor gene ctrA. Our results reveal an evolutionarily conserved, inter-kingdom signaling by which phytopathogenic bacteria intercept a plant hormone signal to promote adaptation to oxidative stress. : How pathogenic bacteria use receptors to recognize the signals of the host plant is unknown. Wang et al. have identified a bacterial receptor histidine kinase that specifically senses the plant hormone cytokinin. Through a four-step phosphorelay, cytokinin perception triggers degradation of a second messenger, c-di-GMP, to activate the bacterial response to oxidative stress. Keywords: histidine kinase, ligand, cytokinin, autokinase activity, phosphorelay, response regulator, two-component signal transduction system, Xanthomonas campestris pv. campestris, virulence, oxidative stress

Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

KAUST Repository

Irving, Helen R.; Kwezi, Lusisizwe; Wheeler, Janet I.; Gehring, Christoph A

2012-01-01

Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

KAUST Repository

Irving, Helen R.

2012-02-01

Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

Dendroaspis natriuretic peptide binds to the natriuretic peptide clearance receptor

International Nuclear Information System (INIS)

Johns, Douglas G.; Ao, Zhaohui; Heidrich, Bradley J.; Hunsberger, Gerald E.; Graham, Taylor; Payne, Lisa; Elshourbagy, Nabil; Lu, Quinn; Aiyar, Nambi; Douglas, Stephen A.

2007-01-01

Dendroaspis natriuretic peptide (DNP) is a newly-described natriuretic peptide which lowers blood pressure via vasodilation. The natriuretic peptide clearance receptor (NPR-C) removes natriuretic peptides from the circulation, but whether DNP interacts with human NPR-C directly is unknown. The purpose of this study was to test the hypothesis that DNP binds to NPR-C. ANP, BNP, CNP, and the NPR-C ligands AP-811 and cANP(4-23) displaced [ 125 I]-ANP from NPR-C with pM-to-nM K i values. DNP displaced [ 125 I]-ANP from NPR-C with nM potency, which represents the first direct demonstration of binding of DNP to human NPR-C. DNP showed high pM affinity for the GC-A receptor and no affinity for GC-B (K i > 1000 nM). DNP was nearly 10-fold more potent than ANP at stimulating cGMP production in GC-A expressing cells. Blockade of NPR-C might represent a novel therapeutic approach in augmenting the known beneficial actions of DNP in cardiovascular diseases such as hypertension and heart failure

Mechanisms for type-II vitellogenesis-inhibiting hormone suppression of vitellogenin transcription in shrimp hepatopancreas: Crosstalk of GC/cGMP pathway with different MAPK-dependent cascades.

Science.gov (United States)

Chen, Ting; Ren, Chunhua; Jiang, Xiao; Zhang, Lvping; Li, Hongmei; Huang, Wen; Hu, Chaoqun

2018-01-01

Vitellogenesis is the process of yolk formation via accumulating vitellin (Vn) with nutrients in the oocytes. Expression of vitellogenin (Vg), the precursor of Vn, is one of the indicators for the start of vitellogenesis. In Pacific white shrimp (Litopenaeus vannamei), the type-II vitellogenesis-inhibiting hormone (VIH-2) effectively suppresses hepatopancreatic Vg mRNA expression. In this study, we demonstrate the increasing transcript levels of hepatopancreatic Vg during L. vannamei ovarian development, suggesting that the hepatopancreas-derived Vg/Vn may also contribute to vitellogenesis in this species. Using a combination of in vivo injections and in vitro primary cell cultures, we provide evidences that the inhibition of VIH-2 on hepatopancreatic Vg gene expression is mediated through a functional coupling of the GC/cGMP pathway with different MAPK-dependent cascades in female shrimp. In VIH-2 signaling, the NO-independent GC/cGMP/PKG cascades were upstream of the MAPKs. Activations of the MAPK signal by VIH-2 include the phosphorylation of JNK and the mRNA/protein expression of P38MAPK. Additionally, the cAMP/PKA pathway is another positive intracellular signal for hepatopancreatic Vg mRNA expression but is independent of its VIH-2 regulation. Our findings establish a model for the signal transduction mechanism of Vg regulation by VIH and shed light on the biological functions and signaling of the CHH family in crustaceans.

Technological progress and challenges towards cGMP manufacturing of human pluripotent stem cells based therapeutic products for allogeneic and autologous cell therapies.

Science.gov (United States)

Abbasalizadeh, Saeed; Baharvand, Hossein

2013-12-01

Recent technological advances in the generation, characterization, and bioprocessing of human pluripotent stem cells (hPSCs) have created new hope for their use as a source for production of cell-based therapeutic products. To date, a few clinical trials that have used therapeutic cells derived from hESCs have been approved by the Food and Drug Administration (FDA), but numerous new hPSC-based cell therapy products are under various stages of development in cell therapy-specialized companies and their future market is estimated to be very promising. However, the multitude of critical challenges regarding different aspects of hPSC-based therapeutic product manufacturing and their therapies have made progress for the introduction of new products and clinical applications very slow. These challenges include scientific, technological, clinical, policy, and financial aspects. The technological aspects of manufacturing hPSC-based therapeutic products for allogeneic and autologous cell therapies according to good manufacturing practice (cGMP) quality requirements is one of the most important challenging and emerging topics in the development of new hPSCs for clinical use. In this review, we describe main critical challenges and highlight a series of technological advances in all aspects of hPSC-based therapeutic product manufacturing including clinical grade cell line development, large-scale banking, upstream processing, downstream processing, and quality assessment of final cell therapeutic products that have brought hPSCs closer to clinical application and commercial cGMP manufacturing. © 2013.

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 ...

Modulation of type II TGF-β receptor degradation by integrin-linked kinase.

Science.gov (United States)

Vi, Linda; Boo, Stellar; Sayedyahossein, Samar; Singh, Randeep K; McLean, Sarah; Di Guglielmo, Gianni M; Dagnino, Lina

2015-03-01

Cutaneous responses to injury, infection, and tumor formation involve the activation of resident dermal fibroblasts and subsequent transition to myofibroblasts. The key for induction of myofibroblast differentiation is the activation of transforming growth factor-β (TGF-β) receptors and stimulation of integrins and their associated proteins, including integrin-linked kinase (ILK). Cross-talk processes between TGF-β and ILK are crucial for myofibroblast formation, as ILK-deficient dermal fibroblasts exhibit impaired responses to TGF-β receptor stimulation. We now show that ILK associates with type II TGF-β receptors (TβRII) in ligand- and receptor kinase activity-independent manners. In cells with targeted Ilk gene inactivation, cellular levels of TβRII are decreased, through mechanisms that involve enhanced ubiquitination and proteasomal degradation. Partitioning of TGF-β receptors into membrane has been linked to proteasome-dependent receptor degradation. We found that interfering with membrane raft formation in ILK-deficient cells restored TβRII levels and signaling. These observations support a model whereby ILK functions in fibroblasts to direct TβRII away from degradative pathways during their differentiation into myofibroblasts.

N-linked oligosaccharides are responsible for rat striatal dopamine D2 receptor heterogeneity

Energy Technology Data Exchange (ETDEWEB)

Clagett-Dame, M.; McKelvy, J.F. (Abbott Laboratories, Abbott Park, IL (USA))

1989-10-01

The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-(p-azido-m-(125I)iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass (Mr). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 Mr; after digestion with peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase, all fractions yielded a single peptide approximately 40,000 Mr. Treatment of photoaffinity labeled membranes with alpha-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000-54,000 Mr. Treatment of the receptor with neuraminidase followed by endo-alpha-N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of approximately 31,000 Mr is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide.

N-linked oligosaccharides are responsible for rat striatal dopamine D2 receptor heterogeneity

International Nuclear Information System (INIS)

Clagett-Dame, M.; McKelvy, J.F.

1989-01-01

The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-(p-azido-m-[125I]iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass (Mr). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 Mr; after digestion with peptide-N4-[N-acetyl-beta-glucosaminyl] asparagine amidase, all fractions yielded a single peptide approximately 40,000 Mr. Treatment of photoaffinity labeled membranes with alpha-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000-54,000 Mr. Treatment of the receptor with neuraminidase followed by endo-alpha-N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of approximately 31,000 Mr is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide

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

Environmental Monitoring Trendy Study for cGMP Tc-99m Production at Nuclear Malaysia for the Year 2014

International Nuclear Information System (INIS)

Bohari Yaacob; Manisah Saedon; Fazliana Mohd Saaya

2015-01-01

Nuclear Malaysia is engaged to produce quality promising Tc-99m generator for the medical usage which is carried out at the production area of block 21, compromise of grade A, C and D clean room. This study applies to the trending of the environmental monitoring of the clean room for the year 2014 in order to ensure that the clean room complies with the cGMP requirement in term of environmental specification which is room temperature, relative humidity, room pressure and particle count. The room temperature specification is 22±2 degree Celsius and result showed that both preparation and pharmaceutical room are within the limit. But it was found that the result from gowning and degowning room temperature was more than 24 degree Celsius. Whereby, result for average percentage relative humidity of the gowning, preparation, pharmaceutical and degowning rooms were found 51 %.±7, 60 %±4, 61 %±5, 61 %±5 and 58 %±4 respectively are out off the range (< 60 %). The target level of pressure differential for all rooms is 1.5 mmH 2 O and only pharmaceutical room was found not within the target with the reading of 3.3 mmH 2 O±0.5. Result for 16 location showed that particle counts which particles size 5.0 μm were within the limit namely ≤ 2000/ m 3 except location number 21 and 18 in the pharmaceutical room and location number 23 in the degowning rooms were exceeding more than 2000/ m 3 for the month of July 2014. However results for all location particles size of 0.5 μm, were within specification (≤350000/ m 3 ). All location of all rooms with class C grade is within limit that is < 50 CFU of microbiology growth except location number 8, 9 and 10 of preparation room whereby this location is a class A grade. Generally, the environmental monitoring result for the clean room are satisfactory for the year 2014 since all the final product of all batches has passed the sterility and endotoxin test. In conclusion, Malaysian Nuclear Agency had fulfills the cGMP requirement

The arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes

KAUST Repository

Meier, Stuart; Ruzvidzo, Oziniel; Morse, Monique; Donaldson, Lara; Kwezi, Lusisizwe; Gehring, Christoph A

2010-01-01

Background: Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3?,5?-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. Principal Findings: We have identified an Arabidopsis receptor type wall associated kinase-like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10431-700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently coexpressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors. Conclusions: We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP. © 2010 Meier et al.

The arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes

KAUST Repository

Meier, Stuart

2010-01-26

Background: Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3?,5?-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. Principal Findings: We have identified an Arabidopsis receptor type wall associated kinase-like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10431-700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently coexpressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors. Conclusions: We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP. © 2010 Meier et al.

Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress.

Directory of Open Access Journals (Sweden)

Christine R Collins

2013-05-01

Full Text Available The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV. Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.

Sviluppo e valutazione di test diagnostici per la sierodiagnosi di brucellosi suina

Directory of Open Access Journals (Sweden)

Tiziana Di Febo

2012-06-01

Full Text Available Sono stati sviluppati una ELISA competitiva (c-ELISA, una ELISA indiretta (i-ELISA e un test immunologico DELFIA (Dissociation-Enhanced Lanthanide Fluorescence Immunoassay per la ricerca di anticorpi verso Brucella suis in sieri di maiale e cinghiale. I tre test prevedono l’utilizzo di un anticorpo monoclonale (MAb 4B5A verso l’LPS di Brucella (c-ELISA e DELFIA e di un anticorpo monoclonale (MAb 10C2G5 verso le IgG suine (i-ELISA. La specificità (Sp e la sensibilità (Se dei tre test sono le seguenti: per la c-ELISA Se e Sp = 100% con un valore di cut-off pari al 61.0% (B/B0%; per la i-ELISA Sp = 99.1% e Se = 100% con un valore di cut-off di 21.7% (PP%; per il DELFIA Sp = 91.0% e Se = 75% ponendo il valore di cut-off al 37.0% (B/B0%. Inoltre sono state valutate le performance, nei confronti di sieri suini, di un test FPA (Fluorescence Polarization Assay commerciale sviluppato per la ricerca di anticorpi anti-Brucella in sieri bovini; la specificità e la sensibilità ottenute sono entrambe del 100% al valore di cut-off di 99.5 (mP. Questi risultati suggeriscono che la combinazione di c-ELISA, i-ELISA e FPA può essere utilizzata per migliorare la diagnosi di brucellosi suina.

When the PilZ don't work

DEFF Research Database (Denmark)

Ryan, Robert P; Tolker-Nielsen, Tim; Dow, J Maxwell

2012-01-01

The second messenger cyclic di-GMP has emerged as a central regulator of many important bacterial processes including biofilm formation and virulence. Although the pathways of cyclic di-GMP synthesis and degradation have been established, the mechanisms by which this second messenger exerts its...... action on diverse cellular functions remain relatively poorly understood. Recent studies report considerable advances in identifying different classes of cyclic di-GMP effectors; these include the PilZ protein domain, transcription factors, proteins involved in RNA processing and riboswitches. Here, we...... review this range of cyclic di-GMP effectors and the biological processes that they govern using examples from several different bacteria....

Spendere meno, spendere meglio: una proposta panottica di J.-C. Guédon

Directory of Open Access Journals (Sweden)

Maria Chiara Pievatolo

2013-06-01

Full Text Available J.-C. Guédon ha commentato la nostra campagna di crowdsourcing in merito alle spese delle biblioteche sulla mailing list Nexa. Offriamo, qui di seguito, la versione italiana delle sue osservazioni – che presuppongono un mondo accademico molto diverso da quello impostoci...

Dicty_cDB: SSC474 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available SS (Link to library) SSC474 (Link to dictyBase) - - - Contig-U07719-1 SSC474P (Link... to Original site) SSC474F 368 SSC474Z 238 SSC474P 606 - - Show SSC474 Library SS (Link to library) Clone ID SSC474 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U07719-1 Original site URL http://dict...logy vs DNA Score E Sequences producing significant alignments: (bits) Value N ( AU071762 ) Dictyostelium di...scoideum slug cDNA, clone SSC474. 448 e-121 1 ( AU060185 ) Dictyostelium discoideum slug cDNA, clone SLA535.

TREX1 deficiency triggers cell-autonomous immunity in a cGAS-dependent manner.

Science.gov (United States)

Ablasser, Andrea; Hemmerling, Inga; Schmid-Burgk, Jonathan L; Behrendt, Rayk; Roers, Axel; Hornung, Veit

2014-06-15

Cytosolic detection of DNA is crucial for the initiation of antiviral immunity but can also cause autoimmunity in the context of endogenous nucleic acids being sensed. Mutations in the human 3' repair exonuclease 1 (TREX1) have been linked to the type I IFN-associated autoimmune disease Aicardi-Goutières syndrome. The exact mechanisms driving unabated type I IFN responses in the absence of TREX1 are only partly understood, but it appears likely that accumulation of endogenous DNA species triggers a cell-autonomous immune response by activating a cytosolic DNA receptor. In this article, we demonstrate that knocking out the DNA sensor cyclic GMP-AMP synthase completely abrogates spontaneous induction of IFN-stimulated genes in TREX1-deficient cells. These findings indicate a key role of cyclic GMP-AMP synthase for the initiation of self-DNA-induced autoimmune disorders, thus providing important implications for novel therapeutic approaches. Copyright © 2014 by The American Association of Immunologists, Inc.

The regulation of aortic endothelial cells by purines and pyrimidines involves co-existing P2y-purinoceptors and nucleotide receptors linked to phospholipase C.

Science.gov (United States)

Wilkinson, G F; Purkiss, J R; Boarder, M R

1993-03-01

1. We have examined the phospholipase C responses in bovine aortic endothelial cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP). 2. The cells responded to purines in a manner consistent with the presence of P2y purinoceptors; both 2-methylthioadenosine 5'-triphosphate (2MeSATP) and adenosine 5'-0-(2-thiodiphosphate) (ADP beta S) were potent agonists (EC50 0.41 microM and 0.85 microM respectively) while beta, gamma-methylene ATP at 300 microM was not. 3. The cells also responded to UTP. The maximal response to UTP was less than that for either 2MeSATP and ADP beta S while adenosine 5'-0-(3-thiotriphosphate) (ATP gamma S) gave the largest maximal response. 4. The concentration-effect curve to UTP was additive in the presence of either 2MeSATP or ADP beta S. However, the concentration-effect curves to ATP gamma S reached the same maximum in the presence or absence of UTP. 5. Suramin, at concentrations between 10 microM and 100 microM was a competitive antagonist for the response to ADP beta S and 2MeSATP but not the response to UTP. 6. The results show that there are two separate, co-existing, receptor populations: P2y-purinoceptors (responding to purines) and nucleotide receptors (responding to both purines and pyrimidines). We conclude that purines such as ATP/ADP may regulate aortic endothelial cells by interacting with two phospholipase C-linked receptors.

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.

Analysis of the selective advantage conferred by a C-E1 fusion protein synthesized by rubella virus DI RNAs

International Nuclear Information System (INIS)

Claus, Claudia; Tzeng, W.-P.; Liebert, Uwe Gerd; Frey, Teryl K.

2007-01-01

During serial passaging of rubella virus (RUB) in cell culture, the dominant species of defective-interfering RNA (DI) generated contains an in-frame deletion between the capsid protein (C) gene and E1 glycoprotein gene resulting in production of a C-E1 fusion protein that is necessary for the maintenance of the DI [Tzeng, W.P., Frey, T.K. (2006). C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage. Virology 356 198-207.]. A BHK cell line stably expressing the RUB structural proteins was established which was used to package DIs into virus particles following transfection with in vitro transcripts from DI infectious cDNA constructs. Packaging of a DI encoding an in-frame C-GFP-E1 reporter fusion protein corresponding to the C-E1 fusion protein expressed in a native DI was only marginally more efficient than packaging of a DI encoding GFP, indicating that the C-E1 fusion protein did not function by enhancing packaging. However, infection with the DI encoding the C-GFP-E1 fusion protein (in the absence of wt RUB helper virus) resulted in formation of clusters of GFP-positive cells and the percentage of GFP-positive cells in the culture following infection remained relatively constant. In contrast, a DI encoding GFP did not form GFP-positive clusters and the percentage of GFP-positive cells declined by roughly half from 2 to 4 days post-infection. Cluster formation and sustaining the percentage of infected (GFP-positive) cells required the C part of the fusion protein, including the downstream but not the upstream of two arginine clusters (both of which are associated with RNA binding and association with mitochondrial p32 protein) and the E1 part through the transmembrane sequence, but not the C-terminal cytoplasmic tail. Among a collection of mutant DI constructs, cluster formation and sustaining infected cell percentage correlated with maintenance during serial passage with wt RUB. We hypothesize that cluster formation and

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.

Analisi dell'efficacia di un programma di prevenzione secondaria del carcinoma orale

Directory of Open Access Journals (Sweden)

S. Iannazzo

2003-05-01

Full Text Available

In Italia i carcinomi del cavo orale (KCO sono diagnosticati
prevalentemente al II-IV stadio TNM con conseguenti basse sopravvivenza a 5 anni e qualità della vita. Una maggior frequenza di diagnosi allo stadio I porterebbe a migliorare ampiamente i valori medi dei suddetti parametri.
Scopo del presente lavoro è stato valutare l’efficacia di un programma di prevenzione secondaria del KCO in termini di aumento di diagnosi allo stadio I e di vite salvate.

Abbiamo stimato la prevalenza di casi istopatologicamente
accertati (livello C3 di leucoplachia, principale lesione precancerosa del KCO, nella ASL/RMA come prodotto tra la prevalenza stimata di leucoplachia diagnosticata clinicamente (livello C2 (2989,2Ĩ13077,7 e la proporzione, scelta arbitrariamente, di C3 tra i C2 (0,50.

Tenendo presente che nel campione studiato nella ASL/RMA la proporzione di KCO allo stadio I tra i C3 era 0,03, abbiamo stimato il numero di KCO.

Abbiamo stimato quanti KCO verrebbero diagnosticati allo stadio I se venisse attuato un programma di prevenzione secondaria, considerando diversi livelli di compliance, e sulla base di una sopravvivenza media a 5 anni, in Italia, del 53% per tutti gli stadi e del 79% per i KCO allo stadio I, abbiamo calcolato il numero di vite salvate. I valori sono stati espressi come IC95.

Le prevalenze di leucoplachia C3 e KCO sono risultate,
rispettivamente, 1494,6Ĩ6538,8 e 44,8Ĩ196,2. Con compliance del 10%, 40% e 75% verrebbero diagnosticati, rispettivamente, 4,5Ĩ19,6, 17,9Ĩ78,5 e 33,6Ĩ147,2 casi di KCO e le vite salvate sarebbero 1,1Ĩ5,1, 4,6Ĩ20,4 e 8,7Ĩ38,3 rispettivamente.
In termini di vite salvate i dati evidenziano la necessita di attuare programmi di screening del KCO, anche nell' eventualità di uno scarso coinvolgimento della popolazione a rischio.

Expansion of microsatellite in the thyroid hormone receptor-alpha1 gene linked to increased receptor expression and less aggressive thyroid cancer

DEFF Research Database (Denmark)

Onda, Masamitsu; Li, Daisy; Suzuki, Shinichi

2002-01-01

PURPOSE: The purpose of this study was to determine whether the length of the THRA1 microsatellite, which resides in a noncoding portion of the thyroid hormone receptor-alpha1 gene, affects receptor expression and is linked to clinicopathological parameters in thyroid cancer. EXPERIMENTAL DESIGN......: In 30 cases of surgically resected sporadic thyroid cancer, the length of the THRA1 microsatellite was determined by DNA sequence analysis, and expression of thyroid hormone receptor-alpha1 was assessed immunohistochemically in thin sections cut from tumor blocks. The length of THRA1 and expression...... of thyroid hormone receptor-alpha1 were also assessed in seven cancer cell lines. Regression analysis was used to gauge the correlation between the size of THRA1 and receptor expression. Multivariate analysis was used to test for links to the clinical parameters of gender, age, histology, stage, nodal...

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...

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.

Effect of cardiopulmonary bypass on beta adrenergic receptor-adenylate cyclase system on surfaces of peripheral lymphocytes.

Science.gov (United States)

Luo, A; Tian, Y; Jin, S

2000-01-01

The experimental results showed that the level of CAMP, the ratio of cAPM to cGMP, IL-2R expression and IL-2 production in vitro in lymphocytes immediate and 2 weeks after cardiopulmonary bypass (CPB) were significantly lower than those before anesthetics in the patients undergoing cardiac surgery with CPB. These findings suggested that CPB could cause serious damage to adrenergic beta receptor-adenylate cyclase system on circulating lymphocytes surfaces, which might be one of the mechanisms resulting in immunosuppression after open heart surgery with CPB.

4-Bromo-N-(di-n-propyl-carbamothioyl)-benzamide.

Science.gov (United States)

Binzet, Gün; Flörke, Ulrich; Külcü, Nevzat; Arslan, Hakan

2009-02-04

The synthesis of the title compound, C(14)H(19)BrN(2)OS, involves the reaction of 4-bromo-benzoyl chloride with potassium thio-cyanate in acetone followed by condensation of the resulting 4-bromo-benzoyl isothio-cyanate with di-n-propyl-amine. Typical thio-urea carbonyl and thio-carbonyl double bonds, as well as shortened C-N bonds, are observed in the title compound. The short C-N bond lengths in the centre of the mol-ecule reveal the effects of resonance in this part of the mol-ecule. The asymmetric unit of the title compound contains two crystallographically independent mol-ecules, A and B. There is very little difference between the bond lengths and angles of these mol-ecules. In mol-ecule B, one di-n-propyl group is twisted in a -anti-periplanar conformation with C-C-C-H = -179.1 (3)° and the other adopts a -synclinal conformation with C-C-C-H = -56.7 (4)°; in mol-ecule A the two di-n-propyl groups are twisted in + and -anti-periplanar conformations, with C-C-C-H = -179.9 (3) and 178.2 (3)°, respectively. In the crystal, the mol-ecules are linked into dimeric pairs via pairs of N-H⋯S hydrogen bonds.

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...

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)

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)

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.

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.

Family C 7TM receptor dimerization and activation

DEFF Research Database (Denmark)

Bonde, Marie Mi; Sheikh, Søren P; Hansen, Jakob Lerche

2006-01-01

The family C seven transmembrane (7TM) receptors constitutes a small and especially well characterized subfamily of the large 7TM receptor superfamily. Approximately 50% of current prescription drugs target 7TM receptors, this biologically important family represents the largest class of drug...... to be fully defined. This review presents the biochemical support for family C 7TM receptor dimerization and discusses its importance for receptor biosynthesis, surface expression, ligand binding and activation, since lessons learnt here may well be applicable to the whole superfamily of 7TM receptors.......-targets today. It is well established that family C 7TM receptors form homo- or hetero-dimers on the cell surface of living cells. The large extra-cellular domains (ECD) have been crystallized as a dimer in the presence and absence of agonist. Upon agonist binding, the dimeric ECD undergoes large conformational...

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...

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)

Gastrin receptor characterization: affinity cross-linking of the gastrin receptor on canine gastric parietal cells

International Nuclear Information System (INIS)

Matsumoto, M.; Park, J.; Yamada, T.

1987-01-01

The authors applied affinity cross-linking methods to label the gastrin receptor on isolated canine gastric parietal cells in order to elucidate the nature of its chemical structure. 125 I-labeled Leu 15 -gastrin and 125 I-labeled gastrin/sub 2-17/ bound to intact parietal cells and their membranes with equal affinity, and half-maximal inhibition of binding was obtained at an incubation concentration of 3.2 x 10 -10 M unlabeled gastrin. 125 I-gastrin/sub 2-17/ was cross-linked to plasma membranes or intact parietal cells by incubation in disuccinimidyl suberate. The membrane pellets were solubilized with or without dithiothreitol and applied to electrophoresis on 7.5% sodium dodecyl sulfate polyacrylamide gels. Autoradiograms revealed a band of labeling at M/sub r/ 76,000 and labeling of this band was inhibited in a dose-dependent fashion by addition of unlabeled gastrin to the incubation mixture. Dithiothreitol in concentrations as high as 100 mM did not later the electrophoretic mobility of the labeled band. After taking into account the molecular weight of 125 I-gastrin/sub 2-17/, the results suggest that the gastrin receptor on parietal cells is a single protein of M/sub r/ 74,000 without disulfide-linked subunits

Deteksi Antibodi terhadap Cysticercus Cellulosae pada Babi Lokal yang Dipotong di Tempat Pemotongan Babi Panjer, Denpasar

Directory of Open Access Journals (Sweden)

Ida Bagus Ngurah Swacita

2016-03-01

Full Text Available Sistiserkosis merupakan penyakit yang bersifat zoonosis yang disebabkan oleh larva cacing pita Taenia solium yang disebut Cysticercus cellulosae. Di Indonesia terdapat tiga provinsi yang berstatus endemik penyakit sistiserkosis, salah satunya adalah provinsi Bali. Penelitian ini bertujuan untuk mendeteksi antibodi terhadap C. cellulosae pada babi lokal yang dipotong di tempat pemotongan babi Panjer, Denpasar. Sampel penelitian adalah 270 sampel serum babi lokal yang dipotong di tempat pemotongan babi Panjer, Denpasar Selatan. Babi lokal ini berasal dari Nusa Penida, Karangasem dan Negara. Sampel serum diuji dengan metode ELISA (Enzyme Linked Immunosorbent Assay indirect. Hasil penelitian menunjukkan bahwa antibodi C. cellulosae terdeteksi pada 33 sampel. Disimpulkan bahwa terdeteksi antibodi C. cellulosae pada 33 sampel dari 270 sampel serum babi lokal yang dipotong di tempat pemotongan babi Panjer, Denpasar.

DMPD: Nuclear receptors in macrophages: a link between metabolism and inflammation. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 18022390 Nuclear receptors in macrophages: a link between metabolism and inflammati...on. Szanto A, Roszer T. FEBS Lett. 2008 Jan 9;582(1):106-16. Epub 2007 Nov 20. (.png) (.svg) (.html) (.csml) Show Nuclear... receptors in macrophages: a link between metabolism and inflammation. PubmedID 18022390 Title Nuclear

Detection of inhibitors of Candida albicans Cdr transporters using a diS-C3(3 fluorescence

Directory of Open Access Journals (Sweden)

Joanna eSzczepaniak

2015-03-01

Full Text Available Candida albicans is a major cause of opportunistic and life-threatening, systemic fungal infections. Hence new antifungal agents, as well as new methods to treat fungal infections, are still needed. The application of inhibitors of drug-efflux pumps may increase the susceptibility of C. albicans to drugs. We developed a new fluorescence method that allows the in vivo activity evaluation of compounds inhibiting of C. albicans transporters. We show that the potentiometric dye 3,3′-dipropylthiacarbocyanine iodide diS-C3(3 is pumped out by both Cdr1 and Cdr2 transporters. The fluorescence labeling with diS-C3(3 enables a real-time observation of the activity of C. albicans Cdr1 and Cdr2 transporters. We demonstrate that enniatin A and beauvericin show different specificities toward these transporters. Enniatin A inhibits diS-C3(3 efflux by Cdr1 while beauvericin inhibits both Cdr1p and Cdr2p.

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.

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

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

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...... channels on the cell surface stimulating synchronized release of SR-calcium and inducing the shift from waves to whole-cell oscillations. The effect of the channel is therefore to couple the processes of the SR with those of the membrane. We hypothesize that the shift in oscillatory mode and the associated...

Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently cross-link to galactosyl receptors on isolated rat hepatocytes

International Nuclear Information System (INIS)

Herzig, M.C.S.; Weigel, P.H.

1989-01-01

The authors have developed chemical affinity reagents for the hepatic galactosyl receptor. Asialoorosomucoid (ASOR) was derivatized with five homobifunctional N-hydroxysuccinimide (NHS) ester cross-linkers. NHS/ASOR derivatives were synthesized, purified, and applied within 10 min to isolated rat hepatocytes at 4 degree C. Specific binding of these 125 I-labeled derivatives was ∼90% in the presence of either EGTA or excess ASOR. Specific cross-linking assessed by the resistance of specifically bound NHS/ 125 I-ASOR to release by EGTA, was 50-75% of the specifically bound ligand. The extent of specific cross-linking correlated with the average number of NHS groups per ASOR and was controlled by varying the molar ratio of cross-linker to ASOR during the synthesis. After being cross-linked with any of the NHS/ 125 I-ASOR derivatives, cells were washed with EGTA, solubilized in Triton X-100, and analyzed by SDA-PAGE and autoradiography. They conclude that all three receptor subunits can cross-link to ligand. They propose a model in which the native receptor is a heterohexamer composed of four subunits of RHL 1 and two subunits of RHL 2 and/or RHL 3

Synthetic water soluble di-/tritopic molecular receptors exhibiting Ca2+/Mg2+ exchange.

Science.gov (United States)

Lavie-Cambot, Aurélie; Tron, Arnaud; Ducrot, Aurélien; Castet, Frédéric; Kauffmann, Brice; Beauté, Louis; Allouchi, Hassan; Pozzo, Jean-Luc; Bonnet, Célia S; McClenaghan, Nathan D

2017-05-23

Structural integration of two synthetic water soluble receptors for Ca 2+ and Mg 2+ , namely 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and o-aminophenol-N,N,O-triacetic acid (APTRA), respectively, gave novel di- and tritopic ionophores (1 and 2). As Mg 2+ and Ca 2+ cannot be simultaneously complexed by the receptors, allosteric control of complexation results. Potentiometric measurements established stepwise protonation constants and showed high affinity for Ca 2+ (log K = 6.08 and 8.70 for 1 and 2, respectively) and an excellent selectivity over Mg 2+ (log K = 3.70 and 5.60 for 1 and 2, respectively), which is compatible with magnesium-calcium ion exchange. While ion-exchange of a single Mg 2+ for a single Ca 2+ is possible in both 1 and 2, the simultaneous binding of two Mg 2+ by 2 appears prohibitive for replacement of these two ions by a single Ca 2+ . Ion-binding and exchange was further rationalized by DFT calculations.

The A-chain of insulin contacts the insert domain of the insulin receptor. Photo-cross-linking and mutagenesis of a diabetes-related crevice.

Science.gov (United States)

Huang, Kun; Chan, Shu Jin; Hua, Qing-xin; Chu, Ying-Chi; Wang, Run-ying; Klaproth, Birgit; Jia, Wenhua; Whittaker, Jonathan; De Meyts, Pierre; Nakagawa, Satoe H; Steiner, Donald F; Katsoyannis, Panayotis G; Weiss, Michael A

2007-11-30

The contribution of the insulin A-chain to receptor binding is investigated by photo-cross-linking and nonstandard mutagenesis. Studies focus on the role of Val(A3), which projects within a crevice between the A- and B-chains. Engineered receptor alpha-subunits containing specific protease sites ("midi-receptors") are employed to map the site of photo-cross-linking by an analog containing a photoactivable A3 side chain (para-azido-Phe (Pap)). The probe cross-links to a C-terminal peptide (residues 703-719 of the receptor A isoform, KTFEDYLHNVVFVPRPS) containing side chains critical for hormone binding (underlined); the corresponding segment of the holoreceptor was shown previously to cross-link to a Pap(B25)-insulin analog. Because Pap is larger than Val and so may protrude beyond the A3-associated crevice, we investigated analogs containing A3 substitutions comparable in size to Val as follows: Thr, allo-Thr, and alpha-aminobutyric acid (Aba). Substitutions were introduced within an engineered monomer. Whereas previous studies of smaller substitutions (Gly(A3) and Ser(A3)) encountered nonlocal conformational perturbations, NMR structures of the present analogs are similar to wild-type insulin; the variant side chains are accommodated within a native-like crevice with minimal distortion. Receptor binding activities of Aba(A3) and allo-Thr(A3) analogs are reduced at least 10-fold; the activity of Thr(A3)-DKP-insulin is reduced 5-fold. The hormone-receptor interface is presumably destabilized either by a packing defect (Aba(A3)) or by altered polarity (allo-Thr(A3) and Thr(A3)). Our results provide evidence that Val(A3), a site of mutation causing diabetes mellitus, contacts the insert domain-derived tail of the alpha-subunit in a hormone-receptor complex.

KANDUNGAN KLOROFIL, KAROTENOID, DAN VITAMIN C BEBERAPA JENIS SAYURAN DAUN PADA PERTANIAN PERIURBAN DI KOTA SURABAYA

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Dwi Iriyani

2015-04-01

Full Text Available Periurban agriculture actually means agriculture that is found surrounding urban boundary. Due to heavy load for various non-agricultural activity and transportation, it necessary to pay attention on the agro-ecological conditions where each crop could grows well. The quality of environment for growing plants in periurban influences on composition of biochemistry in plants’ tissue. The purpose of this study is to determine the chlorophyll, carotenoid, and ascorbic acid contents in three species of vegetables, those are bayam (Amaranthus tricolor, L., kangkung (Ipomoea reptans and sawi (Brassica juncea L. which were cultivated in three periurban agriculture areas of Surabaya. Total contents of chlorophyll and carotenoid was measured by spectrophotometer. Vitamin C contents was analyzed by DCPIP dye method. The results showed that bayam which was cultivated in Bangkingan-Lakarsantri has the highest content of chlorophyll (3.046 mg/g and carotenoid (375.33 μmol/L. The highest content (4.55 μg/g of vitamin C was found on sawi which was cultivated in Wonorejo. There was no significant difference on chlorophyll content, carotenoid content and vitamin C content between organic vegetables labeled and those are cultivated on Bangkingan-Lakarsantri periurban area. Kawasan pertanian periurban merupakan daerah pertanian yang dijumpai di sekitar pinggiran perkotaan. Berkaitan dengan tekanan lingkungan yang berat di kawasan periurban, akibat berbagai kegiatan non pertanian dan transportasi, perlu adanya perhatian terhadap kondisi agro klimat yang mempengaruhi pertumbuhan tanaman. Kualitas lingkungan tempat tumbuh tanaman pada kawasan pertanian periurban berpengaruh terhadap komposisi kandungan biokimia jaringan tanaman. Tujuan penelitian ini adalah untuk menetapkan kadar klorofil, karotenoid, dan vitamin C pada sayuran bayam (Amaranthus tricolor, L., kangkung (Ipomoea reptans dan sawi (Brassica juncea L. yang dibudidayakan di tiga kawasan periurban Kota

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.

Translational PKPD modeling in schizophrenia: linking receptor occupancy of antipsychotics to efficacy and safety

NARCIS (Netherlands)

Pilla Reddy, Venkatesh; Kozielska, Magdalena; Johnson, Martin; Vermeulen, An; Liu, Jing; de Greef, Rik; Groothuis, Genoveva; Danhof, Meindert; Proost, Johannes

2012-01-01

Objectives: To link the brain dopamine D2 receptor occupancy (D2RO) of antipsychotic drugs with clinical endpoints of efficacy and safety to assess the therapeutic window of D2RO. Methods: Pharmacokinetic-Pharmacodynamic (PK-PD) models were developed to predict the D2 receptor occupancy of

[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.

Cloning of the human androgen receptor cDNA

International Nuclear Information System (INIS)

Govindan, M.V.; Burelle, M.; Cantin, C.; Kabrie, C.; Labrie, F.; Lachance, Y.; Leblanc, G.; Lefebvre, C.; Patel, P.; Simard, J.

1988-01-01

The authors discuss how in order to define the functional domains of the human androgen receptor, complementary DNA (cDNA) clones encoding the human androgen receptor (hAR) have been isolated from a human testis λgtll cDNA library using synthetic oligonnucleotide probes, homologous to segments of the human glucocorticoid, estradiol and progesterone receptors. The cDNA clones corresponding to the human glucocorticoid, estradiol and progesterone receptors were eliminated after cross-hybridization with their respective cDNA probes and/or after restriction mapping of the cDNA clones. The remaining cDNA clones were classified into different groups after analysis by restriction digestion and cross-hybridization. Two of the largest cDNA clones from each group were inserted into an expression vector in both orientations. The linearized plasmids were used as templates in in vitro transcription with T7 RNA polymerase. Subsequent in vitro translation of the purified transcripts in rabbit reticulocyte lysate followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) permitted the characterization of the encoded polyeptides. The expressed proteins larger than 30,000 Da were analyzed for their ability to bind tritium-labelled dihydrotestosterone ([ 3 H] DHT) with high affinity and specificity

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.

Adenosine Receptors in Developing and Adult Mouse Neuromuscular Junctions and Functional Links With Other Metabotropic Receptor Pathways.

Science.gov (United States)

Tomàs, Josep; Garcia, Neus; Lanuza, Maria A; Santafé, Manel M; Tomàs, Marta; Nadal, Laura; Hurtado, Erica; Simó-Ollé, Anna; Cilleros-Mañé, Víctor; Just-Borràs, Laia

2018-01-01

In the last few years, we have studied the presence and involvement in synaptogenesis and mature transmitter release of the adenosine autoreceptors (AR) in the mammalian neuromuscular junction (NMJ). Here, we review and bring together the previously published data to emphasize the relevance of these receptors for developmental axonal competition, synaptic loss and mature NMJ functional modulation. However, in addition to AR, activity-dependent mediators originating from any of the three cells that make the synapse (nerve, muscle, and glial cells) cross the extracellular cleft to generate signals in target metabotropic receptors. Thus, the integrated interpretation of the complementary function of all these receptors is needed. We previously studied, in the NMJ, the links of AR with mAChR and the neurotrophin receptor TrkB in the control of synapse elimination and transmitter release. We conclude that AR cooperate with these receptors through synergistic and antagonistic effects in the developmental synapse elimination process. In the adult NMJ, this cooperation is manifested so as that the functional integrity of a given receptor group depends on the other receptors operating normally (i.e., the functional integrity of mAChR depends on AR operating normally). These observations underlie the relevance of AR in the NMJ function.

Adenosine Receptors in Developing and Adult Mouse Neuromuscular Junctions and Functional Links With Other Metabotropic Receptor Pathways

Directory of Open Access Journals (Sweden)

Josep Tomàs

2018-04-01

Full Text Available In the last few years, we have studied the presence and involvement in synaptogenesis and mature transmitter release of the adenosine autoreceptors (AR in the mammalian neuromuscular junction (NMJ. Here, we review and bring together the previously published data to emphasize the relevance of these receptors for developmental axonal competition, synaptic loss and mature NMJ functional modulation. However, in addition to AR, activity-dependent mediators originating from any of the three cells that make the synapse (nerve, muscle, and glial cells cross the extracellular cleft to generate signals in target metabotropic receptors. Thus, the integrated interpretation of the complementary function of all these receptors is needed. We previously studied, in the NMJ, the links of AR with mAChR and the neurotrophin receptor TrkB in the control of synapse elimination and transmitter release. We conclude that AR cooperate with these receptors through synergistic and antagonistic effects in the developmental synapse elimination process. In the adult NMJ, this cooperation is manifested so as that the functional integrity of a given receptor group depends on the other receptors operating normally (i.e., the functional integrity of mAChR depends on AR operating normally. These observations underlie the relevance of AR in the NMJ function.

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.

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.

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.

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.)

Characterization of cholecystokinin receptors on guinea pig gastric chief cell membranes

International Nuclear Information System (INIS)

Matozaki, T.; Sakamoto, C.; Nagao, M.; Nishisaki, H.; Konda, Y.; Nakano, O.; Matsuda, K.; Wada, K.; Suzuki, T.; Kasuga, M.

1991-01-01

The binding of cholecystokinin (CCK) to its receptors on guinea pig gastric chief cell membranes were characterized by the use of 125 I-CCK-octapeptide (CCK8). At 30 degrees C optimal binding was obtained at acidic pH in the presence of Mg2+, while Na+ reduced the binding. In contrast to reports on pancreatic and brain CCK receptors, scatchard analysis of CCK binding to chief cell membranes revealed two classes of binding sites. Whereas, in the presence of a non-hydrolyzable GTP analog, GTP gamma S, only a low affinity site of CCK binding was observed. Chief cell receptors recognized CCK analogs, with an order of potency of: CCK8 greater than gastrin-I greater than CCK4. Although all CCK receptor antagonists tested (dibutyryl cyclic GMP, L-364718 and CR1409) inhibited labeled CCK binding to chief cell membranes, the relative potencies of these antagonists in terms of inhibiting labeled CCK binding were different from those observed in either pancreatic membranes or brain membranes. The results indicate, therefore, that on gastric chief cell membranes there exist specific CCK receptors, which are coupled to G protein. Furthermore, chief cell CCK receptors may be distinct from pancreatic or brain type CCK receptors

Characterization of the somatogenic receptor in rat liver. Hydrodynamic properties and affinity cross-linking

International Nuclear Information System (INIS)

Husman, B.; Haldosen, L.A.; Andersson, G.; Gustafsson, J.A.

1988-01-01

Rat liver somatogenic receptors have been characterized by gel permeation chromatography, sucrose density gradients in H 2 O and D 2 O, and affinity cross-linking using 125 I-bovine growth hormone (bGH) as a specific somatogenic receptor ligand. Cross-linking of 125 I-bovine growth hormone to a Triton X-100-treated low density fraction isolated from livers of late pregnant rats followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions showed three major binders with Mr 95,000, 86,000, and 43,000 and a minor binder of Mr 55,000, after correction for bound ligand assuming a 1:1 binding ratio of ligand-receptor. The Mr 86,000, 55,000, and 43,000 species were recovered in the detergent-soluble supernatant after high-speed centrifugation, whereas the Mr 95,000 species remained Triton X-100 insoluble. Detergent-soluble 125 I-bGH-receptor complexes were further analyzed by sedimentation into sucrose density gradients. The sedimentation coefficient was S20,w = 5.2 S and the partial specific volume v = 0.72 ml/g. Gel permeation chromatography on a Sepharose S-400 column indicated a Stokes radius of 61 A for the 125 I-bGH-receptor-Triton X-100 complex. Based on these figures, the molecular weight of the complex was calculated as 131,100. The molecular weight of the ligand-free receptor-Triton X-100 complex was calculated as Mr 109,100. Affinity cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 61 A peak from Sephacryl S-400 chromatography (cf. above) showed two binding entities, one major and one minor with Mr values 86,000 and 43,000, respectively, in the absence of reductant. When electrophoresis was run in the presence of reductant the Mr 43,000 species was the major binding entity

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.

Dicty_cDB: VSG286 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available VS (Link to library) VSG286 (Link to dictyBase) - - - Contig-U16209-1 VSG286E (Link... Clone ID VSG286 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U16209-1 Original site URL http://dict...846 |BQ096846.1 IfHdk00487 Ictalurus furcatus head kidney cDNA library Ictalurus furcatus cDNA 5' similar to... Ribosomal protein Sa (laminin receptor 1), mRNA sequence. 46 2e-11 4 CK406973 |CK406973.1 AUF_IfLvr_212_m02 Ict...alurus furcatus liver cDNA library Ictalurus furcatus cDNA 5' similar to lami

Phosphodiesterase 2A Inhibitor TAK-915 Ameliorates Cognitive Impairments and Social Withdrawal in N-Methyl-d-Aspartate Receptor Antagonist-Induced Rat Models of Schizophrenia.

Science.gov (United States)

Nakashima, Masato; Imada, Haruka; Shiraishi, Eri; Ito, Yuki; Suzuki, Noriko; Miyamoto, Maki; Taniguchi, Takahiko; Iwashita, Hiroki

2018-04-01

The pathophysiology of schizophrenia has been associated with glutamatergic dysfunction. Modulation of the glutamatergic signaling pathway, including N -methyl-d-aspartate (NMDA) receptors, can provide a new therapeutic target for schizophrenia. Phosphodiesterase 2A (PDE2A) is highly expressed in the forebrain, and is a dual substrate enzyme that hydrolyzes both cAMP and cGMP, which play pivotal roles as intracellular second messengers downstream of NMDA receptors. Here we characterize the in vivo pharmacological profile of a selective and brain-penetrant PDE2A inhibitor, ( N -{(1 S )-1-[3-fluoro-4-(trifluoromethoxy)phenyl]-2-methoxyethyl}-7-methoxy-2-oxo-2,3-dihydropyrido[2,3- b ]pyrazine-4(1 H )-carboxamide) (TAK-915) as a novel treatment of schizophrenia. Oral administration of TAK-915 at 3 and 10 mg/kg significantly increased cGMP levels in the frontal cortex, hippocampus, and striatum of rats. TAK-915 at 10 mg/kg significantly upregulated the phosphorylation of α -amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptor subunit GluR1 in the rat hippocampus. TAK-915 at 3 and 10 mg/kg significantly attenuated episodic memory deficits induced by the NMDA receptor antagonist (+)-MK-801 hydrogen maleate (MK-801) in the rat passive avoidance test. TAK-915 at 10 mg/kg significantly attenuated working memory deficits induced by MK-801 in the rat radial arm maze test. Additionally, TAK-915 at 10 mg/kg prevented subchronic phencyclidine-induced social withdrawal in social interaction in rats. In contrast, TAK-915 did not produce antipsychotic-like activity; TAK-915 had little effect on MK-801- or methamphetamine-induced hyperlocomotion in rats. These results suggest that TAK-915 has a potential to ameliorate cognitive impairments and social withdrawal in schizophrenia. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Human plasmacytoid dentritic cells elicit a Type I Interferon response by sensing DNA via the cGAS-STING signaling pathway.

Science.gov (United States)

Bode, Christian; Fox, Mario; Tewary, Poonam; Steinhagen, Almut; Ellerkmann, Richard K; Klinman, Dennis; Baumgarten, Georg; Hornung, Veit; Steinhagen, Folkert

2016-07-01

Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN) and are important for host defense by sensing microbial DNA via TLR9. pDCs also play a critical role in the pathogenesis of IFN-driven autoimmune diseases. Yet, this autoimmune reaction is caused by the recognition of self-DNA and has been linked to TLR9-independent pathways. Increasing evidence suggests that the cytosolic DNA receptor cyclic GMP-AMP (cGAMP) synthase (cGAS) is a critical component in the detection of pathogens and contributes to autoimmune diseases. It has been shown that binding of DNA to cGAS results in the synthesis of cGAMP and the subsequent activation of the stimulator of interferon genes (STING) adaptor to induce IFNs. Our results show that the cGAS-STING pathway is expressed and activated in human pDCs by cytosolic DNA leading to a robust type I IFN response. Direct activation of STING by cyclic dinucleotides including cGAMP also activated pDCs and knockdown of STING abolished this IFN response. These results suggest that pDCs sense cytosolic DNA and cyclic dinucleotides via the cGAS-STING pathway and that targeting this pathway could be of therapeutic interest. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genotype-Dependent Difference in 5-HT2C Receptor-Induced Hypolocomotion: Comparison with 5-HT2A Receptor Functional Activity

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Darya V. Bazovkina

2015-01-01

Full Text Available In the present study behavioral effects of the 5-HT2C serotonin receptor were investigated in different mouse strains. The 5-HT2C receptor agonist MK-212 applied intraperitoneally induced significant dose-dependent reduction of distance traveled in the open field test in CBA/Lac mice. This effect was receptor-specific because it was inhibited by the 5-HT2C receptor antagonist RS102221. To study the role of genotype in 5-HT2C receptor-induced hypolocomotion, locomotor activity of seven inbred mouse strains was measured after MK-212 acute treatment. We found that the 5-HT2C receptor stimulation by MK-212 decreased distance traveled in the open field test in CBA/Lac, C57Bl/6, C3H/He, and ICR mice, whereas it failed to affect locomotor activity in DBA/2J, Asn, and Balb/c mice. We also compared the interstrain differences in functional response to 5-HT2C and 5-HT2A receptors activation measured by the quantification of receptor-mediated head-twitches. These experiments revealed significant positive correlation between 5-HT2C and 5-HT2A receptors functional responses for all investigated mouse strains. Moreover, we found that 5-HT2A receptor activation with DOI did not change locomotor activity in CBA/Lac mice. Taken together, our data indicate the implication of 5-HT2C receptors in regulation of locomotor activity and suggest the shared mechanism for functional responses mediated by 5-HT2C and 5-HT2A receptors.

The two sides of complement C3d: evolution of electrostatics in a link between innate and adaptive immunity.

Science.gov (United States)

Kieslich, Chris A; Morikis, Dimitrios

2012-01-01

The interaction between complement fragment C3d and complement receptor 2 (CR2) is a key aspect of complement immune system activation, and is a component in a link between innate and adaptive immunities. The complement immune system is an ancient mechanism for defense, and can be found in species that have been on Earth for the last 600 million years. However, the link between the complement system and adaptive immunity, which is formed through the association of the B-cell co-receptor complex, including the C3d-CR2 interaction, is a much more recent adaptation. Human C3d and CR2 have net charges of -1 and +7 respectively, and are believed to have evolved favoring the role of electrostatics in their functions. To investigate the role of electrostatics in the function and evolution of human C3d and CR2, we have applied electrostatic similarity methods to identify regions of evolutionarily conserved electrostatic potential based on 24 homologues of complement C3d and 4 homologues of CR2. We also examine the effects of structural perturbation, as introduced through molecular dynamics and mutations, on spatial distributions of electrostatic potential to identify perturbation resistant regions, generated by so-called electrostatic "hot-spots". Distributions of electrostatic similarity based on families of perturbed structures illustrate the presence of electrostatic "hot-spots" at the two functional sites of C3d, while the surface of CR2 lacks electrostatic "hot-spots" despite its excessively positive nature. We propose that the electrostatic "hot-spots" of C3d have evolved to optimize its dual-functionality (covalently attaching to pathogen surfaces and interaction with CR2), which are both necessary for the formation B-cell co-receptor complexes. Comparison of the perturbation resistance of the electrostatic character of the homologues of C3d suggests that there was an emergence of a new role of electrostatics, and a transition in the function of C3d, after the

The two sides of complement C3d: evolution of electrostatics in a link between innate and adaptive immunity.

Directory of Open Access Journals (Sweden)

Chris A Kieslich

Full Text Available The interaction between complement fragment C3d and complement receptor 2 (CR2 is a key aspect of complement immune system activation, and is a component in a link between innate and adaptive immunities. The complement immune system is an ancient mechanism for defense, and can be found in species that have been on Earth for the last 600 million years. However, the link between the complement system and adaptive immunity, which is formed through the association of the B-cell co-receptor complex, including the C3d-CR2 interaction, is a much more recent adaptation. Human C3d and CR2 have net charges of -1 and +7 respectively, and are believed to have evolved favoring the role of electrostatics in their functions. To investigate the role of electrostatics in the function and evolution of human C3d and CR2, we have applied electrostatic similarity methods to identify regions of evolutionarily conserved electrostatic potential based on 24 homologues of complement C3d and 4 homologues of CR2. We also examine the effects of structural perturbation, as introduced through molecular dynamics and mutations, on spatial distributions of electrostatic potential to identify perturbation resistant regions, generated by so-called electrostatic "hot-spots". Distributions of electrostatic similarity based on families of perturbed structures illustrate the presence of electrostatic "hot-spots" at the two functional sites of C3d, while the surface of CR2 lacks electrostatic "hot-spots" despite its excessively positive nature. We propose that the electrostatic "hot-spots" of C3d have evolved to optimize its dual-functionality (covalently attaching to pathogen surfaces and interaction with CR2, which are both necessary for the formation B-cell co-receptor complexes. Comparison of the perturbation resistance of the electrostatic character of the homologues of C3d suggests that there was an emergence of a new role of electrostatics, and a transition in the function of C3

Link between D1 and D2 dopamine receptors is reduced in schizophrenia and Huntington diseased brain

International Nuclear Information System (INIS)

Seeman, P.; Niznik, H.B.; Guan, H.C.; Booth, G.; Ulpian, C.

1989-01-01

Dopamine receptor types D 1 and D 2 can oppose enhance each other's actions for electrical, biochemical, and psychomotor effects. The authors report a D 1 -D 2 interaction in homogenized tissue as revealed by ligand binding. D 2 agonists lowered the binding of [ 3 H]raclopride to D 2 receptors in striatal and anterior pituitary tissues. Pretreating the tissue with the D 1 -selective antagonist SCH 23390 prevented the agonist-induced decrease in [ 3 H]raclopride binding to D 2 sites in the striatum but not in the anterior pituitary, which has no D 1 receptors. Conversely, a dopamine-induced reduction in the binding of [ 3 H]SCH 23390 to D 1 receptors could be prevented by the D 2 -selective antagonist eticlopride. Receptor photolabeling experiments confirmed both these D 1 -D 2 interactions. The blocking effect by SCH 23390 was similar to that produced by a nonhydrolyzable guanine nucleotide analogue, and SCH 23390 reduced the number of agonist-labeled D 2 receptors in the high-affinity state. Thus, the D 1 -D 2 link may be mediated by guanine nucleotide-binding protein components. The link may underlie D 1 -D 2 interactions influencing behavior, since the link was missing in over half the postmortem striata from patients with schizophrenia and Huntington disease (both diseases that show some hyperdopamine signs) but was present in human control, Alzheimer, and Parkinson striata

Proposal of development of an advanced IORT system; Proposta di sviluppo di un sistema IORT di avanguardia

Energy Technology Data Exchange (ETDEWEB)

Ronsivalle, C [ENEA, Centro Ricerche Frascati, Rome (Italy). Unita tecnico scientifica Tecnologie Fisiche Avanzate; Casali, F [Bologna Univ., Bologna (Italy), Dipartimento di fisica; Colavita, E [Calabria Univ., Arcavacata (Italy). Diparimento di fisica; Lamanna, E [Magna Graecia Univ., Germaneto (Italy). Dipartimento di medicina sperimentale e clinica

2005-07-15

In the last years there has been an increasing interest on IORT (Intraoperative Radiation Therapy), also because of the development of dedicated accelerators. This technique represents a very effective oncological treatment consisting in delivering a single high dose on a tumour bed soon after surgery resection. In the following we present the proposal of development of a last generation IORT system based on the use of a linear accelerator with variable energy in the range 3-15 MeV, operating in C band (5712 MHz). Respect to the accelerator used in the commercial IORT systems operating at a typical frequency of 2998 MHz (S band) limited to a maximum energy of 12 MeV, the use of a higher RF frequency allows an increase of the maximum energy. This extends the use of the IORT technique to a wider field of tumors and an improvement of the system in terms of compactness and weight reduction. In addition the machine will be provided with a devoted absolute dosimetry system that will strongly simplify the procedures of dosimetric characterization. We intend to develop the system by a collaboration between ENEA, some Universities (Bologna, Catanzaro and Cosenza) and the national industry. [Italian] Negli ultimi anni si e sviluppato un sempre maggior interesse intorno alla Radioterapia IntraOperatoria (IORT), una particolare tecnica radioterapica che permette di irradiare la zona interessata da un tumore durante un intervento chirurgico utilizzando un fascio di elettroni prodotto da un acceleratore. La diffusione di questa metodica e stata favorita dalla presenza sul mercato di acceleratori installabili direttamente in sala operatoria. Come conseguenza di questa disponibilita sono emerse sia nuove esigenze legate alle particolari applicazioni cliniche, sia richieste di facilita di utilizzo e perfomances sempre piu sofisticate. Viene qui presentata una proposta di sviluppo di un sistema IORT di ultima generazione basato sull'utilizzo di un acceleratore lineare con energia

Proposal of development of an advanced IORT system; Proposta di sviluppo di un sistema IORT di avanguardia

Energy Technology Data Exchange (ETDEWEB)

Ronsivalle, C. [ENEA, Centro Ricerche Frascati, Rome (Italy). Unita tecnico scientifica Tecnologie Fisiche Avanzate; Casali, F. [Bologna Univ., Bologna (Italy), Dipartimento di fisica; Colavita, E. [Calabria Univ., Arcavacata (Italy). Diparimento di fisica; Lamanna, E. [Magna Graecia Univ., Germaneto (Italy). Dipartimento di medicina sperimentale e clinica

2005-07-15

In the last years there has been an increasing interest on IORT (Intraoperative Radiation Therapy), also because of the development of dedicated accelerators. This technique represents a very effective oncological treatment consisting in delivering a single high dose on a tumour bed soon after surgery resection. In the following we present the proposal of development of a last generation IORT system based on the use of a linear accelerator with variable energy in the range 3-15 MeV, operating in C band (5712 MHz). Respect to the accelerator used in the commercial IORT systems operating at a typical frequency of 2998 MHz (S band) limited to a maximum energy of 12 MeV, the use of a higher RF frequency allows an increase of the maximum energy. This extends the use of the IORT technique to a wider field of tumors and an improvement of the system in terms of compactness and weight reduction. In addition the machine will be provided with a devoted absolute dosimetry system that will strongly simplify the procedures of dosimetric characterization. We intend to develop the system by a collaboration between ENEA, some Universities (Bologna, Catanzaro and Cosenza) and the national industry. [Italian] Negli ultimi anni si e sviluppato un sempre maggior interesse intorno alla Radioterapia IntraOperatoria (IORT), una particolare tecnica radioterapica che permette di irradiare la zona interessata da un tumore durante un intervento chirurgico utilizzando un fascio di elettroni prodotto da un acceleratore. La diffusione di questa metodica e stata favorita dalla presenza sul mercato di acceleratori installabili direttamente in sala operatoria. Come conseguenza di questa disponibilita sono emerse sia nuove esigenze legate alle particolari applicazioni cliniche, sia richieste di facilita di utilizzo e perfomances sempre piu sofisticate. Viene qui presentata una proposta di sviluppo di un sistema IORT di ultima generazione basato sull'utilizzo di un acceleratore lineare con

Inside Job: Viruses Transfer cGAMP between Cells.

Science.gov (United States)

Gulen, Muhammet F; Ablasser, Andrea

2015-09-09

The DNA sensor, cyclic GMP-AMP synthase (cGAS), is essential for the detection of viral infection. In a recent issue of Science, two studies, Bridgeman et al. (2015) and Gentili et al. (2015), report a novel mechanism for propagating an antiviral signal between cells, based on the transfer of the cGAS enzymatic product, cyclic GMP-AMP (cGAMP), in viral particles. Copyright © 2015 Elsevier Inc. All rights reserved.

Cellular targets of nitric oxide in the hippocampus.

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Katalin Bartus

Full Text Available In the hippocampus, as in many other CNS areas, nitric oxide (NO participates in synaptic plasticity, manifested as changes in pre- and/or postsynaptic function. While it is known that these changes are brought about by cGMP following activation of guanylyl cyclase-coupled NO receptors attempts to locate cGMP by immunocytochemistry in hippocampal slices in response to NO have failed to detect the cGMP elevation where expected, i.e. in the pyramidal neurones. Instead, astrocytes, unidentified varicose fibres and GABA-ergic nerve terminals are reported to be the prominent NO targets, raising the possibility that NO acts indirectly via other cells. We have re-investigated the distribution of cGMP generated in response to endogenous and exogenous NO in hippocampal slices using immunohistochemistry and new conditions designed to optimise cGMP accumulation and, hence, its detectability. The conditions included use of tissue from the developing rat hippocampus, a potent inhibitor of phosphodiesterase-2, and an allosteric enhancer of the NO-receptive guanylyl cyclase. Under these conditions, cGMP was formed in response to endogenous NO and was found in a population of pyramidal cell somata in area CA3 and subiculum as well as in structures described previously. The additional presence of exogenous NO resulted in hippocampal cGMP reaching the highest level recorded for brain tissue (1700 pmol/mg protein and in cGMP immunolabelling throughout the pyramidal cell layer. Populations of axons and interneurones were also stained. According with these results, immunohistochemistry for the common NO receptor β1-subunit indicated widespread expression. A similar staining pattern for the α1-subunit with an antibody used previously in the hippocampus and elsewhere, however, proved to be artefactual. The results indicate that the targets of NO in the hippocampus are more varied and extensive than previous evidence had suggested and, in particular, that the

Pharmacology, pharmacokinetics and metabolism of the dopamine receptor agonist 5-hydroxy-6-methyl-2-di-n-propylaminotetralin (DK-118) in the cat

International Nuclear Information System (INIS)

Koons, J.C.

1985-01-01

The dopamine receptor agonist 5-hydroxy-6-methyl-2-di-n-propylaminotetralin (DK-118) lowers blood pressure, heart rat and inhibits tachycardia induced in cats by electrical stimulation of sympathetic nerves innervating the heart. DK-118, unlike most of its chemically related dopaminergic analogs, exhibits a slow onset of activity suggesting that one or more metabolites of the drug may be responsible for its pharmacologic effects. The purpose of the work described in this thesis was to gain information regarding the possible bioactivation of DK-118 in cats. In one series of experiments, cats were pretreated with inhibitors of drug metabolism, metyrapone or SKF 525-A, and alterations of the pharmacologic effects of DK-118 determined. A high-performance liquid chromatography assay-using electrochemical detection was developed to quantify urine and plasma concentrations of DK-118 in control, metyrapone pretreated and SKF 525-A pretreated cats. Urinary metabolites of [ 14 C]DK-118 were identified employing HPLC, GC/MS and FAB/MS. Pharmacologic activity and receptor binding of selected metabolites were determined. Data presented in this thesis are consistent with the hypothesis that metabolites contribute to some of the pharmacologic effects of DK-118

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.

The repertoire of olfactory C family G protein-coupled receptors in zebrafish: candidate chemosensory receptors for amino acids

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Ngai John

2006-12-01

Full Text Available Abstract Background Vertebrate odorant receptors comprise at least three types of G protein-coupled receptors (GPCRs: the OR, V1R, and V2R/V2R-like receptors, the latter group belonging to the C family of GPCRs. These receptor families are thought to receive chemosensory information from a wide spectrum of odorant and pheromonal cues that influence critical animal behaviors such as feeding, reproduction and other social interactions. Results Using genome database mining and other informatics approaches, we identified and characterized the repertoire of 54 intact "V2R-like" olfactory C family GPCRs in the zebrafish. Phylogenetic analysis – which also included a set of 34 C family GPCRs from fugu – places the fish olfactory receptors in three major groups, which are related to but clearly distinct from other C family GPCRs, including the calcium sensing receptor, metabotropic glutamate receptors, GABA-B receptor, T1R taste receptors, and the major group of V2R vomeronasal receptor families. Interestingly, an analysis of sequence conservation and selective pressure in the zebrafish receptors revealed the retention of a conserved sequence motif previously shown to be required for ligand binding in other amino acid receptors. Conclusion Based on our findings, we propose that the repertoire of zebrafish olfactory C family GPCRs has evolved to allow the detection and discrimination of a spectrum of amino acid and/or amino acid-based compounds, which are potent olfactory cues in fish. Furthermore, as the major groups of fish receptors and mammalian V2R receptors appear to have diverged significantly from a common ancestral gene(s, these receptors likely mediate chemosensation of different classes of chemical structures by their respective organisms.

OAS proteins and cGAS: unifying concepts in sensing and responding to cytosolic nucleic acids.

Science.gov (United States)

Hornung, Veit; Hartmann, Rune; Ablasser, Andrea; Hopfner, Karl-Peter

2014-08-01

Recent discoveries in the field of innate immunity have highlighted the existence of a family of nucleic acid-sensing proteins that have similar structural and functional properties. These include the well-known oligoadenylate synthase (OAS) family proteins and the recently identified OAS homologue cyclic GMP-AMP (cGAMP) synthase (cGAS). The OAS proteins and cGAS are template-independent nucleotidyltransferases that, once activated by double-stranded nucleic acids in the cytosol, produce unique classes of 2'-5'-linked second messenger molecules, which - through distinct mechanisms - have crucial antiviral functions. 2'-5'-linked oligoadenylates limit viral propagation through the activation of the enzyme RNase L, which degrades host and viral RNA, and 2'-5'-linked cGAMP activates downstream signalling pathways to induce de novo antiviral gene expression. In this Progress article, we describe the striking functional and structural similarities between OAS proteins and cGAS, and highlight their roles in antiviral immunity.

[¹¹C]-(+)-PHNO PET imaging of dopamine D(2/3) receptors in Parkinson's disease with impulse control disorders.

Science.gov (United States)

Payer, Doris E; Guttman, Mark; Kish, Stephen J; Tong, Junchao; Strafella, Antonio; Zack, Martin; Adams, John R; Rusjan, Pablo; Houle, Sylvain; Furukawa, Yoshiaki; Wilson, Alan A; Boileau, Isabelle

2015-02-01

Dopamine agonist medications with high affinity for the D3 dopamine receptor are commonly used to treat Parkinson's disease, and have been associated with pathological behaviors categorized under the umbrella of impulse control disorders (ICD). The aim of this study was to investigate whether ICD in Parkinson's patients are associated with greater D3 dopamine receptor availability. We used positron emission tomography (PET) radioligand imaging with the D3 dopamine receptor preferring agonist [¹¹C]-(+)-propyl-hexahydro-naphtho-oxazin (PHNO) in Parkinson's patients with (n = 11) and without (n = 21) ICD, and age-, sex-, and education-matched healthy control subjects (n = 18). Contrary to hypotheses, [¹¹C]-(+)-PHNO binding in D3 -rich brain areas was not elevated in Parkinson's patients with ICD compared with those without; instead, [¹¹C]-(+)-PHNO binding in ventral striatum was 20% lower (P = 0.011), correlating with two measures of ICD severity (r = -0.8 and -0.9), which may reflect higher dopamine tone in ventral striatum. In dorsal striatum, where [¹¹C]-(+)-PHNO binding is associated with D2 receptor levels, [¹¹C]-(+)-PHNO binding was elevated across patients compared with controls. We conclude that although D3 dopamine receptors have been linked to the occurrence of ICD in Parkinson's patients. Our findings do not support the hypothesis that D3 receptor levels are elevated in Parkinson's patients with ICD. We also did not find ICD-related abnormalities in D2 receptor levels. Our findings argue against the possibility that differences in D2/3 receptor levels can account for the development of ICD in PD; however, we cannot rule out that differences in dopamine levels (particularly in ventral striatum) may be involved. © 2015 International Parkinson and Movement Disorder Society.

Direct evaluation of Pseudomonas aeruginosa biofilm mediators in a chronic infection model.

Science.gov (United States)

Byrd, Matthew S; Pang, Bing; Hong, Wenzhou; Waligora, Elizabeth A; Juneau, Richard A; Armbruster, Chelsie E; Weimer, Kristen E D; Murrah, Kyle; Mann, Ethan E; Lu, Haiping; Sprinkle, April; Parsek, Matthew R; Kock, Nancy D; Wozniak, Daniel J; Swords, W Edward

2011-08-01

Biofilms contribute to Pseudomonas aeruginosa persistence in a variety of diseases, including cystic fibrosis, burn wounds, and chronic suppurative otitis media. However, few studies have directly addressed P. aeruginosa biofilms in vivo. We used a chinchilla model of otitis media, which has previously been used to study persistent Streptococcus pneumoniae and Haemophilus influenzae infections, to show that structures formed in vivo are biofilms of bacterial and host origin within a matrix that includes Psl, a P. aeruginosa biofilm polysaccharide. We evaluated three biofilm and/or virulence mediators of P. aeruginosa known to affect biofilm formation in vitro and pathogenesis in vivo--bis-(3',5')-cyclic dimeric GMP (c-di-GMP), flagella, and quorum sensing--in a chinchilla model. We show that c-di-GMP overproduction has a positive impact on bacterial persistence, while quorum sensing increases virulence. We found no difference in persistence attributed to flagella. We conclude from these studies that a chinchilla otitis media model provides a means to evaluate pathogenic mediators of P. aeruginosa and that in vitro phenotypes should be examined in multiple infection systems to fully understand their role in disease.

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.

Vitamin C Deficiency Reduces Muscarinic Receptor Coronary Artery Vasoconstriction and Plasma Tetrahydrobiopterin Concentration in Guinea Pigs

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Gry Freja Skovsted

2017-07-01

Full Text Available Vitamin C (vitC deficiency is associated with increased cardiovascular disease risk, but its specific interplay with arteriolar function is unclear. This study investigates the effect of vitC deficiency in guinea pigs on plasma biopterin status and the vasomotor responses in coronary arteries exposed to vasoconstrictor/-dilator agents. Dunkin Hartley female guinea pigs (n = 32 were randomized to high (1500 mg/kg diet or low (0 to 50 mg/kg diet vitC for 10–12 weeks. At euthanasia, coronary artery segments were dissected and mounted in a wire-myograph. Vasomotor responses to potassium, carbachol, sodium nitroprusside (SNP, U46619, sarafotoxin 6c (S6c and endothelin-1 (ET-1 were recorded. Plasma vitC and tetrahydrobiopterin were measured by HPLC. Plasma vitC status reflected the diets with deficient animals displaying reduced tetrahydrobiopterin. Vasoconstrictor responses to carbachol were significantly decreased in vitC deficient coronary arteries independent of their general vasoconstrictor/vasodilator capacity (p < 0.001. Moreover, in vitC deficient animals, carbachol-induced vasodilator responses correlated with coronary artery diameter (p < 0.001. Inhibition of cyclooxygenases with indomethacin increased carbachol-induced vasoconstriction, suggesting an augmented carbachol-induced release of vasodilator prostanoids. Atropine abolished carbachol-induced vasomotion, supporting a specific muscarinic receptor effect. Arterial responses to SNP, potassium, S6c, U46619 and ET-1 were unaffected by vitC status. The study shows that vitC deficiency decreases tetrahydrobiopterin concentrations and muscarinic receptor mediated contraction in coronary arteries. This attenuated vasoconstrictor response may be linked to altered production of vasoactive arachidonic acid metabolites and reduced muscarinic receptor expression/signaling.

Signaling through cGMP-dependent protein kinase I in the amygdala is critical for auditory-cued fear memory and long-term potentiation.

Science.gov (United States)

Paul, Cindy; Schöberl, Florian; Weinmeister, Pascal; Micale, Vincenzo; Wotjak, Carsten T; Hofmann, Franz; Kleppisch, Thomas

2008-12-24

Long-term potentiation (LTP) of inputs relaying sensory information from cortical and thalamic neurons to principal neurons in the lateral amygdala (LA) is thought to serve as a cellular mechanism for associative fear learning. Nitric oxide (NO), a messenger molecule widely implicated in synaptic plasticity and behavior, has been shown to enhance LTP in the LA as well as consolidation of associative fear memory. Additional evidence suggests that NO-induced enhancement of LTP and amygdala-dependent learning requires signaling through soluble guanylyl cyclase (sGC) and cGMP-dependent protein kinase (cGK). Mammals possess two genes for cGK: the prkg1 gene gives rise to the cGK type I isoforms, cGKIalpha and cGKIbeta, and the prkg2 gene encodes the cGK type II. Reportedly, both cGKI and cGKII are expressed in the amygdala, and cGKII is involved in controlling anxiety-like behavior. Because selective pharmacological tools for individual cGK isoforms are lacking, we used different knock-out mouse models to examine the function of cGKI and cGKII for LTP in the LA and pavlovian fear conditioning. We found robust expression of the cGKI specifically in the LA with cGKIbeta as the prevailing isoform. We further show a marked reduction of LTP at both thalamic and cortical inputs to the LA and a selective impairment of auditory-cued fear memory in cGKI-deficient mutants. In contrast, cGKII null mutants lack these phenotypes. Our data suggest a function of cGKI, likely the beta isoform, in the LA, supporting synaptic plasticity and consolidation of fear memory.

diArk – a resource for eukaryotic genome research

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Kollmar Martin

2007-04-01

Full Text Available Abstract Background The number of completed eukaryotic genome sequences and cDNA projects has increased exponentially in the past few years although most of them have not been published yet. In addition, many microarray analyses yielded thousands of sequenced EST and cDNA clones. For the researcher interested in single gene analyses (from a phylogenetic, a structural biology or other perspective it is therefore important to have up-to-date knowledge about the various resources providing primary data. Description The database is built around 3 central tables: species, sequencing projects and publications. The species table contains commonly and alternatively used scientific names, common names and the complete taxonomic information. For projects the sequence type and links to species project web-sites and species homepages are stored. All publications are linked to projects. The web-interface provides comprehensive search modules with detailed options and three different views of the selected data. We have especially focused on developing an elaborate taxonomic tree search tool that allows the user to instantaneously identify e.g. the closest relative to the organism of interest. Conclusion We have developed a database, called diArk, to store, organize, and present the most relevant information about completed genome projects and EST/cDNA data from eukaryotes. Currently, diArk provides information about 415 eukaryotes, 823 sequencing projects, and 248 publications.

Psicoanalisi ed educazione: il lavoro di Vera Schmidt e di Sabina Spielrein nell’asilo sperimentale di Mosca (1921-1925

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Merete Amann Gainotti

2012-12-01

Full Text Available Negli anni 1921-1923 a Mosca, sulla scia delle profonde trasformazioni politiche e sociali innescate dalla Rivoluzione di ottobre si colloca un esperimento educativo originale, promosso da Vera Schmidt, una pedagogista formata alle idee psicoanalitiche, che si proponeva di cercare nuove vie educative per la prima infanzia sulla base delle recenti conquiste e conoscenze fornite dalla teoria psicoanalitica di S. Freud. Obiettivo di questo contributo è di fornire un breve excursus storico relativo alla diffusione della psicoanalisi in Unione sovietica e alla fondazione dell'asilo sperimentale di Mosca, di ricordare le figure delle due studiose Vera Schmidt e Sabina Spielrein che hanno animato l'iniziativa, il cui lavoro scientifico è rimasto in ombra rispetto a quello dei loro più famosi colleghi S. Freud e di C.G. Jung; infine si intende rendere conto dei principi educativi che esse cercarono di mettere in pratica nell'asilo sperimentale di Mosca.

Aspetti di continuità di lungo periodo dell’architettura di Venezia e osservazione sulla tipicità di alcuni processi di stratificazione

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Trovò, Francesco

2010-12-01

Full Text Available In order to interpret the links between Venice and Byzantium, it is firstly described how earliest houses in Venice were decorated by using sculptures coming from trading places. It is described the use of disassembled elements and the replacement of others, such as cornices, windows’ elements, coats of arms, among others. These were used firstly as a revival (Neo-Byzantine, 15th century, and later as reflection of the Venetian society «prudence», related to its behaviours and traditions. This prudence is mainly shown in the Renaissance. Besides this, other aspects such as the type of building and the façades, which are the main transformed surface, are analysed with the aim of researching periodical and characteristic dynamics, mainly in those façades showing stylistic renewal and new elements. Some external façades have maintained specific and easy observable characteristics, so they can be associated to a determined stylistic and morphological period. Other façades are stratified, as it is shown by the presence of elements (windows, cornices, … of different building phases. After tracing the main stratifying processes in about 40 study-cases, thanks to a macroscopic analysis, some ways of new composition elements and different stratified units are described. According to this, it is clear that the main principle is the searching for symmetry. Finally, some ways of intervention on the façades are described, highlighting some significant cases.[it] Nella prima parte dell’articolo viene descritto il modo di adornare le prime case veneziane con sculture importate dai luoghi del commercio, come ricostruzione del legame fra Venezia e Bisanzio. Si descrive l’uso di disaggregare e riposizionare vari elementi, come cornici, elementi di finestre, stemmi, prima come un iniziale revival, sia esso neobizantino o riferito al XV secolo, poi come manifestazione di «prudenza» della società veneziana, legata ai costumi, alle tradizioni. La

Control of a resonant d.c.-link converter for a.c. motor drives

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Astrid Petterteig

1992-10-01

Full Text Available This paper presents the control of the resonant d.c.-link converter for a.c. motor drives. This is a low loss converter with higher efficiency than a conventional PWM converter, but it requires complex control. It needs a special control of the resonant d.c.-link voltage in addition to the discrete control of the a.c. side currents. Simulations show how the control of the a.c. currents, the modulation principle, influences the overall performance of the converter.

Double di oxygenation by mouse 8S-lipoxygenase: Specific formation of a potent peroxisome proliferator-activated receptor α agonist

International Nuclear Information System (INIS)

Jisaka, Mitsuo; Iwanaga, Chitose; Takahashi, Nobuyuki; Goto, Tsuyoshi; Kawada, Teruo; Yamamoto, Tatsuyuki; Ikeda, Izumi; Nishimura, Kohji; Nagaya, Tsutomu; Fushiki, Tohru; Yokota, Kazushige

2005-01-01

Mouse 8S-lipoxygenase (8-LOX) metabolizes arachidonic acid (AA) specifically to 8S-hydroperoxyeicosatetraenoic acid (8S-HPETE), which will be readily reduced under physiological circumstances to 8S-hydroxyeicosatetraenoic acid (8S-HETE), a natural agonist of peroxisome proliferator-activated receptor α (PPARα). Here, we investigated whether 8-LOX could further oxygenate AA and whether the products could activate PPARs. The purified recombinant 8-LOX converted AA exclusively to 8S-HPETE and then to (8S,15S)-dihydroperoxy-5Z,9E,11Z,13E-eicosatetraenoic acid (8S,15S-diHPETE). The k cat /K m values for 8S-HPETE and AA were 3.3 x 10 3 and 2.7 x 10 4 M -1 s -1 , respectively. 8-LOX also dioxygenated 8S-HETE and 15S-H(P)ETE specifically to the corresponding 8S,15S-disubstituted derivatives. By contrast, 15-LOX-2, a human homologue of 8-LOX, produced 8S,15S-diH(P)ETE from 8S-H(P)ETE but not from AA nor 15S-H(P)ETE. 8S,15S-diHETE activated PPARα more strongly than 8S-HETE did. The present results suggest that 8S,15S-diH(P)ETE as well as 8S-H(P)ETE would contribute to the physiological function of 8-LOX and also that 8-LOX can function as a potential 15-LOX

Interferon-gamma up-regulates a unique set of proteins in human keratinocytes. Molecular cloning and expression of the cDNA encoding the RGD-sequence-containing protein IGUP I-5111

DEFF Research Database (Denmark)

Honoré, B; Leffers, H; Madsen, Peder

1993-01-01

AMP (Bt2cAMP), dibutyryl cGMP (Bt2cGMP)] and compounds known to affect keratinocytes [4 beta-phorbol 12-myristate 13-acetate (PMA), retinoic acid, Ca2+, dexamethasone, lipopolysaccharides, foetal calf serum]. Protein IGUP I-5111 was selected for further studies as its level is affected by simian-virus-40......, which migrated with the AMA variant of keratinocyte protein IEF SSP 5111, is novel although it exhibits weak similarity to cytoskeletal proteins. IGUP I-5111 contains the RGD sequence found in many extracellular glycoprotein ligands of the integrin receptor family and it is found at least partially...... in the culture supernatant. Considering the presence of IFN-gamma in psoriatic plaques as well as its putative involvement in the pathophysiology of the disease it was of interest to determine whether the set of proteins was upregulated in these cells. Two-dimensional gel analysis of the protein phenotype of non-cultured...

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

C-Type Lectin Receptors in Asthma

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Sabelo Hadebe

2018-04-01

Full Text Available Asthma is a heterogeneous disease that affects approximately 300 million people worldwide, largely in developed countries. The etiology of the disease is poorly understood, but is likely to involve specific innate and adaptive responses to inhaled microbial components that are found in allergens. Fungal-derived allergens represent a major contributing factor in the initiation, persistence, exacerbation, and severity of allergic asthma. C-type lectin like receptors, such as dectin-1, dectin-2, DC-specific intercellular adhesion molecule 3-grabbing nonintegrin, and mannose receptor, recognize many fungal-derived allergens and other structurally similar allergens derived from house dust mites (HDM. In some cases, the fungal derived allergens have been structurally and functionally identified alongside their respective receptors in both humans and mice. In this review, we discuss recent understanding on how selected fungal and HDM derived allergens as well as their known or unknown receptors shape allergic airway diseases.

Synthesis of [11C]-ohmefentanyl, a novel, highly potent and selective agonist for opiate μ-receptors

International Nuclear Information System (INIS)

Zhu, Y.C.; Academia Sinica, Shangha, SH; Prenant, C.; Crouzel, C.; Comar, D.; Chi, Z.Q.

1992-01-01

We describe the synthesis of 11 C-ohmefentanyl, a novel, highly potent and selective agonist for opiate μ-receptors, to be visualized by Positron Emission Tomography (PET). The unlabelled cis-A-ohmefentanyl was prepared in a nine-step synthesis and two-step fractional crystallization, and the OH-precursor for [ 11 C]-ohmefentanyl labelling was obtained by hydrolysis of the 4-N-propionyl group of cis-A-ohmefentanyl in 6 N hydrochloric acid. The [ 11 C]-propionyl chloride was prepared by carbonation of ethylmagnesium bromide with cyclotron-produced [ 11 C]-carbon dioxide followed by direct treatment of the intermediate complex with phthaloyl dichloride and 2,6-di-t-butylpyridine. Reaction of the OH-precursor with [ 11 C]-propionyl chloride yields [ 11 C]-ohmefentanyl separated by HPLC, with a high specific activity of 300-400 mCi μmol -1 . The keto-precursor prepared by hydrolysis of the 4-N-propionyl group of cis-10 in 8 N hydrochloric acid, was also used for [ 11 C]-ohmefentanyl labelling. Reaction of the [ 11 C]-propionyl chloride with keto-precursor, followed by addition of sodium borohydride, yields [ 11 C]-ohmefentanyl. The [ 11 C]-labelled ohmefentanyl obtained using the OH-precursor is a cis-A form, while that obtained using the keto-precursor is a mixture of cis-A and cis-B forms. (author)

Sphingosine-1-Phosphate and Its Receptors: A Mutual Link between Blood Coagulation and Inflammation

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Shailaja Mahajan-Thakur

2015-01-01

Full Text Available Sphingosine-1-phosphate (S1P is a versatile lipid signaling molecule and key regulator in vascular inflammation. S1P is secreted by platelets, monocytes, and vascular endothelial and smooth muscle cells. It binds specifically to a family of G-protein-coupled receptors, S1P receptors 1 to 5, resulting in downstream signaling and numerous cellular effects. S1P modulates cell proliferation and migration, and mediates proinflammatory responses and apoptosis. In the vascular barrier, S1P regulates permeability and endothelial reactions and recruitment of monocytes and may modulate atherosclerosis. Only recently has S1P emerged as a critical mediator which directly links the coagulation factor system to vascular inflammation. The multifunctional proteases thrombin and FXa regulate local S1P availability and interact with S1P signaling at multiple levels in various vascular cell types. Differential expression patterns and intracellular signaling pathways of each receptor enable S1P to exert its widespread functions. Although a vast amount of information is available about the functions of S1P and its receptors in the regulation of physiological and pathophysiological conditions, S1P-mediated mechanisms in the vasculature remain to be elucidated. This review summarizes recent findings regarding the role of S1P and its receptors in vascular wall and blood cells, which link the coagulation system to inflammatory responses in the vasculature.

Inverse regulatory coordination of motility and curli-mediated adhesion in Escherichia coli.

Science.gov (United States)

Pesavento, Christina; Becker, Gisela; Sommerfeldt, Nicole; Possling, Alexandra; Tschowri, Natalia; Mehlis, Anika; Hengge, Regine

2008-09-01

During the transition from post-exponential to stationary phase, Escherichia coli changes from the motile-planktonic to the adhesive-sedentary "lifestyle." We demonstrate this transition to be controlled by mutual inhibition of the FlhDC/motility and sigma(S)/adhesion control cascades at two distinct hierarchical levels. At the top level, motility gene expression and the general stress response are inversely coordinated by sigma(70)/sigma(FliA)/sigma(S) competition for core RNA polymerase and the FlhDC-controlled FliZ protein acting as a sigma(S) inhibitor. At a lower level, the signaling molecule bis-(3'-5')-cyclic-diguanosine monophosphate (c-di-GMP) reduces flagellar activity and stimulates transcription of csgD, which encodes an essential activator of adhesive curli fimbriae expression. This c-di-GMP is antagonistically controlled by sigma(S)-regulated GGDEF proteins (mainly YegE) and YhjH, an EAL protein and c-di-GMP phosphodiesterase under FlhDC/FliA control. The switch from motility-based foraging to the general stress response and curli expression requires sigma(S)-modulated down-regulation of expression of the flagellar regulatory cascade as well as proteolysis of the flagellar master regulator FlhDC. Control of YhjH by FlhDC and of YegE by sigma(S) produces a fine-tuned checkpoint system that "unlocks" curli expression only after down-regulation of flagellar gene expression. In summary, these data reveal the logic and sequence of molecular events underlying the motile-to-adhesive "lifestyle" switch in E. coli.

Immunization with Tc52 or its amino terminal domain adjuvanted with c-di-AMP induces Th17+Th1 specific immune responses and confers protection against Trypanosoma cruzi.

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Marina N Matos

2017-02-01

Full Text Available The development of new adjuvants enables fine modulation of the elicited immune responses. Ideally, the use of one or more adjuvants should result in the induction of a protective immune response against the specific pathogen. We have evaluated the immune response and protection against Trypanosoma cruzi infection in mice vaccinated with recombinant Tc52 or its N- and C-terminal domains (NTc52 and CTc52 adjuvanted either with the STING (Stimulator of Interferon Genes agonist cyclic di-AMP (c-di-AMP, a pegylated derivative of α-galactosylceramide (αGC-PEG, or oligodeoxynucleotides containing unmethylated CpG motifs (ODN-CpG. All groups immunized with the recombinant proteins plus adjuvant: Tc52+c-di-AMP, NTc52+c-di-AMP, CTc52+c-di-AMP, NTc52+c-di-AMP+αGC-PEG, NTc52+CpG, developed significantly higher anti-Tc52 IgG titers than controls. Groups immunized with c-di-AMP and Tc52, NTc52 or CTc52 showed the highest Tc52-specific IgA titers in nasal lavages. All groups immunized with the recombinant proteins plus adjuvant developed a strong specific cellular immune response in splenocytes and lymph node cells with significant differences for groups immunized with c-di-AMP and Tc52, NTc52 or CTc52. These groups also showed high levels of Tc52-specific IL-17 and IFN-γ producing cells, while NTc52+CpG group only showed significant difference with control in IFN-γ producing cells. Groups immunized with c-di-AMP and Tc52, NTc52 or CTc52 developed predominantly a Th17 and Th1immune response, whereas for NTc52+CpG it was a dominant Th1 response. It was previously described that αGC-PEG inhibits Th17 differentiation by activating NKT cells. Thus, in this work we have also included a group immunized with both adjuvants (NTc52+c-di-AMP+αGC-PEG with the aim to modulate the Th17 response induced by c-di-AMP. This group showed a significant reduction in the number of Tc52-specific IL-17 producing splenocytes, as compared to the group NTc52+c-di-AMP, which has

Spermidine decreases Na⁺,K⁺-ATPase activity through NMDA receptor and protein kinase G activation in the hippocampus of rats.

Science.gov (United States)

Carvalho, Fabiano B; Mello, Carlos F; Marisco, Patricia C; Tonello, Raquel; Girardi, Bruna A; Ferreira, Juliano; Oliveira, Mauro S; Rubin, Maribel A

2012-06-05

Spermidine is an endogenous polyamine with a polycationic structure present in the central nervous system of mammals. Spermidine regulates biological processes, such as Ca(2+) influx by glutamatergic N-methyl-d-aspartate receptor (NMDA receptor), which has been associated with nitric oxide synthase (NOS) and cGMP/PKG pathway activation and a decrease of Na(+),K(+)-ATPase activity in rats' cerebral cortex synaptosomes. Na(+),K(+)-ATPase establishes Na(+) and K(+) gradients across membranes of excitable cells and by this means maintains membrane potential and controls intracellular pH and volume. However, it has not been defined whether spermidine modulates Na(+),K(+)-ATPase activity in the hippocampus. In this study we investigated whether spermidine alters Na(+),K(+)-ATPase activity in slices of hippocampus from rats, and possible underlying mechanisms. Hippocampal slices and homogenates were incubated with spermidine (0.05-10 μM) for 30 min. Spermidine (0.5 and 1 μM) decreased Na(+),K(+)-ATPase activity in slices, but not in homogenates. MK-801 (100 and 10 μM), a non-competitive antagonist of NMDA receptor, arcaine (0.5μM), an antagonist of the polyamine binding site at the NMDA receptor, and L-NAME (100μM), a NOS inhibitor, prevented the inhibitory effect of spermidine (0.5 μM). ODQ (10 μM), a guanylate cyclase inhibitor, and KT5823 (2 μM), a protein kinase G inhibitor, also prevented the inhibitory effect of spermidine on Na(+),K(+)-ATPase activity. Spermidine (0.5 and 1.0 μM) increased NO(2) plus NO(3) (NOx) levels in slices, and MK-801 (100 μM) and arcaine (0.5 μM) prevented the effect of spermidine (0.5 μM) on the NOx content. These results suggest that spermidine-induced decrease of Na(+),K(+)-ATPase activity involves NMDA receptor/NOS/cGMP/PKG pathway. Copyright © 2012 Elsevier B.V. All rights reserved.

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)

Serotonin 2c receptors in pro-opiomelanocortin neurons regulate energy and glucose homeostasis

Science.gov (United States)

Energy and glucose homeostasis are regulated by central serotonin 2C receptors. These receptors are attractive pharmacological targets for the treatment of obesity; however, the identity of the serotonin 2C receptor-expressing neurons that mediate the effects of serotonin and serotonin 2C receptor a...

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

Inventare l’altro. Forme di pseudo-traduzione nella scrittura di Salvatore Di Giacomo e Luigi Capuana.

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Valentina Fulginiti

2014-05-01

Full Text Available Being an extreme case of fictitious representation of linguistic otherness, pseudo-translation challenges the idea of a fatal and exclusive link between language and national ethos, a fundamental notion in the Nineteenth-century linguistic and literary culture. The present article compares two emblematic cases of pseudo-translation in post-Unification Italian culture: Luigi Capuana’s hoax Un poeta danese (published in 1882 and the earliest short stories published by Salvatore di Giacomo in 1878, mistakenly considered a plagiarizing translation from an uncredited German original. Their use of pseudo-translation is marked by opposite goals of parody and stylistic imitation; however, both authors challenge the fundamental assumption underlying the notion of «ethnicity of language». Pseudo-translation thus becomes a space of linguistic elaboration, complementary to the author’s direct involvement in translating major European works into Italian (such as Ibsen’s masterpiece A House of Dolls, which Capuana translated in 1891, and Edmond de Goncourt’s novel Sœur Philomèle, which Di Giacomo translated in 1892. Translation thus provides a free space for authors to experiment with new expressive solutions and challenge commonplaces about language and identity: such reflection on the limits of language and nations represent a direct contribution to the linguistic unification of Italy. Caso estremo di rappresentazione fittizia del- l’alterità linguistica, la pseudo-traduzione chiama in causa l’idea del legame unico e “fatale” fra lingua e nazione – concetto fondamentale nella cultura linguistica del XIX secolo. L’articolo mette a confronto due casi emblematici di pseudo-traduzione nella cultura meridionale post-unitaria: la beffa letteraria di Luigi Capuana Un poeta danese (1882 e le ‘tedescherie’ di Salvatore di Giacomo, gruppo di novelle pubblicate nel 1878 che molti considerarono (a torto un plagio da ignoto autore tedesco

Design and Discovery of Functionally Selective Serotonin 2C (5-HT2C) Receptor Agonists.

Science.gov (United States)

Cheng, Jianjun; McCorvy, John D; Giguere, Patrick M; Zhu, Hu; Kenakin, Terry; Roth, Bryan L; Kozikowski, Alan P

2016-11-10

On the basis of the structural similarity of our previous 5-HT 2C agonists with the melatonin receptor agonist tasimelteon and the putative biological cross-talk between serotonergic and melatonergic systems, a series of new (2,3-dihydro)benzofuran-based compounds were designed and synthesized. The compounds were evaluated for their selectivity toward 5-HT 2A , 5-HT 2B , and 5-HT 2C receptors in the calcium flux assay with the ultimate goal to generate selective 5-HT 2C agonists. Selected compounds were studied for their functional selectivity by comparing their transduction efficiency at the G protein signaling pathway versus β-arrestin recruitment. The most functionally selective compound (+)-7e produced weak β-arrestin recruitment and also demonstrated less receptor desensitization compared to serotonin in both calcium flux and phosphoinositide (PI) hydrolysis assays. We report for the first time that selective 5-HT 2C agonists possessing weak β-arrestin recruitment can produce distinct receptor desensitization properties.

Small molecule inhibition of cGAS reduces interferon expression in primary macrophages from autoimmune mice.

Science.gov (United States)

Vincent, Jessica; Adura, Carolina; Gao, Pu; Luz, Antonio; Lama, Lodoe; Asano, Yasutomi; Okamoto, Rei; Imaeda, Toshihiro; Aida, Jumpei; Rothamel, Katherine; Gogakos, Tasos; Steinberg, Joshua; Reasoner, Seth; Aso, Kazuyoshi; Tuschl, Thomas; Patel, Dinshaw J; Glickman, J Fraser; Ascano, Manuel

2017-09-29

Cyclic GMP-AMP synthase is essential for innate immunity against infection and cellular damage, serving as a sensor of DNA from pathogens or mislocalized self-DNA. Upon binding double-stranded DNA, cyclic GMP-AMP synthase synthesizes a cyclic dinucleotide that initiates an inflammatory cellular response. Mouse studies that recapitulate causative mutations in the autoimmune disease Aicardi-Goutières syndrome demonstrate that ablating the cyclic GMP-AMP synthase gene abolishes the deleterious phenotype. Here, we report the discovery of a class of cyclic GMP-AMP synthase inhibitors identified by a high-throughput screen. These compounds possess defined structure-activity relationships and we present crystal structures of cyclic GMP-AMP synthase, double-stranded DNA, and inhibitors within the enzymatic active site. We find that a chemically improved member, RU.521, is active and selective in cellular assays of cyclic GMP-AMP synthase-mediated signaling and reduces constitutive expression of interferon in macrophages from a mouse model of Aicardi-Goutières syndrome. RU.521 will be useful toward understanding the biological roles of cyclic GMP-AMP synthase and can serve as a molecular scaffold for development of future autoimmune therapies.Upon DNA binding cyclic GMP-AMP synthase (cGAS) produces a cyclic dinucleotide, which leads to the upregulation of inflammatory genes. Here the authors develop small molecule cGAS inhibitors, functionally characterize them and present the inhibitor and DNA bound cGAS crystal structures, which will facilitate drug development.

The Arabidopsis thaliana proteome harbors undiscovered multi-domain molecules with functional guanylyl cyclase catalytic centers

KAUST Repository

Wong, Aloysius Tze

2013-07-08

Background: Second messengers link external cues to complex physiological responses. One such messenger, 3\\',5\\'-cyclic guanosine monophosphate (cGMP), has been shown to play a key role in many physiological responses in plants. However, in higher plants, guanylyl cyclases (GCs), enzymes that generate cGMP from guanosine-5\\'-triphosphate (GTP) have remained elusive until recently. GC search motifs constructed from the alignment of known GCs catalytic centers form vertebrates and lower eukaryotes have led to the identification of a number of plant GCs that have been characterized in vitro and in vivo.Presentation of the hypothesis.Recently characterized GCs in Arabidopsis thaliana contributed to the development of search parameters that can identify novel candidate GCs in plants. We hypothesize that there are still a substantial number (> 40) of multi-domain molecules with potentially functional GC catalytic centers in plants that remain to be discovered and characterized. Testing the hypothesis. The hypothesis can be tested, firstly, by computational methods constructing 3D models of selected GC candidates using available crystal structures as templates. Homology modeling must include substrate docking that can provide support for the structural feasibility of the GC catalytic centers in those candidates. Secondly, recombinant peptides containing the GC domain need to be tested in in vitro GC assays such as the enzyme-linked immune-sorbent assay (ELISA) and/or in mass spectrometry based cGMP assays. In addition, quantification of in vivo cGMP transients with fluorescent cGMP-reporter assays in wild-type or selected mutants will help to elucidate the biological role of novel GCs.Implications of the hypothesis.If it turns out that plants do harbor a large number of functional GC domains as part of multi-domain enzymes, then major new insights will be gained into the complex signal transduction pathways that link cGMP to fundamental processes such as ion transport

CXC chemokine receptor 4 expression and stromal cell-derived factor-1alpha-induced chemotaxis in CD4+ T lymphocytes are regulated by interleukin-4 and interleukin-10

DEFF Research Database (Denmark)

Jinquan, T; Quan, S; Jacobi, H H

2000-01-01

-10. IL-4 and IL-10 up- or down-regulated CXCR4 mRNA expression in CD4+ T lymphocytes, respectively, as detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Scatchard analysis revealed a type of CXCR4 with affinity (Kd approximately 6.3 nM), and approximately 70....... The regulation of CXCR4 expression in CD4+ T lymphocytes by IL-4 and IL-10 could be blocked by a selective inhibitor of protein kinase (staurosporine) or by a selective inhibitor of cAMP- and cGMP-dependent protein kinase (H-8), indicating that these cytokines regulate CXCR4 on CD4+ T lymphocytes via both c......AMP and cGMP signalling pathways. The fact that cyclosporin A or ionomycin were able to independently change the CXCR4 expression and block the effects of IL-4 and IL-10 on CXCR4 expression implied that the capacity of IL-4 and IL-10 to regulate CXCR4 on CD4+ T lymphocytes is not linked to calcium...

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.

Fc Gamma Receptor 3B (FCGR3Bc.233C>A-rs5030738) Polymorphism Modifies the Protective Effect of Malaria Specific Antibodies in Ghanaian Children

DEFF Research Database (Denmark)

Adu, Bright; Jepsen, Micha Phill Grønholm; Gerds, Thomas A

2014-01-01

Immunoglobulin G (IgG) cross-linking with Fc gamma receptor IIIB (FcγRIIIB) triggers neutrophil degranulation, releasing reactive oxygen species with high levels associated with protection against malaria. The FCGR3B-c.233C>A polymorphism thought to influence the interaction between IgG and Fcγ...

Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor - Gi complex

International Nuclear Information System (INIS)

Rollins, T.E.; Siciliano, S.; Kobayashi, S.; Cianciarulo, D.N.; Bonilla-Argudo, V.; Collier, K.; Springer, M.S.

1991-01-01

The authors have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a K d of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the α and β subunits of G i , respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm their earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects

Synthesis, antimicrobial, anti-biofilm evaluation, and molecular modelling study of new chalcone linked amines derivatives.

Science.gov (United States)

El-Messery, Shahenda M; Habib, El-Sayed E; Al-Rashood, Sarah T A; Hassan, Ghada S

2018-12-01

A series of amide chalcones conjugated with different secondary amines were synthesised and characterised by different spectroscopic techniques 1 H NMR, 13 C NMR, and ESI-MS. They were screened for in vitro antibacterial activity. Compounds 36, 37, 38, 42, and 44 are the most active among the synthesised series exhibiting MIC value of 2.0-10.0 µg/ml against different bacterial strains. Compound 36 was equipotent to the standard drug Ampicillin displaying MBC value of 2.0 µg/ml against the bacterial strain Staphylococcus aureus. The products were screened for anti-biofilm activity. Compounds 36, 37, and 38 exhibited promising anti-biofilm activity with IC 50 value ranges from 2.4 to 8.6 µg. Molecular modelling was performed suggesting parameters of signalling anti-biofilm mechanism. AspB327 HisB340 (arene-arene interaction) and IleB328 amino acid residues seemed of higher importance to inhibit c-di-GMP. Hydrophobicity may be crucial for activity. ADME calculations suggested that compounds 36, 37, and 38 could be used as good orally absorbed anti-biofilm agents.

Electrochemical oxidation of 3,5-di-tert-butylcatechol: Synthesis and characterization of the formed ortho-benzoquinhydrone derivative

International Nuclear Information System (INIS)

Nematollahi, D.; Shayani-Jam, H.

2006-01-01

Electrochemical oxidation of 3,5-di-tert-butylcatechol (1) has been studied in ethanol/water mixtures by means of cyclic voltammetry and controlled-potential coulometry. The results revealed that 3,5-di-tert-butyl-1,2-benenzoquinone (2) derived from oxidation of 3,5-di-tert-butylcatechol participate in noncovalently linked interactions with 1 converted to an ortho-benzoquinhydrone (3). The structure of 3 was characterized by MS, IR, 1 H NMR and 13 C NMR. The 1 H NMR studies reveal that the benzoquinhydrone complex 3 is stabilized by one H-bonding interaction. In this work we derived a novel ortho-bezoquinhydrone type complex (3) based on electrochemical oxidation of 3,5-di-tert-butylcatechol at carbon electrode in an undivided cell

Serotonin 2C receptors in pro-opiomelanocortin neurons regulate energy and glucose homeostasis.

Science.gov (United States)

Berglund, Eric D; Liu, Chen; Sohn, Jong-Woo; Liu, Tiemin; Kim, Mi Hwa; Lee, Charlotte E; Vianna, Claudia R; Williams, Kevin W; Xu, Yong; Elmquist, Joel K

2013-12-01

Energy and glucose homeostasis are regulated by central serotonin 2C receptors. These receptors are attractive pharmacological targets for the treatment of obesity; however, the identity of the serotonin 2C receptor-expressing neurons that mediate the effects of serotonin and serotonin 2C receptor agonists on energy and glucose homeostasis are unknown. Here, we show that mice lacking serotonin 2C receptors (Htr2c) specifically in pro-opiomelanocortin (POMC) neurons had normal body weight but developed glucoregulatory defects including hyperinsulinemia, hyperglucagonemia, hyperglycemia, and insulin resistance. Moreover, these mice did not show anorectic responses to serotonergic agents that suppress appetite and developed hyperphagia and obesity when they were fed a high-fat/high-sugar diet. A requirement of serotonin 2C receptors in POMC neurons for the maintenance of normal energy and glucose homeostasis was further demonstrated when Htr2c loss was induced in POMC neurons in adult mice using a tamoxifen-inducible POMC-cre system. These data demonstrate that serotonin 2C receptor-expressing POMC neurons are required to control energy and glucose homeostasis and implicate POMC neurons as the target for the effect of serotonin 2C receptor agonists on weight-loss induction and improved glycemic control.

Vitamin C deficiency reduces muscarinic receptor coronary artery vasoconstriction and plasma tetrahydrobiopterin concentration in guinea pigs

DEFF Research Database (Denmark)

Skovsted, Gry Freja; Tveden-Nyborg, Pernille; Lindblad, Maiken Marie

2017-01-01

Vitamin C (vitC) deficiency is associated with increased cardiovascular disease risk, but its specific interplay with arteriolar function is unclear. This study investigates the effect of vitC deficiency in guinea pigs on plasma biopterin status and the vasomotor responses in coronary arteries...... exposed to vasoconstrictor/-dilator agents. Dunkin Hartley female guinea pigs (n = 32) were randomized to high (1500 mg/kg diet) or low (0 to 50 mg/kg diet) vitC for 10-12 weeks. At euthanasia, coronary artery segments were dissected and mounted in a wire-myograph. Vasomotor responses to potassium......-1 were unaffected by vitC status. The study shows that vitC deficiency decreases tetrahydrobiopterin concentrations and muscarinic receptor mediated contraction in coronary arteries. This attenuated vasoconstrictor response may be linked to altered production of vasoactive arachidonic acid...